Iranian biomedical journal

Publications in this journal

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  Article: Association between HIC1 and RASSF1A promoter hypermethylation with MTHFD1 G1958A polymorphism and clinicopathological features of breast cancer in Iranian patients.

  Mozhgan Rasti, Parastoo Tavasoli, Ahmad Monabati, Mona Entezam
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  ABSTRACT: Ras-associated domain family 1 (RASSF1A) and hypermethylated in cancer (HIC1) genes are methylated more frequently in breast cancer. Genetic factors that alter the DNA methylation levels in normal and tumor tissues could therefore influence the susceptibility to this tumor phenotype. objective: We determined the frequency of aberrant methylation of HIC1 and RASSF1A gene promoters and their association with methylene tetrahydrofolate dehydrogenase (MTHFD1) G1958A polymorphism and major clinical and pathological features of breast cancer in Iranian women. DNA was extracted from 81 primary breast tumors and 100 control blood samples. Gene promoter methylation was analyzed by methylation-specific polymerase chain reaction. Eighty four percent of the breast cancer samples showed total methylation in at least one of two tested loci. We detected HIC1 hypermethylation in 79% of invasive and metastasis tumors and RASSF1A gene hypermethylation in 51% of them. We found no association between HIC1 and RASSF1A gene hypermethylation and MTHFD1 G1958A polymorphism, but a significant correlation between methylation of HIC1 and RASSF1A promoters was indicated (r = 0.24, P = 0.02). There was a combination between hypermethylation of HIC1 locus and nodal involvement in the studied population (p=0.03). We found a significant association between total methylation and nodal involvement (P = 0.01) as well as tumor size more than 2 cm in all cases (P = 0.02). Methylation of HIC1 and RASSF1A promoters can be used as epigenetic markers to detect the malignant progression of breast carcinoma in Iranian women patients.
  Iranian biomedical journal 10/2009; 13(4):199-206.
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  Article: Apoptotic and necrotic effects of pectic acid on rat pituitary GH3/B6 tumor cells.

  Farnoosh Attari, Houri Sepehri, Ladan Delphi, Bahram Goliaei
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  ABSTRACT: Pectin is composed of complex polysaccharides that can inhibit cancer metastasis and proliferation with no evidence of toxicity. In the present study, the apoptotic and necrotic effects of pectic acid (PA) on the rat pituitary GH3/B6 tumor cells has been investigated. GH3/B6 cells were cultured in the Ham's F12 medium enriched with 15% horse serum and 2.5% fetal bovine serum for 3 days. Then, they were treated by various amounts of PA in different periods (6, 24 and 48 hours). Bromocriptine was used as positive control and the cell viability was detected by MTT test. The nuclear morphology of cells was explored by florescent stains including acridine orange (AO)/ethidium bromide (EB). In addition, percentages of apoptotic and necrotic cells were studied with triphosphate nick-end labeling (TUNEL) assay, cell cycle analysis and propidium iodide (PI) staining. Long-term incubation with PA results in increased cell death and DNA damage as detected by MTT assay and AO/EB staining. TUNEL assay showed that PA (100 microg/ml to 1 mg/ml) could induce apoptosis in a dose-dependent manner, while higher concentrations of PA (2.5 and 5 mg/ml) induced necrosis which was confirmed by PI staining. Furthermore, cell cycle analysis indicated that PA induced sub G1 events, and DNA fragmentation was also correlated with the number of the apoptotic cells. It can be concluded that PA is responsible for apoptosis in the rat pituitary tumor cells. Therefore, one may suggest that this group of polysaccharides can be used in treatment of pituitary tumors.
  Iranian biomedical journal 10/2009; 13(4):229-36.
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  Article: The protective effects of N-Acetl-cysteine, oxo-thiazolidine-carboxylate, acetaminophen and their combinations against sulfur mustard cytotoxicity on human skin fibroblast cell line (HF2FF).

