HEY Cells Research Articles

Role of Integrin Receptors for Fibronectin, Collagen and Laminin in the Regulation of Ovarian Carcinoma Functions in Response to a Matrix Microenvironment

Integrins play an important role in cellular matrix interactions requisite for cancer cell adhesion, growth, migration and invasion. In this study, we have investigated the expression of integrin subunits alpha3, alpha6, alphav and beta1 in normal ovaries, benign ovarian tumors and ovarian carcinomas of different pathological grades. The expression of these integrins in ovarian cancer cell lines was also investigated, and their role in sustaining proliferation, adhesion, migration and invasion in cohort with the activation of signaling pathways in response to extracellular matrices (ECM) was evaluated. We demonstrate a differential expression pattern of alpha3, alpha6, alphav and beta1 integrin subunits in ovarian carcinomas compared to normal ovaries and benign ovarian tumors. Ovarian cancer cell lines (Hey, Ovcar3 and Peo.36) demonstrated significantly high expression of alpha3, alpha6, alphav and beta1 integrin subunits. A significant increase in proliferation and adhesion (P<0.05) in response to collagen 1 (Coll) and laminin (LM), ligands for integrin receptor alpha3beta1 and alpha6beta1 was observed in ovarian cancer cell lines. On the other hand, fibronectin (FN), a receptor for alphavbeta1 integrin, increased proliferation in all ovarian cancer cell lines studied but only enhanced adhesion in Hey cell line (P<0.05). Neutralizing antibodies against alpha3, alpha6, alphav and beta1 integrin subunits inhibited ECM-induced proliferation, but increased adhesion to ECM was inhibited by beta1 integrin subunit antibody. No suppression of Coll, LM and FN-induced (Hey cells only) adhesion was observed in the presence of alpha3 or alphav subunit antibodies but LM-induced adhesion was inhibited by blocking alpha6 subunit functions. LM, FN and Coll enhanced chemotactic migration in Hey cells, but direct invasion across ECM was observed only in the presence of LM and Coll. Blocking antibodies against alpha3, alpha6 and beta1 integrin subunits inhibited both chemotactic migration and invasion of Hey cells in response to respective ECM. Adhesion of ovarian cancer cells to FN, Coll and LM activated Ras, Erk and Akt pathways. Neutralizing alphav and beta1 functions did not inhibit FN-induced activation of Ras and Erk pathways but inhibited the Akt pathway. On the other hand, antibodies against alpha6 and beta1 subunits, but not alpha3 subunit, inhibited LM-induced activation of Ras but did not inhibit the downstream Akt pathway. Neutralizing beta1 subunit function however, inhibited LM-induced Erk activation. Coll-induced activation of Ras, Erk and Akt pathways was inhibited by alpha3 and beta1 integrin subunit antibodies. These results indicate that alpha3beta1, alphavbeta1 and alpha6beta1 integrin mediate proliferation, adhesion, migration and invasion of ovarian cancer cells in response to ECM and targeting these integrins to modulate integrin-ECM interactions in tumor cells may be a promising tool to reduce the dissemination of ovarian carcinoma in vivo.

Therapeutic EphA2 Gene Targeting In vivo Using Neutral Liposomal Small Interfering RNA Delivery

Inducing destruction of specific mRNA using small interfering RNA (siRNA) is a powerful tool in analysis of protein function, but its use as a therapeutic modality has been limited by inefficient or impractical delivery systems. We have used siRNA incorporated into the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) for efficient in vivo siRNA delivery. In nude mice bearing i.p. ovarian tumors, nonsilencing siRNA tagged with the fluorochrome Alexa 555 was encapsulated into DOPC liposomes and shown to be taken up by the tumor as well as many major organs. Furthermore, DOPC-encapsulated siRNA targeting the oncoprotein EphA2 was highly effective in reducing in vivo EphA2 expression 48 hours after a single dose as measured by both Western blot and immunohistochemistry. Therapy experiments in an orthotopic mouse model of ovarian cancer were initiated 1 week after injection of either HeyA8 or SKOV3ip1 cell lines. Three weeks of treatment with EphA2-targeting siRNA-DOPC (150 microg/kg twice weekly) reduced tumor growth when compared with a nonsilencing siRNA (SKOV3ip1: 0.35 versus 0.70 g; P = 0.020; HeyA8: 0.98 versus 1.51 g; P = 0.16). When EphA2-targeting siRNA-DOPC was combined with paclitaxel, tumor growth was dramatically reduced compared with treatment with paclitaxel and a nonsilencing siRNA (SKOV3ip1: 0.04 versus 0.22 g; P < 0.001; HeyA8: 0.21 versus 0.84 g; P = 0.0027). These studies show the feasibility of siRNA as a clinically applicable therapeutic modality.

