Liu Bing-Jie, Xu Qi-Ying, Yu Wei-Dong, Li Na, Yao Tian, Zhao Li-Jun, Wang Jian-Liu, Wei Li-Hui, Li Xiao-Ping
Department of Gynecology, Peking University People's Hospital, Beijing, China.
Qinghai University Affiliated Hospital, Xining, China.
Front Med (Lausanne). 2020 Mar 18;7:70. doi: 10.3389/fmed.2020.00070. eCollection 2020.
Radiotherapy, combined regimens as platinum-paclitaxel chemotherapy and/or endocrine therapy is an important adjuvant treatment after surgery for endometrial cancer (EC). While, the resistance to them remain unclear. In our study, to separate the characteristics of side population (SP) cells from EC cell lines, study the mechanism of Taxol-resistance, progestin resistance and radioresistanc, and provide the basic for EC. SP cells from EC cell lines HEC-1A, Ishikawa and RL95-2 were separated by Hoechst 33342 staining and flow cytometry analysis. The expression of breast cancer resistance protein (BCRP) in SP cells and non-SP cells from HEC-1A was examined by immunocytochemistry, and the radiation-resistant and Taxol-resistant characteristics of SP cells and non-SP cells were compared by MTS. Ishikawa, Ishikawa-SP, and Ishikawa-non-SP cells incubated with MPA were selected for cell apoptosis assays by using flow cytometry. The expression of caspase-3 was examined by immunocytochemistry, and autophagy was detected by MDC staining. Small proportions of SP cells, namely, 1.44 ± 0.93%, 2.86 ± 3.09%, and 2.87 ± 1.29%, were detected in HEC-1A, Ishikawa and RL95-2, respectively. There was a stronger clone formation efficiency for the SP cells than for non-SP cells in HEC-1A [(6.02 ± 1.17) vs. (0.53±0.20)%, = 0.001], and there was a significant difference in the rate of tumourigenicity between the SP cells and non-SP cells in HEC-1A (87.5 vs. 12.5%). There were higher levels of BCRP expression ( = 0.001) and resistance to Taxol and radiation ( < 0.05) in the SP cells than in non-SP cells. After MPA treatment, the apoptosis rates were significantly different among the Ishikawa, Ishikawa-SP and Ishikawa-non-SP groups [(4.64 ± 0.18)%, (4.01 ± 0.43)%, and (9.3 ± 0.67)%; ( = 0.05)], and the expression of Caspase-3 in the Ishikawa group was higher than that in Ishikawa-SP group. The autophagic activity of the Ishikawa-SP cells was the strongest, while the autophagic activity of Ishikawa-non-SP was the weakest. There is a significant enrichment in SP cells among different EC cell lines, and these SP cells be more resistant to Taxol, MPA and radiation therapy. The overexpression of BCRP among SP cells may be the cause of resistance to Taxol, progestin and radiotherapy, which may be related to apoptosis and autophagic activity.
放疗、铂类-紫杉醇化疗联合方案及/或内分泌治疗是子宫内膜癌（EC）术后重要的辅助治疗手段。然而，对这些治疗的耐药机制尚不清楚。在我们的研究中，为了分离EC细胞系中的侧群（SP）细胞特征，研究紫杉醇耐药、孕激素耐药和放射抵抗的机制，并为EC研究提供基础。采用Hoechst 33342染色和流式细胞术分析从EC细胞系HEC-1A、Ishikawa和RL95-2中分离SP细胞。通过免疫细胞化学检测HEC-1A中SP细胞和非SP细胞中乳腺癌耐药蛋白（BCRP）的表达，并通过MTS比较SP细胞和非SP细胞的放射抵抗和紫杉醇抵抗特征。选择用甲羟孕酮（MPA）处理的Ishikawa、Ishikawa-SP和Ishikawa-非SP细胞，采用流式细胞术进行细胞凋亡检测。通过免疫细胞化学检测caspase-3的表达，并通过单丹磺酰尸胺（MDC）染色检测自噬。在HEC-1A、Ishikawa和RL95-2中分别检测到小比例的SP细胞，即1.44±0.93%、2.86±3.09%和2.87±1.29%。在HEC-1A中，SP细胞的克隆形成效率高于非SP细胞[（6.02±1.17）%对（0.53±0.20）%，P = 0.001]，且HEC-1A中SP细胞和非SP细胞的致瘤率有显著差异（87.5%对12.5%）。SP细胞中BCRP表达水平较高（P = 0.001），对紫杉醇和放疗的耐药性更强（P < 0.05）。MPA处理后，Ishikawa、Ishikawa-SP和Ishikawa-非SP组的凋亡率有显著差异[（4.64±0.18）%、（4.01±0.43）%和（9.3±0.67）%；P = 0.05]，且Ishikawa组中Caspase-3的表达高于Ishikawa-SP组。Ishikawa-SP细胞的自噬活性最强，而Ishikawa-非SP细胞的自噬活性最弱。不同EC细胞系中SP细胞有显著富集，且这些SP细胞对紫杉醇、MPA和放疗更具耐药性。SP细胞中BCRP的过表达可能是对紫杉醇、孕激素和放疗耐药的原因，这可能与凋亡和自噬活性有关。
