bay-11-7082 and Multiple-Myeloma

To study the effects of BAY-11-7082 on proliferation and apoptosis of U266 cells and its mechanism of action.. Multiple myelomas U266 cells were cultured and divided into control group and gradient-concentration BAY-11-7082 groups (1 μmol/L, 2 μmol/L, 4 μmol/L and 8 μmol/L). Cells in BAY-11-7082 groups were treated with drugs in different concentrations for 4 h, while those in control group were added with an equal volume of solvent. The cell viability was detected via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and lactate dehydrogenase (LDH) release assay was used to detect the cytotoxicity. Furthermore, cells in low-concentration and high-concentration BAY-11-7082 groups were compared with those in control group. The cell proliferation level was evaluated via cell cycle assay, the interleukin-6 (IL-6) level in cells was detected via enzyme-linked immunosorbent assay (ELISA), and the β-catenin protein expression was detected via Western blotting. Moreover, flow cytometry and Hoechst staining were performed to detect the number of apoptotic cells, and the apoptosis level was detected via caspase3 activity and apoptosis-related protein expression. Finally, the levels of p65, p50 and inhibitor kappa B kinase β (IKKβ) were detected via polymerase chain reaction (PCR), and the expressions and changes of phosphorylated (p)-p65 and p-IKKβ were detected via Western blotting.. BAY-11-7082 could reduce the U266 cell viability and increase the cytotoxic effect. Based on gradient concentration, 2 μmol/L was selected as the low concentration, while 4 μmol/L was selected as the high concentration. Compared with those in control group, the number of cells in the S and G2/M phases in drug administration groups was significantly decreased, but that in the G0/G1 phase was significantly increased. Besides, the secretion of IL-6 in cells in drug administration groups was significantly decreased compared with that in control group. The β-catenin protein expression was decreased in drug administration groups, and there was also a difference between high concentration group and low concentration group. Flow cytometry and Hoechst staining showed that the proportion of apoptotic cells in drug administration groups was significantly increased. Western blotting and detection of caspase3 activity revealed that the expression and activation of apoptosis-related protein were increased in drug administration groups. It was found in the detection of nuclear factor-κB (NF-κB) pathway that the NF-κB pathway was inhibited in drug administration groups, and there was also a statistically significant difference between high concentration group and low concentration group.. BAY-11-7082 inhibits the proliferation and induces the apoptosis of U266 cells through inhibiting NF-κB pathway.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Multiple Myeloma; NF-kappa B; Nitriles; Signal Transduction; Sulfones

Multiple myeloma (MM) is a hematological malignancy characterized by the aberrant accumulation of clonal plasma cells in the bone marrow. Despite recent advancement in anti-myeloma treatment, MM remains an incurable disease. This study showed higher intrinsic oxidative stress and higher Trx1 and TrxR1 protein levels in MM cells compared to normal cells. Drug-induced Trx1 (PX-12) and TrxR1 (Auranofin) inhibition disrupted redox homeostasis resulting in ROS-induced apoptosis in MM cells and a reduction in clonogenic activity. Knockdown of either Trx1 or TrxR1 reduced MM cell viability. Trx1 inhibition by PX-12 sensitized MM cells to undergo apoptosis in response to the NF-κβ inhibitors, BAY 11-7082 and curcumin. PX-12 treatment decreased the expression of the NF-κβ subunit p65 in MM cells. Bortezomib-resistant MM cells contained higher Trx1 protein levels compared to the parental cells and PX-12 treatment resulted in apoptosis. Thus, increased Trx1 enhances MM cell growth and survival and exerts resistance to NF-κβ inhibitors. Therefore inhibiting the thioredoxin system may be an effective therapeutic strategy to treat newly diagnosed as well as relapsed/refractory MM.

Topics: Apoptosis; Auranofin; Bortezomib; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Curcumin; Disulfides; Drug Resistance, Neoplasm; Humans; Imidazoles; Leukocytes, Mononuclear; Multiple Myeloma; Nitriles; Oxidative Stress; Reactive Oxygen Species; Sulfones; Thioredoxin Reductase 1; Thioredoxins; Transcription Factor RelA

Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells.

Topics: Amides; Apoptosis; Blotting, Western; Cell Line; Cell Survival; Cyclopentanes; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Kinase; Microscopy, Fluorescence; Multiple Myeloma; NF-kappa B; Nitriles; Pyrimidines; RNA, Small Interfering; Signal Transduction; Sulfones; Thiophenes

B-lymphocyte stimulator (BAFF) is a recently recognized member of the tumor necrosis factor ligand family (TNF) and a potent cell-survival factor expressed in many hematopoietic cells. BAFF regulates B-cell survival, differentiation, and proliferation by binding to three TNF receptors: TACI, BCMA, and BAFF-R. The mechanism involved in BAFF-R gene expression and regulation remains elusive. In this study, we examined BAFF-R gene expression, function, and regulation in multiple myeloma (KM3) cells. It was found that BAFF-BAFF-R induced cell survival by activating NF-κB1 pathway and NF-κB2 pathway. It was also found that NF-κB was an important transcription factor involved in regulating BAFF-R expression through one NF-κB binding site in the BAFF-R promoter, suggesting that inhibiting NF-κB could decrease the expression of BAFF-R mRNA and protein, and promote activity of BAFF-R gene. Our findings indicate that both NF-κB pathways are involved in the regulation of BAFF-R gene and the NF-κB-binding site of BAFF-R may be a new therapeutic target in this disease.

