Quercetin-induced growth inhibition and cell death in prostatic carcinoma cells (PC-3) are associated with increase in p21 and hypophosphorylated retinoblastoma proteins expression

Abstract

Prostate cancer is the major health problem and the leading cause of male cancer death. Quercetin is a novel antitumor and antioxidant, whose molecular mechanism involved in cell cycle arrest in androgen independent prostate cancer cells remains unclear. In this study, we investigated the effects of quercetin on proliferation and cell cycle arrest by modulation of Cdc2/Cdk-1 protein in prostate cancer cells (PC-3). PC- 3 cells are human androgen independent cancer cells and were cultured with quercetin at concentrations of 50 and 100 microM for 24 h. Cell proliferation, apoptosis and cell cycle distribution were analyzed. Expression of Cdc2/Cdk-1, cyclin B1, cyclin A, p21/Cip1, pRb, pRb2/p130, Bcl-2, Bcl-X(L), Bax and caspase-3 proteins were studied with western blot analysis. Addition of quercetin led to substantial decrease in the expression of Cdc2/Cdk-1, cyclin B1 and phosphorylated pRb and increase in p21. Flowcytometric analysis showed that quercetin blocks G2-M transition, with significant induction of apoptosis. Apoptosis markers like Bcl-2 and Bcl-X(L) were significantly decreased and Bax and caspase-3 were increased. From this study, it was concluded that quercetin inhibits prostate cancer cell proliferation by altering the expression of cell cycle regulators and apoptotic proteins.

Fig. 1

Effect of quercetin on PC-3 cell proliferation. 3×10⁴ cells were plated in 24-well plates. After reaching 70–80% confluence, in the presence of 5% FBS, cells were treated with vehicle or with various concentrations of quercetin (25, 50, 75, and 100 μM) for 24, 48 and 72 h. Proliferation of cells was quantitated by [³ H] thymidine incorporation. Experiments were performed in triplicate, values represents % of cpm and SD was less than 10%. a Represents the level of statistical significance at P<0.05 using SNK test between control and quercetin treatment groups

Fig. 2

Effects of quercetin on cell cycle distribution in PC-3 cells. PC-3 cells were treated with quercetin at doses of 50 and 100 μM for 24 h in a 25 cm² flasks. After treatment 1×10⁶ cells were trypsinized and suspended in 1 ml of fluorochrome solution (50 μg/ml propidium iodide, 20 μg/ml RNase A, 1.5% Triton X-100) for at least 1 h in the dark at 4°C. Cell cycle analysis was performed using a Beckman Vantage flow cytometer, and quantitation of cell cycle distribution was performed using Multicycle software (Phoenix Flow Systems, San Diego, CA, USA). Significant decrease in the proportions of cells in G, S and also G₂-M-phases, were evident after exposure to quercetin for 24 h. A concomitant increase of cells in Sub G1-phase was observed in quercetin treated cells, indicating induction of apoptosis (Table 1)

Fig. 3

Western blot analysis of cell cycle regulatory proteins p21/Cip1, cyclin-dependent kinase-1 (Cdc2/Cdk-1), pRb, pRb2/p130, cyclin A, cyclin B1 and β-actin expression in PC-3 cells treated for 24 h with quercetin. Equal volumes of whole-cell extracts containing 50 μg of protein were separated and electrophoretically blotted

Fig. 4

Effects of quercetin on Bcl-2, Bcl-X_L and Bax levels in prostate cancer cell line (PC-3). PC-3 cells were treated with indicated concentrations of quercetin for 24 h. Cell lysates were analyzed by Western blotting. Blots were incubated with anti-Bcl-2, anti-Bcl-x_L and Bax antibodies and then incubated with anti mouse secondary antibody. Bands were developed with ECL kit (Perkin Elmer, USA)

Fig. 5

Effect of quercetin on induction of apoptosis in PC-3 cells. The percentage of apoptotic cells was determined and summarized, generation of free 3′-OH DNA fragments was determined using TUNEL assay. Experiments were performed in triplicate, values represents the percentage of apoptotic cells and SD was less than 10%. a Represents the level of statistical significance at P<0.05 using SNK test between control and quercetin cells

Fig. 6

Effect of quercetin on the expression of caspase-3 in prostate cancer cell line. PC-3 cells were treated with indicated concentration of quercetin. After 24 h incubation, cells were lysed and equal proteins were resolved on SDS polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with the antibodies against caspase-3 and β-actin. Proteins were visualized using ECL detection system. β-actin was used as an internal control