OVCAR-3 Human Ovarian Cancer Cells Research Articles

Bisphenol A induces leptin receptor expression, creating more binding sites for leptin, and activates the JAK/Stat, MAPK/ERK and PI3K/Akt signalling pathways in human ovarian cancer cell

We previously demonstrated that bisphenol A (BPA) promotes proliferation in OVCAR-3 human ovarian cancer cells. This study was designed to investigate the effects of BPA on leptin expression and activity in ovarian cancer. Real-time PCR, Western blot analysis and ELISA assays were used to quantify leptin receptor expression and leptin gene and protein expression after treatment with BPA at doses of 0.2, 2, 8 and 20ng/ml. Our data reveal leptin receptor expression but an absence of leptin gene and protein expression in OVCAR-3 cells. At doses of 8 and 20ng/ml, BPA had stimulatory effects on leptin receptor gene and protein expression. Leptin and BPA alone stimulated cell proliferation but BPA did not potentiate leptin activity. Similarly to leptin, but with different kinetics and duration, BPA induced phosphorylation of Stat3, ERK1/2 and Akt. In co-treatment experiments, the timing of protein phosphorylation represented an additive effect of BPA and leptin treatment.In conclusion, taking into consideration limitation of in vitro study, whether BPA by creating more binding sites for leptin and extending the time of leptin-induced Stat3, ERK1/2 and Akt phosphorylation, can potentiated leptin action in cancer cells, require confirmation by in vivo study.

The apoptosis of OVCAR-3 induced by TNF-α plus IFN-γ co-immobilized polylactic acid copolymers

The cell cycle arrest and apoptosis of human ovarian cancer OVCAR-3 cells, induced by three kinds of co-immobilized polylactic acid copolymer materials, compared with the corresponding control groups and free groups, are investigated in vitro. The three kinds of polylactic acid copolymers are PLGA (poly(lactic-co-glycolic acid)), PDLLA (poly(DL-lactic acid)), and PEG-PLGA (polyethylene glycol-poly(lactic-co-glycolic acid) copolymers). They are respectively photo-immobilized with TNF-α plus IFN-γ as the co-immobilized group, respectively mixed with TNF-α plus IFN-γ as the free group. First, the co-immobilized groups are prepared and examined by Fourier transform attenuated total reflection infrared spectroscopy (ATR-FTIR), atomic force microscopy (AFM), and electron spectroscopy for chemical analysis (ESCA). Second, the cell morphology, cell mortality, and cell cycle arrest of OVCAR-3 cells induced by the co-immobilized group are analyzed by ematoxylin staining and flow cytometry. Third, western immunoblot analysis of the protein expression of p53, Bax, and Bcl-2 and immunofluorescence analysis of the protein location of caspase-3 in the induced OVCAR-3 cells were also performed. The results show that the co-immobilized group shows more visible inhibition activity towards OVCAR-3 cells than the control group and free group, after a time of 48 hours and at a concentration level of 20 ng ml−1. The OVCAR-3 cells mediated by the co-immobilized group are individually arrested in the G1, G2, and S phases. Moreover, the western blotting and immunofluorescence analysis reveals that the treatment of OVCAR-3 cells by the co-immobilized group remarkably enhances the cellular expression of activated p53, Bax, and caspase-3, suggesting that the mitochondrial pathway of apoptosis may be significant. Our data thus provide the microscopic surface characterization and cell biology basis for utilizing the TNF-α plus IFN-γ co-immobilized polylactic acid copolymers as potential antitumorigenic targeted drugs.

Blocking Epidermal Growth Factor Receptor Activation by 3,3′-Diindolylmethane Suppresses Ovarian Tumor Growth In Vitro and In Vivo

