Ovarian Cancer Cell Lines OVCAR3 Research Articles

Anti-Cancer Activities and Interaction of Imiquimod and Flex-Het, SHetA2, in Melanoma and Ovarian Cancer

New strategies are needed to treat cancers that do not respond to chemotherapy or resist chemotherapy after responding initially. The objective of this study was to evaluate non-cytotoxic drugs against two of these cancers, melanoma and ovarian cancer. Imiquimod is an immune stimulant that induces apoptosis in cancer cells. Flexible-Heteroarotinoids (Flex-Hets) are small molecules that regulate growth, differentiation and apoptosis in cancer cells with reduced effects on normal cells. Both imiquimod and SHetA2 inhibited growth and induced apoptosis in the B16 melanoma cell line and cisplatin-sensitive A2780 and cisplatin-resistant OVCAR-3 ovarian cancer cell lines. The growth inhibition was additive in A2780 and B16, and synergistic in OVCAR-3. Both compounds inhibited endothelial tube branching in vitro and exerted an additive effect when combined. Various combinations of imiquimod and SHetA2 did not cause significant differences in the overall survival in the syngeneic B16 murine melanoma model. SHetA2 induced complete tumor regression and a melanoma-free natural life-span in two mice. These cures occurred in one of ten mice treated with oral SHetA2 and one of ten mice intratumorally-injected with SHetA2. Exploratory modeling of the distribution of survival times suggested that the two surviving mice represent rare events. Histologic evaluation of the tumors revealed that imiquimod induced necrosis, SHetA2 induced differentiated architecture and increased cytoplasm, both agents reduced mitotic indices and angiogenesis and neither agent counteracted the effects of the other. No overt toxicities were observed. In conclusion, imiquimod and SHetA2 exhibit complementary anti-cancer activity in vitro and SHetA2 has promise as a single agent.

5-((1-Aroyl-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-diones as potential anticancer agents with anti-inflammatory properties

A series of novel 5-((1-aroyl-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-diones (3a–z) have been evaluated for in vitro cytotoxicity against a panel of 60 human tumor cell lines. Compound 3k exhibited the most potent growth inhibition against melanoma MDA-MB-435 cells (GI50=850nM), against leukemia SR cancer cells (GI50=1.45μM), and OVCAR-3 (GI50=1.26μM) ovarian cancer cell lines. The structurally related compound 3s had a GI50 value of 1.77μM against MDA-MB-435 cells. The N-naphthoyl analogue 3t had GI50 values of 1.30 and 1.91μM against HOP-92 non-small cell lung cancer and MDA-MB-435 melanoma cell lines, respectively. The related analogue 3w had GI50 values of 1.09μM against HOP-92 non-small cell lung cancer cell lines. Interestingly, docking of the two active molecules 3k and 3w into the active site of COX-2 indicates that these compounds are COX-2 ligands with strong hydrophobic and hydrogen bonding interactions. Thus, compounds 3k, 3t, 3s, and 3w constitute a new class of anticancer/anti-inflammatory agents that may have unique potential for cancer therapy.

Zwitterionic Guanidine-Based Oligomers Mimicking Cell-Penetrating Peptides as a Nontoxic Alternative to Cationic Polymers to Enhance the Cellular Uptake of Micelles

