Ovarian Epithelial Cells Research Articles

Abstract 2858: Mirk/dyrk1B kinase inhibition increases ROS and apoptosis in ovarian cancer cells, but has little effect on normal diploid cells

Abstract Quiescent cancer cells in vivo, including stem cell populations, are relatively insensitive to most chemotherapeutic drugs and radiation. Tumor cells in culture can be placed in G0 by serum or nutrient deprivation, and then can be targeted by a small molecule Mirk kinase inhibitor. Mirk/dyrk1B levels are elevated up to 10-fold in quiescent tumor cells where Mirk blocks cell cycling and increases viability by decreasing reactive oxygen species (ROS). In prior studies Mirk was detected in 75% of ovarian cancers, and was amplified in a subset. In contrast, Mirk expression was quite low in most normal cell types and Mirk knockout or depletion had no detectable effects on normal cells. Response to a Mirk kinase inhibitor was compared between two strains of normal diploid fibroblasts, early passage cultures of normal human ovarian epithelial cells, and a series of ovarian cancer cell lines where Mirk had been shown to be an active kinase. Serum-starved normal diploid cells entered a G0 quiescent state and had minimal response to the Mirk inhibitor, but serum-starved cancer cells were kept in cycle by the Mirk inhibitor, which reduced E2F4 sequestration by p130/Rb2 by 2-3fold. Mirk inhibitor treated cancer cultures had several-fold more sub-G0/G1 apoptotic cells and about 30-fold higher levels of the apoptotic proteins cleaved PARP and cleaved caspase 3 compared with normal diploid cells. The Mirk inhibitor increased total ROS levels and superoxide levels only in the cancer cells, in a rapid time-course of 2-3 days. Cancer cell loss after Mirk kinase inhibitor treatment was due at least in part to ROS, since the ROS scavenger N-acetyl cysteine reduced the amount of cleaved PARP and the extent of cancer cell loss. The Mirk inhibitor decreased the EC50 of cisplatin up to 9-fold in ovarian cancer cells. Thus ovarian cancer cells, which exhibit elevated Mirk expression/amplification and active Mirk kinase, were shown to be more susceptible to the Mirk kinase inhibitor than normal diploid cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2858. doi:1538-7445.AM2012-2858

Abstract 3649: Expression of CXCR5 and its natural ligand CXCL13 in ovarian cancer

Abstract Ovarian carcinoma (OvCa) is the most lethal gynecological malignancy among women and its poor prognosis is mainly due to metastasis. Chemokines and their corresponding receptors play an important role in metastasis and organ specific homing of cancer cells including OvCa. CXCL13 is a homeostatic chemokine for lymphocyte- and prostate cancer cell-homing and -positioning within secondary lymphoid tissues. This important chemokine acts through its only known receptor, CXCR5. In this study, we provide the first evidence that in OvCa CXCR5 and CXCL13 are highly elevated in OvCa cell lines and clinical samples. Flow cytometry analysis of OvCa cell lines (OVCAR-3 and SK-OV-3) show significantly higher expression of CXCR5 in comparison to normal adult ovarian epithelial cells. To determine the clinical significance of CXCR5 expressed by OvCa cell lines, ovarian cancer tissue microarrays were stained for CXCR5 and CXCL13. Aperio ScanScope scanning system was used to quantify the immuno-histochemical staining of CXCR5 and CXCL13. Our results show significantly higher expression of CXCR5 and CXCL13 (p < 0.001) in cancerous tissue than compared to non-neoplastic ovarian tissue. Among cancerous tissues, CXCR5 and CXCL13 expression was highest in serous adenocarcinoma followed by serous papillary cystadenoma, endometrioid adenocarcinoma, mucinous adenocarcinoma, cystadenoma, mucinous boderline adenocarcinoma, clear cell carcinoma, granulosa cell tumor, dysgerminoma, transitional cell carcinoma, brenner tumor, yolk sac tumor, adenocarcinoma and fibroma. These data demonstrate biological and clinical significance of CXCR5 and CXCL13 as an OvCa biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3649. doi:1538-7445.AM2012-3649

Abstract 2928: Functional effects of SNPs in non-coding RNAs at the 3q25 ovarian cancer susceptibility locus