Lysophosphatidic acid and endothelin-induced proliferation of ovarian cancer cell lines is mitigated by neutralization of granulin–epithelin precursor (GEP), a prosurvival factor for ovarian cancer

Granulin-epithelin precursor (GEP/progranulin) is an autocrine growth factor for ovarian cancer. We examined the production and function of GEP and report that: (1) GEP production is regulated by endothelin (ET-1), lysophosphatidic acid (LPA), and cAMP; (2) cAMP signals GEP production through exchange protein activated by cAMP (EPAC); (3) ET-1 and cAMP/EPAC induce GEP through ERK1/2; and (4) neutralization of GEP results in apoptosis. Exposure of HEY-A8 and OVCAR3 ovarian cancer cells to LPA and ET-1 yielded GEP production and secretion in a dose- and time-dependent fashion; neither stimulated significant concentrations of cAMP directly. Stimulation of cAMP production with pertussis and cholera toxin, or forskolin induced GEP in a PKA-independent fashion. EPAC, an intracellular cAMP receptor, is activated specifically by the cAMP analog, 8-CPT-2'-O-Me-cAMP (8-CPT); 8-CPT treatment stimulated GEP production and secretion. The MEK inhibitor, U0126, abrogated GEP production in response to ET-1 and 8-CPT, confirming involvement of MAPK. A partial inhibition of basal and stimulated GEP production was observed when cells were treated with a internal calcium chelator, BAPTA. Neutralizing anti-GEP antibody reversed basal as well as LPA, ET-1 and 8-CPT-induced ovarian cancer cell growth and induced apoptosis as demonstrated by caspase-3 and PARP cleavage, DNA fragmentation, and nuclear condensation. These results indicate that GEP is a growth and survival factor for ovarian cancer, induced by LPA and ET-1 and cAMP/EPAC through ERK1/2.

Glyceraldehyde-3-Phosphate Dehydrogenase Binds to the AU-Rich 3′ Untranslated Region of Colony-Stimulating Factor–1 (CSF-1) Messenger RNA in Human Ovarian Cancer Cells: Possible Role in CSF-1 Posttranscriptional Regulation and Tumor Phenotype

The overexpression of the colony-stimulating factor-1(CSF-1) by epithelial ovarian cancer cells enhances invasiveness and metastatic properties, contributing to the poor prognosis of the patients. It has been suggested that CSF-1 3' untranslated region containing AU-rich elements (ARE) could regulate CSF-1 posttranscriptional expression and be responsible for its aberrant abundance in such cancer cells. In this study, normal (NOSE.1) and malignant (Hey) ovarian epithelial cells were used to examine CSF-1 expression and regulation. CSF-1 overexpression in Hey cells was found to associate with increased invasiveness, motility, urokinase activity, and virulence of tumorigenicity, compared with NOSE.1 cells, which expressed little CSF-1. CSF-1 ARE was further found to serve as an mRNA decay element that correlates with down-regulation of protein translation. Moreover, such down-regulation was found more prominent in NOSE.1 than in Hey cells, suggesting differences in posttranscriptional regulation. As a variety of trans-acting factors [AU-binding protein (AUBP)] are known to modulate messenger stability through binding to such elements, we examined the protein content of both cell lines for their ability to bind the CSF-1 ARE. Our results strongly suggested the abundance of such AUBP activity in Hey cells. We isolated a 37-kDa AUBP, which was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To summarize, our study identified GAPDH as an AUBP abundant in Hey cells, where it binds to CSF-1 ARE that imparts mRNA decay. These data suggest that GAPDH binding to CSF-1 ARE sequence prevents CSF-1 mRNA decay and subsequent down-regulation of CSF-1 protein translation, leading to CSF-1 overexpression and increased metastatic properties seen in ovarian cancer.

