17-醋酸甲羟孕酮对人子宫内膜癌干细胞样特性的作用机制研究

[Study of mechanism of medroxyprogesterone 17-acetate on the cancer stem cell-like properties of human endometrial cancer].

作者信息

Liu Bing-jie, Li Xiao-ping, Zhao Li-jun, Wang Jian-liu, Wei Li-hui

机构信息

Department of Gynecology, Peking University People's Hospital, Beijing 100044, China.

Department of Gynecology, Peking University People's Hospital, Beijing 100044, China. Email:

出版信息

Zhonghua Fu Chan Ke Za Zhi. 2013 Oct;48(10):772-7.

PMID:24406136

Abstract

OBJECTIVE

To explore the mechanism resistance of medroxyprogesterone 17-acetate(MPA) on the endometrial cancer side-population(SP) cells.

METHODS

(1) Ishikawa-SP cells from endometrial cancer cell lines Ishikawa were be separated by Hoechst 33342 dyeing method and flow cytometry analysis. The clone formation efficiency between Ishikawa-SP cells and Ishikawa-non-SP cells were performed by clone formation assay. Breast cancer resistance protein (BCRP) was examined by immunocytochemistry method. (2) Ishikawa, Ishikawa-SP, Ishikawa-non-SP cells were treated with various concentrations of MPA at 5, 10, 15, 20 µmol/L. After cultured for 24, 48, and 72 hours, cells growth were measured by methanethiosulfomate (MTS) assay. (3) The groups of Ishikawa, Ishikawa-SP, Ishikawa-non-SP cells incubated with MPA at the half maximal inhibitory concentration (IC50) were selected for cell apoptosis assay by using flow cytometry. After MPA treatment, the expression of caspase-3 was examined by immunocytochemistry method.

RESULTS

(1) There were few proportion of Ishikawa-SP cells in Ishikawa endometrial carcinoma, which were 2.7%. There were stronger clone formation efficiency for Ishikawa-SP cells than that for Ishikawa-non-SP cells in Ishikawa [(6.02 ± 1.17)% vs.(0.53 ± 0.20)%, P = 0.001]. And there were higher level expression of BCRP (P = 0.001) and also more resistant Taxol and radiation between Ishikawa-SP cells and Ishikawa-non-SP cells. (2) The inhibitory effect of MPA was concentration-dependent and time-dependent. (3)After MPA treatment, the apoptosis rates of Ishikawa-SP, Ishikawa-non-SP,Ishikawa were (4.01 ± 0.43) %, (9.30 ± 0.67) %, and (4.64 ± 0.18) %, respectively(P < 0.05). The level expression of caspase-3 in Ishikawa group after MPA treated were higher than that in Ishikawa-SP group.

CONCLUSION

MPA may be inhibit the growth of endometrial cancer, Ishikawa-SP and Ishikawa-non-SP cells, while Ishikawa-SP may be more resistant to MPA than Ishikawa-non-SP, which mechanism of resistance on MPA may be related to the properties of cancer stem-like cells and cell apoptosis.

摘要

目的

探讨醋酸甲羟孕酮（MPA）对子宫内膜癌侧群（SP）细胞的耐药机制。

方法

（1）采用Hoechst 33342染色法和流式细胞术分析从子宫内膜癌细胞系Ishikawa中分离出Ishikawa-SP细胞。通过克隆形成试验检测Ishikawa-SP细胞和Ishikawa非SP细胞之间的克隆形成效率。采用免疫细胞化学方法检测乳腺癌耐药蛋白（BCRP）。（2）用5、10、15、20μmol/L不同浓度的MPA处理Ishikawa、Ishikawa-SP、Ishikawa非SP细胞。培养24、48和72小时后，采用甲硫代磺酸盐（MTS）试验检测细胞生长情况。（3）选择在半数最大抑制浓度（IC50）下用MPA孵育的Ishikawa、Ishikawa-SP、Ishikawa非SP细胞组，采用流式细胞术进行细胞凋亡检测。MPA处理后，采用免疫细胞化学方法检测caspase-3的表达。

结果

（1）Ishikawa子宫内膜癌中Ishikawa-SP细胞比例很少，为2.7%。Ishikawa中Ishikawa-SP细胞的克隆形成效率高于Ishikawa非SP细胞[（6.02±1.17）%对（0.53±0.20）%，P = 0.001]。Ishikawa-SP细胞中BCRP表达水平更高（P = 0.001），并且Ishikawa-SP细胞和Ishikawa非SP细胞之间对紫杉醇和辐射的耐药性更强。（2）MPA的抑制作用呈浓度依赖性和时间依赖性。（3）MPA处理后，Ishikawa-SP、Ishikawa非SP、Ishikawa的凋亡率分别为（4.01±0.43）%、（9.30±0.67）%和（4.64±0.18）%（P < 0.05）。MPA处理后Ishikawa组中caspase-3的表达水平高于Ishikawa-SP组。