摘要
卵巢癌是女性生殖系统常见恶性肿瘤之一,其发病率居妇科肿瘤第三位；然而由于卵巢癌患者以腹水或盆腔包块就诊时,多数已是临床晚期,其死亡率高居妇科恶性肿瘤首位,5年生存率仍然徘徊在30%左右。目前尚缺乏对卵巢癌发生发展的确切分子机制的了解,因此,探索卵巢癌的发病机制,研究参与卵巢癌发生、发展的相关分子,可以为其诊断及靶向治疗奠定理论基础和提供实验依据。
肿瘤发生侵袭和转移是一个多因素参与的极其复杂的过程。研究表明,在诸多影响肿瘤发生、发展及转移的因素中,生长因子介导的细胞内信号转导通路同肿瘤的进展关系密切,一直是近年来肿瘤研究领域人们关注的热点。众多因子在其中发挥调节作用,其中肝细胞生长因子(Hepatocyte growth factor, HGF)是目前已知生物活性最广泛的生长因子之一。研究表明HGF在促进细胞分裂、增殖、分化、运动及形态的发生、血管生成等方面发挥重要的生物学功能。
HGF在细胞内可通过多条信号通路发挥调节作用,其中有丝分裂原活化蛋白激酶途径和3-磷脂酞肌醇激酶途径是最重要的两个生长因子信号转导通路,调节生长、增殖、分化及恶性转化等许多细胞过程,与肿瘤的恶性进展及预后关系密切。HGF作用于受体c-Met,刺激信号传入细胞内,启动系列的级联反应,导致多种底物蛋白磷酸化,从而引起细胞内一系列信号传导,引发下游某些特定分子的表达和生物学效应。然而,HGF发挥作用的分子调控机制尚不清楚,探讨HGF在卵巢癌侵袭生长过程中的作用及其信号传导途径,对于阐明卵巢癌发生发展的分子机制及靶向治疗有着重要意义。
凝血酶敏感蛋白1(Thrombospondin, TSP-1)是一具有多种生物学功能的糖蛋白,在调节肿瘤细胞的生长、粘附、凋亡等生物学行为和肿瘤新生血管形成中发挥重要作用,目前TSP-1与卵巢癌关系的研究仍局限于表达异常方面,TSP-1对卵巢癌细胞生物学功能影响的研究尚未见报道。研究发现TSP-1在细胞中的表达可能受到多种机制的调控,比如,生长因子、癌基因及抑癌基因等。目前有关HGF及TSP-1在卵巢癌的表达及其作用机制中可能存在的相互联系,国内外均未见报道。
本研究旨在探讨卵巢癌发病的分子机制,为研究治疗卵巢癌的方法提供新的特异性靶点。基于HGF及TSP-1在调控细胞凋亡、周期和侵袭生长中起着重要作用,本研究分为三部分：第一部分主要探讨HGF与TSP-1在卵巢癌组织中表达的相互关联；第二部分检测了HGF对卵巢癌SKOV-3细胞中TSP-1表达的调节作用,并构建了TSP-1真核表达载体转染SKOV-3细胞,进一步研究TSP-1在HGF调控下卵巢癌细胞生物学行为中的作用,初步探讨HGF的作用途径及机制；第三部分应用MAPK/ERK及PI3K/AKT信号通路的特异性阻断剂,研究了该信号通路在HGF介导的SKOV-3细胞生物学行为中的作用,为进一步了解HGF在卵巢癌发病进展中的作用机制提供新策略。
第一部分HGF及TSP-1在卵巢癌组织中的表达
【研究目的】
(1)检测HGF及TSP-1在卵巢癌及良性卵巢肿瘤组织中的表达水平,探讨两者与卵巢癌临床病理因素的关系。
(2)研究卵巢癌组织中HGF与TSP-1在蛋白表达水平的相关性,以进一步了解HGF与TSP-1之间存在的关系。
【研究方法】
(1)应用实时荧光定量PCR检测卵巢癌和良性卵巢肿瘤组织中HGF及TSP-1基因mRNA的表达。
(2)免疫组织化学法检测卵巢癌和良性卵巢肿瘤组织中HGF及TSP-1蛋白的定位及表达。
【结果】
(1)HGF及TSP-1 mRNA在卵巢癌和良性卵巢肿瘤组织中的表达：HGF mRNA在卵巢癌组织中的表达水平和阳性表达率均显著高于良性肿瘤组织P＜0.05),卵巢癌中的HGFmRNA的相对表达水平为7.43±0.90,HGF阳性率分别为89.47%及35%。而TSP-1 mRNA在卵巢癌组织中的表达水平和阳性表达率均低于良性肿瘤组织(P＜0.05),卵巢癌中的TSP-1 mRNA的相对表达水平仅为0.34±0.03,阳性率分别为36.84%及75%。
(2)HGF蛋白在卵巢癌和良性卵巢肿瘤组织中的表达：在卵巢良性肿瘤组织中,HGF蛋白在上皮细胞基本无着色,HGF阳性表达率为22.73%。在卵巢癌中,HGF呈现不同程度的高表达,主要表现为肿瘤细胞胞浆着色,阳性表达率为74.29%,显著高于良性肿瘤组织(P＜0.05)。卵巢癌组织中HGF蛋白的阳性表达与患者的年龄、病理分级无关,而与FIGO分期、淋巴结转移情况相关(P＜0.05)。
(3)TSP-1蛋白在卵巢癌和良性卵巢肿瘤组织中的表达：在卵巢良性肿瘤组织中,TSP-1蛋白在上皮细胞中多呈强阳性表达,阳性表达率为77.27%。在卵巢癌中TSP-1蛋白阳性表达部位主要在肿瘤细胞的胞浆内及少量间质,呈弱阳性表达,阳性表达率为40%,与良性肿瘤组织相比明显降低,差异有统计学意义(P＜0.05)。卵巢癌中TSP-1蛋白表达缺失与FIGO分期、病理分级明显相关(P＜0.05),而与年龄及淋巴结转移无关。
(4)HGF及TSP-1蛋白在卵巢癌组织中表达水平的相关性：HGF阳性表达的52例中,TSP-1阳性表达仅为14例；HGF阴性表达的18例中,TSP-1阳性表达14例。两者阳性表达强度在卵巢癌组织中呈负相关(rs=-0.575),且相关有显著性意义(P＜0.01)。
【结论】在卵巢癌组织中,HGF存在过度表达,而TSP-1表达下调,两者具有相关性。
第二部分HGF对卵巢癌细胞TSP-1表达的调控及其生物学行为的影响
【研究目的】
(1)检测外源性HGF对卵巢癌SKOV-3细胞株中TSP-1表达的调节。
(2)探讨TSP-1在HGF调控下的卵巢癌细胞生物学行为中的作用,对HGF调控卵巢癌细胞增殖、凋亡及侵袭的作用途径及可能机制做一初步探索。
【研究方法】
(1)实时定量PCR法及Western blot检测不同浓度HGF作用SKOV-3细胞不同时间对TSP-1mRNA及蛋白水平表达的影响。
(2)将空质粒pEGFP-N1及重组质粒pEGFP-N1-TSP-1分别转染入SKOV-3细胞,在倒置荧光显微镜下观察转染后TSP-1在SKOV-3细胞中的定位与表达,并用Western Blot法检测TSP-1蛋白的表达。
(3)转染后细胞分为如下6组：SKOV-3组;HGF+ SKOV-3组;SKOV-3/pEGFP组；HGF+ SKOV-3/pEGFP组;SKOV-3/TSP-1组;HGF+ SKOV-3/TSP-1组。进行以下SKOV-3细胞生物学行为的检测。
(4)MTT法检测TSP-1对HGF调控的SKOV-3细胞增殖活性的影响。
(5)流式细胞仪检测TSP-1对HGF调控的SKOV-3细胞周期分布及cyclin D1蛋白表达的影响。
(6)流式细胞仪检测TSP-1对HGF调控的SKOV-3细胞凋亡率的影响。
(7) Transwell实验检测TSP-1对HGF调控的SKOV-3细胞侵袭力及MMP-9含量的影响。
【结果】
(1)HGF下调卵巢癌SKOV-3细胞中TSP-1的表达水平：随着HGF作用浓度的增加及作用时间的延长,TSP-1mRNA表达水平逐渐降低(P<0.05), HGF对TSP-1mRNA表达的调控作用呈一定的剂量依赖性及时间依赖性。Western blot结果显示,TSP-1蛋白水平的变化与mRNA水平是一致的。
(2)重组质粒pEGFP-N1-TSP-1瞬时转染SKOV-3细胞：倒置荧光显微镜下显示SKOV-3/pEGFP细胞中,绿色荧光呈整个细胞内的均一弥散分布。而SKOV-3/TSP-1细胞,只在细胞浆中可见绿色荧光。Western blot结果表明,SKOV-3/TSP-1细胞中TSP-1蛋白的表达明显增加(P＜0.05)。
(3)TSP-1转染明显降低了HGF对SKOV-3细胞促增殖活性：细胞生长曲线显示,各组细胞中加入HGF后,细胞增殖明显加快(P＜0.05)。与HGF+SKOV-3/pEGFP组相比,HGF+SKOV-3/TSP-1组细胞增殖显著减弱(P＜0.05)。
(4)TSP-1转染明显改变了HGF调控的SKOV-3细胞周期分布及cyclin D1的蛋白表达：在HGF的刺激作用下,细胞的周期分布出现明显改变,各组S期细胞比例均有显著提高(P<0.05), cyclin D1表达也明显升高(P＜0.05)。HGF+SKOV-3/TSP-1组细胞与HGF+SKOV-3/pEGFP组相比,S期细胞比例及cyclin D1表达明显降低(P＜0.05)。
(5)TSP-1转染对HGF调控的SKOV-3细胞抑制凋亡作用无影响：HGF处理后各组细胞凋亡数明显减少(P＜0.05),但比较HGF+SKOV-3/TSP-1组与HGF+SKOV-3/pEGFP组发现,两组凋亡细胞数无显著差别(P＞0.05)。
(6)TSP-1转染显著减少了HGF对SKOV-3细胞侵袭力及上清液中MMP-9含量的影响：HGF处理后各组细胞透膜细胞数均有增加(P＜0.05),并且诱导MMP-9分泌增加。而比较HGF+SKOV-3/TSP-1组与HGF+SKOV-3/pEGFP组穿透细胞数及MMP-9分泌量可见,HGF+SKOV-3/TSP-1组细胞的侵袭能力及MMP-9含量明显降低(P＜0.05)。
【结论】HGF通过降调TSP-1的表达,促进卵巢癌细胞的增殖及侵袭,其机制可能与cyclin Dl和MMP-9表达上调有关。
第三部分MAPK/ERK信号通路在HGF调控卵巢癌细胞生物学行为中的作用
【研究目的】
探讨MAPK/ERK及PI3K/AKT通路在HGF对SKOV-3细胞中TSP-1的调控表达及诱导产生生物学行为中的作用。
【研究方法】
(1)Western blot检测外源性HGF及信号通路抑制剂对SKOV-3细胞中ERK1/2、AKT磷酸化及TSP-1蛋白表达的影响。
(2)MTT法检测MAPK/ERK信号通路对SKOV-3细胞增殖活性的影响。分组如下：空白对照组；HGF组；HGF+U0126组。
(3)流式细胞仪检测MAPK/ERK信号通路对SKOV-3细胞周期分布及cyclin D1蛋白表达的影响。
(4)流式细胞仪检测MAPK/ERK信号通路对SKOV-3细胞凋亡率的影响。
(5) Transwell体外实验检测MAPK/ERK信号通路对SKOV-3细胞侵袭力及MMP-9含量的影响。
【结果】
(1)HGF诱导SKOV-3细胞MAPK/ERK及PI3K/AKT信号通路的激活：HGF迅速诱导了SKOV-3细胞中p-ERK1/2及p-AKT的表达水平(P＜0.05)。p-AKT与p-ERK1/2的表达类似,在HGF刺激后5min即出现,在15min达到最大效应。
(2)U0126可以阻断HGF对TSP-1下调表达的调控：预先加入U0126或LY294002后,HGF则不能诱导ERK1／2及AKT发生磷酸化；预先加入U0126后,HGF下调TSP-1蛋白表达的作用消失(P<0.05)；而预先加入LY294002后,HGF依然可以下调TSP-1蛋白的表达。
(3)阻断MAPK/ERK信号通路明显降低了SKOV-3细胞增殖活性：U0126的预处理明显减少了HGF诱导的细胞增殖活性,与HGF组之间比较,差异有统计学意义(P＜0.05)。
(4)阻断MAPK/ERK信号通路明显改变了SKOV-3细胞周期分布及cyclin D1表达水平：U0126明显减少了HGF引起的细胞S期比例,与HGF组相比存在显著性差异(P＜0.05)；同时cyclin D1表达水平也明显减弱。
(5)阻断MAPK/ERK信号通路对SKOV-3细胞凋亡率无影响：U0126组与HGF组相比,两组之间凋亡细胞所占百分比无明显差异(P＞0.05)。
(6)阻断MAPK/ERK信号通路降低了SKOV-3细胞侵袭力及MMP-9的含量：U0126显著减少了HGF所诱导的侵袭细胞数量,与HGF组相比存在明显差异(P<0.05)。ELISA结果显示MMP-9水平的变化与侵袭细胞数目一致,HGF组与U0126组细胞上清液中MMP-9的分泌量比较,差异显著(P＜0.05)。
【结论】HGF通过MAPK/ERK信号通路实现对卵巢癌细胞TSP-1的表达调控。
Ovarian cancer is one of the common malignancy in female reproductive system tumors, and its incidence rate ranks third in gynecologic cancer; However, due to ovarian cancer patients with ascites or pelvic mass for treatment, the majority of the patients are clinical late stage already. Its mortality rate is the highest gynecological malignancies, and 5-year survival rate is still hovering at about 30%. There is currently a lack of the understanding of the exact molecular mechanism of occurrence and development in ovarian cancer. Therefore, to explore the pathogenesis of ovarian cancer and research on the related molecules involved in the occurrence and development of ovarian cancer can lay the theoretical foundation and provide experimental evidence for diagnosis and targeted treatment.
