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ABSTRACT: Background: Natural medicines have been recently consid-ered more reasonable for human use most notably due to their safety and tolerance. HESA-A is a marine-originated herbal medicine with a variety of healing effects. However, its exact biological mechanism is not clear. The present study aimed at the evaluation of the HESA-A antioxidant effect. Methods: Chinese hamster ovary (CHO) and human embry-onic kidney (HEK293T) cells were treated with different con-centrations of HESA-A and H 2 O 2 followed by cell prolifera-tion assays. The antioxidant effect of the HESA-A prepara-tions was evaluated by an antioxidant assay kit. Results: The viability of CHO and HEK293T cells were about 89% following their incubation with 100 and 200 ng/ml HESA-A, respectively for 1.5 hrs. However, when the cells were incubated with concentrations of 300 ng/ml or more, the cell viability significantly decreased to 48% compare to the control cells. The cytotoxic effects of H 2 O 2 were observed after 2 hrs of incubation of the HEK293T or CHO cells with 10 mM or 16 mM H 2 O 2 , respectively, while in the presence of HESA-A the cytotoxicity was significantly decreased. Anti-oxidant assay revealed that HESA-A scavenges free radicals. Conclusion: The findings indicate that HESA-A had cytopro-tective effects in vitro, and that such an effect might be due to antioxidant properties. Iran J Med Sci 2012; 37(1): 47-53.
IJMS. 03/2012; 37(1).
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ABSTRACT: The most prominent capabilities of mesenchymal stem cells (MCSs) which make them promising for therapeutic applications are their capacity to endure and implant in the target tissue. However, the therapeutic applications of these cells are limited due to their early death within the first few days following transplantation. Therefore, to improve cell therapy efficacy, it is necessary to manipulate MSCs to resist severe stresses imposed by microenvironment. In this study, we manipulated MSCs to express a cytoprotective factor, nuclear factor erythroid-2 related factor 2 (Nrf2) to address this issue. Full-length human Nrf2 cDNA was isolated and TOPO cloned into TOPO cloning vector and then transferred to gateway adapted adenovirus expression vector by LR recombination reaction. Afterwards, the Nrf2 bearing recombinant virus was prepared in appropriate mammalian cell line and used to infect MSCs. The viability and apoptosis of the Nrf2 expressing MSCs were evaluated following hypoxic and oxidative stress conditions. Transient expression of Nrf2 by MSCs protected them against cell death and the apoptosis triggered by hypoxic and oxidative stress conditions. Nrf2 also enhanced the activity of SOD and HO-1. These findings could be used as a strategy for prevention of graft cell death in MSC-based cell therapy. It also indicates that management of cellular stress responses can be used for practical applications.
Cell Stress and Chaperones 02/2012; 17(5):553-65. · 3.01 Impact Factor
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Iranian Journal of Basic Medical Science 02/2012; · 0.32 Impact Factor
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ABSTRACT: It is proved that testis is sensitive to electromagnetic field (EMF) and its damage results in infertility. Exposure to EMF induces reactive oxygen species production and affects on anti-oxidants defense mechanisms. Metallothionein (MT) is a name for a group of low molecular weight (6-7 kDa), sulfhydryl rich proteins. Expression of MT1 and MT2 genes in testis tissue after EMF exposure was aimed in this study.
Male BALB/c mice (8 weeks old) were exposed to 3 MT EMF for 8 weeks, 4 hours/day. After 8 weeks, the mice were sacrificed and the testis tissue was removed. The testis pieces were stained with hematoxylin and eosin and analyzed under an optical microscope. Assessment of MT1 and MT2 genes and also protein expression was performed by real-time PCR and Western-blot, respectively.
In light microscopic observation, the number of primary spermatocytes was increased significantly in EMF group (P < 0.01). In addition, in interstitial space, the number of leydig cells was increased significantly in EMF group (P < 0.01) and basement membrane thickness was increased as well. MT1 and MT2 genes were down-regulated significantly in testis tissue of mice exposed to EMF both in mRNA and protein level compared to control.
It is clear that MT is mediated in testis development and spermatogenesis. Down-regulation of MT1 and MT2 after EMF in mouse testis might be followed by some consequences that result in infertility.
