Ovarian Epithelial Cells Research Articles

CD24 and Nanog identify stem cells signature of ovarian epithelium and cysts that may develop to ovarian cancer

Ovarian cancer is the most lethal gynecological cancer. There is a general debate whether ovarian cancer is an intrinsic or an imported disease. We investigated whether in normal morphological appearance and in early stages of ovarian tumorgenesis typical cancer cell markers such as CD24 and Nanog are expressed. In 25% of normal appearing ovaries of post-menopausal women there was co-localization of CD24 and Nanog in the walls of the ovarian cysts, leaving the epithelial cells on the surface of these ovaries free of Nanog or CD24 expression. In benign ovarian tumors 37% of specimens were positive to CD24 and Nanog labeling while 26% of them were localized in the cyst walls. In contrast, in serous borderline tumors 79% specimens were labeled with CD24, 42% of them were localized in cysts and in 32% of them showed co-localization with CD24 and Nanog was evident: the rest were labeled in the ovarian epithelial cells. In serous ovarian carcinomas 81% specimens were labeled with CD24 antibodies. In 45% of them co-localization with Nanog was evident in the bulk of the cancerous tissue. In mucinous carcinomas no labeling with CD24 or Nanog was evident. In view of the synergistic effect of CD24 and Nanog expressed in malignant cancer development in other systems, it is suggested that such an analysis can be valuable for early detection of ovarian cancer. Moreover, the abundance of these markers in cysts in the development of ovarian cancer may suggest that they present an intrinsic source of the development of the highly malignant disease. Finally, since CD24 is exposed on the surface of the cancer cells, it may be highly beneficial to target these cells with antibodies to CD24 conjugated to cytotoxic drugs for more efficient treatment of this malignant disease.

CD164 regulates the tumorigenesis of ovarian surface epithelial cells through the SDF-1α/CXCR4 axis

BackgroundCD164 (endolyn), a sialomucin, has been reported to play a role in the proliferation, adhesion, and differentiation of hematopoietic stem cells. The potential association of CD164 with tumorigenicity remains unclear.MethodsThe clinicopathological correlation of ovarian cancer with CD164 was assessed in a 97-patient tumor tissue microarray. Overexpression or silence CD164 was to analyze the effect of CD164 on the proliferation, colony formation and apoptosis via a mouse xenograft and western blotting analysis. The subcellular localization of CD164 was collected in the immunohistochemical and confocal analysis.ResultsOur data demonstrated that higher expression levels of CD164 were identified in malignant ovarian cancer cell lines, such as SKOV3 and HeyA8. The clinicopathological correlation analysis showed that the upregulation of CD164 protein was significantly associated with tumor grade and metastasis. The overexpression of CD164 in human ovarian epithelial surface cells promoted cellular proliferation and colony formation and suppressed apoptosis. These tumorigenicity effects of CD164 were reconfirmed in a mouse xenograft model. We also found that the overexpression of CD164 proteins increased the amounts of CXCR4 and SDF-1α and activated the SDF-1α/CXCR4 axis, inducing colony and sphere formation. Finally, we identified the subcellular localization of CD164 in the nucleus and cytosol and found that nuclear CD164 might be involved in the regulation of the activity of the CXCR4 promoter.ConclusionsOur findings suggest that the increased expression of CD164 is involved in ovarian cancer progression via the SDF-1α/CXCR4 axis, which promotes tumorigenicity. Thus, targeting CD164 may serve as a potential ovarian cancer biomarker, and targeting CD164 may serve as a therapeutic modality in the management of high-grade ovarian tumors.

Abstract C36: Is DHT involved in the action of TGF-β1 in epithelial ovarian cancer?

