The effect of Nrf2 deletion on the proteomic signature in a human colorectal cancer cell line.

BACKGROUND: Colorectal cancer is one of the most common cancer and the third leading cause of death worldwide. Increased generation of reactive oxygen species (ROS) is observed in many types of cancer cells. Several studies have reported that an increase in ROS production could affect the expression of proteins involved in ROS-scavenging, detoxification and drug resistance. Nuclear factor erythroid 2 related factor 2 (Nrf2) is a known transcription factor for cellular response to oxidative stress. Several researches exhibited that Nrf2 could exert multiple functions and expected to be a promising therapeutic target in many cancers. Here, Nrf2 was knocked down in colorectal cancer cell line HT29 and changes that occurred in signaling pathways and survival mechanisms were evaluated. METHODS: The influence of chemotherapy drugs (doxorubicin and cisplatin), metastasis and cell viability were investigated. To explore the association between specific pathways and viability in HT29-Nrf2-, proteomic analysis, realtime PCR and western blotting were performed. RESULTS: In the absence of Nrf2 (Nrf2-), ROS scavenging and detoxification potential were dramatically faded and the HT29-Nrf2- cells became more susceptible to drugs. However, a severe decrease in viability was not observed. Bioinformatic analysis of proteomic data revealed that in Nrf2- cells, proteins involved in detoxification processes, respiratory electron transport chain and mitochondrial-related compartment were down regulated. Furthermore, proteins related to MAPKs, JNK and FOXO pathways were up regulated that possibly helped to overcome the detrimental effect of excessive ROS production. CONCLUSIONS: Our results revealed MAPKs, JNK and FOXO pathways connections in reducing the deleterious effect of Nrf2 deficiency, which can be considered in cancer therapy.

Autophagy stimulation delayed biological aging and decreased cardiac differentiation in rabbit mesenchymal stem cells.

Introduction: Cardiovascular disease (CVD) is a type of disease that affects the function of cardiac-vascular tissues. This study aimed to consider the possible effects of autophagy, as an intrinsic catabolic pathway of cells, on the differentiation and aging process of mesenchymal stem cells (MSCs). Methods: In this study, bone marrow-derived MSCs were obtained from rabbit bone marrow aspirates. The stemness feature was confirmed by using flow cytometry analysis Cells at passage three were treated with 50 µM Metformin and 15µM hydroxychloroquine (HCQ) for 72 hours. The intracellular accumulation of autophagolysosomes was imaged using LysoTracker staining. Protein levels of autophagy (LC3II/I ratio), aging (Klotho, PARP-1, and Sirt-1) effectors, and cardiomyocyte-like phenotype (α-actinin) were studied by western blotting. Results: Based on our findings, flow cytometry analysis showed that the obtained cells expressed CD44 and CD133 strongly, and CD31 and CD34 dimly, showing a typical characteristic of MSCs. Our data confirmed an increased LC3II/I ratio in the metformin-received group compared to the untreated and HCQ-treated cells (P < 0.05). Besides, we showed that the incubation of rabbit MSCs with HCQ increased cellular aging by induction of PARP-1 while Metformin increased rejuvenating factor Sirt-1 comparing with the normal group (P < 0.05). Western blotting data showed that the autophagy stimulation response in rabbit MSCs postponed the biological aging and decreased the differentiation potential to the cardiac cells by diminishing α-actinin comparing with control cells (P < 0.05). Conclusion: In summary, for the informants in this study, it could be noted that autophagy inhibition/stimulation could alter rabbit MSCs aging and differentiation capacity.

Pathophysiological Effects of Sulfur Mustard on Skin and its Current Treatments: Possible Application of Phytochemicals.

BACKGROUND: Sulfur-(SM) and nitrogen (NM)-based mustards are the mutagenic incapacitating compounds which are widely used in vesicating the chemical warfare and cause toxicity in many organs, especially skin. SM, as a potent vesicating agent, contributes to the destruction of skin in dermis and epidermis layers. The progression of the lesion depends on the concentration of SM and the duration of exposure. Body responses start with pruritus, erythema, edema and xerosis, which lead to the accumulation of immune cells in the target sites and recruitment of mast cells and paracrine-mediated activity. Pro-inflammatory effectors are accumulated in the epidermis, hair follicles, and sebaceous glands resulting in the destruction of the basement membrane beneath the epidermis. There is still no satisfactory countermeasure against SM-induced lesions in clinical therapy, and the symptomatic or supportive treatments are routine management approaches. OBJECTIVE: The current review highlights the recent progression of herbal medicines application in SM-induced injuries through the illustrative examples and also demonstrates their efficacies, properties and mechanism of actions as therapeutic agents. CONCLUSION: Phytochemicals and herbal extracts with anti-bacterial, anti-inflammatory and antioxidant properties have been recently shown to hold therapeutic promise against the SM-induced cutaneous complications. The present review discusses the possible application of herbal medicines in the healing of SM-induced injuries.

