沙利霉素对口腔鳞癌细胞增殖和凋亡的影响

doi:10.19723/j.issn.1671-167X.2020.05.018

北京大学学报（医学版） ›› 2020, Vol. 52 ›› Issue (5): 902-906. doi: 10.19723/j.issn.1671-167X.2020.05.018

沙利霉素对口腔鳞癌细胞增殖和凋亡的影响

苏雷震^1,^2,³,陈洁^1,²,李显^1,³,季平^1,^3,^∆()

1.重庆医科大学附属口腔医院口腔颌面外科,重庆 401120
2.口腔疾病与生物医学重庆市重点实验室,重庆 401120
3.重庆市高校市级口腔生物医学工程重点实验室,重庆 401120

收稿日期:2018-09-18 出版日期:2020-10-18 发布日期:2020-10-15
通讯作者: 季平 E-mail:jiping_62@hotmail.com
基金资助:
2017重庆市卫生计生委医学科研项目(2017MSXM075);重庆市渝中区科技计划项目(20170407);重庆市高校市级口腔生物医学工程重点实验室资助项目(CXTDG201602006);2016年重庆高校创新团队建设计划资助项目

Effects of salinomycin on proliferation and apoptosis of oral squamous cell carcinoma

Lei-zhen SU^1,^2,³,Jie CHEN^1,²,Xian LI^1,³,Ping JI^1,^3,^∆()

1. Department of Oral and Maxillofacial Surgery,Stomatological Hospital of Chongqing Medical University, Chongqing 401120, China
2. Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing 401120, China
3. Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing 401120, China

Received:2018-09-18 Online:2020-10-18 Published:2020-10-15
Contact: Ping JI E-mail:jiping_62@hotmail.com
Supported by:
Medical Research Project of Chongqing Health and Family Planning Commission of Chongqing Province(2017MSXM075);Program for Yuzhong District Scientific and Technological Project(20170407);Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education(CXTDG201602006);Program for Innovation Team Building at Institutions of Higher Education in Chongqing in 2016

摘要/Abstract

摘要：

目的:探讨沙利霉素(salinomycin)对口腔鳞癌细胞增殖和凋亡的影响,并初步探讨沙利霉素对信号通路的影响。方法:培养口腔鳞状细胞癌细胞系CAL-27,将1、2、4、8、16、32 μmol/L沙利霉素和1.25、2.5、5、10、20、40、80 μmol/L顺铂与CAL-27细胞共同培养,24 h和48 h后用细胞计数试剂盒-8(cell counting kit-8,CCK-8)法检测沙利霉素和顺铂对CAL-27细胞增殖的影响;0、2、4、8 μmol/L沙利霉素和0、5、10、20 μmol/L顺铂与CAL-27细胞共培养48 h后,通过流式细胞术检测沙利霉素和顺铂对CAL-27细胞周期的影响,蛋白免疫印迹法(Western blot)检测CAL-27细胞中天冬氨酸特异性半胱氨酸蛋白酶-3(cysteine-containing aspartate-specific proteases-3,Caspase-3)、天冬氨酸特异性半胱氨酸蛋白酶-9(cysteine-containing aspartate-specific proteases-9,Caspase-9)、脱氧核糖核酸(deoxyribonucleic acid,DNA)修复酶(poly ADP-ribose polymerase,PARP)、蛋白激酶B(protein kinase B, Akt)和磷酸化蛋白激酶B (phosphorylated protein kinase B,p-Akt)的表达。结果:CCK-8实验表明沙利霉素和顺铂均能显著抑制口腔鳞状细胞癌CAL-27细胞增殖,且抑制作用呈时间依赖性和药物浓度依赖性,但是相对于临床一线化疗药物顺铂而言,沙利霉素对CAL-27细胞增殖的抑制效果更加显著(P<0.001)。细胞周期检测表明,与加入二甲基亚砜(dimethyl sulfoxide,DMSO)的对照组相比,8 μmol/L沙利霉素与CAL-27细胞共同培养48 h后,细胞休眠期/DNA合成前期的CAL-27细胞比例明显升高(40.40%±1.99% vs.64.46%±0.90%,P<0.05), DNA合成期和DNA合成后期/有丝分裂期的CAL-27细胞比例出现降低(24.32%±2.30% vs.18.73%±0.61%,P<0.05,35.01%±1.24% vs.16.54%±1.31%,P<0.05);顺铂对CAL-27细胞周期没有特异性改变。蛋白免疫印迹法结果显示,沙利霉素在上调CAL-27细胞中Caspase-3和 Caspase-9蛋白表达(P<0.05)的同时下调PARP、Akt和p-Akt蛋白的表达(P<0.05)。结论:相对于顺铂而言,沙利霉素对CAL-27细胞增殖有更强的抑制作用,并且能将口腔鳞状细胞癌CAL-27细胞周期阻滞在细胞休眠期/DNA合成前期,同时能够诱导CAL-27细胞发生凋亡,这一机制可能和Akt/p-Akt 信号通路相关。

