All-trans retinoic acid enhances the effect of 5-aza-2′-deoxycytidine on p16INK4a demethylation, and the two drugs synergistically activate retinoic acid receptor β gene expression in the human erythroleukemia K562 cell line

The aim of the current study was to investigate the antineoplastic activities of 5-aza-2'-deoxycytidine (also known as decitabine; DAC) and all-trans retinoic acid (ATRA), administered alone or in combination, in K562 cells in vitro, as well as the effects on the expression of the tumor suppressor genes, p16INK4a (p16) and retinoic acid receptor beta (RAR-beta). Cell growth inhibition, differentiation and apoptosis in K562 cells treated with DAC and/or ATRA were detected. The methylation of the p16 and RAR-beta genes in the K562 cells was detected using the methylation-specific polymerase chain reaction (PCR) method. Quantitative PCR was used for the detection of the mRNA expression of the p16 and RAR-beta genes, and western blot analysis was used to detect protein expression. DAC and ATRA, alone or in combination, had no effect on the growth inhibition, differentiation and apoptosis of the K562 cells. DAC alone induced the demethylation of the p16 gene, and combination of DAC and ATRA demonstrated more evident demethylation of the p16 gene, however, ATRA alone had no effect on methylation. The RAR-beta promoter region was not methylated in the K562 cells. DAC in combination with ATRA appeared to produce a greater activation of the RAR-beta gene, which led to the upregulation of the RAR-beta expression level. ATRA enhanced the effect of DAC on p16 demethylation, and the combination of the two drugs was found to activate RAR-beta expression, which indicated that DAC used in combination with ATRA has clinical potential in the treatment of human erythroleukemia.