Mechanism Of Tanshinone ⅡA Against Lung Cancer Based On The Interaction Of Autophagy And Apoptosis

ObjectiveLung cancer is the most common primary malignancy and the most deadly among all tumors.At present,most of the clinical lung cancer patients were diagnosed in the middle and advanced stage,and the five-year survival rate of lung cancer is still less than 20%.In recent years,more and more attention has been paid to traditional Chinese medicine in tumor treatment.To explore the anti-tumor mechanism of traditional Chinese medicine is of positive significance for guiding clinical drug use and developing new anti-tumor drugs.Salvia miltiorrhiza is a classical traditional Chinese medicine for promoting blood circulation and removing blood stasis.Tanshinone ⅡA(TⅡA)is a diterpenoid quinone that is abundant in the root system of salvia miltiorrhiza.TⅡA can inhibit proliferation,promote apoptosis,inhibit metastasis and other anti-tumor effects on a variety of tumor cells,which has attracted wide attention.This study will be in lung cancer A549,PC-9 on the cell model,study of TⅡA influence on autophagy and apoptosis in lung cancer cells and its mechanism,and preliminarily TⅡA human lung cancer cells induced autophagy and apoptosis are interrelated.This study will clarify the anti-proliferation effect of TⅡA on lung cancer cells,so as to provide a theoretical basis for its application in the treatment of lung cancer.Furthermore,the relationship between autophagy and apoptosis induced by TⅡA in lung cancer cells was preliminarily elucidated,so as to provide new ideas for the treatment of lung cancer.Methods1,Study on the inhibition of tanshinone ⅡA on the proliferation of human non-small cell lung cancer A549 and PC-9 cellsThe effect of TⅡA on the proliferation of human non-small cell lung cancer A549 and PC-9 cells was detected by CCK-8 method.,and the time and concentration of TⅡA were determined.The effect of TⅡA on A549 cell cycle distribution was detected by flow cytometry.The expression level of Cyclin D1 protein in A549 cells was detected by Western Blot.2.Tanshinone ⅡA induced apoptosis of A549 and PC-9 cells in human non-small cell lung cancerFlow cytometry was used to detect the effect of TⅡA on the apoptosis rate of A549 and PC-9 cells in human non-small cell lung cancer.The protein expression levels of Bax,Bcl-2,C-Caspase 3 and C-Caspase 9 in lung cancer cells were detected by Western Blot,and the effect of TⅡA on the apoptosis of lung cancer cells was investigated.3.Tanshinone ⅡA induced autophagy in A549 and PC-9 cells of human non-small cell lung cancerThe effect of TⅡA on the ultrastructure of A549 and PC-9 cells in human non-small cell lung cancer was observed by transmission electron microscope.Western Blot was used to detect the expression of autophagy-related proteins Beclin-1,LC3B and p62 in lung cancer cells,and to observe whether TⅡA could induce autophagy in lung cancer cells.4.Study on the interaction between apoptosis and autophagy induced by TⅡA in lung cancer cellsWestern Blot was used to detect the sequence of TⅡA induced apoptosis and autophagy in human non-small cell lung cancer A549 and PC-9 cells.After pretreatment with autophagy inhibitor 3-MA and autophagy agonist RAPA,the effects of TⅡA on the expression levels of apoptotic protein C-Caspase 3 and autophagy proteins LC3B and p62 in A549 and PC-9 cells of human non-small cell lung cancer were detected by Western Blot.After pretreatment with the apoptotic inhibitor Z-DEVD-FMK and the apoptotic agonist PAC-1,the effects of TⅡA on the expression levels of apoptotic protein C-Caspase 3 and autophagy proteins LC3B and p62 in A549 and PC-9 cells of human non-small cell lung cancer were detected by Western Blot.To explore the interaction mechanism between autophagy and apoptosis induced by TⅡA.5.Effect of tanshinone ⅡA on human non-small cell lung cancer cell A549 tumor-bearing nude mouse model5×107/mL A549 cell suspension was inoculated under the skin of the right axilla near the dorsal part of nude mice in 0.2 mL/mL.Groups of nude mice were divided into control group(normal saline,0.2 mL/10 g)and cisplatin group(2 mg/kg),TⅡA in low dose group(5 mg/kg),TⅡA in middle dose group(10 mg/kg),TⅡA in high dose group(20 mg/kg),each group of eight,each given by intraperitoneal injection of drug dosing 21 days.The body weight and tumor volume of nude mice were observed and recorded.Major organ coefficients were used to evaluate the safety of TⅡA.HE staining was used to observe the effect of TⅡA on the pathological structure of A549 tumor.Results1.TⅡA inhibited the proliferation and cell cycle distribution of A549 and PC-9 cells in human non-small cell lung cancerThe results of cell survival detection by CCK8 showed that TⅡA could significantly inhibit the proliferation of lung cancer A549 cells and PC-9 cells(P<0.01),and the inhibitory effect increased with the up-regulation of drug concentration,and was positively correlated with the time of action.Detection of cell cycle distribution by PI single staining indicated that TⅡA caused significant cell cycle arrest in A549 at G0/G1 phase(P<0.01).The expression level of Cyclin D1 in A549 cells was significantly reduced by Western Blot analysis after TⅡA treatment with different concentrations of 1,2 and 4 μM for 24 h,showing a dosedependent correlation(P<0.01).2.Effect of TⅡA on apoptosis of A549 and PC-9 cells in lung cancerAnnexin