Epithelial Ovarian Carcinoma Cell Lines Research Articles

HDAC1/2 control mesothelium/ovarian cancer adhesive interactions impacting on Talin-1-α5β1-integrin-mediated actin cytoskeleton and extracellular matrix protein remodeling

BackgroundPeritoneal metastasis, which accounts for 85% of all epithelial ovarian carcinoma (EOC) metastases, is a multistep process that requires the establishment of adhesive interactions between cancer cells and the peritoneal membrane. Interrelations between EOC and the mesothelial stroma are critical to facilitate the metastatic process. No data is available so far on the impact of histone acetylation/deacetylation, a potentially relevant mechanism governing EOC metastasis, on mesothelial cells (MCs)-mediated adhesion.MethodsStatic adhesion and peritoneal clearance experiments were performed pretreating mesenchymal-like MCs and platinum—sensitive/resistant EOC cell lines with MS-275—a Histone deacetylase (HDAC)1–3 pharmacological inhibitor currently used in combination trials. Results were acquired by confocal microscopy and were analyzed with an automated Opera software.The role of HDAC1/2 was validated by genetic silencing. The role of α4-, α5-α1 Integrins and Fibronectin-1 was validated using specific monoclonal antibodies.Quantitative proteomic analysis was performed on primary MCs pretreated with MS-275. Decellularized matrices were generated from either MS-275-exposed or untreated cells to study Fibronectin-1 extracellular secretion. The effect of MS-275 on β1 integrin activity was assessed using specific monoclonal antibodies. The role of Talin-1 in MCs/EOC adhesion was analyzed by genetic silencing. Talin-1 ectopic expression was validated as a rescue tool from MS-275-induced phenotype. The in vivo effect of MS-275-induced MC remodeling was validated in a mouse model of peritoneal EOC dissemination.ResultsTreatment of MCs with non-cytotoxic concentrations of MS-275 caused a consistent reduction of EOC adhesion. Proteomic analysis revealed several pathways altered upon MC treatment with MS-275, including ECM deposition/remodeling, adhesion receptors and actin cytoskeleton regulators. HDAC1/2 inhibition hampered actin cytoskeleton polymerization by downregulating actin regulators including Talin-1, impairing β1 integrin activation, and leading to abnormal extracellular secretion and distribution of Fibronectin-1. Talin-1 ectopic expression rescued EOC adhesion to MS-275-treated MCs. In an experimental mouse model of metastatic EOC, MS-275 limited tumor invasion, Fibronectin-1 secretion and the sub-mesothelial accumulation of MC-derived carcinoma-associated fibroblasts.ConclusionOur study unveils a direct impact of HDAC-1/2 in the regulation of MC/EOC adhesion and highlights the regulation of MC plasticity by epigenetic inhibition as a potential target for therapeutic intervention in EOC peritoneal metastasis.

LncRNA PART1 Regulates Ovarian Carcinoma Development via the miR-150-5p/MYB Axis.

Over the past few years, there have been many reports on the abnormal expression and functional relevance of long non-coding RNAs (lncRNAs) in tumors. The role played by lncRNAs in epithelial ovarian carcinoma (EOC) remains poorly understood, however the goal of the present work was to study molecular mechanisms that underlie involvement of prostate androgen-regulated transcript 1 (PART1) lncRNA in EOC development. A total of 25 tumor and 17 normal specimens were obtained from women undergoing surgery between 2015 and 2019 in the Second Affiliated Hospital, Nanjing Medical University. Expression levels for PART1 in EOC tissue and EOC cell lines were assessed using qRT-PCR. Assays for CCK-8, trans-well, colony forming and western blotting were used to investigate PART1, miR-150-5p and MYB (MYB proto-oncogene) for their invovement in EOC cell proliferation, migration and invasion. Luciferase reporter gene assay was also performed to investigate biological functions of PART1, miR-150-5p and MYB in EOC, and an animal xenograft model was employed to test tumorigenicity. PART1 expression was increased in EOC relative to normal cells and correlated with EOC cell proliferation, migration and invasion. PART1 can sponge miR-150, thereby inhibiting growth of EOC by targeting MYB. The xenograft mouse model revealed that PART1 can regulate tumorigenesis in vivo. The PART1/miR-150/MYB axis is involved in EOC pathogenesis and could represent a new target to use in diagnosis and therapy.

