溶血磷脂酸及其受体Edg4、Edg7在卵巢上皮性肿瘤中的表达及其意义
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摘要
背景和目的
卵巢癌是常见的妇科恶性肿瘤之一，由于深居盆腔，缺乏早期症状和体征，75％～80％的患者确诊时已届晚期，由于其发病机制不清，临床上尚缺乏有效的治疗手段，因此，卵巢癌是死亡率最高的妇科恶性肿瘤之一。近年来溶血磷脂酸(lysophosphatidic acid，LPA)的功能以及与卵巢癌的密切关系逐渐成为卵巢癌诊断及基因治疗研究者关注的热点。
LPA是具有多种生物活性的磷脂介质，可以促进肿瘤细胞的增殖、生存、蛋白酶的产生及激活和促进血管生成因子等细胞因子的生成，参与肿瘤的发生、发展、浸润、转移以及化疗耐受等过程。LPA主要由活化的血小板、脂类细胞、血管内皮细胞等分泌，卵巢癌细胞也可产生LPA，并且形成自分泌环，导致卵巢癌患者血浆及腹水中LPA水平明显升高，提示LPA在卵巢癌的发生发展过程中具有重要作用。LPA主要通过LPA受体即G蛋白偶联受体(亦即内皮分化基因蛋白endothelial differentiation gene，Edg2，Edg4，Edg7)介导的多种信号传导通路发挥其生物学效应。其中Edg2在正常细胞中高表达，Edg4和Edg7则在肿瘤细胞中过表达，在正常细胞、永生化的组织细胞中不表达或低表达，LPA与Edg2结合可促进细胞凋亡，具降调节作用；与Edg4、Edg7结合则可促进细胞内DNA合成，诱导多种细胞因子的产生等。因此推测，Edg4和Edg7的表达可能与LPA对肿瘤的作用有关。
本实验首先通过生化的方法测定溶血磷脂酸在卵巢上皮性肿瘤患者血清中的表达情况，并与CA125相比较，探讨LPA对卵巢上皮性癌早期诊断的意义；然后采用免疫组化的方法比较溶血磷脂酸受体Edg4和Edg7在卵巢上皮性肿瘤组织中的表达情况，并观察这两种受体蛋白与卵巢上皮性癌的临床分期、组织学分级等临床和病理参数的关系，探讨LPA促肿瘤生长的作用机制，并为寻找卵巢癌治疗的新靶点，进行后续的卵巢癌基因治疗提供实验依据。
材料和方法
1．选取2005年10月～2006年2月在郑州大学一附院、三附院、郑州市妇幼保健院诊治的卵巢上皮性肿瘤患者70例作为研究对象，其中卵巢上皮性癌50例(浆液性囊腺癌37例，黏液性囊腺癌13例)，包括原发患者30例(Ⅰ期10例、Ⅱ～Ⅳ期20例)和复发患者20例；良性卵巢上皮性肿瘤20例(浆液性囊腺瘤13例、黏液性囊腺瘤6例、子宫内膜样肿瘤1例)，并选择20例正常妇女作为对照，采用生化法检测各组患者血清中LPA表达水平，同时测定血清中CA125水平，并将两者进行比较，探讨LPA对卵巢上皮性癌早期诊断的意义。
2．收集2000年2月～2004年10月郑州大学第一附属医院妇产科住院手术切除的卵巢上皮性肿瘤组织石蜡包埋标本68例，并经两名病理科专家会诊明确诊断。同时收集同期住院的宫颈癌和内膜癌患者手术切除的12例正常卵巢组织标本作为对照组，所有纳入对象于标本采取前均未接受放疗或化疗，临床及病理资料完整。
3．80例标本具体分组为：68例卵巢上皮性肿瘤包括上皮性癌48例(浆液性囊腺癌30例，粘液性囊腺癌11例，子宫内膜样癌7例)，交界性肿瘤10例，良性肿瘤10例，正常卵巢组织12例。卵巢上皮性癌按FIGO(2000年)临床分期：Ⅰ期12例，Ⅱ期16例，Ⅲ期18例，Ⅳ期2例；组织学分级G_1级14例，G_2级16例，G_3级18例。其中有淋巴结转移者22例，无淋巴结转移者26例；腹水≥500ml的30例，腹水＜500ml的18例。年龄在30～60岁之间，平均42.56±4.67岁。
4．采用免疫组化SP法检测80例卵巢上皮性肿瘤组织和正常卵巢组织中Edg4和Edg7蛋白的表达情况，并探讨这两种蛋白的表达与卵巢上皮性癌临床分期、组织学分级等临床和病理参数的关系。
5．统计学处理：采用SPSS13.0统计软件包进行数据分析，计量资料采用单因素方差分析和Fisher精确概率法，多个样本率比较采用行×列表x~2检验和秩和检验，两个样本率的比较采用四格表x~2检验，相关性分析采用Spearman等级相关分析。以α=0.05为检验水准。
结果
1．卵巢上皮性癌各组患者血清LPA表达水平明显升高，与正常妇女和良性卵巢上皮性肿瘤患者相比，其差别均有统计学意义(F=78.386，P＜0.05)；卵巢上皮性癌各组患者血清LPA水平相比无统计学意义(F=2.278，P＞0.05)；正常妇女和良性卵巢上皮性肿瘤患者血清中LPA表达水平相比较，其差别无统计学意义(F=2.563，P＞0.05)。而原发性卵巢癌Ⅱ～Ⅳ期组和复发组患者血清CA125水平明显高于正常妇女组、良性卵巢上皮性肿瘤组和卵巢上皮性癌原发Ⅰ期组，其差别有统计学意义(F=544.416，P＜0.05)。
2．血清LPA水平用于诊断卵巢上皮性癌的敏感性为94.1％(48／51)，特异性为89.5％(17／19)；血清CA125水平用于诊断卵巢上皮性癌的敏感性为87.8％(36／41)，特异性为51.7％(15／29)，而在卵巢上皮性癌原发Ⅰ期组，血清LPA水平为90％(9／10)，血清CA125仅为30％，两者比较有统计学意义(x~2=7.50，P＜0.05)。
3．溶血磷脂酸受体Edg4、Edg7在卵巢上皮性肿瘤组织中的阳性表达率分别为恶性91.7％和95.8％、交界性80％和70％、良性20％和30％；而在正常卵巢组织中阳性表达率仅为16.7％和16.7％。Edg4与Edg7蛋白在卵巢上皮性癌组织中的表达显著高于良性卵巢上皮性肿瘤和正常卵巢组织，差异有统计学意义(Edg4：x~2=38.873；Edg7：x~2=40.449，P＜0.05)。
