cyclin-d1 and Ovarian-Neoplasms

Lysophosphatidic acid (LPA) is a bioactive lipid component of ovarian cancer activating factor, which is present at a high concentration in the ascitic fluid and plasma of patients with ovarian cancer. A group of six lysophosphatidic acid receptors (LPARs), LPAR1 through LPAR6, which belong to the G protein-coupled receptor superfamily (GPCR), mediate cellular activities of LPA and activates a series of downstream molecules and cellular responses, including biological and pathological effects. LPARs are widely expressed in normal ovary, benign tumor, and ovarian cancer tissues and cancer cell lines with a broad range of levels. The LPA/LPAR axis is involved in tumorigenesis and development of ovarian cancer through mediating the cellular responses to LPA and influencing the expression and function of oncogenic molecules. In the present review, the roles of LPARs in ovarian cancer, including the expression, function, and downstream molecules, are summarized, and we discuss the implications for ovarian cancer treatment that targets LPARs.

Topics: AMP-Activated Protein Kinases; Cell Transformation, Neoplastic; Chemokine CXCL1; Cyclin D1; Cyclooxygenase 2; Cytoskeletal Proteins; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukins; Lysophospholipids; Ovarian Neoplasms; Peptide Fragments; Receptors, Lysophosphatidic Acid; Signal Transduction; Urokinase-Type Plasminogen Activator; Vascular Endothelial Growth Factor A

Topics: Age of Onset; Anticarcinogenic Agents; BRCA1 Protein; BRCA2 Protein; Breast Neoplasms; Breast Neoplasms, Male; Carcinoma; Case Management; Cyclin D1; DNA Mutational Analysis; Estrogen Antagonists; Female; Gene Frequency; Genes, BRCA1; Genes, erbB-2; Genetic Counseling; Genetic Testing; Humans; Male; Mammography; Mastectomy; Neoplasm Proteins; Neoplasms, Multiple Primary; Neoplastic Syndromes, Hereditary; Oncogenes; Ovarian Neoplasms; Ovariectomy; Phenotype; Raloxifene Hydrochloride; Retrospective Studies; Risk; Tamoxifen; Transcription Factors

Patients with persistent/recurrent epithelial ovarian cancer/primary peritoneal cancer (EOC/PPC) have limited treatment options. AKT and PI3K pathway activation is common in EOC/PPC, resulting in constitutive activation of downstream mTOR. The GOG conducted a phase II evaluation of efficacy and safety for the mTOR inhibitor, temsirolimus in EOC/PPC and explored circulating tumor cells (CTC) and AKT/mTOR/downstream tumor markers.. Eligible women with measurable, persistent/recurrent EOC/PPC who had received 1-3 prior regimens were treated with 25mg weekly IV temsirolimus until progression or intolerable toxicity. Primary endpoints were progression-free survival (PFS) ≥6-months, tumor response, and toxicity. CellSearch® system was used to examine CTC, and AKT/mTOR/downstream markers were evaluated by archival tumor immunohistochemistry. Kendall's tau-b correlation coefficient (r) and Cox regression modeling were used to explore marker associations with baseline characteristics and outcome.. Sixty patients were enrolled in a two-stage sequential design. Of 54 eligible and evaluable patients, 24.1% (90% CI 14.9%-38.6%) had PFS ≥6 months (median 3.1 months), 9.3% (90% CI 3.7%-23.4%) experienced a partial response. Grade 3/4 adverse events included metabolic (8), gastrointestinal (8), pain (6), constitutional (5) and pulmonary (4). Suggested associations were between cyclin D1 and PFS ≥6 months, PFS or survival; positive CTC pre-treatment and lack of response; and high CTC expression of M30 and PFS ≥6 months/longer PFS.. Temsirolimus appears to have modest activity in persistent/recurrent EOC/PPC; however, PFS is just below that required to warrant inclusion in phase III studies in unselected patients. Cyclin D1 as a selection marker and CTC measures merit further study.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Ovarian Epithelial; Cyclin D1; Disease-Free Survival; Female; Humans; Middle Aged; Neoplasms, Glandular and Epithelial; Neoplastic Cells, Circulating; Ovarian Neoplasms; Peritoneal Neoplasms; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases; Young Adult

The expression of cyclin D1 protein in tumour sections from 81 patients with epithelial ovarian cancer was analysed using immunohistochemistry. The tumours that overexpressed cyclin D1 in more than 10% of neoplastic cells were considered positive. Thus overexpression of cyclin D1 was observed in 72/81 (89%) of the cases examined. Protein was detected in both the nucleus and the cytoplasm in 24/81 (30%) and localized exclusively in the cytoplasm in 48/81 (59%) of the tumours. Cyclin D1 was overexpressed in both borderline and invasive tumours. There was no association between protein overexpression and tumour stage and differentiation. Furthermore, no correlation between cyclin D1 expression and clinical outcome was observed. However, in tumours overexpressing cyclin D1 (n = 72), the proportion displaying exclusively cytoplasmic localization of protein was higher in those with serous compared with non-serous histology (P = 0.004, odds ratio 4.8, 95% confidence interval 1.4-19.1). Western analysis using a monoclonal antibody to cyclin D1 identified a 36 kDa protein in homogenates from seven tumours displaying cytoplasmic only and one tumour demonstrating both nuclear and cytoplasmic immunostaining. Using restriction fragment length polymorphism polymerase chain reaction and PCR-multiplex analysis, amplification of the cyclin D1 gene (CCND1 was detected in 1/29 of the tumours demonstrating overexpression of cyclin D1 protein. We conclude that deregulation of CCND1 expression leading to both cytoplasmic and nuclear protein localization is a frequent event in ovarian cancer and occurs mainly in the absence of gene amplification.

Topics: Blotting, Western; Carcinoma; Cyclin D1; Female; Humans; Immunohistochemistry; Neoplasm Proteins; Ovarian Neoplasms; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Subcellular Fractions; Treatment Outcome

The aim of our study was to investigate the roles and the underlying mechanisms of orthodenticle homolog 1 (OTX1) in ovarian cancer.. OTX1 expression was obtained from TCGA database. OTX1 expression in ovarian cancer cells was detected using qRT-PCR and western blot assay. The cell viability and proliferation was detected by CCK-8 and EdU assays. Cell invasion and migration were detected by transwell assay. Flow cytometry was utilized to determine cell apoptosis and cycle. In addition, western blot assay was used to detect the expression of cell cycle related protein (Cyclin D1 and p21), epithelial-mesenchymal transition (EMT) related protein (E-cadherin, N-cadherin, Vimentin, and Snail), apoptosis related protein (Bcl-2, Bax, and cleaved caspase-3), and JAK/STAT pathway related protein (p-JAK2, JAK2, STAT3, and p-STAT3).. OTX1 was highly expressed in ovarian cancer tissues and cells. OTX1 silencing blocked the cell cycle and repressed cell viability, proliferation, invasion, and migration, while OTX1 silencing facilitated the apoptosis of OVCAR3 and Caov3 cells. OTX1 silencing increased the protein levels of p21, E-cadherin, Bax, and cleaved caspase-3, while the protein levels of Cyclin D1, Bcl-2, N-cadherin, Vimentin, and Snail were decreased by OTX1 silencing. Furthermore, OTX1 silencing suppressed the protein levels of p-JAK2/JAK2 and p-STAT3/STAT3 in OVCAR3 and Caov3 cells. Moreover, overexpression of OTX1 promoted cell proliferation and invasion and inhibited apoptosis in Caov3 cells, but AG490 (an inhibitor of JAK/STAT pathway) reversed the influences on cell biological behavior induced by overexpression of OTX1.. OTX1 silencing repressed ovarian cancer cell proliferation, invasion, and migration and induced cell apoptosis, which might be involved in JAK/STAT signaling pathway. OTX1 may be considered as a novel therapeutic target for ovarian cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Cadherins; Caspase 3; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Janus Kinases; Ovarian Neoplasms; Signal Transduction; STAT Transcription Factors; Vimentin

Ovarian cancer has the highest mortality among all gynecological malignancies; currently, no effective therapeutics are available for its treatment. Naringenin has been shown to inhibit the progression of various cancers, but its inhibitory effect on ovarian cancer remains unknown.. This study aimed to evaluate the inhibitory effects of naringenin on ovarian cancer and elucidate the underlying mechanisms.. Cancer cell proliferation was detected by cell counting kit-8 and crystal violet assays, and the migration capability was determined by wound healing and transwell assays. Western blotting and immunohistochemistry assays were employed to determine the expression levels of the epidermal growth factor receptor, phosphatidylinositol 3-kinase (PI3K) and cyclin D1 in vitro and in vivo, respectively. An ES-2 xenograft nude mouse model was established for the in vivo experiments, and fecal samples were collected for intestinal microbiota analysis by 16S rDNA sequencing.. Naringenin suppressed the proliferation and migration of A2780 and ES-2 cancer cell lines and downregulated PI3K in vitro. In animal experiments, naringenin treatment significantly decreased the tumor weight and volume, and oral administration exhibited greater effects than intraperitoneal injection. Additionally, naringenin treatment ameliorated the population composition of the microbiota in animals with ovarian cancer and significantly increased the abundances of Alistipes and Lactobacillus.. Naringenin suppresses epithelial ovarian cancer by inhibiting PI3K pathway expression and ameliorating the gut microbiota, and the oral route is more effective than parenteral administration.

Topics: Animals; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; DNA, Ribosomal; ErbB Receptors; Female; Flavanones; Gastrointestinal Microbiome; Gentian Violet; Humans; Mice; Mice, Nude; Ovarian Neoplasms; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases

Rab11a is a novel identified tumorigenic factor involved in different cancers.. This study aimed to assess the biological function of Rab11a in ovarian cancer (OC).. GEPIA database and real-time PCR were used to determine Rab11a expression in OC tissues and normal ovarian tissues. CCK-8, cell cycle, wound healing, transwell, and enzyme linked immunosorbent assay were used to detect the effects of Rab11a knockdown or overexpression on the proliferation, migration, and invasion of OC cells. Western blot analysis of autophagy-related markers and immunofluorescence staining of LC3 were performed to determine autophagy induction in Rab11a-silenced or overexpressed OC cells. Moreover, autophagy inhibitor 3-MA was employed to clarify the effects of Rab11a-regulated autophagy on the malignant phenotypes of OC cells.. The mRNA level of Rab11a was increased in tumor tissues from OC patients as compared to the normal ovarian tissues. Knockdown of Rab11a in OVCAR-3 cells inhibited the growth of OC cells and led to cell cycle arrest, accompanied by reduced expression of PCNA and Cyclin D1. Rab11a deficiency suppressed migration and invasion of OC cells, accompanied by decreased secretion of MMP-2 and MMP-9. Silence of Rab11a impeded autophagy induction, as evidenced by decreased LC3 puncta formation, reduced abundance of LC3II and Beclin1, and increased p62 protein expression. In contrast, the ectopic expression of Rab11a in A2780 cells exerted opposite effects. Interestingly, autophagy inhibitor 3-MA abolished the effects of Rab11a overexpression on autophagy, proliferation, migration, and invasion.. Rab11a promotes the malignant phenotypes of OC cells by inducing autophagy.

Topics: Apoptosis; Autophagy; Beclin-1; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Ovarian Neoplasms; Proliferating Cell Nuclear Antigen; rab GTP-Binding Proteins; RNA, Messenger

Ovarian epithelial cancer is the leading cause of deaths associated with gynecologic malignancies. Genistein represents a major type of phytoestrogens widely found in foods and herbal medicines. Although multiple epidemiological studies indicated that the consumption of genistein or other isoflavones is associated with a decreased ovarian cancer risk, the cellular effects and underlying mechanisms are not fully understood. This study focuses on the effect of genistein on the proliferation and cell cycle regulation of ovarian cancer cells.. Ovarian cancer OVCAR-5 cells were treated with genistein in an estrogen-free condition. Cell counting and MTS assays were performed to determine the cell proliferation alterations. Real-time PCR and Western blotting were conducted to examine the expression changes in key cell cycle regulators.. Genistein significantly promoted the proliferation and the viability of OVCAR-5 cells. Upon genistein treatment, cellular mRNA and protein expression levels of PCNA, Cyclin D1 and CDK4 were increased, but those of p21 and p27 were decreased.. In contrary to results of many previous studies, we observed that genistein was able to upregulate the proliferation and G1-S transition of ovarian cancer OVCAR-5 cells. The discrepancy could be caused by diverged experimental conditions and/or different ER expression patterns of cell lines. The findings may provide basic information for in-depth analysis on the role(s) and mechanisms by which genistein confers its effect on ovarian cancer progression.

Topics: Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Female; Genistein; Humans; Ovarian Neoplasms

Cisplatin (DDP) resistance is a major obstacle in the chemotherapeutic efficacy of ovarian cancer. The present study aimed to explore the role of miR‑576‑3p in DDP sensitivity of ovarian cancer cells. Ovarian cancer cell lines SKOV3 and A2780 and DDP‑resistant ovarian cancer cell lines SKOV3/DDP and A2780/DDP were used in the present study.

Topics: B7-H1 Antigen; Cisplatin; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Ovarian Neoplasms; RNA, Neoplasm

15-Hydroxy-8(17),13(E)-labdadiene-19-carboxylic acid (HLCA) isolated from Juniperus foetidissima, has been recently identified as an antiproliferative agent; however, the molecular basis of antiproliferative effects of HLCA remains unknown. To investigate it, the current study has emphasized the hypothesis that HLCA induced cell death is a consequence of intracellular reactive oxygen species (ROS) production followed by cell cycle arrest and apoptosis.. Human ovarian OVCAR-3 and Caov-4 cells were treated with various concentrations of HLCA (48 h) and the measurement of intracellular ROS was considered. Then, the potential of HLCA in promoting apoptosis was investigated via flow cytometry, western blot, and caspase activity assay. Also, the inhibitory effect of HLCA on the cell cycle was evaluated using flow cytometry and western blot analysis.. We found intracellular (ROS) accumulation in HLCA-treated cells. Subsequent observation of the increment in pro-apoptotic Bax as well as the decrement in antiapoptotic Bcl2 revealed that the HLCA-induced cytotoxicity may be triggered by the intrinsic pathway of apoptosis. Our subsequent experiments suggested that caspase-9 and -3 were activated and led the cells to apoptosis during the process. Cell cycle disruption at the G1 phase via down-regulation of cyclin D1 and Cyclin-dependent kinase 4 (CDK4) was another proved mechanism by which HLCA exerts its antiproliferative effects on the ovarian cell lines, OVCAR-3 and Caov-4, especially at relatively lower concentrations.. This is the first study that reveals the apoptotic effects of HLCA, suggesting its therapeutic potential as an effective anti-tumor agent. However, further in vivo studies are required to confirm these effects.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Ovarian Epithelial; Caspase 9; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Diterpenes; Female; Humans; Juniperus; Ovarian Neoplasms; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

Topics: Apoptosis; beta Catenin; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Databases, Genetic; E2F5 Transcription Factor; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hippo Signaling Pathway; Humans; Neoplasm Invasiveness; Ovarian Neoplasms; Protein Serine-Threonine Kinases; Transcription Factors; Wnt Signaling Pathway

Annotation of structural variations (SVs) and base-level karyotyping in cancer cells remains challenging. Here, we present Integrative Framework for Genome Reconstruction (InfoGenomeR)-a graph-based framework that can reconstruct individual SVs into karyotypes based on whole-genome sequencing data, by integrating SVs, total copy number alterations, allele-specific copy numbers, and haplotype information. Using whole-genome sequencing data sets of patients with breast cancer, glioblastoma multiforme, and ovarian cancer, we demonstrate the analytical potential of InfoGenomeR. We identify recurrent derivative chromosomes derived from chromosomes 11 and 17 in breast cancer samples, with homogeneously staining regions for CCND1 and ERBB2, and double minutes and breakage-fusion-bridge cycles in glioblastoma multiforme and ovarian cancer samples, respectively. Moreover, we show that InfoGenomeR can discriminate private and shared SVs between primary and metastatic cancer sites that could contribute to tumour evolution. These findings indicate that InfoGenomeR can guide targeted therapies by unravelling cancer-specific SVs on a genome-wide scale.

Topics: A549 Cells; Breast Neoplasms; Cell Line, Tumor; Chromosome Aberrations; Cyclin D1; DNA Copy Number Variations; Female; Genome, Human; Genomic Structural Variation; Glioblastoma; HeLa Cells; High-Throughput Nucleotide Sequencing; Humans; Karyotyping; Ovarian Neoplasms; Polyploidy; Receptor, ErbB-2; Sequence Analysis, DNA; Whole Genome Sequencing

Topics: 3' Untranslated Regions; Animals; Antineoplastic Agents; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Mice, Inbred BALB C; MicroRNAs; Ovarian Neoplasms; Phthalazines; Piperazines; Xenograft Model Antitumor Assays

Ovarian cancer is known as one of the most common malignancies of the gynecological system, whose treatment is still not satisfactory because of the unclear understanding of molecular mechanism. PSMC2 is an essential component of 19 S regulatory granules in 26 S proteasome and its relationship with ovarian cancer is still not clear. In this study, we found that PSMC2 was upregulated in ovarian cancer tissues, associated with tumor grade and could probably predict poor prognosis. Knocking down the endogenous PSMC2 expression in ovarian cancer cells could decrease colony formation ability, cell motility and cell proliferation rate, along with increasing cell apoptosis rate. Cells models or xenografts formed by cells with relatively lower expression of PSMC2 exhibited weaker oncogenicity and slower growth rate in vivo. Moreover, gene microarray was used to analyze the alteration of gene expression profiling of ovarian cancer induced by PSMC2 knockdown and identify CCND1 as a potential downstream of PSMC2. Further study revealed the mutual regulation between PSMC2 and CCND1, and demonstrated that knockdown of CCND1 could enhance the regulatory effects induced by PSMC2 knockdown and overexpression of CCND1 reverses it. In summary, PSMC2 may promote the development of ovarian cancer through CCND1, which may predict poor prognosis of ovarian cancer patients.

Topics: Animals; Apoptosis; ATPases Associated with Diverse Cellular Activities; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Ovarian Neoplasms; Prognosis; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-akt; Signal Transduction; Up-Regulation

Senescence mechanisms are vital to resistance to long-term olaparib maintenance treatment. Recently, peroxisome proliferator-activated receptor-γ agonists (e.g., rosiglitazone) have been reported to ameliorate the senescence-like phenotype by modulating inflammatory mediator production. This study examined synergistic effects on the anti-tumor activity of rosiglitazone combined with olaparib in ovarian cancer treatment.. A2780 and SKOV3 mouse subcutaneous xenograft models were established for observing anti-tumor effects in living organisms and were randomly split into combination (both olaparib and rosiglitazone), rosiglitazone (10 mg/kg), olaparib (10 mg/kg), control (solvent) groups that received treatment once every 2 or 3 days (n = 6 per group). Cell counting kit-8 (CCK-8) assays were used to test the influences of rosiglitazone and olaparib on cell proliferation. PI and Annexin-V-FITC staining was used with flow cytometry to assess the cell cycle distribution and cell apoptosis. Senescence-associated β-galactosidase (SA-β-Gal) staining was used to observe cellular senescence. We performed quantitative real-time polymerase chain reaction assays to study the senescence-related secretory phenotype (SASP).. Olaparib and rosiglitazone were observed to synergistically retard subcutaneous ovarian cancer growth in vivo, and synergistically suppress ovarian cancer cell proliferation in vitro. Compared with olaparib alone, the percentage of positive cells expressed SA-β-gal and SASP were significantly decreased in the treatment of combination of olaparib and rosiglitazone. Furthermore, olaparib plus rosiglitazone increased the percentage of apoptosis in ovarian cancer cell compared with olaparib alone. In A2780 cells, it showed lower expression of P53, phospho-p53 (Ser15), P21, and P18 protein in combination treatment compared with olaparib alone. While, in SKOV3 cells, it showed lower expression of phosphor-retinoblastoma protein (Rb) (Ser807/811), and higher expression of cyclin D1, P21, and P16 protein in combination treatment compared with olaparib alone.. Rosiglitazone combined with olaparib can help manage ovarian cancer by ameliorating olaparib-induced senescence and improving anti-tumor effects.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Female; Humans; Mice; Mice, Nude; Ovarian Neoplasms; Phthalazines; Piperazines; Rosiglitazone; Tumor Suppressor Protein p53

To review the significance of MDM2 and cyclin D1 expression and loss of p16 expression in malignant and borderline Brenner tumors (BTs) of the ovary.. We describe 2 new cases of ovarian BT, 1 malignant and 1 borderline. We studied MDM2, p16, and cyclin D1 expression by immunohistochemistry in the benign, borderline, and malignant components of these 2 cases and in 5 additional cases of benign BT. We also reviewed and summarized the literature on the clinical, immunohistochemical and molecular characteristics of borderline and malignant BTs (BdBTs and MBTs).. Nuclear expression of MDM2 was seen only in the MBT. Loss of p16 expression was seen in both BdBT and MBT. Cyclin D1 expression was in proportion to the degree of malignancy. Amplification of MDM2, loss of CDKN2A (p16-encoding gene), and amplification of CCND1 (cyclin D1-encoding gene) were confirmed by commercial next-generation sequencing in the case of MBT.. We are the first to report immunohistochemical expression of MDM2 in an MBT. Amplification of MDM2 and loss of p16 expression may have a role in malignant transformation of BT.

Topics: Aged; Biomarkers, Tumor; Brenner Tumor; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Gene Amplification; Humans; Middle Aged; Ovarian Neoplasms; Proto-Oncogene Proteins c-mdm2

The present study aimed to investigate the molecular mechanism and the effect of Saponin from Tupistra chinensis Baker (STCB) on the proliferation and apoptosis of ovarian cancer cells. To investigate the inhibitory effect of STCB on the proliferation of ovarian cancer cells, SKOV3 cells were cultured and the methyl thiazolyl tetrazolium assay was used. Flow cytometry was also used to analyze the cell cycle distribution and apoptotic rate. Ki-67, cyclin D1, cleaved caspase-3, cleaved caspase-9, β-catenin, and c-Myc protein expressions were detected by western blot. Ovarian cancer cells were treated with STCB and Wnt pathway activator lithium chloride (LiCl). These methods were also used to determine the proliferation, cell cycle distribution, and apoptosis of ovarian cancer cells. In STCB-treated group, the proliferation inhibition and apoptosis rate, the proportion of G0-G1 phase, and the expression level of cleaved caspase-3 and 9 of ovarian cancer cells were significantly increased. Similarly, the expression of Ki-67, cyclin D1, β-catenin, and c-Myc were significantly decreased (p < .05). The results also showed that in STCB-LiCl-treated group, while the proliferation inhibition rate of ovarian cancer cells, the proportion of G0-G1 cells, the expression level of cleaved caspase-3 and 9, and the apoptosis rate (p < .05) were significantly decreased, the expression level of Ki-67, cyclin D1, β-catenin, and c-Myc was significantly increased. STCB induced G0-G1 phase arrest, inhibited cell proliferation, and promoted apoptosis of ovarian cancer cells by inhibiting Wnt/β-catenin pathway.

Topics: Apoptosis; Asparagaceae; beta Catenin; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; Ovarian Neoplasms; Proto-Oncogene Proteins c-myc; Saponins; Wnt Signaling Pathway

Ovarian cancer is one of the most common malignant tumors in the female reproductive system with the highest mortality rate. Cul4B participates in the oncogenesis and progression of several malignant tumors. However, the role of Cul4B in ovarian cancer has not been studied.. High expression of intratumor Cul4B was associated with poor patient survival. Cul4B expression was associated with FIGO stage and Cul4B was independent risk factor of ovarian cancer disease-free survival and overall survival. In vitro studies revealed that overexpression of Cul4B promoted tumor proliferation while knockdown of Cul4B significantly inhibited the proliferation capacity of ovarian cancer cells. Mechanistically, Cul4B was found to promotes cell entering S phase from G0/G1 phase by regulating the expression of CDK2 and CyclinD1. Cul4B regulates the expression of CDK2 and CyclinD1 by repressing miR-372.. The results revealed that high expression of Cul4B is associated with poor ovarian cancer prognosis and Cul4B may serve as a potential treating target for an adjuvant therapy.

Topics: Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cullin Proteins; Cyclin D1; Cyclin-Dependent Kinase 2; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Middle Aged; Ovarian Neoplasms; Risk Factors; Up-Regulation

Epithelial ovarian carcinoma (EOC) is the most common cause of gynecological cancer mortality, and poses a threat to women. MicroRNA‑195 (miR‑195) has been reported to induce apoptosis of human OVCAR‑3 cells by inhibiting the VEGFR2/AKT pathway. However, the role of miR‑195 in EOC remains unknown. A previous study reported that cell division cycle 42 (CDC42) can serve as a target gene of miR‑195 and mediate malignant progression of esophageal squamous cell carcinoma (ESCC). The aim of the present study was to investigate the role of miR‑195 in EOC and the regulation in CDC42/CCND1 pathway. Tissues samples and clinical materials were collected from 78 enrolled patients with EOC to analyze the expression and clinical significance of miR‑195, CDC42 and cyclin D1 (CCND1). Human EOC cell lines OVCA420, OVCAR‑3, A2780 and SKOV3 cell lines were used to assess the expression and function of miR‑195, CDC42 and CCND1 in vitro. Cell proliferation, the cell cycle and apoptosis, as well as the cell migratory and invasive abilities were detected in vitro using BrdU incorporation, colony formation, wound healing and Transwell invasion assays, along with flow cytometry. miR‑195 was downregulated, while CDC42 and CCND1 were upregulated in human EOC tissues and cells, and the aberrant expression of both was associated with increased EOC malignancy. Moreover, miR‑195 expression was negatively correlated with CDC42 and CCND1 expression levels, and negatively regulated these expression levels. Thus, it was suggested that miR‑195 functions as a tumor suppressor, but CDC42 and CCND1 act as tumor promoters based their abilities to enhance cell proliferation, cell cycle entry, migration and invasion, as well as decrease apoptosis in OVCAR‑3 cells. the present results demonstrated that miR‑195 inhibited human EOC progression by downregulating CDC42 and CCND1 expression. Furthermore, it was identified that miR‑195, CDC42 and CCND1 may be effective biomarkers for EOC diagnosis and treatment.

Topics: Adult; Apoptosis; Carcinoma, Ovarian Epithelial; cdc42 GTP-Binding Protein; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; MicroRNAs; Middle Aged; Ovarian Neoplasms

Fibroblast growth factor-2 (FGF2) is a protein ligand, which exerts essential roles in development, angiogenesis, and tumor progression via activation of the downstream signaling cascades. Accumulating evidence has demonstrated that FGF2 is involved in the progression of ovarian cancer, providing a novel potential target for ovarian cancer therapy. In this study, we showed that FGF2 is significantly increased in ovarian tumors, and is negatively associated with the overall survival of ovarian cancer by database analysis. A short peptide obtained from a heptapeptide phage display library suppressed FGF2-induced proliferation, migration, and invasion of the p53-null epithelial ovarian cancer (EOC) cells. Further investigations revealed that the short peptide antagonized the effects of FGF2 on G0/G1 to S cell phase promotion, cyclin D1 expression, and MAPK and Akt signaling activation, which might contribute to the mechanism underlying the inhibitory effects of the short peptide on the aggressive phenotype of the ovarian cancer cells triggered by FGF2. Moreover, the short peptide might have the potentials of reversing FGF2-induced resistance to the doxorubicin via downregulation of the antiapoptotic proteins and counteracting of the antiapoptotic effects of FGF2 on p53-null EOC cells. Taken together, the short peptide targeting FGF2 may provide a novel strategy for improving the therapeutic efficiency in a subset of EOC.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Doxorubicin; Drug Resistance, Neoplasm; Female; Fibroblast Growth Factor 2; Humans; Kaplan-Meier Estimate; Ovarian Neoplasms; Peptide Library; Peptides; S Phase; Tumor Suppressor Protein p53

Poly(ADP-ribose) polymerase inhibitors (PARPi) are active in cancer cells that have impaired repair of DNA by the homologous recombination (HR) pathway. Strategies that disrupt HR may sensitize HR-proficient tumors to PARP inhibition. As a component of the core cell cycle machinery, cyclin D1 has unexpected function in DNA repair, suggesting that targeting cyclin D1 may represent a plausible strategy for expanding the utility of PARPi in ovarian cancer.. BRCA1 wildtype ovarian cancer cells (A2780 and SKOV3) were treated with a combination of CCND1 siRNA and olaparib in vitro. Cell viability was assessed by MTT. The effects of the combined treatment on DNA damage repair and cell cycle progression were examined to dissect molecular mechanisms. In vivo studies were performed in an orthotopic ovarian cancer mouse model. Animals were treated with a combination of lentivirus-mediated CCND1 shRNA and olaparib or olaparib plus scrambled shRNA. Molecular downstream effects were examined by immunohistochemistry.. Silencing of cyclin D1 sensitized ovarian cancer cells to olaparib through interfering with RAD51 accumulation and inducing cell cycle G0/G1 arrest. Treatment of lentivirus-mediated CCND1-shRNA in nude mice statistically significantly augmented the olaparib response (mean tumor weight ± SD, CCND1-shRNA plus olaparib vs scrambled shRNA plus olaparib: 0.172 ± 0.070 g vs 0.324 ± 0.044 g, P< 0.05).. Silencing of cyclin D1 combined with olaparib may lead to substantial benefit for ovarian cancer management by mimicking a BRCAness phenotype, and induction of G0/G1 cell cycle arrest.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cyclin D1; DNA Breaks, Double-Stranded; DNA Repair; Female; Genes, BRCA1; Humans; Mice; Mice, Inbred BALB C; Ovarian Neoplasms; Phthalazines; Piperazines; Rad51 Recombinase

The cyclinD1 is an emerging potent therapeutic drug target in the treatment of ovarian cancer. CyclinD1, Cdks phosphorylation regulates cell cycle and controls transcription. For this reason, CyclinD1 have been subject to extensive cell cycle- related research, and consequently various therapeutic inhibitor drugs have been developed to these protein targets. In the present study we identified that the expression levels of Bcl-2, Bax, caspase 8, 9 and cyclinD1 using Q-PCR method in SKOV3 cell lines treated with Isochamanetin. The viability and migratory inhibition ability also studied to know the mode of cell death. Further the expression levels of Bcl-2, caspase8,9, Cytochrome C and CyclinD1 were significantly down regulated in SKOV3 cancer cells treated with isochamanetin, a specific binding molecule to CyclinD1. The therapeutic molecules found by a high through put insilicoscreen of this pocket exhibit cytostatic nature and reduce protein levels of cell cycle. The novel structural site on CyclinD1, which is well conserved and inhibits the SKOV3 cells from G0-G1 to S phase cell cycle progression. The current result suggests that isochamanetin serves as potent binding agent to the cyclinD1.

Topics: Antineoplastic Agents, Phytogenic; Asteraceae; Cell Line, Tumor; Cell Movement; Cell Survival; Chalcones; Cyclin D1; Female; Flavanones; Gene Expression Regulation, Neoplastic; Humans; Magnetic Resonance Spectroscopy; Molecular Docking Simulation; Molecular Structure; Ovarian Neoplasms; Protein Interaction Maps

Topics: Aminopyridines; Animals; Benzimidazoles; Carcinoma, Small Cell; Cell Line, Tumor; Cell Survival; Chromatin Immunoprecipitation; Cyclin D1; DNA Helicases; Female; Humans; Hypercalcemia; Mice; Mice, SCID; Nuclear Proteins; Ovarian Neoplasms; Piperazines; Protein Kinase Inhibitors; Purines; Pyridines; RNA, Small Interfering; Transcription Factors

One of the most common malignancies peculiar to female health with few symptoms, low response to therapy, difficult diagnosis, frequent relapse, and high mortality, is ovarian cancer. Thus, our experiment, using Human amniotic fluid mesenchymal stem cells (hAFMSCs) as a therapeutic tool, aims to find an efficient treatment approach for patients suffering from SKOV3 ovarian cancer.. In this study, we obtained 5 ml amniotic fluid from 16-20 week pregnant women who underwent amniocentesis for routine prenatal diagnosis by karyotyping in Al-Zahra Hospital of Tabriz University of Medical Sciences, Iran. Using trans wells in 24 wells plate, hAFMSCs were isolated from all samples, co-cultured with SKOV3 ovarian cancer cell line, and characterized via flow cytometry and RT-PCR. Human skin fibroblast cells (HSFCs) were isolated and used as a negative control. SKOV3 and HSFCs' viability after 5 days was evaluated by MTT assay. Cell cycle and apoptotic genes were analyzed by real-time PCR.. We successfully isolated and characterized hAFMSCs through it positivity for CD44 and CD90 specific mesenchymal stem cell markers and negativity for CD31 and CD45. Oct4 and NANOG were evaluated by RT-PCR as pluripotency markers, and visualized on 2% gel electrophoresis. We established hAFMS cell lines after 5 days of co-culturing the SKOV3 cells, viability was decreased; however, HSFCs did not show toxicity by MTT assay. The genes indicated upregulation and high expression by a real-time PCR.. Our findings showed that hAFMSCs have natural tumor tropism, and can release soluble factors in a cell culture, which cause an efficient anticancer effect. Thus, we can use hAFMSCs for complete anticancer therapy on SKOV3 cell line at cell culture condition and possibly in vivo in the near future.

Topics: Amniotic Fluid; Cell Line, Tumor; Cell Survival; Coculture Techniques; Cyclin D1; Female; Fibroblasts; Humans; Mesenchymal Stem Cells; Ovarian Neoplasms; Pregnancy; Tumor Suppressor Protein p53

Ovarian cancer is a common malignant cancer among women. Increasing studies have demonstrated that microRNAs function as important regulation factors in the progression of ovarian cancer.. Human ovarian cancer cell lines HO8910 and OVCAR-3 were transfected with miR-934 inhibitor and corresponding negative control (inhibitor control). Cell proliferation and apoptosis were detected by cell counting kit-8 (CCK-8) and TUNEL assay, respectively. The expression levels of proliferation/apoptosis-related genes and BRMS1L were measured by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blotting. Furthermore, the association between miR-934 and BRMS1L was investigated through luciferase reporter assays.. MiR-934 was significantly increased in ovarian cancer cell lines, whereas BRMS1L was significantly decreased. Downregulated miR-934 remarkably inhibited cell proliferation and induced cell apoptosis. Meanwhile, miR-934 could influence the expression levels of Ki67, Cyclin D1, Caspase3, and Bcl-2. In addition, BRMS1L was identified as a target gene of miR-934.. Oncogene miR-934 promotes ovarian cancer cell proliferation and inhibits cell apoptosis through targeting BRMS1L. MiR-934 and BRMS1L may be novel biomarkers or therapeutic targets for ovarian cancer in the future.

