Effects And Mechanism Of Dihydroartemisinin On Proliferation,Apoptosis,Metastasis And Ferroptosis Of Hepatoma HepG2 Cells

Research BackgroundPrimary hepatic carcinoma(PHC)is one of the most common malignant tumors in the world.Hepatocellular carcinoma(HCC)is the main type of liver cancer worldwide,accounting for about 75%of the total.At present,the most commonly used chemotherapy drugs in clinical practice have great toxic and side effects.While killing liver cancer cells,they will also cause damage to normal cells and seriously affect the quality of life of patients.Moreover,the clinical efficacy of existing chemotherapy drugs is limited.Therefore,it is of great significance to actively develop new anti-liver cancer drugs,especially to screen the anti-liver cancer compounds with definite curative effect and little toxic and side effects from the treasure house of traditional Chinese herbal medicine in China.Artemisinin is a kind of colorless needle crystal extracted from the stem and leaf of the compound inflorescence plant Artemisia annul.It is a kind of sesquiterpene lactone compound containing peroxide group,and it is a kind of high efficiency and low toxicity antimalarial drug.In recent years,it has been found that artemisinin and its derivative dihydroartemisinine(DHA)not only has antimalarial effects,but also has significant anti-tumor effects,showing a broad spectrum of anti-tumor activities.Dihydroartemisinin,as an artemisinin derivative,is C15H2405.Compared with artemisinin,dihydroartemisinin has higher water solubility and stronger activity,which can act on tumor cells and has the advantages of high efficiency,low toxicity and selective killing.ObjectiveThe purpose of this study is to investigate the inhibitory effect of DHA on growth,proliferation and migration of hepatocellular carcinoma HepG2 cells and the inducing effect of apoptosis and iron death and its molecular mechanism at cellular and molecular levels.To understand the effect of DHA on proliferation and apoptosis of hepatoma HepG2 cells and the effect of cell metastasis;To reveal the molecular mechanism of DHA inhibiting the growth and metastasis of hepatocellular carcinoma HepG2 cells and inducing their apoptosis and iron death,and to provide a preliminary experimental basis for the application of DHA in clinical treatment of hepatocellular carcinoma.MethodsHepG2 cells were cultured in vitro to logarithmic growth stage,and treated with different doses of DHA in vitro,then the experimental indexes of DHA on cell growth,apoptosis,metastasis and iron death of HepG2 cells were detected by the following experimental methods.1.Use CCK-8(Cell Counting Kit-8)experiment to detect the inhibitory effect of DHA on the proliferation ability of hepatocellular carcinoma HepG2 cells,explore the 50%inhibitory concentration(IC50)of DHA on these two kinds of hepatocellular carcinoma cells,and determine the concentration for subsequent experimental groups based on this result;2.CCK-8 assay was used to detect the inhibitory effect of different concentrations of DHA on HepG2 cells for different periods of time,and the inhibitory rate of DHA on HepG2 cells was calculated statistically.3.The effects of DHA on the growth activity of hepatocellular carcinoma HepG2 cells were detected by plate cloning experiment,the experimental results were counted,and the inhibitory rate of DHA on the growth activity of hepatocellular carcinoma HepG2 cells was calculated.4.The effect of DHA on the apoptotic morphology of hepatoma HepG2 cells was observed by AO/EB fluorescence double staining method,and the observation results were counted and the apoptosis rate of cells was calculated.6.Annexin V-FITC/PI double staining flow cytometry was used to detect the induction effect of DHA on apoptosis of hepatocellular carcinoma HepG2 cells,and the apoptosis rate of hepatocellular carcinoma cells was calculated.7.Western blot assay was used to detect the effects of DHA on the expression of Bax、Bcl-2、Fas、FADD、cleaved-caspase-8、cleaved-caspase-3 and cleaved-caspase-9 proteins in hepatocellular carcinoma HepG2 cells,and their relative expression levels were calculated.9.Cell scratch assay was used to detect the effect of DHA on migration ability of hepatoma HepG2 cells,and the migration inhibition rate of DHA on hepatoma HepG2 