Ascites induces modulation of α6β1 integrin and urokinase plasminogen activator receptor expression and associated functions in ovarian carcinoma

Interactions between cancer cells and the surrounding medium are not fully understood. In this study, we demonstrate that ascites induces selective changes in the expression of integrins and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) in ovarian cancer cells. We hypothesise that this change of integrin and uPA/uPAR expression triggers signalling pathways responsible for modulating phenotype-dependent functional changes in ovarian cancer cells. Human ovarian surface epithelial (HOSE) cell lines and epithelial ovarian cancer cell lines were treated with ascites for 48 h. Ascites induced upregulation of α6 integrin, without any change in the expression of αv, β1 and β4 integrin subunits. Out of the four ovarian cancer cell lines studied, ascites induced enhancement in the expression of uPA/uPAR in the more invasive OVCA 433 and HEY cell lines without any change in the noninvasive OVHS1 and moderately invasive PEO.36 cell lines. On the other hand, no change in the expression of α6 integrin or uPAR, in response to ascites, was observed in HOSE cells. In response to ascites, enhancement in proliferation and in adhesion was observed in all four ovarian cancer cell lines studied. In contrast, no significant increase in proliferation or adhesion by ascites was observed in HOSE cells. Ascites-induced expression of uPA/uPAR correlated with the increased invasiveness of HEY and OVCA 433 cell lines but was not seen in OVHS1, PEO.36 and HOSE cell lines. Upregulation of α6 integrin and uPA/uPAR correlated with the activation of Ras and downstream Erk pathways. Ascites-induced activation of Ras and downstream Erk can be inhibited by using inhibitory antibodies against α6 and β1 integrin and uPAR, consistent with the inhibition of proliferation, adhesion and invasive functions of ovarian cancer cell lines. Based on these findings, we conclude that ascites can induce selective upregulation of integrin and uPA/uPAR in ovarian cancer cells and these changes may modulate the functions of ovarian carcinomas.

Inhibin Resistance Is Associated with Aggressive Tumorigenicity of Ovarian Cancer Cells

Malignant ovarian epithelial tumors have been shown to have decreased inhibin production relative to activin production compared with normal ovarian surface epithelial (OSE) cells and nonmalignant ovarian tumors. Activin stimulates proliferation of many ovarian cancer cell lines. Inhibin antagonizes the action of activin, and inhibin-deficient mice develop gonadal tumors, suggesting that inhibin may be a tumor suppressor. However, its effects on OSE and ovarian cancer cells are unknown. We hypothesize that activin and inhibin are important regulators of biological activity in ovarian cancers. We found that inhibin A decreased murine OSE proliferation, whereas activin A had no effect. Activin A increased the proliferation of four of eight ovarian cancer cell lines (SKOV3, OCC1, OVCAR3, and A2780-s). Inhibin A decreased the proliferation of SKOV3, A2780-s, and OVCAR3 but had no effect on OCC1, ES-2, HEY, A2780-cp, and OVCA429 cells. When injected into nude mice, the inhibin-resistant cancer cell lines resulted in shorter survival time compared with the inhibin-responsive cells. Further investigations on SKOV3 and OCC1 cells showed that activin A increased invasion through Matrigel. Inhibin A decreased both basal and activin-induced proliferation and invasion of SKOV3 but had no effect on OCC1 cells. Reverse transcription-PCR analyses showed that the SKOV3 and OCC1 cells produced activin, but only SKOV3 produced inhibin. Analysis of the activin/inhibin signaling pathways indicated that Smad anchor for receptor activation was elevated in SKOV3 and OCC1 cells and that an up-regulation of the activin receptor expression may explain the inhibin resistance of OCC1 cells. Our results suggest that activin responsiveness may be gained during transformation of OSE cells and that inhibin resistance may contribute to the aggressive behavior of ovarian cancer cells.

Detection of Activity Centers in Cellular Pathways Using Transcript Profiling

We present a new computational method for identifying regulated pathway components in transcript profiling (TP) experiments by evaluating transcriptional activity in the context of known biological pathways. We construct a graph representing thousands of protein functional relationships by integrating knowledge from public databases and review articles. We use the notion of distance in a graph to define pathway neighborhoods. The pathways perturbed in an experiment are then identified as the subgraph induced by the genes, referred to as activity centers, having significant density of transcriptional activity in their functional neighborhoods. We illustrate the predictive power of this approach by performing and analyzing an experiment of TP53 overexpression in NCI-H125 cells. The detected activity centers are in agreement with the known TP53 activation effects and our independent experimental results. We also apply the method to a serum starvation experiment using HEY cells and investigate the predicted activity of the transcription factor MYC. Finally, we discuss interesting properties of the activity center approach and its possible applications beyond the comparison of two experiments.

