Ovarian Cancer Cell Lines OVCAR3 Research Articles

Abstract A01: Trichodermin induces G1/S cell cycle arrest in ovarian cancer cells

Abstract Ovarian cancer is a major health concern for women as it is prominent among gynecologic cancers. Trichodermin is a sesquiterpene isolated from Nalanthamala psidii and found to induce apoptosis in human chondrosarcoma cells. In this study, A2780/CP70 and OVCAR-3 ovarian cancer cell lines were treated with trichodermin to determine whether it had an anti-cancer effect. Our data presented that trichodermin reduced the viability of both ovarian cancer cells by inducing G1/S cell cycle arrest rather than apoptosis. The underlying mechanism might be that trichodermin down-regulated the expression of Cyclin D1 via inhibiting AhR and c-Myc expression. Therefore, trichodermin is promising in ovarian cancer treatment. Citation Format: Yi C. Chen, Ying Gao. Trichodermin induces G1/S cell cycle arrest in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A01.

MicroRNA-1284 Inhibits Cell Viability and Induces Apoptosis of Ovarian Cancer Cell Line OVCAR3.

Ovarian cancer is a malignancy with high mortality among women. Multiple reports show that microRNAs (miRs) act as regulators in ovarian cancer inhibition, while the role of miR-1284 in ovarian cancer is still unknown. This study aimed to investigate the effects of miR-1284 on ovarian cancer cells. Human ovarian cancer cell line OVCAR3 was cultured and transfected with miR-1284 mimics, inhibitors, or control. Viability and apoptosis of transfected cells were then determined by MTT assay, BrdU assay, and flow cytometry. Expression changes of p27, p21, and PI3K/Akt pathway-related proteins were measured by Western blot. Results showed that miR-1284 overexpression suppressed cell viability while increasing the apoptosis in OVCAR3 cells. Moreover, the expression level of p27 was upregulated by miR-1284 overexpression. Furthermore, miR-1284 overexpression and Akt inhibitor GSK690693 downregulated the levels of p-Akt and Bcl-2 while upregulating the levels of Bax and caspase 3. However, miR-1284 suppression attenuated the regulatory effects of GSK690693 on these proteins. In conclusion, miR-1284 could inhibit cell viability via regulating the expression of p27 and induce apoptosis via regulating the PI3K/Akt pathway in OVCAR3 cells.

Epigenetics changes caused by the fusion of human embryonic stem cell and ovarian cancer cells.

To observe the effect of gene expression and tumorigenicity in hybrid cells of human embryonic stem cells (hESCs) and ovarian cancer cells in vitro and in vivo using a mouse model, and to determine its feasibility in reprogramming tumour cells growth and apoptosis, for a potential exploration of the role of hESCs and tumour cells fusion in the management of ovarian cancer. Stable transgenic hESCs (H1) and ovarian cancer cell line OVCAR-3 were established before fusion, and cell fusion system was established to analyse the related indicators. PTEN expression in HO-H1 cells was higher than those in the parental stem cells and lower than those in parental tumour cells; the growth of OV-H1 (RFP+GFP) hybrid cells with double fluorescence expressions were obviously slower than that of human embryonic stem cells and OVCAR-3 ovarian cancer cells. The apoptosis signal of the OV-H1 hybrid cells was significantly higher than that of the hESCs and OVCAR-3 ovarian cancer cells. In vivo results showed that compared with 7 days, 28 days and 35 days after inoculation of OV-H1 hybrid cells; also, apoptotic cell detection indicated that much stronger apoptotic signal was found in OV-H1 hybrid cells inoculated mouse. The hESCs can inhibit the growth of OVCAR-3 cells in vitro by suppressing p53 and PTEN expression to suppress the growth of tumour that may be achieved by inducing apoptosis of OVCAR-3 cells. The change of epigenetics after fusion of ovarian cancer cells and hESCs may become a novel direction for treatment of ovarian cancer.

