3,3'-Diindolylmethane induces a G(1) arrest in human prostate cancer cells irrespective of androgen receptor and p53 status

Abstract

3,3'-Diindolylmethane (DIM) is a potential chemopreventive phytochemical derived from Brassica vegetables. In this study we characterized the effect of DIM on cell cycle regulation in both androgen-dependent LNCaP and androgen receptor negative p53 mutant DU145 human prostate cancer cells. DIM had an anti-proliferative effect on both LNCaP and DU145 cells, as it significantly inhibited [3H]-thymidine incorporation. FACS analysis revealed a DIM-mediated G(1) cell cycle arrest. DIM strongly inhibited the expression of cdk2 and cdk4 protein and increased the expression of the cell cycle inhibitor p27(Kip1) protein in LNCaP and DU145 cells. Promoter deletion studies with p27(Kip1) reporter gene constructs showed that this DIM-mediated increase in p27(Kip1) was dependent on the Sp1 transcription factor. Moreover, using a dominant negative inhibitor of p38 MAPK, we showed that the induction of p27(Kip1) and subsequent G(1) arrest by DIM involve activation of the p38 MAPK pathway in the DU145 cells. Taken together, our results indicate that DIM is able to stop the cell cycle progression of human prostate cancer cells regardless of their androgen-dependence and p53 status, by differentially modulating cell cycle regulatory pathways. The Sp1 and p38 MAPK pathways mediate the DIM cell cycle regulatory effect in DU145 cells.

Fig. 1. Inhibition of prostate cancer cell proliferation and cell cycle by DIM

LNCaP (A) and DU145 (B) cells were treated with 0, 10, 30, or 50 μM DIM for 24, 48 and 72 h. Cell numbers were counted Values were expressed as mean ± SD. LNCaP (C) and DU145 (D) cells were treated as indicated for 24 h. DNA synthesis was determined by measuring thymidine uptake. Values were expressed as mean ± SEM. LNCaP (E) and DU145 (F) were treated with 0, 10, 30, or 50 μM DIM for 24 h. Cells were stained with propidium iodide. Flow cytometric analysis was performed for cell cycle distribution. Values were expressed as mean ± SD. Asterisks denote a significant difference compared with control at a level of P≤ 0.05.

Fig. 2. Effect of DIM on G₁ cell cycle regulators in human prostate cancer cells

Cells were treated with 50 μM DIM for indicated times. LNCaP (A) and DU145 (B) cells were collected, total cell ysates prepared and subjected to SDS-PAGE followed by western immunoblotting. Membranes were probed with indicated antibodies and visualized by ECL detection system. LNCaP (C) cells were treated for indicated times with 50 μM DIM, cells were collected and lysates prepared. Western blotting was performed using an antibody specific to pRb phosphorylated at Thr180/182 or an antibody specific to total pRb protein. LNCaP (D) cells were treated with indicated concentrations of DIM for 24 h and immunoprecipitated cdk2 phosphorylation of pRb was measured ex vivo by kinase assay. Results are representative of data collected from at least three experiments.

Fig. 3. Transcriptional regulation of cell cycle regulators by DIM

LNCaP (A,B,C) and DU145 (D,E,F) cells were treated with 50 μM DIM for indicated times, harvested, RNA isolated and reverse transcription performed. Quantitative PCR was performed on RT samples using primers for indicated genes. Values were expressed as mean ± SD. Asterisk indicates significant difference compared with control at a level of P ≤ 0.05.

Fig. 4. Activation of p27 promoter by DIM

DU145 cells were transfected with the p27 promoter luciferase constructs illustrated. Cells were treated with 0, 10, 30, or 50 μM DIM for 24 hours, harvested, and luciferase assay performed. Values were expressed as mean ± SD. Asterisk indicates significant difference compared with control at a level of P ≤ 0.05.

Fig. 5. DIM activates p38 MAPK pathway in DU145 cells

(A), DU145 cells were treated with or without 50 μM DIM for 24 hours, after which total lysates were prepared and Western blotting conducted. Membranes were probed with indicated antibodies and visualized by ECL detection system. (B), DU145 cells were treated with or without 50 μM DIM and the p38 MAPK inhibitor SB202190 (SB) and subjected to Western blot analysis as in A. (C), DU145 cells transfected with or without dominant negative p38 MAPK (dnp38), were treated with 50 μM DIM and subjected to Western blot analysis as in A.

Fig. 6. The DIM-mediated cell cycle arrest of DU145 is p38 MAPK dependent

DU145 cells were treated with DIM in the absence (solid bars) and presence (dashed bars) of the p38 MAPK inhibitor SB202190 for 24 hours and subjected to FACS analysis to determine percent cell cycle distribution. Results are expressed as mean ± SD (%). Asterisk indicates significant difference from untreated cells P ≤ 0.05. Double asterisk indicates significant difference from respective DIM-dose treated cells in the absence of p38 MAPK inhibitor with P ≤ 0.05.