EphA2/EphrinA1在肾癌中的表达及其在转移中的作用机制研究
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摘要
肾癌是起源于肾小管上皮细胞的恶性肿瘤,占肾脏恶性肿瘤的80%～90%,成人恶性肿瘤的2%～3%。随着生活水平的提高和医学影像学的进步,在早期诊断和早期手术方面均取得满意效果。但对于术后复发和伴有转移灶的晚期肾癌患者目前尚缺乏较好的治疗方案,临床上通常以临床分期和病理分级作为判断预后的依据,有一定的局限性。因此,寻找预测肾癌恶性程度的生物学标记物,已成为肾癌研究的热点,以便为肾癌的生物治疗提供新靶点和新思路。
促红细胞生成素产生肝细胞受体(erythropoietin producinghepatoma receptor,Eph)家族是一类新发现的酪氨酸蛋白激酶受体(receptor tyrosine kinases,RTKs)。EphA2是其中最早被发现的一个成员,在正常上皮细胞中表达,广泛高表达于不同的肿瘤组织(乳腺癌、结肠癌、食管癌、前列腺癌、恶性间皮癌等)和细胞系。EphA2在正常上皮细胞中位于细胞连接处,配体EphrinA1能以较高的亲和力与EphA2结合,两者结合后能导致受体和配体均发生酪氨酸磷酸化而产生双向信号传导,通过其下游信号调节细胞正常的生长、发育,同时也促进EphA2受体自身的降解。在恶性肿瘤细胞中EphA2定位异常,由细胞连接处转变为广泛分布于细胞表面,导致EphA2难以有效的与其配体接触,因而其自身的降解减少,所以在许多侵袭性肿瘤中EphA2和EphrinA1通常是过表达的。有学者通过一种模拟配体EphrinA1-Fc干预肿瘤细胞株(PC-3、293、Cos-7),显示EphA2受体活化、磷酸化程度增强,细胞形态学发生变化。因此,EphA2在肿瘤研究中被学者广泛关注。
有报道,肾癌组织中EphA2高表达可能与肾癌术后无瘤生存期有关,但关于EphA2及配体EphrinA1相互作用的生物学变化及配受体在肾癌中表达和机制研究国内外报道较少。一般认为肿瘤组织类型及恶性程度与疾病的转归和预后有关,在肾癌的不同病理类型中以肾透明细胞癌所占比例较高,为此,本课题通过对EphA2及其配体EphrinA1在肾透明细胞癌组织中的表达及与肾癌临床病理特征、E-cadherin和微血管密度(microvessel density,MVD)关系的研究,探讨EphA2及其配体EphrinA1在肾癌转移中的作用;并通过人工配体EphrinA1-Fc对人肾透明细胞癌786-O细胞系和裸鼠人肾癌细胞移植瘤干预和治疗研究,探讨EphA2与配体EphrinA1结合的生物学效应及其抗肿瘤的分子机制,为EphA2/EphrinA1在肾癌中表达及其在肾癌转移中的作用机制研究提供实验依据。
第一部分EphA2/EphrinA1在肾癌中的表达及生物学意义
目的:通过检测EphA2、EphrinA1在正常肾组织和肾透明细胞癌组织中蛋白和基因表达情况,探讨其在肾癌高表达的机制。
方法:采用半定量逆转录-聚合酶链反应(RT-PCR)和流式细胞术检测22例肾癌组织和12例非肾癌正常肾组织中EphA2和EphrinA1的基因及蛋白表达情况,并对其进行比较和相关分析。
结果:
1 RT-PCR显示:肾癌组织与正常肾组织中均有EphA2和EphrinA1的基因表达,但肾癌组织中EphA2和EphrinA1的mRNA的相对表达量明显高于正常肾组织(1.60±0.80 vs 0.58±0.19,P＜0.01;1.37±0.63vs 0.91±0.40,P＜0.01)。
2流式定量显示:肾癌组织中EphA2和EphrinA1蛋白的表达量明显高于正常肾组织(424.38±43.14 vs 332.44±42.83,P＜0.01;419.44±52.13 vs 325.36±42.33,P＜0.01)。
3相关分析显示,肾癌组织中EphA2、EphrinA1蛋白表达量与其mRNA表达量均无明显相关(r=0.05,P=0.826;r=-0.171,P=0.448)。
小结:EphA2和EphrinA1蛋白高表达于肾癌组织,其机制除与基因表达上调有关外,蛋白质生物学异常也可能是导致其升高的重要因素,提示EphA2和EphrinA1蛋白高表达在肿瘤发病中可能具有重要意义。
第二部分EphA2/EphrinA1在肾癌转移中的作用
目的:通过对EphA2及其配体EphrinA1在肾癌组织中的表达与肾癌患者临床病理特征、E-cadherin和MVD的关系研究,探讨EphA2及其配体EphrinA1在肾癌转移中的作用。
方法:利用免疫组织化学法检测68例肾透明癌组织、24例正常肾组织中EphA2、EphrinA1和E-cadherin的表达;并采用CD34抗体标记微血管内皮细胞,计算微血管密度(microvessel density,MVD)。分析EphA2、EphrinA1蛋白的表达与肾癌临床病理特征、E-cadherin、MVD之间的关系。
结果:
1 EphA2、EphrinA1在肾癌组织中的表达及其与肾癌临床病理特征的关系
免疫组化结果显示:EphA2、EphrinA1蛋白阳性表达在胞浆和胞膜。EphA2和EphrinA1蛋白的表达在肿瘤组织分级不同的各组间有显著性的差异(x~2=12.611,P=0.002;x~2=8.473,P=0.014),随着等级的增加其蛋白表达量也显著增加(r=0.431,P=0.000;r=0.355,P=0.003;);EphA2和EphrinA1蛋白的表达在高分期(Ⅲ、Ⅳ期)组显著高于低分期(Ⅰ、Ⅱ期)组(Z=-2.975,P=0.003;Z=-2.006,P=0.045),淋巴结转移组显著高于无淋巴结转移组的(Z=-2.320,P=0.02;Z=-2.792,P=0.005),肿瘤直径＞7cm与≤7cm两组间、年龄≥45岁与＜45岁两组间、男女性别两组间其蛋白的表达均无显著性差异(P值均＞0.05)。
2 E-cadherin在肾癌组织中的表达及其与肾癌临床病理学特征的关系
免疫组化结果显示:E-cadherin蛋白阳性表达在胞膜,呈棕黄色颗粒。在肾癌组织中,E-cadherin蛋白的表达明显低于正常肾组织(Z=-6.101,P＜0.0001)。
E-cadherin蛋白的表达在肿瘤组织分级不同的各组间有显著性的差异(x~2=8.493,P=0.014),随着等级的增加,其表达量显著降低(r=-0.320,P=0.008);E-cadherin蛋白的表达在高分期组、淋巴结转移组显著低于低分期组、无淋巴结转移组(Z=-2.037,P=0.042;Z=-2.217,P=0.027);肿瘤直径＞7cm与≤7cm两组间、年龄≥45岁与＜45岁两组间、男女性别两组间蛋白的表达均无显著性差异(P值均＞0.05)。
3 MVD在肾癌组织中的表达及其与肾癌临床病理学特征的关系
免疫组化结果显示:CD34主要表达于血管内皮细胞膜。微血管染色呈棕黄色,最密集染色区域即“热点”主要位于癌灶边缘。肾癌组织中MVD显著高于正常肾组织(P＜0.01)。在肿瘤组织分级不同的各组间,MVD有显著性差异(x~2=23.637,P＜0.01),并且随着分级等级的增加MVD也显著增加(r=0.592,P＜0.0001);MVD在肾癌组织的高分期组、淋巴结转移组、肿瘤直径＞7cm组显著高于低分期组、无淋巴结转移组、肿瘤直径≤7cm组(Z=3.735,P＜0.0001;Z=-3.4,P=0.001;Z=-3.522,P＜0.0001)。年龄≥45岁与＜45岁两组间、男女性别不同的两组间MVD均无显著性差异(P值均＞0.05)。
4 EphA2、EphrinA1和E-cadherin蛋白、MVD的相关性
相关分析显示,68例肾癌组织中EphA2和EphrinA1蛋白表达呈显著正相关(r=0.772,P＜0.0001);E-cadherin蛋白的表达与EphA2、EphrinA1蛋白的表达均呈显著负相关(r=-0.378,r=-0.414,P值均＜0.01);EphA2和EphrinA1蛋白的表达与MVD呈显著正相关(r=0.555,r=0.485,P值均＜0.01)。
小结:EphA2和EphrinA1蛋白高表达于肿瘤高分期组、高分级组和淋巴结转移组,与E-cadherin蛋白表达呈显著负相关,与MVD呈显著正相关,提示EphA2和EphrinA1蛋白的高表达预示肿瘤恶性程度的增加,其机制可能通过影响或协同E-cadherin和微血管生成来实现的。
第三部分EphrinA1-Fc对人肾透明癌786-O细胞系抗肿瘤效应机制的研究
目的:通过人工可溶性配体EphrinA1-Fc干预人肾透明细胞癌786-O细胞系抗肿瘤效应及其分子机制的实验研究,探讨EphA2在肿瘤细胞增殖、转移中的作用及机制。
方法:应用可溶性配体EphrinA1-Fc对人肾透明细胞癌786-O细胞系进行体外干预实验,观察不同时间点细胞形态学的变化,对不同时间点细胞内EphA2和ERK1/2的磷酸化程度进行分析,并观察可溶性配体反复刺激786-O后的细胞增殖情况。
结果:
1 EphrinA1-Fc干预后细胞形态学变化
正常条件下培养的肾癌细胞接受可溶性配体EphrinA1-Fc刺激1min,细胞形态即发生改变,细胞由梭形伸展状态开始回缩;5min时部分细胞变圆;30min时90%细胞变圆、与瓶壁结合疏松;40min时部分细胞重新伸展、粘附于瓶壁;60min时90%细胞恢复到刺激前状态。对照组细胞形态学未发生明显变化。
2 EphrinA1-Fc干预对磷酸化EphA2(p-EphA2)的影响
Western blotting结果显示:EphrinA1-Fc干预5min、10min、30min、60min后p-EphA2的相对表达量(p-EphA2/EphA2)分别是0.15±0.04、0.19±0.01、0.21±0.03和0.10±0.00。p-EphA2的相对表达量各组间有显著性差异(F=9.392,P=0.025)。组间两两比较,除5min与60min组、10min与30min组间无显著性差异,余各组间比较均有显著性差异(P值均＜0.05)。EphrinA1-Fc干预前及对照组不同时间点均未见其表达。
3 EphrinA1-Fc干预对磷酸化ERK1/2(p-ERK)的影响
Western blotting结果显示:EphrinA1-Fc干预5min、10min、30min、60min后p-ERK的相对表达量(p-ERN/ERK1/2)分别是0.15±0.07、0.22±0.06、0.26±0.03和0.13±0.04。p-EphA2的相对表达量各组间有显著性差异(F=4.428,P=0.041)。组间两两比较,5min与30min组、60min与30min组间比较均有显著性差异(P值均＜0.05),余各组间无显著差异(P值均＞0.05)。EphrinA1-Fc干预前及对照组不同时间点均未见其表达。
4 EphrinA1-Fc干预对细胞增殖的影响
EphrinA1-Fc干预人肾透明细胞癌786-O细胞株12天,两组细胞形态均未发生改变,但细胞的增殖状况发生了变化。EphrinA1-Fc组细胞数明显低于对照组((1.96±0.29)×10~5vs(3.14±0.49)×10~5,P＜0.01)。
小结:EphrinA1-Fc能抑制786-O细胞株的增殖、降低细胞黏附,其机制可能是通过影响胞内EphA2、ERK磷酸化实现的。
第四部分EphrinA1-Fc对裸鼠人肾癌细胞移植瘤的影响
目的:探讨EphrinA1-Fc对裸鼠人肾癌细胞移植瘤的影响及其作用机制。
方法:将表达EphA2抗原阳性的人肾透明细胞癌786-O细胞株体外培养后接种于裸鼠皮下,建立动物移植瘤模型,观察实验组(瘤周注射EphrinA1-Fc)和对照组(瘤周注射IgG-Fc)移植瘤的生长情况,并采用免疫组化法检测EphA2、E-cadherin及MVD的表达情况。
结果:
1裸鼠人肾癌细胞移植瘤模型的建立
14只裸鼠接种786-O细胞株7周,共8只裸鼠在注射部位发现米粒大小结节,表明人肾癌细胞移植瘤模型建立成功,接种成功率为60%。
2 EphrinA1-Fc对裸鼠人肾癌细胞移植瘤生长影响
接种786-O细胞株8周,移植瘤平均体积为(382.35±122.04)mm~3;干预治疗4周,裸鼠一般状态良好,未见明显的异常反应。
重复测量设计方差分析显示,时间和药物干预因素对移植瘤体积均有影响(F=4.085,P=0.012;F=7.469,P=0.034)。独立样本的非参数检验显示,治疗组干预治疗2周,移植瘤体积较对照组无明显差异[(272.65±55.35)mm~3 vs(535.97±216.07)mm~3,P=0.191)],干预治疗4周,移植瘤体积较对照组明显减小[(115.10±49.16)mm~3 vs(800.00±269.69)mm~3,P=0.021)]。肿瘤抑瘤率为80.62%。
3 EphrinA1-Fc对裸鼠人肾癌细胞移植瘤中EphA2、E-cadherin和MVD的影响
干预治疗4周,治疗组瘤内的EphA2蛋白的表达低于对照组;E-cadherin蛋白的表达高于对照组;治疗组MVD较对照组减少。
小结:表达EphA2阳性的肾透明细胞癌786-O具有致瘤性,但成瘤率低且成瘤时间长;瘤周注射EphrinA1-Fc可抑制肿瘤细胞的生长,其机制可能通过增加E-cadherin蛋白表达和抑制肿瘤血管生成实现的,反证了EphA2和EphrinA1的高表达可能通过促进细胞增殖、降低细胞与细胞黏附、促进血管生成促进人类肾癌的发生。
结论:
1、EphA2/EphrinA1基因和蛋白高表达于肾癌组织。其蛋白高表达的机制可能是自身降解下降、基因表达上调共同参与的结果。
2、EphA2/EphrinA1基因和蛋白高表达促进了肾癌细胞增殖、肿瘤新生血管生成,增强了肿瘤细胞与ECM黏附,削弱了细胞间连接,从而促进人类肾癌的发生和转移。
3、人工可溶性配体EphrinA1-Fc能抑制肾癌细胞的生长,为生物靶向治疗提供了动物实验依据。
4、EphA2和EphrinA1蛋白的高表达预示肾癌细胞侵袭能力强。联合检测EphA2、EphrinA1和E-cadherin三种蛋白的表达对于评价肾癌的恶性程度、判断其转移的危险性,可能具有一定的参考价值。
