内质网应激对蛋白酶体抑制剂诱导乳腺癌MCF-7细胞自噬的作用

发布时间：2018-09-19 15:03

【摘要】：目的:乳腺癌是常见的恶性肿瘤,不仅具有复发率高,转移率高及死亡率高的特点,其发病率也在逐年增加。随着社会的发展科学的进步,近年来乳腺癌的治疗已经取得了一定的进展。虽然乳腺癌外科手术治疗取得了良好的治疗效果,但术后的放疗、化疗及靶向治疗,依然是乳腺癌综合治疗的不可缺少的部分,因此探讨乳腺癌细胞耐受化疗的机制以及开发新的化疗药物一直是乳腺癌研究的重要内容。临床试验表明以蛋白酶体为靶点的药物具有良好的抗肿瘤作用,但是研究显示肿瘤细胞对蛋白酶体抑制剂类化疗药物也会产生耐受治疗的现象,而且这与自噬有着密切的关系。自噬是细胞内由溶酶体介导的蛋白质和细胞器降解过程,在正常情况下,自噬被限制在基本水平范围内,但是在饥饿以及细胞内外环境变化的刺激下,自噬便被激活。虽然研究提示自噬与内质网应激有密切的关系,但以往的研究还很局限,尚未对其中的机制进行研究,因此我们在课题组结合文献提出假设:蛋白酶体抑制剂可能部分通过内质网应激通路激活了自噬;抑制与自噬相关的上游内质网应激信号通路能够增强蛋白酶体抑制对细胞的杀伤效果。我们将通过实验探究内质网应激在蛋白酶体抑制剂诱导的乳腺癌MCF-7细胞自噬的作用。方法:本研究以乳腺癌MCF-7细胞为研究对象,选择蛋白酶体抑制剂MG-132,自噬抑制剂3-MA和内质网应激抑制剂Salubrinal为作用药物进行了实验。本实验分为空白对照组,MG-132组,MG-132+3-MA组和MG-132+Salubrinal组,通过MTT法检测各组对MCF-7细胞活性的影响,采用免疫蛋白印迹法检测各组自噬相关蛋白LC3表达水平,凋亡相关蛋白Bcl-2、Bax和Caspase-3的表达水平及内质网应激相关蛋白Grp-78、GADD153和Caspase-12的表达水平的检测,选择流式细胞检测仪检测各组对MCF-7细胞的凋亡作用的差异,使用RT-PCR法比较各组内质网应激相关基因Grp-78、GADD153和Caspase-12的m RNA水平,从而研究内质网应激在MG-132诱导的MCF-7细胞自噬的作用,探讨自噬与内质网应激在MG-132杀伤MCF-7细胞中的重要调控作用。结果:1MG-132对MCF-7细胞具有活性抑制作用,随着浓度的升高和作用时间的延长作用增强,呈浓度依赖性与时间依赖性,各组浓度抑制作用3小时开始,6小时开始明显,48小时最明显,MG-132浓度为2.5μmol/L作用48小,抑制率为17.4%,当浓度为10μmol/L与40μmol/L作用48小时,抑制率分别达到48.6%与96.3%,通过SPSS17.0软件计算得出MG-132在48小时的IC20值约为2.5μmol/L,。低浓度3-MA对乳腺癌MCF-7细胞没有明显增殖抑制作用,浓度为1mmol/L作用48小时,抑制率为6.1%,3-MA浓度为10mmol/L与20mmol/L作用48小时,抑制率分别达到46.2%与92.4%,呈浓度依赖性,计算得出3-MA在48小时的IC5值约为1mmol/L。低浓度Salubrinal对MCF-7细胞同样没有明显增殖抑制作用,当浓度为5μmol/L与10μmol/L作用48小时,抑制率分别为4.3%与11.5%,当Salubrinal浓度为40μmol/L与80μmol/L作用48小时抑制率分别达到37.9%与84.8%,呈明显浓度依赖性,计算得出Salubrinal在48小时的IC5值约为5μmol/L。2蛋白酶体的糜蛋白酶样活性检测结果显示MG-132对MCF-7细胞蛋白酶体活性有抑制作用,3h开始细胞内蛋白酶体活性受抑制,抑制率为65%,48h时抑制作用最强,呈明显时间依赖性。与上述MTT实验结果对比,当MG-132作用于MCF-7细胞3h时开始出现抑制,6h抑制明显增加,而蛋白酶体活性于3h时抑制率约为65%,呈现明显抑制作用,提示先出现蛋白酶体活性的抑制,再出现细胞增值的抑制。3自噬抑制剂3-MA(1mmol/L)增强MG-132(2.5μmol/L)对细胞的增殖抑制作用,且随着时间的延长作用增强,48小时的抑制作用最明显,呈时间依赖性。Salubrinal(5μmol/L)增强MG-132诱导的MCF-7细胞抑制作用,呈时间依赖性,作用48小时后细胞抑制最明显,与3-MA相比,其细胞增殖抑制作用相对较弱,二者差异48h时最明显。4MG-132(2.5μmol/L)能够诱导MCF-7细胞的凋亡与G2期细胞阻滞,凋亡率为10.6%,G2期细胞百分比为22.40%。3-MA(1mmol/L)和Salubrinal(5μmol/L)增强MG-132诱导的细胞凋亡与G2期细胞阻滞,凋亡率增加至23.3%和24.7%,G2期细胞百分比提高至24.80%和25.76%。5MG-132(2.5μmol/L)诱导MCF-7细胞自噬,自噬抑制剂3-MA(1mmol/L)明显抑制细胞自噬,阻滞LC3-I至LC3-II的转化。内质网应激抑制剂Salubrinal(5μmol/L)降低LC3-I的表达,抑制LC3-I至LC3-II的转化过程,显示自噬抑制作用,但作用弱于3-MA。6与MG-132(2.5μmol/L)组相比,3-MA(1mmol/L)和Salubrinal(5μmol/L)下调了抗凋亡蛋白Bcl-2表达水平,促凋亡蛋白Bax和Caspase-3显示上调,显示良好的增强MG-132诱导的MCF-7细胞凋亡作用。7MG-132(2.5μmol/L)能够诱导MCF-7细胞内质网应激的产生,内质网应激抑制剂Salubrinal(5μmol/L)能够有效抑制该过程,在蛋白与m RNA水平体现抑制作用。与之相似,3-MA(1mmol/L)也表现良好的内质网应激的抑制作用,在蛋白与m RNA表达上具有明显抑制作用,但作用效果弱于Salubrinal。结论:蛋白酶体抑制剂MG-132可能部分通过内质网应激通路激活了自噬;抑制与自噬相关的上游内质网应激信号通路能够增强蛋白酶体抑制剂MG-132对乳腺癌MCF-7细胞的杀伤作用。
