| ObjectiveGastric cancer is the fifth most common malignancy worldwide,after cancers of the lung,breast,colorectum and prostate,and it is the third leading cause of cancer deaths worldwide.More than 70%of gastric cancer cases occur in developing countries,and half of the global total cases occur in Eastern Asia(mainly in China and Japan).H.pylori infection,alcohol,non-steroidal anti-inflammatory drug(NSAID)and improper diets are the major risk factors for gastric cancer.H.pylori infection is a crucial risk factor in the occurrence of gastric adenocarcinoma.H.pylori was designated as a class I carcinogen by the WHO in 1994.Gastric inflammation induced by H.pylori may initiate superficial gastritis,which then progresses to atrophic gastritis,gastric epithelial dysplasia and finally gastric cancer.Artemisinin(ART)and its derivatives attract tremendous attention because of the curative effect on malaria in clinic.And its antitumor activity has been widely researched.Artemisinin and its derivatives can induce apoptosis in several cancer cells,including breast cancer,cervical cancer,hepatocellular cancer,gastric cancer,and colorectal cancer.Moreover,ART and its derivatives also have got great achievements in the field of microbiology.It has been discovered that ART could inhibit the growth of various gram positive and negative bacterias,including carbon maggots coli,staphylococcus epidermidis,neisseria catarrhalis,mycobacterium tuberculosis,pseudemonas aeruginosa,staphylococcus aureus,and various reference strain and clinical strain of H.pylori.Although the effects of ART and its derivates on gastric cancer have been extensively studied,the effects of ART and its derivates on H.pylori-inducing gastric cancer have not been reported.In this study,we will investigate the chemoprevention effects of ART,DHA and ARTS on gastric cancer.And we will focus on Helicobacter pylori-inducing gastric inflammation both in vitro and in vivo.Methods1.Determination of the anti-bacteria activity and cell viabilityThe inhibitory effects of ART(0,5,10,20,40,80 pM),DHA(0,1.25,2.5,5,10,20?M)and ARTS(0,1.25,2.5,5,10,20 ?M)on H.pylori ATCC 43504 were measured by broth microdilution method for 24 h treatment.The effects of ART(0,12.5,25,50,100,200 ?M),DHA(0,6.25,12.5,25,50,100 pM)and ARTS(0,6.25,12.5,25,50,100 ?M)on cell growth in GES-1 cell lines and SGC-7901 cell lines were measured by MTT assay for 24 h.2.Determination of the protein levels in NF-?B signaling pathway and the ROS productions affected by ART,DHA,and ARTS.SGC-7901 cells were treated by ART(5,10,20?M),DHA(5,10,20?M),and ARTS(5,10,20?M)for 24 h,then cells were infected with H.pylori for 24 h.After that,cells were collected and proteins were extracted for subsequent analysis.The protein expression levels of p65,p-p65,I?B-a and p-I?B-a were measured by Western blotting,and the protein expression levels of IL-8 and TNF-a were measured by ELISA.SGC-7901 cells were treated by ART(5,10,20?M),DHA(5,10,20?M),and ARTS(5,10,20?M)for 24 h,then cells were infected with H.pylori for 6 h.The ROS productions were measured by flow cytometry,and the protein expression levels of HO-1 and NQO1 were measured by Western blotting.3.Evaluation of ART,DHA,and ARTS on the antitumor effects in xenograft tumor mice modelWe estabilish nude mice bearing SGC-7901 cells xenograft tumor model to evaluate the preventive effect of ART,DHA and ARTS on human gastric cancer cells,Briefly,SGC-7901 cells were resuspended in 0.2 ml of PBS and subcutaneously inoculated with 2×106/mouse into the right flank of the nude mice.When the average volume of tumor reached approximately 50-100 mm,the mice were divided randomly into eight groups(n =6 for each),including control group(normal saline),CTX treatment groups(25 mg/kg),ART treatment groups(25 mg/kg and 50 mg/kg),DHA treatment groups(25 mg/kg and 50 mg/kg),and ARTS treatment