  Mehdi Saberi, Ali Zaree Mahmodabady
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  ABSTRACT: Using human skin-fibroblast cell line HF2FF, the efficacy of some drugs was evaluated against sulfur mustard (SM) cytotoxicity. The drugs were the sulfhydryl containing molecule including N-acetylcysteine (NAC), 2-oxo-thiazolidine-4-carboxylate (OTC) and acetaminophen as glutathione (GSH) stimulator pathway. The protective effects of NAC (0.1 mM), OTC (1.8 mM), and acetaminophen (25 mM) alone or in combination with each other were evaluated on SM (180 M)-induced cytotoxicity. NAC and OTC were applied with SM simultaneously and acetaminophen 30 min before SM exposure, incubated for 1 h and then were rinsed and incubated with fresh medium. The efficacy was evaluated by determination of cells viability, intracellular GSH level and catalase activity 1 and 24 h post SM exposure or co-treatments. The cells viability was decreased 21.8% and 55.2%, respectively for 1 and 24 h post SM (1 h exposure) incubation. So, the 1-h SM exposure and 24-h treatment incubation were selected for evaluation. While, NAC alone treatment increased the cells viability (25%), GSH level (320%) and catalase activity (18%), the most effective combination was NAC plus OTC and acetaminophen which increased more significantly the cells viability (about 40%), GSH level (470%) and catalase activity (100%). The most effective combination was NAC (0.1 mM) plus OTC (1.8 mM) and acetaminophen (25 mM) which should be used before or concomitant with SM exposure. These drugs may reduce SM toxicity possibly by increment of GSH level and catalase activity. This efficacy needs to be confirmed by in vivo study.
  Iranian biomedical journal 10/2009; 13(4):215-21.
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  Article: Effect of propranolol on angiogenic factors in human hematopoietic cell lines in vitro.

  Fatemeh Hajighasemi, Sakineh Hajighasemi
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  ABSTRACT: Beta-adrenergic blocking agents have been broadly used for treatment of many cardiovascular diseases such as arterial hypertension and ischemic heart failure. Anti-tumoral, anti-inflammatory and anti-angiogenesis effects of propranolol (a non-selective beta-adrenergic blocker) have been shown. Angiogenesis (replenish of the pre-existing vascular networks) plays a critical role in some pathological conditions such as tumor expansion and metastasis. In this study, we investigated the effects of propranolol on vascular endothelial growth factor (VEGF) production and matrix metalloproteinase-2 (MMP-2) activity (two important angiogenic factors) in human leukemic cell lines in vitro. Two human leukemic T (Molt-4 and Jurkat) and one monocyte (U937) cell lines were used in this study. The cells were cultured in complete RPMI medium and then incubated with different concentrations of propranolol (0.3-30 microM) in the presence or absence of phorbol myristate acetate (PMA, 25 ng/ml) for 48 hours. The level of VEGF secreted in the cell culture supernatants was measured with enzyme-linked immunosorbent assay kits (R and D systems) and MMP-2 activity in cell-conditioned media was evaluated by gelatin zymography. Propranolol significantly decreased VEGF production and also MMP-2 activity in PMA-activated human leukemic cell lines Molt-4, Jurkat and U937 at 30 microM concentration of the drug compared to untreated control cells (P<0.05). Propranolol might be a useful anti-angiogenic agent in hematopoietic malignancies. Thus, propranolol along with its chronic long-term usage in cardiac problems may have potential implication in treatment of leukemia.
  Iranian biomedical journal 10/2009; 13(4):223-8.
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  Article: Effects of tamoxifen on morphological and ultrastructural aspects of developing hippocampus of rat.

  Maliheh Nobakht, Nowruz Najafzadeh, Bahareh Kordestani Shargh
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  ABSTRACT: Tamoxifen treatment induced cell death in the hippocampus formation of the prenatal and postnatal rat. The present study delineates the effect of tamoxifen on developing hippocampus in prenatal, postnatal and full term neonate rats received certain doses of the partial antagonist tamoxifen. After perfusion and fixation, the brains were removed and processed for light and electron microscopy. The morphology, ultrastructure and the density of the neurons in different ages (E22, P1, P7 and P21) and in different areas of developing hippocampus including cornu ammonis (CA1 and CA3), dentate gyrus and subiculum were studied. These findings showed that in tamoxifen-treated groups, the cell number of pyramidal neurons of CA1 and subiculum significantly decreased comparing to control groups in E22, P1 and P7 but not in third weeks. The mitochondria of the above mentioned groups also showed a dilated feature with less cristae than control group and most of them were greatly enlarged and swollen into spherical shapes rather than the normal ovoid or rod shape. The present study shows that prenatal exposure to tamoxifen alters neurogenesis in developing rat hippocampus. These results demonstrated the non-neuroprotective roles of tamoxifen.
  Iranian biomedical journal 10/2009; 13(4):237-43.
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  Article: The effect of herpes simplex virus virion host shutoff gene- a new suicide gene- on tumor cells.