Tumor invasion and metastasis is an extremely complex process involved in multi-factors. Research shows that among in the many impact factors, growth factor-mediated intracellular signal transduction pathways closely related with tumor progression, which has been a hot spot of the cancer research field in recent years. A number of factors play regulatory roles, among which hepatocyte growth factor (HGF) is currently the most widely known biological activity of growth factors. Studies have shown that HGF play an important biological functions in promoting cell division, proliferation, differentiation, movement, morphology and angiogenesis, etc.
HGF play a regulatory role through multiple pathways in the cell. Mitogen-activated protein kinase pathway and phosphatidylinositol 3-kinase pathway are the two most important of growth factor signal transduction pathway, which regulate many cellular processes, such as growth, proliferation, differentiation and malignant transformation, and with close relation with the progress and prognosis of malignant tumors. HGF act on the receptor c-Met, stimulate the signals into the cells, start the series of cascade reactions, and lead to a variety of substrate phosphorylation, which led to a series of intracellular signal transduction and certain the expression of the downstream molecules and biological effects. However, the molecular regulation mechanism of HGF is not clear. To explore the role and signal transduction pathway of HGF in ovarian cancer invasion, there is of great significance to clarify the molecular mechanisms and targeted therapy for the occurrence and development of ovarian cancer.
Thrombospondin 1 (TSP-1) is the one glycoprotein with a variety of biological functions, and play an important role in regulating tumor cell growth, adhesion, apoptosis and other biological behaviors and tumor angiogenesis. In the current, the research of TSP-1 in ovarian cancer is still confined to the respect of abnormal expression, and the effect of TSP-1 on biological function of ovarian cancer cells has not been reported. Studies have found that TSP-1 expression in cells may be subject to the regulation of a variety of mechanisms, for example, growth factors, oncogenes and tumor suppressor genes. At present the HGF and TSP-1 expression in ovarian cancer and the possible association existing between each other in mechanism have not been reported.
This study was designed to explore the molecular mechanism of ovarian cancer, and provide new specific target for the study of the treatment for ovarian cancer. Based on the important roles of HGF and TSP-1 in the regulation of cell apoptosis, cycle and invasion, this study is divided into three parts:the first part investigated the relation between the expression of HGF and TSP-1 in ovarian cancer tissues; the second part detected the HGF regulation of TSP-1 expression in ovarian cancer SKOV-3 cells, and TSP-1 was constructed eukaryotic expression vector to transfect into SKOV-3 cells, to further study the role of TSP-1 in the HGF-regulated biological behaviors in ovarian cancer cells, for a preliminary study of the HGF-regulated mechanisms; the third part apply the specific blocking agent of MAPK/ERK and PI3K/AKT signaling pathways, and studied the relevance of HGF and its mediated downstream signaling pathways, to provide new strategies for further understanding of the mechanism of HGF function in ovarian cancer progression.
PARTⅠTHE EXPRESSION OF HGF AND TSP-1 IN OVARIAN CANCER
Objective:
(1) To detect the expression levels of HGF and TSP-1 in ovarian cancer and benign ovarian tumor tissue, and the relationship with the clinical pathological factors.
(2) To study the correlation between HGF and TSP-1 expression at the protein level in ovarian cancer tissue, to a better understanding the relationship of HGF and TSP-1.
Methods:
(1) Real-time quantitative PCR was applied to detect HGF and TSP-1 gene mRNA expression in ovarian cancer and benign ovarian tumor tissue.
(2) Immunohistochemistry was applied to detect the location and expression of HGF and TSP-1 protein in ovarian cancer and benign ovarian tumor tissue.
Results:
(1) The expression of HGF and TSP-1 mRNA in ovarian cancer and benign ovarian tumor tissues:the level and positive expression rate of HGF mRNA in ovarian cancer tissue was significantly higher than benign organizations (P<0.05), which was 7.43±0.90 compared with that in the benign ovarian tumors. HGF-positive rates were 89.47% and 35%, respectively. The level and positive expression rate of TSP-1 mRNA in ovarian cancer tissue was lower than benign organizations (P<0.05), whose positive rates were 75% and 36.84%, respectively. The TSP-1 relative expression level in ovarian cancer was only 0.34±0.03.
(2) The expression of HGF protein in ovarian cancer and benign ovarian tumor tissues: in ovarian benign tumor tissues, HGF protein was almost no color in epithelial cells, and HGF positive expression rate was 22.73%. In ovarian cancer, HGF showed high expression mainly in cytoplasm of tumor cells of varying degrees, and the positive expression rate was 74.29%, significantly higher than benign organizations (P<0.05). The positive expression of HGF protein in ovarian cancer was related with FIGO stage, and lymph node metastasis(P<0.05), but not with patient's age, pathological grading.
