AMPK活化对肿瘤生物学行为和抗肿瘤治疗的作用

发布时间：2018-06-03 00:25

  本文选题：AMPK + LKB1　； 参考：《南昌大学》2016年博士论文

【摘要】：背景和目的:全球癌症死亡人数正在迅猛上升,癌症已成为我国五大致死性疾病之首,严重威胁着我国人民的健康水平。能量代谢的相对平衡是抑制肿瘤细胞过度生长的预防机制,而肿瘤细胞依靠代谢重组(如Warburg效应)获取能量,克服能量供应危机。因此,肿瘤细胞的能量代谢途径已成为肿瘤治疗的潜在靶点。细胞的能量代谢受多条信号通路的调控,其中单磷酸腺苷激酶(AMP-activated protein kinase,AMPK)信号通路是最重要的途径,该通路参与调节Warburg效应,脂肪酸的合成与谷氨酰胺代谢,并与PI3K-Akt信号通路、MAPK信号通路以及多种转录因子有相互作用,共同调节细胞的增殖、生长、凋亡等细胞生物学行为,发挥抑癌作用。肿瘤发生早期,AMPKα1基因的缺失对癌基因诱发的肿瘤有促进作用,有临床病理资料显示肿瘤组织中AMPK活性低于正常组织,AMPK活性不足被认为是恶性肿瘤发生发展的原因之一。鉴于多种恶性肿瘤组织中AMPK的失活,众多学者探索如何利用化学或生物分子激活AMPK以抑制肿瘤生长,并取得良好的效果。例如,二甲双胍、AICAR、A769662、阿斯匹林、水杨酸,黄莲素等化学药物,其中关于降血糖药物二甲双胍的研究最为突出,现已作为抗癌辅助用药进入临床试验。除了化学药物,还发现一些细胞因子和应激压力也能激活AMPK。各种因素激活AMPK方式不尽相同,但AMPK的充分激活都有赖于AMPK激酶(AMPKK)的参与,目前已证实的AMPKK包括:LKB1、CaMKKβ、TAK1等。LKB1作为AMPK最重要的上游激酶,在各种肿瘤组织中表达不一,对二甲双胍等AMPK激活剂的抗癌效果有重要影响,但目前对于LKB1在应激压力激活AMPK的作用尚不清楚。此外,我们的前期研究还发现某些应激压力(细胞因子刺激或化疗药物)激活AMPK的过程中,并未涉及以上AMPKK。例如,可诱导渗透压改变的山梨醇。这些应激压力不仅诱导肿瘤细胞的凋亡,还可以引起AMPK活化,但其具体机制尚不清楚。由此可见,为了提高肿瘤细胞中AMPK的活性,不仅有必要对LKB1-AMPK途径进行深入研究,还要探索其他的AMPK激活途径及其在抗肿瘤治疗的作用。因此,我们进行了以下研究:⑴胃癌及肺癌组织中的p-ampk和lkb1的表达情况;⑵lkb1过表达对胃癌细胞sgc-7901生物学行为的影响;⑶atm和lkb1在抗癌药物依托泊苷激活ampk中的作用;⑷ampk活化是否增加肿瘤细胞对依托泊苷的药物敏感性;⑸lkb1和mlk3对ampk的调节作用。材料与方法:1.本课题的临床标本来自南昌大学第一附属医院手术切除标本(伦理批准号2014〔025〕),收集60例胃癌及其对应癌旁组织,采用免疫组化染色分析检测胃癌及癌旁组织p-ampk(thr172)及lkb1的表达水平;另外,收集了65例不同阶段的肺腺癌标本,采用免疫组化染色分析不同阶段的肺腺癌组织p-ampk(thr172)表达水平;2.在缺乏lkb1蛋白的sgc-7901细胞株中转入功能性野生型lkb1基因,构建稳定表达lkb1的细胞株。以mtt法检测细胞生长和存活,以细胞划痕实验检测细胞迁移,以流式细胞技术检测细胞周期、cd44阳性表达细胞比例、细胞凋亡比率;3.依托泊苷以浓度依赖或时间依赖的方式作用于前列腺癌细胞c4-2后,收集蛋白,以免疫印迹法检测ampk、atm磷酸化水平;另外,sirna干扰c4-2细胞中的atm或lkb1,再给依托泊苷刺激后,检测ampk、atm的磷酸化水平。4.转入ampkdn突变体到c4-2细胞抑制ampk活化(c4-2dn),以空载体为对照(c4-2e),给依托泊苷刺激24小时,以流式细胞仪检测细胞凋亡情况;选择ampk活性不同(c4-2dn及c4-2e)细胞和lkb1表达不同(a549e及a549-lkb1)的细胞,给予依托泊苷刺激不同时间后,以免疫印迹法检测凋亡蛋白表达(cleavedcaspase3\cleavedparp)。5.选择lkb1表达不同(a549及a549-lkb1)细胞,给予各种应激因子刺激后,以免疫印迹技术检测p-ampk与p-jnk,筛选出同时激活ampk与jnk的因子;在hek-293t细胞中转入mlk3质粒,后检测ampk的磷酸化水平;以gsh纯化技术,获得基因重组蛋白mlk3和ampk,以体外酶学实验检测mlk3对ampk的磷酸化;在hek-293t细胞中,转入mlk3质粒后,用gshbeads将gst-mlk3蛋白捕获,以免疫印迹检测沉淀中是否捕获内源性ampkα1或ampkα2蛋白;将myc-ampk?1或?2和gst-mlk3质粒共转染hek-293t细胞,收集细胞裂解液,与gshbeads孵育,离心沉淀,收集gshbeads-mlk3和捕获的蛋白,以anti-gst和anti-myc抗体进行免疫印迹分析。结果1.胃癌及肺癌组织中ampk和lkb1情况。⑴免疫组化结果显示胃癌组织p-ampk(thr172)、lkb1的表达低于癌旁组织(p0.001)。p-ampk(thr172)表达:胃癌中(-)20例,(+)33例,(++)6例,(+++)1例,而癌旁组织中:(-)5例,(+)11例,(++)28例,(+++)16例。lkb1表达:胃癌中(-)23例,(+)25例,(++)12例,(+++)0例,癌旁组织中lkb1表达:(-)9例,(+)13例,(++)27,(+++)11例。⑵不同分期肺癌组织中的ampk活化情况,随着肺腺癌分期的恶性程度增高,p-ampk(thr172)表达渐渐下降,Ⅰ期Ⅱ期Ⅲ期(spearman相关分析=-0.397,p0.05)。Ⅰ期(-)4例,Ⅰ期(+)5例,Ⅰ期(++)10例,Ⅰ期(+++)3例;Ⅱ期(-)5例,Ⅱ期(+)10例,Ⅱ期(++)9例,Ⅱ期(+++)3例;Ⅲ期(-)9例,Ⅲ期(+)8例,Ⅲ期(++)1例,Ⅲ期(+++)0例。2.提高lkb1蛋白表达对胃癌细胞sgc-7901生物学行为的影响在缺乏lkb1的肿瘤细胞sgc-7901中转入lkb1基因,增加lkb1的表达可以抑制细胞的增殖与迁移,减少cd44阳性细胞的比例,提高肿瘤细胞对奥沙利铂、5-氟尿嘧啶和伊立替康三种抗癌药物敏感性。