4．卵巢上皮性癌组织中Edg4和Edg7蛋白在Ⅲ、Ⅳ期阳性表达明显高于在Ⅰ、Ⅱ期组织中的表达，两者比较差异有统计学意义(Edg4：x 2=17.807；Edg7：x~2=15.481，P＜0.05)；G_1级组织中两种蛋白的表达明显低于G_2、G_3级组织，比较结果差异有统计学意义(Edg4：x~2=18.139；Edg7：x~2=20.959，P＜0.05)；有淋巴结转移的癌组织中两种蛋白的表达明显高于无淋巴结转移的癌组织，差异有统计学意义(Edg4：x~2=10.741；Edg7：x~2=6.325，P＜0.05)；腹水≥500ml的癌组织中两种蛋白的表达明显高于腹水＜500ml的癌组织，比较结果差异有统计学意义(Edg4：x~2=5.807；Edg7：x~2=4.434，P＜0.05)。
5．Edg7蛋白在卵巢上皮性癌组织中的表达阳性率高于Edg4蛋白，但两者表达趋势一致，比较的结果，两者的表达呈正相关。(r_s=0.978，P＜0.05)。
结论
1．卵巢上皮性癌患者血清LPA水平明显升高，其敏感性和特异性均高于血清CA125，尤其在卵巢上皮性癌原发Ⅰ期组。LPA有望成为卵巢上皮性癌早期诊断和术后病情监测的生物学标记物。
2．溶血磷脂酸受体Edg4与Edg7蛋白在卵巢上皮性癌组织中高表达，与上皮性癌的恶性程度、临床分期、组织学分级等有关。
3．Edg4和Edg7受体蛋白在卵巢上皮性癌组织中表达程度呈正相关，提示两者在卵巢上皮性癌的发生发展中具有协同作用，并有望成为卵巢癌基因治疗的潜在靶点。
Background and aims:
Ovarian carcinoma is one of common gynecology malignant tumors with its high mortality. Because ovarian cancer spot locates in the deep pelvic bottom absencing of early signs and symptoms, about 75-80% patients of all are final diagnosed to be advanced stage. Due to unknown its pathogenesy, the effective therapy to ovarian carcinoma is still absent. Recently the function of lysophosphatidic acid (LPA) and the relationship with ovarian cancer are gradually regarded as a significant focus to ovarian carcinoma gene therapy.
LPA is a simplest phospholipids, it can mediate multiple functions ranging from growth promotion and cell cycle progression to cell survival. LPA can increase tumor cell proliferation, survival, invasion, protease production and activation, production of cytokines and resistance to chemotherapeutic agents. LPA participates in the occurrence, development, invasion, metastasis of carcinoma. Activated platelet, lipoidcells, vascular endothelialcells, etc. can secrete LPA. The ovarian cancer cells can also produce LPA and induce autocrine loop formation, which leads to high LPA level in the serum and ascites of ovarian cancer patients. It is suggested that LPA may play an important role in the process of ovarian cancer occurrence and development. The effects of LPA are determined by the LPA receptors expressed on the cell surfaces. The LPA receptors belong to members of the Edg family of G protein-coupled receptors, which have been supposed to mediate LPA signaling transduction pathway for multiple biological effection. LPA can promote apoptosis binding to Edg2, which it takes a role of down-regulation; whereas LPA increases cell DNA synthesis, mitochysis prosperity and induces production of many cytokines. Edg2 expresses highly in the normal cells. Edg4 and Edg7 yet overexpresses in the tumor cells, low expression or non-expession in the normal and immortalized cells, which suggests that high expression of Edg4 and Edg7 maybe relate to the effection of LPA in the tumors.