A Novel Ex vivo Model System for Evaluation of Conditionally Replicative Adenoviruses Therapeutic Efficacy and Toxicity

Current animal tumor models are inadequate for the evaluation of toxicity and efficacy of conditionally replicative adenoviruses. A novel model system is needed that will provide insight into the anticipated therapeutic index of conditionally replicative adenoviruses preclinically. We endeavored to show a novel model system, which involves ex vivo evaluation of conditionally replicative adenovirus toxicity and therapeutic efficacy in thin, precision-cut slices of human primary tumor and liver. The Krumdieck thin-slice tissue culture system was used to obtain and culture slices of tumor xenografts of ovarian cancer cell lines, human primary ovarian tumors, and human liver. We determined the viability of slices in culture over a period of 36 to 48 hours by ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxphenyl-2-(4-sulfophenyl)-2H-tetrazolium, inner salt)]) (MTS) assay. In vitro Hey cells, slices of Hey xenografts, and human ovarian tumor or human liver slices were infected with 500vp/cell of either replication competent wild-type adenovirus (Ad5/3wt), conditionally replicative adenovirus (Ad5/3cox-2), or the replication deficient adenovirus (Ad5/3luc1). At 12-, 24-, and 36-hour intervals, the replication of adenoviruses in these slices was determined by quantitative reverse transcription-PCR of adenoviral E4 copy number. Primary tumor slices were able to maintain viability for up to 48 hours after infection with nonreplicative virus (Ad5luc1). Infection of Hey xenografts with Ad5/3cox-2 showed replication consistent with that seen in Hey cells infected in an in vitro setting. Primary tumor slices showed replication of both Ad5/3wt and Ad5/3cox over a 36-hour time period. Human liver slices showed replication of Ad5/3wt but a relative reduction in replication of Ad5/3cox-2 indicative of conditional replication "liver off" phenotype, thus predicting lower toxicity. The thin-slice model system represents a stringent method of ex vivo evaluation of novel replicative adenoviral vectors and allows assessment of human liver replication relative to human tumor replication. This is the first study to incorporate this system for evaluation of therapeutic efficacy and replicative specificity of conditionally replicative adenoviruses. Also, the study is the first to provide a valid means for preclinical assay of potential conditionally replicative adenovirus-based hepatotoxicities, thus providing a powerful tool to determine therapeutic index for clinical translation of conditionally replicative adenoviruses.

447 Retargeting adenoviral gene therapy vectors to bladder tumours over-expressing EGFR and other cell surface markers

Objective. Melanoma differentiation associated gene-7 [mda-7/Interleukin (IL)-24] has been identified as a novel anti-cancer agent, which specifically induces apoptosis in cancer cells but not in normal epithelial, endothelial and fibroblast cells. The objective of this study was to evaluate the anti-tumor effect of adenovirus-mediated mda-7/IL-24 (Ad.mda-7) gene therapy in ovarian carcinoma and further improve anti-tumor effect by enhancing infectivity of Ad.mda-7.Methods. A panel of human ovarian carcinoma cells, OV-4, HEY, SKOV3, SKOV3.ip1 and control normal human mesothelial cells, were infected by a replication deficient recombinant adenovirus encoding mda-7/IL-24 and control virus Ad.CMV.Luc. After 72 h, apoptosis was evaluated by TUNEL and Hoechst staining and further quantified by fluorescent activated cell sorter (FACS) analysis. Infectivity of Ad.mda-7 was enhanced by retargeting it to CD40 or EGF receptors overexpressed on ovarian cancer cells. Subsequently, enhancement in apoptosis of CD40- or epidermal growth factor receptor (EGFR)-retargeted Ad.mda-7 was evaluated.Results. Adenoviral-mediated delivery of mda-7 induces apoptosis ranging from 10–23% in human ovarian cancer cells tested with the highest percentage of apoptosis noted in SKOV3 cells. Minimal apoptosis was noted in normal mesothelial cells. CD40- or EGFR-retargeted Ad.mda-7 increased apoptosis by 10–32% when compared to that achieved with untargeted Ad.mda-7.Conclusion. Ad.mda-7 exhibits ovarian cancer-specific apoptosis, but does not affect normal human mesothelial cells. Infectivity enhanced CD40- and EGFR-retargeted Ad.mda-7 augments apoptosis induction, thus increasing the therapeutic index and translational potential of Ad.mda-7 gene therapy.

