title: Clofarabine, a novel adenosine analogue, reactivates DNA methylation-silenced tumour suppressor genes and inhibits cell growth in breast cancer cells.
abstract: Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine, ClF) is a second-generation 2'-deoxyadenosine analogue that is structurally related to cladribine (2-chloro-2'-deoxyadenosine, 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 ClF 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. ClF demonstrated potent growth inhibitory activity in MCF-7 and MDA-MB-231 cells after 96h treatment with IC50 determined as equal to 640nM and 50nM, respectively. In both breast cancer cell lines, ClF 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 ClF implications in epigenetic regulation of transcriptional activity of selected tumour suppressor genes in breast cancer. It seems to be a new important element of ClF anticancer activity and may indicate its potential efficacy in epigenetic therapy of solid tumours, especially at early stages of carcinogenesis.
authors.affiliation: Department of Biomedical Chemistry, Medical University of Lodz, 6/8 Mazowiecka Street, 92-215 Lodz, Poland. Electronic address: katarzyna.lubecka-pietruszewska@umed.lodz.pl.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Lubecka-Pietruszewska
authors.affiliation: Department of Biomedical Chemistry, Medical University of Lodz, 6/8 Mazowiecka Street, 92-215 Lodz, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Kaufman-Szymczyk
authors.affiliation: Department of Nutrition Science, Purdue University, 700 West State Street, West Lafayette, IN 47907-2059, USA; Department of Pharmacology and Therapeutics, McGill University, 3655 Sir William Osler Promenade, Montreal, QC, Canada H3G 1Y6.
authors.forename: Barbara
authors.initials: B
authors.lastname: Stefanska
authors.affiliation: Department of Experimental Haematology, Medical University of Lodz, 2 Ciolkowskiego Street, 93-510 Lodz, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Cebula-Obrzut
authors.affiliation: Department of Experimental Haematology, Medical University of Lodz, 2 Ciolkowskiego Street, 93-510 Lodz, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Smolewski
authors.affiliation: Department of Biomedical Chemistry, Medical University of Lodz, 6/8 Mazowiecka Street, 92-215 Lodz, Poland.
authors.forename: Krystyna
authors.initials: K
authors.lastname: Fabianowska-Majewska
mesh_terms: D000227:Adenine Nucleotides / Q000494:pharmacology*; D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001087:Arabinonucleosides / Q000494:pharmacology*; D001943:Breast Neoplasms; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D000077866:Clofarabine; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics*; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D019175:DNA Methylation / Q000187:drug effects*; D044127:Epigenesis, Genetic; D005260:Female; D006801:Humans; D051059:PTEN Phosphohydrolase / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: C555337:APC protein, human; D000227:Adenine Nucleotides; D025601:Adenomatous Polyposis Coli Protein; D000964:Antimetabolites, Antineoplastic; D001087:Arabinonucleosides; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D016159:Tumor Suppressor Protein p53; C540966:retinoic acid receptor, beta2, human; D000077866:Clofarabine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human
keywords: Breast cancer; Clofarabine; Epigenetic therapy; Promoter methylation; Tumour suppressor genes