Induction of Apoptosis and Paradoxical Activation of NF-κB by Trichostatin A in Plasma Cell Myeloma Cells.

Abstract

Plasma cell myeloma (PCM) is a mature B cell malignancy characterized by monoclonal gammopathy and destructive bone lesions. Although recent development of therapeutic drugs including thalidomide or proteasome inhibitor may prolong survival time, PCM is still an incurable disease. To improve the treatment outcome of PCM, the development of novel, pathophysiology-based therapies are needed. Histone acetylation plays a role in transcriptional regulation by altering the chromatin structure, which allows the transcription factors to access the DNA. Histone acetyltransferases (HATs) are involved in tumor suppression and inhibition of histone deacetylases (HDACs) results in antitumor effect. HDAC inhibitors have also reported to induce apoptosis or differentiation of tumor cells. Trichostatin A (TSA) is one of the HDAC inhibitors, and it has shown to be effective in vitro against leukemic cells with characteristic chromosomal translocations. Although none of the disease-specific genetic alterations involving aberrant transcription factors in PCM have been reported, previous studies have demonstrated the antitumor effect of HDAC inhibitors on PCM. Accordingly, to address the detailed molecular mechanisms of HDAC inhibitor-induced apoptosis, we examined the in vitro effects of TSA on PCM cell lines. By using flow cytometric analyses, we found that cell cycle arrest at G1 phase and apoptosis were induced in various PCM cell lines, RPMI8226, IM9, and U266 treated with 100nM of TSA for 24 hours. Especially, the viability of TSA-treated RPMI8226 cells was strongly suppressed to approximately 30% compared to the cells without TSA. Nuclear factor (NF) -κB is thought to be a key molecule of cellular growth in PCM; therefore, we studied the functional alterations of NF-κB in PCM cell lines stimulated with TSA. Immunoblot and electrophoretic mobility shift assays demonstrated that TSA augmented the nuclear localization of NF-κB, which resulted in the increased DNA binding activity and upregulation of downstream target gene expression such as ICAM-1. Increased phosphorylation of Iκ-Bα by TSA was observed simultaneously. Transient transfection assays showed that the transcriptional activity of NF-κB was upregulated more than 4-fold by TSA, and the synergistic effects of TSA with TNF-α or PMA were observed. Interestingly, an NF-κB-specific inhibitor, SN50, induced apoptosis of PCM cell lines, and TSA showed a synergism with SN50. These data may provide a notion that increased activity of NF-κB by TSA could be a compensatory response against proapoptotic signals induced by TSA. Additionally, we investigated other molecules responsible for TSA-stimulated apoptosis, and found the downregulation of Mcl-1, Bcl-XL, XIAP proteins, as well as dephosphorylation of Akt and cleavage of Bid proteins in a time-dependent manner. These results suggest that either PI 3-K/Akt pathway or JNK-dependent pathway could be involved in the TSA-induced apoptosis. In conclusion, HDAC inhibitors will be promising agents to improve survival of patients with PCM, and the combination of HDAC inhibitor and NF-κB inhibitor may provide a novel therapeutic strategy against PCM.