The Mechanism Of Dihydroartemisinin Inhibiting The Growth Of Esophageal Squamous Cell Carcinoma In Vitro And In Vivo

BackgroundEsophageal cancer is a serious malignant tumor of digestive tract,and its total morbidity and mortality rank seventh and sixth in the world,respectively.China has a high incidence of esophageal cancer,especially in Linzhou,Henan Province.Esophageal cancer includes two different histological forms:esophageal quamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC),of which esophageal squamous cell carcinoma accounts for about 90%of esophageal cancer.The cause of ESCC is complicated,and most of the patients are in the advanced stage,Surgical resection,radiotherapy and chemotherapy are common methods for the treatment of advanced esophageal squamous cell carcinoma,but due to the limitations of its treatment,the five-year survival rate of esophageal squamous cell carcinoma is still lower.What’s more,many patients have developed resistance to classical chemotherapeutic drugs,and their therapeutic effects have gradually begun to decline down.Early intervention and treatment can effectively inhibit the development of esophageal squamous cell carcinoma,so it is urgent to find new safe and effective drugs for ESCC.Chinese herbal medicine is a precious resource in China,which has a high value of drug utilization.Natural products extracted from Chinese herbal medicine show strong anticancer activity,such as quercetin,emodin,berberine and xpaclitael.Artemisinin is a sesquiterpene lactone chemical component containing peroxide group extracted and isolated from Artemisia annua by Chinese scientist Tu Youyou and others,which can effectively destroy the cellular structure and function of malaria parasite and is a first-line antimalarial drug.In addition to having significant therapeutic effects on malaria,there is growing evidence showing that artemisinins have great potential for the treatment and prevention of cancer.Studies have shown that artemisinin drugs can inhibit the proliferation of breast cancer,colon cancer,gastric cancer,cervical cancer and other cancers,and the mechanism is complex.Artemisinin regulates key factors such as NFkB,Survivin,NOXA and Bim-1,involving multiple signal pathways such as Jak2/STAT3,Wnt/β-catenin,CyclinD1-CDK4-Rb signaling.At present,there are evidence to show that dihydroartemisinin(DHA),one of artemisinin derivatives,can reduce the viability of esophageal cancer cells in a dose-dependent manner,but the detailed mechanism remains to be explored.In this study,we explored the inhibitory effect of dihydroartemisinin on the proliferation of esophageal squamous cell carcinoma in vivo and in vitro,and actively sought its targets and related molecular mechanisms.Methods1.Cytotoxicity assay:Human esophageal cancer cell line KYSE30 and KYSE150 cells were treated with dihydroartemisinin at different concentrations for 24 h and 48 h.Fixed with 4%paraformaldehyde,the number of cells was measured by IN Cell Analyzer 6000 after DAPI staining,and then the cell survival rate at each concentration was calculated.2.Cell growth inhibition assay:KYSE30 and KYSE150 cells were treated with different concentrations of dihydroartemisinin(0,1,5,10 and 15 μM)for 0,24,48,72 and 96 h.After staining with DAPI,the number of cells at each time point was recorded and the growth curve was drawn to evaluate the effect of dihydroartemisinin on the proliferation of esophageal squamous carcinoma cells.3.Soft agar assay:KYSE30 and KYSE150 cells were treated with dihydroartemisinin(0,1,5,10 and 15 μM)in agar gel about 8 d respectively to evaluate the effect of dihydroartemisinin on esophageal squamous carcinoma cell clones forming ability.4.Cell cycle assay:KYSE30 and KYSE150 cells were treated with dihydroartemisinin(0,1,5,10 and 15 μM)for 24 h,and the effect of dihydroartemisinin on esophageal cancer cells cycle was detected by flow cytometry.5.Phosphoproteome analysis:After KYSE30 cells were treated with dihydroartemisinin(0,10 μM)for 12 h,phosphoproteome analysis was performed based on mass spectrometry.6.Human Phospho-Kinase array:After KYSE30 cells were treated with dihydroartemisinin(0,10 μM)for 12 h,phospho-kinase array was performed.7.Western blotting and immunofluorescence array:Western blotting and immunofluorescence array were used to verify the results of phosphoproteome analysis.In addition,Western blotting was performed to detect the total protein levels and phosphorylation levels of mTOR,p70S6K,and RPS6 protein.8.Pull down array:Sepharose 4B beads bound to dihydroartemisinin in advance were incubated with esophageal squamous cell carcinoma cell lysate,then the AKT1 and p70S6K kinase were detected by Western blotting.9.In vitro kinase array:The effect of dihydroartemisinin on the phosphorylation of mTOR by AKT1 and p70S6K kinase was detected in vitro.10.Patient-derived xenografts models assay:Different concentrations of dihydroartemisinin(0,25mg/kg,50mg/kg)were injected intraperitoneally into SCID mice of PDX model,and the effects of different concentrations of dihydroartemisinin on tumor proliferation in mice were examined.11.Immunohistochemical(IHC)staining:The immunohistochemical staining assay were used to detect p-mTOR,p-p70S6K,and p-RPS6 phosphorylation levels of mouse tumors,and the effect of dihydroartemisinin on mTOR,p70S6K,and RPS6 pathways was evaluated.Results1.According to the cell survival rate of each concentration in the cytotoxicity assay,the experimental concentration of dihydroartemisinin was determined as 0,1,5,10 and 15 μM.2.The results of cell growth inhibition assay and soft agar assays showed that with the increase of the concentration,the inhibitory effect of dihydroartemisinin on cell proliferation and soft agar clone formation of esophageal squamous cell carcinoma was enhanced.3.The results of cell cycle assay showed that the cells were blocked at G0/Gi stage with the increasing concentration of dihydroartemisinin.Dihydroartemisinin also increased the expression level of p21 protein and reduced the expression level of CDK2.4.The results of phosphoproteome analysis,phospho-kinase array and Western blotting array showed that dihydroartemisinin may affect the expression of p70S6KT389,p70S6KT421/s424 and RPS6S235/S236 phosphorylated proteins in esophageal squamous cell carcinoma cells and down-regulate mTOR-p70S6K-RPS6 signal pathway.5.Pull down assays and in vitro kinase assays showed that dihydroartemisinin can bind to AKT1 and p70S6K kinases and inhibit the phosphorylation of mTORS2448 by AKT1 and p70S6K kinases,respectively.6.The results of patient-derived xenografts model assay showed that dihydroartemisinin can inhibit the proliferation of human esophageal squamous cell carcinoma in vivo.7.The results of immunohistochemical staining assay showed that dihydroartemisinin can reduce the phosphorylation level of mTOR,p70S6K,RPS6 protein in PDX of SCID mouse tumors in vivo.ConclusionDihydroartemisinin can down-regulate the mTOR-p70S6K-RPS6 signaling pathway by targeting AKT1 and p70S6K kinases to inhibit the proliferation of esophageal squamous cell carcinoma.