مجید صفا

The occurrence of single and double-strand breaks of DNA damage is the major obstacle for proliferation under various environmental factors and, if not repaired, can result in many consequences, including mutation, cell death, and others. So, the present study was conducted to evaluate the damage of DNA and the expression status of DNA repair system genes before and after stem cell proliferation.

The MACS method isolated the umbilical cord blood hematopoietic stem cells (UCB-HSCs). In order to investigate cell death, the study of Annexin V/PI was done by flow cytometry. Comet assay made observation and identification of DNA breaks, and the expression of genes normally involved in the repair of DNA breaks was evaluated by real-time polymerase chain reaction.

The average number of stem cells increased by 1.9-fold after three days of proliferation. The apoptotic percentage of cells was negligible (less than 0.2%), and the purity of the CD34+ cells was reduced by about one-third in three days (67%). By examining the expression of DNA repair genes, including KU70, KU80, RAD51, and XRCC1, their increased fold change was not significant. In a microscopic examination of stem cells in the comet assay, there was no significant difference between DNA damage before (1.33% ± 0.31) and after (2.08% ± 0.92) replication.

In our investigation, neither DNA damage nor changes in the DNA break repair were observed. However, further studies are required to clarify the DNA break repair by recruiting more UCB-HSCs samples.

This study aimed to compare high intensity interval training and continuous endurance training on the gene expression of mir-1 and IGF-1 in cardiomyocytes of diabetic rats. After induction of diabetes, 21 male Wistar rats were randomly divided into three groups (each group 7 subjects): control, continuous endurance training, high intensity interval training). The training program included 5 days a week for 8 weeks. Each endurance session consisted of 30 minutes of running with the intensity of 60% VO2max. Each interval session consisted of 4 intervals, each interval 3 minutes, with the intensity of 90% VO2max and 1 minute of recovery with the intensity of 30% VO2max between each two intervals. Gene expression of IGF-1 and mir-1 of left ventricle tissue was assessed by qRT PCR. The results showed that both types of training significantly reduced the gene expression of mir-1 compared to the control group, but this decline was severer in the high intensity interval group than the endurance group (P≥0.05). Also, the gene expression of IGF-1 significantly increased in both training groups compared to the control group but this increase was severer in high intensity interval group than the endurance group (P≥0.05). It seems that high intensity interval training can be an effective intervention to reduce the complications of diabetic cardiomyopathy by repression of mir-1.

One of the complications of diabetes is muscular atrophy. Considering the role of exercise in controlling diabetes complications, the aim of the study was to evaluate the effect of five weeks of high intensity interval training (HIIT) on miR-23a and Atrogin-1 genes expression in the gastrocnemius muscles of male diabetic rats.
For this purpose, diabetes was induced in 14 Wistar rats with an average weight of 260±10g by injection of Streptozotocin (50mg/kg). They were randomly divided into two groups, controls (n=7) and HIIT (n=7) group. The HIIT program included implementing four 3-minute sets at intensity of 85-90% VO2max and one-minute recovery between each set with intensity of 30 to 35% VO2max. Twenty-four hours after the last training session, animals were anesthetized, gastrocnemius muscles were removed and Mir-23a and Atrogin-1 genes expression was evaluated by quantitative REAL time PCR. Data were analyzed by using the Kolmogorov-Smirnov test and t-test with SPSS software, version 19 and Execle 2007 at a significance level of p≤0.05.
Analysis with independent t-test showed that the HIIT training caused a significant increase in expression of miR-23a and consequently significant decrease in expression of Atrogin-1 gene, compared to controls group.
Evidently high intensity interval training due to decrease of hyperglycemia, change in expression of miR-23a and Atragin-1 can be an effective intervention to reduce diabetes complications such as muscle atrophy.

Prevention, control and treatment of cancer using natural materials currently have been considered as the promising strategy. In this regard, caffeine as a purine alkaloid found in various plants, including coffee, cocoa, cola and tea has recently attracted great interest because of its remarkable multifunctional inhibitory effects on cancers such as leukemia. Regarding the anti-cancer effects of caffeine, we sought to investigate the effectiveness of this methylxanthine alkaloid against NB4 acute promyelocytic leukemia cells. In order to examine the effects of caffeine in APL, NB4 cells were cultured in the presence of different concentrations of caffeine subsequently, MTT, Caspase 3 activity assay, BrdU cell proliferation assay and quantitative real-time PCR were performed to assess the effect of caffeine on cell viability and the possible mechanisms of inducing cell death. Evaluation of DNA synthesis and cell survival using BrdU and MTT methods indicated that caffeine reduces proliferation and survival of NB4 cells in a dose and time dependent manner. The results of this study showed that elevation in the dose of caffeine induces caspase-3 activation and increases Bax and p21 gene mRNA expression levels. Overall, our data illustrate that caffeine inhibits cell proliferation and induces apoptosis in acute promyelocytic leukemia NB4 cells. Thus, it can be concluded that caffeine as a substance in tea may be a useful agent for induction of cell death in malignant cells of patients with acute promyelocytic leukemia.