V-FITC/PI double staining method was used to detect the apoptotic rate of TⅡA on lung cancer A549 and PC-9 cells.The results showed that TⅡA could significantly promote the apoptosis of lung cancer cells and showed a dose-dependent trend(P<0.01).Western Blot method was used to detect the increased Bax/Bcl-2 ratio in A549 and PC-9 cells(P<0.01)and the increased expression levels of C-Caspase 3 and C-Caspase 9 proteins(P<0.01),suggesting that TⅡA may induce apoptosis in A549 and PC-9 cells by regulating the expression levels of apoptosis-related proteins.3.Effect of TⅡA on autophagy of lung cancer A549 and PC-9 cellsTransmission electron microscopy showed that after treatment of lung cancer A549 and PC-9 cells with TⅡA,the autophagosome structure of the double-membrane structure could be observed.Changes in protein levels were detected by Western Blot.Compared with the Control group,LC3-II/LC3-I ratio of A549 and PC-9 cells were significantly increased(P<0.01)and the expression levels of Beclin-1 significantly increased(P<0.01),while the expression levels of p62 significantly decreased(P<0.01),suggesting that TⅡA can induce autophagy in A549 cells.4.TⅡA-induced lung cancer cell autophagy occurs before apoptosis and further initiates apoptosisCompared with the 0 h group,the LC3-Ⅱ/LC3-Ⅰ ratio of A549 cells was significantly increased(P<0.01)after 2 hours of TⅡA treatment,and the difference was statistically significant;however,after about 6 hours of TⅡA treatment,A549 The expression of CCaspase 3 in cells was significantly increased(P<0.01).Compared with the 0 h group,the ratio of LC3-II/LC3-I increased significantly after PCIA cells were treated with TⅡA for 4 h(P<0.01),the difference was statistically significant;and C-Caspase 3 of PC-9 cells The expression of serotonin increased significantly after TⅡA treatment for 6 h(P<0.01).5.Effect of TⅡA on apoptosis of lung cancer cells A549 and PC-9 after promoting or inhibiting autophagyCompared with the TⅡA group,the LC3-II/LC3-I ratio in A549 and PC-9 cells significantly decreased after TⅡA+3-MA treatment(P<0.01),the level of p62 significantly increased(P<0.01),and the expression of C-Caspase 3 significantly decreased(P<0.01).Compared with TⅡA group,LC3-II/LC3-I ratio increase obviously(P<0.01),p62 expression level decreased significantly(P<0.01),while the expression of C-Caspase 3 increased significantly(P<0.01)in TⅡA+RAPA group,showed that 3-MA and RAPA can influence the autophagy induced by TⅡA in lung cancer cells,inhibit the autophagy can obviously recede TⅡA effect on promoting apoptosis of lung cancer cells.6.Effects of TⅡA on autophagy in lung cancer cells A549 and PC-9 after promoting or inhibiting apoptosisCompared with the TⅡA group,the expression of C-Caspase 3 in A549 and PC-9 cells after TⅡA+Z-DEVD-FMK treatment was significantly reduced(P<0.01),while the LC3II/LC3-I ratio and p62 level showed no significant changes(P>0.05).Compared with the TⅡA group,the expression of C-Caspase 3 was significantly increased after TⅡA+PAC-1 treatment(P<0.01),while the LC3-Ⅱ/LC3-Ⅰ ratio and p62 level showed no significant changes(P>0.05),indicating that Z-DEVD-FMK and PAC-1 could affect TⅡA induced apoptosis of lung cancer cells,and the inhibition of this apoptosis had no significant effect on TⅡA induced autophagy in lung cancer cells.7.TⅡA could inhibit the proliferation of transplanted tumor A549 in vivoCompared with Control group,TⅡA administration significantly inhibited the growth of A549 transplanted tumor(P<0.01).Compared with the Control group,there were no statistically significant differences in body weight of nude mice in each TⅡA administration group(P>0.05),and no statistically significant differences in organ coefficients(P>0.05),while body weight of nude mice in the DDP group was significantly reduced(P<0.01),and there were statistically significant differences in liver,spleen,lung and kidney coefficients(P<0.05).HE staining results showed that no significant morphological changes were observed in tumor cells in the Control group.In TⅡA group and DDP group,tumor cell morphology changed,part of the nucleus structure was fuzzy,hyperchromatic,cytoplasm shrank and cytoplasm condensed.WB results showed that compared with the Control group,beclin-1 expression and LC3-Ⅱ/LC3-Ⅰ ratio in the transplanted tumor tissues of nude mice in the TⅡA-M and TⅡA-H groups were significantly increased(P<0.01),p62 level was significantly decreased(P<0.01),Bcl-2/Bax ratio in the DDP group,TⅡA-M group and TⅡAH group was significantly decreased(P<0.01),and c-Caspase 3 expression level was significantly increased(P<0.01).The results showed that TⅡA could induce autophagy and apoptosis of transplanted tumor tissues in nude mice.ConelusionTⅡA can significantly inhibit the proliferation of lung cancer cells A549 and PC-9 in vitro,and has a significant anti-NSCLC effect.In vitro mechanism studies have shown that TⅡA induces autophagy in A549 and PC-9 cells,causing cell cycle arrest.At the same time,TⅡA induced apoptosis in A549 and PC-9 cells.TⅡA induced autophagy preceded apoptosis and further initiated apoptosis.It suggests that the combination of autophagy inhibitor and TⅡA may provide a new idea for the treatment of non-small cell lung cancer.In vivo experiments have shown that TⅡA has a significant therapeutic effect on human non-small cell lung cancer cell A549 tumor-bearing nude mouse model,and the safety is good.