Renal cell carcinoma(RCC) is one kind of malignant tumor from the tubular epithelium of the renal parenchyma,accounting for roughly 80～90% and 2～3%of the renal and adult malignant tumors,respectively.With the improvement of living standards and advance in the medical imaging,early diagnosis and surgical treatment have produced satisfactory results.However, good therapeutic regimens are currently unavailable for the post-operation recurrence and the late stage cases with metastasis.Prediction of the prognosis still depends on the clinical stage and pathological grade,which showed certain limitations.The search of biological markers that may help to predict the degree of the malignance has therefore become a hot topic in the studies of the RCC.Such works may provide new targeting sites and approaches for the biological treatment of the RCC.
The EPH(erythropoietin-producing hepatocyte) receptors represent the largest known family of receptor tyrosine kinases.EphA2 is one of the earliest found and expressed in normal endothelial cells.It is also widely over-expressed in various tumor tissues including the mammary cancer, carcinoma of colon,esophageal cancer,prostatic cancer and mesothelial carcinoma,and cell lines.The EphA2 receptor located in the cell junction in normal epitheliums and,after binding with its EphrinA1 ligand under relatively high affinity and the ensuing tyrosine phosphorylation of both with bidirectional signal conduction,regulates the normal cell growth and development through its downstream signal pathway,and simultaneously promotes the degradation of the EphA2 receptor.The location of the EphA2 receptor is aberrant in the malignant tumors:Instead of the cell junction,it is widely distributed in the cell surface which prevented the effective binding with the EphA2 and reduced the degradation of the receptor per se.The EphA2 and EphrinA1 are thus usually over-expressed in many invasive tumors.By affecting the tumor cell lines(PC-3,293,Cos-7) with EphrinA1-Fc,a simulated ligand,investigators recorded rapid and transient actions of EphA2 receptor activation,enhanced phosphorylation,and morphological changes of the cells.So,EphA2 is now under the spotlight in the tumor research.
It is reported that over-expression of the EphA2 in the RCC may be associated with the tumor free survival after operation.However,biological changes due to the interaction of the EphA2 and its ligand,and EphA2/AphrinA1 in the RCC are rarely studied in and outside China.It is generally considered tissue type and degree of the tumor may predispose to the outcome and prognosis of the disease.Pathologically,renal clear cell carcinoma is proportionally more in the renal tumors.The current work thus examined the role of the EphA2 and its ligand EphrinA1 in the metastasis of the tumor,by studying the expression of the EphA2 and EphrinA1 in the RCC tissue,clinicopathological features,E-cadherin and microvessel density (MVD).Secondly,the biological effects of the EphA2-EphrinA1 binding and the molecular mechanism of its antitumor action were examined.The latter work was aimed to provide experimental evidence for the expression of the Eph/EphrinA1 in the RCC and its mechanism of action in the metastasis of the RCC.
PartⅠ:The effect of EphA2/EphrinA1 in the occurrence of the RCC
Objective:To detect the protein expression of EphA2/EphrinA1 in the normal renal tissue and the RCC,explore its role in the occurrence of RCC.
Methods:Use the Reversed Transcript-Polymerase Chain Reaction (RT-PCR) and flow cytometry to detect mRNA of the EphA2 and EphrinA1, and the protein expression in 22 cases of RCC and 12 cases of normal renal tissue.
Results:
1 RT-PCR results showed that in both normal group and RCC group express EphA2/EphrinA1 gene,but the renal group has express significantly higher EphA2/EphrinA1 mRNA expression than the normal group(P＜0.01),there was a significant difference.(1.60±0.80 vs 0.58±0.19;1.37±0.63vs 0.91±0.40,P＜0.01)
2 The flow cytometry showed that the expression of the EphA2/EphrinA1 was significantly higher in the RCC group(424.38±43.14 vs 332.44±42.83,P＜0.01;419.44±52.13 vs 325.36±42.33,P＜0.01).
3 Correlation analysis showed that in the RCC,EphA2/EphrinA1 protein expression and its mRNA expression were not significantly correlated (r=0.05,P=0.826;r=-0.171,P=0.448).
Conclusion:The expression of the EphA2/EphrinA1 gene and protein in the RCC was higher than those in the normal renal tissue.But in the RCC,EphA2/EphrinA1 protein expression and its mRNA expression were not significantly correlated,suggesting that high protein expression is not only associated with the high gene expression,but also with the post-transcriptional regulation and the biological abnormalities of EphA2/EphrinA1.
PartⅡ:The role of EphA2/EphrinA1 in the invasion and metastasis of the RCC
Objective:To detect the expression of EphA2/EphrinA1 in the RCC and its relationship with the clinicopathological characteristics,E-cadherin and MVD, to explore the role of EphA2/EphrinA1 in the invasion and metastasis of RCC.
Methods:Using immunohistochemistry,we detected EphA2/EphrinA1 and E-cadherin expression in 68 cases of RCC and 24 cases of normal renal tissue, also use CD34 antibody to mark microvascular endothelial cells and calculate microvessel density(MVD).Explore the relationship of EphA2/EphrinA1 expression with clinicopathological features and analyze its correlation with E-cadherin,MVD.
Results:
1 The relationship between clinicopathological features and EphA2/EphrinA1 expression in RCC.
Immunohistochemistry results showed that:In the RCC,different grade have different protein expression of EphA2/EphrinA1(x~2=12.611,P=0.002; x~2=8.473,P=0.014),With the increase in grade levels,the protein expression of EphA2/EphrinA1 increase significantly(r=0.431,P=0.000;r=0.355, P=0.003;);stageⅢandⅣhave significantly higher protein expression of EphA2/EphrinA1 than stageⅠandⅡ(Z=-2.975,P=0.003;Z=-2.006,P=0.045); lymph node metastasis group has significantly higher protein expression of EphA2/EphrinA1 than those without lymph node metastasis (Z=-2.320,P=0.02;Z=-2.792,P=0.005);in tumor diameter≥7cm and＜7cm between the two groups,≥45 years and＜45 years old between the two groups, sex between two groups have no significantly difference about the expression of protein(P＞0.05).
