Cancer

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Marijuana Chemical May Fight Brain Cancer

June 14th, 2010 | Author: Brett

Active Component In Marijuana Targets Aggressive Brain Cancer Cells, Study Says

April 1, 2009 — The active chemical in marijuana promotes the death of brain cancer cells by essentially helping them feed upon themselves, researchers in Spain report.

Guillermo Velasco and colleagues at Complutense University in Spain have found that the active ingredient in marijuana, THC, causes brain cancer cells to undergo a process called autophagy. Autophagy is the breakdown of a cell that occurs when the cell essentially self-digests.

The team discovered that cannabinoids such as THC had anticancer effects in mice with human brain cancer cells and people with brain tumors. When mice with the human brain cancer cells received the THC, the tumor growth shrank.

Two patients enrolled in a clinical trial received THC directly to the brain as an experimental treatment for recurrent glioblastoma multiforme, a highly aggressive brain tumor. Biopsies taken before and after treatment helped track their progress. After receiving the THC, there was evidence of increased autophagy activity.

The findings appear in the April 1 issue of the Journal of Clinical Investigation.

The patients did not have any toxic effects from the treatment. Previous studies of THC for the treatment of cancer have also found the therapy to be well tolerated, according to background information in journal article.

Study authors say their findings could lead to new strategies for preventing tumor growth.

Posted in Articles, Cancer, Glioma/Brain Cancer | Tags: brain, Cancer, cannabinoids, cannabis, glioma, medical marijuana, pot, research, weed | No Comments »

Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells

June 14th, 2010 | Author: Brett

María Salazar,^1,² Arkaitz Carracedo,¹ Íñigo J. Salanueva,¹ Sonia Hernández-Tiedra,¹ Mar Lorente,^1,² Ainara Egia,¹ Patricia Vázquez,³ Cristina Blázquez,^1,² Sofía Torres,¹ Stephane García,⁴ Jonathan Nowak,⁴ Gian María Fimia,⁵ Mauro Piacentini,⁵ Francesco Cecconi,⁶ Pier Paolo Pandolfi,⁷ Luis González-Feria,⁸ Juan L. Iovanna,⁴ Manuel Guzmán,^1,² Patricia Boya,³ and Guillermo Velasco^1,²

¹Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain. ²Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain. ³3D Lab (Development, Differentiation, and Degeneration), Department of Cellular and Molecular Physiopathology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain. ⁴INSERM U624, Campus de Luminy, Marseille, France. ⁵National Institute for Infectious Diseases, IRCCS “L. Spallanzani,” Rome, Italy. ⁶Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia and Department of Biology, University of Rome “Tor Vergata,” Rome, Italy. ⁷Cancer Genetics Program, Beth Israel Deaconess Cancer Center and Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA. ⁸Department of Neurosurgery, University Hospital, Tenerife, Spain.

Address correspondence to: Guillermo Velasco, Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, c/ José Antonio Novais s/n, 28040 Madrid, Spain. Phone: 34-913944668; Fax: 34-913944672; E-mail: gvd@bbm1.ucm.es.

Received November 3, 2008; Accepted February 11, 2009.

This article has been cited by other articles in PMC.

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Abstract

Autophagy can promote cell survival or cell death, but the molecular basis underlying its dual role in cancer remains obscure. Here we demonstrate that Δ⁹-tetrahydrocannabinol (THC), the main active component of marijuana, induces human glioma cell death through stimulation of autophagy. Our data indicate that THC induced ceramide accumulation and eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and thereby activated an ER stress response that promoted autophagy via tribbles homolog 3–dependent (TRB3-dependent) inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis. We also showed that autophagy is upstream of apoptosis in cannabinoid-induced human and mouse cancer cell death and that activation of this pathway was necessary for the antitumor action of cannabinoids in vivo. These findings describe a mechanism by which THC can promote the autophagic death of human and mouse cancer cells and provide evidence that cannabinoid administration may be an effective therapeutic strategy for targeting human cancers.

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Introduction

Macro-autophagy, hereafter referred to as “autophagy,” is a highly conserved cellular process in which cytoplasmic materials — including organelles — are sequestered into double-membrane vesicles called autophagosomes and delivered to lysosomes for degradation or recycling (1). In many cellular settings, triggering of autophagy relies on the inhibition of mammalian target of rapamycin complex 1 (mTORC1), an event that promotes the activation (de-inhibition) of several autophagy proteins (Atgs) involved in the initial phase of membrane isolation (1). Enlargement of this complex to form the autophagosome requires the participation of 2 ubiquitin-like conjugation systems. One involves the conjugation of ATG12 to ATG5 and the other of phosphatidylethanolamine to LC3/ATG8 (1). The final outcome of the activation of the autophagy program is highly dependent on the cellular context and the strength and duration of the stress-inducing signals (2–5). Thus, besides its role in cellular homeostasis, autophagy can be a form of programmed cell death, designated “type II programmed cell death,” or play a cytoprotective role, for example in situations of nutrient starvation (6). Accordingly, autophagy has been proposed to play an important role in both tumor progression and promotion of cancer cell death (2–4), although the molecular mechanisms responsible for this dual action of autophagy in cancer have not been elucidated.

Δ⁹-Tetrahydrocannabinol (THC), the main active component of marijuana (7), exerts a wide variety of biological effects by mimicking endogenous substances — the endocannabinoids — that bind to and activate specific cannabinoid receptors (8). One of the most exciting areas of research in the cannabinoid field is the study of the potential application of cannabinoids as antitumoral agents (9). Cannabinoid administration has been found to curb the growth of several types of tumor xenografts in rats and mice (9, 10). Based on this preclinical evidence, a pilot clinical trial has been recently run to investigate the antitumoral action of THC on recurrent gliomas (11). Recent findings have also shown that the pro-apoptotic and tumor growth–inhibiting activity of cannabinoids relies on the upregulation of the transcriptional co-activator p8 (12) and its target the pseudo-kinase tribbles homolog 3 (TRB3) (13). However, the mechanisms that promote the activation of this signaling route as well as the targets downstream of TRB3 that mediate its tumor cell–killing action remain elusive. In this study we found that ER stress–evoked upregulation of the p8/TRB3 pathway induced autophagy via inhibition of the Akt/mTORC1 axis and that activation of autophagy promoted the apoptotic death of tumor cells. The uncovering of this pathway, which we believe is novel, for promoting tumor cell death may have therapeutic implications in the treatment of cancer.

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Results

Autophagy mediates THC-induced cancer cell death.

As a first approach to gain insight into the morphological changes induced in cancer cells by cannabinoid administration, we performed electron microscopy analysis of U87MG human astrocytoma cells. Interestingly, double membrane vacuolar structures with the morphological features of autophagosomes were observed in THC-treated cells (Figure (Figure1,1

, A–C). The conversion of the soluble form of LC3 (LC3-I) to the lipidated and autophagosome-associated form (LC3-II) is considered one of the hallmarks of autophagy (1), and thus we observed the occurrence of LC3-positive dots as well as the appearance of LC3-II (Figure (Figure1D)1

D) in cannabinoid-challenged cells. In addition, co-incubation with the lysosomal protease inhibitors E64d and pepstatin A, which blocks the last steps of autophagic degradation (14), enhanced THC-induced accumulation of LC3-II (Figure (Figure1E),1

E), confirming that cannabinoids induce dynamic autophagy in U87MG cells. Furthermore, incubation with the cannabinoid receptor 1 (CB1) antagonist SR141716 prevented THC-induced LC3 lipidation and formation of LC3 dots (Figure (Figure1D),1

D), indicating that induction of autophagy by cannabinoids relies on CB1 receptor activation.

Figure 1

Inhibition of autophagy prevents THC-induced cancer cell death.

Since autophagy has been implicated in promotion and inhibition of cell survival, we next investigated its participation in the cancer cell death–inducing action of THC. Pharmacological inhibition of autophagy at different levels (Supplemental Figure 1, A–C; supplemental material available online with this article; doi:10.1172/JCI37948DS1) or selective knockdown of ATG1 (an essential protein in the initiation of autophagy; ref. 1) (Figure (Figure1,1

, F and G), ATG5 (an essential protein in the formation of the autophagosome; ref. 1) (Supplemental Figure 1, D–F), or AMBRA1 (a recently identified beclin-1–interacting protein that regulates autophagy; ref. 15) (Supplemental Figure 1, D–F) strongly reduced cannabinoid-induced autophagy and cell death. Moreover, transformed Atg5-deficient mouse embryonic fibroblasts (MEFs), which are defective in autophagy (16), were more resistant than their wild-type counterparts to THC-induced cell death (Figure (Figure1H)1

H) and did not undergo autophagy upon cannabinoid treatment (Figure (Figure1I).1

I). Taken together, these findings demonstrate that autophagy plays a prominent role in THC-induced cancer cell death.

THC induces autophagy via ER stress–dependent upregulation of p8 and TRB3.

In addition to the presence of autophagosomes, electron microscopy analysis of cannabinoid-treated cells revealed the presence of numerous cells with dilated ER (Figure (Figure2A).2

A). In line with this observation, immunostaining of the ER luminal marker protein disulphide isomerase (PDI) showed a striking dilation in the ER of THC-treated U87MG cells (Figure (Figure2B),2

B), an event that was associated with an increased phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), a hallmark of the ER stress response (17) (Figure (Figure2C).2

C). In addition, THC-induced ER dilation and eIF2α phosphorylation were prevented by pharmacological blockade of the CB1 receptor (Figure (Figure2,2

, B and C).

Figure 2

ER stress precedes autophagy in cannabinoid action.

Time-course analysis of PDI and LC3 immunostaining, eIF2α phosphorylation, and LC3 lipidation of cannabinoid-treated cells revealed that ER stress occurred earlier than autophagy (Figure (Figure2,2

, D and E). Of interest, cannabinoid administration produced similar activation of ER stress and autophagy, as well as cell death, in other human astrocytoma cell lines (Supplemental Figure 2, A–F), a primary culture of human glioma cells (Supplemental Figure 2, G–I), and several human cancer cell lines of different origin, including pancreatic cancer (Supplemental Figure 2, J–L), breast cancer, and hepatoma (data not shown). However, neither ER dilation nor eIF2α phosphorylation or autophagy was evident in normal, nontransformed primary astrocytes (Supplemental Figure 3), which are resistant to cannabinoid-induced cell death (13).

We next investigated whether activation of ER stress is involved in the induction of autophagy in response to cannabinoid treatment of cancer cells. We have previously shown that THC-induced accumulation of de novo–synthesized ceramide, an event that occurs in the ER (18), leads to upregulation of the stress-regulated protein p8 and its ER stress–related downstream targets, ATF4, CHOP, and TRB3, to induce cancer cell death (13). Of importance, incubation with ISP-1 (a selective inhibitor of serine palmitoyltransferase, the enzyme that catalyzes the first step of sphingolipid biosynthesis; ref. 18) prevented ceramide accumulation (Supplemental Figure 4A); THC-induced ER dilation (Supplemental Figure 4B); eIF2α phosphorylation (Figure (Figure3A);3

A); p8, ATF4, CHOP, and TRB3 upregulation (Supplemental Figure 4C); and autophagy (Figure (Figure3B),3

B), supporting that ceramide accumulation is involved in cannabinoid-triggered ER stress and autophagy. We also verified by means of RNA interference that CaCMKKβ — which had been previously implicated in activating autophagy in response to ER stress–associated calcium release (19) — was not involved in THC-induced autophagy and cell death (data not shown). As phosphorylation of eIF2α on Ser51 attenuates general protein synthesis while enhancing the expression of several ER stress response genes (17), we used cells derived from eIF2α S51A knockin mice to test whether eIF2α phosphorylation regulates the expression of p8 and its downstream targets. In agreement with this hypothesis, THC treatment (which promoted ceramide accumulation in both wild-type and eIF2α S51A immortalized MEFs; Supplemental Figure 5A) triggered p8, ATF4, CHOP, and TRB3 upregulation (Figure (Figure3C)3

C) as well as autophagy (Supplemental Figure 5B) in wild-type cells but not in their eIF2α S51A counterparts.

Figure 3

THC induces autophagy via ER stress–evoked p8 and TRB3 upregulation.

We subsequently asked whether p8 and its downstream targets regulate autophagy. Knockdown of p8 or TRB3 prevented THC-induced autophagy (Figure (Figure3,3

, D and E) but not ER dilation (Supplemental Figure 4D) in U87MG cells. Furthermore, THC induced autophagy in p8^+/+ but not p8-deficient transformed MEFs (Figure (Figure3F3

F and Supplemental Figure 5C). Altogether, these findings reveal that THC induces autophagy of cancer cells via activation of an ER stress–triggered signaling route that involves stimulation of ceramide synthesis de novo, eIF2α phosphorylation, and p8 and TRB3 upregulation.

THC inhibits Akt and mTORC1 via TRB3.

Inhibition of mTORC1 is considered a key step in the early triggering of autophagy (6). We therefore tested whether cannabinoid-induced upregulation of the p8 pathway leads to autophagy via inhibition of this complex. THC treatment of U87MG cells reduced the phosphorylation of p70S6 kinase (a well-established mTORC1 substrate) and the ribosomal protein S6 (a well-established p70S6 kinase substrate) (Figure (Figure4,4

, A and C), indicating that mTORC1 is inhibited in cannabinoid-challenged cells. In addition, the cannabinoid-induced decrease in p70S6 kinase and S6 phosphorylation, autophagy, and cell death were not evident in Tsc2^–/– cells, in which mTORC1 is constitutively active (20) (Figure (Figure4B4

B and Supplemental Figure 6, A and B), further supporting a major role for mTORC1 inhibition in the induction of autophagic cell death by cannabinoids.

Figure 4

THC inhibits the Akt/mTORC1 pathway via TRB3.

The protein kinase Akt positively regulates the activity of the mTORC1 complex by phosphorylating and inhibiting TSC2 and PRAS40 (a well-established Akt substrate within the mTORC1 complex). Thus, Akt inhibition decreases mTORC1 activity and promotes autophagy (20). In line with this idea, THC decreased the phosphorylation of Akt, TSC2, and PRAS40 as well as p70S6 kinase and S6 (Figure (Figure4C).4

C). This inhibition of the Akt/mTORC1 pathway was abrogated by incubation with a CB1 receptor antagonist (Supplemental Figure 6C) or a ceramide synthesis inhibitor (Supplemental Figure 6D). Likewise, cells overexpressing a myristoylated (constitutively active) form of Akt were resistant to THC-induced mTORC1 inhibition, autophagy, and cell death (Figure (Figure4D4

D and Supplemental Figure 6, E and F), further supporting that THC induces autophagy via Akt inhibition.

Since TRB3 has been shown to directly interact with and inhibit Akt (21, 22), we investigated whether upregulation of TRB3 was responsible for THC-induced Akt/mTORC1 inhibition. Several observations support that this is indeed the case: (a) THC increased the amount of Akt coimmunoprecipitated with TRB3 from U87MG extracts (Figure (Figure4E),4

E), (b) knockdown of TRB3 prevented the effect of THC on Akt, TSC2, PRAS-40, p70S6 kinase, and S6 phosphorylation (Figure (Figure4F),4

F), and (c) TRB3 overexpression decreased Akt, TSC2, PRAS40, p70S6 kinase, and S6 phosphorylation, enhanced the inhibitory effect of THC on the phosphorylation of these proteins, and promoted autophagy (Figure (Figure4G).4

G). In line with these observations, THC failed to inhibit Akt, p70S6 kinase, and S6 phosphorylation of eIF2α S51A knockin or p8-deficient MEFs, in which TRB3 did not become upregulated upon cannabinoid treatment (Figure (Figure4H4

H and Supplemental Figure 6, G and H). Altogether, these data demonstrate that upregulation of p8 and TRB3 induce autophagy of tumor cells via inhibition of the Akt/mTORC1 pathway.

THC-induced autophagy promotes the apoptotic death of cancer cells.

While analyzing the mechanism of cannabinoid cell-killing action, we observed that incubation with the pan-caspase inhibitor ZVAD-fmk prevented cell death to the same extent as genetic (Figure (Figure5A)5

A) or pharmacological (Supplemental Figure 7) inhibition of autophagy. Furthermore, Bax/Bak double knockout (DKO) immortalized MEFs, which are protected against mitochondrial apoptosis (23), were resistant to THC-induced cell death and apoptosis (Figure (Figure5B)5

B) but underwent eIF2α phosphorylation and autophagy (Figure (Figure5C)5

C) upon THC treatment. We therefore investigated whether cannabinoid-induced autophagy promoted the apoptotic death of cancer cells. Time-course analysis of LC3 and active caspase-3 immunostaining in U87MG cells revealed that autophagy preceded the appearance of apoptotic features in THC-treated cells (Figure (Figure5D).5

D). In addition, selective knockdown of ATG1 (Figure (Figure5D)5

D) as well as of AMBRA1 or ATG5 (Supplemental Figure

prevented THC-induced caspase-3 activation. Moreover, unlike their wild-type counterparts, Atg5-deficient immortalized MEFs did not undergo phosphatidylserine translocation to the outer leaflet of the plasma membrane (Figure (Figure5E),5

E), loss of mitochondrial membrane potential (Figure (Figure5F),5

F), or increased production of reactive oxygen species (Supplemental Figure 9) in response to cannabinoid treatment. These findings indicate that activation of the autophagy-mediated cell death pathway occurs upstream of apoptosis in cannabinoid antitumoral action.

Figure 5

Autophagy is upstream of apoptosis in cannabinoid-induced cancer cell death.

Activation of autophagy is necessary for cannabinoid antitumoral action in vivo.

To determine the in vivo relevance of our findings, we first investigated whether THC promotes the activation of the above-described autophagy-mediated cell death pathway in U87MG cell–derived tumor xenografts, in which we have recently shown that cannabinoid treatment reduces tumor growth (specifically, THC administration for 14 days decreased tumor growth by 50%; ref. 13). Analysis of these tumors revealed that cannabinoid administration increases TRB3 expression and decreases S6 phosphorylation (Figure (Figure6A).6

A). Likewise, formation of LC3 dots as well as increase in LC3-II and active caspase-3 immunostaining were observed in THC-treated, but not vehicle-treated, tumors (Figure (Figure6B).6

B).

Figure 6

THC activates the autophagic cell death pathway in vivo.

To further investigate whether activation of the p8 pathway mediates cannabinoid antitumoral action, we also analyzed tumors derived from p8^+/+ and p8^–/– Ras^V12/E1A-transformed MEFs (in this case, THC administration for 8 days decreased by 45% the growth of p8^+/+ tumors but had no significant effect on p8^–/– tumors; ref. 13). THC treatment increased TRB3 expression, decreased S6 phosphorylation, and increased autophagy as well as TUNEL and active caspase-3 immunostaining in p8^+/+ but not p8^–/– tumors (Figure (Figure6C6

C and Supplemental Figure 10). Moreover, THC treatment enhanced the number of cells with LC3 dots and TUNEL-positive nuclei in p8^+/+ but not in p8^–/– tumors (Figure (Figure6C).6

C).

In order to verify the importance of autophagy for cannabinoid antitumoral action, we next generated tumors with Atg5^+/+ and Atg5^–/– Ras^V12/T-large antigen transformed MEFs. THC administration reduced by more than 80% the growth of tumors derived from wild-type cells but had no significant effect on those tumors generated by autophagy-deficient cells (Figure (Figure7A).7

A). Furthermore, cannabinoid administration increased autophagy, TUNEL (Figure (Figure7B),7

B), and active caspase-3 immunostaining (Supplemental Figure 11) in Atg5^+/+ but not Atg5^–/– tumors. Likewise, cannabinoid administration increased the number of cells with LC3 dots and TUNEL-positive nuclei in Atg5^+/+ but not Atg5^–/– tumors (Figure (Figure7B).7

B). Taken together, these findings demonstrate that activation of the autophagy-mediated cell death pathway is indispensable for cannabinoid antitumoral action.

Figure 7

Autophagy is essential for cannabinoid antitumoral action.

Finally, we analyzed the tumors of 2 patients enrolled in a clinical trial aimed at investigating the effect of THC on recurrent glioblastoma multiforme. The patients were subjected to intracranial THC administration, and biopsies were taken before and after the treatment (11). In the 2 patients, cannabinoid inoculation increased TRB3 immunostaining and decreased S6 phosphorylation (Figure (Figure8A).8

A). Interestingly, the number of cells with autophagic phenotype (Figure (Figure8B)8

B) as well as with active caspase-3 immunostaining (Figure (Figure8C)8

C) was increased in the tumor samples obtained after THC treatment. Although these studies were only conducted in specimens from 2 patients, they are in line with the preclinical evidence shown above and suggest that cannabinoid administration might also trigger autophagy-mediated cell death in human tumors.

