Inflammation-associated gene expression is altered between normal human ovarian surface epithelial cells and cell lines derived from ovarian adenocarcinomas

Ovulation is believed to contribute to the development of ovarian cancers that derive from the ovarian surface epithelium (OSE). The process of ovulation is synonymous with inflammation and inflammatory cytokines such as interleukin-1alpha (IL-1alpha) have recently been shown to induce both inflammatory and anti-inflammatory responses in human OSE (HOSE) cells. In this study we directly compared levels of IL-1alpha-induced gene expression by analysing the levels of 11beta-hydroxysteroid dehydrogenase (11betaHSD) types 1 (11betaHSD-1) and 2 (11betaHSD-2), cyclooxygenase-2 (COX-2), IL-1 receptor (IL-1R) and glucocorticoid receptor alpha (GRalpha) mRNA between normal HOSE cells and cell lines derived from poorly differentiated (SKOV-3, BG-1, PEO-4) and well-differentiated (PEO-14) ovarian adenocarcinoma. In HOSE cell cultures, and to a lesser extent PEO-14 cells, the basal mRNA levels of COX-2 and 11betaHSD-1 were relatively high and further shown to be induced in response to IL-1alpha (for HOSE cells; >20-fold, P<0.05 and PEO-14 cells; >3fold, P<0.05). However, whereas HOSE cells expressed a low level of 11betaHSD-2 mRNA that was only mildly responsive to IL-1alpha (1.3-fold, P<0.001), all cell lines exhibited a higher basal level of 11betaHSD-2 mRNA that was in some cases further stimulated in PEO-4 cells (five-fold; P<0.05) or suppressed in SKOV-3 cells (two-fold; P<0.01) in response to IL-1alpha. All cells tested expressed IL-1R and, with the exception of BG-1, GRalpha. These results indicate that cell lines derived from ovarian cancers have lost the ability to respond normally to inflammatory cytokines such as IL-1alpha. The finding that normal OSE cells, in contrast to cell lines derived from patients with ovarian adenocarcinoma, abundantly express 11betaHSD-1 mRNA but are essentially devoid of 11betaHSD-2 mRNA supports the concept that the pattern of 11betaHSD isoform gene expression is a defining feature of neoplastic cellular transformation, which might have particular relevance to the ovary.     

Differences in expression patterns of the tight junction proteins,claudin 1, 3, 4 and 5, in human ovarian surface epithelium as compared to epithelia in inclusion cysts and epithelial ovarian tumours

Human ovarian surface epithelium (OSE), regarded as the precursor cell of epithelial ovarian adenocarcinoma, is not a fully developed epithelium when situated on the ovarian surface. It lacks epithelial characteristics such as the cell-cell adhesion factor epithelial (E)-cadherin, but as we have shown earlier, this OSE can form functional tight junctions (TJs) in culture. Recent gene-expression data on ovarian adenocarcinoma has pointed out a family of TJ proteins, the claudins, to be highly expressed in malignant compared to benign ovarian tumours. The purpose of this study was to investigate the distribution of claudin 1-5 proteins in cultured OSE (n=4), normal ovarian (n=11), benign (n=8), borderline (n=7) and ovarian cancer (n=22) tissues. We found that a weak or absence of expression of claudin 3 and claudin 4 in surface OSE changed to typical cell-border localisation in OSE of inclusion cysts in the normal ovarian stroma. Semiquantitative estimations of immunoblots showed that claudin 3 was significantly increased in ovarian adenocarcinomas compared to benign and borderline-type tumours. Claudin 4 was significantly increased in both borderline-type and ovarian adenocarcinomas compared to benign tumours, whereas no changes were found for claudin 1 or 5. Concerning relation to Federation for International Gynaecology and Obstetrics (FIGO) grade, claudin 3, but not claudin 4, was significantly increased in moderately, poorly and undifferentiated adenocarcinomas compared to well-differentiated and borderline-type tumours. No significant changes were noticed for any claudin with regard to FIGO stages. We conclude that both claudin 3 and 4, even though they differ in expression during ovarian malignant transformation, might be used as novel markers for ovarian tumours. The observations of lack of claudin 4 and low expression of claudin 3 in OSE strengthen our current knowledge about the biological nature of these cells as an undeveloped epithelium.     

