ABSTRACT
Clofarabine (2chloro2'fluoro2'deoxyarabinosyladenine, CIF), a secondgeneration 2'deoxyadenosine analog, possesses a variety of anticancer activities, including the capacity to modulate DNA methylation marks. Bioactive nutrients, including resveratrol (RSV) and alltrans retinoic acid (ATRA) have been indicated to regulate epigenetic machinery in malignant cells. The purpose of the current study was to evaluate whether the tested phytochemicals, RSV or ATRA, can improve the therapeutic epigenetic effects of CIF in chronic myeloid leukemia (CML) cells. The present study investigates, to the best of our knowledge, for the first time, the influence of CIF in combination with RSV or ATRA on the expression of relevant modifiers of DNA methylation machinery, including DNA Methyltransferase 1 (DNMT1) and Cyclin dependent kinase inhibitor 1A (CDKN1A) in CML cells. Subsequently, the combinatorial effects on promoter methylation and transcript levels of methylationsilenced tumor suppressor genes (TSGs), including phosphatase and tensin homologue (PTEN) and retinoic acid receptor beta (RARB), were estimated using MSRA and qPCR, respectively. The tested TSGs were chosen according to bioinformatical analysis of publicly available clinical data of human DNA methylation and gene expression arrays in leukemia patients. The K562 cell line was used as an experimental CML in vitro model. Following a period of 72 h exposure of K562 cells, the tested combinations led to significant cell growth inhibition and induction of caspase3dependent apoptosis. These observations were accompanied by DNMT1 downregulation and CDKN1A upregulation, with a concomitant enhanced decrease in DNMT1 protein level, especially after ATRA treatment with CIF. Concurrent methylationmediated RARB and PTEN reactivation was detected. The results of the current study demonstrated that CIF that was used in combination with the tested phytochemicals, RSV or ATRA, exhibited a greater ability to remodel DNA methylation marks and promote cell death in CML cells. These results may support the application of CIF combinations with natural bioactive agents in antileukemic epigenetic therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Clofarabine/pharmacology , DNA Methylation/drug effects , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Resveratrol/pharmacology , Tretinoin/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antioxidants/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA (Cytosine-5-)-Methyltransferase 1/genetics , Drug Synergism , Epigenesis, Genetic/drug effects , Gene Expression Regulation, Leukemic/drug effects , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathologyABSTRACT
Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine) is a second generation analogue of 2'-deoxyadenosine connecting biochemical activities of its prototypes: cladribine (2-chloro-2'-deoxyadenosine) and fludarabine (2-fluoro-arabinosyladenine). This new anticancer drug is more effective (in low doses) and indicates higher oral bioavailability in comparison to its congeners. The studies indicated that the molecular mechanism of clofarabine cytotoxic action includes cell apoptosis, which results from inhibition (by the drug triphosphate nucleotides) of ribonucleotide reductase and DNA polymerases. The most recent research demonstrated also that action of the drug may cause up-expression of some genes on mRNA and protein levels. Clofarabine was synthesized in 1992 and in 2004 was approved for treatment of pediatric patients with refractory or relapsed acute lymphoblastic leukemia (ALL). Encouraging results of clinical trials with clofarabine in acute leukemias inclined to present background knowledge about multidirectional biomolecular mechanism of its cytotoxicity.
Subject(s)
Adenine Nucleotides/pharmacology , Antineoplastic Agents/pharmacology , Arabinonucleosides/pharmacology , Adenine Nucleotides/chemistry , Adenine Nucleotides/pharmacokinetics , Adenine Nucleotides/therapeutic use , Adult , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Arabinonucleosides/chemistry , Arabinonucleosides/pharmacokinetics , Arabinonucleosides/therapeutic use , Biotransformation , Child , Clofarabine , DNA Replication/drug effects , Epigenesis, Genetic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Molecular Structure , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Structure-Activity RelationshipABSTRACT
BACKGROUND: In this study, the effect of clofarabine, a new generation 2'-deoxyadenosine analogue, on promoter methylation and transcriptional activity of selected genes (PTEN, APC, RARB2, ZAP70) in K562 cells was assessed. MATERIALS AND METHODS: Promoter methylation was estimated using methylation-sensitive restriction analysis. The mRNA level of the genes was measured with real-time PCR. RESULTS: The inhibitory cytostatic index (IG(50)) for clofarabine in K562 cells cultured for 72 (or 96) h was 8 nM. The drug (20 nM) caused: (i) potent diminution in methylation of PTEN promoter, moderate methylation reduction of APC and RARB2 promoters, and complete methylation of ZAP70 promoter; (ii) significant stimulation of PTEN, APC, RARB, and p21 mRNA expression and (iii) decline in mRNA level of ZAP70 and DNMT1 genes. CONCLUSION: The results indicated that clofarabine is involved in epigenetic regulation of transcriptional activity of the tested tumour suppressor genes and genes encoding proteins involved in DNA methylation process.
