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Hemojuvelin (HJV) enhances signaling to the iron hormone hepcidin and its deficiency causes iron overload, a risk factor for hepatocellular carcinoma (HCC). We utilized Hjv−/− mice to dissect mechanisms for hepatocarcinogenesis. We show that suboptimal treatment with diethylnitrosamine (DEN) triggers HCC only in Hjv−/− but not wt mice. Liver proteomics data were obtained by mass spectrometry. Hierarchical clustering analysis revealed that Hjv deficiency and DEN elicit similar liver proteomic responses, including induction of mitochondrial proteins. Dietary iron overload of wt mice does not recapitulate the liver proteomic phenotype of Hjv−/− animals, which is only partially corrected by iron depletion. Consistent with these data, primary Hjv−/− hepatocytes exhibit mitochondrial hyperactivity, while aged Hjv−/− mice develop spontaneous HCC. Moreover, low expression of HJV or hepcidin (HAMP) mRNAs predicts poor prognosis in HCC patients. We conclude that Hjv has a hepatoprotective function and its deficiency in mice promotes mitochondrial dysfunction and hepatocarcinogenesis.

The aim of this study was to investigate the relationship between p16 methylation and its expression in oral squamous cell carcinoma (OSCC). Also the contribution of clinicopathological factors, HPV infection and smoking in p16 expression and promoter methylation has been investigated. In this study 67 consecutive OSCC patients and 59 normal individuals were enrolled. All patients were candidates for surgery of oral cavity and fresh tumor biopsies were collected and processed for DNA and RNA extraction. Normal gingival tissues were collected from individuals referred to dentistry clinic and considered as controls. All the cases and controls were checked for HPV infection and then promoter methylation and expression of p16 gene were determined using Methylation-specific PCR (MSP) and real-time PCR (QPCR), respectively. Methylation of p16 in tumors and normal tissues were 59.7 and 38.9%, respectively. Most of hypermethylated samples (>82%) were in high grades. P16 methylation was comparable in HPV+ and HPV- patients or smokers. P16 was overexpressed (~3 fold; p = 0.044) in HPV+ tumors, but it was significantly down-regulated in smoker patients (40% of all tumors). Comparison of P16 expression in OSCC tumors with different degrees of promoter methylation further suggest the relationship of methylation rate and down-regulation of P16 expression. The p16 methylation and expression was differentially affected in patients with HPV infection and the smoker cases. Regardless of the influence of environmental factors, it appears that P16 status is useful for classifying patients with OSCC and for influencing treatment strategies in accordance with this classification. Moreover, targeting the upregulation of p16 could be a promising therapeutic option.

Bladder cancer is one of the most prevalent genitourinary cancers responsible for about 150,000 deaths per year worldwide. Currently, several treatments, such as endoscopic and open surgery, appended by local or systemic immunotherapy, chemotherapy, and radiotherapy are used to treat this malignancy. However, the differences in treatment outcome among patients suffering from bladder cancer are considered as one of the important challenges. In recent years, cancer stem cells, representing a population of undifferentiated cells with stem-cell like properties, have been eyed as a major culprit for the high recurrence rate in superficial papillary bladder cancer. Cancer stem cells have been reported to be resistant to conventional treatments, such as chemotherapy, radiation, and immunotherapy, which induce selective pressure on tumoral populations resulting in selection and growth of the resistant cells. Therefore, targeting the therapeutic aspects of cancer stem cells in bladder cancer may be promising. In this study, we briefly discuss the biology of bladder cancer and then address the possible relationship between molecular biology of bladder cancer and cancer stem cells. Subsequently, the mechanisms of resistance applied by cancer stem cells against the conventional therapeutic tools, especially chemotherapy, are discussed. Moreover, by emphasizing the biomarkers described for cancer stem cells in bladder cancer, we have provided, described, and proposed targets on cancer stem cells for therapeutic interventions and, finally, reviewed some immunotargeting strategies against bladder cancer stem cells.

This study was carried out to examine the role of intracellular albumin in the modulation of oxidative damage induced by glutathione modifiers in HepG2 cells. Also, the relationship of albumin synthesis with oxidative stress factors including antioxidants was studied. HepG2 cell culture was supplemented with glutathione modifiers; L-Buthionine-sulfoximine (BSO; 0.1 and 1.0 mM) or N-acetyl cysteine (NAC; 1 and 10 mM) and the cell viability and changes in reduced glutathione (GSH), oxidized glutathione (GSSG), reactive oxygen species (ROS), catalase, and superoxide dismutase were measured. Besides, albumin expression at protein and mRNA levels was determined in cells pretreated with BSO or NAC. Kinetic studies showed that albumin expression in HepG2 cells is correlated with GSH and GSSG levels. Changes in albumin expression at protein and mRNA levels reached their maximum (19% and 55%, respectively) in the cells 6 H after NAC treatments. A substantial decrease in intracellular albumin due to BSO (27%) was associated with a significant increase in the generation of cellular ROS (17%). In contrast, increased albumin synthesis (intracellular and secretory) was associated with inhibition in cellular ROS. Overall data may suggest that albumin expression in coordination with the glutathione redox system is part of the antioxidant defense mechanism in liver cells.

Background A number of complex and rare mutations in epidermal growth factor receptor (EGFR) gene have been identified and the clinical implication of serum EGFR ligands has also been reported. However, the prognostic significance of these mutations and also the serum EGFR and its ligands in Non-Small Cell Lung Carcinoma (NSCLC) has remained a challenging issue. This study is aimed at finding the prognostic importance of EGFR rare mutations and serum EGFR, amphiregulin (AR), and TGF-α (Transforming Growth Factor-alpha) in NSCLC. Materials and method NSCLC patients ( n = 98) with mean age of 59 ± 10.5 were enrolled (M/F: 75/23). DNA was extracted from formalin fixed paraffin embedded tissues. Exons 19 and 21 were amplified using polymerase chain reaction followed by direct sequencing for identification of mutations. Serum EGFR, AR, and TGF-α were measured by ELISA. Results EGFR mutation rate in patients was 37% (exon 19 deletions: 72.2%, exon 21 substitutions: 27.8%). The E872K in exon 21 mutation-positive cases was the most frequent rare mutation detected (90%; 9/10 samples). A significant relationship was found between EGFR exon 21mutations and serum EGFR and TGF-α ( P 3 pg/ml) and TGF-α (> 10.5 pg/ml) were associated with shorter overall survival ( P Conclusions The data clearly show that elevation of serum TGF-α and AR are associated with poor prognosis of NSCLC. In addition to the close relationship between EGFR mutations and serum EGFR, serum TGF-α changes was associated with the gene mutations. These findings could be implicated in clinical decision making related to EGFR-TKIs.