Genetic alterations, including the overexpression of epidermal growth factor receptor (EGFR) (in approximately 70% of ovarian tumors), play a crucial role in the signal transduction pathways that regulate key cellular functions, such as cell survival and proliferation, and are responsible for compromising traditional chemotherapy. 3,3'-Diindolylmethane (DIM) is an indole compound present in Brassica vegetables. In our previous studies, we demonstrated that BR-DIM, a formulated version of DIM, suppressed the growth of ovarian cancer cells by causing cell cycle arrest and apoptosis. In the present study, we delineated the mechanism by which DIM suppressed the growth of SKOV-3, OVCAR-3, and TOV-21G human ovarian cancer cells. DIM treatment caused significant down-regulation of the constitutive EGFR protein level as well as phosphorylation of EGFR at Tyr1068, Tyr992, Tyr845, and Tyr1173 in various ovarian cancer cells. To determine whether DIM suppressed the activation of EGFR by activating phosphorylation, cells were treated with epidermal growth factor. Epidermal growth factor treatment significantly blocked the DIM-mediated inhibition of EGFR activation and apoptosis in both SKOV-3 and OVCAR-3 cells. In addition, DIM treatment drastically reduced the phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK), which are downstream to EGFR, without affecting their protein levels. DIM treatment also inhibited the kinase activity of ERK, as observed by the down-regulation of phospho-E twenty-six like transcription factor 1 (p-ELK1) in all three ovarian cancer cell lines. DIM significantly suppressed the growth of ovarian tumors in vivo. Tumor growth suppressive effects of DIM in SKOV-3 tumor xenografts were associated with reduced phosphorylation of EGFR, MEK, and ERK. These results indicate that DIM induces apoptosis in ovarian cancer cells by inhibiting the EGFR-ERK pathway in vitro and in vivo.

Abstract LB-166: Adoptive immunotherapy for ovarian cancer utilizing T cells engineered to target L1-CAM

Abstract There is pressing need for new therapeutic modalities targeting ovarian cancer, the most lethal gynecologic malignancy. We present in this study a novel immunotherapy strategy against ovarian cancer that utilizing human cytolytic T lymphocytes (CTLs) genetically re-directed to target L1-CAM, a clinically relevant ovarian tumor associated-antigen, as it is over-expressed by patient-derived ovarian tumors and is associated with poor prognosis. L1-CAM-specific targeting is achieved by engineering CTLs genetically to express a HLA-independent chimeric antigen receptor (CAR), which enables CTL to specifically recognize L1-CAM, and initiates T cell cytolytic killing via an intracellular CD3-zeta signaling domain. We report that these genetically re-programmed CTLs specifically kill L1-CAM+ OVCAR-3 human ovarian cancer cells in vitro. Importantly, single dose administration of i.p. L1CAM-CAR+ CTL induced regression of i.p established OVCAR-3 xenograft tumor growth in mice and delayed tumor progression for 6 to 20-days compared to untreated mice. Therapeutic efficacy could be further improved by multiple CTL injections (4 weekly), which delayed tumor progression for 11- to 32-days compared to untreated mice and also conferred a significant survival advantage (median survival days: 62 versus 80.5 days). For the first time, these results demonstrate L1-CAM is amenable to targeting by CAR+ T cells and support the potential of this therapeutic approach in ovarian cancer. However, ultimately tumor recurrence was observed in mice, highlighting the need for further optimization of this therapeutic platform. To investigate involvement of ovarian cancer stem-like cells (CSCs), a population with potent tumor-initiating potential, in this relapse phenomenon, we isolated subpopulations from OVCAR-3 with CSC-like properties including the formation of spheroids in serum-free culture, self-renewal and preferential expression of stem cell markers such as Oct4, Nestin and Bmi-1. Affymetrix microarray revealed these CSCs-like spheroids have a globally distinctive genetic profile compared with serum-differentiated cells, and importantly, one of the most significantly reduced genes in spheroids is L1-CAM (23.6-fold decrease). This decrease in L1-CAM was further validated by quantitative RT-PCR, western blot, confocal microscopy and flow cytometry. Together, these results suggest that an important mechanism for immune escape from L1CAM-CAR+ CTL killing is loss of antigen expression on CSC-like population. Overall, our findings indicate that adoptive transfer of genetically engineered L1-CAM-specific T cells can be a potential therapy for ovarian cancer, and we are conducting further studies to uncover additional antigenic targets that are enriched in the ovarian CSC population. We envision this combined immune approach will have profound implications for opening a new door to treat this deadly disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-166. doi:10.1158/1538-7445.AM2011-LB-166

Hyaluronic acid–paclitaxel: effects of intraperitoneal administration against CD44(+) human ovarian cancer xenografts

