Mohammad Rahmati-Yamchi
1,2*, Nosratollah Zarghami
2, Hojjatollah Nozad Charoudeh
3, Yasin Ahmadi
1,2, Behzad Baradaran
1, Mohammad Khalaj-Kondori
4, Morteza Milani
5, Abolfazl Akbarzadeh
5, Maghsud Shaker
6, Mohammad Pourhassan-Moghaddam
71 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
2 Department of Clinical Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
3 Stem Cell Research center, Tabriz University of Medical Sciences, Tabriz, Iran.
4 Department of Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran
5 Liver and Gastrointestinal disease research center, Tabriz University of Medical Sciences, Tabriz, Iran.
6 Department of Clinical Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
7 Department of Medical biotechnology, Tabriz University of Medical Sciences, Tabriz, Iran.
Abstract
Purpose: 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.
Methods: 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.
Results: 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.
Conclusion: 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.