Mechanism Of The Endophytic Fungi From Ginkgo Biloba L Reverse Cisplatin Resistance In Drug Resistance In Human Ovarian Cancer Cells

Conventional treatment for ovarian cancer includes surgical treatment andchemotherapy..The drug resistance of ovarian cancer make the chemical treatmentnot ideal.The mechanism of drug resistance was studied and some progress wasmade.A new method for the chemotherapy resistance to ovarian cancer,is the hotresearch.Rosoloactone,as a new anticancer drug,can induce the apoptosis of cisplatinresistant cell line SKOV3/DDP and significantly reversed cisplatinresistance.Studying the mechanism of Rosoloactone can not only to develop newanticancer drugs,but may also provide experimental evidence for explaining tumorresistance.P62is a phosphate protein with a multifunctional domain, related to cellreplication and survival. It is found that P62plays its role in keeping cell aliveincluding proliferation,differentiation and induction of apoptosis gene and has anabnormal expression in multiple tumors. It is regarded as the receptor of ubiquitinsubstrate for selective autophagy,regulating the homeostasis and keeping themaintenance of cell survival.It is found that cisplatin resistance is related to the endoplasmic reticulum stressand the unfolded protein response.When the endoplasmic reticulum is stressed, thereis an error folding proteins and unfolded proteins,resulting in unfolded proteinresponse（Unfolded Protein Response，UPR）.Besides the classical endoplasmicreticulum associated protein degradation,there is another way for proteindegradation,ER-activated autophagy.This study mainly takes the human ovarian cisplatin resistant cell lineSKOV3/DDP as the research object,to investigate the role and mechanism ofRosoloactone of reversing cisplatin resistance in human ovarian cancer cells,providing a new direction for clinical treatment for tumor drug resistance.Method(1)Cell viabilityof SKOV3/DDP cells was detected by MTT assay.Themorphology of nucleus was observed by Hoechst33258dyeing.Expression ofCaspase-3Cleaved was detected by Western blot.Apoptosis rate of SKOV3/DDPcells was detected by flow cytometry. Apoptosis of SKOV3/DDP cells was detectedby TUNEL staining.(2)The mitochondrial membrane potential of SKOV3/DDP cells was detected byflow cytometry.The expression of Bcl-2, Bax and Bak was detected by WesternBlot.Expression of mitochondrial and cytochrome C in SKOV3/DDP cells was detected by immunofluorescence assay(3)Endoplasmic reticulum stress associated proteins Grp78and Cleavedcaspase-4was detected by Western blot.(4)Expression of p62and LC3in cells was detected by Westernblot.Colocalization of LC3and p62was detected by indirect immunofluorescenceassay.(5)DCFH-DA method detects the level of ROS. The expression of γ-H2AX wasdetected by western blot. Expression of γ-H2AX was observed byimmunofluorescence technique.Result1. Cisplatin combined with Rosoloactone significantly inhibits the proliferationof cisplatin resistant cell line SKOV3/DDP of human ovarian cancer cells presentingtime dependence and dose dependence. Compared with the control group, cisplatingroup and Rosoloactone group. Cisplatin combined with Rosoloactone acting onSKOV3/DDP cells can induce the apoptosis of cells2. Cisplatin combined with Rosoloactone induced the decrease of mitochondrialmembrane potential of SKOV3/DDP cells,increased the expression of Baksignificantly, highly increased the ratio of Bax/Bcl-2.Cytochrome C was released.Theexpression of Caspase-3Cleaved was increased. The combination of Rosoloactoneand cisplatin may induce apoptosis in SKOV3/DDP cells via mitochondrial pathway.3. The expression of Cleaved caspase-4and Grp78in SKOV3/DDP cells wasenhanced by cisplatin combined with Rosoloactone. Expression of ubiquitin proteinwas increased.The expression of p62protein in SKOV3/DDP cells decreased incisplatin group and Rosoloactone group.The expression level of p62protein wassignificantly lower than that of the control group, also significantly lower thancisplatin and Rosoloactone groups.The LC3II/I ratio were all increased. The p62expression was very weak in the group cisplatin combined with Rosoloactone. Thereis no obvious p62and LC3colocalization detected by indirect immunofluorescencetechnology.The combination of Rosoloactone and cisplatin can induce theendoplasmic reticulum stress mediated apoptosis.4. The expression of ROS in SKOV3/DDP cells was significantly increased bycisplatin combined with Rosoloactone. The expression ofγ-H2AX was increasedsignificantly by cisplatin combined with Rosoloactone.It is suggested that thecombination of cisplatin and Rosoloactone induced DNA damage in SKOV3/DDPcells.ConclusionThe study was based on the cisplatin resistant cell line SKOV3/DDP of humanovarian cancer.SKOV3/DDP cells were acted with different concentrations ofRosoloactone,cisplatin,and cisplatin combined Rosoloactone.It is found that theproliferation of SKOV3/DDP cells can be significantly inhibited by the combination of cisplatin and rosoloactone and was Induced apoptosis.1.Cisplatin combined with Rosoloactone could elevate the ROS levels ofSKOV3/DDP cells and decrease the mitochondrial membrane potential.We canconclude that the increase of mitochondrial membrane permeability in SKOV3/DDPcells caused by cisplatin combined with Rosoloactone could release cytochrome C,resulting to the Caspase family activation and apoptosis.2.Cisplatin combined with Rosoloactone may induce the expression ofendoplasmic reticulum stress associated proteins.We hypothesized that cisplatincombined with Rosoloactone might induce the increasing of the error folding proteinsand induce poptosis by endoplasmic reticence stress.3.The expression of p62was decreased in the cisplatin combined Rosoloactonegroup.The expression of LC3II increased. The results of laser scanning confocalmicroscope show that cisplatin combined with Rosoloactone can decrease thecolocalization of p62and LC3. It is suggested that autophagy decreased while errorfolding pritein increased and endoplasmic reticulum stress happened, resulting tocell’s death.4.Further detection of gamma γ-H2AXdemonstrated that cisplatin combined withRosoloactone may induce the DNA damage and induce the cell death.