Screening and identification of DNA nucleic acid aptamers against F1 protein of Yersinia pestis using SELEX method.

BACKGROUND: Yersinia pestis is a bacterium that causes the disease plague. It has caused the deaths of many people throughout history. The bacterium possesses several virulence factors (pPla, pFra, and PYV). PFra plasmid encodes fraction 1 (F1) capsular antigen. F1 protein protects the bacterium against host immune cells through phagocytosis process. This protein is specific for Y. pestis. Many diagnostic techniques are based on molecular and serological detection and quantification of F1 protein in different food and clinical samples. Aptamers are small nucleic acid sequences that can act as specific ligands for many targets.This study, aimed to isolate the high-affinity ssDNA aptamers against F1 protein. METHODS AND RESULTS: In this study, SELEX was used as the main strategy in screening aptamers. Moreover, enzyme-linked aptamer sorbent assay (ELASA) and surface plasmon resonance (SPR) were used to determine the affinity and specificity of obtained aptamers to F1 protein. The analysis showed that among the obtained aptamers, the three aptamers of Yer 21, Yer 24, and Yer 25 were selected with a KD value of 1.344E - 7, 2.004E - 8, and 1.68E - 8 M, respectively. The limit of detection (LoD) was found to be 0.05, 0.076, and 0.033 µg/ml for Yer 21, Yer 24, and Yer 25, respectively. CONCLUSION: This study demonstrated that the synthesized aptamers could serve as effective tools for detecting and analyzing the F1 protein, indicating their potential value in future diagnostic applications.

The crosstalk between trace elements with DNA damage response, repair, and oxidative stress in cancer.

DNA damage response (DDR) is a regulatory system responsible for maintaining genome integrity and stability, which can sense and transduce DNA damage signals. The severity of damage appears to determine DDRs, which can include damage repair, cell-cycle arrest, and apoptosis. Furthermore, defective components in DNA damage and repair machinery are an underlying cause for the development and progression of various types of cancers. Increasing evidence indicates that there is an association between trace elements and DDR/repair mechanisms. In fact, trace elements seem to affect mediators of DDR. Besides, it has been revealed that oxidative stress (OS) and trace elements are associated with cancer development. In this review, we discuss the role of some critical trace elements in the risk of cancer. In addition, we provide a brief introduction on DDR and OS in cancer. Finally, we will further review the interactions between some important trace elements including selenium, zinc, chromium, cadmium, and arsenic, and DDR, and OS in cancer.

Determining the effects of green chemistry synthesized Ag-nisin nanoparticle on macrophage cells.

Bacteriocins are low molecular weight substances produced through post transcriptional changes. These molecules are easily degraded in mammalian gut by proteolytic enzymes especially protease. Nisin is a peptide with 34 aa and its structure contains a pentacyclic lanthionine and 4 beta metyllanthionine residues. Different formulations have been designed for nisin. Since "green synthesis" is a progressive method to prepare anti-microbial and anti-cancer compounds, this study aimed at green synthesis of nisin metal compounds to be used lower concentration still exerting nisin effects. For this purpose, a 1 mg/ml nisin solution was added to a 1 mM silver nitrate solution and incubated to synthesis nano Ag-nisin, then the optical density of new solution was detected using UV spectroscopy. To determine biomolecules in the Ag-nisin solution, the FTIR method was employed. The size and morphology of Ag-nisin was measured by TEM. The toxicity, inflammatory cytokines production, and intracellular ROS quantity was evaluated using MTT, ELISA and flow-cytometry. XRD pattern indicated the silver crystals in Ag-nisin solution. In addition, FTRI findings showed that the carbonyl groups of amino acid are potently able to bind to metal nanoparticles, cover, and prevent them from particle agglomeration. Treating macrophage cells with 10, 25, 50 and 100 µg/ml of Ag-nisin had no significant effect on the cell viability and intracellular ROS quantity compared to the control group. In addition, different concentrations of Ag-nisin had no effect on the IL-10 and TNF-α levels but caused an increased level of IL-12 in comparison with the control group. In the current study, for the first time, green synthesize was used to prepare Ag-nisin particles. The synthesized nanoparticle is able to induce inflammatory activity via increasing IL-12 without any change in the TNF-α level in macrophage cells.

The inhibitory impacts of Lactobacillus rhamnosus GG-derived extracellular vesicles on the growth of hepatic cancer cells.

