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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 4, 2003 as doi:10.1096/fj.03-0254fje. |
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UCLA Lung Cancer Research Program of the Jonsson Comprehensive Cancer Center and the Division of Pulmonary and Critical Care Medicine, David Geffen School of Medicine at UCLA and VA Greater Los Angeles Healthcare System, Los Angeles, California, USA
2Correspondence: UCLA Lung Cancer Research Program, David Geffen School of Medicine at UCLA, 37-131 Center for Health Sciences, 10833 Le Conte Ave., Los Angeles, CA 90095-1690, USA. E-mail: sdubinett{at}mednet.ucla.edu
SPECIFIC AIM
The ability of methanandamide to modulate murine lung cancer growth was evaluated. We investigated the involvement of the cannabinoid receptors and methanandamide-mediated increased COX-2 and PGE2 production in the observed increased tumor growth.
PRINCIPAL FINDINGS
1. Systemic methanandamide administration increases tumor growth
We have previously shown that systemic THC treatment suppresses the immune response to implanted tumors, resulting in increased tumor growth via CB2 receptor signaling. Because endogenous ligands for the cannabinoid receptors are widely distributed and are elevated in prostate, bladder, and colon carcinoma tissues, we were prompted to determine the effect of endocannabinoids on tumor growth in vivo. We evaluated the effect of methanandamide administration (5 mg/kg, i.p. four times per week) on tumorigenicity in murine lung cancer. We found a significant enhancement of tumor growth in methanandamide-treated mice compared with diluent-treated controls (n=9, P<0.01).
2. Methanandamide-mediated enhancement of tumor growth is cannabinoid receptor independent
Cannabinoid receptors have been implicated in both the enhancement and inhibition of tumor cell proliferation. To determine whether methanandamide-enhanced tumor growth involved the CB1 or CB2 cannabinoid receptors, we used cannabinoid receptor antagonists in combination with methanandamide treatment in an attempt to block methanandamide-enhanced tumor growth. Neither the CB1 receptor antagonist SR141716 (1 mg/kg) nor the CB2 receptor antagonist SR 144528 (1 mg/kg) blocked the increased rate of tumor growth observed with methanandamide treatment, suggesting that methanandamide-enhanced tumor growth is cannabinoid receptor independent.
3. In vivo, methanandamide treatment increases splenocyte and tumoral PGE2 production
Increased production of PGE2 is associated with several different malignancies, including lung cancer. Based on previous studies demonstrating that cannabinoids increase the production of prostaglandins, we determined whether methanandamide treatment increases production of PGE2 in splenocytes and the amount of PGE2 present at the tumor site. We found that methanandamide treatment significantly increased splenocyte PGE2 production and increased the level of PGE2 present at the tumor site compared with diluent-treated controls (Fig. 1
).
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4. In vitro, methanandamide treatment increases murine lung cancer cell COX-2 and PGE2 production in a cannabinoid receptor independent manner
To determine whether tumor cells are one source of the increased PGE2 at the tumor site, we evaluated the ability of methanandamide to directly stimulate tumor cell production of PGE2 and expression of COX-2 in vitro. Methanandamide (13.8 µM, 24 h) -treated L1C2 cells increased PGE2 production and expression of COX-2, suggesting that tumor cells are one source of the increased PGE2 observed in vivo. Because the increased rate of tumor growth observed in methanandamide-treated mice could not be blocked by cannabinoid receptor antagonists, we were prompted to determine whether methanandamide stimulation of tumor cell PGE2 production in vitro was also independent of cannabinoid receptor signaling. L1C2 cells were cultured with methanandamide (13.8 µM, 24 h) in the presence or absence of SR141716 (2 µM) or SR144528 (2 µM) and production of PGE2 was determined in the tumor supernatants. Neither CB1 nor CB2 receptor antagonists blocked methanandamide's capacity to increase PGE2 production, suggesting a cannabinoid receptor-independent mechanism.
5. The ability of methanandamide to increase tumor growth is abrogated by administration of the COX-2 inhibitor SC58236
Elevated COX-2 activity inhibits immune response and increased expression enhances tumor growth. Because methanandamide treatment increases COX-2, we speculated that administration of the COX-2 inhibitor SC58236 would abrogate the ability of methanandamide to increase tumor growth. BALB/c mice (12 mice/group) were treated with methanandamide (5 mg/kg), SC58236 (0.1 mg/kg), methanandamide in combination with SC58236, or diluent control. COX-2 inhibition abrogated methanandamide-mediated increased tumor growth (Fig. 2
).
