Cytotoxic Activity of Histone Deacetylase Inhibitor ITF2357 on Burkitt's Lymphoma Cell Lines Is Associated to Micro-RNA Modulation and Transglutaminase 2 Restoration.

Abstract

Background and Objectives: The significant toxicities associated with current treatments of Burkitt's lymphoma (BL) have fostered the identification of novel targets for effective but less toxic therapies. c-myc overexpression is the hallmark of BL; however it is per se insufficient to cause lymphoma development. Recent studies indicating aberrant expression of microRNAs (miRNAs) in BL have raised the possibility of a c-myc - miRNA interaction within the genesis and the maintenance of the lymphoma phenotype. Myc oncoproteins indeed have been found to inhibit the transcription of tumor suppressor genes. This occurs for instance by recruiting histone deacetylase (HDAC) 1 proteins to target genes, including tissue transglutaminase 2 (TG2), a multifunctional protein that promotes apoptosis and differentiation in normal tissues. Since inhibitors of the epigenetic regulator HDACs are revealing very effective as anticancer agents, we evaluated ITF2357, a new hydroxamate inhibitor of HDAC, with respect to its ability to induce proliferative arrest and apoptosis in BL cell lines by modulating c-myc mRNA, miRNAs and TG2 expression.Methods and Results: Cell growth inhibition by ITF2357 was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in the BL cell lines Namalwa and Raji. ITF2357 IC50 was 200nM after 48h exposure. Most treated Namalwa and Raji cells became respectively late and early apoptotic as assessed by annexin V and propidium iodide staining. Accordingly, ITF235 induced SUBG1 peak formation in Namalwa cells and G1 arrest in Raji cells. Repeated addition of ITF2357 at 24h-intervals enhanced these effects. Array analyses and quantitative real-time PCR of miRNAs indicated a significant decrease in the expression of miR-155 and miR-98, which exert oncogenic activity in MYC-associated tumors, at 24–72 h after treatment. Conversely, the Let-7a miRNA, which targets the c-MYC mRNA stabilizer IMP-1 among its tumor suppressive functions, was up regulated peaking at 24h after drug administration. Finally, immunohistochemical analysis of TG2 expression in ITF2357-treated Raji cells revealed a diffuse cytoplasmic staining, whereas in their untreated counterparts TG2 was sporadically and faintly detectable. Despite ITF2357 restored the expression of different targets of myc, quantitative real-time PCR revealed no parallel decrease in c-myc mRNA suggesting that the epigenetic modulation provided by ITF2357 on BL cell lines did not directly affect myc expression level.Conclusion: Our data indicate potent cytotoxic and growth inhibitory activities of ITF2357 on BL cell lines. These effects might be related to the modulation of both oncogenic and tumor-suppressing miRNAs and to the restoration of the tumor suppressor TG2. Further evidences for the potential candidacy of ITF2357 as a therapeutic agent for BL awaits ongoing analyses in BL primary tumors and in animal models.