The use of low-molecular-weight, nonpeptidic molecules that degrade the interaction between the p53 protein and its negative regulator MDM2 (Murine- double minute colon 2) is a new therapeutic strategy for treatment of various types of cancer. One of these agents is RITA (reactivation of p53 and induction of tumor cell apoptosis) which binds to p53 protein and inhibits formation of p53-MDM2 complex while induces apoptosis in tumor cells by increasing p53 protein levels. The aim of this study was to assess the possible in vitro apoptotic effects of RITA on pre-B ALL NALM-6 cells. NALM-6 cells were treated with different concentrations of RITA at different time intervals. The viability of NALM-6 cells and apoptosis was measured by MTT assay and PI staining, respectively. The level of p53, acetylated p53 as well as cleaved PARP and procaspase-3 were determined by Western blot in RITA-treated cells. The results were analyzed using Paired t test. RITA has cytotoxic effects on NALM-6 cells. Flow cytometry analysis indicated increased apoptotic cells in sub G1 region in treated cells (P < 0.05). The Western blot analysis revealed that protein expression levels of p53, and its acetylation increased in response to RITA. In addition, RITA-induced apoptosis associated with activation of caspase-3 and PARP cleavage. The results of this study showed that RITA induced p53-dependent apoptosis in pre-B ALL NALM-6 cells.

Indole-3-carbinol (I3C)، found in Brassica species vegetables، exhibits antitumor effects. It has been shown that I3C induces apoptosis in various cell types through inactivation of the nuclear factor-kappa B (NF- k B) pathway. Anthracyclines such as doxorubicin، is widely used in the treatment of hematological malignancies، induce apoptosis in tumor cells via DNA damage and activation of p53. However، NF- k B pathway that activated by anthracyclines as a part of DNA damage response can induce chemo resistance. In this study the apoptotic effect of doxorubicin in combination with NF- k B inhibitor I3C was assessed in acute lymphoblastic leukemia cells.

Human pre-B acute lymphoblastic leukemia cell line، NALM-6 cells، were preincubated with various concentrations of I3C for1 hour and then treated with 125nM doxorubicin at 37 ° C for 24 hours. Cellular DNA content assay and Annexin V-FITC staining were performed by flowcytometry for evaluation of apoptosis.

DNA histogram analysis of NALM-6 cells indicates that combination of I3C with doxorubicin synergistically escalated the percentages of sub-G1 population cells (apoptotic cells) as compared to doxorubicin-only treated group. Annexin V-FITC staining showed that cotreatment of NALM-6 cells with I3C and doxorubicin increased the proportion of Annexin-V positive cells (early apoptotic cells) in comparison with the doxorubicin treated cells.

The results of cell culture treatments and cell death analysis by flowcytometry suggest that I3C synergistically potentiates doxorubicin-induced apoptosis in human leukemia NALM-6 cells.

Doxorubicin is a chemotherapeutic agent still in widespread use in hematologic malignancies. A side effect of anthracyclines such as doxorubicin is the activation of nuclear factor-κB (NF-κB), a potent inducer of antiapoptotic genes, which may blunt the therapeutic efficacy of the drugs. In this study, the effect of indole -3-carbinol (I3C) on the activation NF-κB and the anti-apoptotic genes whose expression is regulated by NF-κB was assessed in NALM-6 cells. NALM-6 cells were preincubated with various concentrations of I3C and then treated with doxorubicin. Cellular DNA content assay and Annexin V-FITC staining were performed by flowcytometry for evaluation of apoptosis. For assessing the effect of I3C on the expression of XIAP, survivin, and nuclear p65 proteins, NALM-6 cells were pretreated with I3C and then incubated with doxorubicin. Whole-cell and nuclear extracts were prepared for Western blot analysis. A paired t - test was conducted to evaluate the results. DNA histogram analysis of NALM-6 cells indicates a combination of I3C with doxorubicin significantly escalated the percentages of sub-G1 population cells compared with doxorubicin -only treated group (P< 0.05). Annexin V-FITC staining also showed that cotreatment of NALM-6 cells with I3C and doxorubicin significantly increased the proportion of Annexin-V positive cells in comparison with the doxorubicin treated cells (P <0.05). The western blot analysis indicated I3C significantly inhibits both doxorubicin -induced nuclear translocation of p65 and the expression of doxorubicin-induced NF-κB target. Our results indicated that using natural non-toxic inhibitors of NF- κB such as I3C in combination with anthracyclines might be a rational combination therapy for BCP-ALL cells in which NF- κB is constitutively active.