Radiobiological Effectiveness of Ultrashort Laser-Driven Electron Bunches: Micronucleus Frequency, Telomere Shortening and Cell Viability

Laser-driven electron accelerators are capable of producing high-energy electron bunches in shorter distances than conventional radiofrequency accelerators. To date, our knowledge of the radiobiological effects in cells exposed to electrons using a laser-plasma accelerator is still very limited. In this study, we compared the dose-response curves for micronucleus (MN) frequency and telomere length in peripheral blood lymphocytes exposed to laser-driven electron pulse and X-ray radiations. Additionally, we evaluated the effects on cell survival of in vitro tumor cells after exposure to laser-driven electron pulse compared to electron beams produced by a conventional radiofrequency accelerator used for intraoperative radiation therapy. Blood samples from two different donors were exposed to six radiation doses ranging from 0 to 2 Gy. Relative biological effectiveness (RBE) for micronucleus induction was calculated from the alpha coefficients for electrons compared to X rays (RBE = alpha laser/alpha X rays). Cell viability was monitored in the OVCAR-3 ovarian cancer cell line using trypan blue exclusion assay at day 3, 5 and 7 postirradiation (2, 4, 6, 8 and 10 Gy). The RBE values obtained by comparing the alpha values were 1.3 and 1.2 for the two donors. Mean telomere length was also found to be reduced in a significant dose-dependent manner after irradiation with both electrons and X rays in both donors studied. Our findings showed a radiobiological response as mirrored by the induction of micronuclei and shortening of telomere as well as by the reduction of cell survival in blood samples and cancer cells exposed in vitro to laser-generated electron bunches. Additional studies are needed to improve preclinical validation of the radiobiological characteristics and efficacy of laser-driven electron accelerators in the future.

Abstract 2495: hPaf1/PD2 interacts with OCT3/4 in maintenance of the self-renewal process of ovarian cancer stem cells

Abstract Background: Ovarian cancer (OC) is the most lethal gynecological malignancy among women with more than 85% of the patients manifesting tumor recurrence. Emerging evidence suggests that a small population of cells within the tumor - the ‘cancer stem cells (CSCs)’ is capable to giving rise to the entire histopathology of the tumor and is responsible for mediating drug resistance, recurrence, and disease aggressiveness. Previously, hPaf1(human RNA polymerase II associated factor1)/PD2 (Pancreatic Differentiation2) - a core component of RNA polymerase II associated factor (PAF) complex, was shown to be overexpressed in pancreatic CSCs and involved in the maintenance of mouse ESCs. Hence, we hypothesized that hPaf1 is involved in the maintenance of self-renewal property of ovarian CSCs (OCSCs). In this study, we investigated the functional role of hPaf1 in OCSCs which has not been explored before. Methods: Expression of hPaf1, cancer stem cell marker ESA, and self-renewal protein OCT3/4 was analyzed using confocal microscopy on OC tissue array. Side population (SP) was isolated from two OC cell lines OVCAR3 and A2780 by Hoechst staining using flow-cytometer, and characterized using tumor sphere assay. Immunoblotting was performed on characterized SP and NSP (non-SP) for OCSC and self-renewal markers. Transient knockdown of hPaf1 in SP was performed to understand how hPaf1 affects the CSC phenotype through immunoblotting, confocal microscopy, colony formation assay, and tumor sphere formation assay. To determine the interaction between hPaf1 and OCT 3/4, reciprocal co-immunoprecipitation was performed in SP/OCSCs. Results: There was a significant overexpression and considerable co-localization of hPaf1/PD2 with ESA and OCT3/4 in OC tissues compared to normal ovary tissues. SP from OVCAR3 formed larger and greater number of tumor spheres compared to NSP cells. Moreover, SP isolated from OVCAR3 and A2780 showed higher expression of hPaf1 along with CSC markers (CD44, CD133, ESA, CD24), and self- renewal proteins (β-CATENIN, SOX2, OCT3/4, SHH, and HER2). Transient knockdown of hPaf1 resulted in a significant decrease in expression of CSC markers and self-renewal proteins. In addition, functional characteristics of CSCs such as in vitro tumor formation capacity in non-adherent media and colony formation ability were impaired upon knockdown of hPaf1 suggesting that hPaf1 is involved in maintenance of the CSC phenotype. We also observed that hPaf1 physically interacts with OCT3/4 in OVCAR3 SP cells which provides a mechanism for the maintenance of OCSCs by hPaf1. Conclusion: Altogether, hPaf1/PD2 is overexpressed in OCSCs and its knockdown resulted in loss of OCSC phenotype. Moreover, hPaf1 is responsible for the maintenance of OCSCs through its interaction with OCT3/4. Hence, therapies that are able to abrogate hPaf1 mediated self-renewal of OCSCs represent potential therapeutic avenues to overcome tumor relapse in OC. Citation Format: Saswati Karmakar, Parthasarathy Seshacharyulu, Arokia Priyanka Vaz, Imayavaramban Lakshmanan, Moorthy Palanimuthu Ponnusamy, Surinder Kumar Batra. hPaf1/PD2 interacts with OCT3/4 in maintenance of the self-renewal process of ovarian cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2495.

