Antitumor Activity of 6-(cyclohexylamino)-1, 3-dimethyl-5(2-pyridyl)furo[2,3-d]pyrimidine-2,4(1H,3H)-dione and Its Ti(IV), Zn(II), Fe(III), and Pd(II) Complexes on K562 and Jurkat Cell Lines

(6-(cyclohexylamino)-1,3-dimethyl-5(2-pyridyl)furo[2,3-d]pyrimidine-2,4(1H,3H)-dione) abbreviated as CDP was synthesized and characterized. Ti(IV), Zn(II), Fe(III), and Pd(II) metal complexes of this ligand are prepared by the reaction of salts of Ti(IV), Zn(II), Fe(III), and Pd(II) with CDP in acetonitrile. Characterization of the ligand and its complexes was made by microanalyses, FT-IR, 1H NMR, 13C NMR, and UV-Visible spectroscopy. All complexes were characterized by several techniques using elemental analysis (C, H, N), FT-IR, electronic spectra, and molar conductance measurements. The elemental analysis data suggest the stoichiometry to be 1:1 [M:L] ratio formation. The molar conductance measurements reveal the presence of 1:1 electrolytic nature complexes. These new complexes showed excellent antitumor activity against two kinds of cancer cells that are K562 (human chronic myeloid leukemia) cells and Jurkat (human T lymphocyte carcinoma) cells

The Role of Pomalidomide-Based Epigenetic Effect on DNMT Genes Expression in Myeloma Cell Line

Multiple myeloma (MM) is clonal B-cell malignancy characterized by the progressive proliferation of malignant plasma cells and accumulation of monoclonal immunoglobulin (M-spike) in blood and urine. Pomalidomide is an immunomodulatory agent which has potentially suppressed myeloma cell progression, especially in drug-resistant cases. As epigenetic modifications have an important role in gene regulation and because of the revealing role of DNA-Methyltransferase 1 (DNMT1) overexpression in myeloma pathogenesis, in this study DNMT1, 3a and 3b genes expression of U266 myeloma cell line treated with pomalidomide have been evaluated. In this study after treatment of U266 cells with 1 μM pomalidomide for 48 hours, total RNA extraction and cDNA synthesis was performed. Gene expression of DNMT1, 3a and 3b has been evaluated using real time PCR technique. The result of this study show that pomalidomide can downregulate the expression of DNMT1, 3a, and 3b in 48 hours of treatment as 0.049, 0.058 and 0.055, respectively as comparing with untreated control (P<0.05). Based on these results we conclude that pomalidomide has desired effect on epigenetic modification by downregulation of DNMTs genes expression and has been considered as an effective drug for inhibition of myeloma proliferation

Correlation between Methylation and Expression Level of P15 and P16 Genes during Differentiation of Cord Blood Stem Cells into Erythroid Lineage Mediated by Erythropoietin

Background: Several influential factors such as transcription factors and intracellular signaling components are involved in differentiation of stem cells into a specific lineage. P15 and p16 proteins are among these factors. Accumulating evidences has introduced the epigenetic as a master regulator of these factors during lineage specification. The main objective of this study is to determine the correlation between the expression level and methylation pattern of P15 and P16 genes in erythroid lineage after in vitro differentiation by erythropoietin (EPO).Materials and Methods: The purified and expanded CD34+ cord blood stem cells were differentiated into erythroid lineage in the presence of EPO. DNA was isolated from both cord blood stem cells and differentiated cells. The Real-Time PCR performed using cDNA and the isolated DNA was used in methylation Specific PCR (MSP) reaction for methylation pattern analysis in both pre and post differentiation stages.Results: The study demonstrated that P15 and P16 genes have partial methylation after erythroid differentiation by EPO. The Expression of P15 gene was higher after differentiation and the expression of P16 gene had a slightly decreased level in post differentiation stage.Conclusion: Significant increase in P15 gene expression after differentiation to erythroid lineage, suggests the remarkable efficacy of this gene in erythroid function. According to upregulation of P15 gene after differentiation despite unchanged methylation status and slight down regulation of P16 gene with slight hyper-methylation of the gene it can be suggested that although the methylation can affects the expression level of P16 gene, the P15 gene is not affected by this mechanism during erythroid differentiation mediated by EPO

The Effect of Aligned and Random Electrospun Fibrous Scaffolds on Rat Mesenchymal Stem Cell Proliferation

