Apoptosis Induction of Armeniacae Semen Extractin Human Acute Leukemia (NALM-6 and KG-1) Cells.

Background: Prunusarmeniaca is a member of the Rosacea family. The most important ingredient of this family is amygdalin that is believed to have anti-tumor and analgesic properties. The aim of this study was to evaluate the anti-proliferative effects of Armeniacae semen extract on the acute leukemia, NALM-6, and KG-1 cell lines, and investigate the effect of the extract on apoptosis of these cell lines and caspase-3 gene expression. Materials and Methods: We prepared aqueous, ethyl acetate, and hydro alcoholic extracts of the Armeniacae semen. The NALM-6 and KG-1 cell lines and mononuclear cells (PBMCs) of healthy controls were treated with different doses of the extracts for 48 hours, and then cell viability was investigated with the MTT test. High-Performance Liquid Chromatography was done for amygdalin identification. The percentage of apoptotic cells was determined using the Annexin V-FITC/PI flow cytometric kit, and caspase-3 gene expression was evaluated. Results: MTT test revealed that the strongest Inhibition Concentration (IC50) in KG-1 and NALM-6 cell lines was related to the ethyl acetate extract. This extract did not have toxic effects on PBMCs. Flow cytometric analysis showed that the ethyl acetate extract at its IC50 concentration led to almost 50% apoptosis in both cell lines after 48 hours. In the molecular examination, after treatment, a significant increase was seen in caspase-3 gene expression in NALM6 and KG1 cells compared to the control (P<0.001 and P <0.05, respectively). Conclusion: Our data confirmed that the ethyl acetate extract of Prunusarmeniaca could reduce the proliferation of KG-1 and NALM-6 cell lines probably by activating the apoptotic pathway.

The Prevalence of Allergic Rhinitis, Allergic Conjunctivitis, Atopic Dermatitis and Asthma among Adults of Tehran.

BACKGROUND: Alteration of environmental factors and air pollution affects the trend of allergic diseases especially in cities such as Tehran. This study aimed to determine the prevalence of allergic rhinitis, conjunctivitis, atopic dermatitis and asthma among adults in the capital city of Iran. METHODS: This cross-sectional study was performed between 2013 and 2016 in Tehran, Iran. The participants were adults between 18 and 45 yr of age. A specific questionnaire including demographic data and clinical symptoms was filled out by a trained interviewer. The diagnosis of allergic diseases was performed based on standard questionnaires and criteria. RESULTS: The prevalence of allergic rhinitis, conjunctivitis, asthma and atopic dermatitis were 28.3%, 15.9%, 7.6% and 3.9%, respectively. Allergic rhinitis and conjunctivitis together were reported in 12.3% of the participants. Among patients with asthma, 47.4% had AR. Additionally, 25.7% of atopic dermatitis subjects were reported to have asthma. The subjects with at least one of these allergic diseases were 36.3%. Women showed a higher prevalence of allergic symptoms than men. There was a significant relationship between allergic symptoms and family history of atopic diseases. CONCLUSION: The most common allergic disease was allergic rhinitis. Regarding the comorbidity of asthma and allergic rhinitis, paying more attention to controlling these allergic diseases is deemed necessary.

IgE Sensitization to Inhalant Allergens and Its Association with Allergic Diseases in Adults.

Allergic sensitization to inhalant allergens could be considered as a predictor in allergic diseases. The objective of this study was to assess IgE-mediated sensitization to inhalant allergens in allergic and non-allergic adults as well as the evaluation of its association with allergic diseases. This cross-sectional study was conducted from 2013 to 2016 in 604 allergic and 102 non-allergic adults selected from blood donor volunteers in Tehran, Iran. After taking informed consent, a standard questionnaire was filled to determine asthma, allergic rhinitis and/or conjunctivitis and atopic dermatitis in participants. Specific IgE assay to common inhalant allergens was performed for all subjects. Logistic regression analysis was used to evaluate the impact of IgE sensitization on allergic diseases. A total of 371(61.4%) allergic subjects and 41(40.2%) non-allergic patients were males. The weeds (especially saltwort) and grasses (particularly meadow fescue and ryegrass) were identified as the most common inhalant allergens. The prevalence of IgE sensitization to trees, weeds, and grasses was higher in subjects with allergic rhino-conjunctivitis and trees sensitization was a significant factor in them [OR=2.32, 95% CI (1.58-3.41)]. IgE sensitization to any inhalant allergens could be a predictor for allergic rhinitis, conjunctivitis and rhino-conjunctivitis in adults [OR=2.20, 95% CI (1.54-3.15], [OR=1.81, 95% CI (1.28-2.54)] and [OR=2.55, 95% CI (1.72-3.78)], respectively. With an increase in the sum of specific IgE concentrations, the prevalence of allergic conjunctivitis and rhino-conjunctivitis also increased. Our results showed the association between positive specific IgE and its concentration with some allergic diseases which could help physicians to prevent such diseases by recognizing and treating them, particularly in individuals with a positive family history of allergic diseases.