Topics: Antagomirs; Apoptosis; Base Sequence; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; Repressor Proteins; Sequence Alignment

The purpose of this study was to investigate the antiproliferative effect of active hexose correlated compound (AHCC), derived from basidiomycete mushroom culture, on ovarian cancer cell lines. An in vitro growth inhibition assay was performed using AHCC in ovarian cancer cell lines. Western blotting was performed to investigate the mechanism of the observed antiproliferative effect of AHCC. We identified that ovarian cancer cell viability was significantly reduced through treatment with AHCC compared to that in the control. AHCC inhibited constitutive signal transducer and activator of transcription 3 (STAT3) phosphorylation in ovarian cancer cell lines. In contrast, treatment with pervanadate, a protein tyrosine phosphatase inhibitor, reversed AHCC-induced STAT3 suppression. AHCC treatment induced the expression of SHP-1, a protein tyrosine phosphatase, and suppressed the expression of cyclin D1, Bcl-2, Mcl-1, survivin, and VEGF, which are STAT3-regulated gene products that are associated with cell proliferation or apoptosis. These results suggest that AHCC has an antiproliferative effect on ovarian cancer cell lines, via STAT3 phosphorylation; thus, this compound has the potential to be a complementary and alternative anticancer therapy for the treatment of ovarian cancer.

Topics: Antineoplastic Agents; Caspase 3; Cell Line, Tumor; Cell Survival; Cyclin D1; Drug Screening Assays, Antitumor; Female; Humans; Ovarian Neoplasms; Phosphoric Monoester Hydrolases; Phosphorylation; Polysaccharides; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Proto-Oncogene Proteins c-bcl-2; STAT3 Transcription Factor; Survivin; Vascular Endothelial Growth Factor A

Ovarian cancer is one of the most common malignancies with a high rate of mortality in women. However, current therapies for ovarian cancer treatment are ineffective. Therefore, novel target identification is an urgent requisite. The present study aimed to investigate the role of microRNA-214 (miR-214) in ovarian cancer.. The expression of miR-214, β-catenin, cyclin D1, c-myc, and TCF-1 at the transcriptional level was measured by real-time PCR, while that of β-catenin, Cyclin D1, and c-Myc at the protein level were detected by western blot. Colony formation assay and transwell assay were used to explore the invasion ability of the cancer cells. Cell cycle was measured by flow cytometry.. Real-time PCR showed that miR-214 expression in ovarian cancer cell lines was lower than that in the human normal ovarian epithelial cells, IOSE80. Furthermore, the low expression of miR-214 was correlated with high pathological grade. The rate of colony formation and invasion of miR-214 overexpression in SKOV-3 cells were weaker than that in control cells. Moreover, miR-214 overexpression led to the G0/G1 phase arrest. The expression of β-catenin, Cyclin D1, and c-Myc was suppressed by the overexpression of miR-214.. These results suggested that miR-214 may serve as a tumor suppressor of ovarian cancer by targeting the β-catenin pathway.

Topics: beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; Humans; MicroRNAs; Ovarian Neoplasms; Proto-Oncogene Proteins c-myc; S Phase Cell Cycle Checkpoints; T Cell Transcription Factor 1; Wnt Signaling Pathway

Obesity has been linked to several types of cancers including ovarian cancer. Retinol binding protein 4 (RBP4) is an adipokine that drives the development of hyperinsulinemia and type II diabetes in obesity patients and animals. Previously, we have identified RBP4 as a serum marker for ovarian cancer. Here we further explored the consequence of RBP4 upregulation in ovarian cancer cells and its molecular mechanism.. Our results show that RBP4 is overexpressed in ovarian cancer cells to the same extent as in adipose tissues. The overexpression of RBP4 in ovarian cancer cells promotes cancer cell migration and proliferation. At molecular level, cancer progression factors MMP2 and MMP9 are induced in response to RBP4 overexpression. We further investigated which signaling pathways are utilized by RBP4 to activate ovarian cancer cell migration. We found RhoA/Rock1 pathway is turned on and CyclinD1 is upregulated in RBP4 overexpressed cells. Inhibition of RhoA/Rock1 pathway reduces the RBP4-induced MMP2 and MMP9 expression. The RBP4 action is depend on its associated ligand vitamin A/retinol acid (RA) and possibly involves similar pathways as for conferring insulin resistance. Moreover, we show that knockdown of RBP4 significantly reduce cancer cell migration and proliferation as well as expressions of oncogenic factors.. Our results indicated that RBP4 can drive ovarian cancer cell migration and proliferation via RhoA/Rock1 and ERK pathway. It suggests that RBP4 act as a oncogene in ovarian cancer cells. Thus, RBP4 could be a molecular bridge between obesity and cancers and a potential target for treating obese cancer patients.

Topics: Adipokines; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Humans; Ovarian Neoplasms; Protein Binding; Retinol-Binding Proteins, Plasma; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Tretinoin

BACKGROUND Many findings have shown that pyruvate kinase type M2 (PKM2) plays crucial roles in regulating the occurrence and development of various human cancers; however, its roles in ovarian cancer oncogenesis remain to be determined. MATERIAL AND METHODS The expression intensity of PKM2 in ovarian cancer tissues was examined by immunohistochemistry (IHC), and was then correlated to patient clinicopathologic characteristics. The roles of PKM2 in ovarian cancer cell proliferation, growth, and survival were examined by CCK-8, colony forming, and flow cytometry assays. The potentially involved molecular were then investigated by Western blot analysis. RESULTS IHC results showed that PKM2 was overexpressed in 100 of 114 (87.7%) serous ovarian cancer tissues as compared with 50 cases of non-cancerous ovarian tissues, and was associated with tumor size ≥7.5 cm and <7.5 cm (p<0.05). Overexpression of PKM2 in SKOV3 and HEY ovarian cancer cells by transfection with PKM2 lentivirus vector led to increased cell proliferation, growth, and survival, which may be related with PKM2 being able to increase cell cycle progress: G1 stage decreased, whereas S stage significantly increased. In contrast, all functions of SKOV3 and HEY cells described above were reversed by knocked down PKM2 expression using siRNA. Further data showed that overexpressed PKM2 led to increased CCND1 and decreased CDKN1A expression, whereas underexpressed PKM2 led to decreased CCND1 and increased CDKN1A expression in ovarian cancer cells. CONCLUSIONS PKM2 may play important roles in ovarian cancer development and may be a treatment target for this cancer.

Topics: Apoptosis; Carrier Proteins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Neoplasms, Cystic, Mucinous, and Serous; Ovarian Neoplasms; S Phase; Survival Analysis; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Tumor Stem Cell Assay; Up-Regulation

Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a microtubule-associated proteinrequired for mitosis and spindle assembly. It has been revealed that TPX2 is overexpressedin various human cancers and promotes cancer progression.. The expression of TPX2 was examined in ovarian cancer (OC) tissues and by Western blotting, quantitative real-time reverse transcription PCR (qRT-PCR) and immunohistochemistry. The effects of TPX2 on proliferation and migration of two OC cell lines SKOV3and RMG1 were analyzed using the methylthiazol tetrazolium (MTT) assay, flow cytometry and transwell assay. The mechanisms underlying the effects of TPX2 on OC cells were explored by qRT-PCR and Western blot.. In this study, we found that TPX2 was upregulated in OC tissues. We observed knockdown of TPX2 inhibited the expression of Polo-like kinase 1 (PLK1), which has an important role in the regulation of M phase of the cell cycle, and the activity of Cdc2, induced cell arrested at the G2/M phase and decreased proliferation. Moreover, our data revealed that the levels of PLK1, β-catenin, MMP7 and MMP9 were inhibited following TPX2 knockdown, leading to decrease of cell migration. Finally, we showed that the restoration of PLK1 expression attenuated the anti-proliferation and anti-migration effects of TPX2 knockdown in OC cells.. TPX2 promotes the proliferation and migration of human OC cells by regulating PLK1 expression.

Topics: Adult; Aged; Apoptosis; CDC2 Protein Kinase; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Immunohistochemistry; Microtubule-Associated Proteins; Middle Aged; Nuclear Proteins; Ovarian Neoplasms; Polo-Like Kinase 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; RNA, Small Interfering; Young Adult

Chemoresistance is the major obstacle to effective treatment in patients with serous ovarian carcinoma (SOC), which frequently related to the failure of chemotherapeutic agents to induce apoptosis. In this study, the immunohistochemical expression of Apaf-1, Cyclin D1, and Aquaporin-5 (AQP-5) was studied in 50 paraffin blocks of SOC. Data on overall survival (OS), disease-free survival (DFS) and response to the first-line chemotherapy were collected and then statistically analyzed. Apaf-1 expression was observed in 84% of the SOC cases with a significant down-regulation with higher tumor grade, lymph node metastasis, and advanced FIGO stage. Cyclin D1 expression was found in 70% of the cases with a significant up-regulation with higher tumor grade, lymph node metastasis, and advanced FIGO stage. Positive AQP-5 expression was noted in 84% of the cases with a significant positive association with higher tumor grade, lymph node metastasis, and advanced FIGO stage. During the follow-up period, the Apaf-1 expression had a significant negative association with OS and DFS (p < 0.001 for each), while both Cyclin D1 and AQP-5 expression had a significant positive association with unfavorable OS and DFS. The cases of SOC treated with suboptimal surgery revealed a significant association of low Apaf-1, high Cyclin D1, and strong AQPs with the poor response to the first-line chemotherapy (p = 0.047, p < 0.001, and 0.006 respectively).. Down-regulation of Apaf-1 protein and the overexpression of Cyclin D1 and AQP-5 proteins possibly contribute to an aggressive SOC with a high risk of recurrence and poor response to the first-line chemotherapy.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Apoptotic Protease-Activating Factor 1; Aquaporin 5; Biomarkers, Tumor; Carboplatin; Cyclin D1; Cystadenocarcinoma, Serous; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Middle Aged; Neoplasm Grading; Ovarian Neoplasms; Paclitaxel; Prognosis; Survival Rate; Treatment Outcome

Ovarian cancer (OC) is the most lethal gynecologic malignancy, mainly due to the advanced stage at diagnosis in most patients and high rate of relapse. Thus, it is still essential to elucidate the underlying mechanisms and explore the diagnostic and therapeutic targets of OC. Recent studies have revealed that proline-rich protein 11 (PRR11) is dysregulated in different cancers, participating in their initiation and progression; however, it remains unclear whether PRR11 is involved in OC.. Immunohistochemical staining, quantitative reverse transcription PCR, and western blotting were performed to evaluate PRR11 expression in OC tissues and cells. The relationship between PRR11 expression and the clinicopathologic data of patients were analyzed. We upregulated and downregulated PRR11 expression using a PRR11 overexpression vector and PRR11-specifc small interfering RNA, respectively, to further clarify its role in the malignant biological behavior of OC in vitro.. Overexpression of PRR11 in OC tissues and cells significantly correlated with advanced FIGO stage, lymph node metastasis, and large tumor size. Downregulation of PRR11 inhibited cell proliferation and prevented the invasion and migration of HO-8910 OC cells, whereas opposite results were observed in Caov3 cells upon PRR11 upregulation. Further analyses showed that PRR11 positively regulated cell proliferation-related proteins, including c-myc and cyclin D1, and increased and decreased the expression of matrix metalloproteinase 2 and tissue inhibitor of metalloproteinase 2, respectively. Additionally, our preliminary results demonstrated that PRR11 expression was mediated by the phosphoinositide 3-kinase/AKT/β-catenin signaling pathway.. The results of this study provide evidence that PRR11 plays a critical role in the progression and metastasis of OC, and as such, may serve as a potential prognostic and therapeutic target in OC.

Topics: beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Humans; Lymphatic Metastasis; Matrix Metalloproteinase 2; Middle Aged; Neoplasm Staging; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; RNA Interference; RNA, Small Interfering; Signal Transduction; Tissue Inhibitor of Metalloproteinase-2

BACKGROUND Marsdenia tenacissima extract (MTE) is a traditional Chinese medicine that can be effectively used against various cancers. However, to the best of our knowledge, its role in ovarian cancer is not known. This study investigated the effects of MTE on human ovarian cancer SKOV3 cells. MATERIAL AND METHODS The viability and cell cycle of SKOV3 cells were assessed using the cell counting kit-8 (CCK-8) and propidium Iodide (PI) staining kit, respectively. Cell apoptosis and mitochondrial membrane potential (MMP) were detected by flow cytometry. The expression levels of proliferation-related and apoptosis-related factors were tested by quantitative real-time PCR (qRT-PCR) and Western blot assays, respectively. RESULTS We found that MTE markedly reduced the viability of SKOV3 cells in dose-dependent and time-dependent manners. MTE induced cell cycle arrest by downregulating the levels of cyclin D1and cyclin B1. MTE (10, 20, and 40 mg/mL) markedly increased apoptosis rates (2.77±0.6%, 4.95±0.97%, and 12.16±0.69%, respectively), and enhanced the loss of MMP. MTE obviously downregulated the expression of B cell lymphoma-2 (Bcl-2) and upregulated the expression levels of fibroblast-associated (Fas), Fas ligand (FasL), cleaved cysteinyl aspartate-specific proteinas-3 (caspase-3), and Bcl-2-associated X protein (Bax) compared to the control group. In addition, the expressions of phosphorylated mammalian target of rapamycin (p-mTOR), phosphorylated protein kinase B (p-AKT), and phosphorylated-phosphatidylinositol 3 kinase (p-PI3K) were decreased by MTE. CONCLUSIONS MTE inhibited proliferation and induced apoptosis of SKOV3 cells. The depression of the PI3K/AKT/mTOR pathway may augment the protective effect of MTE. Thus, MTE might be expected to be a new drug for curing ovarian cancer.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; China; Cyclin B1; Cyclin D1; Down-Regulation; Female; Humans; Marsdenia; Ovarian Neoplasms; Plant Extracts; Proto-Oncogene Proteins c-akt; Quinazolines; Signal Transduction; TOR Serine-Threonine Kinases

The long noncoding RNA homeobox (HOX) transcript antisense intergenic RNA (HOTAIR) has been demonstrated to be a vital modulator in the proliferation and metastasis of ovarian cancer cells, but its potential molecular mechanism remains to be elucidated. In the current study, we aimed to uncover the biological role of lncRNA HOTAIR and its underlying regulatory mechanism in the progression and metastasis of ovarian cancer.. HOTAIR expression was detected by quantitative RT-PCR (qRT-PCR) and northern blotting. The SKOV3 ovarian cancer cell line was chosen for the subsequent assays. In addition, the molecular mRNA and protein expression levels were examined by qRT-PCR and western blotting. The competitive endogenous RNA (ceRNA) mechanism was validated by bioinformatics analysis and a dual luciferase reporter gene assay.. HOTAIR expression was significantly higher in ovarian carcinoma tissues and cell lines than in the control counterparts. Both CCND1 and CCND2 were downstream targets of miR-206. The inhibition of HOTAIR elevated the expression of miR-206 and inhibited the expression of CCND1 and CCND2. Moreover, CCND1 and CCND2 were highly expressed in ovarian cancer tissues, and their expression was positively correlated with HOTAIR expression. Finally, the functional assays indicated that the anticancer effects of miR-206 could be rescued by the simultaneous overexpression of either CCND1 or CCND2 in ovarian cancer.. HOTAIR enhanced CCND1 and CCND2 expression by negatively modulating miR-206 expression and stimulating the proliferation, cell cycle progression, migration and invasion of ovarian cancer cells.

Topics: 3' Untranslated Regions; Antagomirs; Base Sequence; Cell Line, Tumor; Cell Movement; Cyclin D1; Cyclin D2; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Middle Aged; Ovarian Neoplasms; RNA Interference; RNA, Long Noncoding; RNA, Small Interfering; Sequence Alignment

Epithelial ovarian carcinoma (EOC) is the most lethal gynecologic malignancy. However, the molecular mechanisms remain unclear. In this study, we found that miR-146b was downregulated in EOC and its expression level was negatively correlated with the pathological staging. Follow-up functional experiments illustrated that overexpression of miR-146b significantly inhibited cell migration and invasion, and increased cell proliferation, but it also improved the response to chemotherapeutic agents. Mechanistically, we demonstrated that miR-146b exerted its function mainly through inhibiting F-box and leucine-rich repeat protein 10 (FBXL10), and upregulated the Cyclin D1, vimentin (VIM), and zona-occludens-1 (ZO-1) expression in EOC. These findings indicate that miR-146b-FBXL10 axis is an important epigenetic regulation pathway in EOC. Low miR-146b may contribute to cancer progression from primary stage to advanced stage, and may be the promising therapeutic target of EOC.

Topics: Adult; Animals; Antineoplastic Agents; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cisplatin; Cyclin D1; Drug Resistance, Neoplasm; Epigenesis, Genetic; F-Box Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Jumonji Domain-Containing Histone Demethylases; Mice; Mice, Nude; MicroRNAs; Middle Aged; Neoplasm Staging; Ovarian Neoplasms; Paclitaxel; RNA, Small Interfering; Signal Transduction; Tumor Burden; Vimentin; Xenograft Model Antitumor Assays; Zonula Occludens-1 Protein

Recently, a large number of studies have focused on the important role of long non-coding RNAs (lncRNAs) in metabolism and development and have found that abnormal lncRNA expression is associated with the pathogenesis and development of many diseases. The lncRNA DLEU1 is involved in many solid tumours and haematological malignancies. However, its role in epithelial ovarian carcinoma (EOC) and the associated molecular mechanisms has not been reported. In this study, quantitative reverse transcription-PCR (qRT-PCR) demonstrated higher lncRNADLEU1 expression in EOC tissues than in normal tissues. Plasmid transfection of DLEU1 to up-regulate its expression in the ovarian cancer cell lines A2780 and OVCAR3 increased cell proliferation, migration, and invasion, while inhibited apoptosis. Nude mouse xenograft assay demonstrated that DLEU1 overexpression promoted tumour growth in vivo. QRT-PCR showed decreased miR-490-3p expression, while Western blotting demonstrated increased its target genes CDK1, cyclinD1 and SMARCD1, as well as matrix metalloproteinase-2 (MMP2), Bcl-xL and P70S6K protein expression, respectively. Short interfering RNA silencing of DLEU1 produced opposite results, where qRT-PCR showed increased miR-490-3p expression. The dual-luciferase reporter assay revealed a direct interaction between DLEU1 and miR-490-3p. MiR-490-3p plays a tumour suppressor role in epithelial ovarian cancer by targeting CDK1 regulation and influencing SMARCD1 and cyclin D1 (CCND1) expressions. Therefore, we suggest that through interaction with miR-490-3p, DLEU1 may influence the expression of CDK1, CCND1 and SMARCD1 protein, subsequently promoting the development and progression of EOC.

Topics: Animals; bcl-X Protein; Carcinogenesis; CDC2 Protein Kinase; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromosomal Proteins, Non-Histone; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 2; Mice; Mice, Nude; MicroRNAs; Neoplasm Transplantation; Ovarian Neoplasms; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Long Noncoding; RNA, Small Interfering; Signal Transduction; Transcription Factors; Tumor Suppressor Proteins

Brenner tumors (BT) are rare ovarian tumors encompassing benign, borderline, and malignant variants. While the histopathology of BTs and their clinical course is well described, little is known about the underlying genetic defects. We employed targeted next generation sequencing to analyze the mutational landscape in a cohort of 23 BT cases (17 benign, 2 borderline, and 4 malignant) and 3 ovarian carcinomas with transitional cell histology (TCC). Copy number variations (CNV) were validated by fluorescence in-situ hybridization (FISH) and quantitative PCR-based copy number assays. Additionally, we analyzed the TERT promotor region by conventional Sanger sequencing. We identified 25 different point mutations in 23 of the analyzed genes in BTs and 10 mutations in 8 genes in TCCs. About 57% percent of mutations occurred in genes involved in cell cycle control, DNA repair, and epigenetic regulation processes. All TCC cases harbored TP53 mutations whereas all BTs were negative and none of the mutations observed in BTs were present in TCCs. CNV analysis revealed recurrent MDM2 amplifications in 3 out of 4 of the malignant BT cases with one case harboring a concomitant amplification of CCND1. No mutations were observed in the TERT promoter region in BTs and TCCs, which is mutated in about 50%-75% of urothelial carcinoma and in 16% of ovarian clear-cell carcinomas. In conclusion, our study highlights distinct genetic features of BTs, and detection of the triplet phenotype MDM2 amplification/TP53 wt/TERT wt may aid diagnosis of malignant BT in difficult cases. Moreover, selected genetic lesions may be clinically exploitable in a metastatic setting.

Topics: Adult; Aged; Aged, 80 and over; Brenner Tumor; Carcinoma; Cyclin D1; DNA Copy Number Variations; Female; Humans; Middle Aged; Ovarian Neoplasms; Point Mutation; Promoter Regions, Genetic; Proto-Oncogene Proteins c-mdm2; Telomerase; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms; Urothelium

Cyclin-dependent kinases (CDKs) are important regulators of the cell cycle; previous studies have shown that misregulation of CDK4 (cyclin-dependent kinase 4) activity can lead to cancer. The present study investigated the anti-tumor effects of a highly selective CDK4 inhibitor fascaplysin in ovarian carcinoma cell lines.. In our study, cell proliferation, cell cycle, cell apoptosis, cell invasion, and cell migration relative assays were performed in ovarian cancer cell lines A2780 and OVCAR3 in the presence of different concentrations of fascaplysin. The protein expression levels of CDK4, cyclin D1, Bcl-2 (B-cell lymphoma-2), and VEGFA (vascular endothelial growth factor A) were determined by western blot.. Our results showed that fascaplysin inhibited ovarian cancer cell proliferation, invasion and migration, as well as inducing S arrest and cell apoptosis. Treatment with fascaplysin also suppressed CDK4, cyclin D1, Bcl-2, and VEGFA expression at protein levels.. Above all, our results showed that fascaplysin has anti-tumor activity against ovarian cancer cell lines through inhibiting CDK4, and may be a therapeutic target for the treatment of ovarian carcinomas.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Female; Gene Expression Regulation, Neoplastic; Humans; Indoles; Neoplasm Invasiveness; Neoplasm Metastasis; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; Vascular Endothelial Growth Factor A

Metformin, which is widely used as an anti-diabetic drug, reduces cancer related morbidity and mortality. However, the role of metformin in cancer is not fully understood. Here, we first describe that the anti-cancer effect of metformin is mediated by cyclin D1 deregulation via AMPK/GSK3β axis in ovarian cancer cells. Metformin promoted cytotoxic effects only in the cancer cells irrespective of the p53 status and not in the normal primary-cultured cells. Metformin induced the G1 cell cycle arrest, in parallel with a decrease in the protein expressions of cyclin D1 without affecting its transcriptional levels. Using a proteasomal inhibitor, we could address that metformin-induced decrease in cyclin D1 through the ubiquitin/proteasome process. Cyclin D1 degradation by metformin requires the activation of GSK3β, as determined based on the treatment with GSK3β inhibitors. The activation of GSK3β correlated with the inhibitory phosphorylation by Akt as well as p70S6K through AMPK activation in response to metformin. These findings suggested that the anticancer effects of metformin was induced due to cyclin D1 degradation via AMPK/GSK3β signaling axis that involved the ubiquitin/proteasome pathway specifically in ovarian cancer cells. © 2016 Wiley Periodicals, Inc.

Topics: AMP-Activated Protein Kinases; Antineoplastic Agents; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Glycogen Synthase Kinase 3 beta; Humans; Hypoglycemic Agents; Metformin; Ovarian Neoplasms; Ovary; Proteolysis; Signal Transduction

Epithelial ovarian cancer is the most common and lethal gynecological cancer in USA and around the world, causing major mortality annually. In the current study, we investigated the potential anti-ovarian cancer activity of WYE-132, a mammalian target of rapamycin (mTOR) complex 1/2 (mTORC1/2) dual inhibitor. Our results showed that WYE-132 potently inhibited proliferation of primary and established human ovarian cancer cells. Meanwhile, WYE-132 induced caspase-dependent apoptosis in ovarian cancer cells. At the molecular level, WYE-132 blocked mTORC1/2 activation and inhibited expression of mTOR-regulated genes (cyclin D1 and hypoxia-inducible factor 1α). Interestingly, introducing a constitutively active AKT (caAKT), which restored mTORC1/2 activation in WYE-132-treated ovarian cancer cells, only mitigated (but not abolished) WYE-132-mediated growth inhibition and apoptosis. Further studies showed that WYE-132 inhibited sphingosine kinase-1 (SphK1) activity, leading to pro-apoptotic ceramide production in ovarian cancer cells. Meanwhile, WYE-132-induced cytotoxicity against ovarian cancer cells was inhibited by sphingosine-1-phosphate (S1P) but was aggravated by SphK1 inhibitor SKI-II or C6 ceramide. In vivo, WYE-132 inhibited ovarian cancer cell growth, and its activity was further enhanced when co-administrated with paclitaxel (Taxol). These results demonstrate that WYE-132 inhibits ovarian cancer cell proliferation through mTOR-dependent and mTOR-independent mechanisms and indicate a potential value of WYE-132 in ovarian cancer treatment.

Topics: Animals; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzyme Inhibitors; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Nude; Middle Aged; Multiprotein Complexes; Neoplasm Transplantation; Ovarian Neoplasms; Paclitaxel; Phenylurea Compounds; Phosphotransferases (Alcohol Group Acceptor); Pyrazoles; TOR Serine-Threonine Kinases

Gankyrin is a regulatory subunit of the 26kD proteasome complex. As a novel oncoprotein, gankyrin is expressed aberrantly in cancers from several different sites and has been shown to contribute to oncogenesis in endometrial and cervical carcinomas. Neither gankyrin's contribution to the development of epithelial ovarian cancer nor its interaction with follicle-stimulating hormone (FSH)-driven proliferation in ovarian cancer has been studied. Here we have found that gankyrin is overexpressed in ovarian cancers compared with benign ovarian cystadenomas and that gankyrin regulates FSH upregulation of cyclin D1. Importantly, gankyrin regulates PI3K/AKT signaling by downregulating PTEN. Prolonged AKT activation by FSH stimulation of the FSH receptor (FSHR) promotes gankyrin expression, which, in turn, enhances AKT activation by inhibiting PTEN. Overexpression of gankyrin decreases hypoxia inducible factor-1α (HIF-1α) protein levels, but has little effect on HIF-1α mRNA levels, which could be attributed to gankyrin mediating HIF-1α protein stability via the ubiquitin-proteasome pathway. Reduction in HIF-1α protein stability led to attenuation of the binding with cyclin D1 promoter, resulted in abolishment of the negative regulation of cyclin D1 by HIF-1α, which promotes proliferation of ovarian cancer cells. Our results document that gankyrin regulates HIF-1α protein stability and cyclin D1 expression, ultimately mediating FSH-driven ovarian cancer cell proliferation.

Topics: Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Follicle Stimulating Hormone; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Ubiquitination; Up-Regulation

In this study, we investigated the expression and role of PSMB4 in human epithelial ovarian cancer(EOC).. Western blot was used to evaluate the expression of PSMB4 in EOC tissues, and immunohistochemical analysis was performed on 115 cases of ovarian cancers. Then, we used Fisher exact test to analyze the correlation between PSMB4 and clinicopathological parameters. Starvation and re-feeding assay was used to assess cell cycle. CCK-8 assay and plate colony formation assay showed the influence of PSMB4 on proliferation of EOC cells.. The expression of PSMB4 in EOC tissues was higher than normal ovary tissues and was significantly associated with clinical pathologic variables. Kaplan-Meier curve showed that high expression of PSMB4 was related to poor prognosis of EOC patients. Starvation and re-feeding assay suggested that PSMB4 played a critical role in EOC cell proliferation. CCK-8 assay and plate colony formation assay showed that EOC cells treated with PSMB4-siRNA reduced cell proliferation of EOC cells. Additionally, PSMB4 knockdown decreased NF-κB activity. PSMB4 also regulated the expression of NF-κB mediated proteins, including cyclin D1, and cyclin E which involved in cell proliferation.. Our findings implied that PSMB4 is involved in the progression of EOC and could serve as potential therapeutical target of EOC. These data suggested that PSMB4 may promote cell proliferation via the NF-κB-target gene in EOC.

Topics: Adult; Biomarkers, Tumor; Carcinoma, Ovarian Epithelial; Cell Proliferation; Cyclin D1; Disease Progression; Female; Humans; Kaplan-Meier Estimate; MicroRNAs; Middle Aged; Neoplasms, Glandular and Epithelial; NF-kappa B; Ovarian Neoplasms; Prognosis; Proteasome Endopeptidase Complex; RNA, Small Interfering

Ovarian cancer (OC) is a deadly disease, and despite improvements in treatment, overall 5-year survival is low. Glycogen synthase kinase (GSK)-3β is a multifunctional serine/threonine kinase. We wished to ascertain if the GSK-3β inhibitor (2Z,3E)-6-bromoindirubin-3'-oxime, known as "BIO," can suppress OC development. The OC cell lines A2780 and OVCAR3 were exposed to BIO. At different time points, cell proliferation, apoptosis, cell cycle, and cell invasion/cell migration assays were carried out. Phalloidin staining was undertaken to observe lamellipodia formation. Real-time reverse transcription-polymerase chain reaction and western blotting were used to assess expression of messenger RNA (mRNA) and protein of GSK-3β, cyclin D1, matrix metalloproteinase (MMP)-9, and p21. BIO suppressed the proliferation, invasion, and migration of OC cells; reduced lamellipodia formation; and induced G1 arrest of the cell cycle. BIO exposure led to a significant downregulation of mRNA and protein expression of cyclin D1 and MMP9 in comparison with untreated control cells. In contrast, BIO exposure upregulated mRNA and protein expression of p21 in comparison with untreated control cells. Besides, GSK-3β small interfering RNA (siRNA) transfection in ovarian cancer cells also downregulated GSK-3β, cyclin D1, and MMP9 protein expression while upregulated p21 expression. These data suggest that BIO, as an inhibitor of GSK-3β, can suppress OC development. Therefore, BIO could be a candidate drug for the treatment of OC.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; Indoles; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Ovarian Neoplasms; Oximes; Phosphorylation; Signal Transduction

In the present study, the underlying apoptotic mechanism of sanggenol L was elucidated in ovarian cancer cells. Sanggenol L showed cytotoxic and antiproliferative effect in A2780, SKOV-3, and OVCAR-3 ovarian cancer cells in a concentration-dependent fashion. Consistently, sanggenol L increased sub-G1 phase population and early and late apoptotic portion in ovarian cancer cells. Also, sanggenol L activated caspase9/3, suppressed the phosphorylation of IκBα and p65 NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), attenuated the expression of Cyclin D1, and cleaved poly(adenosine diphosphate ribose -ribose) polymerase in SKOV-3, A2780, and OVCAR-3 cells. Furthermore, sanggenol L blocked nuclear translocation of NF-κB and also attenuated the expression of NF-κB related genes such as c-Myc, Cyclin D1, and Bcl-X L, Bcl-2, in lipopolysaccharide-treated SKOV-3 cells. Overall, our findings for the first time suggest that sanggenol L induces apoptosis via caspase activation and inhibition of NF-κB/IκBα phosphorylation as a potent chemotherapeutic agent for ovarian cancers.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-X Protein; Caspases; Cell Line, Tumor; Cyclin D1; Female; Flavanones; Humans; I-kappa B Proteins; Morus; NF-KappaB Inhibitor alpha; Ovarian Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Transcription Factor RelA

The most prevalent malignant ovarian neoplasms are epithelial ovarian cancers which is the most common cause of death among all gynecologic malignancies and a result of complex interaction of multiple oncogenes and tumor suppressor genes. The aim of this study was to evaluate expression of survivin and cycline D1 biomarkers in mucinous ovarian neoplasms and their correlations with clinicopathological variables in mucinous ovarian cancers. We analyzed pathological specimens of 98 patients with benign (n = 34), borderline (n = 22) and malignant (n = 42) mucinous ovarian neoplasms. Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded specimens. Immunohistochemical analysis revealed that survivin and cyclin D1 expressions were located primarily in the nucleus of ovarian tumor cells and relatively weaker cytoplasmic staining. Survivin expression was significantly higher in malignant tumors (88.1 %) than those found in borderline (18.2 %) and benign tumors (8.8 %) (p < 0.001). Similarly, higher cyclin D1 expression was observed in malignant tumors (100 %) compared to borderline (36.4 %) and benign tumors (5.9 %) (p < 0.001). Expression of all biomarkers analyzed significantly and gradually increased from benign to borderline and borderline to malignant mucinous tumors. In terms of clinicopathological variables, tumor grade, FIGO stage and lymph node methastasis were associated with the expression of both biomarkers. Whereas age exhibited no different correlations in mucinous ovarian cancers. The expressions of survivin and cycline D1 are positively correlated with the malignant potential of mucinous ovarian neoplasms.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Cell Nucleus; Cyclin D1; Female; Humans; Inhibitor of Apoptosis Proteins; Middle Aged; Neoplasms, Cystic, Mucinous, and Serous; Ovarian Neoplasms; Survivin; Young Adult

Ovarian cancer is the leading cause of death among gynecological malignancies, and high grade serous ovarian carcinoma is the most common and most aggressive subtype. Recently, it was demonstrated that cAMP mediates protein kinase A-independent effects through Epac (exchange protein directly activated by cAMP) proteins. Epac proteins, including Epac1 and Epac2, are implicated in several diverse cellular responses, such as insulin secretion, exocytosis, cellular calcium handling and formation of cell-cell junctions. Several reports document that Epac1 could play vital roles in promoting proliferation, invasion and migration of some cancer cells. However, the expression levels and roles of Epac1 in ovarian cancer have not been investigated. In the present study, we detected the expression levels of Epac1 mRNA and protein in three kinds of ovarian cancer cells SKOV3, OVCAR3 and CAOV3. Furthermore, the effect of Epac1 knockdown on the proliferation and apoptosis of SKOV3 and OVCAR3 cells was evaluated in vitro and in vivo. The results showed that there was higher expression of Epac1 mRNA and protein in SKOV3 and OVCAR3 cells. Epac1 knockdown inhibited the proliferation of SKOV3 and OVCAR3 cells in vitro and in vivo. Decreased proliferation may be due to downregulation of Epac1-induced G1 phase arrest by inactivating the AKT/Cyclin D1/CDK4 pathway, but not to alterations in the MAPK pathway or to apoptosis. Taken together, our data provide new insight into the essential role of Epac1 in regulating growth of ovarian cancer cells and suggest that Epac1 might represent an attractive therapeutic target for treatment of ovarian cancer.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Female; Gene Knockdown Techniques; Guanine Nucleotide Exchange Factors; Heterografts; Humans; Immunohistochemistry; In Vitro Techniques; Mice; Mice, Nude; Ovarian Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction

Mortalin is frequently overexpressed in human malignancies. Previous studies have suggested that mortalin contributes to ovarian cancer development and progression, but further investigation is warranted. The aim of this study is to elucidate the mechanism of mortalin in ovarian cancer development and progression. In this study, lentivirus-delivered mortalin short hairpin RNA (shRNA) was used to knockdown mortalin expression in A2780 and A2780/cis ovarian cancer cell lines, and lentiviral mortalin-pLVX-AcGFP was used to generate mortalin-overexpressing cell lines. The results demonstrated that decreased mortalin expression reduced ovarian cancer cell proliferation, colony formation, migration and invasion by Cell Counting Kit-8 assay, colony formation assay, wounding healing assay and Transwell cell invasion assay, respectively. Flow cytometry results suggested that mortalin promotes the G1 transition, leading to faster restoration of a normal cell-cycle distribution. Cell-cycle proteins, including C-myc and Cyclin-D1, significantly increased, and Cyclin-B1 remarkably decreased upon mortalin down-regulation. Western blot analysis showed that mortalin knockdown significantly decreased p-c-Raf and phospho-extracellular-regulated protein kinases (p-ERK1/2) pathways but not the Jun N-terminal kinase pathway, whereas mortalin overexpression had the opposite effect. Taken together, these results indicate that mortalin is an oncogenic factor, and mitogen-activated protein kinase-ERK signalling pathway activation by mortalin may contribute to ovarian cancer development and progression.