Abstract 2747: PARP inhibitors act synergistically with cisplatin and doxorubicin in BRCA competent cells by compromising chemotherapy-induced replication arrest

Abstract PARP inhibitors as monotherapy have emerged as promising antitumor drugs in particular for malignancies with nonfunctional BRCA1/2 proteins. Furthermore, preclinical data as well as first results from clinical studies suggest that PARP inhibition can potentiate cytotoxic effects of conventional chemotherapy independently of the BRCA status. Mechanisms underlying these combinations have predominantly been attributed to an enhancement of DNA damage through interference with DNA repair. Apart from its central function in DNA damage repair processes the enzymatic function of PARP1/2, namely Poly(ADPribosy)lation (PARylation), PARP has also been implicated in stabilizing replication fork arrest upon Topoisomerase I (TopoI) poisoning. We aimed to determine if the function of PARP1/2 at the stalled replication fork might be important for synergistic effects of PARP inhibitors with widely used chemotherapeutics other than TopoI inhibitors. For this, the BRCA competent ovarian and colon cancer cell lines OVCAR8, OVCAR3, HCT116, and LOVO as well as primary tissue samples from ovarian carcinoma patients were treated with cisplatin, doxorubicin, etoposide, 5-FU, and paclitaxel for one hour in presence or absence of the three commonly used PARP1/2 inhibitors Rucaparib, PJ34, and A966492. DNA fiber spreading analyses, quantification of PARylation and 53BP1/γH2AX spot formation, as well as analysis of DNA strand break repair capacity were performed within the first hours after treatment. Furthermore, cell lines were analyzed for long term survival using colony forming assays. Importantly, PARylation was only observed at early time points after treatment with cisplatin and doxorubicin, but not upon 5-FU, etoposide and paclitaxel. Consequently, PARP inhibition had only synergistic effects on long term survival together with cisplatin and doxorubicin but not with the other tested chemotherapeutics. Inhibition of PARylation caused a significant delay of cisplatin adduct removal and strand break repair upon doxorubicin. More importantly, combination of PARP inhibitors with both cisplatin and doxorubicin led to double strand break formation selectively in S/G2 phase cells as detected by 53BP1/γH2AX spots in Geminin positive cells. This was caused by a complete circumvention of chemotherapy-induced fork slowing in cell lines and primary tumor cells. The inability of initiating replication fork arrest upon chemotherapy in cells with blocked PARP1/2 activity was confirmed by the presence of new origin firing and significantly reduced replication stops. In conclusion our work implicates that PARP inhibitors have significant effects not only in BRCA deficient cells but also in proliferating BRCA competent cells when combined with adduct-forming, replication stress-inducing chemotherapeutics. Citation Format: Lea Schaaf, Matthias Schwab, Simon Heine, Walter E. Aulitzky, Heiko van der Kuip. PARP inhibitors act synergistically with cisplatin and doxorubicin in BRCA competent cells by compromising chemotherapy-induced replication arrest. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2747.

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.

Gene expression profiling of single circulating tumor cells in ovarian cancer – Establishment of a multi-marker gene panel

