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Cancer Research

stabilizes the InBa and/or inhibits the IKK activity, resulting in the inhibition of InBa degradation. In addition, activation of the NF-nB pathway is normally achieved by phosphorylation, polyubiquitina- tion, and subsequent degradation of InBa by the 26S proteasome (15). In general, proteasome activity correlates with a robust NF-nB signaling pathway in the cancer cells; and PC-3 cells are known to have high proteasome activity (26). We showed that capsaicin inhibited proteasome activity in PC-3 cells, suggesting that this inhibition of proteasome function stabilizes InBa expression, preventing NF-nB nuclear localization in PC-3 cells. Overexpression of IKKh, which is the main IKK kinase responsible for the phosphorylation of InB, did not affect capsaicin sensitivity in PC-3 cells, suggesting that the effect of capsaicin on the NF-nB signaling pathway is not mediated through inhibiting IKK but rather through its effect on InBa.

We also showed a profound effect of capsaicin on the levels of the AR-regulated PSA gene as well as AR itself in prostate cancer cells. Capsaicin profoundly inhibited protein levels of PSA and AR. Our reporter gene studies showed that capsaicin inhibited androgen activation of the AREs in the PSA regulatory region. Furthermore, reporter gene experiments revealed that even when AR was exogenously overexpressed in LNCaP cells, capsaicin strongly suppressed the PSA transcriptional activity, suggesting that capsaicin suppressed PSA transcription even when high levels of AR were present. Also, overexpression of AR did not affect the antiproliferative effect of capsaicin, suggesting that down- regulation of AR is not the decisive mechanism of action for capsaicin. By immunofluorescence, we also showed that capsaicin did not inhibit the dihydrotestosterone-induced nuclear transloca- tion of AR. These results suggest that capsaicin acts neither through down-regulation of AR, nor by inhibiting the nuclear translocation of AR. The androgen-AR complex cooperates with various coregulators to modulate its target genes for proper function (27–30). Some of these coregulators contain E3 ligase activity, which regulate AR activity via the ubiquitin-proteasome pathway (27–30). Furthermore, inhibition of the proteasome suppresses AR transactivation and interaction between AR and AR coregulators (31). Our studies revealed that capsaicin suppressed proteasome activity, which may be involved in the disruption of AR activity.

Capsaicin was crucial to the discovery of TRVP1 and the therapeutic effects of the drug in pain disorders are mainly mediated through interaction between the drug and this receptor

Figure 6. Capsaicin inhibits human prostate cancer cells growing in mice in vivo. PC-3 cells (5 106) were injected s.c. into bilateral flanks of 10 male BNX nu/nu mice, forming two tumors per mouse. Capsaicin (5 mg/kg/d; five mice) or diluent (five mice) was given by gavage, 3 days per week for 4 weeks. A, tumor sizes were measured weekly. B, after 4 weeks of therapy, tumors were dissected from all mice and weighted. All statistical tests were two-sided.

(25). However, our experiments using the three TRVP1-inhibitors capsazepine, ruthenium red, and SB366791, did not show any attenuation of the inhibitory activity of capsaicin. Also, concen- trations of capsaicin used to exert the antiproliferative effects in cancer cells are considerably higher than those needed to affect TRVP1 (32). Taken together, our results indicate that the anticancer effects are not mediated through interaction with TRVP1, which is consistent with what others have reported (33). Alternative mechanisms of action that have been associated with capsaicin includes increased production of reactive oxygen species (34), increased NO production (35), and effects mediated through PPARg (36).

In conclusion, we show that capsaicin has potent activity against not only androgen-sensitive, but also androgen-independent prostate cancer cells in vitro and in vivo. Given the clinical tolerability of capsaicin, a clinical trial of this agent seems appropriate in selected individuals with prostate cancer.

Acknowledgments

Received 1/13/2005; revised 11/15/2005; accepted 1/17/2006.

Figure 5. Proteasome activity following exposure to capsaicin. PC3 cells were cultured with capsaicin (2 104 mol/L), harvested at the indicated times and chymotrypsin-like, trypsin-like, and PGPH activities were assayed using appropriate fluorogenic peptides as described in Materials and Methods.

Grant support: NIH grants, George Harrison Trust, and the Parker Hughes Fund. H.P. Koeffler is the holder of the Mark Goodson Endowed Chair of Oncology and is a member of both the Molecular Biology Institute and Jonsson Comprehensive Cancer Center, University of California at Los Angeles.

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.

Cancer Res 2006; 66: (6). March 15, 2006

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