3.atm和lkb1在依托泊苷激活ampk中的作用⑴westernblot结果显示:肿瘤细胞a549受依托泊苷刺激后,p-atm和p-ampk(thr172)逐渐上升,并呈剂量和时间依赖性;⑵sirna干扰c4-2细胞的atm表达后,atm表达下调,给予依托泊苷刺激细胞,westernblot结果显示:p-atm和p-ampk(thr172)无明显上升;而对照组atm表达正常,提示依托泊苷刺激可以同时激活atm和ampk,依托泊苷激活ampk过程中需要atm的参与。⑶sirna干扰c4-2细胞的lkb1表达,lkb1表达下降,给予依托泊苷刺激细胞,westernblot结果显示:p-atm表达逐渐上升,而无法激活ampk;而对照组lkb1表达正常,依托泊苷刺激可以同时激活atm和ampk。提示在缺乏lkb1的条件下,依托泊苷可以激活atm,但无法激活ampk。⑷在缺乏lkb1的a549细胞中,转入lkb1基因,提高lkb1蛋白表达后,给予依托泊苷刺激细胞,westernblot结果显示:可以同时激活atm和ampk;而对照组lkb1表达无变化,依托泊苷刺激p-atm表达逐渐上升,而无法激活ampk。以上结果提示:依托泊苷激活ampk过程中需要atm和lkb1共同参与。4.ampk活化对肿瘤细胞药物敏感性的影响⑴流式细胞分析显示,在依托泊苷刺激下c4-2e细胞(ampk有活性)凋亡细胞的比例明显高于c4-2dn(ampk无活性);⑵依托泊苷刺激后,c4-2e细胞中的p-ampk(thr172)表达、cleavedcaspase3表达和cleavedparp表达明显高于c4-2dn细胞;⑶依托泊苷刺激下,a549-lkb1细胞的p-ampk(thr172)表达、cleavedcaspase3表达和cleavedparp表达明显高于对照组(a549e)。结果提示,ampk活化或提高lkb1表达有助于增强肿瘤细胞的药物敏感性。5.mlk3对ampk的调节作用⑴a549-lkb1和a549-wt细胞受山梨醇、aicar等7种因子刺激,仅在山梨醇所刺激a549-lkb1和a549-wt两种细胞后,p-ampk和p-jnk都升高;提示山梨醇可以同时激活ampk和jnk,并不依赖于lkb1。⑵a549-lkb1和a549-wt细胞受山梨醇刺激,两细胞的p-ampk同时升高,提示山梨醇可能以不依赖lkb1的方式激活ampk;⑶在hek293t细胞中转入mlk3质粒,过表达mlk3蛋白,然后以免疫印迹法检测p-ampk(thr172)水平,结果显示mlk3的异常过表达可提高p-ampk(thr172)水平。⑷体外激活ampk酶学实验显示,单独加入amp或单独加入lkb1蛋白对ampk的磷酸化无明显影响;同时加入amp和lkb1可以引起ampk的磷酸化;单独加入mlk3可以引起明显的ampk磷酸化;同时加入amp和mlk3引起的ampk磷酸化最强。该结果提示mlk3可在缺乏amp的情况下磷酸化ampk,说明ampk是mlk3的催化底物。⑸MLK3与AMPK间的相互结合;通过Pulldown技术发现,转入GST-MLK3质粒的细胞提取物中发现AMPKα1的表达,提示转入表达的MLK3蛋白与细胞内源性AMPKα1亚单位紧密结合;共转染Myc-AMPK?1或?2和GST-MLK3入HEK-293T细胞,然后通过IP技术证明,外源性表达GST-MLK3蛋白能与外源性Myc-AMPK?1蛋白发生特异性结合。结论:1.癌组织中AMPK失活可能是恶性肿瘤发生发展的原因之一,LKB1表达下调可使AMPK失活。在缺乏LKB1表达的肿瘤细胞中,提高LKB1蛋白表达可以抑制细胞的生长、转发移,增强肿瘤细胞的药物敏感性。2.依托泊苷激活AMPK的过程需要ATM和LKB1共同参与;AMPK活化使得肿瘤细胞对依托泊苷诱导凋亡作用更为敏感。3.MLK3能与AMPK?1结合,并以非LKB1方式直接磷酸化AMPK,MLK3有可能是AMPK新的上游激酶。
[Abstract]:Background and purpose: the number of global cancer deaths is rising rapidly. Cancer has become the first of the five fatal diseases in our country. It is a serious threat to the health of our people. The relative balance of energy metabolism is a preventive mechanism to inhibit the overgrowth of tumor cells, and the tumor cells acquire energy depending on the metabolic recombination (such as the Warburg effect). Energy supply crisis. Therefore, the energy metabolism pathway of tumor cells has become a potential target for cancer treatment. The energy metabolism of cells is regulated by multiple signal pathways, in which the AMP-activated protein kinase (AMPK) signal pathway is the most important pathway. The pathway participates in the regulation of the Warburg effect and the synthesis of fatty acids. Metabolism of glutamine, and the interaction with PI3K-Akt signaling pathway, MAPK signaling pathway and various transcription factors to regulate cell biological behavior, such as proliferation, growth, apoptosis and other cell biological behavior, and play the role of inhibiting cancer. Early tumor occurrence, the deletion of AMPK alpha 1 gene can