Abstract Genome-wide association studies have identified six common, low penetrance susceptibility loci for epithelial ovarian cancer (EOC). The functional mechanisms underlying disease susceptibility at these loci are unknown. SNPs at the 3q25 locus confer a 1.2-fold risk of serous EOC (P=7.1x10-8). To identify causal SNP(s) we used the 1000 genomes project to identify 952 correlated SNPs within a 884kb linkage disequilibrium region. Of these, 786 tagging SNPs were genotyped in ∼20,000 invasive EOC cases and 20,000 controls. Fine mapping limited the size of the risk region to ∼130kb and 43 possible causal SNPs. This region contains four protein-coding genes; none of the 43 SNPs was predicted to have a functional effect on the translated protein. Genomic profiling data from The Cancer Genome Atlas analysis of ∼500 serous EOCs indicated one gene in the region (TIPARP) was significantly down regulated compared to normal tissues (P=0.005). Using RT-PCR expression analysis, we found significantly lower TIPARP expression in EOC cell lines (N=37) compared to normal ovarian surface epithelial (OSE) and fallopian tube epithelial (FTE) cell lines (N=39) (P=1.57x10-8). TIPARP was also significantly downregulated in an in vitro model of early stage transformation of OSE cells (P=0.0003). Collectively, these data suggest that TIPARP is a novel tumor suppressor gene (TSG) in EOC development. TIPARP is a poly(ADP-ribose) polymerase known to catalyze histone ribosylation, an epigenetic modification that plays a role in DNA repair. We evaluated its functional significance in more detail using stable lentiviral-mediated shRNA knockdown of TIPARP in normal OSE/FTE cells. TIPARP knockdown induced cellular senescence similar to the effects of many classical TSG knockdowns in normal cells. We hypothesized that TIPARP may be involved in cellular response to DNA damage; when normal OSE/FTE cells were exposed to α-irradiation TIPARP was significantly upregulated in a dose-dependent manner. We examined the functional link between susceptibility SNPs and TiPARP expression. Of 43 putative causal SNPs, four fell within two non-coding RNAs- LOC730091 and TIPARP-AS1. There is no homology between LOC730091 and genes at the 3q25 locus; but TIPARP-ASI is a non-coding RNA with antisense sequence similarity to exon 1 of TIPARP. LOC730091 and TIPARP-AS1 were variably expressed in both normal ovarian and EOC cell lines. We found no correlation between LOC730091 and TIPARP expression; but cell lines expressing TIPARP-AS1 showed significantly reduced TIPARP expression. In summary, fine mapping at the 3q25 has identified multiple possible causal SNPs. Functional studies implicate TIPARP as a plausible target TSG in this region. EOC risk associated SNPs were located in a non-coding RNA that causes reduced expression of TIPARP suggesting a mechanism by which susceptibility SNPs may confer neoplastic transformation of ovarian epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2928. doi:1538-7445.AM2012-2928

Abstract LB-14: Raf-1 in epithelial-mesenchymal transition: a novel therapeutic target for ovarian and endometrial cancers