  Hossein Bannazadeh Baghi, Taravat Bamdad, Hoorieh Soleimanjahi
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  ABSTRACT: The herpes simplex virus (HSV) UL41 gene product, virion host shutoff (Vhs) protein, mediates the rapid degradation of both viral and cellular mRNA. This ability suggests that Vhs protein can be used as a suicide gene in cancer gene therapy applications. The recent reports have shown that the degradation of cellular mRNA during herpes simplex infection is selective. RNA containing AU-rich elements (ARE) in their 3' untranslated ends are the targets for the Vhs protein. RNA that are not subject to Vhs protein-dependent degradation are up-regulated during HSV infection. ARE are frequently found in mRNA that encode proto-oncogenes, nuclear transcription factors, and cytokines. In many human cancers, the AU-rich stretch of proto-oncogenes and regulatory genes has impaired. To investigate whether Vhs protein might be useful for inhibition of tumor cell proliferation, a eukaryotic expression vector containing Vhs protein gene was constructed. Cell degradation and RNA content of HeLa and MRC-5 tumor cells after transfection with the constructed vector were studied. The results showed a strong inhibitory activity in proliferation of transfected tumor cells and a sharp decrease in their RNA content. These data suggest that Vhs protein can be considered as a candidate for suicide cancer gene therapy.
  Iranian biomedical journal 08/2009; 13(3):185-9.
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  Article: In vitro transdifferentiation of bone marrow stromal cells into GABAergic-like neurons.

  Payam Mohammad-Gharibani, Taki Tiraihi, Jalil Arabkheradmand
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  ABSTRACT: Cell therapy of many neurodegenerative diseases using bone marrow stromal cells (BMSC) requires the differentiation of BMSC into neuronal subtype. However, the transdifferentiation of BMSC into GABAergic phenotype requires more investigation. In this study, BMSC of adult female rats were pre-induced into neuroblast-like cells using 1 mM beta-mercaptoethanol (betaME) and 10 microM retinoic acid (RA), followed by 40 mM potassium chloride as inducer. The BMSC were evaluated by fibronectin as well as Oct-4. The percentage of nestin, neurofilaments (NF 68, NF 160, and NF 200) and GABA immuno-reactive cells was used to evaluate the GABAergic differentiation at the pre-induction and induction stages. The statistical analysis was carried out using unpaired student's t-test and ANOVA with Tukey's multiple comparison. The BMSC in the fourth passage expressed fibronectin up to 91.24 +/- 0.82%. The pre-induced cells after 2 days of RA exposure showed the expression of neuroblastic markers of nestin and NF68 (81.56 +/- 2.64% and 82.12 +/- 2.65%, respectively). The yield of GABAergic neurons with beta-ME for 1 h and RA as pre-inducer for 2 days followed by potassium chloride as inducer (40 mM for 3 days) was 60.64% +/- 1.97%. In addition, NF160 and NF200 were detected in the transdifferentiated cells. RT-PCR showed no expression of Oct-4 after the induction and pre-induction stages. GABAergic-like neurons obtained from BMSC can be potentially used in cell transplanting for some neurodegenerative disorders.
  Iranian biomedical journal 08/2009; 13(3):137-43.
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  Article: Human coagulated plasma as a natural and low cost matrix for in vitro angiogenesis.

  Kamran Mansouri, Ali Mostafaei, Manochehr Mirshahi, Hamidreza Mohammadi Motlagh, Ali Maleki, Maryam Keshavarz
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  ABSTRACT: Angiogenesis, the development of new blood vessels, is an important process in tissue development and wound healing, but becomes pathologic when associated with solid tumor growth, proliferative retinopathies, and rheumatoid arthritis. Accurate and reliable qualification of neovascular (angiogenic) response, both in vitro and in vivo, is an essential requirement for the study of new blood vessel growth. The complexity of currently used three-dimensional in vitro angiogenesis systems makes it difficult to approve material in its models. Capillary-like structure occurs on basement membrane components such as collagen and/or laminin, while in other models, CLS formation occurs on transitional matrices such as fibrin. To solve this problem, we were interested in developing an angiogenesis system which allows rapid and reliable quantification of three-dimensional neovessel formation in vitro. Human bone marrow endothelial cells were seeded on gelatin-coated microcarriers and suspended in a solution of platelet-poor plasma which was induced to polymerize by addition of calcium chloride. In this way, microcarriers were entrapped in three-dimensional coagulated plasma. Within a few hours, endothelial cells begin to leave these supporting microcarries and migrate into the coagulated-plasma matrix and formed CLS within 48-72 hours. We developed a convenient angiogenesis in vitro system which allows reliable quantification of capillary formation in a three-dimensional environment.
  Iranian biomedical journal 08/2009; 13(3):179-83.
• Article: Bacterial production of dense granule antigen GRA8 of Toxoplasma gondii.