micro (mi)-RNAs are vital regulators of multiple processes including insulin signaling pathways and glucose metabolism. Pancreatic β-cells function is dependent on some miRNAs and their target mRNA, which together form a complex regulative network. Several miRNAs are known to be directly involved in β-cells functions such as insulin expression and secretion. These small RNAs may also play significant roles in the fate of β-cells such as proliferation, differentiation, survival and apoptosis. Among the miRNAs, miR-7, miR-9, miR-375, miR-130 and miR-124 are of particular interest due to being highly expressed in these cells. Under diabetic conditions, although no specific miRNA profile has been noticed, the expression of some miRNAs and their target mRNAs are altered by posttranscriptional mechanisms, exerting diverse signs in the pathobiology of various diabetic complications. The aim of this review article is to discuss miRNAs involved in the process of stem cells differentiation into β-cells, resulting in enhanced β-cell functions with respect to diabetic disorders. This paper will also look into the impact of miRNA expression patterns on in vitro proliferation and differentiation of β-cells. The efficacy of the computational genomics and biochemical analysis to link the changes in miRNA expression profiles of stem cell-derived β-cells to therapeutically relevant outputs will be discussed as well.

The aim of this study was to investigate the relationship between the H-ras and Cox-2 gene expression in tumors from Iranian Oral Squamous Cell Carcinoma (OSCC) patients. Fresh tumor biopsies removed from oral cavity were collected from 67 new cases. Total RNA was extracted from biopsies and processed for quantification of H-ras and Cox-2 specific RNA expression using real-time PCR (QPCR). In addition, 59 gingival biopsies from apparently normal individuals were processed for QPCR assays. The results showed that Cox-2 expression at mRNA levels was at minimal levels in normal gingival biopsies. However, there was a surge in Cox-2 expression in tumor tissues (11.5 fold, p

Background we recently showed that a hypothesized anti-aging and anti-inflammatory protein, namely Klotho, may contribute to the etiology and/or pathogenesis of multiple sclerosis (MS). In addition, Klotho function and its gene expression are dependent on inflammatory pathways. Accordingly, the aim of this study was to investigate the Klotho gene expression within peripheral blood mononuclear cells (PBMCs) of patients with MS. Methods Altogether, 30 patients with relapsing-remitting MS (RRMS) along with 30 age and sex-matched healthy individuals were enrolled in this study. Blood samples were obtained from all participants and then PBMCs were isolated. The quantitative Real-Time PCR was carried out for Klotho mRNA derived from PBMCs. Results The results showed that klotho gene expression in the PBMCs of patients with RRMS is nearly 2.5-fold less than healthy individuals (P = 0.0006). Conclusion This is the first study demonstrating a possible role of Klotho in the PBMCs of MS patients.

Despite the current guidelines for the management of type 2 diabetes mellitus (T2DM), patients still struggle with the hyperglycemia consequences. Imbalance in zinc homeostasis, in particular, renders diabetic patients more susceptible to the damages of oxidative stress. This study aimed to evaluate the effects of zinc supplementation on the superoxide dismutase gene expression and enzyme activity in overweight individuals with T2DM. Additionally, biochemical parameters, such as fasting blood glucose (FBG), insulin, glycated hemoglobin (HbA1c), homeostasis model of assessment-insulin resistance (HOMA-IR), serum levels of zinc and lipid profile, were assessed.In this randomized, double-blind, placebo-controlled trial, 70 overweight (BMI 25) T2DM patients were selected based on the inclusion criteria. They were divided into two groups for supplementation of daily 50 mg zinc gluconate or placebo for 8 weeks. Blood samples were collected from all the individuals in the zinc group and controls for analysis.The results showed that, in comparison with the control group, zinc supplementation increased both gene expression and enzyme activity of SOD (p 0.01) as well as the levels of insulin (p = 0.02) among the patients in the zinc group. Moreover, there was a meaningful reduction in the levels of FBG, HbA1c and HOMA-IR value (p 0.001), triglycerides and total cholesterol (p 0.05) after the zinc treatment.Taken together, the current study suggests that daily supplementation with 50 mg zinc gluconate could be a useful approach for the management of overweight T2DM.IRCT2015083102.

Sphingosine 1 phosphate (S1P), S1P receptors (S1PRs) and their signaling pathways play an important role in the fate of cancer cells. The expression pattern of S1PR subtypes (S1PR1–S1PR5) may alter in cancer development stages, depending on the origin and the pathologic features of tumors. The present study aimed to examine the relationship between plasma S1P levels and the expression of S1PR subtypes in bladder tumors. These changes were evaluated in terms of the pathologic grades and stages of human bladder cancer samples. For this, tumor biopsies from 41 new bladder cancer patients as well as 26 normal-looking bladder tissues were collected and processed for immunohistochemistry (IHC) and quantitative real-time RT-PCR of S1PR subtypes. Plasma S1P level was measured using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The results show that tissue S1PR1, S1PR2 and S1PR3 are over-expressed in all tumors regardless of their pathological grade (~ 3, ~ 6 and ~ 104 folds, respectively). These results were corroborated by IHC data showing accumulation of S1PR subtypes 1 and 2 in the tissues. Plasma S1P in the plasma samples from patients was in the range of control samples (Controls; 256 ± 47; patients, 270 ± 41). Overexpression of S1PR1, S1PR2 and S1PR3 in bladder tumor biopsies which were corroborated with the pathological grades and stages may suggest that S1PR profile in tumor biopsies is a promising marker in the diagnosis of bladder carcinoma.

The anti-inflammatory role of the active metabolite of vitamin D, namely 1, 25-dihydroxyvitamin D3 (calcitriol), has been reported in multiple sclerosis (MS). Moreover, recent studies have shown that fibroblast growth factor 23 (FGF23) is involved in the regulation of calcitriol biosynthesis. The probable changes of FGF23 and calcitriol concentrations in the CSF and serum of patients with MS were evaluated. Calcitriol concentration in the CSF and serum of MS patients was significantly higher than that in non-MS patients, while FGF23 concentration in MS patients was comparable to controls. We concluded that calcitriol concentration increases in the CSF and serum of MS patients independent of FGF23 status.