Bacterial extracellular vesicles (EVs) have come forth into notice as possible important agent to mediate host-pathogen interactions. In this scientific research, the authors have tried to find out the effect of EVs derived from Lactobacillus rhamnosus GG (LDEVs) on the apoptosis induction in HepG2 cell line. The EVs were purified from the conditioned medium of Lactobacillus rhamnosus GG using ultrafiltration and confirmed by transmission electron microscopy (TEM). The HepG2 cells were treated with different concentrations of purified LDEVs and the cytotoxicity and their effects on the expression of bcl-2 and bax genes were assessed by the MTT assay and semi-quantitative RT-PCR, respectively. The MTT assay showed that only 100 µg/ml of LDEVs had a significant cytotoxic effect on cancer cells (p < 0.05). The apoptotic index (bax/bcl2 expression ratio) was significantly increased after treating with 50 and 100 µg/ml LDEVs (p < 0.05). Increased bax/bcl-2 ratio was led to cancer cell death.

Cellular and molecular mechanisms of acute exposure to sulfur mustard: a systematic review.

CONTEXT: Mustard gas (e.g. sulfur mustard (SM)) has been used as a chemical agent in several battles and is still a potential worldwide menace. Besides local absorption, particularly in the skin, eyes and lungs, systemic spread of the agent also has detrimental effects on gonads, bone marrow and nervous system. Moreover, chronic exposure of SM to respiratory system causes death. Inducing oxidative stress, and disturbing DNA and tissue repair systems, inflammation and cell death signaling pathways have been introduced as molecular mechanisms of the injury. METHODS: In this systematic review, more than 1200 (2000-2014) articles focusing on gross or molecular pathological reports in the acute phase of the respiratory injury after SM exposure were reviewed, followed by two different layers of gross and molecular pathological data (clinic and laboratory) integrated together in a spatio-temporal order. Role of epithelial, neutrophil and macrophage cells and three signaling pathways of inflammation, oxidative stress and cell death are covered in details. RESULTS AND CONCLUSION: Our results propose a critical role of interleukin-17 producing cells in acute and chronic inflammatory responses.

High prevalence of extensively drug-resistant and metallo beta-lactamase-producing clinical Acinetobacter baumannii in Iran.

Acinetobacter species particularly Acinetobacter baumannii (A. baumannii) have been widely reported as broad-spectrum antibiotic resistant pathogens. Expression of various types of metallo beta-lactamases (MBL), classified as Ambler class B, has been associated with carbapenem resistance. Here, we attempted to assess the frequency of extensively drug-resistant (XDR) and MBL-producing A. baumannii among clinical isolates. 86 clinical A. baumannii strains were collected from 2014 to 2015 and their susceptibility to meropenem (10 µg), imipenem (10 µg), azteronem (30 µg), pipracillin (100 µg) tazobactam (110 µg), tobramycin (10 µg), fosfomycin (200 µg), rifampicin (5 µg), colistin (10 µg), tigecycline (15 µg), sulbactam/ampicillin (10 µg + 10 µg) and polymixin B (300 U) was evaluated using disk diffusion method. The MBL-producing isolates were screened using combined disc diffusion method. Furthermore, the presence of blaVIM, blaIMP, blaSPM, blaGIM, blaSIM and blaNDM was detected by PCR. 34.9% of isolates were recovered from bronchoalveolar lavage (BAL). 81 (94.2%) and 62 (71.2%) isolates were multidrug resistance (MDR) and XDR, respectively. 44 (51.2%) and 65 (75.6%) isolates were MBL-producing strains with resistance to imipenem and meropenem, respectively. 2 (2.3%), 13 (15.1%), 2 (2.3%), 4 (4.7%) and 2 (2.3%) isolates carried blaVIM, blaIMP, blaSPM, blaGIM and blaSIM genes, respectively. Our data showed that the rate of XDR and MBL A. baumannii is on the rise.

Staphylococcal enterotoxin B/texosomes as a candidate for breast cancer immunotherapy.

