Correlation of Gankyrin oncoprotein overexpression with histopathological grade in prostate cancer.

Prostate cancer is the second most commonly diagnosed cancer among men worldwide. Identifying new prognostic and predictive biomarkers will help stratification of prostate cancer patients for a better treatment. Gankyrin is a novel oncoprotein which regulates cell cycle and protein degradation. Gankyrin overexpression correlated with the malignant phenotypes and promotes the tumorigenicity and metastasis in many cancers. However, there are not any reports on the role of Gankyrin in prostate cancer. Therefore, this study was designed to investigate the expression of Gankyrin in prostate cancer and analyze its correlation with some clinicopathological characteristics. We characterized the expression of gankyrin in fifty five prostate cancer specimens and twenty non-cancerous tissues by immunohistochemical staining and the results were correlated with clinical characteristics and pathological parameters.Results showed that Gankyrin was expressed in 41 of 55 (74%) prostate cancer patients and its expression was significantly higher than corresponding adjacent normal tissues (p<0.001). Gankyrin overexpression was significantly correlated with histopathological tumor grade, Gleason score and tumor differentiation (P=0.002). These findings showed that Gankyrin is mainly overexpressed in high grade prostate tumors so it may have a significant role in prostate cancer progress and it May serve as a useful biomarker for the identification of aggressive prostate cancers.

Cell-SELEX-based selection and characterization of a G-quadruplex DNA aptamer against mouse dendritic cells.

Targeting of dendritic cells (DCs) by aptamers increases antigen capture and presentation to the immune system. Our aim was to produce aptamers against DC molecules using the cell-SELEX procedure. For this purpose, 18 rounds of cell-SELEX were performed on mouse macrophage J774A.1 and CT26 as target and control cells, respectively. The selected aptamers were truncated and their binding to mouse macrophages, and immature and mature DCs analyzed. Two macrophage-specific aptamers, Seq6 and Seq7, were identified. A truncated form of Seq7, Seq7-4, 33 nucleotides in length and containing the G-quadruplex, bound macrophages and immature DCs with KD values in the nanomolar range. We anticipate that Seq7-4 has potential as a therapeutic tool in targeting of mouse macrophages and immature DCs to efficiently improve different immunotherapy approaches.

The Comparison of Biodistribution, Efficacy and Toxicity of Two PEGylated Liposomal Doxorubicin Formulations in Mice Bearing C-26 Colon Carcinoma: a Preclinical Study.

BACKGROUND: Over the past several years, the considerable attention has been progressively given to liposomal formulations of anthracyclines. SinaDoxosome(®) (Exir Nano Sina Company, Iran) is a pegylated liposomal doxorubicin (DOX) which approved by Food and Drug Administration of IRAN for treatment of some types of cancer. The aim of this study was to compare the biodistribution, efficacy, cardiotoxicity and hepatotoxicity of SinaDoxosome(®) with Caelyx(®) in mice bearing C-26 colon carcinoma. METHODS AND RESULTS: Mice tumor size evaluation during the experimental period (28 days) showed comparable therapeutic efficacy of nano-formulations. The biodistribution studies showed no significant difference in DOX tissue concentration between Caelyx(®) and SinaDoxosome(®). DOX induced-ECG changes were not detected in nano-formulations. No significant alteration was found in biochemical indexes of myocardial injury in mice exposed to nano-formulations in comparison with control mice. The tissue oxidative parameters such as lipid peroxidation, glutathione, catalase and superoxide dismutase was significantly changed as the results of free DOX treatment. However, the oxidative status of Caelyx(®) and SinaDoxosome(®) treated animals did not showed any changes. The experiment also revealed that apoptotic pathway was not activated in the heart of mice exposed to nano-formulations. CONCLUSIONS: Although this investigation showed that Caelyx(®) and SinaDoxosome(®) are similar in terms of biodistribution, efficacy and toxicity, appropriate clinical evaluations in patients should be considered.

Proteomics screening of adenosine triphosphate-interacting proteins in the liver of diazinon-treated rats.