Shift work is associated with sleep deprivation, occupational stress, and increased risk of depression. Depressed patients show increased oxidative stress. During excessive oxidative stress, Malondialdehyde (MDA) increases and total antioxidant capacity (TAC) decreases in body. This cross-sectional study was conducted to determine the serum level of TAC and MDA among depressed rotational shift workers in Shahid Tondooyan Tehran Oil Refinery. 21-item Beck Depression Inventory was used to measure depression level. The level of TAC and MDA was measured by 8 mL fasting blood sample. MDA was determined by thiobarbituric acid reaction. Serum total antioxidants were measured using the ABTS. Results of this study showed that TAC mean and standard deviation concentration was 2.451 (±0.536) mg/dL and MDA was 3.725 (±1.098) mic·mol/L, and mean and standard deviation of depression score and BMI were 14.07 (±3.84) and 24.92 (±3.65) kg/m2, respectively. Depression score had a positive correlation with rotational shift work experience and work experience (r=0.218andr=0.212), respectively, (P<0.05).

Stem cell therapy for degenerative diseases has been established; however there are controversies over the treatment of solid tumors with stem cell transplantation. In the present study, the anti-tumor action of mesenchymal stem cells (MSCs) has been examined in a mouse model of breast cancer with emphasize on tumor growth, angiogenesis and c-Myc expression in breast tumors. For this purpose, MSCs were isolated from bone marrow of Balb/c mice and characterized. A Balb/c mouse model of breast cancer was developed and subjected to cell therapy intra venous (I.V) or intra tumor (I.T) with MSCs. Tumor growth was measured using a digital caliber for until the end of experiment (30days). Then the mice were sacrificed and their tumors were removed and processed for histopathological examination, immunohistochemical assay of CD31 and measuring of c-Myc expression using quantitative PCR. Detection of the labeled-MSCs in tumors following injection of the cells (I.V or I.T) clearly showed the homing of MSCs into tumors. Tumor growth in case of MSC-treated mice by I.V and I.T routes was inhibited by approximately 28% and 34% respectively compared to controls. The suppression of angiogenesis was reflected in Micro Vessel Density (MVD) following I.V or I.T delivery of the MSCs. c-Myc gene expression in tumor tissues of mice treated I.V or IT with MSCs was down-regulated to 28.0% and 16.0% respectively compare to control groups. In conclusion, growth inhibition of breast tumors in mice due to MSC therapy is associated with modulation of c-Myc activation and angiogenesis markers.

Telomerase has been proposed as a novel and potentially selective target in cancer therapy. Silymarin, which is a standardized mixture of flavonolignans from the medical plant Silybum marianum, has potent effects against various types of cancer cells, but its effect on telomerase activity in the human leukemia cell line K562 has not been investigated. The aim of this study was to examine the mechanism of silymarin-induced apoptosis in K562 cells, with particular emphasis on its effect on telomerase activity. The antiproliferation effect of silymarin on K562 cells was evaluated by the MTT assay. To measure apoptosis, Hoechst 33342 staining and flow cytometry were used. The telomerase activity was determined using the telomeric repeat amplification protocol (TRAP)-ELISA assay. The treatment of the K562 cells with silymarin resulted in a significant inhibition of cell growth and telomerase activity. Also, a positive correlation was found between telomerase inhibition and induction of apoptosis in silymarin-treated K562 cells. These results suggest a novel mechanism in the anticancer activity of silymarin in human leukemia K562 cells and may provide a basis for future development of anti-telomerase therapies.

This study compared the sensitivity of differentiated hepatocyte-like cells, their progenitor mesenchymal stem cells (MSCs) and CD34 + stem cells to DNA damage and toxicity induced by aflatoxin B1 (AFB1). The hepatocyte-like cells and their progenitor cells (isolated from umbilical cord blood (UCB)) were each treated with AFB1 on day 15 of differentiation. Cell toxicity and genotoxicity effects were assessed using MTT and alkaline comet assays. AFB1 treatment resulted in a dose- and time-dependent inhibition of cell growth. The IC 50 values of AFB1 for hepatocytes differentiated from CD34 + and MSCs were within the same range (44.7–46.8 μM). The IC 50 calculated for non-differentiated MSCs and CD34 + cells was slightly lower (42.0–43.4 μM) than that calculated for their differentiated counterparts. However, the extent of DNA damage was different in differentiated and non-differentiated cells. The percentages of DNA (% DNA) in comet tails measured in hepatocytes differentiated from MSCs exposed to AFB1 (0, 2.5, 10 and 20 μM) for 24 h were ∼15, 55, 65 and 70%, respectively. In comparison, hepatocytes from CD34 + cells were more resistant to AFB1-induced DNA damage. Hepatocyte-MSCs were most sensitive to DNA damage, followed by UCB-CD34 + cells, then UCB-MSCs and finally hepatocyte-CD34 + cells. These results clearly showed that stem cells from different sources have different sensitivities to DNA damaging agents. These differences can be assigned to the expression levels of cytochrome P450 (CYP) particularly CYP3A4 in non-differentiated and differentiated cells. These data are useful in better understanding the susceptibility/resistance of stem cells in the process of differentiation to environmental toxicants.

Irradiation is an important method of processing herbal drugs, while our understanding of the effects of gamma-irradiation on pharmacological properties of seed products such as caraway essential oils is however still very limited. In this study, caraway seeds were irradiated at dose levels of 0, 10 and 25kGy. After extracting the essential oils, the effects of fresh and gamma-irradiated caraway oils (100mg/kg b.w) on preventing septic-related oxidative liver injury induced by cecal ligation and puncture (CLP) model were investigated by measuring oxidative stress parameters in the liver. CLP operation caused a marked increase in myeloperoxidase (MPO) activity which was readily reversed in rats treated with fresh and irradiated caraway oils. Likewise, thiobarbituric acid reactive substances (TBARS) level in the liver was compensated in rats treated with the fresh and irradiated caraway oils. Moreover, liver GSH which was initially depleted due to CLP was recovered by essential oil treatments. The protective role of oils was further confirmed by showing that liver function tests (ALT/AST) as well as histopathological changes following CLP operation were recovered in rats treated with oils from either fresh or irradiated caraway seeds. These data may suggest that gamma-irradiation to caraway seeds at 10 and 25kGy has no influence on the antioxidative properties of caraway essential oils.