Abstract Ovarian cancer is the ninth most common female cancer worldwide and the fifth leading cause of cancer-related death among women in developed countries. In Chile, ovarian cancer is the second cause of death from gynaecological cancer. This disease is characterized by a silent course, late detection, high angiogenesis, with a poor response to therapy and a low survival. It is postulated that epithelial ovarian cancer (EOC) arises from the ovarian surface epithelium (OSE), but also it has been suggested that epithelial cells from fimbrae of the fallopian tubes could be implicated as the possible site of this malignancy, such as high-grade serous carcinoma. There are several hypotheses that intend to explain the origin of ovarian cancer and one of them postulate that sex steroid hormones have been involved in the aetiology and/or progression of some EOCs. In fact, androgens receptor (AR) is expressed in OSE cells and also in the majority of ovarian cancers. The microenvironment of ovarian cancer cells is enriched in androgens, and high levels of this steroid have also been reported in ascitic fluid; this condition supports the hypothesis that androgens, which are present in the ovarian stromal compartment, can produce neoplastic conversion of OSE and epithelial cells into de inclusion cyst and/or also be involved in ovarian cancer progression. Therefore, it is important to evaluate if dihydrotestosterone (DHT) is involved in the regulation of TGF-β1 receptors and also TGF-β1 signaling pathway, which is a potent inhibitor mechanism of cell proliferation, in EOC. This work was done with two types of experiments: 1) ex-vivo, with tissues of inactive normal ovaries and EOC poorly differentiated obtained from 10 patients from Hospital Clinico Universidad de Chile, with informed consent approved by Ethical Committee of the Institution, in order to evaluate the detection of AR by immunohistotochemistry (IHC). 2) In vitro experiments with human ovarian surface epithelium cell line (HOSE) and human epithelial ovarian cancer cell line (A2780). Both human cell lines are a good model that represents the epithelial cells from inactive normal ovaries and from EOC, respectively. These cells were stimulated with DHT and also with TGF-β1 during 72 h. The AR expression in cell lines was analysed by RT- PCR and immunocytochemistry (ICC). On the other hand, in both cell lines, TGF-β1R1 and TGF-β1R2 were detected by ICC and also in basal condition and with 10 and 100nM of DHT, by western-blot. In addition, we used an AR siRNA in order to know if DHT is involved in the TGF-β1R1 and/or in TGF-β1R2 expression in A2780 cell line. Our results show that AR protein levels in EOC and A2780 are significantly higher than inactive normal ovaries and HOSE cells, respectively (p<0.05). In basal condition, TGF-β1R1 protein levels in HOSE cells were higher than in EOC cells, but no changes were found in TGF-β1R2 protein levels in A2780. When cellular lines were treated with 100nM DHT, TGF-β1R1 and TGF-β1R2 protein levels decreased (p<0.05) with respect to the control condition only in A2780 cells. Besides, a 45% of AR silencing was obtained with AR siRNA (100 pmol) at 72 h in A2780 cells. Then, these cells were stimulated with 100 nM of DHT and it was obtained a 30% protein increase of the TGF-β receptors. These preliminary results suggest that DHT could be involved in TGF-β1 signaling pathway in epithelial ovarian cancer. This work has been support by grant FONDECYT # 1110372 (to CR) Citation Format: Karla Kohan, Cristian Poblete, Margarita Vega, Carmen Romero. Is DHT involved in the action of TGF-β1 in epithelial ovarian cancer? [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C36.

Abstract IA16: Targeting cancer stem cells to overcome chemotherapy resistance

Abstract While most ovarian cancer patients will have a complete response with surgery and chemotherapy, the majority will relapse and die of their disease. The high relapse rate in ovarian cancer after complete clinical response is highly consistent with a cancer stem-like cell (CSC) model. The CSC hypothesis suggests that only rare, inherently chemoresistant cancer cells with stem cell like characteristics are capable of proliferating and differentiating to regenerate the various cell types within a tumor, thereby causing relapse of the disease. In theory and in experimental models, the elimination of CSC can overcome chemotherapy resistance. We are developing novel therapies to specifically target ovarian CSC (OvCSC). Aldehyde Dehydrogenase (ALDH) enzymatic activity was one of the first, and remains one of the best supported CSC markers. ALDH enzymes perform numerous critical functions including the biosynthesis of retinoic acid. Our group and others have found that ALDH identifies a population cancer cells with ovarian CSC (OvCSC) activity. Primary human ALDH+ cells initiate and propagate tumors in mice while ALDH-cells do not. ALDH used in combination with the stem cell marker CD133 enriches CSC isolation. ALDH1A+ or ALDH1A1+CD133+ cells in primary tumor samples predict poor prognosis for ovarian cancer patients [9, 11]. ALDH and CD133 are enriched in tumor specimens immediately following chemotherapy [12]. ALDH1A1 is 100 fold upregulated in chemoresistant ovarian cancer cells and ALDH1A knockdown restores chemosensitivity. Finally, normal ovary epithelial stem cells also express ALDH and CD133, and when mutated for p53 and Rb, these cells gain CSC capacity and initiate serous tumors. Taken together these data strongly support a role for ALDH in serous ovarian CSC. Based on the CSC specific function of ALDH and the function of we generated and screened. We have therefore identified and screened dozens of ALDH inhibitors and molecular analogs of known ALDH inhibitors. Using cell lines with well-defined CSC populations, we have identified several compounds which kill cells expressing CSC markers in vitro. CSC die via necrosis. Lead compounds prevent tumor sphere formation in vitro and restrict tumor initiation in vivo. The novel ALDH inhibitors are highly synergistic with chemotherapy and reverse chemotherapy resistance in primary human tumor xenografts. Taken together our data support ALDH as a CSC specific target and identifies novel compounds as potential new therapeutics. Citation Format: Ronald J. Buckanovich. Targeting cancer stem cells to overcome chemotherapy resistance. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA16.