groups(25 mg/kg and 50 mg/kg).After 24 days,the nude mice were sacrificed,and the tumors were measured.4.The chemoprevention of ART,DHA,and ARTS on C57BL/6 mice gastric cancer model in vivoIn this study,we investigated the chemoprevention of ART,DHA,and ARTS on C57BL/6 gastric cancer model induced by H.pylori plus MNU.After drugs treatment,the C57BL/6 mice were sacrificed.A fraction of gastric tissue was embedded in paraffin.Serial paraffin sections were prepared and stained with hematoxylin and eosin(H&E)for morphological observation.Immunohistochemical analyses were carried out with a mouse monoclonal anti-COX-2 antibody,anti-I?Ba antibody,and anti-p-I?Ba antibody.The serum protein expression levels of TNF-?,IL-6,and IL-1(3 were measured by ELISA.5.Statistical analysisStatistical significance of datas were determined by one-way ANOVA using SPSS 13.0 software.Data were plotted using Graphpad Prism 5.0 and expressed as the mean±SD.Statistical significance was considered at p<0.05.Results1.ART,DHA,and ARTS inhibited the growth of H.pylori and SGC-7901 cellsART,DHA,and ARTS suppressed the growth of H.pylori ATCC 43504.DHA,ARTS could completely inhibit the growth of strain ATCC 43504 at 10?M,20?M treatment respectively.All the tested drugs slightly decreased cell viability after 24 h treatment.Compared with control,the cell viability with ART,DHA,and ARTS treatment were 94.5%±8.2%,76.2%±3.1%,and 67.2%± 3.5%at concentration,of 25 pM,respectively.2.ART,DHA,and ARTS down regulated NF-?B pathway and decreaced the production of ROS in H.pylori-infected SGC-7901 cellsThe H.pylori-infected cells without drug treatment demonstrated a significant increase in ROS production compared to the uninfected cells.Treatment with ART,DHA,and ARTS significantly reduced the amount of ROS in H.pylori-infected SGC-7901 cells.In this study,overexpression of IL-8,TNF-a,and COX-2 were observed in infected cells after co-culture experiment.And this up-regulation of IL-8,TNF-a,and COX-2 was significantly inhibited by the three drugs in a dose dependent manner(p<0.001).3.ART,DHA,and ARTS inhibited the gastric tumor growth in xenograft mice modelOur results showed that 100%tumor formation occurred in nude mice bearing SGC-7901 cells.And ART,DHA,and ARTS at indicated concentration significantly inhibited the tumor growth in xenograft mice.Compared with control group,the growth inhibtion rates were 29.9%,33.9%in treatment of ART,and 19.5%,45.4%in treatment of DHA,and 30.9%,40.7%in treatment of ARTS,at 25 mg/kg and 50 mg/kg group,respectively.4.ART,DHA,and ARTS decreased the incidence of gastric cancer inuced by H.pylori plus MNU in C57BL/6 miceThe gastric cancer induced by H.pylori plus MNU revealed 78.6%tumor formation rates in C7BL/6 mice.And the tumor formation rates of 60 mg/kg ART,DHA,and ARTS treatment groups were 73.3%,43.8%,and 60%,respectively.Immunohistochemistry showed that the protein levels of COX-2 and p-I?Ba were up-regulated,while the levels of I?Ba were down-regulated in model group.Compared with model group,the ART,DHA,and ARTS group significantly down-regulated the protein level of COX-2,and up-regulated the protein level of I?Ba.ELISA showed that the protein level of TNF-a,IL-6,and IL-1? were increased in model group.Compared with model group,the ART,DHA,and ARTS group significantly reduced the protein level of TNF-a,IL-6,and IL-1?.ConclusionsOur findings revealed that ART,DHA,and ARTS could inhibit the growth of H.pylori.And the three drugs also suppress the proliferation of gastric adenocarcinoma cells both in vitro and in vivo.Moreover,ART,DHA,and ARTS inhibit the production of inflammation cytokines and ROS on the H.pylori-infected gastric adenocarcinoma cells.In addition,ART,DHA,and ARTS significantly decreased the incidence of gastric cancer induced by H.pylori plus MNU in vivo. |
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