Iranian biomedical journal 10/2011; 15(4):151-6.
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ABSTRACT: Neutrophil Gelatinase-Associated Lipocalin (NGAL/Lcn2), a member of the lipocalin family, has a variety of functions. There are extensive studies examining the expression of NGAL under harmful conditions. However, its precise function remains poorly understood. Heme Oxygenase 1 (HO-1) is an enzyme with well-established cytoprotective effects. Previous work showed that NGAL induces expression of HO-1. Interestingly, the same stimuli induced the expression of both NGAL and HO-1. The current study was designed to (1) determine whether NGAL exerts its cytoprotective effect through HO-1 and (2) compare NGAL and HO-1 with each other in terms of their protective role against oxidative stress. The current data indicate that NGAL exerts its cytoprotective effect independent of HO-1 and protects cells against oxidative stress more efficiently than HO-1. The data also strongly suggest that induction of NGAL under harmful conditions is a compensatory response to ameliorate oxidative stress-mediated toxicity. These findings may suggest new applications of NGAL, particularly when oxidative stress is a major factor.
Free radical research 07/2011; 45(7):810-9. · 2.22 Impact Factor
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ABSTRACT: Background: Heme oxygenase-1 (HO-1) is a cytoprotective and antiapoptotic enzyme, which has been involved in main-taining cellular homeostasis, and plays an important protective role by modulating oxidative injury. Up-regulation of (HO-1) has contributed to tumorogenicity of some cancers. In this study we investigated the expression pattern of the HO-1, in five different human-derived cancer cell lines with high inci-dence in Iran. Methods: Total cell RNA were extracted from HepG2 (hepato carcinoma), A549 (lung adenocarcinoma), MCF-7 (breast cancer), K562 (myeloid leukemia) and LS174T (colon cancer) cell lines. Human embryonic kidney (HEK293) cell line was used as a control. cDNAs were synthesized and expression of HO-1 was examined using RT-PCR. Results: The expression of HO-1 was not detected in the con-trol cell line (HEK293), but it was observed to express follow-ing ultraviolet (UV) exposure indicating that HO-1 is not con-stantly expressed. The examined cancer cell lines constitutively expressed different variety of HO-1 on mRNA level. Strong expression of HO-1 was observed in HepG2, MCF-7 and A549 cells. A moderate expression of HO-1 was observed in K562 cells, and LS174T cells showed no expression of HO-1. Conclusion: Heme oxygenase-1 could be considered as a new marker in the diagnosis of some cancers, especially hepato-macarcinoma. Our results also suggest that up-regulation of HO-1 may contribute to tumorogenicity of some cancers. Therefore, the combination of gene-silencing effect of HO-1 and chemo-therapy might be considered as a new modality for the treatment of cancers in which the expression HO-1 is up-regulated. Iran J Med Sci 2011; 36(4): 260-265.
IJMS. 02/2011; 36(4).
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ABSTRACT: Leukemia inhibitory factor (LIF) is a 45-56 kDa glycoprotein that has an important role in proliferation and embryo implantation. Its effect on oocyte maturation and how to exert the function remained to be elucidated.
Immature mice superovulated with human menopausal gonadotropin and germinal vesicle (GV) oocytes were obtained from ovary 48 hours after. GV oocytes were cultured in tissue culture medium 199 with 0, 100, 500 and 1,000 U/ml LIF. Cumulus expansion and in vitro maturation (IVM) rate were assessed after culture. For reverse transcriptase PCR, total RNA from GV and metaphase II (MII) oocytes were extracted by Trizol reagent. The quantity and quality of RNA were determined by spectrophotometry and electrophoresis, respectively. Reverse transcription was performed by Super Script III reverse transcriptase with 1 µg of total RNA followed by DNase I treatment and heat inactivation. Expression of gp130 was determined by RT-PCR.
Our results showed that cumulus expansion was improved with 1,000 U/ml in culture medium compared to others. GV breakdown and MII rate in groups with LIF were higher than control group and were dose-dependent. In 1,000 U/ml, LIF rate of MII was significantly higher than control group (P less than 0.05). Our results also showed that gp130 is expressed neither in GV nor in MII oocytes during IVM of mouse oocytes.
gp130 is expressed in human oocyte but not in mouse. Our results suggest that in mouse, LIF could affect the oocyte via another receptor or via cumulus cells; however, further studies are warranted.