The presence of circulating tumor cells (CTCs) in the blood of ovarian cancer patients was shown to correlate with decreased overall survival, whereby CTCs with epithelial–mesenchymal-transition (EMT) or stem-like traits are supposed to be involved in metastatic progression and recurrence. Thus, investigating the transcriptional profiles of CTCs might help to identify therapy resistant tumor cells and to overcome treatment failure. For this purpose, we established a multi-marker panel for the molecular characterization of single CTCs, detecting epithelial (EpCAM, Muc-1, CK5/7), EMT (N-cadherin, Vimentin, Snai1/2, CD117, CD146, CD49f) and stem cell (CD44, ALDH1A1, Nanog, SOX2, Notch1/4, Oct4, Lin28) associated transcripts.First primer specificity and PCR-performance of the multiplex-RT-PCRs were successfully validated on genomic DNA and cDNA isolated from OvCar3 cells. The assay sensitivity of the epithelial panel was evaluated by adding defined numbers of tumor cells into the blood of healthy donors and performing a subsequent immunomagnetic tumor cell enrichment (AdnaTest OvarianCancerSelect), resulting in a 100% concordance for the epithelial markers EpCAM and Muc-1 to the AdnaTest OvarianCancerDetect. Additionally, by processing blood from ovarian cancer patients, high assay sensitivity could be verified. In blood of healthy donors no signals for epithelial markers were detected, for EMT and stem cell markers, however, signals were obtained mainly originating from leukocytes which calls for single cell analysis.To that aim by using the ovarian cancer cell line OvCar3, we successfully established a workflow enabling the characterization of single CTCs. It consists of a density gradient-dependent enrichment for nucleated cells, a depletion of CD45-positive cells of hematopoietic origin followed by immunofluorescent labeling of CTCs by EpCAM and Muc-1. Single CTCs are then isolated by micromanipulation and processed for panel gene expression profiling. Finally, fifteen single CTCs from three ovarian cancer patients were analyzed and found to be positive for stem cell (CD44, ALDH1A1, Nanog, Oct4) and EMT markers (N-cadherin, Vimentin, Snai2, CD117, CD146). Albeit, inter-cellular and intra/inter-patient heterogeneity and co-expression of epithelial, mesenchymal and stem cell transcripts on the same CTC was observed.We have established a robust workflow to perform sensitive single cell panel gene expression analysis without the need of pre-amplification steps. Our data point towards a heterogeneous expression of stem cell and EMT associated transcripts in ovarian cancer CTCs.

Human mesenchymal stem cells are resistant to cytotoxic and genotoxic effects of cisplatin in vitro

Mesenchymal stem cells (MSCs) are known for their important properties involving multilineage differentiation potential., trophic factor secretion and localization along various organs and tissues. On the dark side, MSCs play a distinguished role in tumor microenvironments by differentiating into tumor-associated fibroblasts or supporting tumor growth via distinct mechanisms. Cisplatin (CIS) is a drug widely applied in the treatment of a large number of cancers and is known for its cytotoxic and genotoxic effects, both in vitro and in vivo. Here we assessed the effects of CIS on MSCs and the ovarian cancer cell line OVCAR-3, by MTT and comet assays. Our results demonstrated the resistance of MSCs to cell death and DNA damage induction by CIS, which was not observed when OVCAR-3 cells were exposed to this drug.

Broad-spectrum antiproliferative activity of a series of 6-(4-fluorophenyl)-5-(2-substituted pyrimidin-4-yl)imidazo[2,1-b]thiazole derivatives

This article described the synthesis and in vitro antiproliferative activities a series of 6-(4-fluorophenyl)-5-(2-substituted pyrimidin-4-yl)imidazo[2,1-b]thiazole derivatives. The nine target compounds were tested for in vitro antitumor effect against a panel of 55 cell lines of nine different cancer types at the NCI, and their activities were compared with that of Sorafenib as a reference standard drug. Compounds 1d and 1e possessing terminal arylamide moiety exerted superior potencies than Sorafenib against different cancer cell lines. Both compounds were more potent than Sorafenib against UO-31 renal cancer cell line and MCF7 breast cancer cell line. Compound 1d was also more potent than Sorafenib against COLO 205 colon cancer cell line, and compound 1e showed higher potency than Sorafenib against OVCAR-3 ovarian cancer cell line and DU-145 prostate cancel cell line also. For instance, the IC50 value of compound 1e against DU-145 prostate cancer cell line was 1.04 μM, which is threefold more potent than Sorafenib.

MiR-23b targets cyclin G1 and suppresses ovarian cancer tumorigenesis and progression.

