u-0126 and Ovarian-Neoplasms

Recent studies have revealed that long non‑coding RNAs (lncRNAs) serve important roles in carcinogenesis and that this type of gene may be used as biomarkers in cancer. A high level of lncRNA HOXA distal transcript antisense RNA (HOTTIP) is associated with unfavorable prognosis for patients with ovarian cancer (OC), but the mechanism of HOTTIP involved in OC development remains to be elucidated. The present study aimed to investigate the mechanism of HOTTIP in metastasis‑associated OC cell behaviors. HOTTIP levels in ovarian cells were quantified by reverse transcription‑quantitative PCR, cell proliferation was analyzed by colony formation assay, and apoptosis was assessed by flow cytometry. Cell migratory and invasive abilities were evaluated by wound healing and Transwell assays, respectively. The expression levels of mitogen‑activated protein kinase kinase (MEK)/ERK pathway‑associated proteins were detected by western blotting. The results demonstrated that knockdown of HOTTIP in OC cells significantly reduced the phosphorylation levels of MEK and ERK, inhibited the proliferation and invasion of OC cells and promoted their apoptosis. Furthermore, the effects of HOTTIP on cell migration and invasion were partly associated with the epithelial‑mesenchymal transition (EMT) process. Proliferation, invasion and EMT of OC cells were enhanced following overexpression of HOTTIP; however, these effects were reversed by the MEK/ERK pathway inhibitor U0126. In conclusion, HOTTIP was demonstrated to promote the proliferation, migration and invasion of OC cells by activating the MEK/ERK pathway. Therefore, HOTTIP may serve as a potential therapeutic target for OC.

Topics: Apoptosis; Biomarkers, Tumor; Butadienes; Cell Line, Tumor; Cell Movement; Cell Proliferation; Enzyme Inhibitors; Epithelial-Mesenchymal Transition; Female; Gene Knockdown Techniques; Humans; MAP Kinase Signaling System; Neoplasm Invasiveness; Nitriles; Ovarian Neoplasms; Phosphorylation; RNA, Long Noncoding; Transfection

As widely used in consumer products, perfluorooctanoic acid (PFOA) has become a common environmental pollutant, which has been detected in human serum and associated with cancers. Our previous study showed that PFOA is a carcinogen that promotes endometrial cancer cell migration and invasion through activation of ERK/mTOR signaling. Here, we showed that PFOA (≥100 nM) treatment also stimulated A2780 ovarian cancer cell invasion and migration, which correlated with increased matrix metalloproteinases MMP-2/-9 expression, important proteases associated with tumor invasion and migration. Notably, PFOA treatment induced activation of ERK1/2/ NF-κB signaling. Pre-treatment with U0126, an ERK1/2inhibitor；or JSH-23, a NF-kB inhibitor, can reverse the PFOA-induced cell migration and invasion. Consistent with these results, inhibiting ERK1/2 or NF-κB signaling abolished PFOA-induced up-regulation of MMP-2/-9 expression. These results indicate that PFOA can stimulate ovarian cancer cell migration, invasion and MMP-2/-9 expression by up-regulating ERK/NF-κB pathway.

Topics: Active Transport, Cell Nucleus; Apoptosis; Butadienes; Caprylates; Carcinogens, Environmental; Cell Line, Tumor; Cell Movement; Enzyme Induction; Female; Fluorocarbons; Humans; Kinetics; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Proteins; NF-kappa B; Nitriles; Ovarian Neoplasms; Phenylenediamines; Phosphorylation; Protein Kinase Inhibitors; Protein Processing, Post-Translational

Formononetin is an isoflavone that is extracted from red clovers or soy. It has anti-oxidant, anti-proliferative, and anti-tumor effects against cells in various diseases. Several cohort studies have indicated that phytoestrogen intake, including formononetin, could reduce the risk of various carcinogenesis. In fact, many case-control studies have indicated the potential value of flavonoids as drug supplements in the treatment of many cancer patients. However, the toxic effects and the anti-cancer mechanism of formononetin in ovarian cancer are unknown. We investigated the toxicological mechanism of formononetin in ES2 and OV90 ovarian cancer cells. Formononetin suppressed cell proliferation through sub G0/G1 phase arrest and increased apoptosis in both cell lines. Furthermore, it induced loss of mitochondrial membrane potential and generation of reactive oxygen species in ES2 and OV90 cells. The formononetin-mediated regulation of cell proliferation and apoptosis involved decreased phosphorylation of ERK1/2, P90RSK, AKT, P70S6K, and S6 proteins, and increased phosphorylation of P38 protein in ES2 and OV90 cells. Co-treatment of formononetin with pharmacological inhibitors (LY294002 or U0126) revealed additional anti-proliferative effects on the two human ovarian cancer cell types. Conclusively, the results indicate the potential value of formononetin as an anti-cancer agent in human ovarian cancer.

