ag-490 and Multiple-Myeloma

Accumulating evidences have shown that adipokines secreted from adipocytes contributes to tumor development, especially leptin. However, underlying mechanisms remain unclear. This study aims to explore the effect of leptin on development and chemoresistance in multiple myeloma cells and the potential mechanism. Analysis of levels of adipokines including leptin and adiponectin in 28 multiple myeloma patients identified significantly higher leptin compared with 28 normal controls(P < 0.05), and leptin level was positively correlated with clinical stage, IgG, ER, and ß2MG. Next, by using co-culture system of myeloma and adipocytes, and pharmacologic enhancement of leptin, we found that increased growth of myeloma cells and reduced toxicity of bortezomib were best observed at 50 ng/ml of leptin, along with increased expression of cyclinD1, Bcl-2 and decreased caspase-3 expression. We also found that phosphorylated AKT and STAT3 but not the proteins expression reached peak after 1h and 6h treatment of leptin, respectively. By using AG490, an agent blocking the phosphorylation of AKT and ERK, the proliferation of myeloma cells was inhibited, as well as the phosphorylation of AKT and STAT3, even adding leptin. Taken together, our study demonstrated that up-regulated leptin could stimulate proliferation of myeloma and reduce the anti-tumor effect of chemotherapy possibly via activating AKT and STAT3 pathways, and leptin might be one of the potential therapeutic targets for treating myeloma.

Topics: Adipocytes; Adult; Aged; Cell Proliferation; Drug Resistance, Neoplasm; Female; Humans; Leptin; Male; Middle Aged; Multiple Myeloma; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; STAT3 Transcription Factor; Tyrphostins

We previously reported the establishment and characteristics of a DXM-resistant cell line (7TD1-DXM) generated from the IL6-dependent mouse B cell hybridoma, 7TD1 cell line. After withdrawing DXM from 7TD1-DXM cells over 90 days, DXM significantly inhibited the cell growth and induced apoptosis in the cells (7TD1-WD) compared with 7TD1-DXM cells. Additionally, IL-6 reversed while IL-6 antibody and AG490 enhanced the effects of growth inhibition and apoptosis induced by DXM in 7TD1-WD cells. Our study demonstrates that 7TD1-DXM cells become resensitized to DXM after DXM withdrawal, and IL-6 and JAK2/STAT3 pathways may regulate the phenomenon.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Dexamethasone; Drug Resistance, Neoplasm; Interleukin-6; Janus Kinase 2; Mice; Multiple Myeloma; Signal Transduction; STAT3 Transcription Factor; Tyrphostins

With the goal of developing small molecules as novel regulators of signal transduction and apoptosis, a series of tyrphostin-like compounds were synthesized and screened for their activity against MM-1 (multiple myeloma) cells and other cell lines representing this malignancy. Synthesis was completed in solution-phase initially and then adopted to solid-phase for generating a more diverse set of compounds. A positive correlation was noted between compounds capable of inducing apoptosis and their modulation of protein ubiquitination. Further analysis suggested that ubiquitin modulation occurs through inhibition of cellular deubiquitinase activity. Bulky groups on the sidechain near the α,β-unsaturated ketone caused a complete loss of activity, whereas cyclization on the opposite side was tolerated. Theoretical calculations at the B3LYP/LACV3P(∗∗) level were completed on each molecule, and the resulting molecular orbitals and Fukui reactivity values for C(β) carbon were utilized in developing a model to explain the compound activity.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Humans; Janus Kinase 2; Mice; Mice, Nude; Models, Molecular; Multiple Myeloma; Signal Transduction; STAT3 Transcription Factor; Structure-Activity Relationship; Tyrphostins; Ubiquitin

Even though embelin, an inhibitor of the XIAP, is known to exhibit anti-inflammatory and anti-cancer activities, very little is known about its mechanism of action. Here, we investigated whether embelin mediates its effect through interference with the signal transducer and activator of transcription 3 (STAT3) pathway. We found that embelin inhibited constitutive STAT3 activation in a variety of human cancer cell lines such as U266, DU-145, and SCC4 cells. The suppression of STAT3 was mediated through inhibition of the activation of JAK2 and c-Src. Pervanadate treatment also reversed the embelin-induced down-regulation of STAT3, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that embelin-induced the expression of the tyrosine phosphatase PTEN and deletion of the PTEN gene by small interfering RNA abolished the ability of embelin to inhibit STAT3 activation. Besides, embelin failed to suppress STAT3 activation in PTEN-null PC3 cells, thus indicating that the inhibitory effect of embelin on STAT3 is PTEN-dependent. Embelin down-regulated the expression of STAT3-regulated gene products; this correlated with the suppression of cell proliferation and invasion, and the induction of apoptosis through the activation of caspase-3. Overall, our results indicate that the anti-inflammatory and anti-cancer activities previously assigned to embelin may be mediated in part through the suppression of the STAT3 pathway.

