HRA Cells Research Articles

Differences in the sensitivity of ovarian cancer to photodynamic therapy and the mechanisms for those differences.

Protoporphyrin IX (PpIX) levels are crucial to the antitumor action of photodynamic therapy (PDT). In the present study, the underling molecular mechanisms for the variation in PpIX levels in ovarian cancer cells were investigated. Five ovarian cancer cell lines were subcutaneously grafted onto the backs of nude mice. Once tumors had developed, 5-aminolevulinic acid methyl ester hydrochloride (methyl-ALA) was administered intraperitoneally and the tumor was irradiated twice/week. PpIX levels in the tumor were assayed using high-performance liquid chromatography. Enzymes involved in heme synthesis and degradation were screened using a microarray technique. Expression of the glutathione transferase Omega-1 (GSTO1) gene involved in the conversion of PpIX into heme in cells was quantified using the reverse transcription-quantitative polymerase chain reaction. In HTOA, HRA and DISS cells, PDT resulted in significant tumor shrinkage in comparison with the controls. In MCAS and TOV21G cells, no significant alterations in tumor growth were identified compared with the untreated cells. PpIX levels increased significantly in HTOA, DISS and HRA cells compared with in MCAS and TOV21G cells. A comparison of genetic profiles using PDT-sensitive DISS cells and PDT-resistant MCAS cells indicated that MCAS cells exhibited significantly increased levels of δ-aminolevulinate synthase (a rate-limiting enzyme in heme synthesis), heme oxygenase 2 (an enzyme that degrades heme into biliverdin), and biliverdin reductase B (an enzyme that reduces biliverdin into bilirubin) in comparison with DISS cells. The level of GSTO1 expression in HTOA, HRA and DISS cells was ~2.5-fold that in MCAS and TOV21G cells. Sensitivity to PDT is related to PpIX levels in cells. The results of the present study suggested that PpIX tends not to accumulate in PDT-resistant cells despite active heme synthesis and degradation, and that high levels of GSTO1 expression are associated with increased sensitivity to PDT.

Identification of genes responsible for RelA-dependent proliferation arrest in human mammary epithelial cells conditionally expressing RelA.

The molecular mechanisms responsible for opposing oncogenic and tumor-suppressor activities of NF-kB are obscure. Semi-quantitative immunohistochemistry of primary breast tumors using antibodies to RelA, the pleiotropic NF-kB factor, and Ki67 revealed a negative correlation between RelA levels and Ki67-index among ER +/HER2 − tumors [1]. Similarly, expression of AURKA, a marker for proliferation, negatively correlates with expression of NFKBIA, a surrogate for RelA expression and activity, in ER +/HER2 − tumors analyzed by The Cancer Genome Atlas [2], [3], [4]. Furthermore, conditional expression of RelA using a Tetracycline-inducible system in Human Mammary Epithelial Cells (HRA cells) caused proliferation arrest while withdrawal of Doxycycline (Dox) and suppression of RelA expression in arrested cells restored cell cycle progression [1]. To identify genes responsible for the negative relationship between RelA levels and proliferation, we performed genome-wide gene expression analysis of HRA cells under the following conditions: RelA un-induced, No Dox (ND); Dox induced for 24 h; Dox induced for 72 h; Dox induced for 24 h then Dox withdrawn for 48 h. The expression data was submitted to Gene Expression Ominibus (GEO) and the accession number is GSE65040. Analysis of the data identified cross-talk between basal RelA activity and the Interferon pathway mediated by IRF1, a target of RelA [5]. Activation of the Interferon pathway lead to down-regulation of CDK4 expression resulting in RB1 hypo-phosphorylation and suppression of cell cycle progression. The tumor-suppressor activity of NF-kB, specifically RelA, may stem from cross-talk with the Interferon pathway.

Gene therapy for ovarian cancer using carbonyl reductase 1 DNA with a polyamidoamine dendrimer in mouse models.

