Steroid receptors in epithelial ovarian carcinoma: relation to clinical parameters and survival

Estrogen receptor (ER) and progestin receptor were measured in samples of tumors obtained at first laparotomy from 97 previously untreated patients suffering with a primary ovarian epithelial tumor, for whom a 3-year follow-up was available. The presence or absence of steroid receptors (threshold arbitrarily fixed at 10 fmol/mg of cytoplasmic protein) was determined by the dextran coated charcoal method and related to a number of patient characteristics such as the residual disease (cutoff, 2 cm), histological type, International Federation of Gynecologists and Obstetricians grade and stage, and age. Results were analyzed by univariate and multivariate methods. (a) The tumor ER positivity was associated with better survival; progestin receptor showed a similar trend but did not reach statistical significance. (b) After stratification for residual tumor the association ER positivity/better survival was still statistically significant in the subset of patients with residual tumor greater than 2 cm. (c) When the median survival times were considered it became apparent that progestin receptor absence nullified the effect associated with positive ER. (d) Multivariate analysis confirmed that among the variables considered the main determinants of prognosis were the size of the residual tumor, serous histological type, and positive ER.     

Cytoplasmatic receptors of steroid hormones in malignant epithelial ovarian tumors

The levels of estrogen, progesterone, androgen and glucocorticoid receptors were assayed in 70 malignant epithelial tumors of human ovaries. The percentage of progesterone, androgen and glucocorticoid receptor-positive tumors was significantly higher in reproductive patients than in menopausal ones. Well-differentiated serous cystadenocarcinomas showed a higher level of progesterone receptors than those characterized by poor differentiation of cells. It was found that steroid hormone receptor profile of primary tumor may be determined in its metastases into the greater omentum whenever tumor cannot be removed. In cases of preoperative chemotherapy, the percentage of receptor-positive ovarian tumors was lower.     

caspase-3表达及其与卵巢上皮性肿瘤组织细胞凋亡和增殖的关系

目的 :探讨caspase 3与卵巢上皮性肿瘤发生、发展的关系。方法 :利用免疫组化(SP)法和TdT介导的dUTP缺口末端标记(TUNEL)技术 ,检测 112例卵巢上皮性肿瘤组织中caspase 3和PCNA的表达水平及细胞凋亡情况。结果 :caspase 3在卵巢恶性肿瘤中的表达 ( 4 4 4% ) ,明显低于良性肿瘤 ( 81 8% ,P<0 0 1) ;在低分化卵巢癌中的表达 ( 61 5 % ) ,明显高于高分化卵巢癌 ( 2 3 5 % ,P <0 0 5 ) ;在晚期卵巢癌中的表达 ( 5 7 9% ) ,明显高于早期卵巢癌 ( 2 4 0 % ,P <0 0 5 ) ;在有淋巴结转移癌中的表达 ( 5 6 4% ) ,明显高于无淋巴结转移癌中的表达 ( 3 3 3 % ,P <0 0 5 )。凋亡指数与增殖指数呈正相关 (r =0 467,P <0 0 1) ,与cas pase 3的表达呈正相关 (r =0 5 97,P <0 0 1)。结论 :caspase 3与卵巢上皮性肿瘤的细胞凋亡密切相关 ,参与细胞凋亡的调节机制 ,在卵巢上皮性肿瘤恶变的过程中发挥作用 ,并与恶性程度关系密切 ,有可能成为卵巢癌预后评估的参考。     