Targeting RPL23 restores chemosensitivity of cisplatin-resistant ovarian carcinoma by inhibiting EMT.

Chemoresistance is the inevitable outcome of chemotherapy for epithelial ovarian carcinoma (EOC), and its mechanism is still not fully understood. This study explored the role of ribosomal protein L23 (RPL23) in cisplatin resistance of EOC. WGCNA based on TCGA and GEO was used to screen and analyze target genes related to EOC chemotherapy sensitivity. Clinical samples of cisplatin resistance were collected to detect the expression of target genes. Cisplatin resistance was induced in EOC cell lines A2780 and SKOV3. The cell abilities of invasion, migration and adhesion were observed. Western blotting was used to detect protein expressions. Bioinformatics analysis showed that RPL23 may be related to EOC chemotherapy sensitivity, and was highly expressed in clinical samples and cell lines of cisplatin-resistant. After A2780 and SKOV3 were resistant to cisplatin, the inhibitory abilities of therapeutic dose of cisplatin on their invasion, migration and adhesion were significantly attenuated, and N-cadherin and vimentin were significantly up-regulated while E-cadherin was significantly down-regulated. However, above phenomena were significantly reversed after RPL23 knockdown. Taken together, the overexpressed RPL23 may lead to platinum resistance by inducing epithelial-mesenchymal transition (EMT) in EOC. Targeting knockdown RPL23 would restore the sensitivity of EOC cells to cisplatin by inhibiting EMT, suggesting that RPL23 is a potential therapeutic target for EOC after platinum resistance.

MicroRNA‑195 suppresses cell proliferation, migration and invasion in epithelial ovarian carcinoma via inhibition of the CDC42/CCND1 pathway

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.

Wnt signaling modulator DKK1 as an immunotherapeutic target in ovarian cancer

ObjectivesWnt pathway mutations are a hallmark of endometrioid and clear cell subtypes of epithelial ovarian carcinoma (EOC). However, no drugs targeting the Wnt pathway in EOC are FDA-approved. Dickkopf-related protein 1 (DKK1), a modulator of the Wnt pathway, has emerged as a promising therapeutic target. We aimed to examine the role of DKK1 and the effects of a monoclonal antibody against DKK1 (DKN-01) in vivo and in a murine model of ovarian cancer. MethodsWe examined in vitro the role of DKK1 and the effects of DKK1 inhibition in EOC cell lines. We then studied in vivo the role of DKN-01 and DKK1 overexpression on tumor burden and anti-tumor immune cell populations using the ID8 syngeneic mouse model. ResultsDKN-01 did not phenotypically alter ES2 cells in vitro; however, DKK1 inhibition promoted Wnt signaling. Tumor burden and immune populations were unchanged in ID8 challenged mice treated with mDKN01. Mice challenged with ID8 cells overexpressing DKK1 had tumor burden similar to controls (p = 0.175). However, the overexpression of DKK1 decreased CD45+ leukocyte infiltration into the peritoneum (p = 0.008) and omentum (p = 0.032), reducing both natural killer (NK) and CD8 T cells, and reducing interferon-gamma (IFNγ) expression on activated CD8 T cells. ConclusionsOur results suggest that DKK1 inhibition does not affect tumor growth in the ID8 ovarian cancer model. DKK1 overexpression alters anti-tumor immune populations within the tumor microenvironment. Thus, our findings confirm DKK1 as a new therapeutic target in EOC and suggest that DKK1 inhibition may function best in a combinatorial, immune-modulatory therapy.

Dendritic Cells Loaded with Heat Shock-Conditioned Ovarian Epithelial Carcinoma Cell Lysates Elicit T Cell-Dependent Antitumor Immune Responses In Vitro.

