Synergistic effect of simvastatin and romidepsin on gamma-globin gene induction

Objective: Hemoglobinopathies such as beta-thalassemia and sickle cell disease [SCD] are inherited disorders that are caused by mutations in beta-globin chain. Gamma-globin gene reactivation can ameliorate clinical manifestations of betathalassemia and SCD. Drugs that induce fetal hemoglobin [HbF] can be promising tools for treatment of beta-thalassemia and SCD patients. Recently, it has been shown that Simvastatin [SIM] and Romidepsin [ROM] induce HbF. SIM is a BCL11a inhibitor and ROM is a HDAC inhibitor and both of these drugs are Food and Drug Administration [FDA]-approved for hypercholesterolemia and cutaneous T-cell lymphoma respectively. Our aim was to evaluate the synergistic effects of these drugs in inducing HbF

Materials and Methods: In our experimental study, we isolated CD34+ cells from five cord blood samples that were cultured in erythroid differentiation medium containing ROM and Simvastatin. Then Gamma-globin, BCL11a and HDAC gene expression were evaluated on the 7th and 14th day of erythroid differentiation by real-time polymerase chain reaction [PCR] and immunocytochemistry

Results: Our results showed that combination of SIM and ROM significantly increased Gamma-globin gene expression and inhibit BCL11a and HDAC expression compared to results of using each of them alone. SIM and ROM lead to 3.09- fold increase in HbF production compared to the control group. Also, SIM inhibited BCL11a expression [0.065-fold] and ROM inhibited HDAC1 expression [0.47-fold] as two important inhibitors of HbF production after birth

Conclusion: We propose combination therapy of these drugs may be ameliorate clinical manifestation in beta-thalassemia and SCD with at least side effects and reduce the need for blood transfusion

Significance of inactivated genes in leukemia: pathogenesis and prognosis

Epigenetic and genetic alterations are two mechanisms participating in leukemia, which can inactivate genes involved in leukemia pathogenesis or progression. The purpose of this review was to introduce various inactivated genes and evaluate their possible role in leukemia pathogenesis and prognosis. By searching the mesh words "Gene, Silencing AND Leukemia" in PubMed website, relevant English articles dealt with human subjects as of 2000 were included in this study. Gene inactivation in leukemia is largely mediated by promoter's hypermethylation of gene involving in cellular functions such as cell cycle, apoptosis, and gene transcription. Inactivated genes, such as ASPP1, TP53, IKZF1 and P15, may correlate with poor prognosis in acute lymphoid leukemia [ALL], chronic lymphoid leukemia [CLL], chronic myelogenous leukemia [CML] and acute myeloid leukemia [AML], respectively. Gene inactivation may play a considerable role in leukemia pathogenesis and prognosis, which can be considered as complementary diagnostic tests to differentiate different leukemia types, determine leukemia prognosis, and also detect response to therapy. In general, this review showed some genes inactivated only in leukemia [with differences between B-ALL, T-ALL, CLL, AML and CML]. These differences could be of interest as an additional tool to better categorize leukemia types. Furthermore; based on inactivated genes, a diverse classification of Leukemias could represent a powerful method to address a targeted therapy of the patients, in order to minimize side effects of conventional therapies and to enhance new drug strategies

Expression of hsa-MIR-204, RUNX2, PPAR gamma, and BCL2 in bone marrow derived mesenchymal stem cells from multiple myeloma patients and normal individuals

Objective: Multiple Myeloma [MM] is a heterogeneous cytogenetic disorder in which clonal plasma cells proliferate in the bone marrow [BM] and cause bone destruction. The BM microenvironment plays a crucial role in pathogenesis of this disease, and mesenchymal stem cells [MSCs] are one of the key players. Herein, we propose to investigate the expressions of hsa-MIR-204, runt-related transcription factor 2 [RUNX2], peroxisome proliferator-activated receptor gamma [PPAR gamma], and B-cell lymphoma 2 [BCL2] as factors involved in osteogenesis, adipogenesis, and MSC survival in BM-MSCs from MM patients and normal individuals

