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. 2009 May;150(5):2046-54.
doi: 10.1210/en.2008-1395. Epub 2009 Jan 22.

1Alpha,25-dihydroxyvitamin D3 reduces c-Myc expression, inhibiting proliferation and causing G1 accumulation in C4-2 prostate cancer cells

JoyAnn N Phillips Rohan  1 , Nancy L Weigel
Affiliations

1Alpha,25-dihydroxyvitamin D3 reduces c-Myc expression, inhibiting proliferation and causing G1 accumulation in C4-2 prostate cancer cells

JoyAnn N Phillips Rohan et al. Endocrinology. 2009 May.

Abstract

There is an inverse correlation between exposure to sunlight (the major source of vitamin D) and the risk for prostate cancer, the most common noncutaneous cancer and second most common cause of death from cancer in American men. The active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] acting through the vitamin D receptor decreases prostate cancer cell growth and invasiveness. The precise mechanisms by which 1,25(OH)(2)D(3) inhibits growth in prostate cancer have not been fully elucidated. Treatment with 1,25(OH)(2)D(3) causes an accumulation in the G(0)/G(1) phase of the cell cycle in several prostate cancer cell lines. One potential target known to regulate the G(0)/G(1) to S phase transition is c-Myc, a transcription factor whose overexpression is associated with a number of cancers including prostate cancer. We find that 1,25(OH)(2)D(3) reduces c-Myc expression in multiple prostate epithelial cell lines, including C4-2 cells, an androgen-independent prostate cancer cell line. Reducing c-Myc expression to the levels observed after 1,25(OH)(2)D(3) treatment resulted in a comparable decrease in proliferation and G(1) accumulation demonstrating that down-regulation of c-Myc is a major component in the growth-inhibitory actions of 1,25(OH)2D(3). Treatment with 1,25(OH)(2)D(3) resulted in a 50% decrease in c-Myc mRNA but a much more extensive reduction in c-Myc protein. Treatment with 1,25(OH)(2)D(3) decreased c-Myc stability by increasing the proportion of c-Myc phosphorylated on T58, a glycogen synthase kinase-3beta site that serves as a signal for ubiquitin-mediated proteolysis. Thus, 1,25(OH)(2)D(3) reduces both c-Myc mRNA levels and c-Myc protein stability to inhibit growth of prostate cancer cells.