Abstract Ovarian and endometrial cancers are the 5th and 8th overall leading causes of female cancer related deaths in the US, respectively. According to the American Cancer Society, 21,990 new ovarian cancer cases and 46,470 new endometrial cancer cases were diagnosed in 2011. These cancers share a common mechanism of pathogenesis, epithelial-mesenchymal transition (EMT). EMT increases the invasiveness and progression of epithelial tumor cells into more aggressive and metastatic disease. Amplification of growth factor signaling is also common in malignancy. Epidermal growth factor receptor (EGFR) is amplified in 60-80% of ovarian and endometrial cancer and its overexpression is associated with increased invasiveness and poor prognosis. Studies have shown that specific cytokines, including transforming growth factor-β (TGFβ) and epidermal growth factor (EGF), interact with the intracellular MEK/ERK signaling cascade, a pathway involved in several malignant processes including EMT. Raf-1 is an intracellular MAPK signaling molecule that is recruited to membrane bound Ras upon activation by growth factors. Activation of Raf-1 includes phosphorylation at ser 338, which in turn activates the downstream MEK/ERK signaling cascade. Our data showed that EGF phosphorylates Raf-1 at ser 338 and ERK in endometrial cancer cells, which is inhibited by the specific Raf-1 antagonist GW5074. Little is known on the involvement of Raf-1 in the pathogenesis of ovarian or endometrial cancer. Our studies show that inhibition of Raf-1 by GW5074 significantly decreases TGFβ and EGF induced invasiveness of immortalized endometrial epithelial cells (EECs). EGF and to a lesser degree TGFβ, enhanced the survival of EECs under serum starvation. GW5074 inhibited this effect as evidenced by increased apoptosis and caspase 3/7 activity, suggesting that Raf-1 mediates EEC survival. While EGF had minimal effects on inducing ovarian cancer cell line SKOV3, the addition of GW5074 significantly reduced SKOV3 cell growth by half. Our data shows that the addition of EGF, but not TGF-β, to endometrial and ovarian epithelial cells induced EMT as evidenced by the disruption of cell-cell contact, loss of the characteristic “cobblestone” appearance of epithelial cells, increased motility and induction of the mesenchymal marker vimentin. Treatment with GW5074 of cells exposed to EGF reversed these changes. Our FACS analysis showed that EGF decreases the cell surface detection of EPCAM, an epithelial cell surface adhesion molecule, by more than half, further suggesting that EGF is involved in EMT in endometrial cancer cells. Our data suggests that multiple steps in the pathogenesis of both ovarian and endometrial cancers are mediated by Raf-1. Raf-1's involvement in inducing EMT suggests that Raf-1 may be a novel therapeutic target for ovarian and endometrial cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-14. doi:1538-7445.AM2012-LB-14

Abstract 4800: Quantitative detection of discriminatory proteins in tissue and serum of ovarian cancer patients by LC-MSE

Abstract Purpose: Using label-free UPLC-MSE quantification methods to identifying proteins as well as determining their abundance in serum and microdissected ovarian cancer tissue. Experimental procedures: Serum of six patients with a serous adenocarcinoma of the ovary (stage IIIB or higher) was collected before treatment. Matched control patients with a serous cystadenoma of the ovary were chosen as a control group. In addition, homogeneous regions of cells that exhibited uniform histology were isolated from cancer tissue by laser capture microdissection (LCM), normal ovarian epithelial cell samples and stroma samples were taken from matched controls. We subsequently employed label-free ultra-high pressure liquid chromatography tandem mass spectrometry (UPLC-MSE) to identify proteins and determine their absolute concentrations in serum and tissue lysates. Moderated t-tests were used to identify differentially expressed proteins between patient- and control-group. p-values were adjusted for multiple-testing using Benjamini-Hochberg false discovery rates and considered differentially expressed when <0.05. Summary of the data: In the serum specimens, 13 differentially expressed proteins were identified by LC-MSE profiling. Proteins with different concentrations in patients versus control sera include abundant serum proteins such as apolipoprotein AI and transferrin, both exhibited a lower concentration in serum of cancer patients. Differential expression was also observed for apolipoproteins (APOA IV, APOA II and APOC III) and other proteins that have not been associated with ovarian cancer previously such as C9 and Afamin. In the tissue-lysates we identified 535 Proteins of which 332 could be reliably quantified. Of these 56 were differentially expressed proteins of which 12 had non-overlapping standard deviations. Literature searches confirmed a link between ovarian cancer and 6 of these proteins, suggesting a possible role in disease progression. Of special interest was the identification of prohibitin (PHB) and cofilin-1 (CFL1). Immunohistochemical detection of CFL1 in formalin-fixed paraffin-embedded tissues of our patient group showed heterogeneous expression in 50% of the serous ovarian carcinomas, whereas all the benign lesions were negative. These results again demonstrate the validity of the protein quantitation results obtained by MSE. Interestingly, no overlap was found between the discriminatory proteins in serum specimens and corresponding tissue lysates, illustrating the challenge to identify a single biomarker in the circulation reflecting ovary cancer. Conclusions: Our study revealed several protein changes in serum and tissue of patients suffering from serous adenocarcinoma of the ovary. Further investigation of these proteins is warranted to establish whether they could provide new insights into disease etiology and act as potential new markers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4800. doi:1538-7445.AM2012-4800