The aim of this work is to generate polymer micelles decorated with a synthetic version of cell-penetrating peptides, which are often rich in arginine with its positively charged guanidine group. A methacrylate-based monomer with guanidinium as functional groups was prepared using arginine (M-Arg) as a building block, resulting in a zwitterionic monomer. RAFT (reversible addition-fragmentation chain transfer) polymerization was employed to generate triblock copolymers with poly(methyl methacrylate)-block-poly(polyethylene glycol methyl ether methacrylate) as the first two blocks, which were subsequently chain extended with the guanidine-based monomer to generate micelles with guanidinium functional groups on the surface. To simulate the actual oligoarginine peptide, which only carries cationic charges, the carboxylate group of P(M-Arg) was methylated to convert the zwitterionic polymer into a cationic polymer P(Me-M-Arg). For comparison, micelles based on triblock copolymers with a third block with permanently cationic charges, poly(2-methacryolyloxy ethyl) trimethyl ammonium chloride (PTMA), was prepared. The hydrodynamic diameters of the micelles were approximately 30-40 nm based on DLS and TEM. A direct correlation between surface charge (zeta potential ζ) and cytotoxicity was observed. The micelles based on the zwitterionic P(M-Arg) were nontoxic (ζ = -10 mV at pH = 7), while the methylated version P(Me-M-Arg) with a high cationic charge (ζ = +35 mV at pH = 7) were observed to be toxic. The cellular uptake of the block copolymers by OVCAR-3 ovarian cancer cell lines was found to be relatively fast (about 35% in 3 min) reaching an equilibrium after approximately 30 min. Both micelles, with either P(M-Arg) or P(Me-M-Arg) on the surface, showed an enhanced uptake compared to micelles with P(PEGMEMA) as shell only. In fact, the percentage of uptake was similar, with the difference that cells incubated with micelles with P(M-Arg) (zwitterionic) stayed alive, while P(Me-M-Arg) (cationic) led to significant cell death.

Overexpression of CD157 Contributes to Epithelial Ovarian Cancer Progression by Promoting Mesenchymal Differentiation

Epithelial ovarian carcinoma (EOC) is an aggressive tumor often diagnosed at an advanced stage, when there is little or no prospect of cure. Despite advances in surgical and chemotherapeutic strategies, only marginal improvements in patient outcome have been obtained. Hence, unraveling the biological mechanisms underpinning EOC progression is critical for improving patients’ survival. Recently, we reported that CD157 (an ectoenzyme regulating leukocyte diapedesis) is expressed in EOC and that high expression of the molecule is negatively correlated with the disease outcome in patients. Here, we demonstrate that forced overexpression of CD157 in OVCAR-3, TOV-21G, A2780 and OV-90 ovarian cancer cell lines promotes morphological and phenotypic changes characterized by disruption of intercellular junctions, downregulation of epithelial markers and upregulation of mesenchymal ones. These changes in cell shape and phenotype bring to reduced sensitivity to anoikis, increased anchorage-independent growth, cell motility and mesothelial invasion. Conversely, knockdown of CD157 in OV-90 and OC314 cells reverts the mesenchymal phenotype and reduces the cells’ migratory potential. Transcriptome profiling analysis highlighted 378 significantly differentially expressed genes, representing the signature of CD157-overexpressing OVCAR-3 and OV-90 cells. The modulation of selected genes translates into alteration of protein expression that give cells a highly malignant phenotype. The overall picture deduced from the analysis of the modulated transcripts is that high expression of CD157 strengthens a number of biological processes favoring tumor progression (including development and cell motility), and weakens several biological processes hindering tumor progression (such as apoptosis, cell death and response to stress). Together, these findings implicate CD157 in the progression of EOC to metastatic disease and suggest that CD157 may represent a valuable therapeutic target.

Comparison of the effects of valproic acid and levetiracetam on apoptosis in the human ovarian cancer cell line OVCAR-3