Figure 8

THC administration promotes autophagy in glioblastomas of 2 patients.

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Discussion

In this study we show that cannabinoids, a new family of potential antitumoral agents, induce autophagy of cancer cells and that this process mediates the cell death–promoting activity of these compounds. Several observations strongly support this idea: (a) THC induced autophagy and cell death in different types of cancer cells but not in nontransformed astrocytes, which are resistant to cannabinoid killing action, (b) pharmacological or genetic inhibition of autophagy prevented THC-induced cell death, (c) autophagy-deficient tumors were resistant to THC growth-inhibiting action, and (d) THC administration activated the autophagic cell death pathway in 3 different models of tumor xenografts as well as in 2 human tumor samples.

Depending on the cellular context and the strength and duration of the triggering stimulus, autophagy is involved in the promotion or inhibition of cancer cell survival (4, 5, 24, 25). However, the molecular bases of this dual role of autophagy in cancer remain unknown. Data presented here demonstrate that induction of autophagy by cannabinoids leads to cancer cell death and identify the signaling route responsible for the activation of this cellular process. Thus, our findings suggest that THC — via activation of the CB1 receptor and stimulation of ceramide synthesis de novo — activates an early ER stress response that leads to increased phosphorylation of eIF2α on Ser51. Experiments performed with eIF2α S51A mutant cells have shown that phosphorylation of this residue, which is known to attenuate general protein translation while enhancing the expression of several genes related with the ER stress response (17), is required for the upregulation of the stress protein p8 and its ER stress–related downstream targets ATF4, CHOP, and TRB3 as well as for the induction of autophagy by cannabinoids. Furthermore, we demonstrate that the upregulation of p8 and TRB3, which has been previously implicated in cannabinoid-evoked cell death (13), is a crucial event in the triggering of autophagy. Ceramide accumulation has been proposed to induce ER stress (26, 27) and autophagy (28), and eIF2α phosphorylation has been implicated in the induction of autophagy in response to different situations (29–31). However, the molecular mechanisms responsible for these actions have not been clarified. Findings presented here now suggest that upregulation of the p8-TRB3 pathway constitutes a mechanism by which de novo–synthesized ceramide and eIF2α phosphorylation promote autophagy, thus identifying what we believe is a novel connection between ER stress and autophagy.

Our data also demonstrate that the autophagy-promoting activity of the p8-regulated pathway is based on its ability to inhibit the Akt/mTORC1 axis. Regulation of mTORC1 largely relies on the activity of the prosurvival kinase Akt, whose inhibition leads to mTORC1 inactivation and, in turn, to autophagy (20). Our findings reveal that THC upregulates TRB3, promoting its interaction with Akt and leading to decreased phosphorylation of this kinase as well as of its direct substrates TSC2 and PRAS40, which triggers mTORC1 inhibition and induction of autophagy. TRB3 has been previously shown to inhibit Akt (21, 22), although the precise contribution of this pseudo-kinase to the regulation of Akt activity in different cellular contexts is unclear (32). Here we demonstrate that TRB3 inhibition of the Akt/mTORC1 axis is essential for cannabinoid-induced autophagy of cancer cells. Moreover, we show that this pathway is essential for cannabinoid antitumoral action. Thus, THC administration leads to TRB3 upregulation, mTORC1 inhibition, induction of autophagy, and reduction of tumor growth in different models of tumor xenografts, but not in p8-deficient tumors that are defective in the upregulation of the p8/TRB3 pathway. Furthermore, activation of this pathway was also evident in 2 glioma patients that had been treated with THC. These results thus uncover a role for TRB3 that may be of great importance in the regulation of cancer cell death.

Autophagy has been proposed to protect from apoptosis, act as an apoptosis-alternative pathway to induce cell death, or act together with apoptosis as a combined mechanism for cell death (6, 33). However, very little is known about the role of the interplay between these 2 cellular processes in the control of tumor growth in response to anticancer agents. Our results now clearly demonstrate that induction of autophagy is involved in the mechanism by which cannabinoids promote the activation of the mitochondrial pro-apoptotic pathway. Thus, neither tumors in which the p8-regulated pathway has been ablated (and in which, therefore, THC treatment does not induce autophagy) nor tumors intrinsically deficient in autophagy undergo apoptosis in response to THC, and so they are resistant to THC antitumoral action. These findings reveal that autophagy is required for the activation of apoptosis in response to cannabinoid treatment in vivo.

It is worth noting that the concentrations of THC used in this study are in the same range as those administered intracranially to the patients in which we observed activation of the autophagy-mediated cell death pathway (11) and could be thus considered clinically relevant. Of interest, intraperitoneal administration of THC to U87MG tumor xenografts produces a similar decrease in tumor growth (that occurs in concert with increased autophagy and apoptosis) to that observed when the cannabinoid is administered peritumorally (our unpublished observations). Considering that no signs of toxicity were observed in the clinical trial patients (11) or in tumor-bearing animals treated intracranially, peritumorally, or intraperitoneally with THC (refs. 34 and 35 and data not shown), and that no overt toxic effects have been reported in other clinical trials of cannabinoid use in cancer patients for various applications (e.g., inhibition of nausea, vomiting, and pain) and using different routes of administration (e.g., oral, oro-mucosal) (9, 36), our findings support that safe, therapeutically efficacious doses of THC may be reached in cancer patients.

In summary, in this study we identify what we believe is a new route that links the ER stress response to the activation of autophagy and promotes the apoptotic death of tumor cells (Figure (Figure7C).7

C). The identification of this pathway will help to understand the molecular events that lead to activation of autophagy-mediated cell death by anticancer drugs and may contribute to the design of new therapeutic strategies for inhibiting tumor growth.

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Methods

Cell culture and viability.

Cortical astrocytes were prepared from 24-hour-old mice as previously described (13). Primary cultures of brain tumor cells were prepared and cultured as described in the Supplemental Methods. U87MG, T98G, U373MG, and MiaPaCa2 cells, p8^+/+ and p8^–/– Ras^V12/E1A MEFs, Atg5^+/+ and Atg5^–/– T-large antigen MEFs (provided by Noboru Mizushima, Tokyo Medical and Dental University, Tokyo, Japan), Bax/Bak wild-type and Bax/Bak DKO T-large antigen MEFs (provided by Luca Scorrano, Dulbecco Telethon Institute, Milan, Italy, and Patrizia Agostinis, Catholic University of Leuven, Leuven, Belgium), eIF2α S51S WT and eIF2α S51A T-large antigen MEFs (provided by Richard Kaufman, University of Michigan, Ann Arbor, Michigan, USA, and Cesar de Haro and Juan J. Berlanga, Centro de Biología Molecular Severo Ochoa, Autonoma University, Madrid, Spain), Tsc2^+/+ and Tsc2^–/– p53^–/– MEFs, empty vector (pBABE) and pBABE-myr-Akt MEFs, and Atg5^+/+ and Atg5^–/– Ras^V12/T-large antigen MEFs were cultured in DMEM containing 10% FBS and transferred to medium containing 0.5% FBS (except Ras^V12/E1A-transformed MEFs, which were transferred to medium containing 2% FBS) 18 h before performing the different treatments. p8^+/+ and p8^–/– Ras^V12/E1A MEFs as well as Atg5^+/+ and Atg5^–/– Ras^V12/T-large antigen MEFs correspond to a polyclonal mix of at least 20 different selected clones. Unless otherwise indicated, THC was used at a final concentration of 5 μM. Cell viability was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] test (Sigma-Aldrich).

Flow cytometry.

Briefly, cells (approximately 5 × 10⁵ cells per assay) were trypsinized, divided in 2 tubes, washed, and collected by centrifugation at 1,500 g for 5 min. One aliquot was incubated for 10 min at 37°C with Annexin V–FITC (BD Biosciences). Propidium iodide (1 μg/ml) was added just before cytofluorometric analysis. The other aliquot was simultaneously labeled with 3,3ι-dihexyloxacarbocyanine iodide (DiOC₆[3], 40 nM; Invitrogen) and hydroethidium (5 μM; Invitrogen) for 10 minutes at 37°C, followed by cytofluorometric analysis. Cells (10,000) were recorded in each analysis. Fluorescence intensity was analyzed in an EPICS XL flow cytometer (Beckman Coulter).

Western blot.

Western blot analysis was performed following standard procedures. A list of the antibodies used can be found in Supplemental Methods. Densitometric analysis was performed with Quantity One software (Bio-Rad).

Transfections.

U87MG cells (75% confluent) were transfected with siRNA duplexes using the DharmaFECT 1 Transfection reagent (Dharmacon). Cells were trypsinized and seeded 24 h after transfection, at a density of 5,000 cells/cm². Transfection efficiency was greater than 70% as monitored with a control fluorescent (red) siRNA (siGLO RISC-Free siRNA; Dharmacon). In immunofluorescence experiments, control and selective siRNAs were used in a 1:5 ratio, and cells with red spots were scored as transfected.

Infections with adenoviral vectors.

U87MG cells (75% confluent) were transduced for 1 h with supernatants obtained from HEK293 cells infected with adenoviral vectors carrying EGFP (provided by Javier G. Castro, Hospital Infantil Universitario Niño Jesús, Madrid, Spain), rat HA-tagged TRB3 (donated by Patrick Iynedjian, University of Geneva, Geneva, Switzerland) (32), or human EGFP-LC3 (provided by Aviva Tolkovsky and Christoph Goemans, University of Cambridge, Cambridge, United Kingdom). Infection efficiency was greater than 80% as determined by EGFP fluorescence.

RNA interference.

Double-stranded RNA duplexes were purchased from Dharmacon. A list of sequences can be found in the Supplemental Methods.

RT-PCR analysis.

RNA was isolated using Trizol Reagent (Invitrogen). cDNA was obtained with Transcriptor Reverse transcriptase (Roche Applied Science). Primers and amplification conditions can be found in the Supplemental Methods.

Real-time quantitative PCR.

cDNA was obtained using Transcriptor (Roche Applied Science). Real-time quantitative PCR assays were performed using the FastStart Universal Probe Master mix with Rox (Roche Applied Science), and probes were obtained from the Universal ProbeLibrary Set (Roche Applied Science). Primer sequences can be found in the Supplemental Methods. Amplifications were run in a 7900 HT-Fast Real-Time PCR System (Applied Biosystems). Each value was adjusted by using 18S RNA levels as a reference.

Immunoprecipitation.

U87MG cells were lysed in HEPES lysis buffer (see Supplemental Methods for buffer composition). Lysate (1–4 mg) was precleared by incubating with 5–20 μl of protein G–Sepharose conjugated to pre-immune IgG. The lysate extracts were then incubated with 5–20 μl of protein G–Sepharose conjugated to 5–20 μg of the anti-TRB3 antibody or pre-immune IgG. TRB3 antibody (aminoterminal end, ab50516; Abcam) was covalently conjugated to protein G–Sepharose using dimethyl pimelimidate. Immunoprecipitations were carried out for 1 h at 4°C on a rotatory wheel. The immunoprecipitates were washed 4 times with HEPES lysis buffer, followed by 2 washes with HEPES kinase buffer. The immunoprecipitates were resuspended in 30 μl of sample buffer (not containing 2-mercaptoethanol) and filtered through a 0.22-μm Spin-X filter, and 2-mercaptoethanol was added to a concentration of 1% (vol/vol). Samples were subjected to electrophoresis and immunoblot analysis.

Ceramide levels.

Ceramide levels were determined as previously described (37).

Confocal laser scanning microscopy.

Standard protocols for immunofluorescence microscopy were used (see Supplemental Methods for the antibodies used). To quantify the percentage of cells with LC3 or PDI dots, at least 200 cells per condition were counted in randomly selected fields. In all cases, only those cells with 4 or more prominent dots of either LC3 or PDI were scored positively.

In vivo treatments.

Tumors derived from U87MG cells and p8^+/+ and p8^–/– MEFs were induced and treated as previously described (13). Tumors derived from Atg5^+/+ or Atg5^–/– Ras^V12/T-large antigen MEFs (see Supplemental Methods for the procedure used to generate these cells) were induced in nude mice by subcutaneous injection of 10⁷ cells in PBS supplemented with 0.1% glucose. Tumors were allowed to grow until an average volume of 200–250 mm³, and animals were assigned randomly to the different groups. At this point, vehicle or THC (15 mg/kg/d) in 100 μl of PBS supplemented with 5 mg/ml BSA was administered daily in a single peritumoral injection. Tumors were measured with an external caliper, and volume was calculated as (4π/3) × (width/2)² × (length/2). All procedures involving animals were performed with the approval of the Complutense University Animal Experimentation Committee according to Spanish official regulations.

Human tumor samples.

Tumor biopsies were obtained from 2 recurrent glioblastoma multiforme patients who had been treated with THC. The characteristics of the patients and the clinical study have been described in detail elsewhere (11). Briefly, THC dissolved in 30 ml of physiological saline solution plus 0.5% (wt/vol) human serum albumin was administered intratumorally to the patients. Patient 1 received a total of 1.46 mg of THC for 30 days, while patient 2 received a total of 1.29 mg of THC for 26 days (it was estimated that doses of 6–10 μM THC were reached at the site of administration; ref. 11). Samples were fixed in formalin, embedded in paraffin, and used for immunomicroscopy.

Immunomicroscopy of tumor samples.

Samples from tumor xenografts were dissected, Tissue-Tek (Sakura) embedded, frozen, and, before the staining procedures were performed, fixed in acetone for 10 min at room temperature. Samples from human tumors were subjected to deparaffinization, rehydration, and antigen retrieval before the staining procedures were performed. Standard protocols for immunofluorescence or immunohistochemistry microscopy were used (see Supplemental Methods). Nuclei were counterstained with TOTO-3 iodide (U87MG and human tumor samples; Invitrogen) or Hoechst 33342 (MEF tumors; Invitrogen). Fluorescence images were acquired using Metamorph-Offline 6.2 software (Universal Imaging) and Zeiss Axioplan 2 Microscope.

TUNEL.

Tumor samples were fixed, blocked, and permeabilized, and TUNEL was performed as previously described (13).

Electron microscopy.

Ultrastructural analysis of vehicle- and THC-treated cells was assessed by conventional embedding in the epoxy-resin EML-812 (Taab Laboratories). Ultrathin (20- to 30-nm-thick) sections of the samples were obtained using a Leica-Reichert-Jung ultramicrotome and then stained with saturated uranyl acetate–lead citrate by standard procedures. Ultrathin sections were analyzed in a JEOL 1200-EX II transmission electron microscope operating at 100 kV.

Statistics.

Statistical analysis was performed by ANOVA with a post-hoc analysis using the Student-Neuman-Keuls test. Differences were considered significant when the P value was less than 0.05.

Supplementary Material

Supplemental data

Click here to view.^{(11M, pdf)}

Acknowledgments

This work was supported by grants from the Spanish Ministry of Education and Science (MEC) (HF2005/0021, to G. Velasco; SAF2006/00918, to M. Guzmán; and BFU2006-00508, to P. Boya), Santander-Complutense PR34/07-15856, to G. Velasco), Comunidad de Madrid (S-SAL/0261/2006, to M. Guzmán), and La Ligue contre le Cancer and Canceropole PACA (to J.L. Iovanna). M. Salazar was the recipient of a fellowship from the MEC. A. Carracedo was the recipient of fellowships from Gobierno Vasco, the Federation of European Biochemical Societies, and the European Molecular Biology Organization. M. Lorente and P. Boya have a Juan de la Cierva and a Ramón y Cajal contract from the MEC, respectively. S. Hernández-Tiedra has a technician contract from the Spanish Ministry of Education and the Fondo Social Europeo. The authors thank Dario Alessi (University of Dundee, Dundee, United Kingdom) for donating anti-PRAS40 antibodies and for technical support for immunoprecipitation experiments; Gemma Fabriàs, Josefina Casas, and Eva Dalmau (Instituto de Investigaciones Químicas y Ambientales, Barcelona, Spain) for analyzing ceramide samples; José Lizcano, José Bayascas, María M. Caffarel, and Patrizia Agostinis for their experimental suggestions; and other members of our laboratory for their continual support.

Footnotes

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: Atg, autophagy protein; eIF2α, eukaryotic translation initiation factor 2α; MEF, mouse embryonic fibroblast; THC, Δ⁹-tetrahydrocannabinol; mTORC1, mammalian target of rapamycin complex 1; PDI, protein disulphide isomerase; TRB3, tribbles homolog 3.

Citation for this article: J. Clin. Invest. 119:1359–1372 (2009). doi:10.1172/JCI37948

Arkaitz Carracedo and Ainara Egia’s present address is: Cancer Genetics Program, Beth Israel Deaconess Cancer Center and Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

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Posted in Articles, Cancer, Glioma/Brain Cancer | Tags: brain, Cancer, cannabinoids, cannabis, glioma, medical marijuana, pot, research, weed | No Comments »

Cannabis-Linked Cell Receptor Might Help Prevent Colon Cancer

June 14th, 2010 | Author: Brett

FRIDAY, Aug. 1 (HealthDay News) — A cannabinoid receptor lying on the surface of cells may help suppress colorectal cancer 2 bing Cannabis Linked Cell Receptor Might Help Prevent Colon Cancer , say U.S. researchers. When the receptor is turned off, tumor growth is switched on.

Cannabinoids are compounds related to the tetrahydrocannabinol (THC) found in the cannabis plant.

It’s already known that the receptor, CB1, plays a role in relieving pain and nausea, elevating mood and stimulating appetite by serving as a docking station for the cannabinoid group of signaling molecules. This study suggests that CB1 may offer a new path for cancer prevention 2 bing Cannabis Linked Cell Receptor Might Help Prevent Colon Cancer or treatment.”We’ve found that CB1 expression is lost in most colorectal cancers, and when that happens, a cancer-promoting protein is free to inhibit cell death,” senior author Dr. Raymond Dubois, provost and executive vice president of the University of Texas M.D. Anderson Cancer Center 2 bing Cannabis Linked Cell Receptor Might Help Prevent Colon Cancer , said in a university news release.

In their study of human colorectal tumor specimens, the researchers also found that the drug decitabine can restore CB1 expression.

In addition, mice that are prone to developing intestinal tumors and also have functioning CB1 receptors developed fewer and smaller tumors when treated with a drug that mimics a cannabinoid receptor ligand, the researchers found. Ligands are molecules that function by binding to specific receptors.

“Potential application of cannabinoids as anti-tumor drugs is an exciting prospect, because cannabinoid agonists (synthetic molecules that mimic the action of natural molecules) are being evaluated now to treat the side effects of chemotherapy and radiation therapy,” DuBois said. “Turning CB1 back on and than treating with a cannabinoid agonist could provide a new approach to colorectal cancer treatment or prevention.”

The study was published in the Aug. 1 issue of the journal Cancer Research.

Posted in Articles, Cancer, Colorectal Cancer | Tags: cannabinoids, cannabis, Colon cancer, medical marijuana, pot, weed | No Comments »

Cannabinoid Receptor Activation Induces Apoptosis through Tumor Necrosis Factor α–Mediated Ceramide De novo Synthesis in Colon Cancer Cells

June 14th, 2010 | Author: Brett

+ Author Affiliations

Authors’ Affiliations: Departments of ¹Medical and Surgical Critical Care, ²Experimental Pathology and Oncology, ³Preclinical and Clinical Pharmacology, ⁴Human Pathology and Oncology, and ⁵Excellence Center for Research, Transfer and High Education De Novo Therapies, University of Florence, Florence, Italy; and ⁶Department of Pharmaceutical Sciences, University of Pisa, Pisa, Italy

Requests for reprints:
Sergio Capaccioli, Dipartimento Patologia e Oncologia Sperimentale Medical School, University of Florence, Viale G.B. Morgagni 85, Firenze 50134, Italy. Phone: 3955-4598208; Fax: 3955-4598900; E-mail: sergio@unifi.it.

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Abstract

Purpose: Cannabinoids have been recently proposed as a new family of potential antitumor agents. The present study was undertaken to investigate the expression of the two cannabinoid receptors, CB₁ and CB₂, in colorectal cancer and to provide new insight into the molecular pathways underlying the apoptotic activity induced by their activation.

Experimental Design: Cannabinoid receptor expression was investigated in both human cancer specimens and in the DLD-1 and HT29 colon cancer cell lines. The effects of the CB₁ agonist arachinodyl-2′-chloroethylamide and the CB₂ agonist N-cyclopentyl-7-methyl-1-(2-morpholin-4-ylethyl)-1,8-naphthyridin-4(1H)-on-3-carboxamide (CB13) on tumor cell apoptosis and ceramide and tumor necrosis factor (TNF)-α production were evaluated. The knockdown of TNF-α mRNA was obtained with the use of selective small interfering RNA.