Abstract B50: Preclinical efficacy of combined MEK and Src inhibition: MEK inhibitor reverses resistance to Src inhibitor therapy in ovarian cancer cells

Abstract Introduction: More effective, less toxic treatments for recurrent ovarian cancer are needed. Up to 80% of ovarian cancers show activation of the Src kinase and up to 40% show MEK/MAPK pathway activation. Therapies targeting signaling pathways that drive proliferation have shown promise in ovarian cancers but the development of drug resistance presents a major problem. Saracatinib, is a potent inhibitor of Abl and Src family kinases and selumetinib, is a potent, selective, ATP-uncompetitive inhibitor of MEK 1/2 kinases. Our earlier work has shown that resistance to saracatinib monotherapy emerges within weeks in cell culture and within months in xenograft models. Since oncogenic activation of both Src and MEK pathways has been observed in ovarian cancers and because resistance to Src inhibition rapidly emerges with monotherapy, we hypothesized that blocking both kinases with dual therapy might more effectively decrease ovarian cancer cell proliferation than either drug alone. Experimental Design: Dual Src and MEK blockade effects on cell cycle and survival were assayed in ovarian cancer lines, and a primary human ovarian cancer tumor cultured immediately in vitro. Cells enriched for the stem cell marker, aldeflour dehydrogenase 1 (ALDH1+) were sorted by flow cytometry. Drug effects on tumor sphere formation and on cell cycle profiles of ALDH+ cells and those lacking this activity were analyzed. In vivo experiments are ongoing. Results: Src activity is greater in ovarian cancer lines than normal human ovarian epithelial cells (HOSE). In saracatinib naïve ovarian cancer cells, Src inhibition led to partial growth arrest after 48 hrs of treatment. However, resistance of ovarian cancer cells to saracatinib monotherapy emerged rapidly, over a period of weeks of continuous drug exposure in vitro and in vivo. Activation of the MEK and Akt pathways developed with prolonged saracatinib treatment. While each drug alone caused a partial growth arrest in saracatinib naïve lines, treatment with both saracatinib and the MEK inhibitor drug, selumetinib, caused synergistic cell cycle arrest which was mediated by p27 binding to cylin E/cdk2. Combination treatment decreased Src, Akt and MAPK activities. Combination treatment was required for cell cycle arrest in saracatinib resistant cells. Combination treatment also induced apoptosis by an increase in cleaved PARP and induced autophagy as shown by an increase in LC3-II. Src and MEK activities were increased in ALDH1+ sorted populations compared to the ALDH- population and combination treatment decreased the % of ALDH1+cells and the % of sphere-generating cells. Conclusions: MEK inhibition augments the effects of saracatinib by blocking bypass activation of MEK and Akt pathways, increasing cell cycle arrest, impairing survival and targeting self-renewing subpopulations. These data support further pre-clinical and clinical evaluation of combined saracatinib and MEK inhibition in ovarian cancer. Citation Format: Fiona Simpkins, Diana Azzam, Joyce M. Slingerland. Preclinical efficacy of combined MEK and Src inhibition: MEK inhibitor reverses resistance to Src inhibitor therapy in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B50.

Abstract B27: Melatonin synergistically enhances cisplatin-induced apoptosis via the dephosphorylation of ERK/p90 ribosomal S6 kinase/heat shock protein 27 in SK-OV-3 cells

Abstract To evaluate melatonin's ability to enhance ovarian cancer cells to cisplatin treatment for ovarian cancer, this study was performed. Melatonin by itself had no significant cytotoxicity against SK-OV-3 cells, while cisplatin suppressed the cell viability in a dose-dependent manner. Combined treatment with cisplatin and melatonin synergistically inhibited the viability of SK-OV-3 cells with the synergism between two drugs (1 > combination index). In contrast, melatonin revealed the protective effect against cisplatin-induced cytotoxicity in OSEN normal ovarian epithelial cells. Cotreatment with cisplatin and melatonin increased the sub-G1 DNA contents and TdT-mediated dUTP nick end-labeling (TUNEL)-positive cells compared with cisplatin control in SK-OV-3 cells, suggesting that melatonin augments cisplatin-induced apoptosis. Consistently, combined treatment of cisplatin and melatonin increased the cleavage of caspase-3 and poly-(ADP-ribose) polymerase (PARP). Importantly, melatonin synergistically inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) along with dephosphorylation of 90-kDa ribosomal S6 kinase (p90RSK) and heat shock protein 27 (HSP27) induced by cisplatin. Furthermore, melatonin remarkably blocked the expression and colocalization of p90RSK and HSP27 by combination treatment with cisplatin. Taken together, our findings demonstrate that melatonin enhances cisplatin-induced apoptosis via the inactivation of ERK/p90RSK/HSP27 cascade in SK-OV-3 cells as a potent synergist to cisplatin treatment. Citation Format: Sunghoon Kim, Jihyun Kim, bonglee kim, Junghyo Kim. Melatonin synergistically enhances cisplatin-induced apoptosis via the dephosphorylation of ERK/p90 ribosomal S6 kinase/heat shock protein 27 in SK-OV-3 cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B27.