A new recombinant construct made up of two components, texosomes (TEX) and staphylococcal enterotoxin B (SEB), showed cytostatic properties against several types of tumor cells in vitro. Here, we aimed to assess the construct's antitumor immunogenicity in a murine tumor model. SEB was anchored onto purified texosomes and was used for immunization of mice before challenge with 4T1 cells. Tumor size, survival time, necrosis, metastasis rate, and the levels of IL-2, IL-4, IL-17, IL-12, TNF-α, and IFN-γ were measured. Immunization of the mice with TEX-SEB increased the stimulation index of splenocytes significantly compared with the PBS-treated mice (p < 0.01). In addition, there was a significant increase of TNF-α, IL-2, and IFN-γ secreted from isolated splenocytes of the mice immunized by either TEX-SEB, TEX + SEB, TEX, or SEB in comparison with PBS (p < 0.001, p < 0.001, and p < 0.05, respectively), whereas no significant change of IL-4 secretion was observed in any treated groups. Finding from tumor tissue homogenate testing showed that the level of IL-17 and IFN-γ among mice immunized with TEX-SEB was significantly lower than PBS-treated group (p < 0.05). IL-12, IL-4, and TNF-α levels were not significantly different from PBS- and TEX-SEB-immunized groups except in the SEB-immunized mice. Although TEX-SEB immunization relatively prolonged the survival of the mice, it had no inhibitory impact on tumor size. Pathologic manifestations showed the significant rise of necrosis after immunization with TEX-SEB compared to PBS (p < 0.01). Overall, our findings suggest that the presence of SEB rescues tumorigenesis effects of TEX making the construct an appropriate candidate for tumor immunotherapy.

Comparative transcriptional and translational analysis of heme oxygenase expression in response to sulfur mustard.

Sulfur mustard (SM) is a potent alkylating agent which reacts with nucleophilic groups on DNA, RNA and proteins. It is capable of inducing cellular toxicity and oxidative stress via production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The accumulation of high amounts of the reactive species causes harmful effects such as DNA damage, lipid peroxidation, protein oxidation, inflammation and apoptosis. Although SM (also known as mustard gas) and its derivatives are rapidly removed from the body, long-term damages are much more serious than the short-term effects and may be correlated with the subsequent changes occurred on the genome. In order to defend against oxidative properties of this toxic molecule, cells trigger several anti-oxidant pathways through up-regulating the corresponding genes. Enzymes like heme oxygenase-1, superoxide dismutase and glutathione-S-transferase are the examples of such genes. These enzymes produce anti-oxidant substances that are able to scavenge the reactive species, alleviate their noxious effects and protect the cells. Following SM gas exposure, gene transcription (mRNA levels) of these enzymes are ramped up to help detoxify the cells. Yet, some studies have reported that the up-regulated transcription does not necessarily translate into higher protein expression levels. The exact reason why this phenomenon happens is not clear. Creation of mutations in the genome sequence may lead to protein structure changes. Phosphorylation or other post-translational alterations of proteins upon SM exposure are also considered as possible causes. In addition, alterations in some microRNAs responsible for regulating post-translation events may inhibit the expression of the anti-oxidant proteins in the poisoned cells at translational level.

The effects of sulfur mustard on expression of TGF-ßs variants in lung epithelial cell line.

Sulfur mustard (SM) is a blister-forming agent and can cause damages in various momentous human organs. Previous studies have demonstrated that chemical and mechanical injuries of epithelial cells cause to give rise the secretion of TGF-ß1 and TGF-ß2. These cytokines play a key role in respiratory remodeling due to SM. In this study, we investigated the impact of SM on the expression level of TGF-ß isoforms and their receptors in vitro using reverse transcriptase polymerase chain reaction and western blotting. Our finding revealed the significant increase at concentrations of 25 µl/ml SM for 30 min and 60 min and also 100 µl/ml for 60 min for TGF-ß1, 25, 50 and 100 µl/ml SM for 30 min for TGF-ßr1 and after exposing with 100 µl/ml SM for both 30 and 60 min for TGF-ß2 (p < 0.05). Data from western blotting showed the increase of TGF-ß1 expression at the level of protein as the same pattern as the mRNA level. In vitro short-time exposure of fibroblast to SM can induce the expression of TGF-ß1, TGF-ß2 and TGF-ßR1 denoting that over-expression of TGF-ß isoforms and their receptors leads to differentiation and collagen production, causing in airway remodeling and fibrosis.

Staphylococcal entorotoxin B anchored exosome induces apoptosis in negative esterogen receptor breast cancer cells.