[Abstract]:Objective: Breast cancer is a common malignant tumor, which not only has the characteristics of high recurrence rate, high metastasis rate and high mortality, but also has an increasing incidence year by year. Later radiotherapy, chemotherapy and targeted therapy are still indispensable parts of the comprehensive treatment of breast cancer, so exploring the mechanism of breast cancer cell tolerance to chemotherapy and developing new chemotherapeutic drugs have always been an important part of breast cancer research. Autophagy is a lysosome-mediated process of protein and organelle degradation in cells. Under normal conditions, autophagy is limited to the basic level, but in starvation and in and out of cells. Autophagy is activated under the stimulation of environmental changes. Although studies suggest that autophagy is closely related to endoplasmic reticulum stress, previous studies have been limited and have not yet studied the mechanism of autophagy. Inhibition of autophagy-related upstream endoplasmic reticulum stress signaling pathway enhances the cytotoxicity of proteasome inhibition on breast cancer MCF-7 cells. The preparation MG-132, autophagy inhibitor 3-MA and endoplasmic reticulum stress inhibitor Salubrinal were used as the experimental drugs. The experiment was divided into blank control group, MG-132 group, MG-132+3-MA group and MG-132+Salubrinal group. The effect of each group on the activity of MCF-7 cells was detected by MTT method, and the expression of autophagy-related protein LC3 was detected by Western blot. The levels of Bcl-2, Bax and Caspase-3, and the levels of endoplasmic reticulum stress-related proteins Grp-78, GADD153 and Caspase-12 were detected. The apoptosis of MCF-7 cells was detected by flow cytometry. The endoplasmic reticulum stress-related genes Grp-78, GAD153 and Caspase-12 were compared by RT-PCR. Results: 1MG-132 inhibited the activity of MCF-7 cells in a concentration-dependent manner with the increase of concentration and prolongation of action time. Sexual and time-dependent, the inhibitory effect of MG-132 concentration was less than that of 2.5 micromol/L, the inhibitory rate was 17.4%. When the concentration of MG-132 was 10 micromol/L and 40 micromol/L for 48 hours, the inhibitory rate reached 48.6% and 96.3%, respectively. The IC20 value of MG-132 at 48 hours was calculated by SPSS17.0 software. The inhibitory rate was 6.1% at 1 mmol/L for 48 hours, 46.2% at 10 mmol/L and 92.4% at 20 mmol/L for 48 hours, respectively. The IC5 value of 3-MA at 48 hours was about 1 mmol/L. Salubrina at low concentration was found to be a concentration-dependent inhibitory effect. When the concentration of Salubrinal was 40 micromol/L and 80 micromol/L for 48 hours, the inhibitory rates were 4.3% and 11.5%, respectively. When the concentration of Salubrinal was 40 micromol/L and 80 micromol/L for 48 hours, the inhibitory rates were 