The Effect Of Artesunate Combined With Carboplatin On Cell Proliferation,Apoptosis And Cell Cycle Of A549 Cells In Non-Small Cell Lung Cancer

Objectie: Nowadays,treatment with platinum or drug combined with platinum is still common used for advanced non-small cell lung cancer therapy in the clinical first-line.However when combined with other chemotherapeutic drugs,it is quite easy to induce obvious side reactions,including renal toxicity,gastrointestinal reaction,ototoxicity and blood system toxicity.Aim of this study is to reach a basic understanding of the impact of artesunate(ART)combined with Carboplatin(CBP,a platinum metal complex)on proliferation,apoptosis and cell cycle of A549 cells in non-small cell lung cancer in vitro.In addition,using Carboplatin combined Gemcitabine as control,which is a common chemotherapy drug in clinical therapy,we try to study the feasibility of Artesunate combined with Carboplatin in the treatment of Non-Small Cell Lung Cancer(NSCLC).Such insights will allow us to provide basic research theory and develop new clinical treatment methods for Non-Small Cell Lung Cancer chemotherapy.Methods: Using MTT to detect specific proliferation inhibiting IC50 of Artesunate(ART),Carboplatin(CBP)and Gemcitabine(Gem)on human Non-Small CellLung Cancer A549 cells in vitro.Further,we chose low dose of each drug(the concentration of inhibiting rate<30%)as experimental concentration for combination group to analyze the influence of Artesunate combined with Carboplatin and Gemcitabine combined with Carboplatin on proliferation of A549 cells by MTT method.Therefore,we could evaluate the feasibility of combination groups,and selected the final test groups for the following experiments,including Artesunate group,Carboplatin group,Gemcitabine group,Artesunate combined with Carboplatin group and Gemcitabine combined with Carboplatin group.By cell colony formation assay analysis,we could evaluate the effect of all groups on the proliferation of A549 cells in vitro.In addition,Flow cytometry was used to detect the effects of different groups on apoptosis and cell cycle of A549 cells,while Tunel was used to detect the cell apoptotic morphology.Finally,we also detected the expression levels of cell cycle and apoptosis related proteins on A549 cells using Western Blot.Results: 1.Artesunate(ART)can inhibit the growth of A549 cells in vitro,which depends on the concentration of artesunate to some extent and the IC50 is 22.3μg/ml after analyzed by SPSS 22.0.Further,combining with Carboplatin(CBP)could enhance such effects on A549 cells in vitro.2.Artesunate or Carboplatin inhibits colony formation of A549 cells,and can reduce the cell colony formation by 15.7% and 29.3% compared with control group respectively.However,such inhibition rate is increased to 40.8% significantly whenART combined with CBP is administered compared with control group3.Artesunate mainly induce the cell apoptosis at early stage.However,compared with ART itself,ART combined with CBP can induce more cell apoptosis and apoptotic cells are mainly in the early stage as well.4.ART has block function in cell cycle progression at G2/M period and CBP also has such ability on cell cycle arrest,but mainly in S period.In addition,ART combined with CBP can significantly increase arrested cells at G2/M period compared with negative control group.5.The results of Tunel assay reveals that compared with control group,ART single group and ART combined with CBP group show more apoptotic cells and most cells have phenomenon of nucleus pyknosis.6.Analysis of Western Blot show that compared with control group,ART combined with CBP could increase the expression level of pro-apoptotic proteins,including Bax,P53 and caspase-3 and decrease the expression level of anti-apoptotic proteins,such as Bcl-2 significantly.On contrary,the expression of cell cycle related inhibitory proteins,such as P53 and P21 are increased and decrease Cyclin B1,a cell cycle regulatory protein,expression significantly.Conclusions: Artesunate could inhibit the proliferation of A549 cell significantly and can also enhance carboplatin function on inhibiting proliferation of A549 cells in vitro,which means ART can improve the sensitivity of chemotherapy on A549 cells.In addition,such effects of anti-proliferation may be related to the function of Artesunate orArtesunate combined with Carboplatin on inducing cell apoptosis,up-regulation of cell cycle related inhibitory proteins,such as P53 and P21,down-regulation of cell cycle regulatory protein,such as Cyclin B1 and arresting cell progression at G2/M period.