Investigating The Molecular Mechanisms By Which Thevebioside Inhibits Non-Small Cell Lung Cancer Cell Growth

OBJECTIVE Lung cancer is the the main leading cause of mortality among cancer.Non-small cell lung cancer(NSCLC)accounts for about 80% of all lung cancers,which has low 5-year survival rate and poor prognosis.Thus,effective approaches are urgently needed for treating this malignancy.In this study,it is the first time we investigated the effects of Thevebioside on NSCLC,a monomer isolated from Traditional Chinese medicine(Thevetia peruviana(Pers.)K.Schum.(TPKS).),and identified the mechanisms underlying its potential antitumor activity.METHODS In vitro experiments: the inhibitory effect of Thevebioside on A549 and H460 cells were detected by MTT assay.The colony formation of NSCLC cells treated with Thevebioside was observed.Thevebioside-induced apoptosis of NSCLC cells was evaluated by Annexin V/PI(Propidium Iodide)double staining or Hoechst 33342 staining.Cell cycle was assessed by Flow cytometry.Western Blot was used to detect the changes in protein levels of apoptosis-related pathways.In vivo experiments: nude mice were subcutaneously implanted with A549 cells to observe the tumor growth in tumor-bearing mice.RESULTS In vitro experiment: MTT experiments showed that Thevebioside can effectively inhibit the proliferation of A549 and H460 cells in a dose-and time-dependent manner.The results of clonal formation experiment indicated that the higher the concentration of Thevebioside was used,the stronger the inhibitory effect was observed.Annexin V/PI double staining and Hoechst 33342 revealed that Thevebioside can induce early apoptosis and late apoptosis.The number of viable cells in A549 cells decreased from 97.7% to 49.5%,and the number of early apoptotic cells increased from 1.34% to 24.7% in H460 cells.As the concentration of Thevebioside increased,the number of viable cells decreased from 94.4% to 74.9%,gradually dominated by late apoptosis,and increased from 2.73% to 13.3%.In addition,the cell cycle revealed that Thevebioside induced S phase arrest in NSCLC cells.Finally,western blot analysis of Thevebioside-treated cells revealed the activation of SRC,AKT,caspase-3,PARP,and Bcl-xl,in a dose-dependent manner.In vivo experiments: the tumor volume and weight of tumor-bearing nude mice were observed.Both Thevebioside and digoxin inhibited tumor growth and tumor weight(P<0.05),but Thevebioside had a stronger inhibitory effect on tumor growth of NSCLC xenograft(P<0.01).CONCLUSION Our results showed that Thevebioside induced apoptosis in NSCLC cells by regulating the expression of apoptosis-related proteins,leading to the inhibition of proliferation,and inducing S phase arrest in A549 and H460 cell lines.Therefore,the inhibitory effect of Thevebioside on NSCLC cells suggests that it might be a promising drug for the treatment of NSCLC.