Ovarian cancer (OC) in which carbonyl reductase 1 (CBR1) is highly expressed has good prognosis. The aims of this study were to determine the optimal conditions for delivering CBR1 DNA to OC cells via a polyamidoamine (PAMAM) dendrimer and to examine the therapeutic effectiveness of using a CBR1/PAMAM dendrimer to treat OC. The ratio for mixture of the PAMAM dendrimer and CBR1 plasmid DNA was defined as the ratio of the number of moles of phosphate groups in plasmid DNA to the number of moles of amino groups in PAMAM, which was expressed as N/P ratio. Mice were intraperitoneally injected with OC cells (HRA) to create peritoneal carcinomatosis. CBR1 DNA/PAMAM dendrimer complexes were administered on alternate days after injection of HRA cells. Cells transfected with CBR1 DNA at N/P ratio of 20:1 for 48 h produced the highest level of CBR1 expression. All the mice in control group died prior to day 25. However, all the mice administered the CBR1 DNA/PAMAM dendrimer survived (P<0.001). Use of a PAMAM dendrimer allowed CBR1 DNA to be delivered to cancer cells. The results suggested that CBR1 DNA/PAMAM dendrimer complexes may represent a potent gene therapy for the treatment of advanced OC.

4-Methylumbelliferone inhibits ovarian cancer growth by suppressing thymidine phosphorylase expression.

Background4-Methylumbelliferone (4-MU), a hyaluronan (HA) synthesis inhibitor, has antitumor activity in cancer cells. However, few studies have focused on its effects on ovarian cancer. The aim of this study was to investigate the effects of 4-MU on ovarian cancer and to elucidate its mechanism of action.MethodsThe HRA human ovarian serous adenocarcinoma cell line was used in this study. The effects of 4-MU on cell proliferation, migration, and invasion were determined by using in vitro assays as well as an in vivo rat peritoneal carcinomatosis model. The expression of HA synthase (HAS), CD44 HA receptor, vascular endothelial growth factor (VEGF), and thymidine phosphorylase (TP) mRNA in HRA cells was analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).Results4-MU administration inhibited the growth of peritoneal tumors and significantly prolonged survival. In vitro experiments showed that 4-MU inhibited HRA cell proliferation in a dose-dependent manner, while it did not affect HRA cell invasion and migration. 4-MU significantly decreased TP mRNA expression in HRA cells. On the other hand, since HAS2, CD44, and VEGF endogenous mRNA expression levels were very low in HRA cells, it was impossible to evaluate the effect of 4-MU treatment.ConclusionsThese results suggest that 4-MU exerts its antitumor effect on ovarian cancer through suppressing TP expression.

4-Methylumbelliferone inhibits ovarian cancer growth by suppressing thymidine phosphorylase expression

4-Methylumbelliferone (4-MU), a hyaluronan (HA) synthesis inhibitor, has antitumor activity in cancer cells. However, few studies have focused on its effects on ovarian cancer. The aim of this study was to investigate the effects of 4-MU on ovarian cancer and to elucidate its mechanism of action. The HRA human ovarian serous adenocarcinoma cell line was used in this study. The effects of 4-MU on cell proliferation, migration, and invasion were determined by using in vitro assays as well as an in vivo rat peritoneal carcinomatosis model. The expression of HA synthase (HAS), CD44 HA receptor, vascular endothelial growth factor (VEGF), and thymidine phosphorylase (TP) mRNA in HRA cells was analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). 4-MU administration inhibited the growth of peritoneal tumors and significantly prolonged survival. In vitro experiments showed that 4-MU inhibited HRA cell proliferation in a dose-dependent manner, while it did not affect HRA cell invasion and migration. 4-MU significantly decreased TP mRNA expression in HRA cells. On the other hand, since HAS2, CD44, and VEGF endogenous mRNA expression levels were very low in HRA cells, it was impossible to evaluate the effect of 4-MU treatment. These results suggest that 4-MU exerts its antitumor effect on ovarian cancer through suppressing TP expression.

Targeting aminopeptidase N (APN/CD13) with cyclic-imide peptidomimetics derivative CIP-13F inhibits the growth of human ovarian carcinoma cells

