| Objective:To observe the milk-derived hexapeptide (PGPIPN) enhanceing anti-ovarian cancer of cisplatin (DDP) on proliferation, migration, invasion and apoptosis/n vitro, and explore its possible molecular mechanism.This provides the basis forexploring new anti-ovarian drug.Methods:Primary ovarian cancer cells was cultured by tissue culture method,and the purity of primitive cells identified by immunofluorescence staining The IC50s of DDP on inhibitions of SKOV3 and SKOV3/DDP cells proliferation were assayed by MTS method, and R1 of SKOV3/DDP relative to the SKOV3 was calculated. The effects of different concentration PGPIPN, DDP (IC50) alone and their combination on the inhibitions of human ovarian cancer cell proliferation were detected by MTS method.The cell apoptosis and cycle of cells were detected by flow cytometry.Ability of cell clone forming was detected by Plan clone forming assay.The cell migrations were detected by cell scratch test.The cell invasions and metastasis were detected by Transwell experiment/The expression of Akt1, PI3K, and MDR1 gene were detected by RT-PCR and western Blot.Results:Primary ovarian cancer cells were successfully cultivate by tissue pieces culture method. The result of immunofluorescence showed that the purity of primary ovarian cancer cells reached to 96.8%;By MTS assay,the IC50 of DDP on SKOV3 in 24 h,48 h, 72 h were 10 ug/ml,5.56 ug/ml,3.23 ug/ml respectively, IC50 of DDP on SKOV3/DDP in 24 h,48 h,72 h were 24.9 ug/ml,9.9 ug/ml,7.63 ug/ml respectively, the R1 of SKOV3/DDP relative to SKOV3 in 24 h,48 h,72 h were 2.49,1.78,2.36 respectively.DDP(IC50), PGPIPN and PGPIPN combine with DDP can inhibit the growth of ovarian cancer compared with the control group.PGPIPN combining with DDP group had a significantly higher inhibition compared with the single-drug group. The difference between groups were statistically significant (P<0.05, P<0.01), which exhibitin dose and time dependent.The PGPIPN, DDP(IC50) and combination groups can induce the apoptosis of ovarian cancer cells after 48 h, the apoptosis rate of combination group was increase gradually with the PGPIPN dose. This show that PGPIPN can promote DDP to induce apoptosis of ovarian cancer cells. By flow cytometry, PGPIPN and DDP can arrest cells in G2/M phase.PGPIPN enhances the DDP effect. The treatment group compared with normal control group, cell clone formation inhibition rate increased obviously, in which the combination group compared with the separate DDP group, cloning inhibition rate increased significantly. By cell scratch experiment,cell migration ability of drug group was obviously lower than the control group, in which combining group compared with DDP(IC50) group show better effects.By cells invade experiment, the number of cell through the chamber was reduced with the increase of concentration PGPIPN in combination.The results showed that the PGPIPN can promote DDP to inhibition of cell invasion ability.The RT-PCR results show that expression of akt1, ERCC1, pl3k and MDR1 gens decreased significantly. Statistically significant difference (P< 0.05, P< 0.01) compared with the control group. Western Blot results show that pAKT, PI3K, ERCC1 and P-gp proteins decreased significantly (P< 0.05, P< 0.01) compared with the control group.Conclusion:Primary ovarian cancer cells were successfully cultured and available for the next experiment.PGPIPN can strengthen proliferation inhibition effect of DDP on ovarian carcinoma. PGPIPN can promote DDP induced the apoptosis of ovarian cancer.PGPIPN combining DDP on ovarian cycle arrects G2/M.PGPIPN can strengthen DDP’s inhibition on the invasion and migration of ovarian cancer. PGPIPN can enhance DDP’s inhibitionon cell clone formation.The expresstion of akt1, pI3k,ERCC1 and MDR1 genes were decrease after treating withPGPIPN combining DDP. This result showed the PGPIPN can improve the sensitivity of the DDP on ovarian cancer cells by regulating cell PI3K/AKT pathway. |
PDF Full Text Request