cytochrome-c-t and Multiple-Myeloma

Proteasomes are overexpressed in multiple myeloma (MM) and proteasomal inhibitors (PIs) have been widely used for the treatment of MM. PIs are reported to induce MM cell apoptosis but impair necroptosis. In the present study, we found that PIs MG132 and bortezomib induce MM cell pyroptosis, a novel type of cell death, in a GSDME-dependent manner. Lack of GSDME totally blocks PI-induced pyroptosis. Interestingly, we found that Caspase-3/6/7/9 are all involved in pyroptosis triggered by PIs because the specific inhibitor of each caspase ablates GSDME activation. PIs markedly reduce mitochondrial membrane potential. Moreover, PIs disrupt the interaction of Bcl-2 and BAX, induce cytochrome c release from mitochondria to cytosol and activate GSDME. Furthermore, we found that overexpression of an N-terminal portion of GSDME suffices to release cytochrome c from mitochondria and to activate Caspase-3/9, suggesting N-GSDME might penetrate the mitochondrial membrane. Consistent with Bcl-2 inhibition, BAX can induce MM cell pyroptosis in a GSDME-dependent manner. In accordance with these findings, inhibition of Bcl-2 synergizes with PIs to induce MM cell pyroptosis. Therefore, the present study indicates that PIs trigger MM cell pyroptosis via the mitochondrial BAX/GSDME pathway and provides a rationale for combined treatment of MM with Bcl-2 and proteasome inhibitors to increase therapeutic efficiency via induction of pyroptosis.

Topics: bcl-2-Associated X Protein; Caspase 3; Cytochromes c; Humans; Multiple Myeloma; Proteasome Inhibitors; Pyroptosis

Topics: Aged; Aged, 80 and over; Aniline Compounds; Antineoplastic Agents; bcl-X Protein; Biological Assay; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cytochromes c; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Mitochondria; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Organ Specificity; Peptides; Plasma Cells; Proto-Oncogene Proteins c-bcl-2; Recurrence; Sulfonamides

The prevalence of obesity over the last several decades in the United States has tripled among children and doubled among adults. Obesity increases the incidence and progression of multiple myeloma (MM), yet the molecular mechanisms by which adipocytes contribute to cancer development and patient prognosis have yet to be fully elucidated. Here, we obtained human adipose-derived stem cells (ASCs) from twenty-nine normal (BMI = 20-25 kg/m(2)), overweight (25-30 kg/m(2)), obese (30-35 kg/m(2)), or super obese (35-40 kg/m(2)) patients undergoing elective liposuction. Upon differentiation, adipocytes were co-cultured with RPMI-8226 and NCI-H929 MM cell lines. Adipocytes from overweight, obese and super obese patients displayed increased PPAR-gamma, cytochrome C, interleukin-6, and leptin protein levels, and decreased fatty acid synthase protein. 8226 MM cells proliferated faster and displayed increased pSTAT-3/STAT-3 signaling when cultured in adipocyte conditioned media. Further, adipocyte conditioned media from obese and super obese patients significantly increased MM cell adhesion, and conditioned media from overweight, obese and super obese patients enhanced tube formation and expression of matrix metalloproteinase-2. In summary, our data suggest that adipocytes in the MM microenvironment contribute to MM growth and progression and should be further evaluated as a possible therapeutic target.

Topics: Adipocytes; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Culture Media, Conditioned; Cytochromes c; Disease Progression; Fatty Acid Synthase, Type I; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-6; Leptin; Matrix Metalloproteinase 2; Multiple Myeloma; Neovascularization, Physiologic; Obesity; Paracrine Communication; Phosphorylation; PPAR gamma; Signal Transduction; STAT3 Transcription Factor; Tumor Microenvironment

The natural product tetramethylpyrazine (TMP) and resveratrol have a variety of biologic activities, including anti-cancer effects. However the pharmacological function of CSTMP (a newly designed and synthesized TMP and resveratrol derivative) in cancer have not been elucidated.. In RPMI8226 cells, the cytotoxic effects and apoptosis were detected by MTT and Double staining for Annexin V-FITC and propidium iodide (PI). The protein and mRNA expression levels were detected by Real Time PCR and Western blot, respectively. The localization of cleaved caspase-12 was evaluated by immunofluorescent staining. The activation of caspase were measured by colorimetric assays and Western blot.. CSTMP showed significantly cytotoxic effects and induced apoptosis in RPMI8226 cells. Caspase activation, Cytochrome c release and Bax, Bcl-2 and Bcl-XL levels analyses demonstrated that the anti-cancer effect of CSTMP in RPMI8226 cells was mediated by promoting caspase- and mitochondria-dependent apoptosis. In addition, CSTMP induced the increased expression of endoplasmic reticulum (ER) stress related proteins (CHOP, GRP78, GRP94 and cleaved caspase-12) and the activation of multiple branches of ER stress transducers (PERK-eIF2α, IRE1α and ATF6). Moreover, knockdown of CHOP by siRNA markedly inhibited CSTMP-induced cytotoxic effects, caspases activity and mitochondrial dysfunction in RPMI8226 cells.. Our results indicated that CSTMP could induce apoptosis and mitochondrial dysfunction in RPMI8226 cells via CHOP-dependent ER stress.