BackgroundWe have previously shown that due to its cytotoxic and cytostatic activities, valproic acid (VPA), but not levetiracetam (LEV), may have potential as a drug for treating human ovarian cancer. In the present study, we compare apoptotic mechanisms including gene and protein expression in the human ovarian cancer cell line, OVCAR-3, following exposure to VPA and LEV. MethodsCells were cultured with VPA or LEV at concentrations between 0.1mM and 10mM. Apoptosis was assessed by DNA fragmentation assay and expression of apoptosis-regulatory genes determined by real-time PCR and confirmed by western blotting. Time-dependent effects of VPA and LEV on activity of caspases (-3, -8 and -9) activity were evaluated by fluorescent assay and western blotting. ResultsExposure to VPA at concentrations above 5mM resulted in an increase in DNA fragmentation, modulated expression of genes and proteins associated with apoptosis and activated caspase ca scade. Exposure to LEV, however, did not affect DNA fragmentation and modulation of the mechanisms of apoptosis was not observed in LEV-treated cells at all doses used. ConclusionsExposure to high concentrations of VPA significantly stimulated apoptosis, by modulating the expression of genes and proteins responsible for cell death and also by activation of caspases cascade. Such effects were not observed with LEV. These data suggest that VPA should be seriously evaluated as an anti-cancer drug for ovarian cancer.

Abstract 1090: Impact of CXCL1/CXCR2 signaling in ovarian cancer proliferation

Abstract Introduction: Chemokine network plays a critical role in the development and progression of cancer. Human ovarian tumors express chemokine CXCL1 and its receptor CXCR2, but its role in ovarian cancer development and progression remains unclear. In this study, we investigate whether CXCL1/CXCR2 have functional significance in ovarian cancer cell proliferation. Methods: CXCR2 was overexpressed by stable transfection into the human ovarian surface epithelial cancer cell lines A2780, OVCAR3, and SKOV3. The influence of CXCL1 and CXCR2 on cell proliferation, cell cycle progression, and changes in related proteins and gene expression were determined by using confocal image analysis, MTT assay, Western blotting, flow cytometry, and PCR array. Results: CXCR2 was mainly expressed on the cell surface and intracellular localization, and these expressions were profoundly enhanced in CXCR2 overexpressed cell lines when compared to controls. CXCR2 significantly increased the proliferation of A2780, OVCAR3 and SKOV3 cells in a time-dependent manner, and these increases were abolished by the CXCR2 inhibitor, SD 225002. Furtermore, CXCR2-induced cancer cell proliferation was suppressed by Gefitinib, an inhibitor of epidermal growth factor receptor (EGFR), indicating crosstalk between CXCR2 and EGFR in ovarian cancer cell proliferation. CXCR2 induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and NF-kappa B, probably contributing to ovarian tumor growth and metastatic progression. CXCL1 significantly stimulates the cell proliferation in CXCR2 overexpressed cell lines, and the stimulations were substantially blocked by the ERK1/2 inhibitor, PD 98059, further confirming the involvement of MAPK signaling. CXCR2 reduced cell cycle arrest in the G0-G1 phase and increased the cell population in the S and G2-M phase, and these changes were associated with enhanced expression for CDK6, a G1-S cell cycle transition regulatory gene. Chemokine specific PCR array revealed that CXCR2 enhances proinflammatory chemokines CCL2, CXCL1, CXCL2, CXCL3, and CXCL6, suggesting that CXCR2 accelerates proinflammatory tumor microenvironment to provide the suitable condition for ovarian cancer progression. Conclusions: Taken together, this study demonstrated that CXCL1/CXCR2 signaling plays critical roles in ovarian cancer growth and progression through the activation of ERK1/2 and NF-kappa B. Thus, CXCL1/CXCR2 signaling may be important in the pathogenesis of ovarian cancer, providing a basis for the therapeutic targets in ovarian cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1090. doi:1538-7445.AM2012-1090

Analysis of the characteristics of side population cells in the human ovarian cancer cell line OVCAR-3