The experiment first planed to detect serum LPA expression level of patients with epithelial ovarian tumor by biochemistry assay and CA125, in order to evaluate it's value in diagnosis of ovarian cancer. Then expressions of Edg4 and Edg7 proteins in epithelial ovarian tumor were detected by immunohistochemical assay (SP) and the relationship between the two receptor proteins and the clinical parameters were investigated, such as clinical stage, pathological grade, ascetic fluid quantity, lymphoid node metastasis, etc. The aim was to investigate the mechanism of tumor growth promoted by LPA, exploring new targets for ovarian cancer therapy and to provide the experiment foundation for gene therapy of ovarian cancer.
Material and Methods:
1. 70 patients with epithelial ovarian tumor included 50 malignant cases(37 cases of serous cystadenocarcinoma, 13 cases of mucous cystoadenocarcinoma), 20 benign cases(13 cases of serous cystadenoma, 6 cases of mucous cystadenoma and 1 case of endometrioid tumor). Twenty normal women were studied. 50 malignant cases were divided into three groups: primary I stage, primary II—IV stage and relapsed. All cases were confirmed by pathology. Levels of serum LPA were detected by biochemistry assay and compared with tumor relative antigen CA125 levels to evaluate the value of ovarian cancer early period diagnosis through the measurement of serum LPA levels.
2. 68 cases of epithelial ovarian tumour paraffin imbedding samples and 12 cases of normal ovarian tissues were collected from department of gynaecology and obstetrics between February of 2000 year and October of 2004 year. All samples were diagnosed by two pathologists and all patients had not accepted the radiotherapy and chemotherapy before operation with complete clinical and pathological information.
3. 80 samples of epithelial ovarian tumors included normal(12 cases), benign(10 cases), bord-erline(10 cases) and malignant tumors(48 cases), while 48 malignant cases were dividedinto serous cystadenocarcinoma(30 cases), mucous cystoadenocarcinoma(11 cases) and endometrioid carcinoma(7 cases); FIGO I stage(12 cases), FIGO II stage(16 cases), FIGO III stage(18 cases), FIGO IV stage(2 cases); G_1 grade(14 cases), G_2 grade(16 cases), G_3 grade(18 cases) according to histological grade. The subgroups included lymphoid node metastasis(22 cases) and non-lymphoid node metastasis(26 cases); ascites quantity more than 500ml(30 cases) and ascites quantity less than 500ml(18 cases). The patients' age was from 30 to 60 years old, mean of 42.56±4.67 years.
4. Edg4 and Edg7 proteins expression in epithelial ovarian tumor were detected by immunohistochemical assay (streptomycin avidin-biotin-peroxidase complex staining assay). The relationship between expression of Edg4 and Edg7 proteins and the clinical parameters, such as clinical stage, pathological grade, ascites quantity, lymphoid nodemetastasis, etc. were investigated.
5. The data were analyzed by statistics software SPSS13.0 package. The measurement datawere expressed with X|-±s and dealed with analysis of variance(ANOVA) and Fisher exact probabilistic method. Multi-sample rate comparison used contingency table chi-square test, rank-sum test and two samples rate comparison used fourfold table chi-square test. Spearman rank correlation analysis was used to analyze the relevance. The test strandard wasα=0.05.
Results:
1. Levels of serum LPA in patients with malignant cases were higher significantly than those in normal women and benign cases (F=78.386, P<0.05). Serum LPA level of every epithelial carcinoma group had not statistical significance(F=2.278, P>0.05). The same result to the comparison of normal women and benign cases(F=2.563, P>0.05). CA125 levels in primary ovarian cancer with FIGO II～IV stage and relapsed group were higher than that of primary ovarian cancer with FIGO I stage group, benign tumor group and normal women group(F=544.416, P<0.05).
2. The sensitivity(94.1%) and the specificity(89.5%) of serum LPA in epithelial ovarian carcinoma were superior to CA125 with its sensitivity(87.8%) and specificity(51.7%). Especially in primary ovarian cancer with FIGO I stage LPA positive rate was 90%, which had significant statistical difference comparing with only 30% of CA125. (x~2 = 7.50, P<0.05).
3. The positive expression of Edg4 and Edg7 proteins was 91.7% and 95.8%(malignant), 80% and 70%(borderline), 20% and 30%(benign), 16.7% and 16.7%(nonnal). Edg4 and Edg7 proteins expression in malignant and borderline epithelial ovarian tumor was markedly higher than that of benign tumor and normal tissues (Edg4:x~2=38.873; Edg7:x~2=40.449, P<0.05).
