乳腺癌耐药细胞中c-fos抗凋亡作用的研究  

The anti-apoptotic effect of c-fos in drug-resistant breast cancer cells

作　　者：彭洪薇[1] 师锐赞[2] 袁向飞[3] 熊冬生[3] 魏筱华[1] 

机构地区：[1]南昌大学第一附属医院药学部,江西南昌330006 [2]山西医科大学药理学系,山西太原030001 [3]中国医学科学院,北京协和医学院实验血液学国家重点实验室,天津300020

出　　处：《中国癌症杂志》2014年第8期581-588,共8页China Oncology

摘　　要：背景与目的:乳腺癌是女性最常见的恶性肿瘤之一,且患者死亡率居高不下,其中乳腺癌耐药是导致临床化疗失败的主要原因。该课题组采用已建立的乳腺癌耐药细胞模型MCF-7/ADR及其敏感株MCF-7,旨在探讨c-fos在乳腺癌耐药细胞中的详细作用机制。方法:MTT检测上述细胞对多柔比星的敏感性,并通过实时定量PCR(real-time PCR,RT-PCR)检测上述细胞中mdr-1及c-fos的mRNA表达情况;3μmol多柔比星分别处理MCF-7细胞12、24、36 h后采用RT-PCR检测细胞中c-fos mRNA的表达,以监测化疗药物处理过程中乳腺癌细胞c-fos的表达变化过程;构建相应载体得到了c-fos稳定干扰细胞株及其对照细胞株:MCF-7/ADR/si-fos-8B、MCF-7/ADR/si-fos-3D和MCF-7/ADR/siNC,采用MTT检测干扰c-fos后对5-FU及顺铂的敏感性变化;流式细胞术检测上述药物及γ射线照射处理后细胞凋亡的情况;罗丹明123检测乳腺癌耐药细胞的外排能力;RT-PCR及蛋白质印迹法(Western blot)分别检测c-fos干扰后,凋亡相关基因bax、bcl-2、puma、p53及mdr-1等的表达。结果:与敏感细胞MCF-7相比,MCF-7/ADR细胞中c-fos及mdr-1的表达显著升高,且对多柔比星的耐药倍数为敏感株的近40倍;在3μmol多柔比星处理MCF-7之后c-fos的表达逐渐升高,结果显示,c-fos在乳腺癌的耐药表型形成早期即开始发挥作用;干扰c-fos后,细胞对药物(5-FU、顺铂)的敏感性增高,且经药物处理及γ射线照射后稳定干扰细胞株的凋亡率显著升高,说明干扰c-fos后乳腺癌耐药细胞在受到外界刺激后,更易于发生凋亡;RT-PCR及Western blot检测结果显示,干扰c-fos后,bax、puma、p53的表达明显升高,而bcl-2及mdr-1的表达显著降低。结论:在乳腺癌耐药细胞MCF-7/ADR中发现,c-fos呈异常高表达,其可能通过调节乳腺癌细胞凋亡信号通路蛋白的变化抑制乳腺癌细胞的凋亡,促进了乳腺癌耐药表型的形成。由此,c-fos可作为克服乳腺癌耐药治疗的�Background and purpose：Breast cancer is one of the most common carcinoma among female patients with high mortalities. Drug-resistance is the major reason that leads to chemotherapy failure in clinical practice. MCF-7/ADR is a multi-drug resistant cell line that was established on the basis of breast cancer cell line MCF-7. This research aimed to investigate the anti-apoptotic effect of c-fos in resistant breast cancer cell MCF-7/ADR, and to compare with its sensitive counterpart MCF-7.Methods：Doxorubicin with various concentrations was used to treat MCF-7 as well as its MDR- counterpart MCF-7/ADR. The growth inhibitory rate of MCF-7 and MCF-7/ADR wasdetermined by MTT assay. Additionally, RT-PCR was used to test the expression of P-gp and c-fos mRNA in MCF-7 and MCF-7/ADR; The expression of c-fos mRNA was detected by RT-PCR after 3 μmol doxorubicin treatment;We further established cell lines that stably interfered with c-fos, named MCF-7/ADR/si-fos-8B, MCF-7/ADR/si-fos-3D. Flow cytometry and MTT assay were used to investigate the apoptosis rate and inhibitory rate in these above cells under the treatment of 5-FU, CDDP or γ-radiation. At last, RT-PCR and Western blot analysis were used to detect the expression of bax, bcl-2, puma, p53.Results：The expression of c-fos and P-gp （MDR-1） was up-regulated in MCF-7/ADR, compared with its sensitive counterpart MCF-7. Additionally, the resistant fold of MCF-7/ADR to doxorubicin was nearly 40; The expression of c-fos was gradually up-regulated after 3 μmol doxorubicin treatment; The sensitivity to drugs （5-FU and CDDP） was increased after c-fos interference while the apoptosis rate was also increased after 5-FU, CDDP and γ-radiation treatment. RT-PCR and Western blot analysis indicated that up-regulation ofbax,puma,p53 after c-fos interference while the expression of bcl-2 was down-regulated.Conclusion：c-fos may act as an anti-apoptotic protein in resistant breast cancer cell line MCF-7/ADR by regulating the expression of apoptosis related proteins, and m

关 键 词：乳腺癌耐药 C-FOS 细胞凋亡 

分 类 号：R737.9[医药卫生—肿瘤]