Topics: Carcinogenesis; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Enzyme Activation; Female; HSP70 Heat-Shock Proteins; Humans; MAP Kinase Signaling System; Neoplasm Invasiveness; Oncogenes; Ovarian Neoplasms; Up-Regulation

Long non-coding RNA growth arrest-specific 5 (GAS5) was reported to be aberrantly expressed in various types of cancers. However, the role of GAS5 in the evolution and progression of ovarian cancer remains elusive. In the present study, we aimed to investigate the cellular function and clinical significance of GAS5 in ovarian cancer. GAS5 expression levels in 63 ovarian cancer tissues were detected by quantitative real-time PCR. Cell Counting Kit-8 (CCK-8) assay was performed to analyze the effect of GAS5 on cell proliferation. The effect of GAS5 on cell migration and invasion was detected using Transwell assay. Cell apoptosis was evaluated by flow cytometry and Hoechst staining. SKOV3 cells with stable expression of GAS5 were injected into nude mice to study the effect of GAS5 on tumorigenesis in vivo. Western blotting was used to determine the protein levels of GAS5 potential targets. The results showed that GAS5 was markedly decreased in tumor tissues and a lower expression of GAS5 was detected in tumors with larger size, deeper invasive depth and higher tumor stage. Patients with low GAS5 expression level had poorer disease-free (P<0.0001) and overall survival (P=0.0016) than those with high GAS5 expression. Moreover, overexpression of GAS5 was demonstrated to suppress ovarian cancer cell proliferation in vitro and in vivo. Finally, we found that GAS5 influenced ovarian cancer cell proliferation, partly via regulating cyclin D1, p21 and apoptosis protease activating factor 1 (APAF1) expression. Our findings suggest that lncRNA GAS5 may represent a novel indicator of poor prognosis in ovarian cancer and may be a potential therapeutic target for diagnosis and therapy.

Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Female; Gene Expression; Humans; Kaplan-Meier Estimate; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Ovarian Neoplasms; Prognosis; RNA, Long Noncoding

Panacis Japonici Rhizoma (PJR) is one of the most famous Chinese medical herbs that is known for exhibiting potential anti-cancer effects.. This study aims to isolate and investigate the anti-cancer potential of saponins from PJR in ovarian cancer cells.. The compounds were separated by comprehensive chromatographic methods. By comparison of the 1H- and 13C NMR data, as well as the HR-ESI-MS data, with the corresponding references, the structures of compounds were determined. MTT assay was performed to evaluate cell viability, along with flow cytometry for cell cycle analysis. JC-1 staining, Annexin V-PI double staining as well as Hoechst 33; 342 staining were used for detecting cell apoptosis. Western blot analysis was conducted to determine the relative protein level. Transwell assays were performed to investigate the effect of the saponin on cell migration and invasion and zymography experiments were used to detect the enzymatic activities.. Eleven saponins were isolated from PJR and their anti-proliferative effects were evaluated in human ovarian cancer cells. Chikusetsusaponin IVa methyl ester (1) exhibited the highest anti-proliferative potential among these isolates with the IC50 values at less than 10 µM in both ovarian cancer A2780 and HEY cell lines. Compound 1 induced G1 cell cycle arrest accompanied with an S phase decrease, and down-regulated the expression of cyclin D1, CDK2, and CDK6. Further study showed that compound 1 effectively decreased the cell mitochondrial membrane potential, increased the annexin V positive cells and nuclear chromatin condensation, as well as enhanced the expression of cleaved PARP, Bax and cleaved-caspase 3 while decreasing that of Bcl-2. Moreover, compound 1 suppressed the migration and invasion of HEY and A2780 cells, down-regulated the expression of Cdc42, Rac, RohA, MMP2 and MMP9, and decreased the enzymatic activities of MMP2 and MMP9.. These results provide a comprehensive evaluation of compound 1 as a potential agent for the treatment of ovarian cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase 2; Drug Screening Assays, Antitumor; Drugs, Chinese Herbal; Female; G1 Phase Cell Cycle Checkpoints; Humans; Membrane Potential, Mitochondrial; Molecular Structure; Oleanolic Acid; Ovarian Neoplasms; Saponins

Ovarian cancer is a gynecological malignancy with high mortality rates all over the world. Markers for diagnosis, prognosis and therapy are urgently required to improve the mortality rates. As a key proto-oncogene, CCND1 is known to be amplified in many different carcinomas, including breast cancer, esophageal cancer, bladder cancer, endometrial cancer and ovarian cancer, etc. CCND1 plays an important role in cancer development and progression. However, its function and mechanism have not been completely elucidated in ovarian cancer.. In the present study, we use cisplatin in vitro to inhibit the cell proliferation and promote cell apoptosis in epithelial ovarian cancer cell line SKOV-3. CCND1 expression, cell proliferation and cell cycle analysis were carried out by real-time PCR, CCK-8 and flow cytometry respectively.. Our results demonstrated that cisplatin could inhibit the expression of CCND1 in human epithelial ovarian cancer cell line, which is related to the decreased cell proliferation and increased cell apoptosis.. This study demonstrated that CCND1 is a potential therapeutic target for epithelial ovarian cancer treatment.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cyclin D1; Female; Humans; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Proto-Oncogene Mas

Epithelial ovarian cancer (EOC) is a significant cause of morbidity and mortality. MicroRNAs play important roles in cancer development and progression. The microRNA miR-211 is localized on intron 6 of the Trpm1 gene at 15q13-q14, a locus that is frequently lost in neoplasms. Its function and loss-of-function have been described in normal and cancer cells and tissues. miR-211 is known to be dysregulated in ovarian cancer: however, its function and the downstream effect of its loss-of-function in ovarian cancer have not been described before.. We analyzed miR-211 expression in clinical samples of primary EOC tissues compared to normal epithelial ovarian tissues and in the EOC cell lines: OVCAR3, Caov3, OVCA429, SKOV3 and A2780 compared to human ovarian surface epithelial cells. We then investigated the effect of miR-211 on EOC cell proliferation and apoptosis by counting cell numbers, MTT, colony formation, cell cycle, and PI/Annexin V staining assays. A luciferase reporter system was developed to assess miR-211 regulation of the predicted targets. Expression level of discovered targets and correlation with miR-211 expression were analyzed in EOC tissues. Finally, OVCAR3 stably expressing miR-211 or control cells were injected subcutaneously into mice to determine in vivo effect of miR-211 on tumorigenesis.. We found that the expression of miR-211 is downregulated in EOC tissues and cell lines compared to normal epithelial ovarian tissue and human ovarian surface epithelial cells, respectively. miR-211 was found to arrest cells in the G0/G1-phase, inhibit proliferation and induce apoptosis. Cyclin D1 and CDK6 were found to be direct targets of miR-211, and when overexpressed in miR-211-expressing EOC cells, could restore proliferative ability. Finally, in vitro investigation confirmed that miR-211 is a tumor suppressor that controls Cyclin D1 and CDK6 expression.. Our results demonstrate that miR-211 is a tumor suppressor that controls expression of Cyclin D1 and CDK6, and that its downregulation results in overexpression of Cyclin D1 and CDK6 which increases proliferation ability of EOC cells to proliferate compared to normal cells.

Topics: Apoptosis; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 6; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; MicroRNAs; Middle Aged; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Resting Phase, Cell Cycle

In order to examine new ideas for gene therapy in ovarian cancer, the specific mechanism underlying the effects of the WW domain containing oxidoreductase (WWOX) gene on cell cycle regulation and apoptosis in human ovarian cancer stem cells was investigated. Ovarian cancer stem cells were transfected with a eukaryotic expression vector carrying the WWOX gene in vitro (recombinant plasmid) and cells transfected with the empty plasmid (empty plasmid) or untransfected cells were used as controls. Stably transfected cells were screened and amplified in culture and the WWOX protein was detected by western blot analysis in the three groups of cells. Western blot analysis was performed to detect the expression of cell cycle regulatory proteins cyclin E, cyclin-dependent kinase (CDK) 2, cyclin D1, CDK4 and apoptosis-related protein Wnt-5α and c-Jun N-terminal kinase (JNK), while polymerase chain reaction (PCR) was used to detect alterations in the mRNA expression levels of caspase-3. The results demonstrated that the WWOX protein was stably expressed in cells of the recombinant plasmid group, but was not detected in cells of the empty plasmid group and the control group. Cell proliferation at each time point decreased significantly in the recombinant plasmid group compared with the empty plasmid group and the control group. Flow cytometric analysis demonstrated that the proportion of cells in the G0/G1 phase in the recombinant plasmid group was significantly higher than that of cells in the empty plasmid group and the control group. The rate of apoptosis in the recombinant plasmid group was significantly higher than that of cells in the empty plasmid group and the control group. Western blot analysis demonstrated that the expression levels of cyclin E, CDK2, cyclin D1 and CDK4 in the recombinant plasmid group were significantly lower than those in the empty plasmid group and the control group; however, the expression levels of Wnt-5α and JNK were significantly higher than those in the empty plasmid group and the control group. PCR results demonstrated that the mRNA expression level of caspase-3 in the recombinant plasmid group was significantly higher than that in the empty plasmid group and the control group. In conclusion, the present study demonstrated that the WWOX gene can be stably expressed in ovarian cancer stem cells and that it inhibits the proliferation of ovarian cancer stem cells. The WWOX gene can downregulate the expression levels of cell cycle pro

Topics: Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; Humans; JNK Mitogen-Activated Protein Kinases; Neoplastic Stem Cells; Ovarian Neoplasms; Oxidoreductases; Plasmids; Proto-Oncogene Proteins; Real-Time Polymerase Chain Reaction; Transfection; Tumor Suppressor Proteins; Wnt Proteins; Wnt-5a Protein; WW Domain-Containing Oxidoreductase

Ovarian cancer is a gynecological malignancy with high mortality rates worldwide and novel diagnostic and prognostic markers and therapeutic targets are urgently required. The suppressor of cytokine signaling 1 (SOCS1) and cyclin-dependent kinase inhibitor 1A (p21(KIP)) are known to regulate tumor cell proliferation. However, the mechanisms that regulate these genes have not yet been completely elucidated. In the present study, analysis of a published microarray-based high-throughput assessment (NCBI/E-MTAB-1067) and real-time PCR demonstrated that miR-572 was upregulated in human ovarian cancer tissues and cell lines. Kaplan-Meir analysis indicated that high level expression of miR-572 was associated with poorer overall survival. Ectopic miR-572 promoted ovarian cancer cell proliferation and cell cycle progression in vitro and tumorigenicity in vivo. SOCS1 and p21 were identified as direct targets of miR-572 and suppression of SOCS1 or p21 reversed the inhibiting-function of miR-572-silenced cell on proliferation and tumorigenicity in ovarian cancer cells. Additionally, the expression of miR-572 correlated inversely with the protein expression levels of SOCS1, p21 and positively with Cyclin D1 in ovarian carcinoma specimens. This study demonstrates that miR-572 post-transcriptionally regulates SOCS1 and p21 and may play an important role in ovarian cancer progression; miR-572 may represent a potential therapeutic target for ovarian cancer therapy.

Topics: 3' Untranslated Regions; Animals; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Ovarian Neoplasms; RNA Interference; RNA, Small Interfering; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins

Ovarian cancer represents the most fatal type of gynecological malignancies. Unfortunately, there are still no effective targeted treatment strategies for ovarian cancer. Overexpression of CRM1 has been correlated with poor prognosis of patients with ovarian cancer.. In this study, we investigated the antitumor effects of a novel reversible inhibitor of CRM1 in ovarian cancer cells.. The effects of S109 on proliferation was detected by CCK-8, EdU, clonogenic assay. The protein expression were determined by Western blot. The subcellular localization of RanBP1 was analyzed by immunofluorescence microscopy assay.. We demonstrated that S109 could induce nuclear accumulation of RanBP1, a canonical biomarker for CRM1 inhibition. This effect was clearly reversible in the majority of the cells, whereas the inhibitory effect of LMB could not be reversed. Our data reveal that treatment with S109 results in decrease in proliferation and colonogenic capacity of ovarian cancer cells by arresting cell cycle. Mechanistically, S109 treatment increase the expression of the cyclin-dependent kinase inhibitor p21, while it reduced the expression of cell cycle promoting proteins, Cyclin D1 and Cyclin B. CRM1 level itself was also down-regulated following S109 treatment. Furthermore, the nuclei of cells incubated with S109 accumulated tumor suppressor proteins (Foxo1, p27 and IκB-α). More importantly, Cys528 mutation of CRM1 abolished the ability of S109 to block proliferation of ovarian cancer cells.. Together, our study identifies CRM1 as a valid target in ovarian cancer and provides a basis for the development of S109 in ovarian cancer.

Topics: Aminopyridines; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin B; Cyclin D1; Cyclopentanes; Exportin 1 Protein; Female; Gene Expression Regulation, Neoplastic; Humans; Karyopherins; Ovarian Neoplasms; Receptors, Cytoplasmic and Nuclear

Since our first description of the microcystic stromal tumor (MST) of the ovary, a rare and distinctive neoplasm with a definitional, usually striking microcystic pattern and a CD10+/vimentin+/inhibin-/calretinin- immunophenotype, 3 examples with β-catenin nuclear localization, and CTNNB1 mutation have been reported. We undertook a detailed immunohistochemical study and molecular analysis of CTNNB1 and FOXL2 of 15 cases of MST to further characterize this neoplasm and establish its histogenesis. Diffuse nuclear staining for FOXL2, WT-1, cyclin D1, and β-catenin was present in all tumors tested, and 12/15 were positive for steroidogenic factor-1 (SF-1). Heterozygous missense point mutations in exon 3 of CTNNB1 were detected in 8 of 14 cases, resulting in amino acid changes at codons 32, 34, 35, and 37. There was no correlation between CTNNB1 exon 3 mutation status and tumor immunophenotype. All 14 cases tested showed wild-type FOXL2. Our study establishes that MST of the ovary exhibits a characteristic FOXL2/SF-1/WT-1/cyclin D1/nuclear β-catenin-positive immunohistochemical profile, which may be useful in diagnosis and in the exclusion of histologic mimics. The presence of diffuse nuclear FOXL2 and WT-1 immunostaining in all cases and SF-1 in most supports the classification of MST within the sex cord-stromal category. Aberrant nuclear β-catenin expression, detected in all MSTs, appears to be the result of stabilizing CTNNB1 mutations in 57% of cases, providing further evidence that dysregulation of the Wnt/B-catenin pathway is involved in the tumorigenesis of MST and may involve activation of β-catenin with upregulation of cyclin D1.

Topics: Adult; beta Catenin; Biomarkers, Tumor; Cyclin D1; DNA Mutational Analysis; DNA-Binding Proteins; Female; Forkhead Box Protein L2; Forkhead Transcription Factors; Humans; Immunohistochemistry; Middle Aged; Neoplasms, Cystic, Mucinous, and Serous; Ovarian Neoplasms; RNA Splicing Factors; Sex Cord-Gonadal Stromal Tumors; Transcription Factors; Wnt Signaling Pathway; WT1 Proteins

Fenhexamid and cyprodinil are antifungal agents (pesticides) used for agriculture, and are present at measurable amounts in fruits and vegetables. In the current study, the effects of fenhexamid and cyprodinil on cancer cell proliferation and metastasis were examined. Additionally, the protein expression levels of cyclin D1 and cyclin E as well as cathepsin D were analyzed in BG-1 ovarian cancer cells that express estrogen receptors (ERs). The cells were cultured with 0.1% dimethyl sulfoxide (DMSO; control), 17β-estradiol (E2; 10(-9)M), and fenhexamid or cyprodinil (10(-5)-10(-7)M). Results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that fenhexamid and cyprodinil increased BG-1 cell proliferation about 1.5 to 2 times similar to E2 (5 times) compared to the control. When the cells were co-treated with ICI 182,780 (10(-8)M), an ER antagonist, the proliferation of pesticide-treated BG-1 cells was decreased to the level of the control. A wound healing assay revealed that the pesticides reduced the disrupted area in the BG-1 cell monolayer similar to E2. Protein levels of cyclin D1 and E as well as cathepsin D were increased by fenhexamid and cyprodinil. This effect was reversed by co-treatment with ICI 182,780. In a xenograft mouse model with transplanted BG-1 cells, cyprodinil significantly increased tumor mass formation about 2 times as did E2 (6 times) compared to the vehicle (0.1% DMSO) over an 80-day period. In contrast, fenhexamid did not promote ovarian tumor formation in this mouse model. Cyprodinil also induced cell proliferation along with the expression of proliferating cell nuclear antigen (PCNA) and cathepsin D in tumor tissues similar to E2. Taken together, these results imply that fenhexamid and cyprodinil may have disruptive effects on ER-expressing cancer by altering the cell cycle- and metastasis-related gene expression via an ER-dependent pathway.

Topics: Amides; Animals; Cathepsin D; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Estradiol; Female; Fulvestrant; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Oncogene Proteins; Ovarian Neoplasms; Pyrimidines; Receptors, Estrogen

G protein-coupled estrogen receptor (GPER) is identified as a critical estrogen receptor, in addition to the classical estrogen receptors ERα and ERβ. In ERα-negative ovarian cancer cells, our previous studies have found that estrogen stimulated cell proliferation and metastasis via GPER. However, the ligand-independent function of GPER in ovarian cancer cells is still not clear. Herein, we describe that GPER has a co-expression with ERα and ERβ, which are first determined in SKOV3 ovarian cancer cell line. In the absence of estrogen, GPER depletion by specific siRNA inhibits the proliferation, migration and invasion of SKOV3 cells. Whereas abrogation of ERα or ERβ by specific antagonist MPP and PHTPP has the opposite effects for stimulation of cell growth. Markedly, GPER knockdown attenuates MPP or PHTPP-induced cell proliferation, migration and invasion. Furthermore, GPER modulates protein expression of the cell cycle critical components, c-fos and cyclin D1 and factors for cancer cell invasion and metastasis, matrix metalloproteinase 2 (MMP-2) and MMP-9. These findings establish that GPER ligand-independently stimulates the proliferation, migration and invasion of SKOV3 cells. Knockdown of GPER attenuates the progression of ovarian cancer that caused by functional loss of ERα or ERβ. Targeting GPER provides new aspect as a potential therapeutic strategy in ovarian cancer.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Gene Knockdown Techniques; Humans; Ligands; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Ovarian Neoplasms; Proteolysis; Proto-Oncogene Proteins c-fos; Receptors, Estrogen; Receptors, G-Protein-Coupled; RNA Interference

The purpose of this study was to investigate the expression of A-kinase anchor protein 95 (AKAP95), cell cycle protein E1 (cyclinE1) and D1 (cyclinD1), and gap junction protein connexin 43 (Cx43) in ovarian cancer tissues, the relationship between four proteins and clinicopathologic parameters, and the correlation between these proteins.. The expression of proteins in 54 cases of ovarian cancer tissues was detected by immunohistochemical method.. The positive expression rates of AKAP95, cyclinD1 and cyclinE1 in ovarian cancer tissues were 72.22%, 66.67% and 79.63%, respectively, which were higher than that of ovarian pericarcinoma tissues expressing as 33.33%, 25% and 8.30% (P<0.05). The positive expression rate of Cx43 in ovarian cancer tissues was 40.74%, which was lower than that of ovarian pericarcinoma tissues expressing as 75%; respectively, and the difference was statistically significant between groups (P<0.05). The expression of cyclinD1 in ovarian cancer tissues was related to the histologic type (P<0.05) while it showed no correlation with the degree of differentiation (P>0.05). Additionally, the expression of AKAP95, Cx43 and cyclinE1 in ovarian cancer tissues showed no correlation with the degree of differentiation or the histologic type (P>0.05). Protein expressions of AKAP95, Cx43 and cyclinE1 were correlated with each other (P<0.05), and the expressions of cyclinD1, cyclinE1 and Cx43 were also correlated with each other (P<0.05). However, AKAP95 and cyclinD1 showed no correlation (P>0.05).. AKAP95, cyclinD1 and cyclinE1 play an important role in promoting the process of ovarian cancer formation. The tumor inhibitory effects of Cx43 protein on the pathogenesis of ovarian cancer were weakened. The expression of cyclinD1 in ovarian cancer tissues is related to the histologic type while it shows no correlation with the degree of differentiation. Additionally, the expression of AKAP95, Cx43 and cyclinE1 in ovarian cancer tissues shows no correlation with the degree of differentiation or the histologic type. AKAP95 expression is correlated with Cx43 and cyclinE1 expression; Cx43 expression is correlated with AKAP95, cyclinD1 and cyclinE1 expression; cyclinE1 expression is correlated with AKAP95, Cx43, cyclinD1 expression; cyclinD1 expression is correlated with Cx43 and cyclinE1 expression, while AKAP95 and cyclinD1 show no correlation.

Topics: A Kinase Anchor Proteins; Adult; Biomarkers, Tumor; Cell Differentiation; Connexin 43; Cyclin D1; Cyclin E; Female; G1 Phase; Humans; Immunohistochemistry; Middle Aged; Oncogene Proteins; Ovarian Neoplasms; S Phase

Alterations in the retinoblastoma pathway are frequent in ovarian/tubal high-grade serous cancers, but the mechanism of deregulation and the impact on patient outcome are poorly understood. A cohort of 334 high-grade serous carcinomas was studied by immunohistochemical analysis of RB1, p16, cyclin D1, cyclin E1, and Ki67. Additional detailed analyses including RB1 allelic deletion (n=42), mutation (n=75), methylation (n=31), and SNP array analyses (n=75) were performed on cases with clinical parameters, including age, debulking status, treatment, and clinical outcome. p16/RB1 expression results yielded three distinct clinically relevant subgroups upon multivariable analysis controlling for stage, debulking status, and treatment types: p16 homogeneous/RB1+ with the shortest progression-free survival (median 15 months (95% CI: 13-18); P=0.016) compared with the p16 heterogeneous/RB1+ subgroup (median 22 months (95% CI: 16-32)) and the p16 homogeneous/RB1- subgroup (median 20 months (95% CI: 15-24)). Patients in the p16 homo/RB1- subgroup showed a significant increase in overall survival (>60 months; P=0.013), which suggests an increase in sensitivity to cytotoxic agents. Analyses of Rb pathway mechanistic differences among these groups revealed frequent RB1 genomic alterations such as RB1 allelic loss and/or large spanning deletions (83%) in the p16 homo/RB1- subgroups, also indicating that RB1 deletions are frequent in high-grade serous carcinoma. CCNE1 gene gains/amplifications were frequent in the p16 homogeneous/RB1+ subgroup (68%) and cyclin D1 protein overexpression was predominantly characteristic of the p16 heterogeneous/RB1+ subgroup. These subcategories occur early in tumor progression and are seen with similar frequency in the cancer precursor lesion, serous tubal intra-epithelial carcinoma. Overall, this study uniquely identifies multiple non-synonymous mechanisms of retinoblastoma pathway deregulation that correlate with significantly different clinical outcomes. Furthermore, deregulations identified in precursor lesions suggest a key role of this pathway in serous tumor development. Recognition of these categories may identify patients with increased sensitivity to chemotherapy and new opportunities for novel therapeutics.

Topics: Alleles; Biomarkers, Tumor; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p16; Cystadenocarcinoma, Serous; Disease-Free Survival; Female; Humans; Immunohistochemistry; Middle Aged; Mutation; Oncogene Proteins; Ovarian Neoplasms; Prognosis; Retinoblastoma Protein

Ovarian serous cancer is the most common subtype of epithelial ovarian cancer, and is the leading cause of death from gynecologic cancer. There is an important need for exploration of diagnostic and prognostic markers for this disease. β-catenin and cyclinD1 play central roles in the tumorigenesis for certain cancers. The role of β-catenin and cyclinD1 in diagnosis and prognosis of ovarian serous carcinoma is uncertain. In the present study, the expression of β-catenin and cyclinD1 was examined in 60 ovarian serous carcinomas patients with immunohistochemical staining. The relationship between expression of β-catenin and cyclinD1 and FIGO stage, pathological grade was analyzed. Kaplan-Meier survival function was used to analyze the prognosis. Overexpression of β-catenin is more often detected in patients with FIGO stage III and IV than in those with stage I, and II (P=0.003). No significant relationship was found between expression of β-catenin and pathological grade (P=0.817). Positive expression of β-catenin related to lower survival rate (P=0.034). The expression of cyclinD1 had no relationship with FIGO stage (P=0.829). Overexpression of cyclinD1 was positively to pathological grade (P=0.017) and survival rate (P=0.009). There is a significantly positive relationship between expression of β-catenin and cyclinD1 (P=0.014). No statistical significance was found between expression of β-catenin and cyclinD1 and other pathological parameters.. Expression of β-catenin and cyclinD1 may be used as predict markers for poor prognosis.

Topics: Adult; Aged; beta Catenin; Biomarkers, Tumor; Cyclin D1; Cystadenocarcinoma, Serous; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Middle Aged; Neoplasm Staging; Ovarian Neoplasms; Prognosis; Up-Regulation; Young Adult

The human umbilical cord mesenchymal stem cells (hUCMSCs) have the ability to migrate into tumors and therefore have been considered as an alternative source of mesenchymal progenitors for the therapy of malignant diseases. The present study was aimed to investigate effect of hUCMSCs as vehicles for a constant source of transgenic interleukin-21 (IL-21) on ovarian cancer in vivo.. The hUCMSCs were engineered to express IL-21 via lentiviral vector- designated 'hUCMSCs-LV-IL-21', and then were transplanted into SKOV3 ovarian cancer xenograft-bearing nude mice. The therapeutic efficacy and mechanisms of this procedure on ovarian cancer was evaluated.. The isolated hUCMSCs were induced to differentiate efficiently into osteoblast and adipocyte lineages in vitro. The expressed IL-21 in the supernatant from hUCMSCs-LV-IL-21 obviously stimulated splenocyte's proliferation. The hUCMSCs-LV-IL-21 significantly reduced SKOV3 ovarian cancer burden in mice indicated by tumor sizes compared with control mice. The expressed IL-21 not only regulated the levels of IFN-γ and TNF-α in the mouse serum but also increased the expression of NKG2D and MIC A molecules in the tumor tissues. The down regulation of β-catenin and cyclin-D1 in the tumor tissues may refer to the inhibition of SKOV3 ovarian cancer growth in mice. In addition, hUCMSCs did not form gross or histological teratomas up to 60 days posttransplantation in murine lung, liver, stomach and spleen.. These results clearly indicate a safety and usability of hUCMSCs-LV- IL-21 in ovarian cancer gene therapy, suggesting the strategy may be a promising new method for clinical treatment of ovarian cancer.

Topics: Animals; beta Catenin; Cell Line, Tumor; Cord Blood Stem Cell Transplantation; Cyclin D1; Female; Fetal Blood; Genetic Therapy; HEK293 Cells; Humans; Interleukins; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice, Inbred BALB C; Mice, Nude; Ovarian Neoplasms; Time Factors; Tumor Burden; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

Endometrial endometrioid carcinomas (EECs) account for >80% of endometrial carcinomas (ECs). Continuous stimulation of the endometrium by estrogen is a risk factor for the tumorigenesis of estrogen receptor (ER) α-positive EEC. MicroRNA-22 (miR-22) has been reported to be implicated in the regulation of various types of cancer and directly targets ERα. However, an exact regulatory mechanism between miR-22 and ERα in EEC has yet to be investigated. To the best of our knowledge, the present study demonstrated for the first time that the expression of miR-22 was significantly downregulated in ERα-positive EEC tissues and cell lines, RL95-2 and Ishikawa, when compared with that in normal endometrium and ERα-negative EEC samples. This indicated that miR-22 may be important in ERα-positive EEC, possibly through an estrogen-dependent mechanism. miR-22 mimics were then transfected into RL95-2 and Ishikawa cells, respectively, and revealed that the introduction of miR-22 markedly downregulated the mRNA and protein levels of ERα. Further investigation demonstrated that miR-22 was able to effectively reverse 17β-estradiol (E2)‑induced cell proliferation, cell cycle progression and invasion of ERα-positive RL95-2 and Ishikawa cells, at least partially through inhibiting the expression of Cyclin D1 as well as the secretion of matrix metalloproteinase (MMP)-2 and MMP-9. In conclusion, the present study, to the best of our knowledge, was the first to reveal an inhibitory role of miR-22 in ERα-positive EEC tissues and cells, indicating that miR-22 may be a novel candidate for the endocrine therapy of ERα-positive EEC.

Topics: Carcinoma, Endometrioid; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Neoplasm Invasiveness; Ovarian Neoplasms; Signal Transduction

Loss of cytoglobin is found to be involved in the progression of several human cancers. However, its expression pattern and biological roles in human ovarian cancers are not clear. In this study, we examined cytoglobin expression in 118 archived ovarian cancer specimens using immunohistochemistry. A total of 72 specimens (61.0 %) showed cytoglobin downregulation. cytoglobin downregulation positively correlated with advanced FIGO stage and tumor grade. Cytoglobin plasmid transfection was performed in SKOV3 cell line and siRNA knockdown was carried out in SW626 cell line. MTT, colony formation assay and matrigel invasion assay were carried out to assess the role of cytoglobin on cell proliferation and invasion. Cytoglobin overexpression inhibited cell growth, invasion, cell cycle progression and cyclin D1 expression in SKOV3 cell line and its depletion promoted cell proliferation, invasion, cell cycle transition and cyclin D1 expression. In conclusion, cytoglobin is downregulated in ovarian cancers and associated with advanced stage. Our data provides evidence that cytoglobin regulates the ovarian cancer cell proliferation and invasion.

Topics: Aged; Cell Movement; Cell Proliferation; Cyclin D1; Cytoglobin; Female; Gene Expression Regulation, Neoplastic; Globins; Humans; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Ovarian Neoplasms; RNA, Small Interfering

CARD recruited membrane associated protein 3 (CARMA3) overexpression has been found in several human cancers. However, its expression pattern and biological roles in human ovarian cancers are not clear. In this study, we examined the expression pattern of CARMA3 in 101 ovarian cancer specimens. We found that 52 (51.5 %) showed CARMA3 overexpression. CARMA3 overexpression positively correlated with tumor histology and advanced FIGO stage. CARMA3 depletion in ovarian cancer cell lines A2780 and HO8910 inhibited ovarian cancer cell proliferation and blocked cell cycle progression. CARMA3 depletion also sensitized ovarian cancer cells to cisplatin-induced cytotoxicity. In addition, Western blot showed that CARMA3 depletion downregulated cyclin D1, cyclin E, and Bcl-2 levels. In conclusion, our data provides evidence that CARMA3 is overexpressed in ovarian cancers and associated with advanced stage. CARMA3 regulates the ovarian cancer cell proliferation, cell cycle progression, and chemoresistance.

