Qi Yan-Yu, Liu Kai, Zhang Jie, Li Kai, Ren Jing-Jing, Lin Ping
Division of Geraeology, State Key Laboratory of Biotherapy, West China Hospital, Chengdu, Sichuan University, 610041, P.R. China.
Ai Zheng. 2009 Aug;28(8):861-7. doi: 10.5732/cjc.009.10004.
Na(+)-K(+)ATPase (Na(+)-K(+) pump) is an important cell energy conversion system which is probably associated with tumor metastasis. Expression of its B1 subunit gene-ATP1B1 is high in well differentiated and low in poorly differentiated tumor cells. This study was to investigate the synergic effect of Na(+)-K(+) ATPaseB1 and adriamycin (ADM) on inhibition of cell proliferation and reversal of drug resistance in MCF-7 and MCF-7/ADM cells.
Growth of MCF-7 and MCF-7/ADM cells transfected with PEGFP-ATP1B1 and shATP1B1 were measured by MTT. Intracellular fluorescence intensity of ADM was analyzed by inverted fluorescence microscopy and flow cytometry. ATP enzyme activity was measured by ultramicro-ATP enzyme, and mRNA expression of multi-drug resistance gene MDR1 was detected by RT-PCR and real-time PCR. The expression of P-glycoprotein (P-gp) was analyzed by western blot.
The sensitivity of MCF-7 and MCF-7/ADM cells transfected with pEGFP-ATP1B1 to ADM was higher in comparing to the negative control ADM-C3 (transfected with vector pEGFP-C3) and the control ADM-RPMI-1640 (cultured with RPMI-1640), and the differences between ADM-ATP1B1 and ADM-RPMI-1640 groups were statistically significant at different concentrations of adriamycin (P<0.05). After the B1 subunit was silenced, the sensitivity of cells to ADM in the ADM-shNC group was higher than that in the shATP and ADM-RPMI-1640 groups. The mean fluorescence intensity of ADM in the ADM-ATP1B1 group was higher than that in the ADM-C3 and ADM-RPMI-1640 groups (P<0.05). ATP enzyme activity was significantly higher in ADM-ATP1B1 group comparing to the ADM-RPMI-1640 group (P<0.05). mRNA expression of MDR1 gene and protein expression of P-gp were not significantly different among the ADM-ATP1B1 group and two control groups (P>0.05).
Na(+)-K(+) ATPase B1 can synergize with ADM and reverse drug resistance to ADM in the MCF-7/ADM cell line. This may be related to ATP enzyme activity, but not to influencing the expression of MDR1 gene.
钠钾ATP酶（钠钾泵）是一种重要的细胞能量转换系统，可能与肿瘤转移有关。其B1亚基基因ATP1B1在高分化肿瘤细胞中表达较高，在低分化肿瘤细胞中表达较低。本研究旨在探讨钠钾ATP酶B1与阿霉素（ADM）对MCF-7和MCF-7/ADM细胞增殖抑制及耐药逆转的协同作用。
采用MTT法检测转染PEGFP-ATP1B1和shATP1B1的MCF-7和MCF-7/ADM细胞的生长情况。通过倒置荧光显微镜和流式细胞术分析ADM的细胞内荧光强度。采用超微量ATP酶法测定ATP酶活性，通过RT-PCR和实时PCR检测多药耐药基因MDR1的mRNA表达。采用蛋白质印迹法分析P-糖蛋白（P-gp）的表达。
与阴性对照ADM-C3（转染载体pEGFP-C3）和对照ADM-RPMI-1640（用RPMI-1640培养）相比，转染pEGFP-ATP1B1的MCF-7和MCF-7/ADM细胞对ADM的敏感性更高，在不同浓度阿霉素作用下，ADM-ATP1B1组与ADM-RPMI-1640组之间的差异具有统计学意义（P<0.05）。B1亚基沉默后，ADM-shNC组细胞对ADM的敏感性高于shATP组和ADM-RPMI-1640组。ADM-ATP1B1组ADM的平均荧光强度高于ADM-C3组和ADM-RPMI-1640组（P<0.05）。ADM-ATP1B1组的ATP酶活性显著高于ADM-RPMI-1640组（P<0.05）。ADM-ATP1B1组与两个对照组之间MDR1基因mRNA表达和P-gp蛋白表达无显著差异（P>0.05）。
钠钾ATP酶B1可与ADM协同作用，逆转MCF-7/ADM细胞系对ADM的耐药性。这可能与ATP酶活性有关，而与影响MDR1基因的表达无关。