Tumor suppressive function of microRNA-192 in acute lymphoblastic leukemia.

Non-coding RNAs play a critical role in gene regulation in cancer cells. Reduced expression of microRNA-192 (miR-192) has been detected in many cancers. In this study, we investigated the role of miR-192 in cell proliferation and cell cycle control in NALM-6 cell line, a model of acute lymphoblastic leukemia (ALL). Cell cycle analysis by DNA content using propidium iodide staining and cell apoptosis analysis using Annexin V assay were carried out. Cell proliferation changes were monitored using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, the relative changes in P53, BAX, CASP3, and BCL-2 gene expression were determined by quantitative reverse transcription PCR. Overexpression of miR-192 resulted in cell proliferation arrest in ALL cells. After 72 and 96 hours of transduction, apoptosis was significantly increased in the cells transduced with miR-192-overexpressing virus compared with control cells. The expression of P53, BAX, and CASP3 increased after 48 hours of transduction in miR-192-overexpressing cells, but no change was observed in BCL-2 expression. The G0/S and G1/S ratio changed to 7.5 and 4.5, respectively, in the cells overexpressing miR-192 compared with controls. The results of our study suggest, for the first time, tumor suppressive effects of miR-192 in ALL cells.

Aberrant Methylation of APAF-1 Gene in Acute Myeloid Leukemia Patients.

Background: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder characterized by immature myeloid cell proliferation and bone marrow failure. Various genetic and epigenetic factors have been found to be influential in such patients. Methylation silencing of APAF-1, a putative tumor suppressor gene (TSG), has been found in several human malignancies. In this study, we explored the association of APAF-1 methylation status with AML patients. Materials and Methods: We studied the methylation status of APAF-1 gene in 101 AML patients and 50 healthy subjects as controls. Genomic DNA was extracted from leukocytes in peripheral blood or bone marrow and the methylation status of APAF-1 gene promoter was detectedusing methylation-specific PCR (MSP) method with specific methylated and unmethylated primers. Gene expression was analyzed using real time RT-PCR. Results: The prevalence of methylated (MM) and hemi-methylated (MU) CpG dinucleotides within the APAF-1 gene promoter of AML patients was 12 (11.9%) and 45 (44.6%), respectively, while no methylation was detected in the control samples (p < 0.001). Our results showed a higher frequency of methylated APAF1 in FLT3-ITD mutated cases (p=0.04). APAF1 mRNA expression was significantly lower in methylated cases compared with normal cases. Conclusion: The present study indicated the increased frequency of hypermethylation of APAF-1 gene promoter in AML patients. APAF-1 aberrant CpG island methylation was associated with transcriptional downregulation in AML patients. Therefore, promoter methylation of APAF-1 gene could be considered as an epigenetic factor that contributes to the development of AML.

Frequency of M287T/AS3MT Single Nucleotide Polymorphism in an Iranian Population.

Background: To determine the frequency of the single nucleotide polymorphism M287T in exon 9 of the AS3MT gene in Iranian population and to assess the difference in allele frequencies with other ethnicities. Subjects and Methods: Genotyping analysis was performed on 150 healthy subjects using the PCR-RFLP assay. We used chi-square analysis to check the deviation from Hardy-Weinberg equilibrium and compare of the observed genotype frequencies in various ethnic. The level of statistical significance was considered as p<0.05. Results: The homozygous CC, homozygous TT and heterozygous CT genotypes were observed in 2%, 80% and 18% of participated individuals. The SNP rs11191439 passed the Hardy-Weinberg equilibrium chi-squared test with p-value>0.05 and had a minor allele frequency (MAF)>5%. Conclusion: Iranians are genetically very similar to Caucasian and African individuals and they are considerably different from other East Asians including Koreans, Chinese and Japanese individuals. Due to genetic polymorphisms can contribute to the variability in AS3MT activity; they may contribute to interindividual as well as intra-ethnic differences in response to the detoxification of arsenic.