450 The TGF-beta1 antisense oligonucleotide AP 11014 for the treatment of non-small cell lung, colorectal and prostate cancer: preclinical studies

Objective. Melanoma differentiation associated gene-7 [mda-7/Interleukin (IL)-24] has been identified as a novel anti-cancer agent, which specifically induces apoptosis in cancer cells but not in normal epithelial, endothelial and fibroblast cells. The objective of this study was to evaluate the anti-tumor effect of adenovirus-mediated mda-7/IL-24 (Ad.mda-7) gene therapy in ovarian carcinoma and further improve anti-tumor effect by enhancing infectivity of Ad.mda-7.Methods. A panel of human ovarian carcinoma cells, OV-4, HEY, SKOV3, SKOV3.ip1 and control normal human mesothelial cells, were infected by a replication deficient recombinant adenovirus encoding mda-7/IL-24 and control virus Ad.CMV.Luc. After 72 h, apoptosis was evaluated by TUNEL and Hoechst staining and further quantified by fluorescent activated cell sorter (FACS) analysis. Infectivity of Ad.mda-7 was enhanced by retargeting it to CD40 or EGF receptors overexpressed on ovarian cancer cells. Subsequently, enhancement in apoptosis of CD40- or epidermal growth factor receptor (EGFR)-retargeted Ad.mda-7 was evaluated.Results. Adenoviral-mediated delivery of mda-7 induces apoptosis ranging from 10–23% in human ovarian cancer cells tested with the highest percentage of apoptosis noted in SKOV3 cells. Minimal apoptosis was noted in normal mesothelial cells. CD40- or EGFR-retargeted Ad.mda-7 increased apoptosis by 10–32% when compared to that achieved with untargeted Ad.mda-7.Conclusion. Ad.mda-7 exhibits ovarian cancer-specific apoptosis, but does not affect normal human mesothelial cells. Infectivity enhanced CD40- and EGFR-retargeted Ad.mda-7 augments apoptosis induction, thus increasing the therapeutic index and translational potential of Ad.mda-7 gene therapy.

Infectivity enhanced adenoviral-mediated mda-7/ IL-24 gene therapy for ovarian carcinoma

Objective. Melanoma differentiation associated gene-7 [ mda-7/Interleukin (IL)-24] has been identified as a novel anti-cancer agent, which specifically induces apoptosis in cancer cells but not in normal epithelial, endothelial and fibroblast cells. The objective of this study was to evaluate the anti-tumor effect of adenovirus-mediated mda-7/ IL-24 (Ad.mda-7) gene therapy in ovarian carcinoma and further improve anti-tumor effect by enhancing infectivity of Ad.mda-7. Methods. A panel of human ovarian carcinoma cells, OV-4, HEY, SKOV3, SKOV3.ip1 and control normal human mesothelial cells, were infected by a replication deficient recombinant adenovirus encoding mda-7/ IL-24 and control virus Ad.CMV.Luc. After 72 h, apoptosis was evaluated by TUNEL and Hoechst staining and further quantified by fluorescent activated cell sorter (FACS) analysis. Infectivity of Ad.mda-7 was enhanced by retargeting it to CD40 or EGF receptors overexpressed on ovarian cancer cells. Subsequently, enhancement in apoptosis of CD40- or epidermal growth factor receptor (EGFR)-retargeted Ad.mda-7 was evaluated. Results. Adenoviral-mediated delivery of mda-7 induces apoptosis ranging from 10–23% in human ovarian cancer cells tested with the highest percentage of apoptosis noted in SKOV3 cells. Minimal apoptosis was noted in normal mesothelial cells. CD40- or EGFR-retargeted Ad.mda-7 increased apoptosis by 10–32% when compared to that achieved with untargeted Ad.mda-7. Conclusion. Ad.mda-7 exhibits ovarian cancer-specific apoptosis, but does not affect normal human mesothelial cells. Infectivity enhanced CD40- and EGFR-retargeted Ad.mda-7 augments apoptosis induction, thus increasing the therapeutic index and translational potential of Ad.mda-7 gene therapy.

Expression of platelet-derived growth factor and activated receptor in clinical specimens of epithelial ovarian cancer and ovarian carcinoma cell lines

