Abstract: Ding Min, Xu Song, Li Li, Bi Suyun, Zhou Zhihai, Li Min, Yang Haiping, Chen Xu, Gu Heng Department of Dermatology and Venereology, General Hospital, Tianjin Medical University, Tianjin 300052, China （Ding M, Bi SY, Zhou ZH）; Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing 210042, China （Xu S, Li L, Li M, Chen X, Gu H）; Department of Dermatology, No. 454 Hospital of People′s Liberation Army, Nanjing 210000, China （Yang HP） Corresponding authors: Yang Haiping, Email: 13016990528@163.com; Chen Xu, Email: doctor_chx@126.com 【Abstract】 Objective To evaluate the synergistic effect of everolimus on cisplatin?mediated cytotoxicity against human cutaneous squamous cell carcinoma COLO?16 cells. Methods Cultured COLO?16 cells were divided into several groups to be treated with everolimus at different concentrations of 50, 100 and 200 nmol/L or 25 μmol/L cisplatin for 12 and 24 hours. Acridine orange （AO）?labeled autophagic vesicles combined with lysomal enzyme inhibitors （E64d and pepstatin） were used to detect the levels of autophagy and autophagic flow. Western blot analysis was performed to track the conversion of the autophagosome marker microtubule?associated protein 1 light chain?3 （LC3） ?Ⅰto LC3?Ⅱ, as well as to detect cleavage levels of Caspase 3 and poly?ADP?ribose polymerase （PARP）. Lactate dehydrogenase （LDH） assay was conducted to detect cell death, and Annexin V?EGFP staining to evaluate cell apoptosis. Results The LC3?Ⅱ/ LC3?Ⅰ ratios （LC3?Ⅰ conversion to LC3?Ⅱ） after 12? and 24?hour treatment did not differ among the 50?, 100? and 200?nmol/L everolimus groups （12 hours: 3.52 ± 0.21 vs. 4.03 ± 0.39 vs. 5.05 ± 0.22, P > 0.05; 24 hours: 3.38 ± 0.26 vs. 3.29 ± 0.06 vs. 6.57 ± 0.16, P > 0.05）, but were significantly higher in the three everolimus groups than in the control group receiving no treatment （12 hours: 2.07 ± 0.05, P < 0.05; 24 hours: 2.61 ± 0.16, P < 0.05）. After 12?hour treatment, no significant differences were observed in the ratio of LC3?Ⅱ to β?actin between the 50?nmol/L everolimus + E64d + pepstatin group （1.26 ± 0.40）, 100?nmol/L everolimus + E64d + pepstatin group （1.16 ± 0.34）, 200?nmol/L everolimus + E64d + pepstatin group （1.21 ± 0.39） and E64d + pepstatin group （1.19 ± 0.27, P > 0.05）. Moreover, there was no significant difference in the percentages of autophagic vesicle?positive cells between the 100?nmol/L everolimus + E64d + pepstatin group and E64d + pepstatin group （2.06% ± 0.61% vs. 1.68% ± 0.62%, P > 0.05）. After 24?hour treatment, the everolimus + cisplatin group showed significantly increased rate of cell death compared with the cisplatin alone group （42.58% ± 0.93% vs. 18.20% ± 1.46%）. However, no significant differences were observed in the cleavage levels of Caspase 3 and PARP, the number of annexin V?labelled cells and ratio of LC3?Ⅱ to β?actin between the everolimus + cisplatin group and the cisplatin?alone group （P > 0.05）. Conclusion Everolimus has a synergistic effect on the cisplatin?mediated COLO?16 cell death, and this effect does not depend on cell apoptosis or autophagy.