Ovarian epithelial carcinoma (OEC) is the most frequent ovarian tumor, characterized by a high mortality in advanced stages where conventional therapies are not effective. Based on the role of the immune system in the progression of this disease, immunotherapy using checkpoint blockade has been considered as a therapeutic alternative. Nevertheless, its results do not match up to the positive results in entities like melanoma and other malignancies, suggesting the need to find other therapies to be used alone or in combination. Dendritic cell- (DC-) based vaccines have shown promising results in several types of cancer, such as melanoma, prostate, and lung cancers, due to the essential role played by DCs in the activation of specific T cells, thus using other ways of activating the immune response than immune checkpoint blockade. During the last decade, we have used DC-based vaccines loaded with an allogeneic heat shock-conditioned melanoma cell lysate in the treatment of advanced stage patients in a series of clinical trials. In these studies, 60% of treated patients showed immunological responses which correlated positively with improved survival. Considering the relevance of ovarian cancer and the promising results of our DC-based vaccine, we show here that heat shock-conditioned cell lysates derived from ovarian epithelial carcinoma cell lines have the potential to induce the phenotypic and functional maturation of human DC, which in turn, is able to induce an efficient CD4+ and CD8+ T cell-mediated immune responses against ovarian cancer cell lines in vitro. In summary, OEC heat shock-conditioned cell lysate-loaded DCs may be considered for future combined immunotherapy approaches against ovarian tumors.

Abstract 4296: Identification of cancer specific protein 80 (CSP-80), a novel 80 kDa MAP1a-like protein in ovarian carcinoma cells

Abstract Background: Although interfering with the actions of microtubule-associated proteins (MAP’s) is successful against ovarian epithelial carcinoma (OVCA), knowledge of the MAP’s in OVCA cells is lacking. We sought to identify cancer-specific MAP’s in OVCA cells.Methods: Four human OVCA cell lines, primary OVCA tissue, ascitic cells and metastases from 9 patients and normal ovarian tissue from six women were compared to control human and rat brain tissue, uterine myomas (benign tumors) and rat and human non-neural organs. Modified Western blotting using panels of anti-microtubule-associated protein monoclonal antibodies were used. An in-vitro brain and OVCA mixing and recovery experiment was carried out to assess possible degradation of MAP1a by OVCA. A paclitaxel-driven tubulin assembly assay was performed to determine whether the OVCA protein has microtubule-associated protein properties. The transcriptome of CSP-80 was sequenced using primer extension sequencing. Results: (1) In contradistinction to the 350 kDa MAP1a present in control brain extracts, only an 80 kDa molecule with MAP1a immune-reactivity was found in OVCA cell lines and tissues. No immune-reactivity was present in the negative controls. (2)Following the mixing experiments there was no increase of CSP-80; CSP-80 is not a degradation artifact of MAP1a. (3) The paclitaxel-driven tubulin assembly assay showed that CSP-80 bound to microtubules during tubulin polymerization. (4) No CSP-80 was identified in human or rat non-malignant tissues including normal human and rat brain or normal human ovary, placenta or uterine myomas, or normal rat liver, spleen or lung. (5) cDNA sequencing shows a 2603 bp fragment at a 5’ terminal of p80 to be homologous with the analogous segment of MAP1a. (6) Further testing showed that CSP-80 was present in other reproductive and non-reproductive carcinomas. Again, no 350 kDa MAP1a was present. Studies are underway to determine whether CSP-80 is a mutated protein or the product of post-transcriptional modification. Conclusion: CSP-80 is a novel cancer-related protein that appears to be limited to an 80 kDa sequence of the MAP1a gene, or a post-transcriptionally modified product. Since CSP-80 exhibits tubulin-binding activity, this may explain the efficacy of taxane derivatives. CSP-80 may disclose some aspects of carcinogenesis, be a valuable marker of cancer and therapeutic efficacy, and offer new avenues for cancer treatment. Citation Format: Ahmed Fadiel, Viswam S. Nair, Peter Aceto, Frederick Naftolin. Identification of cancer specific protein 80 (CSP-80), a novel 80 kDa MAP1a-like protein in ovarian carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4296.

Overexpression of thymic stromal lymphopoietin is correlated with poor prognosis in epithelial ovarian carcinoma.

Thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that has been reported to be associated with several malignant tumors. The present study aimed to evaluate its role in epithelial ovarian carcinoma (EOC). The mRNA levels of TSLP in human EOC samples and EOC cell lines were determined. Then, the expression of TSLP was examined in 144 clinical tissue microarray samples and correlated with clinicopathological factors. Finally, the correlation between TSLP overexpression and prognosis of EOC patients was analyzed. Our data show that mRNA levels of TSLP were significantly higher in EOC tissues and cell lines. Chi-square tests revealed that TSLP overexpression in EOC was significantly associated with age, histological type, Federation of Gynecology and Obstetrics (FIGO) stage, histological differentiation, pelvic involvement, and lymphatic metastasis. Kaplan–Meier survival analysis revealed that poor prognosis was significantly correlated with older age, advanced FIGO stage, poor histological differentiation, pelvic involvement, lymphatic involvement, or TSLP overexpression (P<0.05). Additionally, multivariate Cox regression analysis confirmed pelvic involvement and TSLP overexpression as independent prognostic factors for both overall and disease-free survival. Taken altogether, TSLP overexpression reflects a more malignant phenotype and TSLP may be a novel biomarker for EOC.

Elimination of Peroxiredoxin 1 Abrogates Proliferation and Viability in Extracellular Matrix-Detached SKOV3 Ovarian Cancer Cells

Epithelial ovarian carcinoma has a relatively low survival rate due to the fact that this disease is often diagnosed after it has already progressed to stage III or stage IV, thus making the disease more difficult to treat. In order to successfully metastasize to these regional and distant sites, tumor cells must be able to overcome extracellular matrix (ECM)-detachment-induced effects, including combatting increased reactive oxygen species (ROS). Here, we investigate peroxiredoxin 1 (PRDX1) and find that PRDX1 plays an important role in proliferation in ECM-attached and ECM-detached SKOV3 cells, an epithelial ovarian carcinoma cell line. Furthermore, we find that PRDX1 deficiency in SKOV3 cells promotes a substantial decrease in cell viability in ECM detachment contributing to abrogated cell growth in anchorage independence. Thus, our study finds that PRDX1 deficiency uniquely compromises cell viability in ECM-detached cells, suggesting that PRDX1 could be a potential therapeutic target for late-stage epithelial ovarian carcinoma.

Inhibition of ZEB1 leads to inversion of metastatic characteristics and restoration of paclitaxel sensitivity of chronic chemoresistant ovarian carcinoma cells

ZEB1, a member of the zinc-finger E-box binding homeobox family, is considered to play a crucial role in cancer progression and metastasis. In the current study, we investigated the role of ZEB1 in metastasis and chronic chemoresistance of epithelial ovarian carcinoma (EOC) cells. Using several EOC and acquired paclitaxel (PTX)-resistant EOC cell lines, we investigated whether silencing ZEB1 led to a reversal of the chemoresistance and metastatic potential in vitro and in vivo. Subsequently, the expression of ZEB1 in EOC tissues and its association with the oncologic outcome were investigated. According to the immunohistochemical staining of EOC tissues, as the positivity of ZEB1 expression was increased, the overall survival of EOC patients became poorer (P = 0.0022 for trend). Additionally, cell migration and invasion were significantly decreased by ZEB1 silencing in both PTX-sensitive and PTX- resistant cells. Although PTX-sensitivity was not changed by silencing ZEB1 in parental EOC cells, the depletion of ZEB1 made the PTX-resistant EOC cells more sensitive to PTX treatment. In an animal model, mice injected with ZEB1-silencing PTX-resistant cells survived for longer than the control cell-injected mice. Although the intravenous injection of PTX did not affect the tumor weight of shCtrl cells, the tumor weight of shZEB1 cells was significantly reduced by PTX treatment. The current data indicate the possible involvement of ZEB1 in the metastasis and paclitaxel resistance of EOC, and suggest that targeting this molecule may reverse the malignant potential and improve the oncologic outcome for EOC patients.