4. The expression of Edg4 and Edg7 proteins was significantly associated with FIGO stage, pathological grade, ascites quantity and lymphoid node metastasis. The expression of Edg4 and Edg7 proteins in tissues with FIGO III～IV stage was obviously higher than that of FIGO I～II stage(Edg4:x~2=17.807; Edg7:x~2=15.481, P<0.05). The expression of two proteins in the G_1 grade tissues was lower than that of G_2 ,G_3 grade tissues significantly(Edg4: x~2=18.139; Edg7:x~2=20.959, P<0.05). The expression of Edg4 and Edg7 proteins in tissues with lymphoid node metastasis was higher than that of without lymphoid node metastasis (Edg4:x~2=10.741;Edg7: x~2=6.325, P<0.05). While the expression of two receptor proteins in tissues with ascites quantity more than 500ml was higher than that of the ascites quantity less than 500ml (Edg4: x~2=5.807;Edg7:x~2=4.434, P<0.05).
5. The positive expression of Edg7 protein was higher than that of Edg4 protein in epithelial ovarian cancer, but the expression of two proteins was positive correlation.(r_s=0.978, P<0.05).
Conclusions
1. Serum LPA levels in patients with epithelial ovarian cancer were high significantly. The sensitivity and the specificity of LPA in diagnosis epithelial ovarian cancer were superior to CA125, especially for early stage ovarian cancer. LPA may be a potential biomarker for diagnosis of epithelial ovarian cancer and a potential monitoring target after operation.
2. The expression of Edg4 and Edg7 proteins in epithelial ovarian cancer was markedly high. The expression of Edg4 and Edg7 proteins was significantly associated with the clinical stage, pathological grade, ascites quantity and lymphoid node metastasis.
3. The expression degree of Edg4 and Edg7 receptor proteins was positive correlation which suggest that the two proteins had synergistic effect in the occurrence and development of epithelial ovarian cancer. LPA and its receptor would become the potential targets to ovarian cancer gene therapy.
卵巢癌是常见的妇科恶性肿瘤之一，由于深居盆腔，缺乏早期症状和体征，75％～80％的患者确诊时已届晚期，由于其发病机制不清，临床上尚缺乏有效的治疗手段，因此，卵巢癌是死亡率最高的妇科恶性肿瘤之一。近年来溶血磷脂酸(lysophosphatidic acid，LPA)的功能以及与卵巢癌的密切关系逐渐成为卵巢癌诊断及基因治疗研究者关注的热点。
LPA是具有多种生物活性的磷脂介质，可以促进肿瘤细胞的增殖、生存、蛋白酶的产生及激活和促进血管生成因子等细胞因子的生成，参与肿瘤的发生、发展、浸润、转移以及化疗耐受等过程。LPA主要由活化的血小板、脂类细胞、血管内皮细胞等分泌，卵巢癌细胞也可产生LPA，并且形成自分泌环，导致卵巢癌患者血浆及腹水中LPA水平明显升高，提示LPA在卵巢癌的发生发展过程中具有重要作用。LPA主要通过LPA受体即G蛋白偶联受体(亦即内皮分化基因蛋白endothelial differentiation gene，Edg2，Edg4，Edg7)介导的多种信号传导通路发挥其生物学效应。其中Edg2在正常细胞中高表达，Edg4和Edg7则在肿瘤细胞中过表达，在正常细胞、永生化的组织细胞中不表达或低表达，LPA与Edg2结合可促进细胞凋亡，具降调节作用；与Edg4、Edg7结合则可促进细胞内DNA合成，诱导多种细胞因子的产生等。因此推测，Edg4和Edg7的表达可能与LPA对肿瘤的作用有关。
本实验首先通过生化的方法测定溶血磷脂酸在卵巢上皮性肿瘤患者血清中的表达情况，并与CA125相比较，探讨LPA对卵巢上皮性癌早期诊断的意义；然后采用免疫组化的方法比较溶血磷脂酸受体Edg4和Edg7在卵巢上皮性肿瘤组织中的表达情况，并观察这两种受体蛋白与卵巢上皮性癌的临床分期、组织学分级等临床和病理参数的关系，探讨LPA促肿瘤生长的作用机制，并为寻找卵巢癌治疗的新靶点，进行后续的卵巢癌基因治疗提供实验依据。
