Abstract

Pharmacological treatment of cancer cells with demethylating agents and histone deacetylase inhibitors synergistically reactivates the transcription of previously silenced genes.

The aim of this study was to investigate the antileukemic properties of a DNA mehtyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC),an histone deacetylase inhibitor Trichostatin A (TSA) and trans-retinoc acid (ATRA), alone or in combination. The effects of these drugs on apoptosis, cell cycle progression, cell-survival pathways and restoration of proliferation-associated genes silenced by aberrant epigenetic mechanisms were assessed. Four human leukemic cell lines were used: lymphoid cell-lines REH (Tel-Aml1+) and DAUDI (c-myc) and myeloid cell-lines Kasumi-1(Aml1-Eto+) and NB4(Pml-Rara+). The cells were cultured for three days. Cell viability, percentage of apoptosis, cell cycle and apoptosis-controlling proteins were examined by multiparametric flow cytometry, western blotting and a RT-PCR low-density array (LDA)containing 48 probes. Combined 5-Aza-dC and TSA treatment induced a high degree of apoptosis and affected the cell-cycle in all the cell lines analyzed. Increased levels of cleaved PARP were detected after the double treatment and paralleled those of caspase-3 except for DAUDI cell-line where there was no cleavage of procaspase 3 suggesting that this treatment induced a death-pathway independent of caspase-3 activation. Furthermore, ATRA alone has a limited capacity to induce apoptosis in all the tested cell-lines.

The genes upregulated following the combined epigenetic-active treatment in DAUDI were; PRKCG, TNFSF10, TNFRSF10B, MAPK8, CASP9, APAF1, PER1, CDKN1A, CCND1. In the REH cell line were; PRKCG, TNFRSF10A, TNFRSF10B, MAPK8, CASP8, CASP9, APAF1, CASP3, AKT1, PIK3CG, BIK, PRKCG, MCL1, CCND1, CCND2, EZH2, PER1, GCLC, GSTP1. In the KASUMI-1 cell line were; PRKCABP, TNFSF10, TNFRSF10A, TNFRSF10B, FAS, CFLAR, CASP8, APAF1, CEBPA, CCND1, CDC25C, CDKN1A, EZH2.For the NB4 cell-line were: PRKCG, PRKCABP, AKT1, TNFSF10, CFLAR, CASP8 and CASP9. We can conclude that combined treatment with demethylating agents and histone deacetylase inhibitors may be active in a wide range of human leukemias. The potential use of this combination in the commonest form of pediatric ALL warrants further investigation.

Disclosure: No relevant conflicts of interest to declare.

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