Clofarabine, a novel adenosine analogue, reactivates DNA methylation-silenced tumour suppressor genes and inhibits cell growth in breast cancer cells

Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine, CIF) is a second-generation 2'-cleoxyaclenosine analogue that is structurally related to cladribine (2-chloro-2'-deoxyaclenosine, 2CdA) and fludarabine (9-beta-D-arabinosyl-2-fluoroadenine, F-ara-A). It demonstrates potent antitumour activity at much lower doses than parent compounds with high therapeutic efficacy in paediatric blood cancers. Our previous studies in breast cancer cells indicate that 2CdA and F-ara-A are involved in epigenetic regulation of gene transcription. We therefore investigated whether CIF influences methylation and expression of selected tumour suppressor genes, such as adenomatous polyposis coli (APC), phosphatase and tensin homologue (PTEN), and retinoic acid receptor beta 2 (RARbeta2), as well as expression of p53, p21 and DNA methyltransferase 1 (DNMT1) in MCF-7 and MDA-MB-231 breast cancer cell lines with different invasive potential. Promoter methylation and gene expression were estimated using methylation-sensitive restriction analysis (MSRA) and real-time PCR, respectively. CIF demonstrated potent growth inhibitory activity in MCF-7 and MDA-MB-231 cells after 9611 treatment with IC50 determined as equal to 640 nM and 50 nM, respectively. In both breast cancer cell lines, CIF led to hypomethylation and up regulation of APC, PTEN and RARbeta2 as well as increase in p21 expression. Only in non-invasive MCF-7 cells, these changes were associated with down regulation of DNMT1. Our results provide first evidence of CIF implications in epigenetic regulation of transcriptional activity of selected tumour suppressor genes in breast cancer. It seems to be a new important element of CIF anticancer activity and may indicate its potential efficacy in epigenetic therapy of solid tumours, especially at early stages of carcinogenesis. (C) 2013 Elsevier By. All rights reserved