(3) The expression of TSP-1 protein in ovarian cancer and benign ovarian tumor tissues:in ovarian benign tumor tissues, TSP-1 protein showed strong positive expression in epithelial cells, and positive expression rate was 77.27%. In ovarian cancer, TSP-1 protein expressed weakly positive mainly in the cytoplasm of tumor cells and a small amount of mesenchymal, and positive expression rate was 40%, significantly lower than the benign organization (P<0.05). The absence expression of TSP-1 protein in ovarian cancer was significantly related with FIGO stage and histological grade(P<0.05), but not with age and lymph node metastasis.
(4) The correlation between HGF and TSP-1 protein expression in ovarian cancer tissue:among the 52 cases of HGF expression, TSP-1-positive expression was only 14 cases; among the 18 patients with negative HGF expression, TSP-1-positive expression was 14 cases. The intensity of these positive expression in ovarian cancer tissues were significant negatively associated with each other (rs =-0.575) (P<0.01).
Conclusions:In ovarian cancer, there exist HGF over-expression and TSP-1 down-regulation, which are related.
PARTⅡTHE REGULATION OF HGF ON TSP-1 EXPRESSION AND BIOLOGICAL BEHAVIORS IN OVARIAN CANCER CELLS
Objective:
(1) To detect the regulation of exogenous HGF on the expression of TSP-1 in ovarian cancer cell line SKOV-3.
(2) To explore the role of TSP-1 in HGF regulation of the biological behavior in ovarian cancer cells, for a preliminary exploration on the possible mechanism of HGF regulation of proliferation, apoptosis and invasion pathways.
Methods:
(1) Real-time quantitative PCR and Western blot were applied to detect the effects of different concentrations of HGF on the TSP-1 mRNA and protein expression levels in SKOV-3 cells at different time.
(2) Empty vector pEGFP-N1 and the recombinant plasmid pEGFP-N1-TSP-1 were transfected into SKOV-3 cells, observed the expression and localization of TSP-1 in SKOV-3 cells under the inverted fluorescence microscope, and detect TSP-1 protein expression by Western Blot.
(3) After transfection, cells were divided into the following six groups for the following detection of biological behavior:SKOV-3 group; HGF+SKOV-3 group; SKOV-3/pEGFP group; HGF+SKOV-3/pEGFP group; SKOV-3/TSP-1 group; HGF+SKOV-3/TSP-1 group.
(4) Detection the effect of TSP-1 on HGF-regulated proliferation activity in SKOV-3 cells by MTT assay.
(5) Detection the effect of TSP-1 on HGF-regulated cell cycle distribution and cyclin D1 protein expression in SKOV-3 cells by flow cytometry.
(6) Detection the effect of TSP-1 on HGF-regulated apoptosis rates in SKOV-3 cells by flow cytometry.
(7) Detection the effect of TSP-1 on HGF-regulated invasiveness and MMP-9 levels in SKOV-3 cells by transwell experiments and ELISA assay.
Results:
(1) HGF reduced the expression level of TSP-1 in SKOV-3 cells:With the increased concentration of HGF and extension of the stimulation time, TSP-1 mRNA expression levels decreased gradually (P<0.05). HGF regulation of TSP-1 mRNA expression were dose-dependent and time-dependent. Western blot results showed that TSP-1 protein level changes are the same with mRNA level.
(2) The recombinant plasmid pEGFP-N1-TSP-1 was transfected transiently into SKOV-3 cells:in SKOV-3/pEGFP cells, green fluorescence was uniformly dispersed throughout the intracellular distribution; while in SKOV-3/TSP-1 cells, green fluorescence was visible only in the cytoplasm under inverted fluorescence microscope. TSP-1 protein expression was significantly increased in SKOV-3/TSP-1 cells by Western blot (P<0.05).
(3) TSP-1 transfection significantly reduced the effect of HGF on promoting proliferation in SKOV-3 cells:cell growth curve showed that cells in each group of adding HGF, cell proliferation significantly accelerated (P<0.05). Compared with HGF+SKOV-3/pEGFP group, the proliferation of HGF+SKOV-3/TSP-1 cells was significantly reduced (P<0.05).
(4) TSP-1 transfection significantly altered the HGF-regulated cell cycle distribution and cyclin D1 protein expression in SKOV-3 cell:after HGF stimulation, the cell cycle distribution have apparent change, and S-phase fraction of each group were significantly increased (P<0.05). Cyclin D1 expression also increased significantly (P<0.05). Compared HGF+SKOV-3/TSP-1 group with HGF+SKOV-3/pEGFP group, S phase cells percentage and cyclin D1 expression were significantly lower (P<0.05).
(5) TSP-1 transfection had no effect on HGF regulation of apoptosis rate in SKOV-3 cells:HGF treatment significantly reduced the number of apoptosis cells (P<0.05), but we compare HGF+SKOV-3/TSP-1 group and HGF+SKOV-3/pEGFP group and found that the apoptotic cells of two groups has no significant difference (P>0.05).
(6) TSP-1 transfection significantly reduced the effect of HGF on the invasiveness and the supernatant MMP-9 content in SKOV-3 cells:HGF treated increased cell number to pass through membrane in each group (P<0.05), and induce MMP-9 secretion. In comparison penetrate cells and MMP-9 secretion in HGF+SKOV-3/TSP-1 group and HGF+SKOV-3/pEGFP group, we can see that the invasive ability and MMP-9 content were significantly lower in cells of HGF+SKOV-3/TSP-1 group (P<0.05).
Conclusions:HGF down-regulation the expression of TSP-1 may contribute to proliferation and invasion in ovarian cancer cells, and its mechanism may be related to the increase of cyclin D1 and MMP-9 expression.
PARTⅢTHE ROLE OF MAPK/ERK SIGNALING PATHWAY IN HGF-REGULATED BIOLOGICAL BEHAVIORS IN OVARIAN CANCER CELLS
Objective:
To investigate the role of MAPK/ERK and PI3K/AKT pathway in HGF regulation of TSP-1 expression and the induced biological behaviors.
Methods:
(1) Western blot was applied to detect the effect of exogenous HGF and signaling pathway inhibitors on ERK1/2、AKT phosphorylation and TSP-1 protein expression in SKOV-3 cells.
(2) Detection the effect of MAPK/ERK signaling pathway on proliferation activity in SKOV-3 cells by MTT assay. Group are as follows:control group; HGF group; HGF+U0126 group.
(3) Detection the effect of MAPK/ERK signaling pathway on cell cycle distribution and cyclin D1 protein expression in SKOV-3 cells by flow cytometry.
(4) Detection the effect of MAPK/ERK signaling pathway on cell apoptosis rates in SKOV-3 cells by flow cytometry.
(5) Detection the effect of MAPK/ERK signaling pathway on the invasiveness and MMP-9 content in SKOV-3 cells by transwell experiment and ELISA assay.
Results:
(1) HGF induced MAPK/ERK and PI3K/AKT signaling pathway activation in SKOV-3 cells:HGF rapidly induced p-ERK1/2 and p-AKT expression levels in SKOV-3 cells (P<0.05). P-AKT and p-ERK1/2 expression were similar, which appear after the HGF stimulation for 5min and achieve maximum effect at 15 min.
(2) U0126 block HGF down-regulation on TSP-1 expression:after pre-treatment with U0126 or LY294002, HGF can not induce ERK1/2 and AKT phosphorylation; after pre-treatment with U0126, HGF can not downregulate TSP-1 protein expression (P<0.05); while after pre-treatment with LY294002, HGF can still downregulate TSP-1 protein expression.
(3) Blocking MAPK/ERK signaling pathway significantly reduced proliferation activity of SKOV-3 cells:U0126 pretreatment significantly reduced the HGF-induced cell proliferation activity. Compared with HGF group, the difference was statistically significant (P<0.05).
(4) Blocking MAPK/ERK signaling pathway significantly altered cell cycle distribution and cyclin D1 protein expression of SKOV-3 cells:U0126 significantly reduced the HGF-induced S-phase cells fraction, and there was a significant difference compared to HGF group (P<0.05); the expression levels of cyclin D1 was also significantly decreased.
(5) Blocking MAPK/ERK signaling pathway had no effect on apoptosis rate of SKOV-3 cells:compared U0126 group with HGF group, the percentage of apoptotic cells between the two groups has no significant difference (P> 0.05).
(6) Blocking MAPK/ERK signaling pathway reduces the invasiveness and MMP-9 content of SKOV-3 cells:U0126 significantly reduced the HGF-induced invasive cells, and there is significant difference compared with HGF group (P<0.05). ELISA results showed that MMP-9 level was in line with the number of invasive cells. MMP-9 secretion in the supernatant of HGF group and the U0126 group cells, the difference was significant (P<0.05).
Conclusions:HGF activate the MAPK/ERK pathway and regulate TSP-1 expression level in ovarian cancer cells.
引文
1 Tingulstad S, Skjeldestad FE, Halvorsen TB, Hagen B. Survival and prognostic factors in patients with ovarian cancer. Obstet Gynecol 2003; 101:885-91.
2 Maughan K. Specific keynote:in the shadow of illness-supporting women with ovarian cancer. Gynecol Oncol 2003; 88:S129-33; discussion S34-5.
3 Cannistra SA. Cancer of the ovary. N Engl J Med 2004; 351:2519-29.
4 Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100:57-70.
5 Trusolino L, Comoglio PM. Scatter-factor and semaphorin receptors:cell signalling for invasive growth. Nat Rev Cancer 2002; 2:289-300.
6 Miyata Y, Ashida S, Nakamura T, Kanda S. Overexpression of hepatocyte growth factor receptor in renal carcinoma cells indirectly stimulates tumor growth in vivo. Biochem Biophys Res Commun.2003 Mar 21;302(4):892-7.
