| ObjectiveTo observe the inhibiting effect of Qiyu Sanlong(QYSL)formula on cell proliferation and the growth of xenograft tumor of non-small cell lung cancer(NSCLC)and explored its molecular mechanisms based on inducing autophagy by the regulation of Beclin-1/Bcl-2 complex by mTORC1.MethodsPart one:QYSL formula inhibits cell proliferation of A549 cells by inducing cell autophagyForty SD rats were divided into two groups to prepare QYSL serum and normal serum of.A549 cells were cultured in vitro.CCK-8 method was used to detect the effect of QYSL serum on the cell viability of A549 cells,and the optimal action time and concentration were selected for the next experiment.The experiment was divided into control group,normal serum group and QYSL serum group,which were given10%fetal bovine serum,20%normal serum and 20%QYSL serum,respectively.A549 cells in each group were detected as follows after treatment:The cell cycle was detected by Flow cytometry;Cell apoptosis was detected by TUNEL assay;The ultrastructure of A549 cells was observed by transmission electron microscope(TEM);The expression of ATG-7,ATG-13,p62,Beclin-1,ATG-5,LC3-II/LC3-I,were detected by western blot(WB),quantitative real time-PCR(qRT-PCR)and Immunofluorescence(IF).Part two:QYSL formula promotes the dissociation of Beclin-1/Bcl-2 complex by inhibiting mTORC1 to induce cell autophagyA549 cells were pretreated with RAPA(mTORC1 inhibitor and autophagy activator)and MHY1485(mTORC1 activator and autophagy inhibitor)to detect the effect of QYSL serum on autophagy of A549 cells.The experimental groups were as follows:control group,QYSL group,RAPA group,QR(QYSL combined with RAPA)group,MHY1485 group,and QM(QYSL combined with MHY1485)group.A549 cells in each group were detected as follows after treatment:the ultrastructure of A549 cells was observed by TEM;the protein expressions of mTOR,p-mTOR,Beclin-1,p-Bcl-2and LC3-II/LC3-I were detected by WB;the effect of QYSL serum on the interaction between Beclin-1 and Bcl-2 was examined by immunoprecipitation(Co-IP).Beclin-1RNA interference sequence was constructed,the interference efficiency was detected by q RT-PCR,and the optimal interference sequence was selected.The experiment was divided into control group,NC-si RNA group,QYSL group,Beclin-1 si RNA group,and BSQ(Beclin-1 siRNA combined with QYSL)group.The protein expression levels of Beclin-1 and LC3-II/LC3-I were detected by WB.Part three:QYSL formula inhibits NSCLC tumor xenograft growth in nude mice by inducing autophagyA549 cells were subcutaneously implanted into the right flank of BALB/c-nude mice to establish subcutaneous xenograft model.When the tumor volume reached 50-100mm~3,BALB/c-nude mice were randomly divided into five groups:model group,QYSL group,RAPA group,combined group,and DDP group.Model group:0.2ml/10g normal saline was given by intragastric administration;QYSL group:80.48g/kg liquid of QYSL formula was given by intragastric administration.RAPA group:2mg/kg RAPA was intraperitoneal injection.Combined group:80.48g/kg liquid of QYSL formula was given by intragastric administration and 2mg/kg RAPA was intraperitoneal injection.DDP group:0.25mg/ml DDP was intraperitoneal injection.These drugs was given once daily for two weeks.general condition and body weight of nude mice were observed.The tumor volume was measured and tumor growth curve was plotted.The tumor mass was measured and the tumor inhibition rate was calculated.The effect of QYSL formula on the tumor pathology were detected by HE staining.Autophagosomes of lung cancer cells in transplanted tumor tissues were observed by TEM.The expression of mTOR,ATG-7,ATG-13,p62,Beclin-1,p-Bcl-2,ATG-5,LC3-II/LC3-I,were detected by WB,qRT-PCR,and IF.Results Part one:QYSL formula inhibits cell proliferation of A549 cells by inducing cell autophagy1 QYSL formula inhibited the cell proliferation of A549 cellsCCK-8 results showed that QYSL serum could inhibit the cell viability of A549 cells in a concentrate-dependent manner.By calculating the IC50 of QYSL serum at different time points of A549 cells,20%QYSL serum and 24h were finally determined as the optimal concentration and time of action for further study.Flow cytometry showed that QYSL serum could induce cycle arrest of A549 cell in S phase and G2/M phase.TUNEL assay showed that the cell apoptosis rate of A549 cells was significantly increased after QYSL serum intervention,which was significantly better than that of control group and blank serum group,with statistical significance(P<0.01).2 QYSL formula induced autophagy in A549 cellsThe ultrastructure of autophagosomes was observed