Antisense Oligonucleotide Targeting Survivin Reverses Cisplatin-Resistant Ovarian Cancer Cells

Objective: The study was designed to investigate the function of the gene survivin in the cisplatin-resistant and the relationship between gene survivin and other gene correlative to apoptosis, and to investigate the mechanism of the Survivin antisense oligonucleotide on enhancing chemosesitivity to cisplatin. Method: 1. Survivn antisense oligonucleotide was transfected into cisplatin-resistant cell(COC1/DDP), cisplatin-resistant cell(COC1/DDP) and cisplatin-sensitive human ovarian cancer cell(COC1) were treated with different concentration of cisplatin.2. The expression of survivin mRNA and protein were determined with real-time RT-PCR , Western blot analysis and immunofluorescence using fiuorescein isothiocyanate label after treated with different concentration of cisplatin and transfected with survivin antsense oligonucleotide.3. The cell cycle and apoptotic ratios of COC1 and COC1/DDP were measuree with flow cytometry after treated with different concentration of cisplatin and transfected with survivin antisense oligonucleotide.4.An ATP-CVA test were used to determine the drug sensitivity of of COC1 andCOCl/DDP after treated with different concentration of cisplatin and the drug sensitivity of COCl/DDP after transfected with survivin antisense oligonucleotide and cooperated with cisplatin.5. The expression and activity of caspase-3 were determined with Western blotanalysis and caspase-3 colorimetric assay kit after treated with different concentration of cisplatin and transfected with survivin antisense oligonucleotide.6. The expression of p53 and bcl-2 were determined before and after transfected with survivin antsense oligonucleotide.with RT-PCR> Western blot analysis and immunofluorescence using fluorescein isothiocyanate label.Results: 1. The expression of survivin in COCl/DDP cell was significantly higher2 times than that in COC1 cell. There were differents characters between COC1 and COCl/DDP cell after treated with different concentration of cisplatin. The expression of survivin of COC1 cell was enhanced rapidly and higher 3 times than control, whereas the expression of survivin of COCl/DDP cell was enhanced smoothly.2. The expression of protein and mRNA of survivin were descended and then disappeared after treated with high concentration of cisplatin.3. After transfected with survivin antisense oligonucleotide, the expression of survivin protein was inhibited and downregulated 50% and survivin mRNA was inhibited 8.9 times.4. The apoptotic ratios of COC1 and COCl/DDP cell were improved after treated with different concentration of cisplatin. The ratio of S phase and G2M phase of COC1 cell was descend and the ratio of Gi phase was improved, whereas the ratio of Gi phase of COCl/DDP cell was descend and the ratio of G2M phase was improved,and the ratio of S phase was not varied significantly. After transfectedwith survivin antisense oligonucleotide, the apoptotic ratio of COCl/DDP cell was 59.7% and the ratio of S phase was improved from 40% to 50.8% and the ratio of G2M phase also improved from 5.2% to 9.6%, whereas the ratio of d phase was descended from 54.8% to 39.6%.5. The drug sensitivity of COC1 and COCl/DDP cell after treated with different concentration of cisplatin were improved gradually. The drug sensitivity of COCl/DDP cell after transfected with survivin antisense oligonucleotide and cooperated with 0.1 xPPC, 0.5*PPC, l.OxPPC concentration of cisplatin were 32.04%, 41.6% and 72.1% respectively, at the same time the drug sensitivity of COC1 cell treated at the same concentration were 27.2%> 36.07%fP 48.09%.6. The activity of caspase-3 of COC1 and COCl/DDP cell were improved after treated with different concentration of cisplatin,and the activity of caspase-3 of COC1 cell was improve greater than COCl/DDP cell. The caspase-3 of COCl/DDP cell was activated after transfected with survivin antisense oligonucleotide.7. The expression of p53 in COC1 cell was higher than that in COCl/DDP cell, whereas the expression of bcl-2 in COC1 cell was lower than that in COCl/DDP cell. After transfected with survivin antisense oligonucleotide the expression of p53 in COCl/DDP cell was descend little and then improved little, whereas the expression of bcl-2 in COC1 cell was descend obviously.Conclutions: There suggesting that gene survivin was to be responsible for thecisplatin-resistant of COCl/DDP cell. It made COCl/DDP cell to avoid the apoptosis by inhibiting the activity of caspase-3 and had an effect on other apoptotic gene,and at last induced the cisplatin-resistant. Survivin antisense oligonucleotide could down-regulate the expression of survivin and improved theapoptosis of COC1/DDP cell and sensitivity to cisplatin.