子宫内膜癌细胞中干细胞样细胞对醋酸甲羟孕酮的抵抗作用及其机制

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

目的初步探讨子宫内膜癌细胞中干细胞样（sP）细胞对醋酸甲羟孕酮（MPA）的抵抗作用及其机制。方法（1）采用流式细胞仪Hoechst 33342（是一种荧光染色液）染色法分选子宫内膜癌细胞系Ishikawa细胞中的SP细胞（即Ishikawa-SP细胞）和非SP细胞（即Ishikawa-non-SP细胞），采用平板克隆形成实验、免疫组化法分别检On,0Ishikawa-SP和Ishikawa-non-SP细胞的集落形成率及乳腺癌耐药蛋白（BCRP）的表达。（2）不同浓度（分别为5、10、15、20Ixmol／L）的MPA处理Ishikawa-SP、Ishikawa-non-SP、Ishikawa细胞不同时间（分别为24、48、72h）后，采用甲烷硫代磺酸盐（MTS）法检测3种细胞生长的抑制率，选择Ishikawa细胞生长的抑制率最接近50％时MPA的处理浓度和时间（即为10umol／L的MPA处理72h时）用于以下实验。（3）以10umol／L的MPA处理Ishikawa-SP、Ishikawa-non．SP、Ishikawa细胞72h后，采用流式细胞仪检on．03种细胞的凋亡率，采用免疫组化法检测3种细胞中凋亡蛋白--半胱氨酸天冬氨酸蛋白酶3（caspase-3）的表达。结果（1）子宫内膜癌细胞系Ishikawa细胞中存在少量sP细胞，其比例为2．7％；Ishikawa-SP和Ishikawa-non-SP细胞的集落形成率分别为（6．02±1．17）％、（0．53±0．20）％，两者比较，差异有统计学意义（P=0．001）；Ishikawa-SP和Ishikawa-non-SP细胞中BCRP蛋白表达的综合得分分别为（16．3±2．0）、（13．44±1．5）分，两者比较，差异有统计学意义（P=0．001）。（2）Ishikawa-SP、Ishikawa-non-SP及Ishikawa细胞生长的抑制率，随着MPA浓度的增加和处理时间的延长均增加，呈明显的浓度和时间依赖性（P〈0．05）。其中，MPA对Ishikawa-SP细胞的抑制作用最弱。（3）以10umol／L的MPA处理72h后，Ishikawa-SP、Ishikawa-non-SP、Ishikawa细胞的凋亡率分别为（4．01±0．43）％、（9．304±0．67）％、（4．64±0．18）％，3者间比较，差异有统计学意义（P〈0．05）；3种细胞中easpase-3蛋白表达的综合得分分别为（14．9±1．1）、（16．14±1．0）、（15．5±1．2）分，3者间比较，差异有统计学意义（P〈0．05）。结论MPA对Ishikawa、Ishikawa-SP、Ishikawa-non-SP细胞的生长均有抑制作用，但Ishikawa-SP细胞的生长抑制率及凋亡率均明显低于Ishikawa和Ishikawa-norl-SP细胞，显示Ishikawa-SP细胞具有孕激素抵抗的特性，其抵抗机制可能与肿瘤干细胞的耐药性和细胞凋亡有关。

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 umol/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 concentrationdependent and time-dependent. （3） After MPA treatment, the apoptosis rates of Ishikawa-SP, Ishikawa-nonSP, Ishikawa were （4. 01 ± 0. 43 ） %, （ 9. 30± 0. 67 ） % , and （ 4. 64± 0. 18 ） %, respectively （ P 〈 0. 05 ）. The level expression of easpase-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 ceil apoptosis.