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Capsaicin Inhibits the Growth of Prostate Cancer Cells

of NF-nB binding sites attached to the pGL3 luciferase reporter plasmid. Following transfections, cells were incubated with either capsaicin (2 104 mol/L) or diluent control for 24 or 48 hours (Fig. 4A). Luciferase activities were 61% and 81% lower in capsaicin-treated cells compared with control cells after 24 and 48 hours of culture, respectively.

Figure 2. Effect of capsaicin on the transcriptional activity of the PSA promoter/enhancer in LNCaP cells. LNCaP cells were transfected with PSA enhancer-Luciferase (Luc) reporter [ARE sites, darkened rectangles (top right)] and cultured for 24 hours with dihydrotestosterone (108 mol/L) either with or without 2 104 mol/L of capsaicin. PhRLTK vector was cotransfected for normalization. Columns, means of three or more experiments; bars, FSD.

Western blot analysis confirmed that up-regulation of AR occurred in pCMV-AR–transfected cells (data not shown). PSA P/E-Luc reporter activity increased f55-fold in the presence of exogenous AR and dihydrotestosterone. Capsaicin strongly inhibited this activation, suggesting that the ability of capsaicin to inhibit PSA transcriptional activity was not only via down- regulation of AR expression, but also by directly inhibiting the ability of ligand-activated AR to transcriptionally activate the PSA gene. Furthermore, the proliferation of these cells was assessed by performing colony-forming assays in the presence of capsaicin (105 mol/L, 2 104 mol/L). Capsaicin had the same potency to inhibit the growth of LNCaP cells when overexpressing AR (data not shown), suggesting that the growth-inhibitory effect of capsaicin is not mediated via down-regulation of AR expression.

In order to exert its transcriptional effects, AR accumulates in the nucleus. To investigate whether capsaicin affected dihydrotes- tosterone-induced nuclear accumulation of AR, the subcellular localization of AR was studied by immunofluorescence. Figure 3D shows the localization of AR in LNCaP cells after 12 hours of incubation either without dihydrotestosterone (left), with dihy- drotestosterone (108 mol/L; center) or with both dihydrotestos- terone (108 mol/L) and capsaicin (2 104 mol/L; right). In unexposed LNCaP cells, AR is already localized mainly to the nucleus (Fig. 3D, left). However, when cultured with dihydrotes- tosterone, nuclear localization becomes more prominent (Fig. 3D, center), an effect that could not be reversed by capsaicin (Fig. 3D, right), suggesting that the effect of capsaicin on dihydrotestoster- one-induced ARE transcription is not mediated through inhibition of nuclear localization.

Effect of capsaicin on NF-KB promoter activity in PC-3 cells. Previous studies in murine myeloid leukemia cells suggested that capsaicin inhibits the activity of the transcription factor NF-nB by blocking the degradation of InBa (19). NF-nB is constitutively activated in the androgen-independent prostate cancer cell lines PC-3, but not in the androgen-responsive LNCaP cells (13). In order to investigate whether the anti-growth effect of capsaicin on prostate cancer cells is mediated at least in part by NF-nB, PC-3 cells were transfected with NF-nB-Luc, which contains four copies

Activation of NF-nB involves two important steps: (a) degrada- tion of InBa mediated by IKK, resulting in the release of NF-nB, and (b) nuclear translocation of activated NF-nB (15, 16). Initially, therefore, NF-nB status in the PC-3 nucleus was examined. Cells were incubated with capsaicin (2 104 mol/L) for either 2 or 4 hours. After 4 hours of culture with capsaicin, NF-nB levels in the nuclear extracts decreased 84%, whereas NF-nB levels in the whole cell extract decreased 28% of control, suggesting that nuclear translocation of NF-nB was inhibited (Fig. 4B). Subcellular localization of p65, either in the absence or presence of capsaicin (Fig. 4C, left and right panels, respectively), was also studied in these cells by immunofluorescence. Nuclear staining of p65 decreased when PC-3 cells were exposed to capsaicin, confirming that nuclear translocation of NF-nB was inhibited by the compound. Further studies sought to determine whether the inhibitory effect of capsaicin was due to the prevention of InBa degradation. PC-3 cells, either untreated or pretreated with capsaicin (2 104 mol/L, 3 hours, 37jC), and then exposed to TNF (20 ng/mL), were assayed for the amount of cytoplasmatic InBa by Western blot analysis (Fig. 4D). TNF treatment caused InBa to disappear completely by 15 minutes. Pretreatment of these cells with capsaicin, however, prevented the degradation of InBa. Also, PC-3 cells incubated with only with capsaicin (1 or 2 104 mol/L, 2 hours) had an increase in cytoplasmatic levels of InBa (Fig. 4E). Taken together, these results suggest that capsaicin inhibits InBa degradation in the cytoplasm.

Degradation of InBa results from the phosphorylation of InBa by InB kinases, followed by its ubiquitination and degradation by the multicatalytic 26S proteasome (15). To determine whether capsaicin affected the proteasome activity of PC-3 cells, these cells were treated with capsaicin (2 104 mol/L, 24 or 48 hours), followed by proteasome functional assay. Chymotrypsin-like, trypsin-like, and PGPH proteasome activities were down-regulated by capsaicin (Fig. 5). Finally, to study if the anti-proliferative effect of capsaicin could be affected by the overexpression of IKKh, PC-3 cells were transfected with an IKKh expression vector. These cells exhibited significantly increased nuclear localization of p65 but did not affect their sensitivity to capsaicin compared with cells transfected with the control vector (data not shown). These findings suggest that the inhibiting effect of capsaicin on proliferation is not mediated through down-regulation of IKKh and that the effect of capsaicin on InBa degradation is still dominant over IKKh expression.

The antiproliferative effect of capsaicin is not related to TRPV1. Capsaicin exerts its physiologic function in sensory neurons by intracellular binding to TRPV1 (24). Therefore, we studied whether the antiproliferative effect of capsaicin is related to TRPV1. Initially, TRPV1 was confirmed to be expressed in both PC-3 and LNCaP cells, using real-time reverse transcription- PCR (data not shown). To determine the involvement of TRPV1 in the antiproliferative action of capsaicin, LNCaP and PC-3

cell (10 (10 10 s were cultured with the TRPV1 antagonists capsazepine and 104 mol/L), ruthenium red (105 mol/L), or SB366791 mol/L), either with or without capsaicin (107 to 2 mol/L) for 96 hours, and measuring cell viability by MTT 5 5 4



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

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