Clofarabine Has Apoptotic Effect on T 47 D Breast Cancer Cell Line via P 53 R 2 Gene Expression

1 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.


Introduction
After surgery, the standard procedure for treatment of breast cancer is chemotherapy.[4] Clofarabine-3P plays its roles through different mechanisms including: a) it competes with dATP during DNA replication and repair.b) it binds to allostric site of regulatory subunits of Ribonucleotide Reductase (RR) enzyme and inhibts dCTP, dATP formation.Reducing the dCTP level limits DNA synthesis and reactivates deoxyCytidinekinase (dCK) and subsequently increases formation of clofarabine-3P.Also, high clofarabin -3P/dATP concentration ratio results in priority of clofarabine-3P to dATP in competition for DNApolymerase.][4][5] P53R2, a p53-induced peptide, is a homologue of R2 subunit, which can substitute with R2 subunit in RR structure.RR is a tetramer composed of two different homodimers including hRRM1 (R1) and hRRM2 (R2). 6,7P53, R1 and R2 are expressed in a cell cycle dependent manner.R1 and R2 expressed completely during the S-phase. 8,9R2 gene is regulated by cell cycle-associated transcription factors, such as NF-Y and E2F; 10 however, P53R2 is regulated by p53 in response to genotoxic stresses such as irradiation. 11fter DNA damage, p53 not only causes to cell-cycle arrest in the G1 and G2 phases, but also induces P53R2 gene expression.Subsequently, P53R2 accumulates in cell nuclear and increases RR activity. 12,13However, P53R2 activation may not be fast enough to provide dNTPs immidiatly, which can be completed during a few hours after DNA damage.It is shown that ATM phosphorylates P53R2 in response to genotoxic stress.This modification is essential for maintaining P53R2 protein stability against degradation by ubiquitylation. 14ecause of the long half-life, R1 level is constant higher than R2 level throughout cell cycle. 6,15In early G1-phase, R2, but not P53R2, is degraded by cadherin 1/anaphase promoting complex (Cdh1/APC). 126][17] So there are two independent pathways in providing dNTPs: first, R2 provides dNTPs in S-phase and second, P53R2 provides dNTPs for DNA repair in G1 or G2-phase. 12he maximum level of P53R2 has been found at G1/S transition. 6Furthermore, P53R2 up-regulates P21 and down-regulates cyclin D, 15 causing cell cycle arrest in G1 and providing both time and dNTP for the repair of damaged DNA.Also, P53R2 plays an important role in mitochondrial veracity.Based on previous studies, mutations in P53R2 are associated with severe depletion of mitochondrial DNA in both human and mouse cell lines. 18,19Moreover, P53R2 may plays a role in scavenging reactive oxygen species (ROS) and thereby protecting mitochondrial membrane from oxidative stress. 12In addition, P53R2 down-regulates MEK-ERK signal pathway through direct interaction with ERK-Kinase2. 20MEK-ERK signaling pathway regulates various cellular processes including cell cycle progression and cell survival, thus inhibition of this pathway is another anti-cancer property of P53R2. 21,22ince the P53R2 is critical molecule in dNTP formation and DNA repair, we hypothesizes that clofarabine might plays a role in apoptosis and cancer therapy by altering the expression levels of P53R2 gene.To evaluate this hypothesis, T47D breast cancer cell line was treated with several concentrations of clofarabine in different times.The MTT assay, flowcytometry, and qRT-PCR were used for evaluation of cytotoxicity, apoptosis and gene expression, respectively.
Cell culture T47D cells were propagated in RPMI1640 media supplemented with 10% fetal bovine serum (FBS), 2mM of glutamine, penicillin (100 U/ml) and streptomycin (100 g/ml), at 37°C in a 5% CO 2 humidified atmosphere.Then, cells were seeded into plates for MTT assay and into flasks (25 cm 2 ) for gene expression and apoptosis studies, respectively.

