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TRPV6 determines the effect of vitamin D3 on prostate cancer cell growth.

Lehen'kyi V, Raphaël M, Oulidi A, Flourakis M, Khalimonchyk S, Kondratskyi A, Gordienko DV, Mauroy B, Bonnal JL, Skryma R, Prevarskaya N - PLoS ONE (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Inserm, U-1003, Equipe labellisée par la Ligue Nationale contre le cancer, Villeneuve d'Ascq, France.

ABSTRACT
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, enhances 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.

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The effects of 1,25-dihydroxyvitamin D3 on proliferation of LNCaP cells and expression of TRPV6 channel.A, 1,25-dihydroxyvitamin D3 effects on proliferation rate measured by MTS assay of LNCaP cells incubated either with 2% or 10% FCS-containing RPMI medium, * - P<0.05, ** - P<0.01, as compared to their respective controls (DMSO), n = 3. B, The upregulation of TRPV6 mRNA expression by 1,25-dihydroxyvitamin D3 in LNCaP cells cultured in 10% FCS-containing RPMI medium; * - P<0.05, as compared to control (DMSO), n = 3. C, The upregulation of TRPV6 mRNA expression by 1,25-dihydroxyvitamin D3 in LNCaP cells cultured in 2% FCS-containing RPMI medium; * - P<0.05, ** - P<0.01, as compared to control (DMSO), n = 3. D, The time-dependence of TRPV6 expression under 100 µM 1,25-dihydroxyvitamin D3 treatment in LNCaP cells incubated in 10% FCS-containing RPMI medium. * - P<0.05, ** - P<0.01, as compared to control (DMSO), n = 3. E, a western-blotting of TRPV6 protein levels induced by 1,25-dihydroxyvitamin D3 treatment for 3 days in LNCaP cells incubated in 2% FCS-containing RPMI medium. F, A confocal microscopy showing the pattern of TRPV6 protein expression and localisation onto the plasma membrane of LNCaP cells cultivated in 2% FCS-containing RPMI medium. Cholera toxin conjugated to FITC (CTX, green) used to stain the plasma membrane as well as the TRPV6 channel (TRPV6, red), and their respective merge (CTX+TRPV6) are shown.
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pone-0016856-g001: The effects of 1,25-dihydroxyvitamin D3 on proliferation of LNCaP cells and expression of TRPV6 channel.A, 1,25-dihydroxyvitamin D3 effects on proliferation rate measured by MTS assay of LNCaP cells incubated either with 2% or 10% FCS-containing RPMI medium, * - P<0.05, ** - P<0.01, as compared to their respective controls (DMSO), n = 3. B, The upregulation of TRPV6 mRNA expression by 1,25-dihydroxyvitamin D3 in LNCaP cells cultured in 10% FCS-containing RPMI medium; * - P<0.05, as compared to control (DMSO), n = 3. C, The upregulation of TRPV6 mRNA expression by 1,25-dihydroxyvitamin D3 in LNCaP cells cultured in 2% FCS-containing RPMI medium; * - P<0.05, ** - P<0.01, as compared to control (DMSO), n = 3. D, The time-dependence of TRPV6 expression under 100 µM 1,25-dihydroxyvitamin D3 treatment in LNCaP cells incubated in 10% FCS-containing RPMI medium. * - P<0.05, ** - P<0.01, as compared to control (DMSO), n = 3. E, a western-blotting of TRPV6 protein levels induced by 1,25-dihydroxyvitamin D3 treatment for 3 days in LNCaP cells incubated in 2% FCS-containing RPMI medium. F, A confocal microscopy showing the pattern of TRPV6 protein expression and localisation onto the plasma membrane of LNCaP cells cultivated in 2% FCS-containing RPMI medium. Cholera toxin conjugated to FITC (CTX, green) used to stain the plasma membrane as well as the TRPV6 channel (TRPV6, red), and their respective merge (CTX+TRPV6) are shown.