Topics: Apoptosis; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Humans; Mitochondria; Multiple Myeloma; Pyrazines; Resveratrol; Stilbenes; Transcription Factor CHOP; Unfolded Protein Response

Despite great progress made in the treatment of multiple myeloma (MM), it is still incurable. Promising phase II clinical results have been reported recently for oncolytic vaccinia virus (OVV) clinic therapeutics. One reason for this has focused on the critical therapeutic importance of the immune response raised by these viruses. However, few studies have performed their applications as an optimal delivery system for therapeutic gene, especially miRNA in MM. In this study, we constructed two novel OVVs (TK deletion) that express anti-tumor genes, miR-34a and Smac, respectively, in MM cell lines and xenograft model. The results demonstrated that the novel OVV can effectively infect MM cell lines, and forcefully enhance the exogenous gene (miR-34a or Smac) expression. Furthermore, utilization of VV-miR-34a combined with VV-Smac synergistically inhibited tumor growth and induced apoptosis in vitro and in vivo. The underlying mechanism is proposed that blocking of Bcl-2 by VV-miR-34a increases the release of cytochrome c from mitochondria and then synergistically amplifies the antitumor effects of Smac-induced cell apoptosis. Our study is the first to utilize OVV as the vector for miR-34a or Smac expression to treat MM, and lays the groundwork for future clinical therapy for MM.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cytochromes c; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Mice, Inbred BALB C; MicroRNAs; Mitochondria; Mitochondrial Proteins; Multiple Myeloma; Oncolytic Virotherapy; Oncolytic Viruses; Vaccinia virus; Xenograft Model Antitumor Assays

Triptolide, a diterpenoid trioxide purified from the Chinese herb Tripterygium wilfordii Hook F, has been used as a natural medicine in China for hundreds of years. Several reports have demonstrated that triptolide inhibits the proliferation of cancer cells in vitro and reduces the growth of several types of tumors in vivo. To address the potential of triptolide as a novel therapeutic agent for patients with multiple myeloma, we investigated the effects of triptolide on the induction of apoptosis in human multiple myeloma cells in vitro. Triptolide rapidly induces apoptotic cell death in various myeloma cell lines. Triptolide-induced apoptosis in myeloma cells is associated with the loss of mitochondrial transmembrane potential (∆ψm), the release of cytochrome c and Smac/DIABLO from mitochondria into the cytosol, and the activation of caspase-3 and caspase-9. Furthermore, triptolide induces a rapid decline in the levels of Mcl-1 protein that correlates with caspase activation and induction of apoptosis. Inhibition of Mcl-1 synthesis by triptolide occurs at the level of mRNA transcription and is associated with an inhibition of phosphorylation of RNA polymerase II CTD. These results indicate that Mcl-1 is an important target for triptolide-induced apoptosis in myeloma cells that occurs via inhibition of Mcl-1 mRNA transcription coupled with rapid protein degradation through the ubiquitin-proteasome pathway.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Diterpenes; Epoxy Compounds; G1 Phase Cell Cycle Checkpoints; Humans; Intracellular Signaling Peptides and Proteins; Medicine, Chinese Traditional; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Proteins; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Phenanthrenes; Transcription, Genetic

Statins have been demonstrated to effectively inhibit proliferation and induce apoptosis in cancer cells by inhibition of geranylgeranylation, however its novel molecular mechanism remains to be determined. Recently simvastatin has been found to result in the synergistic induction of apoptosis with 7-hydroxystaurosporine (UCN-01) (a Chk1 inhibitor) in myeloma cells. Therefore we hypothesized that Chk1 plays a role in the anti-myeloma effect of simvastatin. Interestingly, we found that simvastatin caused a dose-dependent increase in S phase cell cycle and induced significant apoptosis. The results of western blot showed that simvastatin-induced S-phase cell cycle arrest was associated with activation of Chk1, downregulation of Cdc25A, cyclin A and CDK2 expression. Additionally, simvastatin-induced apoptosis was accompanied by diminished Bcl-2 protein expression, increased cytosolic cytochrome c level, and activation of caspase 9 and caspase 3. Further investigation revealed that silence of Chk1 expression by Chk1 specific siRNA inhibited simvastatin-induced activation of Chk1, downregulation of Cdc25A, cyclin A and CDK2 expression, and diminished S phase cell cycle arrest. Additionally, inhibition of Chk1 expression enhanced simvastatin-induced downregulation of Bcl-2, caspase 9 cleavage and subsequent apoptosis. These results suggested that the Chk1-Cdc25A-cyclin A/CDk2 pathway was involved in simvastatin-induced S-phase cell cycle arrest and apoptosis in multiple myeloma cell lines.

