Patients with hormone-resistant prostate cancer (PCa) have higher biochemical failure rates following radiation therapy (RT)

Patients with hormone-resistant prostate cancer (PCa) have higher biochemical failure rates following radiation therapy (RT). reduction in cyclin D1 protein expression (Figures ?(Figures1A1A PC3 and ?and2A2A 22Rv1). We conducted experiments IL8RA that compared tumorigenicity of the shRNA-cyclin D1- versus shRNA-control-transduced cells. Delay in cell growth, cell cycle analysis, soft agar colony formation-, migration- and invasion-abilities were investigated. Silencing cyclin D1 leds to a delay in growth of PCa cells: PC3- and 22Rv1-Cyclin D1-shRNA transduced cells respectively demonstrated a 5-fold decrease at 10-days and a 2-fold decrease at 12 days in proliferation compared to control-shRNA transduced cells (Figures ?(Figures2B2B and ?and3B).3B). FACS analysis shows that silencing cyclin D1 Daunorubicin increased the proportion of PC3 (Figure ?(Figure1C)1C) and 22Rv1 (Figure ?(Figure2C)2C) cells in G1 phase and up-regulated the p21Waf1 and p27Kip2 cell cycle inhibitor protein expression levels. Figure ?Figure1D1D (PC3) and ?and2D2D (22Rv1) show that silencing cyclin D1 reduced by 80% (PC3) and 82.5% (22Rv1) the ability to form colony in soft agar and by 69% (PC3) and 48% (22Rv1) the colony medium size. Figure ?Figure1E1E (PC3) and ?and2E2E (22Rv1) show that cyclin D1 silencing reduced by 83% (PC3) and 77% (22Rv1) invasion and by 68% (PC3) and 71% (22Rv1) migration abilities. Given the observed effects on invasion and migration, the matrix metallopeptidase 2 and 9 (MMP-2 and -9) activities were assessed by ELISA assay. Figures ?Figures1F1F and ?and2F2F show that cyclin D1 depletion reduced the MMP-2 activity by 81% (PC3) and 82% (22Rv1), the MMP-9 activity by 65% (PC3) and 62% (22Rv1). Open in a separate window Figure 1 Stable and Specific Silencing of cyclin D1 inhibits PC3 onco-phenotype(A) Parental PC3 (PRT), GFP-positive PC3 cells, stably infected with shRNA-cyclin D1 (CD1) vs. shRNA-control (CTR) sequence (CTR), were selected by FACS sorting and examined for cyclin D1 protein expression by immunoblotting. (B) Cell growth assay, (C) cell cycle distribution by FACS and p21waf1, p27KIP2 by immunoblotting, (D) soft agar assay and relative colony size, (E) invasion- and migration-assay and (F) the activation status of MMP-2 and MMP-9 by ELISA assay were performed. The data presented in Figure 1B, 1C, 1D, 1E and 1F represent the mean SD of 3 independent experiments. Statistical analysis was performed using the Student’s 0.01. For immunoblotting, -tubulin expression shows equal loading. Similar results were obtained in = 3 experiments. Open in a separate window Figure 2 Stable and Specific Silencing of cyclin D1 inhibits 22Rv1 onco-phenotype(A) Parental 22Rv1 (PRT), GFP-positive 22Rv1 cells, stably infected with shRNA-cyclin D1 (CD1) vs. shRNA-control (CTR) sequence (CTR), were selected by FACS sorting and examined for cyclin D1 protein expression by immunoblotting. (B) Cell growth assay, (C) cell cycle distribution by FACS and p21waf1, p27KIP2 by immunoblotting, (D) soft agar Daunorubicin assay and relative colony size, (E) invasion- and migration-assay Daunorubicin and (F) the activation status of MMP-2 and MMP-9 by ELISA assay were performed. The data presented in Figure 1B, 1C, 1D, 1E and 1F represent the mean SD of 3 independent experiments. Statistical analysis was performed using the Student’s 0.01. For immunoblotting, -tubulin expression shows equal loading. Similar results were obtained in = 3 experiments. Open in a separate window Figure 3 Silencing cyclin D1 radiosensitizes PC3 and 22Rv1 cell lines 0.01. Figure ?Figure3B3B Lower Panel shows the effects of 4 Gy irradiation on shRNA-cyclin D1- and shRNA-control-transduced PC3 and 22Rv1 cells. Cyclin D1 governs the radioresistant phenotype of PC3 and 22Rv1 cell lines and and experiments, control- and cyclin D1-shRNA transduced cells were treated with several doses (0C8 Gy) of radiation. MTT assay (Figure ?(Figure3A),3A), performed after 24 hrs post irradiation, shows that silencing cyclin D1 significantly reduced the PC3 and 22Rv1 cells survival. Colony formation assay was performed to determine cell reproductive death after treatment with ionizing radiation. Concordant.