Background : The molecular and cellular mechanisms linking chronic inflammation and gastrointestinal malignancy are not known with certainty. Aim : To investigate changes in potential causative factors during progression of esophagus cancer in a population living in high-risk area in Iran. Subjects : Formalin-fixed, paraffin-embedded esophageal specimens ( n =87) from patients with gastroesophageal reflux disease (GERD), Barrett's metaplasia, adenocarcinoma (ADC) and squamous cells carcinoma (SCC) were collected based on their pathological diagnosis. Methods : Immunohistochemical (IHC) technique was used to study tissue accumulation of P53, P21, cyclooxygenase-2 (COX-2), glutathione S-transferase-P (GST-Pi) and nitrotyrosine (NT) in patients and controls. Results : P53 expression was not detected in esophageal tissues from normal and GERD samples, whereas it was found positive in Barrett's, ADC, and SCC samples. P21 positive sample was relatively higher in ADC patients as compared to that in SCC (ADC: 52.6%; SCC: 25%). GST-Pi expression was equally accumulated in all the samples. NT was predominantly expressed in ADC (72.7%). COX-2 expression was significantly higher in Barrett's (60.0%) and ADC (66.6%) as compared to that in GERD, SCC and normal. These data were further confirmed by detecting the scores of immunostainings in all the positive samples. Conclusion : The pathological changes in ADC and SCC samples which were associated with increasing frequency of NT and COX-2 provides further evidence for involvement of these inflammatory factors in progression of esophagus cancer.

The aim of this study was to find out the profile of cellular glutathione (GSH) and GSH S-transferase (GST) in hepatocytes differentiated from adult mesenchymal stem cells (MSC). For this purpose, we have derived functionally active hepatocyte-like cells from normal human multipotent adult MSC. Then the differentiated cells were characterized by specific hepatic markers. The cellular GSH and GST catalytic activity toward 1-chloro-2,4-dinitrobenzene (CDNB) were determined in hepatocyte-like cells differentiated from MSC compared with undifferentiated MSC. Reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting techniques were used to study GST-P1-1 and GST-A1-1 expression in differentiated and undifferentiated cells. The results showed that there is more than threefold increase in GST catalytic activity in hepatocytes recovered by day 14 of differentiation. GST-P1-1 mRNA expression was detected in both differentiated hepatocyte-like cells and their undifferentiated progenitors. Under similar conditions, only differentiated hepatocyte-like cells expressed GST-A1-1 mRNA. These results were further confirmed by showing that the undifferentiated cells expressed both GST-A and GST-P proteins. Unlike GST, the level of cellular GSH was declined (approximately 20%) in hepatocytes derived from MSC as compared to that of undifferentiated cells. These data may suggest that hepatogenic differentiation of human bone marrow MSC is accompanied with the regulation of factors participating in GSH conjugation pathway.

In this study characterization of endothelial cells differentiated from human bone marrow mesenchymal stem cells (hBMCs) was investigated in relation to their capillary network formation potential. Differentiation was performed in presence of vascular endothelial growth factor (VEGF) and insulin like growth factor-1 (IGF-1). A panel of cellular and molecular markers was used for characterization of the endothelial cells. The cells were strongly positive for von Willebrand factor (vWF) and vascular endothelial growth factor receptor 2 (VEGFR2) when measured at protein and mRNA levels. Development of endothelial cells was found to be associated with formation of typical organelles such as Weibel Palade (WP) bodies, Cavealae and pinocytic vesicles. Early vessel growth was also evidenced by showing specific junctions between the cells. The migratory and angiogenic properties of the cells were confirmed by showing capillary network formation in vitro. These results indicate that the capacity of endothelial cells differentiated from hBMSCs in formation of vascular system is consistent with molecular and structural development.

Aim To supporting growth and functional differentiation of adult stem cells into hepatocytes in a well-controlled manner, we performed differentiation of human bone marrow mesenchymal stem cells (hBMSCs) to hepatocytes-like cells on a constructed 3-dimensional (3D) nanofibrous biocompatible scaffold. Methods After characterization of the hBMSCs isolated from human bone marrow, the performance of the cells seeded and their proliferation on the scaffold was evaluated by scanning electron microscopy (SEM) and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Different approaches such as immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), and biochemical assays were used to estimate the ability of hBMSC-derived cells to express hepatocyte-specific markers. Results Scanning electron micrographs and MTT analysis revealed the cells were able to expand and remained biologically and metabolically active for 21 days. Immunocytochemical analysis of albumin and α-fetoprotein showing the accumulation of these markers in differentiated cells was confirmed by RT-PCR. Additional markers such as cytochrome P450 3A4, cytokeratin-18, and cytokeratin-19 detected by RT-PCR showed progressive expression during 3 weeks of differentiation on 3D scaffold. The hepatocyte-like cells displayed several characteristics of metabolic functions as judged by production of albumin, urea, transferrin, serum glutamic pyruvic transaminase (SGPT), and serum oxaloacetate aminotransferase (SGOT). Levels of above-mentioned markers, except SGOT in differentiated cells on scaffold, were found to be significantly greater than in the 2D culture system (pConclusion Overall data suggest that the engineered nanofibrous scaffold is a conductive matrix for functional hBMSC-derived hepatocyte-like cells and is promising for maintenance of hepatocytes suitable for implantation.

