ag-490 and Ovarian-Neoplasms

Carcinoma‑associated fibroblasts (CAFs) are the major components of mesenchymal cells in the inflammatory tumor microenvironment. They are involved in epithelial‑mesenchymal transition (EMT) and chemotherapy resistance by directly contacting with cancer cells or secretory cytokines. In the present study, we examined the role of CAFs in the induction of EMT in ovarian cancer. Primary ovarian cancer cells, CAFs and normal fibroblasts (NFs) were isolated from fresh cancer tissue and cultured for immunohistochemistry studies. Enzyme‑linked immunosorbent assay (ELISA) was used to detect the expression of IL‑6 in the culture supernatants of these cells. The expression of IL‑6 at the mRNA level was examined by RT‑PCR. The expression of IL‑6 at the protein level in ovarian cancer tissues was determined using an immunofluorescence assay in both tissue sections and cell lobes. OVCAR3 cells were treated with the culture supernatants collected from CAFs and NFs. IL‑6 monoclonal antibody (mAb) was employed to neutralize IL‑6. The expression of phosphorylated STAT3 was assessed. Changes in EMT, proliferation, invasion and proapoptotic protein expression were also examined. Flow cytometry was performed to detect the changes in apoptosis resistance of OVCAR3 cells. The JAK2/STAT3 pathway‑specific inhibitor AG490 was used to block this pathway and the β‑TGF inhibitor was used to inhibit EMT. The clinical data of patients treated in our hospital were collected between January 1st, 2009 and June 30th, 2013. The expression of interstitial IL‑6 in paraffin‑embedded tissues was detected by immunohistochemistry. The relationship between the expression of interstitial IL‑6 and the treatment response was examined by linear regression and multiple linear regression analyses. We found that CAFs were the main source of IL‑6 in ovarian cancer tissue. CAFs promoted the phosphorylation of STAT3 in ovarian cancer and enhanced the proliferation, invasion and EMT. Enhanced EMT may lead to apoptosis resistance, inhibitory expression of pro‑apoptotic proteins and paclitaxel resistance. A total of 255 patients were enrolled in this retrospective study. Univariate and multivariate analyses revealed that age, CA125, interstitial IL‑6 expression and cytoreduction satisfaction were closely related to the sensitivity of the TP (docetaxel plus cisplatin or carbopatin) regimen in ovarian cancer (P<0.05). These results demonstrated that CAFs highly secreted IL‑6 and promoted β‑TGF‑mediated EMT in ov

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cells, Cultured; Coculture Techniques; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Fibroblasts; Humans; Interleukin-6; Janus Kinase 2; Neoplasm Grading; Neoplasm Staging; Ovarian Neoplasms; Paclitaxel; Retrospective Studies; STAT3 Transcription Factor; Tyrphostins

B7-H3, a co-stimulatory molecule, has been found expressed in ovarian cancer, but its role and mechanism is not clear. In this study, we further verified the expression of B7-H3 in ovarian carcinoma and normal epithelial ovarian tissues. Three ovarian cancer cell lines, A2780, SKOV3 and HO8910 were selected to explore the effects of B7-H3 on proliferation, apoptosis, migration and invasion. We found that B7-H3 was mainly located in the cytoplasm of ovarian cancer cells as determined by immunofluorescence staining. The ability of cell invasion, migration, proliferation decreased after silencing B7-H3 whereas the apoptosis increased, which was related to the upregulation of Bax, caspase-8, cleaved caspase-8 and the downregulation of Bcl-2, Bcl-xl, matrix metalloproteinase-2 (MMP2) by western blotting. In addition, B7-H3 enhanced the H08910 cell capacities in invasion, migration and proliferation. Expression of the phosphorylation signal transducer and activator of transcription 3 (pStat3) molecules and their upstream molecules phosphorylation Janus kinase 2 (pJak2) were significantly increased. In order to investigate whether B7-H3 plays a role in this pathway, we treated the overexpressed HO8910 cells with AG490 (inhibitors of Jak2). Our findings revealed that B7-H3 affect ovarian cancer progression through the Jak2/Stat3 pathway, indicating that B7-H3 has the potential to be a useful prognostic marker.