2 The expression of the E-cadherin in the normal renal tissue and RCC,and its relationship with clinicopathological features.
Immunohistochemistry results showed that:positive expression of the E-cadherin protein at the membrane as brown particles.The RCC expressed significantly lower E-cadherin protein than the normal renal tissue (Z=-6.101,P＜0.0001).
The expression of E-cadherin has the significant difference(x~2=8.493, P=0.014),and with the increase in grade levels,the expression of E-cadherin decrease significantly(r=-0.320,P=0.008);the expression of E-cadherin in high-stage group and lymph node metastasis group is significantly lower than the low-grade group and without lymph node metastasis group(Z=-2.037, P=0.042;Z=-2.217,P=0.027).In tumor diameter≥7 cm and＜7cm between the two groups,≥45-year-old and＜45 years of age between the two groups, sex between two groups of protein expression were not significantly different (P＞0.05).
3 MVD in the normal kidney and RCC and its relationship with the clinicopathological characteristics of the RCC.
Immunohistochemistry results showed:CD34 was mainly expressed at the vascular endothelial cell membrane.Microvessel staining was brown,the most densely stained region,e.g."hot spots",was mainly located at the border of tumors,cancer and cancer-intensive areas in the nest CD34 expression was significantly less than the cancer and hyperplasia weeks active region.MVD of tumor tissues was significantly higher than the normal renal tissue(P <0.01).In different grade of the tumor group,MVD have the significant difference(x~2=23.637,P<0.01).With the increase in grade levels,MVD increases significantly(r=0.592,P<0.0001).MVD in the RCC in the group of high-grade,high-stage group,with lymph node metastasis was also significantly higher than low-grade group,low stage and without lymph node metastasis group(Z=3.735,P<0.0001;Z=-3.4,P=0.001;Z=-3.522,P<0.0001);≥45-year-old and <45 years of age between the two groups,sex between two groups of MVD were not significantly difference(P>0.05).
4 Correlation of the EphA2,EphrinA1,E-cadherin protein and MVD expression.
Correlation analysis showed that in 68 cases of RCC,the protein expression of the EphA2 and EphrinA1 was significantly correlated(r=0.772, P<0.0001),while the protein expression of the E-cadherin was significantly and negatively correlated with that of the EphA2 and EphrinA1(r=-0.378, r=-0.414,P<0.01);EphA2,EphrinA1 expression and MVD were significantly correlated(r=0.555,r=0.485,P<0.01).
Conclusion:EphA2/EphrinA1 was highly expressed in tumors with high stage,high grade and with lymph nodes metastasis.EphA2/EphrinA1 was negatively correlated with E-cadherin expression and positively correlated with MVD,indicating that high expression of the EphA2/EphrinA1 protein may predict the degree of malignance of the tumor,possibly through the influence of or the coordination with the E-cadherin and the angiogenesis of microvasculatures.
PartⅢ:The anti-cancer effect of the EphrinA1-Fc to the 786-O RCC cells and its molecular mechanism.
Objective:Using EphrinA1-Fc to intervene the 786-O renal clear cell carcinoma in vitro study to examine the role and the possible molecular mechanism of the EphrinA-2.
Methods:Using the soluble ligand EphrinA1-Fc to intervene the 786-O renal carcinoma cells in vitro to observe the morphological changes in the cells at different time points,by the use of different technologies such as cell counting,Western blotting,immunoprecipitation to temporally examine the phosphorylation of EphA2,ERK1/2 and other changes.
Results:
1 The changes in the cell morphology after the EphrinA1-Fc intervention:
Use soluble ligand EphrinA1-Fc to stimulate renal cancer cells.After 1min, the renal cells exhibited morphological change,shrinking from the normal extended shape of spindle;at 5min,part of the cells appeared to be round;at 30min 90%of the cells were round and loosely attached to the flask;at 40min part of the cells re-extended and adhered to the flask;at 60min 90%of the cells recovered to the pre-stimulation shape.In the control group,there were no detectable morphological changes.
2 Impact of the EphrinA1-Fc on the p-EphA2
Western blotting results showed that:EphrinA1-Fc at 5min、10min、30min、60min intervention,p-EphA2 expression(p-EphA2/EphA2)was 0.15±0.04, 0.19±0.01,0.21±0.03和0.10±0.00,there is significant difference in different groups(F=9.392,P=0.025).Compared among the different groups,5 min and 60min between the two groups,10min and 30min between two groups were not significantly different(P＞0.05),other groups have the significant difference(P＜0.05).The pre-intervention group and the control group have no expression of the p-EphA2.
3 Impact of the EphrinA1-Fc on the phosphorylation of the ERK
Western blotting results showed that:EphrinA1-Fc at 5min、10min、30min、60min intervention,p-ERK expression levels were 0.15±0.07,0.22±0.06,0.26±0.03 and 0.13±0.04.p-ERK expression levels were relative,representing a marked increase in the pre-intervention,there was significant difference (F=4.428,P=0.041).Compared among the different groups,5 min and 30min between the two groups,60min and 30min between two groups were significantly different(P＜0.05).The pre-intervention group and the control group have no expression of the p-ERK.
4 Impact of the EphrinA1-Fc on the cell proliferation
After repeatedly intervention of the EphrinA1-Fc in the 786-O RCC cell in 12 days,cell growth but not cell morphology showed significant change:the number of cells decreased significantly in the EphrinA1-Fc group[(1.96±0.29)×10~5 vs(3.14±0.49)×10~5,P＜0.01].
Conclusion:Addition of the EphrinA1-Fc to the 786-O RCC cell could inhibit cell proliferation and reduce cell adhesion,possibly by affecting the phosphorylation of the intracellular EphA2 and ERK.
PartⅣEffects of the EphrinA1-Fe on the nude mice with transplanted human RCC
Objective:To investigate the effects of the EphrinA1-Fc on the nude mice with transplanted human RCC and its possible mechanism of action.
Methods:the animal model of the transplanted tumor is established by subcutaneous inoculation to nude mice of the human RCC cell 786-O,which is positive for the EphA2 antigen and cultivated in vitro.The growth of the transplanted tumor was observed in both the experimental group(injection of the EphrinA1-Fc in the periphery of the tumor) and the control group (injection of the IgG-Fc in the periphery of the tumor).Expression of EphA2, p-ERK,E-cadherin and MVD was examined by immunohistochemistry
Results:
1 The establishment of the nude mice model of transplanted human RCC
7 weeks after the inoculation of the 786-O cells,nodules about the size of grain were found at the injection site in 8 nude mice,indicating the successful establishing of nude mice model of transplanted human RCC(60%of success rate).
2 Effects of the EphrinA1-Fc on the growth of the transplanted RCC in the nude mice
8 weeks after the inoculation,the average tumor volume was(382.35±122.04) mm~3;4 weeks after the application of the EphrinA1-Fc,tumor-bearing mice in each group were in generally good condition with no obvious side effects.
Time and medical intervention could affect the volume of the transplanted tumor(F=4.085,P=0.012;F=7.469,P=0.034,by ANOVA).Nonparametic test of independent samples showed that,in comparison to that of the control group,the volume of the transplanted tumor was significantly reduced at 4 but not 2 weeks after medical intervention[(115.10±49.16) mm~3 vs(800.00±269.69) mm~3,P=0.021 and(272.65±55.35) mm~3 vs(535.97±216.07) mm~3,P= 0.191],respectively,resulting in a inhibition rate of 80.62%.
3 Effects of the EphrinA1-Fc on the EphA2,E-cadherin and MVD in the transplanted RCC in the nude mice.
4 weeks after the medical intervention,protein expression of the EphA2 and MVD was lower,and protein expression of the E-cadherin was higher the treatment group than the corresponding value in the control group.
Conclusion:RCC 786-O of EphA2 positive was oncogenic with low probability of tumor onset and long time of tumor development;the growth of the tumor could be inhibited by the injection of the EphrinA1-Fc in the periphery of the tumor,its possible mechanism was the up-regulation of the protein expression of the E-cadherin and inhibition of the angiogenesis in the tumor.These findings reciprocally indicated that high expression of the EphA2 and EphrinA1 may contribute to the onset of RCC by promoting cell proliferation and decreasing cell adhesion.
Conclusion:
1 The expression of EphA2/EphrinA1 in RCC increases significantly.In additional to the up-regulation of the gene expression,the possible mechanism also includes abnormality in the biological aspects of the protein.
2 High protein expression of the EphA2/EphrinA1 may predict the malignance of the RCC.and joint examination of the EphA2,EphrinA1 and E-cadherin may be useful for the evaluation of malignancy of RCC and the determination of its metastatic potential.
3 EphrinA1-Fc,artificial ligand,was first applied to RCC cell line and nude mice with transplanted RCC,reciprocally indicating that high expression of the EphA2 and EphrinA1 may contribute to the onset of RCC by promoting cell proliferation and increasing cell adhesion.Altered E-cadherin and microvessel growth factor,resulted from the biological abnormality of the EphA2 and EphrinA1,may be one important mechanism enhancing the tumor metastasis.
4 The binding of the EphA2 and EphrinA1 may inhibit the proliferation of the tumor by increasing the phosphorylation of the AphA2 and ERK.