Evaluation of Minimal Residual Disease in Acute Myeloid Leukemia with NPM1 Marker.

BACKGROUND: Minimal residual disease (MRD) tests provide early identification of hematologic relapse and timely management of acute myeloid leukemia (AML) patients. Approximately, 50% of AML patients do not have clonal chromosomal aberrations and categorize as a cytogenetically normal acute myeloid leukemia (CN-AML). About 60% of adult CN-AML has a mutation in exon 12 of NPM1 gene. This mutation is specific for malignant clone and potentially is a good marker of MRD. In this retrospective study, we set up a quantitative test for quantifying NPM1 type A mutation and AML patients carrying this mutation at the time of diagnosis, were followed-up. Materials and Methods : We prepared plasmids containing a cDNA fragment of NPM1 and ABL genes by PCR cloning. The plasmids were used to construct standard curves. Eleven patients were analyzed using established method. Serial PB and/or BM samples (n=71) were taken in 1-3 months intervals (mean 1.5-month intervals) and median follow-up duration after chemotherapy was 11 months (5-28.5 months). RESULTS: In this study, we developed RNA-based RQ-PCR to quantitation of NPM1 mutation A with sensitivities of 10((-5)). The percent of NPMmut/ABL level showed a range between 132 and 757 with median of 383.5 in samples at diagnosis. The median NPMmut transcript level log reduction was 3 logs. Relapse occurred in 54.5% of patients (n=6), all cases at diagnosis demonstrated the same mutation at relapse. In patients who experienced relapse, log reduction levels of NPM1 mRNA transcript after therapy were 4 (n=2), 3 (n=2) and 1 log (n=2). Totally, NPMmut level showed less than 5 log reduction in all of them, whereas this reduction was 5-6 logs in other patients. CONCLUSION: Despite the limitations of this study in terms of sample size and duration of follow-up, it showed the accuracy of set up for detection of mutation and this marker has worth for following-up at different stages of disease. Because of high frequency, stability, specificity to abnormal clone and high sensitivity, NPM1 is a suitable marker for monitoring of NPMc+ AML patients.

Study of SFRP1 and SFRP2 methylation status in patients with de novo Acute Myeloblastic Leukemia.

INTODUCTION: Acute myeloid leukemia (AML) is a heterogeneous group of hematologic malignancies with abundant changeability in the pathogenesis. DNA methylation of CpG islands within the promoters of specific genes may play roles in tumor initiation and progression. Secreted frizzled-related proteins (SFRPs) are negative regulator of the Wnt signaling pathway. In the present study, we examined the methylation status of SFRP1 and SFRP2 genes in patients with AML. METHODS: Isolated DNA from peripheral blood of 43 AML patients and 25 healthy subjects as a control group was treated with sodium bisulfite was treated with sodium bisulfite and analyzed by methylation-specific polymerase chain reaction (MSP) with primers specific for methylated and unmethylated promoter sequences of the SFRP1 & -2 genes. We used Mann-Whitney u-tests to investigate the correlation between SFRP1 and SFRP2 genes hypermethylation and clinical parameters. RESULTS: The frequency of aberrant hypermethylation of SFRP1 and SFRP2 genes in patients with AML was determined 30.2% (13/43) and 20.9% (9/43), respectively. In addition, for all subjects in control group, methylation of SFRP1 and SFRP2 genes were negative. Patients with M0 subtype of FAB-AML had the highest incidence of hypermethylation of SFRP1 (P = 0.028) and SFRP2 (P = 0.004). CONCLUSION: The present study showed that, like many solid tumors, methylation of SFRP genes also occurs in AML. Therefore, the methylation of these genes may play a role in the initiation of leukmogenesis.

Priority Setting for Improvement of Cervical Cancer Prevention in Iran.

BACKGROUND: Cervical cancer is the fourth most common cancer among women worldwide. Organized cervical screening and vaccination against human papilloma virus (HPV) have been successful interventions for prevention of invasive cervical cancer (ICC). Because of cultural and religious considerations, ICC has low incidence in Iran and many other Muslim countries. There is no organized cervical screening in these countries. Therefore, ICC is usually diagnosed in advanced stages with poor prognosis in these countries. We performed a priority setting exercise and suggested priorities for prevention of ICC in this setting. METHODS: We invited experts and researchers to a workshop and asked them to list important suggestions for ICC prevention in Iran. After merging similar items and removing the duplicates, we asked the experts to rank the list of suggested items. We used a strategy grid and Go-zone analysis to determine final list of priorities for ICC prevention in Iran. RESULTS: From 26 final items suggested as priorities for prevention of ICC, the most important priorities were developing national guidelines for cervical screening and quality control protocol for patient follow-up and management of precancerous lesions. In addition, we emphasized considering insurance coverage for cervical screening, public awareness, and research priorities, and establishment of a cervical screening registry. CONCLUSION: A comprehensive approach and implementation of organized cervical screening program is necessary for prevention of ICC in Iran and other low incidence Muslim countries. Because of high cost for vaccination and low incidence of cervical cancer, we do not recommend HPV vaccination for the time being in Iran.