Aminopeptidase N (APN/CD13) is an essential peptidase involved in the process of tumor growth, metastasis and angiogenesis. Inhibition of APN/CD13 may be an effective strategy for cancer treatment. CIP-13F is a cyclic-imide peptidomimetics compound designed to fit the active pockets S1 and S′1 of APN/CD13 that act in tumor proliferation. Our aim in this study was to evaluate the efficacy of CIP-13F as a candidate compound for cancer treatment. The experiments were performed on the human ovarian carcinoma (OVCA) ES-2 and HRA cell lines, which have high and low levels of APN/CD13 respectively. CIP-13F significantly blocked APN/CD13 activity on the surface of ES-2 cells as measured by quantitating the enzymatic cleavage of the substrate l-leucine- p-nitroanilide. CIP-13F effectively inhibited ES-2 cell growth and migration without significant cytotoxic effect. In contrast, CIP-13F did not significantly inhibit HRA cell growth, indicating that CIP-13F may inhibit ES-2 cell growth via suppression of APN/CD13. The suppression of APN/CD13 was also observed by using the assays of flow cytometry and Western blot analysis. Further, the inhibitory effects of CIP-13F on APN/CD13 and on ES-2 proliferation were supported by the induction of ES-2 apoptosis. CIP-13F-treated ES-2 cells resulted apoptotic characteristics, such as induction of externalization of phosphatidylserine and DNA laddering fragment. The activation of caspase-3 and poly ADP-ribose polymerase (PARP) was also enhanced. The inhibitory effects of CIP-13F on APN/CD13 expression and on ES-2 proliferation were confirmed in mice bearing ES-2 xenografts. CIP-13F delayed the growth of ES-2 xenografts in mice after 2 weeks of vena caudalis injection. These results suggest that CIP-13F has a high inhibitory effect on the growth of OVCA cells via decreasing the activity and expression of APN/CD13.

Suppression of Urokinase Receptor Expression by Thalidomide Is Associated with Inhibition of Nuclear Factor κB Activation and Subsequently Suppressed Ovarian Cancer Dissemination

Thalidomide has been used to treat a variety of diseases ranging from alleviation of autoimmune disorders to prevention of metastasis of cancers. It has been shown previously that increased levels of urokinase-type plasminogen activator receptor (uPAR) correlate well with higher invasive phenotype. We examined whether thalidomide is able to suppress the expression of uPAR mRNA and protein in human ovarian cancer cell line HRA and human chondrosarcoma cell line HCS-2/8. Here, we show that: (a) thalidomide suppresses the expression of constitutive and transforming growth factor-beta1 (TGF-beta1)-induced uPAR mRNA and protein; (b) a nuclear factor kappaB (NF-kappaB) activation system (phosphorylation of IkappaB-alpha and degradation of IkappaB-alpha) is necessary for the TGF-beta1-induced increase in uPAR expression, because L-1-tosylamido-2-phenylethyl chloromethyl ketone, a NF-kappaB inhibitor, reduced the uPAR production as well as mRNA expression; (c) thalidomide failed to further strengthen L-1-tosylamido-2-phenylethyl chloromethyl ketone's action; (d) the once-daily i.p. administration of thalidomide (400 microg/g body weight/d) decreased progressive growth of HRA tumors and ascites formation in an in vivo animal model; and (e) the once-daily i.p. administration of thalidomide in combination with paclitaxel (i.p., 100 microg/20 g at days 2 and 5) significantly decreased progressive growth of HRA cells in a synergistic fashion. We conclude that thalidomide down-regulates constitutive and TGF-beta1-stimulated uPAR mRNA and protein expression possibly through suppression of NF-kappaB activation. Furthermore, combination therapy with thalidomide plus paclitaxel may be an effective way to markedly reduce i.p. tumor growth and ascites in ovarian cancer dissemination.

Suppression of Urokinase Expression and Invasion by a Soybean Kunitz Trypsin Inhibitor Are Mediated through Inhibition of Src-dependent Signaling Pathways

A soybean Kunitz trypsin inhibitor (KTI) interacts with cells as a negative modulator of the invasive cells. Using complementary pharmacological and genetic approaches, we provide novel findings regarding mechanisms by which KTI inhibits signaling pathways in ovarian cancer cells leading to invasion. Transforming growth factor-beta1 (TGF-beta1) directly activates Src kinase, which in turn activates ERK-phosphatidylinositol 3-kinase/Akt, the downstream targets of Src, for urokinase-type plasminogen activator (uPA) up-regulation in human ovarian cancer HRA cells. Preincubation of the HRA cells with KTI reduced the ability of TGF-beta1 to trigger the uPA expression at the gene level and at the protein level. To further elucidate the mechanism of the KTI-dependent suppressive effect of TGF-beta1-induced uPA expression and invasion, we investigated which signaling pathway transduced by KTI is responsible for this inhibitory effect. Here, we show that 1) KTI suppressed TGF-beta1-induced phosphorylation of Src, ERK1/2, and Akt by 40-60%; 2) KTI was insensitive to suppress the phosphorylation of ERK1/2 and Akt in the constitutively active (CA)-c-Src (Y529F) cells; 3) uPA expression was up-regulated in TGF-beta1-stimulated HRA cells and in unstimulated Y529F cells; 4) the addition of KTI reduced the TGF-beta1-induced increase of uPA gene and protein expression in the wild-type c-Src-transfected cells (in contrast, KTI could not inhibit uPA expression in the Y529F cells); and 5) CA-c-Src transfection resulted in a 2-fold increase in invasiveness, whereas KTI did not reduce invasion of the Y529F cells. Using additional complementary genetic approaches (CA-MEK1, CA-Akt, or kinase-dead-Akt), we conclude that KTI may suppress uPA expression and promotion of invasion possibly through one or more upstream targets of Src.