promote cancer induced tumor, and there are clinicopathological data. The activity of AMPK in tumor tissue is lower than that of normal tissue, and the insufficiency of AMPK activity is considered to be one of the reasons for the development of malignant tumor. In view of the inactivation of AMPK in many malignant tumor tissues, many scholars have explored how to activate AMPK by using chemical or biological molecules to inhibit the growth of the tumor. For example, metformin, AICAR, A76 9662, aspirin, salicylic acid, Huang Liansu and other chemical drugs, among them, the study of metformin, the hypoglycemic drug, is the most prominent. It has now entered the clinical trial as an adjuvant antitumor drug. Besides chemical drugs, some cytokines and stress stress can also activate AMPK. various factors to activate AMPK methods, but AMPK is sufficient. Activation depends on the participation of AMPK kinase (AMPKK). The current confirmed AMPKK, including LKB1, CaMKK beta, and TAK1 as the most important upstream kinase of AMPK, is expressed differently in various tumor tissues and has an important effect on the anticancer effect of the AMPK activator such as metformin, but the effect of LKB1 on stress activation AMPK is not yet clear. In addition, our previous study also found that some stress stress (cytokine stimulation or chemotherapeutic drugs) activation of AMPK did not involve the above AMPKK., which could induce osmotic pressure change of sorbitol. These stress stresses not only induce apoptosis of tumor cells, but also cause AMPK activation, but the specific mechanisms are not yet clear. Therefore, in order to improve the activity of AMPK in tumor cells, it is not only necessary to study the LKB1-AMPK pathway in depth, but also to explore other AMPK activation pathways and their role in antitumor therapy. Therefore, we have carried out the following studies: (1) the expression of p-ampk and LKB1 in gastric and lung cancer tissues; (2) LKB1 over expression of sGC in gastric cancer cells SGC The effects of -7901 biological behavior; (3) the role of ATM and LKB1 in the activation of etoposide in AMPK; (4) whether AMPK activation increases the drug sensitivity of tumor cells to etoposide; the regulatory effect of LKB1 and mlk3 on AMPK. Materials and methods: 1. clinical specimens from the First Affiliated Hospital of Nanchang University ( Ethical approval number 2014 [025]), 60 cases of gastric cancer and their corresponding para cancerous tissues were collected and immunohistochemical staining analysis was used to detect the expression of p-ampk (thr172) and LKB1 in gastric cancer and para cancer tissues. In addition, 65 specimens of lung adenocarcinoma at different stages were collected, and the expression of p-ampk (thr172) expression in lung adenocarcinoma tissues at different stages by immunohistochemical staining was used. Level; 2. in the SGC-7901 cell line with the lack of LKB1 protein into the functional wild type LKB1 gene to construct a stable LKB1 cell line. The cell growth and survival were detected by MTT assay, cell migration was detected by the cell scratch test, the cell cycle, the proportion of CD44 positive cells, the ratio of apoptosis, and the rate of apoptosis were detected by flow cytometry; 3. etoposide was used. Glucoside acts on the prostate cancer cell C4-2 in a concentration dependent or time dependent manner, collecting protein and detecting AMPK, ATM phosphorylation level by immunoblotting; in addition, siRNA interferes with ATM or LKB1 in C4-2 cells, and after stimulation of etoposide, AMPK, ATM phosphorylation level.4. is transferred to ampkdn mutant to C4-2 cells to inhibit activation. -2dn), using the empty carrier as the control (c4-2e), the apoptosis of etoposide was detected by flow cytometry for 24 hours. The cells with different AMPK activity (c4-2dn and c4-2e) and LKB1 were selected to express different (a549e and a549-lkb1) cells. The expression of apoptotic protein was detected by immunoblotting after the stimulation of etoposide at different time (cleavedcaspase3c). Leavedparp).5. chose LKB1 to express different (A549 and a549-lkb1) cells, and after the stimulation of various stress factors, p-ampk and p-JNK were detected by immunoblotting, and the factors that simultaneously activated AMPK and JNK were screened. The mlk3 plasmids were transferred into hek-293t cells and the phosphorylation level of AMPK was detected. PK was used to detect the phosphorylation of mlk3 to AMPK in vitro. In hek-293t cells, after transferring to mlk3 plasmid, gst-mlk3 protein was captured by gshbeads, and the endogenous AMPK a 1 or AMPK alpha 2 protein was captured by immunoblotting, and myc-ampk? 1 or 2 and gst-mlk3 mass were co transfected to the hek-293t cells, and the cell lysate was collected. Incubation, centrifugation, collection of gshbeads-mlk3 and captured proteins, and immunoblotting with anti-GST and anti-myc antibodies. Results 1. the AMPK and LKB1 in gastric cancer and lung cancer. (1) immunohistochemical results showed p-ampk (thr172) in gastric cancer tissue, and the expression of LKB1 was lower than that in the paracancerous group (p0.001).P-ampk (thr172) expression: 20 cases in gastric cancer (-), (+) 33 (+ + +) 6 cases, (+ + +) 1 cases, and (+) 5 cases, (+) 11 cases, (+ +) 28 cases, (+ + +) 16 cases of.Lkb1 expression: gastric cancer (+ +) in 23 cases, (+ +) 12 cases (+ + +) in 0 cases, (+ + + +) LKB1 expression: (+ + +) 27, (+ + +), AMPK activation in different stages of lung cancer, with lung adenocarcinoma stage increased malignancy, p-ampk (p-ampk ( Thr172) gradually decline, stage I stage II stage III (Spearman correlation analysis =-0.397, P0.05). 