MAL gene overexpression as a marker of high-grade serous ovarian carcinoma stem-like cells that predicts chemoresistance and poor prognosis

BackgroundThe existence of cancer stem cells (CSCs) within a tumor bulk has been demonstrated for many solid tumors including epithelial ovarian carcinoma (EOC). CSCs have been associated to tumor invasion, metastasis and development of chemoresistant recurrences. In this context, we aim to characterize EOC CSCs from the molecular point of view in order to identify potential biomarkers associated with chemoresistance.MethodsWe isolated a population of cells with stem-like characteristics (OVA-BS4 spheroids) from a primary human EOC cell line under selective conditions. OVA-BS4 spheroids were characterized for drug response by cytotoxicity assays and their molecular profile was investigated by microarray and RT-qPCR. Finally, we performed a gene expression study in a cohort of 74 high-grade serous EOC (HGSOC) patients by RT-qPCR.ResultsSpheroids exhibited properties of self-renewal and a pronounced expression of well-known stem cell genes. Moreover, they demonstrated greater resistance towards several anticancer drugs compared to parent cell line, consistent with their higher ABCG2 gene expression. From microarray studies MAL (T-cell differentiation protein) emerged as the most up-regulated gene in spheroids, compared to parent cell line. In HGSOC patients, MAL was significantly overexpressed in platinum-resistant compared to platinum-sensitive patients and resulted as an independent prognostic marker of survival.ConclusionsThis investigation provides an important contribution to the identification of molecular markers of ovarian CSCs and chemoresistance. Successful translation of molecular findings would lead to a better comprehension of the mechanisms triggering chemoresistant recurrences, to the individuation of novel therapeutic targets and to the personalization of treatment regimens.

Capn4 Enhances Osteopontin Expression through Activation of the Wnt/β-Catenin Pathway to Promote Epithelial Ovarian Carcinoma Metastasis

Background and Aim: Increasing evidence shows that the calpain regulatory subunit Capn4 can modulate the proliferation and metastasis of cancer cells, and plays an important role in the development of malignant tumors. However, there is no information on the clinical significance of Capn4 in epithelial ovarian carcinoma (EOC) or the molecular mechanisms by which Capn4 promotes the growth and metastasis of EOC. Therefore, the aim of this study was to clarify the role of Capn4 in EOC. Methods: We evaluated Capn4 and osteopontin (OPN) expression in EOC cell lines and tissues from patients with ovarian cancer by western blotting and immunohistochemical analysis. We then created cell lines with downregulated and upregulated Capn4 expression, using Capn4-targeting small interfering RNA and a pcDNA3.1-Capn4 overexpression vector, respectively, to investigate its function in EOC in vitro. In addition, we investigated the potential mechanism underlying the function of Capn4 by examining the effect of modifying Capn4 expression on Wnt/β-catenin signaling pathway-related genes by western blotting. Results: Capn4 was overexpressed in clinical EOC tissues compared with that in normal ovarian epithelial tissue, and was associated with poor clinical outcomes. Upon silencing or overexpressing Capn4 in EOC cells, we concluded that Capn4 promotes cell proliferation and migration in vitro. Furthermore, Capn4 promoted EOC metastasis by interacting with the Wnt/β-catenin signaling pathway to upregulate OPN expression. Conclusion: Our study indicates that Capn4 plays a critical role in the progression and metastasis of EOC, and could be a potential therapeutic target for EOC management.

Procyanidin-rich extract of natural cocoa powder causes ROS-mediated caspase-3 dependent apoptosis and reduction of pro-MMP-2 in epithelial ovarian carcinoma cell lines.