材料和方法
1．选取2005年10月～2006年2月在郑州大学一附院、三附院、郑州市妇幼保健院诊治的卵巢上皮性肿瘤患者70例作为研究对象，其中卵巢上皮性癌50例(浆液性囊腺癌37例，黏液性囊腺癌13例)，包括原发患者30例(Ⅰ期10例、Ⅱ～Ⅳ期20例)和复发患者20例；良性卵巢上皮性肿瘤20例(浆液性囊腺瘤13例、黏液性囊腺瘤6例、子宫内膜样肿瘤1例)，并选择20例正常妇女作为对照，采用生化法检测各组患者血清中LPA表达水平，同时测定血清中CA125水平，并将两者进行比较，探讨LPA对卵巢上皮性癌早期诊断的意义。
2．收集2000年2月～2004年10月郑州大学第一附属医院妇产科住院手术切除的卵巢上皮性肿瘤组织石蜡包埋标本68例，并经两名病理科专家会诊明确诊断。同时收集同期住院的宫颈癌和内膜癌患者手术切除的12例正常卵巢组织标本作为对照组，所有纳入对象于标本采取前均未接受放疗或化疗，临床及病理资料完整。
3．80例标本具体分组为：68例卵巢上皮性肿瘤包括上皮性癌48例(浆液性囊腺癌30例，粘液性囊腺癌11例，子宫内膜样癌7例)，交界性肿瘤10例，良性肿瘤10例，正常卵巢组织12例。卵巢上皮性癌按FIGO(2000年)临床分期：Ⅰ期12例，Ⅱ期16例，Ⅲ期18例，Ⅳ期2例；组织学分级G_1级14例，G_2级16例，G_3级18例。其中有淋巴结转移者22例，无淋巴结转移者26例；腹水≥500ml的30例，腹水＜500ml的18例。年龄在30～60岁之间，平均42.56±4.67岁。
4．采用免疫组化SP法检测80例卵巢上皮性肿瘤组织和正常卵巢组织中Edg4和Edg7蛋白的表达情况，并探讨这两种蛋白的表达与卵巢上皮性癌临床分期、组织学分级等临床和病理参数的关系。
5．统计学处理：采用SPSS13.0统计软件包进行数据分析，计量资料采用单因素方差分析和Fisher精确概率法，多个样本率比较采用行×列表x~2检验和秩和检验，两个样本率的比较采用四格表x~2检验，相关性分析采用Spearman等级相关分析。以α=0.05为检验水准。
结果
1．卵巢上皮性癌各组患者血清LPA表达水平明显升高，与正常妇女和良性卵巢上皮性肿瘤患者相比，其差别均有统计学意义(F=78.386，P＜0.05)；卵巢上皮性癌各组患者血清LPA水平相比无统计学意义(F=2.278，P＞0.05)；正常妇女和良性卵巢上皮性肿瘤患者血清中LPA表达水平相比较，其差别无统计学意义(F=2.563，P＞0.05)。而原发性卵巢癌Ⅱ～Ⅳ期组和复发组患者血清CA125水平明显高于正常妇女组、良性卵巢上皮性肿瘤组和卵巢上皮性癌原发Ⅰ期组，其差别有统计学意义(F=544.416，P＜0.05)。
2．血清LPA水平用于诊断卵巢上皮性癌的敏感性为94.1％(48／51)，特异性为89.5％(17／19)；血清CA125水平用于诊断卵巢上皮性癌的敏感性为87.8％(36／41)，特异性为51.7％(15／29)，而在卵巢上皮性癌原发Ⅰ期组，血清LPA水平为90％(9／10)，血清CA125仅为30％，两者比较有统计学意义(x~2=7.50，P＜0.05)。
3．溶血磷脂酸受体Edg4、Edg7在卵巢上皮性肿瘤组织中的阳性表达率分别为恶性91.7％和95.8％、交界性80％和70％、良性20％和30％；而在正常卵巢组织中阳性表达率仅为16.7％和16.7％。Edg4与Edg7蛋白在卵巢上皮性癌组织中的表达显著高于良性卵巢上皮性肿瘤和正常卵巢组织，差异有统计学意义(Edg4：x~2=38.873；Edg7：x~2=40.449，P＜0.05)。
4．卵巢上皮性癌组织中Edg4和Edg7蛋白在Ⅲ、Ⅳ期阳性表达明显高于在Ⅰ、Ⅱ期组织中的表达，两者比较差异有统计学意义(Edg4：x 2=17.807；Edg7：x~2=15.481，P＜0.05)；G_1级组织中两种蛋白的表达明显低于G_2、G_3级组织，比较结果差异有统计学意义(Edg4：x~2=18.139；Edg7：x~2=20.959，P＜0.05)；有淋巴结转移的癌组织中两种蛋白的表达明显高于无淋巴结转移的癌组织，差异有统计学意义(Edg4：x~2=10.741；Edg7：x~2=6.325，P＜0.05)；腹水≥500ml的癌组织中两种蛋白的表达明显高于腹水＜500ml的癌组织，比较结果差异有统计学意义(Edg4：x~2=5.807；Edg7：x~2=4.434，P＜0.05)。
5．Edg7蛋白在卵巢上皮性癌组织中的表达阳性率高于Edg4蛋白，但两者表达趋势一致，比较的结果，两者的表达呈正相关。(r_s=0.978，P＜0.05)。
结论
1．卵巢上皮性癌患者血清LPA水平明显升高，其敏感性和特异性均高于血清CA125，尤其在卵巢上皮性癌原发Ⅰ期组。LPA有望成为卵巢上皮性癌早期诊断和术后病情监测的生物学标记物。
2．溶血磷脂酸受体Edg4与Edg7蛋白在卵巢上皮性癌组织中高表达，与上皮性癌的恶性程度、临床分期、组织学分级等有关。
3．Edg4和Edg7受体蛋白在卵巢上皮性癌组织中表达程度呈正相关，提示两者在卵巢上皮性癌的发生发展中具有协同作用，并有望成为卵巢癌基因治疗的潜在靶点。
Background and aims:
Ovarian carcinoma is one of common gynecology malignant tumors with its high mortality. Because ovarian cancer spot locates in the deep pelvic bottom absencing of early signs and symptoms, about 75-80% patients of all are final diagnosed to be advanced stage. Due to unknown its pathogenesy, the effective therapy to ovarian carcinoma is still absent. Recently the function of lysophosphatidic acid (LPA) and the relationship with ovarian cancer are gradually regarded as a significant focus to ovarian carcinoma gene therapy.