4 cases (-), I (+) 5 cases, I phase (+ +) 10 cases, phase I (+ + +) 3 cases, II (-) 5 cases, II (+) 10 cases, II (+ +) 9 cases, II (+ + +) 3 cases, III (+) 9 cases, III (+) 8, + + 1 cases, III phase (+ + +).2. improve the expression of LKB1 protein expression of gastric cancer cell s The effect of gc-7901's biological behavior on the LKB1 gene in the tumor cell SGC-7901 lacking LKB1, increasing the expression of LKB1 can inhibit cell proliferation and migration, reduce the proportion of CD44 positive cells, and increase the three kinds of anti-cancer drug sensitivity.3.atm and LKB1 in the tumor cells activated by oxaliplatin, 5- fluorouracil and erinotecan in etoposide activation The effect of AMPK in Westernblot showed that p-atm and p-ampk (thr172) increased gradually after A549 was stimulated by etoposide, and was dosed and time dependent; and after siRNA interfered with ATM expression of C4-2 cells, ATM expression was down, and etoposide stimulated cells. The expression of ATM in the control group was normal, suggesting that etoposide stimulated the activation of ATM and AMPK simultaneously. ATM was needed during the activation of AMPK by etoposide. (3) siRNA interfered with LKB1 expression in C4-2 cells, LKB1 expression decreased, and etoposide stimulated cells. The Westernblot results showed that p-atm expression increased gradually, but no AMPK was activated; while the control group was not activated. The expression of B1 is normal, and etoposide stimulation can activate both ATM and ampk. at the same time. Etoposide can activate ATM under the condition of lack of LKB1. But it can not activate ampk. (4) in A549 cells lacking LKB1, and turn into the LKB1 gene and increase the expression of LKB1 protein to stimulate the cells by etoposide. The Westernblot result shows that ATM and can be activated at the same time. PK, while the expression of LKB1 in the control group was not changed, and the expression of p-atm increased gradually with etoposide stimulation, but the result of the failure to activate ampk. suggested that the effect of etoposide activation on the AMPK process needed both ATM and LKB1 to participate in the effect of.4.ampk activation on the drug sensitivity of the tumor cells (1) flow cytometric analysis showed that the c4-2e cell (AMPK has the presence of etoposide (AMPK) The proportion of apoptotic cells was significantly higher than that of c4-2dn (AMPK inactive); (2) after stimulation of etoposide, the expression of p-ampk (thr172) in c4-2e cells, cleavedcaspase3 expression and cleavedparp expression were significantly higher than that of c4-2dn