saeed solali

 One of the most common types of leukemia is acute myeloid leukemia (AML). Intrinsic and extrinsic factors may lead to AML. Insulin-like growth factor (IGF) is a mitogenic intermediate from the liver that regulates growth and proliferation in response to GH. In this study, we examined the expression of IGF family genes in bone marrow of AML patients (M3 and Non-M3) and compared them with normal samples.

Forty bone marrow samples from recently diagnosed AML patients along with 15 samples from subjects without hematological malignancies were collected. For molecular tests, RNA extraction and cDNA synthesis were performed. Finally, IGF1, IGF2, IGFBP3, IGF1R, and IGF2R gene expression were examined by Real-Time PCR.

IGF1, IGF1R, and IGFBP3 gene expression were significantly increased in patients with AML. In contrast, IGF2 and IGF2R genes did not show significant expression changes between the two groups.

The expression in this gene family soared in AML patients' bone marrow, compared to normal subjects. This can be caused by malignant cells in the bone marrow. These malignant cells express proteins that increase the number of malignant cells. Moreover, they can be considered as diagnostic biomarkers or therapeutic targets with further research.

EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit), as one of the polycyclic group proteins (PcGs), is an epigenetic regulator that plays a crucial role in the pathophysiology of hematologic malignancies through regulating cell differentiation. Also, it is well known that aberrant expression of specific transcription factors can be involved in the pathogenesis of various cancers. Herein, we aimed to suppress EZH2 expression in MOLT-4 cells, T-ALL (T cell acute lymphoblastic leukemia) cell line, and evaluate the role of EZH2 on the expression of transcription factors that regulate T cell maturation, differentiation, and apoptosis.

EZH2-siRNA was transfected into MOLT-4 cells, and the expression levels of EZH2, NOTCH1, TCF1, IKZF1, and NFATC1 were measured using real-time PCR. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was performed to study the effect of EZH2 knockdown on MOLT-4 cell viability. The apoptosis rate of EZH2-siRNA transfected cells was assessed by flow cytometry. The interaction of mentioned genes was investigated using STRING and GO (gene ontology).

Our results have shown that EZH2-siRNA transfection can substantially decrease EZH2 expression in MOLT-4 cells. Besides, EZH2 suppression can upregulate NOTCH1, TCF1, IKZF1, and NFATC1 expression levels. EZH2 knockdown does not affect the viability and apoptosis of MOLT-4 cells. The most remarkable protein-protein interaction of EZH2 has been with NOTCH1. Besides, GO analysis has demonstrated that EZH2, NOTCH1, TCF1, IKZF1, and NFATC1 were located within nucleoplasm and can regulate RNA polymerase II-mediated transcription.

MOLT-4 cells harbor increased expression of EZH2 in comparison with normal human T cells. EZH2 knockdown can upregulate the expression of the transcription factors involved in T cell differentiation. Thus, EZH2 can halt the differentiation of immature lymphoblastic T cells.

This study evaluated and compared the quantitative expression of Multidrug Resistance-associated Protein 1 (MRP1) and ATP Binding Cassette subfamily G member 2 ( ABCG2), two Multidrug Resistance (MDR) related genes, in 30 CML patients and 27 normal subjects.

Total RNA was extracted from peripheral blood Mononuclear cells (MNCs) using the Trizol method. Then cDNAs were synthesized. Gene expression was quantified with Real-Time PCR System. Relative expression of target genes was calculated using the 2-ΔΔCt method.

High expression of MRP1 and ABCG2 mRNAs were detected in the patient's group. Intra-group comparisons also revealed increased expression of ABCG2 in Accelerated Phase (AP)-Blastic Crisis (BC) patients compared to Chronic Phase (CP) patients. Whereas, increased expression of MRP1 in AP-BC patients was not statistically significant.

Considering the wide spectrum of (ATP Binding Cassette) ABC transporter superfamily substrates, they can play an important role in cell fate determination. High expression of MRP1 and ABCG2 genes can result in the efflux of therapeutic agents, and subsequent reduction in their intracellular concentration. This mechanism finally protects the cells from the therapeutic effects of medications. On the other hand, these transporters are able to export growth factors out of the cell. Such exported molecules may have a growth-inducing effect on adjacent cells. These are the possible mechanisms for the participation of MRP1 and ABCG2 genes in conferring drug resistance to CML cells.

Sepantronium bromide (YM155) is a Survivin inhibitor which recently advanced as an anticancer agent in phase II clinical trials. Survivin belongs to IAP (inhibitor of apoptosis) gene family and is a pivotal target for treatment due to its overexpression and oncogenic function in many malignancies, including acute lymphoblastic leukemia (ALL). Although survivin is a specific target for YM155, recent reports have shown that it has many other crucial targets that regulate its anti-apoptotic effects. The aim of this study was to investigate whether YM155 could have an effect on cell death-inducing genes as well as inducing apoptosis in T-ALL MOLT4- cell line.