Objectives. We determined the expression of platelet-derived growth factor (PDGF), PDGF-receptor (PDGF-R), and phosphorylated PDGF-R (p-PDGF-R) on tumor cells and tumor-associated endothelial cells in clinical specimens of human ovarian carcinoma and human ovarian cancer cells growing in culture and in the peritoneal cavity of nude mice. Methods. Ten specimens of high-grade serous ovarian carcinoma were analyzed using immunohistochemistry (IHC). IHC was used to detect ligand and receptor expression in the human ovarian cancer cells from Hey A8 and SKOV3ip1 growing in culture. Cells from these lines were also implanted orthotopically into the peritoneal cavity of nude mice. IHC was used to determine ligand and receptor expression in tumors that formed in the peritoneal cavity. Results. All 10 evaluable samples expressed both PDGF AA and BB on tumor cells. Tumor cells were positive for PDGF-Rα in 10/10 samples, PDGF-Rβ in 8/10 samples, p-PDGF-Rα in 6/10 samples, and p-PDGF-Rβ in 4/10 samples. p-PDGF-Rα was positive in 4/10 tumor-associated endothelial cell samples and p-PDGF-Rβ was positive in 3/10 samples. Human ovarian cancer cells expressed PDGF, PDGF-R, and p-PDGF-R when growing in culture or in the peritoneal cavity of nude mice. PDGF-R and p-PDGF-R were also present on tumor-associated endothelial cells as demonstrated by simultaneous staining with CD31 antibody. Conclusions. PDGF and the corresponding receptors were expressed in autochthonous human ovarian cancer lesions on both tumor cells and tumor-associated endothelial cells. The ligand and receptor were also present on Hey A8 and SKOV3ip1 human ovarian cancer cells growing in vitro and in the peritoneal cavity of nude mice.

S1P3-mediated Akt activation and cross-talk with platelet-derived growth factor receptor (PDGFR).

Akt plays a pivotal role in cell survival and tumorigenesis. We investigated the potential interaction between sphingosine-1-phosphate (S1P) and platelet-derived growth factor (PDGF) in the Akt signaling pathway. Using mouse embryonic fibroblasts (MEFs) from S1P receptor knockout mice, we show here that S1P3 was required for S473 phosphorylation of Akt by S1P. In addition, S1P-stimulated activation of Akt, but not ERK, was blocked by a PDGF receptor (PDGFR)-specific inhibitor, AG1296, suggesting a S1P3-mediated specific crosstalk between the Akt signaling pathways of S1P and PDGFR in MEFs. We investigated this crosstalk under different conditions and found that both Akt and ERK activation induced by S1P, but not lysophosphatidic acid (LPA), in HEY ovarian cancer cells required PDGFR but not epidermal growth factor receptor (EGFR) or insulin-like growth factor-I receptor (IGFR). Importantly, S1P induced a Gi-dependent tyrosine phosphorylation of PDGFR in HEY cells. This dependence on PDGFR in S1P-induced Akt activation was also observed in A2780, T47D, and HMEC-1 cells (which express S1P3), but not in PC-3 or GI-101A cells (which do not express S1P3), further supporting that S1P3 mediates the crosstalk between S1P and PDGFR. This is the first report demonstrating a unique interaction between S1P3 and PDGFR, in addition to demonstrating a specific role for S1P3 in S1P-induced Akt activation.

Endothelin-1 Decreases Gap Junctional Intercellular Communication by Inducing Phosphorylation of Connexin 43 in Human Ovarian Carcinoma Cells

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.

Vascular Endothelial Growth Factor Immunoneutralization Plus Paclitaxel Markedly Reduces Tumor Burden and Ascites in Athymic Mouse Model of Ovarian Cancer

Ovarian cancer is characterized by rapid growth of solid intraperitoneal tumors and production of large volumes of ascites. Our previous studies of intraperitoneal ovarian carcinoma in an athymic mouse model demonstrated that a monoclonal antibody (mAb) to human vascular endothelial growth factor (VEGF) could prevent ascites formation. Although ascites was almost completely inhibited, tumor burden was variably reduced. To develop more effective therapy, we assessed the combination of a human VEGF mAb plus paclitaxel. Four groups of female athymic nude mice were inoculated intraperitoneally with OVCAR3 cells. Two weeks after inoculation, one group was treated with a human VEGF mAb intraperitoneally twice weekly plus paclitaxel intraperitoneally three times weekly for 6 weeks. The second group was treated with VEGF mAb alone. The third group was treated with paclitaxel alone. The remaining group was treated with vehicle only. Tumor burden in the VEGF mAb plus paclitaxel and paclitaxel alone groups was reduced by 83.3% and 85.7% and 58.5% and 59.5%, respectively, in two separate experiments, compared to controls. VEGF mAb alone caused no significant decrease in tumor burden, nor did treatment of mice inoculated intraperitoneally with HEY-A8 cells, a non-VEGF-secreting ovarian cell line. Virtually no ascites developed in the combined treatment group or the group treated with VEGF mAb alone. Paclitaxel alone reduced ascites slightly, but not significantly. Morphological studies demonstrated that VEGF immunoneutralization enhanced paclitaxel-induced apoptosis in these human ovarian cancers. Thus, combination therapy with inhibitors of VEGF plus paclitaxel may be an effective way to markedly reduce tumor growth and ascites in ovarian carcinoma.