Resistance to oxidative damages in undifferentiated mesenchymal stem cells (MSCs) in comparison with the undifferentiated progenitor cells may differ depending on several factors. This study was carried out to examine the impact of hepatogenic differentiation process of MSCs on oxidative DNA damage markers. Hepatic differentiation of MSCs was carried out using a two-step conventional protocol and the cells were processed for characterization using morphological and biochemical markers. During the course of differentiation cellular levels of reactive oxygen species (ROS), 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and expression of ataxia-telangiectasia mutated (ATM) protein were estimated at time intervals (10, 20 and 30 days). The results showed a decrease in cellular ROS (13%, P

The methylotrophic yeast Pichia pastoris has become a highly popular expression host system for the recombinant production of a wide variety of proteins, such as antibody fragments. Camelids produce functional antibodies devoid of light chains and constant heavy-chain domain (CH1). The antigen binding fragments of such heavy chain antibodies are therefore comprised in one single domain, the so-called VH of the camelid heavy chain antibody (VHH). To test the feasibility of expressing VHHs in the yeast, which on account of their small size and antigen recognition properties would have a major impact on antibody engineering strategies, we constructed two VHH genes encoding the single-domain antibody fragments with specificity for a cancer associated mucin, MUC1. The recombinant strains of the yeast P. pastoris were developed which secrete single-domain antibody fragment to the culture supernatant as a biologically active protein. Supplementation of medium with sorbitol (in pre-induction phase) and casamino acid or EDTA (in induction phase) provided ideal condition of increasing the yield of VHH production compared to culture condition devoid of above recipe. The secreted protein was purified following a 80% ammonium sulfate precipitation step, followed by a affinity chromatography column. The specific activity in enzyme-linked immunosorbant assay (ELISA) of the purified yeast VHH was higher than that of a bacterial periplasmic counterpart. These results reaffirm that the yeast P. pastoris is a suitable host for high level and correctly folded production of VHH antibody fragments with potential in vivo diagnostic and therapeutic applications. This is the first report of expression of VHH in P. pastoris.

To develop an indirect enzyme-linked immunosorbent assay (ELISA) for measuring class-pi glutathione S-transferase (GST) in plasma, and tissue biopsies obtained from upper gastrointestinal cancer (UGI Ca) patients.GST activity and GST-pi concentration were detected in normal human squamous esophageal epithelium, normal gastric cardia and their corresponding malignant tumor biopsies.Plasma GST was significantly higher (p0.05) in UGI Ca patients as compared to those obtained from normal individuals. Plasma GST-pi concentration in normal subjects was 6.6 +/- 1.9 ng/mg protein, whereas it was higher in UGI Ca patients (esophageal, 10.0 +/- 1.8; gastric, 10.7 +/- 1.7 ng/mL, por= 0.01). GST and GST-pi levels were higher in surgically resected tumor biopsies as compared to normal tissues adjacent to a tumor (p0.05). GST-pi was also elevated in malignant esophagus and gastric biopsies taken at endoscopy as compared to normal and normal-appearing esophageal tissues (p0.05). Total GST was also increased significantly in gastric malignant tissues although its activity was within the same range in normal and normal-appearing tissues. A significant correlation between plasma GST-pi with that in malignant tissues was observed. Total GST and GST-pi were also correlated in different malignant tissues.Biopsies obtained at endoscopy can be used satisfactorily to measure tumor markers.

Differentiation of mesenchymal stem cells (MSCs) to hepatocyte-like cells is associated with morphological and biological changes. In this study, the effect of hepatogenic differentiation on fatty acid profile and the expression of proliferator-activated receptors-α (PPAR-α) have been studied. For this purpose, MSCs isolated from human umbilical cord were differentiated into hepatocyte-like cells on selective culture media. The morphological and biochemical changes, PPAR-α expression and reactive oxygen species (ROS) levels were studied during the differentiation process. Besides, the cells were processed to determine changes in fatty acid profile using gas chromatography analysis. The results showed that hepatic differentiation of the MSCs is associated with a decrease in major polyunsaturated fatty acids in mature hepatocytes, whereas there was an increase in the saturated fatty acid (SFA) levels during hepatocyte maturation. The differentiation-dependent shift in the ratio of SFA/USFA was associated with changes in albumin and PPAR-α expression, whereas changes in fatty acid profile were independent of ROS production and lipid peroxidation in differentiating cells. In conclusion, these data may suggest that hepatocyte formation during the stem cell differentiation is associated with a shift in the fatty acid profile that is probably a normal phenomenon in hepatogenic differentiation of the MSCs.

Differentiation of mesenchymal stem cells (MSCs) to hepatocytes-like cells is associated with alteration in the level of reactive oxygen species (ROS) and antioxidant defense system. Here, we report the role of glutathione in the functions of hepatocytes derived from MSCs. The stem cells undergoing differentiation were treated with glutathione modifiers [buthionine sulfoxide (BSO) or N-acetyl cysteine (NAC)], and hepatocytes were collected on day 14 of differentiation and analysed for their biological and metabolic functions. Differentiation process has been performed in presence of glutathione modifiers viz. BSO and NAC. Depending on the level of cellular glutathione, the proliferation rate of MSCs was affected. Glutathione depletion by BSO resulted in increased levels of albumin and ROS in hepatocytes. Whereas, albumin and ROS were inhibited in cells treated with glutathione precursor (NAC). The metabolic function of hepatocytes was elevated in BSO-treated cells as judged by increased urea, transferrin, albumin, alanine transaminase and aspartate transaminase secretions in the media. However, the metabolic activity of the hepatocytes was inhibited when glutathione was increased by NAC. We conclude that the efficiency of metabolic function of hepatocytes is inversely related to the levels of cellular glutathione. These data may suggest a novel role of glutathione in regulation of metabolic function of hepatocytes.

Differentiation of human mesenchymal stem cells (MSCs) to metabolically active hepatocytes depends on different regulatory factors. Trans-differentiation of stem cells into specific cell lineage in the presence of specific stimuli is associated with the molecular and cellular damage. The aim of the present study was to examine the role of P53 in the regulation of cyclooxygenase-2 (COX-2) expression and the generation of protein and lipid oxidation during trans-differentiation of MSCs into hepatocyte-like cells. During the 3-week differentiation process of MSCs to hepatocyte-like cells we found that expression liver-specific markers was associated with increased levels of lipid peroxidation and protein carbonyl formation. Expression of P53 and COX-2 at mRNA and protein levels were evaluated in MSCs before and after differentiation on days 7, 14 and 21. We showed that the up-regulation of COX-2 was associated with augmentation of the rate of cell proliferation, morphological and biochemical changes of hepatocytes-like cells. However, in parallel the P53 at the mRNA level was down-regulated, and at protein levels accumulation in the nuclei was reduced during the hepatogenic differentiation time. Our results may suggest a P53-COX-2 pathway in the regulation of hepatogenic differentiation of stem cells, which is linked to differentiation-dependent molecular oxidative damage.