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CONCLUSIONS AND SIGNIFICANCE
Cannabinoid regulation of prostaglandin production may be one mechanism by which cannabinoids modulate tumor growth. In vitro studies have shown that exposure to cannabinoids can inhibit tumor cell proliferation. In keeping with these in vitro findings, studies in immunocompromised model systems indicate that local or intratumoral injection of THC or CB2 receptor agonists can inhibit tumor growth. In contrast with these reports, we previously showed that systemic administration of THC leads to a CB2 receptor-dependent increase in tumor growth in immune competent mice. Because endocannabinoids are present in many tissues and their receptors are expressed by immune cells, we were prompted to investigate their effect on tumor growth in the context of a functioning immune system. Here, we show that methanandamide administration increases the rate of murine lung cancer growth in vivo.
Cannabinoids exert their biological effects via cannabinoid receptor-dependent and -independent pathways. CB1 and CB2 receptors are expressed on cells of the immune system and thus could mediate cannabinoid regulation of immune responses. The CB2 receptor is the predominant cannabinoid receptor expressed by immune cells, being expressed from 3- to 100-fold the level of the CB1 receptor. We previously reported that THC inhibited the immune response to tumor growth via a CB2 receptor signaling pathway. The present findings suggest that in contrast to THC, methanandamide-mediated augmentation of tumor growth occurs via a cannabinoid receptor-independent pathway.
Because endocannabinoids can stimulate arachidonate metabolism, we assessed PGE2 levels in methanandamide-treated mice. Methanandamide treatment led to an increase in PGE2 production by splenocytes and the amount present at the tumor. To determine the contribution of the tumor to the increased production of PGE2, we evaluated the ability of methanandamide to stimulate tumor cell PGE2 production in vitro. Methanandamide increased tumor cell PGE2 production and COX-2 levels. These findings suggest that the tumor is one source of the increased PGE2. Consistent with our in vivo results demonstrating that methanandamide-enhanced tumor growth is cannabinoid receptor independent, we found that methanandamide-mediated increased tumor cell PGE2 production in vitro was also cannabinoid receptor independent. The increased PGE2 production was dependent on the activity of COX-2, as shown by the ability of COX-2 inhibition to block methanandamide stimulation of PGE2 production. Methanandamide-mediated increased tumor growth was abrogated by administration of the COX-2 inhibitor SC58236. Thus, methanandamide induction of COX-2 could affect multiple mechanistic pathways promoting lung cancer tumorigenicity.
The levels of endocannabinoids are elevated in prostate, colon, and bladder cancer tissues compared with normal adjacent tissue. Therefore, we investigated in vitro the ability of anandamide and 2-arachidonylglycerol (2-AG) to stimulate tumor cell PGE2 production and COX-2 levels. We found that anandamide and 2-AG increased murine tumor PGE2 production and COX-2 protein production. Thus, endocannabinoids present in the tumor environment may enhance tumor progression as a function of heightened COX-2 expression. Ramer et al. recently reported that methanandamide stimulates the production of COX-2 via p38 or p42/44 MAP kinase pathways in human neuroglioma cells. In agreement with these findings, we report here that the p38/MAPK inhibitor SB203508 and the p42/44 MAP kinase inhibitor PD98059 blocked the methanandamide-induced increase in PGE2 production and COX-2 expression in murine lung cancer cells. Further studies are required to determine the significance of these observations and the signaling pathways operative in endocannabinoid regulation of COX-2.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0254fje; doi: 10.1096/fj.03-0254fje 
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5 mg/kg) or diluent (
) as described for assessment of tumor growth. Splenocytes were isolated, cultured at a cellular concentration of 106 cells/mL for 3 days, and production of PGE2 was determined in the culture supernatant. B) Two weeks after implantation of 105 L1C2 cells, tumors were removed and homogenized for PGE2 determination (* P<0.02).
), SC 58236 (0.1 mg/kg, 
), or diluent control (
) four times/wk. 105 L1C2 cells were then implanted s.c., and mice continued to receive treatments throughout the remainder of the experiment. COX-2 inhibition blocked methanandamide-enhanced tumor growth (n=12, P<0.01 for methanandamide vs. methanandamide+SC58236 treatment groups).