trichostatin-a and Multiple-Myeloma

The antiparasitic clioquinol (CQ) represents a class of novel anticancer drugs by interfering with proteasome activity. In the present study, we found that CQ induced blood cancer cell apoptosis by inhibiting histone deacetylases (HDACs). CQ accumulated the acetylation levels of several key proteins including histone H3 (H3), p53, HSP90, and α-tubulin. In the mechanistic study, CQ was found to down-regulate HDAC1, -3, -4, and -5 in both myeloma and leukemia cells. Computer modeling analysis revealed that CQ was well docked into the active pocket of the enzyme, where the oxygen and nitrogen atoms in CQ formed stable coordinate bonds with the zinc ion, and the hydroxyl group from CQ formed an effective hydrogen bond with Asp-267. Moreover, co-treatment with CQ and zinc/copper chloride led to decreased Ac-H3. Furthermore, CQ inhibited the activity of Class I and IIa HDACs in the cell-free assays, demonstrating that CQ interfered with HDAC activity. By inhibiting HDAC activity, CQ induced expression of p21, p27, and p53, cell cycle arrest at G1 phase, and cell apoptosis. This study suggested that the HDAC enzymes are targets of CQ, which provided a novel insight into the molecular mechanism of CQ in the treatment of hematological malignancies.

Topics: Antipruritics; Apoptosis; Chlorides; Clioquinol; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Leukemic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; K562 Cells; Leukemia; Male; Mouthwashes; Multiple Myeloma; Neoplasm Proteins; U937 Cells; Zinc Compounds

BACKGROUND The aim of this study was to explore the impact of Ras homolog C/Rho-associated coiled-protein kinase (Rho/ROCK) signaling pathways intervention on biological characteristics of the human multiple myeloma cell lines RPMI-8226 and U266 cells, and to investigate the expression of RhoC, ROCK1, and ROCK2 in RPMI-8226 and U266 cells. MATERIAL AND METHODS RPMI8226 and U266 cell lines were treated by 5-aza-2-deoxycytidine (5-Aza-Dc), trichostatin A (TSA), RhoA inhibitor CCG-1423, Rac1 inhibitor NSC23766, and ROCK inhibitor fasudil. Cell proliferation was examined by Cell Counting Kit-8 (CCK-8) assay and clone formation. Cell apoptosis was examined by flow cytometry and TUNEL assay. The mRNA and protein expressions of RhoC, ROCK1, and ROCK2 were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot, respectively. RESULTS CCG-1423, NSC23766, and fasudil could significantly inhibit the proliferation of RPMI8226 and U266 cells. The inhibitory effect was dose- and time-dependent within a certain concentration range (P<0.05). After treatment with CCG-1423, NSC23766, and fasudil for 24 hours, the apoptosis rates of RPMI8226 and U266 cells were significantly higher than those of the control group, which were dose-dependent (P<0.05). Compared with the control group, the mRNA and protein expressions of RhoC, ROCK1, and ROCK2 in RPMI8226 and U266 cells were significantly decreased with single 5-Aza-Dc or TSA treatment. However, the effects were obviously stronger after combined treatment of 5-Aza-CdR and TSA (P<0.05). CONCLUSIONS We found that 5-Aza-Dc and TSA can effectively decrease the mRNA and protein expressions of RhoC, ROCK1, and ROCK2. Furthermore, Rho and ROCK inhibitors significantly inhibit cell growth and induce cell apoptosis in the human multiple myeloma cell lines RPMI-8226 and U266.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Apoptosis; Azacitidine; Cell Line, Tumor; Cell Proliferation; Humans; Hydroxamic Acids; Multiple Myeloma; Protein Kinase Inhibitors; rho GTP-Binding Proteins; rho-Associated Kinases; RNA, Messenger; Signal Transduction

A series of bicyclic arylamino/heteroarylamino hydroxamic acids (7-31) have been examined as novel histone deacetylase 6 (HDAC6) inhibitors. One compound (13) exhibits remarkable inhibitory activity of HDAC6 with an IC