37.9% and 84.8%, respectively. It was found that the IC5 value of Salubrinal at 48 hours was about 5 micron. The results of chymotrypsin-like activity of ol/L.2 proteasome showed that MG-132 could inhibit the activity of proteasome in MCF-7 cells. The activity of proteasome in MCF-7 cells was inhibited by MG-132 at 3 h, the inhibition rate was 65%. The inhibition rate was the strongest at 48 h, which was time-dependent. The inhibition rate of proteasome activity was about 65% at 3 h, suggesting that the inhibition of proteasome activity first appeared, and then the inhibition of cell proliferation. 3-MA (1 mmol/L) enhanced the inhibition of MG-132 (2.5 micromol/L) on cell proliferation, and the inhibition increased with the prolongation of time. Salubrinal (5 micromol/L) enhanced the inhibitory effect of MG-132 on MCF-7 cells in a time-dependent manner. After 48 hours, the inhibitory effect was the most obvious. Compared with 3-MA, the inhibitory effect of Salubrinal (5 micromol/L) on the proliferation of MCF-7 cells was relatively weak, and the difference was the most obvious at 48 hours. 4MG-132 (2.5 micromol/L) could induce MCF-7 cells. 3-MA (1 mmol/L) and Sabrina (5 umol/L) enhanced MG-132-induced apoptosis and G2-phase cell arrest, the apoptosis rate increased to 23.3% and 24.7%, the percentage of G2-phase cells increased to 24.80% and 25.76%. 5 MG-132 (2.5 umol/L) induced autophagy and autophagy inhibition of MCF-7 cells. Preparation 3-MA (1mmol/L) significantly inhibited autophagy and inhibited the transformation of LC3-I to LC3-II. Salubrinal (5umol/L), an endoplasmic reticulum stress inhibitor, decreased the expression of LC3-I and inhibited the transformation of LC3-I to LC3-II, showing autophagy inhibition, but the effect was weaker than that of 3-MA.6, compared with MG-132 (2.5umol/L), and decreased the expression of 3-MA (1mmol/L) and Salubrinal (5umol/L). Anti-apoptotic protein Bcl-2, pro-apoptotic protein Bax and Caspase-3 were up-regulated, indicating a good enhancement of MG-132-induced apoptosis in MCF-7 cells. 7MG-132 (2.5 micromol/L) could induce endoplasmic reticulum stress in MCF-7 cells. Salubrinal (5 micromol/L), an endoplasmic reticulum stress inhibitor, could effectively inhibit this process at protein and m RNA levels. Similarly, 3-MA (1mmol/L) also showed a good inhibitory effect on endoplasmic reticulum stress, and had a significant inhibitory effect on the expression of protein and m RNA, but the effect was weaker than that of Salubrinal. Plasma reticulum stress signaling pathway can enhance the killing effect of proteasome inhibitor MG-132 on breast cancer MCF-7 cells.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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