Abstract 29: Functional analysis of PAX8 in high-grade serous ovarian cancer

Abstract High-grade serous ovarian cancers (HGSOC) are the most common and most lethal ovarian cancer subtype. Identifying novel markers of HGSOC development and outcome is critical to reduce mortality. Genomic profiling of ∼500 primary HGSOCs by The Cancer Genome Atlas showed PAX8 overexpression in ∼80% of tumors. In a separate analysis of ∼350 HGSOCs, we correlated PAX8 expression with overall survival in patients. We also found significant PAX8 overexpression in 38 EOC cell lines compared to 48 normal ovarian and fallopian tube cell lines. Together, these data suggest a role for PAX8 as a frequently altered oncogene in HGSOC. To evaluate the functional significance of PAX8 in HGSOC development, we analyzed gene expression in an in vitro model of early stage neoplastic transformation, driven by CMYC and mutant KRAS expression in normal ovarian epithelial cells. PAX8 showed significantly increased expression as cells underwent stepwise transformation (p<0.01). To study the putative role of PAX8 as an oncogene, we used shRNA to stably knock down gene expression in HEY HGSOC cells, and measured the in vitro transformed phenotype after knockdown. Immunofluorescence and western blotting showed a significant reduction of PAX8 expression in PAX8 knockdown cells (HEY-PAX8KD) compared to both parental cells and the scrambled shRNA control (HEY-SCR). HEY-PAX8KD cells were significantly less proliferative, and exhibited 10-fold decreased anchorage independent growth accompanied by reduced colony size. In three dimensional mass culture assays, HEY and HEY-SCR lines aggregated in smooth, dense, regularly sized spheroids; but HEY-PAX8KD cells formed irregular, diffuse spheroids incorporating only ∼50% of cells in suspension. We found no differences in Ki67 or cleaved-caspase 3 expression, suggesting differences in spheroid structure were not due to changes in proliferation or apoptosis. HEY-PAX8KD cells also showed increased migration and invasion, suggesting a novel role for PAX8 in cell-cell/cell-matrix adhesion. Differential expression of cell surface proteins between HEY and HEY-PAX8KD cells supports this hypothesis and suggests several mechanisms by which cell adhesion, migration and invasion are altered by reduced PAX8 expression. We characterized the functional significance of PAX8 in vivo, by generating multiple luciferase labeled HGSOC lines that express tetracyline-inducible PAX8 shRNAmir constructs. PAX8 inducible knockdown cells (HEYLUC-PAX8tet-KD) showed significantly decreased PAX8 expression after 48hrs of doxycycline treatment. Tumorigenicity and chemotherapeutic response with and without doxycycline treatment were evaluated after intra-peritoneal injection of HEYLUC-PAX8tet-KD cells in immunocompromised mice. Together, our data indicate that PAX8 is an essential oncogene in the development of most HGSOCs and may be a therapeutic target when combined with monoclonal antibodies against cell adhesion proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 29. doi:1538-7445.AM2012-29

Effect of XPG down-regulation gene expression towards the proliferation of epithelial ovarian cancer cells and its chemosensitivity to platinum