Topics: Adult; Aged; Antineoplastic Agents; Carcinoma, Ovarian Epithelial; CARD Signaling Adaptor Proteins; Cell Cycle; Cell Line, Tumor; Cisplatin; Cyclin D1; Drug Resistance, Neoplasm; Female; Humans; Middle Aged; Neoplasm Staging; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Ovary; RNA, Small Interfering

Methoxychlor and triclosan are emergent or suspected endocrine-disrupting chemicals (EDCs). Methoxychlor [MXC; 1,1,1-trichlor-2,2-bis (4-methoxyphenyl) ethane] is an organochlorine pesticide that has been primarily used since dichlorodiphenyltrichloroethane (DDT) was banned. In addition, triclosan (TCS) is used as a common component of soaps, deodorants, toothpastes, and other hygiene products at concentrations up to 0.3%. In the present study, the potential impact of MXC and TCS on ovarian cancer cell growth and underlying mechanism(s) was examined following their treatments in BG-1 ovarian cancer cells. As results, MXC and TCS induced BG-1 cell growth via regulating cyclin D1, p21 and Bax genes related with cell cycle and apoptosis. A methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay confirmed that the proliferation of BG-1 ovarian cancer cells was stimulated by MXC (10(-6), 10(-7), 10(-8), and 10(-9)M) or TCS (10(-6), 10(-7), 10(-8), and 10(-9)M). Treatment of BG-1 cells with MXC or TCS resulted in the upregulation of cyclin D1 and downregulation of p21 and Bax transcriptions. In addition, the protein level of cyclin D1 was increased by MXC or TCS while p21 and Bax protein levels appeared to be reduced in these cells. Furthermore, MXC- or TCS-induced alterations of these genes were reversed in the presence of ICI 182,780 (10(-7)M), suggesting that the changes in these gene expressions may be regulated by an ER-dependent signaling pathway. In conclusion, the results of our investigation indicate that two potential EDCs, MXC and TCS, may stimulate ovarian cancer growth by regulating cell cycle- and apoptosis-related genes via an ER-dependent pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Endocrine Disruptors; Female; Gene Expression Regulation, Neoplastic; Humans; Methoxychlor; Ovarian Neoplasms; Receptors, Estrogen; Signal Transduction; Triclosan

Spermatogenesis and oogenesis basic helix-loop-helix (bHLH) transcription factor 2 (Sohlh2) functions as a bhlh transcription factor to regulate mouse germ cell differentiation. Our previous data showed that Sohlh2 was highly expressed in human normal tissues, but low level of Sohlh2 was observed in many cancer cell lines, suggesting a possible role of Sohlh2 in tumorigenesis. In this study, we examined this possibility by using immunohistochemistry, MTT, 5-bromo-2-deoxyuridine, clonogenic assay and tumor xenograft techniques. Our results showed that the expression of Sohlh2 was decreased in epithelial ovarian carcinoma (EOC) tissues compared with benign ovarian tumors and ovarian tumors with low malignant potential. Forced expression of Sohlh2 led to a significant reduction in cancer cell proliferation in vitro and tumorigenesis in nude mice. Conversely, silencing of Sohlh2 enhanced ovarian cancer cell proliferation. Furthermore, Sohlh2 had opposite effects on its two direct targets p21 and cyclin D1: overexpression of Sohlh2 upregulated p21 but downregulated cyclin D1 expression. p21 knockdown could reverse the effects of Sohlh2 overexpression on inhibiting cell proliferation, and cyclin D1 knockdown could reverse the effects of Sohlh2 ablation on promoting cell proliferation. Thus, our data indicate that Sohlh2 likely functions as a tumor suppressor in EOCs, which is achieved by inducing p21 expression but repressing cyclin D1 expression.

Topics: Animals; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Carcinoma, Ovarian Epithelial; Cell Cycle; Cell Proliferation; Chromatin Immunoprecipitation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Fallopian Tubes; Female; Humans; Immunoenzyme Techniques; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Ovary; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Ovarian cancer is the most common cause of death among all gynecologic malignancies and a result of complex interaction of multiple oncogenes and tumor suppressor genes. The aim of this study was to evaluate expression of HER-2/neu (c-erbB2), survivin and cycline D1 biomarkers in serous ovarian neoplasms and their correlations with clinicopathological variables in serous ovarian cancers. We analyzed pathological specimens of 62 patients with benign (n = 25), borderline (n = 14) and malignant (n = 23) serous ovarian neoplasms. Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded specimens. Significantly more immunoreactivity with HER-2/neu was detected in malignant tumors (100 %) compared to borderline (78.6 %) and benign tumors (48 %) (P < 0.01). Survivin expression was significantly higher in malignant tumors (91.3 %) than those found in borderline (71.4 %) and benign tumors (24 %) (P < 0.001). Similarly, higher cyclin D1 expression was observed in malignant tumors (95.6 %) compared to borderline (85.7 %) and benign tumors (48 %) (P < 0.001). Expression of all biomarkers analyzed significantly and gradually increased from benign to borderline and borderline to malignant serous tumors. In terms of clinicopathological variables, only tumor grade was associated with the expression of all biomarkers others exhibited different correlations in serous ovarian cancers. The expressions of HER-2/neu (c-erbB2), survivin and cycline D1 are positively correlated with the malignant potential of serous ovarian neoplasms.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Cyclin D1; Female; Humans; Inhibitor of Apoptosis Proteins; Middle Aged; Neoplasms, Cystic, Mucinous, and Serous; Ovarian Neoplasms; Receptor, ErbB-2; Survivin; Young Adult

Estrogen and estrogen receptor (ER)-mediated signaling pathways play important roles in the etiology and progression of human breast, endometrial, and ovarian cancers. Attenuating ER activities by natural products and their derivatives is a relatively practical strategy to control and reduce breast, endometrial, and ovarian cancer risk. Here, we found 3-butoxy-1,8,9-trihydroxy-6H-benzofuro[3,2-c]benzopyran-6-one (BTB), a new derivative of wedelolactone, could effectively inhibit the 17-estradiol (E2)-induced ER transactivation and suppress the growth of breast cancer as well as endometrial and ovarian cancer cells. Our results indicate that 2.5 μM BTB effectively suppresses ER-positive, but not ER-negative, breast, endometrial, and ovarian cancer cells. Furthermore, our data indicate that BTB can modulate ER transactivation and suppress the expression of E2-mediated ER target genes (Cyclin D1, E2F1, and TERT) in the ER-positive MCF-7, Ishikawa, and SKOV-3 cells. Importantly, this BTB mediated inhibition of ER activity is selective since BTB does not suppress the activities of other nuclear receptors, including glucocorticoid receptor and progesterone receptor, suggesting that BTB functions as a selective ER signaling inhibitor with the potential to treat breast, endometrial, and ovarian cancers.

Topics: Breast Neoplasms; Cell Proliferation; Coumarins; Cyclin D1; Endometrial Neoplasms; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Ovarian Neoplasms; Signal Transduction

The polyphenolic flavonoid silymarin that is the milk thistle extract has been found to possess an anti-cancer effect against various human epithelial cancers. In this study, to explore the regulative effect of silymarin on human ovarian cancer line A2780s and PA-1 cells, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay and flow cytometry were respectively used to determine the inhibitory effect of silymarin on the both cell lines, and to measure their cell cycle progression. Apoptosis induction and mitochondrial membrane potential damage were separately detected by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling assay and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide staining. Additionally, western blotting was applied to determine cytochrome C release and expression levels of p53, p21, p27, p16, CDK2, Bax, Bcl-2, procaspase-9, procaspase-3, cleaved caspase-9 and caspase-3 proteins. The activity of caspase-9 and caspase-3 was measured using Caspase-Glo-9 and Caspase-Glo-3 assay. The results indicated that silymarin effectively suppressed cell growth in a dose- and time-dependent manner, and arrested cell cycle progression at G1/S phase in A2780s and PA-1 cells via up-regulation of p53, p21, and p27 protein expression, and down-regulation of CDK2 protein expression. Additionally, silymarin treatment for 24h at 50 and 100µg/ml resulted in a reduction of mitochondrial membrane potential and cytochrome C release, and significantly induced apoptosis in A2780s and PA-1 cells by increasing Bax and decreasing Bcl-2 protein expression, and activation of caspase-9 and caspase-3. Therefore, silymarin is a possible potential candidate for the prevention and treatment of ovarian cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cytochromes c; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; Humans; Membrane Potential, Mitochondrial; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; Silymarin; Tumor Suppressor Protein p53; Up-Regulation

Products of the SOX gene family play important roles in the life process. One of the members, SOX7, is associated with the development of a variety of cancers as a tumor suppression factor, but its relevance with ovarian cancer was unclear. In this study, we investigated the involvement of SOX7 in the progression and prognosis of epithelial ovarian cancer (EOC) and the involved mechanisms.. Expression profiles in two independent microarray data sets were analyzed for SOX7 between malignant and normal tissues. The expression levels of SOX7 in EOC, borderline ovarian tumors and normal ovarian tissues were measured by immunohistochemistry. We also measured levels of COX2 and cyclin-D1 to examine their possible involvement in the same signal transduction pathway as SOX7.. The expression of SOX7 was significantly reduced in ovarian cancer tissues compared with normal controls, strongly indicating that SOX7 might be a negative regulator in the Wnt/β-catenin pathway in ovarian cancer. By immunohistochemistry staining, the protein expression of SOX7 showed a consistent trend with that of the gene expression microarray analysis. By contrast, the protein expression level of COX2 and cyclin-D1 increased as the tumor malignancy progressed, suggesting that SOX7 may function through the Wnt/β-catenin signaling pathway as a tumor suppressor. In comparison between the protein expression levels of SOX7 with pathological features of the cancer, we found that SOX7 was down-regulated mainly in serous cystadenocarcinoma and advanced stages of the cancers.. The expression of SOX7 correlates with tumor progression as a tumor suppressor, possibly through the Wnt/β-catenin signaling pathway in ovarian cancers, suggesting that SOX7 may be a promising prognostic marker.

Topics: Adult; Aged; Cell Line, Tumor; Cyclin D1; Cyclooxygenase 2; Datasets as Topic; Disease Progression; Down-Regulation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Immunohistochemistry; Middle Aged; Neoplasm Grading; Neoplasm Staging; Ovarian Neoplasms; SOXF Transcription Factors; Wnt Signaling Pathway; Young Adult

We have previously demonstrated that Krüppel-like factor 8 (KLF8) participates in oncogenic transformation of mouse fibroblasts and is highly overexpressed in human ovarian cancer. In this work, we first correlated KLF8 overexpression with the aggressiveness of ovarian patient tumors and then tested if KLF8 could transform human ovarian epithelial cells. Using the immortalized non-tumorigenic human ovarian surface epithelial cell line T80 and retroviral infection, we generated cell lines that constitutively overexpress KLF8 alone or its combination with the known ovarian oncogenes c-Myc, Stat3c and/or Akt and examined the cell lines for anchorage-independent growth and tumorigenesis. The soft agar clonogenic assay showed that T80/KLF8 cells formed significantly more colonies than the mock cells. Interestingly, the cells expressing both KLF8 and c-Myc formed the largest amounts of colonies, greater than the sum of colonies formed by the cells expressing KLF8 and c-Myc alone. These results suggested that KLF8 might be a weak oncogene that works cooperatively with c-Myc to transform ovarian cells. Surprisingly, overexpression of KLF8 alone was sufficient to induce tumorigenesis in nude mice resulting in short lifespan irrespective of whether the T80/KLF8 cells were injected subcutaneously, intraperitoneally or orthotopically into the ovarian bursa. Histopathological studies confirmed that the T80/KLF8 tumors were characteristic of human serous ovarian carcinomas. Comparative expression profiling and functional studies identified the cell cycle regulators cyclin D1 and USP44 as primary KLF8 targets and effectors for the T80 transformation. Overall, we identified KLF8 overexpression as an important factor in human ovarian carcinoma pathogenesis.

Topics: Animals; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Kruppel-Like Transcription Factors; Mice; Mice, Nude; Neoplasm Transplantation; Ovarian Neoplasms; Ovary; Repressor Proteins; Ubiquitin Thiolesterase; Ubiquitin-Specific Proteases

MicroRNAs (miRNAs) are a small class of non‑coding RNAs that negatively regulate gene expression, and are considered as new therapeutic targets for treating cancer. In this study, we performed a gain-of-function screen using miRNA mimic library (319 miRNA species) to identify those affecting cell proliferation in human epithelial ovarian cancer cells (A2780). We discovered a number of miRNAs that increased or decreased the cell viability of A2780 cells. Pro-proliferative and anti-proliferative miRNAs include oncogenic miR-372 and miR-373, and tumor suppressive miR-124a, miR-7, miR-192 and miR-193a, respectively. We found that overexpression of miR-124a, miR-192, miR-193a and miR‑193b inhibited BrdU incorporation in A2780 cells, indicating that these miRNAs affected the cell cycle. Overexpression of miR‑193a and miR-193b induced an activation of caspase 3/7, and resulted in apoptotic cell death in A2780 cells. A genome‑wide gene expression analysis with miR-193a-transfected A2780 cells led to identification of ARHGAP19, CCND1, ERBB4, KRAS and MCL1 as potential miR-193a targets. We demonstrated that miR-193a decreased the amount of MCL1 protein by binding 3'UTR of its mRNA. Our study suggests the potential of miRNA screens to discover miRNAs as therapeutic tools to treat ovarian cancer.

Topics: 3' Untranslated Regions; Apoptosis; Carcinoma, Ovarian Epithelial; Cell Cycle; Cell Proliferation; Cyclin D1; ErbB Receptors; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; GTPase-Activating Proteins; Humans; MicroRNAs; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Receptor, ErbB-4; Transfection; Tumor Cells, Cultured

Tissue transglutaminase (TG2) is a multifunctional enzyme involved in protein cross-linking and cell adhesion to fibronectin (FN). In cancer, TG2 induces an epithelial to mesenchymal transition, contributing to metastasis. Because cadherins bind β-catenin at cell-cell junctions, disruption of adherens junctions destabilizes cadherin-catenin complexes. The goal of the present study was to analyze whether and how TG2 interacts with and regulates β-catenin signaling in ovarian cancer (OC) cells. We observed a significant correlation between TG2 and β-catenin expression levels in OC cells and tumors. TG2 augmented Wnt/β-catenin signaling, as evidenced by enhanced β-catenin transcriptional activity, inducing transcription of target genes cyclin D1 and c-Myc. By promoting integrin-mediated cell adhesion to FN, TG2 physically associates with and recruits c-Src, which in turn phosphorylates β-catenin at Tyr(654), releasing it from E-cadherin and rendering it available for transcriptional regulation. By interacting with FN and enhancing β-catenin signaling, complexed TG2 stimulates OC cell proliferation. In summary, our data demonstrate that TG2 regulates β-catenin expression and function in OC cells and define the c-Src-dependent mechanism through which this occurs.

Topics: beta Catenin; Blotting, Western; Cell Line, Tumor; Cell Proliferation; CSK Tyrosine-Protein Kinase; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Humans; Integrins; Microscopy, Confocal; Ovarian Neoplasms; Phosphorylation; Protein Binding; Protein Glutamine gamma Glutamyltransferase 2; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; src-Family Kinases; Transglutaminases

2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole (5F203, NSC 703786) lysylamide belongs to a novel mechanistic class of antitumor agents. It elicits activity against ovarian, breast, kidney and colorectal cancer models. In sensitive breast cancer cells, 5F203 activates aryl hydrocarbon receptor (AhR) signaling. Herein, we evaluate the role of AhR in 5F203 activity in two ovarian cancer cell lines: IGROV-1 (sensitive to 5F203), SKOV-3 (resistant to this agent). In addition, cancer cells have been isolated from ascites fluid of ovarian cancer patients; sensitivity to 5F203 and concurrent AhR signal transduction has been examined in ascites-isolated ovarian cancer patients' cells. 5F203 induced enhanced CYP1A1 expression, AhR translocation and ROS formation in IGROV-1 cells and ascites-isolated ovarian cancer cells that were sensitive to 5F203. In IGROV-1 cells 5F203-induced ROS formation was accompanied by JNK, ERK and P38MAPK phosphorylation, DNA damage and cell cycle arrest prior to apoptosis. In contrast, 5F203 failed to induce CYP1A1 expression, AhR translocation or oxidative stress in 5F203-resistant SKOV-3 cells, or in ovarian cancer ascites cells inherently resistant to this agent. We propose that AhR may represent a new molecular target in the treatment of ovarian tumors and 5F203 may exemplify a potential novel treatment. Furthermore, putative biomarkers of sensitivity to this agent have been identified.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Caspase 3; Cell Line, Tumor; Cells, Cultured; Cyclin D1; Cytochrome P-450 CYP1A1; DNA Damage; Female; Flow Cytometry; Humans; Microscopy, Confocal; Ovarian Neoplasms; Phosphorylation; Reactive Oxygen Species; Thiazoles

Endocrine disrupting chemicals (EDCs) and estrogens appear to promote development of estrogen-dependent cancers, including breast and ovarian carcinomas. In this study, we evaluated the cell viability effect of BPA on BG-1 human ovarian cancer cells, along with the growth inhibitory effect of resveratrol (trans-3,4,5-trihydroxystilbene; RES), a naturally occurring phytoestrogen. In addition, we investigated the underlying mechanism(s) of BPA and RES in regulating the interaction between estrogen receptor alpha (ERα) and insulin-like growth factor-1 receptor (IGF-1R) signals, a non- genomic pathway induced by 17β-estradiol (E2). BPA induced a significant increase in BG-1 cell growth and up-regulated mRNA levels of ERα and IGF-1R. In parallel with its mRNA level, the protein expression of ERα was induced, and phosphorylated insulin receptor substrate-1 (p-IRS-1), phosphorylated Akt1/2/3, and cyclin D1 were increased by BPA or E2. However, RES effectively reversed the BG-1 cell proliferation induced by E2 or BPA by inversely down-regulating the expressions of ERα, IGF-1R, p-IRS-1, and p-Akt1/2/3, and cyclin D1 at both transcriptional and translational levels. Taken together, these results suggest that RES is a novel candidate for prevention of tumor progression caused by EDCs, including BPA via effective inhibition of the cross-talk of ERα and IGF-1R signaling pathways.

Topics: Antineoplastic Agents, Phytogenic; Benzhydryl Compounds; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Down-Regulation; Estradiol; Estrogen Receptor alpha; Estrogens, Non-Steroidal; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin Receptor Substrate Proteins; Neoplasm Proteins; Ovarian Neoplasms; Phenols; Phosphorylation; Phytoestrogens; Protein Processing, Post-Translational; Receptor, IGF Type 1; Resveratrol; Signal Transduction; Stilbenes

The WAP four-disulfide core domain protein 2 (WFDC2) is frequently overexpressed in epithelial ovarian cancer cells and has been proposed as a potential biomarker. The biological function of WFDC2 in tumor progression remains unclear. In this study, the stable expression of short hairpin RNA (shRNA) against WFDC2 in the human ovarian SKOV3 cell line was established. Cell proliferation in vitro was determined by MTT assay. Cell cycle and apoptosis were analyzed by FACS. The expression of genes related to cell proliferation and survival was detected by real-time RT-PCR and western blotting. In vivo tumor growth assay was performed by establishing WFDC2-knockdown xenografts in nude mice and monitoring tumor growth. The expression of WFDC2, Ki67 and activated caspase-3 was analyzed by immunohistochemistry in order to determine the role of WFDC2 in proliferation and apoptosis. Our results revealed that the silencing of WFDC2 abolished ovarian cancer cell proliferation, suppressing tumor formation and growth in ovarian cancer cells both in vitro and in vivo. The knockdown of WFDC2 induced upregulation of Fasl and the downregulation of cyclin D1 activated caspase-3 and Ki67. These results indicate that WFDC2 plays a crucial role in tumor formation and growth in ovarian cancer cells. WFDC2 may be a potential therapeutic target for epithelial ovarian cancer.

Topics: Animals; Apoptosis; Blotting, Western; Caspase 3; Cell Cycle; Cell Differentiation; Cell Proliferation; Cyclin D1; Fas Ligand Protein; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Mice; Mice, Nude; Ovarian Neoplasms; Proteins; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured; WAP Four-Disulfide Core Domain Protein 2

Gene methylation and other epigenetic modifications of gene regulation have been implicated in the growth of ovarian cancer, but the clinical significance of such modifications in the Notch pathway in high-grade serous ovarian cancer (HGS-OvCa) is not well understood. We used The Cancer Genome Atlas (TCGA) data to study the clinical relevance of epigenetic modifications of Notch superfamily genes.. We analyzed the interaction of DNA methylation and miRNAs with gene expression data for Notch superfamily members with the Spearman rank correlation test and explored potential relationships with overall survival (OS) with the log-rank test. We downloaded clinical data, level 3 gene expression data, and level 3 DNA methylation data for 480 patients with stage II-IV HGS-OvCa from the TCGA data portal. Patients were randomly divided into training and validation cohorts for survival analyses. In each set, patients were grouped into percentiles according to methylation and microRNA (miRNA) or messenger RNA (mRNA) levels. We used several algorithms to predict miRNA-mRNA interaction.. There were significant inverse relationships between methylation status and mRNA expression for PPARG, CCND1, and RUNX1. For each of these genes, patients with a lower methylation level and higher expression level had significantly poorer OS than did patients with a higher methylation level and lower expression level. We also found a significant inverse relationship between miRNAs and mRNA expression for CCND1, PPARG, and RUNX1. By further analyzing the effect of miRNAs on gene expression and OS, we found that patients with higher levels of CCND1, PPARG, and RUNX1 expression and lower expression levels of their respective miRNAs (502-5p, 128, and 215/625) had significantly poorer OS.. Epigenetic alterations of multiple Notch target genes and pathway interacting genes (PPARG, CCND1, and RUNX1) may relate to activation of this pathway and poor survival of patients with HGS-OvCa.

Topics: Core Binding Factor Alpha 2 Subunit; Cyclin D1; Cystadenocarcinoma, Serous; DNA Methylation; Epigenomics; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Ovarian Neoplasms; PPAR gamma; Receptors, Notch; RNA, Messenger; Survival Analysis

Despite the fundamental pathophysiological importance of β-catenin in tumor progression, the mechanism underlying its final transcriptional output has been partially elucidated. Here, we report that β-arrestin-1 (β-arr1) is an epigenetic regulator of endothelin (ET)-1-induced β-catenin signaling in epithelial ovarian cancer (EOC). In response to ET A receptor (ETAR) activation by ET-1, β-arr1 increases its nuclear translocation and direct binding to β-catenin. This in turn enhanced β-catenin nuclear accumulation and transcriptional activity, which was prevented by expressing a mutant β-arr1 incapable of nuclear distribution. β-arr1-β-catenin interaction controls β-catenin target gene expressions, such as ET-1, Axin 2, Matrix metalloproteinase 2, and Cyclin D1, by promoting histone deacetylase 1 (HDAC1) dissociation and the recruitment of p300 acetyltransferase on these promoter genes, resulting in enhanced H3 and H4 histone acetylation, and gene transcription, required for cell migration, invasion and epithelial-to-mesenchymal transition. These effects are abrogated by β-arr1 silencing or by mutant β-arr1, as well as by β-catenin or p300 silencing, confirming that nuclear β-arr1 forms a functional complex capable of regulating epigenetic changes in β-catenin-driven invasive behavior. In a murine orthotopic model of metastatic human EOC, silencing of β-arr1 or mutant β-arr1 expression, as well as ETAR blockade, inhibits metastasis. In human EOC tissues, β-arr1-β-catenin nuclear complexes are selectively enriched at β-catenin target gene promoters, correlating with tumor grade, confirming a direct in vivo β-arr1-β-catenin association at specific set of genes involved in EOC progression. Collectively, our study provides insights into how a β-arr1-mediated epigenetic mechanism controls β-catenin activity, unraveling new components required for its nuclear function in promoting metastasis.

Topics: Animals; Arrestins; Axin Protein; beta Catenin; beta-Arrestin 1; beta-Arrestins; Carcinoma, Ovarian Epithelial; Cell Nucleus; Cyclin D1; Endothelin-1; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase 1; Histones; Humans; Matrix Metalloproteinase 2; Mice, Nude; Mutation; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Promoter Regions, Genetic; Protein Transport; Receptor, Endothelin A; Signal Transduction; Xenograft Model Antitumor Assays

2,4-Dihydroxybenzophenone (benzophenone-1; BP-1) is an UV stabilizer primarily used to prevent polymer degradation and deterioration in quality due to UV irradiation. Recently, BP-1 has been reported to bioaccumulate in human bodies by absorption through the skin and has the potential to induce health problems including endocrine disruption. In the present study, we examined the xenoestrogenic effect of BP-1 on BG-1 human ovarian cancer cells expressing estrogen receptors (ERs) and relevant xenografted animal models in comparison with 17-β estradiol (E2). In in vitro cell viability assay, BP-1 (10(-8)-10(-5)M) significantly increased BG-1 cell growth the way E2 did. The mechanism underlying the BG-1 cell proliferation was proved to be related with the up-regulation of cyclin D1, a cell cycle progressor, by E2 or BP-1. Both BP-1 and E2 induced cell growth and up-regulation of cyclin D1 were reversed by co-treatment with ICI 182,780, an ER antagonist, suggesting that BP-1 may mediate the cancer cell proliferation via an ER-dependent pathway like E2. On the other hand, the expression of p21, a regulator of cell cycle progression at G1 phase, was not altered by BP-1 though it was down-regulated by E2. In xenograft mouse models transplanted with BG-1 cells, BP-1 or E2 treatment significantly increased the tumor mass formation compared to a vehicle (corn oil) within 8 weeks. In histopathological analysis, the tumor sections of E2 or BP-1 group displayed extensive cell formations with high density and disordered arrangement, which were supported by the increased number of BrdUrd positive nuclei and the over-expression of cyclin D1 protein. Taken together, these results suggest that BP-1 is an endocrine disrupting chemical (EDC) that exerts xenoestrogenic effects by stimulating the proliferation of BG-1 ovarian cancer via ER signaling pathway associated with cell cycle as did E2.

Topics: Animals; Antimetabolites; Benzophenones; Blotting, Western; Bromodeoxyuridine; Cell Cycle; Cell Proliferation; Coloring Agents; Cyclin D1; Estrogen Receptor alpha; Female; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Immunohistochemistry; Mice; Mice, Inbred BALB C; Ovarian Neoplasms; p21-Activated Kinases; Pregnancy; Real-Time Polymerase Chain Reaction; RNA, Neoplasm; Signal Transduction; Stimulation, Chemical; Xenograft Model Antitumor Assays

Adenosine is a regulatory molecule with widespread physiological effects in almost every cells and acts as a potent regulator of cell growth. Adenosine has been shown to inhibit cell growth and induce apoptosis in the several cancer cells via caspase activation and Bcl-2/Bax pathway. The present study was designed to understand the mechanism underlying adenosine-induced apoptosis in the OVCAR-3 human ovarian cancer cells. MTT viability, BrdU and cell counting assays were used to study the cell proliferation effect of adenosine in presence of adenosine deaminase inhibitor and the nucleoside transporter inhibitor. Cell cycle analysis, propidium iodide and annexin V staining, caspase-3 activity assay, cyclinD1, Cdk4, Bcl-2 and Bax protein expressions were assessed to detect apoptosis. Adenosine significantly inhibited cell proliferation in a concentration-dependent manner in OVCAR-3 cell line. Adenosine induced cell cycle arrest in G0/G1 phase via Cdk4/cyclinD1-mediated pathway. Adenosine induced apoptosis, which was determined by Annexin V-FITC staining and increased sub-G1 population. Moreover, down-regulation of Bcl-2 protein expression, up-regulation of Bax protein expression and activation of caspase-3 were observed in response to adenosine treatment. The results of this study suggest that extracellular adenosine induced G1 cell cycle arrest and apoptosis in ovarian cancer cells via cyclinD1/ Cdk4 and Bcl-2/Bax pathways and caspase-3 activation. These data might suggest that adenosine could be used as an agent for the treatment of ovarian cancer.

Topics: Adenosine; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Cycle; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Female; Flow Cytometry; Humans; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Vasodilator Agents

The role of estrogen in the growth and survival of ovarian cancer cells is controversial. In this study, we investigated the changes in cell-cycle regulatory proteins in ovarian cancer cell lines after estrogen treatment to explore the role of estrogen in ovarian cancers.. Two ovarian adenocarcinoma cell lines were used for the study: the first, OC-117-VGH, was deficient in estrogen receptors (ER)α and ERβ, and the second, OVCAR3, was positive for ERα and ERβ. Serial concentrations of estrogen were used to evaluate the effects of estrogen on the survival of ovarian cancer cells. The cell-cycle regulatory proteins, including cyclin D1, cyclin E, p16/INK4a, and p27/KIP1, were used to check the possible mechanism of an estrogen effect on survival of the cancer cell line.. Estrogen 0.01-1.0 μM inhibited the growth of both cell lines. There were no differences in cyclin D1 and E expression between the two cell lines after estrogen treatment, but the expression of p16/INK4a and p27/KIP1 was significantly higher in the OC-1170-VGH cell line than in the OVCAR3 cell line.. Although the ER-positive and ER-negative ovarian cancer cell lines were inhibited by estrogen, the influence of cell-cycle regulatory proteins was different between the two, suggesting that the inhibitory effect of estrogen on ovarian cancer cell lines might be mediated through different pathways.

Topics: Adenocarcinoma; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens; Female; Humans; Neoplasm Proteins; Ovarian Neoplasms

Obesity is known to be an important risk factor for many types of cancer, such as breast, prostate, liver and endometrial cancer. Recently, epidemiological studies have indicated that obesity correlates with an increased risk of developing ovarian cancer, the most lethal gynecological cancer in developed countries. Leptin is predominantly produced by adipocytes and acts as a growth factor and serum leptin levels positively correlate with the amount of body fat. In this study, we investigated the effects of leptin on the growth of ovarian cancer cells and the underlying mechanism(s) of action. Our results showed that leptin stimulated the growth of the OVCAR-3 ovarian cancer cell line using MTT assay and trypan blue exclusion. Using western blot analysis, we found that leptin enhanced the expression of cyclin D1 and Mcl-1, which are important regulators of cell proliferation and the inhibition of apoptosis. To investigate the signaling pathways that mediate the effects of leptin, cells were treated with leptin plus specific inhibitors of JAK2, PI3K/Akt and MEK/ERK1/2 and analysis of the phosphorylation state of proteins was carried out by western blot assays. We showed that the activation of the MEK/ERK1/2 and PI3K/Akt signaling pathways were involved in the growth-stimulating effect of leptin on ovarian cancer cell growth and the specific inhibitors of PI3K/Akt and MEK/ERK1/2 revealed that these two pathways interacted with each other. Our data demonstrate that leptin upregulates the expression of cyclin D1 and Mcl-1 to stimulate cell growth by activating the PI3K/Akt and MEK/ERK1/2 pathways in ovarian cancer.

Topics: Apoptosis; Butadienes; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Flavonoids; Humans; Janus Kinase 2; Leptin; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Myeloid Cell Leukemia Sequence 1 Protein; Nitriles; Obesity; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Tyrphostins

The expression of programmed cell death 6 (PDCD6) is known to be down-regulated in cancer cell lines and ovarian cancer tissues compared to normal cells and tissues. In the current study, we characterized the specific function of PDCD6 as a novel pro-apoptotic protein. To define the roles of PDCD6 and cisplatin in tumorigenesis, we either over-expressed PDCD6 or treated it with cisplatin in SKOV-3 ovarian cancer cells. Both PDCD6 and cisplatin respectively inhibited cancer cell proliferation in a dose-dependent manner. The combined treatment of PDCD6 and cisplatin was more effective at suppressing cell growth than with either drug treatment alone, but had no effect with the treatment of caspase-3 and caspase-9 inhibitors. Cleavages of caspase-3, -8, -9, and poly (ADP-ribose) polymerase (PARP) in PDCD6-overexpressing cells were significantly increased after cisplatin treatment. Cell cycle analysis highly correlated with down-regulation of cyclin D1 and CDK4, and the induction of p16 and p27 as a cyclin-dependent kinase inhibitor. Additionally, PDCD6 also suppressed the phosphorylation of signaling regulators downstream of PI3K, including PDK1 and Akt. PDCD6 promotes TNFα-dependent apoptosis through the activation of NF-κB signaling pathways, increasing Bax, p53, and p21 expression, while also down-regulating Bcl-2 and Bcl-xL expression. The p21 and p53 promoter luciferase activities were enhanced by PDCD6, while there was no affect in p53(-/-) and p21(-/-). At the same time, p53 activity was confirmed by UV irradiation and siPDCD6. Taken together, these results provide evidence that PDCD6 can mediate the pro-apoptotic activity of cisplatin or TNFα through the down-regulation of NF-κB expression.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Calcium-Binding Proteins; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Cell Cycle Proteins; Cell Line, Tumor; Cisplatin; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Activation; Female; Humans; NF-kappa B; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Poly(ADP-ribose) Polymerases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Signal Transduction; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

To investigate the effects and possible molecular mechanisms of inhibiting FOXM1 expression on SKOV3 cells by lentiviral vector targeting FOXM1 shRNA.. SKOV3 cells were infected by lentiviral vector targeting FOXM1 shRNA with a multiplicity of infection (MOI) of 20, then growth curve of SKOV3 cells was determined by MTT assay, cell cycle was analysed by flow cytometry(FCM), and the expression of mRNA and protein of FOXM1, Cyclin D1, PLK1 by Real time PCR and Western blot.. Lentiviral vector targeting FOXM1 shRNA with a multiplicity of infection (MOI) of 20 could significantly inhibit the growth of SKOV3 cells. After infected by lentiviral vector targeting FOXM1 shRNA, the G(0);/G(1); phase cells increased and the S-phase cells decreased, and the expression of mRNA and protein of FOXM1, Cyclin D1, PLK1 of SKOV3 cells were significantly down-regulated.. Inhibiting FOXM1 expression has a significantly effect of inhibiting proliferation on SKOV3 cells. Blocking SKOV3 cells in the G(0);/G(1); phase by down-regulating the expression of Cyclin D1, PLK1 protein may be its mechanism.