促红细胞生成素产生肝细胞受体(erythropoietin producinghepatoma receptor,Eph)家族是一类新发现的酪氨酸蛋白激酶受体(receptor tyrosine kinases,RTKs)。EphA2是其中最早被发现的一个成员,在正常上皮细胞中表达,广泛高表达于不同的肿瘤组织(乳腺癌、结肠癌、食管癌、前列腺癌、恶性间皮癌等)和细胞系。EphA2在正常上皮细胞中位于细胞连接处,配体EphrinA1能以较高的亲和力与EphA2结合,两者结合后能导致受体和配体均发生酪氨酸磷酸化而产生双向信号传导,通过其下游信号调节细胞正常的生长、发育,同时也促进EphA2受体自身的降解。在恶性肿瘤细胞中EphA2定位异常,由细胞连接处转变为广泛分布于细胞表面,导致EphA2难以有效的与其配体接触,因而其自身的降解减少,所以在许多侵袭性肿瘤中EphA2和EphrinA1通常是过表达的。有学者通过一种模拟配体EphrinA1-Fc干预肿瘤细胞株(PC-3、293、Cos-7),显示EphA2受体活化、磷酸化程度增强,细胞形态学发生变化。因此,EphA2在肿瘤研究中被学者广泛关注。
有报道,肾癌组织中EphA2高表达可能与肾癌术后无瘤生存期有关,但关于EphA2及配体EphrinA1相互作用的生物学变化及配受体在肾癌中表达和机制研究国内外报道较少。一般认为肿瘤组织类型及恶性程度与疾病的转归和预后有关,在肾癌的不同病理类型中以肾透明细胞癌所占比例较高,为此,本课题通过对EphA2及其配体EphrinA1在肾透明细胞癌组织中的表达及与肾癌临床病理特征、E-cadherin和微血管密度(microvessel density,MVD)关系的研究,探讨EphA2及其配体EphrinA1在肾癌转移中的作用;并通过人工配体EphrinA1-Fc对人肾透明细胞癌786-O细胞系和裸鼠人肾癌细胞移植瘤干预和治疗研究,探讨EphA2与配体EphrinA1结合的生物学效应及其抗肿瘤的分子机制,为EphA2/EphrinA1在肾癌中表达及其在肾癌转移中的作用机制研究提供实验依据。
第一部分EphA2/EphrinA1在肾癌中的表达及生物学意义
目的:通过检测EphA2、EphrinA1在正常肾组织和肾透明细胞癌组织中蛋白和基因表达情况,探讨其在肾癌高表达的机制。
方法:采用半定量逆转录-聚合酶链反应(RT-PCR)和流式细胞术检测22例肾癌组织和12例非肾癌正常肾组织中EphA2和EphrinA1的基因及蛋白表达情况,并对其进行比较和相关分析。
结果:
1 RT-PCR显示:肾癌组织与正常肾组织中均有EphA2和EphrinA1的基因表达,但肾癌组织中EphA2和EphrinA1的mRNA的相对表达量明显高于正常肾组织(1.60±0.80 vs 0.58±0.19,P＜0.01;1.37±0.63vs 0.91±0.40,P＜0.01)。
2流式定量显示:肾癌组织中EphA2和EphrinA1蛋白的表达量明显高于正常肾组织(424.38±43.14 vs 332.44±42.83,P＜0.01;419.44±52.13 vs 325.36±42.33,P＜0.01)。
3相关分析显示,肾癌组织中EphA2、EphrinA1蛋白表达量与其mRNA表达量均无明显相关(r=0.05,P=0.826;r=-0.171,P=0.448)。
小结:EphA2和EphrinA1蛋白高表达于肾癌组织,其机制除与基因表达上调有关外,蛋白质生物学异常也可能是导致其升高的重要因素,提示EphA2和EphrinA1蛋白高表达在肿瘤发病中可能具有重要意义。
第二部分EphA2/EphrinA1在肾癌转移中的作用
目的:通过对EphA2及其配体EphrinA1在肾癌组织中的表达与肾癌患者临床病理特征、E-cadherin和MVD的关系研究,探讨EphA2及其配体EphrinA1在肾癌转移中的作用。
方法:利用免疫组织化学法检测68例肾透明癌组织、24例正常肾组织中EphA2、EphrinA1和E-cadherin的表达;并采用CD34抗体标记微血管内皮细胞,计算微血管密度(microvessel density,MVD)。分析EphA2、EphrinA1蛋白的表达与肾癌临床病理特征、E-cadherin、MVD之间的关系。
结果:
1 EphA2、EphrinA1在肾癌组织中的表达及其与肾癌临床病理特征的关系
免疫组化结果显示:EphA2、EphrinA1蛋白阳性表达在胞浆和胞膜。EphA2和EphrinA1蛋白的表达在肿瘤组织分级不同的各组间有显著性的差异(x~2=12.611,P=0.002;x~2=8.473,P=0.014),随着等级的增加其蛋白表达量也显著增加(r=0.431,P=0.000;r=0.355,P=0.003;);EphA2和EphrinA1蛋白的表达在高分期(Ⅲ、Ⅳ期)组显著高于低分期(Ⅰ、Ⅱ期)组(Z=-2.975,P=0.003;Z=-2.006,P=0.045),淋巴结转移组显著高于无淋巴结转移组的(Z=-2.320,P=0.02;Z=-2.792,P=0.005),肿瘤直径＞7cm与≤7cm两组间、年龄≥45岁与＜45岁两组间、男女性别两组间其蛋白的表达均无显著性差异(P值均＞0.05)。
2 E-cadherin在肾癌组织中的表达及其与肾癌临床病理学特征的关系
免疫组化结果显示:E-cadherin蛋白阳性表达在胞膜,呈棕黄色颗粒。在肾癌组织中,E-cadherin蛋白的表达明显低于正常肾组织(Z=-6.101,P＜0.0001)。
E-cadherin蛋白的表达在肿瘤组织分级不同的各组间有显著性的差异(x~2=8.493,P=0.014),随着等级的增加,其表达量显著降低(r=-0.320,P=0.008);E-cadherin蛋白的表达在高分期组、淋巴结转移组显著低于低分期组、无淋巴结转移组(Z=-2.037,P=0.042;Z=-2.217,P=0.027);肿瘤直径＞7cm与≤7cm两组间、年龄≥45岁与＜45岁两组间、男女性别两组间蛋白的表达均无显著性差异(P值均＞0.05)。
3 MVD在肾癌组织中的表达及其与肾癌临床病理学特征的关系
免疫组化结果显示:CD34主要表达于血管内皮细胞膜。微血管染色呈棕黄色,最密集染色区域即“热点”主要位于癌灶边缘。肾癌组织中MVD显著高于正常肾组织(P＜0.01)。在肿瘤组织分级不同的各组间,MVD有显著性差异(x~2=23.637,P＜0.01),并且随着分级等级的增加MVD也显著增加(r=0.592,P＜0.0001);MVD在肾癌组织的高分期组、淋巴结转移组、肿瘤直径＞7cm组显著高于低分期组、无淋巴结转移组、肿瘤直径≤7cm组(Z=3.735,P＜0.0001;Z=-3.4,P=0.001;Z=-3.522,P＜0.0001)。年龄≥45岁与＜45岁两组间、男女性别不同的两组间MVD均无显著性差异(P值均＞0.05)。
4 EphA2、EphrinA1和E-cadherin蛋白、MVD的相关性
相关分析显示,68例肾癌组织中EphA2和EphrinA1蛋白表达呈显著正相关(r=0.772,P＜0.0001);E-cadherin蛋白的表达与EphA2、EphrinA1蛋白的表达均呈显著负相关(r=-0.378,r=-0.414,P值均＜0.01);EphA2和EphrinA1蛋白的表达与MVD呈显著正相关(r=0.555,r=0.485,P值均＜0.01)。
小结:EphA2和EphrinA1蛋白高表达于肿瘤高分期组、高分级组和淋巴结转移组,与E-cadherin蛋白表达呈显著负相关,与MVD呈显著正相关,提示EphA2和EphrinA1蛋白的高表达预示肿瘤恶性程度的增加,其机制可能通过影响或协同E-cadherin和微血管生成来实现的。
第三部分EphrinA1-Fc对人肾透明癌786-O细胞系抗肿瘤效应机制的研究
目的:通过人工可溶性配体EphrinA1-Fc干预人肾透明细胞癌786-O细胞系抗肿瘤效应及其分子机制的实验研究,探讨EphA2在肿瘤细胞增殖、转移中的作用及机制。
方法:应用可溶性配体EphrinA1-Fc对人肾透明细胞癌786-O细胞系进行体外干预实验,观察不同时间点细胞形态学的变化,对不同时间点细胞内EphA2和ERK1/2的磷酸化程度进行分析,并观察可溶性配体反复刺激786-O后的细胞增殖情况。
结果:
1 EphrinA1-Fc干预后细胞形态学变化
正常条件下培养的肾癌细胞接受可溶性配体EphrinA1-Fc刺激1min,细胞形态即发生改变,细胞由梭形伸展状态开始回缩;5min时部分细胞变圆;30min时90%细胞变圆、与瓶壁结合疏松;40min时部分细胞重新伸展、粘附于瓶壁;60min时90%细胞恢复到刺激前状态。对照组细胞形态学未发生明显变化。
2 EphrinA1-Fc干预对磷酸化EphA2(p-EphA2)的影响
Western blotting结果显示:EphrinA1-Fc干预5min、10min、30min、60min后p-EphA2的相对表达量(p-EphA2/EphA2)分别是0.15±0.04、0.19±0.01、0.21±0.03和0.10±0.00。p-EphA2的相对表达量各组间有显著性差异(F=9.392,P=0.025)。组间两两比较,除5min与60min组、10min与30min组间无显著性差异,余各组间比较均有显著性差异(P值均＜0.05)。EphrinA1-Fc干预前及对照组不同时间点均未见其表达。
3 EphrinA1-Fc干预对磷酸化ERK1/2(p-ERK)的影响
Western blotting结果显示:EphrinA1-Fc干预5min、10min、30min、60min后p-ERK的相对表达量(p-ERN/ERK1/2)分别是0.15±0.07、0.22±0.06、0.26±0.03和0.13±0.04。p-EphA2的相对表达量各组间有显著性差异(F=4.428,P=0.041)。组间两两比较,5min与30min组、60min与30min组间比较均有显著性差异(P值均＜0.05),余各组间无显著差异(P值均＞0.05)。EphrinA1-Fc干预前及对照组不同时间点均未见其表达。
4 EphrinA1-Fc干预对细胞增殖的影响
EphrinA1-Fc干预人肾透明细胞癌786-O细胞株12天,两组细胞形态均未发生改变,但细胞的增殖状况发生了变化。EphrinA1-Fc组细胞数明显低于对照组((1.96±0.29)×10~5vs(3.14±0.49)×10~5,P＜0.01)。
小结:EphrinA1-Fc能抑制786-O细胞株的增殖、降低细胞黏附,其机制可能是通过影响胞内EphA2、ERK磷酸化实现的。
第四部分EphrinA1-Fc对裸鼠人肾癌细胞移植瘤的影响
目的:探讨EphrinA1-Fc对裸鼠人肾癌细胞移植瘤的影响及其作用机制。
方法:将表达EphA2抗原阳性的人肾透明细胞癌786-O细胞株体外培养后接种于裸鼠皮下,建立动物移植瘤模型,观察实验组(瘤周注射EphrinA1-Fc)和对照组(瘤周注射IgG-Fc)移植瘤的生长情况,并采用免疫组化法检测EphA2、E-cadherin及MVD的表达情况。
结果:
1裸鼠人肾癌细胞移植瘤模型的建立
14只裸鼠接种786-O细胞株7周,共8只裸鼠在注射部位发现米粒大小结节,表明人肾癌细胞移植瘤模型建立成功,接种成功率为60%。
2 EphrinA1-Fc对裸鼠人肾癌细胞移植瘤生长影响
接种786-O细胞株8周,移植瘤平均体积为(382.35±122.04)mm~3;干预治疗4周,裸鼠一般状态良好,未见明显的异常反应。
重复测量设计方差分析显示,时间和药物干预因素对移植瘤体积均有影响(F=4.085,P=0.012;F=7.469,P=0.034)。独立样本的非参数检验显示,治疗组干预治疗2周,移植瘤体积较对照组无明显差异[(272.65±55.35)mm~3 vs(535.97±216.07)mm~3,P=0.191)],干预治疗4周,移植瘤体积较对照组明显减小[(115.10±49.16)mm~3 vs(800.00±269.69)mm~3,P=0.021)]。肿瘤抑瘤率为80.62%。
3 EphrinA1-Fc对裸鼠人肾癌细胞移植瘤中EphA2、E-cadherin和MVD的影响
干预治疗4周,治疗组瘤内的EphA2蛋白的表达低于对照组;E-cadherin蛋白的表达高于对照组;治疗组MVD较对照组减少。
小结:表达EphA2阳性的肾透明细胞癌786-O具有致瘤性,但成瘤率低且成瘤时间长;瘤周注射EphrinA1-Fc可抑制肿瘤细胞的生长,其机制可能通过增加E-cadherin蛋白表达和抑制肿瘤血管生成实现的,反证了EphA2和EphrinA1的高表达可能通过促进细胞增殖、降低细胞与细胞黏附、促进血管生成促进人类肾癌的发生。
结论:
1、EphA2/EphrinA1基因和蛋白高表达于肾癌组织。其蛋白高表达的机制可能是自身降解下降、基因表达上调共同参与的结果。
2、EphA2/EphrinA1基因和蛋白高表达促进了肾癌细胞增殖、肿瘤新生血管生成,增强了肿瘤细胞与ECM黏附,削弱了细胞间连接,从而促进人类肾癌的发生和转移。
3、人工可溶性配体EphrinA1-Fc能抑制肾癌细胞的生长,为生物靶向治疗提供了动物实验依据。
4、EphA2和EphrinA1蛋白的高表达预示肾癌细胞侵袭能力强。联合检测EphA2、EphrinA1和E-cadherin三种蛋白的表达对于评价肾癌的恶性程度、判断其转移的危险性,可能具有一定的参考价值。
Renal cell carcinoma(RCC) is one kind of malignant tumor from the tubular epithelium of the renal parenchyma,accounting for roughly 80～90% and 2～3%of the renal and adult malignant tumors,respectively.With the improvement of living standards and advance in the medical imaging,early diagnosis and surgical treatment have produced satisfactory results.However, good therapeutic regimens are currently unavailable for the post-operation recurrence and the late stage cases with metastasis.Prediction of the prognosis still depends on the clinical stage and pathological grade,which showed certain limitations.The search of biological markers that may help to predict the degree of the malignance has therefore become a hot topic in the studies of the RCC.Such works may provide new targeting sites and approaches for the biological treatment of the RCC.