Topics: Apoptosis; Caspase 3; Caspase 9; cdc25 Phosphatases; Cell Line, Tumor; Cell Proliferation; Checkpoint Kinase 1; Cyclin A; Cyclin-Dependent Kinase 2; Cytochromes c; Down-Regulation; Enzyme Activation; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Mitochondria; Multiple Myeloma; Protein Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-bcl-2; S Phase Cell Cycle Checkpoints; Signal Transduction; Simvastatin

Multiple myeloma (MM) is a hematological malignancy characterized by the uncontrolled proliferation of clonal plasma cells in bone marrow in the elderly. Although there have been tremendous advances in the treatment of MM, it remains an incurable disease. Matrine, a main alkaloid of the traditional Chinese herb Sophora flavescens Ait, has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells. The aim of this study was to investigate the possibility of matrine as a novel therapeutic agent for patients with MM. We investigated the effects of matrine for its anti-myeloma activity in vitro, and further examined the mechanisms of apoptosis induced by matrine. Matrine inhibited the proliferation of human myeloma cell lines as well as freshly isolated myeloma cells from patients in a dose- and time-dependent manner. Matrine showed a potent induction of apoptosis of myeloma cells. Mitochondrial membrane potential (Deltapsim) was lost and cytochrome c (cyt c) was released from mitochondria to cytosol in myeloma cells treated by matrine for 24 h in a dose-dependent manner. The ratio of Bcl-2/Bax protein decreased, and the percentage of activated caspase-3 increased in myeloma cells treated by matrine for 48 h, but this matrine-induced activity of caspase-3 was completely canceled by the addition of Z-Asp(O-Me)-Glu(O-Me)-Val-Asp(O-Me) fluoromethyl ketone (Z-DEVD-FMK), a caspase-3 inhibitor. The addition of Z-DEVD-FMK partially blocked the apoptotic effect of matrine on myeloma cells. These data indicated that matrine could exert antiproliferative effects on myeloma cells and induce apoptosis of myeloma cells in vitro. The induction of apoptosis appeared to proceed via the mitochondrial pathway, including down-regulation of Bcl-2/Bax ratio, loss of Deltapsim, release of cyt c from mitochondria to cytosol, and activation of caspase-3. These findings support the view that matrine may be a useful candidate as a chemotherapeutic agent against MM.

Topics: Alkaloids; Anthelmintics; Apoptosis; Caspases; Cell Proliferation; Cytochromes c; Enzyme Activation; Humans; Matrines; Membrane Potential, Mitochondrial; Mitochondria; Multiple Myeloma; Proto-Oncogene Proteins c-bcl-2; Quinolizines; Signal Transduction; Tumor Cells, Cultured

Multiple myeloma (MM) is an incurable hematological malignancy with high incidence in the elderly. The currently used chemotherapeutic drugs show severe side effects, dose-limiting toxicity and development of resistance. In search of novel plant derived anti-cancer agents, Strychnos nux-vomica L. (SN) root extract was screened using the human MM-cell line, RPMI 8226. SN-extract exhibited anti-proliferative activity in a dose and time dependent manner. The morphological assessment of SN-extract treated cells showed significant features associated with apoptosis. Cell cycle analysis using flow cytometry of cells stained with propidium iodide revealed accumulation of cells at sub-G(0)/G(1) phase. In addition, disruption of mitochondrial membrane potential and subsequent leakage of mitochondrial cytochrome c was observed in SN-extract treated myeloma cells. The anti-proliferative and cytotoxic activity could be due to the alkaloids strychnine and brucine, which have been identified by LC-mass spectral analysis of the SN-extract in comparison to the reference standards analyzed under identical conditions.

Topics: Antineoplastic Agents, Phytogenic; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Flow Cytometry; Humans; Membrane Potential, Mitochondrial; Mitochondria; Multiple Myeloma; Plant Extracts; Plant Roots; Strychnine; Strychnos nux-vomica

TP-110, a new proteasome inhibitor, has previously shown potent growth inhibition in various tumor cell lines. In this study, the mechanism of TP-110-induced apoptosis is investigated in a human multiple myeloma cell line. Treatment with TP-110 for 24 h in vitro induced apoptosis in multiple myeloma cell line RPMI8226. Although the expression of Bcl-2, Bcl-xL and Bax was not affected by the treatment of TP-110, cleavage of Bid and release of cytochrome c were enhanced. Interestingly, TP-110 reduced the intrinsic inhibitor of apoptosis proteins (IAPs), cIAP-1 and XIAP, that suppress executioner caspases. The reduction of IAPs was observed not only by TP-110, but also by another proteasome inhibitor, MG-132. These results indicate that proteasome inhibitors reduce the level of IAPs and that the apoptosis induced by TP-110 is correlated with the level of IAPs in leukemia cell lines. Additionally, a reduction of cIAP-1 and XIAP by TP-110 contributes to the sensitization of Fas-mediated apoptosis. Taken together, the alteration of the apoptosis regulatory proteins by a proteasome inhibitor induces apoptosis in tumor cells.

