缺氧条件下雷帕霉素抗人乳腺癌细胞MCF-7细胞增殖能力的研究及其机制的初步探讨
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摘要
目的缺氧是实体肿瘤微环境的基本特征之一,也是造成肿瘤耐药的重要因素,其中缺氧诱导因子-1α(HIF-1α)是介导细胞低氧反应最关键的核转录因子。本研究通过比较新型抗肿瘤药雷帕霉素在常氧和缺氧条件下抗人乳腺癌MCF-7细胞的增殖能力,观察其能否抵抗缺氧造成的耐药,并对其机制进行初步探讨;检测雷帕霉素与目前乳腺癌一线化疗药阿霉素联用的效果,观察有无协同作用。
方法采用密封培养罐快速冲入混合气体(1%的O_2、5%的CO_2、94%的N_2)模拟缺氧环境;根据常用血药浓度设定药物浓度;采用MTT法检测在常氧或缺氧状态下雷帕霉素、阿霉素单独或联合使用时,对MCF-7细胞增殖能力的影响;采用流式细胞仪检测两种药物单独或联合使用对MCF-7细胞细胞周期的影响;Western blot检测两种氧浓度下不同药物处理下的MCF-7细胞HIF-1α蛋白及影响雷帕霉素敏感性的指标(pAkt蛋白)的表达。
结果与阿霉素相反,在缺氧条件下,5ng/ml-40ng/ml的雷帕霉素对MCF-7细胞的增殖抑制能力明显高于常氧条件下。在两种氧浓度下,临床常用浓度的雷帕霉素(10ng/ml)和各个浓度的阿霉素(0.25μg/ml-2.0μg/ml)联合使用均无协同作用。雷帕霉素单独作用于McF-7细胞主要表现为G1期阻滞,与阿霉素联用则表现为G2期细胞增多。缺氧使HIF-1α、pAkt蛋白表达均增加。但10ng/ml的雷帕霉素对HIF-1α、pAkt表达无明显影响。
结论临床常用浓度范围内的雷帕霉素在缺氧状态下抗MCF-7细胞增殖能力增强,其原因除雷帕霉素对细胞的增值抑制作用不受HIF-1α表达的影响外,另一原因可能是缺氧使肿瘤细胞pAkt表达增加而增加了细胞对雷帕霉素的敏感性。
Objective Hypoxia is a basic characteristic of micro-enviroment of solid tumors and also leads to drug resistance.The key factor regulating cellular O_2 homeostastis is hypoxia-inducible factor-1α(HIF-1α).This study was to determine the anti-proliferation effects of using rapamycin, a novel anti-tumor drug,on human breast cancer MCF-7 cells exposed to normal oxygen concentration or hypoxic enviroment and its related mechanism in vitro and to investigate the possibility of anti-hypoxia of this drug and the synergistic possibility of using rapamycin combined with adriamycin.
Methods Hypoxic enviroment is acquired by a modular incubator chamber flushed with an atmosphere saturated with 1%O_2,5%CO_2 and 94%N_2.MTT assay and flow cytometry were used to examine the influence of the drugs on cell proliferation and cell cycle of MCF-7 cells. MCF-7 cells were exposed to normal or hypoxic enviroment and treated with rapamycin or adriamycin alone or co-treated with rapamycin and adriamycin.Western blot analysis was used to assess the expression level of HIF-1αand pAkt,which expression can enhance the sensitivity of rapamycin.
Results Opposite to adriamycin,the effect of rapamycin at 5ng/ml to 40ng/ml on inhibiting cell proliferation of MCF-7 cells was enhanced when exposed to hypoxia compared with the effect under normal oxygen environment.Regardless of oxygen concentrations,no synergistic interaction was observed when rapamycin at clinical normal drug level (10ng/ml)combined with adriamycin at 0.25μg/ml to 2.0μg/ml on MCF-7 cells.The MCF-7 cells treated with rapamycin alone were arrested at G1,while co-treated with rapamycin and adriamycin were arrested at G2.Hypoxia enhanced the expression levels of HIF-1αand pAkt,while rapamycin at 10ng/ml had no influence on the expression levels of HIF- 1αand pAkt.