TIRAP对上皮性卵巢癌细胞增殖和凋亡能力的影响

目的:研究TIRAP在上皮性卵巢癌细胞中的表达情况,及其对上皮性卵巢癌细胞增殖和凋亡能力的影响。方法:通过实时荧光定量PCR（qRT-PCR）和Western blot检测各组细胞中TIRAP的表达。利用CCK-8法、流式细胞技术探究TIRAP的表达对人上皮性卵巢癌细胞增殖和凋亡的影响。利用Western blot、ELISA法探究TIRAP的表达对p-NF-κB p65及下游免疫分子IL-6、TNF-α水平的影响。结果:与正常卵上皮细胞（IOSE-80）相比,上皮性卵巢癌细胞（SKOV-3,OVCAR3,A2780）中TIRAP的mRNA和蛋白表达显著升高。转染TIRAP siRNA 后,SKOV-3细胞中TIRAP的表达显著降低;CCK-8实验显示:转染培养第96 h时,si-TIRAP组OD值（1.85±0.06）较Blank组（2.42±0.16）及Scramble control组（2.50±0.10）明显降低;细胞凋亡检测显示:si-TIRAP组凋亡率明显高于对照组;Western blot实验显示:LPS+si-TIRAP组p-NF-κB p65的表达与对照组相比显著降低;ELISA实验显示:LPS+si-TIRAP组IL-6、TNF-α的含量与对照组相比显著下降。结论:TIRAP在上皮性卵巢癌细胞中的表达升高,可能是上皮性卵巢癌细胞增殖的机制之一;下调TIRAP的表达可以抑制上皮性卵巢癌细胞增殖,促进细胞凋亡,并且通过抑制NF-κB通路的活化降低其下游免疫分子的表达。     

Survivin expression and its correlation with cell proliferation and prognosis in epithelial ovarian tumors

Survivin is a new member of the inhibitors of apoptosis proteins (IAP) family, selectively overexpressed in common human cancers but not in normal adult tissues, and associated with aggressiveness of the disease and unfavorable outcomes. Recent study also found that survivin expression is associated with cell proliferation. In order to gain insight into the role of survivin in ovarian tumors, we investigated the expression of survivin in a group of epithelial ovarian tumors, and examined the relationship of its expression with cell proliferation and clinical outcome. Immunohistochemical analysis was performed in 103 cases of epithelial ovarian tumors. Twenty-six of the 103 cases were evaluated by Western blot analysis. The results showed that survivin overexpression was detected in 21.2% (7 of 33) of benign tumors, 47.8% (11 of 23) of borderline tumors, and 51.1% (24 of 47) of ovarian carcinomas. The positive ratio was significantly higher in malignant or borderline tumors than in benign tumors, and the overexpression of survivin was significantly correlated with the size of residual disease. A positive correlation between survivin expression and proliferative activity of tumor cell measured by PCNA index was found. Kaplan-Meier analysis demonstrated that the patients with survivin overexpression have a short overall survival. These findings suggest that survivin overexpression may play a pivotal role in the progression of ovarian tumors and may provide an important prognostic implication for epithelial ovarian carcinomas.     

Effect of reproductive hormones on ovarian epithelial tumors: I. Effect on cell cycle activity

We examined the effects of the 4 major female reproductive hormones, estradiol (E2), progesterone (P4), follicle stimulating hormone (FSH), and luteinizing hormone (LH) on thymidine incorporation in benign and malignant ovarian epithelial tumors cultured in vitro. Treatment of these tumors with E2, FSH and LH resulted in increased thymidine incorporation while treatment with P4 inhibited growth as well as thymidine incorporation. The inhibitory effect of progesterone could not be reproduced by treating the cells with ligands for other steroid hormone receptors known to interact with P4 such as the mineralocorticoid and glucocorticoid receptors. All cells lines expressed at least the PR-A isoform of the progesterone receptor. ORG2058, R5020, RU486, and ZK98299 acted as progesterone receptor agonists with regard to their effect on thymidine incorporation. P4 down-regulated cyclin Bl expression and cdkl activity and up-regulated the p21 and p27 proteins. Expression of a reporter gene downstream to an AP-1 responsive element in a plasmid construct transfected into ovarian epithelial tumor cells was induced by P4 and inhibited by RU486. We conclude that P4 inhibits cell cycle activity in ovarian epithelial tumors, in part via down-regulation of the cdkl/cyclin B complex. This inhibitory effect may have therapeutic utility against ovarian epithelial tumors.     

Isoflavones inhibit intestinal epithelial cell proliferation and induce apoptosis in vitro.