Topics: Apoptosis; Caspases; Cytochromes c; Down-Regulation; Humans; Immunoblotting; Inhibitor of Apoptosis Proteins; Multiple Myeloma; Oligopeptides; Poly(ADP-ribose) Polymerases; Proteasome Inhibitors; Tumor Cells, Cultured

Dietary flavonoids have many health-promoting actions, including anticancer activity via proteasome inhibition. Bor-tezomib is a dipeptide boronate proteasome inhibitor that has activity in the treatment of multiple myeloma but is not effective in chronic lymphocytic leukemia (CLL). Although CLL cells are sensitive in vitro to bortezomib-induced apoptosis when cultured in medium, the killing activity was blocked when cultured in 50% fresh autologous plasma. Dietary flavonoids, quercetin and myricetin, which are abundant in plasma, inhibited bortezomib-induced apoptosis of primary CLL and malignant B-cell lines in a dose-dependent manner. This inhibitory effect was associated with chemical reactions between quercetin and the boronic acid group, -RB(OH)2, in bortezomib. The addition of boric acid diminished the inhibitory effect of both quercetin and plasma on bortezomib-induced apoptosis. The protective effect was also reduced when myeloma cell lines, but not B-cell lines, were preincubated with quercetin, indicating a direct effect of quercetin on myeloma cells. At high doses, quercetin itself induced tumor cell death. These data indicate that dietary flavonoids limit the efficacy of bortezomib, whereas supplemental inorganic boric acid is able to reverse this. The complex interactions between quercetin, tumor cells, and bortezomib mean caution is required when giving dietary advice to patients.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Boric Acids; Boronic Acids; Bortezomib; Cell Line, Transformed; Cell Line, Tumor; Cytochromes c; Diet; Flavonoids; Free Radical Scavengers; Humans; In Vitro Techniques; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Multiple Myeloma; Protease Inhibitors; Pyrazines; Quercetin

Interferon-alpha (IFN-alpha) has been used for the last 20 years in the maintenance therapy of multiple myeloma (MM), though it is only effective in some patients. Congruent with this, IFN-alpha induces apoptosis in some MM cell lines. Understanding the mechanism of IFN-alpha-induced apoptosis could be useful in establishing criteria of eligibility for therapy. Here we show that IFN-alpha-induced apoptosis in the MM cell lines U266 and H929 was completely blocked by a specific inhibitor of Jak1. The mTOR inhibitor rapamycin mitigated apoptosis in U266 but potentiated it in H929 cells. IFN-alpha induced PS exposure, DeltaPsi(m) loss and pro-apoptotic conformational changes of Bak, but not of Bax, and was fully prevented by Mcl-1 overexpression in U266 cells. IFN-alpha treatment caused the release of cytochrome c from mitochondria to cytosol and consequently, a limited proteolytic processing of caspases. Apoptosis induced by IFN-alpha was only slightly prevented by caspase inhibitors. Levels of the BH3-only proteins PUMA and Bim increased during IFN-alpha treatment. Bim increase and apoptosis was prevented by transfection with the siRNA for Bim. PUMA-siRNA transfection reduced electroporation-induced apoptosis but had no effect on apoptosis triggered by IFN-alpha. The potentiating effect of rapamycin on apoptosis in H929 cells was associated to an increase in basal and IFN-alpha-induced Bim levels. Our results indicate that IFN-alpha causes apoptosis in myeloma cells through a moderate triggering of the mitochondrial route initiated by Bim and that mTOR inhibitors may be useful in IFN-alpha maintenance therapy of certain MM patients.

Topics: Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Caspases; Cell Line, Tumor; Cell Nucleus; Cytochromes c; Drug Synergism; Enzyme Activation; Gene Silencing; Glutathione; Humans; Interferon-alpha; Janus Kinase 1; Membrane Proteins; Mitochondria; Multiple Myeloma; Protein Kinase Inhibitors; Protein Structure, Tertiary; Protein Transport; Proto-Oncogene Proteins; Sirolimus

Lycorine is a natural anti-tumor alkaloid extracted from Amaryllidaceae and has various biological effects on malignant cells. The present study explores the effects of lycorine on the human multiple meyloma cell line, KM3, and the possible mechanisms of these effects. An MTT assay showed that lycorine had significant inhibitory activity on KM3 cells. The growth rates of the KM3 cells exposed to lycorine evidently slowed down. Cell fluorescent apoptotic morphological changes, DNA degradation fragments, and a sub-G1 peak were detected, indicating the occurrence of cell apoptosis after lycorine treatment. Furthermore, the release of mitochondrial cytochrome c, the augmentation of Bax with the attenuation of Bcl-2, and the activation of caspase-9, -8, and -3 were also detected, suggesting that the mitochondrial pathway and the death acceptor pathway were also involved. The results also showed that lycorine was able to block the cell cycle at the G0/G1 phase through the downregulation of both cyclin D1 and CDK4. In summary, lycorine can suppress the proliferation of KM3 cells and reduce cell survival by arresting cell cycle progression as well as inducing cell apoptosis.