The EPH(erythropoietin-producing hepatocyte) receptors represent the largest known family of receptor tyrosine kinases.EphA2 is one of the earliest found and expressed in normal endothelial cells.It is also widely over-expressed in various tumor tissues including the mammary cancer, carcinoma of colon,esophageal cancer,prostatic cancer and mesothelial carcinoma,and cell lines.The EphA2 receptor located in the cell junction in normal epitheliums and,after binding with its EphrinA1 ligand under relatively high affinity and the ensuing tyrosine phosphorylation of both with bidirectional signal conduction,regulates the normal cell growth and development through its downstream signal pathway,and simultaneously promotes the degradation of the EphA2 receptor.The location of the EphA2 receptor is aberrant in the malignant tumors:Instead of the cell junction,it is widely distributed in the cell surface which prevented the effective binding with the EphA2 and reduced the degradation of the receptor per se.The EphA2 and EphrinA1 are thus usually over-expressed in many invasive tumors.By affecting the tumor cell lines(PC-3,293,Cos-7) with EphrinA1-Fc,a simulated ligand,investigators recorded rapid and transient actions of EphA2 receptor activation,enhanced phosphorylation,and morphological changes of the cells.So,EphA2 is now under the spotlight in the tumor research.
It is reported that over-expression of the EphA2 in the RCC may be associated with the tumor free survival after operation.However,biological changes due to the interaction of the EphA2 and its ligand,and EphA2/AphrinA1 in the RCC are rarely studied in and outside China.It is generally considered tissue type and degree of the tumor may predispose to the outcome and prognosis of the disease.Pathologically,renal clear cell carcinoma is proportionally more in the renal tumors.The current work thus examined the role of the EphA2 and its ligand EphrinA1 in the metastasis of the tumor,by studying the expression of the EphA2 and EphrinA1 in the RCC tissue,clinicopathological features,E-cadherin and microvessel density (MVD).Secondly,the biological effects of the EphA2-EphrinA1 binding and the molecular mechanism of its antitumor action were examined.The latter work was aimed to provide experimental evidence for the expression of the Eph/EphrinA1 in the RCC and its mechanism of action in the metastasis of the RCC.
PartⅠ:The effect of EphA2/EphrinA1 in the occurrence of the RCC
Objective:To detect the protein expression of EphA2/EphrinA1 in the normal renal tissue and the RCC,explore its role in the occurrence of RCC.
Methods:Use the Reversed Transcript-Polymerase Chain Reaction (RT-PCR) and flow cytometry to detect mRNA of the EphA2 and EphrinA1, and the protein expression in 22 cases of RCC and 12 cases of normal renal tissue.
Results:
1 RT-PCR results showed that in both normal group and RCC group express EphA2/EphrinA1 gene,but the renal group has express significantly higher EphA2/EphrinA1 mRNA expression than the normal group(P＜0.01),there was a significant difference.(1.60±0.80 vs 0.58±0.19;1.37±0.63vs 0.91±0.40,P＜0.01)
2 The flow cytometry showed that the expression of the EphA2/EphrinA1 was significantly higher in the RCC group(424.38±43.14 vs 332.44±42.83,P＜0.01;419.44±52.13 vs 325.36±42.33,P＜0.01).
3 Correlation analysis showed that in the RCC,EphA2/EphrinA1 protein expression and its mRNA expression were not significantly correlated (r=0.05,P=0.826;r=-0.171,P=0.448).
Conclusion:The expression of the EphA2/EphrinA1 gene and protein in the RCC was higher than those in the normal renal tissue.But in the RCC,EphA2/EphrinA1 protein expression and its mRNA expression were not significantly correlated,suggesting that high protein expression is not only associated with the high gene expression,but also with the post-transcriptional regulation and the biological abnormalities of EphA2/EphrinA1.
PartⅡ:The role of EphA2/EphrinA1 in the invasion and metastasis of the RCC
Objective:To detect the expression of EphA2/EphrinA1 in the RCC and its relationship with the clinicopathological characteristics,E-cadherin and MVD, to explore the role of EphA2/EphrinA1 in the invasion and metastasis of RCC.
Methods:Using immunohistochemistry,we detected EphA2/EphrinA1 and E-cadherin expression in 68 cases of RCC and 24 cases of normal renal tissue, also use CD34 antibody to mark microvascular endothelial cells and calculate microvessel density(MVD).Explore the relationship of EphA2/EphrinA1 expression with clinicopathological features and analyze its correlation with E-cadherin,MVD.
Results:
1 The relationship between clinicopathological features and EphA2/EphrinA1 expression in RCC.
Immunohistochemistry results showed that:In the RCC,different grade have different protein expression of EphA2/EphrinA1(x~2=12.611,P=0.002; x~2=8.473,P=0.014),With the increase in grade levels,the protein expression of EphA2/EphrinA1 increase significantly(r=0.431,P=0.000;r=0.355, P=0.003;);stageⅢandⅣhave significantly higher protein expression of EphA2/EphrinA1 than stageⅠandⅡ(Z=-2.975,P=0.003;Z=-2.006,P=0.045); lymph node metastasis group has significantly higher protein expression of EphA2/EphrinA1 than those without lymph node metastasis (Z=-2.320,P=0.02;Z=-2.792,P=0.005);in tumor diameter≥7cm and＜7cm between the two groups,≥45 years and＜45 years old between the two groups, sex between two groups have no significantly difference about the expression of protein(P＞0.05).
2 The expression of the E-cadherin in the normal renal tissue and RCC,and its relationship with clinicopathological features.
Immunohistochemistry results showed that:positive expression of the E-cadherin protein at the membrane as brown particles.The RCC expressed significantly lower E-cadherin protein than the normal renal tissue (Z=-6.101,P＜0.0001).
The expression of E-cadherin has the significant difference(x~2=8.493, P=0.014),and with the increase in grade levels,the expression of E-cadherin decrease significantly(r=-0.320,P=0.008);the expression of E-cadherin in high-stage group and lymph node metastasis group is significantly lower than the low-grade group and without lymph node metastasis group(Z=-2.037, P=0.042;Z=-2.217,P=0.027).In tumor diameter≥7 cm and＜7cm between the two groups,≥45-year-old and＜45 years of age between the two groups, sex between two groups of protein expression were not significantly different (P＞0.05).
3 MVD in the normal kidney and RCC and its relationship with the clinicopathological characteristics of the RCC.
Immunohistochemistry results showed:CD34 was mainly expressed at the vascular endothelial cell membrane.Microvessel staining was brown,the most densely stained region,e.g."hot spots",was mainly located at the border of tumors,cancer and cancer-intensive areas in the nest CD34 expression was significantly less than the cancer and hyperplasia weeks active region.MVD of tumor tissues was significantly higher than the normal renal tissue(P <0.01).In different grade of the tumor group,MVD have the significant difference(x~2=23.637,P<0.01).With the increase in grade levels,MVD increases significantly(r=0.592,P<0.0001).MVD in the RCC in the group of high-grade,high-stage group,with lymph node metastasis was also significantly higher than low-grade group,low stage and without lymph node metastasis group(Z=3.735,P<0.0001;Z=-3.4,P=0.001;Z=-3.522,P<0.0001);≥45-year-old and <45 years of age between the two groups,sex between two groups of MVD were not significantly difference(P>0.05).