Hyaluronan (HA)-receptors (mainly CD44 and RHAMM) are overexpressed in a wide variety of cancers including ovarian tumors, and HA-bioconjugates have been developed to enhance selective entry of cytotoxic drugs into HA receptor-expressing cancerous cells. Here, we evaluated the potential application of a new HA-paclitaxel bioconjugate, ONCOFID-P, for intraperitoneal (IP) treatment of ovarian cancer. In vitro cytotoxic effect of ONCOFID-P was first assessed on CD44(+) OVCAR-3 and SKOV-3 human ovarian cancer cell lines. Studies were performed in female Balb/c athymic mice IP implanted with OVCAR-3 or SKOV-3 and treated with IP ONCOFID-P, and IP and intravenous (IV) free paclitaxel, at their maximum tolerated dose (MTD 168, 80 and 80mg/kg, total dose, respectively). The potential detrimental effect of the IP ONCOFID-P and IP free paclitaxel on hematopoiesis was also assessed on peripheral blood, bone marrow and spleen. Results show that ONCOFID-P cytotoxicity against both OVCAR-3 and SKOV-3 cell lines was somewhat less effective than free paclitaxel. Conversely, in in vivo experiments, IP treatment with ONCOFID-P was overall more effective than IV and IP free paclitaxel in inhibiting intra-abdominal tumor dissemination, abrogating ascites, prolonging survival and curing mice. ONCOFID-P and IP free paclitaxel were equivalent in terms of myelotoxicity, although the former was administered at a two-fold higher dose. Present data strongly support the development of ONCOFID-P for locoregional treatment of ovarian cancer.

Abstract 383: Down-regulation of tumoral Hypoxia-Inducible Factor-1α expression in OVCAR-3 tumors by albendazole

Abstract Purpose: To determine if albendazole (ABZ) interferes with the tumoral production of HIF-1α and VEGF proteins. Procedures: Human ovarian cancer OVCAR-3 cells were exposed to hypoxic conditions and the affect of ABZ on HIF-1α and VEGF expression was determined. In vivo, female nude mice bearing intraperitoneal tumors were treated with an acute dose of ABZ (150 mg/kg, i.p.), and then euthanased at various time points (1, 6, 24, 48 and 72 h). Excised tumors were analysed for HIF-1α and VEGF expression. Results: ABZ blocked the hypoxia-induced surge in HIF-1α protein. In mice, tumoral expression of HIF-1α and VEGF proteins were highly suppressed by ABZ in a reversible time-dependent manner. In drug treated mice, tumor VEGFmRNA levels were also profoundly suppressed. Conclusion: The results show that the anti-VEGF effect is mediated through the inhibition of HIF-1α. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 383.

Abstract 3781: 3,3′-diindolylmethane(DIM)-induced apoptosis in ovarian cancer cells is mediated by inhibition of EGFR-ERK pathway

Abstract Ovarian cancer is one of the leading causes of gynaecological deaths in the United States and several other countries. Genetic alterations including overexpression of EGFR (about 70% of ovarian tumors) play a crucial role in the signal transduction pathways that regulate key cellular functions such as cell survival and proliferation and are responsible for comprising traditional chemotherapy. Several epidemiological studies suggested a decreased risk of ovarian cancer in the population consuming Brassica vegetables. 3,3′-diindolylmethane (DIM) is an indole compound present in Brassica vegetables. In our previous studies, we have demonstrated that BR-DIM, a formulated version of DIM suppress the growth of ovarian cancer cells by causing cell cycle arrest. However, the exact mechanism by which DIM suppress the growth of ovarian cancer cells was not clear. In the present study, we delineated the mechanism of DIM in SKOV3, OVCAR-3 and TOV-21G human ovarian cancer cells. Our results show that DIM treatment induces apoptosis in all the cell lines in a dose-dependent manner, as analyzed by flow cytometry. Our western blot analysis reveals that DIM treatment causes significant down regulation of constitutive EGFR and HER2 protein level as well as phosphorylation of EGFR at Tyr992, Tyr845 and Tyr1173 in SKOV-3 and OVCAR-3 cells. To determine whether DIM can suppress the activation of EGFR by activating phosphorylation, cells were treated with EGF after DIM treatment. Our results reveal that DIM treatment block the activation of EGFR induced by EGF in OVCAR-3 and TOV-21G cells. In addition, DIM treatment drastically reduces the phosphorylation of ERK without affecting the protein level. DIM treatment also inhibits the kinase activity of ERK as observed by the down regulation of p-ELK in all the three ovarian cancer cell lines. ERK is known to be regulated by EGFR. Taken together our results indicate that DIM induces apoptosis in ovarian cancer cells by inhibiting EGFR-ERK pathway. Further mechanistic studies are in progress. [Supported in part by RO1 grants CA 106953 and CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3781.