Topics: Animals; Antineoplastic Agents; Caco-2 Cells; Cell Proliferation; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Humans; Multiple Myeloma; Quinolines; Rats

This study was aimed to investigate the effects of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) and histone deacetylase inhibitor trichostatin A (TSA) on DLC-1 gene transcription regulation and molecular biological behaviours in the human multiple myeloma RPMI-8226 cells. The cells were treated respectively with 5-Aza-CdR and TSA alone, or the both combination; the cell proliferation and apoptosis, DLC-1 expression, the protein expression of Ras homolog family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac1) were examined by CCK-8 method, RT-PCR and ELISA, respectively. The results showed that the 5-Aza-CdR and TSA had cell growth inhibitory and apoptosis-inducing effects in dose-dependent manner (P < 0.05). Compared with a single drug (5-Aza-CdR or TSA alone), the effects were significantly enhanced after treatment with the combination of 5-Aza-CdR and TSA (P < 0.05). DLC-1 was weakly expressed in the control group; the treatment with 5-Aza-CdR alone enhanced its re-expression dose-dependently (P < 0.05). Compared with 5-Aza-CdR alone, 5-Aza-CdR plus TSA enhanced DLC-1 re-expression significantly.Compared with the control, 5-Aza-CdR and TSA significantly decreased RhoA and Rac1 protein expression (P < 0.05). It is concluded that 5-Aza-CdR and TSA can effectively reverse DLC-1 expression of RPMI-8226 cells; TSA has a synergistic effect on its re-expression. 5-Aza-CdR and TSA have significant cell growth inhibitory and apoptosis-inducing effects on RPMI-8226 cells. These effects may be related to the inhibition of Rho/Rho kinase signalling pathway.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Azacitidine; Cell Line, Tumor; Cell Proliferation; Decitabine; Gene Expression; GTPase-Activating Proteins; Humans; Hydroxamic Acids; Multiple Myeloma; Tumor Suppressor Proteins

This study evaluates the role of scavenger receptor class A member 3 (SCARA3) in multiple myeloma (MM). SCARA3 expression was induced upon treatment with oxidative stressors (ionizing radiation and chemotherapeutic drugs). An epigenetic inactivation of SCARA3 was noted in MM.1S myeloma cells. Myeloma cell killing by dexamethasone and bortezomib was inhibited by up-regulation of SCARA3 while SCARA3 knockdown sensitized myeloma cells to the drugs. Clinical samples showed an inverse correlation between SCARA3 gene expression, myeloma progression, and favorable clinical prognosis. In MM, SCARA3 protects against oxidative stress-induced cell killing and can serve as predictor of MM progression and therapeutic response.

Topics: Antineoplastic Agents; Azacitidine; Boronic Acids; Bortezomib; Cell Line; Cell Line, Tumor; Decitabine; Dexamethasone; Disease Progression; Drug Resistance, Neoplasm; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Heat-Shock Proteins; Humans; Hydrogen Peroxide; Hydroxamic Acids; Multiple Myeloma; Oligonucleotide Array Sequence Analysis; Oxidants; Prognosis; Pyrazines; Radiation, Ionizing; Reverse Transcriptase Polymerase Chain Reaction; Scavenger Receptors, Class A

Multiple myeloma (MM) is still a fatal plasma cell cancer. Novel compounds are currently clinically tested as a single agent in relapsing patients, but in best cases with partial response of a fraction of patients, emphasising the need to design tools predicting drug efficacy. Histone deacetylase inhibitors (HDACi) are anticancer agents targeting epigenetic regulation of gene expression and are in clinical development in MM.. To create a score predicting HDACi efficacy, five MM cell lines were treated with trichostatin A (TSA) and gene expression profiles were determined.. The expression of 95 genes was found to be upregulated by TSA, using paired supervised analysis with Significance Analysis of Microarrays software. Thirty-seven of these 95 genes had prognostic value for overall survival in a cohort of 206 newly diagnosed MM patients and their prognostic information was summed up in a histone acetylation score (HA Score); patients with the highest HA Score had the shorter overall survival. It is worth noting that MM cell lines or patients' primary MM cells with a high HA Score had a significant higher sensitivity to TSA, valproic acid, panobinostat or vorinostat.. In conclusion, the HA Score allows identification of MM patients with poor survival, who could benefit from HDACi treatment.