Materials and Methods: In this experimental study, we isolated MSCs from BM aspirates of MM patients and healthy donors. Total RNA were extracted before and after co-culture with L363 myeloma cells. Gene expressions of RUNX2, PPAR gamma, BCL2, and hsa-MIR-204 were assessed by quantitive real time polymerase chain reaction [qRT-PCR]

Results: Higher levels of RUNX2, PPAR gamma, and hsa-MIR-204 expressions existed in MM-MSCs compared to normally derived [ND]-MSCs. BCL2 expression decreased in MM-MSCs. We observed different results in the co-culture model

Conclusion: In general, the MM-MSCs gene expression profile differed compared to ND-MSCs. Upregulation of RUNX2, PPAR gamma, and hsa-MIR-204 in MM-MSCs compared to ND-MSCs would result in formation of bone defects. Downregulation of BCL2 would lead to MM-MSC cell death

MicroRNA expression in beta-Thalassemia and sickle cell disease: a role in the induction of fetal hemoglobin

Today the regulatory role of microRNAs [miRs] is well characterized in many diverse cellular processes. MiR-based regulation is categorized under epigenetic regulatory mechanisms. These small non-coding RNAs participate in producing and maturing erythrocytes, expressing hematopoietic factors and regulating expression of globin genes by post-transcriptional gene silencing. The changes in expression of miRs [miR-144/-320/-451/-503] in thalassemic/sickle cells compared with normal erythrocytes may cause clinical severity. According to the suppressive effects of certain miRs [miR-15a/-16-1/-23a/-26b/-27a/-451] on a number of transcription factors [myeloblastosis oncogene [MYB], B-cell lymphoma 11A [BCL11A], GATA1, Krüppel-like factor 3 [KLF3] and specificity protein 1 [Sp1]] during beta globin gene expression, It has been possible to increasing ? globin gene expression and fetal hemoglobin [HbF] production. Therefore, this strategy can be used as a novel therapy in infusing HbF and improving clinical complications of patients with hemoglobinopathies

Effect of the receptor activator of nuclear factor [ka]B and rank ligand on in vitro differentiation of cord blood CD133[+] hematopoietic stem cells to osteoclasts

Objective: receptor activator of nuclear factor-kappa B ligand [RANKL] appears to be an osteoclast-activating factor, bearing an important role in the pathogenesis of multiple myeloma. Some studies demonstrated that U-266 myeloma cell line and primary myeloma cells expressed RANK and RANKL. It had been reported that the expression of myeloid and monocytoid markers was increased by co-culturing myeloma cells with hematopoietic stem cells [HSCs]. This study also attempted to show the molecular mechanism of RANK and RANKL on differentiation capability of human cord blood HSC to osteoclast, as well as expression of calcitonin receptor [CTR] on cord blood HSC surface

Materials and Methods: in this experimental study, CD133[+] hematopoietic stem cells were isolated from umbilical cord blood and cultured in the presence of macrophage colony-stimulating factor [M-CSF] and RANKL. Osteoclast differentiation was characterized by using tartrate-resistant acid phosphatase [TRAP] staining, giemsa staining, immunophenotyping, and reverse transcription-polymerase chain reaction [RT-PCR] assay for specific genes

Results: hematopoietic stem cells expressed RANK before and after differentiation into osteoclast. Compared to control group, flow cytometric results showed an increased expression of RANK after differentiation. Expression of CTR mRNA showed TRAP reaction was positive in some differentiated cells, including osteoclast cells

Conclusion: presence of RANKL and M-CSF in bone marrow could induce HSCs differentiation into osteoclast

Advances in hematopoietic stem cell mobilization and peripheral blood stem cell transplantation

Hematopoietic stem/progenitor cells [HSPCs] which give rise to different blood cell types are present within the bone marrow microenvironment, especially in flat bones such as skull, vertebrae, pelvis and chest. Interacting factors such as stromal derived factor-1/CXCR4, very late antigen-4/vascular cell adhesion molecule-1, Lymphocyte function-associated antigen-1/ intercellular adhesion molecule-1 retain the cells in the microenvironment. Any factor affecting these links may lead to migration and mobilization of HSPCs into peripheral blood. Several factors are involved in hematopoietic stem cells [HSC] mobilization such as granulocyte-colony stimulating factor, sphingosine-1-phosphate, hepatocyte growth factor, complement system, plasminogen system and matrix metalloproteinases. In bone marrow transplantation, HSC is transferred to the recipient from bone marrow of the donor, which can be performed in two ways. In the first method, Jamshidi needle is used for aspiration of bone marrow to extract hematopoietic cells usually from the hip. The second method uses mobilizer factors such as granulocyte-colony stimulating factor and granulocyte-macrophage colony-stimulating factor to mobilize the HSC into peripheral blood. Mobilized hematopoietic stem cells are suitable for the bone marrow transplantation in leukemias such as chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocyte leukemia, Hairy cell leukemia, etc

