siRNA-Mediated Silencing of CIP2A Enhances Docetaxel Activity Against PC-3 Prostate Cancer Cells

Purpose: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an identified human oncoprotein which modulates malignant cell growth. It is overexpressed in human prostate cancer and in most of the human malignancies. The aim of this study was to investigate the effects of CIP2A silencing on the sensitivity of PC-3 prostate cancer cells to docetaxel chemotherapy. Methods: PC-3 cells were transfected using CIP2A siRNA. CIP2A mRNA and protein expression were assessed after CIP2A gene silencing using q-RT PCR and Western blotting. Proliferation and apoptosis were analyzed after treatment with docetaxol using MTT assay, DAPI staining, and flow cytometry, respectively. Results: Silencing of CIP2A enhanced the sensitivity of PC-3 cells to docetaxel by strengthening docetaxel induced cell growth inhibition and apoptosis against PC-3 cells. Conclusion: Silencing of CIP2A may potentiate the cytotoxic effects of docetaxel and this might be a promising therapeutic approach in prostate cancer treatment.


Introduction
Prostate cancer is the common malignancy among men and it accounts for the second cause of cancer-related death in men. 1,2 In spite of significant efforts in the treatment of prostate cancer, conventional therapies could not successfully treat the tumors. Therefore, most of the patients will develop castration resistant prostate cancer (CRPC) for the duration of 18-24 months and this is associated with poor prognosis. 3,4 Chemotherapy, using docetaxel as a taxan family member, is the first choice for the treatment of CRPC. 5,6 The anti-tumor effects of docetaxel depend on its ability to promote microtubules polymerization and stabilization, which leads to cell cycle arrest and apoptosis. 7,8 The toxicity and undesirable events in docetaxel based treatment reduce the therapeutic efficacy and limit its tolerated dose. 9 Lack of efficient treatments shows the importance of additional means to develop effective therapeutic approaches in the treatment of prostate cancer. One of the feasible approaches consists of combining a chemotherapy agent with silencing specific proteins involved in proliferation and survival of the cancer cells using RNA interference technology due to RNAi's high specificity, noticeable efficacy, and low toxicity when compared with the other reverse genetic technologies. [10][11][12] Cancerous inhibitor of protein phosphatase 2A (CIP2A), a human oncoprotein, is generally overexpressed in most human malignancies and its overexpression is closely associated with poor outcome in patients. 13,14 It promotes malignant cell growth and tumor progression. 15 It is overexpressed in prostate cancer samples and cell lines. 16,17 Since, the effect of CIP2A suppression on docetaxel induced cytotoxicity against prostate cancer cells has not been reported, this study investigated the effects of CIP2A silencing on the sensitivity of PC-3 cells to docetaxel chemotherapy.
CIP2A siRNA, the cells at 2×10 5 /well density were seeded in 6-well plate and cultured overnight before transfection using CIP2A siRNA according to the manufacturer's instruction. Briefly, siRNA transfection medium was used to dilute siRNAs and siRNA transfection reagent. The diluted solutions were mixed gently and incubated at room temperature for 15 to 30 min. Then, the complexes were added to each well and were incubated in a humidified CO2 incubator at 37°C for 6 h. 1 ml of RPMI-1640 medium containing 20% FBS was added to each well. After 24, 48, and 72 h, down-regulation of CIP2A was monitored using quantitative RT-PCR (qRT-PCR) and Western blotting.

Combination effect analysis
To study the interaction between CIP2A siRNA and docetaxel, combination effect analysis was performed based on Chou and Talalay principles. 18

Apoptosis assay by Annexin-V FITC/PI staining
Forty eight hours after CIP2A siRNA transfection, the cells were exposed to 3.6 nM of docetaxel for 24 h. After the exposure, the cells were harvested and resuspended in cold PBS for analysis. Annexin-V FITC/PI staining kit (Roche Diagnostics, Germany) was used to stain the cells for additional analysis of cell death according to the manufacturer's instructions. Data were collected using a BD FACS Calibur flow cytometer (San Jose, CA, USA) and were analyzed with Flowing software.

Statistical analysis
All data were presented as mean ± standard deviation (SD). Statistical differences between groups were analyzed using T test and Two-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons with GraphPad Prism software, La Jolla California USA, http://www.graphpad.com. P value less than 0.05 was considered statistically significant.