cells; (3) the expression of p-ampk (thr172) in a549-lkb1 cells under the stimulation of etoposide, cleavedcaspase3 expression and expression clearly expressed. Significantly higher than the control group (a549e), the results suggest that AMPK activation or enhancement of LKB1 expression helps to enhance the drug sensitivity of tumor cells to the regulatory effect of.5.mlk3 on AMPK (1) a549-lkb1 and a549-wt cells are stimulated by 7 factors, such as sorbitol, AICAR and so on. Only after sorbitol stimulates the two cells of a549-lkb1 and a549-wt, p-ampk and p-JNK are elevated. Sorbitol can simultaneously activate AMPK and JNK, which does not depend on lkb1. (lkb1.) a549-lkb1 and a549-wt cells stimulated by sorbitol, and the p-ampk increases at the same time, suggesting that sorbitol may activate AMPK in the manner that does not depend on LKB1; (3) the mlk3 plasmid is transferred into the HEK293T cell, the mlk3 protein is overexpressed, and then the Western blot method is used to detect the p-ampk. The results showed that the abnormal overexpression of mlk3 could increase the level of p-ampk (thr172). 4. In vitro activation of AMPK enzymology experiments showed that adding amp alone or adding LKB1 protein alone had no obvious effect on the phosphorylation of AMPK; meanwhile, AMP and LKB1 could cause phosphorylation of AMPK. The results suggested that the phosphorylation of AMPK caused by mlk3 is the strongest. The results suggest that mlk3 can phosphorylate AMPK under the absence of AMP, indicating that AMPK is a catalytic substrate for mlk3, the binding between MLK3 and AMPK; by Pulldown technology, the expression of AMPK alpha 1 is found in the cell extracts of the GST-MLK3 plasmid, indicating the transfer of the expressed protein to the cell source. Sexual AMPK alpha 1 subunits were tightly combined; CO transfected Myc-AMPK? 1 or 2 and GST-MLK3 into HEK-293T cells, and then IP technique proved that exogenous GST-MLK3 protein could be associated with exogenous Myc-AMPK? 1 protein specific binding. Conclusion: AMPK deactivation in 1. cancer tissues may be one of the causes of malignant tumor development, LKB1 expression downregulation can make AMP K inactivation. In the tumor cells lacking LKB1 expression, increasing the expression of LKB1 protein can inhibit cell growth, transfer and enhance the drug sensitivity of tumor cells..2. etoposide activates AMPK, which requires the joint participation of ATM and LKB1; AMPK activation makes the tumor cells more sensitive to the apoptosis effect of etoposide and AMPK? 1 In combination with direct phosphorylation of AMPK in non LKB1 mode, MLK3 may be a new upstream kinase of AMPK.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.5
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