Shi Lijun, Yu Hongjing, Zhang Wei, Li Li, Wang Qi
Ministry of Education Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor, Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, China.
Ministry of Education Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor, Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, China. Email:
Zhonghua Fu Chan Ke Za Zhi. 2014 Jul;49(7):523-30.
To establish a platinum resistance nude mice model of epithelial ovarian cancer (EOC) and investigate its resistance to cisplatin (DDP) biological characteristics, so as to provide evidences for exploring chemoresistence mechanisms and screening for reversal targets in vivo micro-environment.
The resistance model was produced by repeating a crossover subcutaneous injection of human ovarian cancer SKOV3 cells labelled green fluorescent protein (GFP) and transplatation of tumor fragment into nude mice. Two kinds of cancer cell lines of SKOV3/DDP I and SKOV3/DDP II were induced with acquired resistence to DDP. The chemosensitivities of EOC cells to DDP were tested and half maximal inhibitory concentration (IC50) was measured by methyl thiazolyl tetrazolium (MTT) and flow cytometry (FCS). Dynamic analysis among the concentration of DDP treatment and cell apoptosis, cell cycle phase distribution and intracellular DDP concentration. The expression of PTEN, STAT5, XIAP, BRCA1 and MDR1 were examined by real time quantitative reverser transcription PCR (qRT- PCR) in vivo.
IC50 value of cisplatin for SKOV3/DDP II were 2.83 ± 0.12 and 3.82±0.19 folds than those for SKOV3/GFP by MTT and flow cytometry, separately. SKOV3/DDP I were 2.20 ± 0.16 and 3.40 ± 0.20 folds. The apoptosis rate of SKOV3/DDP II and SKOV3/DDP I were decreased significantly at 29.7 and 39.6 µmol/L DDP when treatment for 36 hours, which were lower than that of SKOV3/GFP cells [(57.0 ± 1.4)% vs (37.6 ± 4.36)% vs (83.1 ± 2.71)%, P = 0.024; (74.4 ± 2.3)% vs (50.5 ± 3.4)% vs (87.4 ± 4.0)%, P = 0.001]. SKOV3/DDP I and SKOV3/DDP II was positively related with cisplatin processing time. Intracellular DDP accumulation of SKOV3/DDP II and SKOV3/DDP I were lower than SKOV3-GFP in dynamic processes(P < 0.05). Besides intracellular DDP accumulation of SKOV3/DDP II also lower than SKOV3/DDP I in dynamic processes (P < 0.05). Transplanted tumor of SKOV3/GFP appeared organelle degradation and nuclear membrane imcompleted after five times DDP injection with concentration of 4 mg/kg. SKOV3/DDP II and SKOV3/DDP I did not generate these phenomenon untill eighth DDP injections with concentration of 4 mg/kg. STAT5 and BRCA1 of SKOV3/DDP II were increased with DDP treatment at concentration of 4 mg/kg. Expression of XIAP from SKOV3/DDP II was positive correlated with injection times. STAT5, XIAP and BRCA1 of SKOV3/DDP II were up-regulated 3.86, 28.1 and 14.6 folds than those in SKOV3/GFP cells after eighth DDP treatment, separately. While PTEN of SKOV3/DDP II was decreased 3.77 folds.
We have successfully established platinum-resistent EOC mice model, which provides a new platform for further study on chemoresistant reversal and individualized clinical treatment. The results shown that potential mechanisms of SKOV3/DDP II DDP-resistance included over-expressed BRCA1 gene may be promote DNA damage repair, elevate XIAP gene to decrease cell apoptosis, up- regulated STAT5 gene and decrease PTEN gene to stimulate proliferation.
建立铂耐药上皮性卵巢癌（EOC）裸鼠模型，研究其对顺铂（DDP）耐药的生物学特性，为探索体内微环境中的化疗耐药机制及筛选逆转靶点提供依据。
通过重复交叉皮下注射绿色荧光蛋白（GFP）标记的人卵巢癌SKOV3细胞并将肿瘤片段移植到裸鼠体内来建立耐药模型。用SKOV3/DDP I和SKOV3/DDP II两种癌细胞系诱导获得对DDP的耐药性。采用甲基噻唑基四氮唑（MTT）法和流式细胞术（FCS）检测EOC细胞对DDP的化疗敏感性并测定半数最大抑制浓度（IC50）。对DDP处理浓度与细胞凋亡、细胞周期阶段分布及细胞内DDP浓度进行动态分析。通过实时定量逆转录PCR（qRT-PCR）检测体内PTEN、STAT5、XIAP、BRCA1和MDR1的表达。
MTT法和流式细胞术检测显示，顺铂对SKOV3/DDP II的IC50值分别是SKOV3/GFP的2.83±0.12倍和3.82±0.19倍。SKOV3/DDP I分别是2.20±0.16倍和3.40±0.20倍。当用29.7和39.6μmol/L DDP处理36小时时，SKOV3/DDP II和SKOV3/DDP I的凋亡率显著降低，低于SKOV3/GFP细胞[（57.0±1.4）%对（37.6±4.36）%对（83.1±2.71）%，P = 0.024；（74.4±2.3）%对（50.5±3.4）%对（87.4±4.0）%，P = 0.001]。SKOV3/DDP I和SKOV3/DDP II与顺铂处理时间呈正相关。在动态过程中，SKOV3/DDP II和SKOV3/DDP I的细胞内DDP积累低于SKOV3-GFP（P < 0.05）。此外，在动态过程中SKOV3/DDP II的细胞内DDP积累也低于SKOV3/DDP I（P < 0.05）。用浓度为4mg/kg的DDP注射5次后，SKOV3/GFP移植瘤出现细胞器降解和核膜不完整。用浓度为4mg/kg的DDP注射8次后，SKOV/DDP II和SKOV3/DDP I才出现这些现象。浓度为4mg/kg的DDP处理后，SKOV3/DDP II的STAT5和BRCA1增加。SKOV3/DDP II的XIAP表达与注射次数呈正相关。第8次DDP处理后，SKOV3/DDP II的STAT5、XIAP和BRCA分别比SKOV3/GFP细胞上调3.86倍、28.1倍和14.6倍。而SKOV3/DDP II的PTEN降低了3.77倍。
我们成功建立了铂耐药EOC小鼠模型，为进一步研究化疗耐药逆转和个体化临床治疗提供了新平台。结果表明，SKOV3/DDP II对DDP耐药的潜在机制包括BRCA1基因过表达可能促进DNA损伤修复、XIAP基因升高导致细胞凋亡减少、STAT5基因上调和PTEN基因降低刺激增殖。