Epidermal growth factor increases lysophosphatidic acid production in human ovarian cancer cells: roles for phospholipase D2 and receptor transactivation

Lysophosphatidic acid (LPA), is a lipid mediator that binds to G-protein coupled receptors. Epidermal growth factor (EGF), a polypeptide growth factor, binds to the EGF receptor (EGFR), a receptor tyrosine kinase. Both LPA and EGF induce responses in tumor cells that include proliferation, migration, metastasis, and induction of angiogenesis. LPA has the potential to act as an autocrine/paracrine factor and can transactivate the EGFR. This study explores the role of phospholipase D2 (PLD2) activation in LPA production, as well as cross-talk between EGF and LPA receptors. We demonstrate that EGF and LPA both stimulate production of LPA by OVCAR3 and SKOV3 human ovarian cancer cell lines. PD158780, an EGFR-selective tyrosine kinase inhibitor, blocks LPA production in response to both EGF and LPA in OVCAR3 and SKOV3 cells. Pertussis toxin, an inhibitor of LPA receptor signaling, inhibits LPA production in response to both EGF and LPA. Similar results were observed for the LPA receptor antagonist, Ki16425. Overexpression of PLD2 increases LPA production, while knockdown of PLD2 blocks EGF-induced LPA production. A phospholipase A2 (PLA2) inhibitor also blocks LPA- and EGF-induced LPA production. These results indicate that EGF stimulates LPA production in a manner that requires PLD2, and suggest that cross-talk can occur bidirectionally between EGF and LPA receptors.

Molecular profile of breast versus ovarian cancer cells in response to treatment with the anticancer drugs cisplatin, carboplatin, doxorubicin, etoposide and taxol

We assessed changes in the apoptosis-related genes BCL2, BAX, BCL2L12, FAS and CASPASE-3 in OVCAR-3 human ovarian cancer cells and BT-20 human breast cancer cells to provide an insight into the molecular mechanisms involved in the response of these cells to treatment with anticancer drugs and to assess their value as potential biomarkers of chemotherapy response in breast and ovarian cancer. Cells were treated with different chemotherapeutic drugs (cisplatin, carboplatin, doxorubicin, etoposide and taxol) and assessed for changes in the expression of apoptosis-related genes at the mRNA level. Total RNA was extracted, reverse-transcribed into cDNA and amplified by PCR using gene-specific primers. GAPDH was used as a housekeeping gene. Cytotoxicity was assessed by MTT assay. Both cancer cell lines responded differentially at the molecular level to the drug treatments. OVCAR-3 cells showed more pronounced sensitivity and changes compared to BT-20 cells at the mRNA level for different apoptosis-related genes, leading to cell and cancer type dependence in conjunction with drug dependence.

Sheep stromal‐epithelial cell interactions and ovarian tumor progression

Previous studies suggest that underlying ovarian stromal cues may regulate the ovarian surface epithelium. However, little is known about the interaction between ovarian stromal cells (OSC) and ovarian surface epithelial cells (OSE) under normal physiologic and pathologic conditions, largely because of the lack of a suitable model. In the current study, the OSC obtained from a sheep were immortalized with SV-40 T/t antigen (designated IOSC) and telomerase reverse transcriptase (designated IOSCH), followed by transfection with the oncogenic allele of the human H-Ras oncogene (designated IOSChR). IOSC cells transfected with H-Ras before immortalization with telomerase were designated IOSCRH. These sheep OSCs were used in both in vitro and in vivo model systems to evaluate mechanisms by which OSCs influence ovarian tumor progression. Normal sheep OSCs were found to inhibit the growth of SKOV3 and OVCAR3 human ovarian cancer cells, but not normal sheep OSE and human OSE cells (hOSE137 cells). In contrast, IOSChR and IOSCRH cells stimulated the growth of normal sheep and human OSE cells, as well as cancer cells. These findings were confirmed by in vivo studies. Our data provide compelling support for the importance of stromal-epithelial cell interactions during tumor progression, and show for the first time that immortalized and transformed OSCs promote growth of ovarian epithelial tumors.

