Abstract:

Objective   To investigate the effects of retigeric acid B (RB) on cell cycle arrest in human prostate carcinoma LNCaP cells. Methods   After exposed to RB, changes in cell cycle were monitored by flow cytometry assays. Cell proliferation was measured by incorporation of 5-Bromo-2′-deoxyuridin (BrdU) into DNA. Expressions of p53, p21CIP1(p21) and cell cycle related proteins in cells exposed to RB were determined by Western blotting. The mRNA level of p21 was tested by quantitative RT-PCR. Activity of p21 promoter by RB was measured by luciferase reporter assays. The effect of p53 on RB-induced p21 was assessed through interference of p53 activity with mutant p53 expression vector in cells following transfection. The role of p21 in RB-mediated cell cycle arrest was determined by knockdown of p21 with small interfering RNA (siRNA). Results   RB treatment led to the accumulation of LNCaP cells in the G0/G1 phase. Accordingly, the block of G0/G1 phase was accompanied with decreases in Cyclin D, Cyclin E, Cdk 4 and p-Rb in cells exposed to RB in a time-dependent manner. RB significantly induced p53 expression, and promoted its nuclear location, accompanied with the increased p21 as evidenced by increases in the mRNA and protein levels. Interference of p53 activity resulted in downregulation of p21 protein level and suppression of p21 promoter activity in RB-treated cells, suggesting that overexpression of p21 by RB was, at least in part, p53-dependent. Depletion of p21 by specific targeting siRNA in RB-treated cells led to the accumulation of G2/M cells, and a corresponding decrease in G0/G1-phase fraction as compared to the scramble siRNA control, indicating the importance of RB-induced p21 in cell cycle arrest. Conclusion    RB could inhibit cell proliferation via the promotion of p53/p21, overexpression of p21 in turn leading to the accumulation of LNCaP cells in the G0/G1 phase, which could contribute to RB-mediated cell proliferation suppression.

Key words: Retigeric acid B； G0/G1 arrest； p21 protein； p53 protein； Prostate cancer cells