There have been many reports that high soya-based diets reduce the risk of certain types of cancer. This effect may be due to the presence of high levels of isoflavones derived from the soya bean, particularly genistein which has been shown to be a protein tyrosine kinase (PTK) inhibitor and have both oestrogenic and anti-oestrogenic properties. We have examined the effect of genistein and a number of novel synthetic analogues on both normal (IEC6, IEC18) and transformed (SW620, HT29) intestinal epithelial cell lines. Responses were compared to those elicited by oestradiol, the anti-oestrogen tamoxifen, and the tyrosine kinase inhibitor tyrphostin. Genistein and tamoxifen were potent inhibitors of cell proliferation. Of seven novel isoflavones tested, none were more potent inhibitors than genistein, and all displayed similar relative activities across the different cell lines. In addition to inhibiting cell proliferation, cell death via apoptosis was observed when the cells were exposed to the isoflavones and all but one exhibited PTK inhibitory activity. These data suggest that by reducing proliferation and inducing apoptosis, possibly due in part to PTK inhibition, isoflavones may have a role in protecting normal intestinal epithelium from tumour development (reducing the risk) and may reduce colonic tumour growth.     

人鼻咽癌细胞株自发性凋亡与放射敏感性的研究

  目的   探讨鼻咽癌细胞自发性凋亡对放射敏感性的预测意义及自发性凋亡水平差异的分子学基础。   方法   克隆形成实验测定人鼻咽高分化鳞癌细胞株(CNE-1)、人鼻咽低分化鳞癌细胞株(CNE-2)接受Varian加速器单野照射(6 MV X线)的存活分数, 单击多靶和线性二次模型拟合辐射剂量存活曲线并求出放射生物学参数; 流式细胞术检测自然生长2、4、6、8d细胞凋亡情况; RT-PCR、Western blot检测凋亡相关基因Bcl-2、Bcl-xl、Bcl-w、Bax、Bad、Bid、Bak转录及蛋白表达水平。   结果   函数模型参数显示CNE-2较CNE-1具有更高的辐射敏感性; 相同培养天数下CNE-2早期及晚期凋亡率显著高于CNE-1(P < 0.05);CNE-2中Bcl-2、Bcl-xl、Bcl-w、Bax、Bad、Bid、Bak基因转录及蛋白表达水平均高于CNE-1(P < 0.05)。   结论   自发性凋亡可能成为放射敏感性的预测指标, 自发性凋亡水平差异可能与凋亡相关基因差异性表达有关。      

Cytogenetic studies on an epithelial cell line derived from poorly differentiated nasopharyngeal carcinoma

An epithelial cell line, CNE-2, has been recently established from a poorly differentiated nasopharyngeal carcinoma, and it represents the first of its kind. Using chromosome banding techniques, cytogenetic analysis of the cell line was carried out. It was demonstrated that the chromosome numbers of the CNE-2 cells varied from 87 to 107 and the modal number was 104-103. All cells contained a series of structurally abnormal chromosomes, and most of them were either consistent or frequently found. Among these chromosomes there were two giant markers which, by banding pattern analysis, proved to be distinct from the so-called giant group A marker chromosomes previously found in many lymphoblastoid cell lines from NPC. Comparison between the CNE-2 and CNE, another epithelial cell line, which was established from well-differentiated squamous NPC, showed that while they were quite different in many cytogenetic aspects, they had three marker chromosomes in common, namely, an iso8q, a t(?;3q) and a small acrocentric one. The question of whether chromosome markers specific for NPC exist is discussed in the light of the data presented.     