Over the last four centuries, cocoa and chocolate have been described as having potential medicinal value. As of today, Theobroma cacao L. (Sterculiaceae) and its products are consumed worldwide. They are of great research interest because of the concentration dependent antioxidant as well as pro-oxidant properties of some of their polyphenolic constituents, specially procyanidins and flavan-3-ols such as catechin. This study was aimed at investigating the cellular and molecular changes associated with cytotoxicity, caused due pro-oxidant activity of cocoa catechins and procyanidins, in ovarian cancer cell lines. Extract of non-alkalized cocoa powder enriched with catechins and procyanidins was used to treat human epithelial ovarian cancer cell lines OAW42 and OVCAR3 at various concentrations ≤1000μg/mL. The effect of treatment on intracellular reactive oxygen species (ROS) levels was determined. Apoptotic cell death, post treatment, was evaluated microscopically and using flow cytometry by means of annexin-propidium iodide (PI) dual staining. Levels of active caspase-3 as a pro-apoptotic marker and matrix metalloproteinase 2 (MMP2) as an invasive potential marker were detected using Western blotting and gelatin zymography. Treatment with extract caused an increase in intracellular ROS levels in OAW42 and OVCAR3 cell lines. Bright field and fluorescence microscopy of treated cells revealed apoptotic morphology and DNA damage. Increase in annexin positive cell population and dose dependent upregulation of caspase-3 confirmed apoptotic cell death. pro-MMP2 was found to be downregulated in a dose dependent manner in cells treated with the extract. Treated cells also showed a reduction in MMP2 activity. Our data suggests that cocoa catechins and procyanidins are cytotoxic to epithelial ovarian cancer, inducing apoptotic morphological changes, DNA damage and caspase-3 mediated cell death. Downregulation of pro-MMP2 and reduction in active MMP2 levels imply a decrease in invasive potential of the cells. Apoptosis and MMP2 downregulation appear to be linked to the increase in intracellular ROS levels, caused due to the prooxidant effect of cocoa procyanidin extract.

Effect of procyanidin-rich extract from natural cocoa powder on cellular viability, cell cycle progression, and chemoresistance in human epithelial ovarian carcinoma cell lines

Effect of procyanidin-rich extract from natural cocoa powder on cellular viability, cell cycle progression, and chemoresistance in human epithelial ovarian carcinoma cell lines

Effect of Procyanidin-rich Extract from Natural Cocoa Powder on Cellular Viability, Cell Cycle Progression, and Chemoresistance in Human Epithelial Ovarian Carcinoma Cell Lines.

Background:Over the last 400 years, cocoa and chocolate have been described as having potential medicinal value, being consumed as a beverage or eaten as food. Concentration–dependant, antiproliferation, and cytotoxic effects of some of their polyphenolic constituents have been demonstrated against various cancers. Such an effect remains to be demonstrated in ovarian cancerObjective:To investigate the effect of cocoa procyanidins against ovarian cancer in vitro using OAW42 and OVCAR3 cell lines.Materials and Methods:Cocoa procyanidins were extracted and enriched from non alkalized cocoa powder. The polyphenolic content and antioxidant activity were determined. Effect on cell viability was determined after the treatment with ≤1000 μg/mL cocoa procyanidin-rich extract on OAW42 and OVCAR3 and normal human dermal fibroblasts. Similarly, chemosensitization effect was determined by pretreating cancer cell lines with extract followed by doxorubicin hydrochloride treatment. The effect of treatment on cell cycle and P-glycoprotein (P-gp) expression was determined using flow cytometry.Results:The cocoa extract showed high polyphenolic content and antioxidant activity. Treatment with extract caused cytotoxicity and chemosensitization in OAW42 and OVCAR3 cell lines. Normal dermal fibroblasts showed an increase in cell viability post treatment with extract. Treatment with extract affected the cell cycle and an increasing percentage of cells in hypodiploid sub-G1/G0 phase was observed. Treatment of OVCAR3 with the extract caused reduction of P-gp expression.Conclusion:Cocoa procyanidins were found to be selectively cytotoxic against epithelial ovarian cancer, interfered with the normal cell cycle and sensitized cells to subsequent chemotherapeutic treatment. Chemosensitization was found to be associated with P-gp reduction in OVCAR3 cells.SUMMARY Among the naturally occurring flavonoids, procyanidins have been shown to be effective against cancersNon alkalized cocoa powder is one of the richest sources of procyanidinsCocoa procyanidin-rich extract (CPRE) caused cytotoxicity and chemosensitization in ovarian carcinoma cell lines OAW42 and OVCAR3CPRE affected normal cell cycle progressionCPRE also downregulated P-glycoprotein, which mediates chemoresistance in multidrug-resistant OVCAR3 cell line. Abbreviations used: P-gp: P-glycoprotein, CPRE: Cocoa procyanidin rich extract, DMAC: 4-dimethylaminocinnamaldehyde, DPPH: Diphenylpicrylhydrazyl, ABTS: 2,2’;-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), PI: Propidium iodide, FITC: Fluorescein isothiocyanate, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, TLC: Thin layer chromatography, HPTLC: High-performance thin layer chromatography.