Topics: Acetylation; Cohort Studies; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Male; Multiple Myeloma; Neoplasm Grading; Predictive Value of Tests; Transcriptome; Up-Regulation

A disintegrin‑like and metalloprotease with thrombospondin type Ⅰ motifs (ADAMTS) are a family of 19 secreted mammalian metalloproteases. ADAMTS9 was reported to be a novel tumor suppressor gene and is downregulated in various types of human cancer due to hypermethylation of promoter CpG islands. In the present study, the silencing mechanism of the ADAMTS9 gene was analyzed in the multiple myeloma (MM) cell lines, KM3 and RPMI‑8226. Control and MM samples were obtained by conventional bone marrow (BM) biopsy of normal and MM adult BM, respectively. RT‑PCR revealed a high expression of the ADAMTS9 gene in normal samples and RPMI‑8226 cells while marked gene silencing of ADAMTS9 was observed in MM patients and KM3 cells. Promoter methylation of ADAMTS9 was detected in the KM3 cell line and 66% (37/56) MM patients by methylation‑specific PCR. In addition, the DNA demethylating agent, 5‑aza‑2'‑deoxycytidine and trichostatin A restored ADAMTS9 expression by suppressing promoter methylation in KM3 cells. Ectopic expression of ADAMTS9 in ADAMTS9‑silenced MM cells was found to significantly suppress cell colony formation and proliferation. In the present study, DNA methylation was found to play a key role in ADAMTS9 gene silencing and the biological behavior of myeloma cells. The results demonstrate that ADAMTS9 silencing by methylation may be a novel tumor marker for MM and the applicability of demethylating agents in the treatment of MM.

Topics: ADAM Proteins; ADAMTS9 Protein; Adult; Aged; Azacitidine; Cell Line, Tumor; Cell Proliferation; CpG Islands; Decitabine; DNA Methylation; Down-Regulation; Female; Humans; Hydroxamic Acids; Male; Middle Aged; Multiple Myeloma; Promoter Regions, Genetic; RNA Interference; RNA, Small Interfering; S Phase Cell Cycle Checkpoints

Bortezomib is a proteasome inhibitor important to the therapy of multiple myeloma (MM), though a number of patients show resistance to this drug. To study the cellular basis of this resistance we have generated a MM cell line displaying enhanced (5-6-fold) resistance to bortezomib by serial cultivation of RPMI 8226 cells with increasing concentrations of this drug. Bortezomib-resistant cells (8226/7B) became bigger in size than parental cells and nearly doubled the amount of DNA per cell, evolving from hypotriploidy to near-tetraploidy. 8226/7B displayed lowered Noxa accumulation and reduced caspase-3 activation in response to bortezomib. Resistant 8226/7B cells overexpressed the PSMβ5 proteasome subunit, the molecular target of bortezomib, both at the mRNA and protein level. No mutations were detected in the PSMβ5 gene. Bortezomib-resistant cells were roughly as sensitive as parental cells to other chemotherapeutic drugs, including doxorubicin, melphalan, vincristine, BMS-214662 and BMS-345541. 8226/7B cells showed partial and high cross-resistance to the proteasome inhibitors epoxomicin and MG-132, respectively. Co-treatment with the histone deacetylase inhibitor trichostatin A (TSA) potentiated bortezomib-induced apoptosis in parental RPMI 8226 cells but did not revert bortezomib resistance in 8226/7B cells. Therefore, treatment of bortezomib-refractory myeloma with drugs targeting molecular structures other than proteasome seems to be the more suitable therapeutic strategy to overcome bortezomib resistance.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Boronic Acids; Bortezomib; Caspase 3; Cell Line, Tumor; Drug Resistance, Neoplasm; Flow Cytometry; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Multiple Myeloma; Polyploidy; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Here, we focus on epigenetic changes in leukaemia and MM (multiple myeloma) cells. We show how the histone signature, DNA methylation and levels of select tumour-suppressor proteins can be affected by inhibitors of HDACs (histone deacetylases) and Dnmts (DNA methyltransferases). Both inhibitors, TSA (trichostatin A) and 5-AZA (5-azacytidine), have the ability to change the histone signature in a tumour-specific manner. In MM cells, we observed changes in H3K4 methylation, while in leukaemia cells, H3K9 methylation was especially affected by select inhibitors. Compared with normal peripheral blood lymphocytes, tumour cell samples were characterized by increased H3K9 acetylation, increased H3K4me2, H3K9me2 and HP1α (heterochromatin protein 1α) levels and specific changes were also observed for DNA methylation. Additionally, we showed that the tumour suppressor pRb1 (retinoblastoma protein) is more sensitive to epigenetic-based anti-cancer stimuli than p53. We have found significant decrease in the levels of pRb1 and p53 in both myeloma and leukaemia cells after HDAC inhibition.