Toll‐like receptor 9 expression in breast and ovarian cancer is associated with poorly differentiated tumors

Toll‐like receptor 9 (TLR9) activates the innate immune response when exposed to non‐methylated CpG‐DNA. TLR9 was recently shown to be expressed by cancer cells which have been previously characterized by global hypomethylation. We set out to examine the expression and molecular activity of TLR9 in breast and ovarian cancer cells. Firstly, we confirmed higher levels of hypomethylated DNA in the serum of patients with metastatic breast cancer (n = 18) versus age‐matched tumor‐free women (n = 18). In breast cancer cell lines and tissues, TLR9 mRNA expression was associated with estrogen‐receptor (ER) status (n = 124, P = 0.005). Expression also correlated with increasing tumor grade in both breast (P = 0.03) and ovarian cancer specimens (n = 138, P = 0.04). Immunohistochemical analysis of formalin‐fixed paraffin‐embedded (FFPE) breast cancer tissues revealed higher TLR9 protein expression in hormone‐receptor (HR)‐negative specimens (n = 116, P < 0.001). Using an in vitro scratch assay, we observed that cell lines transfected to overexpress TLR9 demonstrated increased cellular migration when stimulated with CpG‐DNA. When assessing the molecular activity of TLR9 in breast cancer, we found a strong positive correlation of nuclear factor‐kappa B (NF‐κB) activity with TLR9 mRNA expression (correlation coefficient r = 0.7, P < 0.001). Finally, immunofluorescence analysis of BT‐20 and Hs578T breast cancer cell lines showed partial colocalizations of CpG‐DNA with TLR9, which diminished when the cells were exposed to methylated CpG‐DNA (mCpG‐DNA) or control GpC‐DNA. In summary we demonstrate that TLR9 expression is associated with poor differentiation in breast and ovarian cancer specimens, and that TLR9 overexpression and stimulation with hypomethylated DNA augments the migratory capacity of cancer cell lines. (Cancer Sci 2010; 101: 1059–1066)     

Effect of reproductive hormones on ovarian epithelial tumors: II. Effect on angiogenic activity

Menstrual cycle activity predisposes to ovarian epithelial tumors based on numerous epidemiological studies. We showed that the hormones involved in menstrual cycle regulation modulate cell cycle activity in these tumors in an accompanying paper. We investigated whether such hormones could also influence angiogenesis, an important determinant of tumor progression, in the same tumors. Treatment with progesterone (P4) stimulated VEGF protein secretion in 4 of 5 ovarian carcinoma cell lines examined. Northern blot analyses performed in MCV50 cells showed that this effect was accompanied by increased VEGF mRNA levels. P4 also stimulated VEGF promoter activity in these cells. Estradiol (E2) showed a similar, but substantially smaller effect on VEGF secretion which was additive to that of P4. Conditioned medium from P4-treated cells strongly stimulated angiogenesis on chicken chorio-allantoic membranes. Incubating the conditioned medium with a neutralizing anti-VEGF antibody, but not with non-specific immunoglobulins abolished this effect. Angiogenic activity was not altered by treatment of the membranes with P4 directly. We conclude that P4 can stimulate angiogenic activity via induction of VEGF secretion in some ovarian epithelial tumors. Therapeutic use of progestins may be most effective when administered in combination with an anti-angiogenic agent, at least against a subset of ovarian carcinomas.     

Flow cytometric analysis of DNA and cell proliferation in ovarian tumors

DNA content in tumor cells from 50 patients with ovarian tumors was analysed by flow cytometry (FCM). Solid tissue samples were processed to obtain monodispersed cells. Staining for DNA analysis was achieved with ethidium bromide and mithramycin. Peripheral blood lymphocytes were used as reference diploid cell population. All benign ovarian tumors exhibited only diploid cells. DNA aneuploid cell lines were found 66.6% of serous carcinomas and in 80% of malignant granulosa cell tumors. The S-phase fraction of DNA diploid cells in benign ovarian tumors (S = 2.4 +/- 1.2%) was smaller than those of malignant tumors (S = 8.2 +/- 5.2%). DNA aneuploid cell populations in serous carcinomas display a higher S-phase fraction (S = 19.2 +/- 9.3%) than DNA diploid cells (S = 11.7 +/- 3.2%). No major differences were obtained between primary ovarian tumors and their metastases, as far as degree of aneuploidy and S-phase fraction are concerned. A high degree of correlation was established between the grade of differentiation of ovarian tumors and the DNA ploidy abnormalities.     