Histone Trimethylation of the p53 Gene by Expression of a Constitutively Active Prolactin Receptor in Prostate Cancer Cells

Prolactin (PRL) is a pituitary polypeptide hormone characterized by multiple biological actions including stimulation of growth in the prostate, breast and ovarian epithelial cells. A sizable body of reports has presented evidences to indicate the involvement of PRL in the pathogenic process of cancers of the reproductive system, such as prostate and breast cancers. PRL exerts its effects by dimerizing its receptor (PRLR) on the plasma membrane, and initiating cellular Jak-Stat signal pathway. We have previously cloned from prostate cancer cells a natural variant of PRLR in which the S2 subdomain of the extracellular domain is missing (ΔS2). Our preliminary data showed that ΔS2 PRLR was able to dimerize and to constitutively activate the β-casein promoter (in the absence of its ligand, PRL) in breast and prostate epithelial cells. Enhancer of zeste homologue 2 (EZH2), an important histone-modifying enzyme, is able to trimethylate histone 3 on lysine 27 (H3K27Me3), consequently leading to gene silencing, especially silencing of tumor suppressor genes such as p53. We hypothesized that ΔS2 PRLR played an important pathogenic role in prostate cancer through, at least partly, alterations in the expression of EZH2 and the trimethylation of histone 3 on lysine 27. In the present study, overexpression of ΔS2 PRLR in prostate epithelial cells was achieved by infection with an adenoviral vector carrying the cDNA. The viable cell number overexpressing ΔS2 PRLR was assessed using MTS reagent. Western blot, chromatin immunoprecipitation (ChIP) assay and acid histone extraction were applied to detect expression of EZH2 as well as trimethylation of histone 3, respectively. In prostate epithelial cells, overexpression of ΔS2 PRLR increased the levels of EZH2 methyltransferase mRNA and protein, induced EZH2 methyltransferase recruitment to chromatin, increased the trimethylation of histone 3 on lysine 27, and decreased expression of the p53 gene. We concluded that ΔS2 PRLR plays an important pathogenic role in prostate cancer through epigenetic covalent modification leading to chromatin remodeling. Hypertrimethylation on H3K27 of the p53 gene promoter region due to elevated expression of ΔS2 PRLR by alternative splicing of the pre-mRNA in its full-length form might serve as a new mechanism underlying human prostate cancer.

Ghrelin inhibits ovarian epithelial carcinoma cell proliferation in vitro

The only orexigenic peptide, ghrelin, which is primarily produced by the gastrointestinal tract, has been implicated in malignant cell proliferation and invasion. Ghrelin is a natural ligand of the growth hormone secretagogue receptor 1a (GHSR1a). However, the role of ghrelin in ovarian epithelial carcinoma remains unknown since the expression of GHSR1a in ovary is not confirmed. The aim of the present study was to assess expression of ghrelin and its receptor in human ovarian epithelial carcinoma and to examine the effect of ghrelin on carcinoma cell proliferation. Frozen sections of ovarian samples and the human ovarian epithelial carcinoma cell line, HO-8910, were used to characterize the expression of ghrelin/GHSR1a axis and the effect of ghrelin on proliferation. We found that ghrelin and GHSR1a are expressed in ovarian epithelial carcinoma in vivo and in vitro. Ghrelin inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, and this inhibition may be abolished by the ghrelin receptor antagonist D-Lys-3-GH-releasing peptide-6 and ghrelin neutralizing antibody. Ghrelin enhances HO-8910 cell apoptosis and autophagy. The activation of mammalian target of rapamycin (mTOR) signaling pathway blocks the effects of ghrelin-induced autophagy and apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation induced by ghrelin. In conclusion, the present study demonstrates that ghrelin inhibits the proliferation of human HO-8910 ovarian epithelial carcinoma cells by inducing apoptosis and autophagy via the mTOR signaling pathway. This study provides a novel regulatory signaling pathway of ghrelin-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy.

PAX8: a sensitive and specific marker to identify cancer cells of ovarian origin for patients prior to neoadjuvant chemotherapy

BackgroundNeoadjuvant chemotherapy followed by cytoreduction surgery has been used where an accurate cytologic or pathologic diagnosis is usually required before the initiation of neoadjuvant chemotherapy. However, it is difficult to make definitive diagnosis of presence of cancer cells, particularly gynecologic versus non-gynecologic origin, from those ascites specimens due to the absence of specific biomarkers of gynecologic cancers. In the present study, we evaluated if, in addition to the routine morphologic diagnosis, the biomarker PAX8 could be useful in recognition of ovarian epithelial cancer cells prior to the neoadjuvant chemotherapy.MethodsTwo hundred and two cytology specimens including 120 pretreatment ovarian cancer samples, 60 benign controls, and 22 malignant non-gynecologic cases were studied. All cytology slides were morphologically reviewed in a blinded fashion without knowing corresponding pathology diagnosis, if present. A total of 168 cytology specimens with a cell block were stained with PAX8 and Calretinin. These included patients with potential for ovarian cancer neoadjuvant chemotherapy (n = 96), metastatic cancers (n = 22), and benign controls (n = 50).ResultsAmong the 96 ascitic samples prior to neoadjuvant chemotherapy, 76 (79%) showing morphologic features consistent with cancers of ovarian primary were all PAX+/Calretinin-. The remaining 20 (21%) cases were positive for adenocarcinoma, but morphologically unable to be further classified. Among the 22 metastatic cancers into the pelvis, one case with PAX8+/Calretinin- represented a renal cell carcinoma and the remaining 21 PAX8-/Calretinin- metastatic cancers were either breast metastasis (n = 4) and the metastasis from gastrointestinal tract (n = 17). Among the 50 benign control pelvic washing cases, 5 PAX8+/Calretinin-cases represented endosalpingiosis (n = 4) and endometriosis (n = 1), 25 PAX8-/Calretinin + cases showed reactive mesothelial cells, and the remaining 20 specimens with PAX8-/Calretinin- phenotype typically contained inflammatory or blood cells without noticeable diagnostic epithelia.ConclusionsPAX8 identifies all Müllerian derived benign or malignant epithelia. When combining with Calretinin, PAX8 is a sensitive marker to diagnose the carcinomas of ovarian origin, which will be ideal to be used for those patients with a possible advanced ovarian cancer prior to receiving neoadjuvant chemotherapy.