Preparation, characterization and in vitro cytotoxicity of paclitaxel-loaded sterically stabilized solid lipid nanoparticles

In an effort to develop an alternative formulation of paclitaxel suitable for parenteral administration, paclitaxel-loaded sterically stabilized solid lipid nanoparticles (SLNs) were prepared, characterized and examined for in vitro cytotoxicity. The SLNs, comprising trimyristin (TM) as a solid lipid core and egg phosphatidylcholine and pegylated phospholipid as stabilizers, were prepared using a hot homogenization method. Regardless of paclitaxel loading, the particle sizes and zeta potentials of the prepared SLNs were around 200 nm and −38 mV, respectively, suggesting that they would be suitable as a parenteral formulation. Cryo-scanning electron microscopy showed that the SLNs were homogeneous and spherical in shape, while differential scanning calorimetry measurement of the melting peak revealed that the TM exists as a solid in our formulation. Paclitaxel was loaded to the solid cores at a w/w ratio of 6%. Gel column chromatography showed that paclitaxel co-eluted with the phospholipids, indicating that paclitaxel was incorporated in the SLNs. An in vitro drug release study showed that paclitaxel was released from the SLNs in a slow but time-dependent manner. Furthermore, treatment of the OVCAR-3 human ovarian cancer cell line and the MCF-7 breast cancer cell line with paclitaxel-loaded SLNs yielded cytotoxicities comparable to those of a commercially available Cremophor EL-based paclitaxel formulation. These results collectively suggest that our optimized SLN formulation may have a potential as alternative delivery system for parenteral administration of paclitaxel.

Desialylation of human cancer cells leading apoptosis by treatment with purified and overexpressed nanI cloned from Clostridium perfringens ATCC 10543

The effect of NanI, overexpressed nanI gene cloned from Clostridium perfringens ATCC 10543, on NIH:OVCAR-3 cancer cells and the mediators relaying the apoptosis induced by NanI were investigated. Overexpressed NanI was purified by Ni-NTA affinity column and eluted by 250 mM imidazole in 10 mM phosphate buffer, pH 7.4. The homogeneously purified NanI was added to the culture medium of OVCAR-3 human ovarian cancer cells at indicated time. The experiments included flow cytometry for glycosidic linkage determinations, in vitro invasion assay for examining cancer cell invasiveness; TUNEL/PI reactions as well as Western blot analysis for cell apoptosis detections. NanI hydrolyzed the terminal sialic acids of glycoconjugates on cancer cell surfaces and predominantly hydrolysed the α 2,6 glycosidic linkages of glycoconjugates on OVCAR-3 cell surfaces. In vitro invasion assays demonstrated that NanI reduced the number of cells; the percentage of invaded cells was reduced to 12.39 ± 3.44% when OVCAR-3 cells were treated with 0.6 U/ml of NanI. NanI suppressed the growth rate of OVCAR-3 cells efficiently. The apoptotic effects were exhibited by positive fluorescence image of TUNEL plus PI assay. Western blot analysis demonstrated down regulation of Bcl-2, releases of cytochrome c, as well as activation of caspase-9 and -3. PARP was found to be cleaved into 85 kDa after the treatment of OVCAR-3 cells with NanI. The effects of 0.6 U/ml NanI were abrogated with the addition of 10 mM of NanI inhibitor DANA. The results delineated that the induction of apoptosis in NanI-treated OVCAR-3 cells is through the mitochondria-dependent pathway. Predominant desialylation of α 2,6 linkages of glycoconjugates on cancer cell surfaces renders cells more susceptible to the membrane damage, which may suppress the invasiveness and trigger apoptosis of OVCAR-3 cells. It is anticipated that potential applications of NanI catalytic domain contained in liposome conjugated with specific monoclonal antibody to target on the cancer cell surfaces may open up a new modality of treating cancers.