Iranian biomedical journal 07/2010; 14(3):103-7.
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ABSTRACT: Recombinant coagulation factor VII (FVII) is used as a potential therapeutic intervention in hemophilia patients who produce antibodies against the coagulation factors. Mammalian cell lines provide low levels of expression, however, the Spodoptera frugiperda Sf9 cell line and baculovirus expression system are powerful systems for high-level expression of recombinant proteins, but due to the lack of endogenous vitamin K-dependent carboxylase, expression of functional FVII using this system is impossible. In the present study, we report a simple but versatile method to overcome the defect for high-level expression of the functional recombinant coagulation FVII in Sf9 cells. This method involves simultaneous expression of both human gamma-carboxylase (hGC) and human FVII genes in the host. It may be possible to express other vitamin K-dependent coagulation factors using this method in the future.
Biotechnology Letters 03/2010; 32(6):803-9. · 1.68 Impact Factor
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ABSTRACT: Objective(s)Neutrophil gelatinase-associated lipocalin (NGAL/Lcn2), comprise a group of small extracellular proteins with a common β-sheet-dominated 3-dimensional structure. In the past, it was assumed that the predominant role of lipocalin was acting as transport proteins. Recently it has been found that oxidative stress induces Lcn2 expression. It has been also proved that electromagnetic field (EMF) produces reactive oxygen species (ROS) in different tissues. Expression of Lcn2 following exposure to electromagnetic field has been investigated in this study. Materials and MethodsBalb/c mice (8 weeks old) were exposed to 3 mT, 50 HZ EMF for 2 months, 4 hr/day. Afterwards, the mice were sacrificed by cervical dislocation and livers were removed. The liver specimens were stained with Haematoxylin- Eosin (H&E) and analyzed under an optical microscope. Total RNA was extracted from liver and reverse transcription was performed by SuperScript III reverse transcriptase with 1 µg of total RNA. Assessment of Lcn2 expression was performed by semiquantitative and real time- PCR.ResultsThe light microscopic studies revealed that the number of lymphocyte cells was increased compared to control and dilation of sinosoids was observed in the liver. Lcn2 was up-regulated in the mice exposed to EMF both in mRNA and protein levels.ConclusionTo the extent of our knowledge, this is the first report dealing with up-regulation of Lcn2 in liver after exposure to EMF. The up-regulation might be a compensatory response that involves cell defense pathways and protective effects against ROS. However, further and complementary studies are required in this regards.
Iranian Journal of Basic Medical Sciences. 01/2010;
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ABSTRACT: Factor VII (FVII) is a plasma glycoprotein that participates in the coagulation process leading to the generation of fibrin. The aim of this study was to construct, express and purify recombinant FVII fused to a polyhistidine (his) tag using Gateway technology.
To construct the entry clone, blunt-end FVII cDNA and subsequent polymerase chain reaction (PCR) product isolated from a HepG2 cell line was TOPO-cloned into a pENTR TOPO vector. To construct the expression clone, a LR recombination reaction was carried out between the entry clone and destination vector, pDEST26. Chinese hamster ovary (CHO) cells were transfected with 1 microg of DNA of PDEST26-FVII using the FuGENE HD transfection reagent. Two cell lines that permanently expressed recombinant FVII were established. The expression of recombinant FVII was confirmed by reverse transcriptase PCR and enzyme-linked immunosorbent assay. Culture medium containing his-tagged FVII was added to the nickel-nitrilotriacetic acid resin column and bound protein was eluted. The purified protein was detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The biological activity of the recombinant FVII was determined by a prothrombin time assay using FVII-depleted plasma.
The results showed that human recombinant FVII was successfully cloned and the accuracy of the nucleotide sequence of the gene and its frame in the vector were confirmed by DNA sequencing. Stable clones transfected with the construct expressed FVII mRNA and related protein but no expression was detected in the CHO cells containing an empty vector. A protein of about 52 KDa was detected in SDS-PAGE and was further confirmed by western blot analysis. A three-fold decrease in clotting time was observed using this recombinant FVII.