关键词: 沙利霉素, 口腔鳞状细胞癌, 细胞周期, 凋亡

Abstract:

Objective: To investigate the effects of salinomycin on the proliferation and apoptosis of oral squamous carcinoma cells and to further understand the mechanisms of these effects. Methods: The human oral squamous carcinoma cell line CAL-27 was cultured in different concentrations of salinomycin and cisplatin. After co-culture with 0, 1, 2, 4, 8, 16 and 32 μmol/L salinomycin or 0, 1.25, 2.5, 5, 10, 20, 40 and 80 μmol/L cisplatin for 24 hours and 48 hours, the proliferation of oral squamous carcinoma cells were detected by cell counting kit-8(CCK-8) assay. After being exposed to 0, 2, 4, 8 μmol/L salinomycin and 0, 5, 10, 20 μmol/L cisplatin for 48 hours, the cell cycle of oral squamous carcinoma cells was detected by flow cytometry assay, and Western blot analysis was performed to analyze the expressions of cysteine-containing aspartate-specific proteases-3(Caspase-3), cysteine-containing aspartate-specific proteases-9(Caspase-9), poly ADP-ribose polymerase (PARP), protein kinase B (Akt) and phosphorylated protein kinase B (p-Akt) protein in oral squamous carcinoma cells. Results: Both salinomycin and cisplatin significantly inhibited the proliferation of oral squamous cell carcinoma CAL-27 cells in a time- and dose-dependent manner. However, compared with the first-line chemotherapeutic drug cisplatin, salinomycin showed stronger anti-proliferation activity in oral squamous carcinoma cells than cisp-latin (P<0.001). After being exposed to 8 μmol/L salinomycin, CAL-27 cells exhibited markedly higher proportion in quiescent/ first gap phases (40.40%±1.99% vs. 64.46%±0.90%, P<0.05), and had a significantly lower proportion in synthesis phases and second gap / mitosis phases (24.32%±2.30% vs. 18.73%±0.61%, P<0.05; 35.01%±1.24% vs. 16.54%±1.31%, P<0.05) compared with the dimethyl sulfoxide control group; moreover cisplatin didn’t show cell-cycle specific effect on CAL-27. Western blot proved that salinomycin could up-regulate the expressions of Caspase-3 and Caspase-9 protein in oral squamous cell carcinoma CAL-27 cells (P<0.05). At the same time, the levels of PARP, Akt and p-Akt protein were down-regulated (P<0.05). Conclusion: Compared with cisplatin, salinomycin has a better inhibitory effect on the proliferation of oral squamous carcinoma cells and blocks the cell cycle process at the quiescent / first gap phase. At the same time, salinomycin could trigger apoptosis of oral squamous carcinoma cells and the mechanism is associated with the Akt/p-Akt signaling pathway.

Key words: Salinomycin, Oral squamous cell carcinoma, Cell cycle arrest, Apoptosis

中图分类号:

R739.8

苏雷震,陈洁,李显,季平. 沙利霉素对口腔鳞癌细胞增殖和凋亡的影响[J]. 北京大学学报（医学版）, 2020, 52(5): 902-906.

Lei-zhen SU,Jie CHEN,Xian LI,Ping JI. Effects of salinomycin on proliferation and apoptosis of oral squamous cell carcinoma[J]. Journal of Peking University (Health Sciences), 2020, 52(5): 902-906.

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沙利霉素对口腔鳞癌细胞增殖和凋亡的影响

Effects of salinomycin on proliferation and apoptosis of oral squamous cell carcinoma

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