BackgroundIt has been proposed that cyclin G1 (CCNG1) participates in p53-dependent G1–S and G2 checkpoints and might function as an oncogenic protein in the initiation and metastasis of ovarian carcinoma. MicroRNA 23b (miR-23b) is a critical regulatory factor in the progression of many cancer cell types that targets the relevant genes.MethodsMiR-23b expression in ovarian tissues was quantified by quantitative reverse transcription–PCR. The ovarian cancer cell lines OVCAR3, HO8910-PM, and SKOV3/DDP were transfected with miR-23b, after we assayed the cell phenotype and expression of the relevant molecules. Dual-luciferase reporter assay and a xenograft mouse model were used to examine the expression of miR-23b and its target gene CCNG1.ResultsMIR23B mRNA expression was significantly lower in epithelial ovarian carcinoma and borderline tumors than in normal ovarian tissues and benign tumors, and miR-23b expression among ages and pathological subtypes was significantly different. CCNG1 mRNA expression was significantly lower in normal ovarian tissues than in benign tumors, borderline tumors, and ovarian carcinomas, and expression among pathological subtypes was significantly different. MiR-23b overexpression inhibited ovarian cancer cell proliferation, invasion, and migration, and induced apoptosis. Dual-luciferase reporter assay showed that miR-23b bound with the 3′ untranslated region of CCNG1. MiR-23b overexpression significantly downregulated CCNG1, urokinase, survivin, Bcl-xL, P70S6K, and matrix metallopeptidase-9 (MMP9) mRNA and protein expression. Furthermore, miR-23b inhibited tumor growth and suppressed CCNG1 expression in vitro.ConclusionsOur findings show that miR-23b may inhibit ovarian cancer tumorigenesis and progression by downregulating CCNG1 and the expression of the relevant genes. MiR-23b is a potentially novel application for regulating ovarian carcinoma progression.

The effect of salinomycin on ovarian cancer stem-like cells.

ObjectiveThe identification of cancer stem-like cells is a recent development in ovarian cancer. Compared to other cancer cells, cancer stem-like cells present more chemo-resistance and more aggressive characteristics. They play an important role in the recurrence and drug resistance of cancer. Therefore, the target therapy of cancer stem-like cell may become a promising and effective approach for ovarian cancer treatment. It may also help to provide novel diagnostic and therapeutic strategies.MethodsThe OVCAR3 cell line was cultured under serum-free conditions to produce floating spheres. The CD44+CD117+ cell line was isolated from the human ovarian cancer cell line OVCAR3 by using immune magnetic-activated cell sorting system. The expression of stemness genes such as OCT3/4, NANOG and SOX2 mRNA were determined by reverse transcription polymerase chain reaction. OVCAR3 parental and OVCAR3 CD44+CD117+ cells were grown in different doses of paclitaxel and salinomycin to evaluate the effect of salinomycin. And growth inhibition of OVCAR3 CD44+CD117+ cells by paclitaxel combined with salinomycin was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay.ResultsTumor spheroids generated from the OVCAR3 cell line are shown to have highly enriched CD44 and CD117 expression. Treatment with a combination of paclitaxel and salinomycin demonstrated growth inhibition of OVCAR3 CD44+CD117+ cells.ConclusionThe present study is a detailed investigation on the expression of CD44 and CD117 in cancer stem cells and evaluates their specific tumorigenic characteristics in ovarian cancer. This study also demonstrates significant growth inhibition of cancer stem-like cells by paclitaxel combined with salinomycin. Identification of these cancer stem-like cell markers and growth inhibition effect of salinomycin may be the next step to the development of novel target therapy in ovarian cancer.

MicroRNA-433 inhibits migration and invasion of ovarian cancer cells via targeting Notch1.

We aimed to determine the effects of miR-433 on the malignant behaviors of ovarian cancer cells, as well as to elucidate the possible mechanisms of ovarian cancer development. A total of 9 ovarian cancer tissues and 9 matched normal ovary tissues were obtained, and the expression levels of miR-433 and Notch1 were then determined by real-time PCR. Human ovarian cancer cell lines SKOV3 and OVCAR3 were transfected with miR-433 mimics, negative miR-control and Notch1 siRNA. The expression of Notch1 protein in transfected cells was determined by western blot. In addition, the proliferation, migration and invasion of SKOV3 and OVCAR3 cells in vitro were then evaluated using Cell Counting Kit 8, wound healing assay and Transwell invasion assay, respectively. Besides, bioinformatics methods and luciferace reporter assay were performed to confirm whether Notch1 was a direct target of miR-433. The expression of miR-433 was markedly down-regulated while Notch1 expression was significantly up-regulated in ovarian cancer tissues compared with matched normal ovary tissues. Overexpression of miR-433 significantly inhibited the migration and invasion of ovarian cancer cells, but had not significant effects on cell proliferation. In addition, Notch1 was a direct target of miR-433. Besides, down-regulation of Notch1 inhibited the invasion of ovarian cancer cells. Our findings indicate that miR-433 may inhibit cell migration and invasion in the development of ovarian cancer via down-regulation of Notch1. Notch1 may serve as a potential target in cancer therapy.

