Effects Of Ginkgol C17:1 On Autophagy And Apoptosis In Liver Cells With Chemotherapy

With liver cancer patients tend to be younger,liver cancer has become a major global health problem.Surgery,radiofrequency ablation therapy and chemotherapy can effectively remove or reduce tumor tissue.There is a certain progress in the treatment of liver cancer in the recent years.However,the ability to prevent metastasis and recurrence is limited.For the vast majority of patients with advanced cancer,chemotherapy is necessary as it is the most effective and most important treatment for cancer.Due to the side effects of cisplatin,its anticancer efficacy is limited,therefore finding a novel auxiliary chemotherapy drugs with less toxic effects or attenuated role is an imperative strategy for cancer treatment at present.Now numerous studies have confirmed that some Chinese herbal medicine composition had anti-cancer effect,and the tumor-suppressor of ginkgo phenol have been reported.Ginkgo phenol can significantly affect the cell survival,proliferation,invasion and infection,but the tumor-suppressor mechanism of ginkgo phenol is not clear.Autophagy and apoptosis are two important regulatory mechanisms in cancer cell,which involved in processes of cell growth,reproduction,and metastasis,invasion and metabolism.This study aim to discuss the effects of Ginkgol C17:1 on autophagy and apoptosis with chemotherapy in hepatoma carcinoma cells,normal hepatocyte cells and tumor-burdened body.Experimental contents and results of this study:(1)Ginkgol C17:1 can inhibit autophagy and promote apoptosis in HepG2 cells with chemotherapy,through regulating different signaling pathway.MTT method determined HepG2 cell activity,RFP-GFP-LC3 adenovirus transfection experiment detected LC3 autophagosome and the autophagy flux,Western blotting method detected the expression of autophagy protein(Beclin1,LC3 Ⅰ/Ⅱ,p62),apoptosis protein(Bax/Bcl-2,Cleaved caspase3)and pathway proteins,PCR method detected the gene expression of autophagy protein Beclin1,Hoechst staining detected the cell nucleus distortion,Immunofluorescence method detected the expression of apoptosis protein cleaved caspase3.The results show that the Ginkgol C17:1 and cisplatin all inhibited the growth of liver cancer cell HepG2.With combination,Ginkgol C17:1 further enhanced the antitumor activity of cisplatin.In HepG2 cells,Ginkgol C17:1 and cisplatin all induced the autophagy,but compared with cisplatin alone,co-treatment reduced autophagy.In addition,the results confirmed that Ginkgol C17:1 and cisplatin all induced apoptosis,and compared with cisplatin treatment,the co-treatment further promoted apoptosis in HepG2 cells.In conclusion,the Ginkgol C17:1 can suppress the protective autophagy induced by cisplatin,and enhance the apoptosis induced by cisplatin,as result to enhance the anticancer effect of cisplatin in HepG2 cells.The study of signaling pathway found Ginkgol C17:1 regulated autophagy through by AMPK/ULK1 pathway,regulated apoptosis through by PI3K/AKT/mTOR pathway in HepG2 cells with chemotherapy,and the regulation of AMPK was related to the activity of p53.(2)Ginkgol C17:1 can enhance autophagy,reduce apoptosis and activate the Nrf2 pathway in L02 cells with chemotherapy.MTT method determined L02 cell activity,RFP-GFP-LC3 adenovirus transfection experiment detected LC3 autophagosome and the autophagy flux,Western blotting method detected the expression of autophagy protein(Beclin1,LC3 Ⅰ/Ⅱ,p62),apoptosis protein(Bax/Bcl-2,Cleaved caspase3)and pathway proteins,PCR method detected the gene expression of autophagy protein Beclin1 and NADPH oxidase,Hoechst staining detected the cell nucleus distortion,Immunofluorescence method detected the expression of apoptosis protein cleaved caspase3.Results show that cisplatin significantly inhibited the growth activity of normal liver cells L02.However,the effect of ginkgol C17:1 on L02 cells was not obvious at low dose.Obvious inhibitory effect only was showed at high dose.With comnination,low dose Ginkgol C17:1 weakened the inhibitory effect of cisplatin,medium dose Ginkgol C17:1 has no effect on cisplatin effect,high dose Ginkgol C17:1 further enhances the inhibition of cisplatin in L02 cells.Ginkgol C17:1 induced the autophagy,but cisplatin did not.In comparison with treatment with cisplatin alone,autophagy was enhanced in co-treatment of cisplatin and Ginkgol C17:1.Cisplatin could induce apoptosis in L02 cells.Compared with cisplatin treatment,co-treatment did not merely enhance apoptosis and also show a downward trend.In conclusion,the ginkgolC17: induced protective autophagy in chemotherapy cell,and reduced the cisplatin-induced apoptosis,further to repair the damage induced by cisplatin to protect the normal liver cell.At the same time,we found Ginkgol C17:1 also regulated autophagy through by AMPK/ULK1 pathway,regulated apoptosis through by PI3K/AKT/mTOR pathway in L02 cells with chemotherapy.In addition,the Ginkgol C17:1 activated the Nrf2 protective pathway of normal liver cells.Through starting the expression of downstream antioxidant components NADPH oxidase,Ginkgol C17:1 protected the cells against the damage of cisplatin.(3)Ginkgol C17:1 can enhance the anticancer effect of chemotherapy in heps tumor-burdened mice,and inhibit autophagy and promote apoptosis in cancer tissue with chemotherapy.Kunming mice were inoculated with heps cells to prepare subcutaneous tumor-bearing mice model and carcinoma in situ model respectively.According to the requirements of the experimental group,the drugs wese used.Eyeball blood obtained from tumor-burdened mice before death,ELISA kit detected the levels of serum cytokines;Tumor was obtained,weighed and record analyzed after mice death;Western blotting and IHC method detected the expression of autophagy and apoptosis related proteins;HE staining detected the tumor tissue change.The experimental results show that all drugs shown significantly inhibitory effect for tumor growth.In combination group,inhibitory effect was obvious higher compared to the group with alone drug.Tumor-burdened mice had better mental state treated with combination.In addition,the detection of immune organs and immune-related cytokines confirmed that Ginkgol C17:1 improved the immunity of tumor-burdened mice with chemotherapy.The detection of tumor tissue protein found that Ginkgol C17:1 reduced autophagy induced by cisplatin,enhanced the apoptosis induced by cisplatin and inhibited cancer metastasis,those conclusions were consistent with the experimental results in vitro.The experiments have confirmed that,in the process of combination therapy of cisplatin and ginkgol C17:1,ginkgol C17:1 can enhance the antitumor activity of cisplatin and immunity ability,to protect normal liver cells from the damage caused by cisplatin in vivo and vitro.The research provided the certain theory basis for the mechanism study of auxiliary chemotherapy drugs.