【作者】 佟敬山；

【导师】 盛军； 付学奇；

【作者基本信息】 吉林大学 ， 细胞生物学， 2011， 博士

【摘要】 雌激素受体(estrogen receptor, ER)是固醇类核受体超家族成员之一。广泛存在于各种组织中,在正常的组织发育和肿瘤发生中起重要作用。雌激素受体分为ER-α和ER-β两种亚型。ER-α36是最近发现的新的α亚型,基因定位于6号染色体q24.2-25.3,cDNA全长5.4kb,编码分子量约为36kDa的蛋白。ER-a36 mRNA的转录起始点位于ER-α66基因的第一个内含子中。与ER-α66不同,ER-α36缺少氨基端转录激活域AF-1和羧基端转录激活域AF-2,但保留了DNA结构域和部分配体结合域。在ER-α36羧基末端有27个特有的氨基酸残基,代替了ER-α66基因第7和第8个外显子编码的位于氨基末端的138个氨基酸残基。本研究的主要目的是探讨ER-α36在子宫内膜癌细胞系Ishikawa中的功能及其调节细胞增殖的机制。激光共聚焦的结果表明,ER-α36主要定位于人子宫内膜癌细胞系Ishikawa的细胞膜上和细胞质中,而ER-α66主要定位于人子宫内膜癌细胞系Ishikawa的细胞核中。PKC信号通路是细胞中常见的激酶信号通路,参与各种细胞功能,包括细胞增殖,分化,衰老,凋亡以及肿瘤的形成。子宫内膜癌是常见的妇科恶性肿瘤之一,近年来发病率明显增高。实验和流行病学研究发现PKC在子宫内膜癌的发生中起着重要的作用,但具体的机制并不是十分清楚。虽然,在先前的研究中,PKCδ的主要功能是参与细胞凋亡的过程,然而,有不少研究表明,在一部分细胞系中PKCδ起着促进细胞增殖的作用。本实验中,我们发现用雌激素(E2)和偶联BSA的雌激素(E2-BSA)处理子宫内膜癌细胞系Ishikawa后都能激活PKCδ. RNA干扰ER-α36后能废除E2激活的PKCδ,这些结果表明,雌激素通过ER-α36调节的膜起始信号通路迅速激活PKCδ。通常情况下,PKCα活性的提高与肿瘤细胞的迁移和增殖相关。此外,PKCα表现出抑制或减轻肿瘤细胞的细胞凋亡特征。本实验中,我们发现雌激素不能激活PKCα,表明PKCα并不参与雌激素依赖的子宫内膜癌增殖。cAMP是一个第二信使,在各种刺激的情况下起信号传导的作用,在真核细胞中cAMP的主要功能是激活cAMP依赖性蛋白激酶(PKA)。本实验中,只有E2能激活PKA,E2-BSA并不能激活PKA。表明膜受体并不参与雌激素依赖的PKA激活。E2和E2-BSA能激活ERK1/2,并且这个过程需要ER-α36和PKCδ的参与。同时我们的结果表明,ER-α36介导了膜起始的E2诱导的cyclin D1/cdk4的表达水平的提高。以上结果表明ER-a36通过调节膜起始的PKCS/ERK通路参与雌激素调节的子宫内膜癌的增殖。水合淫羊藿素(Icaritin)是一种中药淫羊藿的提取物,研究表明其具有选择性雌激素受体调节活性和抗肿瘤效应。Icaritin具有多种生物学活性,如刺激神经元和心脏细胞分化,提高成骨细胞和抑制破骨细胞的分化,预防激素相关的骨坏死以及通过ERK1/2信号通路诱导前列腺癌细胞细胞凋亡。先前的报道表明Icairitin在低浓度处理雌激素受体阳性的乳腺癌细胞MCF-7时表现出雌激素样活性。然而,在微摩尔浓度时抑制前列腺癌细胞PC-3的增殖。这些结果表明Icaritin在不同浓度时表现出雌激素样和抗雌激素样活性并且作为一种选择性雌激素受体调节剂调剂细胞的增殖。但目前国内外并没有Icaritin在子宫内膜癌细胞中研究的报道。丝裂原活化蛋白激酶(MAPK)参与多种细胞功能,如细胞增殖、分化、迁移和死亡。MAPK主要包括三个亚族,ERK1/2, JNK和p38蛋白激酶。JNK和p38激活后主要参与细胞凋亡和分化。先前的研究表明短暂的激活ERK1/2在细胞增殖中起作用,但持续激活ERK1/2诱导细胞周期阻滞和分化。在本研究中,我们检测了Icaritin对子宫内膜癌细胞HeclA增殖的影响,研究发现Icaritin能有效抑制HeclA细胞的生长,并同时抑制细胞周期蛋白cyclin D1和细胞周期蛋白依赖性激酶cdk4的表达,上调细胞周期抑制蛋白WAF1/p21和KIP1/p27的蛋白表达。接下来,研究发现Icaritin能诱导Hec1 A细胞凋亡,这个过程伴随着caspase 9和caspase 3的激活,PARP的裂解和细胞色素c的释放。如果我们提前加入广谱的caspase抑制剂z-VAD-fmk,则会抑制Icaritin诱导的细胞凋亡。Icaritin处理Hec1 A细胞后,抗凋亡蛋白Bcl-2蛋白表达水平下降,同时促凋亡蛋白Bax的表达水平上升。我们进一步的研究表明,在子宫内膜癌细胞系Hecl A中Icaritin能持续激活ERK1/2,并且ERK1/2的抑制剂U0126能抑制Icaritin激活ERK1/2,同时抑制了Icaritin诱导的Hecl A细胞的生长抑制和细胞凋亡。以上结果表明,Icaritin能通过线粒体途径诱导子宫内膜癌Hecl A细胞凋亡,并且持续激活ERK1/2在此凋亡过程中起着非常重要的作用,从而表明Icaritin有可能成为治疗子宫内膜癌的有效药物。更多还原