Topics: B7 Antigens; Biomarkers, Tumor; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Janus Kinase 2; Middle Aged; Neoplasm Invasiveness; Ovarian Neoplasms; Signal Transduction; STAT3 Transcription Factor; Tyrphostins

Resistance to chemotherapy is a major factor that limits the postsurgical survival of ovarian cancer patients. Janus-activated kinase 2 (JAK2) has been implicated in cancer cell survival and the development of drug resistance in ovarian cancers. In the present study, we sought to determine whether inhibition of JAK2 reverses drug resistance in OC3/TAX300 cells, a paclitaxel-resistant human ovarian cancer cell line previously established in our laboratory.. OC3/TAX300 cells were transduced with lentivirus expressing small interference RNA (siRNA) against JAK2 and treated with JAK2 kinase inhibitor AG490.. Treatment with JAK2-siRNA markedly decreased the messenger RNA and protein of JAK2 as determined by real-time polymerase chain reaction and Western blot analysis. OC3/TAX300 cells treated with JAK2-siRNA exhibited stalled growth, increased cell cycle arrest in G2/M phase, and enhanced apoptosis in response to paclitaxel. In keeping with this, JAK2-siRNA also inhibited the expression of multidrug resistance protein 1. To determine whether JAK2 promotes paclitaxel resistance via phosphorylation of signal transducer and activator of transcription 3 (STAT3), a transcription factor known to be involved in resistance to chemotherapy, we treated OC3/TAX300 cells with JAK2 kinase inhibitor AG490. Of note, AG490 reduced the level of p-STAT3 and inhibited the expression of multidrug resistance protein 1 in a dose-dependent manner.. Collectively, we conclude that the JAK2-STAT3 pathway promotes the development of paclitaxel resistance via upregulating the expression of prosurvival and antiapoptotic genes. Targeting this pathway may be effective in reversing resistance to chemotherapy in ovarian cancers.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Proliferation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Janus Kinase 2; M Phase Cell Cycle Checkpoints; Ovarian Neoplasms; Paclitaxel; Phosphorylation; RNA, Messenger; RNA, Small Interfering; Signal Transduction; STAT2 Transcription Factor; Transduction, Genetic; Tyrphostins; Up-Regulation

Ovarian cancer is the major cause of cancer death among female genital malignancies, and requires developing novel therapeutic measures. Immune escape and acquisition of tolerance by tumor cells are essential for cancer growth and progression. An immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) overexpression in tumors is essential for host immune tolerance. Janus-activated kinase-signal transducer and activator of transcription (JAK-STAT) pathway is involved in various kinds of tumor biology. Thus, we examined the effects of STAT1 inhibition by AG490 (a JAK2 inhibitor) on ovarian cancer progression in mice. In vitro study, IFN-γ treatment up-regulated Ido mRNA expression with STAT1 activation in OV2944-HM-1 cells, whereas AG490 treatment significantly inhibited this effect with the suppression of STAT1 phosphorylation. In vivo model, OV2944-HM-1 cells were intraperitoneally/subcutaneously transplanted into syngeneic immunocompetent female mice. AG490 treatment significantly suppressed subcutaneous tumor growth, compared with control. Consistently, in mice intraperitoneally inoculated HM-1 cells, the same treatment significantly improved survival rate with the reduced number of intraperitoneal tumors. Actually, intratumoral IDO expression was significantly suppressed with the reduction of STAT1 activation in AG490-treated mice. Moreover, in tumor microenvironment of mice treated with AG490, the accumulation of anti-tumor leukocytes such as CD8(+) T-cells, M1 macrophages, and NK cells was apparently exaggerated with the reciprocal reduction of regulatory T cells. Furthermore, intratumoral expression of anti-tumor cytokines such as IL-1α, IL-1β and IL-12 expression was significantly enhanced in mice treated with AG490. Collectively, JAK/STAT signal pathways may be good molecular target for immunotherapy of ovarian cancer.