Topics: Analysis of Variance; Antineoplastic Agents; Apoptosis; Butadienes; Cell Line, Tumor; Cell Proliferation; Chromones; Drug Synergism; Enzyme Inhibitors; Female; G1 Phase; Humans; Imidazoles; Isoflavones; MAP Kinase Signaling System; Matrix Metalloproteinases; Membrane Potential, Mitochondrial; Morpholines; Nitriles; Ovarian Neoplasms; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyridines; Reactive Oxygen Species; Resting Phase, Cell Cycle; Signal Transduction

Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme involved in tumor malignancy. However, the regulatory mechanism underlying its involvement remains largely uncharacterized. The present study aimed to investigate the hypothesis that NK4, an antagonist of hepatocyte growth factor (HGF), can regulate IDO and to characterize the signaling mechanism involved. Following successful transfection of the human ovarian cancer cell line SKOV-3 (which constitutively expresses IDO) with an NK4 expression vector, we observed that NK4 expression suppressed IDO expression; furthermore, NK4 expression did not suppress cancer cell growth in vitro [in the absence of natural killer (NK) cells], but did influence tumor growth in vivo. In addition, NK4 enhanced the sensitivity of cancer cells to NK cells in vitro and promoted NK cell accumulation in the tumor stroma in vivo. In an effort to clarify the mechanisms by which NK4 interacts with IDO, we performed investigations utilizing various biochemical inhibitors. The results of these investigations were as follows. First, c-Met (a receptor of HGF) tyrosine kinase inhibitor PHA-665752, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 both suppress IDO expression. Second, enhanced expression of PTEN (a known tumor suppressor) via negative regulation within a PI3K-AKT pathway, inhibits IDO expression. Conversely, neither the MEK1/2 inhibitor U0126 nor the STAT3 inhibitor WP1066 affects IDO expression. These results suggest that NK4 inhibits IDO expression via a c-Met-PI3K-AKT signaling pathway.

Topics: Animals; Butadienes; Cell Line, Tumor; Chromones; Female; Hepatocyte Growth Factor; Heterografts; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Indoles; Mice; Mice, Inbred BALB C; Mice, Nude; Morpholines; Nitriles; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-met; PTEN Phosphohydrolase; Pyridines; Signal Transduction; Sulfones; Tyrphostins

Notch3 is implicated in chemoresistance of ovarian cancer, yet the molecular mechanism underlying Notch3-mediated drug resistance remains to be elucidated. Here, we investigated the role of Notch3 in carboplatin-induced apoptosis in ovarian cancer cells.. Ovarian cancer cell line OVCA429 cells were stably transduced with an empty vector or a retroviral vector expressing the Notch3 intracellular domain (NICD3, the constitutively active form of Notch3) to generate OVCA429/vector and OVCA429/NICD3 cells. Epithelial-mesenchymal transition (EMT) was determined by morphological change and expression of the EMT markers. Carboplatin-induced cytotoxicity was determined by the neutral red uptake assay. Apoptosis was determined by Annexin V staining and Western blotting. Carboplatin-induced phosphorylation of extracellular signal-regulated kinase (ERK) was identified by a phospho-kinase array and confirmed by Western blotting.. Activation of Notch3 in OVCA429 cells causes a spindle and fibroblast-like morphology, induces the expression of smooth muscle α-actin, Slug and Snail, but decreases the expression of E-cadherin, indicating that Notch3 activation induces EMT in OVCA429 cells. Furthermore, Notch3 activation renders OVCA429 cells more resistant to carboplatin-induced cytotoxicity and attenuates carboplatin-induced apoptosis in these cells. Our results indicate that phosphorylation of ERK is a positive regulator of carboplatin-induced apoptosis in OVCA429 cells. Interestingly, carboplatin-induced ERK phosphorylation is inhibited by Notch3 activation.. Notch3 activation induces EMT and attenuates carboplatin-induced apoptosis in OVCA429 cells. ERK phosphorylation plays a pro-apoptotic role in carboplatin-induced apoptosis in OVCA429 cells. Interestingly, Notch3 activation attenuates carboplatin-induced ERK phosphorylation in these cells.