Results: We show that the CB₁ receptor was mainly expressed in human normal colonic epithelium whereas tumor tissue was strongly positive for the CB₂ receptor. The activation of the CB₁ and, more efficiently, of the CB₂ receptors induced apoptosis and increased ceramide levels in the DLD-1 and HT29 cells. Apoptosis was prevented by the pharmacologic inhibition of ceramide de novo synthesis. The CB₂ agonist CB13 also reduced the growth of DLD-1 cells in a mouse model of colon cancer. The knockdown of TNF-α mRNA abrogated the ceramide increase and, therefore, the apoptotic effect induced by cannabinoid receptor activation.

Conclusions: The present study shows that either CB₁ or CB₂ receptor activation induces apoptosis through ceramide de novo synthesis in colon cancer cells. Our data unveiled, for the first time, that TNF-α acts as a link between cannabinoid receptor activation and ceramide production.

Translational Relevance

The present study shows that the antitumor actions of cannabinoid receptor agonists on colon cancer cells may be exerted either via the CB₁ receptor or, more efficiently, via the CB₂ receptor. The fact that selective targeting of CB₂ receptor results in colorectal tumor growth inhibition is of potential clinical interest for future cannabinoid-based anticancer therapies because the use of CB₂-selective ligands is not linked to the typical marijuana-like psychoactive effects of CB₁ activation. Moreover, we showed that only the CB₂ receptor is expressed by tumor cells in colorectal cancer human specimens and, thus, it is likely that only compounds with high selectivity for this receptor may be effective as anticancer agents in humans. The recent synthesis of new, highly selective CB₂ agonists opens the very attractive clinical possibility of using these compounds as adjuvants to conventional chemotherapeutic regimens for the treatment of colorectal cancer.

Cannabinoids, the active components of Cannabis sativa and their derivates, exert a wide spectrum of central and peripheral actions through the activation of specific receptors that are normally bound by a family of endogenous ligands, the endocannabinoid anandamide and 2-arachidonoylglycerol (1, 2). Two cannabinoid receptors have been characterized and cloned thus far: CB₁ (3) and CB₂ (4). CB₁ receptors are particularly abundant in discrete areas of the brain, where they mediate cannabinoid psychoactivity, and are also expressed in peripheral nerve terminals and various extraneural sites, such as the testis, uterus, eye, and spleen (1–4). In contrast, CB₂ receptors are believed to be almost exclusively expressed in the cells and organs of the immune system and are unrelated to cannabinoid psychoactivity (4). Nevertheless, they have also been recently shown in cells from other origins, especially tumor cells (5, 6).

Experimental evidence has shown that cannabinoid administration can inhibit the growth of several models of tumor xenografts in rats and mice (7–11). This antitumor action of cannabinoids relies on the ability of these drugs to inhibit tumor angiogenesis (12) or directly induce apoptosis or cell cycle arrest in neoplastic cells (7–11). Cannabinoid receptors have been shown to modulate several signaling pathways involved in the control of cell survival, including extracellular signal-regulated kinase (ERK; ref. 13), c-Jun-NH2-kinase (14), p38 mitogen-activated protein kinase (MAPK; ref. 15), and the ceramide pathway (16, 17). These findings point to the potential application of cannabinoids as antitumor agents (5, 6). However, the majority of these studies have been done with the use of psychoactive cannabinoids, in particular δ⁹-tetrahydrocannabinol, the main active component of marijuana, and WIN, 55,212-2, a nonselective synthetic cannabinoid agonist. It would be preferable, instead, to have cannabinoid-based therapeutic strategies that are devoid of typical CB₁ receptor–mediated psychotropic side effects. Hence, the recent synthesis of new, highly selective CB₂ agonists (18) opens the very attractive clinical possibility of targeting this receptor selectively.

Ceramide is a ubiquitous sphingolipid messenger that plays an important role in the control of tumor cell fate (19). CB₁ receptor activation has been shown to induce sphingomyelin hydrolysis and acute ceramide production within minutes in both primary astrocytes (20) and C6 glioma cells (16). The functional coupling of CB₁ receptors to sphingomyelinases might involve different adaptor proteins, one of which is the factor associated with neutral sphingomyelinase activation (FAN). FAN binds to a cytoplasmatic nine-amino-acid motif of the 55-kDa tumor necrosis factor (TNF) receptor, the neutral sphingomyelinase–activating domain, thereby coupling the receptor to sphingomyelin breakdown (21). On the contrary, sustained ceramide accumulation through enhanced de novo synthesis seems to play a major role in CB₂ receptor activation–induced apoptosis in tumor cells, including glioma (8, 16, 22), leukemia (23), and pancreatic cancer (9) cells. However, the precise molecular mechanisms involved in the CB₂ receptor–mediated generation of ceramide are still unknown.

TNF-α is one of the most pleiotropic cytokines acting as a host defense factor in a number of immunologic responses and antitumor activity (24, 25). The deregulation of TNF-α signaling results in a wide spectrum of human diseases, including sepsis, multiple sclerosis, rheumatoid arthritis, and cancer (26). It has been shown that TNF-α exerts a cytostatic or cytotoxic effect in several types of cells, and this effect is mediated, at least in part, by the stimulated production of ceramide (27–29). A large body of evidence has shown that the cannabinoid system is involved in the regulation of the cytokine network (30). However, the effects of cannabinoids on cytokines and, in particular, TNF-α production are often conflicting and can be either stimulatory or inhibitory, depending on the experimental model system and the type of cell investigated. To our knowledge, the influence of cannabinoid receptor activation on TNF-α production in cancer cells has not yet been addressed.

Although it is well known that the endogenous cannabinoid system and cannabinoid receptors regulate gastrointestinal functions, such as gastric emptying, secretion, and intestinal motility (31, 32), few studies have investigated the expression and role of CB₁ and/or CB₂ receptors in normal (33) or neoplastic (15, 34) epithelial cells of the colon in humans. The reported results have shown that both inhibition of proliferation (34) and induction of apoptosis (15) in colon cancer cells are mediated by CB₁ receptor activation whereas little or no activity has been observed for the CB₂ receptor.

The aim of this study was to investigate whether even CB₂ receptor activation is involved in the antitumor action of cannabinoids in colon cancer cells and to identify the possible molecular mechanisms underlying this effect. In particular, we explored the hypothesis that cannabinoid receptor activation might induce a proapoptotic effect through a TNF-α–mediated increase in ceramide production.

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Materials and Methods

Cell culture and drugs. DLD-1, HT29, LoVo, HCT8, SW480, HCA7, and HCT15 colon cancer cells were purchased from Interlab Cell Line Collection. The cells were cultured as previously described (35). The CB₁ receptor agonist arachinodyl-2′-chloroethylamide (ACEA), the CB₁ antagonist AM251, and the CB₂ antagonist AM630 were purchased from Tocris Bioscience. The newly synthesized CB₂ agonist N-cyclopentyl-7-methyl-1-(2-morpholin-4-ylethyl)-1,8-naphthyridin-4(1H)-on-3-carboxamide (CB13) was supplied by C.M. (18). The ceramide synthase inhibitor fumonisin B1 was purchased from Cayman Chemical Co.

Human tumor samples. Tissue samples were obtained from 24 patients (18 males, 6 females; median age: 68.5 y; age range: 58-77 y) who had undergone surgical resections for primary sporadic colorectal adenocarcinoma. All patients were thoroughly informed about the aims of the study and gave their written consent for the investigation in accordance with the ethical guidelines of our University. The tumor distribution was as follows: 10 (41.6%) in the proximal colon, 7 (29.2%) in the distal colon, and 7 (29.2%) in the rectum. The tumors were classified into four stages according to the American Joint Committee on Cancer staging system (36): stage I (T₁-T₂, N₀, M₀; n = 3), stage II (T₃-T₄, N₀, M₀; n = 14), stage III (any T, N_1-2, M₀; n = 4), and stage IV (any T, any N, M₁; n = 3).

Cancer tissue (from the edge of the tumor) and adjacent normal mucosa (at least 10 cm from the tumor) were excised from each surgical specimen. The samples were flash frozen in liquid nitrogen for PCR analysis and frozen at −80°C for Western blot analysis. Other samples were fixed in 4% formaldehyde and embedded in paraffin for immunohistochemical analysis.

Western blot analysis. The total proteins from the tissue samples were obtained as described previously (37). DLD-1, HT29, and LoVo cells were grown to subconfluence and starved for 24 h in 0.1% FCS-supplemented media. After incubation in the absence or presence of drugs, the cells were washed in PBS and lysed with radioimmunoprecipitation assay buffer [0.9% NaCl, 20 mmol/L Tris-HCl (pH 7.6), 0.1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, and 0.01% leupeptin]. The total proteins (70 μg) as evaluated with the use of a bicinchoninic acid protein assay from tissue or cultured cells were subjected to Western blotting and immunoblotting analysis as previously described (35). The loading and transfer of equal amounts of proteins were ascertained by either reblotting the membrane with an antitubulin antibody or staining the membrane with Ponceau S. The primary antibodies used were anti-CB₁ and anti-CB₂ rabbit polyclonal antibody (1:250; Alexis Biochemicals), anti–caspase-3 (1:500; Santa Cruz Biotechnology, Inc.), and antitubulin goat polyclonal antibody (1:1,000; Santa Cruz Biotechnology, Inc.). The binding of each primary antibody was determined by the addition of suitable peroxidase-conjugated secondary antibodies (anti-mouse and anti-rabbit antibodies 1:5,000, and anti-goat antibody 1:10,000; Amersham). Human spleen tissue served as CB₁ and CB₂ positive control. Densitometric analysis was done with the ImageJ software.

Reverse-transcriptase PCR. The total RNA was extracted from the tissue samples and cells with the use of the RNAeasy kit (Qiagen) according to the manufacturer’s instructions. One microgram of total RNA was retrotranscribed to cDNA with the Immprom-II Reverse Transcriptase kit (Promega) and amplified with the use of the GoTaq (Promega) according to the manufacturers’ instructions. Cannabinoid receptor mRNA was detected with the QuantiTect Primer Assay (QT00998823 and QT00012376 for CB₁ and CB₂ receptor, respectively; Qiagen) in nonquantitative assays. For glyceraldehydes-3-phosphate dehydrogenase (GAPDH), the primer sequences were 5′-ACCACCATGGAGAAGGCTGG-3′ (forward) and 5′AAGTTGTCGCTGTGGGTG-3′ (reverse). The size of CB₁, CB₂, and GAPDH reverse transcription-PCR products was 148, 87, and 196 bp, respectively.

Immunofluorescence analysis. The DLD-1 and HT29 cells (2 × 10⁵) were seeded onto glass coverslips (15 × 15 mm). After 24 h, the cells were washed twice with 1 mL of cold PBS and fixed for 20 min in 3.7% paraformaldehyde in PBS. After three washes for 2 min with PBS, the cells were permeabilized with 1 mL 0.25% Triton X-100 in PBS for 5 min at room temperature and washed thrice for 5 min in PBS. All the following treatments were done in the dark. After the staining of nuclei with Hoechst 33258 (blue fluorescence; Sigma-Aldrich) diluted 1:1000 in PBS for 30 min at 37°C, the cells were washed thrice for 5 min with 1 mL PBS at room temperature and incubated in 1 mL of blocking buffer (3% bovine serum albumin, 0.1% Triton X-100 in PBS) for 1 h at room temperature and then incubated with the primary rabbit CB₁ or CB₂ polyclonal antibody (Cayman Chemical Co.) diluted 1:200 in blocking buffer overnight at 4°C. The next day, the cells were washed thrice for 15 min each in washing buffer (0.1% Triton X-100 in PBS) and incubated with the secondary Rodamine-conjugated anti-rabbit antibody (red fluorescence; Santa Cruz Biotechnology, Inc.) diluted 1:800 for 60 min at room temperature, washed thrice with 1 mL of washing buffer for 5 min at room temperature, dried, mounted onto glass slides, and examined with a Nikon Eclipse TE2000-U (Nikon J.P.) confocal microscopy. Confocal images (1024 × 768 pixels) were obtained with the use of an objective lens (magnification, ×63).

Immunohistochemistry. Four-micrometer-thick sections were cut from formalin-fixed and paraffin-embedded tissue blocks and processed as described previously (37). For CB₁/CB₂ receptor detection, the sections were incubated in 10 mmol/L citrate buffer (pH 6) in a microwave oven for 5 min. The sections were blocked in 5% bovine serum albumin in TBS (pH 9) for 1 h before the application of primary antibodies. CB₁ and CB₂ antibodies (Cayman Chemical Co) at 1:200 dilution in TBS (pH 9) were incubated overnight at 4°C. The slices were then washed again in PBS and incubated with secondary antibody (rabbit anti-goat IgG horseradish peroxidase conjugated; Zymed Laboratories) for 25 min. For ceramide detection, the slices were washed in PBS (0.1 mol/L; pH 7.4) and then incubated with the primary antibody (MID 15B4; Sigma-Aldrich) at 1:10 concentration for 18 h overnight at 4°C. The slices were then washed again in PBS and incubated with secondary antibody (anti-mouse IgG peroxidase conjugate 1:300; Sigma-Aldrich) for 30 min. In all cases, the detection of the antibody complex was done with the use of 3,3′-diaminobenzidine tetrahydrochloride–plus kit substrate for horseradish peroxidase (Zymed Laboratories). As a negative control for CB₁, CB₂, and ceramide staining, the tissue sections were treated with normal serum instead of each primary antibody.

The extent of CB₁ and CB₂ staining in tumor samples was recorded through a three-grade system based on the percentage of tumor epithelial cells stained: grade 0 = 1% to 20%, grade 1 = 21% to 70%, and grade 2 = more than 70%. The ceramide staining in tumor xenografts was graded on the basis of the intensity of positive cells: none/weak and moderate/strong.

Caspase-3 activity determination. The activity of caspase-3 was determined with the use of a fluorescent substrate according to the method previously described (37). The determinations were done in quintuplicate. The data were expressed as arbitrary units per milligram of proteins.

Flow cytometric analysis. The cells were seeded at 5 × 10⁵ per well in six-well plates and treated with test drugs. For the determination of apoptosis, they were washed in Annexin binding buffer containing 125 mmol/L NaCl, 10 mmol/L HEPES/NaOH (pH 7.4), and 5 mmol/L CaCl₂. The cells were then stained with the combination of Annexin V FITC and 7-amino-actinomycin D (7-AAD; Beckman Coulter, Inc.). The samples were measured by flow cytometric analysis on a Coulter XL flow cytometer (Coulter XL; Beckman Coulter) with the use of the EXPO 32ADC Software (Beckman Coulter). The determinations were done in triplicate, and the data on apoptotic cells were expressed as percentage of total cells counted.

The ceramide content was determined according to the method previously described (37). The determinations were done in triplicate, and the data were expressed as mean fluorescence intensity of positive stained cells.

Cytotoxic assay. The sulforhodamine-B protein staining assay (Sigma-Aldrich) was used for the measurement of in vitro cytotoxicity. The cells were seeded in 96-microwell plates (10⁵ cells in 200 μL for each well). After 24-h preincubation, increasing concentrations of CB13 were added to the plates, which were incubated for another 48 h. As the end point measurement, the sulforhodamine-B test was done according to the procedure described by Skehan et al. (38). Briefly, the cells were fixed with 80% trichloroacetic acid for 1 h at 4°C and then washed five times with distilled water. The trichloroacetic acid–fixed cells were stained for 30 min with 0.4% sulforhodamine B dissolved in 1% acetic acid. At the end of the staining period, the sulforhodamine B was removed, and the wells were rinsed four times with 1% acetic acid. Bound dye was dissolved with 10 mmol/L unbuffered Tris base (pH 10.5) for 5 min on a shaker. The absorbance was measured at 564 nm with a multilabel plate counter (Wallace Victor2; Perkin-Elmer). The determinations were done in triplicate, and the data were expressed as percentage cell survival compared with vehicle treatment regarded as 100%.

Tumor xenografts. Tumors were induced by s.c. flank injection of 2 × 10⁶ DLD-1 or HT29 cells in PBS supplemented with 0.1% glucose in immunodeficient nude (BALB/c) mice. When the tumors reached an average size of 100 mm³, the animals were assigned randomly to one of the two groups (n = 8 for each group) and injected peritumorally for 12 d with CB13 (2.5 mg/kg/d) or vehicle in 100 μL of PBS supplemented with 5 mg/mL defatted and dialyzed bovine serum albumin. The tumors were measured with an external caliper, and volume was calculated as (4π/3) × (width/2)² × (length/2).

TNF-α measurement. The levels of TNF-α in the DLD-1 and HT29 cells, and in the tumor xenografts were measured with the use of a commercial ELISA kit (Cayman Chemical Co.) according to the protocol provided by the manufacturer. The determinations were done in quadruplicate and were expressed as picograms per microgram of protein.

RNA interference and transfections. DLD-1 and HT29 cells (7 × 10⁵) were seeded onto 6-well plates and, after 24 h, treated with either a small interfering RNA (siRNA; Qiagen) directed against the TNF-α or a siRNA (Qiagen) directed against the GFP gene as a control (40 nmol/L). The TNF-α sequences targeted by siRNA are siRNA 1: 5′-AACCCAAGCTTAGAACTTTAA-3′ and siRNA 2: 5′-CCGACTCAGCGCTGAGATCAA-3′. The GFP sequence targeted by the siRNA is siRNA 3: 5′-CGGCAAGCTGACCCTGAAGTTCAT-3′. Forty-eight hours post siRNA transfection, the supernatant was collected to analyze the basal TNF-α levels, or the cells were treated with test drugs.

Statistical analysis. The caspase-3 activity, flow cytometric determination of cell apoptosis and ceramide synthesis, percentage cell survival, TNF-α production, and tumor volumes in mice were expressed as mean ± SE. The differences in these parameters were compared through the paired-value Wilcoxon test or the Mann-Whitney test as appropriate. The differences in ceramide immunostaining intensity were compared with the use of the χ² test. Statistical analysis was done with the Stata Statistic software (release 5.0; Stata Corp.). All the P resulted from the use of two-sided statistical tests; a P < 0.05 was considered statistically significant.

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Results

Colorectal cancer cells express cannabinoid receptors. First, we determined the expression of cannabinoid receptors in the DLD-1, HT29, and LoVo human colon cancer cells by Western blot analysis. CB₁ and CB₂ cannabinoid receptor proteins were expressed in all three cell lines (Fig. 1A ). Because the HT29 and DLD-1 cells showed, respectively, the highest levels of either CB₁ or CB₂ cannabinoid receptor protein expression (Fig. 1A), subsequent experiments were done on these two cell lines. Reverse transcription-PCR showed detectable levels of mRNA for both CB₁ and CB₂ receptors in the same cells (Fig. 1B) whereas immunofluorescence analysis confirmed their expression at a protein level (Fig. 2A ). CB₁ and CB₂ cannabinoid receptor protein and mRNA were also expressed in both human tumor and normal mucosa specimens (Fig. 1A and B). However, Western blot experiments showed a higher amount of CB₁ receptor protein in the normal mucosa than in the tumor tissue in 20 paired specimens (Fig. 1A). In 4 patients, very low levels of CB₁ protein were found in both normal and neoplastic specimens. Unlike CB₁, the expression of CB₂ had increased in 22 of the 24 tumor specimens when compared with paired normal mucosa (Fig. 1A). No CB₂ protein expression was noted in two paired normal mucosa and tumor specimens. Immunohistochemistry confirmed this different epithelial expression profile of the two cannabinoid receptors; CB₁ receptor was mainly expressed by absorptive crypt epithelium in normal mucosa whereas its immunoreactivity was very slight in neoplastic epithelial cells: 19 tumors (79.1%) were grade 0, 4 (16.7%) were grade 1, and 1 (4.2%) was grade 2 (Fig. 2B). On the contrary, CB₂ staining was weak in normal colon epithelium whereas it was intense in colorectal cancer cells: 5 tumors (20.8%) were grade 0, 12 (50.0%) were grade 1, and 7 (29.2%) were grade 2 (Fig. 2B). CB₁ and CB₂ positive staining was also found, respectively, in the subepithelial smooth muscle cells and the subepithelial interstitial cells, most likely macrophages (Fig. 2B). The extent of CB₁ and CB₂ receptor expression in the tumor samples did not significantly vary with respect to the tumor site or stage.

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Fig. 1.