7 Maulik G, Shrikhande A, Salgia R. Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition. Cytokine Growth Factor Rev.2002 Feb;13(1):41-59.
8 Wong AS, Leung PC, Auersperg N. Hepatocyte growth factor promotes in vitro scattering and morphogenesis of human cervical carcinoma cells. Gynecol Oncol. 2000 Aug;78(2):158-65.
9 Michalopoulos GK, DeFrances MC. Liver regeneration. Science.1997 Apr 4;276(5309):60-6.
10 Bottaro DP, Rubin JS, Aaronson S A. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science.1991 Feb 15;251(4995):802-4.
11 Royal I, Fournier TM, Park M. Differential requirement of Grb2 and PI3-kinase in HGF/SF-induced cell motility and tubulogenesis. J Cell Physiol.1997 Nov; 173(2):196-201.
12 Jones NC, Fedorov YV, Olwin BB. ERK1/2 is required for myoblast proliferation but is dispensable for muscle gene expression and cell fusion. J Cell Physiol.2001 Jan;186(1):104-15.
13 Nakamura T, Komiya M, Mori N. Discrimination between phosphotyrosine-mediated signaling properties of conventional and neuronal Shc adapter molecules. Oncogene.2002 Jan 3;21(1):22-31.
14 Ueoka Y, Kato K, Wake N. Hepatocyte growth factor modulates motility and invasiveness of ovarian carcinomas via ras mediated pathway. Mol Cell Endocrinol. 2003 Apr 28;202(1-2):81-8.
15 Werner JA, Rathcke IO, Mandic R. The role of matrix metalloproteinases in squamous cell carcinomas of the head and neck. Clin Exp Metastasis. 2002;19(4):275-82.
16 Roberts DD. Regulation of tumor growth and metastasis by thrombospondin-1. FASEB J 1996; 10:1183-91.
17 Iruela-Arispe ML, Luque A, Lee N. Thrombospondin modules and angiogenesis. Int J Biochem Cell Biol 2004; 36:1070-8.
18 Michalopoulos G, Houck KA, Dolan ML, Leutteke NC. Control of hepatocyte replication by two serum factors. Cancer Res.1984 Oct;44(10):4414-9.
19 Chau GY, Lui WY, Wu CW. Significance of serum hepatocyte growth factor levels in patients with hepatocellular carcinoma undergoing hepatic resection. Eur J Surg Oncol.2008 Mar;34(3):333-8.
20 Wang P, Nishitani MA, Kanayama HO. Bladder cancer cell invasion is enhanced by cross-talk with fibroblasts through hepatocyte growth factor. Urology.2007 Apr;69(4):780-4.
21 Maulik G, Kijima T, Johnson BE. Modulation of the c-Met/hepatocyte growth factor pathway in small cell lung cancer. Clin Cancer Res.2002 Feb;8(2):620-7.
22 Seiden-Long IM, Tsao MS. Transcriptional targets of hepatocyte growth factor signaling and Ki-ras oncogene activation in colorectal cancer. Oncogene.2006 Jan 5;25(1):91-102.
23 Baykal C, Demirtas E, Ayhan A. Comparison of HGF (hepatocyte growth factor) levels of epithelial ovarian cancer cyst fluids with benign ovarian cysts. Int J Gynecol Cancer.2003 Nov-Dec;13(6):771-5.
24 Sowter HM, Corps AN, Smith SK. Hepatocyte growth factor (HGF) in ovarian epithelial tumour fluids stimulates the migration of ovarian carcinoma cells. Int J Cancer 1999; 83:476-80.
25 Parrott JA, Skinner MK. Expression and action of hepatocyte growth factor in human and bovine normal ovarian surface epithelium and ovarian cancer. Biol Reprod. 2000 Mar;62(3):491-500.
26 Toi M, Taniguchi T, Tominaga T. Significance of circulating hepatocyte growth factor level as a prognostic indicator in primary breast cancer. Clin Cancer Res.1998 Mar;4(3):659-64.
27 Olivero M, Rizzo M, Di Renzo MF. Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas. Br J Cancer 1996; 74:1862-1868.
28 Nagy J, Curry GW, George WD. Hepatocyte growth factor/scatter factor expression and c-met in primary breast cancer. Surg Oncol 1996; 5:15-21.
29 Dong G, Chen Z, Li ZY, Van Waes C. Hepatocyte growth factor/scatter factor-induced activation of MEK and PI3K signal pathways contributes to expression of proangiogenic cytokines interleukin-8 and vascular endothelial growth factor in head and neck squamous cell carcinoma. Cancer Res.2001 Aug 1;61(15):5911-8.
30 Bharti A, Ma PC, Salgia R. Haptoglobin alpha-subunit and hepatocyte growth factor can potentially serve as serum tumor biomarkers in small cell lung cancer. Anticancer Res.2004 Mar-Apr;24(2C):1031-8.
31 Ren Y, Cao B, Law S, Vande Woude G, Wong J. Hepatocyte growth factor promotes cancer cell migration and angiogenic factors expression:a prognostic marker of human esophageal squamous cell carcinomas. Clin Cancer Res.2005 Sep 1;11(17):6190-7.
32 Bertin N, Clezardin P, Kubiak R, Frappart L. Thrombospondin-1 and -2 messenger RNA expression in normal, benign, and neoplastic human breast tissues:correlation with prognostic factors, tumor angiogenesis, and fibroblastic desmoplasia. Cancer Res. 1997 Feb 1;57(3):396-9.
33 Tanaka K, Sonoo H, Yamamoto S. Inhibition of infiltration and angiogenesis by thrombospondin-1 in papillary thyroid carcinoma. Clin Cancer Res.2002 May;8(5):1125-31.
34 Kwak C, Jin RJ, Lee SE. Thrombospondin-1, vascular endothelial growth factor expression and their relationship with p53 status in prostate cancer and benign prostatic hyperplasia. BJU Int.2002 Feb;89(3):303-9.
35 Maeda K, Nishiguchi Y, Hirakawa K. Expression of vascular endothelial growth factor and thrombospondin-1 in colorectal carcinoma. Int J Mol Med.2000 Apr;5(4):373-8.
36 Karavasilis V, Malamou-Mitsi V, Pavlidis N. Clinicopathologic study of vascular endothelial growth factor, thrombospondin-1, and micro vessel density assessed by CD34 in patients with stage Ⅲ ovarian carcinoma. Int J Gynecol Cancer.2006 Jan-Feb;16 Suppl 1:241-6.
37 Alvarez AA, Axelrod JR, Whitaker RS, Isner PD, Bentley RC, Dodge RK, et al. Thrombospondin-1 expression in epithelial ovarian carcinoma:association with p53 status, tumor angiogenesis, and survival in platinum-treated patients. Gynecol Oncol 2001; 82:273-8.
38 Grossfeld GD, Ginsberg DA, Stein JP, Bochner BH, Esrig D, Groshen S, et al. Thrombospondin-1 expression in bladder cancer:association with p53 alterations, tumor angiogenesis, and tumor progression. J Natl Cancer Inst 1997; 89:219-27.
39 Raghavan D. Molecular targeting and pharmacogenomics in the management of advanced bladder cancer. Cancer.2003 Apr 15;97(8 Suppl):2083-9.
40 Yan M, Schneider J, Heffelfinger SC. Expression of angiogenic factors is upregulated in DMBA-induced rat mammary pathologies. Pathobiology. 2004;71(5):253-60.
41 Rofstad EK, Galappathi K, Mathiesen B. Thrombospondin-1 treatment prevents growth of dormant lung micrometastases after surgical resection and curative radiation therapy of the primary tumor in human melanoma xenografts. Int J Radiat Oncol Biol Phys.2004 Feb 1;58(2):493-9.
42 Straume O, Akslen LA. Strong expression of ID1 protein is associated with decreased survival, increased expression of ephrin-A1/EPHA2, and reduced thrombospondin-1 in malignant melanoma. Br J Cancer.2005 Oct 17;93(8):933-8.
1 Watnick RS, Cheng YN, Weinberg RA. Ras modulates Myc activity to repress thrombospondin-1 expression and increase tumor angiogenesis. Cancer Cell.2003 Mar;3(3):219-31.
2 Janz A, Sevignani C, Thomas-Tikhonenko A. Activation of the myc oncoprotein leads to increased turnover of thrombospondin-1 mRNA. Nucleic Acids Res.2000 Jun 1;28(11):2268-75.
3 Slack JL, Bornstein P. Transformation by v-src causes transient induction followed by repression of mouse thrombospondin-1. Cell Growth Differ.1994 Dec;5(12):1373-80.
4 Mettouchi A, Cabon F, Binetruy B. SPARC and thrombospondin genes are repressed by the c-jun oncogene in rat embryo fibroblasts. EMBO J.1994 Dec 1;13(23):5668-78.
5 Harada H, Nakagawa K, Ohnishi T. Introduction of wild-type p53 enhances thrombospondin-1 expression in human glioma cells. Cancer Lett.2003 Feb 28;191(1):109-19.
6 Chandrasekaran L, He CZ, Roberts DD. Cell contact-dependent activation of alpha3betal integrin modulates endothelial cell responses to thrombospondin-1. Mol Biol Cell.2000 Sep;11(9):2885-900.
7 Postovit LM, Seftor EA, Seftor RE, Hendrix MJ. Influence of the microenvironment on melanoma cell fate determination and phenotype. Cancer Res.2006 Aug 15;66(16):7833-6.
8 Van Golen CM, Schwab TS, Feldman EL. Insulin-like growth factor-Ⅰ receptor expression regulates neuroblastoma metastasis to bone. Cancer Res.2006 Jul 1;66(13):6570-8.