Role of BCR-ABL P190 in Diagnosis and Prognosis of ALL patients

Acute lymphoblastic leukemia[ALL] is due to early stage arrest of lymphoblast development. The translocation of Philadelphia [Ph] chromosome occurs as a result of the BCR-ABL fusion gene, which constitutively produced activated tyrosine kinase. This gene fusion is an important indicator for prognosis in ALL and is associated with poor overall survival and remission duration. BCR-ABL could interfere in establishment of ALL. Therefore, in this study, we will try to investigate most pathological aspects involved in BCR-ABL fusion. Strategies for genetic alterations in B-ALL pathogenesis are discussed. Then, the main cytogenetic changes and genetic subtypes for ALL are highlighted. Moreover, intermediate reactions between cancer stem cells [CSC] related to ALL, its niche and microenvironment is discussed. The main objective in this review is to understand the principle prognosis in ALL to introduce new approaches and treatment alternatives

Molecular aspects of bone resorption in beta-thalassemia major

Beta-thalassemia is the most common single gene disorder worldwide, in which hemoglobin beta-chain production is decreased. Today, the life expectancy of thalassemic patients is increased because of a variety of treatment methods; however treatment related complications have also increased. The most common side effect is osteoporosis, which usually occurs in early adulthood as a consequence of increased bone resorption. Increased bone resorption mainly results from factors such as delayed puberty, diabetes mellitus, hypothyroidism, ineffective hematopoiesis as well as hyperplasia of the bone marrow, parathyroid gland dysfunction, toxic effect of iron on osteoblasts, growth hormone [GH] and insulin-like growth factor-1 [IGF-1] deficiency. These factors disrupt the balance between osteoblasts and osteoclasts by interfering with various molecular mechanisms and result in decreased bone density. Given the high prevalence of osteopenia and osteoporosis in thalassemic patients and complexity of their development process, the goal of this review is to evaluate the molecular aspects involved in osteopenia and osteoporosis in thalassemic patients, which may be useful for therapeutic purposes

STATs: an old story, yet mesmerizing

Signal transducers and activators of transcription [STATs] are cytoplasmic transcription factors that have a key role in cell fate. STATs, a protein family comprised of seven members, are proteins which are latent cytoplasmic transcription factors that convey signals from the cell surface to the nucleus through activation by cytokines and growth factors. The signaling pathways have diverse biological functions that include roles in cell differentiation, proliferation, development, apoptosis, and inflammation which place them at the center of a very active area of research. In this review we explain Janus kinase [JAK]/STAT signaling and focus on STAT3, which is transient from cytoplasm to nucleus after phosphorylation. This procedure controls fundamental biological processes by regulating nuclear genes controlling cell proliferation, survival, and development. In some hematopoietic disorders and cancers, overexpression and activation of STAT3 result in high proliferation, suppression of cell differentiation and inhibition of cell maturation. This article focuses on STAT3 and its role in malignancy, in addition to the role of microRNAs [miRNAs] on STAT3 activation in certain cancers

Differentiation potential of O Bombay human-induced pluripotent stem cells and human embryonic stem cells into fetal erythroid-like cells