  Jalal Babaie, Mehrak Zare, Ghazaleh Sadeghiani, Mohammad Lorgard-Dezfuli, Zohre Aghighi, Majid Golkar
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  ABSTRACT: Dense granule antigens (GRA antigens) of Toxoplasma gondii induce strong antibody response in humans and are considered as useful diagnostic antigens. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags such as glutathione-S-transferase. The present study aimed to produce soluble full length immunogenic GRA8 in bacteria. GRA8 complementary DNA (cDNA), encoding amino acids 24 to 258, was amplified from tachyzoites of RH strain and cloned in prokaryotic expression vector pET-28b(+). Expression of recombinant GRA8 (rGRA8) was analyzed by SDS-PAGE. Antigenicity and immunogenicity of the protein were evaluated by Western-blotting. The cloned gene fragment exhibited complete similarity with the published sequence of gra8 gene by sequence analysis. rGRA8 was expressed in Escherichia coli in fusion with a very small tag and the soluble protein was purified by immobilized metal ion affinity chromatography. In immunoblot, serum sample from a rabbit immunized with rGRA8 recognized a single antigen of T. gondii tachyzoite at the expected molecular weight of native GRA8. Sera from acutely-infected pregnant women strongly reacted with rGRA8 in Western-blotting, while sera from chronically-infected or T. gondii-negative women failed to recognize the protein. The full length soluble rGRA8 was successfully produced in E. coli and shown to be a highly immunogenic protein. As a result it could be used in immunological as well as molecular biology experiments.
  Iranian biomedical journal 08/2009; 13(3):145-51.
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  Article: Repair of spinal cord injury by co-transplantation of embryonic stem cell-derived motor neuron and olfactory ensheathing cell.

  Mohammad Salehi, Parichehr Pasbakhsh, Masoud Soleimani, Mehdi Abbasi, Gholamreza Hasanzadeh, Mohammad Hossein Modaresi, Aligholi Sobhani
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  ABSTRACT: The failure of regeneration after spinal cord injury (SCI) has been attributed to axonal demyelination and neuronal death. Cellular replacement and white matter regeneration are both necessary for SCI repair. In this study, we evaluated the co-transplantation of olfactory ensheathing cells (OEC) and embryonic stem (ES) cell-derived motor neurons (ESMN) on contused SCI. OEC cultured from olfactory nerve rootlets and olfactory bulbs. ESMN was generated by exposing mouse ES cells to retinoic acid and sonic hedgehog. Thirty female rats were used to prepare SCI models in five groups. Control and medium-injected groups was subjected to induce lesion without cell transplantation. OEC or ESMN or both were transplanted into the site of the lesion in other groups. The purity of OEC culture was 95%. Motor neuron progenitor markers (Olig2, Nkx6.1 and Pax6) and motor neuron markers (Isl1, Isl2 and Hb9) were expressed. Histological analysis showed that significantly more (P<0.001) spinal tissue was spared in OEC, ESMN and OEC+ ESMN groups but the OEC+ ESMN group had a significantly greater percentage of spared tissue and myelination than other groups (P< 0.05). The numbers of ESMN in co-transplanted group were significantly higher than ESMN group (P<0.05). A significant (P<0.05) recovery of hindlimb function was observed in rats in the transplanted groups. We found that the co-transplantation of ESMN and OEC into an injured spinal cord has a synergistic effect, promoting neural regeneration, ESMN survival and partial functional recovery.
  Iranian biomedical journal 07/2009; 13(3):125-35.
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  Article: Transplantation and homing of mouse embryonic stem cells treated with erythropoietin in spleen and liver of irradiated mice.