Soluble Flt-1 gene delivery in acute myeloid leukemic cells mediating a nonviral gene carrier.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor involved in angiogenesis-mediated progression of acute myeloid leukemia (AML). Studies have reported the role of soluble form of fms-like tyrosine kinase (sFlT-1) delivery as an antitumor agent by inhibiting VEGF. This study investigates the outcome of delivery of a VEGF165 antagonist, soluble vascular endothelial growth factor receptor, namely sFLT-1, mediating lipofectamine 2000 in acute myeloid leukemic cells. A recombinant plasmid expressing sFLT-1 was constructed and transfected into the K562 and HL60 cells using lipofectamine 2000 transfection reagent. sFLT-1 expression/secretion in pVAX-sFLT-1 transfected cells was verified by RT-PCR and western blot. MTS assay was carried out to evaluate the effect of sFLT-1 on human umbilical vein endothelial cells and K562 and HL60 cells in vitro. Treatment with pVAX-sFLT-1 showed no association between sFLT-1 and proliferation of infected K562 and HL60 cells, while it demonstrated a significant inhibitory impact on the proliferation of HUVECs. The results of the current study imply that the combination of nonviral gene carrier and sFLT-1 possesses the potential to provide efficient tool for the antiangiogenic gene therapy of AML.

Detection of BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia on imatinib.

BCR-ABL tyrosine kinase domain mutations are the most important factor contributing to imatinib-resistance in patients with chronic myeloid leukemia. We used a semi-nested reverse transcriptase-polymerase chain reaction followed by bidirectional sequencing to detect mutations in a cohort of 110 chronic myeloid leukemia patients. In total, 34 mutations in 19 distinct codons were identified in 32 patients, of which D276N and E279A were novel. The most commonly mutated region was drug-binding site (29%) followed by P-loop region (26%) and most patients bearing them were in accelerated phase and blastic phase. This report expands the spectrum of BCR-ABL mutations and stresses the use of mutation testing in imatinib-resistant patients for continuation of treatment procedure.

Evaluation of T315I mutation frequency in chronic myeloid leukemia patients after imatinib resistance.

The occurrence of resistance mutations in the Abl kinase domain plays a central role in drug treatment failure in chronic myeloid leukemia (CML) patients. Among them, the T315I mutation at the gatekeeper position affects a common Abl kinase contact residue and confers complete resistance to all known ATP-competitive BCR-ABL inhibitors. In the present study, an allele-specific oligonucleotide reverse transcriptase polymerase chain reaction assay was used to detect T315I mutation in a cohort of 60 imatinib-resistant CML patients. In terms of disease phase, 43 patients (71%) were in late chronic phase, 4 (7%) in accelerated phase, and 13 (22%) in blastic phase. The prevalence of the T315I mutation was found to be 7% (4/60). All four patients with mutation were in advance phases and had previously lost all their responses. The results of the study confirmed that this method is low cost and easy tool to operate for T315I mutation screening and direct sequencing should be performed in positive cases for confirmation.

Induced in vitro differentiation of neural-like cells from human exfoliated deciduous teeth-derived stem cells.