The relationship between ANT1 and NFL with autophagy and mitophagy markers in patients with multiple sclerosis.

Multiple sclerosis (MS) is the most common inflammatory neurodegenerative disease. Neurofilament light chain (NFL) is a novel adverting biomarker of axonal damage that suggested as a useful assistant in the monitoring of MS patients. It has been shown that the auto/mitophagy associated with MS pathogenesis. In this study, we aimed to study correlation between ATG5 and Parkin, as markers of autophagy and mitophagy respectively, with NFL and ANT1 in serum and cerebrospinal fluid (CSF) in MS subjects. ATG5, Parkin, NFL, and ANT1 levels were measured in a cross-sectional study of 40 MS patients compared with gender, age and BMI matching healthy volunteers. Based on our results, levels of ATG5, Parkin, and NFL significantly were elevated in both serum and CSF of MS patients comparing control individuals (p < 0.0001) but ANT1 levels significantly was decreased in both serum and CSF of MS patients comparing control individuals (p < 0.0001). The correlation indices between NFL, ANTI1, ATG5 and Parkin in both case and control groups showed a direct and moderate the correlation between ANTI1 and ATG5 in the CSF level of the control group (r = 0.554, P = 0.011). Our data support the feasibility of quantifying of NFL as a sensitive and clinically meaningful serum/CSF biomarker to follow-up nerve tissue injury in MS condition.

Current status of cardiac regenerative medicine; An update on point of view to cell therapy application.

Cardiovascular diseases (CVDs) are the leading cause of death globally. Because of the economic and social burden of acute myocardial infarction and its chronic consequences in surviving patients, understanding the pathophysiology of myocardial infarction injury is a major priority for cardiovascular research. MI is defined as cardiomyocytes death caused by an ischemic that resulted from the apoptosis, necrosis, necroptosis, and autophagy. The phases of normal repair following MI including inflammatory, proliferation, and maturation. Normal repair is slow and inefficient generally so that other treatments are required. Because of difficulties, outcomes, and backwashes of traditional therapies including coronary artery bypass grafting, balloon angioplasty, heart transplantation, and artificial heart operations, the novel strategy in the treatment of MI, cell therapy, was newly emerged. In cell therapy, a new population of cells has created that substitute with damaged cells. Different types of stem cell and progenitor cells have been shown to improve cardiac function through various mechanisms, including the formation of new myocytes, endothelial cells, and vascular smooth muscle cells. Bone marrow- and/or adipose tissue-derived mesenchymal stem cells, embryonic stem cells, autologous skeletal myoblasts, induced pluripotent stem cells, endothelial progenitor cells, cardiac progenitor cells and cardiac pericytes considered as a source for cell therapy. In this study, we focused on the point of view of the cell sources.

Distinct Effects of Royal Jelly on Human Endothelial Cells Under High Glucose Condition.

To assess different effects of royal Jelly in protecting the human endothelial cells from high glucose level, human umbilical vein endothelial cells were exposed to various concentrations of royal jelly, from 0.625 to 10 mg/mL, at the presence of 5 and 30 mM glucose contents over a course of 72 h. In addition to cell viability assessment by conventional MTT assay, we also analyzed the feature of stemness by expression of Sox-2 and CD133 factors. Moreover, fatty acid profile, the expression of autophagy-related factor, namely microtubule-associated protein light chain 3 and activity of metalloproteinase 2 and 9 and were investigated. Royal jelly supplementation at the concentrations lower than 2.5 mg/mL did not influence the survival rate of cells and partially blunted the cytotoxic effects of 30 mM glucose. The expression of CD133 and Sox-2 factors were increased by royal jelly alone. Interestingly, an up-regulated expression of Sox-2 (58.8 ± 4%) coincided with a reduction in the levels of CD133 (15.1 ± 8.3%) in the combined treatment. We notified that the contents of palmitate and trans-palmitate as well as linoleate decreased by 30 mM glucose content while cis-palmitate levels increased when RJ returned them to near-normal levels (p < 0.05). The expression of autophagy marker was prominently induced in the presence of royal jelly in both conditions (p < 0.05). The glucose-induced activity of metalloproteinases was also reduced. Royal jelly is able to attenuate the abnormal status of 30 mM glucose condition in endothelial cells by different mechanisms.

