抑制自噬对三阴性乳腺癌EGFR靶向治疗的影响研究

发布时间：2018-05-24 12:07

本文选题：乳腺肿瘤 + 三阴性　；参考：《济南大学》2017年硕士论文

【摘要】：目的与背景:乳腺癌处于女性恶性肿瘤的首要位置。三阴性乳腺癌(triple negative breast cancer,TNBC)属于乳腺癌luminal分型中的一种特殊的分子亚型,其特点为雌激素受体、孕激素受体及表皮生长因子受体2均阴性表达。TNBC侵袭性高,具有首发年龄小、病情进一步发展时间快、影像学表现各不相同、非常容易复发转移等特点。据相关的研究表明在中国女性中,TNBC的发病率大约占到了乳腺癌发病率的17%。由于相关受体均阴性表达,TNBC没有内分泌和HER-2靶向治疗的机会,目前化疗是TNBC主要的治疗方法。TNBC恶性程度很高、病人预后很差,这些特点与表皮生长因子受体(Epidermal growth factor receptor,EGFR)在TNBC患者中高度表达密切相关。但是最近发现EGFR靶向治疗的相关研究并没有取得理想的疗效,疗效不理想的原因主要和EGFR分子靶向治疗过程中耐药性的产生相关。如何增强TNBC靶向治疗敏感性成为目前研究的新热点。有报道表明在EGFR异常表达的肿瘤中,可以检测到自噬表达水平发生了明显改变。自噬是真核生物所特有生理病理现象,它可被外界多种刺激(如放疗、化疗、靶向治疗)激活。在肿瘤形成过程中,自噬的激活能够为瘤体提供必需的营养及能量供给,使肿瘤组织免于治疗损伤而得以继续存活,这也是肿瘤发生耐药的重要机制。阻断肿瘤细胞在靶向治疗过程中增加的自噬水平,可以显著提高肿瘤治疗的敏感性。目前相关研究表明,在异常表达EGFR的非小细胞肺癌细胞中,抑制自噬的表达水平,可以明显增强非小细胞肺癌细胞对吉非替尼治疗的敏感性。然而,在TNBC细胞中,自噬与EGFR靶向治疗的研究目前缺乏相关研究报道。方法:本研究通过吉非替尼单独用药或者联合两种作用机制不同的自噬抑制剂3甲基腺嘌呤(3-methyladenine,3MA)和巴弗洛霉素A1(Bafilomycin A1,BAF)处理TNBC细胞系MDA-MB-468和MDA-MB-231细胞,采用MTT实验方法、克隆形成实验方法检测肿瘤细胞的增殖抑制率与克隆形成率,通过应用流式细胞仪检测肿瘤细胞的周期分布、Western blot免疫印迹法检测与DNA损伤相关的标志性蛋白和线粒体凋亡相关标志性蛋白的表达,进一步验证在TNBC细胞系MDA-MB-468和MDA-MB-231中,自噬抑制剂对EGFR抑制剂吉非替尼治疗敏感性的具体机制。通过ROS染色测定实验明确自噬抑制剂与吉非替尼联合用药时导致的凋亡与线粒体凋亡的关系。建立BALB/c裸鼠移植瘤模型,通过裸鼠体内实验验证自噬抑制剂对吉非替尼治疗敏感性的影响,免疫组化方法检测移植瘤中Cleaved-CASP 3的表达。结果:吉非替尼+3MA组和吉非替尼+BAF组MDA-MB-468细胞的IC50分别为(4.1±0.2)μmol/L和(3.8±0.3)μmol/L,均明显低于吉非替尼组[(7.0±0.2)μmol/L,均P0.05]。吉非替尼+3MA组和吉非替尼+BAF组MDA-MB-231细胞的IC50分别为(9.7±0.1)μmol/L和(7.7±0.2)μmol/L,均明显低于吉非替尼组[(14.7±0.1)μmol/L,均P0.05]。克隆形成实验显示,吉非替尼与自噬抑制剂联合用药组MDA-MB-468细胞克隆形成率分别为(29.85±5.54)%和(33.58±6.31)%,显著低于单用吉非替尼组(63.46±8.32)%(Z=-2.309,P0.017)。吉非替尼与自噬抑制剂联合用药组MDA-MB-231细胞克隆形成率分别为(22.63±6.27)%和(31.54±5.81)%,显著低于单用吉非替尼组(49.35±6.56)%(Z=-2.309,P0.017)。吉非替尼联两种作用机制不同的自噬抑制剂3MA和BAF,诱导了TNBC细胞系MDA-MB-468和MDA-MB-231细胞DNA发生损伤,诱导了TNBC细胞发生G0/G1期细胞周期阻滞。吉非替尼联合3MA和BAF处理TNBC细胞,导致细胞氧自由基ROS的表达水平明显增加。Western Blot免疫印迹实验发现线粒体凋亡相关蛋白细胞色素C、BAX、cleaved CASP 3表达水平明显增加。裸鼠移植瘤模型实验发现自噬抑制剂联合吉非替尼处理后,MDA-MB-468细胞肿瘤的生长受到明显的抑制。结论:本研究表明自噬抑制剂可以增强TNBCMDA-MB-468和MDA-MB-231细胞对EGFR靶向治疗的敏感性。并且这种作用与线粒体凋亡相关途径的激活有关。
[Abstract]:Objective and background: breast cancer is the primary position of female malignant tumor. Three triple negative breast cancer (TNBC) belongs to a special molecular subtype in the luminal classification of breast cancer, which is characterized by estrogen receptor, progesterone receptor and epidermal growth factor receptor 2 negative expression of.TNBC, which has high invasiveness and is the first one. In Chinese women, the incidence of TNBC is about 17%. due to negative expression of related receptors, and TNBC does not have the opportunity for endocrine and HER-2 targeting therapy, and chemotherapy is present. It is the main treatment of TNBC.TNBC with high malignancy and poor prognosis. These characteristics are closely related to the high expression of Epidermal growth factor receptor (EGFR) in TNBC patients. However, the recent discovery of the related research on EGFR targeting therapy has not achieved an ideal effect. It is related to the production of drug resistance in the course of EGFR molecular targeted therapy. How to enhance the sensitivity of TNBC targeting therapy has become a new focus of research. It is reported that the level of autophagy can be detected in the abnormal expression of EGFR. Autophagy is a special physiological pathological phenomenon in eukaryotes. Stimulation (such as radiotherapy, chemotherapy, targeting therapy) activation. In the process of tumor formation, activation of autophagy can provide the necessary nutrition and energy supply for the tumor and keep the tumor tissue from treatment damage and continue to survive, which is an important mechanism