Based On Mitochondrial Fusion Protein (Mfn2), The Study Of Shenqi Fuzheng Injection Combined With Cisplatin Induced Apoptosis And Cell Cycle Arrest In A549/DDP Cells

Purpose: Shen-qi-Fu-zheng Injection(SFI)is a recently developed Chinese traditional medicine extract that has been composed of Radix Astragali and Radix Codonopsis prepared as a clinical treatment.Excepted antithrombosis,returned blood volume,antihypertensive effects and immunomodulatory,SFI posseses the efficacy of SFI-systematic chemotherapy combination in sensitizing tumors and reducing the toxicity of standard chemotherapy.However,the potential role of whether SFI as a chemo-resistance reversal agent or the underlying mechanisms of SFI in increasing chemotherapy sensitivity are still unknown.In this study,respectively using immunohistochemistry,Western blot and flow cytometry technology,we investigated whether SFI could reverse chemo-resistance in the cisplatin-resistant lung carcinoma A549/DDP cell line.The exploration of integrated Chinese and Western therapy of non-small-cell lung cancer(NSCLC)provides the experiental basis which will greatly improve the efficacy of NSCLC therapeutics.Methods: 1.Experimental materials The A549 human lung adenocarcinoma cell lines(cisplatin-susceptible cells)and the A549/DDP variant cell lines(cisplatin-resistant cells)were obtained from the Cancer Hospital at the Chinese Academy of Medical Sciences(Beijing,China).The SFI(Livzon Pharmaceutics Ltd,146195,250 m L,Zhuhai,China).Cisplatin for Injection(freeze-dried)(Qilu pharmaceutical Ltd,2030132,10 mg,Shandong,China).2.Experimental methods The A549 cells and the A549/DDP cells were cultured in RPMI 1640 media containing 10% fetal bovine serum(FBS)and 1% penicillin and streptomycin with 0.25% trypsin enzyme digesting in a humid atmosphere of 5% CO2/95% air.A549/DDP cells are divided into 5 groups: control group(PBS),cisplatin group(40 ?g/m L),cisplatin + low concentration of SFI group(40 ?g/m L + 2 mg/m L),cisplatin + midium concentration of SFI group(40 ?g/m L + 3.78 mg/m L),cisplatin + high concentration of SFI group(40 ?g/m L + 35.18 mg/m L).To maintain the drug-resistant phenotype,2 ?g/m L cisplatin was supplemented in the A549/DDP media.(1)A Cell Counting Kit-8(CCK-8)was used to evaluate the chemo-resistance capacity of the two cell lines.A Western blotting analysis and a Immunocytochemical(IHC)assay evaluate the Mfn2 chemo-resistance capacity diffrence of the two cell lines.(2)We exposed the A549/DDP cells of low Mfn2 expression,which were incubated with various concentrations of SFI: 1)The cell viability of SFI or co-treatment of cisplatin with SFI on A549/DDP cells was determined by counting viable cells with CCK-8 assay;2)To determine the cell cycle progression index induced by SFI on cisplatin-resistant A549/DDP cells: A549/DDP cells were treated with the SFI(2 mg/m L as the low intervention dose;3.78 mg/m L as the medium intervention dose;35.18 mg/m L as the high intervention dose respectively,and then exposed to cisplatin(40 ?g/m L).Flow cytometry assay was used to detect cell cycle and DNA contents and Western blotting analysis was determined by cyclin expression of p53 and p21.3)To determine the apoptosis index induced by SFI in cisplatin-resistant A549/DDP cells: A549/DDP cells were co-treated with cisplatin + SFI(the low,medium,high intervention dose)and subsequently subjected to Hoechst staining followed by immunofluorescence staining.The apoptosis was further evaluated by double staining with Annexin V-FITC and propidium iodide(PI)by flow cytometry assay.The protein expression of apoptotic proteins Caspase-3,PARP,Bax and Bcl-2 was examined by Western blotting.4)To determine the mechanisms of the cell cycle progression and apoptosis induced by SFI in cisplatin-resistant A549/DDP cells:we further investigated Mfn2 protein expression by Western blotting,mitochondrial membrane potential(MMP)and reactive oxidative species(ROS)by flow cytometry assay in cisplatin-resistant A549/DDP cells induced by co-treatment with cisplatin and the low,medium,high concentrations of SFI.Results : 1.Cisplatin-susceptible A549 cells had a higher level of Mfn2 expression,while cisplatin-resistant A549/DDP cells had a lower than that of A549 cells.2.SFI reversed the chemo-resistance of A549/DDP cells to cisplatin.3.Cisplatin + SFI co-treatment induced G2/M cell cycle arrest in cisplatin-resistant cells A549/DDP followed by increase in p53 and p21 protein expression,which suggested that SFI reversed cisplatin-resistance of A549/DDP cells.4.Apoptosis of cisplatin-resistant A549/DDP cells induced by cisplatin in combination with SFI was mediated via increasing the protein levels of Bax and reducing protein expressions of Bcl-2.5.Cisplatin + SFI co-treatment suppressed the proliferation of cisplatin-resistant A549/DDP cells,halted cell cycle and induced cell apoptosis through up-regulation of Mfn2,which were all involved with subsequent activation of the mitochondrial apoptotic pathway and up-regulation of ROS.Conclusion: In summary,our findings suggest the effect of SFI in increasing chemotherapy sensitivity in cisplatin-resistance of NSCLC is via cell cycle arrest and induction of mitochondrial apoptosis involved with up-regulation of Mfn2 expression.