Topics: Apoptosis; Benzoquinones; Caspase 3; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; DNA, Neoplasm; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Humans; Janus Kinase 2; Male; Multiple Myeloma; Neovascularization, Pathologic; Phosphorylation; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Protein Tyrosine Phosphatases; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Transfection; Tyrphostins

Serenoa repens, a palm species native to the Southeastern United States, is one of the widely used phytotherapeutic agents in benign prostatic hyperplasia. In this study, we found for the first time that Serenoa repens induced growth arrest of a variety of human leukemia cells including U266 and RPMI 8226 multiple myeloma cells as measured by mitochondrial-dependent conversion of the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. TUNEL assays showed that Serenoa repens induced apoptosis of U266 cells in a time- and dose-dependent manner. Serenoa repens also increased the expression of cleaved-PARP or p27 protein in different human leukemia cell lines. In addition, we found that Serenoa repens down-regulated basal level of phosphorylated form of signal transducer and activator of transcription 3 (STAT 3) and Interleukin-6 induced level of phosphorylated form of STAT 3 and extracellular signal-related kinase (ERK) were also reduced after Serenoa repens treatment in U266 cells. Furthermore, we found that inhibition of STAT 3 signaling by Serenoa repens or Janus family of tyrosine kinase (JAK) inhibitor of AG490 enhanced the ability of docetaxel to inhibit the growth of U266 and RPMI 8226 cells, as measured by trypan blue exclusion test. These results indicate that Serenoa repens might be useful for the treatment of individuals with multiple myeloma.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Docetaxel; Extracellular Signal-Regulated MAP Kinases; Humans; Interleukin-6; Intracellular Signaling Peptides and Proteins; Multiple Myeloma; Phosphorylation; Phytotherapy; Serenoa; Signal Transduction; STAT3 Transcription Factor; Taxoids; Tyrphostins

Interleukin-6 (IL-6) and the subsequent Janus-activated kinase (JAK)-dependent signaling pathways play a critical role in the pathogenesis of multiple myeloma. Here, we compared the sensitivity and specificity of a novel pan-JAK inhibitor, tetracyclic pyridone 6 (P6), with that of AG490 in a panel of myeloma-derived cell lines. P6 induced growth arrest and subsequent apoptosis of the IL-6-dependent hybridoma and myeloma-derived cell lines (B9 and INA-6) grown either in IL-6-containing medium or in the presence of bone marrow-derived stromal cells (BMSC) using much lower concentrations of drug and with significantly faster kinetics than AG490. Myeloma-derived cell lines, which either express constitutively activated JAK/signal transducers and activators of transcription (STAT) 3 (U266) or are IL-6 growth stimulated (KMS11), are partially growth inhibited by P6. However, P6 does not inhibit the growth of myeloma-derived cell lines lacking activated JAKs/STATs nor does it inhibit mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase activity compared with AG490, which led to activation of ERK and induced robust apoptosis of all the examined cell lines. Finally, P6 inhibited the growth of primary myeloma patient samples grown in the presence of BMSCs. Thus, P6 is a more sensitive and specific inhibitor of JAK-STAT3 activity compared with AG490 and potently inhibited the growth of primary myeloma cells and myeloma-derived cell lines grown on BMSCs.

Topics: Animals; Benzimidazoles; Cell Line, Tumor; Coculture Techniques; Drug Screening Assays, Antitumor; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Hybridomas; Interleukin-6; Janus Kinases; MAP Kinase Signaling System; Mice; Multiple Myeloma; Neoplasm Proteins; Phosphorylation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Pyridones; STAT3 Transcription Factor; Stromal Cells; Tyrphostins