As far as we are aware, this is the first report of expression of recombinant FVII fused with a his-tag through Gateway technology. The next steps, including large scale expression, purification, activation and stabilisation, are underway.
Blood transfusion = Trasfusione del sangue 10/2009; 7(4):305-12. · 2.10 Impact Factor
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Mehryar Habibi Roudkenar,
Raheleh Halabian, Amaneh Mohammadi Roushandeh,
Mohammad Reza Nourani,
Nasser Masroori,
Majid Ebrahimi,
Mahin Nikogoftar,
Mehdi Rouhbakhsh,
Parisa Bahmani,
Ali Jahanian Najafabadi,
Mohammad Ali Shokrgozar
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ABSTRACT: Environmental temperature variations are the most common stresses experienced by a wide range of organisms. Lipocalin 2 (Lcn2/NGAL) is expressed in various normal and pathologic conditions. However, its precise functions have not been fully determined. Here we report the induction of Lcn2 by thermal stresses in vivo, and its role following exposure to cold and heat stresses in vitro. Induction of Lcn2 in liver, heart and kidney was detected by RT-PCR, Western blot and immunohistochemistry following exposure of mice to heat and cold stresses. When CHO and HEK293T cells overexpressing NGAL were exposed to cold stress, cell proliferation was higher compared to controls. Down-regulatrion of NGAL by siRNA in A549 cells resulted in less proliferation when exposed to cold stress compared to control cells. The number of apoptotic cells and expression of pro-apoptotic proteins were lower in the NGAL overexpressing CHO and HEK293T cells, but were higher in the siRNA-transfected A549 cells compared to controls, indicating that NGAL protects cells against cold stress. Following exposure of the cells to heat stress, ectopic expression of NGAL protected cells while addition of exogenous recombinant NGAL to the cell culture medium exacerbated the toxicity of heat stress specially when there was low or no endogenous expression of NGAL. It had a dual effect on apoptosis following heat stress. NGAL also increased the expression of HO-1. Lcn2/NGAL may have the potential to improve cell proliferation and preservation particularly to prevent cold ischemia injury of transplanted organs or for treatment of some cancers by hyperthermia.
Experimental Cell Research 10/2009; 315(18):3140-51. · 3.58 Impact Factor
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ABSTRACT: The aim of this study was to assess effects of cysteamine as an anti-oxidant on rate of in vitro maturation of oocyte and determination of its effects on spindle size and shape.
Pre-mature mice were primed with pregnant mare stimulating gonadotrophin (PMSG) and germinal vesicle (GV) stage oocytes were obtained 48 h after. The oocytes were cultured in tissue culture medium (TCM 199) with 50, 100, 200 and 500 microM cysteamine. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and human chorionic gonadotrophin. For spindle immuno-cytochemistry of metaphase II (MII), oocytes alpha and beta tubulin antibody were performed. Chromosome staining was performed with Hoechst.
Our results showed that the rate of GV breakdown (GVBD) and MII increased in all cysteamine groups except in group cultured with 500 microM cysteamine. Immuno-cytochemistry analysis showed that spindle area in all in vitro Maturation oocytes increased when compared to in vivo oocytes. Spindle shape and size (spindle area) in 100 microM cysteamine in comparison to in vivo group was insignificant (P > 0.05).
Our results revealed that cysteamine improved IVM rate in a dose dependant manner. The size and shape of spindle in the presence of given concentration of cysteamine was similar to in vivo. Therefore, cysteamine improved microtubule organization, rate of GVBD and MII development.
Iranian biomedical journal 05/2009; 13(2):73-8.
