【作者】 刘振宇；

【导师】 范志民；

【作者基本信息】 吉林大学 ， 外科学， 2017， 博士

【摘要】 目的:已有文献报道肥胖是乳腺癌的高危因素并与乳腺癌患者较差的治疗疗效和预后相关。同时还有研究表明肥胖所引起的脂肪因子分泌异常与乳腺癌的发生和发展相关。抵抗素是一种新发现的脂肪因子,其在乳腺癌患者血清中分泌增多并能促进乳腺癌细胞的生长、入侵和迁移。本实验的目的在于检测抵抗素是否会影响阿霉素(Doxorubicn,Dox)的治疗疗效,其是乳腺癌化学治疗中效果最好的蒽环类药物之一,并深入探讨抵抗素的作用机制。方法:1.应用Annexin V/PI双染色法检测乳腺癌细胞系MCF-7和MDA-MB-231经过阿霉素和/或抵抗素处理后细胞凋亡率;并收集细胞提取蛋白进行Western Blot,检测各处理组细胞细胞色素c(cytochrome c)、裂解的半胱天冬酶9(cleaved caspase-9)和裂解的聚腺苷二磷酸-核糖聚合酶(cleaced poly-ADP-ribose polymerase,cleaved PARP)蛋白表达水平,进一步验证细胞凋亡水平。2.乳腺癌细胞系MCF-7和MDA-MB-231经抵抗素处理后,应用免疫荧光染色法检测细胞LC3的表达变化,从而反映细胞自噬水平的变化。应用Western Blot法检测细胞中自噬相关蛋白(如BECN1,SQSTM1,LC3B-I/II,LAMP1)的表达水平,并定量检测LC3B-II/LC3B-I比例,进一步验证细胞自噬水平的变化。3.应用细胞自噬特异性抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)抑制细胞自噬后,乳腺癌细胞系MCF-7和MDA-MB-231经阿霉素和/或抵抗素处理后,应用流式细胞术检测各组细胞凋亡率,并用Western Blot法检测细胞Cytochrome c、cleaved Caspase-9和cleaved PARP蛋白表达水平。进一步应用sh RNA特异性敲除细胞自噬相关基因5(Atg5)后,应用Annexin V/PI双染色法检测的细胞凋亡水平的变化。4.乳腺癌细胞系MCF-7和MDA-MB-231经抵抗素处理后,应用Western Blot法检测AMP激活的蛋白激酶(AMP-activated protein kinase,AMPK)/雷帕霉素靶蛋白(m TOR)和c-Jun氨基末端激酶(JNK)信号通路相关蛋白的表达变化。细胞用AMPK抑制剂compound C和JNK抑制剂SP600125处理后,应用Western Blot法检测LC3B-I/LC3B-II表达变化,同时应用Annexin V/PI双染色法细胞凋亡水平的变化。结果:首先我们证明了阿霉素可以有效地诱导人乳腺癌细胞MCF-7和MDA-MB-231的细胞凋亡。并且抵抗素可以明显地以剂量依赖方式和时间依赖方式降低阿霉素所诱导的细胞凋亡。Western Blot结果进一步证实了抵抗素能下调阿霉素诱导的细胞凋亡相关蛋白的表达,如cytochrome c、cleaved caspase-9和cleaved PARP。其次免疫荧光染色结果表明抵抗素显著地增加了MCF-7和MDA-MB-231细胞的LC3表达量,同时Western Blot结果也进一步证实了抵抗素能增加细胞内LC3B-Ⅱ,BECN1,LAMP1的蛋白表达量和LC3B-Ⅱ/LC3B-I的表达比例,而降低SQSTM1的蛋白表达量。这些结果均证明抵抗素能激活乳腺癌细胞的细胞自噬。为了进一步证明抵抗素是通过细胞自噬抑制阿霉素诱导的细胞凋亡,我们用3-MA和基因敲除Atg5两种方法抑制细胞自噬,流式细胞术以及Western Blot的结果均表明抑制细胞自噬后,抵抗素不再对阿霉素诱导的细胞凋亡起到保护作用。最后,Western Blot的结果显示抵抗素能以剂量依赖的方式增加p-AMPK的蛋白表达,同时降低p-m TOR和p-ULK1 Ser757的蛋白表达。并且,抵抗素可以以剂量依赖的方式使p-JNK和p-Bcl-2的蛋白表达增加。这些结果证明抵抗素能激活乳腺癌细胞中AMPK/m TOR/ULK1和JNK信号通路。抑制剂Compound C和SP600125可以分别有效地抑制这两条信号通路的活化,并且两者联合应用能拮抗抵抗素诱导的细胞自噬和抑制细胞凋亡的作用。结论:(1)抵抗素可以对阿霉素诱导的人类乳腺癌细胞MCF-7和MDA-MB-231的凋亡起到保护作用;(2)抵抗素可以激活人类乳腺癌细胞MCF-7和MDA-MB-231的细胞自噬效应;(3)抵抗素活化的细胞自噬可以抑制阿霉素诱导的人类乳腺癌细胞MCF-7和MDA-MB-231的凋亡;(4)抵抗素通过AMPK/m TOR/ULK1和JNK信号通路激活抗凋亡细胞自噬效应。我们的实验首次提出了脂肪因子抵抗素可以通过诱导乳腺癌细胞自噬拮抗阿霉素所引起的细胞凋亡。这些发现提示了在乳腺癌患者中抵抗素水平的升高可以促进肿瘤细胞耐药发生。我们的研究揭示了抵抗素在乳腺癌中新的作用,同时也为克服乳腺癌治疗中的药物抵抗提供了可能的作用靶点。更多还原

