| Backgrounds and Objectives:Lung cancer is a malignant lung tumor characterized by uncontrolled cell growth in one or both lungs.It is one of the malignant tumors with the highest increase in morbidity and mortality and the greatest threat to human health and life.According to pathological classification,lung cancer can be divided into Small Cell Lung Cancer(Small Cell Lung Cancer,SCLC)and Non-Small Cell Lung Cancer(Non-Small Cell Cancer,NSCLC).Among them,85%of the cases are NSCLC patients.NSCLC is one of the most common malignant tumors given its frequent tumor ’driver gene’EGFR or Ras mutation.Dihydroartemisinin(DHA)is a derivative of artemisinin and belongs to sesquiterpene compounds.DHA has a powerful and rapid killing effect on malaria parasite.DHA has been found its anti-malaria,anti-inflammatory,anti-oxidation,anti-diabetes and anti-tumor effects.Recent research found that DHA have good anti-tumor effects in lung cancer,colon cancer,ovarian cancer,pancreatic cancer,liver cancer and other malignant cancer types.It has many advantages like low toxic and side effects,multiple targets and preferred to tumor cells.However,DHA alone is limitedly inhibits tumor types,thus combined treatment seems to be a more promising option in NSCLC.ABT-263(Navitoclx)is a potent inhibitor of Bcl-2,Bcl-xL and Bcl-w developed by Abbott laboratories.A large number of experiments show that ABT-263 induces cell apoptosis in vitro.Because navitoclax inhibits Bxl-xL,it reduces platelet lifespan,which caused thrombocytopenia,and this makes it dose-limiting.Therefore,we explored DHA combined with low-concentration ABT-263 to treat EGFR or Ras mutant lung cancer and its molecular mechanisms.Methods and Results:1.DHA enhanced ABT-263 induced anti-cancer effect in EGFR or Ras mutant NSCLC cells(1)Using high content real time observes cell growth in EGFR mutant H1975 and Ras mutant A549 cells,we found DHA or ABT-263 treatment alone caused only limited cell growth inhibition,while the coadministration significantly triggered cell death and cell growth inhibition.(2)Flow cytometry and statistical results demonstrated that DHA synergistically enhances ABT-263-induced NSCLC cell apoptosis with time-and dose-dependent manners.NSCLC cells with EGFR or Ras mutations were also treated with single or combination drugs,respectively.The combination treatment resulted NSCLC cells significant apoptosis and had little side effects on immortalized lung fibroblast.Knocking down Bax significantly resuced combination induced cell apoptosis,indicating that DHA combine ABT-263 induced intrinsic apoptotic pathway.(3)Colony formation experiments confirmed DHA synergistic ABT-263 induced cell growth inhibition.2.The molecular mechanism of DHA synergistically enhaced ABT-263 induced apoptosis(1)Immunobolting and shRNA interference experiments found that DHA inhibited the expression level of pro-survival member Mcl-1,thus enhanced ABT-263 induced cell apoptosis.(2)Screening of Bcl-2 family members by western blotting showed that DHA both inhibited the expression of Survivin and increased the upregulation of pro-apoptotic member Bim with a time-and dose-dependent manner.(3)Protein kinase array results showed that the phosphorylation of Signal Transducer and Activator of Transcription 3(STAT3)is inhibited by DHA with a time-and dose-dependent manner.(4)Using shRNA or small molecule inhibitor Stattic to mimic the DHA inhibitory function of STAT3,we found that DHA inhibited the phosphorylation of STAT3 and significantly enhanced the role of ABT-263.Constructive active STAT3 partially protected combination induced cell apoptosis.The above results demonstrate that DHA significantly enhances the effect of ABT-263 by inhibiting the phosphorylation of STAT3.(5)Immunoblotting screening found that JAK2 is an upstream molecule of STAT3.Inhibition of JAK2 by AZD1480 significantly enhanced ABT-263 induced cell apoptosis.(6)Inhibition and Overexpression of STAT3 found Mcl-1 and Survivin are the downstream of STAT3.The results of dual luciferase reporter assay showed that DHA inhibited the transcriptional activity of STAT3.Q-RT-PCR tests showed DHA downregulate Mcl-land Survivin transcriptional level.3.The role of dual drug combination in vivo(1)Xenograft studies in nude mice show that cotreatment of DHA and ABT-263 significantly inhibits tumor growth in vivo.(2)Western blot results consistent with the in vitro studies.DHA inhibited STAT3,Mcl-1,Survivin expression but increased the apoptotic number Bim expression levels,thus combined with ABT-263 induced caspase 3 activation.Conclusion and significance:In this study,we investigated the interaction between DHA and a BH3-mimetic ABT-263 in NSCLC cell lines carrying oncogenic EGFR or RAS mutation.We demonstrated that cotreatment of these cells with DHA and ABT-263 synergistically triggered apoptosis in vitro and reduced their tumorigenicity in vivo.Mechanistically,DHA inhibited the JAK2-STAT3 pathway to control the expression of the Mcl-1 and Survivin.Our study provides an effective drug combination and theoretical data for cancer treatment. |