9 Sattler M, Salgia R. c-Met and hepatocyte growth factor:potential as novel targets in cancer therapy. Curr Oncol Rep.2007 Mar;9(2):102-8.
10 Scarpino S, Di Napoli A, Ruco LP. Hepatocyte growth factor (HGF) downregulates thrombospondin 1 (TSP-1) expression in thyroid papillary carcinoma cells. J Pathol.2005 Jan;205(1):50-6.
11 Horiguchi H, Jin L, Lloyd RV. Regulation of VEGF-A, VEGFR-I, thrombospondin-1,-2, and -3 expression in a human pituitary cell line (HP75) by TGFbeta1, bFGF, and EGF. Endocrine.2004 Jul;24(2):141-6.
12 Corps AN, Sowter HM, Smith SK. Hepatocyte growth factor stimulates motility, chemotaxis and mitogenesis in ovarian carcinoma cells expressing high levels of c-met. Int J Cancer 1997; 73:151-5.
13 Recio JA, Merlino G. Hepatocyte growth factor/scatter factor activates proliferation in melanoma cells through p38 MAPK, ATF-2 and cyclin D1. Oncogene. 2002 Feb 7;21(7):1000-8.
14 Paumelle R, Tulasne D, Fafeur V. Hepatocyte growth factor/scatter factor activates the ETS1 transcription factor by a RAS-RAF-MEK-ERK signaling pathway. Oncogene.2002 Apr 4;21(15):2309-19.
15 Peter M, Herskowitz I. Joining the complex:cyclin-dependent kinase inhibitory proteins and the cell cycle. Cell.1994 Oct 21;79(2):181-4.
16 Opitz OG, Suliman Y, Rustgi AK. Cyclin D1 overexpression and p53 inactivation immortalize primary oral keratinocytes by a telomerase-independent mechanism. J Clin Invest.2001 Sep;108(5):725-32.
17 Ruhul Quddus M, Latkovich P, Miranda RN. Expression of cyclin Dl in normal, metaplastic, hyperplastic endometrium and endometrioid carcinoma suggests a role in endometrial carcinogenesis. Arch Pathol Lab Med.2002 Apr;126(4):459-63.
18 Derksen PW, de Gorter DJ, Pals ST. The hepatocyte growth factor/Met pathway controls proliferation and apoptosis in multiple myeloma. Leukemia.2003 Apr;17(4):764-74.
19 Takami K, Takuwa N, Takizawa H. Interferon-gamma inhibits hepatocyte growth factor-stimulated cell proliferation of human bronchial epithelial cells:upregulation of p27(kip1) cyclin-dependent kinase inhibitor. Am J Respir Cell Mol Biol.2002 Feb;26(2):231-8.
20 Wang Y, Wang S, Sheibani N. Enhanced proangiogenic signaling in thrombospondin-1-deficient retinal endothelial cells. Microvasc Res.2006 May;71 (3):143-51.
21 Wang X, Zhou Y, Kim HP, Song R, Zarnegar R, Ryter SW, et al. Hepatocyte growth factor protects against hypoxia/reoxygenation-induced apoptosis in endothelial cells. J Biol Chem 2004; 279:5237-43.
22 Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev.1999 Aug 1;13(15):1899-911.
23 Shore GC, Viallet J. Modulating the bcl-2 family of apoptosis suppressors for potential therapeutic benefit in cancer. Hematology Am Soc Hematol Educ Program. 2005:226-30.
24 Srivastava M, Ahmad N, Gupta S, Mukhtar H. Involvement of Bcl-2 and Bax in photodynamic therapy-mediated apoptosis. Antisense Bcl-2 oligonucleotide sensitizes RIF 1 cells to photodynamic therapy apoptosis. J Biol Chem.2001 May 4;276(18):15481-8.
25 Nakagami H, Morishita R, Ogihara T. Hepatocyte growth factor prevents endothelial cell death through inhibition of bax translocation from cytosol to mitochondrial membrane. Diabetes.2002 Aug;51(8):2604-11.
26 Nakagami H, Morishita R, Ogihara T. Phosphorylation of p38 mitogen-activated protein kinase downstream of bax-caspase-3 pathway leads to cell death induced by high D-glucose in human endothelial cells. Diabetes.2001 Jun;50(6):1472-81.
27 Nor JE, Mitra RS, Polverini PJ. Thrombospondin-1 induces endothelial cell apoptosis and inhibits angiogenesis by activating the caspase death pathway. J Vasc Res.2000 May-Jun;37(3):209-18.
28 Lee CH, Wu CL, Shiau AL. Systemic administration of attenuated Salmonella choleraesuis carrying thrombospondin-1 gene leads to tumor-specific transgene expression, delayed tumor growth and prolonged survival in the murine melanoma model. Cancer Gene Ther.2005 Feb; 12(2):175-84.
29 Nakagawa T, Lan HY, Sautin YY. Role of ERK1/2 and p38 mitogen-activated protein kinases in the regulation of thrombospondin-1 by TGF-betal in rat proximal tubular cells and mouse fibroblasts. J Am Soc Nephrol.2005 Apr;16(4):899-904.
30 Bruel A, Touhami-Carrier M, Thomaidis A, Legrand C. Thrombospondin-1 (TSP-1) and TSP-1-derived heparin-binding peptides induce promyelocytic leukemia cell differentiation and apoptosis. Anticancer Res.2005 Mar-Apr;25(2A):757-64
31 Graf R, Freyberg M, Friedl P. Mechanosensitive induction of apoptosis in fibroblasts is regulated by thrombospondin-1 and integrin associated protein (CD47). Apoptosis.2002 Dec;7(6):493-8.
32 Jimenez B, Volpert OV, Bouck N. Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nat Med.2000 Jan;6(1):41-8.
33 Jimenez B, Volpert OV, Bouck N. c-Jun N-terminal kinase activation is required for the inhibition of neovascularization by thrombospondin-1. Oncogene.2001 Jun 7;20(26):3443-8.
34 Gallagher PG, Bao Y, Prorock A, Fox JW. Gene expression profiling reveals cross-talk between melanoma and fibroblasts:implications for host-tumor interactions in metastasis. Cancer Res.2005 May 15;65(10):4134-46.
35 Yasui W, Oue N, Aung PP, Nakayama H. Molecular-pathological prognostic factors of gastric cancer:a review. Gastric Cancer.2005;8(2):86-94.
36 Fidler IJ. The pathogenesis of cancer metastasis:the 'seed and soil' hypothesis revisited. Nat Rev Cancer.2003 Jun;3(6):453-8.
37 Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases:structure, function, and biochemistry. Circ Res 2003; 92:827-39.
38 Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer.2002 Mar;2(3):161-74.
39 Curran S, Murray GI. Matrix metalloproteinases in tumour invasion and metastasis. J Pathol.1999 Nov;189(3):300-8.
40 DeClerck YA. Interactions between tumour cells and stromal cells and proteolytic modification of the extracellular matrix by metalloproteinases in cancer. Eur J Cancer. 2000 Jun;36(10):1258-68.
41 Davidson B, Goldberg I, Nesland JM. The prognostic value of metalloproteinases and angiogenic factors in ovarian carcinoma. Mol Cell Endocrinol 2002; 187:39-45.
42 Gong R, Rifai A, Tolbert EM, Centracchio JN, Dworkin LD. Hepatocyte growth factor modulates matrix metalloproteinases and plasminogen activator/plasmin proteolytic pathways in progressive renal interstitial fibrosis. J Am Soc Nephrol 2003; 14:3047-60.
43 Zhou HY, Wong AS. Activation of p70S6K induces expression of matrix metalloproteinase 9 associated with hepatocyte growth factor-mediated invasion in human ovarian cancer cells. Endocrinology 2006; 147:2557-66.
44 Wong AS, Roskelley CD, Pelech S, Miller D, Leung PC, Auersperg N. Progressive changes in Met-dependent signaling in a human ovarian surface epithelial model of malignant transformation. Exp Cell Res 2004; 299:248-56.
45 Scarpino S, Stoppacciaro A, Colarossi C, Cancellario F, Marzullo A, Marchesi M, et al. Hepatocyte growth factor (HGF) stimulates tumour invasiveness in papillary carcinoma of the thyroid. J Pathol 1999; 189:570-5.
46 Monvoisin A, Bisson C, Rosenbaum J. Involvement of matrix metalloproteinase type-3 in hepatocyte growth factor-induced invasion of human hepatocellular carcinoma cells. Int J Cancer.2002 Jan 10;97(2):157-62.
47 Hanzawa M, Shindoh M, Higashino F, Yasuda M, Inoue N, Hida K, et al. Hepatocyte growth factor upregulates E1AF that induces oral squamous cell carcinoma cell invasion by activating matrix metalloproteinase genes. Carcinogenesis 2000; 21:1079-85.
48 Rodriguez-Manzaneque JC, Lane TF, Iruela-Arispe ML. Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor. Proc Natl Acad Sci U S A.2001 Oct 23;98(22):12485-90.
49 Ghoneim C, Soula-Rothhut M, Blanchevoye C, Martiny L, Antonicelli F, Rothhut B. Activating transcription factor-1-mediated hepatocyte growth factor-induced down-regulation of thrombospondin-1 expression leads to thyroid cancer cell invasion. J Biol Chem 2007; 282:15490-7.
1 Wu CJ, O'Rourke DM, Greene MI. The tyrosine phosphatase SHP-2 is required for mediating phosphatidylinositol 3-kinase/Akt activation by growth factors. Oncogene. 2001 Sep 20;20(42):6018-25.