Effect of sodium butyrate and other differentiation inducers on poorly differentiated human ovarian adenocarcinoma cell lines

We have studied the effects of sodium butyrate, retinoic acid, and dimethyl sulfoxide on two human ovarian carcinoma cell lines PE04 and PE01. PE04 cells, after treatment with sodium butyrate at cytostatic doses (2-3 mM for 4 days), exhibited phenotypic changes including induction of alkaline phosphatase and determinants recognized by the monoclonal antibodies 123C3 and 123A8. These effects are not simply the result of cytostasis as they were not produced by dimethyl sulfoxide or retinoic acid. Other markers are also modified by sodium butyrate including lipid, acid mucin, and glycogen. Retinoic acid modulated expression of lipid and CA125, while dimethyl sulfoxide reduced expression of CA125. Other short chain fatty acids such as propionic acid and valeric acid (in addition to butyric acid) also induced alkaline phosphatase and the determinants recognized by 123C3 and 123A8 in PE04 cells. Other differentiation inducers and cytotoxic agents studied did not induce these markers at cytostatic concentrations. The effects of sodium butyrate (and related short chain fatty acids) thus appear to be relatively specific for this cell line.     

Risk of epithelial ovarian cancer in relation to benign ovarian conditions and ovarian surgery

Objective  Some forms of ovarian neoplasms may be preventable through the removal of precursor lesions. We assessed the risk associated with a prior diagnosis of, and ovarian surgery following, ovarian cysts and endometriosis, with a focus on characterizing risk among tumor subgroups. Methods  Information was collected during in-person interviews with 812 women with ovarian cancer diagnosed in western Washington State from 2002 to 2005 and 1,313 population-based controls. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Results  The risk of a borderline mucinous ovarian tumor associated with a history of an ovarian cyst was increased (OR=1.7, 95% CI: 1.0–2.8), but did not vary notably according to receipt of subsequent ovarian surgery. While risk of invasive epithelial ovarian cancer was slightly increased among women with a cyst who had no subsequent ovarian surgery, it was reduced when a cyst diagnosis was followed by surgery (OR = 0.6, 95% CI: 0.4–0.9). This reduction in risk was most evident for serous invasive tumors. Women with a history of endometriosis had a threefold increased risk of endometrioid and clear cell invasive tumors, with a lesser risk increase among women who underwent subsequent ovarian surgery. Conclusions  Our results suggest differences in the relation of ovarian cysts and endometriosis with risk of specific subtypes of ovarian cancer as well as the possibility that ovarian surgery in women with these conditions may lower the risk of invasive disease. Funding for this work was provided by the National Institutes of Health (RO1 CA87538).     

Six new monoclonal antibodies to serous, mucinous, and poorly differentiated ovarian adenocarcinomas

Six monoclonal antibodies directed against ovarian adenocarcinoma were generated by use of 100,000 x g supernatants of Triton-X-100 solubilized extracts of ovarian serous adenocarcinoma as the antigen source. Immunoperoxidase preparation of frozen-sections and routinely processed paraffin section specimens revealed a highly restricted reactivity of these antibodies when tested with adult (n = 2) and fetal (n = 3) tissue types. Coreactivities were occasionally observed with epithelia of the kidney, mammary gland, and pancreas. One monoclonal antibody, Ki-OC I-6-2, cross-reacted only with epididymal epithelia. No coreaction occurred with normal tissue of the ovary, Fallopian tube, or uterus. All antibodies were additionally tested on 74 cases of nonovarian malignancies, 15 cases of ovarian metastases of nonovarian carcinomas, and 114 specimens of ovarian neoplasms other than carcinomas. Ki-OC I-6-2 had no cross-reactivity with these tumors except for one case of renal cell carcinoma. This monoclonal antibody recognized serous, mucinous, and poorly differentiated adenocarcinoma cell types. None of the six antibodies reacted with clear cell or endometrioid carcinoma. All were found to be of the IgG-1 subclass. The tumor antigen to which Ki-OC I-6-2 immunoreacted was estimated to have a molecular weight of 80 kilodaltons (KD).     