MiR-494 suppresses tumor growth of epithelial ovarian carcinoma by targeting IGF1R.

A growing body of evidence suggests that microRNA-494 (miR-494) could act as tumor-suppressive or oncogenic microRNAs (miRNAs) in different types of tumors. However, the biological roles and underlying mechanisms of miR-494 remain unknown in human epithelial ovarian carcinoma (EOC). Therefore, the aims of this study were to investigate the miR-494 expression and the significance of its clinical diagnosis in patients suffering EOC and to analyze its role and underlying molecular mechanism on the carcinogenesis of EOC. Here, we found that miR-494 was significantly decreased in EOC cell lines and tissues and its expression was negatively correlated with advanced International Federation of Gynecology and Obstetrics (FIGO) stage, high pathological grade, and lymph node metastasis (all P < 0.01). Functional studies showed that overexpression of miR-494 in EOC cells could remarkably inhibit proliferation, colony formation, migration, and invasion and induce cell apoptosis, G0/G1 phase arrest. An in vivo analysis revealed that the overexpression of miR-494 suppressed tumor growth in a nude mouse xenograft model system. Bioinformatic assay and dual-luciferase assay confirmed that insulin-like growth factor 1 receptor (IGF1R) was as a direct target of miR-494 in EOC cells. Western blot assay showed that overexpression of miR-494 inhibited IGF1R expression and its downstream signal protein expression. In addition, downregulation of IGF1R has similar effects with miR-494 overexpression on EOC cells and overexpression of IGF1R effectively rescued the inhibition of overexpressed miR-494 in EOC cells. These data suggested that miR-494 functions as a tumor suppressor in EOC by targeting IGF1R.

MiR-30a inhibits endothelin A receptor and chemoresistance in ovarian carcinoma.

Drug resistance remains the major clinical barrier to successful treatment in epithelial ovarian carcinoma (EOC) patients, and the evidence of microRNA involvement in drug resistance has been recently emerging. Endothelin-1 (ET-1)/ETA receptor (ETAR) axis is aberrantly activated in chemoresistant EOC cells and elicits pleiotropic effects promoting epithelial-to-mesenchymal transition (EMT) and the acquisition of chemoresistance. However, the relationship between ETAR and miRNA is still unknown. Hence, in this study we evaluated whether dysregulation of miRNA might enhance ETAR expression in sensitive and resistant EOC cells. Based on bioinformatic tools, we selected putative miRNA able to recognize the 3′UTR of ETAR. An inverse correlation was observed between the expression levels of miR-30a and ETAR in both EOC cell lines and tumor samples. miR-30a was found to specifically bind to the 3′UTR of ETAR mRNA, indicating that ETAR is a direct target of miR-30a. Overexpression of miR-30a decreased Akt and mitogen activated protein kinase signaling pathway activation, cell proliferation, invasion, plasticity, EMT marker levels, and vascular endothelial growth factor release. Interestingly, ectopic expression of miR-30a re-sensitized platinum-resistant EOC cells to cisplatinum-induced apoptosis. Consistently, resistant EOC xenografts overexpressing miR-30a resulted in significantly less tumor growth than controls. Together our study provides a new perspective on the regulatory mechanism of ETAR gene. Interestingly, our findings highlight that blockade of ETAR regulatory axis is the mechanism underlying the tumor suppressor function of miR-30a in chemoresistant EOC cells.

Whole Genome Expression Profiling Analysis of Metastasis and Drug-resistance-related Genes in Epithelial Ovarian Cancer Cells