Chronic Exposure of Human Endothelial Progenitor Cells to Diabetic Condition Abolished the Regulated Kinetics Activity of Exosomes.

By virtue of lifestyle change, incidence of diabetes mellitus type 2 is increasingly being raised with different up-surging pathologies. It was showed that endothelial progenitor cells (EPCs) were disqualified in neo-angiogenesis induction. Besides, to an aborted differentiation property, malfunctioned paracrine activities worsen off vascular abnormality. Nano-scaled exosomes play essential roles in reciprocal cell-cell crosstalk via bioactive molecules. To address the effect of diabetic serum on exosome secretion capacity, EPCs were exposed to diabetic condition for seven days. In addition to in-vitro tubulogenesis, migration and LDL uptake assessment, exosome release capacity, and expression profiles of three genes participating in exosome kinetics, including CD63, Alix and Rab27a, revealed by Real-time PCR method. Data showed diabetic sera not only abolished the in-vitro tubulogenesis, migration and LDL uptake properties but also decreased exosome release and expression of related genes. This study sheds lights on the adverse effect of diabetic condition on exosome kinetics in EPCs.

Telomerase activity and telomere on stem progeny senescence.

The end of linear chromosomes is formed of a special nucleoprotein heterochromatin structure with repetitive TTAGGG sequences called telomere. Telomere length is regulated by a special enzyme called telomerase, a specific DNA polymerase that adds new telomeric sequences to the chromosome ends. Telomerase consists of two parts; the central protein part and the accessory part which is a RNA component transported by the central part. Regulation of telomere length by this enzyme is a multi-stage process. Telomere length elongation is strongly influenced by the level of telomerase and has a strong correlation with the activity of telomerase enzyme. Human Telomerase Reverse Transcriptase (hTERT) gene expression plays an important role in maintaining telomere length and high proliferative property of cells. Except a low activity of telomerase enzyme in hematopoietic and few types of stem cells, most of somatic cells didn't showed telomerase activity. Moreover, cytokines are secretory proteins that control many aspects of hematopoiesis, especially immune responses and inflammation. Also, the induction of hTERT gene expression by cytokines is organized through the PI3K/AKT and NF/kB signaling pathways. In this review we have tried to talk about effects of immune cell cytokines on telomerase expression/telomere length and the induction of telomerase expression by cytokines.

Copper sulfate pentahydrate reduced epithelial cytotoxicity induced by lipopolysaccharide from enterogenic bacteria.

The over usage of multiple antibiotics contributes to the emergence of a whole range of antibiotic-resistant strains of bacteria causing enterogenic infections in poultry science. Therefore, finding an appropriate alternative natural substance carrying an antibacterial capacity would be immensely beneficial. It has been previously discovered that the different types of cupric salts, especially copper sulfate pentahydrate (CuSO4·5H2O), to carry a potent bactericidal capacity. We investigated the neutralizing effect of CuSO4·5H2O (6.25µg/ml) on the reactive oxygen species generation, and expression of MyD88, an essential adaptor protein of Toll-like receptor, and NF-κB in three intestinal epithelial cell lines exposed to 50ng/ml lipopolysaccharide. In order to find the optimal cupric sulfate concentration without enteritis-inducing toxicity, broiler chickens were initially fed with water containing 0.4, 0.5, and 1mg/l during a period of 4days. After determination of appropriate dosage, two broiler chickens and turkey flocks with enteritis were fed with cupric compound for 4days. We found that cupric sulfate can lessen the cytotoxic effect of lipopolysaccharide by reducing the reactive oxygen species content (p<0.05). Additionally, the expression of MyD88 and NF-κB was remarkably down-regulated in the presence of lipopolysaccharide and cupric sulfate. The copper sulfate in doses lower than 0.4mg/ml expressed no cytotoxic effect on the liver, kidney, and the intestinal tract while a concentration of 0.5 and 1mg/ml contributed to a moderate to severe tissue injuries. Pearson Chi-Square analysis revealed the copper cation significantly diminished the rate of mortality during 4-day feeding of broiler chicken and turkey with enteritis (p=0.000). Thus, the results briefed above all confirm the potent anti-bactericidal feature of cupric sulfate during the course of enteritis.

Endothelial cells' biophysical, biochemical, and chromosomal aberrancies in high-glucose condition within the diabetic range.