Akt activation induced by lysophosphatidic acid and sphingosine-1-phosphate requires both mitogen-activated protein kinase kinase and p38 mitogen-activated protein kinase and is cell-line specific.

The signaling pathways that lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) use to activate Akt in ovarian cancer cells are investigated here. We show for the first time, with the use of both pharmacological and genetic inhibitors, that the kinase activity and S473 phosphorylation of Akt induced by LPA and S1P requires both mitogen-activated protein (MAP) kinase kinase (MEK) and p38 MAP kinase, and MEK is likely to be upstream of p38, in HEY ovarian cancer cells. The requirement for both MEK and p38 is cell type- and stimulus-specific. Among 12 cell lines that we tested, 11 respond to LPA and S1P and all of the responsive cell lines require p38 but only nine of them require MEK. Among different stimuli tested, platelet-derived growth factor stimulates S473 phosphorylation of Akt in a MEK- and p38-dependent manner. However, epidermal growth factor, thrombin, and endothelin-1-stimulated Akt S473 phosphorylation require p38 but not MEK. Insulin, on the other hand, stimulates Akt S473 phosphorylation independent of both MEK and p38 in HEY cells. T308 phosphorylation stimulated by LPA/S1P requires MEK but not p38 activation. MEK and p38 activation were sufficient for Akt S473 but not T308 phosphorylation in HEY cells. In contrast to S1P and PDGF, LPA requires Rho for Akt S473 phosphorylation, and Rho is upstream of phosphatidylinositol 3-kinase (PI3-K). LPA/S1P-induced Akt activation may be involved in cell survival, because LPA and S1P treatment in HEY ovarian cancer cells results in a decrease in paclitaxel-induced caspase-3 activity in a PI3-K/MEK/p38-dependent manner.

Endothelin-1 promotes proteolytic activity of ovarian carcinoma.

Endothelin-1 (ET-1) is a potent mitogenic and angiogenic factor for ovarian carcinoma cell lines, which acts selectively through the ET(A) receptor (ET(A)R). A previous study demonstrated that ET-1 is present at high concentrations in ovarian cancer ascites, indicating a direct role in the progression and metastasis of ovarian carcinoma. In this study, we investigated whether ET-1 could induce production and activation of tumour-associated proteinases in ovarian carcinoma cells. As demonstrated by ELISA, we found that the secretion of matrix metalloproteinase (MMP)-2 and MMP-9, urokinase-type plasminogen activator and plasminogen activator inhibitor type-1 and -2 was upregulated by ET-1 in a dose-dependent manner in the HEY cell line. In addition, the MMPs in ET-1-treated cells are consistently active, as shown by MMP gelatinase activity assay. Finally, we demonstrated that BQ-123, an antagonist of ET(A)R, inhibited the ET-1-induced tumour protease secretion and activity, suggesting that ET-1/ET(A)R may play an important role in the progression and metastasis of ovarian carcinoma, activating multiple proteinase cascades.

Malignant ascites fluid (MAF), including ovarian-cancer-associated MAF, contains angiostatin and other factor(s) which inhibit angiogenesis.

The aim was to determine whether human malignant ascites fluid (MAF) associated with abdominal cancer, including ovarian cancer, contained factors which inhibit angiogenesis as well as others which stimulate this process. MAF was collected from six patients, four with ovarian cancer, one with gastric cancer, and one with liver metastases. Using the chick chorioallantoic membrane (CAM) the effect of MAF on 7-day-old CAM capillaries was examined for 48 h. Vascular endothelial growth factor (VEGF) was evaluated by ELISA. Five samples of MAF were fractionated by lysine-Sepharose chromatography and the lysine-bound and -unbound fractions were eluted by epsilon-amino-n-hexanoic acid. Whole MAF, the lysine-bound and -unbound fractions, and human angiostatin were subjected to SDS-PAGE/Western blot analysis and immunostained after exposure to anti-human plasminogen. Human plasminogen was exposed to conditioned medium from ovarian epithelial cancer (HEY) cells and subjected to similar Western blot analysis. Despite containing VEGF, each MAF sample examined caused a loss of capillaries from the CAM; a similar response was seen using purified human angiostatin. Whole MAF and the lysine-bound fraction contained plasminogen (90 kDa) and a 55-kDa protein which migrated in a similar manner to human angiostatin on Western blot. Both the lysine-bound and -unbound fractions caused a loss of capillaries in the CAM. Human plasminogen exposed to conditioned medium from HEY cells yielded a fragment which was similar in size to angiostatin. MAF from patients with various clinical presentations contains angiostatin and VEGF as well as other factors which are capable of inhibiting angiogenesis.