Topics: Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Lentivirus; Ovarian Neoplasms; Polo-Like Kinase 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; RNA, Small Interfering; Transduction, Genetic

Phytochemicals constitute a heterogeneous group of substances with an evident role in human health. Their properties on cancer initiation, promotion and progression are well documented. Particular attention is now devoted to better understand the molecular basis of their anticancer action. In the present work, we studied the effect of resveratrol on the ovarian cancer cell line OVCAR-3 by a proteomic approach. Our findings demonstrate that resveratrol down-regulates the protein cyclin D1 and, in a concentration dependent manner, the phosphorylation levels of protein kinase B (Akt) and glycogen synthase kinase-3β (GSK-3β). The dephosphorylation of these kinases could be responsible for the decreased cyclin D1 levels observed after treatment. We also showed that resveratrol reduces phosphorylation levels of the extracellular signal-regulated kinase (ERK) 1/2. Chemical inhibitors of phosphatidylinositol 3-kinase (PI3K) and ERK both increased the in vitro therapeutic efficacy of resveratrol. Moreover, resveratrol had an inhibitory effect on the AKT phosphorylation in cultured cells derived from the ascites of ovarian cancer patients and in a panel of human cancer cell lines. Thus, resveratrol shows antitumor activity in human ovarian cancer cell lines targeting signalling pathway involved in cell proliferation and drug-resistance.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Microscopy, Confocal; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Proteomics; Proto-Oncogene Proteins c-akt; Resveratrol; Signal Transduction; Stilbenes

To investigate the effect of Lewis y overexpression on the expression of proliferation-related factors in ovarian cancer cells.. mRNA levels of cyclins, CDKs, and CKIs were measured in cells before and after transfection with the α1,2-fucosyltransferase gene by real-time PCR, and protein levels of cyclins, CDKs and CKIs were determined in cells before and after gene transfection by Western blot.. Lewis y overexpression led to an increase in both mRNA and protein expression levels of cyclin A, cyclin D1 and cyclin E in ovarian cancer cells, decrease in both mRNA and protein expression levels of p16 and p21, and decrease of p27 at only the protein expression level without change in its mRNA level. There were no differences in proteins and the mRNA levels of CDK2, CDK4 and CDK6 before and after gene transfection. Anti-Lewis y antibody, ERK and PI3K pathway inhibitors PD98059 and LY294002 reduced the difference in cyclin and CKI expression caused by Lewis y overexpression.. Lewis y regulates the expression of cell cycle-related factors through ERK/MAPK and PI3K/Akt signaling pathways to promote cell proliferation.

Topics: Antibodies; Cell Line, Tumor; Chromones; Cyclin A; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Extracellular Signal-Regulated MAP Kinases; Female; Flavonoids; Fucosyltransferases; Humans; Lewis Blood Group Antigens; Morpholines; Ovarian Neoplasms; Proto-Oncogene Proteins c-akt; RNA, Messenger; Signal Transduction

To explore the expression of cyclinD1 in ovarian carcinoma cell 3AO and analyze its relationship with the proliferation of ovarian cancer cell.. Human ovarian epithelium and ovarian cancer cells 3AO were cultured in vitro. CyclinD1 genes and proteins were detected by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry before and after the activation of cell 3AO by cis-platinum. Also the cell activity and cell cycle were observed.. Abnormal gene amplification and over-expressions of cyclinD1 were found in ovarian cancer cell while the expression of cyclinD1 was negative in normal ovarian epithelium. Under cis-platinum, different expressions of cyclinD1 genes were found in 3AO by RT-PCR. The higher the concentrations of cisplatin, the lower expressions of cyclinD1 genes. By flow cytometry, it was also found that there were lower expressions of cyclinD1 protein in 3AO under cisplatin than without it. With the rising concentrations of cisplatin, the low expressions of cyclinD1 protein in 3AO were detected. The mean numbers were 105.9, 15.42 and 8.59, the cell apoptotic rates 0.63%, 9.08% and 27.41% and the proliferation index (PI) numbers 38.83%, 44.54%, 37.31%. The differences were statistically significant (P < 0.01).. The up-regulation of cyclinD1 is detected in ovarian cancer cell. A positive correlation is found between the lower expressions of cyclinD1 and the concentration of cisplatin. There is a close relationship between the expressions of cyclinD1 and ovarian cancer cell activity.

Topics: Carcinoma, Ovarian Epithelial; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms

The ubiquitin-proteasome system (UPS) is usurped by many if not all cancers to regulate their survival, proliferation, invasion, angiogenesis and metastasis. Bioflavonoids curcumin and chalcones exhibit anti-neoplastic selectivity through inhibition of the 26S proteasome-activity within the UPS. Here, we provide evidence for a novel mechanism of action of chalcone-based derivatives AM146, RA-9 and RA-14, which exert anticancer activity by targeting deubiquitinating enzymes (DUB) without affecting 20S proteasome catalytic-core activity. The presence of the α,β-unsaturated carbonyl group susceptible to nucleophilic attack from the sulfhydryl of cysteines in the active sites of DUB determines the capacity of novel small-molecules to act as cell-permeable, partly selective DUB inhibitors and induce rapid accumulation of polyubiquitinated proteins and deplete the pool of free ubiquitin. These chalcone-derivatives directly suppress activity of DUB UCH-L1, UCH-L3, USP2, USP5 and USP8, which are known to regulate the turnover and stability of key regulators of cell survival and proliferation. Inhibition of DUB-activity mediated by these compounds downregulates cell-cycle promoters, e.g., cyclin D1 and upregulates tumor suppressors p53, p27(Kip1) and p16(Ink4A). These changes are associated with arrest in S-G 2/M, abrogated anchorage-dependent growth and onset of apoptosis in breast, ovarian and cervical cancer cells without noticeable alterations in primary human cells. Altogether, this work provides evidence of antitumor activity of novel chalcone-based derivatives mediated by their DUB-targeting capacity; supports the development of pharmaceuticals to directly target DUB as a most efficient strategy compared with proteasome inhibition and also provides a clear rationale for the clinical evaluation of these novel small-molecule DUB inhibitors.

Topics: Antineoplastic Agents; Apoptosis; Benzylidene Compounds; Boronic Acids; Bortezomib; Breast Neoplasms; Catalytic Domain; Cell Cycle Checkpoints; Cell Proliferation; Cell Survival; Chalcone; Cyclin D1; Cysteine; Dose-Response Relationship, Drug; Endopeptidases; Female; HeLa Cells; Humans; Ovarian Neoplasms; Phenotype; Piperidones; Protease Inhibitors; Proteasome Endopeptidase Complex; Pyrazines; Tumor Suppressor Protein p53; Ubiquitin Thiolesterase; Ubiquitins

It has been shown that IL-8 is elevated in ovarian cyst fluid, ascites, serum, and tumor tissue from ovarian cancer (OVCA) patients, and increased IL-8 expression correlates with poor prognosis and survival. However, the exact role that IL-8 plays in this malignancy or whether IL-8 can regulate malignant behavior has not been established. Here we demonstrate that overexpression of IL-8 in non-IL-8-expressing A2780 cells (by transfecting with plasmid encoding for sense IL-8) increases anchorage-independent growth, proliferation, angiogenic potential, adhesion and invasion while depletion of endogenous IL-8 expression in IL-8-overexpressing SKOV-3 cells (by transfecting with plasmid encoding for antisense IL-8) decreases the above effects. Further investigation indicates that IL-8-stimulated cell proliferation correlates with alteration of cell cycle distribution by increasing levels of cell cycle-regulated Cyclin D1 and Cyclin B1 proteins as well as activation of PI3K/Akt and Raf/MEK/ERK, whereas IL-8-enhanced OVCA cell invasive correlates with increased MMP-2 and MMP-9 activity and expression. Our data suggest that IL-8 secreted by OVCA cells promotes malignant behavior of these cells via inducing intracellular molecular signaling. Therefore, modulation of IL-8 expression or its related signaling pathway may be a promising strategy for controlling the progression and metastasis of OVCA.

Topics: Cell Adhesion; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin B1; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neovascularization, Pathologic; Ovarian Neoplasms; Phenotype; RNA, Messenger; Transfection; Vascular Endothelial Growth Factor A

Paired-like homeodomain 2 (PITX2) is a bicoid homeodomain transcription factor which plays an essential role in maintaining embryonic left-right asymmetry during vertebrate embryogenesis. However, emerging evidence suggests that the aberrant upregulation of PITX2 may be associated with tumor progression, yet the functional role that PITX2 plays in tumorigenesis remains unknown.. Using real-time quantitative RT-PCR (Q-PCR), Western blot and immunohistochemical (IHC) analyses, we demonstrated that PITX2 was frequently overexpressed in ovarian cancer samples and cell lines. Clinicopathological correlation showed that the upregulated PITX2 was significantly associated with high-grade (P = 0.023) and clear cell subtype (P = 0.011) using Q-PCR and high-grade (P<0.001) ovarian cancer by IHC analysis. Functionally, enforced expression of PITX2 could promote ovarian cancer cell proliferation, anchorage-independent growth ability, migration/invasion and tumor growth in xenograft model mice. Moreover, enforced expression of PITX2 elevated the cell cycle regulatory proteins such as Cyclin-D1 and C-myc. Conversely, RNAi mediated knockdown of PITX2 in PITX2-high expressing ovarian cancer cells had the opposite effect.. Our findings suggest that the increased expression PITX2 is involved in ovarian cancer progression through promoting cell growth and cell migration/invasion. Thus, targeting PITX2 may serve as a potential therapeutic modality in the management of high-grade ovarian tumor.

Topics: Animals; beta Catenin; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Genes, myc; Homeobox Protein PITX2; Homeodomain Proteins; Humans; Mice; Neoplasm Invasiveness; Ovarian Neoplasms; Transcription Factors; Up-Regulation; Wnt Proteins

δ-Catenin is found to be involved in the progression of several human cancers. However, its expression pattern and biological roles in human ovarian cancers are not clear. In this study, we examined the expression pattern of δ-catenin in 149 ovarian cancer specimens. We also depleted and overexpressed δ-catenin expression in ovarian cancer cell lines and investigated its role in cell proliferation and invasion.. δ-Catenin expression was analyzed in 149 archived ovarian cancer specimens using immunohistochemistry. siRNA knockdown and plasmid transfection were performed in SKOV3, SW626, and OVCAR3 cell lines. MTT, colony formation assay, soft agar colony assay, and matrigel invasion assay were carried out to assess the role of δ-catenin in cell proliferation and invasion. We also performed cell cycle analysis in δ-catenin depleted and overexpressed cells. In addition, we examined the level of several cell cycle-related molecules using Western blot.. Of the 149 patients in the study, 104 (69.7 %) showed δ-catenin overexpression. δ-catenin overexpression positively correlated with advanced FIGO stage. δ-Catenin depletion in ovarian cancer cell lines inhibited ovarian cancer cell proliferation and invasion. Depletion of δ-catenin also blocked cell cycle progression and downregulated cyclin D1 expression in ovarian cancer cells. Overexpression of δ-catenin enhanced cell proliferation, invasion, and upregulated cyclinD1 expression.. δ-Catenin is overexpressed in ovarian cancers and associated with advanced stage. Our data provide evidence that δ-catenin regulates the ovarian cancer cell proliferation, invasion, and cell cycle. δ-Catenin thus has potential as a therapeutic target.

Topics: Adult; Aged; Catenins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Delta Catenin; Down-Regulation; Female; Humans; Middle Aged; Neoplasm Invasiveness; Ovarian Neoplasms; Up-Regulation; Young Adult

BRCA1 plays an important role in DNA damage and repair, homologous recombination, cell-cycle regulation and apoptosis. BRCA-mutated ovarian cancer often presents at an advanced stage, however, tend to have better response to platinum-based chemotherapy as compared with sporadic cases of epithelial ovarian cancer (EOC). In spite of this, most patients will develop a recurrence and eventually succumb to the disease. Preclinical studies are currently investigating natural compounds and their analogs for tumor-directed targets in ovarian cancer. The aim of this study is to investigate whether the STAT3 inhibitor HO-3867, a novel curcumin analog, has a therapeutic effect on BRCA1-mutated ovarian cancer. Our novel agent, HO-3867 and a commercial STAT3 inhibitor, STATTIC, significantly inhibited BRCA-mutated ovarian cancer cells in vitro in a dose- and time-dependent manner. BRCA-mutated ovarian cancer cells treated with HO-3867 exhibited a significant degree of apoptosis with elevated levels of cleaved caspase-3, caspase-7 and PARP. HO-3867 treatment induced more reactive oxygen species (ROS) in BRCA-mutated cells compared with wild-type cells, however, there was no increased ROS when benign ovarian surface epithelial cells were treated with HO-3867. BRCA1-mutated cancer cells had higher expression of Tyrosine-phosphorylated STAT3 (pTyr705) as compared with other STAT proteins. Furthermore, treatment of these cells with HO-3867 resulted in decreased expression of pTyr705 and its downstream targets cyclin D1, Bcl-2 and survivin. In addition, overexpression of STAT3 cDNA provided resistance to HO-3867-induced apoptosis. Our results show that HO-3867, a potent STAT3 inhibitor, may have a role as a biologically targeted agent for BRCA1-mutated cancers either as an adjunct to cytotoxic chemotherapy or as a single agent.

Topics: Antineoplastic Agents; Apoptosis; BRCA1 Protein; BRCA2 Protein; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclic S-Oxides; Cyclin D1; Drug Screening Assays, Antitumor; Female; Humans; Ovarian Neoplasms; Phosphorylation; Piperidones; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; STAT3 Transcription Factor

The present study was designed to investigate the effects of cyclooxygenase (COX) inhibitors in combination with taxol on the expression of cyclin D1 and Ki-67 in human ovarian SKOV-3 carcinoma cells xenograft-bearing mice. The animals were treated with 100 mg/kg celecoxib (a COX-2 selective inhibitor) alone, 3 mg/kg SC-560 (a COX-1 selective inhibitor) alone by gavage twice a day, 20 mg/kg taxol alone by intraperitoneally (i.p.) once a week, or celecoxib/taxol, SC-560/celecoxib, SC-560/taxol or SC-560/celecoxib/taxol, for three weeks. To test the mechanism of the combination treatment, the index of cell proliferation and expression of cyclin D1 in tumor tissues were determined by immunohistochemistry. The mean tumor volume in the treated groups was significantly lower than control (p < 0.05), and in the three-drug combination group, tumor volume was reduced by 58.27% (p < 0.01); downregulated cell proliferation and cyclin D1 expression were statistically significant compared with those of the control group (both p < 0.01). This study suggests that the effects of COX selective inhibitors on the growth of tumors and decreased cell proliferation in a SKOV-3 cells mouse xenograft model were similar to taxol. The three-drug combination showing a better decreasing tendency in growth-inhibitory effect during the experiment may have been caused by suppressing cyclin D1 expression.

Topics: Animals; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Celecoxib; Cell Proliferation; Cyclin D1; Cyclooxygenase Inhibitors; Female; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Mice; Mice, Nude; Ovarian Neoplasms; Paclitaxel; Pyrazoles; Sulfonamides; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Ovarian epithelial cancer is the leading cause of death among gynecological malignancies. FSH may increase the risk of ovarian malignancy and play an important role in ovarian carcinogenesis. Our previous studies showed that FSH increases the expression of VEGF through survivin. In this study, the function and mechanism of FSH in ovarian cancer were further explored. We found that FSH promoted proliferation and prevented apoptosis of ovarian cancer cells by activating survivin through the SAPK/JNK and PI3K/AKT pathways. FSH also down-regulated the expression of programmed cell death gene 6 (PDCD6) and death receptor 5 (DR5), two molecules required for induction of apoptosis. RNA interference was applied to knock down survivin and PDCD6 expression, and we found that the blockage of survivin reversed the effects of FSH on apoptosis and proliferation, whereas knock down of PDCD6 enhanced these effects. The expression of DR5, cyclin D1, and cyclin E correlated with survivin expression, but PDCD6 did not. Using immunohistochemical staining, we further showed that ovarian serous cystadenocarcinoma samples had higher expression of survivin than did benign ovarian cystadenoma and borderline cystadenoma samples (P<0.01). Furthermore, survivin expression in the ovarian serous cystadenocarcinoma specimens was correlated with disease stage (P<0.05). Our results suggest that FSH promotes ovarian cancer development by regulating the expression of survivin, PDCD6, and DR5. Greater understanding of the molecular mechanisms of FSH in ovarian epithelial carcinogenesis and development will ultimately help in the development of a novel targeted therapy for ovarian cancer.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Calcium-Binding Proteins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Female; Follicle Stimulating Hormone; Humans; Inhibitor of Apoptosis Proteins; JNK Mitogen-Activated Protein Kinases; Mice; Mice, Inbred BALB C; Microtubule-Associated Proteins; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; Survivin

OVCA1, a tumor suppressor gene, is deleted or lower expressed in about 80% of ovarian cancer. Over expression of OVCA1 in human ovarian cancer A2780 cells inhibits cell proliferation and arrests cells in G1 stage. However, the fact that the molecular mechanism of OVCA1 inhibits cell growth is presently elusive. Here we investigated the potential signaling pathway induced by over-expression of OVCA1. Our results show that over-expression of human OVCA1 in ovarian cancer cells A2780 leads to down-regulation of cyclin D1, and up-regulation of p16, but no effect on the expression of NF-κB. It indicates that OVCA1 could inhibit the proliferation of ovarian cancer cell A2780 by p16/cyclin D1 pathway, but not by NF-κB.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Minor Histocompatibility Antigens; Neoplasm Proteins; NF-kappa B; Ovarian Neoplasms; Recombinant Fusion Proteins; Transcription, Genetic; Tumor Suppressor Proteins

Overexpression of the HER2 oncogene contributes to tumor cell invasion, metastasis and angiogenesis and correlates with poor prognosis. Magnolol has been reported to exhibit anti-tumor activities. However, the molecular mechanism of action of magnolol has not been investigated in HER2-positive cancer cells. Therefore, we examined the anti-cancer effects of magnolol on HER2-overexpressing ovarian cancer cells. Magnolol treatment caused a dose-dependent inhibition of HER2 gene expression at the transcriptional level, potentially in part through suppression of NF-κB activation. Treatment of HER2-overexpressing ovarian cancer cells with magnolol down-regulated the HER2 downstream PI3K/Akt signaling pathway, and suppressed the expression of downstream target genes, vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP2) and cyclin D1. Consistently, magnolol-mediated inhibition of MMP2 activity could be prevented by co-treatment with epidermal growth factor. Migration assays revealed that magnolol treatment markedly reduced the motility of HER2-overexpressing ovarian cancer cells. Furthermore, magnolol-induced apoptosis in HER2-overexpressing ovarian cancer cells was characterized by the up-regulation of cleaved poly(ADP-ribose) polymerase (PARP) and activated caspase 3. These findings suggest that magnolol may act against HER2 and its downstream PI3K/Akt/mTOR-signaling network, thus resulting in suppression of HER2-mediated transformation and metastatic potential in HER2-overexpressing ovarian cancers. These results provide a novel mechanism to explain the anti-cancer effect of magnolol.

Topics: Biphenyl Compounds; Caspase 3; Cell Growth Processes; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Genes, erbB-2; Humans; Lignans; Neoplasm Metastasis; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Signal Transduction; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

Ovarian clear cell adenocarcinoma (OCCA) is an aggressive ovarian malignancy with a poor prognosis. The role of estrogen receptor β (ERβ) in the development of OCCA remains to be clarified. To investigate the action of ERβ in the proliferation and invasion of OCCA cells, the ES-2 cell line was stably transfected with full-length human ERβ cDNA, and clones were screened and identified using RT-PCR and western blot assay. ERβ stable transfectants, referred to as ESβ1 and ESβ2 cells, were compared with mock transfectant ESVE and parental ES-2 cells with respect to their growth, motility and ability to activate target genes. ESβ1 and ESβ2 cells expressed ERβ mRNA and protein, whereas ES-2 and ESVE cells were ERβ negative. ERβ transfectants exhibited distinct characteristics from ES-2 and ESVE cells including proliferative properties and the ability to express cyclin D1 in the presence of 17β-estradiol (E2). ERβ inhibited ES-2 cell proliferation, which was determined using the MTT assay, BrdU labeling method and by the down-regulation of cyclin D1 gene expression. Moreover, exogenous ERβ expression resulted in a significant inhibition of ES-2 cell motility in an in vitro invasion assay. ERβ reduced the expression of MMP2 mRNA and the activity of MMP2 enzymatic activity in a ligand-dependent manner. In summary, ERβ may inhibit the proliferation and invasion of ES-2 cells and may be an important regulator in OCCA carcinogenesis.

Topics: Adenocarcinoma, Clear Cell; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Estrogen Receptor beta; Female; Gene Expression; Humans; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Ovarian Neoplasms; Recombinant Proteins

To investigate the expression of the nucleostemin (NS) gene in ovarian tumors and its correlation with the expression of cyclin D1.. The expression levels of nucleostemin and cyclin D1 proteins were measured by immunohistochemical staining in ovarian tumors and normal ovarian tissues, and the relationship between their levels was analyzed.. Nucleostemin gene and cyclin D1 expressions were detected in 28 and 27 specimens of malignant ovarian tumors (93.7% and 90.0%), 4 and 8 specimens of ovarian borderline tumors (40.0% and 80.0%), and in both specimens of benign ovarian tumors, respectively. The expression of NS gene was seen in 5, 13, and 10 specimens of malignant ovarian tumors of high, moderate, and low grade, respectively. The expression of the nucleostemin gene was significantly related to the tumor grade (r = 0.786, P < 0. 05), and a significant relationship between nucleostemin and cyclin D1 expression was found (r = 0.834, P < 0.05). The expression of the nucleostemin gene was not detected in normal ovarian tissue.. The expression of the nucleostemin gene in ovarian tumors is closely correlated with origination, progression, and grading of tumors and may serve as a marker in estimating the malignancy of ovarian tumors. The overexpression of cyclin D1 might be correlated with nucleostemin expression. Nucleostemin may have an impact on the passage of cells through the G1/S checkpoint, and thus cell cycle progress, by regulating cyclin D1 expression.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma; Cyclin D1; Female; GTP-Binding Proteins; Humans; Middle Aged; Nuclear Proteins; Ovarian Neoplasms; Ovary; Young Adult

Mechanisms of human Vγ2Vδ2 T cell-mediated tumor immunity have yet to be fully elucidated.. At least some tumor cell recognition is mediated by NKG2D-MICA interactions. Herein, by using MTT assay and PI-BrdU co-staining and Western-blot, we show that these Vγ2Vδ2 T cells can limit the proliferation of ovarian tumor cells by down regulation of apoptosis and cell cycle related molecules in tumor cells. Cell-to-cell contact is critical. γδ T cell-resistant, but not susceptible ovarian tumor cells escape γδ T cell-mediated immune recognition by up-regulating pErk1/2, thereby decreasing surface MICA levels. Erk1/2 inhibitor pretreatment or incubation prevents this MICA decrease, while up-regulating key cell cycle related molecules such as CDK2, CDK4 and Cyclin D1, as well as apoptosis related molecules making resistant tumor cells now vulnerable to γδ T cell-mediated lysis.. These findings demonstrate novel effects of γδT cells on ovarian tumor cells.

Topics: Apoptosis; Cell Communication; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Down-Regulation; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Histocompatibility Antigens Class I; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Ovarian Neoplasms; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocytes; Up-Regulation

To evaluate the prognostic relevance of key cell cycle regulatory proteins p53, p16(INK4a), pRb and Cyclin D1 expression, the presence of high risk HPVs and their association with clinicopathological parameters and the clinical follow up in ovarian cancer patients.. 53 cases of primary ovarian serous carcinomas were immunohistochemically examined for the expression of p53, p16(INK4a), pRb and Cyclin D1 proteins. Tumor DNA was extracted from paraffin blocks and subjected to HPV 16 and 18 testing. The association between HPV 16 and 18 E6 oncoprotein and cell cycle proteins expression in ovarian carcinomas also was evaluated by immunohistochemistry.. We demonstrated that a majority of moderately and poorly differentiated ovarian carcinomas are characterized by strong expression of p53 and p16(INK4a) proteins. In contrast, strong staining with cyclin D1 antibody was observed in well differentiated tumors. The correlation between strong p53, pRb, Cyclin D1 and clinical stages of disease was also observed. We show that patients with high positivity for p53, p16(INK4a) and Cyclin D1 had a poor prognosis and reduced overall survival. The presence of HPV 16/18 DNA was detected in 17% of ovarian carcinomas. The tumor tissues that reacted positively to HPV E6 antibody in focal and diffuse manners had also significantly low p53 expression profile.. These findings suggest that p53, p16(INK4a) and Cyclin D1 expression and HPV infection may represent a promising tool toward the identification of ovarian cancer patients with poorer prognosis and shorter survival who might therefore need a more aggressive therapy and HPV screening.

Topics: Adolescent; Adult; Aged; Alphapapillomavirus; Antibodies, Monoclonal; Biomarkers, Tumor; Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; DNA-Binding Proteins; Female; Human papillomavirus 16; Human papillomavirus 18; Humans; Immunohistochemistry; Middle Aged; Oncogene Proteins, Viral; Ovarian Neoplasms; Papillomavirus Infections; Prognosis; Repressor Proteins; Retinoblastoma Protein; Tumor Suppressor Protein p53

Endometrioid type adenocarcinoma sometimes occupies both endometrium and ovary and in some cases the origin cannot be determined.. In this study, we established a formula to distinguish ovarian endometrioid cancer (EOC) from endometrioid type endometrial cancer (EEC), based on our previous report of cyclin and KI67 expression pattern by immunohistochemistry of 36 EECc and 37 OECc by the logistic regression. We calculated the diagnostic accuracy using 92 test samples retrospectively and finally could diagnose the origin of 16 cases in whom endometrioid type adenocarcinoma arose in both ovary and endometrium and could be determined by Scully's criteria, and 15 cases in whom endometrioid type adenocarcinoma arose in both ovary and endometrium and Scully's criteria were not useful retrospectively.. The estimated formula is as follows: Logit(Prob(EOC))=-1.1437-0.0853 CNA+0.0423 CNB+0.173 CND1+0.0129 CNE+0.0224 CNF+0.0508 KI67, where Prob(EOC) is the probability that a clinical sample is EOC. If Prob(EOC) is larger than 0.5, the diagnosis is ovarian cancer; if less than 0.5 it is endometrial cancer. Finally, using the formula, 37 of 48 EECs (77.1%) and 33 of 44 EOCs (75.0%) were correctly classified, with an accuracy of 76.1% (p<0.0001), retrospectively. In 12 of the 16 cases (75%) who could be determined by Scully's criteria, the origin determined by Scully's criteria was concordant with the origin determined by the formula retrospectively. In the other 15 cases, 12 cases were judged as ovary/ovary, 2 cases were judged as uterus/uterus and 1 case was judged as uterus/ovary.. The formula we established was thought to be useful to distinguish the origin of the cases in whom endometrioid type adenocarcinoma arises in both ovary and endometrium.

Topics: Adult; Aged; Carcinoma, Endometrioid; Cyclin A; Cyclin B1; Cyclin D1; Cyclin E; Cyclins; Endometrial Neoplasms; Endometrium; Female; Humans; Ki-67 Antigen; Middle Aged; Ovarian Neoplasms; Retrospective Studies; Sensitivity and Specificity

Signal transducer and activator of transcription 3 (STAT3) are constitutively activated in a variety of cancers and it is a common feature of ovarian cancer. Thus, STAT3 represents a promising molecular target for tumor therapy. We applied a DNA vector-based STAT3-specific RNA interference approach which specifically blocks over-activated STAT3, to treat human ovarian cancer cells, and evaluated the cellular proliferation ability and investigated the molecular mechanisms in vitro.. A DNA vector-based RNA interference approach was used to knockdown STAT3 expression in human ovarian cancer cells in vitro.. The STAT3 siRNA down-regulated the expression of cyclin D1, survivin, and VEGF in ovarian cancer cells both at transcription and translation levels. Inhibition of STAT3 and its related genes was accompanied by growth suppression and induction of apoptosis in cancer cells in vitro.. These data indicate that STAT3 signaling is a promising molecular target for ovarian cancer therapy.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Humans; Inhibitor of Apoptosis Proteins; Mice; Microtubule-Associated Proteins; Ovarian Neoplasms; RNA, Small Interfering; STAT3 Transcription Factor; Survivin; Transfection; Vascular Endothelial Growth Factor A

Development of chemoresistance limits the clinical efficiency of platinum-based therapy. Although many resistance mechanisms have been demonstrated, genetic/molecular alterations responsible for drug resistance in the majority of clinical cases have not been identified. We analyzed three pairs of testicular germ cell tumor cell lines using Affymetrix expression microarrays and revealed a limited number of differentially expressed genes across the cell lines when comparing the parental and resistant cells. Among them, CCND1 was the most significantly differentially expressed gene. Analysis of testicular germ cell tumor clinical samples by quantitative reverse transcription PCR analysis revealed that overall expression of CCND1 was significantly higher in resistant cases compared with sensitive samples (P < 0.0001). We also found that CCND1 was dramatically overexpressed both in induced and intrinsically resistant samples of ovarian and prostate cancer. Finally combined CCND1 knockdown using small-interfering RNA and cisplatin treatment inhibited cell growth in vitro significantly more effectively than any of these single treatments. Therefore, deregulation of CCND1 may be a major cause of cisplatin resistance in testicular germ cell tumors and may also be implicated in ovarian and prostate cancers. CCND1 could be potentially used as a marker for treatment stratification and as a molecular target to improve the treatment of platinum-resistant tumors.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Comparative Genomic Hybridization; Cyclin D1; Drug Resistance, Neoplasm; Female; Gene Expression Profiling; Humans; Male; Microarray Analysis; Neoplasms, Germ Cell and Embryonal; Ovarian Neoplasms; Prostatic Neoplasms; RNA, Small Interfering; Testicular Neoplasms

Rab5a is a regulatory guanosine triphosphatase that is associated with the transport and fusion of endocytic vesicles, and participates in regulation of intracellular signaling pathways embraced by cells to adapt to the specific environment. Rab5a is also correlated with lung, stomach, and hepatocellular carcinomas. Here, we detected Rab5a in paraffin-embedded samples of 20 ovarian cysts, 20 benign cystadenomas, and 39 ovarian cancers by immunohistochemistry, and observed that Rab5a expression was significantly higher in ovarian cancer (P = 0.0001). By setting up stable HO-8910 cell lines expressing Rab5a or dominant negative Rab5a (Rab5a:S34N), we found that Rab5a overexpression enhanced the cell growth by promoting G1 into S phase. In contrast, Rab5a:S34N inhibited this process. Additionally, APPL1 (adaptor protein containing PH domain, PTB domain, and Leucine zipper motif), a downstream effector of Rab5a, was also involved in promoting HO-8910 cell cycle progress. But this function was blocked by Rab5a:S34N. Laser scanning confocal microscopy represented the colocalization of APPL1 and Rab5a in the plasmolemma, which changed with the time of epidermal growth factor (EGF) stimulation. We also found APPL1 could transfer from the membranes into the nucleus where it interacted with NuRD/MeCP1 (the nucleosome remodeling and histone deacetylase multiprotein complex). NuRD is reported to be involved in the deacetylation of histone H3 and H4 to regulate nuclear transcription. So Rab5a promoted proliferation of ovarian cancer cells, which may be associated with the APPL1-related epidermal growth factor signaling pathway.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Aged, 80 and over; Cell Cycle; Cell Proliferation; Cyclin D1; Epidermal Growth Factor; Female; Gene Expression Regulation; Humans; Middle Aged; Ovarian Neoplasms; rab5 GTP-Binding Proteins; Signal Transduction

Hypoxia, which is commonly observed in many solid tumors, is a major impediment to chemo- or radiation therapy. Hypoxia is also known to overexpress/activate signal transducer and activator of transcription 3 (STAT3) leading to tumor progression as well as drug resistance. We hypothesized that increased oxygenation of the hypoxic tumor may have an inhibitory effect on STAT3 activation and hence tumor-growth inhibition. Mice containing human ovarian cancer xenograft tumor were exposed to hyperbaric oxygen (HBO; 100% oxygen; 2 atm; 90-min duration) daily, for up to 21 days. Mice exposed to HBO showed a significant reduction in tumor volume, with no effect on body weight. STAT3 (Tyr 705) activation and cyclin-D1 protein/mRNA levels were significantly decreased up on HBO exposure. Interestingly, HBO exposure, in combination with weekly administration of cisplatin, also significantly reduced the tumor volume; however, this group of mice had drastically reduced body weight when compared to other groups. While conventional wisdom might suggest that increased oxygenation of tumors would promote tumor growth, the results of the present study indicated otherwise. Hyperoxia appears to inhibit STAT3 activation, which is a key step in the ovarian tumor progression. The study may have important implications for the treatment of ovarian cancer in the clinic.

Topics: Animals; Antineoplastic Agents; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Cisplatin; Combined Modality Therapy; Cyclin D1; Down-Regulation; Drug Resistance, Neoplasm; Female; Humans; Hyperbaric Oxygenation; Mice; Ovarian Neoplasms; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; STAT3 Transcription Factor; Xenograft Model Antitumor Assays

Epithelial ovarian cancer is one of the most malignant cancers in women because metastasis occurs in the most of patients by the time of diagnosis. Cancer cells have strong capacity to form angiogenesis or vasculogenic mimicry, which plays the major role in its malignant phenotype. Vasculogenic mimicry might contribute to the failure of the angiogenesis-targeted therapy strategies. Under the microenvironment of the tumor, hypoxia is the most common phenomena because of the vast energy and oxygen consuming. In the present study, the endothelial-like cells induced by hypoxia from SKOV-3 and ES-2 ovarian cancer cells were harvested to investigate the changes in their biological behaviors.. The endothelial-like cells from SKOV-3 and ES-2 cells were harvested by laser capture microdissection. The biological behaviors of the endothelial-like cells, including proliferation, cell cycle, apoptosis, invasion and telomerase activity were determined by MTT, FCM, Transwell chamber and TRAP-ELISA methods. HIF-1α is the most important factor for the behavior changes under hypoxic condition. Some other genes relative to biological behaviors are also changes following the changes of HIF-1α. In order to elucidate the underlying mechanisms for these changes by hypoxia, the relative genes expressions including HIF-1α, CyclinD1, Flk-1, VEGF, p53 and V-src were determined by real-time PCR.. SKOV-3 and ES-2 cells were resistant to hypoxia by adoption of proliferation, apoptosis, differentiation and invasion. Combined with other studies, the more poorly cancer cells differentiate, the more strongly cells are resistant to hypoxia, the more possible to form vasculogenic mimicry. The changes in the expression of HIF-1α, and HIF-1α-dependent VEGF, Flk-1, Cyclin D1, and HIF-1α-independent p53 have been involved in this process.. HIF-1α took an important role in the behavioral changes of SKOV-3 and ES-2 cells by hypoxia. At the same time, other mechanisms were also involved in this process.