Topics: Amaryllidaceae Alkaloids; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cytochromes c; Gene Expression Regulation, Neoplastic; Humans; Multiple Myeloma; Phenanthridines; Plant Extracts; Proto-Oncogene Proteins c-bcl-2

Bcl family members Bcl-2, Bcl-x(L), and Mcl-1, are frequently expressed and implicated in the survival of myeloma cells. Obatoclax (GX015-070) is a novel, small-molecule antagonist of the BH3-binding groove of the Bcl family of proteins. We show that GX015-070 inhibits the binding of Bak to Mcl-1, up-regulates Bim, induces cytochrome c release, and activates capase-3 in human myeloma cell lines (HMCLs), confirming the predicted mechanism of action. Consequently, GX015-070 potently inhibited the viability of 15 of 16 HMCLs (mean IC(50) of 246 nM), including those resistant to melphalan and dexamethasone. In combination studies, GX015-070 enhanced the antimyeloma activity induced by melphalan, dexamethasone, or bortezomib. Sensitivity to GX015-070 correlated with the absence or near absence of Bcl-x(L). Coculture with interleukin-6 or adherence to bone marrow stroma conferred modest resistance; however, it did not overcome GX015-070-induced cytotoxicity. Of importance, GX015-070 as a single agent induced potent cytotoxic responses against patient-derived tumor cells. GX015-070 inhibited normal bone marrow-derived colony formation; however, cytotoxicity to human blood lymphocytes was not observed. Taken together, these studies describe a novel BH3 mimic with selectivity for Mcl-1, and support the therapeutic application of GX015-070 for diverse neoplasias including multiple myeloma.

Topics: Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; Bcl-2-Like Protein 11; Caspase 3; Cell Line, Tumor; Cytochromes c; Drug Screening Assays, Antitumor; Humans; Indoles; Membrane Proteins; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Up-Regulation

The effectiveness of IFN-alpha2b for human multiple myeloma has been variable. TRAIL has been proposed to mediate IFN-alpha2b apoptosis in myeloma. In this study we assessed the effects of IFN-alpha2b signaling on the apoptotic activity of TRAIL and human myeloma cell survival. While TRAIL was one of the most potently induced proapoptotic genes in myeloma cells following IFN-alpha2b treatment, less than 20% of myeloma cells underwent apoptosis. Thus, we hypothesized that an IFN-stimulated gene (ISG) with prosurvival activity might suppress TRAIL-mediated apoptosis. Consistent with this, IFN-alpha2b stabilized mitochondria and inhibited caspase-3 activation, which antagonized TRAIL-mediated apoptosis and cytotoxicity after 24 hours of cotreatment in cell lines and in fresh myeloma cells, an effect not evident after 72 hours. Induced expression of G1P3, an ISG with largely unknown function, was correlated with the antiapoptotic activity of IFN-alpha2b. Ectopically expressed G1P3 localized to mitochondria and antagonized TRAIL-mediated mitochondrial potential loss, cytochrome c release, and apoptosis, suggesting specificity of G1P3 for the intrinsic apoptosis pathway. Furthermore, RNAi-mediated downregulation of G1P3 restored IFN-alpha2b-induced apoptosis. Our data identify the direct role of a mitochondria-localized prosurvival ISG in antagonizing the effect of TRAIL. Curtailing G1P3-mediated antiapoptotic signals could improve therapies for myeloma or other malignancies.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Cytochromes c; Gene Expression Regulation; Gene Expression Regulation, Leukemic; Humans; Interferon alpha-2; Interferon-alpha; Mitochondria; Mitochondrial Proteins; Multiple Myeloma; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand

Despite major advances, multiple myeloma (MM) remains an incurable malignancy. Recently we have found that disease stabilization was achieved in 64% of patients with advanced MM treated with the farnesyltransferase inhibitor R115777 (Zarnestra) in a phase 2 clinical trial. In order to enhance R115777 antitumor activity in MM, we examined the combination of this novel agent with other anticancer drugs in MM cell lines. In this study, R115777 was found to synergize with paclitaxel and docetaxel, but not with other chemotherapy agents, including doxorubicin, 5-fluorouracil, cisplastin, melphalan, mitoxantrone, and dexamethasone. R115777 synergized with paclitaxel to inhibit MM cell proliferation and to induce apoptosis. Synergism in the induction of apoptosis was accompanied by increase in cytochrome c release and caspase-3 activation. Furthermore, flow cytometry analysis also showed that paclitaxel and R115777 synergized to induce G(2)/M cell-cycle arrest. Importantly, synergism was observed in taxane- and R115777-resistant MM cells. In the human severe combined immunodeficient (SCID-hu) bone model of myeloma growth, the ability of paclitaxel to inhibit tumor growth in vivo was enhanced by R115777. Combination of paclitaxel or docetaxel with R115777 in the treatment of MM cells from patients with multiple myeloma was more beneficial than treatment with single agents. Our results provide the basis for combination therapy clinical trials with paclitaxel or docetaxel with R115777 in MM patients.