Topics: Apoptosis; B-Cell Activating Factor; B-Cell Activation Factor Receptor; Binding Sites; Cell Line, Tumor; Cell Survival; Gene Expression Regulation, Neoplastic; Humans; Molecular Targeted Therapy; Multiple Myeloma; NF-kappa B; NF-kappa B p52 Subunit; Nitriles; Promoter Regions, Genetic; Signal Transduction; Sulfones; Transcription Factor RelA; Transcription, Genetic

Multiple myeloma (MM) is a common malignant tumor, characterized by unlimited proliferation of abnormal plasmocytes in bone marrow. Considering the biological function of B-Lymphocyte stimulator (BLyS) and its receptors in B cell, we examined BLyS and its receptors expression in MM cells. Our studies confirmed that BLyS and its receptors are expressed in MM cells, including KM3, CZ-1, and primary MM cells, playing an important role in the survival and proliferation of MM cells. Additionally, we provide evidence that BLyS protein is located in the MM cell plasma membrane. We also found that IFN-gamma and IL-6 can induce BLyS expression on MM cells, while after the treatment of BAY11-7082, an IkB-alpha phosphorylation inhibitor, IFN-gamma induced up regulation of BLyS was completely inhibited, suggesting that nuclear factor kappaB (NF-kappaB) might be involved in the mechanism of the regulation of BLyS levels in response to cytokines. Finally, linear correlation analysis of the Lactate Dehydrogenase concentration and beta 2-microglobulin level with BLyS, and expressions of BLyS mRNA in MM patients revealed a significant correlation between them (P < 0.01 in all case), showing that BLyS could be a biomarker for the diagnosis and treatment of MM.

Topics: Adult; Aged; B-Cell Activating Factor; B-Lymphocytes; Biomarkers; Bone Marrow Cells; Cell Division; Cells, Cultured; DNA Primers; Female; Flow Cytometry; Humans; Lymphocyte Activation; Male; Middle Aged; Monocytes; Multiple Myeloma; NF-kappa B; Nitriles; Reference Values; Reverse Transcriptase Polymerase Chain Reaction; Sulfones; Tumor Necrosis Factor-alpha

Interactions between pharmacologic NF-kappaB inhibitors (eg, Bay 11-7082, SN-50) and the checkpoint abrogator UCN-01 have been examined in human multiple myeloma (MM) cells. Exposure of U266 cells to Bay 11-7082 (Bay) in combination with UCN-01 resulted in the abrogation of NF-kappaB/DNA binding activity and the synergistic induction of apoptosis. Comparable synergism was observed in other MM cell lines and patient-derived CD138+ cells and between an inhibitory peptide of NF-kappaB (SN50) and UCN-01. Bay/UCN-01-mediated lethality involved mitochondrial dysfunction, caspase cleavage, and poly adenosine diphosphate-ribose polymerase (PARP) degradation. Although Bay modestly blocked UCN-01-induced extracellular signal-regulated kinase (ERK) phosphorylation, coadministration activated c-Jun N-terminal kinase (JNK) and cdc2/cdk1 and down-regulated Mcl-1, XIAP, and Bcl-xL. Transfection with a constitutively activated mitogen-activated protein kinase kinase (MEK1)/green fluorescent protein (GFP) construct failed to block apoptosis induced by Bay/UCN-01 but significantly attenuated MEK inhibitor (U0126)/UCN-01-induced lethality. Inhibiting JNK activation with SP600125 or D-JNKI1 peptide markedly reduced Bay/UCN-01-mediated mitochondrial dysfunction and apoptosis and the down-regulation of Mcl-1, XIAP, and Bcl-xL but not of cdc2/cdk1 activation. Stable transfection of cells with dominant-negative caspase-9 dramatically diminished Bay/UCN-01 lethality without altering JNK or cdc2/cdk1 activation. Neither interleukin-6 (IL-6)- nor fibronectin-mediated adherence conferred resistance to Bay/UCN-01-induced apoptosis. Together, these findings suggest that a strategy combining UCN-01 with disruption of the IkappaB kinase (IKK)/IkappaB/NF-kappaB pathway warrants attention in MM.

Topics: Antigens, CD; Antineoplastic Agents; Apoptosis; Cell Adhesion; Cell Line, Tumor; Humans; Intracellular Membranes; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Membrane Glycoproteins; Membrane Potentials; Mitochondria; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Multiple Myeloma; NF-kappa B; Nitriles; Organic Chemicals; Proteoglycans; Staurosporine; Sulfones; Syndecan-1; Syndecans