【Abstract】 Aim:Clear evidence has linked obesity to a high risk of incidence as well as poor clinical outcome of breast cancer.It has been proven that changes in the levels of adipokines caused by obesity are associated with the initiation and progression of breast cancer.Resistin is a novel adipokine that is upregulated in breast cancer patients and promotes breast cancer cell growth,invasion,and migration.The aim of the study was to investigate whether resistin affected the efficacy of doxorubicin(Dox),one of the most effective anthracycline chemotherapeutic agents in the treatment of breast cancer.Methods:1.Human breast cancer cell lines MCF-7 and MDA-MB-231 were treated with Dox and/or resistin.Annexin V binding assay was used to evaluate apoptosis of the treated cells.Western blot analysis was used for determining the expression levels of Cytochrome c,cleaved-Caspase-9 and cleaved-PARP to further confirm apoptotic level.2.Human breast cancer cell lines MCF-7 and MDA-MB-231 were cultured with or without resistin.Immunofluorescence staining was used to demonstrate LC3 expression to reflect autophagy.Western blot analysis was used to test the expression levels of autophagy-related proteins(BECN1,SQSTM1,LC3B-I/II,LAMP1).And quantitative analysis of LC3B-II/LC3B-I ratio was used to further confirm the autophagy level.3.Human breast cancer cell lines MCF-7 and MDA-MB-231 were pretreated with autophagy inhibitor 3-methyladenine(3-MA)and then cultured in the media containing Dox and/or resistin.Flow cytometry and Western Blot were used to test the apoptosis.Then,the cells were infected by lentivirus containing Atg5-sh RNA toknockdown the expression of Atg5.Annexin V binding assay was used to test apoptosis.4.Human breast cancer cell lines MCF-7 and MDA-MB-231 were treated with or without resistin.Western Blot analysis was used to test the expression levels of components of AMPK/m TOR and JNK signaling pathways.Western Blot analysis for LC3B-I/LC3B-II and annexin V binding assay for apoptosis were used when cells were treated with Compound C and SP600125,which are inhibitors of AMPK/m TOR and JNK signaling pathways respectively.Results:First,we demonstrated that Dox effectively induced the apoptosis of both MCF-7and MDA-MB-231 cells.And we found that the addition of resistin significantly decreased Dox-induced apoptosis of breast cancer cells in a dose-dependent and time-dependent manner.As expected,MCF-7 and MDA-MB-231 cells treated with Dox alone had significantly higher levels of cytochrome c,cleaved caspase-9,and cleaved PARP than untreated cells,while addition of resistin significantly decreased the levels of these proteins in the presence of Dox.Next,we detected the accumulation of LC3,a hallmark of mammalian autophagy,by immunofluorescence.Addition of resistin resulted in a remarkable increase in LC3 dots in MCF-7 and MDA-MB-231 cells.It was also confirmed by Western Blot results showing that addition of resistin dramatically increased the expression of LC3B-Ⅱ,BECN1,LAMP1,and the ratio of LC3B-Ⅱ to LC3B-Ⅰ,and decreased the expression of SQSTM1 in a dose-dependent manner.These results suggest that resistin activates autophagy in breast cancer cells.To further confirm that resistin-induced resistance to Dox was mediated through activated autophagy,we used 3-MA(a specific autophagy inhibitor)and a lentiviral vector containing sh RNA of Atg5 inhibit autophagy.Annexin V binding assay and Western blot analysis both confirmed that inhibition of autophagy in breast cancer cells abrogated the protective effect of resistin in Dox-treated cells.Western blot analyses showed that addition of resistin significantly upregulated the expression of p-AMPK and downregulated the expression of p-m TOR and p-ULK1 Ser757 in a dose-dependent manner.Resistin was also found to upregulate the expression of p-JNK and p-Bcl-2 in a dose dependent manner.Meanwhile,autophagy induced by resistin was abrogated by inhibitors of the two pathways,Compound Cand SP600125.These data indicate that activation of AMPK/m TOR/ULK1 and JNK signalings are responsible for pro-survival autophagy induced by resistin.Conclusions:(1)Resistin protects human breast cancer cell lines MCF-7 and MDA-MB-231 against Dox-induced apoptosis;(2)Resistin activates autophagy in human breast cancer cell lines MCF-7 and MDA-MB-231;(3)Autophagy induced by resistin confers Dox resistance in breast cancer cell lines MCF-7 and MDA-MB-231;(4)Resistin induces autophagy via AMPK/m TOR and JNK signaling.Our data firstly propose that adipokine resistin is able to attenuate Dox-induced apoptosis through autophagy induction.This finding suggests that upregulated levels of resistin in breast cancer patients could facilitate the chemoresistance of tumor cells.Thus,our study reveals a new role of resistin in breast cancer,and provides a potential molecular target to overcome chemoresistance in the treatment of breast cancer.更多还原