Topics: Alkyl and Aryl Transferases; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Bone Marrow Cells; Caspase 3; Caspases; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Proliferation; Cisplatin; Clinical Trials as Topic; Cytochromes c; Deoxycytidine; Docetaxel; Dose-Response Relationship, Drug; Doxorubicin; Drug Synergism; Enzyme Activation; Farnesyltranstransferase; Flow Cytometry; Fluorouracil; G2 Phase; Gemcitabine; Homozygote; Humans; In Situ Nick-End Labeling; Inhibitory Concentration 50; Mice; Mice, SCID; Mitosis; Multiple Myeloma; Paclitaxel; Quinolones; Taxoids; Tetrazolium Salts; Thiazoles

The purpose of this study was to characterize interactions between the farnesyltransferase inhibitor L744832 and the checkpoint abrogator UCN-01 in drug-sensitive and drug-resistant human myeloma cell lines and primary CD138+ multiple myeloma cells.. Wild-type and drug-resistant myeloma cell lines were exposed to UCN-01 +/- L744832 for 24 hours, after which mitochondrial injury, caspase activation, apoptosis, and various perturbations in signaling and survival pathways were monitored.. Simultaneous exposure of myeloma cells to marginally toxic concentrations of L744832 and UCN-01 resulted in a synergistic induction of mitochondrial damage, caspase activation, and apoptosis, associated with activation of p34cdc2 and c-Jun-NH2-kinase and inactivation of extracellular signal-regulated kinase, Akt, GSK-3, p70(S6K), and signal transducers and activators of transcription 3 (STAT3). Enhanced lethality for the combination was also observed in primary CD138+ myeloma cells, but not in their CD138- counterparts. L744832/UCN-01-mediated lethality was not attenuated by conventional resistance mechanisms to cytotoxic drugs (e.g., melphalan or dexamethasone), addition of exogenous interleukin-6 or insulin-like growth factor-I, or the presence of stromal cells. In contrast, enforced activation of STAT3 significantly protected myeloma cells from L744832/UCN-01-induced apoptosis.. Coadministration of the farnesyltransferase inhibitor L744832 promotes UCN-01-induced apoptosis in human multiple myeloma cells through a process that may involve perturbations in various survival signaling pathways, including extracellular signal-regulated kinase, Akt, and STAT3, and through a process capable of circumventing conventional modes of myeloma cell resistance, including growth factor- and stromal cell-related mechanisms. They also raise the possibility that combined treatment with farnesyltransferase inhibitors and UCN-01 could represent a novel therapeutic strategy in multiple myeloma.

Topics: Alkyl and Aryl Transferases; Antineoplastic Agents; Apoptosis; Blotting, Western; CDC2 Protein Kinase; Cell Line, Tumor; Cytochromes c; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Synergism; Farnesyltranstransferase; Glycogen Synthase Kinase 3; Humans; JNK Mitogen-Activated Protein Kinases; Methionine; Multiple Myeloma; Phosphorylation; Ribosomal Protein S6 Kinases, 70-kDa; STAT3 Transcription Factor; Staurosporine; Trans-Activators

The precise molecular mechanism underlying arsenic trioxide (As(2)O(3))-induced apoptosis is a subject of extensive study. Here, we show that clinically relevant doses of As(2)O(3) can induce typical apoptosis in IM-9, a multiple myeloma cell line, in a Bcl-2 inhibitable manner. We confirmed that As(2)O(3) directly induced cytochrome c (cyto c) release from isolated mouse liver mitochondria via the mitochondrial permeability transition pore, and we further identified the voltage-dependent anion channel (VDAC) as a biological target of As(2)O(3) responsible for eliciting cyto c release in apoptosis. First, pretreatment of the isolated mitochondria with an anti-VDAC antibody specifically prevented As(2)O(3)-induced cyto c release. Second, in proteoliposome experiments, VDAC by itself was sufficient to mediate As(2)O(3)-induced cyto c release, which could be specifically inhibited by Bcl-X(L). Third, As(2)O(3) induced mitochondria membrane potential (DeltaPsim) reduction and cyto c release only in the VDAC-expressing, but not in the VDAC-deficient yeast strain. Finally, we found that As(2)O(3) induced the increased expression and homodimerization of VDAC in IM-9 cells, but not in Bcl-2 overexpressing cells, suggesting that VDAC homodimerization could potentially determine its gating capacity to cyto c, and Bcl-2 blockage of VDAC homodimerization represents a novel mechanism for its inhibition of apoptosis.