under TEM,and the effects of QYSL formula on the autophagy related molecules of A549 cells were detected by WB,qRT-PCR and IF method.TEM showed that,compared with the control group and the blank serum group,the number of autophagosomes and autophagy vesicles in A549 cells of the QYSL serum group increased,and autophagy lysosomes were observed,which were wrapped by unit membrane and contained residual organelles.The results of WB,qRT-PCR and IF method showed that compared with the control group and the blank serum group,the expression levels of ATG-7,ATG-13,Beclin-1and ATG-5 in QYSL serum group were significantly increased,while the expression level of p62 was significantly decreased,with statistical significance(P<0.05 or P<0.01).Part two:QYSL formula promotes the dissociation of Beclin-1/Bcl-2 complex by inhibiting mTORC1 to induce cell autophagy1 QYSL formula induced autophagy by inhibiting m TORC1 in A549 cellsTEM examination of the ultrastructure of A549 cells in each group showed that there were rare or no autophagosomes or autophagolysosomes in the control group and the MHY1485 group,while the number of autophagosomes or autophagolysosomes in the QYSL group and RAPA group increased significantly,and the number of autophagosomes in the QYSL combined with RAPA(QR)group was more than QYSL group and RAPA group.The number of autophagosomes in QYSL combined with MHY1485(QM)group was significantly higher than that in the control group and MHY1485 group.Protein expression of mTOR and p-mTOR:WB analysis showed that compared with the control group,the protein expression level of p-mTOR/mTOR in QYSL group,RAPA group and QR group was decreased(P<0.01),and the expression level of p-mTOR/mTOR in MHY1485 group was significantly increased(P<0.01),while the expression level of p-mTOR/mTOR in QM group was significantly decreased compared with MHY1485group(P<0.01).Protein expression of LC3-II/LC3-I:Compared with the control group,the expression level of LC3-II/LC3-I in QYSL group,RAPA group and QR group was significantly increased(P<0.01),and there was no statistical difference between MHY1485 group and the control group(P>0.05).Compared with MHY1485 group,the expression level of LC3-II/LC3-I in QM group was significantly increased(P<0.01).2 QYSL formula promoted the dissociation of Beclin-1/Bcl-2 complex by inhibiting mTORC1Protein expression of Beclin-1:Compared with the control group,the expression level of Beclin-1 in QYSL group,RAPA group and QR group was significantly increased(P<0.01),and the expression level of Beclin-1 in MHY1485 group was significantly decreased(P<0.01).Compared with MHY1485 group,the expression level of Beclin-1 in QM group was significantly increased(P<0.05).Protein expression of p-Bcl-2:Compared with the control group,the expression level of p-Bcl-2 in QYSL group,RAPA group and QR group was significantly increased(P<0.01 or P<0.05),and the expression level of p-Bcl-2 in MHY1485 group was significantly decreased(P<0.01).Compared with MHY1485 group,the expression level of p-Bcl-2 in QM group was significantly increased(P<0.05).The effect of QYSL serum on the interaction between Beclin-1 and Bcl-2 was examined by Co-IP:Beclin-1 was immunoprecipitated from cell lysate(IP),and Bcl-2and Beclin-1(IB)in immunoprecipitated cells were detected by WB.Compared with the control group,Bcl-2/Beclin-1 binding was significantly decreased in QYSL group and RAPA group(P<0.05 or P<0.01),while Bcl-2/Beclin-1 binding was significantly increased in MHY1485 group(P<0.01).Compared with the MHY1485 group,Bcl-2/Beclin-1 protein binding was significantly reduced in the QM group(P<0.01).The results showed that QYSL serum could promote the dissociation of Bcl-2 and Beclin-1,while MHY1485 could reverse this effect.3 QYSL formula counteracted autophagy inhibition induced by Beclin-1silencingBeclin-1 siRNA-3 was screened out by qRT-PCR as the best interference sequence for the next experiment.Beclin-1 protein expression:Compared with the control group,the protein expression of Beclin-1 in QYSL group was significantly increased(P<0.01),and the protein expression of Beclin-1 in Beclin-1 si RNA group was significantly decreased(P<0.01).Compared with Beclin-1 siRNA group,the protein expression of Beclin-1 in BSQ group was significantly increased(P<0.01).LC3-II/LC3-I protein expression:Compared with the control group,the protein expression of LC3-II/LC3-I in the QYSL group was significantly increased(P<0.01),and the protein expression of LC3-II/LC3-I in the Beclin-1 siRNA group was significantly decreased(P<0.01).Compared with beclin-1 si RNA group,the protein expression of