There is constant difficulty in obtaining adequate supplies of blood components, as well as disappointing performance of "universal" red blood cells. Advances in somatic cell reprogramming of human-induced pluripotent stem cells [hiPSCs] have provided a valuable alternative source to differentiate into any desired cell type as a therapeutic promise to cure many human disease. In this experimental study, we examined the erythroid differentiation potential of normal Bombay hiPSCs [B-hiPSCs] and compared results to human embryonic stem cell [hESC] lines. Because of lacking ABO blood group expression in B-hiPSCs, it has been highlighted as a valuable source to produce any cell type in vitro. Similar to hESC lines, hemangioblasts derived from B-hiPSCs expressed approximately 9% KDR+CD31+ and approximately 5% CD31+CD34+. In semisolid media, iPSC and hESC-derived hemangioblast formed mixed type of hematopoietic colony. In mixed colonies, erythroid progenitors were capable to express CD71+GPA+HbF+ and accompanied by endothelial cells differentiation. Finally, iPS and ES cells have been directly induced to erythropoiesis without hemangioblast formation that produced CD71+HbF+erythroid cells. Although we observed some variations in the efficiency of hematopoietic differentiation between iPSC and ES cells, the pattern of differentiation was similar among all three tested lines

Potential uses for cord blood mesenchymal stem cells

Stem cell therapy is a powerful technique for the treatment of a number of diseases. Stem cells are derived from different tissue sources, the most important of which are the bone marrow [BM], umbilical cord [UC] blood and liver. Human UC mesenchymal stem cells [hUC-MSCs] are multipotent, non-hematopoietic stem cells that have the ability to self-renew and differentiate into other cells and tissues such as osteoblasts, adipocytes and chondroblasts. In a number of reports, human and mouse models of disease have hUC-MSCs treatments. In this article, we review studies that pertain to the use of hUC-MSCs as treatment for diseases

Osteogenic inhibition in multiple Myeloma

Multiple myeloma [MM] is a plasma cell malignancy where plasma cells are increased in the bone marrow [BM] and usually do not enter peripheral blood, but produce harmful factors creating problems in these patients [e.g. malignant plasma cells over activate osteoclasts and inhibit osteoblasts with factors like RANKL and DKK]. These factors are a main cause of bone lesion in MM patients. Recently SOST gene which responsible to encodes the sclerostin protein was identify. This protein specifically inhibits Wnt signaling in osteoblasts [inhibition of osteoblast differentiation and proliferation] and decrease bone formation and can also cause bone lesion in MM patients. In this experimental study, human myeloma cell lines [U266 b1] were purchased from Pasteur Institute of Iran. Samples consisted of BM aspirates from the iliac crest of MM patients. BM with more than 70% plasma cell were selected for our study [6 patients] and one healthy donor. RNA extraction was done with Qiagen kit. was undertaken on mRNA of samples and cell lines. Also we purchased unrestricted somatic stem cells from Bonyakhte Company to evaluate the effect of soluble factors from myeloma cell lines on osteogenic differentiation medium. Our results showed that SOST is expressed significantly in primary myeloma cells derived from MM patients and myeloma cell lines. In other words, patients with more bone problems, express SOST in their plasma cells at a higher level. In addition, myeloma cells inhibit osteoblast differentiation in progenitor cells from umbilical cord blood stem cell [UCSC] in osteogenic inducing medium. There are many osteoblast maturation inhibitory factors such as DKK, Sfrp and Sclerostin that inhibit maturation of osteoblast in bone. Among osteoblast inhibitory agents [DKK, Sfrp, Sclerostin] sclerostin has the highest specificity and therefore will have less side effect versus non-specific inhibitory agents. Our results also show that based on SOST expression in MM, there is a potential to inhibit sclerostin with antibody or alternative methods and prevent bone lesion in MM patients with the least side effect

High expression of cyclin D1 and p21 in N-nitroso-N-methylurea-induced breast cancer in wistar albino female rats

N-nitroso-N-methylurea [NMU] induces breast cancer in rodents, particularly in rats. This model of breast cancer is very similar to human breast cancer. As a continuation of our recent work, we investigated the expressions of cyclin D1 and p21 in NMU-induced breast cancer of Wistar Albino rats. In this experimental study, mammary carcinoma was induced in female Wistar Albino rats by a new protocol which included the intraperitoneal injection of NMU [50 mg/kg] at 50, 65, and 80 days of the animal's age. The animals were weighed weekly and palpated in order to record the numbers, location, and size of tumors. Subsequently tumor incidence [TI], latency period [LP], and tumor multiplicity [TM] were reported. About four weeks after the tumor size reached 1.5 cm[3], rats were sacrificed. Cyclin D1 and p21 expressions in tumors and normal mammary glands from normal rats were measured by reverse-transcription polymerase chain reaction [RT- PCR] and Western blot analysis. Statistical analysis of the data was performed using SPSS software version 16.0. The efficiency of tumor induction was 65%, LP was 150 days, and a TM of 1.43 +/- 0.53 per rat was noted. RT-PCR and Western blot data indicated significant [p<0.05] induction of both cyclin D1 and p21 expressions in rat mammary tumors compared with normal tissue from the control group. These results indicate an efficient mammary tumor induction protocol for this type of rat, which is accompanied by an increase in cyclin D1 and p21 expressions

