Study On Synergistic Inhibitory Effect Of Dihydroartemisinin And Dasatinib On Hepatocarcinoma Cells

Purpose:To investigate dihydroartemisinin(DHA)synergistic inhibitory effect with dasatinib(Das)on human hepatocellular carcinoma cells(HCC)and explore the molecular mechanism by which DHA can reduce the chemoresistance of HCC and provide a safe and effective treatment option for patients with advanced HCC.Method:CCK8 assay was performed for drug(DHA or/and Das)cytotoxicity to HepG2.HepG2 cells were tested for proliferation and clonality by plate clone formation assay.Scratch assay was used to detect the migration of HepG2 cells.Flow cytometry was used to detect the cell cycle changes,apoptosis of HepG2 cells,the content of intracellular superoxide anion ROS and mitochondrial membrane potential(??m).In addition,the apoptosis of the cells was observed and detected by morphological observation and acridine orange / ethidium bromide(AO/EB)staining.In order to further study the mechanism of DHA increasing HepG2 cells chemosensitivity,Western blot was used to detect the expression of some related proteins.To investigate the relationship between chemosensitization of DHA and FOXC1 gene,the expression of FOXC1 mRNA was detected by real-time fluorescent quantitative PCR(RT-qPCR).CCK8 assay was used to determine the cell viability of co-treated(DHA and Das)cells after treating with bortezomib(the proteasome inhibitor),as well as the expression of FOXC1 protein were detected by western blot.Result:DHA(0~50 ?mo L/L)and Das(0 ~ 200 nmoL/L)were toxic to HepG2 cells in a concentrationdependent manner.Concentrations of DHA(10 ?moL/L)and Das(70 nmo L/L)which had rather small cytotoxicity to HepG2(decreased cell viability was about 20%)and compared with the control group,the cell viability was significantly different.And moreover the combination treatment of them showed significant synergistic antitumor activity(decreased cell viability was 55.02%±2.53%).The result of the colony formation experiment showed that the co-treatment group had a small number of cell clones and small colonies,and the scratch test result showed that co-treatment almost completely inhibited cell migration.In addition,the results of flow cytometry showed that co-treatment of DHA and Das for 48 h significantly induced G0/G1 phase arrest in HepG2 cell cycle(DHA: Das: Co-treated=49.59%±0.96%: 54.55%±0.97%: 61.12%±1.74%)and significantly decreased ??m(36h)and induced more apoptosis(DHA: Das: Cotreated=15.81%±2.72%: 14.65%±1.21%: 60.24%±2.57%).The results of morphological observation and AO/EB staining were consistent with the results of flow cytometry.Simultaneously,it was found by flow cytometry(dihydroethidium staining)that DHA specifically promoted production of intracellular ROS.Co-treatment significantly down-regulated the expression of c-Myc,Cyclin E1 and E2F1 proteins and anti-apoptotic proteins Bcl-2 and Poly ADP-ribose polymerase(PARP),and significantly upregulated the pro-apoptotic proteins Bim,Cleaved-Caspase 9 and Cleaved-PARP.Among them,DHA could specifically up-regulated the expression of Bim,a pro-apoptotic protein,and Cleaved-PARP,an inactivating DNA repair enzyme.In addition,co-treatment significantly down-regulated the expression of the cancer drug resistance-associated proteins Nrf2 and FOXC1,with DHA specifically down-regulating the expression of Nrf2 and FOXC1.The result of RT-qPCR showed that the expression level of FOXC1 mRNA in HepG2 cells decreased slightly(97.76%±0.63% relative to the control group)after DHA treatment.On the contrary,bortezomib(10 nmoL/L)significantly increased the activity of the co-treatment group cells and the FOXC1 protein expression was significantly increased(relative to the co-treatment group was 279.40% ± 4.90%).Conclusion:DHA can increase the chemosensitivity of HepG2 to Das,synergize with Das up-regulate proapoptotic protein expression and down-regulate the expression of anti-apoptotic protein and drug resistance-related protein in cancer cells,promote intracellular ROS production,decrease ??m and reduce the ability of cancer cells to repair DNA damage,activate the mitochondrial apoptotic pathway in cancer cells to induce cell cycle arrest and apoptosis of Hep G2 cells,and inhibit the proliferation,clonogenicity and migration of HepG2 cells.Thereinto,the chemosensitization of DHA is closely related to its promotion of the degradation of FOXC1 protein by proteasome in HepG2 cells.