LPA is a simplest phospholipids, it can mediate multiple functions ranging from growth promotion and cell cycle progression to cell survival. LPA can increase tumor cell proliferation, survival, invasion, protease production and activation, production of cytokines and resistance to chemotherapeutic agents. LPA participates in the occurrence, development, invasion, metastasis of carcinoma. Activated platelet, lipoidcells, vascular endothelialcells, etc. can secrete LPA. The ovarian cancer cells can also produce LPA and induce autocrine loop formation, which leads to high LPA level in the serum and ascites of ovarian cancer patients. It is suggested that LPA may play an important role in the process of ovarian cancer occurrence and development. The effects of LPA are determined by the LPA receptors expressed on the cell surfaces. The LPA receptors belong to members of the Edg family of G protein-coupled receptors, which have been supposed to mediate LPA signaling transduction pathway for multiple biological effection. LPA can promote apoptosis binding to Edg2, which it takes a role of down-regulation; whereas LPA increases cell DNA synthesis, mitochysis prosperity and induces production of many cytokines. Edg2 expresses highly in the normal cells. Edg4 and Edg7 yet overexpresses in the tumor cells, low expression or non-expession in the normal and immortalized cells, which suggests that high expression of Edg4 and Edg7 maybe relate to the effection of LPA in the tumors.
The experiment first planed to detect serum LPA expression level of patients with epithelial ovarian tumor by biochemistry assay and CA125, in order to evaluate it's value in diagnosis of ovarian cancer. Then expressions of Edg4 and Edg7 proteins in epithelial ovarian tumor were detected by immunohistochemical assay (SP) and the relationship between the two receptor proteins and the clinical parameters were investigated, such as clinical stage, pathological grade, ascetic fluid quantity, lymphoid node metastasis, etc. The aim was to investigate the mechanism of tumor growth promoted by LPA, exploring new targets for ovarian cancer therapy and to provide the experiment foundation for gene therapy of ovarian cancer.
Material and Methods:
1. 70 patients with epithelial ovarian tumor included 50 malignant cases(37 cases of serous cystadenocarcinoma, 13 cases of mucous cystoadenocarcinoma), 20 benign cases(13 cases of serous cystadenoma, 6 cases of mucous cystadenoma and 1 case of endometrioid tumor). Twenty normal women were studied. 50 malignant cases were divided into three groups: primary I stage, primary II—IV stage and relapsed. All cases were confirmed by pathology. Levels of serum LPA were detected by biochemistry assay and compared with tumor relative antigen CA125 levels to evaluate the value of ovarian cancer early period diagnosis through the measurement of serum LPA levels.
2. 68 cases of epithelial ovarian tumour paraffin imbedding samples and 12 cases of normal ovarian tissues were collected from department of gynaecology and obstetrics between February of 2000 year and October of 2004 year. All samples were diagnosed by two pathologists and all patients had not accepted the radiotherapy and chemotherapy before operation with complete clinical and pathological information.
3. 80 samples of epithelial ovarian tumors included normal(12 cases), benign(10 cases), bord-erline(10 cases) and malignant tumors(48 cases), while 48 malignant cases were dividedinto serous cystadenocarcinoma(30 cases), mucous cystoadenocarcinoma(11 cases) and endometrioid carcinoma(7 cases); FIGO I stage(12 cases), FIGO II stage(16 cases), FIGO III stage(18 cases), FIGO IV stage(2 cases); G_1 grade(14 cases), G_2 grade(16 cases), G_3 grade(18 cases) according to histological grade. The subgroups included lymphoid node metastasis(22 cases) and non-lymphoid node metastasis(26 cases); ascites quantity more than 500ml(30 cases) and ascites quantity less than 500ml(18 cases). The patients' age was from 30 to 60 years old, mean of 42.56±4.67 years.
4. Edg4 and Edg7 proteins expression in epithelial ovarian tumor were detected by immunohistochemical assay (streptomycin avidin-biotin-peroxidase complex staining assay). The relationship between expression of Edg4 and Edg7 proteins and the clinical parameters, such as clinical stage, pathological grade, ascites quantity, lymphoid nodemetastasis, etc. were investigated.
5. The data were analyzed by statistics software SPSS13.0 package. The measurement datawere expressed with X|-±s and dealed with analysis of variance(ANOVA) and Fisher exact probabilistic method. Multi-sample rate comparison used contingency table chi-square test, rank-sum test and two samples rate comparison used fourfold table chi-square test. Spearman rank correlation analysis was used to analyze the relevance. The test strandard wasα=0.05.