Cytotoxicity assay
Before doing MTT assay, the count and viability of cultured cells were determined u8sing a hemocytometer.Then, for MTT assay, the cells were seeded at 5×10 3 cells per well in 96-well culture plates and were settled at 37°C for 24 h, followed by treatment with different concentrations of clofarabine (1.5 -8μM).The concentration of clofarabine that inhibited cell proliferation by 50% (IC 50 s) was determined from MTT assay data after 24, 48 and 72h treatments.Each experiment was done in triplicate manner.

P53R2 gene expression
The cells were seeded in 7 flasks with concentration of 1×10 6 cells/flask.Then 3 flasks were treated with IC 50 of 48h exposure time for 24, 48 and 72h exposure, and other three flasks were treated with IC 50 of 72h exposure time for 24, 48 and 72h exposure times; and last flask was served as control group.Total RNA was extracted from cultured cells using RNX Plus kit and 1μg of total RNA was used as substrate for reverse transcription using Thermo Scientific cDNA synthesis kit according to the manufacturer's protocols.Quantification of genes expression was performed with master mix from Thermo scientific company using thermo-cycler Corbet 2000 real-time PCR mashine.PCR primers were 5'-AGG CTC GCT TCT ATG GC-3' (forward) and5'-TCT GCT ATC CAT CGC AAG GC-3' (reverse) for P53R2.Gene expression levels in each cDNA sample were normalized to the internal β-actin gene expression level (Forward primer: 5´ACCGTGAAAAGATGACCCAG3´ and Reverse primer: 5´CCATACCCAAGAAGGAAGGC3´). The experiments were done in triplicate manner for each sample.Apoptosis assay T74D cells were seeded at concentration of 1×10 6 cells/ flask.One of these flasks was used as control and other two flasks were used for 48 and 72h treatment with clofarabine.The 24 h time exposure was not included in the experiments, because after treatments the 24 h exposure did not show significant cytotoxic effects on T47D cell line.After treatments, apoptosis detection kit (CALBIOCHEM, Germany) was used for apoptosis evaluation according to the manufacturer ' s protocol.

Statistical analysis
The alteration of P53R2 gene expression was analyzed by ∆∆Ct method and version 16 of SPSS (with p<0.05) was used for statistical analyses.The results of apoptosis experiment were analyzed by Flowjo software.

Clofarabine has cytotoxicity effect on T47D cell line
Clofarabine induces DNA damage and therefore it can impose cytotoxicity effect on T47D cells.After treatment of T47D cell line with different concentrations of clofarabine for 24, 48 and 72h, clofarabine IC 50 was calculated as 3µM for 48h treatment and 2.5µM for 72h treatment.In this study, after 24h treatment, clofarabine did not show cytotoxic effect on T47D cell line (Figures 1 and 2).These results show that clofarabine at the longer treatment duration has a stronger cytotoxic effect on T47D cell line.

Clofarabine induces P53R2 gene expression
Clofarabine inhibits RR complex that results in gradual reduction of dNTPs pool.Low level of dNTPs inhibits or defects repair of damaged DNA which induces p53 and p53R2 proteins,subsequently.Upon binding of P53R2 to R1, they form an active RR complex that provides dNTPs for repair of DNA.P53R2 expression after 24, 48 and 72h treatments with two clofarabine concentrations equal to acquired IC 50 s (2.5 and 3µM) has shown in Table 1 and Figure 3. Based on the results, the higher concentration of clofarabine and longer treatment duration induce p53R2 gene expression more efficiently, but it seems that the effect of treatment duration is more significant than the effect of concentration (Figure 3).In other words, in longer treatment duration, the P53R2 gene expression was increased regularly.The level of P53R2 gene expression after treatment with different clofarabine concentrations (3 µM and 2.5µM), shows that clofarabine induces P53R2 gene expression and it's level is directly related to treatment duration so that in treatment of 24h the gene expression is after the lowest level and after treatment of 72h is in the highest level .