Expression of galectin-1 in normal human thyroid gland and in differentiated and poorly differentiated thyroid tumors

We previously reported that galectin-l gene expression increases up to 100-fold in oncogene-transformed rat thyroid cells compared with their normal counterparts and that the relative mRNA levels correlate with the degree of malignancy. In the present study we investigated whether galectin-l is differentially expressed in human thyroid neoplasms, which range from well-differentiated tumors to undifferentiated ana-plastic carcinomas. We analyzed 74 human thyroid specimens of neoplastic, hyperproliferative and normal tissues and several tumor cell lines. Galectin-l mRNA and protein levels were higher in 6 thyroid carcinoma-derived cell lines than in normal thyroid primary cultures and adenoma cells. Galectin-l mRNA levels increased in 28/40 papillary carcinomas and in 6/7 anaplastic carcinomas compared with normal or hyperplastic thyroid. Conversely, galectin-l expression was unaffected in follicular carcinomas and benign adenomas. Immunohistochemi-cal analysis of normal thyroid and papillary carcinoma sections revealed a higher content of galectin-l protein in neoplastic follicular cells than in normal cells. © 1995 Wiley-Liss, Inc.     

Poorly differentiated carcinoma and germ cell tumors.

Although many questions remain unanswered, recent clinical and pathologic studies have shed considerable light on the subject of carcinoma of unknown primary site. It is now clear that some patients in this group have extragonadal germ cell tumors. This is suggested by the superior treatment results in patients with clinical features of extragonadal germ cell tumor and is confirmed by the finding of the diagnostic chromosome abnormality in tumor cells of some patients. These patients have tumors that are unrecognizable using all available pathologic techniques other than molecular genetic analysis; most patients also do not have elevated serum tumor marker levels. Young men with poorly differentiated carcinoma located predominantly in the mediastinum or retroperitoneum should be strongly suspected of having germ cell tumors; chromosomal analysis should be obtained if possible, and these patients should be treated as for germ cell tumor. It is clear that some responsive patients with poorly differentiated carcinoma do not have extragonadal germ cell tumors. A few patients initially thought to have poorly differentiated carcinoma actually have non-Hodgkin's lymphoma. With the widespread availability of immunoperoxidase staining for LCA, this diagnostic error should be minimized. Other responsive patients have poorly differentiated neuroendocrine tumors. The nature and spectrum of neuroendocrine tumors is still being defined; however, our initial documentation of cisplatin responsiveness has been confirmed, even in poorly differentiated neuroendocrine tumors with a known primary site. It is likely that additional responsive subgroups also exist but have not yet been identified. With the availability of a diagnostic chromosomal marker, the answers to other questions regarding the relationship of poorly differentiated carcinoma and germ cell tumors will soon be forthcoming.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroid hormone receptors in dimethylhydrazine-induced rat colonic tumors

The presence of steroid hormone receptors was investigated in rat colonic tumors, experimentally induced by 1,2-dimethylhydrazine, and in adjacent normal tissues. Estrogen, progesterone, glucocorticoid, and 5-alpha-dihydrotestosterone receptor levels were measured by the dextran charcoal competitive binding assay. Estrogen receptors were present in 25% of tumors, although not in adjacent normal tissue. Higher levels of glucocorticoid binding in tumor versus adjacent normal tissue were demonstrated in 5 out of 8 cases. Progesterone and 5-alpha-dihydrotestosterone levels in tumor versus adjacent normal tissue were not significantly different. The results reflect the possible endocrine dependence of colonic tumors.     

Steroid receptors in tumors of nerve sheath origin

The present study dearly demonstrates that neurofibromas and neurofibrosarcomas are devoid of cytosol receptor for steroid. These observations are in sharp contrast to the significant incidence of receptors in soft tissue and osseous sarcomas and raise doubts that steroids exert a direct effect on the growth of these tumors.