LC3-II/LC3-I in BSQ group was significantly increased(P<0.01).Part three:QYSL formula inhibits NSCLC tumor xenograft growth in nude mice by inducing autophagy1 The effect of QYSL formula on the general condition of nude miceThe mental state of the nude mice in the DDP group was the worst,the activity of nude mice decreased.The mental state of nude mice in model group was general,and the food intake was acceptable.The mental state of nude mice in QYSL group was always good,which was obviously better than that in DDP group.The general condition of nude mice in RAPA group and combined group was worse than that in QYSL group,but significantly better than that in DDP group.Before administration,there was no statistical significance in body weight of nude mice among all groups(P>0.05).With the extension of time,the body weight of nude mice in model group had a slight trend of decline,but it was not obvious.During the treatment,no significant weight loss was observed in nude mice in the QYSL group,the RAPA group and the combined group,with no statistical difference(P>0.05),which indicated that the nude mice were well tolerated to the treatment of QYSL formula and had no obvious toxicity.However,from the second day of administration,the body weight of nude mice in the DDP group showed a continuous downward trend,indicating that DDP had a great effect on the reduction of body weight of nude mice and the tolerance of the drug was poor in the nude mice.2 QYSL formula inhibited the growth of subcutaneous transplanted tumor in nude miceBefore administration,there was no significant difference in tumor volume among all groups(P>0.05).With the extension of time,the volume of the transplanted tumor in each group increased gradually,but compared with the model group,the tumor volume in each treatment group increased slowly,and the difference was statistically significant(P<0.05).Before the sampling,the final tumor volume was measured,and the tumor tissues were stripped,weighed and photographed.Compared with model group,the tumor volume and weight in QYSL group,RAPA group,combined group and DDP group were all reduced(P<0.01).Compared with QYSL group and RAPA group,the tumor volume and weight in the combined group were significantly reduced(P<0.01 or P<0.05).Compared with the DDP group,the tumor volume and weight in the QYSL group were higher than those in the DDP group,and the difference was statistically significant(P<0.01).According to the tumor weight,the tumor inhibition rates in each treatment group were calculated,and the tumor inhibition rates of QYSL group,RAPA group,combination group and DDP group were 13.46%,30.43%,46.00%and 31.50%,respectively.HE staining was used to observe the necrosis of tumors in each group.Compared with the model group,cell necrosis of tumors appeared in each treatment group.The degree of necrosis was as follows:combined group>DDP group>RAPA group>QYSL group.3 The effect of QYSL formula on Beclin-1/Bcl-2 complex regulated by mTORC1and autophagy in transplanted tumor tissues of nude miceTEM showed that there were rare or no autophagosomes in the model group,and the number of autophagosomes or autophagolysosomes was increased in each treatment groups,among which the number of autophagosomes or autophagolysosomes was the largest in the combined group.WB,qRT-PCR and IF results showed that compared with model group,the expression levels of ATG-7,ATG-13,Beclin-1,p-Bcl-2,ATG-5and LC3-II/LC3-I were increased in all treatment groups,while the expression levels of mTOR and p62 were decreased,with statistical significance(P<0.05 or P<0.01).Compared with QYSL group or RAPA group alone,the expression levels of ATG-7,ATG-13,Beclin-1,p-Bcl-2,ATG-5 and LC3-II/LC3-I in combined group were relatively increased,while the expression levels of mTOR and p62 were relatively decreased,with statistical significance(P<0.05 or P<0.01).Conclusions(1)QYSL formula can inhibit NSCLC A549 cell viability and induce cell cycle arrest,inhibit the growth of the nude mice subcutaneous transplantation tumor,promote autophagosome formation,increase autophagy molecular ATG-7,ATG-13,Beclin-1,p-bcl-2,ATG-5,LC3-II/LC3-I expression,decrease the expression of the p-m TOR/mTOR and p62,which show that QYSL formula inhibit the proliferation of A549 cells and the growth of the transplantation tumor may be associated with induction of cell autophagy;(2)QYSL formula may be used as an mTORC1 inhibitor and autophagy agonist to promote the dissociation of complex Beclin-1/Bcl-2 by inhibiting mTORC1,thus inducing autophagy in NSCLC cells. |
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