Exosomes (EXO) are acellular vehicles used for cancer immunotherapy due to their immune inducing properties. To identify whether designed structure based on tumoral EXO have a cytotoxic effect together with a potent immunological property, we synthesized a novel structure based on EXO and staphylococcal entrotoxin B (SEB), two immune inducer substances, and surveyed its cytostatic effect on the breast cancer cell line. EXO were purified from tumor cells and SEB was anchored on it by protein transfer method. To determine the cytotoxic and apoptosis inducing effect of this structure, treated cells with different concentrations of EXO/SEB were examined by MTT assay and Hoechst staining method. In addition, the expression rate of bcl-2, bax, bak, fas, bcl-xl and the activity of caspase-3 and caspase-9 were assessed. We observed that EXO/SEB significantly decreased the cell proliferation and stimulated apoptosis (P < 0.001) at all concentration after 24 h (P < 0.001). Furthermore, EXO/SEB raised the expression rate of bax and bak (P < 0.001) but no impact on fas and bcl-xl after 48 h. We observed reducing effect of EXO/SEB on the mRNA expression of bcl-2. After 24 h of exposing the cell with the EXO/SEB, a significant increase was found in the activity of caspase at the concentration of 2.5, 5 and 10 µg/100 µl for caspase-9 and at all concentrations for caspase-3 (P < 0.001). Our designed structure, the EXO/SEB, is a novel model for apopto-immunotherapy being able to induce apoptosis in ER(-) breast cancer cells.

Exosome/staphylococcal enterotoxin B, an anti tumor compound against pancreatic cancer.

PURPOSE: Exosomes (EXOs) are acellular vehicles used for cancer immunotherapy due to their immune-inducing properties. We synthesized a novel structure based on EXOs and staphylococcal enterotoxin B (SEB) and surveyed its cytostatic effect on a pancreatic cell line. METHODS: EXOs were purified from tumor cells and SEB was anchored on it by protein transfer method. To determine the cytotoxic and apoptosis-inducing effect of this structure, treated cells with different concentrations of EXO/SEB were examined by MTT assay and Hoechst staining method. In addition, the expression rate of bcl-2, bax, bak, fas, bcl-xl and the activity of caspase-3 and caspase-9 were assessed. RESULTS: We observed that 0.5 and 2.5 µg/100µl of EXO/ SEB significantly (p<0.001) stimulated apoptosis after 24 hrs. The concentrations of 0.5 and 2.5µg/100µl of EXO/SEB raised the expression rate of bax, bak, fas (p<0.001) but had no impact on bcl-2 and bcl-xl after 48 hrs. Furthermore, it was shown that 0.5, 2.5 and 5 µg/100µl of EXO/SEB only increased the activity of caspase-3 after 48 hrs (p<0.001). CONCLUSION: Our designed structure, the EXO/SEB, is a novel model being able to induce apoptosis.

Exercise Training and Probiotic Lacticaseibacillus rhamnosus GG Reduce Tetracycline-Induced Liver Oxidative Stress and Inflammation in Rats with Hepatic Steatosis.

Lifestyle modification with regular exercise can improve metabolic diseases by reducing lipid profile and inflammation. Probiotics have been recently recommended not only for gastrointestinal diseases but also for metabolic and even degenerative diseases. Therefore, in the present study, the effect of high-intensity interval training (HIIT) and Lacticaseibacillus rhamnosus strain GG (LGG) as a probiotic on tetracycline-induced hepatic steatosis in an animal model was evaluated. Eighty male Wistar rats were randomly divided into eight groups (n = 10 in each group): control, LGG, HIIT, LGG + HIIT, tetracycline-induced (TTC), TTC + LGG, TTC + HIIT, and TTC + LGG + HIIT. The rats are treated by intraperitoneal injection (IP) with 140 mg kg-1 tetracycline, an antibiotic previously known to induce steatosis. The exercise training groups performed HIIT 5 days/week for 5 weeks, and 107 CFU/ml of Lacticaseibacillus rhamnosus GG was gavaged for the LGG groups 5 days/week for 5 weeks. Fatty droplets in the hepatocyte were considered with Oil Red staining. TTC-receiving rats have more lipid accumulation and larger lipid droplets in the liver compared to healthy animals. The two-way ANOVA showed that the interaction of LGG and HIIT significantly decreased LDL, cholesterol, and triglyceride in the healthy rats (p < 0.05). In TTC-receiving rats, the interaction of LGG and HIIT significantly increased HDL and SOD and significantly decreased triglyceride, ALP, AST, and ALT (p < 0.05). The consumption of probiotic LGG, along with HIIT with control of lipid profile and liver enzymes and improvement of the oxidative capacity, neutralizes the damage of TTC to liver tissue. Therefore, this protocol can be recommended for people with hepatic steatosis.