Topics: Apoptosis; Cell Cycle; Cell Differentiation; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Shape; Cyclin D1; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Genes, src; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Lasers; Microdissection; Neoplasm Invasiveness; Ovarian Neoplasms; Oxygen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Telomerase; Time Factors; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

The report aims to investigate the relationship between the expression of cyclin D1 and Cyclooxgenase-2 (COX-2), thus to explore the molecular mechanisms of the antitumor efficacy of Celecoxib, a COX-2 inhibitor. Human ovarian SKOV-3 carcinoma cell xenograft-bearing mice were treated with Celecoxib by infusing gaster (i.g.) twice/day for 21 days. The mRNA levels of COX-2 and cyclin D1 were determined by RT-PCR. The expression of cyclin D1 at the protein level was detected by immunohistochemistry, while COX-2 protein expression was determined by Western blot. A high-dose of Celecoxib (100 mg/kg) significantly inhibited tumor growth (P < 0.05), and the expression of cyclin D1 was reduced by 61%. Celecoxib decreased the proliferation cell index by 40% (P < 0.001) and increased apoptotic index by 52% (P < 0.05) in high-dose Celecoxib treated group. Our results suggest that the antitumor efficacy of Celecoxib against ovarian cancer in mice may in part be mediated through suppression of cyclin D1, which may contribute to its ability to suppress proliferation.

Topics: Animals; Antineoplastic Agents; Apoptosis; Celecoxib; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Female; Humans; Mice; Neoplasms, Experimental; Ovarian Neoplasms; Pyrazoles; RNA, Messenger; Sulfonamides; Xenograft Model Antitumor Assays

The epidermal growth factor receptor (EGFR) has previously been detected in the nucleus of cancer cells and primary tumors. We have reported that EGFR translocates from the plasma membrane to the nucleus. Accumulation of nuclear EGFR is linked to increased DNA synthesis and proliferation; however, the pathological significance of nuclear EGFR is not completely understood. Here, we sought to determine the predictive value of EGFR for the survival of ovarian cancer patients, through the examination of 221 cases of ovarian cancer tissues by immunohistochemical analysis to determine nuclear EGFR expression. In addition, we also examined cyclin D1 and Ki-67 through immunohistochemisty. Furthermore, we examined nuclear EGFR levels in ovarian cancer cell lines treated with EGF, and primary ovarian tumor tissue using immunofluorescence analysis. Nuclear fractions extracted from serum-starved cells treated with or without EGF were subjected to SDS-PAGE and Western blot analyses. We found that 28.3% of the cohort had high levels of nuclear EGFR, while 22.5% had low levels of nuclear EGFR, and 49.2% were negative for nuclear EGFR. Importantly, there was an inverse correlation between high nuclear EGFR, cyclin D1, and Ki-67 with overall survival (P < 0.01, P < 0.09, P < 0.041). Additionally, nuclear EGFR correlated positively with increased levels of cyclin D1 and Ki-67, both indicators for cell proliferation. Our findings indicate a pathological significance of nuclear EGFR that might be important for predicting clinical prognosis of ovarian cancer patients.

Topics: Blotting, Western; Cell Nucleus; Cyclin D1; Electrophoresis, Polyacrylamide Gel; ErbB Receptors; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Ovarian Neoplasms; Prognosis; Survival Analysis

Ovarian surface epithelium (OSE) is considered to give rise to epithelial ovarian carcinomas (EOCs). To elucidate early processes contributing to the development of EOCs from the OSE, two batches of primary human OSE cells were transduced with non-viral human genes (mutant Cdk4, cyclinD1 and hTERT) so as to efficiently establish normal diploid OSE cells without chromosomal instability. Then defined genetic alterations frequently observed in EOCs were transduced into the OSE cells. A combination of p53 inactivation and oncogenic Kras transduction did not confer tumor-forming ability in immunodeficient mice, though additional transduction of Akt or combined transduction of c-myc with bcl-2 did result in tumor formation. In the latter case, tumors demonstrated phenotypes reminiscent of human EOCs, including cytokeratin expression, a highly aggressive phenotype, metastatic behavior and formation of ascites. These results indicate that inactivation of p53 and activation of the Ras pathway play critical roles in ovarian carcinogenesis in co-operation with the Akt or c-myc pathways. This first in vitro model system faithfully recapitulating the development of EOCs using normal human OSE cells should greatly facilitate further studies of EOCs.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Chromosomal Instability; Cyclin D1; Cyclin-Dependent Kinase 4; Epithelial Cells; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Mutation; Oncogenes; Ovarian Neoplasms; Ovary; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; ras Proteins; Telomerase; Transduction, Genetic; Tumor Suppressor Protein p53

Polymorphisms in genes critical to cell cycle control are outstanding candidates for association with ovarian cancer risk; numerous genes have been interrogated by multiple research groups using differing tagging single-nucleotide polymorphism (SNP) sets. To maximize information gleaned from existing genotype data, we conducted a combined analysis of five independent studies of invasive epithelial ovarian cancer. Up to 2,120 cases and 3,382 controls were genotyped in the course of two collaborations at a variety of SNPs in 11 cell cycle genes (CDKN2C, CDKN1A, CCND3, CCND1, CCND2, CDKN1B, CDK2, CDK4, RB1, CDKN2D, and CCNE1) and one gene region (CDKN2A-CDKN2B). Because of the semi-overlapping nature of the 123 assayed tagging SNPs, we performed multiple imputation based on fastPHASE using data from White non-Hispanic study participants and participants in the international HapMap Consortium and National Institute of Environmental Health Sciences SNPs Program. Logistic regression assuming a log-additive model was done on combined and imputed data. We observed strengthened signals in imputation-based analyses at several SNPs, particularly CDKN2A-CDKN2B rs3731239; CCND1 rs602652, rs3212879, rs649392, and rs3212891; CDK2 rs2069391, rs2069414, and rs17528736; and CCNE1 rs3218036. These results exemplify the utility of imputation in candidate gene studies and lend evidence to a role of cell cycle genes in ovarian cancer etiology, suggest a reduced set of SNPs to target in additional cases and controls.

Topics: Adult; Aged; Alleles; Case-Control Studies; Cell Cycle; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Female; Genotype; Humans; Logistic Models; Markov Chains; Middle Aged; Minnesota; Neoplasm Invasiveness; North Carolina; Oncogene Proteins; Ovarian Neoplasms; Polymorphism, Single Nucleotide; Registries; Risk

To investigate the inhibitory effects of an antisense PC cell derived growth factor (PCDGF) vector on proliferation and invasion of highly malignant ovarian cancer cell lines Sw626 and A2780 cells, and preliminarily explore the related mechanisms.. MTT assay and Boyden chamber in vitro invasion assay were employed to detect the changes of proliferation and invasion ability in the Sw626 and A2780 cells transfected with anti-sense PCDGF. The expression levels of cyclin D1 and CDK4 proteins before and after transfection were detected by Western blotting. The effects on the expression and activity of MMP-2 were evaluated by quantitative RT-PCR and zymography, respectively.. Comparing with the blank group, the proliferation inhibition rate of the Sw626 and A2780 cells transfected with anti-sense PCDGF was 72.9% and 70.9%, respectively, and the invasion ability was inhibited by 62.9% and 59.0%, respectively. The levels of cyclin D1 and CDK4 protein expression in antisense PCDGF transfected cells were 0.38 +/- 0.08 and 0.37 +/- 0.13, respectively, all significantly lower than 0.84 +/- 0.11 and 0.64 +/- 0.11, respectively, in the blank group (P < 0.01). The MMP-2 mRNA expression level in antisense PCDGF transfected cell group was 0.66 +/- 0.11, not significantly decreased in comparison with 0.89 +/- 0.09 in the blank group (P > 0.05), but the activity of MMP-2 was inhibited significantly.. The antisense PCDGF vector may inhibit markedly the proliferation and invasion of highly malignant ovarian cancer cells, and partially reverses their malignant phenotype. It seems to be related with down-regulating the expression of cyclin D1 and CDK4 and inhibiting the activity of MMP-2. Our findings indicate that PCDGF may become a new target for antisense gene therapy of ovarian cancer.

Topics: Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; DNA, Antisense; Down-Regulation; Female; Genetic Vectors; Humans; Intercellular Signaling Peptides and Proteins; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Ovarian Neoplasms; Progranulins; RNA, Messenger; Transfection

We selected 5 oncogenes with well-established roles in carcinogenesis -- CCND1, ErbB1, ErbB2, c-myc and ZNF217 -- to investigate the coexistence of their copy imbalances in relation to the clinico-pathological characteristics of ovarian tumors.. Fluorescence in situ hybridization for the 5 genes was applied to a preexisting tissue microarray. 38 ovarian tumors were successfully analyzed for copy number changes of the 5 genes.. At least one of these oncogenes was gained/amplified in 27 out of 38 tumors (71.1%). We report the highest frequency of c-myc genetic gain/amplification since it affected 42.1% of the ovarian tumors. We observed sequential involvement of copy number alterations of the other genes in the presence of c-myc disruption. The incidence of copy number changes of the 5 oncogenes -- both single and combinatorial -- was higher in high-grade tumors. All double aberrations in the serous group comprised c-myc and ZNF217copy number increases.. Our results revealed a combination between copy number increases of c-myc and ZNF217, associated with serous histology. The data from this combined analysis of the 5 oncogenes could be used as a basis in considering the combined approach in molecular-based therapy of ovarian cancer.

Topics: Adaptor Proteins, Signal Transducing; Bulgaria; Cyclin D1; Female; Gene Dosage; Genetic Predisposition to Disease; Humans; Incidence; Oncogene Proteins v-erbB; Ovarian Neoplasms; Proto-Oncogene Proteins c-myc; Trans-Activators

D- and E-type cyclins mediate G(1)-S phase cell cycle progression through activation of specific cyclin-dependent kinases (cdk) that phosphorylate the retinoblastoma protein (pRb), thereby alleviating repression of E2F-DP transactivation of S-phase genes. Cyclin D1 is often overexpressed in a variety of cancers and is associated with tumorigenesis and metastasis. Loss of cyclin D can cause G(1) arrest in some cells, but in other cellular contexts, the downstream cyclin E protein can substitute for cyclin D and facilitate G(1)-S progression. The objective of this study was to determine if a flexible heteroarotinoid anticancer compound, SHetA2, regulates cell cycle proteins and cell cycle progression in ovarian cancer cells. SHetA2 induced cyclin D1 phosphorylation, ubiquitination, and proteasomal degradation, causing G(1) arrest in ovarian cancer cells despite continued cyclin E2 expression and independently of p53 and glycogen synthase kinase-3beta. Cyclin D1 loss inhibited pRb S780 phosphorylation by cyclin D1-cdk4/6 and released p21 from cyclin D1-cdk4/6-p21 protein complexes to form cyclin E2-cdk2-p21 complexes, which repressed phosphorylation of pRb S612 by cyclin E2-cdk2 and ultimately E2F-DP transcriptional activity. G(1) arrest was prevented by overexpression or preventing degradation of cyclin D1 but not by restoration of pRb S612 phosphorylation through p21 knockdown. In conclusion, we show that loss of cyclin D1 in ovarian cancer cells treated with SHetA2 is sufficient to induce G(1) cell cycle arrest and this strategy is not impeded by the presence of cyclin E2. Therefore, cyclin D1 is a sufficient therapeutic target in ovarian cancer cells.

Topics: Apoptosis; Cell Proliferation; Chromans; Cyclin D1; Cyclins; Drug Evaluation, Preclinical; Female; G1 Phase; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Ovarian Neoplasms; Phosphorylation; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Thiones; Transfection; Tumor Cells, Cultured; Ubiquitination

Our previous study has suggested that the cell cycle-related kinase (CCRK) is a putative candidate oncogene in glioblastoma tumorigenesis. The potential oncogenic role of CCRK and its clinical/prognostic significance, however, in ovarian carcinoma are unclear. In this study, CCRK expression was examined by immunohistochemistry in a series of ovarian carcinoma tissues. Overexpression of CCRK was detected in 53% of the ovarian carcinomas, and it was positively correlated with an ascending histological grade and/or advanced clinical stage of the disease (p < 0.05). In addition, overexpression of CCRK in ovarian carcinoma was determined to be a strong and an independent predictor of short overall survival (p < 0.05). In ovarian carcinoma cells, CCRK knockdown by RNAi led to a G1 phase cell cycle arrest, while CCRK overexpression by stable transfection of CCRK-containing plasmid pcDNA-CCRK promoted cell proliferation in vitro and tumor growth in vivo. In addition, CCRK knockdown was found to reduce cyclin D1 expression. Consistently, CCRK overexpression increased cyclin D1 expression, and furthermore, a significant correlation between expression of CCRK and cyclin D1 in ovarian carcinomas was observed (p < 0.001). These findings suggest a potential important role of CCRK in the control of cell proliferation via regulation of cyclin D1 expression, and the overexpression of CCRK, as detected by immunohistochemistry, is an independent molecular marker for shortened survival time of patients with ovarian carcinoma.

Topics: Adult; Aged; Animals; Apoptosis; Blotting, Western; Case-Control Studies; Cell Proliferation; Cohort Studies; Cyclin D1; Cyclin-Dependent Kinase-Activating Kinase; Cyclin-Dependent Kinases; Cystadenocarcinoma, Serous; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Mice; Mice, Inbred BALB C; Middle Aged; Ovarian Neoplasms; Ovary; Phosphorylation; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Survival Rate; Tissue Array Analysis; Tumor Cells, Cultured

Little is known about the molecules that contribute to tumor progression of epithelial ovarian cancer (EOC), currently a leading cause of mortality from gynecological malignancies. Glucocorticoid-Induced Leucine Zipper (GILZ), an intracellular protein widely expressed in immune tissues, has been reported in epithelial tissues and controls some of key signaling pathways involved in tumorigenesis. However, there has been no report on GILZ in EOC up to now. The objectives of the current study were to examine the expression of GILZ in EOC and its effect on tumor cell proliferation.. GILZ expression was measured by immunohistochemical staining in tissue sections from 3 normal ovaries, 7 benign EOC and 50 invasive EOC. GILZ was not detected on the surface epithelium of normal ovaries and benign tumors. In contrast, it was expressed in the cytoplasm of tumor cells in 80% EOC specimens. GILZ immunostaining scores correlated positively to the proliferation marker Ki-67 (Spearman test in univariate analysis, P < 0.00001, r = 0.56). They were also higher in tumor cells containing large amounts of phosphorylated protein kinase B (p-AKT) (unpaired t test, P < 0.0001). To assess the effect of GILZ on proliferation and AKT activation, we used the BG-1 cell line derived from ovarian tumor cells as a cellular model. GILZ expression was either enhanced by stable transfection or decreased by the use of small interfering (si) RNA targeting GILZ. We found that GILZ increased cell proliferation, phospho-AKT cellular content and AKT kinase activity. Further, GILZ upregulated cyclin D1 and phosphorylated retinoblastoma (p-Rb), downregulated cyclin-dependent kinase inhibitor p21, and promoted the entry into S phase of cell cycle.. The present study is the first to identify GILZ as a molecule produced by ovarian cancer cells that promotes cell cycle progression and proliferation. Our findings clearly indicate that GILZ activates AKT, a crucial signaling molecule in tumorigenesis. GILZ thus appears as a potential key molecule in EOC.

Topics: Adult; Aged; Aged, 80 and over; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Activation; Epithelial Cells; Female; Gene Silencing; Humans; Middle Aged; Ovarian Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Transcription Factors

The malignant biological behaviors are enhanced in human ovarian cancer cell line RMG-1 after transfection of alpha1,2-fucosyl transferase (alpha1,2-FT) gene. This study was to investigate the influence of alpha1,2-FT gene transfection on cancer-related gene expression profile of RMG-1 cells.. Gene expressing vector pcDNA3.1-HFT-H and empty vector pcDNA3.1 were transfected into RMG-1 cells to produce RMG-1-H and RMG-1-C cells separately. Gene expression profiles of these two cell lines were detected by gene chip assay. The acquired data were inquired on the GoMiner online database.. After transfection, comparing with those in RMG-1-C cells, 88 differentially expressed genes were identified in RMG-1-H cells: 60 were up-regulated and 28 were down-regulated. These genes are involved in protein binding, nucleotide binding, cell proliferation, DNA-dependent regulation of transcription, signal transduction, protein amino acid phosphorylation, transcription, cell adhesion, and so on.. The transfection of alpha1,2-FT gene causes the changes of gene expression profile in ovarian cancer RMG-1 cells.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Fucosyltransferases; Galactoside 2-alpha-L-fucosyltransferase; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Oligonucleotide Array Sequence Analysis; Ovarian Neoplasms; RNA, Messenger; Signal Transduction; Transfection

Deregulated signaling through the epidermal growth factor receptor (EGFR) is involved in chemoresistance. To identify the molecular determinants of sensitivity to the EGFR inhibitor gefitinib (Iressa, ZD1839) in chemoresistance, we compared the response of matched chemosensitive and chemoresistant glioma and ovarian cancer cell lines. We found that chemoresistant cell lines were 2- to 3-fold more sensitive to gefitinib growth-inhibitory effects, because of decreased proliferation rather than survival. Sensitivity to gefitinib correlated with overexpression and constitutive phosphorylation of HER2 and HER3, but not EGFR, altered HER ligand expression, and enhanced activation of EGF-triggered EGFR pathway. No activating mutations were found in EGFR. Gefitinib fully inhibited EGF-induced and constitutive Akt activation only in chemoresistant cells. In parallel, gefitinib downregulated constitutively phosphorylated HER2 and HER3, and activated GSK3beta with a concomitant degradation of cyclin D1. Ectopically overexpressed HER2 on its own was insufficient to sensitize chemonaive cells to gefitinib. pHER3 coimmunoprecipitated with p85-PI3K in chemoresistant cells and gefitinib dissociated these complexes. siRNA-mediated inhibition of HER3 decreased constitutive activation of Akt and sensitivity to gefitinib in chemoresistant cells. Our study indicates that in chemoresistant cells gefitinib inhibits both an enhanced EGF-triggered pathway and a constitutive HER3-mediated Akt activation, indicating that inhibition of HER3 together with that of EGFR could be relevant in chemorefractory tumors. Furthermore, in combination experiments gefitinib enhanced the effects of coadministered drugs more in chemoresistant than chemosensitive ovarian cancer cells. Combined treatment might be therapeutically beneficial in chemoresistant tumors from ovary and likely from other tissues.

Topics: Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cisplatin; Cyclin D1; DNA, Complementary; Down-Regulation; Drug Resistance, Neoplasm; ErbB Receptors; Female; Gefitinib; Gene Expression Regulation, Neoplastic; Glioma; Humans; Immunoblotting; Immunoprecipitation; Neoplasms; Ovarian Neoplasms; Phosphorylation; Polymerase Chain Reaction; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Receptor, ErbB-2; Receptor, ErbB-3; Repressor Proteins; RNA, Small Interfering; Sequence Analysis, DNA; Signal Transduction

The pathophysiological mechanisms that drive the development and progression of epithelial ovarian cancer remain obscure. Recently, we identified TCEAL7 as a transcriptional regulatory protein often downregulated in epithelial ovarian cancer. However, the biological significance of such downregulation in cancer is not currently known. Here, we show that TCEAL7 is downregulated frequently in many human cancers and that in immortalized human ovarian epithelial cells this event promotes anchorage-independent cell growth. Mechanistic investigations revealed that TCEAL7 associates with cyclin D1 promoter containing Myc E-box sequence and transcriptionally represses cyclin D1 expression. Moreover, downregulation of TCEAL7 promotes DNA-binding activity of Myc-Max, and upregulates the promoter activity of c-Myc-target gene, ornithine decarboxylase (ODC), whereas enhanced expression of TCEAL7 inhibits Myc-induced promoter activity of ODC. Our findings suggest that TCEAL7 may restrict ovarian epithelial cell transformation by limiting Myc activity. These results also suggest a potential, alternative mechanism by which c-Myc activity may be deregulated in cancer by the downregulation of TCEAL7.

Topics: Biological Phenomena; Cell Line, Transformed; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Genes, myc; Genes, Tumor Suppressor; HeLa Cells; Humans; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Ovarian Neoplasms; Proto-Oncogene Proteins c-myc

Hus1 and Rad9 are proteins involved in DNA damage checkpoint regulation, which is required for the maintenance of genomic stability. In addition to checkpoint activation, mammalian cells also use apoptosis to eliminate cells with severe DNA damage. Interestingly, Rad9 was shown to be directly involved in apoptosis as well. Despite the knowledge of molecular mechanisms on how Hus1 and Rad9 act in response to DNA damage, little is known about the role of these 2 proteins in cancer progression. In this study, we analyzed the expression of Rad9 and Hus1 in epithelial ovarian tumors and correlated them to clinopathological parameters and apoptotic biomarkers (p53, Bcl-2, and Bax). Histological sections from 114 primary ovarian epithelial tumors were stained with antibodies using the streptavidin-biotin method. In addition, mitotic and apoptotic indices (both hematoxylin-eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling assay) were also measured. We found that Rad9 expression correlated closely to significance only with the apoptotic and mitotic indices (P = 0.056 and 0.059, respectively). Hus1 levels correlated significantly with the clinicopathologic factors of bad prognosis, including FIGO (International Federation of Gynecology and Obstetrics) stage (P < 0.002) and with the p53 expression (P < 0.001), Bax expression (P < 0.008), mitotic index (P < 0.001), and apoptotic index (P < 0.003).

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; bcl-2-Associated X Protein; Biomarkers, Tumor; Cell Cycle Proteins; Cyclin D1; Female; Gene Expression; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Kaplan-Meier Estimate; Middle Aged; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Prognosis; Tumor Suppressor Protein p53

A few highly aggressive and malignant tumor cells could acquire identities by turning on genes expressed by endothelial cells and recruit blood vessels to sustain tumor growth. Hypoxia was reported recently to play an essential role in these events. These 'plastic' tumor-cell phenotypes and the exact mechanism driving transendothelial differentiation by hypoxia-inducible factor (HIF)-1alpha is unclear. In this study, epithelial ovarian carcinoma cells were exposed to hypoxia and the tumor cells were transformed into endothelial cells-like (ECs-like). Typical endothelial features such as cell markers and uptaking of acetylated low density lipoprotein were identified constantly. Small interference RNA was used to block the expression of HIF-1alpha. Analysis revealed that hypoxia promotes transendothelial differentiation through stimulating HIF-1-dependent transcriptional expression of vascular endothelial growth factor (VEGF), VEGF receptor-2 (Flk-1) and P53, and through decreasing HIF-1-independent transcriptional expression of Cyclin D1. These results demonstrate that ECs-like derived from epithelial ovarian cancer cells are similar to endothelial progenitor cells rather than endothelial cells. HIF-1alpha is crucial but not unique in alternation of tumor cells towards ECs-like.

Topics: Cell Differentiation; Cyclin D1; Endothelial Cells; Endothelium, Vascular; Epithelial Cells; Female; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Microscopy, Fluorescence; Ovarian Neoplasms; RNA, Small Interfering; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor Receptor-2

To study the clinicopathological features and expression of cyclin D1 and p53 in epithelial ovarian tumors, and to investigate the correlation between pathogenesis of ovarian cancer and epithelial borderline tumors.. Fifty four cases of ovarian borderline tumors and 45 cases of ovarian carcinomas from the People's Hospital, Peking University were reviewed retrospectively. The clinical data and pathological findings were analyzed. Immunohistochemical study of cyclin D1 and p53 was performed in all 99 cases.. (1) In borderline tumors, the age of patients ranged from 14 - 82 (mean age = 42.5) years. International Federation of Gynecology and Obstetrics (FIGO) stage of borderline tumors was stage I in 48 cases, stage II in 3 cases, and stage III in 3 cases. In ovarian carcinomas, the age of patients ranged from 26 - 80 (mean age = 53.5) years. FIGO stage of carcinoma was stage I in 6 cases, stage II in 8 cases, stage III in 26 cases, and stage IV in 5 cases. In follow-up of 54 cases with borderline tumors the 5-year survival rate was 98% and of 45 cases with carcinomas a 5-year survival rate of 51% was noted. (2) In 54 cases of borderline tumors, mucinous types accounted for 56% (30/54) and serous types accounted for 30% (16/54). There were 5 cases with micropapillary pattern, 3 cases with peritoneal implants, 3 cases with lymph node involvement, 6 cases with microinvasion, one case with intraepithelial carcinoma, and one case with mural nodules. In 45 cases of carcinomas, serous carcinoma was the most (49%, 22/45). The remainder included 3 cases of mucinous types, 8 cases of endometrioid types, 6 cases of transitional cell types, 3 cases of mixed phenotype and 3 cases of undifferentiated types. (3) Overexpression of cyclin D1 and p53 was observed in 31% (14/45) and 56% (25/45) of ovarian carcinomas, respectively. There was a significant association between p53 overexpression and tumor grade. In the borderline tumor group, 69% (37/54) had overexpression of cyclin D1 and 6% (3/54) had overexpression of p53. There were significant differences in expression of cyclin D1 and p53 between conventional serous borderline tumors and high-grade serous carcinomas (cyclin D1: 91% vs 26%; p53: 0 vs 58%). However, micropapillary serous borderline tumors and low-grade serous carcinomas showed remarkably similar expression of cyclin D1 and p53.. Epithelial ovarian borderline tumors are distinct from ovarian cancer in clinical progress and prognosis, and histological types. Overexpression of cyclin D1 is common in ovarian borderline tumors and low grade carcinomas. And overexpression of p53 is more common in high grade ovarian carcinomas. Conventional serous borderline tumors are distinct from high-grade serous carcinomas in pathogenesis. Micropapillary serous borderline ovarian tumors may be closely related to low grade serous carcinomas.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Cyclin D1; Cystadenocarcinoma, Mucinous; Cystadenocarcinoma, Serous; Female; Humans; Immunohistochemistry; Middle Aged; Neoplasm Staging; Ovarian Neoplasms; Tumor Suppressor Protein p53

To study the clinicopathologic features and biologic behavior of pediatric immature teratoma.. The clinical data, pathologic features, immunohistochemical findings (for cyclin D1, P27 and Ki-67) and follow-up information of 39 cases of pediatric immature teratoma were analyzed.. Amongst the 39 cases studied, 12 arose in the sacrococcygeal region, 12 in testis, 5 in retroperitoneum, 4 in ovary, 4 in abdomen and 2 in mediastinum. Histologically, 16 cases were of grade 1, 8 cases of grade 2 and 15 cases of grade 3. Seven of the cases contained foci of yolk sac tumor. Immature neuroepithelial features used in histologic grading included the presence of primitive neural tubules, immature rosettes, undifferentiated neuroblastoma cells and primitive neuroectodermal structures. Immunohistochemical study showed that cyclin D1 was positive in 3 cases of grade 1 tumors, 4 cases of grade 2 tumors and 9 cases of grade 3 tumors. The positivity rates for p27 were 8, 3 and 6 cases respectively, while those for Ki-67 were 3, 4 and 13 cases respectively. Follow-up data were available in 30 cases. Three of them, including 2 cases with histologic grade 3 (with or without yolk sac tumor component), recurred after operation.. The expression of cyclin D1 and Ki-67 is a useful adjunct in histologic grading. On the other hand, p27 overexpression shows little correlation with tumor grade. The prognosis of immature teratoma in children is different from that in adults. Sacrococcygeal immature teratoma occurring in patients younger than 1 year old and with low histologic grade do not require postoperative chemotherapy if the tumor is completely excised. Similarly, for testicular immature teratoma occurring in patients below 1 year of age, regardless of tumor grading, need no adjunctive therapy. On the other hand, ovarian immature teratoma with high histologic grade requires postoperative chemotherapy, regardless of age of the patients. The presence of microscopic foci of yolk sac tumor is a useful predictor of recurrence in pediatric immature teratoma.

Topics: Adolescent; alpha-Fetoproteins; Cyclin D1; Endodermal Sinus Tumor; Female; Follow-Up Studies; Humans; Infant; Infant, Newborn; Ki-67 Antigen; Male; Mediastinal Neoplasms; Neoplasm Recurrence, Local; Neoplasm Staging; Ovarian Neoplasms; Proliferating Cell Nuclear Antigen; Retroperitoneal Neoplasms; Sacrococcygeal Region; Survival Rate; Teratoma; Testicular Neoplasms

In this study, we analyzed the PTEN expression in a large collection of clear cell adenocarcinomas of the ovary. Furthermore, we analyzed the expression of cyclin D1 and p27, and investigated the correlation among all these variables.. Totally, 40 clear cell adenocarcinomas were included in this study. The protein expression of PTEN, cyclin D1 and p27 was investigated by immunohistochemistry.. Of 40 clear cell adenocarcinomas, 15 (37.5%) lost all PTEN immunoreactivity. There was no significant correlation between PTEN expression and clinical stage. Cyclin D1 expression and loss of p27 expression were detected in 16/40 (40.0%) and 14/40 (35.0%) clear cell adenocarcinoma cases. There was no significant correlation between PTEN expression and cyclin D1 or p27 protein expression.. Loss of PTEN expression is relatively common and both cyclin D1 and p27 expressions are not related with PTEN inactivation in clear cell adenocarcinoma of the ovary.

Topics: Adenocarcinoma, Clear Cell; Adult; Aged; Aged, 80 and over; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Immunohistochemistry; Middle Aged; Ovarian Neoplasms; PTEN Phosphohydrolase

The insulin-like growth factor receptor (IGF-IR) and insulin receptor are either overactivated and/or overexpressed in a wide range of tumor types and contribute to tumorigenicity, proliferation, metastasis, and drug resistance. Here, we show that BMS-554417, a novel small molecule developed as an inhibitor of IGF-IR, inhibits IGF-IR and insulin receptor kinase activity and proliferation in vitro, and reduces tumor xenograft size in vivo. In a series of carcinoma cell lines, the IC50 for proliferation ranged from 120 nmol/L (Colo205) to >8.5 micromol/L (OV202). The addition of stimulatory ligands was unnecessary for the antiproliferative effect in MCF-7 and OV202 cells. BMS-554417 treatment inhibited IGF-IR and insulin receptor signaling through extracellular signal-related kinase as well as the phosphoinositide 3-kinase/Akt pathway, as evidenced by decreased Akt phosphorylation at Ser473. At doses that inhibited proliferation, the compound also caused a G0-G1 arrest and prevented nuclear accumulation of cyclin D1 in response to LR3 IGF-I. In Jurkat T-cell leukemia cells, this agent triggered apoptotic cell death via the mitochondrial pathway. BMS-554417 was orally bioavailable and significantly inhibited the growth of IGF1R-Sal tumor xenografts in vivo. BMS-554417 is a member of a novel class of IGF-IR/insulin receptor inhibitors that have potential clinical applications because of their antiproliferative and proapoptotic activity in vitro and in vivo.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspases; Cell Growth Processes; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Dose-Response Relationship, Drug; Enzyme Activation; Female; G1 Phase; Humans; Insulin-Like Growth Factor I; Mice; Mice, Nude; Mitochondria; Mitogen-Activated Protein Kinase Kinases; Ovarian Neoplasms; Phosphorylation; Piperazines; Proto-Oncogene Proteins c-akt; Pyridones; Receptor, IGF Type 1; Receptor, Insulin; S Phase

Although glycogen synthase kinase-3 (GSK-3) might act as a tumor suppressor since its inhibition is expected to mimic the activation of Wnt-signaling pathway, GSK-3beta may contribute to NF-kappaB activation in cancer cells leading to increased cancer cell proliferation and survival. Here we report that GSK-3beta activity was involved in the proliferation of human ovarian cancer cell both in vitro and in vivo. Inhibition of GSK-3 activity by pharmacological inhibitors suppressed proliferation of the ovarian cancer cells. Overexpressing constitutively active form of GSK-3beta induced entry into the S phase, increased cyclin D1 expression and facilitated the proliferation of ovarian cancer cells. Furthermore, GSK-3 inhibition prevented the formation of the tumor in nude mice generated by the inoculation of human ovarian cancer cells. Our findings thus suggest that GSK-3beta activity is important for the proliferation of ovarian cancer cells, implicating this kinase as a potential therapeutic target in ovarian cancer.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzyme Inhibitors; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lithium Chloride; Mice; Ovarian Neoplasms

1,1-Bis(3'-indolyl)-1-(p-t-butylphenyl)methane (DIM-C-pPhtBu) is a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, and treatment of SKOV3 ovarian cancer cells with this compound (5 micromol/L) inhibits cell proliferation, whereas up to 15 micromol/L rosiglitazone had no effect on cell growth. DIM-C-pPhtBu also inhibits G0-G1 to S phase cell cycle progression and this is linked, in part, to PPARgamma-dependent induction of the cyclin-dependent kinase inhibitor p21. DIM-C-pPhtBu induces PPARgamma-independent down-regulation of cyclin D1 and we therefore further investigated activation of receptor-independent pathways. DIM-C-pPhtBu also induced apoptosis in SKOV3 cells and this was related to induction of glucose-related protein 78, which is typically up-regulated as part of the unfolded protein response during endoplasmic reticulum (ER) stress. Activation of ER stress was also observed in other ovarian cancer cell lines treated with DIM-C-pPhtBu. In addition, DIM-C-pPhtBu induced CCAAT/enhancer binding protein homologous protein through both ER stress and c-jun NH2-terminal kinase-dependent pathways, and CCAAT/enhancer binding protein homologous protein activated death receptor 5 and the extrinsic pathway of apoptosis. These results show that DIM-C-pPhtBu inhibits growth and induces apoptosis in ovarian cancer cells through both PPARgamma-dependent and PPARgamma-independent pathways, and this complex mechanism of action will be advantageous for future clinical development of these compounds for treatment of ovarian cancer.