BAD (Bcl-2-associated death promoter) is a proapoptotic protein which in phosphorylated state is inactivated in terms of its apoptotic function while in dephosphorylated status causes proceeding of apoptosis via inhibition of BCL-XL. cAMP elevating agents attenuate Doxorubicin inducedapoptosis. The aim of this study was to assess the effects of cAMP elevating agents via Forskolin and IBMX on phosphorylation of BAD in Pre-B acute lymphoblastic leukemia (pre-B ALL) cells. NALM-6 cells were incubated with cAMP increasing agents along with two concentrations of Doxorubicin 250 and 500 for 24 hours. Assessment of cell death was performed by Trypan blue staining and apoptosis was assessed by Annexin V via flowcytometry. Western blot was used for evaluating the expression of BAD protein and phosphorylation changes. Data was analyzed by Paired t-test. The flowcytometry analysis of Annexin V revealed that cAMP increasing agents in combination with Doxorubicin lead to decreased rate of apoptosis in comparison to Doxorubicin alone. Analysis of western blotting showed that cAMP increasing agents cause phosphorylate serine residues of BAD. The study also indicated that Doxorubicin alone is unable to alter the phosphorylation status of BAD. Results of this study indicate that cAMP increasing agents attenuate doxorubicin-induced apoptosis via phosphorylation of BAD in NALM-6 cells.

Acute lymphoblastic leukemia (ALL) is the most common type of cancer in children. Currently, chemotherapy is the most effective method of leukemia cancer treatmentwhich has many side effects. New strategies in cancer therapy utilizecompounds that specifically target aberrant signaling pathways in order to reduce toxic sideeffects Indole-3-carbinl (I3C) found in vegetables has multiple anti-cancer properties because of its ability to modulate multiple cellular signaling pathways. In this study the molecular mechanism of the action of indole-3-carbinol on pre- B ALL cells was investigated. In current study, NALM-6 cells were treated with different concentrations of I3C at specific times. Analysis of cellular DNA content was performed by flow cytometry for evaluation of cell cycle status. The protein expression of p21, p53 as well as c-Myc proteins was determined by Western blot in I3C-treated cells. Cell cycle histogram analysis showed that I3C significantly increased the percentage of G1 cells compared with non-treated cells (control)(p<0.05). The western blot analysis also indicated I3C significantly up regulated p21, p53 expression and down regulated c-Myc expression (p<0.05). The G1 arrest induced by I3C is associated with down-regulation of c-Myc and up-regulation of p53 and its downstream target p21.

Given the prevalence of breast cancer in Iran، the increased incidence of this malignancy during the past two decades، the high mortality rate of patients، and according to oral consumption، low cost and easy access to the public extracts of milk thistle، in this study we aimed to investigate the effectiveness of this herb on MCF-7 breast cancer cells. In order to investigate the effect of silibinin in breast cancer، MCF-7 cells were cultured in the presence of different concentrations of the drug. MTT assay، Immunoblotting، and quantitative real-time PCR were used to assess the effect of the drug on cell viability and the expression levels of mRNA and protein of Bax and Bcl-2 genes. Evaluation of cell survival using the MTT assay showed that silibinin reduced the viability of MCF-7 cells in a time and dose dependent manner. The results of this study showed that silibinin significantly increased levels of Bax mRNA expression، while the amount of Bcl-2 gene expression was not affected. As expected according to the results of real-time PCR، expression level of Bax protein was also increased significantly، whereas the Bcl-2 protein level was not changed. Overall، the results of this study confirm the efficacy of this herb against breast cancer. Therfore، the milk thistle as a natural herb supplement with low cost and public access، could be effective in breast cancer therapy.

Knowledge of allele groups and specific alleles present in individuals has important implications in organ and stem cell transplantation and in disease association studies. In organ transplantation application of molecular HLA typing allowed to improve typing quality, leading to a more precise matching assessment with better clinical outcomes. In this study, we have developed the reverse Dot-Blot (RDB) hybridization technique as a rapid and simple method for detection of HLA-DRB1*01 group alleles. For high-resolution typing of HLA-DRB1*01 group alleles, we performed amplification of the alleles of this group using allele-specific primers. Productive DNA amplification occurs if an allele perfectly be complementary to the two primers chosen is present. This technique is often referred to as PCR-SSP. Then for specific discrimination between these alleles (HLA-DRB1*0101, 0102, 0103, 0104), we hybridized PCR product, labeled with biotinylated primers during the amplification, with spots of immobilized probes on a membrane. Variety of immobilization methods could be used. We developed a covalent attachment between 5´-amino probes and carboxylated membrane surfaces. The presence of the PCR product bound to the specific probes at specific locations is detected using streptavidin-AP conjugate and NBT/BCIP as a chromogenic substrate. The presence of each specific allele was detected by the appearance of a dot on the membrane. Obtained results were compatible with those of expected for standard allele samples. Furthermore, results obtained for several other samples which were typed by our technique, were confirmed using sequencing. Our results suggest that HLA typing by RDB method has proved to be a high-resolution, high specificity, rapid and accurate, suitable for clinical application with a greater precision than serological techniques.