Bikunin down‐regulates heterodimerization between CD44 and growth factor receptors and subsequently suppresses agonist‐mediated signaling

We provided evidence previously that bikunin, a Kunitz-type protease inhibitor, can disrupt dimerization of CD44 proteins, which may result in suppression of receptor-mediated MAP kinase signaling. However, to what extent dimerization may alter ligand-induced signaling has not been documented. Given the recent recognition that some growth factor receptors can form heterodimers with CD44, the present study was undertaken to determine whether the CD44 and growth factor receptors (e.g., EGFR, FGFR, HGFR, VEGFR, TGF-betaRI, or TGF-betaRII) can form heterodimers in cancer cells and, if so, to investigate the potential functional consequences of such heterodimerization. We also examined whether bikunin can abrogate these heterodimerizations and inhibit CD44/growth factor-dependent signaling. Here, we show direct evidence for heterodimerization of CD44-FGFR and CD44-TGF-betaRI in human chondrosarcoma HCS-2/8 cells, CD44-EGFR complex in human glioma U87MG cells, and CD44-TGF-betaRI heterodimer in human ovarian cancer HRA cells. Coupling of CD44 and growth factor receptor may be selective, depending on a cell type. Bikunin does not alter the ligand binding, whereas functionally reduces heterodimerization between CD44 and growth factor receptors. The disruption of heterodimerization substantially reduces receptor-induced tyrosine phosphorylation and ERK1/2 activation. Taken together, our data suggest that bikunin-mediated suppression of heterodimerization between CD44 and growth factors may inhibit the agonist-promoted activation of the signaling pathway.

Suppressing effects of dietary supplementation of soybean trypsin inhibitor on spontaneous, experimental and peritoneal disseminated metastasis in mouse model.

The modifying effects of a Kunitz trypsin inhibitor (KTI) and a Bowman-Birk trypsin inhibitor (BBI), purified from soybean trypsin inhibitor, as dietary supplements on experimental and spontaneous pulmonary metastasis of murine Lewis lung carcinoma 3LL cells as well as peritoneal disseminated metastasis model in human ovarian cancer HRA cells were investigated in i.v., s.c. and i.p. injection models in mice. Seven groups of female C57BL/6 or nude mice were fed a basal diet (control group) or the basal diet supplemented with KTI or BBI (5, 15, or 50 g/kg). Here we show that, in an in vivo spontaneous metastasis assay, the diet supplementation with KTI (15 and 50 g/kg), but not with BBI, for 28 days immediately after s.c. tumor cell inoculation significantly inhibited the formation of lung metastasis in C57BL/6 mice in a dose-dependent manner. The inhibition of lung metastasis was not due to direct antitumor effects of KTI. In an in vivo experimental metastasis assay, the diet supplementation with KTI or BBI for 21 days after i.v. tumor cell inoculation did not reduce the number of lung tumor colonies. In addition, KTI (15 or 50 g/kg) treatment in a peritoneal disseminated metastasis model of HRA cells resulted in a 40% reduction in total tumor burden when compared with control animals. Immunoblot analysis revealed that KTI specifically reduced expression of uPA protein as well as phosphorylation of MAP kinase and PI3 kinase proteins in the cells stimulated with agonists (G-CSF for 3LL cells or TGF-beta1 for HRA cells). These results suggest that dietary supplementation of KTI more efficiently regulates the mechanism involved in the entry into vascular circulation of tumor cells (intravasation) than in extravasation during the metastatic process. KTI treatment may also be beneficial for ovarian cancer patients with or at risk for peritoneal disseminated metastasis; it greatly reduces tumor burden in part by inhibiting phosphorylation of MAP kinase and PI3 kinase, leading to suppression of uPA expression.