Small molecule inhibitor of c-Met (PHA665752) suppresses the growth of ovarian cancer cells and reverses cisplatin resistance.

c-Met as a tyrosine-kinase receptor plays a major role in tumorigenesis, invasion, and metastatic spread of human tumors, including ovarian cancer. Expressing high levels of c-Met proteins is often associated with resistance to chemotherapy and an adverse prognosis. In this study, we have determined the effect of PHA665752, a small molecule inhibitor of c-Met proteins, with and without cisplatin and the role of c-Met in several ovarian cancer cell lines having high c-Met expression. The methyl thiazolyl tetrazolium (MTT) assay was used to detect cell proliferation, and apoptosis was evaluated by flow cytometry. Western blotting was carried out to determine protein expression levels. Gene silencing was used to detect the influence of c-Met gene silence on the resistance to cisplatin. Compared to more sensitive ovarian cancer cell lines SKOV3 and 3AO, we found that the expression of c-Met was significantly increased in SKOV3(DDP), OVCAR3, and OV-90 ovarian cancer cell lines, which were resistant to cisplatin. Our data indicated that cisplatin sustained activated phosphor-Met in SKOV3(DDP), OVCAR3, and OV-90 cell lines. We also observed a significant transient activation of c-Met phosphorylation in SKOV3 and 3AO cells. Treatment with PHA665752 inhibited c-Met expression inhibited cell growth, induced apoptosis, and enhanced cisplatin-induced proliferation inhibition and apoptosis in c-Met over-expressed cell lines. In addition, blocking c-Met expression with small interfering RNA (siRNA) overcame the resistance of cancer cells to cisplatin. Thus, blocking c-Met expression presents a promising therapeutic approach for ovarian cancer.

Histone deacetylase 4 increases progressive epithelial ovarian cancer cells via repression of p21 on fibrillar collagen matrices.

Histone deacetylase (HDAC) 4 is an emerging target in cancer therapeutics, but little is known about the function of HDAC4 in gynecologic malignancies. Therefore we investigated the mechanism of HDAC4 promoting the proliferation of epithelial ovarian cancer cells (OV). In this study, we observed that the proliferation of cells with HDAC4 inhibitor Trichostatin A (TSA) treatment was markedly decreased, Further, we showed that epithelial ovarian cancer tissues with stage III/IV had higher HDAC4 expression, compared to that with stage I/II. We examined first that the HDAC4 expression was increased in response to fibrillar collagen matrices. In addition, we found that HDAC4 was retained in the nucleus by regulation of PP1α, which regulated HDAC4 cellular fraction via phosphorylation of HDAC4. In addition, we found that HDAC4 bound to Sp1 in epithelial ovarian cancer cells. Finally, ovarian cancer cell line OVCAR3 was evaluated via gain/loss-of-function of HDAC4 by either overexpression of HDCA4 or knock-down of HDAC4 with shRNA. We examined both protein and mRNA of p21 by western blotting and qPCR. We performed analysis of colony formation in matrigel and migration by ECIS. Our results suggest that the accumulation of HDAC4 induced by fibrillar collagen matrices in the nucleus via co-localization of PP1α, leads to repression of the mRNA/protein of p21 and in turn promotes the proliferation and migration of epithelial ovarian cancer cells.

Association between the rs11614913 variant of miRNA-196a-2 and the risk of epithelial ovarian cancer.