Topics: Antineoplastic Agents; Apoptosis; Butadienes; Carboplatin; Cell Line, Tumor; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Ovarian Neoplasms; Phosphorylation; Protein Kinase Inhibitors; Receptor, Notch3; Receptors, Notch

Ovarian cancer is a leading cause of cancer death in women in the United States. While the majority of ovarian cancers are serous, some rarer subtypes (i.e. clear cell) are often associated with endometriosis, a benign gynecological disease. Iron is rich in the cyst fluid of endometriosis-associated ovarian cancers and induces persistent oxidative stress. The role of iron, an essential nutrient involved in multiple cellular functions, in normal ovarian cell survival and ovarian cancer remains unclear. Iron, presented as ferric ammonium citrate (FAC), dramatically inhibits cell survival in ovarian cancer cell types associated with Ras mutations, while it is without effect in immortalized normal ovarian surface epithelial (T80) and endometriotic epithelial cells (lacking Ras mutations). Interestingly, FAC induced changes in cytoplasmic vacuolation concurrently with increases in LC3-II levels (an autophagy marker); these changes occurred in an ATG5/ATG7-dependent, beclin-1/hVps34-independent, and Ras-independent manner. Knockdown of autophagy mediators in HEY ovarian cancer cells reversed FAC-induced LC3-II levels, but there was little effect on reversing the cell death response. Intriguingly, transmission electron microscopy of FAC-treated T80 cells demonstrated abundant lysosomes (confirmed using Lysotracker) rich in iron particles, which occurred in a Ras-independent manner. Although the mitogen-activated protein kinase (MAPK) inhibitor, U0126, reversed FAC-induced LC3-II/autophagic punctae and lysosomes in a Ras-independent manner, it was remarkable that U0126 reversed cell death in malignant ovarian cells associated with Ras mutations. Moreover, FAC increased heme oxygenase-1 expression in H-Ras-overexpressing T80 cells, which was associated with increased cell death when overexpressed in T80 cells. Disruption of intracellular iron levels, via chelation of intracellular iron (deferoxamine), was also detrimental to malignant ovarian cell survival; thus, homeostatic intracellular iron levels are essential for cell survival. Collectively, our results implicate iron in modulating cell death in a Ras- and MAPK-dependent manner in ovarian cancer cells.

Topics: Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Beclin-1; Butadienes; Cell Line, Tumor; Cell Survival; Class III Phosphatidylinositol 3-Kinases; Female; Ferric Compounds; Heme Oxygenase-1; Humans; Lysosomes; Membrane Proteins; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinases; Nitriles; Ovarian Neoplasms; Quaternary Ammonium Compounds; ras Proteins; Ubiquitin-Activating Enzymes

Transcriptional co-activator with PDZ-binding motif (TAZ), a downstream effector of the Hippo pathway, has been reported to regulate organ size, tissue homeostasis, and tumorigenesis by acting as a transcriptional co-activator. Lysophosphatidic acid (LPA) is a bioactive lipid implicated in tumorigenesis and metastasis of ovarian cancer through activation of G protein-coupled receptors. However, the involvement of TAZ in LPA-induced tumorigenesis of ovarian cancer has not been elucidated.. In order to demonstrate the role of TAZ in LPA-stimulated tumorigenesis, the effects of LPA on TAZ expression and cell migration were determined by Western blotting and chemotaxis analyses in R182 human epithelial ovarian cancer cells.. Treatment of R182 cells with the LPA receptor inhibitor Ki16425 blocked LPA-induced cell migration. In addition, transfection of R182 cells with small interfering RNA specific for LPA receptor 1 resulted in abrogation of LPA-stimulated cell migration. LPA induced phosphorylation of ERK and p38 MAP kinase in R182 cells and pretreatment of cells with the MEK-ERK pathway inhibitor U0126, but not the p38 MAPK inhibitor SB202190, resulted in abrogation of LPA-induced cell migration. Pretreatment of R182 cells with U0126 attenuated LPA-induced mRNA levels of TAZ and its transcriptional target genes, such as CTGF and CYR61, without affecting phosphorylation level of YAP. These results suggest that MEK-ERK pathway plays a key role in LPA-induced cell migration and mRNA expression of TAZ in R182 cells, without affecting stability of TAZ protein. In addition, small interfering RNA-mediated silencing of TAZ expression attenuated LPA-stimulated migration of R182 cells. These results suggest that TAZ plays a key role in LPA-stimulated migration of epithelial ovarian cancer cells.

Topics: Butadienes; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cells, Cultured; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Isoxazoles; Lysophospholipids; Neoplasms, Glandular and Epithelial; Nitriles; Ovarian Neoplasms; Propionates; Protein Stability; Trans-Activators; Transcription Factors; Transcriptional Coactivator with PDZ-Binding Motif Proteins

