Iranian Biomedical Journal
Volume:13 Issue: 4, Oct 2009

Evidence is accumulating to support disruption of tissue architecture as a powerful event in tumor formation. For the past four decades, intensive cancer research with the premise of “cancer as a cell based-disease” focused on finding oncogenes or tumor suppressor genes. However, the role of the tissue architecture was neglected. Three dimensional (3D) cell cultures which can recapitulate major aspects of the microenvironment are appropriate models for exploring cancer. For the first time in Iran, we have launched Matrigel based non-malignant, tumorigenic and reverted breast 3D cell cultures. Non-tumorigenic MCF-10A and tumorigenic MCF-7 breast cell lines were cultured on plastic and Matrigel. MCF-7 cell lines were reverted to normal phenotype via AIIB2 and LY 294002 inhibitors against β1 integrin and class I phosphatidylinositol 3-kinase, respectively. MCF-10A acini were distinguishably different from MCF-7 on Matrigel. MCF-10A formed organized hollow spherical structures which were in stark contrast to the MCF-7 disorganized cluster of cells. Matrigel allowed visual monitoring of MCF-7 cells treated with inhibitors. After treatment of MCF-7 cells, we observed reversion of MCF-7 phenotype toward normal, comparable to MCF-10A acini. The 3D culture provides a microenvironment which allows malignant and non-malignant cells to demonstrate near physiological behavior and this can distinguish nonmalignant from malignant cells. The 3D culture also allows visual monitoring of malignant phenotypereversion to organized spheres.

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. 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.

The combustion of sulfur-rich fossil fuels leads to release of sulfur oxide pollution in the environment. In biodesulfurization process, an organism is able to remove sulfur from fossil fuels without decreasing the caloric value of those substrates. The main aim of this research was to design a recombinant microorganism to remove the highest amount of sulfur compounds in fossil fuels. Three genes (dszA,B,C) from dsz operon are responsible for the 4S pathway (biodesulfurization pathway) in Rhodococcus erythropolis IGTS8 were inserted into the chromosome of a novel indigenous Pseudomonas putida. The reaction catalyzed by products of dszA,B,C genes require FMNH2 supplied by dszD enzyme. Thus, pVLT31vector harboring dszD gene was transferred into this recombinant strain. The results demonstrated a higher biodesulfurization activity when the flavin reductase gene was transferred into recombinant P. putida harboring dszA,B,C. These results were approved by the Gibbs test and HPLC analysis. These analyses showed that this novel indigenous engineered P. putida could be a promising candidate for an industrial and environmental application for Biodesulfurization process. Iran. Biomed.

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 mM)-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

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 µM) 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 µM 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.

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 µg/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.

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.

The therapy of leishmania infection is difficult and each year 1.5 million new cases of cutaneous leishmaniasis and 500,000 new cases of visceral leishmaniasis are estimated, therefore, there is a need for an effective vaccine. Monoclonal antibody (mAb) is one of the suitable methods for isolation and purification of leishmania antigens. In this report, we produced several mAb against leishmania infantum antigens for antigen purification to be used as candidate vaccine. BALB/c mice were injected with freeze-thawed promastigote twice together with Freund adjuvant. Three days before fusion, antigen in saline was injected into the tail vain and then mice were killed and the spleen lymphocytes were fused with myeloma SP2/0. Five mAb against promastigote form of Leishmania infantum parasite were obtained. Western-blot analysis showed that these mAb recognize a band of 57- kDa protein either in parasite lysate or on whole L. infantum, L. tropica, L. major and L. donovani. It seems that the 57 kDa-protein is the major surface leishmania antigen (gp63) that is neither stage-specific nor differentially regulated. These mAb do not recognize the recombinant gp63 antigen and seems recognizing only the native form of a gp63 isoform. The IgG1 mAb was purified by affinity column and was used to purify 57 kDa antigens from Leishmania lysate. Since these antibodies recognizing one specific protein band in 4 different strains of leishmania, they could be used for leishmania diagnostic kits and also for purification of antigen to be tested for its protective effect against leishmania infection.