Experimental Study On Effects Of Arsenic Trioxide On Histone Deacetylase Of Multiple Myeloma

Multiple myeloma (MM) is a neoplasm of malignant plasma cells, characterized by accumulation of malignant plasma cells in the bone marrow, production of a monoclonal protein, reduction in uninvolved immunoglobulins and lytic bone lesions. It accounted for 10% of hematological malignancies and 1% of all cancers. It represents approximately 2% of all cancer deaths.The American Cancer Sosiety has estimated 20,580 new cancer cases of MM in the United States in 2009, including 11,680 cases in men and 8900 cases in women, with an estimated 10,580 deaths. MM accounted for estimated 19,920,19,900, 15,980 new cancer cases in the United States in the year 2008,2007,2005, respectively. It was responsible for approximately 10,690,10,790,11,300 cancer deaths in 2008,2007, 2005, respectively. Its incidence also appears to be increasing in our country. MM is the second most prevalent blood cancer after non-Hodgkin's lymphoma. The treatments of MM include conventional chemotherapy, hematopoietic stem cell transplantation and new drugs. These new targeted drugs have recently been important approaches in MM treatment, but the precise mechanism of action of these drugs have not been well defined. Therefore, further study on their molecular biology mechanism is needed. Further understanding of the mechanism of these drugs will create the rationale for clinical application; help to design new combinations of therapies to raise clinical response.Arsenic trioxide (As2O3), as a drug, has been used for more than several thousand years for the treatment of human diseases. It has been applied as a cancer therapeutic for more than 100 years.In traditional Chinese medicine, several prescriptions have effect on leukemia, whose active component is identified as arsenic compound. In the 1990s, As2O3 is discovered as an efficient drug for the treatment of acute promyelocytic leukemia, without severe myelosuppression, which dramatically improves the treatment of acute promyelocytic leukemia patients. Recent years, more study has suggested As2O3 have antimyeloma efficacy; can be used for the treatment of relapse and refractory patients.The mechanism of action of As2O3 focuses on several areas:1.inhibiting proliferation of MM cells and inducing apoptosis:interleukin-6(IL-6) is a key factor of MM cells, mediating proliferation of MM cells and rescuing MM cells from dexamethasone-induced apoptosis. As2O3 not only blocks protection conferred by IL-6, but also abrogates the production of IL-6 triggered by MM cell to bone marrow stroma cell binding. Inhibition of proliferation of MM cells by As2O3 also was associated with p21, cyclin-dependent kinase inhibitor factors.2. Inducing production of reactive oxygen species (ROS):the survival and growth of cells require energy supplied by ATP, while synthesis of ATP will demand lots of oxygen, resulting in production of ROS, such as H2O2.If accumulation of ROS is not resolved by anti-oxidant system, it will cause damage to cells. ROS can enhance As2O3-mediated cytotoxicity.3. Immunologic enhancement:As2O3 may exert its antimyeloma activity via immunologic enhancement of cells.4 Angiogenesis:previous reports show As2O3 inhibits growth of MM cells in the bone marrow microenvironment. As2O3 also abrogates adhesion of MM cell to bone marrow stroma cell, inhibiting production vascular endothelial growth factor (VEGF).However, the precise antimyeloma efficacy remains unclear. Trivalent arsenicals, including sodium arsenite and the more soluble arsenic trioxide, inhibit many enzymes by reacting with biological ligands that possess available sulfur groups. The biological effects of arsenic (principally the trivalent forms) may be mediated by reactions with closely spaced cysteine residues on critical cell proteins. Several proteins with a high cysteine content and accessible thiol groups are candidates for interactions with arsenic.Transcription in eukaryotic cells is influenced by the manner in which DNA is packaged. In resting cells, DNA is tightly compacted to prevent accessibility of transcription factors. DNA is packaged into chromatin. Local chromatin architecture is now generally recognized as an important factor in the regulation of gene expression. This architecture of chromatin is strongly influenced by posttranslational modifications of the histones. These posttranslational modifications include methylation, phosphorylation, acetylation and so on. The acetylation levels of histones are modified by histone acetyltransferase (HAT) and histone deacetylase (HDAC). In general, increased levels of histone acetylation (hyperacetylation) are associated with increased transcriptional activity, whereas decreased levels of acetylation (hypoacetylation) are associated with repression of gene expression. HDAC is a key protease in the regulation of gene expression and aberrant function of HDAC is associated with oncogenesis and development.Previous studies demonstrate several HDACs contain cysteine-rich region. HDAC6 possesses a cysteine/histidine-rich domain in its C-terminal part. HDAC2 contains 2 cysteines at positions 262 and 274. The cysteine-S-nitrosylation correlated with a significant reduction in the enzymatic activity of HDAC2. Since the biological effects of As2O3 may be mediated by reactions with proteins with