To identify the presence of side population (SP) cells in human ovarian cancer cell line OVCAR-3 and to investigate whether SP cells have the characteristics of cancer stem cells. SP and non-SP (NSP) cells from OVCAR-3 were isolated by fluorescence-activated cell sorting after being stained by DNA-binding dye Hoechst 33342. Limiting dilution transplantation assay, real-time PCR, and drug sensitivity assay were performed to compare the tumorigenic ability, differentiation ability in vivo, the mRNA expression of "stemness" marker (Oct-4, Klf4, and Nanog) and ATP-binding cassette (ABC) transporter (ABCG2, ABCB1, and ABCC2), and response to multiple drugs (cisplatin, paclitaxel, doxorubicin, and mitoxantrone) between SP and NSP cells. A few of SP cells [(1.13 ± 0.39)%] which were sensitive to reserpine were identified in OVCAR-3 cells. The injection of as few as 10(2) SP cells initiated tumors in two of five mice. Tumor latency was 52 - 61 days. However, the NSP cells did not generate any tumors in mice until 10(4) NSP cells were injected (two of five mice). Tumor latency was 64 - 98 days. Tumorigenicity of SP cells was enhanced by at least 100-fold than that of NSP cells. The SP cells regenerated both SP [(2.09 ± 0.73)%] and NSP populations in vivo with a fraction size that was comparable to the original population. The mRNA expression of "stemness" genes Oct-4, Klf4 and ABC transporters ABCG2, ABCC2 genes were elevated in SP cells compared to NSP cells, the fold changes were 1.95 ± 0.41 (P < 0.05), 4.26 ± 0.63 (P < 0.01), 3.22 ± 0.36 (P < 0.01), and 1.76 ± 0.26 (P < 0.01), respectively. The relative activity of SP and NSP cells were 0.757 ± 0.105 versus 0.474 ± 0.035 (P < 0.01), 0.521 ± 0.092 versus 0.384 ± 0.073 (P < 0.05), 0.742 ± 0.051 versus 0.526 ± 0.088 (P < 0.01), and 0.690 ± 0.096 versus 0.466 ± 0.112 (P < 0.01) when they exposed to 0.25 µg/ml cisplatin, 0.01 µmol/L paclitaxel, 0.25 µmol/L doxorubicin, and 0.05 µg/ml mitoxantrone, respectively. SP cells from OVCAR-3 have enhanced self-renewal, differentiation, and tumor-initiating capacity compared to NSP cells. The mRNA expression of stemness genes and ABC transporters are markedly elevated in SP cells, which showed resistance to multiple chemotherapeutic drugs and have characteristics of cancer stem-like cells. Therefore, SP phenotype could be used as a marker to isolate the cancer stem-like cells in ovarian cancer.

Minocycline inhibits growth of epithelial ovarian cancer

ObjectiveThese studies were designed to determine whether minocycline inhibits ovarian cancer growth in vitro and in vivo and the molecular mechanisms involved. Materials and methodsThe effect of minocycline on ovarian cancer cell proliferation, cell cycle progression and apoptosis was assessed using human ovarian cancer cell lines OVCAR-3, SKOV-3 and A2780. Then, the capacity of minocycline to inhibit growth of OVCAR-3 xenografts in female nude mice was examined. ResultsMinocycline inhibited cell proliferation and colony formation, down-regulated cyclins A, B and E leading to arrest of cells in the G0 phase of the cycle and suppression of DNA synthesis. Furthermore, exposure of these cells to minocycline led to DNA laddering, activation of caspase-3 and cleavage of PARP-1. In nude mice bearing sub-cutaneous tumors, minocycline suppressed tumor proliferation index, angiogenesis and tumor growth. ConclusionThese findings provide the initial basis for further evaluation of minocycline in the treatment of ovarian cancer.

Effects of decabrominated diphenyl ether (PBDE-209) in regulation of growth and apoptosis of breast, ovarian, and cervical cancer cells.