cells was calculated.10.Transwell invasion assay was used to detect the effect of DHA on the invasion ability of hepatoma HepG2 cells,and the inhibition rate of DHA on the invasion ability of hepatoma HepG2 cells was calculated.13.Western blot was used to detect the effects of DHA on the expression of e-cadherin,N-cadherin and protein in HepG2 cells,and calculate their relative expression levels.14.DCFH-DA flow cytometry was used to detect the effect of DHA on ROS content in hepatocellular carcinoma HepG2 cells.15.Western blot was used to detect the effect of DHA on GPX4 protein expression in HepG2 cells and calculate its relative expression level.Result1.IC50 of CCK-8 assay showed that the IC50 of DHA in HepG2 cells was 172 μM for 24 h in vitro.2.CCK-8 assay showed that compared with the Control group(Control),the growth and proliferation of hepatoma HepG2 cells treated with DHA at different concentrations for 24 h and 48 h in vitro were significantly inhibited(*P<0.05),and the inhibition rate of dihydroartemisinin on cell growth and proliferation increased with the increase of concentration and time,presenting a dose and time dependent effect.3.Compared with the Control group,the growth activity of HepG2 cells treated with DHA at different concentrations for 24 h in vitro was significantly inhibited(*P<0.05),and its inhibition rate increased with the increase of concentration.4.The results of AO and EB fluorescence double staining showed that compared with the Control group,the proportion of apoptotic cells in HepG2 cells treated with DHA at different concentrations for 24 h in vitro increased,and the apoptotic rate was significantly different(*P<0.05),and the apoptosis rate increased with the increase of concentration.6.Flow cytometry results of Annexin V-FITC/PI double staining showed that compared with the Control group(Control),the proportion of apoptotic cells in HepG2 cells treated with different concentrations of DHA for 24 h and 48 h in vitro increased significantly,and the apoptotic rate was significantly different(*P<0.05),and the apoptosis rate increased with the increase of drug concentration and time.7.The results of cell scratch experiment showed that,compared with the Control group,the scratch healing of hepatoma HepG2 cells treated with DHA at all concentrations for 24 h in vitro was significantly inhibited by the dose-supplemented groups,and the healing rate was significantly different(*P<0.05),and its healing rate decreased with the increase of concentration.8.The results of Transwell experiment showed that compared with the Control group,after treated with DHA at all concentrations for 24 h in vitro,DHA in all dose groups could significantly inhibit the passage of tumor cells through the compartment,and the passage inhibition rate was significantly different(*P<0.05),and the crossing inhibition rate increased with increasing concentration.9.Western blot results showed that different doses of DHA could act on hepatoma HepG2 cells in vitro 24 After h,compared with the Control group,the relative expression levels of Bax,Fas,FADD,cleaved-caspase-8,cleaved-caspase-3,cleaved-caspase-9,e-cadherin and GPX4 were significantly increased,while the expression levels of Bcl-2 and N-cadherin were significantly decreased.(P<0.05).Conclusion1.Dihydroartemisinin can significantly inhibit the growth and proliferation of hepatocellular carcinoma HepG2 cells cultured in vitro,presenting a dose and time dependent effect.2.Dihydroartemisinin significantly induced apoptosis of hepatocellular carcinoma HepG2 cells cultured in vitro and presented a dose-dependent effect.3.Dihydroartemisinin significantly inhibited the metastasis of hepatocellular carcinoma HepG2 cells in vitro and presented a dose-dependent effect.4.The potential mechanism of dihydroartemisinin inducing apoptosis of HepG2 cells may be related to the activation of death receptor and mitochondrial pathway signal;The potential molecular mechanism of dihydroartemisinin inhibiting the metastasis of hepatocellular carcinoma HepG2 cells may be related to the inhibition of EMT.The mechanism that dihydroartemisinin can induce iron death in HepG2 cells may be related to the accumulation of reactive oxygen species(ROS)and the accumulation of lipid peroxides by promoting the inactivation of GPX4.