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ABSTRACT: Background: The effects of electromagnetic field (EMF) onreproductive system have been of critical concern for a longtime. It has been shown that the EMF can adversely affect testicularcells and tissue and decrease male fertility. The mostimportant determinants of male fertility are sperm developmentand motility, which are affected by changes in several factorsincluding lipocalin 2 proteins. In the present study, we investigatedthe effects of exposure to EMF on testis tissue and expressionof lipocalin 2 gene.Methods: Male BALB/c mice (8 weeks old) were exposed to 3mT EMF for 8 weeks, 4 hours/day. Control group (10 mice)did not receive EMF exposure. After the experimental period,the mice were sacrificed, and their testis tissues were examinedby using light microscopy after hematoxylin-eosin staining.Additionally, total RNA and proteins were extracted from testistissue and used to study the lipocalin 2 expression by realtime RT-PCR and Western blot analysis.Results: The histological changes observed in the testes of experimentalgroup included increased number of spermatocytesand Leydig cells, and increased thickness of basement membranecompared with the control group. The mRNA and proteinstudies showed that expression of lipocalin 2 gene wasdown regulated in testes of the mice exposed to EMF.Conclusion: Our study showed that EMF down regulates theexpression of lipocalin 2, a cytoprotective molecule, in testis tissue.This down regulation can be one of the mechanisms that contributeto the decreased fertility observed after exposure to EMF
Iranian Journal of Medical Sciences. 01/2009;
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ABSTRACT: Lipocalin 2 (Lcn2, NGAL) is a member of the lipocalin superfamily for which a variety of functions have been reported. However, the precise biological roles of NGAL are not fully known. We have investigated the ability of NGAL to prevent H(2)O(2) toxicity, which is considered to be the classical inducer of oxidative stress caused by ROS generation in an in vitro model.
NGAL cDNA was isolated from HepG2 cell line and cloned to pcDNA3.1(+) vector. The construct was transfected to CHO cell line. Stable clones were generated, and the expression of NGAL was determined by RT-PCR, Western blot analysis and ELISA. NGAL gene in A549 cell line was downregulated with the siRNA. CHO and A549 cells were intoxicated with H(2)O(2) and cell proliferation was performed by MTT assay. Apoptotic cells were detected by flow cytometry.
Cell proliferation was higher in CHO expressing NGAL in doses of 5 and 10 mM H(2)O(2) after 2h compared with the control. H(2)O(2) was also more toxic in the presence of NGAL siRNA compared with the control in A549 cell. Our results also revealed that NGAL protect cells from apoptosis.
Overall, our results revealed for the first time a new function for NGAL/Lcn2: acting as a protective factor against H(2)O(2) toxicity. In the future, NGAL may have the potential application to ameliorate the toxicity induced by oxidative stress conditions.
Archives of Medical Research 09/2008; 39(6):560-6. · 1.88 Impact Factor
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ABSTRACT: One of the major consequences in beta- thalassemia is iron overload. Oxidative statuses have been reported in beta-thalassemia patients by several studies. It has been proven that iron plays a critical role in the formation of reactive oxygen species (ROS). More recently, we have found the induction of Lcn2/NGAL expression under oxidative stress condition. In this study, it was assumed that NGAL should be upregulated in beta-thalassemia patients because of oxidative stress condition.
Assessment of NGAL expressions in 25 adult beta-thalassemia and 9 pediatric patients was performed by semiquantitative RT-PCR, real-time RT-PCR and ELISA.
Adult beta-thalassemia patients upregulated NGAL expression compared with the normal samples but no upregulation was observed in pediatric patients.
Upregulation may play an important role in decreasing ROS or iron in beta-thalassemia patients.
Archives of Medical Research 06/2008; 39(4):402-7. · 1.88 Impact Factor
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ABSTRACT: Immunotoxins are comprised of both the cell targeting and the cell killing moieties. We previously established a new immunotoxin, i.e. Shiga toxin granulocyte macrophage-colony stimulating factor (StxA1-GM-CSF), comprises of catalytic domain of Stx, as a killing moiety and GM-CSF, as a cell targeting moiety. In this study, the ability of the immunotoxin to induce apoptosis and double strand breaks (DSB) on different cell lines was investigated.
The recombinant hybrid protein was expressed in bacterial expression system and purified with nickel-nitrilotriacetate acid resin. The K562 (erythroid leukemia) cell line and LS174 (colon carcinoma) were used in this study. The neutral comet assay was carried out for the detection of DSB and Hoechst staining was performed for apoptosis.
