Aberrant DNA methylation is a critical epigenetic process involved in gene expression of
tumor cells. Diverse
DNA methyltransferase inhibitors are being studied as potential anticancer drugs, and there is interest in developing novel and more effective DNMTIs. We evaluated
zebularine, a stable and low-toxic
cytidine analog, effects on human promyelocytic
leukemia cell lines, NB4 and KG1.
Zebularine caused a dose- and time-dependent NB4 and KG1 cell growth inhibition, did not induce myeloid differentiation but triggered concentration-dependent apoptosis as manifested by
procaspase-3 and PAR-1 cleavage and the occurrence of early apoptosis detected by
Annexin-V-
propidium iodide.
Zebularine co-treatment with
all-trans retinoic acid (RA) at pharmacological dose (1 μM for NB4 cells) and higher (3 μM for KG1 cells) increased granulocytic differentiation in both cell lines. Pretreatment for 24 or 48 h with
zebularine before the treatment with different doses of RA alone or RA with
histone deacetylase inhibitors, phenyl
butyrate, and
BML-210, resulted in significant acceleration and enhancement of differentiation and cell cycle arrest at G0/1.
Zebularine alone or in sequential combination with RA decreased expression of DNMT1, caused fast and time-dependent expression of pan-
cadherin and partial demethylation of
E-cadherin but not
tumor suppressor p15. When used in combination with RA,
zebularine increased expression of both genes transcript and
protein.
Zebularine induced regional chromatin remodeling by local
histone H4 acetylation and
histone H3-K4 methylation in promoter sites of methylated
E-cadherin and also in the promoter of unmethylated p21 as evidenced by
chromatin immunoprecipitation assay. Our results extend the spectrum of
zebularine effects and the evaluation its utility in
acute myeloid leukemia therapy based on epigenetics.