nutlin-3a and Multiple-Myeloma

To investigate the role of p53 on ran transcription in myeloma cells.. Using real-time fluorescence quantitative PCR, the ran transcription level was measured in 8 human myeloma cell lines such as OPM-2, RPMI-8226, U-266, KAS6/1, ANML-6, H-929, MM1.S and MOLP-8. The ran transcription level and P53 expression were detected by Q-PCR in MM1.S treated with Nutlin-3a for 24, 48 and 72 hours, respectively. The Western blot was used to detect the expression levels of ran and P53 proteins, and ran expression level after transfection of MM1.S cells using different concentration of plasmids which express the P53 luciferase reporter.. H-929 and MM1.S cells showed the highest ran transcription level among the above-mentioned 8 cell lines (P<0.05). After treatment with Nutlin-3a, ran transcription level in MM1.S cells decreased (P<0.05), (r=-1.00, P=0.04) and P53 expression increased (r=1.00, P=0.06) in time-dependence manner. The detection by p53 luciferase reporter showed that the ran transcription decreased and the plasmid increased to 25 ng (P<0.05).. This study demonstrated that ran is a target gene regulated by P53 in myeloma cells for the first time.

Topics: Cell Line, Tumor; Humans; Imidazoles; Multiple Myeloma; Piperazines; ran GTP-Binding Protein; Tumor Suppressor Protein p53

Myeloma cells are sensitive to TRAIL through the two death receptors DR4 and DR5. Because p53 directly modulates expression of death receptors, we investigated here whether p53 can modulate myeloma sensitivity to TRAIL. We found that p53 affects the sensitivity of myeloma cells to the DR5 agonistic human antibody lexatumumab but not the DR4 antibody mapatumumab. TP53 wild-type myeloma cells overexpressed DR5 in correlation with sensitivity to lexatumumab. Both nongenotoxic (nutlin-3a) and genotoxic (melphalan) p53-inducing stresses increased DR5 expression only in TP53 wild-type cells and synergistically increased lexatumumab efficiency yet did not increase DR4 expression, nor sensitivity to mapatumumab. Silencing of p53 strongly decreased DR5 expression and induced resistance to nutlin-3a and lexatumumab but did not modulate DR4 expression or sensitivity to mapatumumab. Increase of lexatumumab efficiency induced by nutlin-3a was related to a p53-dependent increase of DR5 expression. In primary myeloma cells, nutlin-3a increased DR5 expression and lexatumumab efficiency but did not increase mapatumumab efficiency. Taken together, our findings indicate that p53 controls the sensitivity of myeloma through DR5 but not DR4 and suggest that a subset of patients with multiple myeloma may benefit from DR5 therapy.

Topics: Antibodies, Monoclonal; Caspase 8; Cell Death; Cell Line, Tumor; Chromosome Deletion; Chromosomes, Human, Pair 17; Drug Resistance, Neoplasm; Enzyme Activation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Imidazoles; Multiple Myeloma; Piperazines; Receptors, TNF-Related Apoptosis-Inducing Ligand; Stress, Physiological; Tumor Suppressor Protein p53

Multiple myeloma (MM) is an incurable plasma cell malignancy in which p53 is rarely mutated. Thus, activation of the p53 pathway by a small molecule inhibitor of the p53-MDM2 interaction, nutlin, in MM cells retaining wild type p53 is an attractive therapeutic strategy. Recently we reported that nutlin plus velcade (a proteasome inhibitor) displayed a synergistic response in MM. However, the mechanism of the p53-mediated apoptosis in MM has not been fully understood. Our data show that nutlin-induced apoptosis correlated with reduction in cell viability, upregulation of p53, p21 and MDM2 protein levels with a simultaneous increase in pro-apoptotic targets PUMA, Bax and Bak and downregulation of anti-apoptotic targets Bcl2 and survivin and activation of caspase in MM cells harboring wild type p53. Nutlin-induced apoptosis was inhibited when activation of caspase was blocked by the caspase inhibitor. Nutlin caused mitochondrial translocation of p53 where it binds with Bcl2, leading to cytochrome C release. Moreover, blocking the transcriptional arm of p53 by the p53-specific transcriptional inhibitor, pifithrin-α, not only inhibited nutlin-induced upregulation of p53-transcriptional targets but also augmented apoptosis in MM cells, suggesting an association of transcription-independent pathway of apoptosis. However, inhibitor of mitochondrial translocation of p53, PFT-μ, did not prevent nutlin-induced apoptosis, suggesting that the p53 transcription-dependent pathway was also operational in nutlin-induced apoptosis in MM. Our study provides the evidence that nutlin-induced apoptosis in MM cells is mediated by transcription-dependent and -independent pathways and supports further clinical evaluation of nutlin as a novel therapeutic agent in MM.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Benzothiazoles; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Immunoblotting; Multiple Myeloma; Piperazines; Protein Binding; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sulfonamides; Toluene; Transcription, Genetic; Tumor Suppressor Protein p53