Expression of cannabinoid receptors in colon cancer cells and human specimens. A, Western blot analysis. Expression of CB₁ and CB₂ receptor in the LoVo, DLD-1, and HT29 colon cancer cells, and in representative paired adenocarcinoma and adjacent normal mucosa from two patients. The samples were normalized for protein loading (70 μg) by reblotting the membrane-bound protein with an antitubulin antibody. The densitometric histograms of relative band intensities are shown. Human spleen was used as a positive control for CB₁ and CB₂ receptor expression. N, normal mucosa; T, tumor; kDa, kilodaltons. B, qualitative reverse transcription-PCR. Expression of CB₁ and CB₂ receptor mRNA in the DLD-1 and HT29 colon cancer cells (top), and in representative paired adenocarcinoma and adjacent normal mucosa from two patients (middle). The housekeeping gene GAPDH was used as an internal control (bottom). RNase-treated samples were used as negative controls. N, normal mucosa; T, tumor.

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Fig. 2.

Expression of cannabinoid receptors in colon cancer cells and human specimens. A, immunofluorescence analysis of CB₁ and CB₂ receptor expression in the DLD-1 and HT29 colon cancer cells. Representative (×63) images of cannabinoid receptor–stained colon cancer cells. Red, cannabinoid receptors; blue, nuclei. B, immunohistochemical analysis of CB₁ and CB₂ receptors in two representative paired human cancer specimens and adjacent normal colon mucosa. Hematoxylin counterstain. Original magnification, ×200.

Cannabinoid receptor agonists induce apoptosis in colon cancer cells. We tested the functionality of cannabinoid receptors in the control of colon cancer cell growth by using the synthetic cannabinoid CB₁ agonist ACEA and the CB₂ agonist CB13. We first evaluated whether ACEA and CB13 were involved in inducing the early events of the apoptotic process (i.e., caspase-3 activation). The treatment of both DLD-1 and HT29 cells with 100 nmol/L ACEA and CB13 resulted in a decrease in procaspase-3 levels (Supplementary Fig. S1) and, as a consequence, in a significant increase in caspase-3 activation (Fig. 3A ). Flow cytometric detection of cell apoptosis confirmed these findings; the treatment of both DLD-1 and HT29 cells with either ACEA or CB13 produced a significant increase in the number of apoptotic cells when compared with vehicle treatment (Fig. 3B). In both experiments, CB13 administration was significantly more efficient than that of ACEA (Fig. 3A and B). ACEA-mediated and CB13-mediated apoptosis were prevented by the administration of the CB₁ antagonist AM251 or the CB₂ antagonist AM630, respectively, thus indicating the receptor specificity of the two types of response (Fig. 3A and B).

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Fig. 3.

Effects of 100 nmol/L ACEA, 100 nmol/L CB13, ACEA + 100 nmol/L AM251, CB13 + 100 nmol/L AM630, ACEA + 10 μmol/L FB1 and CB13 + FB1 on caspase-3 activity (A) and cell apoptosis (B) in the DLD-1 and HT29 colon cancer cells transfected with either control or TNF-α–selective siRNA (siRNA TNF-α). Columns, mean of three different experiments; bars, SE. *, significant increase compared with vehicle treatment (P < 0.05); #, significant decrease compared with treatment with ACEA or CB13 (P < 0.05); §, significantly different compared with treatment with ACEA (P < 0.05). FB1, fumonisin B1.

Cytotoxic effect of CB₂ agonist in colon cancer cells. To investigate whether CB13 could have a growth inhibitory effect on other colon cancer cells, HCT8, SW480, HCA7, and HCT15 cells were treated with increasing concentrations of the compound. The four cell lines have been shown to express the CB₂ receptor in a previously published study (15). The sulforhodamine-B protein staining assay was used to determine cytotoxicity. The results show that the growth of all cell lines was inhibited in a dose-dependent manner. CB13 had similar cytotoxic effects against the cell lines tested with significant loss of viability at concentrations >50 nmol/L (Supplementary Fig. S2).

Antitumor effect of CB₂ agonist in colon cancer models in vivo. To confirm the novel findings of CB₂-mediated proapoptotic effects in vitro, we investigated the antitumor activity of CB13 in vivo. We first generated tumor xenografts by s.c. injection of either DLD-1 or HT29 cells in immunodeficient mice. Western blot analysis showed that tumors obtained with DLD-1 cells showed a higher expression of CB₂ receptor than those obtained with HT29 (Fig. 4A ). Therefore, we tested the tumor growth inhibitory effect of CB13 on DLD-1 colon cancer models. As shown in Fig. 4B and C, peritumoral treatment with CB13 significantly reduced the growth of the established colon tumors.

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Fig. 4.

In vivo antitumor activity of CB13. A, Western blot analysis. Expression of CB₂ receptor in tumors generated by s.c. injection of either DLD-1 (tumors 1, 11, and 12) or HT29 (tumors 4, 5, and 11) colon cancer cells in BALB/c mice. The samples were normalized for protein loading (70 μg) by reblotting the membrane-bound protein with an antitubulin antibody. The densitometric histograms of relative band intensities are shown. Columns, mean of three different experiments; bars, SE. B, tumors generated by s.c. injection of DLD-1 cells in BALB/c mice. The animals were treated with either vehicle or CB13 (2.5 mg/kg/d) for up to 12 d (n = 8 for each experimental group). The tumor volume was monitored during the treatment. *, significantly different from vehicle-treated tumors at the corresponding day of treatment (P < 0.05). C, photographs of a representative vehicle-treated and CB13-treated tumor.

De novo synthesized ceramide mediates the proapoptotic effect induced by cannabinoid receptor activation. As shown in Fig. 5A and Supplementary Fig. S3A and B, incubation with ACEA or CB13 led to ceramide accumulation in the DLD-1 and HT29 cells, and this effect was prevented by the administration of the ceramide synthase inhibitor fumonisin B1. The ceramide increase was significantly higher after CB13 treatment than ACEA treatment. Moreover, treatment with fumonisin B1 prevented the ACEA/CB13-mediated procaspase-3 decrease (Supplementary Fig. S1), activation of caspase-3 (Fig. 3A), and induction of apoptosis (Fig. 3B). We also investigated the immunohistochemical expression of ceramide in tumors generated in mice. The administration of CB13 increased ceramide expression in tumor epithelial cells when compared with treatment with only vehicle (Fig. 5B). Altogether these findings indicate that the de novo synthesized ceramide is involved in CB₁/CB₂ receptor–induced apoptosis in colon cancer cells.

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Fig. 5.

Ceramide production. A, variations in ceramide production as determined by flow cytometry, after treatment with 100 nmol/L ACEA, 100 nmol/L CB13, ACEA + 10 μmol/L FB1 and CB13 + FB1 in the DLD-1 and HT29 cells transfected with either control or TNF-α–selective siRNA (siRNA TNF-α). Columns, mean of three determinations of ceramide-positive cells given in mean fluorescence intensity of stained cells; bars, SE. *, significant increase compared with vehicle treatment (P < 0.05); #, significant decrease compared with ACEA or CB13 treatment (P < 0.05); §, significantly different compared with treatment with ACEA (P < 0.05). B, ceramide immunostaining intensity in tumors generated by s.c. injection of DLD-1 cells in BALB/c mice after treatment with either vehicle or CB13 (left) and representative images of ceramide immunostaining in tumor xenografts treated with either vehicle (none/weak intensity) or CB13 (moderate/strong intensity; right). Hematoxylin counterstain. Original magnification, ×200.

Cannabinoid receptor activation stimulates TNF-α production. The administration of either 100 nmol/L ACEA or CB13 determined a significant increase in TNF-α synthesis in both DLD-1 and HT29 cells (Fig. 6A ). The TNF-α induction was maximal after 48 hours of treatment with the agonists. The TNF-α levels were significantly higher after treatment with CB13 than with ACEA (Fig. 6A). The administration of either the CB₁ antagonist AM251 or the CB₂ antagonist AM630 prevented the TNF-α increase induced by ACEA or CB13, respectively (Fig. 6A). In tumor xenografts, the TNF-α levels were significantly higher in neoplastic nodules treated with CB13 than those treated with vehicle (Fig. 6B).

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Fig. 6.

TNF-α measurement. A, variations in TNF-α concentration in the DLD-1 and HT29 cells after 48 h of treatment with 100 nmol/L ACEA, 100 nmol/L CB13, ACEA + 100 nmol/L AM251 and CB13 + 100 nmol/L AM360. Columns, mean of five determinations; bars, SE. *, significant increase compared with vehicle treatment (P < 0.05); #, significant decrease compared with ACEA or CB13 treatment (P < 0.05); §, significantly different compared with treatment with ACEA (P < 0.05). B, variations in TNF-α concentration in tumors generated by s.c. injection of DLD-1 cells in BALB/c mice after treatment with CB13 (2.5 mg/kg/d). Columns, mean of five determinations; bars, SE. *, significant increase compared with vehicle treatment (P < 0.05). C, control of TNF-α knockdown after 48 h of treatment with CB13. Columns, mean of four determinations; bars, SE; #, significant decrease compared with control cells (P < 0.05).

TNF-α mediates the increase in ceramide production induced by cannabinoid receptor activation. We tested the involvement of TNF-α in mediating the stimulation of ceramide synthesis induced by CB₁ and CB₂ agonists. The knockdown of TNF-α mRNA was obtained by transfecting cancer cells with a TNF-α–selective siRNA. The prevention of the CB13-stimulated increase in TNF-α levels in TNF-α–selective siRNA-transfected cells confirmed the knockdown of TNF-α mRNA after 48 hours of treatment (Fig. 6C). The lack of TNF-α function prevented the increase in ceramide production induced by ACEA or CB13 (Fig. 5A; Supplementary Fig. S3A and B). The administration of fumonisin B1 had no effect on the production of ceramide in the DLD-1 and HT29 cells transfected with TNF-α–selective siRNA and treated with CB₁/CB₂ receptor agonists (Fig. 5A). This clearly means that TNF-α is the main mediator of ceramide de novo synthesis following cannabinoid receptor activation in the DLD-1 and HT29 colon cancer cells. As a consequence, the lack of TNF-α function in colon cancer cells prevented the ACEA/CB13-mediated activation of caspase-3 (Fig. 3A) and induction of apoptosis (Fig. 3B).

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Discussion

There is growing evidence that cannabinoids may selectively target tumor cells by the activation of their membrane receptors, CB₁ and CB₂. However, the mechanisms underlying the antitumor effects of this activation are still not well understood, and experimental data suggest that these effects may be cell-type specific. The regulation of the RAS–mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and the phosphatidylinositol 3-kinase–AKT pathways and the stimulation of ceramide synthesis are among the mechanisms proposed to explain the antitumor effects of cannabinoids in different types of human cancer (reviewed in refs. 5, 6). Indeed, these pathways have been reported to be differently triggered depending on the tumor cell type investigated. In the present study, we report that both CB₁ and CB₂ cannabinoid receptor activation induces apoptosis in colon cancer cells, and this is mediated by the de novo synthesis of ceramide. Interestingly, we show for the first time that signaling through CB₁/CB₂ receptor increases ceramide production via a mechanism that involves TNF-α.

To our knowledge, only two previously published studies have investigated the expression of cannabinoid receptors in colorectal cancer and their involvement in mediating the antitumor effect of either the endocannabinoids anandamide/2-arachidonoylglycerol (34) or δ⁹-tetrahydrocannabinol (15). Ligresti et al. (34) found significant levels of CB₁ and CB₂ mRNA and protein expression in both normal and colorectal cancer tissue and showed that the antiproliferative effect of anandamide and 2-arachidonoylglycerol in the Caco-2 and DLD-1 colon cancer cells is mediated by the activation of the CB₁ receptor. However, an antiproliferative role, even for CB₂ receptors in the DLD-1 cells, which express both cannabinoid receptors, has been proposed in the same study. Recently, Greenhough et al. (15) showed that δ⁹-tetrahydrocannabinol can induce apoptosis in colorectal cancer cells by selective targeting of the CB₁ receptor through a mechanism involving the inhibition of the RAS-MAPK/ERK and phosphatidylinositol 3-kinase (P13K)-AKT cell survival pathways and the increased expression of the proapoptotic protein BAD. These experiments were done on the SW480 colon cancer cell line, which expressed equal levels of CB₁ and CB₂ receptor expression. However, δ⁹-tetrahydrocannabinol has been shown to behave as a partial agonist at both CB₁ and CB₂ receptors, with less efficacy at the CB₂ ones (2). Therefore, these findings cannot be considered definitive and do not exclude a possible antitumor activity of highly selective CB₂ receptor ligands.

In the present study, we confirm that both human colorectal cancer specimens and the corresponding normal colonic mucosa express CB₁ and CB₂ receptors at the mRNA and protein levels as reported in previously published studies (15, 34). However, Western blot and immunohistochemical analyses showed a different expression pattern of the two receptors in either normal or neoplastic colon tissue. Indeed, the CB₁ receptor was mainly expressed by normal epithelial cells whereas CB₂ immunoreactivity was strongly positive in tumor epithelium. Because an anti-inflammatory role has been suggested for CB₂ receptor overexpression in colonic epithelium during the acute phase of inflammatory bowel diseases (33), it might be hypothesized that the induction of this receptor may represent a defensive mechanism of the colonic epithelium under pathologic conditions, such as chronic inflammation or malignant transformation. These data have to be taken into account when considering potential therapeutic applications of cannabinoids because only compounds with high selectivity for CB₂ receptor are likely to be effective as anticancer agents in humans.

To investigate the possible effects of CB₁ and CB₂ receptor activation on the inhibition of colon cancer growth, we used highly selective receptor ligands. This permits to maximally reduce any interference due to receptor-independent activities previously reported for both endogenous and natural cannabinoids (39). Moreover, we used these drugs at nanomolar concentrations, which are most likely comparable with those potentially detected in human serum after drug treatment, whereas natural cannabinoids such as δ⁹-tetrahydrocannabinol have been shown to induce cell death only at concentrations of 2.5 μM and above (15, 40). We tested for the first time on tumor cells a newly synthesized CB₂ agonist, CB13, which has been shown to have higher CB₂ selectivity than the commercially available CB₂ agonists (18). We found that ACEA and, more efficiently, CB13 administration to DLD-1 and HT29 cells could induce a significant increase in caspase-3 activity and the number of tumor apoptotic cells. Moreover, we showed a significant cytotoxic effect of CB13 in four other colon cancer cells, HCT8, SW480, HCA7, and HCT15. To the best of our knowledge, this is the first report that signaling through the CB₂ receptor plays a major role in inducing apoptosis in colon cancer cells and exerts a remarkable growth-inhibiting effect in models of colon cancer in vivo. Previous observations have shown that the CB₂ receptor is involved in the antitumor effect of cannabinoids in other types of human tumors, such as gliomas (8), skin carcinomas (10), lymphomas (41), and pancreatic cancer (9). Altogether, these data may be of potential clinical importance because a new therapeutic approach to cancer might be on the basis of the use of nonpsychoactive cannabinoid ligands (i.e., compounds without typical marijuana-like collateral effects due to CB₁ activation).

Ceramide is a well known proapoptotic lipid which has been shown to act as a second messenger of cannabinoid action (16, 17). CB₁ receptor activation induces ceramide accumulation in primary astrocytes and glioma cells through both sphingomyelin hydrolysis and ceramide de novo synthesis, thus playing an important role in regulating cell fate in neural disease and malignancy (16, 17). Recently, even CB₂ receptor activation has been shown to induce cell apoptosis through the stimulation of ceramide de novo synthesis in a number of human tumors, such as glioma (8, 16, 22), leukemia (23), and pancreatic cancer (9). In the present study we show that both CB₁ and CB₂ receptor activation stimulated ceramide synthesis in colon cancer cells, and its abrogation by the ceramide synthase inhibitor fumonisin B1 prevented the induction of apoptosis that occurred after the administration of ACEA and CB13. Moreover, ceramide expression was significantly higher in tumor xenografts treated with CB13 than those treated with vehicle. Therefore, these data point to ceramide as an important mediator of antitumor activity of cannabinoids even in colorectal cancer.

Some of the downstream targets of ceramide involved in cannabinoid-induced apoptosis have been recently identified. Carracedo et al. (9, 22) have shown that cannabinoid-induced ceramide synthesis in glioma and pancreatic cancer cells led to cell apoptosis through the up-regulation of the stress-regulation protein p8 and the endoplasmic reticulum stress-related genes ATF-4 and TRB3. On the contrary, little is known about the signaling pathways underlying the promotion of ceramide synthesis through cannabinoid receptor activation. Experimental studies have provided evidence for the pivotal role of ceramide in transmitting some of the functional responses induced by TNF-α in adipocytes and neural cells (27, 28, 42). Moreover, it has been shown that the cannabinoid system can modulate TNF-α production, with either suppressing (43, 44) or stimulating effects (45–48), depending on the type of cells investigated. In particular, the CB₁ receptor antagonist rimonabant can decrease the level of TNF-α in hepatic (45) and intestinal cells (46) whereas the agonist-specific stimulation of the CB₂ receptor has been shown to trigger the up-regulation of TNF-α mRNA in the promyelocytic cell line HL-60 (47) and in monocytes treated with Echinacea alkylamides (48). In the present study, we show for the first time that CB₁ and, more efficiently, CB₂ receptor activation induced TNF-α production in colon cancer cells and tumors generated in mice. Importantly, the knockdown of TNF-α function in the same cells abrogates the synthesis of ceramide and, consequently, the effects induced by CB₁ and CB₂ agonists on the induction of apoptosis. Therefore, TNF-α most likely plays a key role in the initialization of the antitumor activity induced by cannabinoid receptor activation through the induction of ceramide de novo synthesis.

In conclusion, the present study shows that the activation of the CB₁ and, more efficiently, of the CB₂ receptors exerts apoptotic effects in colorectal cancer. The lipid second messenger ceramide seems to play a major role in this process. Our data have unveiled, for the first time, that TNF-α acts as a link between cannabinoid receptor activation and ceramide production. The fact that selective targeting of CB₂ receptor results in colorectal tumor growth inhibition is of potential clinical interest for future cannabinoid-based anticancer therapies because the use of CB₂-selective ligands is not linked to the typical marijuana-like psychoactive effects of CB₁ activation.

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Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

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Footnotes

Grant support: Italian Ministry of University, Scientific and Technological Research, the Ente Cassa di Risparmio di Firenze, and the Associazione Italiana Ricerca sul Cancro.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
- Accepted July 28, 2008.
- Received March 27, 2008.
- Revision received July 25, 2008.

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Inhibition of Cancer Cell Invasion by Cannabinoids via Increased Expression of Tissue Inhibitor of Matrix Metalloproteinases-1

June 14th, 2010 | Author: Brett

Robert Ramer, Burkhard Hinz Affiliation of authors: Institute of Toxicology and Pharmacology, University of Rostock, Rostock, Germany

Correspondence to: Burkhard Hinz, PhD, Institute of Toxicology and Pharmacology, University of Rostock, Schillingallee 70, Rostock D-18057, Germany (e-mail: burkhard.hinz@med.uni-rostock.de).

	ABSTRACT

Top Abstract Context and Caveats Materials and Methods Results Role of cb1, cb2,… Discussion Funding References

Background: Cannabinoids, in addition to having palliative benefits in cancertherapy, have been associated with anticarcinogenic effects.Although the antiproliferative activities of cannabinoids havebeen intensively investigated, little is known about their effectson tumor invasion.

Methods: Matrigel-coated and uncoated Boyden chambers were used to quantifyinvasiveness and migration, respectively, of human cervicalcancer (HeLa) cells that had been treated with cannabinoids(the stable anandamide analog R(+)-methanandamide [MA] and thephytocannabinoid ${Delta}$ ⁹-tetrahydrocannabinol [THC]) in the presenceor absence of antagonists of the CB₁ or CB₂ cannabinoid receptorsor of transient receptor potential vanilloid 1 (TRPV1) or inhibitorsof p38 or p42/44 mitogen–activated protein kinase (MAPK)pathways. Reverse transcriptase–polymerase chain reaction(RT-PCR) and immunoblotting were used to assess the influenceof cannabinoids on the expression of matrix metalloproteinases(MMPs) and endogenous tissue inhibitors of MMPs (TIMPs). Therole of TIMP-1 in the anti-invasive action of cannabinoids wasanalyzed by transfecting HeLa, human cervical carcinoma (C33A),or human lung carcinoma cells (A549) cells with siRNA targetingTIMP-1. All statistical tests were two-sided.

Results: Without modifying migration, MA and THC caused a time- and concentration-dependentsuppression of HeLa cell invasion through Matrigel that wasaccompanied by increased expression of TIMP-1. At the lowestconcentrations tested, MA (0.1 µM) and THC (0.01 µM)led to a decrease in invasion (normalized to that observed withvehicle-treated cells) of 61.5% (95% CI = 38.7% to 84.3%, P< .001) and 68.1% (95% CI = 31.5% to 104.8%, P = .0039),respectively. The stimulation of TIMP-1 expression and suppressionof cell invasion were reversed by pretreatment of cells withantagonists to CB₁ or CB₂ receptors, with inhibitors of MAPKs,or, in the case of MA, with an antagonist to TRPV1. Knockdownof cannabinoid-induced TIMP-1 expression by siRNA led to a reversalof the cannabinoid-elicited decrease in tumor cell invasivenessin HeLa, A549, and C33A cells.