To investigate the effect of XPG down-regulation gene expression towards the proliferation of epithelial ovarian cancer cells and its chemosensitivity to platinum. The small interference RNA (siRNA)-XPG fragments were designed and tranfected into SKOV3/DDP cell lines by lipofectamine transiently for choosing the best siRNA-XPG fragment to silence XPG gene expression. The pGPU6/GFP/Neo vector was used to construct the siRNA-XPG vectors, which was transfected into SKOV3/DDP cell line with expression of XPG gene. Real-time PCR and western blot were employed to confirm the silencing efficacy of siRNA-XPG. The growth curve of cells, cell cycle, the drug-resistance index of cells and intracellular drug concentration were measured by 4-methyl-thiazolyl-tetrazolium (MTT), flow cytometer (FCM) and high performance liguid chromatograph respectively. (1) Real-time PCR results showed that XPG mRNA expression copy number in SKOV3/DDP tranfected with siRNA-XPG-733 fragment was 1.050 ± 0.023, which was significantly lower than that in SKOV3/DDP tranfected with other siRNA-XPG fragments (P < 0.05, respectively), and was chosed to construct the siRNA-XPG vectors. The XPG mRNA expression was down-regulated in short hairpin RNA (shRNA)-XPG-733-SKOV3/DDP cell lines that confirmed by western blot. (2) The growth curve showed that growth velocity of shRNA-XPG-733-SKOV3/DDP cell lines was lower than that of shRNA-GAPDH and shRNA-NC cell lines (P < 0.05, respectively). The results of FCM also showed that 34.0% of cells in shRNA-XPG-733-SKOV3/DDP cell lines were in S + G(2)/M phase, while only 58.7% and 51.3% in shRNA-GAPDH and shRNA-NC cell lines respectively (P < 0.05, respectively). (3) The drug-resistance index of shRNA-XPG-733-SKOV3/DDP cell lines [50% inhibiting concertration (IC(50)): (13.79 ± 0.06) µg/ml] was lower than that in shRNA-GAPDH and shRNA-NC cell lines [IC(50): (27.84 ± 0.34) µg/ml and (28.32 ± 0.42) µg/ml, respectively] statistically significant (P < 0.05, respectively); but there was not statistically significant difference in intracellular drug concentration between shRNA-XPG-733-SKOV3/DDP cell lines [(0.026 ± 0.005) µg/ml] and shRNA-GAPDH [(0.024 ± 0.003) µg/ml] and shRNA-NC cell lines [(0.025 ± 0.007) µg/ml] after treated by cisplatin in vitro(P > 0.05, respectively). The down-regulating of XPG gene resulted in slowing growth velocity and descending the drug-resistance index of shRNA-XPG-733-SKOV3/DDP cell lines, which may be related with descending in capability of DNA excision repair in cells.

Luteinizing hormone facilitates angiogenesis in ovarian epithelial tumor cells and metformin inhibits the effect through the mTOR signaling pathway

High levels of gonadotropin are a risk factor for ovarian cancer development. Aberrant gonadotropin levels benefit tumor angiogenesis, but the detailed mechanism is not clear. Therefore, the aim of this study was to investigate the molecular mechanism of high levels of luteinizing hormone (LH) on the promotion of tumor angiogenesis and to outline a feasible therapeutic strategy. Western blotting and immunofluorescence staining were used to determine the effect of LH on VEGF and slit2 expression and examine the signaling pathway involved in regulating the expression of both molecules. Real-time PCR was used to investigate the effect of metformin on LH induction of VEGF and slit2 expression. It was found that 50 mIU/ml LH significantly upregulated VEGF and slit2 expression, and activated the PI3K/AKT-mTOR signaling pathway. However, metformin inhibited the mTOR signaling pathway and further blocked LH-induced VEGF and slit2 expression. In conclusion, high levels of LH promote angiogenesis in ovarian cancer via the PI3K/AKT-mTOR pathway. However, metformin could inhibit tumor angiogenesis by blocking the mTOR signaling pathway.

The Cell Surface Glycoprotein CUB Domain-containing Protein 1 (CDCP1) Contributes to Epidermal Growth Factor Receptor-mediated Cell Migration

Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.

WNT7A Regulates Tumor Growth and Progression in Ovarian Cancer through the WNT/β-Catenin Pathway

Abnormal activation the WNT/β-catenin signaling pathway has been associated with ovarian carcinomas, but a specific WNT ligand and pertinent downstream mechanisms are not fully understood. In this study, we found abundant WNT7A in the epithelium of serous ovarian carcinomas, but not detected in borderline and benign tumors, normal ovary, or endometrioid carcinomas. To characterize the role of WNT7A in ovarian tumor growth and progression, nude mice were injected either intraperitoneally or subcutaneously with WNT7A knocked down SKOV3.ip1 and overexpressed SKOV3 cells. In the intraperitoneal group, mice receiving SKOV3.ip1 cells with reduced WNT7A expression developed significantly fewer tumor lesions. Gross and histologic examination revealed greatly reduced invasion of WNT7A knockdown cells into intestinal mesentery and serosa compared with the control cells. Tumor growth was regulated by loss or overexpression of WNT7A in mice receiving subcutaneous injection as well. In vitro analysis of cell function revealed that cell proliferation, adhesion, and invasion were regulated by WNT7A. The activity of the T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter was stimulated by overexpression of WNT7A in ovarian cancer cells. Cotransfection with WNT7A and FZD5 receptor further increased activity, and this effect was inhibited by cotransfection with SFRP2 or dominant negative TCF4. Overexpression of WNT7A stimulated matrix metalloproteinase 7 (MMP7) promoter, and mutation of TCF-binding sites in MMP7 promoter confirmed that activation of MMP7 promoter by WNT7A was mediated by β-catenin/TCF signaling. Collectively, these results suggest that reexpression of WNT7A during malignant transformation of ovarian epithelial cells plays a critical role in ovarian cancer progression mediated by WNT/β-catenin signaling pathway.

