Abstract
Clofarabine, a purine nucleoside analogue and inhibitor of Ribonucleotide Reductase (RR), is used for treatment of leukemia. Clofarabine-induced defect in DNA replication, induces p53 and subsequently P53R2 genes as subunit of RR. clofarabine deregulated P53R2 gene expression leading to the elevated levels of P53R2 which impose resistance to DNA damaging drugs. In this study the apoptotic and cytotoxic effects of clofarabine has been investigated on breast cancer cell line. Cofarabine cytotoxicity on T47D cells has been studied by MTT assay. T47D cells were exposed to the different concentrations of clofarabine for 24, 48 and 72 hours intervals. Relative expression of P53R2 gene has been studied using real-time PCR. Moreover, after treating with clofarabine the apoptotic and necrotic cells were detected using Annexin V and propodium iodide (PI) reagents by flowcytometry technique. MTT assay results showed that the clofarabine IC50 on T47D cell line were 3 and 2.5µM after 48 and 72 h exposure, respectively. Clofarabine did not show any significant cytotoxic effect after 24 h exposure. The analysis of qRT-PCR showed a significant increase in P53R2 gene expression in treated cells with both 2.5 and 3 μM doses and also, the results of flowcytometry revealed 26.91 and 74.46 percent apoptosis induction in 48 and 72h treatments respectively in comparison to the control groups. Our results showed that apoptotic and cytotoxic effects of clofarabine on T47D cell line were in time and dose dependent manner; therefore it could be considered a new candidate in breast cancer therapy.
Highlights
IntroductionThe standard procedure for treatment of breast cancer is chemotherapy
After surgery, the standard procedure for treatment of breast cancer is chemotherapy
Our results showed that apoptotic and cytotoxic effects of clofarabine on T47D cell line were in time and dose dependent manner; it could be considered a new candidate in breast cancer therapy
Summary
The standard procedure for treatment of breast cancer is chemotherapy. Clofarabin, one of the compounds that are used in chemotherapy, is a purine nucleoside analogue that has a strong inhibitory effect on wide-spread leukemia and some of solid tumors such as ovary, prostate, kidney and breast.[1,2,3] In the unphosphorylated form, clofarabine passes through the cell membrane using several transporting system including diffusion, facilitated diffusion and active transportation, and it is phosphorylates by intracellular kinases to its mono, di and three phosphates forms.[1] From these forms, only clolfarabine-3 phosphate is therapeutically active, and the remaining forms act as intracellular clofarabine reservoir.[2,3,4]. Clofarabine-3P plays its roles through different mechanisms including: a) it competes with dATP during DNA replication and repair. Reducing the dCTP level limits DNA synthesis and reactivates deoxyCytidinekinase (dCK) and subsequently increases formation of clofarabine-3P.
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