Background: Polybrominated diphenyl ethers (PBDEs), commonly used in building materials, electronics, plastics, polyurethane foams, and textiles, are health hazards found in the environment.Objective: In this study we investigated the effects of PBDE-209, a deca-PBDE, on the regulation of growth and apoptosis of breast, ovarian, and cervical cancer cells as well as the underlying protein alterations.Methods: We used MCF-7 and MCF-7/ADR (multidrug-resistant MCF-7) breast cancer cell lines, the HeLa cervical cancer cell line, the OVCAR-3 ovarian cancer cell line, and the normal CHO (Chinese hamster ovary) cell line to assess the effects of PBDE-209 using cell viability, immunofluorescence, and flow cytometric assays. Western blot assays were used to detect changes in protein expression. To assess the effects of PBDE-209 on apoptosis, we used the protein kinase Cα (PKCα) inhibitor Gö 6976, the extracellular signal-regulated kinase (ERK) inhibitor PD98059, and tamoxifen.Results: Our data indicate that PBDE-209 increased viability and proliferation of the tumor cell lines and in CHO cells in a dose- and time-dependent manner. PBDE-209 also altered cell cycle distribution by inducing the S phase or G2/M phase. Furthermore, PBDE-209 partially suppressed tamoxifen-induced cell apoptosis in the breast cancer cell lines (MCF-7 and MCF-7/ADR) but suppressed Gö 6976- and PD98059-induced apoptosis in all cell lines. At the molecular level, PBDE-209 enhanced PKCα and ERK1/2 phosphorylation in the cell lines.Conclusions: Our data demonstrate that PBDE-209 is able to promote proliferation of various cancer cells from the female reproductive system and normal ovarian CHO cells. Furthermore, it reduced tamoxifen, PKCα, and ERK inhibition-induced apoptosis. Finally, PBDE-209 up-regulated phosphorylation of PKCα and ERK1/2 proteins in tumor cells and in CHO cells.

Macromolecular ruthenium complexes as anti-cancer agents

Ruthenium complexes have shown promise as anti-cancer therapeutics. Among them, RAPTA-C [RuCl2(p-cymene)(PTA)] with the water-soluble 1,3,5-triaza-7-phosphaadamantane (PTA) ligand holds great promise as an alternative to platinum drugs. This paper explores synthetic pathways by which RAPTA-C can be attached to polymer moieties to create a water-soluble macromolecular drug that can potentially display an enhanced permeation and retention (EPR) effect. A reactive scaffold for RAPTA-C conjugation, poly(2-chloroethyl methacrylate) [PCEMA], was prepared via RAFT polymerisation. To increase the reactivity of the substrate, poly(2-iodoethyl methacrylate) [PIEMA] was prepared from PCEMA via the Finkelstein reaction. Two pathways were employed to conjugate RAPTA-C to the polymer, either by attachment of PTA first followed by reaction with the RuCl2(p-cymene) dimer, or by the direct conjugation of RAPTA-C to the polymer. Prior to the study with the polymer, both routes were tested using n-butyl iodide as a model compound. Several NMR experiments, namely 31P, 1H, [1H–31P] HMBC, [1H–13C] HMBC and [1H–15N] HMBC confirmed that both routes lead to the complete reaction of n-butyl iodide and that only the desired product was obtained. However, the direct reaction of RAPTA-C with the polymer required elevated temperature, which led to the loss of the p-cymene ligand. The two-step process at ambient temperature led to the successful formation of the product, albeit the amount of iodide reacted was now only 50% due to steric hindrance. Subsequently, a copolymer based on 2-hydroxypropyl methacrylamide [HPMA] and IEMA was synthesised. The two-step reaction with PTA and RuCl2(p-cymene) dimer led to a water-soluble polymer with an intense orange colour, indicative of the pendant RAPTA-C complex. The monomodal molecular weight distribution monitored using UV-VIS detection of the macromolecular ruthenium complex confirmed drug attachment and indicates the absence of crosslinking events during drug conjugation. A cytotoxicity profile for the copolymer–RAPTA-C product was measured on the ovarian cancer cell line OVCAR-3 and compared with the profile of the lone RAPTA-C drug.

