The survival rate was calculated as a ratio to the control group (untreated cells). 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 combination. 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 SR 146131 the anti-proliferation activity of TRAIL alone, and increased the expressions of DR4 and DR5. Conclusion The synergistic SR 146131 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. 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 Western blotting analyses, as described previously . Apoptosis assay Apoptosis was determined using an annexin V-FITC/PI apoptosis detection kit from DOJINDO (Shanghai, China). A schematic plot was used to display the results: the lower left quadrant represents live cells; the lower right and upper right quadrants represent early and late apoptotic cells, respectively; the upper left quadrant represents 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 drugs for 24?h. Later, 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 represented the nucleus, while the red represented 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 negative siRNA were purchased from Ruibo Biotechnology (Guangzhou, China) and dissolved in RNase-free water as a 20?mol/L stock. Negative 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 Cxcl12 to addition to the cells (final concentration: 30?nmol/L). Experiments with test drugs started 24?h after the transfection. Efficiency of transfection was verified with Western blotting. Colon cancer xenograft All animal experiments were performed in accordance with relevant guidelines and regulations. Briefly, HCT-116 cells (1??107 cells in 200-L PBS) were injected into the right armpits of 6-week-old female BALB/c nude mice (SPF Biotechnology Co., Ltd., Beijing, China). At 21?days after the inoculation, tumors were removed and cut into 2?m??2?m??2?m prisms, and transplanted into the right flanks of other mice through a trocar. Seven SR 146131 days later, mice were randomized to receive azithromycin (50?mg/kg/day, via oral administration, for 3 consecutive days in a week) or TRAIL (10?mg/kg, via the tail vein, once a week). Tumor volumes and body weights were monitored once every 2?days. The tumor volume was calculated by the following formula: test for independent samples. Statistical significance was set at P?0.05..