Polymorphisms in microRNA (miR) genes and their target sites are a distinct classification of variation in the human genome, which are rapidly being identified and investigated in human cancer. A polymorphism in the miR-196a-2 locus has demonstrated significant associations with various types of cancer, including lung, breast, esophageal and gastric tumors. However, miR-196a-2 has not been fully explored in ovarian cancer, which shares similar biological characteristics with other types of cancer. Therefore, the present study aimed to elucidate the association between a single nucleotide polymorphism (SNP) in the mature sequence of miR-196a-2 (rs11614913, T/C) and the clinical features of 479 Chinese patients with epithelial ovarian cancer (EOC). In addition, the biological significance of this polymorphism was investigated in the OVCAR3 ovarian cancer cell line. Risk association was evaluated in 479 cases of EOC patients and 431 controls. SNPs were analyzed by using polymerase chain reaction based restriction fragment length polymorphism assay. miR-196a expression was evaluated with reverse transcription polymerase chain reaction. The influence of miR-196a-2 rs11614913 T/C on EOC cell migration and invasion ability was further investigated in vitro. The results revealed significant differences in the homozygous CC genotype distribution in patients with EOC (n=479), compared with that of the control subjects (n=431; P=0.026). Analysis of the association between genotype and the risk of EOC revealed that individuals who carried the homozygous CC genotype were 1.34-fold more susceptible to EOC, compared with those carrying the wild-type TT and heterozygous CT genotypes [odds ratio, 1.34; 95% confidence interval, 1.04-2.17; P=0.023]. In addition, the role of this polymorphism in the production of mature miR-196a was investigated. Significantly enhanced production of mature miR-196a was revealed in the C-allelic compared with that of the T-allelic miR-196a-2 precursor (P<0.05). Further examination indicated that miR-196a significantly promoted cell migration and invasion ability in the human OVCAR3 ovarian cell line (P<0.05). In conclusion, the results indicated that the miR-196a-2 rs11614913 CC genotype may increase the risks of ovarian cancer by affecting the expression of mature miR-196a and enhancing cell migration/invasion. The current results provided evidence that the T>C polymorphism in the miR-196a-2 precursor may influence tumorigenesis and metastasis in EOC, and suggested that the functional SNP rs11614913 in the promoter region of pri-miR-196a-2 may be a potential indicator of EOC susceptibility in the population analyzed.

Effects of shRNA-mediated silence of metastasis-associated lung adenocarcinoma transcript 1 on the invasion and metastasis of ovarian carcinoma cell line OVCAR3

Objective To evaluate the expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) in ovarian cancer cell lines, and to investigate the biological effects of down-regulated MALAT-1 on OVCAR3 cells. Methods qRT-PCR analysis was used to examine the expression level of MALAT-1 gene in ovarian cancer cells, including ES-2, A2780, SKOV3 and OVCAR3 cell lines. For functional research, four shRNA oligos specially targeting MALAT-1 and a empty vector were designed and constructed into pGPU6/GFP/Neo, then transfected into OVCAR3 cells. qRT-PCR was used to confirm the effective suppression of MALAT-1. Changes of proliferation and adhesion of cells were analyzed by CCK-8 and adhesion assays. Wound-healing, transwell migration and invasion assays were used to examine migration and invasion of MALAT-1-silencing cells in vitro. Results The expression of MALAT-1 gene in OVCAR3 cells was high, and qRT-PCR results confirmed successfully the knockdown of MALAT-1 after transient transfection. After successful suppression of MALAT-1, the proliferation, wound-healing and adhesion ability in vitro were inhibited to some degree. In transwell migration assay, the number of migration cells in MALAT-1-silencing group was 52.17 ± 4.48, which is much less than that in the negative and control groups (286.50 ± 12.23 and 295.67±6.96, respectively). In invasion assay, the number of invasion cells passing the transwell membrane in MALAT-1-silencing group (37.33±2.40) was also decreased significantly, compared to that in the negative and control groups (239.00±15.72 and 222.67±20.85, P< 0.05). Conclusions shRNA-mediated silence of MALAT-1 can effectively inhibit the proliferation, adhesion, migration and invasion abilities of ovarian cancer cell line OVCAR3 in vitro, indicating MALAT-1 is expected to be a target gene for the treatment of ovarian cancer. Key words: Ovarian neoplasms; Metastasis-associated lung adenocarcinoma transcript 1; Neoplasm invasiveness; Neoplasm metastasis; RNA interference