Follicle-stimulating hormone inhibits apoptosis in ovarian cancer cells by regulating the OCT4 stem cell signaling pathway

OCT4, a stem cell marker, is overexpressed in several types of human cancer and can induce resistance to chemotherapy and inhibition of apoptosis. We previously demonstrated that human follicle stimulating hormone (FSH) can inhibit ovarian cancer cell apoptosis. However, the role of OCT4 in FSH-induced inhibition of apoptosis has not been reported in detail. Here, we profiled OCT4 protein expression in ovarian epithelial cancer (OEC) with benign cystadenoma, borderline tumor and carcinoma tissues as well as different ovarian cancer cell lines and normal ovarian epithelial cells. Furthermore, the effects of FSH on OCT4 expression and related signaling pathways were evaluated. The overexpression of OCT4 in ovarian carcinoma and OEC cell lines suggest that OCT4 plays a critical role in OEC carcinogenesis. Moreover, FSH-induced apoptosis inhibition was confirmed and FSH stimulation induced the expansion of CD44+CD117+ cells with a stem cell-like phenotype. Re-expression of OCT4 enhanced the expression of Notch, Sox2 and Nanog molecules that play critical roles in cancer stem cell proliferation and differentiation. FSH upregulated the expression of Notch, Sox2 and Nanog and these effects were abolished by knocking down OCT4, suggesting that several cancer stem cell pathways are involved in FSH regulation. We also examined OCT4 expression in surgical specimens of ovarian cancer. Immunohistostaining revealed that OCT4 expression was increased in ovarian carcinoma compared with benign cystadenomas and borderline tumors, and OCT4 expression was significantly correlated with histological grade. Staining for OCT4 was increased in serous cystadenocarcinoma, when compared with clear cell carcinoma. In summary, the OCT4 cancer stem cell signaling pathway may mediate FSH-induced inhibition of apoptosis and could provide a target for treatment of ovarian cancer.

Association of myeloperoxidase with ovarian cancer

Myeloperoxidase (MPO) is an oxidant generating enzyme normally restricted to myeloid cells, however aberrant MPO expression has been found to occur in non-myeloid cells in some disease states. The functional -463GA promoter polymorphism alters MPO expression levels. The -463G is within an SP1 binding site and is associated with higher gene expression. The G allele is most frequent with ~62% of European populations being GG homozygotes. The GA polymorphism has been associated with risk or survival in a variety of cancers including lung and breast cancer. In this study we determined the frequency of the -463G/A polymorphism in 230 ovarian cancer patients, 75 patients with borderline ovarian tumors, and 299 healthy controls. The GG genotype was found to be overrepresented in patients with early stage ovarian cancer (83.3% GG, p = 0.008) as compared to healthy controls (62% GG), suggesting that MPO oxidants may increase risk. Immunohistochemical analysis revealed MPO expression in a subset of columnar ovarian epithelial carcinoma cells in early stage carcinomas.

Enrichment of ovarian cancer stem‐like cells is associated with epithelial to mesenchymal transition through an miRNA‐activated AKT pathway

Evidence has indicated that ovarian epithelial cancer-type cells under serum-free culture conditions can form spheroid cells and exhibit characteristics expected of cancer stem-like cells (CSCs). However, the mechanism by which differentiated ovarian cancer cells acquire stem-cell properties during CSC enrichment has needed to be elucidated. Recent studies have demonstrated that induction of epithelial to mesenchymal transition (EMT) can generate CSCs and be associated with tumour aggressiveness and metastasis. Ovarian epithelial cancer cell lines, SKOV3 and HO8920, were cultured for spheroid cells and adherent cells. CSC enrichment was investigated using MTT assay, flow cytometery and qRT-PCR and expression level of PI3K/AKT pathway components was analysed by western blotting. Compared to adherent cells, the spheroid cells expressed mesenchymal markers highly and exhibited significantly more motility; we also observed increases in phosphate AKT1 levels in the spheroid cells. Moreover, transfection of miR-20a or miR-200c led to corresponding reduction in endogenous PTEN protein, while AKT1 and phosphate AKT1 levels were upregulated in miRNAs-transfected cells. Finally, PI3K/AKT pathway inhibitor LY294002 reduced expressions of mesenchymal markers and stem-cell gene activity in spheroid cells, enhancing sensitivity of spheroid cells to paclitaxel treatment. Our findings demonstrate that EMT contributed to enrichment of ovarian CSCs in vitro, making EMT targeting in epithelial ovarian cancer a novel therapeutic option.