Obesity is known to be an important risk factor for many types of cancer, such as breast, prostate, liver and endometrial cancer. Recently, epidemiological studies have indicated that obesity correlates with an increased risk of developing ovarian cancer, the most lethal gynecological cancer in developed countries. Leptin is predominantly produced by adipocytes and acts as a growth factor and serum leptin levels positively correlate with the amount of body fat. In this study, we investigated the effects of leptin on the growth of ovarian cancer cells and the underlying mechanism(s) of action. Our results showed that leptin stimulated the growth of the OVCAR-3 ovarian cancer cell line using MTT assay and trypan blue exclusion. Using western blot analysis, we found that leptin enhanced the expression of cyclin D1 and Mcl-1, which are important regulators of cell proliferation and the inhibition of apoptosis. To investigate the signaling pathways that mediate the effects of leptin, cells were treated with leptin plus specific inhibitors of JAK2, PI3K/Akt and MEK/ERK1/2 and analysis of the phosphorylation state of proteins was carried out by western blot assays. We showed that the activation of the MEK/ERK1/2 and PI3K/Akt signaling pathways were involved in the growth-stimulating effect of leptin on ovarian cancer cell growth and the specific inhibitors of PI3K/Akt and MEK/ERK1/2 revealed that these two pathways interacted with each other. Our data demonstrate that leptin upregulates the expression of cyclin D1 and Mcl-1 to stimulate cell growth by activating the PI3K/Akt and MEK/ERK1/2 pathways in ovarian cancer.

Topics: Apoptosis; Butadienes; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Flavonoids; Humans; Janus Kinase 2; Leptin; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Myeloid Cell Leukemia Sequence 1 Protein; Nitriles; Obesity; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Tyrphostins

RAB25 belongs to the Rab family of small GTPases and is implicated in the development of various types of human cancer. To evaluate the role of RAB25 in ovarian cancer, RAB25 was knocked down by siRNA in HEY and ES‑2 human ovarian cancer cells. Autophagy, cell growth and cell apoptosis were evaluated. The results showed that knockdown of RAB25 increased acidic vesicle organelles and GFP-microtubule-associated protein 1 light chain 3 punctate fluorescence in ovarian cancer cells. Autophagy that promoted by knockdown of RAB25 was not observed in cells where the ERK1/2 signaling pathway had been inhibited by U0126. Knockdown of RAB25 reduced cell cycle progression and cell growth. Apoptosis of ovarian cancer cells could be induced by knockdown of RAB25. These results support the tumorigenic role of RAB25 in ovarian cancer cells.

Topics: Apoptosis; Autophagy; Butadienes; Cell Line, Tumor; Cell Proliferation; Female; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Ovarian Neoplasms; rab GTP-Binding Proteins; RNA Interference; RNA, Small Interfering; Signal Transduction

Cisplatin is the first-line chemotherapy for the treatment of several cancers. However, the development of cisplatin resistance represents a major clinical problem, and the mechanisms of acquired resistance are not fully understood. Here we show that degradation of the Bcl-2 homology 3-only proapoptotic protein Bim plays an important role in cisplatin resistance in ovarian cancer. Specifically, we show that treatment of ovarian cancer cells with cisplatin caused Bim phosphorylation and subsequent degradation and that its degradation is associated with cisplatin resistance. We also show that cisplatin treatment caused the activation of ERK, which correlated with Bim phosphorylation and degradation. By inhibiting ERK phosphorylation with the MEK inhibitor and knocking down ERK expression with siRNA, we show that Bim phosphorylation and degradation were blocked, which suggests that Bim is phosphorylated by ERK and that such phosphorylation is responsible for cisplatin-induced Bim degradation. We show that ERK was activated in cisplatin-resistant OV433 cells as compared with their counterpart parental OV433 cells. We also show that Bim was phosphorylated and degraded in cisplatin-resistant OV433 cells but not in the parental OV433 cells. Importantly, we show that inhibition of Bim degradation by the proteasome inhibitor MG132 sensitized resistant OV433 cells to cisplatin-induced death. Taken together, our data indicate that degradation of Bim via ERK-mediated phosphorylation can lead to cisplatin resistance. Therefore, these findings suggest that cisplatin resistance can be overcome by the combination of cisplatin and the proteasome inhibitors in ovarian cancer cells.

Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Butadienes; Cell Death; Cell Line, Tumor; Cell Proliferation; Cisplatin; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Female; Gene Knockdown Techniques; Humans; Membrane Proteins; Mitogen-Activated Protein Kinases; Nitriles; Ovarian Neoplasms; Phosphorylation; Proteasome Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins; RNA, Small Interfering