Contributions of stromal metalloproteinase-9 to angiogenesis and growth of human ovarian carcinoma in mice.

The expression level of several matrix metalloproteinases (MMPs), including MMP-2 and MMP-9, in ovarian cancer cells is directly associated with their invasive and metastatic potentials. MMP-9 is also expressed in stromal cells adjacent to the tumor. To investigate the contribution of MMP-9 expression in stromal cells to ovarian tumor growth, we examined angiogenesis and progressive growth of human ovarian cancer cells implanted into mice with and without the MMP-9 gene. Human ovarian cancer cells SKOV3.ip1 and HEY-A8 were implanted into the peritoneal cavities of nude mice that lacked the gene for MMP-9 (MMP-9(-/-)) or were wild type for MMP-9 (MMP-9(+/+)) (10 mice of each genotype per cell line). Tumor incidence, tumor size, and volume of ascites fluid were recorded for each mouse at 30 and 45 days after HEY-A8 and SKOV3.ip1 cell injections, respectively. Blood vessel density and macrophage infiltration into the lesions were analyzed in excised tumors by immunohistochemistry and double immunofluorescence. Tumor growth was also studied in MMP-9(-/-) nude mice that had been reconstituted with spleen cells collected from either MMP-9(+/+) or MMP-9(-/-) nude mice. All statistical tests were two-sided. HEY-A8 cells expressed high levels of MMP-9, and SKOV3.ip1 cells expressed low levels. Nevertheless, tumor incidence and growth were statistically significantly lower in MMP-9(-/-) mice than in MMP-9(+/+) mice injected with cells from either line (for tumor size, P =.006 and.042 for HEY-A8 and SKOV3.ip1 cells, respectively). Compared with MMP-9(+/+) mice injected with human ovarian cancer cells, MMP-9(-/-) mice injected with human ovarian cancer cells displayed decreased microvessel density and decreased macrophage infiltration into the lesions. Compared with MMP-9(-/-) mice that received spleen cells (a rich source of macrophages) from MMP-9(-/-) mice, those that received spleen cells from MMP-9(+/+) mice before cancer cell injections displayed increased angiogenesis and tumorigenicity of the cancer cells. The growing tumors contained MMP-9-expressing macrophages. Host-derived MMP-9 expression, most likely in tumor-infiltrating macrophages, appears to play a critical role in angiogenesis and progressive growth of human ovarian tumors in mice.

Cyclic AMP in Ovarian Cancer Cells Both Inhibits Proliferation and Increases c-KIT Expression

C-KIT encodes a tyrosine kinase receptor (KIT) that, when activated by its ligand (KL), stimulates proliferation, differentiation, migration, and survival. Greater than 70% of epithelial ovarian cancers coexpress c-KIT and KL. C-KIT and KL expression levels have been shown to be up-regulated by cAMP in some cell types. Additionally, cAMP is well-recognized for its anti-proliferative effects in cancer cells. The goal of these experiments was to investigate these seemingly contradictory consequences of cAMP treatment by: (1) confirming the growth inhibitory actions of cAMP on ovarian cancer cells; (2) investigating the ability of cAMP to affect c-KIT and KL expression in these cells; and (3) examining the possible role of endogenous and/or cAMP-regulated c-KIT and KL expression in ovarian cancer cell proliferation. HEY cells, an ovarian cancer cell line which expresses c-KIT and KL, were treated with dibutyryl cyclic AMP (dbcAMP), 8-bromo-cAMP, and cholera toxin over a range of concentrations. With all treatments, stimulation of cAMP signaling caused a dose-dependent inhibition of HEY cell proliferation by up to 40, 62, and 38%, respectively. This inhibition of proliferation correlated with a dose-dependent increase in c-KIT mRNA expression, yielding 4- to 7-fold elevations in transcript abundance; there were no changes in steady-state levels of KL transcripts. In order to determine whether KIT expression/activity was responsible for the observed decrease in proliferation, dbcAMP-treated HEY cells were exposed either to anti-KIT neutralizing antibodies or to the KIT inhibitor STI571. These experiments demonstrated that KIT inhibition did not alter the growth rate of cells or reverse the dbcAMP-induced inhibition of proliferation. These results suggest that cAMP signaling pathways regulate both cell proliferation and c-KIT expression in ovarian cancer cells; however, KIT is not assuming its well-established role as a growth factor.