Mechanisms Of Induction Of Apoptosis In Myeloma Cells By Arsenic Trioxide

Multiple myeloma (MM) is one of the common hematopoietic malignancies, so far, it is incurable. In order to find a new valid drug to MM, we studied the feasibility of arsenic trioxide (As₂O₃) on MM, which has special effect on acute promyelocytic leukemia in recent years, and explored its mechanism on MM. Using MTT bioassay, we found that As₂O₃ could inhibit the proliferation of five myeloma cell lines, U266, SKO-007, LP-1, HS-Sultan and KM3 in dose-and time-dependency, the concentration of 50% growth inhibition (IC_{50}) was 1.4±0.27μmol/L, 1.0±0.13μmol/L, 1.8±0.08μmol/L, 4.1±0.57μmol/L and 5.3±0.38μmol/L respectively. We also examed the synergistic or antagonistic effects of menadione (vitamin K3, VK3), N-acetyl- cysteine (NAC) and reduced glutathione (GSH) combined with As₂O₃, experiments showed that oxidant VK3 had significant synergistic effect with As₂O₃, besides its own cytotoxity to U266 cells, meanwhile, antioxidant NAC and GSH could partly blocked the growth inhibition of As₂O₃. Morphology, DNA electrophoresis and DNA flow cytometric analysis indicated that As₂O₃ induced apoptosis of MM cells characterized by apoptosis body, DNA ladder and sub-G0/G1 cells. DNA flow cytometric analysis also showed that As₂O₃ induced most of a G0/G1 phase and small of a G2/M phase arrest in U266 cell, and apoptosis happened mainly in S phase. Using colorimetric assay, we determined the cellular GSH levels and found that GSH contents in five MM cell lines were correlated with its IC_{50} significantly (r=0.87, p<0.05), As₂O₃, VK3, NAC and exogenous GSH could affect the cellular GSH contents: both of As₂O₃ and VK3 decreased the GSH contents, NAC and GSH increased them. By using methylation-specific primer PCR(MSP-PCR) technique,we found that the CpG islands in the 5' promoter regions of P15 and P16 genes in U266 cell line were completely methylated, it resulted in the transcriptional silence of thetow genes, 1.0μmol/L As₂O₃ could restore their transcriptions after exposed 24 or 48 hours detected by reverse transcriptase PCR (RT-PCR), expression of P21 mRNA was elevated by As₂O₃ also. In conclusion, As₂O₃ had therapeutic function on MM, oxidant VK3 could potentiate its effect. The antiproliferation and apoptosis effects on MM cells may be mediated by decreasing the cellular GSH level and activating the expression of cyclin dependent kinase inhibitors (CDKIs).