4 Correlation of the EphA2,EphrinA1,E-cadherin protein and MVD expression.
Correlation analysis showed that in 68 cases of RCC,the protein expression of the EphA2 and EphrinA1 was significantly correlated(r=0.772, P<0.0001),while the protein expression of the E-cadherin was significantly and negatively correlated with that of the EphA2 and EphrinA1(r=-0.378, r=-0.414,P<0.01);EphA2,EphrinA1 expression and MVD were significantly correlated(r=0.555,r=0.485,P<0.01).
Conclusion:EphA2/EphrinA1 was highly expressed in tumors with high stage,high grade and with lymph nodes metastasis.EphA2/EphrinA1 was negatively correlated with E-cadherin expression and positively correlated with MVD,indicating that high expression of the EphA2/EphrinA1 protein may predict the degree of malignance of the tumor,possibly through the influence of or the coordination with the E-cadherin and the angiogenesis of microvasculatures.
PartⅢ:The anti-cancer effect of the EphrinA1-Fc to the 786-O RCC cells and its molecular mechanism.
Objective:Using EphrinA1-Fc to intervene the 786-O renal clear cell carcinoma in vitro study to examine the role and the possible molecular mechanism of the EphrinA-2.
Methods:Using the soluble ligand EphrinA1-Fc to intervene the 786-O renal carcinoma cells in vitro to observe the morphological changes in the cells at different time points,by the use of different technologies such as cell counting,Western blotting,immunoprecipitation to temporally examine the phosphorylation of EphA2,ERK1/2 and other changes.
Results:
1 The changes in the cell morphology after the EphrinA1-Fc intervention:
Use soluble ligand EphrinA1-Fc to stimulate renal cancer cells.After 1min, the renal cells exhibited morphological change,shrinking from the normal extended shape of spindle;at 5min,part of the cells appeared to be round;at 30min 90%of the cells were round and loosely attached to the flask;at 40min part of the cells re-extended and adhered to the flask;at 60min 90%of the cells recovered to the pre-stimulation shape.In the control group,there were no detectable morphological changes.
2 Impact of the EphrinA1-Fc on the p-EphA2
Western blotting results showed that:EphrinA1-Fc at 5min、10min、30min、60min intervention,p-EphA2 expression(p-EphA2/EphA2)was 0.15±0.04, 0.19±0.01,0.21±0.03和0.10±0.00,there is significant difference in different groups(F=9.392,P=0.025).Compared among the different groups,5 min and 60min between the two groups,10min and 30min between two groups were not significantly different(P＞0.05),other groups have the significant difference(P＜0.05).The pre-intervention group and the control group have no expression of the p-EphA2.
3 Impact of the EphrinA1-Fc on the phosphorylation of the ERK
Western blotting results showed that:EphrinA1-Fc at 5min、10min、30min、60min intervention,p-ERK expression levels were 0.15±0.07,0.22±0.06,0.26±0.03 and 0.13±0.04.p-ERK expression levels were relative,representing a marked increase in the pre-intervention,there was significant difference (F=4.428,P=0.041).Compared among the different groups,5 min and 30min between the two groups,60min and 30min between two groups were significantly different(P＜0.05).The pre-intervention group and the control group have no expression of the p-ERK.
4 Impact of the EphrinA1-Fc on the cell proliferation
After repeatedly intervention of the EphrinA1-Fc in the 786-O RCC cell in 12 days,cell growth but not cell morphology showed significant change:the number of cells decreased significantly in the EphrinA1-Fc group[(1.96±0.29)×10~5 vs(3.14±0.49)×10~5,P＜0.01].
Conclusion:Addition of the EphrinA1-Fc to the 786-O RCC cell could inhibit cell proliferation and reduce cell adhesion,possibly by affecting the phosphorylation of the intracellular EphA2 and ERK.
PartⅣEffects of the EphrinA1-Fe on the nude mice with transplanted human RCC
Objective:To investigate the effects of the EphrinA1-Fc on the nude mice with transplanted human RCC and its possible mechanism of action.
Methods:the animal model of the transplanted tumor is established by subcutaneous inoculation to nude mice of the human RCC cell 786-O,which is positive for the EphA2 antigen and cultivated in vitro.The growth of the transplanted tumor was observed in both the experimental group(injection of the EphrinA1-Fc in the periphery of the tumor) and the control group (injection of the IgG-Fc in the periphery of the tumor).Expression of EphA2, p-ERK,E-cadherin and MVD was examined by immunohistochemistry
Results:
1 The establishment of the nude mice model of transplanted human RCC
7 weeks after the inoculation of the 786-O cells,nodules about the size of grain were found at the injection site in 8 nude mice,indicating the successful establishing of nude mice model of transplanted human RCC(60%of success rate).
2 Effects of the EphrinA1-Fc on the growth of the transplanted RCC in the nude mice
8 weeks after the inoculation,the average tumor volume was(382.35±122.04) mm~3;4 weeks after the application of the EphrinA1-Fc,tumor-bearing mice in each group were in generally good condition with no obvious side effects.
Time and medical intervention could affect the volume of the transplanted tumor(F=4.085,P=0.012;F=7.469,P=0.034,by ANOVA).Nonparametic test of independent samples showed that,in comparison to that of the control group,the volume of the transplanted tumor was significantly reduced at 4 but not 2 weeks after medical intervention[(115.10±49.16) mm~3 vs(800.00±269.69) mm~3,P=0.021 and(272.65±55.35) mm~3 vs(535.97±216.07) mm~3,P= 0.191],respectively,resulting in a inhibition rate of 80.62%.
3 Effects of the EphrinA1-Fc on the EphA2,E-cadherin and MVD in the transplanted RCC in the nude mice.
4 weeks after the medical intervention,protein expression of the EphA2 and MVD was lower,and protein expression of the E-cadherin was higher the treatment group than the corresponding value in the control group.
Conclusion:RCC 786-O of EphA2 positive was oncogenic with low probability of tumor onset and long time of tumor development;the growth of the tumor could be inhibited by the injection of the EphrinA1-Fc in the periphery of the tumor,its possible mechanism was the up-regulation of the protein expression of the E-cadherin and inhibition of the angiogenesis in the tumor.These findings reciprocally indicated that high expression of the EphA2 and EphrinA1 may contribute to the onset of RCC by promoting cell proliferation and decreasing cell adhesion.
Conclusion:
1 The expression of EphA2/EphrinA1 in RCC increases significantly.In additional to the up-regulation of the gene expression,the possible mechanism also includes abnormality in the biological aspects of the protein.
2 High protein expression of the EphA2/EphrinA1 may predict the malignance of the RCC.and joint examination of the EphA2,EphrinA1 and E-cadherin may be useful for the evaluation of malignancy of RCC and the determination of its metastatic potential.
3 EphrinA1-Fc,artificial ligand,was first applied to RCC cell line and nude mice with transplanted RCC,reciprocally indicating that high expression of the EphA2 and EphrinA1 may contribute to the onset of RCC by promoting cell proliferation and increasing cell adhesion.Altered E-cadherin and microvessel growth factor,resulted from the biological abnormality of the EphA2 and EphrinA1,may be one important mechanism enhancing the tumor metastasis.
4 The binding of the EphA2 and EphrinA1 may inhibit the proliferation of the tumor by increasing the phosphorylation of the AphA2 and ERK.
