COX-2通路中PGE2-EP4对口腔鳞状细胞癌生物学特性的作用及机制

发布时间：2018-03-28 08:52

本文选题：口腔鳞状细胞癌　切入点：前列腺素E2　出处：《南方医科大学》2017年博士论文

【摘要】：口腔鳞状细胞癌(Oral Squamous Cell Carcinoma,OSCC)是临床上常见的恶性肿瘤之一,其发病率在全球呈现逐年上升的趋势。在过去的20年中,尽管针对OSCC的治疗方法不断发展,但OSCC的五年生存率无明显改善,因此发病机制、诊断及治疗仍是OSCC的研究热点。近年来,肿瘤微环境中炎症与恶性肿瘤关系的研究备受关注,尤其是环氧化酶(Cyclooxygenase,COX)通路在介导炎症和癌症关系中的作用值得深入探究。研究表明,COX-2过表达与口腔癌的发生、侵袭以及化疗药物的耐药性相关,而针对COX-2的非类固醇消炎药(Nonsteroidal anti-inflammatory drugs,NSAIDs)可以抑制恶性肿瘤的发生发展,但它的长期使用会产生诸多副作用如胃溃疡和心肌梗死等,临床应用受限。前列腺素E2(Prostaglandin E2,PGE2)是COX-2通路的下游产物。研究表明,PGE2能促进多种恶性肿瘤的增殖及侵袭,其机制可能为抑制局部免疫反应。然而,PGE2及其受体4亚型(E-prostaglandin,EP4)对OSCC生物学特性的影响及机制尚未完全明了。为此,我们通过在OSCC细胞株中外源性添加PGE2及相应的拮抗剂来研究PGE2对OSCC细胞增殖及凋亡的影响并探讨其可能的机制,从而寻找OSCC治疗的潜在靶点。(1)我们采用cellcounting kit 8(CCK8)法检测外源源性PGE2对OSCC细胞株Tca8113增殖活性的影响,发现在作用48 h时,PGE2能显著地促进细胞增殖,效应呈现剂量依赖趋势,且在PGE2浓度为1.0μmol/L时出现峰值。(2)采取Western-blot(WB)的方法研究PGE2促Tca8113细胞增殖的作用机制,我们发现,外源性添加PGE2可激活ERK的磷酸化,而磷酸化过程可被MEK的抑制剂U0126拮抗;通过CCK8检测Tca8113细胞增殖,我们发现,外源性的U0126能拮抗PGE2对Tca8113细胞增殖的促进作用;上述结果表明PGE2可通过激活ERK的磷酸化促进Tca8113细胞的增殖。(3)WB法检测显示,4种类型的EP受体在Tca8113细胞中均有表达。(4)为确定与PGE2结合的EP受体类型,我们分别加入EP1、EP2及EP4受体拮抗剂,结果表明,EP4拮抗剂L-161,982可拮抗PGE2对Tca8113细胞的增殖作用,并呈现剂量和时间依赖性;进一步采用WB法检测,我们发现,L-161,982通过抑制PGE2对ERK磷酸化的促进作用,从而发挥拮抗作用,这也同样验证了 PGE2是通过激活ERK的磷酸化来促进细胞增殖。(5)为了进一步验证PGE 2是否通过结合受体EP4而起作用,我们在细胞中加入3种类型的EP受体激动剂,结果发现,仅有EP4激动剂PGE1-OH可以恢复被塞来昔布预处理的Tca8113细胞的增殖,并具有浓度依赖性趋势;进一步采用WB法,我们发现,PGE1-OH通过激活ERK磷酸化来促进细胞增殖,而MEK的抑制剂U0126可抑制此作用,提示PGE2可能通过结合受体EP4来促进细胞增殖的。(6)流式细胞仪检测显示,L-161,982可以诱导Tca8113细胞凋亡,并通过阻断s期来影响细胞周期,其作用具有浓度依赖趋势;WB法进一步检测发现,L-161,982通过上调Bax和下调Bcl-2蛋白水平来诱导细胞凋亡,同时通过上调P21和下调CDK2及CyclinA2的蛋白表达影响细胞周期。综上所述,本研究发现PGE2可通过结合受体EP4来介导ERK磷酸化诱导的细胞增殖,并影响细胞凋亡及周期,因此,EP4受体可能成为OSCC的治疗靶点。
[Abstract]:Oral squamous cell carcinoma (Oral Squamous Cell Carcinoma, OSCC) is one of the common malignant tumor, its incidence shows a rising trend in the world. In the past 20 years, although the treatment method for the continuous development of OSCC, but the five year survival rate of OSCC without obvious improvement, so the pathogenesis. Study on the diagnosis and treatment of OSCC is still hot. In recent years, the research of inflammation and tumor in the tumor microenvironment has attracted much attention, especially cyclooxygenase (Cyclooxygenase, COX) pathway mediated role in the relationship between inflammation and cancer in the intermediate value to explore. The research results show that the overexpression of COX-2 and oral cancer development, invasion the related and drug resistance, and for non steroidal anti-inflammatory drugs (COX-2 Nonsteroidal anti-inflammatory drugs, NSAIDs) can inhibit the occurrence and development of malignant tumors, but its long-term use will have many Side effects such as gastric ulcer and myocardial infarction, clinical application of prostaglandin E2 (Prostaglandin E2 limited., PGE2) is the downstream products of the COX-2 pathway. The results show that PGE2 can promote the proliferation and invasion of malignant tumors, the possible mechanism is inhibiting the local immune response. However, PGE2 and its receptor subtype 4 (E-prostaglandin, EP4) influence