Effect Of Resveratrol On Apoptosis And Autophagy Induced By Cisplatin In A549 Cells

Lung cancer is one of the most common malignant tumors in our country.Data obtained from the National Cancer Center in 2015 showed that the 5-year incidence of lung cancer in China(2006-2011)was 130.2(1/10 million).In China,the incidence of lung cancer is increasing year by year,of which about 85%of lung cancer is non small cell lung cancer(NSCLC).Despite the developing of lung cancer treatment,5 years survival is still less than 20%.The study showed that 57%of lung cancer patients had distant metastases when the diagnosis was ensured.Cisplatin(DDP)play an important role in chemotherapy of advanced NSCLC.However,acquired drug resistance and side effects are the main reasons for the failure of DDP treatment in clinical.A number of studies have shown that the treatment of chemotherapy combined with traditional Chinese medicine can improve the sensitivity of chemotherapeutic drugs.Resveratrol(RSV)is a natural polyphenol compound.Various researches showed that RSV has many kinds of biological activities,such as anti-inflammation,protect the cardiovascular system.In recent years,more and more studies pay attention to the anti tumor effect of RSV.RSV can enhance the activity of other antitumor drugs through one or many ways,and have a good application potential in clinical.The aim of this study was to investigate whether RSV could enhance the anti-tumor effect of cisplatin on human non small cell lung cancer cell line A549 and explore the possible mechanism,which provide a new strategy for the treatment of lung cancer in clinical.Methods:human NSCLC cell line A549 were cultured in RPMI-1640 with 10%FBS,100 U/ml and 100 mg/L penicillin streptomycin,after adherent to the bottom,A549 cells were treated with different concentration of DDP(3.125,6.25,12.5,25,50 μM);RSV(2.5,5,10,20,40 μM);2.5 μM RSV combined with DDP(3.125,6.25,12.5,25,50 μM)for 48 h,cell viability were detected by CCK-8 method.A549 cells were divided into control group(blank medium),RSV group(final concentration 2.5μM,),DDP group(final concentration 20 μM)and combination group(final concentration RSV 2.5 μM combined with DDP 20 μM)and treated for 24 h.Apoptosis was detected by Hoechst staining and flow cytometry.Autophagy was detected by immunofluorescence assay and transmission electron microscopy(TEM).Bax,Bcl-2,P-AKT,AKT,A549,LC3-Ⅰ/Ⅱ and P62 protein levels were detected by Western blot.The relationship between apoptosis and autophagy was detected by using the autophagy inhibitor 3-methyladenine(3-MA).Results:1.The results obtained from CCK-8 showed that RSV,DDP and RSV in combination with DDP inhibited A549 cell viability in a manner of concentration dependent,and RSV combined with DDP had a synergistic effect on A549 cells.2.Hoechst staining showed that RSV in combination with DDP induced more apoptosis.3.Flow cytometry showed that compared with the control group,DDP group and combination group significantly increased A549 cell apoptosis(3.30 ±0.61%vs 12.98%± 0.84%,17.12%± 1.10%,p＜0.05),compared with DDP group,RSV combined with DDP increased apoptosis of A549 cells(12.98%± 0.84%vs 17.12%± 1.10%,p＜0.05).4.Transmission electron microscopy indicated that RSV combined with DDP induced autophagy in A549 cells.5.Cell immunofluorescence demonstrated that compared with control group,RSV combined with DDP improved the activity of autophagy.6.Western blot showed that compared with the control group,the levels of Bcl-2,P-Akt,P62 proteins were significantly decreased in DDP and RSV group,compared with DDP group,RSV in combination with DDP decreased more,on the contrary,compared with the control group the levels of Bax and LC3-Ⅱ proteins were increased in DDP and combination group,compared with DDP group,RSV in combination with DDP increased more(P<0.05).7.Autophagy inhibitor 3-MA could significantly inhibited apoptosis induced by RSV combined with DDP in A549 cells(P<0.05).Conclusion:RSV could synergistically enhance anti-tumor effect of DDP on A549 cells.On the one hand,they can increase the level of Bax while decrease the level of bcl-2,on the other hand they through increasing LC3 and decreasing p62.In a word,RSV combined with DDP has a synergistic anti-tumor effect on A549 cells through modulating autophagy and apoptosis.