Study of growth, invasion and metastasis on ovarian epithelial cancer cell line with CCL18 over-expression by mediated in vitro

To investigate the effect of growth, invasion and metastasis on ovarian cancer cell line SKOV3 with chemokine (C-C motif) ligand 18 (CCL18) over-expression by mediated in vitro. The restructuring plasmid of CCL18 expression was constructed and SKOV3 cells was transfected with plasmid DNA in vitro. The growth curve, cell cycle, cell migration, invasion and adhesion capacity in SKOV3-CCL18 cells and control cells were detected by methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, transwell chamber, migration invasion and fibronectin adhesion method, respectively. (1) CCL18-pEGFP-N1 plasmid was successfully constructed and transmitted to the SKOV3 cells, the stable growth of the subculture SKOV3-CCL18 cells was screening and completed. (2) Compared with SKOV3-CCL18 cells and SKOV3 or SKOV3-pEGFP-N1 cells, there was not differences statistically significant in growth curve (all P > 0.05); but the rate of the SKOV3-CCL18 cells in a proliferative field (S+G2+M) was significantly higher than that in SKOV3 or SKOV3-pEGFP-N1 cells (32.80% versus 27.06%, 32.80% versus 26.98%, respectively; all P < 0.05). (3) The invasion, migration and adhesion capacity of SKOV3-CCL18 cells (being 0.49 ± 0.18, 1.16 ± 0.25 and 0.39 ± 0.10, respectively) in vitro were significantly higher than those in SKOV3 (being 0.23 ± 0.13, 0.36 ± 0.10 and 0.16 ± 0.03, respectively) or SKOV3-pEGFP-N1 cells (being 0.19 ± 0.05, 0.38 ± 0.23 and 0.13 ± 0.11, respectively; all P < 0.05). The CCL18 over-expression in epithelial ovarian cancer SKOV3 cells could lead to strengthen ability of invasion, migration and adhesion in vitro.

An HLA-modified ovarian cancer cell line induced CTL responses specific to an epitope derived from claudin-1 presented by HLA-A*24:02 molecules

In an attempt to induce cytotoxic T lymphocytes (CTLs) that react to ovarian cancer cells, we isolated a CTL clone that specifically recognizes claudin-1 in an HLA-A*24:02-restricted manner. Naïve CD8(+) T lymphocytes were obtained from a healthy adult donor and stimulated twice in vitro with HLA-modified TOV21G cells that were originally derived from an ovarian clear-cell carcinoma line. The TOV21G modification involved RNAi-mediated gene silencing of intrinsic HLA molecules and lentiviral transduction of a synonymously mutated HLA-A*24:02. Then, cDNA library construction using mRNA extracted from the parental TOV21G cells and subsequent expression cloning were conducted. These experiments revealed that a CTL clone obtained from the bulk culture recognized a minimal epitope peptide RYEFGQALF, which was derived from an autoantigen claudin-1 presented by HLA-A*24:02 molecules. This clone exhibited cytolytic activities against three ovarian cancer cell lines and normal bronchial epithelial cells in an HLA-A*24:02-restricted manner. Our data indicate that HLA-modified cancer cells can be used as an artificial antigen-presenting cell to generate antigen-specific CTLs in a manner restricted by an HLA allele of interest.

Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients.

Proteomic analyses of serous and endometrioid epithelial ovarian cancers – Cases studies – Molecular insights of a possible histological etiology of serous ovarian cancer

Epithelial ovarian carcinogenesis may occur de novo on the surface of ovarian mesothelial epithelial cells or from cells originating in other organs. Foreign Müllerian cell intrusion into the ovarian environment has been hypothesized to explain the latter scenario. In this study, MALDI MS profiling technology was used to provide molecular insights regarding these potentially different mechanisms. Using MALDI MS profiling, the molecular disease signatures were established in their anatomical context. MALDI MS profiling was used on serous and endometrioid cancer biopsies to investigate cases of epithelial ovarian cancer. We then applied bioinformatic methods and identification strategies on the LC-MS/MS analyses of extracts from digested formalin-fixed, paraffin-embedded tissues. Extracts from selected regions (i.e. serous ovarian adenocarcinoma, fallopian tube serous adenocarcinoma, endometrioid ovarian cancer, benign endometrium, and benign ovarian tissues) were performed, and peptide digests were subjected to LC-MS/MS analysis. Comparison of the proteins identified from benign endometrium or three ovarian cancer types (i.e. serous ovarian adenocarcinoma, endometrioid ovarian adenocarcinoma, and serous fallopian tube adenocarcinoma) provided new evidence of a possible correlation between the fallopian tubes and serous ovarian adenocarcinoma. Here, we propose a workflow consisting of the comparison of multiple tissues in their anatomical context in an individual patient. The present study provides new insights into the molecular similarities between these two tissues and an assessment of highly specific markers for an individualized patient diagnosis and care.