【Abstract】 Estrogen receptor (ER) belongs to the steroid hormone family of the nuclear receptor superfamily and expressed in various tissues.ER plays an important role in tissue development and tumor occurrence.ER consists of ER-a and ER-(3. Previously, we identified and cloned a novel variant of ER-a. ER-a36.ER-a36 gene is localized on chromososme 6q24.2-25.3 and contains a 5.4 kb cDNA, which encode a 310 amino acid open-deading frame and a a protein with a molecular weight of 36 kDa that is transcribed from previously unidentified promoter located in the first intron of the original 66 kDa ER-a (ER-a66) gene.ER-a36 lacks both transcriptional activation domains of ER-a66 (AF-1 and AF-2).but it retains the DNA-binding domain and partial ligand-binding domain. It possesses a unique 27 amino acid domain that replaces the last 138 amino acids encoded by exons 7 and 8 of the ER-a66 gene.The purpose of this study was to investigate the function and the underlying mechanisms of ER-a36 in growth regulation of endometrial Ishikawa cancer cells. The cellular localization of ER-a36 and ER-a66 were determined by immunofluorescence in the Ishikawa cells.Immunofluorescence staining of Ishikawa cells demonstrated that ER-a36 was expressed mainly on the plasma membrane and in the cytoplasm, while ER-a66 was predominantly localized in the cell nucleus. PKC isoforms are involved in a variety of cellular functions, including growth, differentiation, tumor promotion, aging, and apoptosis.Although early studies of the effect of PKC8 on cell proliferation suggested that PKC8 suppresses proliferation. However, several reports have demonstrated that PKC5 could act as a positive regulator of cell proliferation. Here, we found that both E2 and E2-BSA stimulated the activation of PKC5 signaling pathway in Ishikawa cells, and knockdown of ER-a36 expression with the shRNA abrogated E2-induced PKC5 phosphorylation. These indicated that ER-a36 mediates membrane-initiated PKC8 pathway induced by E2. In general, increased PKCαactivity is associated with increased motility and proliferation of cancer cells. In addition, PKCαhas been shown to inhibit or facilitate apoptosis of cancer cells. Here, we found that E2 was unable to induce PKCa phosphorylation. cAMP is a second messenger that plays a role in intracellular signal transduction of various stimuli. A major function of cAMP in eukaryotes is activation of cAMP-dependent protein kinase (PKA). In the present study, only E2 was able to induce cAMP-dependent protein kinase A (PKA) phosphorylation. E2-and E2-BSA-induced ERK phosphorylation required ER-α36 and PKCδ. Furthermore, E2 enhances cyclin D1/cdk4 expression via ER-α36.Our results demonstrated that E2 activates the PKCδ/ERK pathway and enhances cyclin D1/cdk4 expression via the membrane-initiated signaling pathways mediated by ER-α36, suggesting a possible involvement of ER-α36 in E2-dependent growth-promoting effects in endometrial cancer cells.Icaritin, a compound from Epimedium Genus, Icaritin exhibits many pharmacological and biological activities, such as stimulation of neuronal and cardiac differentiation, enhancement of osteoblastic and suppressed osteoclastic differentiation and activity, prevention of steroid-associated osteonecrosis, induction of human prostatic smooth muscle cells apoptosis via ERK1/2 pathway. Previously, it was reported that icaritin exhibits estrogen-like activity in estrogen receptor-positive breast cancer MCF-7 cells at sub-micromolar concentrations. At micromolar range, however, icaritin inhibited growth of prostate cancer PC-3 cells. These results indicated that icaritin has both agonist and antagonist activities depending on concentrations and may function as an estrogen receptor modulator to regulate cell growth. However, there are no reports on activity of Icaritin against endometrial cancer.Mitogen-activated protein (MAP) kinases participate in diverse cellular functions such as cell proliferation, cell differentiation, cell motility, and cell death. There are three major MAPK family subgroups:extracellular signal-regulated kinase1/2 (ERK1/2),c-Jun N-terminal of stress-activated protein kinasesl/2 (JNK1/2) and the p38 protein kinases. The signaling cascades involving JNK and p38, activated by extracellular stress signals, are involved in cell differentiation and apoptosis. Previous studies have demonstrated that transient activation of ERK1/2 plays a pivotal role in cell proliferation and that sustained ERK1/2 activation induces cell cycle arrest and differentiation.Here,we examined Icaritin effect on cell growth of human endometrial cancer Hecl A cells and found that Icaritin potently inhibited proliferation of Hecl A cells. Icaritin-inhibited cell growth was associated with increased levels of p21 and p27 expression and reduced cyclinDl and cdk 4 expression. Icaritin also induced cell apoptosis accompanied by activation of caspases as evidenced by the cleavage of endogenous substrate Poly (ADP-ribose) polymerase (PARP) and cytochrome c release, which was abrogated by pretreatment with the pan-caspase inhibitor z-VAD-fmk. Icaritin treatment also induced expression of pro-apoptotic protein Bax with a concomitant decrease of Bcl-2 expression. Furthermore, Icaritin induced sustained phosphorylation of extracellular signal-regulated kinase 1/2 (the MAPK/ERK1/2) in Hecl A cells and U0126, a specific MAP kinase kinase (MEK1/2) inhibitor, blocked the ERK activation by Icaritin and abolished the Icaritin-induced growth inhibition and apoptosis. Our results demonstrated that Icaritin induced sustained ERK activation and inhibited growth of endometrial cancer Hecl A cells, and provided a rational for preclinical and clinical evaluation of Icaritin for endometrial cancer therapy.更多还原