Stem cells from human exfoliated deciduous teeth (SHED) are highly proliferative, clonogenic and multipotent stem cells with a neural crest cell origin. Additionally, they can be collected with minimal invasiveness in comparison with other sources of mesenchymal stem cells (MSCs). Therefore, SHED could be a desirable option for potential therapeutic applications. In this study, SHEDs were established from enzyme-disaggregated deciduous dental pulp obtained from 6 to 9 year-old children. The cells had typical fibroblastoid morphology and expressed antigens characteristic of MSCs, STRO1, CD146, CD45, CD90, CD106 and CD166, but not the hematopoietic and endothelial markers, CD34 and CD31, as assessed by FACS analysis. Differentiation assessment revealed a strong osteogenic and adipogenic potential of SHEDs. In order to further evaluate the in vitro differentiation potential of SHED into neural cells, a simple short time growth factor-mediated induction was used. Immunofluorescence staining and flow cytometric analysis revealed that SHED rapidly expressed nestin and b-III tubulin, and later expressed intermediate neural markers. In addition, the intensity and percentages of nestin and b-III tubulin and mature neural markers (PSA-NCAM, NeuN, Tau, TH, or GFAP) increased significantly following treatment. Moreover, RT-PCR and Western blot analyses showed that the neural markers were strongly up-regulated after induction. In conclusion, these results provide evidence that SHED can differentiate into neural cells by the expression of a comprehensive set of genes and proteins that define neural-like cells in vitro. SHED cells might be considered as new candidates for the autologous transplantation of a wide variety of neurological diseases and neurotraumatic injuries.

The efficiency of CD40 down regulation by siRNA and antisense ODN: comparison of lipofectamine and FuGENE6.

BACKGROUND: Dendritic cells (DCs) are ideal accessory cells in the field of gene therapy. Delivery of DNA and siRNA into mammalian cells is a useful technique in treating various diseases caused by single gene defects. Selective gene silencing by small interfering RNAs (siRNAs) and antisense oligodeoxynucleotides (ODN)s is an efficient method for the manipulation of cellular functions. An efficient, functional delivery system with no toxicity problems would be attractive. OBJECTIVE: We compared two commercially available cationic lipids, Lipofectamine and FuGENE6, in the delivery of both siRNA and antisense ODNs into mice spleen-derived DCs. METHODS: Cellular uptake was measured by the means of fluorescein-labelled non-silencing siRNA and antisense ODNs as a model system using flow cytometry. Cytotoxicity of the two delivery systems was compared with propidium iodide and annexin-V staining, and quantified with flow cytometry. The efficiency of our oligonucleotide delivery systems was compared by measuring CD40 expression by flow cytometry. RESULTS: CD40 expression in DCs was 38%. After siRNA transfection by Lipofectamine, CD40 expression decreased to 13%, and after transfection by FuGENE6, it decreased to 18%. The difference was statistically significant. CD40 down regulation in DCs transfected with the two different antisense sequences by Lipofectamine was 21% and 23%, and down regulation after transfection by FuGENE6 was 19% and 18%, respectively. The differences were not statistically significant. The effects of siRNA and antisense ODNs were specific. CONCLUSION: Lipofectamine was a more potent delivery system in siRNA effect, followed by FuGENE6. There was no significant difference between Lipofectamine and FuGENE6 as a delivery system of antisense ODNs.

Multidrug resistance inhibition by antisense oligonucleotide against MDR1/mRNA in P-glycoprotein expressing leukemic cells.

INTRODUCTION: Acute myeloblastic leukemia (AML) is the most common form of acute leukemia in adults. One major problem in this disease is the emergence of leukemic blast cells that are resistant to anticancer drugs. This phenomenon is termed multidrug resistance (MDR). One cause of MDR is the expression of the MDR1 gene and its product, P-glycoprotein (Pgp). AIM: In the present study, we tried to inhibit the MDR phenotype with MDR1/mRNA/Pgp in leukemic cells using different antisense sequences and two non-viral vectors. MATERIALS AND METHODS: The Pgp expressing cell line was established from a parental K562 (Erythroleukemia) cell line with increasing concentrations of doxorubicin, and named KDI/20. In order to reverse the MDR phenotype due to Pgp expression, four different sequences of sense, antisense and one random sequence with phosphorothioate (PTO) modification (PS-ODN) against MDR1/mRNA were synthesized. They were used on the KDI/20 cells in combination with two non-viral vectors: (1) Fugene 6 transfection reagent (cationic lipid) and (2) polyethylenimine (cationic polymer). The effect of PS-ODN was assessed at the cellular level by flow cytometry (for Pgp detection), and Rhodamine 123 assay (for functional assessment of Pgp) at the molecular level by RT-PCR (for MDR1/mRNA detection) and MTT assay in order to assess the sensitivity of cell to doxorubicin. RESULTS: The results showed a decrease in the percentage of Pgp protein and MDR1/mRNA expression and an increase in the accumulation of Rh123 and drug sensitivity of cells to doxorubicin by antisense I and III. The reduction of MDR1/mRNA was more significant than its protein reduction. Therefore, our data showed that antisense can reverse the MDR phenotype at the transcription level and the PEI vector is more efficient than cationic lipid.