Effect of lysophosphatidylglycerol on several signaling molecules in OVCAR-3 human ovarian cancer cells: Involvement of pertussis toxin-sensitive G-protein coupled receptor

In this study, we observed that lysophosphatidylglycerol (LPG) stimulated intracellular calcium ([Ca 2+] i ) increase in OVCAR-3 human ovarian cancer cells. LPG-stimulated [Ca 2+] i increase was inhibited by U-73122 but not by U-73343, suggesting that LPG stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca 2+] i increase by LPG, indicating the activation of PTX-sensitive G-proteins. LPG-induced [Ca 2+] i increase was only observed in OVCAR-3 ovarian cancer cells and SK-OV3 ovarian cancer cells among tested several cell types. LPG also induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation in OVCAR-3 ovarian cancer cells. Pertussis toxin did not affect the LPG-induced activation of ERK and Akt phosphorylation. We also found that LPG failed to stimulate NF-κB-driven luciferase activity in exogenously LPA 1, LPA 2, or LPA 3-transfected HepG2 cells. Taken together we suggest that LPG stimulates a membrane bound receptor which is different from well-known LPA receptors (LPA 1, LPA 2, and LPA 3), resulting in at least two different signaling cascades; one involves a pertussis toxin-sensitive and phospholipase C-dependent [Ca 2+] i increase, and the other involves a pertussis toxin-insensitive activation of ERK and Akt in ovarian cancer cells.

Lysophosphatidic Acid (LPA) Protects against Calpain‐mediated Degradation of Focal Adhesion Kinase (FAK) in Human Prostate and Ovarian Cancer Cells

Focal adhesion kinase (FAK) is a tyrosine kinase that associates with focal adhesion complexes upon agonist stimulation. FAK plays a role in diverse cellular responses including cell adhesion, migration, proliferation, and cytoskeletal reorganization. FAK can phosphorylate paxillin and other substrates and recruit effectors such as calpain to focal adhesion complexes. Calpain, a Ca2+-dependent protease, sequentially cleaves 125 kDa FAK into 90 and 45 kDa fragments. Lysophosphatidic acid (LPA) serves as a lipid mediator for various cellular responses including proliferation and survival. We tested the hypothesis that LPA can exert a protective effect on FAK-related signaling events. Ionomycin, a calcium ionophore, was incubated with serum-starved PC3 (human prostate cancer) or OVCAR3 (human ovarian cancer) cells. Western blots were performed to identify intact and degraded FAK in whole-cell extracts. FAK exhibited significant proteolysis when incubated with ionomycin, as compared to untreated control cells. When cells were briefly pre-incubated with LPA and then subsequently exposed to ionomycin, FAK degradation was significantly reduced. The ability of LPA to protect against ionomycin-induced FAK degradation was blocked by Ki16425, an LPA receptor antagonist, and by pertussis toxin. In conclusion, LPA provides transitory protection against calpain-mediated FAK degradation. This effect, which is mediated by LPA receptors, may contribute to the ability of LPA to enhance survival of cancer cells. (Supported by DAMD17-01-1-0730).

P38 Mitogen-activated protein kinases is required for counteraction of 2-methoxyestradiol to estradiol-stimulated cell proliferation and induction of apoptosis in ovarian carcinoma cells via phosphorylation Bcl-2

2-Methoxyestradiol (2ME2), a natural endogenous product of estradiol (E2) metabolism, has been shown to be a selective apoptotic agent for cancer cells but not for normal cells. In this study, we determined that 2ME2 counteracts E2-stimulated cell growth and induces apoptosis in ovarian carcinoma cells. In addition, we demonstrate that 2ME2 induces apoptosis via p38 and phospho-Bcl2 pathway. 2ME2 and/or E2 were administered to the OVCAR-3 (human ovarian cancer) cell line. Cell growth inhibition was analyzed by [3H] Thymidine incorporation assay and DNA fluorometric assay. Cell apoptosis was tested by DNA fragmentation analysis and FACS. The signaling pathway was determined by a series of biochemical assays. 2ME2 inhibited estradiol-stimulated cell growth and induced apoptosis in an ovarian carcinoma cell line. MAPK and p38, but not JNK, were found to be critical mediators in this process. Expression of a dominant negative mutant of p38 kinase or p38 specific inhibitor, SB 203580, almost completely blocked the process. Furthermore, Bcl-2 phosphorylation was required for 2ME2-induced effects. Our data suggest that 2ME2 inhibits E2-stimulated proliferation and induces apoptosis in ovarian carcinoma cells. Furthermore, activation of p38 and phosphorylation of Bcl-2 plays a critical role in the mechanism. 2ME2 therefore, may have a clinical application for the treatment of ovarian cancer.