Multiple myeloma is a fatal B cell malignancy characterized by the accumulation of plasma cells within the bone marrow. IL-6 is a major survival factor for myeloma cells. Bcl-2 protein family regulates pathways to apoptosis that are activated upon growth factor deprivation. Pro-apoptotic proteins that have only a single Bcl-2 homology domain, BH3-only, are potent inducers of apoptosis. In myeloma cells, Mcl-1 has been shown to be a major anti-apoptotic protein that appears to regulate cell survival through the JAK/STAT pathway. In this study, we examined the regulation of the BH3-only protein Bim and its interaction with Mcl-1. The three major Bim isoforms are expressed in myeloma cells and are negatively regulated by IL-6. Blockade of IL-6 signaling induces an up-regulation of Bim concomitant to Mcl-1 down-regulation. Of major interest, Bim is found strongly associated with Mcl-1 in viable myeloma cells while this interaction is disrupted under apoptosis induction. Of note, while Bim is also found strongly associated to Bcl-2, this interaction is not changed under apoptosis induction. Thus, in myeloma cells, Mcl-1 neutralizes Bim through complex formation and therefore prevents apoptosis. Under apoptosis induction, the disappearance of Mcl-1 allows Bim to exercise its pro-apoptotic function and to activate Bax.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Carrier Proteins; Cell Line, Tumor; Cell Survival; Cycloheximide; Dactinomycin; Humans; Interleukin-6; Janus Kinase 2; Membrane Proteins; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Protein Isoforms; Protein Synthesis Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Transcription, Genetic; Tyrphostins

Hematopoietic malignancies have been shown to depend on cytokine growth factor autocrine/paracrine loops for growth and differentiation. This results in the constitutive activation of cytokine-mediated transcription factors like signal transducer and activators of transcription (STAT) 3 in non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). Recent evidence demonstrates that cytokines also contribute to a drug-resistant phenotype in many tumor cell types. We hypothesized that inhibitors of the STAT3 pathway would sensitize drug-resistant and endogenous cytokine-dependent NHL and MM tumor cells to the cytotoxic effects of chemotherapeutic drugs. We examined an AIDS-related NHL cell line, 2F7, known to be dependent on interleukin (IL)-10 for survival and an MM cell line, U266, known to be dependent on IL-6 for survival. IL-10 and IL-6 signal the cells through the activation of Janus kinase (JAK)1 and JAK2, respectively. Thus, we investigated the effect of two chemical STAT3 pathway inhibitors, namely, piceatannol (JAK1/STAT3 inhibitor) and tyrphostin AG490 (JAK2/STAT3 inhibitor), on the tumor cells for sensitization to therapeutic drugs. We demonstrate by phosphoprotein immunoblotting analysis and electrophoretic mobility shift analysis that piceatannol and AG490 inhibit the constitutive activity of STAT3 in 2F7 and U266, respectively. Furthermore, piceatannol and AG490 sensitize 2F7 and U266 cells, respectively, to apoptosis by a range of therapeutic drugs including cisplatin, fludarabine, Adriamycin, and vinblastine. The specificity of the inhibitors was corroborated in experiments showing that piceatannol had no effect on U266 and, likewise, AG490 has no effect on 2F7. The sensitization observed by these inhibitors correlated with the inhibition of Bcl-2 expression in 2F7 and Bcl-xL expression in U266. Altogether, these results demonstrate that STAT3 pathway inhibitors are a novel class of chemotherapeutic sensitizing agents capable of reversing the drug-resistant phenotype of cytokine-dependent tumor cells.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; bcl-X Protein; Carrier Proteins; Cytokines; DNA Fragmentation; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Humans; Immunoblotting; Interleukin-10; Interleukin-6; Janus Kinase 1; Janus Kinase 2; Lymphoma, Non-Hodgkin; Models, Biological; Multiple Myeloma; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; STAT3 Transcription Factor; Stilbenes; Time Factors; Trans-Activators; Tumor Cells, Cultured; Tyrphostins; Vinblastine

