TRPV6 Determines the Effect of Vitamin D3 on Prostate Cancer Cell Growth

Despite remarkable advances in the therapy and prevention of prostate cancer it is still the second cause of death from cancer in industrialized countries. Many therapies initially shown to be beneficial for the patients were abandoned due to the high drug resistance and the evolution rate of the tumors. One of the prospective therapeutical agents even used in the first stage clinical trials, 1,25-dihydroxyvitamin D3, was shown to be either unpredictable or inefficient in many cases. We have already shown that TRPV6 calcium channel, which is the direct target of 1,25-dihydroxyvitamin D3 receptor, positively controls prostate cancer proliferation and apoptosis resistance (Lehen'kyi et al., Oncogene, 2007). However, how the known 1,25-dihydroxyvitamin D3 antiproliferative effects may be compatible with the upregulation of pro-oncogenic TRPV6 channel remains a mystery. Here we demonstrate that in low steroid conditions 1,25-dihydroxyvitamin D3 upregulates the expression of TRPV6, enchances the proliferation by increasing the number of cells entering into S-phase. We show that these pro-proliferative effects of 1,25-dihydroxyvitamin D3 are directly mediated via the overexpression of TRPV6 channel which increases calcium uptake into LNCaP cells. The apoptosis resistance of androgen-dependent LNCaP cells conferred by TRPV6 channel is drastically inversed when 1,25-dihydroxyvitamin D3 effects were combined with the successful TRPV6 knockdown. In addition, the use of androgen-deficient DU-145 and androgen-insensitive LNCaP C4-2 cell lines allowed to suggest that the ability of 1,25-dihydroxyvitamin D3 to induce the expression of TRPV6 channel is a crucial determinant of the success or failure of 1,25-dihydroxyvitamin D3-based therapies

The effects of 1,25-dihydroxyvitamin D3 on androgen-independent cell lines.

<p><b>A</b>, <b>B</b>, The effects of 1,25-dihydroxyvitamin D3 on androgen receptor-deficient DU-145 cell line in both 2 and 10% FCS-containing RPMI medium (<b>A</b> and <b>B</b>, respectively), * - P<0.05 (as compared to control), n = 3. <b>C, D</b>, The effects of 1,25-dihydroxyvitamin D3 on androgen-insensitive LNCaP C4-2 cell line in both 2 and 10% FCS-containing RPMI medium (<b>C</b> and <b>D</b>, respectively), * - P<0.05 (as compared to control), n = 3. <b>E</b>, the relative expression levels of TRPV6 channel in DU-145 cells treated with 100 µM 1,25-dihydroxyvitamin D3 for 3 days in 2 and 10% FCS-containing RPMI medium, * - P<0.05 (as compared to control), n = 3. <b>F</b>, the expression of TRPV6 channel induced by 100 nM 1,25-dihydroxyvitamin D3 for 3 days in LNCaP cells in steroid-deprived RPMI medium (LNCaP-ST), n = 3.</p

Primers and siRNA.

<p>Primers and siRNA.</p

The effects of 1,25-dihydroxyvitamin D3 on proliferation and apoptosis resistance of LNCaP cells are mediated via TRPV6 channel.

<p><b>A</b>, LNCaP cells proliferation in 2% FCS-containing RPMI medium treated with 1,25-dihydroxyvitamin D3 (100 nM, applied at D1), siRNA-TRPV6 (siTRPV6, 80 nM, transfected at D0), the combined treatment of 1,25-dihydroxyvitamin D3 and siTRPV6 specified above, and siRNA-AR (siAR, 80 nM, transfected at D0) as a positive control. * - P<0.05, ** - P<0.01, as compared to control, n = 4; <b>B</b>, a cell cycle assay of LNCaP cells (incubated with 2% FCS-containing RPMI medium) for the same conditions as in MTS assay (<b>A</b>) (D3 equals 100 nM 1,25-dihydroxyvitamin D3), carried out by flow cytometry of the cells stained with propidium iodide. * - P<0.05, ** - P<0.01, § - P<0.05 vs. Vitamin D3; n = 3. <b>C</b>, a western-blotting of proliferating cell nuclear antigen (PCNA) in the conditions indicated above as compared to β-actin. <b>D</b>, an apoptosis assay carried out by flow cytometry as a subG1 population of LNCaP cells cultured in 2% FCS-containing RPMI medium stained with propidium iodide. * - P<0.01 vs. control; n = 3.</p