Expressions and significance of cyclinD1 in epithelial ovarian cancer cell 3AO

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.

Bmi-1 siRNA inhibited ovarian cancer cell line growth and decreased telomerase activity

ABSTRACTThe knockdown of Bmi-1 could effectively suppress cancer cell proliferation and tumourigenicity in several cancers. This study aims to investigate whether or not Bmi-1 plays a causative role in the proliferation of ovarian epithelial cancer cells and telomerase activity. The messenger RNA (mRNA) and protein expression levels of Bmi-1 in the human ovarian carcinoma cell line OVCAR-3 were down-regulated by Bmi-1 siRNA, as confirmed by real-time polymerase chain reaction (PCR) and Western blot. Cell viability was analysed by MTT assay, and telomerase activity was analysed by a modified telomeric repeat amplification protocol. Targeting Bmi-1 with siRNA inhibited Bmi-1 mRNA over five-fold compared with the control cells, and inhibited Bmi-1 protein expression over three-fold compared with control cells. The viability of the OVCAR-3 ovarian cancer cell line was reduced by Bmi-1 mRNA compared to control cells. Telomerase activity was decreased 22.73% (from 0.33 to 0.255) by Bmi-1 siRNA treatment compared to control cells. As Bmi-1 siRNA depressed telomerase activity, cell immortalisation may be prevented; thus, silencing Bmi-1 may be a potential therapy to manage ovarian cancer.

Investigation of stem cells in adult and prepubertal mouse ovaries

The possible presence of oocyte and granulosa cells originated from stem cells in the adult mammalian ovaries was claimed by some studies which will lead to major changes in reproductive biology and infertility treatments. Purpose of this research is to investigate the possible existence and the location of the potential stem cells in mouse ovaries. In this study, the ovaries from 2-week (pre-puberty) and 8-week (adult) old BALB-C mice were used. For the investigation of the presence of possible stem cells, the expression profiles of three well known stem cell markers, Oct-4, Nanog and Sox2 were determined in the ovaries of two different age groups by real time quantitative RT-PCR (qRT-PCR). Protein expression levels and their localization in the ovary cells were immunohistochemically evaluated on fresh-frozen ovary tissue sections by using monoclonal antibodies specific to Sox2, Nanog and Oct-4. The gene expression levels of Oct-4 and Nanog were found to be significantly differentiated between 2-week old and 8-week old mice whereas no significant difference was observed in the expression level of Sox2 between two age groups. Immunohistochemistry results showed the presence of both Sox2 and Oct-4 protein in the cytoplasm of ovarian epithelial cells, granulosa cells, oocytes and theca cells. Nanog protein was observed only in the nucleus of the oocytes and furthermore the expression of Nanog was higher in eight weeks old samples compared to two weeks old ones according to qRT-PCR results. These results suggest for the first time that Nanog protein is expressed both in adult and pre-puberty mouse ovaries and locate at the nucleus of the oocytes and to the best of our knowledge this is the first study that shows the differential expression of Oct-4, Nanog and Sox2 in pre-puberty and adult mouse ovaries by qRT-PCR. Collectively, our results may suggest that both pre-puberty and adult mice ovaries accommodate cells carrying stem cell features.

Initial report on differential expression of sprouty proteins 1 and 2 in human epithelial ovarian cancer cell lines.

Sprouty (Spry) proteins, modulators of receptor tyrosine kinase signaling pathways, have been shown to be deregulated in a variety of pathological conditions including cancer. In the present study we investigated the expression of Spry1 and Spry2 isoforms in a panel of human ovarian cancer cell lines in vitro. Our western blot analysis showed nonuniform patterns of Spry expression in the cancer cells, none of which conformed to the pattern observed in the normal ovarian epithelial cells employed as the control. Among the seven cancer cell lines studied, Spry1 was expressed lower in four cell lines and higher in one as compared with the control. As for Spry2, four cell lines showed lower and two exhibited higher expression. Results from RT-PCR assay raised the possibility that Spry protein levels may not necessarily correspond with its expression at mRNA level. Our immunostaining study revealed that Spry2 was predominantly distributed within the whole cytoplasm in vesicular structures whereas Spry1 was found in both the cytoplasm and nucleus. This might provide clues to further investigation of Spry mode of action and/or function. Collectively, our study unveiled the differential expression of Spry1 and Spry2 proteins in various ovarian cancer cell lines.