Epithelial mesenchymal transition (EMT) and cancer stem cells (CSC) have been associated with resistance to chemotherapy. Eighty percent of ovarian cancer patients initially respond to platinum-based combination therapy but most return with recurrence and ultimate demise. To better understand such chemoresistance we have assessed the potential role of EMT in tumor cells collected from advanced-stage ovarian cancer patients and the ovarian cancer cell line OVCA 433 in response to cisplatin in vitro. We demonstrate that cisplatin-induced transition from epithelial to mesenchymal morphology in residual cancer cells correlated with reduced E-cadherin, and increased N-cadherin and vimentin expression. The mRNA expression of Snail, Slug, Twist, and MMP-2 were significantly enhanced in response to cisplatin and correlated with increased migration. This coincided with increased cell surface expression of CSC-like markers such as CD44, α2 integrin subunit, CD117, CD133, EpCAM, and the expression of stem cell factors Nanog and Oct-4. EMT and CSC-like changes in response to cisplatin correlated with enhanced activation of extracellular signal-regulated kinase (ERK)1/2. The selective MEK inhibitor U0126 inhibited ERK2 activation and partially suppressed cisplatin-induced EMT and CSC markers. In vivo xenotransplantation of cisplatin-treated OVCA 433 cells in zebrafish embryos demonstrated significantly enhanced migration of cells compared to control untreated cells. U0126 inhibited cisplatin-induced migration of cells in vivo, suggesting that ERK2 signaling is critical to cisplatin-induced EMT and CSC phenotypes, and that targeting ERK2 in the presence of cisplatin may reduce the burden of residual tumor, the ultimate cause of recurrence in ovarian cancer patients.

Topics: Butadienes; Cell Line, Tumor; Cisplatin; Epithelial-Mesenchymal Transition; Female; Humans; Matrix Metalloproteinase 2; Mitogen-Activated Protein Kinase 1; Neoplasms, Glandular and Epithelial; Neoplastic Stem Cells; Nitriles; Nuclear Proteins; Ovarian Neoplasms; Snail Family Transcription Factors; Transcription Factors; Tumor Cells, Cultured; Twist-Related Protein 1

Forkhead box M1 (FOXM1) is a proliferation-associated transcription factor essential for cell cycle progression. Numerous studies have documented that FOXM1 has multiple functions in tumorigenesis and its elevated levels are frequently associated with cancer progression. Here, we characterized the role of ERK/FOXM1 signaling in mediating the metastatic potential of ovarian cancer cells. Immunohistochemical (IHC), immunoblotting and semi-quantitative RT-PCR analyses found that both phospho-ERK and FOXM1 were frequently upregulated in ovarian cancers. Intriguingly, the overexpressed phospho-ERK (p<0.001) and FOXM1 (p<0.001) were significantly correlated to high-grade ovarian tumors with aggressive behavior such as metastasized lymph node (5 out of 6). Moreover, the expressions of phospho-ERK and FOXM1 had significantly positive correlation (p<0.001). Functionally, ectopic expression of FOXM1B remarkably enhanced cell migration/invasion, while FOXM1C not only increased cell proliferation but also promoted cell migration/invasion. Conversely, inhibition of FOXM1 expression by either thiostrepton or U0126 could significantly impair FOXM1 mediated oncogenic capacities. However, the down-regulation of FOXM1 by either thiostrepton or U0126 required the presence of p53 in ovarian cancer cells. Collectively, our data suggest that over-expression of FOXM1 might stem from the constitutively active ERK which confers the metastatic capabilities to ovarian cancer cells. The impairment of metastatic potential of cancer cells by FOXM1 inhibitors underscores its therapeutic value in advanced ovarian tumors.

Topics: Blotting, Western; Butadienes; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Nitriles; Ovarian Neoplasms; Phosphoproteins; Protein Isoforms; Receptors, Urokinase Plasminogen Activator; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Thiostrepton; Tumor Suppressor Protein p53

Hepatocyte growth factor (HGF) plays an important role in stimulating tumor invasion and metastasis of tumor cells. However, the exact mechanisms of HGF regulation of cell invasion are largely unknown. In this study, we demonstrated that the MAPK specific inhibitor U0126 prevented the HGF-induced invasion of SKOV-3 cells. HGF stimulation of cell invasion and enhancement of MMP-9 expression were partially suppressed by TSP-1 overexpression. Furthermore, the MAPK signaling pathway is predominantly responsible for HGF-induced TSP-1 downregulation. We conclude that HGF is a potential downregulator of TSP-1, through MAPK signaling pathways, leading to the induction of MMP-9 expression and subsequent invasion of SKOV-3 cells. This study enhances our understanding of the mechanisms of signaling transduction in the HGF-induced invasion and progression of ovarian cancer.

