中华皮肤科杂志 ›› 2017, Vol. 50 ›› Issue (10): 739-741.doi: 10.3760/cma.j.issn.0412-4030.2017.10.010

• 论著 • 上一篇    下一篇

依维莫司与顺铂协同损伤人皮肤鳞状细胞癌COLO-16细胞机制的初步研究

丁敏 徐松 李莉 毕素云 周之海 李岷 陈旭 顾恒   

  1. 300070 天津医科大学总医院皮肤性病科（丁敏、毕素云、周之海）；中国医学科学院北京协和医学院皮肤病研究所江苏省皮肤病与性病分子生物学重点实验室（徐松、李莉、李岷、陈旭、顾恒）

  • 收稿日期:2017-05-03 修回日期:2017-04-13 发布日期:2017-09-29
  • 通讯作者: 陈旭 E-mail:doctor_chx@126.com
  • 基金资助:
    国家自然科学基金（81371755、81673083、81773342）；教育部博士点基金（20131106120046）；江苏省临床医学科技专项（BL2012003）；重大疾病创新研究（2016ZX320014）；医学创新工程（2016?12M?1?005）

Mechanisms underlying the synergistic damage to human squamous cell carcinoma cell line COLO-16 by everolimus and cisplatin: a preliminary study

Ding Min, Xu Song, Li Li, Bi Suyun, Zhou Zhihai, Li Min, Chen Xu, Gu Heng#br#   

  1. 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）
  • Received:2017-05-03 Revised:2017-04-13 Published:2017-09-29
  • Supported by:
    National Natural Science Foundation of China（81371755, 81673083, 81773342）;Doctoral Fund of Ministry of Education of China（20131106120046）; Jiangsu Provincial Special Program of Medical Science（BL2012003）; Innovation Research on Critical Diseases（2016ZX320014）; CAMS Initiative for Innovative Medicine（2016?12M?1?005）

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摘要： 目的 探讨依维莫司与顺铂联合损伤人皮肤鳞状细胞癌COLO?16细胞的分子机制。方法 依维莫司处理细胞后的信号通路实验分为对照组、50、100、200 nmol/L依维莫司处理组4组。依维莫司与顺铂联合处理的机制实验分为对照组、50 nmol/L依维莫司、25 μmol/L顺铂、50 nmol/L依维莫司 + 25 μmol/L顺铂处理细胞组4组。通过Western印迹进行mTOR、Akt、DNA损伤相关信号以及Chk通路分析。结果 50、100、200 nmol/L依维莫司处理COLO?16细胞12、24 h后，mTOR 2448位点和2481位点的磷酸化水平降低，Rictor 1135位点磷酸化水平也降低。然而，下游信号ULK1蛋白的ser757位点的磷酸化水平、P70 S6激酶的thr389位点的磷酸化水平以及PKCα的thr638/641位点磷酸化水平的变化并不显著。依维莫司处理对Akt的总蛋白水平和磷酸化水平无明显影响。顺铂处理COLO?16细胞12、24 h后，Rictor的thr1135位点磷酸化水平和Chk1的ser345位点磷酸化水平明显升高，但依维莫司单独处理无类似效应。当依维莫司与顺铂联合处理后12和24 h，相对于顺铂单独处理的细胞，上调的Chk1和Rictor磷酸化水平受到明显抑制。结论 COLO?16细胞mTOR信号对依维莫司处理敏感，但其下游信号并非同步产生级联反应。依维莫司可能通过抑制检测点激酶1的激活增强顺铂诱导COLO?16细胞死亡，但无加重顺铂所导致的DNA损伤。

关键词: 顺铂, 细胞凋亡, COLO?16细胞, 依维莫司, 检测点激酶1

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