Resveratrol downregulates Akt/GSK and ERK signalling pathways in OVCAR-3 ovarian cancer cells

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

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

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

Loss of breast cancer metastasis suppressor 1 promotes ovarian cancer cell metastasis by increasing chemokine receptor 4 expression

Breast cancer metastasis suppressor 1 (BRMS1) is a predominantly nuclear protein that differentially regulates the expression of multiple genes, leading to suppression of metastasis without affecting orthotopic tumor growth. It has been demonstrated that BRMS1 may be correlated with advanced ovarian cancer. The aim of this study was to investigate the mechanisms of BRMS1 involvement in ovarian cancer metastasis. We constructed a plasmid containing a short hairpin RNA (shRNA) against BRMS1 and transfected it into the ovarian cancer cell line OVCAR3. Real-time reverse transcription polymerase chain reaction (real-time PCR) and Western blot analyses demonstrated that BRMS1 expression was efficiently downregulated. Stable suppression of BRMS1 significantly enhanced cell adhesion, migration, invasion and angiogenesis. We also found that chemokine receptor 4 (CXCR4) was upregulated at both the mRNA and protein levels. When approaching for the mechanism, we discovered that activation of the nuclear factor-κB (NF-κB) signaling pathway mediated CXCR4 upregulation, as demonstrated by the electrophoretic mobility shift assay (EMSA). Collectively, these results suggest that attenuation of BRMS1 may play a critical role in promoting migration, invasion and angiogenesis of ovarian cancer cells and BRMS1 may regulate the metastatic potential at least in part through upregulation of CXCR4 via NF-κB activation. Restoration of BRMS1 function is thus a potential new strategy for treating human ovarian cancer.

Role of hCG in Vasculogenic Mimicry in OVCAR-3 Ovarian Cancer Cell Line

Vasculogenic mimicry (VM) is induced by hypoxia in 3-dimensional culture of ovarian cancer cells. By using this 3D model system, we explored the expression of human chorionic gonadotropin (hCG) and its effects on VM formation in ovarian cancer cell line OVCAR-3 both under normoxic and hypoxic conditions. Vasculogenic mimicry was identified by morphological observation and detection of vascular cell marker expressed by OVCAR-3. Potential formation of tumor channels was observed by light microscopy and scanning electron microscopy. Expression of vascular cell marker CD31, vascular endothelial growth factor, and Factor VIII were detected by flow cytometry, immunochemistry, and Western blot. Expression of hCG was investigated by enzyme-linked immunosorbent assay, chemiluminescence immunoassay, real-time polymerase chain reaction (PCR), and Western blot. Expression of hypoxia-inducible factor-1α (HIF-1α) was detected by real-time PCR, Western blot, and blocked by small interference RNA. Incubation of OVCAR-3 with recombinant hCG was used to evaluate its effect on VM formation. The specificity of the effect of hCG was assessed by inhibition with the neutralizing anti-hCG antibody. OVCAR-3 cells formed vessel-like network structures and expressed vascular marker significantly under hypoxia in 3D. The expression level of hCG under hypoxia was significantly higher than that under normoxia. Attenuating hypoxia-inducible factor (HIF)-1α expression via small interference RNA resulted in a significantly decreased hCG expression in OVCAR-3, which indicated that the effect of hypoxia on hCG expression was mediated through HIF-1α. Treatment of OVCAR-3 with 5000 mU/mL hCG resulted in the presence of tumor cell-lined vasculature and significant elevation in vascular marker expression, even under normoxia. Expression level of vascular marker and HIF-1α in OVCAR-3 increased in response to hCG treatment in a dose-dependent manner. The effect of hCG was inhibited by the neutralizing anti-hCG antibody. Human chorionic gonadotropin has the potential to induce VM in OVCAR-3. Human chorionic gonadotropin might have synergistic hypoxia-induced effect on vascular marker and HIF-1α expression.