Mentions: The effect of 1,25-dihydroxyvitamin D3 on prostate cancer cell proliferation has been studied in two experimental conditions: 2% and 10% foetal calf serum (FCS)-supplemented RPMI medium. The growth of androgen-dependent LNCaP cell line was surprisingly increased by 100 nM 1,25-dihydroxyvitamin D3 in 2% FCS supplemented medium and suppressed in 10% FCS (Fig. 1A). We have already demonstrated the role of TRPV6 channel in proliferation of prostate cancer cells [15], and therefore we sought to investigate the regulation of TRPV6 channel expression by 1,25-dihydroxyvitamin D3. Since it has been shown that trpv6 is a VDR-regulated gene [17], we have studied the regulation of TRPV6 expression by 1,25-dihydroxyvitamin D3 in LNCaP cells in different steroid content of the media (Fig. 1B, C). 1,25-dihydroxyvitamin D3 appears to directly activate the trpv6 gene in LNCaP cells, though in 10% FCS medium its effects were not that significant (Fig. 1B) than in 2% FCS (Fig. 1C). 1,25-dihydroxyvitamin D3 significantly dose-dependently increased TRPV6 mRNA expression in 2% FCS-containing RPMI medium (Fig. 1C). To check whether the diminished effects of 1,25-dihydroxyvitamin D3 were due to FCS content and not to the optimal effect time we performed the time curve using the maximal concentration of 100 nM over three days at different time intervals (Fig. 1D). To confirm the significant induction of TRPV6 protein by 1,25-dihydroxyvitamin D3 in 2% FCS containing RPMI medium obtained by real time quantitative PCR a western-blotting was performed. It showed a considerable increase in TRPV6 protein level upon activation with 100 nM 1,25-dihydroxyvitamin D3 (Fig. 1E). Immunocytochemistry using TRPV6 specific antibody showed the expression of TRPV6 channels in LNCaP cells (Fig. 1F) as well as its localisation on the plasma membrane using Cholera toxin (CTX) conjugated with FITC labelling specifically G2M lipids in the membrane. Hence, the effects of 1,25-dihydroxyvitamin D3 on the growth of androgen-dependent LNCaP cells depend on the relative steroid content. Besides, 1,25-dihydroxyvitamin D3 significantly increases the expression of TRPV6 channel in low-steroid conditions.


TRPV6 determines the effect of vitamin D3 on prostate cancer cell growth.

Lehen'kyi V, Raphaël M, Oulidi A, Flourakis M, Khalimonchyk S, Kondratskyi A, Gordienko DV, Mauroy B, Bonnal JL, Skryma R, Prevarskaya N - PLoS ONE (2011)

The effects of 1,25-dihydroxyvitamin D3 on proliferation of LNCaP cells and expression of TRPV6 channel.A, 1,25-dihydroxyvitamin D3 effects on proliferation rate measured by MTS assay of LNCaP cells incubated either with 2% or 10% FCS-containing RPMI medium, * - P<0.05, ** - P<0.01, as compared to their respective controls (DMSO), n = 3. B, The upregulation of TRPV6 mRNA expression by 1,25-dihydroxyvitamin D3 in LNCaP cells cultured in 10% FCS-containing RPMI medium; * - P<0.05, as compared to control (DMSO), n = 3. C, The upregulation of TRPV6 mRNA expression by 1,25-dihydroxyvitamin D3 in LNCaP cells cultured in 2% FCS-containing RPMI medium; * - P<0.05, ** - P<0.01, as compared to control (DMSO), n = 3. D, The time-dependence of TRPV6 expression under 100 µM 1,25-dihydroxyvitamin D3 treatment in LNCaP cells incubated in 10% FCS-containing RPMI medium. * - P<0.05, ** - P<0.01, as compared to control (DMSO), n = 3. E, a western-blotting of TRPV6 protein levels induced by 1,25-dihydroxyvitamin D3 treatment for 3 days in LNCaP cells incubated in 2% FCS-containing RPMI medium. F, A confocal microscopy showing the pattern of TRPV6 protein expression and localisation onto the plasma membrane of LNCaP cells cultivated in 2% FCS-containing RPMI medium. Cholera toxin conjugated to FITC (CTX, green) used to stain the plasma membrane as well as the TRPV6 channel (TRPV6, red), and their respective merge (CTX+TRPV6) are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037935&req=5