Aberrant regulation of argininosuccinate synthetase by TNF-alpha in human epithelial ovarian cancer

The pro-inflammatory cytokine, tumour necrosis factor-alpha, TNF-alpha, is dysregulated in malignant compared with normal ovarian surface epithelium (OSE). Several epidemiological studies have associated inflammation with ovarian tumorigenesis, with TNF-alpha playing a key role in modulating invasion, angiogenesis and metastasis. Here, we show that TNF-alpha also induces expression of arate-limiting enzyme in arginine synthesis, argininosuccinate synthetase (AS), thereby linking inflammation with several arginine-dependent metabolic pathways, implicated in accelerated carcinogenesis and tumour progression. Having identified AS mRNA induction in TNF-alpha-treated IGROV-1 ovarian cancer cells, using RNA-arbitrarily primed-PCR, we then observed differential regulation of AS mRNA and protein in malignant, compared with normal, OSE cells. A cDNA cancer profiling array with matched normal ovarian and ovarian tumour samples revealed increased expression of AS mRNA in the latter. Moreover, AS protein co-localised with TNF-alpha in ovarian cancer cells, with significantly higher levels of AS in malignant compared with normal ovarian tissue. Increased co-expression of AS and TNF-alpha mRNA was also observed in 2 other epithelial tumours, non-small cell lung and stomach cancer, compared with normal corresponding tissues. In summary, high levels of AS expression, which may be required for several arginine-dependent processes in cancer, including the production of nitric oxide, proline, pyrimidines and polyamines, is regulated by TNF-alpha and may provide an important molecular pathway linking inflammation and metabolism to ovarian tumorigenesis.     

人卵巢上皮癌微血管内皮细胞分离培养与鉴定

目的:建立人卵巢上皮癌微血管内皮细胞的原代分离纯化及培养方法。方法:收集2012-03-01-2012-10-31山东省肿瘤医院妇瘤科手术切除的卵巢上皮癌标本21例,采用胶原酶消化法分离人卵巢上皮癌微血管内皮细胞,免疫磁珠法提取纯化内皮细胞。采用形态学观察,免疫荧光技术及自发管腔形成实验对提取的内皮细胞进行鉴定;流式细胞仪检测细胞纯度;MTT法测定细胞增殖并绘制细胞生长曲线。结果:提取人卵巢微血管内皮细胞呈圆形或类圆形,周围有抗CD31免疫磁珠附着,活细胞计数测定>95%,流式细胞仪检测纯度>95%,细胞贴壁后呈长梭形、星形或多角形,胞内含有CD31磁珠,3～6d细胞间互相融合呈单层片状生长,铺满后呈典型铺路石样特征,荧光显微镜下观察表达内皮细胞特异性标志vWF,管腔形成实验证实体外有二维管腔形成。通过观察发现,内皮细胞24h内贴壁,7～11d可完全融合并出现接触抑制现象。通过MTT法测定内皮细胞生长分为增殖期、对数增长期和平台期3个阶段,生长曲线为"S"型。结论:应用免疫磁珠法成功建立了一种操作简单,且可获得纯度高和活性好的人卵巢上皮癌微血管内皮细胞分离及原代培养体系。     

人卵巢癌细胞系OV1228的建立及其生物学特性

目的用人卵巢癌组织建立新的卵巢癌细胞系,为探讨卵巢癌细胞的发病机制和治疗药物的筛选提供可靠的材料。方法选用人复发卵巢癌组织,进行肿瘤细胞原代培养,通过克隆稀释法,培养建立卵巢癌细胞系,进行体外传代,并通过细胞形态学、生长动力学以及致瘤性来分析其生物学特性。结果该卵巢癌细胞系已在体外培养生存18个月以上,传代超过80余代,被命名为OV1228。其生物学特性显示：倍增时间为29．39h;软琼脂集落形成率为17．5％;接种至裸鼠后具有较高的致瘤性;染色体核型分析为多倍体,众数为21～61条;透射电镜下可观察到卵巢癌细胞表面有少量短的细胞突起,部分细胞间可见发育比较差的桥粒样结构,具有明显的恶性卵巢癌细胞特征。结论OV1228经体外长期培养后已形成永生化卵巢癌细胞系,并具有明显的恶性特征,为开展人类卵巢癌的基础及临床研究提供了理想的实验材料。     