Topics: Adenocarcinoma; Butadienes; Cell Line, Tumor; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Proteins; Nitriles; Ovarian Neoplasms; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Thrombospondin 1; Transfection

Aberrant overexpression of growth factor receptor-bound protein 7 (GRB7) and its variant GRB7v has been found in numerous human cancers. The goal of this study was to characterize the functions of GRB7 and GRB7v in the ovarian carcinogenesis and to investigate the differential roles of GRB7 and GRB7v in the modulation of signaling pathways.. Quantitative reverse transcription-PCR, Western blot, and immunohistochemical analyses were used to evaluate the levels of GRB7 and GRB7v. The cellular localization, functions, and signaling pathways regulated by GRB7 and GRB7v were investigated by enforced expression of GRB7 and GRB7v.. Quantitative reverse transcription-PCR and Western blot analyses showed that GRB7 and GRB7v were frequently upregulated in ovarian cancer samples. The overexpressed GRB7 (P = 0.009) and GRB7v (P = 0.017) were significantly correlated with high-grade ovarian cancer. Immunohistochemical analysis on ovarian cancer tissue array confirmed that the upregulated GRB7 was significantly correlated with high-grade ovarian cancer (P = 0.001). Confocal microscopy analysis showed that GRB7 and GRB7v predominately localized in cytoplasm of ovarian cancer cells, consistent with their roles as signaling adaptors. Enforced expression of GRB7 promoted cell proliferation, migration, and invasion, whereas GRB7v only increased cell proliferation and anchorage-independent growth ability. With the treatment of specific kinase inhibitors, we showed that both GRB7 and GRB7v promoted cell proliferation through activating extracellular signal-regulated kinase signaling, whereas GRB7 enhanced cell migration/invasion by activating c-Jun NH(2) terminal kinase signaling.. Our studies implicate that the overexpressed GRB7 and GRB7v are associated with high-grade tumors and exert distinct tumorigenic functions through regulating different signaling pathways in ovarian cancer cells.

Topics: Alternative Splicing; Anthracenes; Blotting, Western; Butadienes; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Female; Gene Expression Regulation, Neoplastic; GRB7 Adaptor Protein; Green Fluorescent Proteins; Humans; Immunohistochemistry; Microscopy, Confocal; Middle Aged; Mitogen-Activated Protein Kinases; Neoplasm Staging; Nitriles; Ovarian Neoplasms; Protein Isoforms; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transfection

Lysophosphatidic acid (LPA) is a bioactive phospholipids and involves in various cellular events, including tumor cell migration. In the present study, we investigated LPA receptor and its transactivation to EGFR for cyclooxygenase-2 (COX-2) expression and cell migration in CAOV-3 ovarian cancer cells. LPA induced COX-2 expression in a dose-dependent manner, and pretreatment of the cells with pharmacological inhibitors of Gi (pertussis toxin), Src (PP2), EGF receptor (EGFR) (AG1478), ERK (PD98059) significantly inhibited LPA- induced COX-2 expression. Consistent to these results, transfection of the cells with selective Src siRNA attenuated COX-2 expression by LPA. LPA stimulated CAOV-3 cell migration that was abrogated by pharmacological inhibitors and antibody of EP2. Higher expression of LPA2 mRNA was observed in CAOV-3 cells, and transfection of the cells with a selective LPA2 siRNA significantly inhibited LPA-induced activation of EGFR and ERK, as well as COX-2 expression. Importantly, LPA2 siRNA also blocked LPA-induced ovarian cancer cell migration. Collectively, our results clearly show the significance of LPA2 and Gi/Src pathway for LPA-induced COX-2 expression and cell migration that could be a promising drug target for ovarian cancer cell metastasis.

Topics: Butadienes; Cell Line, Tumor; Cell Movement; CSK Tyrosine-Protein Kinase; Cyclooxygenase 2; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Flavonoids; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Lysophospholipids; Nitriles; Ovarian Neoplasms; Pertussis Toxin; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Pyrimidines; Quinazolines; Receptors, Lysophosphatidic Acid; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP2 Subtype; Signal Transduction; src-Family Kinases; Transcriptional Activation; Tyrphostins

The aim of this study was to elucidate the role of ERK1/2 on cisplatin resistance in human ovarian cancer cells.. The relationship between nuclear levels of ERK2 and cisplatin-induced apoptosis in human ovarian carcinoma cell line, OVCAR-3, and in cells of the cisplatin-resistant subclone, OVCAR-3/CDDP, was examined using immunoblotting and immunocytochemistry.. Cisplatin treatment resulted in the activation of ERK2, both in OVCAR-3 and OVCAR-3/CDDP cells. However, considerable levels of activated ERK2 existed in the nuclei of OVCAR-3/CDDP cells during serum starvation and in the early period (1-3 h) after cisplatin treatment. Conversely, phospho-ERK2 was marginally detected in the nuclei of OVCAR-3 cells prior to cisplatin treatment. These phenomena were confirmed by immunofluorescence staining of the phosphorylated ERK2 in the nuclei of both cells. High basal phospho-ERK2 in the nuclei of OVCAR-3/CDDP cells contributed to cisplatin resistance, and was supported by several observations; (1) treatment of U0126, an inhibitor of MEK/ERK signaling pathway, partially sensitized OVCAR-3/CDDP cells to cisplatin; (2) pretreatment of OVCAR-3 cells with phorbol 12-myristate 13-acetate (PMA), an activator of ERK, induced nuclear translocation of activated ERK2, which led to the suppression of cisplatin-induced apoptosis.. These results collectively indicate that prelocalization of activated ERK2 in the nuclei contribute to cisplatin resistance in OVCAR-3/CDDP cells.

