Study On The Effect And Mechanism Of Oleanolic Acid Derivative SZC015 On The Induction Of Apoptosis And Autophagy In Non-small-cell Lung Cancer

Objective: Oleanolic acid(3?-hydroxyolean-12-en-28-oic acid,OA)is a ubiquitous multifunctional pentacyclic triterpenoid,which has been widely found in several natural plants whereas it possesses poor pharmacological activity and low bioavailability,greatly limiting its further study and application.In the present study,we tried to select the optimal derivative from OA derivatives,SZC009,SZC014 and SZC015,and then to explore the anticancer effect of SZC015 in non-small-cell lung cancer in vitro and elucidate the underlying mechanisms.Methods:(1)MTT method was used to detect the effects of different concentrations of OA and its derivatives SZC009,SZC014 and SZC015 on the cell viability of human non-small-cell lung cancer(A549,H1299 and H322),as well as the normal human bronchial epithelial cells(HBE).(2)The morphological changes of apoptosis and autophagy of H322 cells induced by SZC015 were observed by optical microscope and electron transmission microscope.(3)The morphological changes of apoptosis induced by SZC015 were observed by an inverted fluorescence microscope.(4)The effects on the percentage of H322 cell apoptosis induced by SZC015 were detected by flow cytometry AV-PI double staining method.(5)The effect of SZC015 inH322 cell cycle arrest was detected by PI flow cytometry staining.(6)The intracellular reactive oxygen species(Reactive oxygen species,ROS)of H322 was assayed with flow cytometry DCFH-DA fluorescent staining after the treatment with SZC015.(7)Western blotting method was used for the determination of intracellular Procaspase3,Procaspase9,Bcl-2,Bax,LC3 B,Akt,p-Akt,p-I?B?,P65 and p-P65 protein expression level.(8)The nuclear translocation of P65 protein induced by SZC015 in H322 cells was detected by immunofluorescence staining.Results:(1)Compared with the control group,SZC015 could significantly inhibit human non-small-cell lung cancer A549,H1299 and H322 cell proliferation activity in time and dose dependent manner,however,the inhibitory effect on human bronchial epithelial cells of normal HBE growth was lower compared with another human non-small-cell lung cancer cells,indicating that SZC015 could selectively inhibit the proliferation of non-small-cell lung cancer.(2)Compared with the control group,SZC015 could significantly alter the cell morphology,showing that the occurence of cell apoptosis was increased significantly in a dose-dependent manner,suggesting that SZC015 could induce apoptosis of H322 cells.(3)Compared with the control group,H322 cells group treated with SZC015 could significantly improve the percentage of the apoptosis,the Bax/Bcl-2 protein ratio and reduce the expression of procaspase-3 and procaspase-9 protein,suggesting that SZC015 could induce apoptosis of H322 cells,while after H322 cells group treated with OA,the procaspase-3 protein expression of it was higher than that of SZC015,as a result of the effect of apoptosis being more obvious,the expression of procaspase-3 being lower,further suggesting that the effect of SZC015 on apoptosis of H322 cells may be better than that of OA.(4)Compared with the control group,H322 cells group treated with SZC015 under the observation by transmission electron microscopy,it showed obvious autophagosomes.In addition,the transformation of LC3B-I to LC3B-II also was increased in a dose-dependent manner,demonstrating that SZC015 could induce H322 cell autophagy.Compared with OA,the expression of LC3B-II/I protein induced by SZC015 in H322 cells was increased,as a result of the effect of autophagy being more obvious,the transformation of LC3B-I toLC3B-II being higher,further suggesting that SZC015 induced autophagy may be better than that of OA.(5)Compared with control group,SZC015 significantly decreased the expression of Akt,p-Akt,p-I?B?,P65 and p-P65 and inhibited the nuclear translocation of P65 in H322 cells,showing that SZC015 possessed antitumor effect by the regulation of the Akt/NF-?B signaling pathway.(6)Compared with SZC015 group alone,the procaspase-3 protein expression was significantly decreased in SZC015 group by the addition of chloroquine(Chloroquine,CQ),suggesting that autophagy could inhibit apoptosis.(7)Compared with control group,the level of ROS in H322 cells was markedly increased after the treatment with SZC015,indicating that SZC015 could promote the formation of ROS in H322 cells.The procaspase-3 and Akt protein expressions in SZC015 group were increased after the addition of active oxygen scavengers(N-Acetyl-L-Cysteine,NAC),while the expression of LC3B-II/I protein was reduced in H322 cells.Conclusions: Our findings provide novel insights into SZC015-induced the inhibition of NSCLC cell proliferation via the induction of apoptosis and autophagy and highlight Akt/NF-?B signaling as a novel therapeutic target for the treatment of NSCLC.These results suggest that SZC015 may have the potential as NSCLC-preventing agent.