2 Day RM, Cioce V, Bottaro DP. Differential signaling by alternative HGF isoforms through c-Met:activation of both MAP kinase and PI 3-kinase pathways is insufficient for mitogenesis. Oncogene.1999 Jun 3;18(22):3399-406.
3 Arbabi S, Maier RV. Mitogen-activated protein kinases. Crit Care Med.2002 Jan;30(1 Supp):S74-S79.
4 Park EM, Joh TH, Cho S. A neuroprotective role of extracellular signal-regulated kinase in N-acetyl-O-methyldopamine-treated hippocampal neurons after exposure to in vitro and in vivo ischemia. Neuroscience.2004; 123(1):147-54.
5 Martelli AM, Faenza I, Cocco L. Intranuclear 3'-phosphoinositide metabolism and Akt signaling:new mechanisms for tumorigenesis and protection against apoptosis? Cell Signal.2006 Aug; 18(8):1101-7.
6 Hu L, Zaloudek C, Jaffe RB. In vivo and in vitro ovarian carcinoma growth inhibition by a phosphatidylinositol 3-kinase inhibitor (LY294002). Clin Cancer Res. 2000 Mar;6(3):880-6.
7 Tsuboi Y, Ichida T, Sugitani S, Aoyagi Y. Overexpression of extracellular signal-regulated protein kinase and its correlation with proliferation in human hepatocellular carcinoma. Liver Int.2004 Oct;24(5):432-6.
8 Oka N, Nakahara S, Takenaka Y,, Raz A. Galectin-3 inhibits tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by activating Akt in human bladder carcinoma cells. Cancer Res.2005 Sep 1;65(17):7546-53.
9 Aoki K, Ogawa T, Ito Y, Nakashima S. Cisplatin activates survival signals in UM-SCC-23 squamous cell carcinoma and these signal pathways are amplified in cisplatin-resistant squamous cell carcinoma. Oncol Rep.2004 Feb;11(2):375-9.
10 Wang X, DeFrances MC, Zarnegar R. A mechanism of cell survival:sequestration of Fas by the HGF receptor Met. Mol Cell.2002 Feb;9(2):411-21.
11 Merkulova-Rainon T, England P, Tobelem G The N-terminal domain of hepatocyte growth factor inhibits the angiogenic behavior of endothelial cells independently from binding to the c-met receptor. J Biol Chem.2003 Sep 26;278(39):37400-8.
12 Wiesenauer CA, Yip-Schneider MT, Wang Y, Schmidt CM. Multiple anticancer effects of blocking MEK-ERK signaling in hepatocellular carcinoma. J Am Coll Surg. 2004 Mar; 198(3):410-21.
13 Liebmann C. Regulation of MAP kinase activity by peptide receptor signalling pathway:paradigms of multiplicity. Cell Signal.2001 Nov;13(11):777-85.
14 Lowes VL, Ip NY, Wong YH. Integration of signals from receptor tyrosine kinases and g protein-coupled receptors. Neurosignals.2002 Jan-Feb;11(1):5-19.
15 Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature.2001 Mar 1;410(6824):37-40.
16 Chong H, Vikis HG, Guan KL. Mechanisms of regulating the Raf kinase family. Cell Signal.2003 May;15(5):463-9.
17 Henson ES, Gibson SB. Surviving cell death through epidermal growth factor (EGF) signal transduction pathways:implications for cancer therapy. Cell Signal. 2006 Dec;18(12):2089-97.
18 Katso R, Okkenhaug K, Waterfield MD. Cellular function of phosphoinositide 3-kinases:implications for development, homeostasis, and cancer. Annu Rev Cell Dev Biol.2001;17:615-75.
19 Cantrell DA. Phosphoinositide 3-kinase signalling pathways. J Cell Sci.2001 Apr;114(Pt 8):1439-45.
20 Alessi DR, Cohen P. Mechanism of activation and function of protein kinase B. Curr Opin Genet Dev.1998 Feb;8(1):55-62.
21 Hemmings BA. Akt signaling:linking membrane events to life and death decisions. Science.1997 Jan 31;275(5300):628-30.
22 Chan TO, Rittenhouse SE, Tsichlis PN. AKT/PKB and other D3 phosphoinositide-regulated kinases:kinase activation by phosphoinositide-dependent phosphorylation. Annu Rev Biochem.1999;68:965-1014.
23 Kumar CC, Diao R, Xiao L. Expression, purification, characterization and homology modeling of active Akt/PKB, a key enzyme involved in cell survival signaling. Biochim Biophys Acta.2001 Jun 15;1526(3):257-68.
24 Zhang YW, Su Y, Volpert OV, Vande Woude GF. Hepatocyte growth factor/scatter factor mediates angiogenesis through positive VEGF and negative thrombospondin 1 regulation. Proc Natl Acad Sci U S A 2003; 100:12718-23.
25 Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science.2002 Dec 6;298(5600):1911-2.
26 Bild AH, Yao G, Chang JT, Wang Q. Oncogenic pathway signatures in human cancers as a guide to targeted therapies. Nature.2006 Jan 19;439(7074):353-7.
27 Hecquet C, Lefevre G, Mascarelli F. Activation and role of MAP kinase-dependent pathways in retinal pigment epithelial cells:ERK and RPE cell proliferation. Invest Ophthalmol Vis Sci.2002 Sep;43(9):3091-8.
28 Lunghi P, Tabilio A, Bonati A. Downmodulation of ERK activity inhibits the proliferation and induces the apoptosis of primary acute myelogenous leukemia blasts. Leukemia.2003 Sep; 17(9):1783-93.
29 Osada S, Kanematsu M, Sugiyama Y. Evaluation of extracellular signal regulated kinase expression and its relation to treatment of hepatocellular carcinoma. J Am Coll Surg.2005 Sep;201(3):405-11
30 Talarmin H, Rescan C, Baffet G. The mitogen-activated protein kinase kinase/extracellular signal-regulated kinase cascade activation is a key signalling pathway involved in the regulation of G(1) phase progression in proliferating hepatocytes. Mol Cell Biol.1999 Sep;19(9):6003-11.
31 Osada S, Saji S, Osada K. Critical role of extracellular signal-regulated kinase phosphorylation on menadione (vitamin K3) induced growth inhibition. Cancer.2001 Mar 15;91(6):1156-65.
32 Nam SY, Jung GA, Lee BL.Upregulation of FLIP(S) by Akt, a possible inhibition mechanism of TRAIL-induced apoptosis in human gastric cancers. Cancer Sci.2003 Dec;94(12):1066-73.
33 Kakazu A, Chandrasekher G, Bazan HE. HGF protects corneal epithelial cells from apoptosis by the PI-3K/Akt-1/Bad- but not the ERK 1/2-mediated signaling pathway. Invest Ophthalmol Vis Sci.2004 Oct;45(10):3485-92.
34 Xiao GH, Jeffers M, Bellacosa A, Testa JR. Anti-apoptotic signaling by hepatocyte growth factor/Met via the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways. Proc Natl Acad Sci U S A.2001 Jan 2;98(1):247-52.
35 Reddy KB, Nabha SM, Atanaskova N. Role of MAP kinase in tumor progression and invasion. Cancer Metastasis Rev 2003; 22:395-403.
36 Smalley KS. A pivotal role for ERK in the oncogenic behaviour of malignant melanoma? Int J Cancer 2003; 104:527-32.
37 McCawley LJ, Li S, Wattenberg EV, Hudson LG. Sustained activation of the mitogen-activated protein kinase pathway. A mechanism underlying receptor tyrosine kinase specificity for matrix metalloproteinase-9 induction and cell migration. J Biol Chem 1999; 274:4347-53.
38 Simon C, Hicks MJ, Boyd D. PD 098059, an inhibitor of ERK1 activation, attenuates the in vivo invasiveness of head and neck squamous cell carcinoma. Br J Cancer.1999 Jul;80(9):1412-9.
1. Maughan K. Specific keynote:in the shadow of illness-supporting women with ovarian cancer. Gynecol Oncol 2003; 88:S129-33; discussion S34-5.
2. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100:57-70.
3. Corps AN, Sowter HM, Smith SK. Hepatocyte growth factor stimulates motility, chemotaxis and mitogenesis in ovarian carcinoma cells expressing high levels of c-met. Int J Cancer 1997; 73:151-5.
4. Baykal C, Demirtas E, Al A, Ayhan A, Yuce K, Tulunay G, et al. Comparison of hepatocyte growth factor levels of epithelial ovarian cancer cyst fluids with benign ovarian cysts. Int J. Gynecol Cancer 2004; 14:152-6.
5. Walter-Yohrling J, Cao X, Callahan M, Weber W, Morgenbesser S, Madden SL, et al. Identification of genes expressed in malignant cells that promote invasion. Cancer Res 2003; 63:8939-47.
6. Nakamura T, Nishizawa T, Hagiya M, Seki T, Shimonishi M, Sugimura A, et al. Molecular cloning and expression of human hepatocyte growth factor. Nature 1989; 342:440-3.
7. Trusolino L, Comoglio PM. Scatter-factor and semaphorin receptors:cell signalling for invasive growth. Nat Rev Cancer 2002; 2:289-300.
8. Day RM, Cioce V, Breckenridge D, Castagnino P, Bottaro DP. Differential signaling by alternative HGF isoforms through c-Met:activation of both MAP kinase and PI 3-kinase pathways is insufficient for mitogenesis. Oncogene 1999; 18:3399-406.
