Xing Hui, Gao Qing-Lei, Yang Xiao-Kui, Li Jing, Gao Chun, Wu Jian-Hong, Lu Yun-Ping, Ma Ding
Department of Obstetrics &Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei,430030, PR China.
Ai Zheng. 2003 Aug;22(8):826-30.
BACKGROUND & OBJECTIVE: Cytotoxic anticancer drugs are less effective in killing tumor cells grown as multicellular spheroids than monolayer cell cultures. The aim of this study was to investigate the molecular mechanism of chemoresistance.
Ovarian cancer A2780, CAOV3 multicellular spheroids were obtained from three-dimensional culture. Expression of P-glycoprotein (P-gp) was determined using Western blot analysis and flow cytometry (FCM). The subcellular distribution of P-gp was also determined using laser confocal microscope. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to detect the mdr1 mRNA of both monolayer cells and multicellular spheroids. Cell cycle profiles and apoptosis were also analyzed using FACS. The resistance was detected with trypan blue exclusion testing.
Compared with control cells, no expression of P-gp was detected in monolayer cells, but expression of P-gp in aggregate cells was significantly elevated(P< 0.05). The mdr1 mRNA positive nodes were confirmed by RT-PCR in the aggregate cells. The percentage of cells increased in G(0)-G(1) phase and decreased in S and G(2)-M phase significantly in spheroids cells. Spheroids cells showed higher cell viability than monolayer cells (P(A2780)=0.003, P(CAOV3)=0.015). More apoptotic cells were induced by Taxol in MCS cells than in monolayer cells.
Ovarian cancer A2780, CAOV3 multicellular spheroids cultures induced cell resistance to Taxol. High expression of P-gp was induced in ovarian multicellular spheroids and the cells were arrested in G(0)-G(1) phase.
细胞毒性抗癌药物在杀死以多细胞球体形式生长的肿瘤细胞方面比单层细胞培养效果更差。本研究旨在探讨化疗耐药的分子机制。
从三维培养中获得卵巢癌A2780、CAOV3多细胞球体。使用蛋白质印迹分析和流式细胞术（FCM）测定P-糖蛋白（P-gp）的表达。还使用激光共聚焦显微镜测定P-gp的亚细胞分布。采用逆转录聚合酶链反应（RT-PCR）检测单层细胞和多细胞球体的mdr1 mRNA。也使用流式细胞仪分析细胞周期谱和细胞凋亡。用台盼蓝排斥试验检测耐药性。
与对照细胞相比，单层细胞中未检测到P-gp表达，但聚集细胞中P-gp表达显著升高（P<0.05）。RT-PCR在聚集细胞中证实了mdr1 mRNA阳性节点。球体细胞中G(0)-G(1)期细胞百分比显著增加，S期和G(2)-M期细胞百分比显著降低。球体细胞比单层细胞显示出更高的细胞活力（P(A2780)=0.003，P(CAOV3)=0.015）。紫杉醇诱导多细胞球体中的凋亡细胞比单层细胞更多。
卵巢癌A2780、CAOV3多细胞球体培养诱导细胞对紫杉醇耐药。卵巢多细胞球体中诱导了P-gp的高表达，且细胞停滞在G(0)-G(1)期。