Inhibition of Tumor Invasion by Genomic Down-regulation of Matriptase through Suppression of Activation of Receptor-bound Pro-urokinase

Urokinase-type plasminogen activator (uPA) degrades the extracellular matrix and plays critical roles in tumor invasion and metastasis. Matriptase, a membrane-bound serine protease, was shown to activate uPA in a uPA receptor-free, solution-based study. We now investigate whether matriptase affects activation of receptor-bound uPA and contributes to the invasiveness of HRA human ovarian cancer cells in vitro and tumor behavior in nude mice. Here we show the following. 1) uPA expression was effectively stimulated by TGF-beta1 in HRA cells. 2) Antisense (AS)-matriptase transfection achieved a marked inhibition of receptor-bound pro-uPA activation without altering expression of uPA and uPA receptor mRNA and proteins, irrespective of whether cells were stimulated with TGF-beta1. 3) Tumor cell receptor-bound pro-uPA could be efficiently cleaved by matriptase to generate enzymatically active two-chain uPA. Thus, matriptase can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active uPA. 4) The AS-matriptase-treated cells had a decreased ability to invade an extracellular matrix layer, as compared with control cells. 5) When the AS-matriptase-treated cells were injected intraperitoneally into nude mice, the mice developed smaller tumors. Our data identify a novel role for matriptase for activation of receptor-bound uPA.

Bikunin plus paclitaxel markedly reduces tumor burden and ascites in mouse model of ovarian cancer.

Our previous studies of intraperitoneal ovarian carcinoma in a mouse model demonstrated that bikunin gene transfection could prevent ascites formation and intraperitoneal disseminated metastasis. Although ascites was almost completely inhibited, tumor burden was variably reduced. Several reports have indicated that bikunin may be involved in tumor survival. In the present study, the effectiveness of exogenous bikunin and the biodistribution characteristics of (125)I-bikunin were initially examined in a mouse model of human ovarian cancer HRA cells. The once-daily i.p. administration of bikunin significantly decreased progressive growth of HRA tumors and ascites formation in a dose-dependent manner. Maximal radioisotope tumor uptake peaked at 7.4% injected dose/g at 3 hr. Bikunin binding specificity was demonstrated by reduced tumor uptake after coinjection of excess nonradioactive bikunin. Bikunin was rapidly excreted renally. The bikunin therapy produced the significant inhibition in expression of the proteolysis (uPA and uPAR) and angiogenesis-related molecules (VEGF and bFGF). The second purpose of our study was to optimize the antimetastatic activity of bikunin in combination with paclitaxel against HRA cells growing orthotopically in mice. The once-daily i.p. administration of bikunin (25 microg/g body weight/day) in combination with paclitaxel (i.p., 100 microg/20 g at days 2 and 5) significantly decreased progressive growth of HRA cells in a synergistic fashion. In conclusion, combination therapy with bikunin plus paclitaxel may be an effective way to markedly reduce i.p. tumor growth and ascites in ovarian carcinoma possibly through suppression of uPA, uPAR, VEGF and bFGF expression.

Genetic Down-regulation of Phosphoinositide 3-Kinase by Bikunin Correlates with Suppression of Invasion and Metastasis in Human Ovarian Cancer HRA Cells

Using a cDNA microarray analysis, we previously found that exposure of a highly invasive ovarian cancer cell line HRA with bikunin, a Kunitz-type protease inhibitor, or bikunin gene overexpression markedly reduced phosphoinositide kinase (PI3K) p85 gene expression, demonstrating that PI3K may be a candidate bikunin target gene. To clarify how reduced levels of PI3K may confer repressed invasiveness, we transfected HRA cells with PI3K p85 antisense-oligodeoxynucleotide (AS-ODN) and compared the properties of the transfected cells with those of parental cells and sense (S)-ODN cells. We have also demonstrated previously that transforming growth factor-beta1 (TGF-beta1) stimulates urokinase-type plasminogen activator (uPA)-dependent invasion and metastasis of HRA cells. Here, we show that 1) TGF-beta1 induced a rapid increase of the PI3K activity that was accompanied by increased expression (5-fold) of the uPA mRNA; 2) pharmacological inhibition of PI3K or AS-PI3K ODN transfection inhibited TGF-beta1-stimulated Akt phosphorylation; 3) both PI3K pharmacological inhibitors and forced expression of AS-PI3K ODN reduced TGF-beta1-stimulated uPA mRNA and protein expression by approximately 70% compared with controls; 4) concentrations of PI3K inhibitors, sufficient to inhibit uPA up-regulation, inhibited TGF-beta1-dependent HRA cell invasion; 5) the AS-PI3K ODN cells had a decreased ability to invade the extracellular matrix layer as compared with controls; and 6) when the AS-PI3K ODN cells were injected intraperitoneally into nude mice, the mice developed smaller intraperitoneal tumors and showed longer survival. We conclude that PI3K plays an essential role in promoting uPA-mediated invasive phenotype in HRA cells. Our data identify a novel role for PI3K as a bikunin target gene on uPA up-regulation and invasion.