The role of mesenchymal stem cell in cellular therapy is the subject of interest for many researchers. The differentiation potential of MSCs and abilities in modulations of the recipient's immune system makes them important cells in tissue regenerative studies. MSCs by releasing the proinflammatory cytokines play important role in immunomodulatory systems; however the signaling pathways for releasing of these mediators are not well understood. Glutathione has been shown to play a role in modulation of cytokines in hepatogenic differentiation.In the current study we aimed to investigate the effects of buthionine sulfoximine (BSO, inhibitor for glutathione synthesis) and N-acetylecystin (NAC, an inhibitor for ROS generation) on proinflammatory cytokines production in a hepatogenic differentiation model.BSO and NAC significantly decreased IL-6 and TNF-α levels at 14 days of differentiation, whereas, NAC decreased the levels of IL-8 at days 2 and 14 of differentiation. Moreover, intracellular glutathione level during the differentiation was depleted.Our current study suggests a novel role of GSH as an immunopharmacological regulatory molecule during hepatogenic differentiation. Finally, this information may shed some light on the understanding of MSCs responses in transplantation and cell therapy in diseases such as chronic hepatic diseases.

Asthma and Chronic Obstructive Pulmonary Disease (COPD) are two important lung and airways diseases which affect the lives of ~500 million people worldwide. Asthma is a heterogeneous disease that is broadly defined as a clinical syndrome characterized by altered lung function, mucus hypersecretion, peribronchial inflammation and hyperresponsiveness In contrast, the effect of inhalation of toxic particles and gases on the innate and adaptive inflammatory immune systems underlie the pathogenesis of COPD. In the last decade, knowledge concerning the pathophysiologic mechanisms underlying asthma and COPD has risen tremendously and current dogma suggests that the pathogenesis of both diseases is driven by the chronic inflammation present in the airways of these patients. Thus, understanding the mechanisms for the persistence of inflammation may lead to new therapeutic approaches. In this review, we provide an overview of the main signal transduction pathways implicated in asthma and COPD pathophysiology focusing on inflammasome signaling in various cells types which result in altered inflammatory mediator expression.

There are several issues to be considered to reduce the risk of rejection and minimize side effects associated with liver cell transplantation in chronic liver diseases. The source and the condition of stem cell proliferation and differentiation ex vivo and the transplantation protocols are important safety considerations for cell based therapy. The biochemical and molecular markers are important tools for safety evaluation of different processes of cell expansion and transplantation. Studies show that hepatocytes differentiated from adult and embryonic stem cells exhibit biochemical and metabolic properties resembling mature hepatocytes. Therefore these assays can help to assess the biological and metabolic performance of hepatocytes and progenitor stem cells. The assays also help in testing the contribution of transplanted hepatocytes in improving the repair and function of damaged liver in the recipient. Here we review the biochemical and metabolic markers, which are implicated in evaluation of safety issues of stem cells used for therapeutic purposes in chronic liver diseases and regeneration of damaged liver. We also highlight application of biochemical tests for assessment of liver cell transplantation.

Silymarin, a mixture of flavonolignans, is extracted from milk thistle (Silybum marianum) and has a strong antioxidant activity and exhibits anticarcinogenic, anti-inflammatory, and cytoprotective effects. In this study we attempted to determine whether silymarin and the glutathione modifiers, buthionine sulfoxamine (BSO) and N-acetylcysteine (NAC), are involved in regulation of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in proliferating mesenchymal stem cells (MSCs). Cellular glutathione was manipulated during a 14-day culture using BSO, NAC and silymarin. At intervals of 2, 7 and 14 days, cells were collected and COX-2 and iNOS levels were measured. In parallel, generation of cellular H(2)O(2) and glutathione were measured. Supplementation of the culture media with BSO caused a dose-dependent decrease in MSC proliferation, whereas NAC or silymarin elevated the proliferation (p0.05). Treatment of MSC with NAC or silymarin caused a significant decrease in COX-2 levels. However, COX-2 levels in cells treated with high levels of NAC (1.0 mM) were significantly lower than those in MSCs treated with high levels of silymarin (100 μM). BSO (1.0 and 5.0 μM) caused a significant increase in COX-2 on days 2, 7 and 14. BSO caused a significant increase in iNOS, whereas NAC or silymarin decreased cellular iNOS. Overall result show that glutathione, iNOS and COX-2 in proliferating MSCs are affected by silymarin treatment. It appears that glutathione is the main target of silymarin, and in consequence iNOS and COX-2 are affected in response to silymarin treatment.

Carcinoma antigen 125 (CA 125) is overexpressed in ovarian cancer and antibodies against it are widely employed for diagnostic purposes. The rarity of CA 125 antigenic domains and its highly glycosylated structure, however, is a problem that may prevent immunized mice from developing a diversified population of anti-CA 125 antibodies. In this study a prime-boost strategy, which potentially could augment the humoral immune responses against rare and poorly immunogenic determinants, was used for immunization of mice and monoclonal antibodies (mAbs) were produced by hybridoma technology. Reactivity of mAbs was then assessed by ELISA, western blotting, immunoprecipitation, immunohistochemistry and immunofluorescence staining of OVCAR-3 cell line. Altogether, 10 clones were produced, 3 of which had IgG isotype and the rest were IgM. Two-third of clones recognized cognate antigen in fixed and living cells and had strong immunoreactivity in IHC staining. In Western blotting, our antibodies recognized CA 125 as high molecular weight antigen mostly migrated in the 3% stacking gel. Immunoprecipitation of OVCAR-3 cell lysate by mAbs resulted in a very similar migration pattern that reconfirmed their specificities. The mAbs produced in this study are invaluable tools in diagnosis and research fields for assessment of CA 125 expression in cancerous ovarian tissues.

Monoclonal antibodies (mAbs) are essential tools for many molecular immunology investigations, epitope mapping and molecular modelling, clinical laboratory diagnostic tests and immunotherapy. Humoral immune response of immunized animals largely depends on the nature of antigen and the immunization technique. Polysaccharides and heavily-glycosylated proteins are very elusive targets incapable of mounting long-lasting, high affinity antibody responses. Carcinoma antigen 125 (CA 125), a well known tumor marker of ovarian cancer, is a mucin type antigen consisting of repetitive units of heavily glycosylated moieties which render production of mAbs very difficult.To evaluate the efficacy of heterologous antigen preparations as a way of mouse immunization in the production of anti-CA 125 mAb.Two different protocols of immunization were used for priming of NMRI mice. In the first method, mice conventionally immunized by three intraperitoneal injections of purified CA 125 and boosted by the antigen three days before fusion. In the second approach, mice were primed by three intraperitoneal injections of living CA 125 positive cells of OVCAR-3 cell line, and boosted by intravenous injection of the purified extracellular domain of CA 125. Production of mAb was performed by standard hybridoma technology and mAbs were characterized by different immunoassays.The first method failed to produce stable clones despite six time fusion. A total of ten stable clones, however, were produced in the second approach. Some of the clones were characterized and found to have excellent immunoreactivity when tested by ELISA assay, western blotting, intracellular and surface immunofluorescent staining of OVCAR-3 cell line and immunohistochemical staining of ovarian cancer tissues.Altogether the results of the present study clearly showed that heterologous antigen preparation is the method of choice for immunization when production of monoclonal antibody against highly glycosylated poorly immunogenic antigens is concerned.