9. Comoglio PM. Pathway specificity for Met signalling. Nat Cell Biol 2001; 3:E161-2.
10. Fan S, Wang JA, Yuan RQ, Ma YX, Meng Q, Erdos MR, et al. BRCA1 as a potential human prostate tumor suppressor:modulation of proliferation, damage responses and expression of cell regulatory proteins. Oncogene 1998; 16:3069-82.
11. Stoker M, Gherardi E, Perryman M, Gray J. Scatter factor is a fibroblast-derived modulator of epithelial cell mobility. Nature 1987; 327:239-42.
12. Scarpino S, Stoppacciaro A, Colarossi C, Cancellario F, Marzullo A, Marchesi M, et al. Hepatocyte growth factor (HGF) stimulates tumour invasiveness in papillary carcinoma of the thyroid. J Pathol 1999; 189:570-5.
13. Zhang YW, Su Y, Volpert OV, Vande Woude GF. Hepatocyte growth factor/scatter factor mediates angiogenesis through positive VEGF and negative thrombospondin 1 regulation. Proc Natl Acad Sci U S A 2003; 100:12718-23.
14. Scarpino S, Di Napoli A, Taraboletti G, Cancrini A, Ruco LP. Hepatocyte growth factor (HGF) downregulates thrombospondin 1 (TSP-1) expression in thyroid papillary carcinoma cells. J Pathol 2005; 205:50-6.
15. Tingulstad S, Skjeldestad FE, Halvorsen TB, Hagen B. Survival and prognostic factors in patients with ovarian cancer. Obstet Gynecol 2003; 101:885-91.
16. Cannistra SA. Cancer of the ovary. N Engl J Med 2004; 351:2519-29.
17. Vasey PA. Resistance to chemotherapy in advanced ovarian cancer:mechanisms and current strategies. Br J Cancer 2003; 89 Suppl 3:S23-8.
18. Parrott JA, Skinner MK. Expression and action of hepatocyte growth factor in human and bovine normal ovarian surface epithelium and ovarian cancer. Biol Reprod 2000; 62:491-500.
19. Sowter HM, Corps AN, Smith SK. Hepatocyte growth factor (HGF) in ovarian epithelial tumour fluids stimulates the migration of ovarian carcinoma cells. Int J Cancer 1999; 83:476-80.
20. Wang X, Zhou Y, Kim HP, Song R, Zarnegar R, Ryter SW, et al. Hepatocyte growth factor protects against hypoxia/reoxygenation-induced apoptosis in endothelial cells. J Biol Chem 2004; 279:5237-43.
21. Bardella C, Dettori D, Olivero M, Coltella N, Mazzone M, Di Renzo MF. The therapeutic potential of hepatocyte growth factor to sensitize ovarian cancer cells to cisplatin and paclitaxel in vivo. Clin Cancer Res 2007; 13:2191-8.
22. Rasola A, Anguissola S, Ferrero N, Gramaglia D, Maffe A, Maggiora P, et al. Hepatocyte growth factor sensitizes human ovarian carcinoma cell lines to paclitaxel and cisplatin. Cancer Res 2004; 64:1744-50.
23. Coltella N, Rasola A, Nano E, Bardella C, Fassetta M, Filigheddu N, et al. p38 MAPK turns hepatocyte growth factor to a death signal that commits ovarian cancer cells to chemotherapy-induced apoptosis. Int J Cancer 2006; 118:2981-90.
24. Kikuchi T, Maemondo M, Narumi K, Matsumoto K, Nakamura T, Nukiwa T. Tumor suppression induced by intratumor administration of adenovirus vector expressing NK4, a 4-kringle antagonist of hepatocyte growth factor, and naive dendritic cells. Blood 2002; 100:3950-9.
25. Saga Y, Mizukami H, Suzuki M, Urabe M, Kume A, Nakamura T, et al. Expression of HGF/NK4 in ovarian cancer cells suppresses intraperitoneal dissemination and extends host survival. Gene Ther 2001; 8:1450-5.
26. Wong AS, Roskelley CD, Pelech S, Miller D, Leung PC, Auersperg N. Progressive changes in Met-dependent signaling in a human ovarian surface epithelial model of malignant transformation. Exp Cell Res 2004; 299:248-56.
27. Maggiora P, Lorenzato A, Fracchioli S, Costa B, Castagnaro M, Arisio R, et al. The RON and MET oncogenes are co-expressed in human ovarian carcinomas and cooperate in activating invasiveness. Exp Cell Res 2003; 288:382-9.
28. Yabkowitz R, Mansfield PJ, Dixit VM, Suchard SJ. Motility of human carcinoma cells in response to thrombospondin:relationship to metastatic potential and thrombospondin structural domains. Cancer Res 1993; 53:378-87.
29. Roberts DD. Regulation of tumor growth and metastasis by thrombospondin-1. FASEB J 1996; 10:1183-91.
30. Iruela-Arispe ML, Luque A, Lee N. Thrombospondin modules and angiogenesis. Int J Biochem Cell Biol 2004; 36:1070-8.
31. Sheibani N, Frazier WA. Thrombospondin 1 expression in transformed endothelial cells restores a normal phenotype and suppresses their tumorigenesis. Proc Natl Acad Sci U S A 1995; 92:6788-92.
32. Bleuel K, Popp S, Fusenig NE, Stanbridge EJ, Boukamp P. Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization. Proc Natl Acad Sci U S A 1999; 96:2065-70.
33. Guo N, Zabrenetzky VS, Chandrasekaran L, Sipes JM, Lawler J, Krutzsch HC, et al. Differential roles of protein kinase C and pertussis toxin-sensitive G-binding proteins in modulation of melanoma cell proliferation and motility by thrombospondin 1. Cancer Res 1998; 58:3154-62.
34. Alvarez AA, Axelrod JR, Whitaker RS, Isner PD, Bentley RC, Dodge RK, et al. Thrombospondin-1 expression in epithelial ovarian carcinoma:association with p53 status, tumor angiogenesis, and survival in platinum-treated patients. Gynecol Oncol 2001; 82:273-8.
35. Grossfeld GD, Ginsberg DA, Stein JP, Bochner BH, Esrig D, Groshen S, et al. Thrombospondin-1 expression in bladder cancer:association with p53 alterations, tumor angiogenesis, and tumor progression. J Natl Cancer Inst 1997; 89:219-27.
36. Zabrenetzky V, Harris CC, Steeg PS, Roberts DD. Expression of the extracellular matrix molecule thrombospondin inversely correlates with malignant progression in melanoma, lung and breast carcinoma cell lines. Int J Cancer 1994; 59:191-5.
37. Campbell SC, Volpert OV, Ivanovich M, Bouck NP. Molecular mediators of angiogenesis in bladder cancer. Cancer Res 1998; 58:1298-304.
38. Morandi V, Cherradi SE, Lambert S, Fauvel-Lafeve F, Legrand YJ, Legrand C. Proinflammatory cytokines (interleukin-1 beta and tumor necrosis factor-alpha) down regulate synthesis and secretion of thrombospondin by human endothelial cells. J Cell Physiol 1994; 160:367-77.
39. Bouck N, Stellmach V, Hsu SC. How tumors become angiogenic. Adv Cancer Res 1996; 69:135-74.
40. Ghoneim C, Soula-Rothhut M, Blanchevoye C, Martiny L, Antonicelli F, Rothhut B. Activating transcription factor-1-mediated hepatocyte growth factor-induced down-regulation of thrombospondin-1 expression leads to thyroid cancer cell invasion. J Biol Chem 2007; 282:15490-7.
41. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases:structure, function, and biochemistry. Circ Res 2003; 92:827-39.
42. Bar JK, Grelewski P, Popiela A, Noga L, Rabczynski J. Type IV collagen and CD44v6 expression in benign, malignant primary and metastatic ovarian tumors: correlation with Ki-67 and p53 immunoreactivity. Gynecol Oncol 2004; 95:23-31.
43. Davidson B, Goldberg I, Gotlieb WH, Kopolovic J, Ben-Baruch G, Nesland JM, et al. The prognostic value of metalloproteinases and angiogenic factors in ovarian carcinoma. Mol Cell Endocrinol 2002; 187:39-45.
44. Gong R, Rifai A, Tolbert EM, Centracchio JN, Dworkin LD. Hepatocyte growth factor modulates matrix metalloproteinases and plasminogen activator/plasmin proteolytic pathways in progressive renal interstitial fibrosis. J Am Soc Nephrol 2003; 14:3047-60.
45. Zhou HY, Wong AS. Activation of p70S6K induces expression of matrix metalloproteinase 9 associated with hepatocyte growth factor-mediated invasion in human ovarian cancer cells. Endocrinology 2006; 147:2557-66.
46. Trusolino L, Cavassa S, Angelini P, Ando M, Bertotti A, Comoglio PM, et al. HGF/scatter factor selectively promotes cell invasion by increasing integrin avidity. FASEB J 2000; 14:1629-40.
47. Hanzawa M, Shindoh M, Higashino F, Yasuda M, Inoue N, Hida K, et al. Hepatocyte growth factor upregulates E1AF that induces oral squamous cell carcinoma cell invasion by activating matrix metalloproteinase genes. Carcinogenesis 2000; 21:1079-85.
48. Bein K, Simons M. Thrombospondin type 1 repeats interact with matrix metalloproteinase 2. Regulation of metalloproteinase activity. J Biol Chem 2000; 275:32167-73.
49. Qian X, Wang TN, Rothman VL, Nicosia RF, Tuszynski GP. Thrombospondin-1 modulates angiogenesis in vitro by up-regulation of matrix metalloproteinase-9 in endothelial cells. Exp Cell Res 1997; 235:403-12.