Transforming growth factor-beta1-dependent urokinase up-regulation and promotion of invasion are involved in Src-MAPK-dependent signaling in human ovarian cancer cells.

Urokinase-type plasminogen activator (uPA) has been implicated in tumor cell invasion and metastasis. We reported previously that transforming growth factor (TGF)-beta1 induces a dose- and time-dependent up-regulation of uPA mRNA and protein in highly invasive human ovarian cancer cell line HRA, leading to invasion. To further elucidate the mechanism of the invasive effect of TGF-beta1, we investigated which signaling pathway transduced by TGF-beta1 is responsible for this effect. Here, we show that 1) nontoxic concentrations of TGF-beta1 activated Src kinase; 2) TGF-beta1 rapidly phosphorylates ERK1/2 and Akt, but not p38; 3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment reduced TGF-beta1-induced phosphorylation of ERK1/2 and Akt by 85-90% compared with controls; 4) pharmacological inhibition of MAPK by PD98059 abrogated TGF-beta1-mediated Akt stimulation, whereas TGF-beta1-induced ERK1/2 stimulation was not inhibited by PI3K inhibitor LY294002 or AS-PI3K ODN transfection; 5) up-regulation of uPA mRNA in response to TGF-beta1 was almost totally blocked by PP2 and PD98059 and partially ( approximately 55%) by LY294002; 6) TGF-beta1-induced uPA mRNA up-regulation was inhibited by treatment with AS ODNs to c-Src or PI3K by 90 or 60%, respectively, compared with control ODN treatment; and 7) blockade of the release of the transcription factor NF-kappaB by pyrrolidinedithiocarbamate reduced the TGF-beta1-induced activation of the uPA gene by approximately 65%. In addition, curcumin, a blocker of the transcriptional factor AP-1, partially (35%) canceled this effect. Taken together, these data support a role for TGF-beta1 activation of two distinct pathways (Src-MAPK-PI3K-NF-kappaB-dependent and Src-MAPK-AP-1-dependent) for TGF-beta1-dependent uPA up-regulation and promotion of invasion.

Suppression of cell migration in ovarian cancer cells mediated by PTEN overexpression

Peritoneal dissemination is the major progression pathway of ovarian cancer, and its control is important for improvement of the prognosis. PTEN is a tumor suppressor gene, and is known to inhibit cancer cell growth and migration. To investigate the possibility of gene therapy using PTEN for ovarian cancer, we introduced PTEN cDNA into an ovarian cancer cell line HRA carrying wild-type PTEN, and examined the effects in vitro and in vivo. Using PTEN cDNA cloned from a human liver cDNA library, a PTEN expression vector was constructed. This vector was introduced into HRA cells by the standard calcium phosphate precipitation method, and an HRA cell line overexpressing PTEN (HRA/PTEN) was established. On the cell migration test by in vitro scratch wound healing assay, the number of migrating cells was 6.3+/-0.9 cells/mm(2) in HRA/PTEN, which was significantly smaller than that in the control (39.7+/-3.2 cells/mm(2)) (p<0.01). No significant differences were observed in the in vitro cell growth or in vivo tumor growth between HRA/PTEN and the control. The findings described above, show that enhanced expression of PTEN inhibits ovarian cancer cell migration, suggesting that gene therapy approaches using PTEN for control of peritoneal dissemination of ovarian cancer are possible.