引文
1 吴阶平,主编.吴阶平泌尿外科学.济南:山东科技技术出版社,2004,889-917
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7 Zantek ND,Walker-Daniels J,Stewart J,et al.MCF-10A-NeoST:a new cell system for studying cell-ECM and cell-cell interactions in breast cancer.Clin Cancer Res,2001,7(11):3640-3648
8 张水军,张弓,赵永福,等.EphA2在原发性肝细胞癌中的表达及临床意义.中华实验外科杂志,2006,23(3):269-270
9 Walker-Daniels J,Riese DJ,2nd,Kinch MS.C-Cb1-dependent EphA2protein degradation is induced by ligand binding.Mol Cancer Res,2002,1(1):79-87
10 Miao H,Bumett E,Kinch M,et al.Activation of EphA2 kinase suppresses integrin function and causes focal adhesion kinase dephosphorylation.Nat Cell Biol,2002,2(2):62-69
11 Nakamura R,Kataoka H,Sato N,et al.EPHA2/EFNA1 expression in human gastric cancer.Cancer Sci,2005,96(1):42-47
12 Nasreen N,Mohammed K A.,Lai Y,et al.Receptor EphA2 activation with ephrinAl supresses growth of malignant mesothelioma(MM).Cancer Letters,2007,258:215-222
13 Brantley-Sieders DM,Fang WB,Hwang Y,et al.Ephrin-A1 facilitates mammary tumor metastasis through an angiogenesis-dependent mechanism mediated by EphA receptor and vascular endothelial growth factor in mice.Cancer Res,2006,66(21):10315-10324
14 Hess AR,Seftor EA,Gruman LM,et al.VE-cadherin regulates EphA2 in aggressive melanoma cells through novel signaling pathway:implications for vasculogenic mimicry.Cancer Biol Ther,2006,5(2):228-233
15 Ogawa K,Pasqualini R,Lindberg RA,et al.The ephrinAl ligand and its receptor EphA2 are expressed during tumor neovascularization.Oncogene,2000,19(11):6043-6052
16 Pandey A,Shao H,Marks RM,et al.Role of B61,the ligand for the Eck receptor tyrosine kinase,in TNF-alpha-induced angiogenesis.Science,1995,268(5210):567-569
1 Zhou R.The Eph family receptors and ligands.Pharmacol Ther,1988,77(3):151-181
2 Bruckner K,Pasquale EB,Klein R.Tyrosine phosphorylation of transmembrane ligands for Eph receptors.Science,1997,275(5306):1640-1643
3 Davy A,Gale NW,Murray EW,et al.Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion.Genes Dev,1999,13(23):3125-3135
4 Klein R.Excitatory Eph receptors and adhesive ephrin ligands.Curr Opin Cell Biol,2001,13(2):196-203
5 Holder N,Klein R.Eph receptors and ephrins:effectors of morphogenesis.Development,1999,126(10):2033-2044
6 Knoll B,Zarbalis K,Wurst W,et al.A role for the EphA family in the topographic targeting of vomeronasal axons.Development,2001,128(6):895-906
7 Walker-Daniels J,Hess AR,Hendrix MJ,et al.Differential regulation of EphA2 in normal and malignant cells.Am J Pathol,2003,162(4):1037-1042
8 Davis S,Gale NW,Aldrich TH,et al.Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity.Science,1994,266(5186):816-819
9 Holman LB,Marks RM,Dixit VM.A novel immediate-early response gene of endothelium is induced by cytokines and encodes a secreted protein.Mol Cell Biol,1990,10(11):5830-5838
10 Andres AC,Reid HH,Zurcher G,et al.Expression of two novel eph-related receptor protein tyrosine kinases in mammary gland development and carcinogenesis.Oncogene,1994,9:1461-1467
11 Rosenberg IM,Goke M,Kanai M,et al.Epithelial cell kinase-B61:an autocrine loop modulating intestinal epithelial migration and barrier function.Am J Physiol,1997,273:824-832
12 Pandey A,Shao H,Marks RM,et al.Role of B61,the ligand for the Eck receptor tyrosine kinase,in TNF-alpha-induced angiogenesis.Science,1995,268(5210):567-569
13 Pandy A,Lazar DF,Saltiel AR,et al.Activation of the Eck receptor protein tyrosine kinase stimulates phosphatidylinositol 3-kinase activity.J Biol Chem,1994,269:30154-30157
14 Ganju P,Shigemoto K,Brennan J,et al.The Eck receptor tyrosine kinase is implicated in pattern formation during gastrulation,hindbrain segmentation and limb development.Oncogene,1994,9:1613-1624
15 Walker-Daniels J,Riese DJ,2nd,Kinch MS.C-Cbl-dependent EphA2 protein degradation is induced by ligand binding.Mol Cancer Res,2002,1(1):79-87
16 Miao H,Burnett E,Kinch M,et al.Activation of EphA2 kinase suppresses integrin function and causes focal adhesion kinase dephosphorylation.Nat Cell Biol,2002,2(2):62-69
17 Pratt RL,Kinch MS.Activation of the EphA2 tyrosine kinase stimulates the MAP/ERK kinase signaling cascade.Oncogene,2002,21:7690-7699
18 Pandey A,Duan H,Dixit VM.Characterization of a novel src-like adapter protein that associates with the Eck receptor tyrosine kinase.J Biol Chem,1995,270:19201-19204
19 Zantek ND,Walker-Daniels J,Stewart J,et al.MCF-lOA-NeoST:a new cell system for studying cell-ECM and cell-cell interactions in breast cancer.Clin Cancer Res,2001,7(11):3640-3648
20 Zelinski DP,Zantek ND,Stewart JC,et al.EphA2 overexpress causes tumorigenesis of mammary epithelial cells.Cancer Res,2001,61(5):2301-2306
21 Rosenberg M,Goke M,Kanai M,et al.Epithelial cell kinaseB61:an autocrine loop modulating intestinal epithelial migration and barrier function.Am J Physiol,1997,273(4pt1):G824-G832
22 蒋萍,李景和,罗庚求,等.EphA2对结肠癌细胞株HCT116中VEGF和MMP9蛋白表达的影响.中南大学学报(医学版),2007,32(4):679-683
23 Miyazaki T,Kato H,Fukuchi M,et al.EphA2 overexpression correlates with poor prognosis in esophageal squamous cell carcinoma.Cancer,2003,103(5):657-663
24 Walker-Daniels J,Coffman K,Azimi M,et al.Overexpression of the EphA2 tyrosine kinase in prostate cancer.Prostate,1999,41(4):275-280
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28 Nasreen N,Mohammed KA.,Lai Y,et al.Receptor EphA2 activation with ephrinA1 supresses growth of malignant mesothelioma(MM).Cancer Letters,2007,258:215-222
29 Wu D,Suo Z,Kristensen GB,et al.Prognostic value of EphA2 and EphrinA1 in squamous,cell cervical carcinoma.Gynecol Oncol,2004,94(2):312-319
30 Tanker PH,Deavers M,Celestino J,et al.EphA2 expression is associated with aggressive features in ovarian carcinoma:Clin Cancer Res,2004,10(15):5145-5150
31 韩丽萍,董子明,乔玉环,等.卵巢癌组织中EphA2受体蛋白的表达.郑州大学学报(医学版),2005,40(2):308-311
32 赵瑞皎,吴凯彦,刘欣,等.乳腺癌组织中EphA2蛋白的表达及意义. 山东医药,2008,48(12):55-56
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43 张水军,张弓,赵永福,等.EphA2在原发性肝细胞癌中的表达及临床意义.中华实验外科杂志,2006,23(3):269-270
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53 Brantley DM,Fang WB,Hwang Y,et al.Ephrin-Al facilitates mammary tumor metastasis through an angiogenesis-dependent mechanism mediated by EphA receptor and vascular endothelial growth factor in mice.Cancer Res,2006,66(21):10315-10324
54 Hess AR,Seftor EA,Gruman LM,et al.VE-cadherin regulates EphA2 in aggressive melanoma cells through novel signaling pathway:implications for vasculogenic mimicry.Cancer Biol Ther,2006,5(2):228-233
55 Ogawa K,Pasqualini R,Lindberg RA,et al.The ephrinAl ligand and its receptor EphA2 are expressed during tumor neovascularization.Oncogene, 2000,19(11):6043-6052
56 Herrem CJ,Tatsumi T,Olson KS,et al.Expression of EphA2 is prognostic of disease-free interval and overall survival in surgically treated patients with renal cell carcinoma.Clin Cancer Res,2005,11(1):226-23
2 Valladares A,Hernandezn G,Gomez S,et al.Genetic expression profiles and chromosomal alterations in sporadic breast cancer in Mexican women.Cancer Genet Cytogenet,2006,170(2):147-15
3 Yamaguch IS,Tatsum IT,Takehara T,et al.Immunotherapy of murine colon cancer using receptor tyrosine kinase EphA2-derived peptide-pulsed dendritic cell vaccines.Cancer,2007,110(7):1469-1477
4 Abraham S,Knapp DW,Chen GL,et al.Expression of EphA2 and EphrinA-1 in carcinoma of the urinary bladder.Clin Cancer Res,2006,12(2):353-360
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6 Hess AR,Seftor EA,Gruman LM,et al.VE-cadherin regulates EphA2 in aggressive melanoma cells through a novel signaling pathway:implications for vasculogenic mimicry.Cancer Biol Ther,2006,5(2):228-233
7 Wu D,Suo Z,Kristensen GB,et al.Prognostic value of EphA2 and EphrinA-1 in squamous cell cervical carcinoma.Gynecol Oncol,2004,94(2):312-319
8 Meade-toll NL,Martnez JD.Loss of p53 and overexpression of EphA2predict poor prognosis for ovarian cancer patients.Cancer Biol Ther,2007,6(2):288-289
9 Nasreen N,Mohammed KA,Antony VB,et al.Silencing the receptor EphA2 suppresses the growth and haptotaxis of malignant mesothelioma cells.Cancer,2006,107(10):2425-2435
10 Nakamura R,Kataoka H,Sato N,et al.EPHA2/EFNA1 expression in human gastric cancer.Cancer Sci,2005,96(1):42-47
11 Zhou R.The Eph family receptors and ligands.Pharmacol Ther, 1988,77(3):151-181
12 Zelinski DP,Zantek ND,Walker-Daniel J,et al.Estrogen and Myc negatively regulate expression of the EphA2 tyrosine kinase.Cell Biochem,2002,85:714-720
13 Zantek ND,Azimi M,Fedor-Chaiken M,et al.E-cadherin regulates the function of the EphA2 receptor tyrosine kinase.Cell Growth Differ,1999,10:629-638
14 Zou JX,Wang B,Kalo MS,et al.An Eph receptor regulates integrin activity through R-Ras.Proc Natl Acad Sci USA,1999,96:13813-13818
1 Valladares A,Hernandezn G,Gomez S,et al.Genetic expression profiles and chromosomal alterations in sporadic breast cancer in Mexican women.Cancer Genet Cytogenet,2006,170(2):147-151
2 Yamaguch IS,Tatsum IT,Takehara T,et al.Immunotherapy of murine colon cancer using receptor tyrosine kinase EphA2-derived peptide-pulsed dendritic cell vaccines.Cancer,2007,110(7):1469-1477
3 Abraham S,Knapp DW,Chen GL,et al.Expression of EphA2 and EphrinA-1 in carcinoma of the urinary bladder.Clin Cancer Res,2006,12(2):353-360
4 Cho IK,Creighton CJ,Stiver SD,et al.Transcriptional profiling of non-small cell lung cancer cells with activating EGFR somatic mutations.Plos One,2007,2(11):el 226
5 Hess AR,Seftor EA,Gruman LM,et al.VE-cadherin regulates EphA2 in aggressive melanoma cells through a novel signaling pathway:implications for vasculogenic mimicry.Cancer Biol Ther,2006,5(2):228-233
6 Wu D,Suo Z,Kristensen GB,et al.Prognostic value of EphA2 and EphrinA-1 in squamous cell cervical carcinoma.Gynecol Oncol,2004,94(2):312-319
7 Meade-toll NL,Martnez JD.Loss of p53 and overexpression of EphA2 predict poor prognosis for ovarian cancer patients.Cancer Biol Ther,2007,6(2):288-289
8 Nasreen N,Mohammed KA,Antony VB,et al.Silencing the receptor EphA2 suppresses the growth and haptotaxis of malignant mesothelioma cells.Cancer,2006,107(10):2425-2435
9 Abraham S,Knapp DW,Cheng L,et al.Expression of EphA2 and EphrinAl in carcinoma of the urinary bladder.Clin Cancer Res,2006,15,12(2):353-360
10 Coffman KT,Hu M,Carles-Kinch K,et al.Differential EphA2 epitope display on normal versus malignant cells.Cancer Res,2003,63(22):7907-7912
11 Brantley-Sieders DM,Fang WB,Hicks DJ,et al.Impaired tumor n microenvironment in EphA2-deficient mice inhibits tumor angiogenesis and metastatic progressoion.FASEB J,2005,19(13):1884-1886
12 武海盐,陈大氢,唐英姿,等.小儿卵黄囊瘤临床病理及免疫组化分析.中国当代儿科杂志,2003,5:348-350
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14 Zantek ND,Azimi M,Fedor-Chaiken M,et al.E-cadherin regulates the function of the EphA2 receptor tyrosine kinase.Cell Growth Differ.1999,10(9):629-38
15 Weider N,Folkman J,Pozza F,et al.Tumor angiogenesis:a new siginificant and independent prognostic indicator in early-stage breast carcinoma.Natl Cancer Inst,1992,84:1875-1887
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17 Tanker PH,Deavers M,Celestino J,et al.EphA2 expression is associated with aggressive features in ovarian carcinoma.Clin Cancer Res,2004,10(15):5145-5150
18 赵瑞皎,吴凯彦,刘欣,等.乳腺癌组织中EphA2蛋白的表达及意义.山东医药,2008,48(12):55-56
19 Zelinski DP,Zantek ND,Stewart JC,et al.EphA2 overxepression causes tumorigenesis of mammary epithelial cell.Cancer Res,2001,61(5):2301-2306.