Topics: Animals; Annexin A5; Arsenic Trioxide; Arsenicals; Cytochromes c; Flow Cytometry; Humans; Intracellular Membranes; Ion Channel Gating; Membrane Potentials; Mice; Mice, Inbred BALB C; Mitochondria; Mitochondria, Liver; Multiple Myeloma; Oxides; Permeability; Porins; Proteolipids; Tumor Cells, Cultured; Voltage-Dependent Anion Channels

Bortezomib (PS-341), a selective inhibitor of proteasomes, induces apoptosis in multiple myeloma (MM) cells; however, prolonged drug exposure may result in cumulative toxicity and the development of chemoresistance. Here we show that combining PK-11195 (PK), an antagonist to mitochondrial peripheral benzodiazepine receptors (PBRs), with bortezomib triggers synergistic anti-MM activity even in doxorubicin-, melphalan-, thalidomide-, dexamethasone-, and bortezomib-resistant MM cells. No significant cytotoxicity was noted in normal lymphocytes. Low-dose combined PK and bortezomib treatment overcomes the growth, survival, and drug resistance conferred by interleukin-6 or insulin growth factor within the MM bone marrow milieu. The mechanism of PK + bortezomib-induced apoptosis includes: loss of mitochondrial membrane potential; superoxide generation; release of mitochondrial proteins cytochrome-c (cyto-c) and Smac; and activation of caspases-8/-9/-3. Furthermore, PK + bortezomib activates c-Jun NH2 terminal kinase (JNK), which translocates to mitochondria, thereby facilitating release of cyto-c and Smac from mitochondria to cytosol. Blocking JNK, by either dominant-negative mutant (DN-JNK) or cotreatment with a specific JNK inhibitor SP600125, abrogates both PK + bortezomib-induced release of cyto-c/Smac and induction of apoptosis. Together, these preclinical studies suggest that combining bortezomib with PK may enhance its clinical efficacy, reduce attendant toxicity, and overcome conventional and bortezomib resistance in patients with relapsed refractory MM.

Topics: Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Caspases; Cell Survival; Cytochromes c; Drug Resistance, Neoplasm; Drug Synergism; GABA-A Receptor Antagonists; Humans; Insulin-Like Growth Factor I; Interleukin-6; Isoquinolines; JNK Mitogen-Activated Protein Kinases; Lymphocytes; Membrane Potentials; Mitochondria; Multiple Myeloma; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Stromal Cells; Superoxides; Tumor Cells, Cultured

Interactions between the cyclin-dependent kinase inhibitor flavopiridol (FP) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L), were examined in human leukemia cells (U937 and Jurkat). Coexposure of cells to marginally toxic concentrations of TRAIL and FP (24 h) synergistically increased mitochondrial injury (eg, cytochrome c, AIF, Smac/DIABLO release), cytoplasmic depletion of Bax, activation of Bid as well as caspase-8 and -3, PARP cleavage, and apoptosis. Coadministration of TRAIL markedly increased FP-induced apoptosis in leukemic cells ectopically expressing Bcl-2, Bcl-x(L), or a phosphorylation loop-deleted form of Bcl-2 (DeltaBcl-2), whereas lethality was substantially attenuated in cells ectopically expressing CrmA, dominant-negative-FADD, or dominant-negative-caspase-8. TRAIL/FP induced no discernible changes in FLIP, DR4, DR5, Mcl-1, or survivin expression, modest declines in levels of DcR2 and c-IAP, but resulted in the marked transcriptional downregulation of XIAP. Moreover, cells stably expressing an XIAP-antisense construct exhibited a pronounced increase in TRAIL sensitivity comparable to degrees of apoptosis achieved with TRAIL/FP. Conversely, enforced XIAP expression significantly attenuated caspase activation and TRAIL/FP lethality. Together, these findings suggest that simultaneous activation of the intrinsic and extrinsic apoptotic pathways by TRAIL and FP synergistically induces apoptosis in human leukemia cells through a mechanism that involves FP-mediated XIAP downregulation.