  Mandana Beigi Boroujeni, Mojdeh Salehnia, Mojtaba Rezazadeh Valojerdi, Mehdi Forouzandeh Moghadam
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  ABSTRACT: The present study was designed to evaluate the homing potential of mouse embryonic stem cells (ESC) treated with erythropoietin (EPO) in hematopoietic organs such as spleen and liver after transplantation using morphological and immuno-histochemical techniques. Day-four embryoid body (EB)-derived cells were dissociated and re-plated in medium in the presence and absence of EPO for three days. The EPO- and untreated differentiated cells were labeled with 5-bromo-2 deoxyuridine (BrdU) before transplantation and analyzed using flow cytometry and reverse transcription-PCR methods. BrdU-labeled cells were injected via the tail vein into irradiated adult mice in both groups. The spleen colony-forming unit assay (CFU-S) was performed 12 days after transplantation. Immuno-histochemistry was also carried out to trace transplanted cells. The percentage of CD34 positive cells was 5.51 +/- 1.06% in the EPO-treated group and 1.63 +/- 0.225% in untreated group. The RT-PCR analysis showed that the EPO-treated cells expressed epsilon globin, betaH1 globin, RUNX1 and EPO receptor genes, but the beta-major globin gene was not expressed. The number of colonies formed in the spleens of treated group (17.33 +/- 4.726) was significantly different from the control group (6 +/- 1). The population of BrdU positive cells in spleen of EPO-treated cell-transplanted group was higher than that of the control group. Also, BrdU positive cells were observed in the central vein of the liver sections of EPO-treated and control groups but were not observed in the liver parenchyma. There were not BrdU positive cells in the spleen and liver sections of the sham group. Our results confirm that ESC have the ability to home and form colonies in spleen after transplantation and EPO-treated EB-derived cells caused an increase in the number of colonies in spleen after CFU-S.
  Iranian biomedical journal 05/2009; 13(2):87-94.
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  Article: Pharmacogenomic profiling of the PI3K/PTEN pathway in sporadic breast cancer.

  Issar Nassiri, Mehri Faghihi, Manoochehr Tavassoli
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  ABSTRACT: Pharmacogenomics is the study of genetic variations among individuals to predict the probability that a patient will respond to single or multidrug chemotherapy. Breast cancer is one of the most common cancers among women worldwide. Treatment of breast cancer by application of biological rationales gives us the ability to match the correct pharmacology to individual tumour genetic profiles. The breast cancers exhibit multiple anomalies in phosphatidylinositol 3 kinase pathways, such as phosphatase and tensin homolog deleted on chromosome TEN loss that can be put in context of therapy with rapamycin analogues. Considering the high incidence of breast cancer in Iran, the potential role of tumor suppressor PTEN/MMAC1gene was investigated in Isfahanian breast cancer patients. In this study, PTEN was evaluated by means of polymerase chain reaction, single strand conformation polymorphism, Heteroduplex mobility assay and direct DNA sequencing in 72 breast cancer tumors for detection and characterization of mutations. According to the results of this research, nucleotide substitutions were found in 5/72 (7%) of samples. The sporadic breast cancer patient was found to be heterozygote for the p.D92N, p.C105W, p.D107N, p.A121P and p.R130Q mutations. One novel mutation, p.D107N, was found in this study. Loss of PTEN function in breast cancer can occur either by mutation or reduction of PTEN expression in almost half of sporadic breast tumors. This rate of mutations is an important consideration for novel therapeutic in which biological efficacy is influenced by the activity of PTEN.
  Iranian biomedical journal 05/2009; 13(2):79-86.
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  Article: Focal adhesion kinase (FAK) involvement in human endometrial remodeling during the menstrual cycle.

  Mahmoud Orazizadeh, Iran Rashidi, Jamileh Saremi, Mahmoud Latifi
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  ABSTRACT: Endometrial remodeling occurs during each menstrual cycle in women. Reports have shown that, in a variety of cell types, processes such as proliferation, signaling complex formation and extra cellular matrix remodeling require a cytoplasmic tyrosine kinase, focal adhesion kinase (FAK). The present study has focused on the expression pattern of FAK in human endometrium during the menstrual cycle. The purpose of this study was to ascertain the probable function of FAK in menstrual cycle changes and the role of FAK in tissue repair and tissue remodeling in vivo. Formalin-fixed paraffin-embedded endometrial samples were obtained from 400 pre-menopausal, non-pregnant women, who underwent hysterectomy and biopsy for benign diseases. Forty six samples with no tissue abnormalities were studied and ABC staining method of immuno-histochemistry methods was applied. Positive staining of FAK by different cell types of human endometrium was scaled and compared with each other by using histologic score method. All different cell types of endometrium showed various patterns of FAK expression in different stages of menstruation. FAK in glandular and luminal epithelial cells is up-regulated during the early proliferative (EP) to mid-secretory (MS) phases. FAK in stromal cells is up-regulated during the EP, early and MS phases in comparison to the late secretory (LS) phase. FAK expression in endothelial cells is up-regulated during the EP and MS phases in comparison to LS phase. This study showed that endometrial FAK expression is a phase-dependent manner during the menstrual cycle. It appears that up-regulation of FAK during the proliferative phases is responsible for endometrial regeneration and high expression of FAK in the EP and MS phases may associate with the implantation. Down-regulation of FAK during the LS phase may facilitate apoptosis in human endometrium. It seems that FAK as a key kinase plays a critical role in endometrial remodeling that it may regulate by steroid hormones.
  Iranian biomedical journal 05/2009; 13(2):95-101.