人卵巢癌永生化细胞系BUPH:OVSC-2的建立及其生物学特性研究

背景与目的：利用永生化技术建立预期表型的细胞系,已成为建设系的重要手段之一,而人卵巢癌永生化细胞系的建立国内外少有报道。本文介绍人卵巢肉瘤样癌永生化细胞系的建立,以及对其生物学特性的研究。方法：以手术切除的卵巢肉瘤样癌腹壁转移组织为材料,进行体外培养。将永生化基因SV40T抗原基因转染第10代细胞,经筛选、抗性克隆扩大培养,得到永生化细胞系。通过光学显微镜、电子显微镜、生化曲线测定、染色体分析、双层软琼脂培养、裸鼠接种、免疫组化等,研究其生物学特性,并与其来源细胞的生物学特性进行比较。结果：建立一株人卵巢肉瘤样癌永生化细胞系,命名为BUPH：OVSC－2,现已传至90年代。其生物学特性为：形态学观察细胞呈肉瘤样细胞形态,超微结构证实为上皮起源;细胞生长旺盛;具有恶性细胞的特征,恶性度高。通过比较,与其来源细胞的生物学特性无明显差别。结论：BUPH：OVSC－2为一株恶性度高的人卵巢肉瘤样癌永生化细胞系,保留了其来源细胞的生物学特性,可作为卵巢肉瘤样癌研究的实验模型。     

腹水来源卵巢癌细胞系的建立及其生物学特性

背景与目的:体外建立卵巢癌细胞系有利于深入了解卵巢癌干细胞的生物学特性及抗癌药物的筛选.本研究中我们以人卵巢癌患者腹水为材料建立卵巢癌细胞系,并进一步探讨其基本生物学特性.方法:体外分离卵巢癌患者腹水中的肿瘤细胞,纯化并传代培养,观察细胞形态学变化,绘制细胞生长曲线,计算细胞倍增时间,进行染色体核型分析.收集体外培养上清,进行放射免疫法检测相关激素状况和肿瘤标志物表达.动物实验观察细胞成瘤性以及瘤体的病理形态.结果:该卵巢癌细胞系已在体外培养与传代传110余代,历时近2年,透射电镜下可观察到卵巢癌细胞有显著的异形核,体外生长的倍增时间为40.8 h,染色体核型分析为超二倍体,染色体数目分布为63～120条.第80代细胞培养上清中雌二醇为45.0 pg/mL、睾酮0.03 ng/mL、孕酮未测出,CA125为4.49 U/mL,CA19-9为4.09 U/mL.第51代细胞接种于裸鼠后有皮下成瘤性,为低分化腺癌.结论:腹水来源的卵巢癌细胞在体外培养已是永生化细胞系,并具有明显的恶性细胞的生物学特征.     

Establishment of a cisplatin-resistant human ovarian cancer cell line

A cisplatin-resistant cell line was established by use of KF-1 cells derived from human serous cystadenocarcinoma of the ovary. This resistant cell line, designated "KFr," was capable of proliferating in the presence of 1.0 micrograms cisplatin/ml. It had doubling times of 24.8 and 27.2 hours in the presence of 0.5 and 1.0 micrograms cisplatin/ml, respectively. The KFr cells had lactate dehydrogenase (LDH) activities about fourfold higher than those of the parent KF-1 cells. Analysis of the isozymes of the KFr cells revealed that they had a faint LDH-3 band in addition to LDH-4, the only stained band in the parent KF-1 cells. The morphologic characteristics of the KFr cells were an enlarged nucleus and prominent nucleoli, unlike the nucleus and nucleoli of the parent KF-1 cells. The degree of resistance to cisplatin of the KFr cells was about twenty-fold higher than that of the KF-1 cells, with regard to the concentrations of cisplatin required for 50% inhibition of cell proliferation. However, when 5 microM calmodulin antagonists [N-(6-aminohexyl)-1-naphthalenesulfonamide or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] were added in the presence of concentrations of cisplatin that hardly inhibited cell proliferation, the KFr cell proliferation was markedly inhibited. These results suggest that cisplatin resistance can be overcome by calmodulin antagonists.     