on the biological characteristics of OSCC and the mechanism is not yet fully understood. Therefore, we passed in OSCC cells by exogenous addition of PGE2 antagonists and corresponding to the effects of PGE2 on proliferation and apoptosis of OSCC cells and to explore its possible mechanism, in order to find a potential target for the treatment of OSCC. (1) we use cellcounting kit 8 (CCK8) effect analysis of exogenous derived PGE2 on proliferation of Tca8113 OSCC cells, found in 48 h, PGE2 can significantly promote cell proliferation, the effect showed a dose dependent trend, and concentrated in PGE2 As the peak of 1 mol/L. (2) to Western-blot (WB) mechanism, study the method of PGE2 to promote the proliferation of Tca8113 cells we found that exogenous addition of PGE2 can activate the phosphorylation of ERK and phosphorylation by MEK inhibitor U0126 antagonized by CCK8; proliferation of Tca8113 cells was detected, we found that exogenous U0126 can promote the role of antagonist PGE2 on the proliferation of Tca8113 cells; the results show that PGE2 can be activated by phosphorylation of ERK promote the proliferation of Tca8113 cells. (3) WB assay showed that the 4 types of EP receptors were expressed in Tca8113 cells. (4) to determine the binding of EP and PGE2 receptor types, we added EP1, EP2 and EP4 receptor antagonist, the results show that the EP4 antagonist L-161982 can antagonize the effect of PGE2 on proliferation of Tca8113 cells, and showed a dose and time dependent; further with WB assay, we found that L-161982 PGE2 inhibition of ERK phosphorylation promotes, and thus play antagonism, it also proves that PGE2 is to promote cell proliferation by activating the phosphorylation of ERK. (5) in order to verify whether PGE 2 act by binding to the receptor EP4 in the cell, we added 3 types of EP receptor agonists the results, found that only EP4 agonist PGE1-OH can be recovered by celecoxib pretreatment of the proliferation of Tca8113 cells in a concentration dependent manner; further using WB method, we found that PGE1-OH through activation of ERK phosphorylation to promote cell proliferation, while MEK inhibitor U0126 inhibited this effect, suggesting that PGE2 might to promote cell proliferation by binding to a receptor of EP4. (6) flow cytometry showed that L-161982 could induce apoptosis of Tca8113 cells, and by blocking s can influence the cell cycle, and its effect is concentration dependent trend; WB method Found a step detection, L-161982 through upregulation of Bax and downregulation of Bcl-2 protein levels to induce cell apoptosis, and expression of cell cycle effects through upregulation of P21 and downregulation of CDK2 and CyclinA2 protein. In summary, this study found that PGE2 by combining the receptor EP4 mediated ERK phosphorylation induced cell proliferation, apoptosis and cell cycle and influence therefore, the EP4 receptor may become a therapeutic target for OSCC.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.8

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