Conclusion: Increased expression of TIMP-1 mediates an anti-invasive effectof cannabinoids. Cannabinoids may therefore offer a therapeuticoption in the treatment of highly invasive cancers.

	CONTEXT AND CAVEATS

Top Abstract Context and Caveats Materials and Methods Results Role of cb1, cb2,… Discussion Funding References

Prior knowledgeTreatment with cannabinoids had been shownto reduce the invasiveness of cancer cells, but the cellularmechanisms underlying this effect were unclear.

Study design

Cancercells treated with combinations of cannabinoids, antagonistsof cannabinoid receptors, and siRNA to tissue inhibitor of matrixmetalloproteinases-1 (TIMP-1) were assessed for invasiveness,protein expression, and activation of signal transduction pathways.

Contribution

Theexpression of TIMP-1 was shown to be stimulated by cannabinoidreceptor activation and to mediate the anti-invasive effectof cannabinoids.

Implications

Clarification of the mechanismof cannabinoid action may help investigators to explore theirtherapeutic benefit.

Limitations

The relevance of the findingsto the behavior of tumor cells in vivo remains to be determined.

Although cannabinoids are currently used to palliate wasting,emesis, and pain in cancer patients, there is increasing evidenceto suggest that these compounds may be useful for the inhibitionof tumor cell growth through their modulation of several cellsurvival pathways [for review see Bifulco et al. (1)]. For example,in animals, cannabinoid administration has been shown to induceregression of lung adenocarcinomas (2), gliomas (3,4), thyroidepitheliomas (5), lymphomas (6), and skin carcinomas (7). Furthermore,several in vitro studies have confirmed proapoptotic and antiproliferativeeffects of cannabinoids on cancer cells by mechanisms that involvede novo synthesis of ceramide (3,8) and/or activation of mitogen-activatedprotein kinases (MAPKs) (3,9). Moreover, antiangiogenic effectssuch as cannabinoid-attenuated expression of vascular endothelialgrowth factor have been described (10). The majority of theseand other cannabinoid effects are mediated by two G_i/o protein-coupledreceptors, CB₁ and CB₂. There are also experimental data thatsuggest a stimulatory effect of the endocannabinoid anandamideon transient receptor potential vanilloid 1 (TRPV1), a nonselectivecation channel (11,12). However, several cannabinoid effects,including induction of apoptosis and cell death in several celltypes (8,13,14), release of arachidonic acid and intracellularcalcium (15), stimulation of MAPKs (16,17), and inhibition ofinterleukin 2 release (18) have been associated with molecularevents that are independent of either CB₁/CB₂ or TRPV1 activation.

Although the mechanisms underlying the proapoptotic and antiproliferativeactions of cannabinoids have been studied extensively, thereare only a few reports of anti-invasive properties of thesecompounds (7,19,20), and the mechanism that leads to decreasedinvasiveness of cancer cells exposed to cannabinoids has notbeen clarified. Cancer cell invasion is one of the crucial eventsin local spreading, growth, and metastasis of tumors. Matrixmetalloproteinases (MMPs) have emerged as a group of enzymesthat exert an important function during tumor invasion, thatis, degradation of extracellular matrix components such as collagensand proteoglycans (21,22). Tissue inhibitors of MMPs (TIMPs)have been shown to inhibit the proteolytic activity of tumortissues by binding noncovalently with 1:1 stoichiometry to theactive forms of these enzymes and thereby inhibiting proteolyticactivity. Among the four distinct members of the TIMP family,the 28.5-kDa glycoprotein TIMP-1 has been demonstrated to bea potent MMP inhibitor that suppresses vascular tumor growthand angiogenesis in xenographic animal models (23). Furthermore,several studies have demonstrated a correlation between highcancer invasiveness and decreased TIMP-1 expression (24,25).Consistent with this finding, the anti-invasive action of severalanticarcinogenic substances has been associated with elevatedTIMP-1 levels (26–30).

To better understand the mechanism by which cannabinoids exertinhibitory effects on cancer progression, we studied the effectof the hydrolysis-stable endocannabinoid analog R(+)-methanandamide(MA) and the plant-derived cannabinoid ${Delta}$ ⁹-tetrahydrocannabinol(THC) on the expression of TIMP-1 and cancer cell invasiveness.In view of recent studies demonstrating p38 and p42/44 MAPKactivation as intracellular signaling events that lead to inductionof TIMP-1 (31,32) and findings showing cannabinoid receptor–dependentactivation of MAPKs (3,9,33), we also assessed a possible roleof both MAPKs in cannabinoid-modulated invasion and TIMP-1 expression.

	Materials and Methods

Top Abstract Context and Caveats Materials and Methods Results Role of cb1, cb2,… Discussion Funding References

Materials

MA was purchased from Calbiochem (Bad Soden, Germany), AM-251,AM-630, capsazepine, PD98059, and SB203580 from Alexis DeutschlandGmbH (Grünberg, Germany), and THC from Sigma (Steinheim,Germany). Dulbecco’s Modified Eagle’s medium (DMEM) with 4 mML-glutamine and 4.5 g/L glucose was from Cambrex Bio ScienceVerviers S.p.r.l. (Verviers, Belgium). Fetal calf serum (FCS)and penicillin-streptomycin were obtained from PAN Biotech (Aidenbach,Germany) and Invitrogen (Karlsruhe, Germany), respectively.

Cell Culture

The highly invasive cervical cancer cell line HeLa (12,34) aswell as additional human cervical (C33A) and lung carcinoma(A549) cell lines were used to study the anti-invasive actionof cannabinoids. HeLa, C33A, and A549 were maintained in DMEMsupplemented with 10% heat-inactivated FCS, 100 U/mL penicillin,and 100 µg/mL streptomycin. The cells were grown in ahumidified incubator at 37°C and 5% CO₂. All incubationswere performed in serum-free medium. Phosphate-buffered salinewas used as a vehicle for the tested substances with a finalconcentration of 0.1% (v/v) ethanol (for MA und THC) or 0.1%(v/v) dimethyl sulfoxide (DMSO) (for AM-251, AM-630, capsazepine,PD98059, and SB203580).

Matrigel Invasion and Migration Assays

The effect of test substances on the invasiveness of cells wasdetermined using a modified Boyden chamber technique with Matrigel-coatedmembranes according to the manufacturer’s instructions (BD Biosciences,Oxford, UK). In this assay, tumor cells must overcome a reconstitutedbasement membrane by a sequential process of proteolytic degradationof the substrate and active migration. In brief, the upper sidesof the transwell inserts (8 µm pore size) were coatedwith 28.4 µg Matrigel per insert in 24-well plates. Trypsinizedand pelleted cells were suspended to a final concentration of5 x 10⁵ cells in 500 µL serum-free DMEM in each insertand treated with MA and THC or ethanol vehicle for various times.To address the role of cannabinoid receptors, TRPV1, and MAPKsp38 and p42/44, specific antagonists (AM-251, AM-630, capsazepine,PD98059, SB203580) were tested vs DMSO vehicles. DMEM containing10% FCS was used as a chemoattractant in the companion plate.Following incubation in a humidified incubator at 37°C and5% CO₂ for the indicated times, the noninvading cells on theupper surface of the inserts were removed with a cotton swab,and the viability of the cells on the lower surface was measuredby the colorimetric WST-1 test (Roche Diagnostics, Mannheim,Germany). This cell viability test is based on the cleavageof the tetrazolium salt WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1.6-benzenedisulfonate) by mitochondrial succinate-tetrazolium-reductasein metabolically active cells. For calculation of migration,the viability of cells on the lower side of uncoated invasionchambers was determined by the WST-1 test. Invasion was expressedas the invasion index, which is calculated as the absorbanceat 490 nm of cells that invaded through Matrigel-coated Boydenchambers divided by absorbance of cells that migrated throughuncoated control inserts with equal treatment ([invasion/migration]x 100%). For corroboration of the calculated invasion indices,HeLa cells that had invaded through Matrigel-coated membraneswere fixed and stained with Diff-Quick (Medion Diagnostics GmbH,Düdingen, Switzerland) and visualized using a microscopeat x200 magnification.

To exclude the possibility that the effect of cannabinoids oninvasion was an unspecific cytotoxicity-related phenomenon,cell viability was analyzed after cannabinoid exposure in quadruplicate.For this purpose, cells were seeded into 48-well plates at 5x 10⁵ cells per well to match conditions of invasion assaysor 2.5 x 10⁵, 1 x 10⁵, 0.5 x 10⁵, and 0.1 x 10⁵ cells per wellfor testing lower cell densities in a volume of 500 µLDMEM per well and treated with 10 µM MA and 1 µMTHC or ethanol vehicles for 72 hours. Viability was measuredsubsequently using the WST-1 test.

Quantitative Reverse Transcriptase–Polymerase Chain Reaction Analysis

HeLa cells were seeded into 48-well plates at a density of 5x 10⁵ cells per well. Following incubation of cells with therespective test compounds or their vehicles for the indicatedtimes, supernatants were removed, and cells were lysed as previouslydescribed (17) for subsequent RNA isolation using the RNeasytotal RNA Kit (Qiagen GmbH, Hilden, Germany). β-actin (internalstandard) and TIMP-1 mRNA levels were determined by quantitativereal-time RT-PCR as described (35). Primers and probe for humanTIMP-1 was an Assay-on-demand product (Applied Biosystems, Darmstadt,Germany).

Western Blot Analysis

For determination of TIMP-1, MMP-2, TIMP-2, and MMP-9 proteinlevels, cells grown to confluence in 6-well plates were incubatedwith test substances or vehicles for the indicated times. Afterward,cell culture supernatants were centrifuged and concentratedusing Microcon YM-10 centrifugal filter units (Millipore GmbH,Schwalbach, Germany) with a 10-kDa cutoff as described (35).In some instances, TIMP-1 was determined in supernatants collectedfrom the upper Boyden chambers. Total protein was measured usingthe bicinchoninic acid assay (Pierce, Rockford, IL).

For Western blot analysis of p38, phospho-p38, p42/44, and phospho-p42/44,cells that had been grown to confluence in 6-well plates wereincubated with test substance or vehicle for the indicated times.Afterward, cells were washed, harvested, lysed in solubilizationbuffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v)Triton X-100, 10% (v/v) glycerol, 1 mM phenylmethylsulfonylfluoride, 1 µg/mL leupeptin, and 10 µg/mL aprotinin),homogenized by sonication, and centrifuged at 10000 x g for5 minutes. Supernatants were used for Western blot analysis.

All proteins were separated on a 12% sodium dodecyl sulfate-polyacrylamidegel. Following transfer to nitrocellulose and blocking of themembranes with 5% milk powder, blots were probed with specificantibodies raised to TIMP-1, MMP-2, TIMP-2, MMP-9 (diluted with1% milk powder to 1:1000 for TIMP-1, MMP-2, TIMP-2 and 1:500for MMP-9; all antibodies from Oncogene Research Products, SanDiego, CA) or p38, phospho-p38, p42/44, and phospho-p42/44 (dilutedwith 5% milk powder to 1:500 for p38 and phospho-p38 and 1:1000for p42/44 and phospho-p42/44; all antibodies from New EnglandBioLabs GmbH, Frankfurt, Germany). Membranes were probed withhorseradish peroxidase–conjugated Fab-specific anti-mouseIgG for detection of TIMPs and MMPs (diluted with 1% milk powderto 1:1000) or anti-rabbit IgG for analysis of MAPKs (dilutedwith 5% milk powder to 1:1000; both antibodies from New EnglandBioLabs GmbH). Antibody binding was visualized by enhanced chemiluminescencewestern blotting detection reagents (Amersham Biosciences, Freiburg,Germany).

To ensure that equal amounts of protein in cell culture supernatantsused for protein analysis of TIMPs and MMPs had been transferredto the membrane, proteins on Western blot membranes were stainedwith the fluorescent dye Roti-Green (Carl Roth, Karlsruhe, Germany).As a corresponding standard, a band with a size of about 65kDa that appeared unregulated was chosen as a loading controlfor protein analysis of supernatants. Nonphosphorylated MAPKbands were chosen as loading control for MAPK activation. Vehiclecontrols were defined as 100% for evaluation of changes in proteinexpression. Densitometric analysis of all protein band intensities(normalized to respective loading controls) was performed usingan optical scanner and the Multi-Analyst program, version 1.1(Bio-Rad Laboratories, Hercules, CA).

For determination of cellular levels of cannabinoid receptorand TRPV1, membrane fractions of proteins were obtained as describedpreviously (17). The blots were probed with antibodies raisedto the CB₁ receptor (Becton Dickenson GmbH, Heidelberg, Germany),CB₂ receptor (Calbiochem), or TRPV1 (Chemicon International,Temecula, CA) (all diluted with 1% milk powder to 1:1000). Subsequently,membranes were probed with anti-rabbit IgG (diluted 1:1000 with1% milk powder).

SiRNA Transfections

HeLa, C33A, and A549 cells were transfected with siRNA targetingthe indicated sequences using RNAiFect as the transfection reagent(Qiagen GmbH, Hilden, Germany) or negative control RNA (Eurogentec,Seraing, Belgium; Cat. No. OR-0030-neg). The target sequencesof siRNAs (Qiagen GmbH) were as follows: 5′-tcccatctttcttccggacaa-3′for TIMP-1, 5′-acccatttacacctacaccaa-3′ for MMP-2, and 5′-aacctttgagggcgacctcaa-3′for MMP-9. A BLAST search revealed that the sequences selecteddid not show any homology to other known human genes. Transfectionswere performed according to the manufacturer’s instructions.For invasion assays, cells grown to confluence were transfectedwith 0.25 or 1 µg/mL siRNA or nonsilencing siRNA as negativecontrol with an equal ratio (w/v) of RNA to transfection reagentfor 24 hours in DMEM supplemented with 10% FCS. Subsequently,cells were treated with trypsin for 3 minutes at 37°C ina humidified incubator, centrifuged at 200 x g, resuspendedto a final density of 5 x 10⁵ cells in 500 µL of serum-freeDMEM containing the same amounts of siRNA or nonsilencing siRNAto provide constant transfection conditions, and seeded forinvasion analysis as described above.

Statistical Analyses

Differences in invasion, migration, mRNA levels, protein levels,and viability between groups were analyzed with a two-sidedunpaired Student’s t test by use of GraphPad Prism 3.00 (GraphPadSoftware, San Diego, CA). Results were considered to be statisticallysignificant at P < .05.

	Results

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Time Course and Concentration Dependence of the Inhibitory Effect of Cannabinoids on HeLa Cell Invasion

HeLa cells incubated with the cannabinoids MA (10 µM)or THC (1 µM) showed diminished invasion through a reconstitutedbasement membrane (Matrigel) after 24 hours, and invasivenesswas diminished further after 72 hours incubation (Fig. 1, A).Moreover, MA and THC treatment led to statistically significantand concentration-dependent decreases of invasion through Matrigeleven at concentrations as low as 0.1 µM for MA (decreasein invasion index relative to that of vehicle-treated cells= 61.5%, 95% CI = 38.7% to 84.3%, P < .001) and 0.01 µMfor THC (decrease = 68.1%, 95% CI = 31.5% to 104.8%, P = .0039[Fig. 1, B]).

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Fig. 1. Influence of cannabinoids on HeLa cell invasion. Cell invasion indices were analyzed as previously described (30). Briefly, cells that were able to pass through the Matrigel-coated (invasion) or uncoated (migration) membranes toward the chemoattractant (10% FCS) in the companion plate were quantified using the WST-1 test after removal of cells at the upper part of the transwell chambers. The invasion index was calculated as the absorbance at 490 nm of cells that invaded through Matrigel-coated Boyden chambers divided by absorbance of cells that migrated through uncoated control inserts with equal treatment ([invasion/migration] x 100%). Percent control represents comparison with vehicle-treated cells (100%) in the absence of test substance. A) Time dependence of invasion (left) and migration (right) after treatment of cells with R(+)-methanandamide (MA) and ${Delta}$ ⁹-tetrahydrocannabinol (THC). Cells were incubated with cannabinoids (10 µM MA or 1 µM THC) or vehicle for the indicated times. P values for difference in invasion (cannabinoid-treated cells vs vehicle-treated control) were .554, .418, .030, .002, and *P<.001 for MA after 4, 12, 24, 48, 72 hours, respectively, and .060 (after 4 hours), .704 (after 12 hours), and *P<.001 (after 24, 48, 72 hours) for THC (Student’s t test). P values for migration were .931, .174, .172, .234, .314 for MA and .253, .116, .255, .368, 1.00 for THC after 4, 12, 24, 48, 72 hours, respectively, vs corresponding vehicle control. B) Concentration-dependence of the anti-invasive action of MA (0.1, 1, or 10 µM; left graph) and THC (0.01, 0.1, or 1 µM; right graph) after a 72-hour incubation. P = .0039 for 0.01 µM THC and P = .0015 for 0.1 µM THC, *P<.001, vs corresponding vehicle control (Student’s t test). C) Effect of a 1-hour pretreatment with the CB₁ antagonist AM-251 (1 µM), the CB₂ antagonist AM-630 (1 µM), or the TRPV1 antagonist capsazepine (Capsa; 1 µM) on invasiveness of cells that were treated with MA (10 µM) or THC (1 µM) for 72 hours. P<.001 for MA- or THC-treated vs vehicle controls, P = .0019 for MA + AM-251 vs MA, *P<.001 for cannabinoid and AM-251–or AM-630–treated vs cannabinoid alone (Student’s t test). Values are means with the upper or lower 95% confidence interval (error bar) from n = 4 (A, B), n = 16 (C, left), or n = 12 (C, right) independent experiments.

Reduced invasion was not associated with decreased migrationthrough membranes that were not coated with Matrigel (Fig. 1, A).To rule out the possibility that decreased Matrigel invasionby cells that were treated with cannabinoids was an unspecificcytotoxicity-related phenomenon, cellular viability was measuredfollowing exposure to 10 µM MA or 1 µM THC underexperimental conditions of the invasion assays (5 x 10⁵ cellsper well of a 48-well plate in 500 µL serum-free DMEM,72-hour incubation). Incubation with MA or THC at these concentrationshad no statistically significant effect on viability (percentageof viable MA-treated to viable vehicle-treated cells = 104.4%,95% CI = 94.4% to 114.5%, P = .553, and percentage of viableTHC-treated to viable vehicle-treated cells = 105.9, 95% CI= 102.4% to 109.4%, P = .391).However, toxic effects of both cannabinoids were observed whenlower cell densities were used in viability assays. The percentageof viable cells after treatment with 10 µM MA to viablecells after treatment with vehicle for 72 hours was 81.1% (95%CI = 46.1% to 116.1%, P = .117) when 2.5 x 10⁵ cells were seededper well, 76.1% (95% CI = 60.2% to 92.0%, P = .012) for 1 x10⁵ cells per well, 69.4% (95% CI = 63.0% to 75.8%, P = .002)for 0.5 x 10⁵ cells per well, and 17.4% (95% CI = 11.0% to 23.8%,P < .001) for 0.1 x 10⁵ cells per well. Similarly, decreasingcell density was associated with increased cell death by 1 µMTHC with viabilities relative to controls (100%) of 106.4% (95%CI = 93.7% to 119.1%, P = .202) for 2.5 x 10⁵ cells per well,68.2% (95% CI = 55.5% to 80.9%, P < .001) for 1 x 10⁵ cellsper well, 73.1% (95% CI = 57.2% to 89.0%, P = .002) for 0.5x 10⁵ cells per well, and 32.0% (95% CI = 19.3% to 44.7%, P< .001) for 0.1 x 10⁵ cells per well (all groups vs vehicle[100%] at n = 4; Student’s t test).