Background: There is significant interest in using nanofibers in tissue engineering from stem cells. The transdifferentiation of mesenchymal stem cells into the hepatic lineage in a nanofibrous structure has not been reported. In this study, a three dimensional nanofibrous scaffold is introduced for differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs) into hepatocytes. Methods: A scaffold composed of Poly (e-caprolactone), collagen and polyethersulfone was fabricated by the electrospinning technique. After characterization of isolated hBMSCs, the performance of the cells on the scaffold was evaluated by Scanning Electron Microscopy (SEM) and MTT assay. Cytological, molecular and biochemical markers were measured to confirm differentiation potential of hBMSCs into hepatocytes. Results: The isolated cells possessed the basic properties of mesenchymal stem cells (MSCs). Based on scanning electron microscope (SEM) analysis and MTT assay, it was shown that the cells adhere, penetrate and proliferate on the nanofibers. Cultured cells on the nanofibers differentiated into hepatocyte-like cells and expressed hepatocyte specific markers such as albumin, α-fetoprotein, cytokeratin-18, cytokeratin-19 and cytochrome P450 3A4 at mRNA levels. Appearance of a considerable number of albumin-positive cells cultivated on the scaffold (47 ± 4%) as compared to the two-dimensional culture system (28 ± 6%) indicates the supporting role of the scaffold. The efficiency of the cells to produce albumin, urea, transferrin, serum glutamic pyruvic transaminase and serum oxaloacetate aminotransferase in hepatocytes on the scaffold further attest to the functionality of the cells. Conclusion: The data presented in this study show that the engineered nanofibrous scaffold is a conductive matrix which supports and enhances MSC development into functional hepatocyte-like cells.

The aim of this study was to find out substitution effect of fetal bovine serum (FBS) with human platelet releasate (HPR) as a major growth factor source during expansion and differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) into hepatocytes.Propagation and differentiation potential of hBMSCs into hepatocyte-like cells in a medium fortified with HPR instead of FBS were investigated with morphological, cytochemical and molecular experiments.Multiplex analysis showed that HPR was more efficient than FBS in supporting hBMSC outgrowth. The proliferation rate of MSC in presence of HPR (derived from 10(9) platelets/ml) was about threefold greater than that of FBS (P0.001). Despite the differences in MTT value, hBMSCs-driven HPR or FBS did not differ in terms of gross morphology, immunophenotype and osteogenic differentiation potential. Hepatic differentiation of hBMSCs was successfully performed in the media enriched with HPR. Immunoreactivity of cells with monoclonal antibodies against for albumin and alpha-fetoprotein (AFP) was even more positive in hepatocytes differentiated in presence of HPR as compared to that of FBS. The gene expression of albumin, AFP and cytokeratin-18 at mRNA levels in differentiated cells attest to supporting role of HPR in hepatic differentiation media. These findings were further confirmed with greater urea production (approximately twofold) in the culture media of cells differentiated under HPR compared to that in FBS (P0.001).Human platelet releasate is an efficient and safe substitute for FBS in culture media used for expansion and differentiation of hBMSCs to hepatocyte.

This study was to investigate the effect of sodium selenite (SS) on in vitro maturation of mouse preantral follicles.The isolated preantral follicles were cultured in TCM 199 medium supplemented with different concentrations (0, 5, 10, 15 ng/ml) of SS and 3 mg/ml bovine serum albumin (BSA) or 5% Fetal Bovine Serum (FBS). The ovulation was induced by addition of 1.5 IU/ml human chorionic gonadotropin. The size and development of follicles and oocytes were assessed by calibrated eyepiece.The survival rates of follicles in FBS supplemented groups containing 5 and 10 ng/ml SS (88.23%, 90.83%) were higher than other groups (P0.05 and P0.001 respectively). The mean diameter of follicles (199.84 +/- 15.58 microm) and the percentage of MII oocyte (33.08%) were higher in FBS supplemented group containing 10 ng/ml SS (P0.001).The sodium selenite and FBS improve the in vitro growth and maturation of mouse preantral follicles.

Neuroserpin, a member of the Serine Proteinase Inhibitor (Serpin) superfamily, is known to be a neuroprotective factor in the focal ischemic stroke followed by reducing the microglial activation. Neuroserpin is a protein rich of methionine residues that can scavenge the free radical species which may increase its neuroprotective effect. On the other hand, the oxidative modifications of the amino acid residues in neuroserpin may lead to changes in its conformation and function. In this study, it was investigated the changes in the conformation and the function of the oxidized neuroserpin.Neuroserpin expressed in E. coli, BL21 or M15 harboring plasmid pQE81L containing neuroserpin cDNA. Expressed neuroserpin was purified by resin sulfopropyl A50 precharged with 0.1 M NiSO4 under denaturing condition. Neuroserpin was oxidized under oxidative stress condition in the presence of different concentration of hydrogen peroxide. The oxidation of neuroserpin was conveniently detected by a carbonyl content assay using 2, 4 dinitrophenylhydrazine. Changes in tertiary structure of neuroserpin were monitored by spectrofluorimeter to study the alteration of intrinsic fluorescence and also fluorescence of 8-anilinonaphthalin-1 sulfonic acid (ANS) in native and oxidized form of neuroserpin.Total expressed neuroserpin was estimated 4-5 mg/lit in 2XYT culture media. SDS-PAGE analysis of purified neuroserpin showed a single band which reflects the efficiency of the resin SP A50 for purification of the proteins containing 6xHis tag. Carbonyl content of oxidized and native neuroserpin was estimated 12.3 +/- 0.3 and 0.45 +/- 0.05, respectively. The inhibitory activity of oxidized neuroserpin decreased up to 40-60% as compared with native form of neuroserpin. Intrinsic fluorescence and also the emission of ANS bind to the hydrophobic region of the protein altered from 380 to 85 and in the case of ANS from 105 to 150 in oxidized and native form of neuroserpin, respectively.The decreased intrinsic fluorescence intensity, an enhancement in the fluorescence of ANS, and loss of the inhibitory activity up to 40-60% in neuroserpin, all suggested a conformational modification in the protein under the oxidative stress condition. Remaining the inhibitory activity of neuroserpin reflects that the protein tolerates the oxidative stress condition effectively.

