| Objective Because the pathogenesis of pancreatic cancer is not well understood,because early symptoms can be atypical,and because of its insensitivity to radiotherapy and chemotherapy,its fatality rate is extremely high.The 5-year survival rate is only 9%.In the United States,the Pancreatic Cancer Action Network and the Anderson Cancer Center,both located in the United States,estimated pancreatic cancer would become second only to lung cancer among cancer deaths in the country by 2030.The term pancreatic cancer covers a class of malignant tumors that pose a serious threat to human life,and finding new treatments for it has become a top priority.In a previous study,we found RBBP4 to be over-expressed in a variety of tumor cells,such as human lung carcinoma cells,gastrointestinal carcinoma cells,and cervical carcinoma cells.Down-regulation of RBBP4 has been found to effectively inhibit the proliferation of these cells,suggesting that it might be a suitable anti-tumor target.The present study targets RBBP4 to find a new treatment for pancreatic cancer.Methods The expression of RBBP4 in human pancreatic cancer and para-cancerous tissues was assessed with immunohistochemical staining;RNAi was used to down-regulate target protein;MTT assay and plate clone formation assay detected cell proliferation;cell cycle was examined by flow cytometry(FCM)analysis;the β-galactosidase test was used to detect cell senescence.Protein expression was evaluated by Western blot.Lentiviral transfection was used to construct stable high-RBBP4,high-CyclinD1,and high-CDC25C cell lines.Results The results of immunohistochemical staining differed significantly between the two groups(P<0.001).There were 51(71.83%)cases with moderate to strong staining in human pancreatic cancer tissue samples,and 26(36.62%)cases with para-cancerous tissue samples.Expression of RBBP4 was found to be reduced by siRBBP4.After the administration of siRBBP4,the proliferation rates of Panc-1,Bxpc-3,and Aspc-1 were 65.16±5.04%,75.98±3.89%,and 75.38±6.84%(P<0.01).The clone numbers were 25.33±3.51,87.00±3.65,and 45.75±7.76,which were significantly lower than 56.67±4.04,197.25±8.02,and 117.25±10.5 in the control groups(P<0.01).The use of siRBBP4 increased the sensitivity of gemcitabine and cisplatin.In the siRBBP4 groups,the IC50 values of gemcitabine on pancreatic carcinoma cells decreased from 56.90±1.19 to 5.18±1.31 nM(P<0.001),from 0.044±0.012 to 0.012±0.003 nM(P<0.001),and from 2.36±1.18 to 0.38±0.14 nM(P<0.001).The IC50 values of cisplatin on pancreatic carcinoma cells decreased from 8.54±0.52 to 4.09±0.69 μg/mL(P<0.001),from 1.10±0.06 to 0.22±0.11 μg/mL(P<0.001),and from 20.40±2.12 to 11.85±2.40 μg/mL(P<0.001).G2/M cell-cycle arrest occurred in the siRBBP4 groups.The G2/M phase rates rose from 22.31±0.54%to 39.31±4.69%(96 h,P<0.01),from 19.85±2.10%to 27.22±3.33%(24 h,P<0.01),and from 10.85±2.32%to 18.07±1.24%(72 h,P<0.01).p-galactosidase staining showed that after down-regulation RBBP4 expression,the cells resumed aging state.Study of mechanism found that after transfection of siRBBP4 for 2 days,Cyclin D1 and CDC25C were down-regulated as 29.2±1.42%and 34.8±1.29%of basal levels in Panc-1 cells(P<0.001),as 88.5±3.83%and 76.9±4.10%of basal levels in Bxpc-3 cells(P<0.01),and as 85.8±2.11%and 65.7±3.82%of basal levels in Aspc-1 cells(P<0.01).To further elucidate the role of Cyclin D1 and CDC25C in the siRBBP4 inhibition of pancreatic cancer proliferation,we constructed high-Cyclin D1 and high-CDC25C pancreatic cell lines.The results showed that the overexpression of Cyclin D1 showed partially antagonistic effects on the inhibitory effect of siRBBP4,but overexpression of CDC25C significantly antagonized the inhibitory effect of siRBBP4 on cells.Conclusions RBBP4 protein is overexpressed in about 70 percent of human pancreatic cancer cases.Down-regulation of RBBP4 can arrest the G2/M cell-cycle arrested,promote the development of senescence,inhibit the proliferation of pancreatic cancer cells and increase the sensitivity of gemcitabine and cisplatin.The mechanism of action is mainly involves inhibiting the expression of CDC25C. |
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