The Anti-Adhesion Effect of Nisin as a Robust Lantibiotic on the Colorectal Cancer Cells.

Background: Bacteriocins are a type of antimicrobial peptide that are produced by probiotics. They have been studied as possible therapeutic drugs and have been used to suppress bacterial development in foods. Nisin is a potent bacteriocin having the anti-microbial and anti-cancer characteristics produced by Lactococcus lactis. The aim of the present paper is to evaluate the influence of Nisin on cell adhesion and its two related genes, mmp-2 and mmp-9, in the colorectal cancer cell line. Materials and Methods: For this purpose, HT-29 cells were treated with various concentrations of Nisin and the cell cytotoxicity, cell adhesion, and gene expression were evaluated using the MTT assay, cell adhesion assay, and real-time PCR. Results: Our findings showed that 32 to 1024 µg/ml of Nisin resulted in a significant reduction in cell viability (P < 0.05). Furthermore, 128 and 256 µg/ml of Nisin significantly reduced the cell adhesion, and mmp-2 and mmp-9 gene expressions (P < 0.05). Conclusion: Our findings suggested that Nisin could prevent metastasis and cancer progression.

Screening and Identification of DNA Nanostructure Aptamer Using the SELEX Method for Detection of Epsilon Toxin.

Background: Epsilon toxin (ETX), produced by Clostridium perfringens, is one of the most potent toxins known, with a lethal potency approaching that of botulinum neurotoxins. Epsilon toxin is responsible for enteritis. Therefore, the development of rapid and simple methods to detect ETX is imperative. Aptamers are single-stranded oligonucleotides that can bind tightly to specific target molecules with an affinity comparable to that of monoclonal antibodies (mAbs). DNA aptamers can serve as tools for the molecular identification of organisms, such as pathogen subspecies. Objectives: This study aimed to isolate high-affinity single-stranded DNA (ssDNA) aptamers against ETX. Methods: This study identified aptamers using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, enzyme-linked apta-sorbent assay (ELASA), and surface plasmon resonance (SPR) to determine the affinity and specificity of the newly obtained aptamers targeting ETX. Results: Several aptamers obtained through the SELEX process were studied. Among them, 2 aptamers, ETX clone 3 (ETX3; dissociation constant (Kd = 8.4 ± 2.4E-9M) and ETX11 (Kd = 6.3 ± 1.3E-9M) had favorable specificity for ETX. The limits of detection were 0.21 and 0.08 µg/mL for ETX3 and ETX11, respectively. . Conclusions: The discovered aptamers can be used in various aptamer-based rapid diagnostic tests for the detection of ETX.

Evaluation of anti-biofilm activity of Lactobacillus rhamnosus GG and Nisin on the expression of aap, ica-A and ica-D as biofilm-associated genes of Staphylococcus epidermidis.

Background and Objectives: In the present study, the anti-biofilm activity of Lactobacillus rhamnosus GG and Nisin was investigated on biofilm-forming abilities of Staphylococcus epidermidis strains and the expression of the biofilm-associated genes. Materials and Methods: In this study, the standard strain of L. rhamnosus GG (ATCC 53103) and Nisin were used to assess their anti-microbial and anti-biofilm effects on S. epidermidis (RP62A). Results: The MIC and MBC analysis showed that Nisin at 256 µg/mL and 512 µg/mL, and L. rhamnosus GG at 1×107 CFU/mL and 1×108 CFU/mL have anti-microbial activity compared to the negative control respectively. L. rhamnosus GG bacteria and Nisin inhibited the biofilm formation of S. epidermidis based on optical density of at 570 nm (P <0.001). The relative mRNA expression of aap, icaA, and icaD genes was significantly reduced compared to the negative control after treating S. epidermidis with sub-MIC of Nisin (0.44, 0.25 and 0.6 fold, respectively) (P>0.05). In addition, the relative expression of aap and icaA genes, but not icaD (P>0.05), was significantly lower than the negative control (0.62 and 0.7 fold, respectively) (P>0.05), after exposure to the sub MIC of L. rhamnosus GG. Conclusion: Nisin and L. rhamnosus GG exhibit potent activity against biofilm-forming abilities of S. epidermidis and these agents could be utilized as an anti-biofilm agents against S. epidermidis infections.