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Figures

Figure 1
Figure 1
1,25(OH)2D3 treatment reduces cell growth in C4-2 cells and c-Myc expression in normal and malignant prostate epithelial cell lines. A, C4-2 cells were treated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) for the indicated times, harvested, and counted using a Coulter counter. Data are expressed as the mean ± sem. *, Significance at P < 0.05 with respect to vehicle-treated cells within the same time frame; **, significance at P < 0.005 with respect to vehicle-treated cells within the same time frame. B, LNCaP, C4-2, and RWPE-1 cells were treated with vehicle (−) or 100 nm 1,25(OH)2D3 (1,25D; +) for 3 d. Cells were harvested and c-Myc and actin protein levels were detected by Western blotting. C, C4-2 cells were treated with vehicle (−) or 100 nm 1,25(OH)2D3 (1,25D; +) for the indicated times. Cells were harvested, extracts run on an SDS-PAGE gel, and proteins (c-Myc and actin) detected by Western blotting.
Figure 2
Figure 2
Proliferation in the C4-2 prostate cancer cell line is reduced by c-Myc siRNA or 1,25(OH)2D3 treatment. A, C4-2 cells were treated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) or transiently transfected with 75 pm of control or c-Myc siRNA no. 1 for 2 d. Cells were harvested, extracts run on an SDS-PAGE gel, and c-Myc and actin protein levels detected by Western blotting. Representative westerns corresponding to the samples in B are shown. B, C4-2 cells were treated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) or transiently transfected with 75 pm of control or c-Myc siRNA no. 1 for 2 d. Proliferation was measured by [3H]thymidine incorporation. Data are expressed as the mean ± sem. *, Significance at P < 0.005 with respect to vehicle or control siRNA. C, C4-2 cells were transiently transfected with 150 pm control or c-Myc siRNA no. 2. C4-2 cells transfected with control siRNA were further treated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) for 2 d. c-Myc mRNA was measured by qPCR and normalized with 18S RNA. Results from two separate experiments, each performed in triplicate, were pooled and analyzed. Data are expressed as the mean normalized to control ± sem. *, Significance at P ≤ 0.001 with respect to control siRNA plus vehicle. D, C4-2 cells were transiently transfected with 150 pm control or c-Myc siRNA no. 2. C4-2 cells transfected with control siRNA were further treated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) for 2 d. Proliferation was measured by [3H]thymidine incorporation. Data are expressed as the mean ± sem. Results from two separate experiments, each performed in triplicate, were pooled and analyzed. *, Significance at P ≤ 0.001 with respect to control siRNA plus vehicle. 1,25(OH)2D3, 1,25D.
Figure 3
Figure 3
c-Myc siRNA or 1,25(OH)2D3 induces G0/G1 accumulation. A, C4-2 cells were transiently transfected with 150 pm control or c-Myc siRNA. Control siRNA-transfected cells were treated with vehicle (+Veh) or 100 nm 1,25(OH)2D3 (+1,25D) for 2 d. Cells were then stained with propidium iodide (PI) for total DNA content and analyzed by flow cytometry using FlowJo. G0/G1, S, and G2/M phases of a representative experiment performed in triplicate are indicated. Data are expressed as the mean ± sem for each treatment group. *, Significance at P ≤ 0.05 with respect to vehicle or control siRNA. B, cdc25A, E2F, and c-Myc mRNA were measured by qPCR and normalized with 18S RNA. Results from a representative experiment performed in triplicate are shown. Data are expressed as the mean ± sem. *, Significance at P ≤ 0.05 with respect to vehicle or control siRNA.
Figure 4
Figure 4
Reducing β-catenin modestly reduces c-Myc protein. A, C4-2 cells were transiently transfected with 80 pm control siRNA or β-catenin siRNA. Two days after transfection, C4-2 cells were harvested for protein analysis and extracts run on an SDS-PAGE gel. β-Catenin, c-Myc, and tubulin protein levels were detected by Western blotting. B, C4-2 cells were transiently transfected with 80 pm control siRNA or β-catenin siRNA. Three days after transfection, C4-2 cells were harvested for protein analysis and extracts run on an SDS-PAGE gel. β-Catenin protein levels were detected by Western blotting. In parallel, proliferation was measured after 3 d of treatment with siRNA by [3H]thymidine incorporation. Data are expressed as the mean ± sem.
Figure 5
Figure 5
1,25(OH)2D3 reduces c-Myc mRNA and protein. A, C4-2 cells were treated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) for 3 d. Cells were harvested for protein analysis, extracts run on an SDS-PAGE gel, and phosphotyrosine residues in proteins detected by Western blotting. Arrows indicate reduction in phosphotyrosine residues after 1,25D treatment in a subset of proteins. B, C4-2 cells were treated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) for 3 d. c-Myc mRNA was measured by qPCR and normalized with 18S. Data are expressed as the mean ± sem. B, C4-2 cells were treated with vehicle (−) or 100 nm 1,25(OH)2D3 (1,25D; +) for 3 d. Cells were harvested for protein analysis, extracts run on an SDS-PAGE gel, and proteins (c-Myc and actin) detected by Western blotting. Intensity of signals was measured with a densitometer. Densitometric values represent c-Myc/actin normalized to vehicle (−).
Figure 6
Figure 6
1,25(OH)2D3 treatment reduces stability of c-Myc protein in C4-2 cells. A, C4-2 cells were pretreated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) for 3 d. Before harvesting, cells were treated with CHX for 0–60 min. Cells were harvested for protein analysis at the indicated times, extracts run on an SDS-PAGE gel, and c-Myc and actin protein levels detected by Western blotting. A representative western is shown. B, Percent of time 0 was calculated from the Western blot after normalization to actin. Percentage reductions are graphed on a log scale. The line indicates 50% of maximal signal. C, Graph indicating mean values of 50% of maximum signal from five independent experiments for each treatment group. *, Significance at P ≤ 0.005 with respect to vehicle-treated cells. D, C4-2 cells were treated with vehicle, BIO, or GSK-3β I for 24 h. Cells were harvested for protein analysis, extracts run on an SDS-PAGE gel, and c-Myc and tubulin protein levels detected by Western blotting. E, Cells were pretreated with vehicle (−) or 100 nm 1,25(OH)2D3 (1,25D; +) for 3 d. Then cells were treated with vehicle (−) or BIO (+) for 6 h. Twice as much protein extract (2 times) compared with 1 time were run on an SDS-PAGE gel to enhance signal in selected cases. c-Myc and phosphorylated c-Myc (T58/S62) were detected by Western blotting. The signals were measured with a densitometer. Results are presented as ratios of phosphorylated c-Myc to total c-Myc normalized to the ratio for the control sample (arbitrarily set at 1).
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