Suppression of proliferation and oxidative stress by extracts of Ganoderma lucidum in the ovarian cancer cell line OVCAR-3

Lingzhi (LZ) is a medical mushroom also known as Ganoderma lucidum, which has been used in traditional Chinese medicine for more than 4,000 years and moreover, due to its presumed health benefits and apparent absence of side-effects it has also been widely consumed as a dietary supplement by cancer patients and by individuals diagnosed with various chronic diseases. The reported benefits of Ganoderma lucidum may be largely ascribed to its biologically active constituent polysaccharides and triterpenes known as ganoderic acids having structural similarity to steroid hormones. Laboratory studies have shown that Ganoderma lucidum enhances immune functions and also inhibits growth of various cancer cells both in vitro and invivo. However, the mechanism by which Ganodermalucidum exerts its chemopreventive activities remains unknown. In this study, we investigated whether Ganoderma lucidum elicits its anti-tumor effects by suppressing cell growth and inducing antioxidative/detoxification activity in human ovarian OVCAR-3 cells. The results showed that Ganoderma lucidum inhibits cell growth and disruption of cell cycle progression via down regulation of cyclinD1. Chemopreventive activities elicited by Ganodermalucidum were demonstrated by the induction of antioxidant SOD and catalase as well as the phaseII detoxification enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase P1 (GSTP1) via the Nrf2 mediated signaling pathway known to provide chemoprotection against carcinogenicity. These findings indicate that Ganoderma lucidum possesses chemopreventive potential contributing to its overall health effects and further suggest that Ganoderma lucidum may have clinical applications as an adjunct supplementary agent in chemotherapy.

Indole-3-carbinol에 의한 OVCAR-3 인체 난소암세포의 침윤 억제

In the present study, we investigated the effect of indole-3-carbinol (I3C), a natural compound present in vegetables, on the cell migration and invasion of OVCAR-3 ovarian cancer cells. Our results indicated that I3C inhibited the proliferation of OVCAR-3 cells, a process which was associated with inhibition of cell motility as determined by wound healing experiments and cell invasion studies. I3C treatment increased the tightness of the tight junctions (TJs), which was demonstrated by an increase in transepithelial electrical resistance and a decrease in paracellular permeability. The RT-PCR and immunoblotting results indicated that I3C repressed the levels of claudin-3 as well as claudin-4, proteins that comprise a major part of TJs and play a key role in the control and selectivity of paracellular transport. Furthermore, the activities of matrix metalloproteinase (MMP)-2 and MMP-9 were also decreased by treatment with I3C, which was connected with the down-regulation of their mRNAs and protein expression. The results suggest that I3C may be expected to inhibit cancer cell metastasis and invasion by restoring TJs and decreasing MMP activity in ovarian cancer cell line OVCAR-3.

Effect of BRMS1 on the invasion and metastasis of ovarian cancer cells by RNA interference

Objective To study the effect of BRMS1 on the invasion and metastasis of ovarian cancer cells. Methods BRMS1 small interfering RNA (BRMS1-siRNA) was transfected into human ovarian cancer cell-line OVCAR3 by liposome transfection method. The cells were divided into 3 groups: experimental group with BRMSl-siRNA, negative control group with siRNA that did not impact any gene, blank control group without any transfection. Changes of invasion and migration in the cells were per-formed using transwell invasion and migration assay. Results BRMS1 gene was silenced in OVCAR3 cell line successfully detecting by quantitative real-time RT-PCR and immunoblotting.In transwell invasion assay,the numbers of cells in lower chamber passing through the membrane in transfected group were more than negative control group and blank control group (190±8.5, 144±7.8 and 146±6.8 respectively) (t=8.747, t=8.869, P=0.000), while in transwell migration assay, the numbers of cells in lower chamber passing through the membrane in transfected group were more than negative control group and blank control group were (231 ±8.9, 177±9.7 and 182±7.9 respectively) (t=9.314, t=9.224, P=0.000), both with significant differences among the 3 groups. Conclusion BRMS1 gene could suppress the invasion and metastasis of ovarian cancer cells. Key words: Ovarian neoplasms; Neoplasm metastasis; Breast cancer metastasis suppressor 1