Expression and subcellular localization of a novel gene &amp;lt;i&amp;gt;Bm-X&amp;lt;/i&amp;gt; of the silkworm, &amp;lt;i&amp;gt;Bombyx mori&amp;lt;/i&amp;gt;

According to the large-scale sequencing of cDNA library from silkworm pupae, the cDNA of a novel gene with blank research background was identified and temporarily named Bm-X. The length of this cDNA is 778 bp. We obtained its ORF for further study by bioinformatics analysis. It is 444 bp and encodes 147 amino acid residues, with a predicted molecular weight (MW) of 16.4 kD and an isoelectric point (pI) of 3.69. In this study, we successfully constructed a recombinant plasmid pET-28a(+)-Bm-X and expressed it in Escherichia coli. We used the fusion protein rBm-X which purified by Niaffinity chromatography to produce polyclonal antibodies against Bm-X for Western blot analysis. The analysis revealed that Bm-X was expressed in the larval midgut, the epidermis and the silk gland. In addition, the subcellular localization analysis of silkworm ovary epithelial cells (BmN cells) showed that Bm-X protein was located both in cytoplasm and nucleus, and the signal was stronger in cytoplasm than in nucleus. Our findings indicate that Bm-X gene is a novel species-specificity gene and its expression product can be detected in tissues of the fifth silkworm instar larvae and BmN cells.

Ovarian Cancer: Opportunity for Targeted Therapy

Ovarian cancer is a common cause of cancer mortality in women with limited treatment effectiveness in advanced stages. The limitation to treatment is largely the result of high rates of cancer recurrence despite chemotherapy and eventual resistance to existing chemotherapeutic agents. The objective of this paper is to review current concepts of ovarian carcinogenesis. We will review existing hypotheses of tumor origin from ovarian epithelial cells, Fallopian tube, and endometrium. We will also review the molecular pathogenesis of ovarian cancer which results in two specific pathways of carcinogenesis: (1) type I low-grade tumor and (2) type II high-grade tumor. Improved understanding of the molecular basis of ovarian carcinogenesis has opened new opportunities for targeted therapy. This paper will also review these potential therapeutic targets and will explore new agents that are currently being investigated.

Overexpression of dedicator of cytokinesis I (Dock180) in ovarian cancer correlated with aggressive phenotype and poor patient survival

Dedicator of cytokinesis I (Dock180) is a novel guanine nucleotide exchange factor for Rho guanosine triphosphates (GTPases) important for cell migration. The aim of this study was to evaluate the role of Dock180 in ovarian carcinogenesis. Using immunohistochemistry, real-time polymerase chain reaction and Western blotting, overexpression of Dock180 RNA and protein was demonstrated in the nucleus and cytoplasm of ovarian cancer cell lines (n = 5) and clinical samples of ovarian borderline tumours (n = 21) and invasive cancers (n = 108) when compared with ovarian epithelial cell lines (n = 3) and benign cystadenomas (n = 10) (P < 0.05). High Dock180 cytoplasmic expression in ovarian cancer (n = 108) was associated significantly with serous histological type, high-grade cancer and advanced stage (P < 0.05), as well as poor overall and disease-free survival (P = 0.004). Using multivariate progression analysis, high Dock180 cytoplasmic expression and advanced cancer stage were found to be independent prognostic factors for short overall survival and disease-free survival (P < 0.05). Exogenous expression of Dock180 by transient transfection enhanced cancer cell migration and invasion, whereas knockdown of Dock180 by an siRNA approach retarded cancer cell migration and invasion in association with down-regulation of matrix metalloproteinase 2. Our findings suggest that Dock180 contributes to ovarian carcinogenesis and dissemination and is a potential prognostic marker and therapeutic target.