Effect of extracellular vesicles of Lactobacillus rhamnosus GG on the expression of CEA gene and protein released by colorectal cancer cells.

Background and Objectives: Almost all living cells secret nano-sized structures enclosed by the lipid bilayer called extracellular vesicles (EVs) into their extracellular milieu. These EVs play important roles in several physiological processes as a cargo delivery system. In probiotics, EVs are the main communication tool with the host. The present study aimed to assess the effect of EVs originated from Lactobacillus rhamnosus GG on the Carcinoembryonic antigen (cea) gene expression and protein (CEA) synthesis in the SW480 and HT-29 cell lines. Materials and Methods: Different concentrations of Lactobacillus rhamnosus GG EVs were applied on the SW480 and HT-29 cell lines. The MTT assay, Real-Time PCR, and ELISA analysis methods were exploited to explore the cell viability and the expression level of the cea gene in comparison with the ß-actin gene as the control. Results: The two concentrations of 80 and 100 µg/ml of Lactobacillus rhamnosus GG EVs considerably affected the anti-proliferation and increased the amount of both CEA mRNA and protein (p < 0.05). Conclusion: Our findings showed that EVs of Lactobacillus rhamnosus GG could induce the gene expression and protein synthesis of CEA. Also, they reduced the cell proliferation of HT29 and SW480. Thus, probiotics such as EVs of Lactobacillus rhamnosus GG could be useful for preventing colorectal cancer.

Targeting the EGFR in cancer cells by fusion protein consisting of arazyme and third loop of TGF-alpha: an in silico study.

The anticancer effects of arazyme, a bacterial metalloprotease, have been revealed in previous studies. Because of the overexpression of epidermal growth factor receptor (EGFR) in tumor cells, targeting this receptor is one of the approaches to cancer therapy. In the present study, we designed fusion protein by using a non-mitogenic binding sequence of TGFα, arazyme, and a suitable linker. The I-TASSER and Robetta web servers were employed to predict the territory structures of the Arazyme-linker-TGFαL3, and TGFαL3-linker-Arazyme. Then, models were validated by using PROCHECK, ERRAT, ProSA, and MolProbity web servers. After docking to EGFR, Arazyme-linker-TGFαL3 showed a higher binding affinity and was selected to be optimized through 100 ns Molecular dynamic (MD) simulation. Next, the stability of ligand-bound receptor was examined utilizing MD simulation for 100 ns. Furthermore, the binding free energy calculation and free energy decomposition were carried out employing MM-PBSA and MM-GBSA methods, respectively. The root mean square deviation and fluctuation (RMSD, RMSF), the radius of gyration, H-bond, and binding free energy analysis revealed the stability of the complex during simulation time. Finally, linear and conformational epitopes of B cells, as well as MHC class I and MHC class II were predicted by using different web servers. Meanwhile, the potential B cell and T cell epitopes were identified throughout arazyme protein sequence. Collectively, this study suggests a novel chimera protein candidate prevent cancer cells potentially by inducing an immune response and inhibiting cell proliferation. Communicated by Ramaswamy H. Sarma.

Positive Association of Matrix Proteins Alteration with TAZ and The Progression of High-Grade Bladder Cancer.

OBJECTIVE: Bladder cancer is the 9th leading cause of human urologic malignancy and the 13th cause of death worldwide. Increased collagen cross-linking, NIDOGEN1 expression and consequently stiffness of extracellular matrix (ECM) may be responsible for the mechanotransduction and regulation of transcriptional co-activator with PDZ-binding motif (TAZ) and transforming growth factor ß1 (TGF-ß1) signaling pathways, resulting in progression of tumorigenesis. The present study aimed to assess whether type 1 collagen expression is associated with TAZ nuclear localization. MATERIALS AND METHODS: In this case-control study, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemical analysis were performed to evaluate the activation of the TAZ pathway in patients with bladder cancer (n=40) and healthy individuals (n=20). The ELISA method was also conducted to measure the serum concentrations of TGF-ß1. Masson's trichrome staining was carried out to histologically evaluate the density of type 1 collagen. RESULTS: Our findings that the expression levels of COL1A1, COL1A2, NIDOGEN1, TAZ, and TGF-ß1 genes were overexpressed in patients with bladder cancer, and their expression levels were positively associated with the grade of bladder cancer. The immunohistochemical analysis demonstrated that the nuclear localization of TAZ was markedly correlated with high-grade bladder cancer. We also found that TAZ nuclear localization was substantially higher in cancerous tissues as compared with normal bladder tissues. Masson's trichrome staining showed that the tissue density of type I collagen was considerably increased in patients with bladder cancer as compared with healthy subjects. CONCLUSION: According to our findings, it seems the alterations in the expression of type I collagen and NIDOGEN1, as well as TAZ nuclear localization influence the progression of bladder cancer. The significance of TGF-ß1 and TAZ expression in tumorigenesis and progression to high-grade bladder cancer was also highlighted. However, a possible relationship between TGF-ß1 expression and the Hippo pathway needs further investigations.