Involvement of Cannabinoid Receptors and TRPV1 in Anti-invasive Action of Cannabinoids

To investigate whether cannabinoid receptors and TRPV1 are involvedin cannabinoid-mediated reduction of HeLa cell invasiveness,the effect of antagonists of the CB₁ receptor (AM-251), theCB₂ receptor (AM-630), and TRPV1 (capsazepine) on cannabinoidaction was tested. These inhibitors were all used at a concentrationof 1 µM, which is within the range of concentrations ofthese substances that have been reported to inhibit responsesof cells to activation of the cognate receptors (36,37). MA-inducedinhibition of cancer cell invasion was completely preventedby 1-hour incubation with the CB₂ antagonist (invasion indices[relative to control] of MA- and MA + AM-630–treated cells= 33.9% and 106.2%, respectively; difference = 72.3%, 95% CI= 48.8% to 95.9%, P < .001 [Fig. 1, C]). Preincubation ofcells for 1 hour with antagonists to both CB₁ and CB₂ furtherincreased the invasion index of cells treated with MA (33.9%vs 129.3%; difference = 95.4%, 95% CI = 73.2% to 117.7%, P <.001). Preincubation of cells with the CB₁ antagonist AM-251led to a partial reconstitution of invasion (invasion indexfor MA and MA + AM-251 = 33.9% and 72.5%, respectively; difference= 38.6%, 95% CI = 15.5% to 61.8%, P = .0019). Preincubationof cells for 1 hour with the TRPV1 antagonist capsazepine alsorestored invasiveness in the presence of MA (invasion indicesfor MA and MA + capsazepine = 33.9% and 68.9%, respectively;difference = 35.0%, 95% CI = 16.7% to 53.3%, P < .001 [Fig. 1, C]),suggesting that TRPV1 activity contributes to the anti-invasiveaction of MA.

THC-induced inhibition of cancer cell invasion was also preventedby 1-hour incubation with the CB₁ antagonist (invasion indices[relative to control] of THC- and THC + AM-251–treatedcells = 11.8% and 83.2%, respectively; difference = 71.4%, 95%CI = 54.2% to 88.6%, P < .001 [Fig. 1, C]). Preincubationof cells with the CB₂ antagonist AM-630 led to a reconstitutionof invasion (invasion indices [relative to control] for THCand THC + AM-630 = 11.8% and 72.6%, respectively; difference= 60.8%, 95% CI = 47.5% to 74.1%, P < .001). As noted forcells treated with MA preincubation of THC-treated cells for1 hour with antagonists to both CB₁ and CB₂ further increasedthe invasion index of cells treated with THC (invasion indices[relative to control] of THC- and THC + AM-251 + AM-630–treatedcells = 11.8% vs 97.9%; difference = 86.1%, 95% CI = 69.0% to103.3%, P < .001 [Fig. 1, C]).

Effect of Cannabinoids on the Expression of TIMP-1

To investigate a causal link between modulation of invasivenessand cannabinoid-induced release of proteolytic enzymes intothe cell culture microenvironment, supernatants and lysatesof HeLa cells were analyzed for changes in MMP and TIMP expressionafter stimulation with cannabinoids (Fig. 2, B). Treatment ofcells with 10 µM MA or 1 µM THC led to inductionof TIMP-1 mRNA and protein expression after a 12-hour incubationperiod (mean mRNA expression in cells treated with 10 µMMA or 1 µM THC as a percentage of that of vehicle-treatedcells = 181%, 95% CI = 163% to 199%, P = .0033 and 119%, 95%CI = 109% to 129%, P = .0499, respectively; mean TIMP-1 proteinexpression in cells treated with 10 µM MA or 1 µMTHC as a percentage of that of vehicle-treated cells = 119%,95% CI = 89% to 149%, or 140%, 95% CI = 69% to 211%, respectively[Fig. 2, A]).

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Fig. 2. Time- and concentration-dependent action of cannabinoids on the expression of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) and other matrix metalloproteinases (MMPs). A) Time course of TIMP-1 mRNA (right graph) and protein expression (left panel) after treatment of cells with cannabinoids. Cells treated with cannabinoids (10 µM R(+)-methanandamide [MA] or 1 µM ${Delta}$ ⁹-tetrahydrocannabinol [THC]) or vehicle for the indicated times were solubilized, and total RNA was used to determine TIMP-1 mRNA levels by quantitative real-time reverse transcriptase–polymerase chain reaction. Proteins separated from the cell culture supernatant were transferred to nitrocellulose membranes and probed with an antibody to TIMP-1. Bound secondary antibodies were visualized by chemiluminescence. Densitometric analyses were obtained from n = 3 blots. For mRNA values, means and upper 95% confidence intervals of n = 4 independent experiments are shown. P values were .003 for MA and .0499 for THC vs corresponding vehicle after 12 hours, *P < .001 for MA and .034 for THC vs corresponding vehicle after 24 hours, .001 for MA and .003 for THC vs corresponding vehicle after 48 hours (Student’s t test). B) Concentration-dependent effect of MA and THC on expression of the TIMP-1, MMP-2, TIMP-2 and MMP-9 proteins. Cells were treated with the indicated concentrations of MA or THC for 48 hours. C) Effect of a 1-hour pretreatment with AM-251 (1 µM), AM-630 (1 µM), or capsazepine (Capsa; 1 µM) on TIMP-1 and MMP-2 levels of cells incubated with 10 µM MA or 1 µM THC for 48 hours. Densitometric analyses were obtained from n = 4–5 (B) or n = 4–6 blots (C). A band corresponding to a protein of about 65 kDa whose expression was not affected by treatment with cannabinoids or receptor antagonists was used as the loading control (LC) and internal standard for densitometric analyses.

Increased TIMP-1 levels in cannabinoid-treated HeLa cells weredetected at concentrations as low as 1 µM for MA (meanexpression relative to vehicle = 223%, 95% CI = 139% to 307%)and 0.1 µM for THC (mean expression relative to vehicle= 206%, 95% CI = 137% to 275% [Fig. 2, B]).We observed no alteration of TIMP-2 and MMP-9 levels in cellstreated with MA or THC (Fig. 2, B). By contrast, concentrationsof MMP-2 were decreased upon treatment of cells with increasingconcentrations of MA or THC (mean MMP-2 expression of cellstreated with 1 µM MA or 0.1 µM THC as a percentof expression in vehicle-treated cells = 42%, 95% CI = 15% to69% or 45%, 95% CI = –5% to 95% [Fig. 2, B]).

Effect of Cannabinoid Receptor and TRPV1 Antagonists on Cannabinoid-Elicited TIMP-1 Induction

Cells were preincubated with cannabinoid receptor and TRPV1antagonists to determine the receptors of MA and THC that mediatedchanges in TIMP-1 and MMP-2 expression. The TIMP-1 expressionthat was observed upon induction with MA was substantially reducedby treatment of cells with 1 µM AM-251, an antagonistof the CB₁ receptor (decrease = 110%, 95% CI = 57% to 162%,P = .0013), or 1 µM AM-630, an antagonist of the CB₂ receptor(decrease = 99%, 95% CI = 35% to 163%, P = .0073), or treatmentwith both antagonists at a 1 µM concentration (decrease= 142%, 95% CI = 92% to 192%, P < .001 [Fig. 2, C]). At thesame concentrations, these antagonists exerted similar effectson THC-stimulated TIMP-1 expression. Furthermore, MA-inducedTIMP-1 expression was prevented by treatment with 1 µMcapsazepine (decrease = 103%, 95% CI = 29% to 176%, P = .0123[Fig. 2, C]). By contrast, the decrease in MMP-2 levels in responseto MA or THC treatment was not antagonized by cannabinoid receptorantagonists or capsazepine (Fig. 2, C).

Involvement of p38 and p42/44 MAPK Pathways in Cannabinoid Effects on HeLa Cell Invasion and TIMP-1 Induction

TIMP-1 has been described as a target of p38 and p42/44 MAPKs(31,32). To investigate the possible role of p38 and p42/44MAPKs in cannabinoid-mediated suppression of invasion, HeLacells were pretreated with 10 µM SB203580 or PD98059,which are inhibitors of p38 and p42/44 MAPK activity, respectively.Treatment of HeLa cells with these inhibitors prevented theeffects of MA and THC on invasiveness to the extent that levelsof invasiveness were indistinguishable from those of cells treatedwith vehicle alone (Fig. 3, A). Consistent with these data,SB203580 and PD98059 decreased TIMP-1 protein expression incells treated with MA to levels observed when cells were treatedwith vehicle alone [Fig. 3, B]). Similar effects of these inhibitorson TIMP-1 expression were observed in cells that had been treatedwith THC.

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Fig. 3. Role of p38 and p42/44 mitogen–activated protein kinase (MAPK) pathways in the effects of cannabinoids on HeLa cell invasion and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) expression. A) Effect of a 1-hour pretreatment with 10 µM SB203580 (SB; inhibitor of p38 MAPK activity) and 10 µM PD98059 (PD; inhibitor of p42/44 MAPK activation) on the anti-invasive action of R(+)-methanandamide (MA; 10 µM) and ${Delta}$ ⁹-tetrahydrocannabinol (THC; 1 µM) after a 72-hour incubation period. Values are means + the upper 95% confidence interval (error bar) of n = 8 independent experiments. P < .001 vs vehicle control, *P < .001 vs MA or THC (Student’s t test). B) Effect of SB203580 and PD98059 on TIMP-1 protein levels in cell culture supernatants after a 48-hour incubation with both MA and THC. Densitometric analyses were performed on 4–5 experiments standardized to loading control (LC), a band corresponding to a protein of about 65 kDa whose expression was not affected by treatment with cannabinoids or MAPK inhibitors. C) Effect of a 1-hour pretreatment with AM-251 (1 µM), AM-630 (1 µM), and capsazepine (Capsa; 1 µM) on phosphorylation of p38 and p42/44 MAPKs after a 48-hour incubation with 10 µM MA or 1 µM THC. To analyze MAPK phosphorylations, immunoblots were probed with antibodies directed against the phosphorylated form of p38 or p42/44. Equal loading of lysates is ensured by probing membranes with antibodies against the nonphosphorylated forms of p38 and p42/44 MAPKs. Densitometric values were obtained from n = 3 experiments and were standardized to the nonphosphorylated forms of p38 or p42/44.

We assessed the relationship between receptor and MAPK activationas detected with antibodies to the phosphorylated (active) formsof the kinases as previously described (17). MA-induced activationof p38 (mean increase in phosphorylated protein = 274%, 95%CI = 206% to 342%) was decreased by CB₁ and CB₂ antagonistsAM-251 and AM-630 (decrease = 122%, 95% CI = 59% to 185%, P= .006, and 150%, 95% CI = 85% to 215%, P = .0031, respectively),whereas blockade of TRPV1 with 1 µM capsazepine left phosphorylationof p38 virtually unaltered (Fig. 3, C). Similar effects of AM-251and AM-630 were observed on p42/44 phosphorylation (activation)when cells were treated with MA (decrease = 100%, 95% CI = 30%to 170%, P = .0167, and 143%, 95% CI = 74% to 211%, P = .0044,respectively). Simultaneous treatment with both cannabinoidreceptor antagonists did not further decrease MAPK activationcompared with treatment with a single antagonist. Capsazepinetreatment caused a small decrease in p44/42 phosphorylationthat was not statistically significant. Similar effects of thereceptor antagonists on MAPK activation were observed in cellstreated with THC (Fig. 3, B).Effect of TIMP-1 Knockdown on the Anti-invasive Action of Cannabinoids

To confirm a causal link between cannabinoid-mediated TIMP-1induction and decreased invasion, the expression of TIMP-1 wasblocked by transfecting cells with TIMP-1 siRNA. siRNA to TIMP-1at concentrations of 0.25 and 1.0 µg/mL decreased TIMP-1expression in vehicle-treated cells by 69% and 87%, respectively(Fig. 4, A). Because the higher siRNA concentration interferedwith the basal level of invasion (Fig. 4, A, upper panel), thelower concentration was used.

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Fig. 4. Involvement of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in cannabinoid-modulated invasiveness and role of MMP-2 and MMP-9 in basal HeLa cell invasion. A) Effect of TIMP-1 siRNA (0.25 and 1 µg/mL) on basal HeLa cell invasion. HeLa cells were transfected with TIMP-1 siRNA (si) or nonsilencing siRNA (nonsi) for 24 hours. Subsequently, cells were placed into invasion chambers, retransfected with the indicated siRNA or suspension buffer for an additional 72 hours before determination of the invasion index and preparation of cell culture supernatants from the upper Boyden chambers for Western blot analyses of TIMP-1. Invasion index values are means + the upper 95% confidence interval (CI) (error bar) of n = 4 independent experiments. P = .671 for vehicle vs 0.25 µg/mL TIMP-1 siRNA and *P < .001 vs vehicle (Student’s t test). B) Effect of TIMP-1 siRNA (0.25 µg/mL) and cannabinoids on cell invasion. In addition to transfection with 0.25 µg/mL TIMP-1 siRNA (A), HeLa cells were subsequently coincubated with vehicle, 10 µM R(+)-methanandamide (MA) or 1 µM ${Delta}$ ⁹-tetrahydrocannabinol (THC) for 72 hours. P < .001 vs vehicle; *P < .001 vs MA or THC for n = 4 independent experiments. C) Microscopy of HeLa cells from the different treatment groups in B that invaded through Matrigel-coated membranes. Cells were stained with Diff-Quick^® (Medion Diagnostics GmbH, Büdingen, CH). Magnification of photomicrographs is x200. D) Effect of MMP-2 or MMP-9 siRNA (each at 0.25 or 1 µg/mL) on basal HeLa cell invasion. P = .226 for 0.25-µg/mL MMP-2 siRNA vs vehicle, P = .063 for 1.0-µg/mL MMP-2 siRNA vs vehicle, and P = .007 for 0.25-µg/mL MMP-9 siRNA vs vehicle, *P < .001 vs vehicle. TIMP-1, MMP-2, and MMP-9 protein levels were determined in supernatants collected from the upper Boyden chamber. Invasion index values are means + the upper 95% CI (error bar) of n = 4 independent experiments. Densitometric analysis were obtained from n = 6–7 (A) and n = 4 (D) blots standardized to loading control (LC), a band corresponding to a protein of about 65 kDa whose expression was not affected by treatment with siRNA, transfection agent, or cannabinoids.

Knockdown of TIMP-1 expression with 0.25 µg/mL TIMP-1siRNA led to a statistically significant abrogation of the MA-and THC-mediated decrease of invasion, restoring the invasionindex to that observed in vehicle-treated cells (Fig. 4, B).A nonsilencing siRNA had no effect on the invasiveness of vehicle-,MA-, or THC-treated cells. Monitoring of TIMP-1 secretion intothe culture medium of the upper Boyden chamber confirmed aninhibition of cannabinoid-induced TIMP-1 expression to controllevels in samples transfected with TIMP-1 siRNA (Fig. 4, B).The effect of TIMP-1 siRNA transfection on tumor cell invasionwas also confirmed by staining cells that had invaded throughMatrigel-coated membranes at the lower surface. A lower numberof cells invaded in the presence of cannabinoids, and this effectwas not seen when cells were also treated with 0.25 µg/mLTIMP-1 siRNA (Fig. 4, C). Knockdown of TIMP-1 also led to aninhibition of the effect of cannabinoids on invasion in A549and C33A cells (Supplementary Fig. 1, available online).Requirement of MMP-2 and MMP-9 for HeLa Cell Invasion

TIMP-1 is known to form inhibitory complexes with MMP-2 andMMP-9 (23). To determine whether MMP-2 and MMP-9 are essentialfor HeLa cell invasion, cells were transfected with 0.25 and1.0 µg/mL of siRNA corresponding to the genes encodingthese proteins (Fig. 4, D). As measured by densitometric analysis,MMP-2 protein levels in cells treated with 0.25 µg/mLand 1 µg/mL siRNA relative to that observed when cellswere treated with vehicle alone were 34% (95% CI = 22% to 46%)and 19% (95% CI = 8% to 30%), respectively. Similarly, MMP-9levels decreased to 28% (95% CI = 23% to 33%) and 15% (95% CI= 6% to 24%) of control level after transfection with 0.25 µg/mLand 1 µg/mL MMP-9 siRNA, respectively. Inhibition of MMP-9expression by 0.25 µg/mL or 1.0 µg/mL MMP-9 siRNAdecreased invasion of HeLa cells through Matrigel (decrease= 48.0%, 95% CI = 18.6% to 77.4%, P = .0071 or 95.7%, 95% CI= 78.5% to 112.9%, P < .001, respectively), whereas MMP-2silencing caused a decrease in invasion of only 15% that wasnot statistically significant. Thus, MMP-9, but not MMP-2, isessential for HeLa cell invasion in our experimental system.

	Role of CB₁, CB₂, and TRPV1 in Cannabinoids’ Anti-invasive Action and TIMP-1 Induction in Other Tumor Cell Lines

Top Abstract Context and Caveats Materials and Methods Results Role of cb1, cb2,… Discussion Funding References

To exclude the possibility that the observed cannabinoid effectsare restricted to HeLa cells, experiments were also performedin another human cervical carcinoma cell line (C33A) as wellas in human lung carcinoma cells (A549). Like HeLa cells, bothcell lines express CB₁ and CB₂ receptors as well as TRPV1, withC33A having a lower concentration of the latter protein as comparedwith HeLa and A549 cells (Supplementary Fig. 2 A, availableonline). Addition of cannabinoids to these cells resulted ina statistically significant inhibition of invasion through Matrigelaccompanied by increased TIMP-1 secretion, and both events weresuppressed by antagonists of CB₁ and CB₂ receptors and, in thecase of MA, by a TRPV1 antagonist (Supplementary Fig. 2 B,C).Knockdown of TIMP-1 led to an inhibition of the effect of cannabinoidson invasion in A549 cells and C33A cells (Supplementary Fig.1).

	Discussion

Top Abstract Context and Caveats Materials and Methods Results Role of cb1, cb2,… Discussion Funding References

There is considerable evidence to suggest an important rolefor cannabinoids in conferring anticarcinogenic activities.In this study, we identified TIMP-1 as a mediator of the anti-invasiveactions of MA, a hydrolysis-stable analog of the endocannabinoidanandamide, and THC, a plant-derived cannabinoid.

Both cannabinoids decreased HeLa cell invasion in a time- andconcentration-dependent manner. Following a 72-hour incubation,the decrease of invasiveness by MA and THC was statisticallysignificant at concentrations as low as 0.1 µM and 0.01µM (at these concentrations we observed 61.5% inhibitionof invasion by MA and 68.1% inhibition by THC). In humans, averagepeak plasma concentrations of THC of 0.03 µM and 0.045µM could be achieved with oral doses of 15 and 20 mg,respectively (38) and were associated with a statistically significantreduction of cancer pain (39,40). Thus, effects of THC on cellinvasion occurred at therapeutically relevant concentrations.

The possibility that decreased invasion by cannabinoids wasan unspecific cytotoxicity-related phenomenon was ruled outby an analysis of cellular viability that revealed no statisticallysignificant cytotoxicity by either MA or THC under experimentalconditions very similar to those used for invasion assays. However,MA and THC did lead to increasing and statistically significanttoxicity when cellular density was decreased. To our knowledge,cell density–dependent toxicity has not been describedfor cannabinoids before but is well documented as the “inoculumeffect” for several chemotherapeutics, including tamoxifen (41),doxorubicin, and vincristine (42). In the study of vincristinetoxicity, measurements of cellular drug levels revealed thatat high densities, cells accumulate much smaller amounts ofchemotherapeutics, resulting in impaired availability of thedrug at its intracellular binding sites (42). A similar patternmay occur when cells are exposed to MA or THC given that bothcannabinoids cause receptor-independent apoptosis (8). In thecase of MA, this effect probably involves a lipid raft–dependentintracellular uptake of the compound (43). However, high celldensities do allow considerable binding of cannabinoids to theirextracellular membrane receptors, as revealed by the profoundreceptor-dependent anti-invasive action observed here.

Our finding that reduced invasion was not associated with decreasedcellular motility suggested that the reduction in invasivenessthat was observed when cells were treated with cannabinoidswas a specific effect that was dependent on the modulation ofmatrix-degrading enzymes. Although this result rules out a decisiverole of migration in mediating the anti-invasive action of cannabinoidsin our system, others have reported antimigrative propertiesof cannabinoids that suggest that these substances affect migrationin a cell type–specific and/or chemoattractant-dependentmanner. For example, in human breast cancer cells, cannabinoidtreatment inhibits adhesion and migration on type IV collagen,possibly via decreased tyrosine phosphorylation of focal adhesionkinase (44). Furthermore, a cannabinoid receptor–independentmechanism was proposed to underlie the antimigrative actionof cannabidiol on human glioma cells (45).