Nonsteroidal antiinflammatory drugs and progestins synergistically enhance cell death in ovarian epithelial cells

There is growing evidence that progestins and nonsteroidal antiinflammatory drugs (NSAIDs) may prevent ovarian cancer. Because both induce apoptosis, we investigated the potential for synergistic impact of combined drug treatment on cell death. Using normal and malignant human ovarian epithelial cells and an NSAID-sensitive human colon cancer cell line, we evaluated the effects of progestins and NSAIDs alone and in combination on apoptosis. Both progestins and NSAIDs dose dependently inhibited cell growth (P < .0001). Doses of NSAIDs or progestins that independently reduced cell viability by less than 30% synergistically reduced cell viability by 70-95% when combined. Similarly, the NSAID/progestin combination conferred 4- to 18-fold (P < .05) increased apoptosis over either treatment alone. Our results suggest it may be possible to combine progestins and NSAIDs to achieve ovarian cancer prevention at lower doses of each than are required for single administration, thereby lessening the risk of side effects posed by these agents.

Glucocorticoid Regulation of SLIT/ROBO Tumour Suppressor Genes in the Ovarian Surface Epithelium and Ovarian Cancer Cells

The three SLIT ligands and their four ROBO receptors have fundamental roles in mammalian development by promoting apoptosis and repulsing aberrant cell migration. SLITs and ROBOs have emerged as candidate tumour suppressor genes whose expression is inhibited in a variety of epithelial tumours. We demonstrated that their expression could be negatively regulated by cortisol in normal ovarian luteal cells. We hypothesised that after ovulation the locally produced cortisol would inhibit SLIT/ROBO expression in the ovarian surface epithelium (OSE) to facilitate its repair and that this regulatory pathway was still present, and could be manipulated, in ovarian epithelial cancer cells. Here we examined the expression and regulation of the SLIT/ROBO pathway in OSE, ovarian cancer epithelial cells and ovarian tumour cell lines. Basal SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 expression was lower in primary cultures of ovarian cancer epithelial cells when compared to normal OSE (P<0.05) and in poorly differentiated SKOV-3 cells compared to the more differentiated PEO-14 cells (P<0.05). Cortisol reduced the expression of certain SLITs and ROBOs in normal OSE and PEO-14 cells (P<0.05). Furthermore blocking SLIT/ROBO activity reduced apoptosis in both PEO-14 and SKOV-3 tumour cells (P<0.05). Interestingly SLIT/ROBO expression could be increased by reducing the expression of the glucocorticoid receptor using siRNA (P<0.05). Overall our findings indicate that in the post-ovulatory phase one role of cortisol may be to temporarily inhibit SLIT/ROBO expression to facilitate regeneration of the OSE. Therefore this pathway may be a target to develop strategies to manipulate the SLIT/ROBO system in ovarian cancer.

Expression of c-Kit and PDGFRα in epithelial ovarian tumors and tumor stroma

The purpose of this study was to investigate the expression of c-Kit and platelet-derived growth factor receptor α (PDGFRα) in epithelial ovarian tumor cells and tumor stroma. The expression of c-Kit and PDGFRα in 71 malignant or benign epithelial ovarian tumor tissues and 20 normal ovarian tissues was evaluated by immunohistochemical staining. The expression of c-Kit and PDGFRα in 71 malignant epithelial ovarian tumors and tumor stroma tissue samples was analyzed. A significant increase (P<0.01) of c-Kit expression was observed in malignant ovarian tumors (50.7%) when compared to normal ovarian tissues (10.0%) or benign ovarian tumors (20.0%). The PDGFRα expression rate in malignant ovarian tumors (63.4%) was also significantly higher (P<0.01) than that in normal ovarian tissues (15.0%) or benign ovarian tumors (25.0%). c-Kit was expressed in only 4.2% of the tumor stroma samples, which was significantly lower than the expression of malignant ovarian tumors (P<0.01), whereas the PDGFRα expression in tumor stroma (87.3%) was significantly higher than that of the malignant ovarian tumors (P<0.01). The expression levels of c-Kit and PDGFRα are higher in the malignant ovarian tumors than in the benign ovarian tumors or normal tissues. In the malignant ovarian tumor stroma, c-Kit expression is low and PDGFRα expression is high, and the differential changes of c-kit and PDGFRα suggest distinct roles in ovarian cancer.