ARA-linker-TGFαL3: a novel chimera protein to target breast cancer cells.

Targeted cancer therapies based on overexpressed receptors and the fractions containing immunotoxins and bacterial metabolites are one of the well-known methods to overcome the chemotherapy resistance of cancer cells. In this paper, we designed ARA-linker-TGFαL3, using Arazyme, a Serratia proteamaculans metabolite, and a third loop segment of TGFα to target EGFR-expressing breast cancer cells. After cloning in pET28a (+), the expression of recombinant protein was optimized in Escherichia coli strain BL21 (DE3). MDA-MB-468 (EGFR positive) and MDA-MB-453 (EGFR negative) breast cancer cell lines were employed. Also, the chemotherapeutic drug, Taxotere (Docetaxel), was employed to compare cytotoxicity effects. Cell ELISA assessed the binding affinity of recombinant proteins to the receptor, and the cytotoxicity was detected by MTT and lactate dehydrogenase release assays. The interfacing with cancer cell adhesion was evaluated. Furthermore, the induction of apoptosis was examined utilizing flow cytometric analysis, and caspase-3 activity assay. Moreover, RT-PCR was conducted to study the expression of apoptosis (bax, bcl2, and casp3), angiogenesis (vegfr2), and metastasis (mmp2 and mmp9) genes. ARA-linker-TGFαL3 revealed a higher binding affinity, cytotoxicity, and early apoptosis induction in MDA-MB-468 cells compared to the effects of Arazyme while both recombinant proteins showed similar effects on MDA-MB-453. In addition, the Taxotere caused the highest cytotoxicity on cancer cells through induction of late apoptosis. Meanwhile, the expression of angiogenesis and metastasis genes was decreased in both cell lines after treatment with either ARA-linker-TGFαL3 or Arazyme. Our in vitro results indicated the therapeutic effect of ARA-linker-TGFαL3 on breast cancer cells.

A new scfv-based recombinant immunotoxin against EPHA2-overexpressing breast cancer cells; High in vitro anti-cancer potency.

Immunotoxin therapy is one of the immunotherapy strategies providing a new, effective and high potency treatment against various cancers. Breast cancer is the most common cancer among women in many countries. The EPH receptors are a large part of tyrosine kinase receptors family and play an effective role in tumor development and angiogenesis. Among EPH receptors, EPHA2 is more commonly well-known and widely expressed in many cancers like breast cancer. In this study, we evaluated the specification of a designed immunotoxin formed by EPHA2-specific scfv linked with PE38KDEL on EPHA2-overexpressing breast cancer cell line. This new scfv-based recombinant immunotoxin was studied in terms of features such as binding potency, cytotoxicity effects, apoptosis induction ability, and internalization. The flow cytometry results showed that the immunotoxin can significantly (approximately 99%) bind to EPHA2-overexpressing breast cancer cell line (MDA-MB-231) in a low concentration (2.5 ng/ul) while cannot significantly bind to the normal cell line (HEK-293) or even EPHA2-very low expressing cell line (MCF-7). Using the MTT assay and Annexin V/Propidium iodide (PI) double staining method by flow cytometry, we observed significant killing and apoptosis induction of the MDA-MB-231 cells at different concentrations. Immunotoxin tracking by confocal microscopy at 2 h and 6 h revealed a massive presence of immunotoxin in the cytoplasm. Finally, given the in vitro results, it seems that this immunotoxin is competent enough to serve as a good candidate for in vivo studies to further explore the possibility of breast cancer treatment.