50. Qian X, Rothman VL, Nicosia RF, Tuszynski GP. Expression of thrombospondin-1 in human pancreatic adenocarcinomas:role in matrix metalloproteinase-9 production. Pathol Oncol Res 2001; 7:251-9.
51. Ahmed N, Pansino F, Baker M, Rice G, Quinn M. Association between alphavbeta6 integrin expression, elevated p42/44 kDa MAPK, and plasminogen-dependent matrix degradation in ovarian cancer. J Cell Biochem 2002; 84:675-86.
52. Lee KH, Hyun MS, Kim JR. Growth factor-dependent activation of the MAPK pathway in human pancreatic cancer:MEK/ERK and p38 MAP kinase interaction in uPA synthesis. Clin Exp Metastasis 2003; 20:499-505.
53. McCawley LJ, Li S, Wattenberg EV, Hudson LG.Sustained activation of the mitogen-activated protein kinase pathway. A mechanism underlying receptor tyrosine kinase specificity for matrix metalloproteinase-9 induction and cell migration. J Biol Chem 1999; 274:4347-53.
54. Gum R, Wang H, Lengyel E, Juarez J, Boyd D. Regulation of 92 kDa type Ⅳ collagenase expression by the jun aminoterminal kinase-and the extracellular signal-regulated kinase-dependent signaling cascades. Oncogene 1997; 14:1481-93.
55. McCawley LJ, Li S, Benavidez M, Halbleib J, Wattenberg EV, Hudson LG. Elevation of intracellular cAMP inhibits growth factor-mediated matrix metalloproteinase-9 induction and keratinocyte migration. Mol Pharmacol 2000; 58:145-51.
56. Zhou HY, Wan KF, Ip CK, Wong CK, Mak NK, Lo KW, et al. Hepatocyte growth factor enhances proteolysis and invasiveness of human nasopharyngeal cancer cells through activation of PI3K and JNK. FEBS Lett 2008; 582:3415-22.
57. Zeigler ME, Chi Y, Schmidt T, Varani J. Role of ERK and JNK pathways in regulating cell motility and matrix metalloproteinase 9 production in growth factor-stimulated human epidermal keratinocytes. J Cell Physiol 1999; 180:271-84.
58. Reddy KB, Nabha SM, Atanaskova N. Role of MAP kinase in tumor progression and invasion. Cancer Metastasis Rev 2003; 22:395-403.
59. Smalley KS. A pivotal role for ERK in the oncogenic behaviour of malignant melanoma? Int J Cancer 2003; 104:527-32.
60. Suthiphongchai T, Promyart P, Virochrut S, Tohtong R, Wilairat P. Involvement of ERK1/2 in invasiveness and metastatic development of rat prostatic adenocarcinoma. Oncol Res 2003; 13:253-9.
61. Choo MK, Sakurai H, Koizumi K, Saiki I. Stimulation of cultured colon 26 cells with TNF-alpha promotes lung metastasis through the extracellular signal-regulated kinase pathway. Cancer Lett 2005; 230:47-56.
62. Liu Z, Klominek J. Regulation of matrix metalloprotease activity in malignant mesothelioma cell lines by growth factors. Thorax 2003; 58:198-203.
1 Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics,2009. CA Cancer J Clin.2009 Jul-Aug;59(4):225-49.
2 Van Golen CM, Schwab TS, Feldman EL. Insulin-like growth factor-I receptor expression regulates neuroblastoma metastasis to bone. Cancer Res.2006 Jul 1;66(13):6570-8.
3 Miyata Y, Ashida S, Nakamura T, Kanda S. Overexpression of hepatocyte growth factor receptor in renal carcinoma cells indirectly stimulates tumor growth in vivo. Biochem Biophys Res Commun.2003 Mar 21;302(4):892-7.
4 Chau GY, Lui WY, Wu CW. Significance of serum hepatocyte growth factor levels in patients with hepatocellular carcinoma undergoing hepatic resection. Eur J Surg Oncol.2008 Mar;34(3):333-8.
5 Maulik G, Shrikhande A, Salgia R. Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition. Cytokine Growth Factor Rev.2002 Feb;13(1):41-59.
6 Wong AS, Leung PC, Auersperg N. Hepatocyte growth factor promotes in vitro scattering and morphogenesis of human cervical carcinoma cells. Gynecol Oncol. 2000 Aug;78(2):158-65.
7 Iruela-Arispe ML, Luque A, Lee N. Thrombospondin modules and angiogenesis. Int J Biochem Cell Biol 2004; 36:1070-8.
8 Yabkowitz R, Mansfield PJ, Dixit VM, Suchard SJ. Motility of human carcinoma cells in response to thrombospondin:relationship to metastatic potential and thrombospondin structural domains. Cancer Res 1993; 53:378-87.
9 Sheibani N, Frazier WA. Thrombospondin 1 expression in transformed endothelial cells restores a normal phenotype and suppresses their tumorigenesis. Proc Natl Acad Sci U S A 1995; 92:6788-92.
10 Bleuel K, Popp S, Fusenig NE, Stanbridge EJ, Boukamp P. Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization. Proc Natl Acad Sci U S A 1999; 96:2065-70.
11 Zhou HY, Wong AS. Activation of p70S6K induces expression of matrix metalloproteinase 9 associated with hepatocyte growth factor-mediated invasion in human ovarian cancer cells. Endocrinology 2006; 147:2557-66.
12 Trusolino L, Comoglio PM. Scatter-factor and semaphorin receptors:cell signalling for invasive growth. Nat Rev Cancer 2002; 2:289-300.
13 Gallagher PG, Bao Y, Prorock A, Fox JW. Gene expression profiling reveals cross-talk between melanoma and fibroblasts:implications for host-tumor interactions in metastasis. Cancer Res.2005 May 15;65(10):4134-46.
14 Parrott JA, Skinner MK. Expression and action of hepatocyte growth factor in human and bovine normal ovarian surface epithelium and ovarian cancer. Biol Reprod. 2000 Mar;62(3):491-500.
15 Ren Y, Cao B, Law S, Vande Woude G, Wong J. Hepatocyte growth factor promotes cancer cell migration and angiogenic factors expression:a prognostic marker of human esophageal squamous cell carcinomas. Clin Cancer Res.2005 Sep 1;11(17):6190-7.
16 Toi M, Taniguchi T, Tominaga T. Significance of circulating hepatocyte growth factor level as a prognostic indicator in primary breast cancer. Clin Cancer Res.1998 Mar;4(3):659-64.
17 Baykal C, Demirtas E, Al A, Ayhan A, Yuce K, Tulunay G, et al. Comparison of hepatocyte growth factor levels of epithelial ovarian cancer cyst fluids with benign ovarian cysts. Int J Gynecol Cancer 2004; 14:152-6.
18 Sowter HM, Corps AN, Smith SK. Hepatocyte growth factor (HGF) in ovarian epithelial tumour fluids stimulates the migration of ovarian carcinoma cells. Int J Cancer 1999; 83:476-80.
19 Nagy J, Curry GW, George WD. Hepatocyte growth factor/scatter factor expression and c-met in primary breast cancer. Surg Oncol 1996; 5:15-21.
20 Olivero M, Rizzo M, Di Renzo MF. Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas. Br J Cancer 1996; 74:1862-1868.
21 Roberts DD. Regulation of tumor growth and metastasis by thrombospondin-1. FASEB J 1996; 10:1183-91.
22 Alvarez AA, Axelrod JR, Whitaker RS, Isner PD, Bentley RC, Dodge RK, et al. Thrombospondin-1 expression in epithelial ovarian carcinoma:association with p53 status, tumor angiogenesis, and survival in platinum-treated patients. Gynecol Oncol 2001; 82:273-8.
23 Karavasilis V, Malamou-Mitsi V, Pavlidis N. Clinicopathologic study of vascular endothelial growth factor, thrombospondin-1, and micro vessel density assessed by CD34 in patients with stage Ⅲ ovarian carcinoma. Int J Gynecol Cancer.2006 Jan-Feb;16 Suppl 1:241-6.
24 Grossfeld GD, Ginsberg DA, Stein JP, Bochner BH, Esrig D, Groshen S, et al. Thrombospondin-1 expression in bladder cancer:association with p53 alterations, tumor angiogenesis, and tumor progression. J Natl Cancer Inst 1997; 89:219-27.
25 Zabrenetzky V, Harris CC, Steeg PS, Roberts DD. Expression of the extracellular matrix molecule thrombospondin inversely correlates with malignant progression in melanoma, lung and breast carcinoma cell lines. Int J Cancer 1994; 59:191-5.
26 Bertin N, Clezardin P, Kubiak R, Frappart L. Thrombospondin-1 and -2 messenger RNA expression in normal, benign, and neoplastic human breast tissues:correlation with prognostic factors, tumor angiogenesis, and fibroblastic desmoplasia. Cancer Res. 1997 Feb 1;57(3):396-9.
27 Tanaka K, Sonoo H, Yamamoto S. Inhibition of infiltration and angiogenesis by thrombospondin-1 in papillary thyroid carcinoma. Clin Cancer Res.2002 May;8(5):1125-31.
28 Kwak C, Jin RJ, Lee SE. Thrombospondin-1, vascular endothelial growth factor expression and their relationship with p53 status in prostate cancer and benign prostatic hyperplasia. BJU Int.2002 Feb;89(3):303-9.
29 Maeda K, Nishiguchi Y, Hirakawa K. Expression of vascular endothelial growth factor and thrombospondin-1 in colorectal carcinoma. Int J Mol Med.2000 Apr;5(4):373-8.