Bioactive Peptide (PGPIPN) From Casein Reduced The Resistance Of Human Ovarian Cancer Cells To Cisplatin By HSP Signaling Pathway In Vitro

Objective:1. To investigate the decrease of bioactive peptide (PGPIPN) from casein on the resistance of human ovarian cancer cells to cisplatin.2. To explore the possible molecular mechanism of PGPIPN on reducing the resistance of human ovarian cancer cell line to cisplatin by HSP signaling pathway.Methods:1. The primary human ovarian cancer cells were cultureed and its purities were identified.2. We conducted CCK8 experiments to compare the inhibition of human ovarian cancer lines COC1, COC1/DDP and primary human ovarian cancer cells proliferation and select the best time of drug action by using cisplatin only and cisplatin combining different concentrations of PGPIPN.3. The effect were detected on apoptosis and cell cycle of COC1, COC1/DDP and primary human ovarian cancer cells with different drug concentrations by flow cytometry(FCM).4. The expressions of relevant HSF1, HSP70, ERCC1,MDR1 gene were tested by RT-PCR.5. The expressions of relevant HSF1, HSP70, ERCC1, MDR1 protain were tested by Western blot.Result:1. Human ovarian cancer cells were successfully cultured and passed, and their purities were over 80% after identification by immunofluorescence.2. The CCK8 experiments showed that compared with the normal group, the group with cisplatin alone, groups combined with different concentrations of PGPIPN of COC1 cells and COCl/DDP cells, human ovarian cancer cell inhibition rates increased and the cell inhibition rate was reached 76.8% at 48h by the IC50 of cisplatin combined the highest concentration PGPIPN. For the COC1 group, the inhibition rate also increased, but less than COC1/DDP cells inhibition rate, and the maximum inhibition rate was 62.3% at 48h. The cell inhibition rate reached 87.6% of primary human ovarian cancer at 48h by the IC50 of cisplatin combined the highest concentration PGPIPN.3. By flow cytometry, apoptosis of COC1 cells treaded with cisplatin combined different concentrations PGPIPN were slightly higher than that of cisplatin alone, and the maximum apoptosis rate was 45.4%. The apoptosis of COCl/DDP cells treaded with cisplatin combining different concentration PGPIPN were also higher than that of cisplatin alone, even slightly higher than COC1 group,and the maximum apoptosis rate was 67.4%. The maximum apoptosis rate was 61.4% of primary human ovarian cancer cells. Meanwhile, PGPIPN conbining cisplatin can significantly arrest G2/M phase on cell cycle, and influence was more apparent with the increase of concentrations of PGPIPN conbining cisplatin.4. The expressions of hsf1, hsf, ERCC1 and MDR1 were decreased in COC1, COC1/DDP cells treaded PGPIPN combining cisplatin, which were dose and time-dependment. The expression of ERCC1 and MDR1 in COC1 cell was weak, and theirs decreased expression were not obvious with PGPIPN increased. While the expression of ERCC1 and MDR1 were significantly reduced in COC1/DDP cell with the increase of concentrations of PGPIPN.5. The expressions of proteins about hsf1, hsf, ERCC1 and MDR1 were decreased inCOC1, COCl/DDP cells treaded PGPIPN combining cisplatin, which were dose and time- dependment. The expression of ERCC1 and MDR1 in COC1 cell was weak. Meanwhile, the expression of ERCC1 and MDR1 were significantly reduced in COC1/DDP cell with the increase of concentrations of PGPIPN.Conclusion:1. PGPIPN can reduce the cisplatin of the human ovarian cancer cells.2. The mechanism is that during carcinogenesis, the activation of HSF1 is inhibited, and the expression of HSP70 is reduced, thereby reducing the resistance of cells expressing ERCC1 and MDR1.