辛伐他汀联合紫杉醇靶向作用Hippo信号通路对乳腺癌干细胞的影响
发布时间：2018-07-15 14:52
【摘要】：研究背景:乳腺癌是威胁全球女性健康的杀手,其术后复发和化疗效果不佳,是乳腺癌死亡率高的主要原因之一。乳腺癌干细胞(Breast cancer stem cells,BCSCs)被认为是其复发的根源,能够抵抗放射疗法且影响化疗的效果。乳腺癌干细胞因其具有自我更新能力、多向分化性、高致瘤性和对放化疗耐药等特性。成为近年来乳腺癌研究的热点。乳腺癌干细胞成为潜在治疗靶点。Hippo信号通路最初被发现是抑制增殖和促进凋亡来限制器官大小的信号通路。然而,许多证据表明Hippo信号通路可以调节干细胞和祖细胞自我更新和扩增。在乳腺癌、肝癌、非小细胞肺癌和子宫内膜癌发生进展中起作用。Hippo信号通路经过激酶级联反应,最终作用其核心转录因子TAZ/YAP,磷酸化的TAZ/YAP与14-3-3蛋白相互作用被阻断在细胞质中。并被泛素依赖的蛋白酶体降解。未被磷酸化的TAZ/YAP转移到细胞核中,与TEAD1-4或其他转录因子Smad、sp73等相互作用,诱导基因CTGF、AX1、BMP4等的表达,其中,以Hippo信号通路中核心因子TAZ/YAP表现最为明显。而在乳腺癌中,TAZ在影响乳腺癌干细胞成球、自我更新、分化则更加明显。辛伐他汀是HMG-Co A抑制剂,近几年研究表明辛伐他汀可以作为抗癌药对肿瘤起到抑制作用。研究表明辛伐他汀可以抑制Hippo-TAZ从胞质向胞核的转运,从而抑制TAZ在细胞核操控下游转录因子发挥生物学作用。紫杉醇是一种抗微管药物,促进微管蛋白聚合,抑制解聚,保持微管蛋白均一稳定,抑制癌细胞有丝分裂过程,作为乳腺癌、卵巢癌临床化疗的一线药物已经有很多年历史了,近年来,其药物效能减弱及耐药现象增加,辛伐他汀是否可以协同紫杉醇,增加其对肿瘤的药效还未曾报道。本研究选用辛伐他汀和紫杉醇对乳腺癌细胞MDA-MB-231细胞侵袭、转移、EMT过程及对乳腺癌干细胞的影响,并探讨其有关作用机制。方法:实验分为四组:空白处理组、辛伐他汀处理组、紫杉醇处理组、辛伐他汀+紫杉醇处理组。(1)cck8检测肿瘤抑制率:各处理组的胰酶消化后将MDA-MB-231细胞按5×103/孔接种,24h后换用不同浓度药物梯度培养基(梯度),培养48h,CCK8避光加入,避光37℃孵育2h,用酶标仪测出OD值,应用Gradpad计算药物IC50。加入两种药物与之前单独测IC50时浓度梯度相同(两种药物添加比例1:1)48h后,测OD值。(2)划痕实验检测MDA-MB-231细胞迁移能力的变化:各处理组的MDA-MB-231细胞无血清培养48h,分别于0h以及48h时取样拍照,观察细胞迁移距离。采用Transwell实验检测各组MDA-MB-231细胞侵袭和转移能力的变化:各处理组细胞分别培养24h后取样拍照,并计数小室膜细胞数量。采用明胶酶谱检测各处理组MDA-MB-231细胞内MMP-2表达水平的变化。(3)Western blot技术检测MDA-MB-231细胞内蛋白水平的变化。检测指标包括:Hippo信号通路关键因子TAZ,干细胞标志物-OCT4、ALDH1。金属基质蛋白酶-2—MMP-2,E-钙粘素—E-cadherin,波形蛋白—Vimentin,凋亡通路相关蛋白—Bax、P53、Caspsase3、Bcl-2。免疫细胞化学技术观察细胞内TAZ、OCT4及E-cadherin、Vimentin蛋白表达情况。(4)流式细胞仪检测CD44+/CD24-染色检测各处理组干细胞比例。(5)干细胞成球实验检测各处理组成球能力及数量。(6)各组细胞药物处理48h后,每孔种植相同数量细胞培养,两周后,结晶紫色,观察克隆细胞群大小和数量。结果:(1)划痕及侵袭实验结果显示:辛伐他汀联合紫杉醇可以效地抑制其迁移距离及侵袭及转移数量。Western blot技术及明胶酶谱技术检测MMP-2的表达情况,结果显示:与空白对照组和单独加药组相比,辛伐他汀联合紫杉醇处理组MMP-2表达更显著下降。(2)通过对EMT标记物的检测发现:与空白对照组和单独加药组相比,辛伐他汀联合紫杉醇组升高E-cadherin的蛋白含量,降低细胞内Vimentin的蛋白含量。(3)凋亡相关信号通路的标记物检测结果显示:与空白对照组和单独加药组相比,辛伐他汀联合紫杉醇组,P53、Bcl-2、Caspsase3表达明显下降,Bax表达明显升高,克隆形成实验:(4)Western blot技术及免疫荧光化学技术检测结果显示:与空白对照组和单独加药组相比,辛伐他汀联合紫杉醇组,TAZ、OCT4、ALDH1蛋白表达明显下降,(5)流式细胞仪CD44+/CD24-染色检测干细胞比例及干细胞成球实验结果显示:空白对照组和单独加药组相比,辛伐他汀联合紫杉醇组更明显抑制乳腺癌干细胞比例及其成球能力。结论:辛伐他汀与紫杉醇联用可以抑制MDA-MB-231转移、侵袭,促进凋亡、抑制增殖。辛伐他汀与紫杉醇联用对乳腺癌干细胞通过下调Hippo-TAZ及其下游靶点Oct4对增殖与自我更新能力均有影响,或许可以对临床化疗提供新思路。
[Abstract]:Background: breast cancer is a killer that threatens the health of women in the world. The recurrence and chemotherapy of breast cancer are one of the main causes of high mortality in breast cancer. Breast cancer stem cells (BCSCs) is considered to be the root cause of its recurrence. It can resist radiation therapy and affect the effect of chemotherapy. The stem cells of breast cancer are caused by the cancer stem cells. It has the characteristics of self renewal, multidifferentiation, high tumorigenicity, and chemoradiotherapy. It has become a hot spot in the research of breast cancer in recent years. The.Hippo signal pathway of breast cancer stem cells has been found to be a signal pathway that inhibits proliferation and promotes apoptosis to limit the size of the organ. However, a lot of evidence suggests that Hippo signals are used. The pathway regulates the self renewal and amplification of stem cells and progenitor cells. In the progression of breast cancer, liver cancer, non-small cell lung cancer and endometrial cancer, the.Hippo signaling pathway is cascaded through kinase cascade, which ultimately acts as the core transcription factor TAZ/YAP, and the interaction of phosphorylated TAZ/YAP and 14-3-3 protein is blocked in the cytoplasm. The ubiquitin dependent proteasome is degraded. The non phosphorylated TAZ/YAP is transferred to the nucleus, interacting with TEAD1-4 or other transcription factors Smad, sp73 and so on, inducing gene CTGF, AX1, BMP4 and so on. Among them, the core factor TAZ/YAP in Hippo signaling pathway is most obvious. In breast cancer, TAZ is affecting the stem cells of breast cancer. The ball, self-renewal, and differentiation is more obvious. Simvastatin is a HMG-Co A inhibitor. Recent studies have shown that simvastatin can inhibit the tumor as an anticancer drug. The study shows that simvastatin