氯喹增强口腔癌CAL-27细胞对顺铂化疗敏感性的观察

上海口腔医学 ›› 2015, Vol. 24 ›› Issue (1): 30-36.

氯喹增强口腔癌CAL-27细胞对顺铂化疗敏感性的观察

全海英^1,2,周丽佳¹,李昂迪¹,张泽兵¹

1.吉林大学口腔医院病理科,吉林长春 130021;
2.吉林医药学院附属医院口腔科,吉林吉林 132013

收稿日期:2014-02-17 出版日期:2015-02-20 发布日期:2015-07-24
通讯作者: 张泽兵,Tel:0431-85579328,E-mail:zebing@jlu.edu.cn
作者简介:全海英（1987-）,女,硕士研究生,医师,E-mail:hanbingsui111@163.com
基金资助:
吉林省科技厅基金(201105101); 吉林省卫生厅基金(20132004)

Mechanism of chloroquine in promoting sensitivity of chemotherapeutics in oral squamous cell carcinoma CAL-27 cell line to cisplatin

QUAN Hai-ying^1,2,ZHOU Li-jia¹,LI Ang-di¹,Zhang Ze-bing¹

1.Department of Pathology, Stomatological Hospital, Jilin University. Changchun 130021;
2.Department of Stomatology, the Affiliated Hospital of Jilin Medical College. Jilin 132013, Jilin Province, China

Received:2014-02-17 Online:2015-02-20 Published:2015-07-24
Supported by:
Research Fund of Department of Science and Technology of Jilin Province (20115101) and Department of Health of Jilin Province (20132004)

摘要/Abstract

摘要： 目的利用氯喹（chloroquine,CQ）及顺铂（cisplatin,DDP）作用于CAL-27细胞,探讨自噬在口腔癌化疗中的作用及机制,为临床增强口腔癌化疗敏感性提供理论依据。方法应用四甲基偶氮唑蓝（MTT）比色法比较药物对CAL-27细胞的生长抑制作用,采用激光共聚焦显微镜观察自噬特异性蛋白LC3-Ⅱ的表达,AnnexinV/PI双染色流式细胞仪检测细胞凋亡,流式细胞术观察细胞周期的分布。采用SPSS17.0软件包对数据进行统计学分析。结果不同浓度氯喹及顺铂处理CAL-27细胞不同时间后,细胞生存率逐渐降低,呈浓度和时间依赖性。5 mg/L氯喹联合顺铂在IC₅₀浓度（5 mg/L）处理后,与单独顺铂处理相比,显著降低了CAL-27细胞的生存率（P<0.05）。氯喹或顺铂作用于CAL-27细胞48 h后,自噬在细胞中分布清晰,顺铂组（DDP组）细胞平均荧光强度高于对照组、氯喹处理组（CQ组）及氯喹与顺铂联合处理组（CQ+DDP组）（P<0.05）;氯喹组细胞平均荧光强度显著低于其他3组（P<0.05）。氯喹或顺铂作用于CAL-27细胞48 h后,与对照组相比,DDP组和CQ+DDP组细胞凋亡率显著提高（P<0.05）;与顺铂单独作用相比,CQ+DDP组细胞凋亡率显著提高（P<0.05）。氯喹和顺铂作用于CAL-27细胞48 h后,DDP组和CQ+DDP组G1期细胞明显增多,出现G1期阻滞,CQ+DDP组G1期细胞数显著高于DDP组（P<0.05）。结论抑制自噬能够提高CAL-27细胞对顺铂的化疗敏感性,CAL-27细胞本身的自噬是产生化疗耐药的重要机制。自噬抑制剂有望成为口腔癌化疗的增敏剂。

关键词: 细胞自噬, 顺铂, 氯喹, 细胞凋亡, 口腔鳞状细胞癌

Abstract: PURPOSE: To study the role and mechanism of autophagy in chemotherapy of oral squamous cell carcinoma, and provide theoretical evidence to improve chemotherapeutic efficacy of oral squamous cell carcinoma patients. METHODS: The cell survival rate changes induced by cisplatin (DDP) and chloroquine (CQ) in CAL-27 cells were assayed by methyl thiazolyl tetrazolium method(MTT). The LC3-Ⅱ expression level was detected by laser scanning confocal microscope; The apoptotic rate was determined by flow cytometry. SPSS17.0 software package was used for statistical analysis. RESULTS: MTT results showed that compared with the control group, the cell survival rate reduced with the increasing time of DDP and CQ treatment; The optimal concentration of CAL-27 cells was 5 mg/L after treatment with CQ. IC₅₀ of the CAL-27 cells was 5 mg/L after treatment with DDP; MTT results showed that the cell survival rate of CQ+DDP group was significantly lower than control group, CQ group and DDP group (P<0.05). With the action of CQ and DDP to CAL-27 cells for 48 hours, immunofluorescence results showed that the average fluorescence intensity of DDP group was significantly higher than the other 3 groups (P<0.05), while it was significantly lower in CQ group than the other 3 groups (P<0.05). With the action of CQ and DDP to CAL-27 cells for 48 hours, flow cytometry results showed that the cell apoptosis rate of DDP group and CQ+DDP group were significantly higher than control group and CQ group. The cell apoptosis rate of CQ+DDP group was significantly higher than DDP group (P<0.05). With the action of CQ and DDP to CAL-27 cells for 48 hours, cells in G1 phase of DDP group and CQ+DDP group increased, indicating G1 phase blockage. The cell count in G1 phase of CQ+DDP group was significantly higher than DDP group (P<0.05). CONCLUSIONS: Inhibition of autophagy can enhance the chemotherapeutic sensitivity of DDP in CAL-27 cells. Autophagy in CAL-27 cells is an important mechanism for chemotherapy resistance of oral squamous cell carcinoma. Autophagy inhibitor may have significant potential to be a novel chemotherapeutic sensitizer for oral squamous cell carcinoma.

Key words: Cell autophagy, Cisplatin, Chloroquine, Cell apoptosis, Oral squamous cell carcinoma

中图分类号:

R739.8

全海英,周丽佳,李昂迪,张泽兵. 氯喹增强口腔癌CAL-27细胞对顺铂化疗敏感性的观察[J]. 上海口腔医学, 2015, 24(1): 30-36.

QUAN Hai-ying,ZHOU Li-jia,LI Ang-di,Zhang Ze-bing. Mechanism of chloroquine in promoting sensitivity of chemotherapeutics in oral squamous cell carcinoma CAL-27 cell line to cisplatin[J]. Shanghai Journal of Stomatology, 2015, 24(1): 30-36.

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