Transforming Growth Factor-β1 Produced by Ovarian Cancer Cell Line HRA Stimulates Attachment and Invasion through an Up-regulation of Plasminogen Activator Inhibitor Type-1 in Human Peritoneal Mesothelial Cells

The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator (uPA) and its specific inhibitor (plasminogen activator inhibitor type-1 (PAI-1)) could be involved in the pathogenesis of peritoneal dissemination. We showed previously that expression of uPA and PAI-1 in the human ovarian cancer cell line HRA can be down-regulated by exogenous bikunin (bik), a Kunitz-type protease inhibitor, via suppression of transforming growth factor-beta1 (TGF-beta1) up-regulation and that overexpression of the bik gene can specifically suppress the in vivo growth and peritoneal dissemination of HRA cells in an animal model. We hypothesize that the plasminogen activator system in mesothelial cells can be modulated by HRA cells. To test this hypothesis, we used complementary techniques in mesothelial cells to determine whether uPA and PAI-1 expression are altered by exposure to culture media conditioned by HRA cells. Here we show the following: 1) that expression of PAI-1, but not uPA, was markedly induced by culture media conditioned by wild-type HRA cells but not by bik transfected clones; 2) that by antibody neutralization the effect appeared to be mediated by HRA cell-derived TGF-beta1; 3) that exogenous TGF-beta1 specifically enhanced PAI-1 up-regulation at the mRNA and protein level in mesothelial cells in a time- and concentration-dependent manner, mainly through MAPK-dependent activation mechanism; and 4) that mesothelial cell-derived PAI-1 may promote tumor invasion possibly by enhancing cell-cell interaction. This represents a novel pathway by which tumor cells can regulate the plasminogen activator system-dependent cellular responses in mesothelial cells that may contribute to formation of peritoneal dissemination of ovarian cancer.

Thymidine phosphorylase-mediated angiogenesis regulated by thymidine phosphorylase inhibitor in human ovarian cancer cells in vivo

Metastasis or progression of ovarian cancer cells is known to be due to the action of various angiogenic factors. We determined the expression of thymidine phosphorylase/platelet-derived endothelial cell growth factor (TP/PD-ECGF) and vascular endothelial growth factor (VEGF) in cell lines established from 3 serous adenocarcinomas, 3 clear cell carcinomas and 2 mucinous carcinomas of the human ovary. TP activity and the TP mRNA level were much higher in the serous adenocarcinoma cells than in the clear cells and mucinous carcinoma cells, and TP expression was extremely low in the clear cell carcinoma cells. Expression of VEGF mRNA was variable, but not significantly different between the 3 histological types of ovarian cancer. In vivo angiogenesis in the ovarian cancer cells was evaluated by the dorsal air sac assay and revealed that SHIN-3 and HRA serous adenocarcinoma cells, which have high levels of TP expression, induced angiogenesis, while KK clear cell carcinoma cells with low TP expression, did not. The degree of ovarian-cancer-induced angiogenesis seemed to be independent of expression of VEGF in the cells. To confirm that the serous adenocarcinoma-induced angiogenesis is dependent on TP levels, a potent and specific inhibitor of TP was administered orally to mice implanted with a chamber containing SHIN-3 or HRA cells. The TP inhibitor significantly inhibited the angiogenesis induced by the serous adenocarcinoma cells. These results suggest that the angiogenic potency of ovarian cancer cells differs with the histological type and is controlled by expression of TP/PD-ECGF, not by VEGF, and that TP-mediated angiogenesis may be the main factor responsible for progression or metastasis of ovarian serous adenocarcinomas.

Bikunin Target Genes in Ovarian Cancer Cells Identified by Microarray Analysis

Bikunin, a Kunitz-type protease inhibitor, could potentially suppress tumor cell invasion and metastasis. Our previous study revealed that overexpression of bikunin in a human ovarian cancer cell line, HRA, resulted in a down-regulation in uPA and uPAR gene expression. For identifying the full repertoire of bikunin-regulated genes, a cDNA microarray hybridization screening was conducted using mRNA from bikunin-treated or bikunin-transfected HRA cells. A number of bikunin-regulated genes were identified, and their regulation was confirmed by Northern blot analysis. Our screen identified 11 bikunin-stimulated genes and 29 bikunin-repressed genes. The identified genes can indeed be classified into distinct subsets. These include transcriptional regulators, oncogenes/tumor suppressor genes, signaling molecules, growth/cell cycle, invasion/metastasis, cytokines, apoptosis, ion channels, extracellular matrix proteins, as well as some proteases. This screen identified suppression of several genes such as CDC-like kinase, LIM domain binding, Ets domain transcription factor, Rho GTPase-activating protein, tyrosine phosphorylation-regulated kinase, hyaluronan-binding protein, matriptase, and pregnancy-associated plasma protein-A (PAPP-A), which have previously been implicated in enhancing tumor promotion. Northern blot analysis confirmed that several genes including matriptase and PAPP-A were down-regulated by bikunin by approximately 9-fold. Further, genetic inhibition of matriptase or PAPP-A could lead to diminished invasion. These results show that bikunin alters the pattern of gene expression in HRA cells leading to a block in cell invasion.