Results:
1. Levels of serum LPA in patients with malignant cases were higher significantly than those in normal women and benign cases (F=78.386, P<0.05). Serum LPA level of every epithelial carcinoma group had not statistical significance(F=2.278, P>0.05). The same result to the comparison of normal women and benign cases(F=2.563, P>0.05). CA125 levels in primary ovarian cancer with FIGO II～IV stage and relapsed group were higher than that of primary ovarian cancer with FIGO I stage group, benign tumor group and normal women group(F=544.416, P<0.05).
2. The sensitivity(94.1%) and the specificity(89.5%) of serum LPA in epithelial ovarian carcinoma were superior to CA125 with its sensitivity(87.8%) and specificity(51.7%). Especially in primary ovarian cancer with FIGO I stage LPA positive rate was 90%, which had significant statistical difference comparing with only 30% of CA125. (x~2 = 7.50, P<0.05).
3. The positive expression of Edg4 and Edg7 proteins was 91.7% and 95.8%(malignant), 80% and 70%(borderline), 20% and 30%(benign), 16.7% and 16.7%(nonnal). Edg4 and Edg7 proteins expression in malignant and borderline epithelial ovarian tumor was markedly higher than that of benign tumor and normal tissues (Edg4:x~2=38.873; Edg7:x~2=40.449, P<0.05).
4. The expression of Edg4 and Edg7 proteins was significantly associated with FIGO stage, pathological grade, ascites quantity and lymphoid node metastasis. The expression of Edg4 and Edg7 proteins in tissues with FIGO III～IV stage was obviously higher than that of FIGO I～II stage(Edg4:x~2=17.807; Edg7:x~2=15.481, P<0.05). The expression of two proteins in the G_1 grade tissues was lower than that of G_2 ,G_3 grade tissues significantly(Edg4: x~2=18.139; Edg7:x~2=20.959, P<0.05). The expression of Edg4 and Edg7 proteins in tissues with lymphoid node metastasis was higher than that of without lymphoid node metastasis (Edg4:x~2=10.741;Edg7: x~2=6.325, P<0.05). While the expression of two receptor proteins in tissues with ascites quantity more than 500ml was higher than that of the ascites quantity less than 500ml (Edg4: x~2=5.807;Edg7:x~2=4.434, P<0.05).
5. The positive expression of Edg7 protein was higher than that of Edg4 protein in epithelial ovarian cancer, but the expression of two proteins was positive correlation.(r_s=0.978, P<0.05).
Conclusions
1. Serum LPA levels in patients with epithelial ovarian cancer were high significantly. The sensitivity and the specificity of LPA in diagnosis epithelial ovarian cancer were superior to CA125, especially for early stage ovarian cancer. LPA may be a potential biomarker for diagnosis of epithelial ovarian cancer and a potential monitoring target after operation.
2. The expression of Edg4 and Edg7 proteins in epithelial ovarian cancer was markedly high. The expression of Edg4 and Edg7 proteins was significantly associated with the clinical stage, pathological grade, ascites quantity and lymphoid node metastasis.
3. The expression degree of Edg4 and Edg7 receptor proteins was positive correlation which suggest that the two proteins had synergistic effect in the occurrence and development of epithelial ovarian cancer. LPA and its receptor would become the potential targets to ovarian cancer gene therapy.
引文
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49 Usatyuk PV, Cummings R, Saatian B, et al. Lipid phosphate phosphatase-1 regulates lysophosphatidic acid-induced calcium release, NF-kappaB activation and interleukin-8 secretion in human bronchial epithelial cells. Biochem J.2005,385(Pt2):493-502
50 Goppelt-Struebe M, Fickel S, Reiser CO. The platelet-derived growth-factor receptor, not the epidermal-growth-factor receptor, is used by lysophosphatidic acid to activate p42/44 mitogen-activated protein kinase and to induce prostaglandin G/H synthase-2 in mesangial cells.J.Biochem,2000,345(Pt2):217-224
51 Burnett C, Makridou P, Hewlett L, Howard K. Lipid phosphate phosphatases dimerise, but this interaction is not required for in vivo activity. BMC Biochem,2004,5(16):2-7
52 Yue J, Yokoyama K, Balazs L, et al. Mice with transgenic overexpression of lipid phosphate phosphatase-1 display multiple organotypic deficits without alteration in circulating lysophosphatidate levels.Cell Signal,2004,16(3):385-399
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54 Long JS, Yokoyama K, Tigyi G, et al. Lipid phosphate phosphataseol regulates lysophosphatidic acid- and platelet-derived growth factor-induced cell migration.J Biochem,2006,394(Pt2):495-500
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56 Carman GM, Hart GS. Roles of phosphatidate phosphatase enzymes in lipid metabolism. Trends Biochem Sci,2006,31(12):694-699