Because the role of TIMP-1 in reducing invasiveness is wellestablished (24–30), we assessed the role of this endogenousMMP inhibitor in the context of the anti-invasive effects ofcannabinoids on HeLa cells. Our results suggest a causal linkbetween cannabinoid receptor activation, TIMP-1 induction, anddecreased invasiveness of HeLa cells. Consistent with the hypothesisthat TIMP-1 is a mediator of the effects of cannabinoids oncell invasion, TIMP-1 induction first became evident after a12-hour incubation period with both cannabinoids and a decreasein invasion appeared between 12 and 24 hours after cannabinoidexposure. Furthermore, inhibitors of the cannabinoid receptorsCB₁ and CB₂ and TRPV1 that caused a profound reduction of cannabinoid-inducedTIMP-1 expression reversed cannabinoid-mediated effects on invasion.The most convincing evidence for a crucial role of TIMP-1 incannabinoid-mediated decreased invasion was our finding thattransfection of cells with siRNA targeting TIMP-1 markedly suppressedcannabinoid-mediated decreases in invasion. We confirmed a TIMP-1–dependentanti-invasive effect of cannabinoid treatment in another humancervical cell line (C33A) as well as in human lung carcinomacells (A549), suggesting that increased expression of TIMP-1is part of a general anti-invasive mechanism of cannabinoids.

Our results also suggest that MAPKs are targets of cannabinoidreceptor signaling that are upstream of TIMP-1. In support ofthis idea, preincubation of cannabinoid-treated cells with theinhibitor of p38 MAPK activity SB203580 and the inhibitor ofp42/44 MAPK activation PD98059 led to impaired induction ofexpression of TIMP-1 by MA and THC. Moreover, inhibitor experimentswith AM-251 and AM-630 revealed a CB₁ and CB₂ receptor–mediatedMAPK activation consistent with previous studies that demonstrateda mediating role of MAPKs in cannabinoid receptor-elicited effects(3,9,33). These results suggest that both p38 and p42/44 MAPKsare mediators of cannabinoid receptor activation and subsequentTIMP-1 regulation. Treatment of cells with the TRPV1 antagonistcapsazepine had virtually no effect on MA-mediated activationof p38 and p42/44 MAPKs, suggesting that TRPV1 activation increasesTIMP-1 expression by a mechanism that bypasses MAPKs.

Specific inhibition of MMP expression by siRNA suggested a decisiverole of MMP-9 but not MMP-2 in basal HeLa cell invasion. Thus,the decreased expression of MMP-2 mediated by cannabinoids weobserved does not appear to contribute to their anti-invasiveaction in our experimental system. The lack of a role for MMP-2in modulating the anti-invasive effects of cannabinoids is furthersupported by the finding that the suppressive effect of cannabinoidson MMP-2 expression was not mediated by CB₁ or CB₂. Thus, iflowering MMP-2 was responsible for the inhibitory effect ofcannabinoids on tumor cell invasion, treatment of cells withreceptor antagonists should be expected to elicit only partialsuppression of invasiveness. However, the anti-invasive actionof cannabinoids was of fully reversed when both CB₁ and CB₂receptors were blocked.

Our study has some limitations. First, it is not known to whatextent the principal finding of this study can be generalizedto cell types other than those examined in this study. Moreover,further studies will be required to examine the relevance ofour findings to in vivo tumors. Finally, we did not identifythe mechanism underlying receptor-independent decreased expressionof MMP-2. It remains to be determined whether lipid raft microdomainsthat have been recently proposed to be an initial target ofthe endocannabinoid analog MA in eliciting receptor-independentinduction of ceramide synthesis, MAPK activation, and cyclo-oxygenase-2expression (43) are involved in this response.

In conclusion, our results suggest that there exists a signalingpathway by which the binding of cannabinoids to specific receptorsleads via intracellular MAPK activation to induction of TIMP-1expression and subsequent inhibition of tumor cell invasion.To our knowledge, this is the first report of TIMP-1–dependentanti-invasive effects of cannabinoids. This signaling pathwaymay play an important role in the antimetastatic action of cannabinoids,whose potential therapeutic benefit in the treatment of highlyinvasive cancers should be addressed in clinical trials.

	Funding

Top Abstract Context and Caveats Materials and Methods Results Role of cb1, cb2,… Discussion Funding References

Deutsche Krebshilfe e.V., Bonn, Germany (Project #107209); DeutscheForschungsgemeinschaft (SFB 539, Project BI.6); Johannes undFrieda Marohn Stiftung, Erlangen, Germany (Hin/2005).

The study sponsors had no role in the design of the study orin the collection, analysis, or interpretation of the data.The authors take full responsibility for the study design, datacollection, analysis and interpretation of the data, the decisionto submit the manuscript for publication, and the writing ofthe manuscript.

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Manuscript received April 24, 2007; revised September 24, 2007; accepted November 16, 2007.

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Posted in Articles, Cancer, Cervical Cancer | Tags: cannabinoids, cannabis, Cervical Cancer, medical marijuana, pot, weed | No Comments »

Marijuana use and cervical HPV/neoplasia

June 14th, 2010 | Author: Brett

G D’Souza¹, R Burk², Y Zhong², H Minkoff³, LS Massad⁴, K Anastos⁵, A Levine⁶, M Moxley⁷, X Xue⁵, J Palefsky⁸ and HD Strickler²

¹Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA

²Department of Epidemiology & Social Medicine, Albert Einstein College of Medicine, Bronx, New York, USA

³Maimonides Medical Center and SUNY Downstate, Brooklyn, New York, USA

⁴Southern Illinois University School of Medicine, Springfield, Illinois, USA

⁵Albert Einstein College of Medicine, Bronx, New York, USA

⁶University of Southern California, Los Angeles, California, USA

⁷Georgetown University Medical Center, Washington, DC, USA

⁸University of California at San Francisco, California, USA

email ca Marijuana use and cervical HPV/neoplasia corresponding author email

from 11th International Conference on Malignancies in AIDS and Other Acquired Immunodeficiencies (ICMAOI): Basic, Epidemiologic, and Clinical Research
Bethesda, MD, USA. 6–7 October 2008

Infectious Agents and Cancer 2009, 4(Suppl 2):P15doi:10.1186/1750-9378-4-S2-P15

The electronic version of this abstract is the complete one and can be found online at: http://www.infectagentscancer.com/content/4/S2/P15

Published:

17 June 2009

Poster presentation

Recent research suggests that marijuana use may be positively associated with risk of human papillomavirus (HPV)-associated oropharyngeal cancer, and the immunomodulatory effects of cannabinoids are considered a possible mechanism. Were marijuana to have systemic effects on HPV-associated tumorigenesis it would be of major concern, and might be especially harmful in immunologically susceptible populations such as HIV-positive women.

We studied the effect of marijuana use on cervical HPV natural history and cervical squamous intraepithelial lesions (SIL) among HIV-positive and HIV-negative women in the Women’s Interagency HIV Study (WIHS), a large prospective cohort study. HPV DNA testing by PCR and Pap smears were conducted semi-annually. Prevalent HPV and cervical SIL were analyzed using logistic regression with GEE. Persistence of HPV was analyzed using Cox regression.

Of the 3,499 women in the WIHS, 1,414 reported ever using marijuana. There were no associations between HPV prevalence and either current (OR = 0.98 95% CI = 0.90–1.07), frequent (current daily use: OR = 0.98 95 CI = 0.90–1.07), or sustained (daily use for more than 3 years: OR = 1.03, 95% CI = 0.90–1.19) marijuana use, controlling for age, race, HIV status, CD4 cell count, number of sexual partners in the last 6 months, tobacco use and cervical treatment. Nor were there effects on prevalence of SIL, or on the persistence of cervical HPV infection. Results were similar among HIV-negative and HIV-positive women.

Our data do not support an effect of marijuana on cervical HPV infection, persistence or risk of cervical dysplasia. If there is an effect of marijuana use on oral HPV-associated tumorigenesis, it may be a local effect.

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JunD is involved in the antiproliferative effect of Delta(9)-tetrahydrocannabinol on human breast cancer cells.

June 14th, 2010 | Author: Brett

It has been recently shown that cannabinoids, the active components of marijuana and their derivatives, inhibit cell cycle progression of human breast cancer cells. Here we studied the mechanism of Delta(9)-tetrahydrocannabinol (THC) antiproliferative action in these cells, and show that it involves the modulation of JunD, a member of the AP-1 transcription factor family. THC activates JunD both by upregulating gene expression and by translocating the protein to the nuclear compartment, and these events are accompanied by a decrease in cell proliferation. Of interest, neither JunD activation nor proliferation inhibition was observed in human non-tumour mammary epithelial cells exposed to THC. We confirmed the importance of JunD in THC action by RNA interference and genetic ablation. Thus, in both JunD-silenced human breast cancer cells and JunD knockout mice-derived immortalized fibroblasts, the antiproliferative effect exerted by THC was significantly diminished. Gene array and siRNA experiments support that the cyclin-dependent kinase inhibitor p27 and the tumour suppressor gene testin are candidate JunD targets in cannabinoid action. In addition, our data suggest that the stress-regulated protein p8 participates in THC antiproliferative action in a JunD-independent manner. In summary, this is the first report showing not only that cannabinoids regulate JunD but, more generally, that JunD activation reduces the proliferation of cancer cells, which points to a new target to inhibit breast cancer progression.Oncogene advance online publication, 5 May 2008; doi:10.1038/onc.2008.145.

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Inhibition of Breast Cancer Aggressiveness by Cannabidiol

June 14th, 2010 | Author: Brett

Initial Award Abstract (2006)
An anti-cancer agent with a low toxicity profile that can both inhibit cancer cell growth and metastasis would be extremely valuable clinically. We have discovered that cannabidiol (CBD), a non-psychotropic cannabinoid constituent of the plant Cannabis sativa, can inhibit the growth, migration and invasion of aggressive breast cancer cells in culture. Cannabinoid compounds, in general, have low toxicity profiles. Furthermore, our preliminary research demonstrated that CBD is a novel inhibitor of a protein whose activity has been closely linked to the aggressiveness of human breast cancers; called inhibitor of DNA binding-1 (Id-1). Whether CBD can inhibit the spread of metastatic breast cancer in vivo (in the body), compared to cell culture conditions, has not been determined. However, CBD has been demonstrated to inhibit aggressive human brain cancers in vivo. Understanding the mechanisms behind the anti-cancer activity of CBD may lead to the validation of new biological targets for diagnostics and therapies for breast cancer. To study the effects of CBD on the growth and spread of aggressive breast cancer, we will use time lapse microscopy to capture detailed changes in cell growth/death, migration, and morphology. Classical biochemical measures of cell growth/death and invasion will also be used in combination with novel compounds to identify receptors that CBD interacts with to produce its anti- breast cancer effects. A mouse model of breast cancer metastasis will be used to determine whether CBD effectively reduces the spread of aggressive breast cancer in vivo. At the intracellular level, we will study whether CBD, (1) regulates Id-1 in an extracellular signal-regulated kinase-dependent manner, and (2) serves to activate a pro-apoptotic (cell death) pathway in a caspase-dependent manner. Novel and effective non-toxic therapies for aggressive breast cancers are urgently required. Plant cannabinoids are compounds that are well tolerated during chronic (long-term) administration. CBD, and compounds based on its structure, may be the basis for the treatment of metastatic breast cancer. If successful, our studies will set the stage for additional translational work to develop CBD’s eligibility for clinical trials.

Final Report (2008)
We discovered that cannabidiol (CBD), a non-psychotropic compound from the plant Cannabis sativa, can inhibit the process of breast cancer cells that allow them to grow and spread (metastasis). CBD can also inhibit breast cancer metastasis in a mouse model. The research carried out in our CBCRP proposal demonstrated that CBD is a novel inhibitor of a gene whose activity is intimately linked to the aggressiveness of human breast cancers; this gene has been termed Id-1. Notably, our findings also indicated that Id-1 was a key gene whose expression needed to be reduced in order for CBD to inhibit aggressive breast cancer. One of the most significant high risk components of the initial application was to determine if CBD had appreciable efficacy against breast cancer in vivo (i.e., animal models). This high risk component was not pursued, since an independent group showed CBD was able to inhibit metastasis of MDA-MB231 cells to the lung of nude mice. Building of the previous findings, we made small structural changes to CBD that are expected to produce drugs that are much more active than CBD at inhibiting Id-1 and corresponding aggressive breast cancers. CBD has a low toxicity profile. An anticancer agent with a low toxicity profile that can both inhibit cancer cell growth and metastasis would be extremely valuable clinically. Understanding the mechanisms behind the anticancer activity of CBD may also lead to the discovery of new biological targets for the development of diagnostic tools and additional therapies for the treatment of cancer. In this project we found portions of the CBD structure essential to its biological activity for breast cancer cell growth inhibition. We are in the process of filing a patent on these discoveries. In addition, we studies the moleculr mechanisms that underlie CBD activity, and found that sustained upregulation of Erk (extracellular signal-regulated kinases, a type of protein kinase intracellular signaling molecules) is key to the ability of CBD to regulate the metastasis-specific inhibition of the Id-1 transcription factor.

Symposium Abstract (2007)
The spread (metastasis) of aggressive breast cancer cells to other parts of the body is the final and fatal step during cancer progression. Clinically, there are still limited therapeutic interventions for aggressive breast cancers available. Clearly, effective and non-toxic therapies are urgently required. The Id-1 gene, a helix-loop-helix type transcription factor, has recently been shown to be a key regulator of the metastatic potential of breast and additional cancers. We previously determined that aggressive breast cancer cells became significantly less invasive in vitro (in culture) and less metastatic in vivo (in mice) when Id-1 expression was reduced using a technique called gene antisense therapy. It is not possible at this point, however, to use this technology to reduce Id-1 expression in patients with metastatic breast cancer. In our search for a non-toxic drug that could inhibit Id-1 expression, a potential candidate agent was discovered. Here we report that cannabidiol (CBD), a compound extracted from cannabis and with a low toxicity profile, can down-regulate Id-1 expression in aggressive human breast cancer cells. The CBD concentrations effective at inhibiting Id-1 expression correlated with those used to inhibit the proliferative and invasive characteristics of aggressive breast cancer cells. CBD was able to inhibit Id-1 expression at the mRNA and protein level in a concentration-dependent fashion. Most importantly, constitutive expression of Id-1 in breast cancer cells abolished the effects of CBD on cell invasiveness. This suggests that Id-1 is indeed a key factor whose expression needs to be down-regulated in order to observe the effects of CBD on the reduction of breast cancer cell aggressiveness. In conclusion, CBD represents the first non-toxic drug that can significantly decrease Id-1 expression in metastatic breast cancer cells leading to reduction of tumor aggressiveness. CBD and additional analogs based off its structure could be used as inhibitors of Id-1 and might be of benefit for patients with breast cancers. Cannabinoids are already being used in clinical trials for purposes unrelated to their anticancer activity and these compounds have been reported to be well tolerated. We expect that using CBD as a template will lead to the discovery of more potent and efficacious drugs. This research could lead to a new area of investigation in the treatment of aggressive forms of breast cancer with novel cannabinoid compounds.

Posted in Articles, Breast Cancer, Cancer | Tags: Breast Cancer, cannabinoids, cannabis, medical marijuana, pot, weed | No Comments »

Medical Marijuana helps Breast Cancer

June 14th, 2010 | Author: Brett

Cannabinoids have been reported to possess antitumorogenic activity. Not much is known, however, about the effects and mechanism of action of synthetic nonpsychotic cannabinoids on breast cancer growth and metastasis. We have shown that the cannabinoid receptors CB1 and CB2 are overexpressed in primary human breast tumors compared with normal breast tissue. We have also observed that the breast cancer cell lines MDA-MB231, MDA-MB231-luc, and MDA-MB468 express CB1 and CB2 receptors. Furthermore, we have shown that the CB2 synthetic agonist JWH-133 and the CB1 and CB2 agonist WIN-55,212-2 inhibit cell proliferation and migration under in vitro conditions. These results were confirmed in vivo in various mouse model systems. Mice treated with JWH-133 or WIN-55,212-2 showed a 40% to 50% reduction in tumor growth and a 65% to 80% reduction in lung metastasis. These effects were reversed by CB1 and CB2 antagonists AM 251 and SR144528, respectively, suggesting involvement of CB1 and CB2 receptors. In addition, the CB2 agonist JWH-133 was shown to delay and reduce mammary gland tumors in the polyoma middle T oncoprotein (PyMT) transgenic mouse model system. Upon further elucidation, we observed that JWH-133 and WIN-55,212-2 mediate the breast tumor-suppressive effects via a coordinated regulation of cyclooxygenase-2/prostaglandin E2 signaling pathways and induction of apoptosis. These results indicate that CB1 and CB2 receptors could be used to develop novel therapeutic strategies against breast cancer growth and metastasis. [Mol Cancer Ther 2009;8(11):3117–29]

Posted in Articles, Breast Cancer, Cancer | Tags: Breast Cancer, cannabinoids, cannabis, medicinal marijuana, pot, weed | No Comments »

Prostate Cancer and Medical Marijuana

June 14th, 2010 | Author: Brett

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

Endocannabinoids have been implicated in cancer. Increasingendogenous 2-arachidonoylglycerol (2-AG) by blocking its metabolisminhibits invasion of androgen-independent prostate cancer (PC-3and DU-145) cells. Noladin ether (a stable 2-AG analog) andexogenous CB1 receptor agonists possess similar effects. Conversely,reducing endogenous 2-AG by inhibiting its synthesis or blockingits binding to CB1 receptors with antagonists increases thecell invasion. 2-AG and noladin ether decrease protein kinaseA activity in these cells, indicating coupling of the CB1 receptorto downstream effectors. The results suggest that cellular 2-AG,acting through the CB1 receptor, is an endogenous inhibitorof invasive prostate cancer cells.

	Introduction

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

2-Arachidonoylglycerol (2-AG) is a monoacylglycerol derivativeof arachidonic acid (AA) that can be synthesized from AA-enrichedmembrane phospholipids such as phosphatidylinositol and phosphatidicacid via the sequential actions of phospholipase C or D anddiacylglycerol lipase (1) . Fatty acid amide hydrolase (2) and monoacylglycerol lipase (3 , 4) can hydrolyze 2-AG to AAand glycerol. 2-AG is an endogenous ligand (endocannabinoid)for the central cannabinoid (CB1) receptors (5) and for theperipheral cannabinoid (CB2) receptors (6) in some cells. Studieshave implicated endocannabinoids and cannabinoid receptors intumorigenesis. 2-AG inhibits proliferation of rat C6 gliomacells (7) and prolactin-induced DU-145 cells (8) . It reducesthe growth of colon cancer (9) . These 2-AG activities in DU-145cells and colon cancer involve the CB1 receptors. 2-AG inducesa rapid, transient elevation of intracellular Ca²⁺ concentrationin neuroblastoma x glioma hybrid NG108-15 cells (10) , similarto the cannabinoid agonist WIN55,212-2, and the cannabinoidCB1 receptor antagonist SR141716 blocks this effect. On theother hand, exogenous 2-AG stimulates migration of CB2-overexpressingmyeloid leukemia cells and normal splenocytes (11) . 2-AG alsoinduces the migration of microglial cells through CB2 receptors(12) . In these studies, 2-AG was applied as a chemoattractantin the bottom compartment of the Transwell. A different mechanismcould be responsible for 2-AG functions in these cells thanother cells. Whereas exogenous 2-AG inhibits proliferation ofprolactin-induced DU-145 cells, the effects of endogenous 2-AGhave not been studied. We investigated the roles of endogenous2-AG and cannabinoid receptors in regulating invasion of humanprostate cancer cells. Androgen-independent (PC-3 and DU-145)and androgen-dependent (LNCaP) prostate cancer cells were usedto determine whether 2-AG functions differently in hormone-refractoryprostate cancer cells.

	Materials and Methods

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

Cells and Cell Culture.
Androgen-independent (PC-3 and DU-145) and androgen-dependent(LNCaP) human prostate cancer cells were obtained from AmericanType Culture Collection (Manassas, VA). Cells were maintainedin Eagle’s minimal essential medium (RPMI 1640) supplementedwith 10% fetal bovine serum, L-glutamine (2 mmol/L), streptomycin(100 µg/mL), and penicillin (100 units/mL). Cells weregrown in 75-cm² polystyrene tissue culture flasks at 37°Cin 5% CO₂ to about 60% to 70% confluence before use.

