Immunomodulating Peptide Inhibits The Drug Resistance Of Ovarian Cancer Cell Through The Ubiquitin-proteasome Pathway

Objective:1.To investigate the effect of immunomodulating peptide?PGPIPN?on the sensitization of human ovarian cancer cells to the cisplatin?DDP?.2.To investigate the possible molecular mechanism of reducing the drug resistance of human ovarian cancer cells by combination of PGPIPN and DDP.Methods:1.Primary human ovarian cancer cells and normal human ovarian cell were cultured and the purity of cells were assessed by immunofluorescence.2.MTT assay was used to detect the cell survival rate in 48h on normal human ovarian cell by PGPIPN.3.MTT assay was used to detect the half inhibitory concentration?IC50?and cell proliferation inhibition rate of DDP in 48h on human ovarian cancer sensitive cell line?SKOV₃ and COC1?,drug-resistant cell line?SKOV₃/DDP and COC1/DDP?and primary ovarian cancer cells.4.MTT assay was used to detect the cell proliferation inhibition rate of human ovarian cancer cells line?SKOV₃,SKOV₃/DDP,COC1,COC1/DDP and primary human ovarian cancer cells?by the combination of PGPIPN and DDP in 48h.5.The expression of genes related to the ubiquitin-proteasome pathway?UPP?was examined by RT-PCR treated with PGPIPN and DDP in human ovarian cancer cells?SKOV₃,SKOV₃/DDP,COC1,COC1/DDP and primary human ovarian cancer cells?.6.The expression of genes related to UPP was examined by western blot treated with PGPIPN and DDP in human ovarian cancer cells?SKOV₃,SKOV₃/DDP,COC1,COC1/DDP and primary human ovarian cancer cells?.Results:1.The primary ovarian cancer cells and normal human ovarian cell were cultured with tissue pieces culture method,and the purity were more than 94%by immunofluorescence.2.MTT results showed that the low concentration of PGPIPN had no significant inhibitory effect on human normal ovarian cells.3.MTT results showed that DDP inhibited human ovarian cancer cells?SKOV₃,SKOV₃/DDP,COC1,COC1/DDP and primary ovarian cancer cells?.Compared with the control group,the inhibitory effect is more obvious with the increase of DDP concentration.The average IC50 of DDP on human ovarian cancer cells?SKOV₃,SKOV₃/DDP,COC1,COC1/DDP and primary human ovarian cancer cells?were?3.759±0.790??mol/L??9.092±1.738??mol/L??4.997±0.437??mol/L??21.819±2.644??mol/L??17.266±7.454??mol/L in 48h.4.The concentration of DDP in IC50 was associated with low,medium and high concentrations of PGPIPN in human ovarian cancer cells?SKOV₃,SKOV₃/DDP,COC1,COC1/DDP and primary human ovarian cancer cells?.Compared with the control group,the cell proliferation inhibition rate of the combination was significantly higher than that of the DDP group and PGPIPN group.With the increase concentration of PGPIPN,the inhibition rate also increased,and the inhibitory rate of drug-resistant cell line changed more than the sensitive cell.5.PCR results showed that compared with the blank control group,the mRNA expression of SIAH and PSMA1 in UPP were increased and the mRNA expression of?-catenin was decreased.Compared with DDP and PGPIPN group alone,with the increase of PGPIPN concentration,mRNA expression of SIAH and PSMA1 in the combination group increased and the mRNA expression of?-catenin decreased.6.Western blot results showed that compared with the blank control group,the protein expression of SIAH and PSMA1 in UPP was increased and the protein expression of?-catenin was decreased.Compared with DDP and PGPIPN group alone,with the increase of PGPIPN concentration,protein expression of SIAH and PSMA1 in the combination group increased and the protein expression of?-catenin decreased.Conclusion:1.DDP can inhibit the proliferation of ovarian cancer cells.PGPIPN combined with DDP can improve the sensitivity of ovarian cancer cells to cisplatin.Low concentration of PGPIPN had no significant inhibitory effect on normal ovarian cells.2.After the combination of drugs,the gene and protein expression of SIAH and PSMA1increased,and the gene and protein expression of?-catenin decreased.It is suggested that PGPIPN raises the sensitivity of ovarian cancer cells to cisplatin.This process may be related to UPP.