Numerous reports suggest that IL-6 promotes survival and proliferation of multiple myeloma (MM) cells through the phosphorylation of a cell signaling protein, STAT3. Thus, agents that suppress STAT3 phosphorylation have potential for the treatment of MM. In the present report, we demonstrate that curcumin (diferuloylmethane), a pharmacologically safe agent in humans, inhibited IL-6-induced STAT3 phosphorylation and consequent STAT3 nuclear translocation. Curcumin had no effect on STAT5 phosphorylation, but inhibited the IFN-alpha-induced STAT1 phosphorylation. The constitutive phosphorylation of STAT3 found in certain MM cells was also abrogated by treatment with curcumin. Curcumin-induced inhibition of STAT3 phosphorylation was reversible. Compared with AG490, a well-characterized Janus kinase 2 inhibitor, curcumin was a more rapid (30 min vs 8 h) and more potent (10 micro M vs 100 micro M) inhibitor of STAT3 phosphorylation. In a similar manner, the dose of curcumin completely suppressed proliferation of MM cells; the same dose of AG490 had no effect. In contrast, a cell-permeable STAT3 inhibitor peptide that can inhibit the STAT3 phosphorylation mediated by Src blocked the constitutive phosphorylation of STAT3 and also suppressed the growth of myeloma cells. TNF-alpha and lymphotoxin also induced the proliferation of MM cells, but through a mechanism independent of STAT3 phosphorylation. In addition, dexamethasone-resistant MM cells were found to be sensitive to curcumin. Overall, our results demonstrated that curcumin was a potent inhibitor of STAT3 phosphorylation, and this plays a role in the suppression of MM proliferation.

Topics: Active Transport, Cell Nucleus; Cell Line; Cell Line, Tumor; Cell Membrane Permeability; Cell Nucleus; Curcumin; DNA-Binding Proteins; Growth Inhibitors; Humans; Interferon-alpha; Interleukin-6; Milk Proteins; Multiple Myeloma; Oligopeptides; Phosphorylation; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; STAT5 Transcription Factor; Trans-Activators; Tyrphostins

Cytokines of the gp130 family, particularly interleukin 6 (IL-6), play a central role in the growth and survival of malignant plasma cells. Recently, novel neurotrophin-1 (NNT-1)/B cell-stimulating factor-3 (BSF-3), also reported as cardiotrophin-like cytokine (CLC), was identified as a cytokine belonging to the gp130 family. BSF-3, similar to IL-6, exerts regulatory effects on normal B cell functions, but its functional significance in haematological malignancies has not been defined. The purpose of this study was to evaluate the biological effects and signalling pathways that are induced by BSF-3 in malignant plasma cells. Recombinant human BSF-3 was found to have growth stimulatory activity on plasmacytoma cell lines and primary tumour cells. In addition, BSF-3 was able to protect from Dexamethasone (Dex)-induced apoptosis. BSF-3 stimulated cell growth could not be inhibited by neutralizing anti-IL-6 or anti-IL-6 receptor antibodies, but was abrogated by anti-gp130 antibodies. In INA-6.Tu11 cells, a subline of the IL-6-dependent human plasma cell line INA-6 expressing gp130 and the receptor for leukaemia inhibitory factor (LIF), stimulation with BSF-3 induced tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3). AG490, an inhibitor of Janus kinases, decreased BSF-3 induced cell growth in a dose-dependent manner. This correlated with a reduction of STAT3 phosphorylation levels, while p44/42 mitogen-activated protein kinase (MAPK) phosphorylation was not affected. In conclusion, BSF-3 is a novel myeloma growth and survival factor with a potential role in the pathophysiology of the disease.

Topics: Antibodies, Monoclonal; Antigens, CD; Antineoplastic Agents; Apoptosis; Cell Division; Cytokine Receptor gp130; Cytokines; Dexamethasone; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Membrane Glycoproteins; Multiple Myeloma; Phosphorylation; Recombinant Proteins; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Tumor Cells, Cultured; Tyrphostins

Interleukin 6 (IL-6) is the major survival factor of myeloma cells. In this study, we demonstrate that IL-6, oncostatin M (OSM) and leukemia inhibitory factor (LIF) upregulate membrane IL-6 receptor alpha (IL-6Ralpha) on OPM-2 myeloma cell line at transcriptional level. In OPM-2 cells, IL-6, OSM and LIF induce both signal transducers and activators of transcription (STAT), mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI 3-K) activation. We show that the cytokine-induced upregulation of IL-6Ralpha can be abolished by a janus kinase (JAK)-2 specific inhibitor, i.e. AG490, suggesting an involvement of the JAK/STAT pathway in this process. Finally, IL-6Ralpha upregulation was also inhibited by wortmannin, an inhibitor of the PI 3-kinase pathway. In conclusion, IL-6 can upregulate its own receptor on OPM-2 cells probably through the JAK/STAT and PI 3-kinase pathways.