Topics: Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Immunoblotting; Multiple Myeloma; Piperazines; Protein Binding; Protein Transport; Proto-Oncogene Proteins c-mdm2; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transcription, Genetic; Tumor Suppressor Protein p53

p53 is inactivated in many human malignancies through missense mutations or overexpression of the human homologue of Mdm2 (Hdm2), an E3 ubiquitin ligase that ubiquitinates p53, thereby promoting its proteasomal degradation. The cis-imidazoline nutlin-3 can disrupt the p53-Hdm2 interaction and activate p53, inducing apoptosis in vitro in many malignancies, including multiple myeloma (MM).. We hypothesized that suppression of Hdm2-mediated p53 ubiquitination may augment sequelae of p53 accumulation caused by proteasomal inhibition. We compared the response of MM cells versus several epithelial cancer models to the proteasome inhibitor bortezomib in combination with nutlin-3.. The combination of sublethal concentrations of bortezomib plus nutlin-3 induced additive cytotoxicity against bortezomib-sensitive MM cell lines. Importantly, however, in breast, prostate, colon, and thyroid (papillary, follicular, anaplastic, and medullary) carcinoma cell lines, this combination triggered synergistic cytotoxicity, and increased expression of p53, p21, Hdm2, Bax, Noxa, PUMA, and cleavage of caspase-3 and poly ADP ribose polymerase. Coculture with bone marrow stromal cells attenuated MM cell sensitivity to nutlin-3 monotherapy and was associated with evidence of suppression of p53 activity in MM cells, whereas combined bortezomib-nutlin-3 treatment maintained cytotoxicity even in the presence of bone marrow stromal cells.. This differential response of MM versus epithelial carcinomas to combination of nutlin-3 with bortezomib sheds new light on the role of p53 in bortezomib-induced apoptosis. Concurrent Hdm2 inhibition with bortezomib may extend the spectrum of bortezomib applications to malignancies with currently limited sensitivity to single-agent bortezomib or, in the future, to MM patients with decreased clinical responsiveness to bortezomib-based therapy.

Topics: Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Survival; Humans; Imidazoles; Multiple Myeloma; Mutation, Missense; Neoplasms, Glandular and Epithelial; Piperazines; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-mdm2; Pyrazines; Tumor Suppressor Protein p53

Mutation of p53 is a rare event in multiple myeloma, but it is unknown if p53 signaling is functional in myeloma cells, and if targeted nongenotoxic activation of the p53 pathway is sufficient to kill tumor cells. Here, we demonstrate that treatment of primary tumor samples with a small-molecule inhibitor of the p53-murine double minute 2 (MDM2) interaction increases the level of p53 and induces p53 targets and apoptotic cell death. Significantly, given the importance of the bone marrow microenvironment for the support and drug resistance of myeloma cells, tumor cells undergo effective apoptosis also in the presence of stromal cells, which themselves appear to tolerate exposure to nutlin-3. The in vitro toxicity of nutlin-3 was similar to that of the genotoxic drug melphalan. Because nutlin-mediated p53 activation is not dependent on DNA damage, MDM2 antagonists may help to avoid or reduce the severe genotoxic side effects of chemotherapeutic agents currently used to treat multiple myeloma. Therefore, MDM2 antagonists may offer a new treatment option for this disease.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Cell Line, Tumor; DNA Damage; Humans; Imidazoles; Melphalan; Mice; Multiple Myeloma; Mutagens; Piperazines; Protein Binding; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Stromal Cells; Tumor Suppressor Protein p53