Topics: Antineoplastic Agents; Apoptosis; Butadienes; Caspase 3; Cell Line, Tumor; Cell Nucleus; Cisplatin; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Female; Humans; Mitogen-Activated Protein Kinase 1; Nitriles; Ovarian Neoplasms; Phosphorylation; Tetradecanoylphorbol Acetate

Synuclein-gamma is aberrantly expressed in more than 70% of stage III/IV breast and ovarian carcinomas. Ectopic overexpression of synuclein-gamma enhanced MDA-MB-435 cell migration in vitro and metastasis in a nude mouse model. However, the mechanism of how synuclein-gamma promotes cell motility is not clear. In our previous studies, we showed that synuclein-gamma overexpression activates ERK. In the present study, we overexpressed synuclein-gamma in several breast and ovarian cancer cell lines and evaluated the effect of synuclein-gamma on the activity of small G-protein RHO family members. We found that at least one of the RHO/RAC/CDC42 GTPases showed a higher level of the GTP-bound active form. Consistent with their role in regulating the intracellular motile machinery, inhibition of the RHO/RAC/CDC42 by C. difficile Toxin B blocked cell migration in both parental cells and synuclein-gamma overexpressing cells. The ERK inhibitor U0126 also blocked the cell migration in both parental cells and synuclein-gamma overexpressing cells. Collectively, our data indicate that synuclein-gamma might be involved in late stage breast and ovarian cancer metastasis by enhancing cell motility through activation of the RHO family small-GTPases and ERK.

Topics: Bacterial Proteins; Bacterial Toxins; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Movement; Extracellular Signal-Regulated MAP Kinases; Female; gamma-Synuclein; Humans; Nitriles; Ovarian Neoplasms; Protein Kinase Inhibitors; rho GTP-Binding Proteins; Transcriptional Activation; Transfection

A combination of paclitaxel (Taxol) and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK/Erk) inhibitor represents a rational new approach to chemotherapy. We performed Affymetrix microarray analysis to understand the global effects of this combination in lung carcinoma. Genes involved in cell cycle control, apoptosis, adhesion, proliferation, invasion, and metastasis were modulated. We observed similar patterns of gene modulation in ovarian and melanoma cell lines, indicating the general applicability of these findings. Functional genomic analysis identified two genes as new targets of drug-induced tumor apoptosis. The MGSA/Gro1 gene, important in melanoma growth, was induced by paclitaxel and reduced by MEK inhibition. Blockage of paclitaxel-induced melanoma growth stimulatory activity significantly reduced melanoma growth. Additionally, the expression of topoisomerase III beta, which exhibited a clear pattern of gene reduction by a combination of the two drugs, was significantly increased (5.7-fold) in primary lung cancers but not adjacent tissues. These findings provide potential new biomarkers and gene targets for the development of improved cancer treatment.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Butadienes; Chemokine CXCL1; Chemokines; Chemokines, CXC; Chemotactic Factors; Dactinomycin; Enzyme Inhibitors; Female; Gene Expression Profiling; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Melanoma; Mitogen-Activated Protein Kinase Kinases; Nitriles; Oligonucleotide Array Sequence Analysis; Ovarian Neoplasms; Paclitaxel

Our laboratory showed that bikunin, a Kunitz-type protease inhibitor, suppresses 4beta-phorbol 12-myristate 13-acetate (PMA)- or tumor necrosis factor-alpha (TNFalpha)-induced urokinase-type plasminogen activator (uPA) expression in different cell types. In addition to its effects on protease inhibition, bikunin could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we examined whether bikunin was able to suppress the expression of uPA receptor (uPAR) mRNA and protein in a human chondrosarcoma cell line, HCS-2/8, and two human ovarian cancer cell lines, HOC-I and HRA. The present study showed that (a) bikunin suppresses the expression of constitutive and PMA-induced uPAR mRNA and protein in a variety of cell types; (b) an extracellular signal-regulated kinase (ERK) activation system is necessary for the PMA-induced increase in uPAR expression, as PD098059 and U0126, which prevent the activation of MEK1, reduce the uPAR expression; (c) bikunin markedly suppresses PMA-induced phosphorylation of ERK1/2 at the concentration that prevents uPAR expression, but does not reduce total ERK1/2 antigen level; (d) bikunin has no ability to inhibit overexpression of uPAR in cells treated with sodium vanadate; and (e) we further studied the inhibition of uPAR expression by stable transfection of HRA cells with bikunin gene, demonstrating that bikunin secretion is necessary for inhibition of uPAR expression. We conclude that bikunin downregulates constitutive and PMA-stimulated uPAR mRNA and protein possibly through suppression of upstream targets of the ERK-dependent cascade, independent of whether cells were treated with exogenous bikunin or transfected with bikunin gene.