high cysteine content or accessible thiol groups, HDACs own the region that may be candidates for interactions with As2O3. In this experiment, we will explore the effect of As2O3 on HDAC; attempt to find out whether As2O3 can inhibit the enzymatic activity of HDAC. We will study the effect of As2O3 on substrates of HDAC, protein-protein interaction, downstream changes. These studies have not been reported before domestic and overseas. Further study on action of As2O3 on MM will provide important theoretical and practical significance for the treatment of MM.Therefore, we studied whether As2O3 can inhibit the enzymatic activity of HDAC. Myeloma cell line NCI-H929 was used in this study. NCI-H929 cells and primary MM cells were incubated with various concentrations of As2O3 and for different time, we observed the change of acetylation level of a-tubulin which was a substrate of HDAC in cytoplasma; we also detected the change of acetylation level of heat shock protein 90 (HSP90) which was another cytoplasma substrate of HDAC; explored the effect of As2O3 on Hsp90 chaperon activity and total amount of IκB kinase a (IKKa) protein. This study included the following three parts:PartⅠStudy on Effects of Arsenic Trioxide on Histone DeacetylaseObjective To explore the effect of As2O3 on histone deacetylase.Methods Human MM cell line NCI-H929 was treated with As2O3. The concentration of total proteins extraction from cultured cells was determined by the Bradford assay method. HDAC activity in equivalent protein extraction from each treated group was measured using the colorimetric HDAC assay. The effect of As2O3 on HDAC standard was detected using HDAC inhibitor drug screening kit.Results After the treatment with different concentration of As2O3, the HDAC activity in human MM cell line NCI-H929 was analyzed. At the concentration of 0.1μmol/L, the HDAC activity in treated cells was 83.5% of control cells, while no statistical difference was observed (P>0.05). After the incubation with 0.5μmol/L, 1μmol/L,2μmol/L and 4μmol/L As2O3, the HDAC activity in treated cells was decreased to 76.8%,72.3%, 69.1% and 62.8%, respectively (P<0.05). After treatment with 2μmol/L As2O3 for 24 and 48 hours, the HDAC activity in treated cells were statistically lower than in the control cells, which were 74.7% and 66.3%, respectively. (P<0.05)。The effect of As2O3 on HDAC standard was detected. At the concentration of 0.5μmol/L, 1μmol/L and 2μmol/L, the HDAC activity were 95.96%,95.71% and 95.67%, respectively, no statistical difference was observed (P>0.05). After the incubation with 4μmol/L,8μmol/L, 16μmol/L,32μmol/L,64μmol/L As2O3, the HDAC activity in treated cells was decreased to 91.08%,81.21%,65.54%,52.45% and 25.89%, respectively. (P<0.05)。Conclusion These results suggested that HDAC activity was inhibited by As2O3. Incubation with As2O3 revealed a concentration-and time-dependent decrease of HDAC activity.PartⅡStudy on Effects of Arsenic Trioxide on Acetylation Level ofα-tubulinObjective To identify the role of As2O3 in the change of acetylation level of a-tubulin which was a substrate of HDAC 6 in cytoplasma.Methods Human MM cell line NCI-H929, primary myeloma cells and bortezomib-resistant NCI-H929 cell line were treated with As2O3. The acetylation level ofα-tubulin which was a substrate of HDAC 6 in cytoplasma was detected by Western blot.Results Treating MM cell line NCI-H929, with different concentration of As2O3 (1μmol/L,2.5μmol/L,5μmol/L,7.5μmol/L and 10μmol/L) for 48h, resulted in concentration-dependent accumulation of acetylated a-tubulin. The level of acetylatedα-tubulin at 1μmol/L was statistically higher than in the control cells (P＜0.05). After treatment with 2.5μmol/L As2O3 for 24 and 48 hours, time-dependent accumulation of acetylated a-tubulin was detected. The level of acetylated a-tubulin was statistically higher after incubation with 2.5μmol/L As2O3 for 24 hours than in the control cells (P<0.05). Treating bortezomib-resistant NCI-H929 cell line with As2O3, concentration-and time-dependent accumulation of acetylatedα-tubulin was also detected. Primary myeloma cells were treated with 5μmol/L As2O3 for 12 hours, the level of acetylatedα-tubulin was higher than control.Conclusion Incubation with As2O3 revealed a concentration-and time-dependent accumulation of acetylated a-tubulin.PartⅢStudy on Effects of Arsenic Trioxide on Molecular Chaperon Heat Shock Protein 90Objective To identify the effect of As2O3 on the level of acetylated HSP90, discuss the role of As2O3 in chaperon activity of HSP90 and the protein level of client protein.Methods Human MM cell line NCI-H929 was treated with different concentration of As2O3. The acetylation level of HSP90 and HSP90-IKKαchaperone complex were detected by immunoprecipitation. The total amount of IKKαprotein was detected by western blot.Results Treating MM cell line NCI-H929 with different concentration of As2O3 (2.5μmol/L and 5μmol/L) for 48h resulted in accumulation of acetylated HSP90. HSP90 chaperone function was affected by the acetylation status of HSP90. Acetylation of HSP90 inhibited chaperone association with client protein IKKa. The protein level of IKKa decreased.Conclusion Incubation with As2O3 revealed accumulation of acetylated HSP90. HSP90 chaperone function was inhibited by the acetylation status of HSP90. Protein level of client protein was affected by As2O3.