20 Miyazaki T,Kato H,Fukuchi M,et al.EphA2 overexpression correlates with poor prognosis in esophageal squamous cell carcinoma.Cancer,2003,103(5):657-663
21 Saito T,Masuda N,Miyazaki T,et al.Expression of EphA2 and E-cadherin in colorectal cancer:correlation with cancer metastasis.Oncol Rep,2004,11(3):605-611
22 Zantek ND,Walker-Daniels J,Stewart J,et al.MCF-10A-NeoST:a new cell system for studying cell-ECM and cell-cell interaction in breast cancer.Clin Cancer Res,2002,7(11):3640-3648
23 Cheng N,Brantley DM,Liu H,et al.Blockade of EphA receptor tyrosine kinase activeation inhibits vascular endothelial cell growth factor-induced angiogenesis.Mol Cancer Res,2002,1(1):2-11
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25 刘广华,李汉忠,夏溟,等.多中心肾癌发生机制探讨.临床泌尿外科杂志,2006,21(8):975-978
26 Herrem CJ,Tatsumi T,Olson KS,et al.Expression of EphA2 is prognostic of disease-free interval and overall survival in surgically treated patients with renal cell carcinoma.Clin Cancer Res,2005,11(1):226-23
1 Nasreen N,Mohammed K A.,Lai Y,et al.Receptor EphA2 activation with ephrinA1 supresses growth of malignant mesothelioma(MM).Cancer Letters,2007,258,:215-222
2 Nakamura R,Kataoka H,Sato N,et al.EPHA2/EFNA1 expression in Human gastric cancer.Cancer Sci,2005,96(1):42-47
3 Miao H,Bumett E,Kinch M,et al.Activation of EphA2 kinase suppresses integrin function and causes focal adhesion kinase dephosphorylation.Nat Cell Biol,2000,2(2):62-69
4 Carles-Kinch K,Klipatrick KE,Stewart JC,et al.Antibody targeting of the EphA2 receptor tyrosine kinase on malignant carcinomas.Cancer Res,2001,62:2840-2847
5 Zelinski DP,Zantek ND,Stewart JC,et al.EphA2 overexpression causes tumorigenesis of mammary epithelial cells.Cancer Res,2001,61:2301-2306.
6 Seftor EA,Meltzer PS,Kirschmann DA,et al.Molecular determinants of human uveal melanoma invasion and metastasis.Clin Exp Metastasis,2002,19:233-246
7 Zantek ND,Azimi M,Fedor-Chaiken M,et al.E-cadherin regulates the function of the EphA2 receptor tyrosine kinase.Cell Growth Differ,1999,10:629-638 Carles-Kinch K,Klipatrick KE,Stewart JC,et al.Antibody targeting of the EphA2 receptor tyrosine kinase on malignant carcinomas.Cancer Res,2001,62:2840-2847
2 Zelinski DP,Zantek ND,Stewart JC,et al.EphA2 overexpression causes tumorigenesis of mammary epithelial cells.Cancer Res,2001,61:2301-2306.
3 郑宏祥,毛立军,郝林等.3种建立人肾皮下荷瘤鼠模型方法比较.徐州医学院报,2007,27(4):216-218
4 Soffer SZ,Moore JT,Kin E,et al.Combination antiangiogenic therapy in creased effincacy in amurine model of With stumor.J Pediatr Surg,2001,36(8):1177-1181.
5 Pukkaneu K J Pakkinen JJ.Kettunen MI,et al.Characterization of a new animal model for human an renal cell carcinoma.In Vivo,2000,14(3):393-400
6 Zantek ND,Azimi M,Fedor-Chaiken M,et al.E-cadherin regulates the function of the EphA2 receptor tyrosine kinase.Cell Growth Differ,1999,10(9):629-638
7 Zantek ND,Walker-Daniels J,Stewart J,et al.MCF-10A-NeoST:a new cell system for studying cell-ECM and cell-cell interactions in breast cancer.Clin Cancer Res,2001,7(11):3640-3648
8 张水军,张弓,赵永福,等.EphA2在原发性肝细胞癌中的表达及临床意义.中华实验外科杂志,2006,23(3):269-270
9 Walker-Daniels J,Riese DJ,2nd,Kinch MS.C-Cb1-dependent EphA2protein degradation is induced by ligand binding.Mol Cancer Res,2002,1(1):79-87
10 Miao H,Bumett E,Kinch M,et al.Activation of EphA2 kinase suppresses integrin function and causes focal adhesion kinase dephosphorylation.Nat Cell Biol,2002,2(2):62-69
11 Nakamura R,Kataoka H,Sato N,et al.EPHA2/EFNA1 expression in human gastric cancer.Cancer Sci,2005,96(1):42-47
12 Nasreen N,Mohammed K A.,Lai Y,et al.Receptor EphA2 activation with ephrinAl supresses growth of malignant mesothelioma(MM).Cancer Letters,2007,258:215-222
13 Brantley-Sieders DM,Fang WB,Hwang Y,et al.Ephrin-A1 facilitates mammary tumor metastasis through an angiogenesis-dependent mechanism mediated by EphA receptor and vascular endothelial growth factor in mice.Cancer Res,2006,66(21):10315-10324
14 Hess AR,Seftor EA,Gruman LM,et al.VE-cadherin regulates EphA2 in aggressive melanoma cells through novel signaling pathway:implications for vasculogenic mimicry.Cancer Biol Ther,2006,5(2):228-233
15 Ogawa K,Pasqualini R,Lindberg RA,et al.The ephrinAl ligand and its receptor EphA2 are expressed during tumor neovascularization.Oncogene,2000,19(11):6043-6052
16 Pandey A,Shao H,Marks RM,et al.Role of B61,the ligand for the Eck receptor tyrosine kinase,in TNF-alpha-induced angiogenesis.Science,1995,268(5210):567-569
1 Zhou R.The Eph family receptors and ligands.Pharmacol Ther,1988,77(3):151-181
2 Bruckner K,Pasquale EB,Klein R.Tyrosine phosphorylation of transmembrane ligands for Eph receptors.Science,1997,275(5306):1640-1643
3 Davy A,Gale NW,Murray EW,et al.Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion.Genes Dev,1999,13(23):3125-3135
4 Klein R.Excitatory Eph receptors and adhesive ephrin ligands.Curr Opin Cell Biol,2001,13(2):196-203
5 Holder N,Klein R.Eph receptors and ephrins:effectors of morphogenesis.Development,1999,126(10):2033-2044
6 Knoll B,Zarbalis K,Wurst W,et al.A role for the EphA family in the topographic targeting of vomeronasal axons.Development,2001,128(6):895-906
7 Walker-Daniels J,Hess AR,Hendrix MJ,et al.Differential regulation of EphA2 in normal and malignant cells.Am J Pathol,2003,162(4):1037-1042
8 Davis S,Gale NW,Aldrich TH,et al.Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity.Science,1994,266(5186):816-819
9 Holman LB,Marks RM,Dixit VM.A novel immediate-early response gene of endothelium is induced by cytokines and encodes a secreted protein.Mol Cell Biol,1990,10(11):5830-5838
10 Andres AC,Reid HH,Zurcher G,et al.Expression of two novel eph-related receptor protein tyrosine kinases in mammary gland development and carcinogenesis.Oncogene,1994,9:1461-1467
11 Rosenberg IM,Goke M,Kanai M,et al.Epithelial cell kinase-B61:an autocrine loop modulating intestinal epithelial migration and barrier function.Am J Physiol,1997,273:824-832
12 Pandey A,Shao H,Marks RM,et al.Role of B61,the ligand for the Eck receptor tyrosine kinase,in TNF-alpha-induced angiogenesis.Science,1995,268(5210):567-569
13 Pandy A,Lazar DF,Saltiel AR,et al.Activation of the Eck receptor protein tyrosine kinase stimulates phosphatidylinositol 3-kinase activity.J Biol Chem,1994,269:30154-30157
14 Ganju P,Shigemoto K,Brennan J,et al.The Eck receptor tyrosine kinase is implicated in pattern formation during gastrulation,hindbrain segmentation and limb development.Oncogene,1994,9:1613-1624
15 Walker-Daniels J,Riese DJ,2nd,Kinch MS.C-Cbl-dependent EphA2 protein degradation is induced by ligand binding.Mol Cancer Res,2002,1(1):79-87
16 Miao H,Burnett E,Kinch M,et al.Activation of EphA2 kinase suppresses integrin function and causes focal adhesion kinase dephosphorylation.Nat Cell Biol,2002,2(2):62-69
17 Pratt RL,Kinch MS.Activation of the EphA2 tyrosine kinase stimulates the MAP/ERK kinase signaling cascade.Oncogene,2002,21:7690-7699
18 Pandey A,Duan H,Dixit VM.Characterization of a novel src-like adapter protein that associates with the Eck receptor tyrosine kinase.J Biol Chem,1995,270:19201-19204
19 Zantek ND,Walker-Daniels J,Stewart J,et al.MCF-lOA-NeoST:a new cell system for studying cell-ECM and cell-cell interactions in breast cancer.Clin Cancer Res,2001,7(11):3640-3648
20 Zelinski DP,Zantek ND,Stewart JC,et al.EphA2 overexpress causes tumorigenesis of mammary epithelial cells.Cancer Res,2001,61(5):2301-2306
21 Rosenberg M,Goke M,Kanai M,et al.Epithelial cell kinaseB61:an autocrine loop modulating intestinal epithelial migration and barrier function.Am J Physiol,1997,273(4pt1):G824-G832
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