can inhibit the transport of Hippo-TAZ from the cytoplasm to the nucleus, thus inhibiting the biological role of TAZ in the nuclear control of the downstream transcription factors. Paclitaxel is an anti microtubule drug that promotes microtubule polymerization, inhibits depolymerization, keeps microtubule protein stable and inhibits the process of mitosis in cancer cells. As breast cancer, the clinical chemotherapy of ovarian cancer has a history of many years. In recent years, its drug efficacy and drug resistance have increased. The effects of simvastatin and paclitaxel on the invasion, metastasis, EMT process and the effect on breast cancer stem cells of breast cancer cell MDA-MB-231 were selected in this study. Methods: the experiment was divided into four groups: the blank treatment group, the simvastatin treatment group, the paclitaxel treatment group. Simvastatin + paclitaxel treatment group. (1) CCK8 detection of tumor inhibition rate: after trypsin digestion, MDA-MB-231 cells were inoculated after trypsin digestion in each treatment group, and MDA-MB-231 cells were inoculated at 5 x 103/ holes. After 24h, different concentrations of drug gradient medium (gradient) were changed, 48h, CCK8 were incubated to avoid light, and 2H was incubated at 37 degrees, and two species were added with Gradpad to calculate IC50.. After the same concentration gradient (two medications added to 1:1) 48h before IC50, the drug was measured. (2) the change of migration ability of MDA-MB-231 cells was detected by scratch test: the MDA-MB-231 cells in each treatment group were serum-free to culture 48h, and were taken and photographed at 0h and 48h, respectively, to observe the migration distance of the cells. Transwell test was used to detect the MD of each group. Changes in the invasion and metastasis ability of A-MB-231 cells: the cells in each treatment group were cultured for 24h to take pictures and count the number of small cell membrane cells. The changes in the level of MMP-2 expression in the MDA-MB-231 cells of each treatment group were detected by gelatinase spectrum. (3) the Western blot technique was used to detect the changes in the level of the intracellular protein in the MDA-MB-231 cells. The detection index included: Hippo Key signal pathway factor TAZ, stem cell marker -OCT4, ALDH1. metal matrix proteinase -2 MMP-2, E- cadherin E-cadherin, vimentin Vimentin, apoptotic pathway related proteins - Bax, P53, Caspsase3, Bcl-2. immunocytochemical technique observation cell expression. (4) flow cytometry The ratio of stem cells in each treatment group was measured by CD44+/CD24- staining. (5) the ability and quantity of each treatment was detected by the stem cell formation test. (6) after the treatment of 48h, the cells were cultivated for the same number of cells in each hole. After two weeks, the size and quantity of the cloned cell group were observed. Results: (1) the results of scratch and invasion test showed octyl. Statins combined paclitaxel could effectively inhibit the migration distance, invasion and metastasis number.Western blot technique and gelatinase spectrum technique to detect the expression of MMP-2. The results showed that compared with the blank control group and the single addition group, the expression of MMP-2 in the combined paclitaxel treatment group was significantly decreased. (2) the detection of EMT markers was detected. It was found that: compared with the blank control group and the single addition group, simvastatin combined paclitaxel group increased the protein content of E-cadherin and decreased the protein content of Vimentin in the cell. (3) the detection results of apoptosis related signal pathway markers showed that compared with the blank control group and the single addition group, the simvastatin combined paclitaxel group, P53, Bcl-2, Caspsa The expression of Se3 was obviously decreased, the expression of Bax was obviously increased and the cloning experiment was found. (4) the results of Western blot and