AIM: Diazinon (DZN) is one of the most important organophosphorus compounds used to control pests in agriculture in many countries. Several studies have shown that exposure to DZN may alter protein expression in the liver. In order to further investigate the mechanism of DZN toxicity, differentially expressed ATP-interacting proteins, following subacute exposure to toxin, were separated and identified in rat liver. MAIN METHODS: Male rats were equally divided into four groups: control (corn oil) and DZN (15 mg/kg) by gavage once a day for 4 weeks. After homogenization of liver tissue, lysates were incubated ATP-sepharose beads. After several washes, ATP-interacting proteins were eluted and separated on 2-D polyacrylamide gels. Deferentially expressed proteins were cut and identified using matrix-assisted laser desorption/ionization/time-of-flight and Mascot database. Identified proteins were classified according to their biological process using protein analysis through evolutionary relationships (PANTHER) Web site. KEY FINDING: In this work, we showed that several key proteins involved in biological processes such as antioxidant system, oxidative stress, apoptosis, and metabolism were differentially expressed after subacute exposure to DZN.

Identification of possible cytotoxicity mechanism of polyethylenimine by proteomics analysis.

Polyethylenimine (PEI) is a polycation widely used for successful gene delivery both in vitro and in vivo experiments. However, different studies showed that PEI could be cytotoxic to transfected cells, and the mechanism of toxicity is poorly understood. Identification of PEI-interacting proteins may help in understanding the toxicity pathways. In this study, we investigated proteins that could interact with PEI in human colorectal adenocarcinoma cells (HT29). In order to identify the proteins interacting with PEI, PEI was immobilized to sepharose beads as solid matrix. The HT29 cell lysate were passed through the matrix. PEI-bound proteins were isolated, and further separation was performed by two-dimensional gel electrophoresis. After gel digestion, proteins were identified by matrix-assisted laser desorption/ionization-time-of-flight (TOF)/TOF mass spectrometry. Our data indicated that most of the identified PEI-interacting proteins such as shock proteins, glutathione-S-transferases, and protein disulfide isomerase are involved in apoptosis process in cells. Thus, although this is a preliminary experiment implicating the involvement of some proteins in PEI cytotoxicity, it could partly explain the mechanism of PEI cytotoxicity in cells.

Evaluation of Protein Ubiquitylation in Heart Tissue of Rats Exposed to Diazinon (an Organophosphate Insecticide) and Crocin (an Active Saffron Ingredient): Role of HIF-1α.

INTRODUCTION: Organophosphate compounds, such as diazinon (DZN), are widely used in agriculture and can lead to formation of reactive oxygen species (ROS) in cardiovascular system. ROS are highly toxic since they can cause serious changes in proteins including ubiquitylation. Crocin (a carotenoid isolated from saffron), has protective effects against DZN cardiotoxicity. In this study level of total protein ubiquitylation as markers of oxidative stress and level of ubiquitin-HIF-1α and P53, known substrates of ubiquitylation, in rat hearts exposed to DZN and crocin were evaluated. METHODS: Rats were divided into 7 groups: corn oil (control), DZN (15 mg/kg/day, gavage), crocin (12.5, 25, 50 mg/kg/day, i. p.) plus DZN, vitamin E (200 IU/kg, i. p., 3 days a week) plus DZN and crocin (50 mg/kg/day, i. p.). Treatments were continued for 4 weeks. Total protein ubiquitylation, total HIF-1α and P53 were analyzed by western blotting. Total HIF-1α and P53 were purified by immunoprecipitation (IP) and ubiquitin- HIF-1α and P53 were analyzed by western blotting. RESULTS: Higher protein ubiquitylation levels were observed in DZN treated rats. Decrease in ubiquitin-HIF-1α was also shown, and leads to higher HIF-1α protein levels in DZN group. Crocin (50 mg/kg) and vit. E protected cells against DZN protein ubiquitylation. Significant differences were not observed between the ubiquitin - P53 and total P53 protein levels. CONCLUSION: Our results showed that ubiquitylation could be considered as a marker of oxidative stress in rats exposed to DZN. Increase in level of HIF-α may compensate adverse effect of DNZ in rat heart.

Antidepressant Effect of Crocus sativus Aqueous Extract and its Effect on CREB, BDNF, and VGF Transcript and Protein Levels in Rat Hippocampus.