Topics: Androstadienes; Antibodies, Monoclonal; Blotting, Northern; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Flow Cytometry; Growth Inhibitors; Humans; Immunoblotting; Interleukin-6; Janus Kinase 2; Leukemia Inhibitory Factor; Lymphokines; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Multiple Myeloma; Oncostatin M; Peptides; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Interleukin-6; Signal Transduction; Tyrphostins; Up-Regulation; Wortmannin

Our previous work demonstrated that the Janus kinase (JAK)-Stat3 pathway regulates expression of Bcl-x(L) in the U266 human multiple myeloma cell line and prevents Fas-mediated apoptosis. Inhibition of this pathway by the JAK selective kinase inhibitor AG490 or dominant-negative Stat3 protein results in down-regulation of Bcl-x(L) expression and enhanced sensitivity to Fas-mediated apoptosis. Because Bcl-x(L) has also been implicated in resistance to chemotherapeutic drugs, we investigated whether inhibition of the JAK-Stat3 pathway and subsequent reduction in Bcl-x(L) expression would also enhance cytotoxic drug activity. Contrary to this prediction, pretreatment of U266 myeloma cells with AG490, followed by exposure to topoisomerase II- inhibiting agents, antagonized drug-induced apoptosis. This effect correlated with reduced cyclin D1 expression and cell cycle arrest. The cell cycle arrest following AG490 pretreatment further correlated with reduced mitoxantrone-induced DNA double-strand breaks and reduced cell death, findings consistent with the critical requirement of DNA damage for drug cytotoxicity. These studies demonstrate that inhibition of the JAK-Stat3 pathway can result in paradoxical effects relative to cytotoxic drug response. These paradoxical responses may be explained by the findings that JAK-Stat3 signaling regulates the expression of multiple genes involved in controlling cell proliferation and apoptosis. Thus, understanding the cellular context of inhibiting signal transduction pathways is essential for the design of novel combination therapies for cancer.

Topics: Antineoplastic Agents; Apoptosis; bcl-X Protein; Cell Survival; Enzyme Inhibitors; fas Receptor; Gene Expression Regulation, Neoplastic; Humans; Janus Kinase 1; Janus Kinase 2; Janus Kinase 3; Multiple Myeloma; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Topoisomerase II Inhibitors; Transfection; Tumor Cells, Cultured; Tyrphostins

Cytokines of the interleukin 6 (IL-6) family, which activates the signal transducer gp130, are major survival and growth factors for human multiple myeloma (MM) cells. The signal transduction of gp130 involves the Janus tyrosine kinases (JAK) JAK1, JAK2 and Tyk2 and then the downstream effectors comprising the signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) pathways. We evaluated the effects of the JAK2 inhibitor tyrphostin AG490 on MM cells. We found that AG490 suppressed cell proliferation and induced apoptosis in IL-6-dependent MM cell lines. JAK2 kinase activity, ERK2 and STAT3 phosphorylation were inhibited. These results suggest that the chemical blocking of the gp130 signalling pathway at the JAK level could be a relevant therapeutic approach to MM.

Topics: Antigens, CD; Apoptosis; Cell Division; Cytokine Receptor gp130; Depression, Chemical; DNA-Binding Proteins; Enzyme Inhibitors; Flow Cytometry; Humans; Immunoblotting; Interleukin-6; Janus Kinase 2; MAP Kinase Signaling System; Membrane Glycoproteins; Mitogen-Activated Protein Kinase 1; Multiple Myeloma; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Signal Transduction; STAT2 Transcription Factor; STAT3 Transcription Factor; Trans-Activators; Tumor Cells, Cultured; Tyrphostins

Interleukin 6 (IL-6) is the major survival factor for myeloma tumor cells and induces signaling through the STAT proteins. We report that one STAT family member, Stat3, is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in the IL-6-dependent human myeloma cell line U266. Moreover, U266 cells are inherently resistant to Fas-mediated apoptosis and express high levels of the antiapoptotic protein Bcl-xL. Blocking IL-6 receptor signaling from Janus kinases to the Stat3 protein inhibits Bcl-xL expression and induces apoptosis, demonstrating that Stat3 signaling is essential for the survival of myeloma tumor cells. These findings provide evidence that constitutively activated Stat3 signaling contributes to the pathogenesis of multiple myeloma by preventing apoptosis.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Apoptosis; bcl-X Protein; DNA-Binding Proteins; Gene Expression Regulation; Humans; Immunity, Innate; Mice; Multiple Myeloma; Promoter Regions, Genetic; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-2; Receptors, Interleukin-6; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Tyrphostins