Topics: Animals; Apoptosis; Cell Cycle; Cell Growth Processes; Cyclin D1; Down-Regulation; Endoplasmic Reticulum; Female; Humans; Indoles; JNK Mitogen-Activated Protein Kinases; Ovarian Neoplasms; p21-Activated Kinases; PPAR gamma; Protein Serine-Threonine Kinases; Rabbits; Receptors, TNF-Related Apoptosis-Inducing Ligand; Transcription Factor CHOP

Activation of mitogen-activated protein kinase (MAPK) occurs in response to various growth stimulating signals and as a result of activating mutations of the upstream regulators, KRAS and BRAF, which can be found in many types of human cancer. To investigate the roles of MAPK activation in tumors harboring KRAS or BRAF mutations, we inactivated MAPK in ovarian tumor cells using CI-1040, a compound that selectively inhibits MAPK kinase, an upstream regulator of MAPK and thus prevents MAPK activation. Profound growth inhibition and apoptosis were observed in CI-1040-treated tumor cells with mutations in either KRAS or BRAF in comparison with the ovarian cancer cells containing wild-type sequences. Long serial analysis of gene expression identified several differentially expressed genes in CI-1040-treated MPSC1 cells harboring an activating mutation in BRAF (V599L). The most striking changes were down-regulation of cyclin D1, COBRA1, and transglutaminase-2 and up-regulation of tumor necrosis factor-related apoptosis-induced ligand, thrombospondin-1, optineurin, and palladin. These patterns of gene expression were validated in other CI-1040-treated tumor cells based on quantitative PCR. Constitutive expression of cyclin D1 partially reversed the growth inhibitory effect of CI-1040 in MPSC1 cells. Our findings indicate that an activated MAPK pathway is critical in tumor growth and survival of ovarian tumors with KRAS or BRAF mutations and suggest that the CI-1040 induced phenotypes depend on the mutational status of KRAS and BRAF in ovarian tumors.

Topics: Apoptosis; Benzamides; Calcium-Calmodulin-Dependent Protein Kinases; Cell Cycle; Cyclin D1; Cystadenocarcinoma, Serous; Female; Gene Expression Profiling; Genes, ras; Humans; Mitogen-Activated Protein Kinases; Mutation; Ovarian Neoplasms; Proto-Oncogene Proteins B-raf; Signal Transduction; Tumor Cells, Cultured

Although the chemopreventive and antitumorigenic activities of nonsteroidal anti-inflammatory drug (NSAID) against colorectal cancer are well established, the molecular mechanisms responsible for these properties in ovarian cancer have not been elucidated. Therefore, there is an urgent need to develop mechanism-based approaches for the management of ovarian cancer. To this end, the effect of several NSAIDs on ovarian cancer cells was investigated as assessed by the induction of NAG-1/MIC-1/GDF-15, a proapoptotic gene belonging to the transforming growth factor-beta superfamily. Sulindac sulfide was the most significant NSAID activated gene 1 (NAG-1) inducer and its expression was inversely associated with cell viability as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. This growth suppression by sulindac sulfide was recovered by transfection of NAG-1 small interfering RNA. These results indicate that NAG-1 is one of the genes responsible for growth suppression by sulindac sulfide. Furthermore, we observed down-regulation of p21 WAF1/CIP1 by introduction of NAG-1 small interfering RNA into sulindac sulfide-treated cells. In addition, to elucidate other potential molecular mechanisms involved in sulindac sulfide treatment of ovarian cancer cells, we did a membrane-based microarray experiment. We found that cyclin D1, MMP-1, PI3KR1, and uPA were down-regulated by sulindac sulfide. In conclusion, a novel molecular mechanism is proposed to explain the experimental results and provide a rationale for the chemopreventive activity of NSAIDs in ovarian cancer.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Blotting, Western; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Coloring Agents; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cytokines; Down-Regulation; Female; Growth Differentiation Factor 15; Humans; Luciferases; Oligonucleotide Array Sequence Analysis; Ovarian Neoplasms; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Sulindac; Tetrazolium Salts; Thiazoles; Time Factors; Up-Regulation

To assess the relationship between the expression of cell cycle and apoptotic proteins and the morphological appearance of the surface epithelium in non-neoplastic ovaries.. The subjects for this study were 79 women who had undergone oophorectomy for benign conditions at the North Middlesex Hospital, London, and Royal Free Hospital, London, and whose ovaries had been reported on routine histology as entirely normal or containing physiological cysts or endometriosis. The epithelial morphology was reassessed on haematoxylin and eosin-stained paraffin wax sections using nine cytological and architectural parameters associated with premalignant intraepithelial changes. A 'score' was obtained for each ovary. Expression of p53, Ki67, cyclin D1 and Bcl-2 in the surface, cystic and endometriotic epithelium was assessed in corresponding sections using standard immunohistochemistry.. The median score for the morphological changes was significantly higher in the sections, which expressed p53 compared to those which did not. This difference remained significant in a subanalysis of the sections, which did not contain endometriosis. No relationship was identified between the morphological score and the expression of Ki67, Bcl-2 and cyclin D1.. Increased intraepithelial abnormality as assessed by an epithelial morphological score of ovarian sections is associated with expression of the p53 cell cycle protein. This lends credence to the hypothesis that the ovarian surface or cystic epithelium goes through an identifiable precursor or "premalignant" phase before the development of invasive disease. Further work is required to characterise the changes that take place before the development of malignancy in ovarian epithelium.

Topics: Apoptosis; Cell Cycle; Cyclin D1; Endometriosis; Endometrium; Epithelial Cells; Female; Humans; Ki-67 Antigen; Ovarian Neoplasms; Ovary; Precancerous Conditions; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

Ovarian cancer is one of the most common cancers among women. Recent studies demonstrated that the gene encoding the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K) is frequently amplified in ovarian cancer cells. PI3K is involved in multiple cellular functions, including proliferation, differentiation, antiapoptosis, tumorigenesis, and angiogenesis. In this study, we demonstrate that the inhibition of PI3K activity by LY-294002 inhibited ovarian cancer cell proliferation and induced G(1) cell cycle arrest. This effect was accompanied by the decreased expression of G(1)-associated proteins, including cyclin D1, cyclin-dependent kinase (CDK) 4, CDC25A, and retinoblastoma phosphorylation at Ser(780), Ser(795), and Ser(807/811). Expression of CDK6 and beta-actin was not affected by LY-294002. Expression of the cyclin kinase inhibitor p16(INK4a) was induced by the PI3K inhibitor, whereas steady-state levels of p21(CIP1/WAF1) were decreased in the same experiment. The inhibition of PI3K activity also inhibited the phosphorylation of AKT and p70S6K1, but not extracellular regulated kinase 1/2. The G(1) cell cycle arrest induced by LY-294002 was restored by the expression of active forms of AKT and p70S6K1 in the cells. Our study shows that PI3K transmits a mitogenic signal through AKT and mammalian target of rapamycin (mTOR) to p70S6K1. The mTOR inhibitor rapamycin had similar inhibitory effects on G(1) cell cycle progression and on the expression of cyclin D1, CDK4, CDC25A, and retinoblastoma phosphorylation. These results indicate that PI3K mediates G(1) progression and cyclin expression through activation of an AKT/mTOR/p70S6K1 signaling pathway in the ovarian cancer cells.

Topics: Antibiotics, Antineoplastic; Blood Proteins; Cell Division; Chromones; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Enzyme Inhibitors; Female; G1 Phase; Humans; Mitogen-Activated Protein Kinases; Morpholines; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Retinoblastoma Protein; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

A limited number of studies have looked at premalignant lesions of ovaries and the results are conflicting. Our goal is to identify, histologically and by immunohistochemistry, any features that may represent premalignant changes in the ovaries.. Our cases included 29 patients with unilateral ovarian cancer. These were compared to 19 controls that had unilateral benign ovarian pathology and to 39 controls with bilateral normal ovaries. Tissue sections from the contralateral normal ovary were examined. Analysis of histological features and immunohistochemical staining for the apoptosis inhibitor Bcl-2, the proliferation marker Ki-67 and the tumor suppressor gene p53 was performed.. Epithelial stratification, nuclear atypia, and inclusion cysts were more often seen in the cases than in the two control groups. Epithelial stratification and nuclear atypia was statistically significantly more common among the cases than the normal controls. Inclusion cysts were present in more of the cases (P = 0.017) and in higher numbers than in the normal controls. Bcl-2 overexpression was statistically more commonly seen in the cases with contralateral ovarian cancer (39%) than in the normal controls (15%), while it was present in 28% of cases with contralateral benign pathology.. Epithelial alterations and Bcl-2 overexpression was seen in all three groups studied. However, the epithelial alterations and Bcl-2 overexpression was more commonly seen in the contralateral ovary of women with unilateral ovarian cancer. This suggests an association between these changes and ovarian cancer. Although it is tempting to label the above changes premalignant, women with the above changes are at possibly higher risk of developing ovarian cancer rather than having acquired an oncogenic change that would inevitably lead to ovarian cancer.

Topics: Apoptosis; Cyclin D1; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Middle Aged; Ovarian Neoplasms; Precancerous Conditions; Tumor Suppressor Protein p53

In order to assess the prognostic role of the cell-cycle regulator cyclin D1 in epithelial ovarian cancer, 70 patients have been studied during an observation period of 8 years.. The cyclin D1 protein content was analyzed by Western blotting, and classed as negative, positive and highly positive by densitometric scanning. The relationship between cyclin D1 expression and clinicopathological variables was determined. Univariate and multivariate survival analyses were also carried out.. Patients with highly positive cyclin D1 tumors had shorter overall survival than patients with positive cyclin D1 (median survival 31 vs. 49 months; p = 0.058). Furthermore, in patients with stage III/IV tumors and residual disease greater than 2 cm, cyclin D1 expression significantly influenced clinical outcome (p = 0.047 and 0.040, respectively). In the Cox's regression model, cyclin D1 expression and residual disease were identified as the most important predictors of survival (p = 0.016 and 0.002, respectively). In patients with high cyclin D1 expression and residual disease after debulking surgery greater than 2 cm, the relative risks of death were to 2.48 and 3.7, respectively, compared to their correspondent counterparts.. The overexpression of cyclin D1 is significantly related to a more aggressive tumor phenotype and poor prognosis in ovarian carcinoma.

Topics: Analysis of Variance; Biomarkers, Tumor; Blotting, Western; Carcinoma; Cyclin D1; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Ovarian Neoplasms; Predictive Value of Tests; Prognosis; Survival Analysis; Up-Regulation

Dysregulation of cell cycle control, in particular G(1)-S-phase transition, is implicated in the pathogenesis of most human cancers, including epithelial ovarian cancer (EOC). However, the prognostic significance of aberrant cell cycle gene expression in EOC remains unclear.. The expression of selected genes from the pRb pathway that regulates G(1)-S-phase progression, including cyclin D1, p16(Ink4a), cyclin E, p27(Kip1), p21(Waf1/Cip1), and p53, was examined in a consecutive series of 134 serous EOC using immunohistochemistry and the results correlated to disease outcome.. Molecular markers predictive of reduced overall survival in univariate analysis were overexpression of cyclin D1 (P = 0.03) and p53 (P = 0.03) and reduced expression of p27(Kip1) (P = 0.05) and p21(Waf1/Cip1) (P = 0.02), with the latter three also being prognostic for a shorter progression-free interval. In addition, patients displaying overexpression of p53 with concurrent loss of p21(Waf1/Cip1) had a significantly shorter overall (P = 0.0008) and progression-free survival (P = 0.0001). On multivariate analysis, overexpression of cyclin D1 and combined loss of p21(Waf1/Cip1) in the presence of p53 overexpression were independent predictors of overall survival. Similarly, the combination of p21(Waf1/Cip1) loss and p53 overexpression was independently predictive of a shorter progression-free interval. Overexpression of p53 and cyclin E and reduced expression of p27(Kip1) and p21(Waf1/Cip1) were significantly associated with increasing tumor grade.. This study confirms that dysregulation of cell cycle genes is common in EOC, and that aberrant expression of critical cell cycle regulatory proteins can predict patient outcome in serous EOC.

Topics: Biomarkers, Tumor; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p21; Cystadenocarcinoma, Serous; Disease Progression; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Odds Ratio; Ovarian Neoplasms; Proportional Hazards Models; Risk; Time Factors; Treatment Outcome; Tumor Suppressor Protein p53

ARHI, an imprinted putative tumor suppressor gene, is expressed in normal ovarian epithelial cells, but its expression is down-regulated or lost in most ovarian cancer cell lines. Reexpression of ARHI in cancer cells induces p21(WAF1/CIP1), down-regulates cyclin D1 promoter activity and inhibits growth in cell culture and in heterografts. To determine the relevance of these observations to clinical cancer, we have now measured ARHI expression in normal, benign and malignant ovarian tissues using immunohistochemistry and in situ hybridization.. Paraffin embedded tissues from 7 normal ovaries, 22 cystadenomas and 42 borderline lesions were analyzed using standard immunoperoxidase and in situ hybridization techniques to assess ARHI expression. In addition, immunohistochemistry against ARHI was performed on a tissue microarray containing 441 consecutive cases of ovarian carcinoma.. Strong ARHI expression was found in normal ovarian surface epithelial cells, cysts and follicles using immunohistochemistry and in situ hybridization. Reduced ARHI expression was observed in tumors of low malignant potential as well as in invasive cancers. ARHI expression was down-regulated in 63% of invasive ovarian cancer specimens and could not be detected in 47%. When immunohistochemistry and in situ hybridization were compared, ARHI protein expression could be down-regulated in the presence of ARHI mRNA. ARHI expression was correlated with expression of p21(WAF1/CIP1) (P = 0.0074) but not with cyclin D1 and associated with prolonged disease free survival (P = 0.001). On multivariate analysis, ARHI expression, grade and stage were independent prognostic factors. ARHI expression did not correlate with overall survival.. Persistence of ARHI expression in epithelial ovarian cancers correlated with prolonged disease free survival and expression of the cyclin dependent kinase inhibitor p21(WAF1/CIP1).

Topics: Adult; Aged; Aged, 80 and over; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Immunohistochemistry; In Situ Hybridization; Middle Aged; Multivariate Analysis; Neoplasm Staging; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; rho GTP-Binding Proteins; RNA, Messenger; Time Factors

To clarify the mechanisms underlying cell cycle promotion in malignant germ cell tumours of the ovary (MGCTOs), beta-catenin and components of the pRB pathway, cyclin D1 and p16, were analysed in relation to cell proliferation. Immunohistochemically, p16 protein was not expressed in a number of MGCTOs (9 of 42 tumours: 21.4%) and was associated with p16 gene (INK4A) promoter 5'-CpG islands methylation. Amplification of the cyclin D1 gene (CCND1) was detected in a small number of MGCTOs (5 of 42 tumours: 13.5%). Reduced expression of p16 due to promoter methylation correlated significantly with increased cell proliferation as evidenced by Ki-67 labelling index (p < 0.001) and mitotic index (p < 0.01). In some tumour types, nuclear localization of beta-catenin has been reported to be associated with beta-catenin gene (CTNNB1) mutation, cyclin D1 overexpression, and increased cell proliferation. Nuclear localization of beta-catenin, which was observed in MGCTOs other than dysgerminoma, was not associated with cyclin D1 expression and increased cell proliferation, but appeared to be related to tumour differentiation. Furthermore, CTNNB1 mutations were not detected in any of the MGCTOs examined. Our results suggest that reduced expression of p16 due to INK4A promoter methylation is one of the principal factors that promote cell proliferation in MGCTOs. Thus, p16 may be a novel target for gene therapies to treat MGCTOs.

Topics: Adolescent; Adult; beta Catenin; Cell Cycle Proteins; Cell Transformation, Neoplastic; Child; Child, Preschool; CpG Islands; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cytoskeletal Proteins; DNA, Neoplasm; Female; Genes, p16; Germinoma; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Methylation; Mutation; Neoplasm Proteins; Ovarian Neoplasms; Polymerase Chain Reaction; Promoter Regions, Genetic; Trans-Activators

Molecular markers enabling the prediction of sensitivity/resistance to rapamycin may facilitate further clinical development of rapamycin and its derivatives as anticancer agents. In this study, several human ovarian cancer cell lines (IGROV1, OVCAR-3, A2780, SK-OV-3) were evaluated for susceptibility to rapamycin-mediated growth inhibition. The differential expression profiles of genes coding for proteins known to be involved in the mTOR signaling pathway, cell cycle control and apoptosis were studied before and after drug exposure by RT-PCR. In cells exposed to rapamycin, we observed a dose-dependent downregulation of CCND1 (cyclin D1) and CDK4 gene expression and late G1 cell cycle arrest. Among these cell lines, SK-OV-3 cells resistant to both rapamycin and RAD001 were the sole to show the expression of the anti-apoptotic gene Bcl-2. Bcl-2/bclxL-specific antisense oligonucleotides restored the sensitivity of SK-OV-3 cells to apoptosis induction by rapamycin and RAD001. These results indicate that baseline Bcl-2 expression and therapy-induced downexpression of CCND1 and CDK4 may be regarded as molecular markers enabling the prediction and follow-up of the cellular effects on cell cycle and apoptosis induction of rapamycin in ovarian cancer. Furthermore, strategies to down regulate Bcl-2 in ovarian cancer may prove useful in combination with rapamycin or RAD001 for ovarian cancer.

Topics: Antibiotics, Antineoplastic; Blotting, Western; Carcinoma; Cell Death; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Everolimus; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Oligonucleotides, Antisense; Ovarian Neoplasms; Polymerase Chain Reaction; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Sirolimus; Transfection

Lysophosphatidic acid (LPA), at concentrations present in ascitic fluid, indirectly stimulates the growth of malignant ovarian tumors by increasing the expression of vascular endothelial growth factor (VEGF) in ovarian cancer cells. We investigated whether LPA could also directly promote ovarian tumor growth by increasing the level of cyclin D1, a key G1-phase checkpoint regulator, which thereby increases cell proliferation.. Expression of cyclin D1 and LPA receptors (EDG4 and EDG7) was determined in six ovarian cancer cell lines (including OVCAR-3 cells) and immortalized ovarian surface epithelial cells (IOSE-29). Cyclin D1 promoter activity was measured in LPA-treated OVCAR-3 cells cotransfected with cyclin D1 promoter-driven luciferase constructs and cDNA expression plasmids for IkappaBalphaM (a nuclear factor kappaB [NFkappaB] super-repressor).. Four of six cancer cell lines, including OVCAR-3, overexpressed cyclin D1 protein relative to levels in IOSE-29 cells. LPA treatment increased cyclin D1 protein in a dose- and time-dependent manner in OVCAR-3 cells but not in IOSE-29 cells. LPA stimulated cyclin D1 promoter activity (3.0-fold, 95% confidence interval [CI] = 2.7-fold to 3.3-fold). Mutation of the NFkappaB-binding site in the cyclin D1 promoter to block NFkappaB binding and expression of IkappaBalphaM, which binds NFkappaB and inhibits its binding to the promoter, markedly diminished LPA stimulation of cyclin D1 promoter activity (activity stimulated only 1.4-fold, 95% CI = 1.1-fold to 1.7-fold, and 0.7-fold, 95% CI = 0.6-fold to 0.8-fold, respectively). EDG4 was overexpressed in all cancer cell lines studied relative to that in IOSE-29 cells, but EDG7 was overexpressed in only two lines.. Dual mechanisms are probably involved in LPA stimulation of ovarian tumor growth in vivo. In addition to the previously characterized indirect mechanism that increases angiogenesis via VEGF, LPA may directly increase the level of cyclin D1 in ovarian cancer cells, increasing their proliferation.

Topics: Blotting, Northern; Blotting, Western; Carcinoma; Cell Division; Cyclin D1; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Proteins; Luciferases; Lysophospholipids; Mutation; NF-kappa B; Ovarian Neoplasms; Promoter Regions, Genetic; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; Receptors, Vascular Endothelial Growth Factor; RNA, Messenger; RNA, Neoplasm; Serum Response Element; Time Factors; Transfection; Tumor Cells, Cultured; Up-Regulation

Flavopiridol, a cyclin-dependent kinase (cdk) inhibitor, can cause cell cycle arrest, induce apoptosis in cancer cells, and inhibit tumor cell growth in vivo. The present study investigated the in vitro radiosensitizing effect of flavopiridol and the underlying molecular mechanisms in a murine ovarian cancer cell line, OCA-I. Flavopiridol inhibited cell growth in a dose-dependent manner and enhanced cell radiosensitivity assessed by the clonogenic cell survival assay. A flavopiridol dose of 300 nM, given for 1 day, enhanced radiosensitivity by a factor of 2.1. Clonogenic cell survival after split-dose radiation showed that flavopiridol inhibited repair from radiation damage. In addition, flavopiridol treatment (300 nM, 1 day) resulted in decreased levels of Ku70 and Ku86 proteins that play a role in DNA repair processes, suggesting that DNA repair processes may have been disrupted by this agent. Flow cytometry analysis showed that flavopiridol (300 nM, 1 day) accumulated the cells in G(1) and G(2) phases, with a significant reduction in the S phase component. This cell cycle redistribution is likely another mechanism underlying flavopiridol-induced cell radiosensitivity. Flavopiridol down-regulated cyclin D1 and cyclin E protein levels and also inhibited phosphorylation of retinoblastoma protein, which is inconsistent with the observed cell cycle arrest. Among the cdks tested, cdk-9, the catalytic subunit of positive transcription elongation factor b, was significantly down-regulated by flavopiridol, suggesting that flavopiridol may modulate cellular transcription processes. Furthermore, flavopiridol on its own induced apoptosis in the OCA-I cells, whereas in combination with radiation, exerted no additional increase in apoptosis. Taken together, our data show that flavopiridol strongly augmented the response of ovarian carcinoma cells to radiation and that the underlying mechanisms included inhibition of sublethal DNA damage repair and cell cycle redistribution. At the molecular level, transcriptional regulation by flavopiridol may have been involved.

Topics: Animals; Antigens, Nuclear; Antineoplastic Agents; Apoptosis; Caspase 3; Caspases; Cell Cycle; Cyclin D1; Cyclin E; Cyclin-Dependent Kinases; DNA Helicases; DNA Repair; DNA-Binding Proteins; Drug Screening Assays, Antitumor; Enzyme Activation; Enzyme Inhibitors; Female; Flavonoids; Gamma Rays; Ku Autoantigen; Mice; Neoplasm Proteins; Ovarian Neoplasms; Piperidines; Radiation Tolerance; Radiation-Sensitizing Agents

Amplification of chromosomal regions leads to an increase of DNA copy numbers and expression of oncogenes in many human tumors. The identification of tumor-specific oncogene targets has potential diagnostic and therapeutic implications. To identify distinct spectra of oncogenic alterations in ovarian carcinoma, metaphase comparative genomic hybridization (mCGH), array CGH (aCGH), and ovarian tumor tissue microarrays were used in this study. Twenty-six primary ovarian carcinomas and three ovarian carcinoma cell lines were analyzed by mCGH. Frequent chromosomal overrepresentation was observed on 2q (31%), 3q (38%), 5p (38%), 8q (52%), 11q (21%), 12p (21%), 17q (21%), and 20q (52%). The role of oncogenes residing in gained chromosomal loci was determined by aCGH with 59 genetic loci commonly amplified in human tumors. DNA copy number gains were most frequently observed for PIK3CA on 3q (66%), PAK1 on 11q (59%), KRAS2 on 12p (55%), and STK15 on 20q (55%). The 11q13-q14 amplicon, represented by six oncogenes (CCND1, FGF4, FGF3, EMS1, GARP, and PAK1) revealed preferential gene copy number gains of PAK1, which is located at 11q13.5-q14. Amplification and protein expression status of both PAK1 and CCND1 were further examined by fluorescence in situ hybridization and immunohistochemistry using a tissue microarray consisting of 268 primary ovarian tumors. PAK1 copy number gains were observed in 30% of the ovarian carcinomas and PAK1 protein was expressed in 85% of the tumors. PAK1 gains were associated with high grade (P < 0.05). In contrast, CCND1 gene alterations and protein expression were less frequent (10.6% and 25%, respectively), suggesting that the critical oncogene target of amplicon 11q13-14 lies distal to CCND1. This study demonstrates that aCGH facilitates further characterization of oncogene candidates residing in amplicons defined by mCGH.

Topics: Carcinoma; Chromosome Mapping; Chromosomes, Human, Pair 11; Cyclin D1; Female; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Metaphase; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Ovarian Neoplasms; p21-Activated Kinases; Protein Serine-Threonine Kinases; Tumor Cells, Cultured

Malignant transformation of endometriosis, an uncommon phenomenon, can occur in gonadal and extragonadal sites and results in a wide histological range of tumors. Published series reporting malignant transformation of endometriosis have largely been confined to clinical and histopathological discussions with no studies reporting oncoprotein expression and genetic alterations. We report three cases of carcinomas arising in ovarian endometriosis: a serous cystadenocarcinoma, an endometrioid carcinoma with squamous differentiation, and a pure squamous cell carcinoma. Each tumor was analyzed immunohistochemically to compare oncoprotein expression (p53, bcl2, cyclin D1, and c-erb B2) between the tumors and the endometriotic tissue as well as with comparative genomic hybridization (CGH) to compare genetic alterations. All three tumors expressed nuclear p53, in contrast to the endometriotic tissue in which no p53 expression was found. Both endometrial and tumor tissue expressed bcl-2. No expression of cyclin D1 or c-erb B2 was detected in endometriotic or tumoral tissues. The CGH analysis revealed one or two chromosomal aberrations in each of the three tumors with gains on chromosomes 1q, 8q, and 13q, and losses on chromosome 10p. The endometriotic tissue, as expected, showed a normal genetic profile. These results suggest that p53 protein abnormalities and chromosomal aberrations may be involved in malignant transformation of endometriosis in the ovary. However, our results are limited by the number of cases examined and a definite conclusion on the pathogenesis of this process should be followed by future studies with a larger number of cases.

Topics: Adult; Carcinoma, Endometrioid; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Chromosome Aberrations; Cyclin D1; Cystadenocarcinoma, Serous; DNA; Endometriosis; Female; Humans; Immunohistochemistry; Middle Aged; Nucleic Acid Hybridization; Ovarian Diseases; Ovarian Neoplasms; Receptor, ErbB-2; Tumor Suppressor Protein p53

We immunohistochemically analyzed cellular apoptosis susceptibility (CAS) protein expression and compared it with 20q13.2 copy number and the expression of cell cycle-associated proteins retinoblastoma (Rb), cyclin D1, and p53 and prognosis on paraffin-embedded tissue from 69 ovarian carcinomas (OCs). CAS protein reactivity was present in 100%, Rb in 54%, cyclin D1 in 47%, and p53 in 49%. Significant reciprocal correlation was observed between high levels of CAS and histologic type, FIGO (International Federation of Obstetrics and Gynecology) stage III and grade 3, residual tumor (>2 cm), 20q13.2 (ZNF217 gene) amplification (>4 copies in >20% cells), and high expression of cyclin D1 (all P < .05). No association was found between cyclin D1, p53, or Rb levels with clinicopathologic factors. In univariate analysis, residual tumor, FIGO stage and grade, ZNF217 amplification, and CAS levels predicted outcome (all P < .05). In multivariate analysis, stage, grade, amount of residual tumor, and ZNF217 amplification showed independent prognostic value (all P < .05). In OC, alteration of CAS and ZNF217 genes, both located at 20q13, is frequent and relevant prognostically. Cyclin D1, Rb, and p53 seem to have a secondary role.

Topics: Apoptosis; Cellular Apoptosis Susceptibility Protein; Chromosomes, Human, Pair 20; Cyclin D1; Female; Gene Dosage; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Ovarian Neoplasms; Prognosis; Retinoblastoma Protein; Survival Analysis; Trans-Activators; Tumor Suppressor Protein p53

In various cancers, inactivating mutations in the adenomatous polyposis coli or Axin tumor suppressor proteins or activating mutations in beta-catenin's amino-terminal domain elevate beta-catenin levels, resulting in marked effects on T-cell factor (TCF)-regulated transcription. Several candidate beta-catenin/TCF-regulated genes in cancer have been proposed. Expression of a few of these genes has been studied in primary human cancers, but most studies have focused on colon cancers and not on other cancer types that harbor mutational defects in adenomatous polyposis coli, AXIN, or beta-catenin. Mutations leading to beta-catenin deregulation are found in nearly half of ovarian endometrioid adenocarcinomas (OEAs). We report here on the expression of 6 candidate beta-catenin/TCF-regulated genes in a panel of 44 primary OEAs, more than a third of which carry demonstrable defects in beta-catenin regulation. Using quantitative assays of gene expression, we found significantly elevated expression of the MMP-7, CCND1 (Cyclin D1), CX43 (Connexin 43), PPAR-delta, and ITF2 genes in OEAs with deregulated beta-catenin. This correlation was not observed for c-myc, another putative beta-catenin/TCF-regulated gene. Immunohistochemical studies confirmed that overexpression of cyclin D1 and MMP-7 was highly associated with nuclear accumulation of beta-catenin and mutational defects of the Wnt/beta-catenin/TCF-signaling pathway. Our findings indicate cyclin D1, MMP-7, connexin 43, PPAR-delta, and ITF-2, likely play important roles in the pathogenesis of those OEAs that manifest defects in beta-catenin regulation.

Topics: Adult; Aged; beta Catenin; Carcinoma, Squamous Cell; Cyclin D1; Cytoskeletal Proteins; Female; Gene Expression; Gene Expression Regulation; Genes, Neoplasm; Humans; Immunohistochemistry; Matrix Metalloproteinase 7; Middle Aged; Ovarian Neoplasms; Trans-Activators; Transcription Factors

Although early stage ovarian cancer can be effectively treated with surgery and chemotherapy, the majority of cases present with advanced disease, which remains essentially incurable. Unfortunately, little is known about the genes important for the development and progression of this disease. In this study, the expression of 68 phosphatases was determined in immortalized ovarian epithelial cells (IOSE) and compared to ovarian cancer cell lines. CL100, a dual specificity phosphatase, displayed 10-25-fold higher expression in normal compared to malignant ovarian cell lines. Immunohistochemical staining of normal ovaries and 68 ovarian cancer specimens confirmed this differential expression. Re-expression of CL100 in ovarian cancer cells decreased adherent and non-adherent cell growth and induced phenotypic changes including loss of filopodia and lamellipodia with an associated decrease in cell motility. Induced expression of CL100 in ovarian cancer cells suppressed intraperitoneal tumor growth in nude mice. These results show for the first time that CL100 expression is altered in human ovarian cancer, that CL100 expression changes cell morphology and motility, and that it suppresses intraperitoneal growth of human ovarian epithelial cancer. These data suggest that down-regulation of CL100 may play a role in the progression of human ovarian cancer.

Topics: Adenocarcinoma, Papillary; Animals; Blotting, Northern; Blotting, Western; Cell Adhesion; Cell Cycle Proteins; Cell Differentiation; Cell Movement; Cyclin D1; Cystadenocarcinoma, Serous; DNA Primers; Down-Regulation; Dual Specificity Phosphatase 1; Epithelial Cells; Female; Humans; Immediate-Early Proteins; Immunoenzyme Techniques; Luciferases; Mice; Mice, Nude; Ovarian Neoplasms; Phosphoprotein Phosphatases; Phosphoric Monoester Hydrolases; Polymerase Chain Reaction; Protein Phosphatase 1; Protein Tyrosine Phosphatases; RNA; Tumor Cells, Cultured

PTEN (MMAC1/TEP1), a tumor suppressor gene on chromosome subband 10q23.3, is variably mutated and/or deleted in a variety of human cancers. Germline mutations in PTEN, which encode a dual-specificity phosphatase, have been implicated in at least two hamartoma tumor syndromes that exhibit some clinical overlap, Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome. Among several series of ovarian cancers, the frequency of loss of heterozygosity (LOH) of markers flanking and within PTEN, is approximately 30 to 50%, and the somatic intragenic PTEN mutation frequency is <10%. In this study, we screened primary adenocarcinomas of the ovary for LOH of polymorphic markers within and flanking the PTEN gene and for intragenic mutations of the PTEN gene and compared them to PTEN expression using immunohistochemistry. Furthermore, we sought to detect the expression of the presumed downstream targets of PTEN, such as P-Akt, p27, and cyclin D1 by immunohistochemistry. LOH at 10q23 was observed in 29 of 64 (45%) cases. Of the 117 samples, 6 somatic intragenic PTEN mutations, 1 germline mutation, and 1 novel polymorphism were found in 7 (6%) patients. Immunostaining of 49 ovarian cancer samples revealed that 13 (27%) were PTEN immunostain-negative, 25 (51%) had reduced staining, and the rest (22%) were PTEN expression-positive. Among the 44 informative tumors assessed for 10q23 LOH and PTEN immunostaining, there was an association between 10q23 LOH and decreased or absent staining (P = 0.0317). Of note, there were five (11%) tumors with neither mutation nor deletion that exhibited no PTEN expression and 10 (25%) others without mutation or deletion but had decreased PTEN expression. Among the 49 tumors available for immunohistochemistry, 28 (57%) showed P-Akt-positive staining, 24 (49%) had decreased p27 staining, and cyclin D1 was overexpressed in 35 (79%) cases. In general, P-Akt expression was inversely correlated with PTEN expression (P = 0.0083). These data suggest that disruption of PTEN by several mechanisms, allelic loss, intragenic mutation, or epigenetic silencing, all contribute to epithelial ovarian carcinogenesis, and that epigenetic silencing is a significant mechanism. The Akt pathway is prominently involved, but clearly not in all cases. Surprisingly, despite in vitro demonstration that p27 and cyclin D1 lies downstream of PTEN and Akt, there was no correlation between p27 and cyclin D1 expression and PTEN or P-Akt status. Thus, in vivo, although PTEN and

Topics: Carcinoma; Cyclin D1; Female; Genes, Tumor Suppressor; Humans; Immunohistochemistry; Loss of Heterozygosity; Microfilament Proteins; Muscle Proteins; Mutation; Ovarian Neoplasms; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Tumor Suppressor Proteins

The search for new prognostic indicators is especially important in the diagnosis and treatment of ovarian cancer because clinicopathologic criteria currently used to predict survival are largely inadequate. We examined 2 groups of patients with epithelial ovarian cancer, 1 group of long-term survivors (>5 years), and 1 group of short-term survivors (<2 years) for levels of expression of the cell cycle regulators p57(KIP2), cyclin D1, and cyclin E and their relationship with survival. Our findings show that p57(KIP2) is not associated with prognosis, in contrast to p27(KIP1) expression, which is previously shown to be positively associated with long-term survival in univariate analysis (P =.001). Cyclin E expression, in contrast to cyclin D1 expression, is marginally associated with short-term survival in univariate analysis for a group of 53 women. Among the short-term survivors, 15 (65%) of 23 were positive for cyclin E expression, compared with only 11 (37%) of 30 long-term survivors (P = 0.054). This association remained significant (P =.04) in a logistic regression analysis adjusted simultaneously for performance status and extent of residual disease, the 2 strongest predictors of survival in our study. We also found a significant difference in the frequency of the cyclin E staining pattern between nonserous and serous ovarian tumor subtypes (P =.0002). Immunostaining for levels of cyclin E and p27(KIP1) expression may have potential as prognostic markers in the management of ovarian cancer.