Objective To investigate the genes associated with higher ability of metastasis and chemotherapic resistance in epithelial ovarian carcinoma (EOC) by using Agilent whole genome oligonucleotide gene chip,with an attempt to further investigate the molecular mechanism of metastasis and chemotherapic resistance of EOC. Methods Oligonucleotide microarrays were used to determine whether gene expression profile might differentiate EOC cell lines (RMG-1-C,COC1 and HO8910) from their sub-lines (RMG-1-H,COC1/DDP and HO8910/PM) with higher ability of metastasis and chemotherapic resistance. Quantitative real-time polymerase chain reaction and immunohistochemical staining validated the microarray results. Results Gene expression profile identified 49 differentially expressed genes that showed≥2.0 fold change. All these differentially expressed genes were involved mainly in gene expression and biopolymer biosynthesis. Interaction network analysis predicted 21 genes participating in the regulatory connection. Highly differential expression of GCET2,CFTR,FOXP1 and GARS genes was validated by quantitative realtime polymerase chain reaction in all cell line samples,and the Results were consistent with microarray findings. Conclusion The change in the metastasis and chemotherapic resistance-associated gene expression profiles may provide a theoretical basis for studies on the molecular mechanisms of metastasis and chemotherapic resistance in EOC.

The expression and function of toll-like receptors (TLRs) in ovarian cancer cell lines

Meeting abstracts This study is to investigate which Toll Like Receptors (TLRs) are expressed on human ovarian cancer cell lines and to evaluate the effect of TLR expression on the growth of ovarian cancer cells. Four human ovarian epithelial carcinoma cell lines SKOV3 (clear), OVCAR3 (serous

Abstract 3677: Enhanced targeted delivery of paclitaxel to tumor cells of epithelial and neuroectodermal origin using chlorotoxin-chitosan nanodelivery system

Abstract We report a development of a novel localized nanodelivery system featuring an enhanced targeted and controlled paclitaxel (PTX) delivery to epithelial and neuroectodermal origin tumor cells. The presented drug delivery system consists of a chitosan hydrogel embedded with PTX loaded non-ionic surfactant vesicles (PTX-niosomes) and chlorotoxin (CTX). We found a higher accumulation of chitosan on the surface of ovarian epithelial carcinoma cells known to overexpress MUC1 antigen as compared to normal ovarian epithelial cells. We therefore hypothesized that besides serving as a localized drug delivery platform, chitosan hydrogel can actively target MUC1 overexpressing tumor cells. To further improve the tumor-specific delivery of PTX, we have incorporated CTX, a 36-amino acid peptide, which binds to tumor cells of neuroectodermal origin, but not to non-transformed cells. Using a High Performance Liquid Chromatography (HPLC) assay, we have measured the in-vitro release profiles of PTX from chitosan hydrogel embedded with PTX-niosomes and CTX. We have assessed the morphology of CTX and PTX-nisomes as well as chitosan hydrogel embedded with PTX-niosomes and CTX using Transmission Electron Microscopy (TEM). We have used Attenuated Total Reflectance-Fourier Transform Infra-Red (ATR-FTIR) spectroscopy to investigate the possible molecular interactions between chitosan hydrogel and various tumor cells. To better understand the mechanism involved in interactions between chitosan and specific tumor cells, we have developed a new Cell based Enzyme Linked Immunosorbent Assay (Cell ELISA) allowing us to quantify the expression level of MUC1 in epithelial and neuroectodermal origin tumor cells. Our Cell ELISA results revealed higher expression level of MUC1 (around 1.5 fold) in ovarian epithelial carcinoma cell line (OV2008) when compared to normal ovarian epithelial cell line (MCC3). The ATR-FTIR results showed a specific interaction of chitosan with OV2008 cells implying the mucoadhesive property of chitosan and its potential in targeting of MUC1 overexpressing tumor cells. We observed a sustained PTX release from chitosan hydrogel embedded with PTX-niosomes (&amp;lt; 40% of cumulative release occurred within two weeks). Release studies in cell-free system also indicated that embedding CTX along with the niosomes does not have any adverse effect on the controlled release of PTX from the chitosan network. In fact, attachment of CTX to the surface of PTX-niosomes shown by TEM imaging improves the stability of PTX-niosomes resulting in extended release rates. Together these findings indicate that the proposed drug delivery system with the distinctive tumor targeting features and extended release profiles would improve the specific delivery of PTX to epithelial and neuroectodermal origin tumor cells. Citation Format: Rana Falahat, Eva Williams, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Enhanced targeted delivery of paclitaxel to tumor cells of epithelial and neuroectodermal origin using chlorotoxin-chitosan nanodelivery system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3677. doi:10.1158/1538-7445.AM2015-3677