Regardless of these limitations, the synergistic action between azithromycin and TRAIL is definitely apparent. focuses on tumor cells without damaging healthy cells. Cyclosporine In the present study, we examined whether azithromycin is definitely 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 combination. A sulforhoddamine B assay was used to examine cell survival. Apoptosis was examined using Cyclosporine 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 transporting HCT-116 xenografts. Results Azithromycin decreased the proliferation of HCT-116 and SW480 cells inside a dose-dependent manner. Combination of azithromycin and TRAIL inhibited tumor growth in a manner that could not become explained by additive effects. Azithromycin improved the expressions of DR4, DR5, p62 and LC-3B proteins and potentiated induction of apoptosis by TRAIL. Knockdown of DR4 and DR5 with siRNAs improved cell survival rate and decreased the manifestation of cleaved-PARP induced from the combination of azithromycin and TRAIL. LC-3B siRNA and CQ potentiated the anti-proliferation activity of TRAIL only, and improved the expressions of DR4 and DR5. Summary The synergistic antitumor effect of azithromycin and TRAIL primarily relies on the up-regulations of DR4 and DR5, which in turn result from LC-3B-involved autophagy inhibition. Electronic supplementary material The online version of this article (10.1186/s40880-018-0309-9) contains supplementary material, which is available to authorized users. for 15?min at 4?C, prior to European blotting analyses, as described previously . Apoptosis assay Apoptosis was identified using an annexin V-FITC/PI apoptosis detection kit from DOJINDO (Shanghai, China). A schematic storyline was used to display the results: the lower left quadrant signifies live cells; the lower right and upper right quadrants Gpc4 symbolize early and past due apoptotic cells, respectively; the top left quadrant signifies necrotic cells. Cell death refers to the sum of early and late apoptotic and necrotic cells. Acridine orange (AO) staining HCT-116 and SW480 cells were plated into 6-well plates and treated with medicines for 24?h. Later on, cells were washed by PBS twice and stained with 700?L/well AO (1?g/mL) for 15?min at 37?C in the dark. Then, the cells were washed by PBS twice. Watching the images under a fluorescence microscope through a 490?nm band-pass excitation filter and a 515?nm long-pass barrier filter. The green color displayed the nucleus, while the reddish displayed the acidic vesicles. siRNA transfection DR4 siRNA (sense: 5-AACGAGATTCTGAGCAACGCA-3, anti-sense: 3-TTGCTCTAAGACTCGTTGCGT-5), DR5 siRNA (sense: 5-AAGACCCTTGTGCTCGTTGTC-3, anti-sense: 3-TTCTGGGAACACGAGCAACAG-5), LC-3B siRNA (sense: 5-GGTGTATGAGAGTGAGAAA-3, anti-sense: 3-CCACATACTCTCACACTTT-5) and bad siRNA were purchased from Ruibo Biotechnology (Guangzhou, China) and dissolved in RNase-free water like a 20?mol/L stock. Bad siRNA was designed by Ruibo biotechnology and belonged to scrambled control. Cells were transfected with siRNAs using the Ruibo FECT? CP transfection kit, plated in 96-well or 6-well plates and incubated at 37?C for 24?h. siRNAs were diluted in transfection reagent and incubated for 15?min at room temperature to allow the formation of transfection complexes prior to addition to the cells (final concentration: 30?nmol/L). Experiments with test medicines started 24?h after the transfection. Effectiveness of transfection was verified with Western blotting. Colon cancer xenograft All animal experiments were performed in accordance with relevant recommendations and regulations. Briefly, HCT-116 cells (1??107 cells in 200-L Cyclosporine PBS) were injected into the right.