Topics: Animals; Cell Line, Tumor; Cytokines; Female; Indoleamine-Pyrrole 2,3,-Dioxygenase; Janus Kinase 2; Mice; Ovarian Neoplasms; STAT1 Transcription Factor; Tyrphostins

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

Lysophosphatidic acid (LPA) is a biolipid that stimulates tumor cell invasion and metastasis. In this report, we determined the role of signal transducers and activators of transcription 3 (STAT3) and the effect of a chemopreventive agent, curcumin, on LPA-induced ovarian cancer cell motility. LPA phosphorylated STAT3 in a dose-dependent manner. Treatment of cells with a JAK/STAT inhibitor, AG490, inhibited LPA-induced cell motility. In contrast, transfection of a constitutively active form of STAT3 induced ovarian cancer cell motility. LPA also stimulated interleukin (IL)-6 and IL-8 secretion, which results in STAT3 phosphorylation. Treatment of the cells with curcumin inhibited LPA-induced IL-6 and IL-8 secretion and STAT3 phosphorylation, leading to blocked ovarian cancer cell motility. Collectively, the present study shows the critical role of STAT3 in ovarian cancer cell motility and that this process can be prevented by curcumin.

Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Movement; Cell Survival; Curcumin; Dose-Response Relationship, Drug; Female; Humans; Interleukin-6; Interleukin-8; Janus Kinases; Lysophospholipids; Neoplasm Invasiveness; Ovarian Neoplasms; Phosphorylation; Protein Kinase Inhibitors; Signal Transduction; STAT3 Transcription Factor; Transfection; Tyrphostins

Ovarian cancer is the leading cause of death from gynecologic malignancies in the United States. In an attempt to develop drugs that suppress ovarian cancer cells, we examined the effect of selective inhibitors of protein tyrosine kinases-tyrphostins, which are likely to play a role in ovarian cancer cells.. We examined the cellular and biochemical effects of tyrphostins AG1478, PP2, AGL2592, and AG490 from four different families on the ovarian carcinoma cell line OV1063.. We found that the AG1478, PP2, AGL2592, and AG490 tyrphostins suppressed cell proliferation and altered cell cycle distribution of the OV1063 cells in a dose- and time-dependent manner. Immunoblotting analysis indicated that AG1478 effectively inhibited epidermal growth factor receptor autophosphorylation, that AG490 decreased the level of Jak2 and phosphorylated Stat3, and that PP2 decreased the level of pp60Src protein. AGL2592 decreased the level of constitutive activated epidermal growth factor receptor and pStat3, but its molecular targets have not been identified completely.. The growth-arresting properties of these tyrphostins identify them as possible candidates for signal transduction therapy.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma; Catechols; Cell Cycle; Cell Division; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Humans; Nitriles; Ovarian Neoplasms; Protein Kinase Inhibitors; Pyrimidines; Quinazolines; Time Factors; Tumor Cells, Cultured; Tyrphostins

Signal transducers and activators of transcription (STATs) are transcription factors activated in response to cytokines and growth factors. Constitutively active Stat3 has been shown to mediate oncogenic transformation in cultured cells and induce tumor formation in mice. An increasing number of tumor-derived cell lines as well as samples from human cancer have been reported to express constitutively active Stat3 protein. We previously demonstrated that ovarian cancer cell lines express high levels of constitutively active Stat3. In this study, we show that inhibition of the Stat3 signaling pathway using the Janus Kinase-selective inhibitor, AG490, and a dominant negative Stat3 (Stat3beta) significantly suppresses the growth of ovarian and breast cancer cell lines harboring constitutively active Stat3. In the ovarian cancer cell lines, AG490 also diminished the phosphorylation of Stat3, Stat3 DNA binding activity, and the expression of Bcl-x(L). Further, AG490 induced significant apoptosis in ovarian and breast cancer cell lines expressing high levels of constitutively active Stat3 but had a less profound effect on normal cells lacking constitutively active Stat3. AG490 also enhanced apoptosis induced by cisplatin in ovarian cancer cells. These results suggest that inhibition of Stat3 signaling may provide a potential therapeutic approach for treating ovarian and breast cancers.

Topics: Apoptosis; bcl-X Protein; Blotting, Western; Breast Neoplasms; Cell Division; Cell Line; Cell Size; Cisplatin; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Epithelial Cells; Female; Fibroblasts; Genes, Dominant; Humans; Janus Kinase 3; Mutation; Ovarian Neoplasms; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; Time Factors; Trans-Activators; Transfection; Tumor Cells, Cultured; Tyrphostins