Expression of multiple human endogenous retrovirus surface envelope proteins in ovarian cancer

Individual classes of human endogenous retrovirus (HERV) genes and proteins are expressed in cancer, but expression of more than one type of HERV is rare. We report here the expression of multiple HERV genes and proteins in ovarian cell lines and tissues. Expression of HERV-K env mRNA was greater in ovarian epithelial tumors than in normal ovarian tissues (N = 254). The expression of this protein on the surface and in the cytoplasm of ovarian cancer cells was confirmed using anti-HERV-K specific antibody by flow cytometric analysis. The frequency of expression of HERV-K env protein in multitissue microarrays (N = 641) was determined by immunohistochemistry and a significant correlation with tumor histotype was found. A significantly increased expression of HERV-K was observed in tumors with low malignant potential and low grade, relative to expression in normal ovarian tissues. The increase in expression of HERV-K env protein took place in a stepwise fashion in serous papillary adenocarcinoma. Interestingly, we found that other classes of HERV env mRNAs, including ERV3 and HERV-E, are expressed in the same ovarian cancer tissues that expressed HERV-K. Furthermore, anti-HERV antibodies including anti-ERV3 (30%), anti-HERV-E (40%) and anti-HERV-K (55%) were detected in patients with ovarian cancer, but not in normal female controls. HERV env proteins are frequently transcribed and translated in ovarian epithelial tumors, and multiple HERV families are detectable in ovarian cancer. HERV env proteins, and especially those expressed on the cell surface, may serve as novel tumor targets for detection, diagnosis and immunotherapy of ovarian cancer.     

Overexpression of follicle-stimulating hormone receptor facilitates the development of ovarian epithelial cancer

We previously showed that the expressing level of FSH receptor (FSHR) increased from ovarian epithelial inclusions (OEIs) to benign ovarian epithelial tumors (OETs) and to borderline OETs, whereas FSHR levels decreased with an increase in carcinoma grade. The aim of this study was to investigate the role of FSHR in OET development. MCV152 cells with FSHR overexpression showed an increased cellular proliferation and invasive capacity, which was associated with reduced levels of prohibitin and RII-β expression and increased levels of HER-2/neu, c-Myc, and EGFR expression. Overexpression of FSHR may be associated with an elevated level of OET cell proliferation via an enhanced activity of potential oncogenic pathways. Therefore, the findings in this study suggest that overexpression of FSHR may play a role in OET development.     

Genetic alterations in a telomerase-immortalized human esophageal epithelial cell line: Implications for carcinogenesis

Ectopic expression of viral oncoproteins disrupts cellular functions and limits the value of many existing immortalization models as models for carcinogenesis, especially for cancers without definitive viral etiology. Our newly established telomerase-immortalized human esophageal epithelial cell line, NE2-hTERT, retained nearly-diploid and non-tumorigenic characteristics, but exhibited genetic and genomic alterations commonly found in esophageal cancer, including progressive loss of the p16^INK4a alleles, upregulation of anti-apoptotic proteins, epithelial-mesenchymal transition, whole-chromosome 7 gain and duplicated 5q arm. Our data also revealed a novel positive regulation of p16^INK4a on cyclin D1. These findings probably represent early crucial events and mechanisms in esophageal carcinogenesis.     

Methylation status of oestrogen receptor-alpha gene promoter sequences in human ovarian epithelial cell lines

We have determined the methylation status of the CpG island of the oestrogen receptor alpha gene in seven human ovarian cell lines. Cell lines expressing oestrogen receptor alpha showed no evidence of hypermethylation. In three of four cell lines that produced no detectable oestrogen receptor alpha protein, hypermethylation was observed at the NotI site of the CpG island. These results indicate that aberrant hypermethylation may be responsible for a significant proportion of epithelial ovarian tumours in which oestrogen receptor alpha expression is lost.     

