缺氧对人乳腺癌细胞微球体生长及化疗耐药性的影响

Influence of hypoxia on human breast cancer mammospheres culture and resistance to chemotherapy

目的研究缺氧对人乳腺癌细胞微球体(mammospheres,MSs)培养效率及其化疗耐药性的影响。方法分别在常氧及缺氧条件下对人乳腺癌MDA-MB-231细胞进行无血清悬浮培养,观察记录MSs的生长速率及体积变化。MTT比色法检测各组人乳腺癌微球体细胞(mammospheres-derived cells,MSDCs)及MDA-MB-231细胞在不同浓度、不同药物作用下的存活率(survive rate,SR)。免疫组织荧光化学法检测HIF-2α、ABCG2在各组MSDCs中的表达。Western blot检测乳腺癌干细胞标志物CD44及ALDH1在各组MSDCs中的表达。Western blot及Real-time RT-PCR检测HIF-2α、ABCG2、P-gp及MDR1在各组MSDCs中的表达。结果缺氧组MSs生长速率及球体体积优于常氧组。缺氧组MSDCs在不同化疗药物作用下的SR高于常氧组MSDCs及MDA-MB-231细胞(P<0.05)。免疫组织荧光化学检测发现HIF-2α及ABCG2在缺氧组MSDCs中的表达强于常氧组。Western blot检测发现CD44与ALDH1在缺氧组MSDCs中表达量高于常氧组。缺氧组MSDCs中HIF-2α、ABCG2及P-gP蛋白表达量均高于在常氧组MSDCs及MDA-MB-231细胞中的表达(P<0.05)。Real-time RT-PCR检测发现缺氧组MSDCs中HIF-2α、ABCG2及MDR1 mRNA的表达量均高于常氧组MSDCs及MDA-MB-231细胞(P<0.05)。结论以HIF-2α为启动因子,ABCG2、MDR1及P-gP等为效应因子的生物化学反应可能是缺氧状态加快MSs的成球速度、提高其球体体积、增强其化疗耐药能力的可能机制。

Objective To study the effect of hypoxia on human breast cancer mammospheres （MSs） culture and resistance to chemotherapy. Methods MDA-MB-231 cells were cultured in serum-free medium supplemented with growth factors under normoxia and hypoxia conditions, respectively. The culture time and volume of the MSs were recorded. The survival rates （SR） of mammos-pheres-derived cells （MSDCs） and MDA-MB-231 cells incubated with various chemotherapy drugs with various concentrations were detected by MTT assay. The expression of hypoxia inducible factor 2α （HIF-2α） and ATP-binding cassette super family G2 （ABCG2） in the MSs were observed by immunofluorescence technique. The expression levels of CD44 and ABCG2 were detected by Western blotting. The expression levels of HIF-2α, ABCG2, glycoprotein P （P-gp） and multi-drug resistance gene 1 （MDR1） in the MSDCs were detected by Western blotting and real-time RT- PCR. Results The MSs of the hypoxia group had shorter culture time and greater volume than those of the normoxia group. The SR of the MSDCs incubated with various chemotherapy drugs with various concentrations in the hypoxia group were significantly higher than that of the MSDCs and MDA-MB-231 cells in the normoxia group （P 〈 0. 05 ）. Immunofluorescence showed that the fluorescence of HIF-2α and ABCG2 in the MSDCs of the hypoxia group was stronger than that of the normoxia group. The expression levels of CD44 and ALDH1 inthe MSDCs of the hypoxia group were higher than those of the normoxia group. The protein expression levels of HIF-2α, ABCG2 and P-gp in the MSDCs of the hypoxia group were significantly higher than those in the MSDCs and MDA-MB-231 cells of the normoxia group （P 〈 0. 05 ）. The mRNA expression levels of HIF-2α, ABCG2 and MDR1 in the MSDCs of the hypoxia group were higher than those of the other groups. Conclusion Hypoxia can shorten the culture time for MDA-MB-231 cells forming MSs and increase the size of MSs. The pathway of HIF-2α, ABCG2, P-gp and MDR1 may be involved in the mechanism of the above phenomenon.