Resistin Promotes the Expression of Vascular Endothelial Growth Factor in Ovary Carcinoma Cells

Resistin is a novel hormone that is secreted by human adipocytes and mononuclear cells and is associated with obesity, insulin resistance and inflammation. Recently, resistin has been postulated to play a role in angiogenesis. Here, we investigated the hypothesis that resistin regulates ovary carcinoma production of vascular endothelial growth factor (VEGF) and the angiogenic processes. We found that in human ovarian epithelial carcinoma cells (HO-8910), resistin (10–150 ng/mL) enhanced both VEGF protein and mRNA expression in a time- and concentration-dependent manner, as well as promoter activity. Furthermore, resistin enhanced DNA-binding activity of Sp1 with VEGF promoter in a PI3K/Akt-dependent manner. PI3K/Akt activated by resistin led to increasing interaction with Sp1, triggering a progressive phosphorylation of Sp1 on Thr453 and Thr739, resulting in the upregulation of VEGF expression. In an in vitro angiogenesis system for endothelial cells (EA.hy926) co-cultured with HO-8910 cells, we observed that the addition of resistin stimulated endothelial cell tube formation, which could be abolished by VEGF neutralizing antibody. Our findings suggest that the PI3K/Akt-Sp1 pathway is involved in resistin-induced VEGF expression in HO-8910 cells and indicates that antiangiogenesis therapy may be beneficial treatment against ovarian epithelial carcinoma, especially in obese patients.

Abstract 3730: 2-Methoxyestradiol induces differentiation of epithelial ovarian cancer-initiating cells sensitizing them to chemotherapy.

Abstract Ovarian cancer is the most deadly gynecological malignancy. Its lethality is mainly due to late diagnosis and acquisition of resistance to available therapies. Current evidence considers “tumor cell heterogeneity” as a cancer hallmark. A subset of cancer cells, named "cancer-initiating cells" (CICs), possesses stem cell-like capabilities allowing self-renewal, differentiation and dedifferentiation into other cell types, some of them evolved to a more invasive and resistant phenotype. Thus, the existence of CICs allows tumor to overcome therapy and regrowth as resistant kind over time. Finding therapeutic strategies effective against this subset of cells remain a major goal in cancer therapeutics. Previously we have shown that 2-methoxyestradiol (2-ME), an endogenous estrogen metabolite, induces cell death in ovarian cancer cells. Objective: In the present work we investigated the potential effects of 2-ME on CICs isolated from either ovarian cancer cell lines or primary tissue cultures obtained from ovarian cancer patients. Methods: CICs were isolated from both ovarian cancer cell lines (SKOV3, UCI 101, HEY) as well as primary tissue cultures established from ascites samples. Stemness, epithelial-mesenchymal transition (EMT), and anti-apoptotic markers were studied by immunoblotting (W-B), RT-PCR and immunohistochemistry in CICs cultured under stem-selection medium/non-attachment conditions and after treatment with different concentrations of 2-ME (5 to 10 uM, up to 24h). CICs treated under similar conditions were then exposed to different concentrations of chemotherapeutic reagents (i.e. paclitaxel, 0,5-5 uM, for up to 48 h). Cell death was measured by Tunel assay and PARP cleavage (measured by W-B). Results: Isolated CICs grow forming ovospheres that express higher levels of Oct-4, Nanog, Snail, N-Cadherin, CD44, CD133, survivin, and lower levels of E-cadherin compared with non-selected cell population (whole cell population, WP). In contrast to WP, CICs did not undergo apoptosis upon 2-ME exposure (5 uM for 24 h) measured by PARP cleavage. However, 2-ME treatment results in a decrease of Nanog, N-cadherin, Snail, Survivin and an increase in E-cadherin protein expression levels. In addition, a decrease in mRNA levels of Twist, Zeb2 (but not Zeb1) is observed under similar conditions (5 uM for 6 h). 2-ME pretreatment increases paclitaxel-induced cell death in CICs compared to those treated with paclitaxel alone, measured by Tunel assay. Conclusions: 2-ME induces the loss of stemness and EMT markers in epithelial ovarian CICs. This differentiating effect induced by 2-ME results in sensitization of this subset of resistant cells to chemotherapeutic agents. Further studies might be conducted to assess the clinical usefulness of this combination. (FONDECYT 1120292, and BMRC CTU06) Citation Format: Mauricio A. Cuello, Sumie Kato, Katherine Hormazábal, Lorena Abarzua, Loreto Bravo, Isabel Barriga, Nicanor Barrena, Jorge Brañes, Erasmo Bravo, Roger Gejman, Enrique Castellón, Gareth Owen. 2-Methoxyestradiol induces differentiation of epithelial ovarian cancer-initiating cells sensitizing them to chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3730. doi:10.1158/1538-7445.AM2013-3730

Abstract 1735: Obatoclax enhances CDDP and trail-induced apoptosis in epithelial ovarian adenocarcinoma cells.