Conclusions Hypoxia increase the sensitivity of rapamycin at clinical normal drug level on MCF-7 cells and may be caused by increasing pAkt expression subsequent to hypoxia.
方法采用密封培养罐快速冲入混合气体(1%的O_2、5%的CO_2、94%的N_2)模拟缺氧环境;根据常用血药浓度设定药物浓度;采用MTT法检测在常氧或缺氧状态下雷帕霉素、阿霉素单独或联合使用时,对MCF-7细胞增殖能力的影响;采用流式细胞仪检测两种药物单独或联合使用对MCF-7细胞细胞周期的影响;Western blot检测两种氧浓度下不同药物处理下的MCF-7细胞HIF-1α蛋白及影响雷帕霉素敏感性的指标(pAkt蛋白)的表达。
结果与阿霉素相反,在缺氧条件下,5ng/ml-40ng/ml的雷帕霉素对MCF-7细胞的增殖抑制能力明显高于常氧条件下。在两种氧浓度下,临床常用浓度的雷帕霉素(10ng/ml)和各个浓度的阿霉素(0.25μg/ml-2.0μg/ml)联合使用均无协同作用。雷帕霉素单独作用于McF-7细胞主要表现为G1期阻滞,与阿霉素联用则表现为G2期细胞增多。缺氧使HIF-1α、pAkt蛋白表达均增加。但10ng/ml的雷帕霉素对HIF-1α、pAkt表达无明显影响。
结论临床常用浓度范围内的雷帕霉素在缺氧状态下抗MCF-7细胞增殖能力增强,其原因除雷帕霉素对细胞的增值抑制作用不受HIF-1α表达的影响外,另一原因可能是缺氧使肿瘤细胞pAkt表达增加而增加了细胞对雷帕霉素的敏感性。
Objective Hypoxia is a basic characteristic of micro-enviroment of solid tumors and also leads to drug resistance.The key factor regulating cellular O_2 homeostastis is hypoxia-inducible factor-1α(HIF-1α).This study was to determine the anti-proliferation effects of using rapamycin, a novel anti-tumor drug,on human breast cancer MCF-7 cells exposed to normal oxygen concentration or hypoxic enviroment and its related mechanism in vitro and to investigate the possibility of anti-hypoxia of this drug and the synergistic possibility of using rapamycin combined with adriamycin.
Methods Hypoxic enviroment is acquired by a modular incubator chamber flushed with an atmosphere saturated with 1%O_2,5%CO_2 and 94%N_2.MTT assay and flow cytometry were used to examine the influence of the drugs on cell proliferation and cell cycle of MCF-7 cells. MCF-7 cells were exposed to normal or hypoxic enviroment and treated with rapamycin or adriamycin alone or co-treated with rapamycin and adriamycin.Western blot analysis was used to assess the expression level of HIF-1αand pAkt,which expression can enhance the sensitivity of rapamycin.
Results Opposite to adriamycin,the effect of rapamycin at 5ng/ml to 40ng/ml on inhibiting cell proliferation of MCF-7 cells was enhanced when exposed to hypoxia compared with the effect under normal oxygen environment.Regardless of oxygen concentrations,no synergistic interaction was observed when rapamycin at clinical normal drug level (10ng/ml)combined with adriamycin at 0.25μg/ml to 2.0μg/ml on MCF-7 cells.The MCF-7 cells treated with rapamycin alone were arrested at G1,while co-treated with rapamycin and adriamycin were arrested at G2.Hypoxia enhanced the expression levels of HIF-1αand pAkt,while rapamycin at 10ng/ml had no influence on the expression levels of HIF- 1αand pAkt.
Conclusions Hypoxia increase the sensitivity of rapamycin at clinical normal drug level on MCF-7 cells and may be caused by increasing pAkt expression subsequent to hypoxia.
引文
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