Cell Invasion Assay.
Cells were incubated overnight with [methyl-³H]thymidine (1µCi/mL; Applied Biosystems, Foster City, CA) in mediacontaining 10% fetal bovine serum. Cells were rinsed once withfresh complete medium to remove unbound [methyl-³H]thymidine,and fresh complete medium was added to each flask. Cells werethen detached from the flask by using Pucks buffer (150 mmol/LNaCl, 5 mmol/L KCl, 10 mmol/L HEPES, 5 mmol/L NaHCO₃, 1 mmol/Lacid-free EDTA, 5 mmol/L glucose, and 0.25% trypsin) at 37°Cfor 5 minutes. The cells were centrifuged at 200 x g and resuspendedin 3 mL of serum-free RPMI 1640. The experiments were performedin 24-well plates containing Transwells with 8.0-µm porepolyvinylpyrrolidone-free polycarbonate filters (Corning Inc.,Corning, NY) coated with Matrigel (BD Biosciences, Bedford,MA) on the top compartments. Cell suspension (100 µL;50,000 cells) containing the vehicle control or pharmacologicalagents [2-AG and noladin ether (Cayman Chemical, Ann Arbor,MI), methyl arachidonyl fluorophosphonate (MAFP; Tocris, Ballwin,MO), diazomethylarachidonyl ketone (DAK; synthesized in ourlaboratory; ref. 13 ), RHC-80267 (Calbiochem, San Diego, CA),SR141716 and SR144528 (Research Triangle Institute, ResearchTriangle Park, NC), WIN55,212-2 (Sigma, St. Louis, MO), (R)-(+)-methanandamide(RBI, Natick, MA)] at various concentrations was added to eachupper compartment. Fibroblast conditioned-media (400 µL)was added in the bottom compartment of the well as a chemoattractant.Normally, 6 wells per treatment were performed for each batchof experiments. An additional 6 wells without Matrigel and Transwellbut containing the identical number of cells and pharmacologicalagents were used for the “control counts” of [methyl-³H]thymidine.Cells were incubated at 37°C in the incubator for 5 hours.Plates were centrifuged at 1,450 x g at 22°C for 30 minutes.The cells passed into the lower compartment media were detachedwith Pucks buffer containing 0.75% trypsin and placed in vials.The bottom side of the filter and the lower compartment wererinsed with 400 µL of Pucks buffer, incubated at 37°Cfor 20 minutes to remove adherent cells, combined in the correspondingvials, and counted for radioactivity. Each treatment was performedat least three times. The invasion was reported as the percentageof the invasion of the control cells.

Determination of 2-Arachidonoylglycerol in Prostate Cancer Cells by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry.
Cells were grown in T-75 flasks, rinsed with 5 mL of HEPES buffer(pH 7.4), and treated with RHC-80267 (100 nmol/L) or vehiclein HEPES buffer at 37°C for 15 minutes (14) . The cellswere lysed, scraped, and transferred into 15-mL tubes for samplepreparation by solid phase extraction. An aliquot of 100 µLwas saved for protein determination using Bio-Rad (Hercules,CA) protein assay. [²H₈]2-AG (15 ng; Cayman Chemical) was addedto the samples as an internal standard. The samples were extractedby solid phase extraction as described previously (14) , redissolvedin 20 µL of acetonitrile, and analyzed or kept at –80°C.Samples were analyzed by using liquid chromatography-electrosprayionization-mass spectrometry (Agilent 1100 LC-MSD, SL model;ref. 14 ). Briefly, the samples were separated on a reversephase C₁₈ column (Kromasil; 250 x 2 mm; Phenomenex) using water/acetonitrilecontaining 0.005% acetic acid as a mobile phase at a flow rateof 0.2 mL/min. The gradient started at 35% acetonitrile, increasedlinearly to 100% acetonitrile in 35 minutes, and held at 100%acetonitrile for 10 minutes. Drying gas flow of the electrospraychamber was 12 L/min, drying gas temperature was 350°C,nebulizer pressure was 35 psig, vaporizer temperature was 350°C,and fragmentor voltage was 90 V. The detection was made in thepositive mode. For quantitative measurement, m/z 379 and 387were used for 2-AG and [²H₈]2-AG, respectively. The concentrationsof 2-AG were calculated by comparing their ratios of peak areasto the standard curves. The results were normalized to the proteincontent.

Determination of 2-OG Hydrolysis.
Cells were grown in T-75 flasks to 60% to 70% confluence. Then,the medium was replaced by 100 µL of 100 mmol/L PBS (pH7.4), after cells were washed two times with PBS buffer. Cellswere homogenized by pulling them through a 27-gauge needle afew times. The membrane proteins were separated by centrifugationat 20,000 x g at 4°C for 30 minutes, and cytosolic proteinswere separated by centrifugation at 60,000 x g at 4°C for60 minutes. The membrane protein pellet was resuspended in 150µL of PBS buffer. The hydrolysis of 2-oleoyl-[³H] glycerol(2-OG) by cytosolic and membrane proteins (96 µg each)was determined according to the previously described method(4) , with modifications. The final volume of the assay was0.5 mL of 100 mmol/L PBS buffer (pH 7.4) in the absence andpresence of MAFP (100 nmol/L) or DAK (1 µmol/L). 2-Oleoyl-[³H]glycerol(20 Ci/mmol; American Radiolabeled Chemical, St. Louis, MO)at 10,000 dpm was used as a substrate. Incubations were carriedout at 37°C for 30 minutes.

Western Blot Analysis of Cannabinoid Receptors.
Cells were lysed as described above in the presence of a proteaseinhibitor. Membrane proteins (50 µg) were separated bySDS-PAGE (Ready Gels) and transferred to a 0.7-µm nitrocellulose(Bio-Rad) membrane. Proteins were incubated with the CB1 andCB2 antibodies. Anti-CB1 and anti-CB2 receptor antibodies raisedin rabbit from Cayman Chemical and Affinity BioReagents (Golden,CO) were used. Then, goat antirabbit IgG-horseradish peroxidase(Zymed Laboratories Inc., South San Francisco, CA) was usedto complex with the primary antibodies. The detection was madeby using Western Lightning Chemiluminescence Reagent (Perkin-Elmer,Boston, MA) and captured by Fuji X-ray film (Tokyo, Japan).

Determination of Protein Kinase A Activity.
The activity of cAMP-dependent protein kinase A (PKA) was determinedby using the PepTag assay kit (Promega, Madison, WI). Cellswere treated with serum-free media for 24 hours before incubationwith vehicle, 2-AG (1 µmol/L), noladin ether (10 µmol/L),MAFP (1 µmol/L), or MAFP (1 µmol/L) with SR141716(500 nmol/L) at 37°C for 30 minutes. The PKA activity assayprotocol was as described in the kit. The fluorescent peptidesubstrate that is highly specific for PKA was used. The phosphorylatedpeptide and nonphosphorylated peptide were then electrophoreticallyseparated on a 0.8% agarose gel at 100 V for 15 (or 25) minutes.Phosphorylated peptide migrated toward the anode, and nonphosphorylatedpeptide migrated toward the cathode. The gel was then visualizedand photographed by an ultraviolet transilluminator (FotodyneInc., Hartland, WI). The band intensity was analyzed by UN-SCAN-ITsoftware (Silk Scientific, Inc., Orem, UT). The intensity ofphosphorylated peptide bands represents the activity of PKA.

	Results and Discussion

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

Cellular Synthesis of 2-Arachidonoylglycerol and Its Effects on Cell Invasion.
Prostate cancer PC-3, DU-145, and LNCaP cells produced 2-AGat high concentrations (in the ng/mg protein range), and RHC-80267(100 nmol/L), a diacylglycerol lipase inhibitor, significantlyinhibited 2-AG production in these three cell lines (Fig. 1A) .Treatment of cells with RHC-80267 (10, 100 nmol/L) increasedthe invasion of PC-3 and DU-145 cells (Fig. 1B) . Of the twocell lines, PC-3 cells (4.2-fold increase) were more sensitiveto RHC-80267 (100 nmol/L) than DU-145 cells (2.0-fold increase).RHC-80267 did not affect invasion of LNCaP cells. These resultssuggest that 2-AG is a potential endogenous inhibitor of invasionof androgen-independent prostate cancer cells. To test the inhibitoryeffect of 2-AG, cells were incubated with exogenous 2-AG (1µmol/L), and cell invasion was measured. However, exogenous2-AG did not inhibit invasion of PC-3, DU-145, and LNCaP cells,but it reversed the cell invasion increase by RHC-80267 in PC-3and DU-145 cells to a level similar to that of the control cells(without RHC–80267). Analysis of radiolabeled speciesobtained after incubation of these cells with [¹⁴C]2-AG for15 minutes indicates that the added [¹⁴C]2-AG was rapidly convertedto [¹⁴C]AA (data not shown). These results suggest that exogenous2-AG is rapidly metabolized by these cells before it binds tocannabinoid receptors and induces a signaling cascade. Therefore,noladin ether (a nonhydrolyzable analog of 2-AG; ref. 15 ),which is a ligand of CB1 receptors (16) and weakly binds toCB2 receptors, was studied. However, noladin ether is less potentthan 2-AG in many systems studied. Noladin ether significantlyinhibited invasion in a concentration-dependent manner of PC-3(67.58 ± 3.15% and 28.89 ± 0.97% invasion at 10and 50 µmol/L, respectively) and DU-145 cells (70.19 ±6.13% at 50 µmol/L), but not LNCaP cells (Fig. 1C) . Inparallel with the effects of RHC-80267, PC-3 cells were moresensitive to the effects of noladin ether than DU-145 cells.

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Fig. 1. 2-AG synthesis and invasion of PC-3, DU-145, and LNCaP cells. A, 2-AG concentrations in PC-3, DU-145, and LNCaP cells. RHC-80267 (100 nmol/L) significantly inhibited 2-AG synthesis in these cells. B, effect of RHC-80267 on invasion of PC-3, DU-145, and LNCaP cells. Invasion was normalized to the control cells for each cell line. Effects of (C) noladin ether, (D) MAFP, and (E) DAK on invasion of PC-3, DU-145, and LNCaP cells. F, effects of MAFP (1 µmol/L), 2-AG (1 µmol/L), and MAFP (1 µmol/L) and 2-AG (1 µmol/L) on invasion of PC-3 cells. Values are mean ± SE (n = 12–18). *, significantly different from control, P < 0.005; #, significantly different from control, P < 0.01; **; significantly different from MAFP treatment, P < 0.05.

Hydrolysis of 2-OG.
Because 2-AG can be metabolized to AA and glycerol and is lesseffective in inhibiting cell invasion than noladin ether, theeffects of inhibition of endogenous 2-AG metabolism on cellinvasion were investigated. Two characterized hydrolases, fattyacid amide hydrolase and monoacylglycerol lipase (17) , andother less well-defined esterases can metabolize 2-AG to AAand glycerol. This is a terminating step of the action of endocannabinoids(3 , 17) . To investigate the inhibitors of 2-AG hydrolysis,the hydrolysis of 2-oleoyl-[³H]glycerol (a readily availableradiolabeled analog of 2-AG) by cytosolic and membrane proteinfractions of each cell type was determined. 2-OG hydrolysisoccurred with both the cytosolic and membrane fractions of eachcell type studied (Table 1) . Interestingly, the relative hydrolysis(expressed as a percentage of conversion of 2-OG) in each fractiondisplayed a different pattern in the three cells: in PC-3 cells,the activity was similar in both fractions (30.45 ± 1.94%in the cytosolic fraction and 21.75 ± 0.90% in the membranefraction); in DU-145 cells, the activity was greater in themembrane fraction (76.06 ± 1.97%) than in the cytosolicfraction (53.19 ± 1.97%); and in LNCaP cells, the activitywas much greater in the membrane fraction (84.55 ± 0.71%)than in the cytosolic fraction (21.40 ± 3.76%). In allof the cells, the nonselective serine esterase/amidase inhibitors,MAFP (100 nmol/L) and DAK (1 µmol/L), significantly reducedbut did not completely eliminate the hydrolysis of 2-OG in thecytosolic and membrane fractions. These concentrations of MAFPand DAK are the concentrations that gave the inhibition at nearthe maximum invasion (see Fig. 1D and E ). Again, the hydrolysisactivity of the membrane fractions in DU-145 and LNCaP cellsremained high in the presence of MAFP and DAK at these concentrations.DAK (25 µmol/L) inhibited 2-OG hydrolysis in these cellsslightly more than DAK (1 µmol/L).

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Table 1 Hydrolysis of 2-OG by cytosolic and membrane proteins of prostate cancer cells in the absence and presence of enzyme inhibitors (100 nM MAFP and 1 µM DAK)

These results suggest that the localization of enzymes may beimportant for 2-AG hydrolysis. Furthermore, these cells maycontain other enzymes such as esterases that can hydrolyze 2-AG.The smaller hydrolysis activity in PC-3 cells suggests thatit may be one of the factors responsible for the greater sensitivityof PC-3 cells to treatment with MAFP and DAK as compared withother cells.

Effects of Inhibition of 2-Arachidonoylglycerol Metabolism on Cell Invasion.
Cells were incubated with MAFP and DAK to inhibit 2-AG metabolism,and invasion was determined. Both MAFP (Fig. 1D) and DAK (Fig.1E) inhibited invasion of PC-3 and DU-145 cells but were withouteffect in LNCaP cells. Exogenous 2-AG alone did not inhibitinvasion of PC-3 cells (probably due to its rapid metabolism).However, when PC-3 cells were incubated with 2-AG (1 µmol/L)in the presence of MAFP (1 µmol/L), 2-AG augmented theinhibition of cell invasion by MAFP (Fig. 1F ; 61.87 ±2.85% for MAFP and 46.54 ± 2.90% for MAFP and 2-AG).These results indicate that the blockade of 2-AG metabolisminhibits cell invasion, and they further support the hypothesisthat endogenous 2-AG is a negative regulator of invasion inandrogen-independent prostate cancer cells.

Cannabinoid Receptors and Cell Invasion.
To investigate whether 2-AG regulation of cell invasion is CBreceptor dependent, the expression of CB receptors was determinedin these cells. Antibodies against CB1 and CB2 receptors fromtwo different sources were used with identical results. Westernblot analysis indicates that all three prostate cancer celllines examined here express CB1 and CB2 receptor proteins (at55 and 50 kDa, respectively; Fig. 2A and B ) as reported previously(8 , 18) . CB1 receptor expression, as compared with ß-actin,is higher in PC-3 cells (0.13) than in DU-145 (0.07) cells andLNCaP cells (0.06), whereas CB2 receptor expression in PC-3cells (0.11) is similar to that in DU-145 cells (0.12) but higherthan that in LNCaP cells (0.06).

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Fig. 2. Expression of CB receptors and effects of CB agonists/antagonists on invasion of PC-3, DU-145, and LNCaP cells. A, Western blots depicting immunoreactive bands corresponding to CB1 receptor (at 55 kDa) with rat cerebellum as a positive control. B, Western blots depicting immunoreactive bands corresponding to CB2 receptor (at 50 kDa) with rat spleen as a positive control. The intensity of immunoreactive bands was normalized to the intensity of ß-actin. (C) Effect of WIN55,212-2, a CB receptor agonist, (D) effect of (R)-(+)-methanandamide, a CB1 selective agonist, and (E) effect of SR141716, a CB1 selective antagonist, on invasion of PC-3, DU-145, and LNCaP cells. Values are mean ± SE (n = 12–18). F, effects of MAFP (1 µmol/L) and MAFP (1 µmol/L) with SR141716 (SR1; 500 nmol/L) on invasion of PC-3 cells. Values are mean ± SE (n = 6). *, significantly different from control, P < 0.005; #, significantly different from control, P < 0.01.

To further characterize the pharmacological response of theCB receptors on cell invasion, cells were treated with WIN55,212-2(a nonselective CB agonist), (R)-(+)-methanandamide (a selectiveCB1 agonist), SR141716

(a selective CB1 antagonist), and SR144528

(a selective CB2 antagonist), and cell invasion was measured.Both WIN55,212-2 (Fig. 2C) and (R)-(+)-methanandamide (Fig.2D) inhibited cell invasion in a concentration-dependent manner,whereas SR141716

(Fig. 2E) increased invasion of PC-3 and DU-145cells. These CB agonists and antagonists did not significantlyaffect the invasion of LNCaP cells. SR144528

did not affectthe invasion of any of the cell lines (data not shown). Furthermore,SR141716

(500 nmol/L) reversed the inhibition of cell invasionby MAFP (1 µmol/L) in PC-3 cells (Fig. 2F ; 63.18 ±2.75% for MAFP and 98.76 ± 3.98% for MAFP and SR141716

).These results suggest that the inhibition of cell invasion ofandrogen-independent prostate cancer cells by 2-AG involvesa CB1 receptor pathway. A higher relative expression of CB1receptors in PC-3 cells may be one of the reasons that thiscell line is more sensitive to endogenous 2-AG, noladin ether,and CB agonists than other cells.

Protein Kinase A Activity.
All prostate cancer cells investigated express CB1 receptors,but the inhibition of cell invasion was observed only in androgen-independentcells. Activation of the CB1 receptor leads to inhibition ofadenylyl cyclase activity (19) , which may result in a disruptionof downstream signaling transduction pathway(s) that regulatescell invasion (20) . To determine whether CB1 receptor activationinhibited adenylyl cyclase in these prostate cancer cells, cAMP-dependentPKA activity was determined in cells treated with 2-AG and MAFPor with noladin ether. Treatment with 2-AG (1 µmol/L)+ MAFP (1 µmol/L) or with noladin ether (10 µmol/L)decreased PKA activity in PC-3 and DU-145 cells but slightlyincreased the activity in LNCaP cells (Fig. 3A) . The averageof PKA activities (n = 4) of cells treated with 2-AG + MAFPor with noladin ether were 62.0 ± 6.0% and 55.3 ±10.4% of control, respectively, for PC-3 cells; 79.2 ±12.8% and 61.8 ± 3.6% of control, respectively, for DU-145cells; and 138.9 ± 10.1% and 118.1 ± 12.3% ofcontrol, respectively, for LNCaP cells (Fig. 3B) . Furthermore,MAFP (1 µmol/L) inhibited PKA activity (73.43 ±3.93% of control) in PC-3 cells, and SR141716 reversed thisinhibition (111.71 ± 4.37% of control; Fig. 3C and D ).These results suggest that inhibition of PKA activity by 2-AGand noladin ether may be one of the inhibition effects 2-AGhas on androgen-independent prostate cancer cell invasion.

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Fig. 3. Effects of 2-AG and noladin ether on PKA activity. A, representative photographs of nonphosphorylated bands and phosphorylated bands of PKA-specific substrate peptide (30-minute reaction time) in PC-3, DU-145, and LNCaP cells incubated with vehicle (Lane 1), 2-AG (1 µmol/L) and MAFP (1 µmol/L; Lane 2), and noladin ether (10 µmol/L; Lane 3) [15-minute electrophoretic run time]. B, the average of relative intensity of phosphorylated bands in these cells. The intensity was normalized to the control cells for each gel (as 100%). Values are mean ± SE (n = 4). C, representative photographs of nonphosphorylated bands and phosphorylated bands of PKA-specific substrate peptide (45-minute reaction time) in PC-3 cells incubated with vehicle (Lane 1), MAFP (1 µmol/L; Lane 2), and MAFP (1 µmol/L) plus SR141716 (SR1; 500 nmol/L; Lane 3) [25-minute electrophoretic run time]. D, the average relative intensity of phosphorylated bands. The intensity was normalized to the control cells for each gel (as 100%). Values are mean ± SE (n = 4). *, significantly different from control, P < 0.005; #, significantly different from control, P < 0.01.

In conclusion, prostate cancer cells produce 2-AG. A stableanalog of 2-AG, blockade of 2-AG metabolism, and binding ofagonists to CB1 receptors inhibited the invasion of PC-3 andDU-145 cells. Conversely, inhibition of 2-AG synthesis and blockadeof CB1 receptors increased their cell invasion. Taken together,the following three observations may be responsible for a differentinhibition of cell invasion in these prostate cancer cells:(a) a different activity for glycerol hydrolysis in membraneand cytosolic fractions; (b) a different relative expressionof CB1 receptors; and (c) a different effect of 2-AG on PKAactivity in these cells. The results from this study suggestthat endogenous 2-AG possesses an anti-invasive effect in androgen-independentprostate cancer cells through a CB1-dependent pathway by inhibitingadenylyl cyclase and decreasing the activity of PKA and resultsin a further downstream signaling cascade that inhibits cellinvasion. These results potentially lead to the understandingof mechanisms involved in metastasis and resistance to hormonetherapy in prostate cancer. Furthermore, specific inhibitorsof enzymes degrading 2-AG and CB1 agonists may be importanttherapeutic agents for hormone-refractory prostate cancer.

	ACKNOWLEDGMENTS

We thank Craig T. Roelke and Blythe B. Holmes for excellenttechnical assistance.

	FOOTNOTES

Grant support: Grants from the Medical College of WisconsinCancer Center and the National Institute of Health (DA 09155).

The costs of publication of this article were defrayed in partby the payment of page charges. This article must thereforebe hereby marked advertisement in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

Requests for reprints: Kasem Nithipatikom, Department of Pharmacologyand Toxicology, Medical College of Wisconsin, 8701 WatertownPlank Road, Milwaukee, WI 53226. Phone: 414-456-8605; Fax: 414-456-6545;E-mail: kasemn@mcw.edu.

Received 8/31/04. Revised 10/ 7/04. Accepted 10/29/04.

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