immunofluorescence chemical technique showed that compared with the blank control group and the single addition group, the expression of TAZ, OCT4, ALDH1 protein in the group of simvastatin combined paclitaxel, TAZ, OCT4 and ALDH1 was markedly decreased, and (5) flow cytometry CD44+/CD24- staining was used to detect the proportion of stem cells The results of the stem cell formation test showed that the ratio of simvastatin combined with paclitaxel group was more obviously inhibited by the combination of simvastatin and paclitaxel than in the single addition group. Conclusion: the combined use of simvastatin and paclitaxel can inhibit MDA-MB-231 metastasis, invasion, apoptosis and inhibition of proliferation. Breast cancer stem cells may influence the proliferation and self-renewal ability by downregulating Hippo-TAZ and its downstream target Oct4, which may provide new ideas for clinical chemotherapy.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
本文编号：2124440
[Abstract]:Background: breast cancer is a killer that threatens the health of women in the world. The recurrence and chemotherapy of breast cancer are one of the main causes of high mortality in breast cancer. Breast cancer stem cells (BCSCs) is considered to be the root cause of its recurrence. It can resist radiation therapy and affect the effect of chemotherapy. The stem cells of breast cancer are caused by the cancer stem cells. It has the characteristics of self renewal, multidifferentiation, high tumorigenicity, and chemoradiotherapy. It has become a hot spot in the research of breast cancer in recent years. The.Hippo signal pathway of breast cancer stem cells has been found to be a signal pathway that inhibits proliferation and promotes apoptosis to limit the size of the organ. However, a lot of evidence suggests that Hippo signals are used. The pathway regulates the self renewal and amplification of stem cells and progenitor cells. In the progression of breast cancer, liver cancer, non-small cell lung cancer and endometrial cancer, the.Hippo signaling pathway is cascaded through kinase cascade, which ultimately acts as the core transcription factor TAZ/YAP, and the interaction of phosphorylated TAZ/YAP and 14-3-3 protein is blocked in the cytoplasm. The ubiquitin dependent proteasome is degraded. The non phosphorylated TAZ/YAP is transferred to the nucleus, interacting with TEAD1-4 or other transcription factors Smad, sp73 and so on, inducing gene CTGF, AX1, BMP4 and so on. Among them, the core factor TAZ/YAP in Hippo signaling pathway is most obvious. In breast cancer, TAZ is affecting the stem cells of breast cancer. The ball, self-renewal, and differentiation is more obvious. Simvastatin is a HMG-Co A inhibitor. Recent studies have shown that simvastatin can inhibit the tumor as an anticancer drug. The study shows that simvastatin can inhibit the transport of Hippo-TAZ from the cytoplasm to the nucleus, thus inhibiting the biological role of TAZ in the nuclear control of the downstream transcription factors. Paclitaxel is an anti microtubule drug that promotes microtubule polymerization, inhibits depolymerization, keeps microtubule protein stable and inhibits the process of mitosis in cancer cells. As breast cancer, the clinical chemotherapy of ovarian cancer has a history of many years. In recent years, its drug efficacy and drug resistance have increased. The effects of simvastatin and paclitaxel on the invasion, metastasis, EMT process and the effect on breast cancer stem cells of breast cancer cell MDA-MB-231 were selected in this study. Methods: the experiment was divided into four groups: the blank treatment group, the simvastatin treatment group, the paclitaxel treatment group. Simvastatin + paclitaxel treatment group. (1) CCK8 detection of tumor inhibition rate: after trypsin digestion, MDA-MB-231 cells were inoculated after trypsin digestion in each treatment group, and MDA-MB-231 cells were inoculated at 5 x 103/ holes. After 24h, different concentrations of drug gradient medium (gradient) were changed, 48h, CCK8 were incubated to avoid light, and 2H was incubated at 37 degrees, and two species were added with Gradpad to calculate IC50.. After the same concentration gradient (two medications added to 1:1) 48h before IC50, the drug was measured. (2) the change of migration ability of MDA-MB-231 cells was detected by scratch test: the MDA-MB-231 cells in each treatment group were serum-free to culture 48h, and were taken and photographed at 0h and 48h, respectively, to observe the migration distance of the cells. Transwell test was used to detect the MD of each group. Changes in the invasion and metastasis ability of A-MB-231 cells: the cells in each treatment group were cultured for 24h to take pictures and count the number of small cell membrane cells. The changes in the level of MMP-2 expression in the MDA-MB-231 cells of each treatment group were detected by gelatinase spectrum. (3) the Western blot technique was used to detect the changes in the level of the intracellular protein in the MDA-MB-231 cells. The detection index included: Hippo Key signal pathway factor TAZ, stem cell marker -OCT4, ALDH1. metal matrix proteinase -2 MMP-2, E- cadherin E-cadherin, vimentin Vimentin, apoptotic pathway related proteins - Bax, P53, Caspsase3, Bcl-2. immunocytochemical technique observation cell expression. (4) flow cytometry The ratio of stem cells in each treatment group was measured by CD44+/CD24- staining. (5) the ability and quantity of each treatment was detected by the stem cell formation test. (6) after the treatment of 48h, the cells were cultivated for the same number of cells in each hole. After two weeks, the size and quantity of the cloned cell group were observed. Results: (1) the results of scratch and invasion test showed octyl. Statins combined paclitaxel could effectively inhibit the migration distance, invasion and metastasis number.Western blot technique and gelatinase spectrum technique to detect the expression of MMP-2. The results showed that compared with the blank control group and the single addition group, the expression of MMP-2 in the combined paclitaxel treatment group was significantly decreased. (2) the detection of EMT markers was detected. It was found that: compared with the blank control group and the single addition group, simvastatin combined paclitaxel group increased the protein content of E-cadherin and decreased the protein content of Vimentin in the cell. (3) the detection results of apoptosis related signal pathway markers showed that compared with the blank control group and the single addition group, the simvastatin combined paclitaxel group, P53, Bcl-2, Caspsa The expression of Se3 was obviously decreased, the expression of Bax was obviously increased and the cloning experiment was found. (4) the results of Western blot and immunofluorescence chemical technique showed that compared with the blank control group and the single addition group, the expression of TAZ, OCT4, ALDH1 protein in the group of simvastatin combined paclitaxel, TAZ, OCT4 and ALDH1 was markedly decreased, and (5) flow cytometry CD44+/CD24- staining was used to detect the proportion of stem cells The results of the stem cell formation test showed that the ratio of simvastatin combined with paclitaxel group was more obviously inhibited by the combination of simvastatin and paclitaxel than in the single addition group. Conclusion: the combined use of simvastatin and paclitaxel can inhibit MDA-MB-231 metastasis, invasion, apoptosis and inhibition of proliferation. Breast cancer stem cells may influence the proliferation and self-renewal ability by downregulating Hippo-TAZ and its downstream target Oct4, which may provide new ideas for clinical chemotherapy.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
【参考文献】
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1 Binhua P.Zhou;;Epithelial-mesenchymal transition in breast cancer progression and metastasis[J];癌症;2011年09期
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