A Kunitz-type Protease Inhibitor, Bikunin, Inhibits Ovarian Cancer Cell Invasion by Blocking the Calcium-dependent Transforming Growth Factor-β1 Signaling Cascade

Bikunin is a Kunitz-type protease inhibitor, acting at the level of tumor invasion and metastasis. The goal of this study was to investigate the effect of bikunin-dependent signal transduction involved in the expression of a plasminogen activator (PA) system and invasion. We report here the following. 1) The human ovarian cancer cell line HRA produced secreted and cell-associated urokinase-type PA (uPA) and PA inhibitor type 1 (PAI-1). The plasma membrane of the cells showed enzymatically active uPA even in the presence of high level of PAI-1, as measured by zymography, Western blot, chromogenic assay, enzyme-linked immunosorbent assay, and Northern blot. 2) HRA cells leading to invasion are induced through up-regulation of uPA expression. 3) HRA cells specifically released transforming growth factor-beta type 1 (TGF-beta1) participating in an autocrine/paracrine regulation of cell invasion. 4) Elimination of endogenous TGF-beta1 could induce change in uPA/PAI-1 expression, which could in turn modify the invasive behavior of the cells. 5) The constitutive expression of TGF-beta1 as well as up-regulation of the PA system observed in HRA cells was inhibited by preinoculation of the cells with bikunin or calcium channel blocker SK&F 96365 but not with nifedipine or verapamil, with an IC(50) of approximately 100 nm for bikunin or approximately 30 microm for SK&F 96365, respectively, as measured by enzyme-linked immunosorbent assay. Bikunin showed no additive effect on SK&F 96365-mediated suppression of TGF-beta1 expression. 6) The ability of TGF-beta1 to elevate free intracellular Ca(2+), followed by activation of Src and ERK, was reduced by preincubation of the cells with bikunin. In conclusion, bikunin could inhibit the constitutive expression of TGF-beta1 and TGF-beta1-mediated, Src- and ERK-dependent, PA system signaling cascade, at least in part, through inhibition of a non-voltage-sensitive calcium channel.

Suppression of invasion and peritoneal carcinomatosis of ovarian cancer cell line by overexpression of bikunin

Bikunin (bik), a Kunitz-type protease inhibitor, also known as urinary trypsin inhibitor, is proposed as a main participant in the inhibition of tumor cell invasion and metastasis, possibly through the direct inhibition of cell-associated plasmin activity and suppression of urokinase-type plasminogen activator (uPA) mRNA expression. In the present study, we transfected the human ovarian carcinoma cell line HRA, highly invasive cells, with an expression vector harboring a cDNA encoding for human bik. Our study was designed to investigate the effect of bik overexpression and changes in tumor cell phenotype and invasiveness in the stably transfected clones. Bik gene transfection of HRA gave the following results: 1) transfection of HRA with the bik cDNA resulted in 5 variants stably expressing functional bik; 2) bik(+) clones exhibited a significantly reduced uPA mRNA expression as compared to the parental cells; 3) bikunin negatively regulates the ERK1/2 activity; 4) secretion-blocking treatments of bik(+) clones abrogated bik-mediated suppression of ERK1/2 activation and uPA expression; 5) the regulation of invasion seen in the HRA cells is mainly mediated by the uPA-plasmin-MMP-2 system; 6) transfection of HRA with the bik gene significantly reduced invasion, but not proliferation, adhesion, or migration relative to the parental cells; and 7) animals with bik(+) clones induced reduced peritoneal dissemination and long term survival. We conclude that transfection of HRA cells with the bik cDNA constitutively suppresses ERK1/2 activation, which results in inhibition of uPA expression and subsequently reduces dissemination of bik(+) clones.