Background:Azithromycin is a member of macrolide antibiotics,and has been reported to inhibit the proliferation of cancer cells.However,the underlying mechanisms are not been fully elucidated.Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)selectively targets tumor cells without damaging healthy cells.In the present study,we examined whether azithromycin is synergistic with TRAIL,and if so,the underlying mechanisms in colon cancers.Methods:HCT-116,SW480,SW620 and DiFi cells were treated with azithromycin,purified TRAIL,or their combina-tion.A sulforhoddamine B assay was used to examine cell survival.Apoptosis was examined using annexin V-FITC/PI staining,and autophagy was observed by acridine orange staining.Western blot analysis was used to detect protein expression levels.In mechanistic experiments,siRNAs were used to knockdown death receptors(DR4,DR5)and LC-3B.The anticancer effect of azithromycin and TRAIL was also examined in BALB/c nude mice carrying HCT-116 xenografts.Results:Azithromycin decreased the proliferation of HCT-116 and SW480 cells in a dose-dependent manner.Combination of azithromycin and TRAIL inhibited tumor growth in a manner that could not be explained by additive effects.Azithromycin increased the expressions of DR4,DR5,p62 and LC-3B proteins and potentiated induction of apoptosis by TRAIL.Knockdown of DR4 and DR5 with siRNAs increased cell survival rate and decreased the expression of cleaved-PARP induced by the combination of azithromycin and TRAIL.LC-3B siRNA and CQ potentiated the anti-proliferation activity of TRAIL alone,and increased the expressions of DR4 and DR5.Conclusion:The synergistic antitumor effect of azithromycin and TRAIL mainly relies on the up-regulations of DR4 and DR5,which in turn result from LC-3B-involved autophagy inhibition.