Serum and intraperitoneal pharmacokinetics of cisplatin within intraoperative intraperitoneal chemotherapy: influence of protein binding

Intraperitoneal (i.p.) chemotherapy is a promising therapeutic method to improve the effectiveness of cisplatin in patients with ovarian cancer and peritoneum involvement. Intraperitoneal treatment can be intraoperatively performed just after a complete surgical resection of peritoneal tumor nodules. However, little is known regarding the pharmacokinetics of platinum during intraoperative i.p. chemotherapy (IIC). Serum and i.p. measurements of total and ultrafilterable platinum were performed to determined pharmacokinetic parameters in 11 consecutive patients who received a 2-h IIC with 50 mg/m cisplatin. Protein concentrations were determined in serum and peritoneal liquid at the same times. The cisplatin concentration required to kill OVCAR-3 human ovarian cancer cells and evaluation of cisplatin binding to proteins were determined in vitro. Platinum i.p. concentration decreased rapidly and quickly came under the cytotoxicity threshold (10 mg for 2 h). About 85% of i.p. and serum cisplatin was ultrafilterable during IIC. Platinum concentrations were closely related to protein concentrations. Due to the very low level of serum protein (almost 25 g/l), serum cisplatin binding during chemotherapy was very low (almost 25%), but increased with protein concentration recovery. These pharmacokinetic data show that a sufficient concentration to kill human ovarian cancer is not reached with a single i.p. bath containing 50 mg/m cisplatin for 2 h. A new protocol with a renewed bath and a higher cisplatin concentration is under investigation.

Evidence for a Role of p38 Kinase in Hypoxia-inducible Factor 1-independent Induction of Vascular Endothelial Growth Factor Expression by Sodium Arsenite

Recently we have demonstrated that sodium arsenite induces the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) protein and vascular endothelial growth factor (VEGF) in OVCAR-3 human ovarian cancer cells. We now show that arsenic trioxide, an experimental anticancer drug, exerts the same effects. The involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) pathways in the effects of sodium arsenite was investigated. By using kinase inhibitors in OVCAR-3 cells, both effects of sodium arsenite were found to be independent of phosphatidylinositol 3-kinase and p44/p42 MAPKS but were attenuated by inhibition of p38 MAPK. A role for p38 in the regulation of HIF-1alpha and VEGF expression was supported further by analysis of activation kinetics. Experiments in mouse fibroblast cell lines, lacking expression of c-Jun N-terminal kinases 1 and 2, suggested that these kinases are not required for induction of HIF-1alpha protein and VEGF mRNA. Unexpectedly, sodium arsenite did not activate a HIF-1-dependent reporter gene in OVCAR-3 cells, indicating that functional HIF-1 was not induced. In agreement with this hypothesis, up-regulation of VEGF mRNA was not reduced in HIF-1alpha(-/-) mouse fibroblast cell lines. Altogether, these data suggest that not HIF-1, but rather p38, mediates induction of VEGF mRNA expression by sodium arsenite.

Vascular Endothelial Growth Factor-165 Overexpression Stimulates Angiogenesis and Induces Cyst Formation and Macrophage Infiltration in Human Ovarian Cancer Xenografts