Topics: Antineoplastic Agents; Azacitidine; Chromobox Protein Homolog 5; DNA Methylation; DNA Modification Methylases; Epigenesis, Genetic; Gene Silencing; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Leukemia; Multiple Myeloma; Retinoblastoma Protein; Tumor Suppressor Protein p53

Real time PCR is a powerful tool for studying the expression of genes involved in the pathophysiology of human diseases. Recent studies of the RAN (6p21), ZHX-2 (8q24.3), CHC1L (13q14.3) loci highlight the importance of these genes in multiple myeloma (MM) prognosis and therapeutic applications. Here, we described a detailed Real-Time PCR method for the detection of RAN, ZHX-2, and CHC1L expression, which could be applied in clinical situations. The expression profiles of these genes were studied in peripheral blood lymphocytes of healthy individuals, patients suffering from MM, and in the myeloma cell line, MOLP-8. Low expression levels of RAN, ZHX-2, and CHC1L were observed in myeloma patients, compared with peripheral blood lymphocytes and MOLP-8 cells. An inhibitor of histone deacetylases (TSA) had the ability to decrease expression of CHC1L and ZHX-2 in MOLP-8 cells, while expression of RAN was relatively stable in peripheral blood lymphocytes, control MOLP-8, and TSA- or 5-azacytidine treated MOLP-8 cells. In myeloma patients, we observed significant decreases in the expression of selected genes, but it was patient-specific. Our experiments illustrate new methodological approaches and troubleshooting for conducting gene expression studies in clinical laboratories.

Topics: Azacitidine; Cell Line, Tumor; Histone Deacetylase Inhibitors; Homeodomain Proteins; Humans; Hydroxamic Acids; Multiple Myeloma; Neoplasm Proteins; ran GTP-Binding Protein; Transcription Factors