To develop a RT-PCR technique coupled with Enzyme Linked Immunosobant Assay (ELISA) i.e. RT-PCR-ELISA for measurement of class-Pi glutathione S-transferase (GST-P)-specific mRNA in esophageal diseases.In this study, 66 esophageal tissue biopsies diagnosed as non-erosive reflux disease (NERD), gastroesophageal reflux disease (GERD), adenocarcinoma (ADC), and squamous cell carcinoma (SCC) were used. Standardization of the RT-PCR-ELISA was carried out using specific GST-Pi and beta-actin primers, biotin labeled probe, DIG-labeling RT-PCR and anti-DIG-HRP conjugate.The results of RT-PCR-ELISA based on OD ratio of GST-Pi mRNA/beta-actin showed that there was no significant difference in GST-Pi expression in normal, NERD and GERD samples. Overexpression of GST-Pi in malignant tissues (ADC and SCC) was distinguishable. The OD ratio of GST-Pi mRNA expression to beta-actin mRNA was 1.17+/-0.13 and 1.3+/-0.13 in ADC and SCC samples, respectively, which is significantly higher (P0.05) than matching control (0.78+/-0.06 and 0.85+/-0.07).RT-PCR-ELISA showed that GST-Pi expression was not altered in GERD and NERD esophagus, whereas, in ADC and SCC samples, it was significantly higher (P0.05) as compared to inflamed and normal tissues.

In contrast to the murine and human VHs, camels' single domain antibodies (sdAb) have sufficient solubility. These antigen-specific fragments are expressed well in Escherichia coli. Here, we report high expression and purification of sdAbs against MUC1 mucin. MUC1 is a high molecular weight glycoprotein with an aberrant expression profile in various malignancies. The sdAb genes were sub-cloned into a pET32a(+) vector to overexpress the protein coupled with fusion tags in E. coli BL21(DE3). The expressed single domain antibodies were purified by immobilized metal affinity chromatography and antigen affinity chromatography. Analysis by SDS-PAGE and Western blotting demonstrated the integrity of the sdAbs-tags, while ELISA results confirm that the activity of these molecules compare favorably with that of the parent recombinant antibodies. Enterokinase treated sdAb showed a band at the molecular weight around 12 kDa which demonstrated the naked protein in its natural structure with activities comparable to that of native protein. The high binding activity to MUC1 antigen purified from ascitic fluid (of patients with small-cell lung aggressive carcinoma and metastasis to peritoneum) and the very close similarity of these molecules to human VHs illustrated the potential application of these novel products as an immunodiagnostic and immunotherapeutic reagent.

Objectives: Persistent hepatitis B virus (HBV) infection often leads to the development of chronic hepatitis and cirrhosis. The role of host genetic factors in chronic HBV infection is not fully understood. We studied the influence of glutathione S -transferase (GST) M1, T1, and P1 polymorphisms in patients with different stages of HBV infections. Methods: The sample population included 41 HBV normal carriers, 37 patients with chronic hepatitis, and 38 patients with cirrhosis (infected with HBV) compared to a control group ( n = 59). PCR-based procedures were performed in the studied populations to confirm the genotypes of GSTT1, M1, and P1. Odds ratio analysis tests were used for statistical evaluation. Results: We found that the frequency of GSTP1-Val (105)/Val (105) genotype was significantly higher in patients with liver cirrhosis (27%) than HBV normal carriers (2.4%; OR 14.8, 95% CI 1.8–122.5) and the frequency GSTP1-Val (105)/Ile (105) genotype was significantly higher in patients with liver cirrhosis (59.5%) than HBV normal carriers (19.5%; OR 6.1, 95% CI 2.1–16.7). The genotype GSTP1-Val (105)/Val (105) was more frequent in patients with chronic hepatitis (19.4%) than HBV normal carriers (2.4%; OR 9.65, 95% CI 1.1–82.8). Patients with cirrhosis also had a higher frequency of the GSTM1 null genotype (71.1%) than HBV normal carriers (27.5%; OR 6.5, 95% CI 2.4–17.4) and the GSTM1 null genotype was more frequent in patients with chronic hepatitis (64.9%) than HBV normal carriers (27.5%OR 4.9, 95% CI 1.8–12.8). The frequency of GSTT1 genotype was similar in all groups. Conclusion: These results suggest that in HBV infection, inheritance of the null GSTM1 and GSTP1-Val (105) polymorphisms involves a host genetic factor that is relevant to disease progression.

Background: It has been reported that the activity of glutathione S-transferase (GST) is over-expressed in plasma and esophagus biopsies in Iranian patients suffering from esophageal squamous cell carcinoma (SCC). The aim of this study was to find out the frequency of GST-P genotypes in these patients. Moreover, the association of GST-P genotypes with p53 protein accumulation in esophageal epithelium was investigated. Materials and Methods: DNA isolated from paraffin-embedded tissue biopsies from patients suffering from esophageal SCC (n = 56) were collected. polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using Alw261 enzyme was applied to determine GST-P genotypes (Ile 105 Val). All the samples were also subjected to immunohistochemistry (IHC) for p53. Results: The frequency of GST-P genotypes in Iranian esophagus SCC patients for Ile/Ile, Ile/Val and Val/Val was 73.2, 21.5 and 5.3%. There was no association between GST-P polymorphism and p53 accumulation in esophageal epithelial cells. Conclusions: The frequency of GST-P polymorphism was not associated with p53 protein accumulation in esophagus epithelium. The frequency of polymorphic variants of GST-P, Ile/Ile, Ile/Val and Val/Val in SCC patients may suggest that Ile to Val substitution in GST-P gene dose not represent susceptibility to SCC in high-risk Iranian population.