Topics: Bone Neoplasms; Butadienes; Carcinoma; Chondrosarcoma; Depression, Chemical; Enzyme Activation; Enzyme Inhibitors; Female; Flavonoids; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase 1; MAP Kinase Signaling System; Membrane Glycoproteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Nitriles; Ovarian Neoplasms; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Recombinant Fusion Proteins; RNA, Messenger; Tetradecanoylphorbol Acetate; Transfection; Trypsin Inhibitor, Kunitz Soybean; Tumor Cells, Cultured

Altered expression of alphav integrins plays a critical role in tumor growth, invasion, and metastasis. In this study, we show that normal human epithelial ovarian cell line, HOSE, and ovarian cancer cell lines, OVCA 429, OVCA 433, and OVHS-1, expressed alphav integrin and associated beta1, beta3, and beta5 subunits, but only ovarian cancer cell lines OVCA 429 and OVCA 433 expressed alphavbeta6 integrin. The expression of alphavbeta6 in OVCA 429 and OVCA 433 was far higher than alphavbeta3 and alphavbeta5 integrin and correlated with high p42/p44 mitogen activated protein kinase (MAPK) activity and high secretion of high molecular weight urokinase plasminogen activator (HMW-uPA), pro-metalloproteinase 2 and 9 (pro-MMP-9 and pro-MMP-2). In contrast to HOSE and OVHS 1, OVCA 433 and OVCA 429 exhibited approximately 2-fold more plasminogen-dependent [3H]-collagen type IV degradation. Plasminogen-dependent [3H]-collagen IV degradation was inhibited by inhibitor of uPA (amiloride) and MMP (phenanthroline) and by antibodies against uPA or MMP-9 or alphavbeta6 integrin, indicating the involvement of alphavbeta6 integrin, uPA and MMP-9 in the process. The alphavbeta6 correlated increase in HMW-uPA and pro-MMP secretion could be inhibited by tyrosine kinase inhibitor genistein or the MEK 1 inhibitor U0126, consistent with a role of active p42/44 MAPK in the elevation of uPA, MMP-9, and MMP-2 secretion. Under similar conditions, genistein and U0126 inhibited plasminogen-dependent [3H]-collagen type IV degradation. These data suggest that sustained elevation of p42/44 MAPK activity may be required for the co-expression of alphavbeta6 integrin, which in turn may regulate the malignant potential of ovarian cancer cells via proteolytic mechanisms.

Topics: Antigens, Neoplasm; Butadienes; Collagen Type IV; Culture Media, Conditioned; Enzyme Inhibitors; Enzyme Precursors; Epithelial Cells; Female; Genistein; Humans; Integrins; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitriles; Ovarian Neoplasms; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Cell adhesion to the extracellular matrix appears to trigger a cascade of intracellular signalings. We have previously shown that treatment of ovarian cancer cells, NOM1, with fibronectin (FN) stimulated matrix metalloproteinase (MMP)-9 secretion and thereby activated the invasiveness of cells via the FAK/Ras signaling pathway. By use of chemical inhibitors, we investigated the downstream effectors critical for FN-dependent secretion of MMP-9. Treatment of cells with MEK1 inhibitors, U0126 and PD98059, dramatically suppressed the secretion of MMP-9 activated by FN. Similarly, P1-3 kinase inhibitors, Wortmannin and LY294002, strongly suppressed the FN-dependent secretion of MMP-9 together with the inhibition of Akt activation. In contrast, a specific PKC inhibitor (GF109203X) showed no inhibitory effect on the FN-dependent MMP-9 secretion. Moreover, we found that both the MEK1 inhibitor and the P13-K inhibitor, but not the PKC inhibitor, strongly suppressed the invasiveness of NOM1 cells. Taken together, our results suggest that activation of dual signaling pathways, MEKI-MAPK and P13K-Akt, is required for the FN-dependent activation of MMP-9 secretion. Our results suggest the importance of these signaling molecules as a chemotherapeutic target for cancer.

Topics: Androstadienes; Butadienes; Chromones; Enzyme Inhibitors; Female; Fibronectins; Flavonoids; Humans; Indoles; Maleimides; MAP Kinase Kinase 1; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinase Kinases; Morpholines; Neoplasm Invasiveness; Nitriles; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Tumor Cells, Cultured; Wortmannin