57 Kishore KW, Joseph OH. Anti-lipid phosphate phosphohydrolase-3 (LPP3) antibody inhibits bFGF-and VEGF-induced capillary morphogenesis of endothelial cells.Cell Communication and Signaling.2005,3(10):9-19
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59 Coon M, Ball A, Pound J, et al. Inhibition of lysophosphatidic acid acyltransferase-beta disrupts proliferative and survival signals in normal cells and induces apoptosis of tumor cells.Mol Cancer Ther,2003,2(10):1067-1078
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15 Hu YL, Albanese C, Pestell RG, et al. Dual mechanisms for ysophosphatidic acid stimulation of human ovarian carcinoma cells. Natl Cancer Inst,2003,95(10):733～740
16 Pustilnik TB, Estrella V, Wiener JR, et al. Lysophosphatidic acid induces urokinase secretion by ovarian cancer cells. Clin Cancer Res,1999,5(11):3704～3710
17 Fishman DA, Liu Y, Ellerbroek SM, et al. Lysophosphatidic acid promotes matrix metalloproteinase(MMP) activation and MMP-dependent invasion in ovarian cancer cells. Cancer Res,2001,61(7):3194～3199
18 Sawada K, Morishige K, Tahara M, et al. Lysophosphatidic acid induces focal sdhesion assembly through Rho/Rho-associated kinase pathway in human ovarian cancer cells. Gynecol Oncol,2002,87(3):252～259
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20 Gadducci A, Viacava P, Cosio S, et al. Vascular endothelial growth factor (VEGF) expression in primary tumors and peritoneal metastases from patients with advanced ovarian carcinoma. Anticancer Res, 2003, 23(3C) :3001～3008
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24 Frankel A, Mills GB. Peptide and lipid growth factors decrease cisdiminedichloroplatinum-induced cell death in human ovarian cancer. Clin Cancer Res,1996,2:1307～1313
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47 Diana EA, Lidia H, Hervé LS, et al. The lipid phosphatase LPP3 regulates extra-embryonic vasculogenesis and axis patterning.Development,2003,130(19): 4623-4637
48 Susan P, Kok-Choi K, Darroch PI. Lysophosphatidic acid and sphingosine 1-phosphate biology:the role of lipid phosphate phosphatases.Seminars in Cell & Developmental Biology, 2004,15 (5):491-501
49 Usatyuk PV, Cummings R, Saatian B, et al. Lipid phosphate phosphatase-1 regulates lysophosphatidic acid-induced calcium release, NF-kappaB activation and interleukin-8 secretion in human bronchial epithelial cells. Biochem J.2005,385(Pt2):493-502
50 Goppelt-Struebe M, Fickel S, Reiser CO. The platelet-derived growth-factor receptor, not the epidermal-growth-factor receptor, is used by lysophosphatidic acid to activate p42/44 mitogen-activated protein kinase and to induce prostaglandin G/H synthase-2 in mesangial cells.J.Biochem,2000,345(Pt2):217-224
51 Burnett C, Makridou P, Hewlett L, Howard K. Lipid phosphate phosphatases dimerise, but this interaction is not required for in vivo activity. BMC Biochem,2004,5(16):2-7
52 Yue J, Yokoyama K, Balazs L, et al. Mice with transgenic overexpression of lipid phosphate phosphatase-1 display multiple organotypic deficits without alteration in circulating lysophosphatidate levels.Cell Signal,2004,16(3):385-399
53 Smyth SS, Sciorra VA, Sigal YJ, et al. Lipid phosphate phosphatases regulate lysophosphatidic acid production and signalling in platelets: studies using chemical inhibitors of lipid phosphate phosphatase activity. J Biol Chem,2003,278(44):43214-43223
54 Long JS, Yokoyama K, Tigyi G, et al. Lipid phosphate phosphataseol regulates lysophosphatidic acid- and platelet-derived growth factor-induced cell migration.J Biochem,2006,394(Pt2):495-500
55 Pilquil C, Dewald J, Chemey A, et al. Lipid phosphate phosphatase-1 regulates lysophosphatidate-induced fibroblast migration by controlling phospholipase D2-dependent phosphatidate generation.Biol Chem, 2006,281(50):38418-38429
56 Carman GM, Hart GS. Roles of phosphatidate phosphatase enzymes in lipid metabolism. Trends Biochem Sci,2006,31(12):694-699
57 Kishore KW, Joseph OH. Anti-lipid phosphate phosphohydrolase-3 (LPP3) antibody inhibits bFGF-and VEGF-induced capillary morphogenesis of endothelial cells.Cell Communication and Signaling.2005,3(10):9-19
58 Hideshima T, Chauhan D, Hayashi T, et al. Antitumor activity of lysophosphatidic acid acyltransferase-beta inhibitors,a novel class of agents, in multiple myeloma. Cancer Res,2003,63(23):8428-8436
59 Coon M, Ball A, Pound J, et al. Inhibition of lysophosphatidic acid acyltransferase-beta disrupts proliferative and survival signals in normal cells and induces apoptosis of tumor cells.Mol Cancer Ther,2003,2(10):1067-1078
60 Rocconi RP, Numnum TM, Stoff-Khalili M, et al. Targeted gene therapy for ovarian cancer. Curr Gene Ther,2005,5(6):643-653
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