Topics: Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspases; Cell Cycle; Complement Membrane Attack Complex; Complement System Proteins; Cytochromes c; Down-Regulation; Drug Interactions; Drug Synergism; Flavonoids; Flavoproteins; Glycoproteins; HL-60 Cells; Humans; Intracellular Signaling Peptides and Proteins; Jurkat Cells; Leukemia; Membrane Glycoproteins; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Multiple Myeloma; Phosphorylation; Piperidines; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; TNF-Related Apoptosis-Inducing Ligand; Transcription, Genetic; Tumor Necrosis Factor-alpha; U937 Cells; X-Linked Inhibitor of Apoptosis Protein

2-(8-Hydroxy-6-methoxy-1-oxo-1Eta-2-benzopyran-3-yl)propionic acid (NM-3) is a small molecule isocoumarin derivative that has recently entered clinical trials as an orally bioavailable anticancer agent. NM-3 induces lethality of human carcinoma cells by both apoptotic and nonapoptotic mechanisms and potentiates the effects of cytotoxic chemotherapeutic agents. The present studies have evaluated the effects of NM-3 on human multiple myeloma (MM) cells. The results demonstrate that NM-3 potentiates dexamethasone-induced killing of both dexamethasone-sensitive MM1.S and dexamethasone-resistant RPMI8226 and U266 MM cells. We show that NM-3 enhances dexamethasone-induced release of the mitochondrial apoptogenic factors cytochrome c and Smac/DIABLO. The results also demonstrate that NM-3 enhances dexamethasone-induced activation of the intrinsic caspase-9->caspase-3 apoptotic pathway. In concert with these results, NM-3 potentiates dexamethasone-induced apoptosis of MM1.S cells. Moreover, NM-3 acts synergistically with dexamethasone in inducing apoptosis of the dexamethasone-resistant RPMI8226 and U266 MM cells. These findings indicate that NM-3 may be effective in combination with dexamethasone in the treatment of MM.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Carrier Proteins; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Coumarins; Cytochromes c; Dexamethasone; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; Isocoumarins; Mitochondria; Mitochondrial Proteins; Multiple Myeloma; Reactive Oxygen Species

Interactions between the cyclin-dependent kinase inhibitor flavopiridol and the small-molecule Bcl-2 antagonist HA14-1 were examined in human multiple myeloma cells. Whereas individual treatment of U266 myeloma cells with 10 micromol/L HA14-1 or 100 nmol/L flavopiridol had little effect, exposure of cells to flavopiridol (6 hours) followed by HA14-1 (18 hours) resulted in a striking increase in mitochondrial dysfunction (cytochrome c and Smac/DIABLO release; loss of mitochondrial membrane potential), activation of the caspase cascade, apoptosis, and diminished clonogenic survival. Similar findings were noted in other myeloma cell lines (e.g., MM.1S, RPMI8226, and NCI-H929) as well as in those resistant to dexamethasone and cytotoxic agents (e.g., MM.1R, 8226/Dox40, and 8226/LR5). Combined exposure to flavopiridol and HA14-1 was associated with down-regulation of Mcl-1 and Bcl-xL, Bid cleavage, and mitochondrial translocation of Bax. Flavopiridol/HA14-1-treated cells also exhibited a pronounced activation of Jun NH2-terminal kinase, a modest activation of p38 mitogen-activated protein kinase, and down-regulation of cyclin D1. Flavopiridol/HA14-1-induced apoptosis was associated with a marked increase in reactive oxygen species generation; moreover,both events were attenuated by the antioxidant N-acetyl-l-cysteine. Finally, in contrast to dexamethasone, flavopiridol/HA14-1-induced lethality was unaffected by exogenous interleukin-6 or insulin-like growth factor-I. Together, these findings indicate that flavopiridol and the small-molecule Bcl-2 antagonist HA14-1 cooperate to trigger oxidant injury, mitochondrial dysfunction, caspase activation, and apoptosis in human multiple myeloma cells and suggest that this approach may warrant further evaluation as an antimyeloma strategy.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Benzopyrans; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Cyclin D1; Cyclin-Dependent Kinases; Cytochromes c; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Free Radicals; Humans; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Nitriles; p38 Mitogen-Activated Protein Kinases; Piperidines; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

Mitochondria play central roles in cellular metabolism and apoptosis and are a major source of reactive oxygen species (ROS). We investigated the role of ROS and mitochondria in radiation-induced apoptosis in multiple myeloma cells. Two distinct levels of ROS were generated following irradiation: a small increase observed early, and a pronounced late increase, associated with depletion of reduced glutathione (GSH) and collapse of mitochondrial membrane potential (deltapsi(m)). Exogenous ROS and caspase-3 induced deltapsi(m) drop and cytochrome c release from mitochondria, which could be prevented by molecular (dominant-negative caspase-9) and pharmacologic (zVAD-fmk) caspase inhibitors and overexpression of Bcl-2. Exogenous ROS also induced mitochondrial permeability transition (PT) pore opening and cytochrome c release in isolated mitochondria, which could be blocked by inhibition of PT with cyclosporin A. These results indicate that the late ROS production is associated with increased PT pore opening and decreased deltapsi(m), and GSH, events associated with caspase activation and cytochrome c release.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 3; Caspase 9; Caspases; Cell Death; Cell Line, Tumor; Cell-Free System; Chromatography, High Pressure Liquid; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; Genes, Dominant; Glutathione; Humans; Immunoblotting; Membrane Potentials; Mitochondria; Multiple Myeloma; Oxidative Stress; Reactive Oxygen Species; Subcellular Fractions; Time Factors