Clofarabine induces apoptosis in T47D cell line
As mentioned earlier, clofarabine causes DNA damage by RR complex inhibition.And it can induce P53 gene expression that induces.
As shown in Table 2, after 48 and 72h treatments, clofarabine induced apoptosis in T47D cell line about 26.91% and 74.46%, respectively.These results show that treatment in longer duration is more effective for induction of apoptosis.The flowcytometry results for control, 48 and 72h treatments are shown in Figures 4-6.The results of flowcytometry (the percentage of live cell, early apoptotic, late apoptotic, and necrotic cells) after treatment of T47D cell line with 3μM clofarabine.The number of dead cells in the treated samples after 48 and 72h is remarkably more than the control sample.
On the other hand, the number of apoptotic cells after 72h treatment is more than 48h treatment which suggests that clofarabine induces more apoptosis in longer duration treatment time.*: apoptosis induction in the treated cells is significant in comparison to the control group.

Discussion
Clofarabine is a therapeutic agent in treatment of leukemias, also, has a good inhibitory effect up on several solid tumors such as ovary and colon cancers. 23,24he results of the present study show that clofarabine inhibits T47D cell growth.It means that clofarabine might have anti-cancer effects on breast cancer.It has been shown that clofarabine had anti-cancer effect by induction of p53 and P21 in MDA-MB-231 breast cancer cell line. 25Considering the results of the present study, in T47D breast cancer cell line, clofarabine induces DNA damage and P53R2 gene expression.Therefore, clofarabine could plays cytotoxic effect by induction of DNA damage and gene expression on T47D cells line.
Based on the results of flowcytometry, clofarabine can induces apoptosis in T47D cell line through induction of DNA damage and subsequent activation of internal apoptosis pathway.Deregulated expression of P53R2 has been shown in various cancer types. 20P53R2 repairs DNA and acts as scavenger of ROS, maintaining cell survival of both cancerous and non-cancerous cells.Also, other studies showed apposite effects and in some cancers elevated P53R2 level can causes cancer progression through exerting resistance to DNA damaging therapies such as clofarabine and radiotherapy.Therefore, it is possible that clofarabine-induced P53R2 gene expression, attenuates cytotoxic effect of clofarabine on T47D cell and other solid cancer, and interesting neglable cytotoxic effect of clofarabine on T47D cells during 24 h exposure confirms anti-apoptotic behavior of P53R2 protein in breast cancer.

Conclusion
Clofarabine possess cytotoxic effect on T47D cell line and induces its apoptosis.On the other hand, it acts as an inducer for P53R2 gene expression.Some studies have reported that P53R2 can improve cancer cell response to therapy.Regarding to anti-growth effect of clofarabine on T47D cell line as in vitro model of breast cancer, further studies are required to study anti-cancer efficacy of clofarabine on breast cancer animal models, before using it in the clinical trial studies.

Figure 1 .
Figure 1.The results of MTT assay after 48h treatment with different concentrations of clofarabine.3µM clofarabine is cytotoxic to half of cellular population (IC50 of 48h).

Figure 2 .
Figure 2. The results of MTT assay after 72h treatment with different concentrations of clofarabine.2.5µM clofarabine induces cell death in half of cellular population (IC50 of 72h).

Figure 3 .
Figure 3.The clofarabine effect on P53R2 gene expression.As this figure shows, in higher clofarabine concentrations and treatment durations, the expression of P53R2 increases.P53R2 expression is more dependent on treatment duration than clofarabine concentration (C1=3μM amd C2=2.5μM).*: increasing in P53R2 gene expression is significant in comparison to the control group.

Figure 4 .
Figure 4.The flowcytometry result for the control sample.It shows there is an acceptable amount of alive cells in the control sample (%97.24), on the other hand the number of necrotic and apoptotic cell are not remarkable.

Figure 5 .
Figure 5.The flowcytometry result after 48h treatment with 3μM of clofarabine.After treatment there is a significant cell death.

Figure 6 . 5
Figure 6. the flowcytometry result for 72h treatment with 3μM of clofarabine.The number of dead cells has increased significantly in comparison with control and treatment of 48h samples.

Table 1 .
The effect of clofarabine on P53R2 gene expression in T47D cell line

Table 2 .
The apoptotic effect of clofarabine on T47D cell line