Topics: Adenocarcinoma; Biomarkers, Tumor; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p57; Disease-Free Survival; Female; Humans; Immunoenzyme Techniques; Middle Aged; Nuclear Proteins; Ovarian Neoplasms; Retrospective Studies; Survival Rate

The aim of this study was to investigate the occurrence of (pre)neoplastic lesions in overtly normal Fallopian tubes from women predisposed to developing ovarian carcinoma. The presence of (pre)neoplastic lesions was scored in histological specimens from 12 women with a genetically determined predisposition for ovarian cancer, of whom seven tested positive for a germline BRCA1 mutation. A control group included 13 women. Immunohistochemistry was used to determine the expression of p21, p27, p53, cyclin A, cyclin D1, bcl-2, Ki67, HER-2/neu, and the oestrogen and progesterone receptors. Loss of heterozygosity (LOH) analysis on the BRCA1 locus was also assessed on dysplastic tissue by PCR studies. Of the 12 women with a predisposition for ovarian cancer, six showed dysplasia, including one case of severe dysplasia. Five harboured hyperplastic lesions and in one woman no histological aberrations were found in the Fallopian tube. No hyperplastic, dysplastic or neoplastic lesions were detected in the Fallopian tubes of control subjects. In the cases studied, morphologically normal tubal epithelium contained a higher proportion of Ki67-expressing cells (p=0.005) and lower fractions of cells expressing p21 (p<0.0001) and p27 (p=0.006) than in the control group. Even higher fractions of proliferating cells were found in dysplastic areas (p=0.07) and accumulation of p53 was observed in the severely dysplastic lesion. Expression patterns of other proteins studied, including the hormone receptors, were similar in cases and controls. One subject, a germline BRCA1 mutation carrier, showed loss of the wild-type BRCA1 allele in the severely dysplastic lesion. In conclusion, the Fallopian tubes of women predisposed to developing ovarian cancer frequently harbour dysplastic changes, accompanied by changes in cell-cycle and apoptosis-related proteins, indicating an increased risk of developing tubal cancer.

Topics: Adult; Aged; Case-Control Studies; Cyclin A; Cyclin D1; Fallopian Tubes; Female; Genes, bcl-2; Genes, BRCA1; Humans; Ki-67 Antigen; Loss of Heterozygosity; Middle Aged; Ovarian Neoplasms; Polymerase Chain Reaction; Precancerous Conditions; Proliferating Cell Nuclear Antigen; Receptors, Estrogen; Receptors, Progesterone; rho GTP-Binding Proteins; Tumor Suppressor Protein p53

Recent studies have shown that in vitro steady-state expression of the tumor susceptibility gene TSG101 is important for maintenance of genomic stability and cell cycle regulation. To determine the contribution of TSG101 expression in neoplastic formation, expression of TSG101 protein levels were evaluated in primary ovarian and endometrial adenocarcinoma tumors. Expression of TSG101 was also examined in various tumor cell lines (PA-1, AN3CA, HeLa, HS578T, HCT116). Full-length TSG101 protein was detected in these tumors and cell lines indicating that intragenic deletions were not characteristic of TSG101. In addition, TSG101 protein levels were compared with aberrations of prominent cell cycle regulatory molecules such as cyclin D1, cyclin E, p16 and p53. Reduced TSG101 protein was observed in 36% (8/22) of ovarian and 17% (1/6) of endometrial adenocarcinoma. Aberrant levels of p53, p16, cyclin D or E were comparable to published studies indicating that the clinicopathological distribution of these cases did not favor advanced stage tumors. Altogether, these findings suggest that a down-regulation of TSG101 is associated with tumorigenesis in a subgroup of gynecological tumors.

Topics: Adenocarcinoma; Blotting, Western; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p16; DNA-Binding Proteins; Endometrial Neoplasms; Endosomal Sorting Complexes Required for Transport; Female; HeLa Cells; Humans; Ovarian Neoplasms; RNA, Messenger; Transcription Factors; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Defects of the 'Rb/cyclin D1/p16 pathway' have been shown to play a critical role in the development of virtually all human malignancies assessed. To determine the contribution of G1 phase cell cycle defects to ovarian tumorigenesis, we have examined a panel of normal and tumor ovarian tissues and ovarian cancer cell lines for the expression of Rb, p16 and cyclin D1 proteins. Unlike most types of human cancer whose development involves the loss of either Rb or p16 expression, we observed the coexpression of Rb, p16 and cyclin D1 in 82% of ovarian cancer tissues and cell lines. Furthermore, the growth and cell cycle distribution profiles of three ovarian cancer cell lines (ES-2, PA-1 and NIH OVCAR-3) that coexpressed Rb and p16, were found to be unaffected by adenoviral-mediated overexpression of functional p16 protein, indicating the existence of a defect(s) downstream from p16 in these cells. By contrast overexpression of ectopic p16 in the one ovarian cancer cell line (SK-OV-3) that expressed Rb but lacked p16 protein, resulted in a G1 growth arrest. These data suggest that defects of the 'Rb/cyclin D1/p16 pathway', other than the loss of Rb or p16, may play a major role in the development of ovarian cancer.

Topics: Adenoviridae; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Ovarian Neoplasms; Retinoblastoma Protein; Tumor Cells, Cultured

Inheritance of a mutant allele of the breast cancer susceptibility gene BRCA1 confers increased risk of developing breast and ovarian cancers. Likewise, inheritance of a mutant allele of the retinoblastoma susceptibility gene (RB1) results in the development of retinoblastoma and/or osteosarcoma, and both alleles are often mutated or inactivated in sporadic forms of these and other cancers. We now demonstrate that the product of the RB1 gene, Rb, regulates the expression of the murine Brca1 and human BRCA1 genes through its ability to modulate E2F transcriptional activity. The Brca1 gene is identified as an in vivo target of E2F1 in a transgenic mouse model. The Brca1 promoter contains E2F DNA-binding sites that mediate transcriptional activation by E2F1 and repression by Rb. Moreover, ectopic expression of cyclin D1 and Cdk4 can stimulate the Brca1 promoter in an E2F-dependent manner, and this is inhibited by coexpression of the p16(INK4a) cyclin-dependent kinase inhibitor. The human BRCA1 promoter also contains a conserved E2F site and is similarly regulated by E2F1 and Rb. This functional link between the BRCA1 and Rb tumor suppressors may provide insight into the mechanism by which BRCA1 inactivation contributes to cancer development.

Topics: Animals; BRCA1 Protein; Breast Neoplasms; Carrier Proteins; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Female; Gene Expression Regulation, Neoplastic; Keratinocytes; Mice; Mice, Transgenic; Ovarian Neoplasms; Promoter Regions, Genetic; Proto-Oncogene Proteins; Repressor Proteins; Retinoblastoma; Retinoblastoma Protein; Retinoblastoma-Binding Protein 1; RNA, Messenger; Skin; Transcription Factor DP1; Transcription Factors; Transcriptional Activation

In ovarian carcinomas, alterations of the chromosomal region 20q13 and the cyclin D1 gene have been described. This study has sought to determine their prognostic significance. Fluorescence in situ hybridization (FISH) on dissociated nuclei and paraffin sections with DNA probes for 20q13.2 and cyclin D1, as well as immunohistochemistry (cyclin D1), were applied to formalin-fixed tissue of 69 invasive ovarian carcinomas, mainly of serous type. On dissociated nuclei 33/47 cases (70%) and on tissue sections 13/66 cases (20%) demonstrated an increase of 20q13.2 copies. The presence of > or =4 copies per nucleus (isolated nuclei) and > or =3 copies per nucleus (sections) was associated with an adverse prognosis (Kaplan-Meier for FIGO stage III after stratification for residual tumour: p=0.0049 and p=0.03, respectively). Thirty-four out of 47 cases (72%) showed an increase of cyclin D1 copies. Kaplan-Meier analysis for FIGO stage III after stratification for residual tumour>2 cm or < or =2 cm revealed an unfavourable outcome for cases with more than two cyclin D1 copies (p=0.04). No correlation was seen between FISH and immunohistochemistry. Multivariate analysis identified residual tumour (p=0.0002), 20q13.2 gain (p=0.0004) and cyclin D1 gain (p=0.0343) as independent prognostic factors. It is concluded that gains of chromosomal region 20q13.2 and the cyclin D1 gene are frequent and biologically important events, with prognostic relevance, in advanced ovarian carcinomas.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Chromosomes, Human, Pair 20; Cyclin D1; DNA, Neoplasm; Female; Follow-Up Studies; Gene Amplification; Humans; In Situ Hybridization, Fluorescence; Middle Aged; Neoplasm Proteins; Ovarian Neoplasms; Ploidies; Prognosis; Survival Rate

Stat 3 functions in transducing signals from the cell's surface to its nucleus and activation of gene transcription. Aberrations of Stat 3 in breast cancer have raised the possibility of its contribution to oncogenesis. Our goal was to examine ovarian cancer cell lines to determine whether Stat 3 plays a relevant role in ovarian carcinogenesis.. Protein lysates were extracted from normal ovarian surface epithelial cells and malignant cells. Western blotting techniques were performed with phosphorylation-independent or phosphorylation-specific Stat 3 (tyrosine 705) antibody. Confirmation of Stat 3 activation was determined by a luciferase reporter driven by a promoter containing Stat 3-specific binding sites. Bcl-x(L) and cyclin D(1) were also analyzed by Western blotting.. MDAH 2774, OV-1063, Caov-3, and O.C. 22819 expressed high levels of phosphorylated Stat 3. In contrast, A2780 and normal ovarian surface epithelial cells had little Stat 3 phosphorylation recognized. Confirmation of persistent activation of Stat 3 activity was shown by transfection of cells with a Stat 3 luciferase reporter. Potential downstream mediators of Stat 3 including Bcl-x(L) and cyclin D(1) were also evaluated. In cells expressing activated Stat 3, high levels of both Bcl-x(L) and cyclin D(1) were detected, whereas in A2780 cells, which did not express activated Stat 3, only low levels of Bcl-x(L) and cyclin D(1) were expressed.. Constitutive activation of Stat 3 is present in ovarian cancer lines but not in normal ovarian surface epithelial cells. Activation of Stat 3 is a common event during oncogenic transformation upstream to both Bcl-x(L) and cyclin D(1). The relationship of this aberrancy of ovarian carcinoma harboring activated Stat 3 deserves further investigation.

Topics: bcl-X Protein; Cell Transformation, Neoplastic; Cyclin D1; DNA-Binding Proteins; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Ovarian Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Transcriptional Activation; Tumor Cells, Cultured

Trimidox (3,4,5-trihydroxybenzohydroxamidoxime), a recently synthesized inhibitor of ribonucleotide reductase (RR), was shown to exert anti-proliferative activities in HL-60 and K562 human leukemia cell lines and to prolong the life span of mice inoculated with L1210 mouse leukemia cells. Here we test whether trimidox also exhibits anti-neoplastic properties in ovarian carcinoma cells. Since the mode of action of trimidox on cell fate has not been investigated so far, we addressed this unresolved item and find that this polyhydroxybenzoic acid derivative induces apoptosis of N.1 human ovarian carcinoma cells when tested in growth factor deprived medium. Utilizing an improved analysis, based on Hoechst 33258/propidium iodide double staining, apoptosis is quantified and discriminated from necrosis. Trimidox induces c-myc expression, which is indispensible for apoptosis of N.1 cells, and expression of plasminogen activator/urokinase type (upa), which supports the apoptotic process under more physiological conditions. Surprisingly, trimidox does not block dNTP synthesis in N.1 cells at the concentrations tested and, therefore, trimidox induces apoptosis independent of RR-inhibition. Like TNFalpha or benzamide riboside, which are also inducers of apoptosis of N.1 cells, trimidox also down-regulates the G1 cell cycle phosphatase cdc25A, whereas cyclin D1 becomes up-regulated. This report shows that trimidox destroys human ovarian carcinoma cells by inducing them to undergo apoptosis as well as corroborating previous investigations which demonstrated that apoptosis of these cells depends on c-myc over-expression when survival factors are withdrawn.

Topics: Antineoplastic Agents; Apoptosis; Benzamidines; cdc25 Phosphatases; Cyclin D1; Deoxyribonucleotides; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Female; Gene Expression; Genes, cdc; Genes, myc; HL-60 Cells; Humans; Ovarian Neoplasms; Ribonucleotide Reductases; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Using differential display PCR, we have identified a gene [NOEY2, ARHI (designation by the Human Gene Nomenclature Committee)] with high homology to ras and rap that is expressed consistently in normal ovarian and breast epithelial cells but not in ovarian and breast cancers. Reexpression of NOEY2 through transfection suppresses clonogenic growth of breast and ovarian cancer cells. Growth suppression was associated with down-regulation of the cyclin D1 promoter activity and induction of p21(WAF1/CIP1). In an effort to identify mechanisms leading to NOEY2 silencing in cancer, we found that the gene is expressed monoallelically and is imprinted maternally. Loss of heterozygosity of the gene was detected in 41% of ovarian and breast cancers. In most of cancer samples with loss of heterozygosity, the nonimprinted functional allele was deleted. Thus, NOEY2 appears to be a putative imprinted tumor suppressor gene whose function is abrogated in ovarian and breast cancers.

Topics: Amino Acid Sequence; Breast Neoplasms; Carcinoma; Chromosomes, Human, Pair 1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Genes, ras; Genes, Tumor Suppressor; Genomic Imprinting; Germ-Line Mutation; Growth Inhibitors; Humans; Loss of Heterozygosity; Molecular Sequence Data; Mothers; Ovarian Neoplasms; Point Mutation; Polymorphism, Single-Stranded Conformational; rho GTP-Binding Proteins; Sequence Homology, Amino Acid

Mammalian cell-cycle progression is regulated by the combined action of cyclins/cyclin-dependent kinases (cdks) and cdk inhibitors. Abnormal expression as well as interaction of these proteins may result in malignant transformation of cells. To further address alterations and roles of these cell-cycle proteins in the development of epithelial ovarian carcinomas, we analyzed the expression of the p27(kip1), cyclin D1, cyclin E, and cdk2. A panel of 79 epithelial ovarian tumors was selected. Immunohistochemical staining of serial paraffin sections was performed using antibodies to p27(kip1), cyclin D1, cyclin E, and cdk2. The results showed that p27(kip1) and cyclin D1 were concurrently expressed in epithelial ovarian tumors, and the expression was down-regulated in ovarian carcinomas. There was an inverse relationship between the expression level of p27(kip1) and cyclin D1 and the histological tumor grades. On the other hand, the expression of cyclin E and cdk2 was enhanced in ovarian carcinomas. The results suggest that low expression of p27(kip1) and cyclin D1 as well as high expression of cyclin E and cdk2 promotes the development of ovarian tumors. p27(kip1) and cyclin D1 expression are negatively correlated with the malignant degree of epithelial ovarian tumors. Thus, the ovarian tumors with high p27(kip1) and cyclin D1 expression may generally have a somewhat better prognosis, while those with low p27(kip1) and cyclin D1 expression may have a worse prognosis.

Topics: CDC2-CDC28 Kinases; Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Microtubule-Associated Proteins; Ovarian Neoplasms; Protein Serine-Threonine Kinases; Tumor Suppressor Proteins

Alterations in the expression of cyclin D1 have been reported frequently in several human cancers, but their significance in the multistep model of carcinogenesis has been scantly described. To define the pattern of cyclin D1 expression in the development of ovarian cancer and clinical outcome, 55 cases of benign ovarian tumors, 12 borderline cases, and 37 ovarian carcinomas (32 primary and 5 recurrent carcinomas) were studied. Analyses were carried out on fresh tumor specimens by Western blotting and reverse transcription-PCR and provided significant superimposable results (P = 0.00001). Cyclin D1 abundance was classed according to the densitometric values as undetectable, detectable, well detectable, and highly detectable. A significant increase (P < 0.000001) in median cyclin D1 values was observed from benign (0.038; range, 0.001-0.705) to borderline (0.226; range, 0.001-0.623) to malignant (0.347; range, 0.027-2.330) to recurrent (0.887; range, 0.309-2.2260) tumors. In addition, higher median cyclin D1 values were reported in serous carcinomas (P = 0.058) and advanced-stage diseases (P = 0.003). Survival analyses carried out in the 32 primary carcinomas showed no significant difference in overall survival between detectable versus well/highly detectable cyclin D1 neoplasms. Conversely, a significant relationship between cyclin D1 expression and progression-free survival was found (P = 0.031). These results may elucidate the function of altered cyclin D1 expression in ovarian tumorigenesis and provide a basis for additional studies on its prognostic role.

Topics: Biomarkers, Tumor; Cyclin D1; Female; Humans; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Ovarian Neoplasms; Survival Rate

Using a semiquantitative telomeric repeat amplification protocol assay, telomerase-positive frequencies and enzyme levels were measured. Out of 95% of 49 human ovarian tumors, the highest level of telomerase activity was observed in malignant tumors. Furthermore, by immunohistochemical staining of cell cycle regulatory proteins (pRB, p16, cyclin D1, cyclin E and p53) at the G1 checkpoint, we evaluated the relation between each protein alterations and the levels of telomerase activity. We could not demonstrate a clear relation with each molecule except for cyclin E, but suggesting that aberrant accumulation of these proteins was considered as a reason for telomerase deregulation, which may play an essential role in the pathway of telomerase regulation.

Topics: Adult; Aged; Aged, 80 and over; Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p16; Female; G1 Phase; Humans; Immunohistochemistry; Middle Aged; Neoplasm Staging; Ovarian Neoplasms; Polymerase Chain Reaction; Telomerase; Tumor Suppressor Protein p53

We have previously reported on the development of an in vitro model system for studying the effect of hypoxia on ovarian carcinoma cell proliferation and invasion (Krtolica and Ludlow, 1996). These data indicate that the cell division cycle is reversibly arrested during the G1 phase. Here, we have continued this study to include the proliferation properties of both aerobic and hypoxic human ovarian carcinoma cells at the molecular level. The growth suppressor product of the retinoblastoma susceptibility gene, pRB, appears to be functional in these cells as determined by SV40 T-antigen binding studies. Additional G1-to-S cell cycle regulatory proteins, cyclins D and E, cyclin-dependent kinases (cdks) 4 and 2, and cdk inhibitors p27 and p18, also appear to be intact based on their apparent molecular weights and cell cycle stage-specific abundance. During hypoxia, there is a decrease in abundance of cyclins D and E, with an increase in p27 abundance. cdk4 activity towards pRB and cdk2 activity towards histone H1 are also decreased. Co-precipitation studies revealed an increased amount of p27 complexing with cyclin E-cdk2 during hypoxia than during aerobic cell growth. In addition, pRB-directed phosphatase activity was found to be greater in hypoxic than aerobic cells. Taken together, a model is suggested to explain hypoxia-induced cell cycle arrest in SKA human ovarian carcinoma cells.

Topics: Aerobiosis; Antigens, Polyomavirus Transforming; CDC2-CDC28 Kinases; Cell Cycle; Cell Division; Cell Hypoxia; Cyclin A; Cyclin D1; Cyclin D2; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; Female; G1 Phase; Humans; Microfilament Proteins; Muscle Proteins; Ovarian Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Retinoblastoma Protein; Simian virus 40; Tumor Cells, Cultured

A significant positive association has been reported between p16 expression and clinical outcome for epithelial ovarian cancer patients. However, there is a reciprocal correlation between genetic alterations of single members of the p16-cyclin D1/CDK4-pRb pathway (G1 pathway). Simultaneous evaluation of these four elements may produce a better prognostic factor than p16 alone. We studied the prognostic significance of the G1 pathway in 59 epithelial ovarian cancer patients undergoing surgery and platinum-based chemotherapy by immunohistochemical technique. Abnormal expression of p16 or pRb was defined by negative nuclei staining, and that of CDK4 and cyclin D1 was defined by 50% nuclear staining. An abnormal G1 pathway was indicated in cases that have at least one abnormality among these four elements. Abnormal expression of p16, pRb, and cyclin D1/CDK4 was observed in 33.9, 3.4, and 15.3% of studied cases, respectively. Abnormal G1 pathway was detected in 49.2% (29 of 59) of all cases. The patients with normal G1 pathway tended to achieve a higher complete response rate (81.0%) to chemotherapy, compared with patients with abnormal G1 pathway (55.0%); however, there was no significant difference (P = 0.1001) between the two groups. Univariate analyses identified advanced stage [hazards ratio (HR), 3.665; P = 0.0218], histological low grade (HR, 3.625; P = 0.0066), and abnormal G1 pathway (HR, 2.935; P = 0.03) as prognostic factors for overall survival. The G1 pathway might help as a prognostic factor to select high-risk patients.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cisplatin; Combined Modality Therapy; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Cyclophosphamide; Doxorubicin; Female; Humans; Immunohistochemistry; Middle Aged; Ovarian Neoplasms; Prognosis; Proto-Oncogene Proteins; Retinoblastoma Protein; Survival Analysis; Treatment Outcome

The scheduled expressions of cyclins are observed in the normal cells or the tumor cells whose phenotype is characterized by scheduled expression of cyclins, while unscheduled expression of cyclins were reported in several leukemic and solid tumor cell lines by anti-cancer drugs. We studied the effects of cytotoxic concentrations of Taxol (TXL) on cyclin D1 and B1 expression on human ovarian cancer cell lines. In KFr13 cells, the control group showed low degree of cyclin D1 and moderate degree of cyclin B1 expression in all cell cycles, while 1 microM TXL exposure resulted remarkable cyclin D1 and B1 expression in G2+M phase cells. OVCAR-3 cells showed relatively high degree of cyclin D1 expression and mild to moderate degrees of cyclin B1 expression in control group. 1 microM TXL showed no significant changes in cyclin D1 expression, while decreased expression cyclin B1 in G0+1 and S and moderate degree of expression in G2+M.

Topics: Antineoplastic Agents; Cell Cycle; Cells, Cultured; Cyclin B; Cyclin B1; Cyclin D1; Cyclins; Female; Humans; Ovarian Neoplasms; Paclitaxel; Tumor Cells, Cultured

Amplification and overexpression of the cell cycle-related gene cyclin D1 have been demonstrated in several human malignancies and have been shown to be directly oncogenic in breast epithelium and lymphocytes. Overexpression of the gene can occur in the absence of gene amplification. We have investigated whether cyclin D1 is overexpressed in a panel of 43 sporadic epithelial ovarian cancers using immunohistochemistry. Cyclin D1 was overexpressed in 26% of these tumors. Overexpression of cyclin D1 is associated with borderline or well-differentiated, grade 1 tumors but does not correlate with a particular histological type, overexpression of the c-erb-B2 oncogene, or presence of estrogen receptors. It is suggested that overexpression of cyclin D1 may contribute to the pathogenesis of epithelial ovarian cancers, including a subset of tumors different from those overexpressing the c-erb-B2 oncogene.

Topics: Cyclin D1; Cyclins; Cystadenoma; Female; Gene Expression Regulation, Neoplastic; Genes, erbB-2; Humans; Oncogene Proteins; Ovarian Neoplasms; Receptors, Estrogen

Cyclin D1 is a cell cycle regulator of G1 progression that has been suggested to play a relevant role in the pathogenesis of several human cancer types. In the current study, the expression of cyclin D1 has been investigated in a series of 33 patients, with benign (10 patients), borderline (five patients) and malignant (18 patients) ovarian disease. Cyclin D1 protein and mRNA content were analysed by Western blotting and reverse transcriptase polymerase chain reaction respectively. The levels of cyclin D1 protein were undetectable in patients with benign disease, detectable in the majority of patients with borderline disease and elevated in those with ovarian carcinomas, being significantly related to the degree of malignancy (carcinoma vs benign, P = 0.0001; benign vs borderline, P = 0.0238). A significant relationship between cyclin D1 expression and tumour proliferative activity was also found (P = 0.000001). Moreover, eight benign lesions, two borderline tumours and 11 carcinomas proved to be suitable for the analysis of cyclin D1 transcript, and emerging data demonstrated significant agreement between protein abundance and mRNA expression. Results from the current study suggest that cyclin D1 expression is associated with the degree of transformation and most probably plays a role in the early development of ovarian malignancy.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Blotting, Western; Carcinoma; Cell Division; Cell Transformation, Neoplastic; Cyclin D1; Cyclins; DNA Primers; Female; Humans; Middle Aged; Oncogene Proteins; Ovarian Neoplasms; Polymerase Chain Reaction; RNA, Messenger; Thymidine; Tumor Cells, Cultured

We analyzed the expression and amplification of cyclin D1 and CDK4 genes in ovarian carcinomas. Northern blot analysis revealed overexpression of cyclin D1 in 12 of 65 (18%) ovarian carcinomas while CDK4 was overexpressed in 7 of 48 cases (14%). None of the tumors showed amplification of any of the 2 genes. Overexpression of cyclin D1 and CDK4 transcripts was correlated, suggesting a role of both genes in altered growth control of ovarian cancer cells. Elevated levels of cyclin D1 were significantly associated with a well-moderately differentiated grade (G1-G2) (p < 0.005). No significant association was found between cyclin D1 expression and estrogen receptor, progesterone and epidermal growth factor receptor content. Cyclin D1 expression does not appear to be associated with clinical outcome in human ovarian cancer, although a longer follow-up period and screening of other molecules involved in the same pathway would be necessary to assess this hypothesis.

Topics: Age Factors; Biomarkers, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; Disease Progression; Epithelium; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Neoplasm Staging; Oncogene Proteins; Ovarian Neoplasms; Predictive Value of Tests; Proto-Oncogene Proteins; Receptors, Estrogen; Receptors, Progesterone; Recurrence; Regression Analysis; Statistics, Nonparametric; Tumor Cells, Cultured

Down-regulation of oncogene expression is one of the hallmarks of the process whereby transformed cells are forced into differentiation and/or growth arrest by potent inducers and therefore can represent an interim end point in cancer treatment. The differentiation inducer sodium butyrate (NaB) arrested growth of N.1 ovarian carcinoma cells and repressed expression of cyclin D1/prad1 and the invasiveness-related protease plasminogen activator-urokinase (plau). This was accompanied by the acquisition of a differentiated morphology, all of which characteristics were maintained as long as N.1 cells were exposed to the inducer. In accordance with a differentiated phenotype was the finding that fibronectin expression was increased significantly. Recently, it was shown that NaB represses the transcription factor c-myc by blocking Ca2+ signals and modulating serine threonine kinase activity. We wanted to investigate NaB-mediated interference on signals contributing to the expression on prad1, plau and growth arrest-specific 6 (gas6). Protein kinase A (PKA) inactivation de-repressed prad1 and plau transcript levels. NaB had onlygeneral but no specific influence on PKA-modulated prad1 and plau expression however. Protein kinase C activation up-regulated plau transcript levels, but not that of prad1. Prad1 expression seemed to depend on Ca2+-triggered signals. Constitutive plau expression was insensitive to additional Ca2+-mediated signals, but it became responsive upon NaB treatment.

Topics: Adenylyl Cyclases; Butyrates; Butyric Acid; Cell Division; Cell Line; Colforsin; Cyclic AMP-Dependent Protein Kinases; Cyclin D1; Cyclins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; Isoquinolines; Neoplasm Invasiveness; Oncogene Proteins; Oncogenes; Ovarian Neoplasms; Protein Kinase C; Proteins; RNA, Messenger; Signal Transduction; Sulfonamides; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

The alteration of cyclin D1 gene expression is a common feature of malignancies of diverse histogenesis. Our recent results showed that over expression of cyclin D1 protein is frequently found in human ovarian cancer. In this study, we investigated the effect of cyclin D1 antisense oligodeoxynucleotides on cell growth and gene expression in PA-1 ovarian cancer cells. Our results demonstrated that cyclin D1 antisense oligodeoxynucleotides indeed functioned as an antiproliferative agent. More strikingly, we found that different cyclin D1 antisense oligodeoxynucleotides exert different efficacy on the inhibition of cell growth and gene expression. We suggest that selection and characterization of high affinity oligodeoxynucleotides is strictly required before the application of antisense technology.

Topics: Base Sequence; Cell Cycle; Cell Division; Cell Line; Cyclin D1; Cyclins; Female; Flow Cytometry; Gene Expression; Humans; Molecular Sequence Data; Oligonucleotides, Antisense; Oncogene Proteins; Ovarian Neoplasms; RNA, Messenger; Suppression, Genetic; Thionucleotides; Tumor Cells, Cultured

Evidence of the involvement of cyclin genes in genetic alterations in human cancer is growing. In the present study, we investigated the amplification, in human breast and ovarian cancer, of 5 cyclin genes; cyclin A, cyclin D1, cyclin D2, cyclin D3 and cyclin E. For this purpose, a series of 1,171 breast and 237 ovarian tumors tested for DNA amplification by Southern blotting and a subset of 132 breast and 22 ovarian cancers were analyzed for RNA expression levels by slot-blot and Northern blotting. In breast tumors, only cyclin D1 was found to be activated in a sizeable fraction of the tumors (amplification 12.6%, overexpression 19%). Cyclin A, D2, D3, and E genes never, or only on rare occasions, showed increased DNA copy numbers and were never found overexpressed at the RNA level. Amplification of cyclin D1 correlated with ER+ breast cancer and the presence of lymph-node metastasis. Interestingly, we were also able to determine an association with invasive lobular carcinoma. Our data suggest that cyclin D1 activation determines the evolution of a particular subset of estrogen-responsive tumors. Data obtained in ovarian tumors contrasted with observations in breast cancer. Cyclin D1 DNA amplification was much less frequent in ovarian than in breast tumors (3.3% vs. 12.6%), whereas cyclin E amplification and overexpression were observed in a significant number of cases (12.5% and 18.0% respectively). Cyclin A, cyclin D2 and D3 rarely showed anomalies at the DNA level and were never overexpressed. No clear correlation could be observed between amplification of the cyclin E gene and tumor type, stage or grade in ovarian cancer. Data presented here suggest distinct pathways of cyclin activation in human breast and ovarian cancer.

Topics: Blotting, Northern; Blotting, Southern; Breast Neoplasms; Cyclin D1; Cyclins; DNA, Neoplasm; Female; Gene Amplification; Gene Expression; Humans; Oncogene Proteins; Ovarian Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; RNA, Messenger; Tumor Cells, Cultured

The expression of cyclins, cyclin-dependent kinases (cdk), and cdk inhibitors was evaluated in clones from a human ovarian cancer cell line transfected with a temperature-sensitive mutant of p53, after treatment with the anticancer agents doxorubicin (DX) and AMSA. The two drugs were selected on the basis of their activity in these clones, since AMSA is equally active in cells expressing mutated or wild-type (wt) p53, while DX was much less cytotoxic in cells expressing wt p53. In untreated cells, the expression of wt p53 induced an accumulation of cells in the G2 and perhaps also the G1 phase of the cell cycle. Concomitantly cyclin B1 and cdc2 increased. Cyclin E and particularly D1 levels were also raised by wt p53 expression. Treatment of mutated p53-expressing cells (SK23a cells kept at 37 degrees C) with DX or, more so, with AMSA, resulted in a strong accumulation of cyclin B1 and cdc2, in accordance with their ability to block cells in G2 phase of the cell cycle. Wt p53-expressing cells (SK23a cells kept at 32 degrees C) treated with the drugs showed an increase in p21 expression and consequently decreased kinase activity after immunoprecipitation with p21 antibodies. Cdc2-associated kinase activity was also reduced in these conditions. We could also observe a decrease in the percentage of cells in G1 and G2 phases and an accumulation of cells in S phase after both DX and AMSA. Cdk2, retinoblastoma, and p27 levels did not change significantly. Treatment with DX or AMSA caused similar effects, suggesting that p53-induced changes in cyclin, cdk, and cdk inhibitors after DNA damage are not responsible for the marked reduction in the cytotoxicity of DX we observed in wt p53-expressing cells.

Topics: Amsacrine; Antibiotics, Antineoplastic; Antineoplastic Agents; Blotting, Northern; CDC2 Protein Kinase; Cyclin B; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinases; Cyclins; DNA Damage; DNA, Neoplasm; Doxorubicin; Female; Gene Expression Regulation, Neoplastic; Humans; Mutation; Oncogene Proteins; Ovarian Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The expression of cyclin D1 gene product in human ovarian tumors was studied. We found that cyclin D1 is expressed at high levels in several ovarian cancer cell lines. Immunohistochemical study also showed that a significant proportion of primary ovarian tumor tissues overexpressed cyclin D1 gene product. Clear nuclear staining of cyclin D1 protein was detected in 28% of the cases. We also characterized the expression of c-Ki-ras gene product in ovarian cancer cell lines and tumor tissues. Amplification or overexpression of this proto-oncogene has been reported in ovarian tumors from Taiwan. These results show that c-Ki-ras is strongly expressed in PA-1 and NIH:OVCAR-3 cells in which cyclin D1 also expressed at high levels. Specific cytoplasmic staining of c-Ki-ras protein was detected in 11 tumors (52%). Statistical analyses show a strong positive correlation between cyclin D1 and c-Ki-ras immunoexpression. Thus, these data support the ideas that cyclin D1 may be involved in the pathogenesis of ovarian cancer, and coactivation of cyclin D1 and c-Ki-ras gene expression may represent one of the major pathways that lead to the development of ovarian cancer in Taiwan.

Topics: Carcinoma; Cyclin D1; Cyclins; Female; Humans; Immunohistochemistry; Oncogene Proteins; Ovarian Neoplasms; Proto-Oncogene Mas; Proto-Oncogene Proteins p21(ras); Staining and Labeling; Tumor Cells, Cultured