三氧化二砷联合塞来昔布对上皮性卵巢癌细胞的抗肿瘤效应及相关机制研究

目的:研究对上皮性卵巢癌采用三氧化二砷（Arsenic Trioxide,AS2O3）与环氧化酶-2（Cyclooxygenase-2,COX-2）抑制剂塞来昔布（Celecoxib）联合用药的有效性及其机制。方法:以上皮性卵巢癌细胞株OVCAR-3为研究对象,采用MTT体外药敏试验、Hoechest33258凋亡染色、流式细胞仪检测凋亡及细胞周期、蛋白质印迹法检测环氧化酶-2、凋亡相关蛋白等方法在细胞水平检验AS2O3和Celecoxib的单药及联合用药的有效性及可能的分子机制。结果:AS2O3和Celecoxib对OVCAR-3细胞株均具有剂量依赖性的生长抑制作用,两种药物单药的IC50值分别为4.72μmol/L和42.58μmol/L。两药联合后呈现明显的协同抗肿瘤效应,联合后的增殖抑制率和细胞凋亡率均大于任一单药作用,P<0.05,联合用药组的q值分别为3.59和2.16,联合用药具有明显的G2/M期阻滞效应。蛋白质印记法结果显示,两药联合作用后,COX-2、bcl-2表达均低于单药组,bax和caspase-3表达均高于单药组。结论:AS2O3和COX-2抑制剂Celecoxib对上皮性卵巢癌细胞株单药有效,联合后具有显著的协同抗肿瘤效应。     

短发卡状RNA抑制人Bubl基因表达及其对人卵巢癌A2780细胞药物敏感性和周期的影响

背景与目的:既往研究提示,紫杉醇类药物作用的发挥有赖于功能完整的纺锤体检查点,而Bubl是纺锤体检查点的重要组成部分.本研究旨在探讨Bubl短发卡状RNA真核表达载体稳定转染对人卵巢癌A2780细胞紫杉醇敏感性及细胞周期的影响.方法:构建Bubl基因短发卡状RNA真核表达载体pEGFP-Bubl-shRNA,以脂质体Lipofectamine~(TM)2000包裹空质粒转染体外培养的人卵巢癌细胞株A2780后进行稳定筛选.应用RT-PCR、Western印迹法分析目的基因mRNA及其蛋白水平的表达情况:MTT法、流式细胞术等方法检测转染前后,A2780细胞对紫杉醇敏感性及其细胞周期的变化;Hoechst33342染色法检测细胞分裂指数.结果:与对照组pEGFP-Cl/A2780及A2780组相比,pEGFP-Bubl-ShRNA/A2780组细胞中mRNA和蛋白水平的表达均明显下降,其差异具有统计学意义(P＜0.05);MTT检测结果提示,转染pEGFP-Bubl-shRNA质粒后细胞对紫杉醇的敏感性明显降低,与对照组相比,差异具有统计学意义(P＜0.05);流式细胞术检测结果显示,紫杉醇1 μ mol/L处理细胞24及48 h后,与pEGFP-Cl/A2780及A2780组相比,该组细胞凋亡率明显降低(P＜0.05),G_2/M期细胞比例下降,差异具有统计学意义(P＜0.05);Hoechst33342染色提示,与对照组相比,pEGFP-Bubl-shRNA/A2780组的分裂指数(MI)明显降低,差异具有统计学意义(P＜0.05).结论:Bubl基因的下调可导致人卵巢癌A2780细胞对紫杉醇敏感性及G_2/M期细胞比例下降.pEGFP-Cl-shBubl质粒的成功构建,为研究Bubl基因的功能及定位的研究提供了进一步的实验条件.     

他莫昔芬和甲孕酮对人卵巢癌细胞系增殖和凋亡的影响

目的：研究他莫昔芬和甲孕酮对人卵巢癌细胞增殖和凋亡的影响。方法：用不同剂量的他莫昔芬和甲孕酮与人卵巢癌细胞系ＨＯ－８９１０体外培养９６ｈ,用苔盼蓝活细胞拒染法计活细胞数,免疫组化ＳＡＢＣ法检测癌细胞增殖核抗原（ＰＣＮＡ）表达情况,ＤＮＡ缺口原位末端标记方法（ＴＵＮＥＬ）检测细胞凋亡情况。结果：他莫昔芬和甲孕酮在０．１、１、１０ｕｍｏｌ均可使ＨＯ－８９１０活细胞数显著减少（Ｐ〈０．０１）;通过诱导细