Effect of small interfering RNA targeting hypoxia-inducible factor-1α on radiosensitivity of PC3 cell line.

OBJECTIVE To evaluate the effect of silencing hypoxia-inducible factor-1 alpha (HIF-1 alpha) expression by small interfering RNA (siRNA) on the radiosensitivity of the PC3 cell line. METHODS The expression of HIF-1 alpha in PC3, a p53-null and androgen-independent prostate cancer cell line, was knocked down by siRNA. Irradiation was performed at 48 hours after transfection. The cells were divided into 3 groups: the PC3 group, control group (transfected with scramble siRNA), and HIF-1 alpha silence group. HIF-1 alpha expression was determined using real-time polymerase chain reaction and Western immunoblotting. A clonogenic assay and the cell counting kit-8 assay were performed to determine the radiosensitivity. Flow cytometry was used to assess apoptosis and cell cycle distribution. RESULTS HIF-1 alpha siRNA downregulated HIF-1 alpha expression in PC3 cells on the mRNA level and protein level, and its silencing effect on mRNA level was evident at 24-72 hours. The HIF-1 alpha silence group had a low final slope of exponential part of a radiation survival curve, survival fraction of 2 Gy, quasi-threshold dose, and extrapolation number, and the sensitizing enhancement ratio was 1.24. The cell counting kit-8 assay showed decreased cellular viability (24 hours, F = 139.74, P < .01; 48 hours, F = 495.49, P < .01; 72 hours, F = 426.89, P < .01; 96 hours, F = 471.11, P < .01) in the HIF-1 alpha silence group. Silencing HIF-1 alpha also induced more apoptosis (PC3, 17.9% +/- 1.65%; control group, 18.6% +/- 1.37%; HIF-1 alpha silence group, 29.1% +/- 2.16%; F = 169.9, P < .01) and cell cycle arrest at the S, G(2)/M phase. CONCLUSION The suppression of HIF-1 alpha in PC3 cells sensitizes the PC3 cells to irradiation. We have shown that HIF-1 alpha inhibition attenuates repair of postradiation injury, with an increase in both interphase death and reproductive death after irradiation, apoptotic potential, and cell cycle arrest at the proliferative phase. UROLOGY 79: 744.e17-744.e24, 2012. (C) 2012 Elsevier Inc.