StxA1-GM-CSF effectively induced apoptosis on K562 cell line and DNA Double Strand Break (DSB) were observed on colon cancer cell line treated with StxA1-GM-CSF.
This novel action i.e. DNA damage might be a relevant mechanism of action for StxA1-GM-CSF that is designed to act as immunotoxin, although further investigation is required.
Iranian biomedical journal 02/2008; 12(1):1-6.
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ABSTRACT: Lipocalin 2 (Lcn2, NGAL) is a member of the lipocalin superfamily with diverse functions such as the transport of fatty acids and the induction of apoptosis. Previous reports indicated that expression of Lcn2 is induced under harmful conditions. However, the mechanisms of the induction of Lcn2 expression remain to be elucidated. In this report, we intended to identify the factor or factors that induce Lcn2 expression. Up-regulation of Lcn2 expression after X-ray exposure was detected in the heart, the kidney and especially in the liver. Primary culture of liver component cells revealed that this up-regulation in the liver was induced in hepatocytes. Up-regulation of Lcn2 expression was also detected in HepG2 cells after the administration of X-rays or H(2)O(2). Interestingly, up-regulation of Lcn2 expression after H(2)O(2) treatment was canceled by the addition of the anti-oxidants, dimethylsulfoxide or cysteamine. These results strongly suggest that Lcn2 expression is induced by reactive oxygen species. Therefore, Lcn2 could be a useful biomarker to identify oxidative stress both in vitro and in vivo.
Journal of Radiation Research 02/2007; 48(1):39-44. · 1.68 Impact Factor
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ABSTRACT: AbstractBackground: Preparation of oocytes is one of the critical factors that determine the developmental competence of embryos produced by in vitro fertilization (IVF). Objective: In this study, the effect of cysteamine, type of media and glutathione (GSH) level on blastocysts development after in vitro maturation of mouse oocytes were investigated. Materials and Methods: Premature female mice were primed with pregnant mare stimulating gonadotrophin (PMSG), and germinal vesicle (GV) stage oocytes were obtained 45 hr later. GV oocytes were cultured in presence of 0, 50, 100, 200 and 500 µm cysteamine in TCM199 and MEME media. After IVM, MII oocytes were in vitro fertilized (IVF) and in vitro cultured (IVC) in order to observe embryo development. A group of In Vivo Ovulated (IVO) oocytes after priming with PMSG and HCG also were included in this study. 5,5-Dithio-bis (2nitrobenzoic acid) DTNB-recycling protocol was used for GSH assay. Results: Rate of IVM and IVF were improved in all oocytes treated with cysteamine in the two medium except 500 µm (81% MII rate in TCM and 64% MII in MEME). Rate of blastocyst in 100 µm cysteamine in TCM1199 and 200 µm in MEME was higher compared to control groups (In TCM 45% and in MEME 35%). In vivo MII and GV oocytes represented the highest and lowest GSH level respectively. Conclusion: Our results revealed that the media and concentration of cysteamine can affects on IVM, IVF and rate of blastocysts development on dose dependant manner.
Iranian Journal of Reproductive Medicine. 01/2007;
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ABSTRACT: To investigate the selective cytotoxic effect of constructed hybrid protein on cells expressing granulocyte macrophage colony stimulating factor (GM-CSF) receptor.
HepG2 (human hepatoma) and LS174T (colon carcinoma) were used in this study. The fused gene was induced with 0.02% of arabinose for 4 h and the expressed protein was detected by Western blotting. The chimeric protein expressed in E.coli was checked for its cytotoxic activity on these cells and apoptosis was measured by comet assay and nuclear staining.
The chimeric protein was found to be cytotoxic to the colon cancer cell line expressing GM-CSFRs, but not to HepG2 lacking these receptors. Maximum activity was observed at the concentration of 40 ng/mL after 24 h incubation. The IC(50) was 20+/-3.5 ng/mL.
Selective cytotoxic effect of the hybrid protein on the colon cancer cell line expressing GM-CSF receptors (GM-CSFRs) receptor and apoptosis can be observed in this cell line. The hybrid protein can be considered as a therapeutic agent.
World Journal of Gastroenterology 05/2006; 12(15):2341-4. · 2.47 Impact Factor