We treated MOLT-4 cells with increasing concentrations of YM155 and then cell viability was determined using MTT (methyl thiazolyl tetrazolium) assay. Also, the rate of induction of apoptosis in MOLT-4 cells and the target genes expression levels were evaluated by Annexin V/PI and real-time PCR, respectively.

YM155 inhibited cell growth in MOLT-4 cells. This outcome is achieved by inducing apoptosis and a significant increase in the expression level of P53, MiR-9, caspase 3 and decreasing the mRNA expression levels of survivin, Sirtuin1(SIRT1), member of anti-apoptotic proteins family (Bcl-2), and epithelial-to-mesenchymal transition (EMT) initiating factors Snail1and Zeb2.

The results showed that use of YM155 can be a potential drug therapy in T-ALL patients with promising effects on apoptosis induction.

One of the most significant problems in the treatment of leukemia is the expansion of resistance to chemotherapeutic agents. Therefore, assessing the drug resistance and especially the drug resistance genes of leukemic cells is important in any treatment. The impact of Mesenchymal Stem Cells (MSCs) and hypoxic condition have been observed in the biological performance of majority of leukemic cells.
MOLT-4 cells were co-cultured with MSCs in the hypoxic condition induced by Cobalt Chloride (CoCl2) for 6 and 24 hr. Then, apoptosis of cells was analyzed using annexin-V/PI staining and expression of the drug resistance genes including MDR1, MRP, and BCRP along with apoptotic and anti-apoptotic genes, including BAX and BCL2, was evaluated by real-time PCR.
The hypoxic condition for MOLT-4 cells co-cultured with MSCs could significantly increase the expression of MDR1 and BCRP genes (p These effects can demonstrate the important role of hypoxia and MSCs on the biological behavior of Acute Lymphoblastic Leukemia (ALL) cells that may lead to particular treatment outcomes.

DNA methylation is a well-studied epigenetic mechanism that is a potent arm of the gene expression controlling machinery. Since the hypoxic situation and the various cells of bone marrow microenvironment, e.g. mesenchymal stem cells, play a role in the in vivo and in vitro biology of leukemic cells, we decided to study the effects of hypoxia and mesenchymal stem cells (MSCs) on the promoter methylation pattern of BAX and BCL2 genes.
In this experimental study, the co-culture of MOLT-4 cells with MSCs and treatment with CoCl2 was done during 6, 12, and 24 hour periods. Total DNA was extracted using commercial DNA extraction kits, and sodium bisulfite (SBS) treatment was performed on the extracted DNA. Methylation specific polymerase chain reaction (MSP) was used to evaluate the methylation status of the selected genes’ promoter regions.
The BAX and BCL2 promoters of untreated MOLT-4 cells were in partial methylated and fully unmethylated states, respectively. After incubating the cancer cells with CoCl2 and MSCs, the MSP results after 6, 12, and 24 hours were the same as untreated MOLT-4 cells. In other words, the exposure of MOLT-4 cells to the hypoxia-mimicry agent and MSCs in various modes and different time frames showed that these factors have exerted no change on the methylation signature of the studied fragments from the promoter region of the mentioned genes.
Hypoxia and MSCs actually have no notable effect on the methylation status of the promoters of BAX and CL2 in the specifically studied regions. DNA methylation is probably not the main process by which MSCs and CoCl2 induced hypoxia regulate the expression of these genes. Finally, we are still far from discovering the exact functional
mechanisms of gene expression directors, but these investigations can provide new insights into this field for upcoming tudies.

Mesenchymal stem cells (MSCs) are a group of adult stem cells naturally found in the body. These cells were reported from many sources. Bone marrow and adipose tissue are two main sources of human MSCs. MSCs have attracted considerable attention in the fields of cell and gene therapy due to their intrinsic characteristics and ability to differentiate into multiple lineages. In this study MSCs were isolated from adipose tissue and the harvested cells were characterized, using minimal criteria defined by International Society for Cellular Therapy (ISCT) for human MSCs. Adipose tissue derived MSCs (AD-MSCs) were adherent to plastic surface of culture flask. In the culture with defined differentiation condition, these cells differentiated to adipocyts and osteoblasts. Also immunophenotyping using flow cytometry showed CD90 (96.3%), CD73 (97.7%) positive and CD34 (0.99%), CD45 (0.34%), CD31 (0.41%) negative cells. Adipose tissue due to its easy accessibility and less aggressiveness in compare to other organs, such as bone marrow, seems to be an ideal source for human MSCs. Besides, frequency of MSCs in adipose tissue is higher than bone marrow and adipose-MSCs can easily be isolated by tissue digestion. It is anticipated that adipose tissue would become a popular source for MSC studies in future.