Objective: The development of combining mesenchymal stem cells (MSCs) with surface modified three-dimensional (3D) biomaterial scaffold provides a desirable alternative for replacement of damaged and diseased tissue. Nanofibrous scaffolds serve as suitable environment for cell attachment and proliferation due to their similarity to the physical dimension of the natural extracellular matrix. In this study the properties of plasma treated poly-C-caprolactone nanofiber scaffolds (p-PCL) and unaltered PCL scaffolds were compared,and then p-PCL scaffolds were evaluated for MSC culture.Materials and Methods: Aligned and random PCL nanofibrus scaffolds were fabricatedby electrospining and their surface modified with O2 plasma treatment to enhanceMSC proliferation, adhesion and interaction. Chemical and mechanical characterizationswere carried out using scanning electron microscopy (SEM), water contact angle and tensile testing. Cell adhesion and morphology were evaluated using SEM 1 day after culture. Statistical analysis was carried out using one way analysis of variance(ANOVA).Results: The proliferation of MSCs were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-DiphenyltetrazoliumBromide(MTT) assay on day 1, 3, and 5 after cell culture. Results showed that the numbers of cells that had grown on PCL nanofibrous scaffolds were significantly higher than those of control surfaces without nanofibers. Furthermore, the proliferation of MSCs on random nanofiber was significantly higher compared to that on aligned nanofiber.Conclusion: This study showed that while both aligned and random plasma treated PCL nanofibrous scaffold are more suitable substrates for MSC growth than tissue culture plates, random nanofiber best supported the proliferation of MSCs

Regulation of bone formation and resorption by Beta Adrenergic system

Background Following initial studies reporting the presence of &beta;eta adrenergic receptors on bone cells, several studies have investigated the role of the Beta adrenergic system in the formation and resorption of bone. The goal of this report was to review the data supporting the role of the&nbsp; beta adrenergic system and osteo-neuromediators in the regulation of bone homeostasis. Materials and Methods: In this review, more than 100 published articles were reviewed for the evidence of the adrenergic effect on bone remodeling and focused on the latest advances in this area. Results: Based on a variety of pharmacologic, genetic and clinical studies focused on b-adrenergic receptor (bAR) signaling in bone cells, The adrenergic activity of the sympathetic nervous system has been shown to be a negative regulator of bone mass; adrenergic signaling inhibits osteoblast proliferation and promotes osteoclastogenesis . Conclusion: Altogether, these observations and linked findings are of great significance since they improve our understanding of bone physiology., and uncovered new potential therapeutic strategies for the design of bone anabolic drugs. Certainly, knowledge about downstream factors of beta-adrenergic system can be helpful in making decisions about appropriate therapeutic interventions

Efficient Expansion of SALL4–Transduced Umbilical Cord Blood Derived CD133+Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) were characterized by self-renewal and multilineage potential. Umbilical cord blood-derived (UCB) as an alternative source of HSCs is widely used especially in children for stem cells transplant (SCT). The main limitation in using UCB for transplantation especially in adults is low cell dose. To overcome this limitation besides using double dose UCB, ex vivo expansion is the most important way to increase cell number for transplantation. HSCs are mainly isolated using CD133 or CD34. CD133, as the most primitive marker, shows important physiological role in maintenance and expansion of HSCs. SALL4 plays crucial role in the development and maintaining the pluripotency and self-renewal ability of embryonic stem cells (ESCs) as well as HSCs. Moreover, SALL4 act as a regulator of HSCs expansion, normal hematopoiesis, and hematological malignancies. In the present study, CD133+ cells positively selected and ex vivo expanded in SALL-4 and GFP-transduced group. CD133 expression assessed using flow cytometry at day 0, 7 and 10. Moreover, multilineage differentiation and proliferation potential of expanded cells in both groups evaluated using colony forming unit (CFU) assay, and cells count assay. Karyotyping analysis was performed to assess any chromosomal instability after 7 days of expansion. Obtained results demonstrated that SALL-4 transduced cells showed significant increase in cell number compared to control group. Moreover, immunophenotyping results showed higher expression level of CD133 at day 7 and 10 following expansion in SALL-4 transduced (62 % and 42%) compared to control group (51% and 20.6%). Our results illustrated that SALL4 could act as a positive factor for the expansion of CD133+ derived UCB cells besides maintaining self-renewal and differentiation ability of expanded cell without any numerical and structural chromosomal aberrations

Effect of The Receptor Activator of Nuclear Factor кB 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

Osteogenic Inhibition in Multiple Myeloma

Objective: 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.Materials and Methods: 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.Results: 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.Conclusion: 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