Dihydroartemisinin And Cisplatin Synergistically Induced Apoptosis In Human Lung Adenocarcinoma H1299 Cells

Objectives: The incidence and related mortality rate of lung cancer occupies the highest percentage of all cancers in the wrold.Non-small cell lung cancer(NSCLC)accounts for about 80% of lung cancer.The clinical effectiveness of first line platinum-based chemotherapy is only~30%.Thus,novel chemotherapy regimens to improve efficacy are urgently needed.Artemisinin and its derivatives have shown a potent anti-neoplastic activity in both drug-sensitive and resistant cancer cell lines in several studies.Dihydroartemisinin(DHA),a kind of artemisinin derivatives,has been extensively investigated alone or together with other drugs for anti-cancer therapy in recent years.DHA displayed anti-cancer effects on glioma,breast,colon,lung,ovarian and pancreatic cancer cells.Researches show that deletion of p53 is associated with resistance to chemotherapy in some tumor cells.However,the effect of DHA and cisplatin(Cis,an anti-cancer drug)in treatment of human lung adenocarcinoma H1299 cells was unknown.This study was designed to explore the potential synergism effect of DHA and Cis to human lung adenocarcinoma H1299 cells.Methods:1 The cells were respectively treated by DHA,Cis and DHA plus Cis.No treated cells were used as control group.Cell inhibition were evaluated by MTT assays.2 A549 cell line with normal expression of p53 was taken as control,to compare the effect of DHA and Cis on A549 and H1299 cell lines respectively in the same drug treatment conditions by MTT assay.3 Flow cytometry was used to detect the DHA induced cell apoptosis.4 Nuclear staining with Hoechst 33258 showed morphology of the nucleus treated with DHA.5 The apoptotic-associated proteins and genes were assessed by Western blotting and RT-qPCR.Results:1 MTT assays and nuclear staining with Hoechst 33258 showed that DHA and Cis pro-apoptotic activity of alone and combined therapy enhanced as drug concentration increased or elongation of time.Another MTT assay showed that the inhibition ratio of A549 cells and H1299 cells were significantly different when different doses of Cis acting upon the two kinds of cells at the same time,respectively.The difference was that DHA had similar effects on two cells;2 Flow cytometry revealed that DHA treated induced a obviously increase in the percentage of cells with phosphatidyl-serine externalization from 9.09%(control)to 17.07 %(24h)and 24.31 %(48h)at 20 μM concentration,while from 9.09 %(control)to 17.07 % at 20 μM concentration and 22.5 % at 40 μM concentration for 24 h;3 The result of RT-qPCR revealed that the expression of Caspase-3,-8,-9 mRNA in H1299 cell were up-regulated when incubated with 20 μM DHA,12.5 μM Cis and combination of the two drugs for 24 h and 48 h.Drug combinations of 2-(35)(35)Ctvalue about caspase-3,-8,-9 were significantly higher than monotherapy and the monotherapy of 2-(35)(35)Ctvalue about caspase-3,-8,-9were higher than control group;4 Western blot revealed that the combined treatment with two drugs significantly enhanced caspase-8,-9,-3,-6,-7 and PARP activation.we observed the expressions of caspase-3,-8,-9 after H1299 cells were pre-incubated with caspase-3,-8,and-9 inhibitors(zDQMD-fmk,zIETD-fmk,zLEHD-fmk)1h.The result indicated that inhibition of caspase-3 by pre-incubated with zDQMD-fmk,obviously decreased the expression of cleave-caspase-3,but the effect on cleave-caspase-8 and-9 was not great.In addition,inhibition of caspase-8 by pre-incubated with zIETD-fmk,obviously decreased the expression of cleave-caspase-8 and partialy decreased the expression of cleave-caspase-3 and-9.Inhibition of caspase-9 by pre-incubated withzLEHD-fmk,obviously decreased the expression of cleave-caspase-9 and partialy decreased the expression of cleave-caspase-3,but it had little effect on cleave-caspase-8.Conclusion:1 Dose-and time-dependent effects of DHA induced cytotoxicity.2 DHA induced dose-and time-denpendent cell apoptosis.3 Synergistic effect of pro-apoptosis in H1299 cells by DHA and Cis.4 The caspase-8 participated in death receptor signaling pathway and the caspase-9 participated in mitochondrial signaling pathway may played improtant roles respectively in the synergistic effect of the combination treatments.5 The expressions of Caspase-3,-8,-9 mRNA in H1299 cells were up-regulated when incubated with DHA,Cis and combination of the two drugs.In general,this study revealed the obvious synergistic action of the DHA and Cis in inducing apoptosis of H1299 cells by the death receptor and mitochondrial apoptosis pathway.