Abstract: Ding Min, Xu Song, Li Li, Bi Suyun, Zhou Zhihai, Li Min, 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 STI, Nanjing 210042, China （Xu S, Li L, Li M, Chen X, Gu H） Corresponding authors: Chen Xu, Email: doctor_chx@126.com; Zhou Zhihai, Email: zhouzh65@sohu.com 【Abstract】 Objective To investigate molecular mechanisms underlying the synergistic damage to the human squamous cell carcinoma cell line COLO-16 by everolimus and cisplatin. Methods In the signaling pathway experiment, COLO-16 cells were divided into 4 groups: control group receiving no treatment, 50, 100 and 200 nmol/L everolimus groups treated with 50, 100 and 200 nmol/L everolimus respectively. In the combined experiment, COLO-16 cells were divided into another 4 groups: control group, 50 nmol/L everolimus group, 25 mol/L cisplatin group, and 50 nmol/L everolimus + 25 mol/L cisplatin group. Western blot analysis was performed to analyze changes in mammalian target of rapamycin （mTOR） pathway, Akt pathway, DNA damage-related pathway and Csk homologous kinase （Chk） pathway. Results After the treatment with everolimus at different concentrations of 50, 100 and 200 nmol/L for 12 and 24 hours, the phosphorylation levels of mTOR at ser2448 and ser2481 as well as Rictor at thr1135 in COLO-16 cells were all decreased compared with the control group. However, there were no significant changes in the phosphorylation levels of downstream signals ULK1 at ser757, p70 S6 at thr389 and PKCα at thr638/64. The treatment with everolimus did not change the total protein level and phosphorylation of Akt. After the treatment with cisplatin for 12 and 24 hours, the phosphorylation levels of Rictor at thr1135 and Chk1 at ser345 were significantly increased, but the treatment with everolimus alone showed no such effects. After the combined treatment with everolimus and cisplatin for 12 and 24 hours, the upregulation of Chk1 and Rictor phosphorylation were significantly inhibited compared with the cisplatin alone group. Conclusions mTOR signaling is sensitive to everolimus in COLO-16 cells, but its targeted pathway is not regulated simultaneously to develop a cascade reaction. Everolimus may increase the cisplatin-induced death of COLO-16 cells by inhibiting the activation of Chk1, but can not aggravate DNA damage induced by cisplatin.

Key words: Cisplatin, Apoptosis, COLO?16 cells, Everolimus, Chk1