Vascular endothelial growth factor (VEGF) is suggested to be an important regulator of angiogenesis in ovarian cancer. We have evaluated the effects of VEGF overexpression on the histology and growth rate of human ovarian cancer xenografts. OVCAR-3 human ovarian cancer cells were stably transfected with an expression vector encoding the 165-amino acid isoform of VEGF. As subcutaneous xenografts, moderately and highly VEGF(165)-overexpressing OVCAR-3 cells formed tumors with large cysts. Immunohistochemistry demonstrated an increase in the number of CD31-positive microvessels, some of which were larger in diameter than those in the parental tumors, as well as extensive vascular rimming around the cysts. Weakly VEGF(165)-overexpressing tumors also contained an increased number of CD31-positive microvessels and occasional vascular rimming, but cysts were not present. Immunohistochemistry further revealed the presence of monocytes and macrophages in both parental and VEGF(165)-overexpressing xenografts. Interestingly, the number of monocytes/macrophages was greatly increased in moderately and highly VEGF(165)-overexpressing xenografts and large areas populated with monocytes/macrophages were detected within the tumor stroma. Although the higher number of CD31-positive cells would suggest a better vascularization pattern in VEGF(165)-overexpressing xenografts, tumor growth rates were not increased when compared with that of parental xenografts. These data provide functional evidence for a role of VEGF(165) in cyst formation and monocyte/macrophage infiltration.

CONTAMINANTS WITHIN BACTERIAL PLASMID PREPARATIONS TRIGGER APOPTOSIS IN LIPOSOME TRANSFECTED OVCAR3, BUT NOT IN SKOV3 OR AZ224 HUMAN OVARIAN CANCER CELLS

Toxicity associated with plasmid/liposome transfection of eucaryote cells has been attributed to the inherent toxicity of cationic lipid formulations and also to bacterial contaminants of plasmid DNA preparations, such as lipopolysaccharides (LPS). Certain plasmid preparations were observed to trigger apoptosis in DNA/liposome transfected OVCAR3 human epithelial ovarian cancer cells. In contrast, AZ224 and SKOV3 cells were unaffected under the same conditions. Agarose gel electrophoresis with recovery of the plasmid DNA removed the toxic component, but not purification by phenol/chloroform extraction or isopicnic CsCl ultracentrifugation. The toxicity of individual preparations correlated with the concentration of bacterial LPS. However, polymixin B could not neutralise the toxicity and neither could the effect be reproduced by the addition of bacterial LPS to non-toxic plasmid preparations. Surprisingly, the conditioned medium of OVCAR3 cells undergoing apoptosis was found to kill non-transfected OVCAR3 cells but not AZ224 or SKOV3 cells. This observation illustrates the possibility that unpredictable contaminants of bacterial plasmid preparations are able to cause cell death in the context of plasmid/liposome transfection in a cell-type specific way. It emphasizes the importance of achieving maximal plasmid DNA purity when performing DNA transfection experiments that focus on cell survival.

Efficacy of intraperitoneal adenovirus-mediated p53 gene therapy in ovarian cancer.

The purpose of this study was to determine the efficacy of adenovirus-based p53 gene therapy in the treatment of ovarian cancer using an intraperitoneal microscopic tumor animal model system. Adenovirus-mediated wild-type p53 gene was introduced into the NIH:OVCAR-3 human ovarian cancer cell line in vitro and in vivo. In order to study microscopic intraperitoneal tumor, athymic nude mice were inoculated intraperitoneally (i.p.) with 1 x 107 OVCAR-3 cells and observed for tumor growth. Three days after inoculation with OVCAR-3 cells, the mice were divided into 3 treatment groups. One group received three daily i.p. injections of 1 x 108 pfu Ad-CMV-p53, a second group received three daily i.p. injection of 1 x 108 pfu of the control adenovirus construct expressing beta galactosidase (Ad-CMV-betagal) and a third group received three daily i.p. injections of normal saline. Adenovirus-mediated introduction of the wild-type p53 gene in the ovarian cancer cell line resulted in transient high levels of p53 protein for 24-48 h. Cell cycle analysis revealed G1 arrest, as well as the appearance of apoptosis. In vitro cell growth assays showed growth inhibition of cancer cells infected with Ad-CMV-p53 compared to cells infected with Ad-CMV-betagal or normal saline. There was a significant increase in survival in the Ad-CMV-p53 adenovirus treated animals compared to the PBS treated animals (P = 0.004). Likewise, the survival in Ad-CMV-p53 treated mice was also significantly greater than mice treated with Ad-CMV-betagal (P < 0.0001). These results demonstrated that Ad-CMV-p53 treatment is effective in inhibiting tumor growth and prolonging survival in this microscopic cancer xenograft model. The results of this study constitute a step in translating promising in vitro and in vivo data from an adenovirus-based gene therapeutic model system into practical and scientifically based human cancer therapeutic trials.