Crocus sativus L., commonly known as saffron, is a perennial stemless herb in Iridaceae family. It has been used in traditional medicine as well as in modern pharmacological studies for variety of conditions including depression. Recent studies have suggested brain-derived neurotrophic factor (BDNF), VGF Neuropeptide, Cyclic-AMP Response Element Binding Protein (CREB) and phospho-CREB (p-CREB) may play roles in depression. In this research the molecular mechanism of antidepressant effect of aqueous extract of saffron and its effect on the levels of BDNF, VGF, CREB and p-CREB in rat hippocampus, were investigated. The aqueous extract of saffron (40, 80 and 160 mg/kg/day) and imipramine 10 mg/kg/day were injected intraperitoneally (i.p.) for 21 days to rats. The FST (forced swimming test) was performed on the days 1(st) and 21(st). The protein expression and transcript levels of BDNF, VGF CREB and phospho-CREB in rat hippocampus, were evaluated using western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results of FST showed that saffron reduced the immobility time. The protein levels of BDNF, CREB and p-CREB were significantly increased in saffron treated rats. VGF protein expression was also increased, but not significantly. The transcript levels of BDNF significantly increased. No significant changes in CREB and VGF transcript levels were observed. It was concluded that aqueous extract of saffron has antidepressant effects and the mechanism of its antidepressant effect may be due to increasing the levels of BDNF, VGF, CREB and P-CREB in rat hippocampus.

Conformational switch of insulin-binding aptamer into G-quadruplex induced by K⁺ and Na⁺: an experimental and theoretical approach.

Guanine-rich sequences can form the G-quadruplex structure in the presence of specific metal ions. Here, circular dichroism, UV-vis absorption, fluorescence, and molecular dynamics simulation studies revealed that insulin-binding aptamer (IBA) could form an intramolecular G-quadruplex structure after binding K(+). Circular dichroism (CD) spectra demonstrated that IBA could fold into a parallel G-quadruplex with a strong positive peak at 263 nm. Analysis of equilibrium titration data revealed that cation binding was cooperative with the Hill coefficient of 2.01 in K(+) and 1.90 in Na(+). Thermal denaturation assays indicated that K(+)-induced G-quadruplex is more stable than Na(+)-induced structure. Folding of IBA into G-quadruplex leading to the contact quenching occurs as a result of the formation of a nonfluorescent complex between donor and acceptor. Based on fluorescence quenching of IBA folding, a potassium-sensing aptasensor in the range of 0-1.4 mM was proposed. Since the quenching process was predominantly static, the binding constant and the number of binding sites were determined. In this research, based on the experimental data, the initial model of IBA G-quadruplex was constructed by molecular modeling method. The modeling structure of IBA is an intramolecular parallel-strand quadruplex conformation with two guanine tetrads. The extended molecular dynamics simulation for the model indicated that the G-quadruplex maintains its structure very well in aqueous solution in presence of K(+) in the central cavity. In contrast, it was demonstrated that the G-quadruplex structure of model in the water collapses in absence of this cation.

Crocin improves lipid dysregulation in subacute diazinon exposure through ERK1/2 pathway in rat liver.

INTRODUCTION: Diazinon Yis one of the most broadly used organophosphorus insecticides in agriculture. It has been shown that exposure to diazinon may interfere with lipid metabolism. Moreover, the hypolipidemic effect of crocin has been established. Earlier studies revealed the major role of Extracellular signal-regulated kinase (ERK) pathways in low-density lipoprotein receptor (LDLr) expression. The aim of this study was to evaluate changes in the regulation of lipid metabolism, ERK and LDLr expression in the liver of rats exposed to subacute diazinon. Furthermore ameliorating effect of crocin on diazinon induced disturbed cholesterol homeostasis was studied. METHODS: 24 Rats were divided into 4 groups and received following treatments for 4 weeks; Corn oil (control), diazinon (15mg/kg per day, orally) and crocin (12.5 and 25mg/kg per day, intraperitoneally) in combination with diazinon (15 mg/kg). The levels of cholesterol, triglyceride and LDL in blood of rats were analyzed. Moreover mRNA levels of LDLr and ERK1/2 as well as protein levels of total and activated forms of ERK1/2 in rat liver were evaluated by Western blotting and quantitative real time polymerase chain reaction analysis. RESULTS: Our data showed that subacute exposure to diazinon significantly increased concentrations of cholesterol, triglyceride and LDL. Moreover diazinon decreased ERK1/2 protein phosphorylation and LDLr transcript. Crocin reduced inhibition of ERK activation and diazinon-induced hyperlipemia and increased levels of LDLr transcript. CONCLUSIONS: Crocin may be considered as a novel protective agent in diazinon-induced hyperlipemia through modulating of ERK pathway and increase of LDLr expression.