miR-155 down Regulation by LNA inhibitor can reduce cell growth and proliferation in PC12 cell line

MicroRNAs [miRNAs] are a class of small non coding regulatory RNAs that have key functions in multiple cell processes. Deregulation of these tiny miRNAs are involved in various human diseases. MiR-155 is one of the multifunctional miRNA that its over-expression has been found to be associated with different kinds of cancer such as leukemia, breast and colon cancers. It is thought that deregulation and over-expression of this microRNA may be associated with PC12 cell proliferation. So, the aim of this study was to investigate the role of miR-155 expression on PC12 cell growth. For this reason, PC12 cells were cultured and transfected by 3 different concentration [25, 50 and 75 nmol] of either LNA anti-miR-155 or scramble antisense in 24-well plate. Then, total RNA was extracted from transfected cells. miRNA cDNAs were synthesized from isolated total RNA. In the second step, miR-155 expression level was analyzed using the quantitative real-time polymerase chain reaction [QRT-PCR]. MTT test was performed to evaluate cell viability. In the next step, apoptosis assay was assessed to investigate anti miR-155 effect on PC12 cells death. Obtained results were analyzed with t-test. MTT test revealed that cell viability of transfected cells with 75 nM of anti-miR- 155 to be reduced by half of the control and scramble groups [0.5 vs. 0.97 and 0.94]. Our data suggest that miR-155 over-expression is associated with PC12 cell growth. So, miR-155 down regulation by anti-miR-155 could open up new ways to restrain brain tumor growth, as anti-miR-155 causes PC12 cells to repress

Neoplastic bone marrow niche: hematopoietic and mesenchymal stem cells

The neoplastic niche comprises complex interactions between multiple cell types and molecules requiring cell-cell signaling as well as local secretion. These niches are important for both the maintenance of cancer stem cells and the induction of neoplastic cells survival and proliferation. Each niche contains a population of tumor stem cells supported by a closely associated vascular bed comprising mesenchyme- derived cells and extracellular matrix. Targeting cancer stem cells and neoplastic niche may provide new therapies to eradicate tumors. Much progress has been very recently made in the understanding of the cellular and molecular interactions in the microenvironment of neoplastic niches. This review article provides an overview of the neoplastic niches in the bone marrow. In addition to highlighting recent advances in the field, we will also discuss components of the niche and their signaling pathways

high yield expansion and megakaryocytic differentiation of human umbilical cord blood CD133[+] cells

Despite of many benefits, umbilical cord blood [UCB] hematopoietic stem cell [HSC] transplantation is associated with low number of stem cells and slow engraftment; in particular of platelets. So, expanded HSCs and co-transfusion of megakaryocyte [MK] progenitor cells can shorten this period. In this study, we evaluated the cytokine conditions for maximum expansion and MK differentiation of CD133[+] HSCs. In this experimental study, The CD133[+] cells were separated from three cord blood samples by magnetic activated cell sorting [MACS] method, expanded in different cytokine combinations for a week and differentiated in thrombopoietin [TPO] for the second week. Differentiation was followed by the flow cytometry detection of CD41 and CD61 surface markers. Colony forming unit [CFU] assay and DNA analysis were done for colonogenic capacity and ploidy assay. CD133[+] cells showed maximum expansion in the stem span medium with stem cell factor [SCF] + FMS-like tyrosine kinase 3-ligand [Flt3-L] + TPO but the maximum differentiation was seen when CD133[+] cells were expanded in stem span medium with SCF + Interleukin 3 [IL-3] + TPO for the first and in TPO for the second week. Colony Forming Unit-MK [CFU-MK] was formed in three sizes of colonies in the mega-cult medium. In the DNA analysis; 25.2 +/- 6.7% of the cells had more than 2n DNA mass. Distinct differences in the MK progenitor cell count were observed when the cells were cultured in stem span medium with TPO, SCF, IL-3 and then the TPO in the second week. Such strategy could be applied for optimization of CD133[+] cells expansion followed by MK differentiation