of tumor resistance. Leveling can significantly increase the sensitivity of tumor therapy. Current studies have shown that inhibition of autophagy expression in non small cell lung cancer cells with abnormal expression of EGFR can significantly enhance the sensitivity of non small cell lung cancer cells to gefitinib treatment. However, there is a lack of research on autophagy and EGFR targeting therapy in TNBC cells. Methods: This study treated TNBC cell lines MDA-MB-468 and MDA-MB-231 cells by using gefitinib alone or combined with two different mechanisms of autophagic inhibitor 3-methyladenine (3MA) and buffalamycin A1 (Bafilomycin A1, BAF). The experimental method of MTT was used to clone and form an experimental method for detection of swelling. The proliferation inhibition rate and clone formation rate of tumor cells were detected by flow cytometry, and the expression of marker proteins associated with DNA damage and mitochondrial apoptosis related proteins were detected by Western blot immunoblotting, and the autophagy inhibitor was further verified in the TNBC cell line MDA-MB-468 and MDA-MB-231. A specific mechanism for the sensitivity of EGFR inhibitor gefitinib in the treatment of gefitinib. The relationship between apoptosis and apoptosis induced by autophagy inhibitor and gefitinib was determined by ROS staining. A BALB/c nude mouse transplanted tumor model was established, and the effects of autophagy inhibition on gefitinib sensitivity were tested in nude mice. Immunohistochemical method was used to detect the expression of Cleaved-CASP 3 in the transplanted tumor. Results: the IC50 of MDA-MB-468 cells in gefitinib group +3MA and gefitinib group +BAF were respectively (4.1 + 0.2) mu mol/L and (3.8 + 0.3) mu mol/L, all significantly lower than that of gefitinib Group [(7 + 0.2) Mu mol/L, all P0.05]. gefitinib +3MA group and gefitinib group MDA-MB-231 cells 50 (9.7 + 0.1) mu mol/L and (7.7 + 0.2) mu mol/L were significantly lower than that of gefitinib Group [(14.7 + 0.1) mol/L. All P0.05]. clone formation experiments showed that the MDA-MB-468 cell clone formation rate of gefitinib combined with autophagy inhibitor group was (29.85 + 5.54)% and (33.58 + 9.7)% respectively, significantly lower than that of gefitinib group (63.46 + 8.32)% (63.46 + 8.32)%, respectively. Z=-2.309, P0.017). The rate of MDA-MB-231 cell clone formation in gefitinib combined with autophagic inhibitor group was (22.63 + 6.27)% and (31.54 + 5.81)% respectively, significantly lower than (49.35 + 6.56)% (Z=-2.309, P0.017) in the single use gefitinib group (Z=-2.309, P0.017). The different autophagy inhibitors, 3MA and BAF, were made by gefitinib, and TNBC cell line MDA-MB-468 was induced. The damage of MDA-MB-231 cell DNA induced the cell cycle arrest of TNBC cells at G0/G1 stage. Gefitinib combined with 3MA and BAF to treat TNBC cells, which resulted in a significant increase in the expression level of oxygen free radical ROS in the cell, and the.Western Blot immunoblotting experiment found that the mitochondrial apoptosis related protein cytochrome C was found. BAX, 3 expression level was obvious. Increase. Nude mice transplantation tumor model experiment found that the growth of MDA-MB-468 cell tumor was significantly inhibited after the autophagy inhibitor combined with gefitinib. Conclusion: This study suggests that autophagy inhibitors can enhance the sensitivity of TNBCMDA-MB-468 and MDA-MB-231 cells to EGFR targeting therapy. The activation is related.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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