Bisphenol A modulates leptin receptor expression and action in OVCAR-3 ovarian cancer cell line

Bisphenol A modulates leptin receptor expression and action in OVCAR-3 ovarian cancer cell line

EN2: A candidate antigen for the development of targeted therapies in ovarian cancer.

e15528 Background: Ovarian cancer remains the most lethal gynaecologic tumour in the Western world. Stimulation of the immune system to consolidate response to chemotherapy can potentially be beneficial however so far none of the vaccination strategies have offered survival advantage. Thus identifying and targeting clinically relevant antigens for immunotherapy continues to be an important research strategy. We have evaluated Engrailed-2 (EN2) as a potential target for vaccine strategy. EN2 is a homeodomain-containing transcription factor with a multifunctional role in neural development. There is evidence that over-expression of EN2 protein maybe linked to tumour development. Methods: Ovarian cancer cell lines were analysed by FACS for EN2 cell surface expression. EN2 expression in ovarian cancer tissue arrays were done by immunohistochemistry. A serum analysis (ELISA) was done to evaluate the presence of antibodies to EN2 in ovarian cancer patients and age-matched controls. A set of potentially immunogenic HLA-A2 restricted epitopes from the EN2 protein was identified using a computer algorithm SYFPEITHI. These peptides have been tested on HLA-A2 positive ovarian cancer patients’ PBMC using an in vitro culture method. The specificity of these T cell lines was analysed against T2 target cells loaded with or without EN2 peptides Results: Cell surface expression of EN2 was observed in ovarian cancer cell lines OVCAR3, OV90, CaOV-3, ES-2 and SKOV-3 of which ES-2 and SKOV3 showed strong expression. EN2 was also present in approximately 80% of ovarian cancer tissues whereas EN-2 expression was very low (<10%) or absent in normal tissues. Out of the 67 ovarian cancer patients 20.9% (14/67) had antibodies against EN2 compared to 2.4% (1/42) of age-matched female controls. 4 of the identified EN2 epitopes were able to generate peptide specific cytotoxic T lymphocytes in the ovarian cancer patients tested. Conclusions: The over-expression and immunogenicity of EN2 in ovarian cancer makes it a credible antigen to exploit as a novel target for ovarian cancer immunotherapy.

Induction of a menopausal state alters the growth and histology of ovarian tumors in a mouse model of ovarian cancer

Ovarian cancer is often diagnosed in women after menopause when the levels of the serum gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are increased because of the depletion of growing follicles within the ovary. The ability of FSH and LH to modulate the disease has not been well studied owing to a lack of physiologically relevant models of ovarian cancer. In this study, 4-vinylcyclohexene diepoxide (VCD) was used to deplete ovarian follicles and increase the levels of circulating FSH and LH in the tgCAG-LS-TAg mouse model of ovarian cancer. VCD-induced follicle depletion was performed either before or after induction of the oncogene SV40 large and small T-antigens in the ovarian surface epithelial cells of tgCAG-LS-TAg mice, which was mediated by the intrabursal delivery of an adenovirus expressing Cre recombinase (AdCre). tgCAG-LS-TAg mice injected with AdCre developed undifferentiated ovarian tumors with mixed epithelial and stromal components and some features of sex cord stromal tumors. Treatment with VCD before or after AdCre injection yielded tumors of similar histology, but with the unique appearance of Sertoli cell nests. In mice treated with VCD before the induction of tumorigenesis, the ovarian tumors tended to grow more slowly. The human ovarian cancer cell lines SKOV3 and OVCAR3 responded similarly to increased levels of gonadotropins in a second model of menopause, growing more slowly in ovariectomized mice compared with cycling controls. These results suggest that follicle depletion and increased gonadotropin levels can alter the histology and the rate of growth of ovarian tumors.