[Quantitative evaluation of imatib efflux and influx transporters expression in chronic myeloid leukemia [CML] patients]

In this study quantitative expression of MDR1 and hOCT1 genes in CML patients and normal people were measured using Real-Time PCR. To study quantitative expression of these genes by real-time PCR, mastermix with syber green was used. Peripheral blood samples from 30 CML patients and 27 normal persons were harvested. Real-time PCR results were analyzed with relative quantification method. This study showed that in the patients group who were under treatment with Imatib, MDR1 gene expression was increased which was statistically significant. This increase has a direct relation with disease progress. Gene expression in AP and BP patients was also higher than CP patients. In contrast, hOCT1 expression in patients group in comparison with normal group was not statistically significant. MDR1 increase in leukemic cell membrane results in the reduction of intra-cellular drug concentration. Thus, optimal concentration of drug for inhibition of BCR-ABL tyrosine kinase is not achieved which culminated in disease progression to AP and BP phases. Moreover changes in hOCT1 gene expression as an influx transporter of Imatib could affect intracellular concentration of drug and finally determine therapy outcome. However, in this study hOCT1 gene expression was variable and was not statistically significant

[ role of myeloma cells in the in-vitro diffrentiation of cord blood hematopoietic stem cell to osteoclast]

Average Age of population in the industrial countries has increased. Because of aging the percent of the diseases related to the oldness such as multiple myeloma have also increased. It has both common and unique symptoms and effects. The unique effects include wide bone reabsorption. It seems necessary to understand the structure of Bone Marrow Niche and the effects of Myeloma cells on adjacent hematopoietic Stem cells with a new approach. We have studied the differentiating effect induced by the Myeloma cells through co-culturing the Myeloma cells and hematopoietic stem cells, extracted out of cord blood. In this investigation we also cocultured myeloma cells with the monoblastic cell line [U937] in order to evaluate the effect of myeloma cells on monoblastic cells differentiation. Our findings show that increased expression of myeloid and monocytoid markers in coculturing of myeloma cells and HSCs. Moreover following monoblastic and myeloid cells coculturing, we observed probably TRAP positive osteoclastic like cells. Our findings show that presence of myeloma cells in Bone Marrow play essential role in HSCs differentiation to monocytoid [osteoclastic] lineage

[Evaluation of Morpholino antisense oligos' role on BCR-ABL gene silencing in the K562 cell line]

Chronic myeloid leukemia [CML] develops when a hematopoietic stem cell acquires the BCR/ABL fusion gene. This causes these transformed hematopoietic cells to have a greater than normal proliferation rate. Scientists attempt to improve the CML treatment process by silencing the BCR/ABL oncogene. In this work, we used morpholino antisense oligos to silence the BCR/ABL oncogene. In this study, the K562 was used as a BCR/ABL fusion-gene positive cell line and the Jurkat cell line as a control. We explored the inhibiting capacity of morpholino antisense oligos in the the expression of the BCR/ABL oncogene and studied their p210 BCR/ABL suppression, inhibition of cell proliferation and stimulation of apoptosis in the K562 cells after 24 and 48 hours. Endo-Porter was used for delivery of morpholino antisense oligos into cell cytosols. Meanwhile, flow cytometric analysis was performed in order to determine the appropriate concentration of morpholino antisense oligos. Prolonged exposure of the K562 cell line to the morpholino antisense oligos targeted against the BCR-ABL gene showed proliferation inhibition as its main feature. After western blotting, we found that complete silencing of BCR/ABL was achieved, but flow cytometric analysis showed no broad apoptosis. The results indicate that the Morpholino antisense oligo is able to inhibit p210 BCR/ABL; however, it cannot induce broad apoptosis due to co-silencing of BCR