To identify epigenetically silenced cancer-related genes and to determine molecular effects of 5-aza-2'-deoxycytidine (Aza-dC) and/or trichostatin A (TSA) in multiple myeloma (MM), we analyzed global changes in gene expression profiles of three MM cell lines by microarray analysis. We identified up-regulation of several genes whose epigenetic silencing in MM is well known. However, much more importantly, we identified a large number of epigenetically inactivated cancer-related genes that are involved in various physiologic processes and whose epigenetic regulation in MM was unknown thus far. In addition, drug treatment of MM cell lines resulted in down-regulation of several MM proliferation-associated factors (i.e., MAF, CCND1/2, MYC, FGFR3, MMSET). Ten Aza-dC and/or TSA up-regulated genes (CPEB1, CD9, GJA1, BCL7c, GADD45G, AKAP12, TFPI2, CCNA1, SPARC, and BNIP3) were selected for methylation analysis in six MM cell lines, 24 samples from patients with monoclonal gammopathy of undetermined significance (MGUS), and 111 samples from patients with MM. Methylation frequencies of these genes ranged between 0% and 17% in MGUS samples and between 5% and 50% in MM samples. Interestingly, methylation of SPARC and BNIP3 was statistically significantly associated with a poor overall survival of MM patients (P = 0.003 and P = 0.017, respectively). Moreover, SPARC methylation was associated with loss of SPARC protein expression by immunostaining in a subset of MM patients. In conclusion, we identified new targets for aberrant methylation in monoclonal gammopathies, and our results suggest that DNA methyltransferase and histone deacetylase inhibition might play an important role in the future treatment of patients with MM.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Decitabine; DNA Methylation; DNA Modification Methylases; DNA, Neoplasm; Enzyme Inhibitors; Epigenesis, Genetic; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Genome, Human; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Multiple Myeloma; Transcription, Genetic; Tumor Cells, Cultured

Deleted in liver cancer-1 (DLC-1) is a tumour suppressor gene that is inactive in liver carcinogenesis. It encodes a rho-guanosine triphosphatase-activating protein (rho-GAP) and maps to one of the deleted regions (8p21.3-22). Little is known, however, about the methylation status of the DLC-1 promoter in myeloma cells.. To identify whether methylation of DLC-1 was associated in pathogenesis of multiple myeloma.. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect DLC-1 transcripts in RPMI 8226, U266, OPM-2 and XG-2 cell lines. The methylation status was determined by methylation-specific PCR followed by bisulphite DNA sequencing in these four cell lines and in the bone marrow of 14 patients with multiple myeloma and 4 normal patients. DLC-1 mRNA expression in cells with or without treatment with 5-aza-deoxycytidine (5-aza-CdR) or trichostatin A (TSA) was investigated by real-time RT-PCR.. RPMI 8226 and U266 showed complete methylation and XG-2 showed partial methylation. DLC-1 was expressed only in OPM-2 cell lines that showed no methylation. DLC-1 methylation was shown in 11 of 14 (78%) patients with multiple myeloma and none of the normal controls. The exposure of cell lines to 5-aza-CdR or TSA resulted in the up regulation of DLC-1 gene expression.. DLC-1 methylation is often present in multiple myeloma and has a key role in DLC-1 silencing.

Topics: Azacitidine; Base Sequence; Bone Marrow; Decitabine; DNA Methylation; DNA Modification Methylases; Enzyme Inhibitors; Female; GTPase-Activating Proteins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Middle Aged; Molecular Sequence Data; Multiple Myeloma; Neoplasm Proteins; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Tumor Suppressor Proteins; Up-Regulation

Histone deacetylase (HDAC) inhibitors cause growth arrest and apoptosis of cancer cells by both p21-dependent and independent mechanisms. Decreased expression of growth factor receptors may be a key factor in the p21-independent mechanism, although this has not been directly tested. We have tested the effects of sodium butyrate and trichostatin A on human myeloma cell lines and have observed G1 arrest and apoptosis associated with increased expression of p21(WAF1), Bax, Rb dephosphorylation, and decreased IL-6 receptor (IL-6R) expression. Experiments to determine the role of disruption of IL-6 signaling as a result of decreased IL-6 receptor expression in mediating these effects were conducted using a stable transfectant of the OPM-2 line which constitutively expressed the IL-6 receptor. Our results indicated that decreased IL-6R expression was not required for induction of p21(WAF1) or apoptosis. Thus, HDAC inhibitors appear to activate multiple cellular pathways, leading to growth arrest and apoptosis, and their use in the treatment of myeloma, particularly in combination with other agents, warrants further investigation.

Topics: Apoptosis; bcl-2-Associated X Protein; Butyrates; Cell Cycle; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Multiple Myeloma; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Interleukin-6; Tumor Cells, Cultured