Abstract Epithelial ovarian cancer comprises the majority of malignant ovarian tumors in adult women. Ovarian cancer is the fifth cause of death in women and annually 13,850 deaths are reported in United States. Effective screening strategies are lacking and the majority of cases are diagnosed with advanced stage disease. Chemo is the conventional therapy after surgery but the initial response is between 60 to 80%, however, some patients that initially responded to the therapy suffer of recurrent disease or become resistant to the treatment. As a consequence, the overall 5 year survival is only about 30%. It is important to identify novel therapies for ovarian cancer to treat or sensitize tumor cells to the existence therapy. It has been suggested that the tumor cells become resistance due to an increased expression of antiapoptotic proteins especially from the Bcl2 family (Bcl-2, Bcl-XL, Mcl-1). Obatoclax is a small molecule that mimics the BH3 domain of the BH3 only family members and inhibits the effect of antiapoptotic Bcl-2 members. The objective of this study was to assess the susceptibility of ovarian cancer cell lines to chemo or immuno therapy mediated apoptosis by obatoclax and also to elucidate the mechanism. Four different epithelial ovarian adenocarcinomas cell lines (SKOV3, NIH-OVCAR-3, TOV112D and TOV21G) were treated with different concentrations of Obatoclax (nM-μM), CDDP (5-50μg/ml) or TRAIL (50-1200ng/ml) alone or with a combination of treatment to assess the viability, measured by the XTT kit (Roche). Basal and cell treated total protein expressions were assessed by western. Induction of apoptosis was measured by TUNEL. As a single agent, Obatoclax or CDDP inhibited the viability of the cells in a dose response form; TRAIL did not show an evident response. The combination of the treatment after selecting the IC50 and IC20 response showed an increased inhibition of the viability. TUNEL positive cells were measured and the induction of apoptosis was mediated by a caspase dependent manner and PARP cleavage. Our data suggest that obatoclax in combination with CDDP or TRAIL can enhance the induction of apoptosis in resistance cell lines, suggesting to be an optional treatment of patients with ovarian cancer. Citation Format: Melisa A. Martinez-Paniagua, Luis A. Romero-Gonzalez, Yu Mei Anguiano-Hernandez, Carolina Gonzalez-Torres, Mario I. Vega. Obatoclax enhances CDDP and trail-induced apoptosis in epithelial ovarian adenocarcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1735. doi:10.1158/1538-7445.AM2013-1735

Abstract 4523: Targeted delivery to tumor cells by using tunable nano-delivery system with chlorotoxin.

Abstract The aim of this research is to develop the Tunable Nano-Delivery System (TNDS) for specific targeting of the tumor cells using Chlorotoxin (CTX). We present preliminary findings for the TNDS localized drug delivery system comprising of non-ionic surfactant vesicles (niosomes) with CTX embedded in a biodegradable and temperature sensitive chitosan network. The system demonstrates a new approach in the treatment of cancers, through the controlled and targeted delivery to tumor cells (the release time and dosage can be accurately controlled) while sparing normal cells. CTX is a scorpion-derived peptide that binds specifically to neuroectodermal tumors, e.g., glioma, but not to normal tissues. Previously, we have shown that there is a specific interaction between chitosan and Mucin1 (MUC1) receptors over expressed in epithelial ovarian carcinoma resulting in higher chitosan accumulation on the cancer cell surface than on normal ovarian epithelial cells. The current research addresses the incorporation of CTX embedded in Chitosan network to enhance specific targeting, which already benefits from the intrinsic interaction of MUC1 with Chitosan. Our current studies incorporating CTX in chitsoan resulting in TNDS-CTX complex are preformed in cell-free system. First, the release rates and release kinetics of TNDS-CTX are being measured in cell-free system using High Performance Liquid Chromatography (HPLC) and Attenuated Total Reflectance- Fourier Transform Infra-Red (ATR-FTIR) spectroscopy. Results are being compared to our established TNDS delivery system to better determine CTX specific chemical binding sites along with the sites already determined to be specific for MUC1. Next, we will be testing TNDS-CTX in vitro in glioma, epithelial ovarian carcinoma, and normal ovarian epithelial and astrocyte cells. The level of fluorescence of normal cells and tumor cells exposed to the TNDS-CTX-fluorescently labeled are evaluated in in vitro by confocal microscopy. The studies in cell-free system showed that the release rates could be finely controlled depending on the specific design of the TNDS-CTX e.g., ratio of niosomes to chitosan and amount of crosslinker for the chitosan network. We are currently evaluating TNDS-CTX containing benchmark chemotherapeutics such as Paclitaxel, and it is expected that the presence of CTX will enhance cellular uptake of chemotherapeutics when compared to the noisome-chitosan delivery system without CTX. Based on our preliminary data, it is anticipated that CTX will perform as a targeting ligand improving the TNDS-CTX's anti-tumor efficacy owing to enhanced tumor targeting and its tunable and localized delivery. Citation Format: Rana Falahat, Eva Christabel Williams, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Targeted delivery to tumor cells by using tunable nano-delivery system with chlorotoxin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4523. doi:10.1158/1538-7445.AM2013-4523