pone-0016856-g001: The effects of 1,25-dihydroxyvitamin D3 on proliferation of LNCaP cells and expression of TRPV6 channel.A, 1,25-dihydroxyvitamin D3 effects on proliferation rate measured by MTS assay of LNCaP cells incubated either with 2% or 10% FCS-containing RPMI medium, * - P<0.05, ** - P<0.01, as compared to their respective controls (DMSO), n = 3. B, The upregulation of TRPV6 mRNA expression by 1,25-dihydroxyvitamin D3 in LNCaP cells cultured in 10% FCS-containing RPMI medium; * - P<0.05, as compared to control (DMSO), n = 3. C, The upregulation of TRPV6 mRNA expression by 1,25-dihydroxyvitamin D3 in LNCaP cells cultured in 2% FCS-containing RPMI medium; * - P<0.05, ** - P<0.01, as compared to control (DMSO), n = 3. D, The time-dependence of TRPV6 expression under 100 µM 1,25-dihydroxyvitamin D3 treatment in LNCaP cells incubated in 10% FCS-containing RPMI medium. * - P<0.05, ** - P<0.01, as compared to control (DMSO), n = 3. E, a western-blotting of TRPV6 protein levels induced by 1,25-dihydroxyvitamin D3 treatment for 3 days in LNCaP cells incubated in 2% FCS-containing RPMI medium. F, A confocal microscopy showing the pattern of TRPV6 protein expression and localisation onto the plasma membrane of LNCaP cells cultivated in 2% FCS-containing RPMI medium. Cholera toxin conjugated to FITC (CTX, green) used to stain the plasma membrane as well as the TRPV6 channel (TRPV6, red), and their respective merge (CTX+TRPV6) are shown.
Mentions: The effect of 1,25-dihydroxyvitamin D3 on prostate cancer cell proliferation has been studied in two experimental conditions: 2% and 10% foetal calf serum (FCS)-supplemented RPMI medium. The growth of androgen-dependent LNCaP cell line was surprisingly increased by 100 nM 1,25-dihydroxyvitamin D3 in 2% FCS supplemented medium and suppressed in 10% FCS (Fig. 1A). We have already demonstrated the role of TRPV6 channel in proliferation of prostate cancer cells [15], and therefore we sought to investigate the regulation of TRPV6 channel expression by 1,25-dihydroxyvitamin D3. Since it has been shown that trpv6 is a VDR-regulated gene [17], we have studied the regulation of TRPV6 expression by 1,25-dihydroxyvitamin D3 in LNCaP cells in different steroid content of the media (Fig. 1B, C). 1,25-dihydroxyvitamin D3 appears to directly activate the trpv6 gene in LNCaP cells, though in 10% FCS medium its effects were not that significant (Fig. 1B) than in 2% FCS (Fig. 1C). 1,25-dihydroxyvitamin D3 significantly dose-dependently increased TRPV6 mRNA expression in 2% FCS-containing RPMI medium (Fig. 1C). To check whether the diminished effects of 1,25-dihydroxyvitamin D3 were due to FCS content and not to the optimal effect time we performed the time curve using the maximal concentration of 100 nM over three days at different time intervals (Fig. 1D). To confirm the significant induction of TRPV6 protein by 1,25-dihydroxyvitamin D3 in 2% FCS containing RPMI medium obtained by real time quantitative PCR a western-blotting was performed. It showed a considerable increase in TRPV6 protein level upon activation with 100 nM 1,25-dihydroxyvitamin D3 (Fig. 1E). Immunocytochemistry using TRPV6 specific antibody showed the expression of TRPV6 channels in LNCaP cells (Fig. 1F) as well as its localisation on the plasma membrane using Cholera toxin (CTX) conjugated with FITC labelling specifically G2M lipids in the membrane. Hence, the effects of 1,25-dihydroxyvitamin D3 on the growth of androgen-dependent LNCaP cells depend on the relative steroid content. Besides, 1,25-dihydroxyvitamin D3 significantly increases the expression of TRPV6 channel in low-steroid conditions.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Inserm, U-1003, Equipe labellisée par la Ligue Nationale contre le cancer, Villeneuve d'Ascq, France.

ABSTRACT
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, enhances 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.

Show MeSH
Related in: MedlinePlus