CIP2A siRNA down-regulated CIP2A expression in PC-3 cells
To investigate the effect of CIP2A siRNA on CIP2A expression in PC-3 cells, the CIP2A expression in PC-3 cells was knocked down by specific CIP2A siRNA for 24, 48, and 72 h with 60 pmol of CIP2A siRNA. The CIP2A expression level was analyzed using qRT-PCR. The relative expression of CIP2A mRNA for 24, 48 and 72 h after transfection were 0.71, 0.52 and 0.34, respectively ( Figure 1A). Then, to setup the optimum dose of CIP2A siRNA, the cells were transfected using 40, 60, and 80 pmol of siRNA for 72 h. qRT-PCR and Western blot analysis were used to determine the expression of CIP2A at mRNA and protein levels following transfection using different doses. The relative CIP2A mRNA expression level for doses 40, 60, and 80 pmol of CIP2A siRNA was 0.50, 0.35, and 0.01, while the relative CIP2A protein expression levels were %73, %42, and 5% of the control, respectively. The results showed that CIP2A expression was remarkably reduced in cells transfected using 80 pmol of CIP2A siRNA as compared to the control. The optimum knockdown dose and time were achieved on 80 pmol of siRNA, 72 h post transfection ( Figure 1B, 1C and 1D). Accordingly, the following tests were performed using the same condition. No significant differences were found in CIP2A mRNA and protein levels in the control groups (un-treated cells and negative siRNA group; p > 0.05). Therefore, the results showed that silencing CIP2A with specific siRNA significantly reduced the expression of CIP2A in a time and dose dependent manner.

siRNA mediated CIP2A down-regulation inhibited the proliferation of PC-3 cells
In order to analyze the effect of CIP2A down-regulation on the proliferation and viability of PC-3 cells, MTT assay was used at different time points after CIP2A siRNA transfection. As shown in Figure 2, compared to the control group, CIP2A siRNA significantly decreased the proliferation and cell viability of P-C3 cells at 24, 48 and 72 h after transfection in a time dependent manner (p<0.0001). 24 h after CIP2A siRNA transfection, the cell viability was reduced to 81% and later dropped to 70% at 72 h. Also, no significant differences in cell viability were observed between the NC siRNA transfected cells and the un-treated control group (p>0.05). Therefore, CIP2A plays a key role in the proliferation of prostate cancer cells.

CIP2A siRNA synergistically enhanced the cytotoxic effects of docetaxel
Sequential treatment regime was designed to explore whether CIP2A silencing could enhance the chemosensitivity of PC-3 cells to docetaxel. PC-3 cells were pre-treated with CIP2A siRNA for 48 h followed by 0.39 to 50 nM of docetaxel for 24 h and the effects of the mentioned treatment were evaluated using MTT assay. As shown in Figure 3, CIP2A siRNA significantly decreased the cell survival rate to 70% and the combination of docetaxel and CIP2A siRNA significantly decreased the cell survival rate to 45% as compared to the control. The IC50 value of docetaxel was reduced from 3.59 to 1.97 nM after CIP2A siRNA transfection (Figures 3 and 4). Also, the CDI values were less than 1 in all the concentrations of docetaxel which pointed to the synergistic effect between CIP2A siRNA and docetaxel (Table 2). Thus, it is suggested that CIP2A down-regulation could sensitize PC-3 cells to docetaxel.

Silencing of CIP2A increased apoptosis induced by docetaxel
To determine whether CIP2A suppression could enhance docetaxel induced apoptosis, DAPI and Annexin-V FITC/PI staining were used. Distinctive apoptosis-related morphological changes such as nuclear condensation were observed and nuclear fragmentation in the cells was treated using CIP2A siRNA, docetaxel, and both of them on the florescence micrographs when compared with NC siRNA treated and un-treated control groups ( Figure 5). These changes, such as nuclear condensation and fragmentation, are in line with the apoptosis. The apoptotic effect of CIP2A siRNA and docetaxel on PC-3 cells was further confirmed by Annexin-V/PI staining.   Figure 6 shows that the apoptosis percentage in PC-3 cells treated with CIP2A siRNA + 3.6 nM docetaxel was significantly higher than CIP2A siRNA treated cells and docetaxel treated cells alone (p<0.0001). However, no significant change was observed for NC siRNA treated group in the apoptosis rate when compared with the untreated cells. Therefore, the knockdown of CIP2A enhanced the apoptosis induction effect by docetaxel on PC-3 cells. Fang et al. showed that down-regulation of CIP2A enhanced the paclitaxel induced apoptosis in human ovarian cancer cells. 19 Also, Zhang et al. showed that the depletion of CIP2A sensitized ovarian cancer cells to cisplatin. 20 These findings are fully in agreement with the results of similar studies, which further verify the important role of CIP2A in the survival and proliferation of cancer cells.

Conclusion
Conclusively, the present study showed that silencing of CIP2A could enhance the antitumor effects of docetaxel against PC-3 cells. Therefore, targeting of CIP2A can be considered as a novel strategy for targeted prostate cancer synergy therapy.