MicroRNA activity profile in the ovarian cancer cell line OVCAR3 identifies a proapoptotic effect of miR-23a

MicroRNA activity profile in the ovarian cancer cell line OVCAR3 identifies a proapoptotic effect of miR-23a

Abstract POSTER-THER-1429: Selection of DNA aptamers for an ovarian cancer cell line using high-throughput sequencing

Abstract Humanized antibodies have been extensively investigated as therapeutic as well as diagnostic agents. While the antigen specificity offered by antibodies makes them very attractive for such theranostic applications, their large-scale synthesis can be challenging and expensive. We are therefore investigating alternate strategies to develop agents that can be used for in vivo monitoring as well as for treatment of epithelial ovarian tumors. One approach is to develop Single Stranded DNA aptamers that selectively bind to ovarian cancer cells. The ssDNA aptamers can be synthesized using template-driven or de novo chemical synthetic approaches to manufacture agents at a large scale and low cost. The challenge however, is to develop aptamers that are specific to ovarian cancer cells. In the current study, we report a streamlined approach that incorporates the cell-based Systematic Evolution of Ligands by Exponential Enrichment (cell-SELEX) with DNASeq technology to select aptamers that recognize ovarian cancer cells. An ssDNA aptamer library composed of ~1015 sequences was subjected to ten iterative rounds of selection against the ovarian cancer cell line OVCAR-3. Aptamers from each round were amplified by asymmetric PCR and subjected to high-throughput sequencing. Eight ssDNA aptamers enriched through the selection process were identified by DNASeq and subsequent bioinformatics analysis and their selectivity and affinity for OVCAR-3 cells was determined by flow cytometry. Two of these aptamers (Apt-1 and Apt-8) showed significant binding to OVCAR-3 cells with Kd of 24 and 28 nM, respectively. Secondary structure analysis using mfold indicated that Apt-1 and Apt-8 had defined secondary structures resulting from ordered base pairing of the ssDNA. The inclusion of high-throughput sequencing techniques has therefore allowed rapid identification of theranostic aptamers from an large randomized library of ssDNA sequences. Our ongoing experiments are focused on coupling of the ovarian cancer cell-specific ssDNA aptamers to contrast agents or cytotoxic drugs. The ssDNA aptamers coupled to contrast agents are specifically being investigated for in vivo imaging of ovarian cancer masses in the peritoneum whereas the aptamers coupled to drugs can be used for the treatment of ovarian cancer. Citation Format: Rebecca J. Whelan, Arvinder Kapur, Mildred Felder, Jamie Shallcross, Jeff Nie, Manish S. Patankar. Selection of DNA aptamers for an ovarian cancer cell line using high-throughput sequencing [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1429.

Antitumoral Activity Of Nitric Oxide-Releasing Compounds

Despite significant improvements in the conventional anti-ovarian cancer therapies, tumor cell resistance to various cytostatic drugs remains a relevant problem. Therefore, the new cancer treatment strategies are being developed. Among many agents that have been studied for their potential anti-cancer activity, the most promising are the nitric oxide (NO) donors-syntethic compounds that release NO in vivo and/or in vitro. We have evaluated the effect of NO donors on the SK-OV-3 and OVCAR-3 ovarian cancer cell lines. We assessed some of the cancer cells' specific features: the uncontrolled proliferation, over-activation of particular signaling proteins, high resistance to therapeutics and elevated expression and secretion of invasiveness/metastatic factors. Two members of NONOates family were used: SPER/NO and DETA/NO. Cancer cell lines were cultured with different concentrations of NO donors. The cytotoxic, pro-apoptotic activity of NO donors and their impact on the phosphorylation status of STAT-3 and AKT in cells were determined. The expression of VEGF-A, MMPs and TGF-β was also evaluated. NO donors inhibited ovarian cancer cells growth making them also more susceptible to the cisplatin cytotoxic activity. Moreover, both NO donors induced apoptosis of cells and decreased activity of signaling proteins (STAT3 and AKT). Similarly, SPER/NO and DETA/NO lowered the secretion of pro-metastatic factors, responsible for cancer cells invasiveness. The obtained results show that both NO donors demonstrated a wide range of action on both ovarian cancer cell lines. Therefore, they have a high potential of being a supporting compounds in the cancer therapies.