To date, many studies have been conducted to find out the underlying mechanisms of hyperglycemia-induced complications in diabetes mellitus, attributed to the cellular pathologies of different cells-especially endothelial cells. However, there are still many ambiguities and unresolved issues to be clarified. Here, we investigated the alteration in biophysical and biochemical properties in human umbilical vein endothelial cells exposed to a high-glucose concentration (30mM), comparable to glucose content in type 2 diabetes mellitus, over a course of 120 hours. In addition to a reduction in the rate of cell viability and induction of oxidative stress orchestrated by the high-glucose condition, the dynamic of the fatty acid profile-including polyunsaturated, monounsaturated, and saturated fatty acids-was also altered in favor of saturated fatty acids. Genetic imbalances were also detected at chromosomal level in the cells exposed to the abnormal concentration of glucose after 120 hours. Moreover, the number of tip cells (CD31+ /CD34+ ) and in vitro tubulogenesis capability negatively diminished in comparison to parallel control groups. We found that diabetic hyperglycemia was associated with a decrease in the cell-cell tight junction and upregulation in vascular endothelial cadherin and zonula occludens (ZO)-1 molecules after 72 and 120 hours of exposure to the abnormal glucose concentration, which resulted in a profound reduction in transendothelial electrical resistance. The surface plasmon resonance analysis of the human umbilical vein endothelial cells immobilized on gold-coated sensor chips confirmed the loosening of the cell to cell intercellular junction as well as stable attachment of each cell to the basal surface. Our findings highlighted the disturbing effects of a diabetic hyperglycemia on either biochemical or biophysical properties of endothelial cells.

Type 2 Diabetes Inhibited Human Mesenchymal Stem Cells Angiogenic Response by Over-Activity of the Autophagic Pathway.

The current study aimed to address the impact of serum from type 2 diabetes patients on the angiogenic properties of human bone marrow mesenchymal stem cells and its relationship to autophagy signaling. Human primary stem cells were enriched and incubated with serum from diabetic and normal subjects for 7 days. Compared to data from the control group, diabetic serum was found to induce a higher cellular death rate (P < 0.001) and apoptotic changes (P < 0.01). We also showed that diabetic condition significantly abolished angiogenesis tube formation on Matrigel substrate, decreased cell chemotaxis (P < 0.01) in response to SDF-1α, and inhibited endothelial differentiation rate (P < 0.0001). Western blotting showed autophagic status by high levels of P62 (P < 0.0001), beclin-1 (P < 0.0001), and increase in LC3II/I ratio (P < 0.001). In vivo Matrigel plug assay revealed that supernatant conditioned media prepared from cells exposed to diabetic serum caused a marked reduction in the recruitment of VE-cadherin- (P < 0.01) and α-SMA-positive (P < 0.0001) cells 7 days after subcutaneous injection. PCR expression array analysis confirmed the overexpression of autophagy and apoptosis genes in cultured cells in response to a diabetic condition (P < 0.05). Using bioinformatic analysis, we noted a crosstalk network between DM2, angiogenesis, and autophagy signaling. DM2 could potently modulate angiogenesis by the interaction of IL-1ß with downstream insulin receptor and upstream androgen receptor. Corroborating to data, diabetic serum led to abnormal regulation of P62 during the angiogenic response. These data demonstrate that diabetic serum decreased human mesenchymal stem cell angiogenic properties directly on angiogenesis pathways or by the induction of autophagy signaling. J. Cell. Biochem. 118: 1518-1530, 2017. © 2016 Wiley Periodicals, Inc.

A reversal of age-dependent proliferative capacity of endothelial progenitor cells from different species origin in in vitro condition.

Introduction: A large number of cardiovascular disorders and abnormalities, notably accelerated vascular deficiencies could be related to aging changes and increased length of life. During the past decades, the discovery of different stem cells facilitates ongoing attempts for attenuating many disorders, especially in vascular beds. Endothelial progenitor cells (EPCs) are a subtype of stem cells that have potent capacity to differentiate into mature endothelial cells (ECs). However, some documented studies reported an age-related decline in proliferation and function of many stem cells. There is no data on aging effect upon proliferation and morphological feature of EPCs. Methods: To show aging effect on EPCs proliferation and multipotentiality, bone marrow samples were provided from old and young cases in three different species; human, mouse and dog. After 7 days of culture, the cell morphology and clonogenic capacity were evaluated. We also calculated the mean number of colonies both in bone marrow samples from old and young subjects. To confirm the cell phenotype, isolated cells were immune-phenotyped by a panel of antibodies against Tie-2, CD133 and CD309 markers. Results: Our results showed that EPCs exhibited prominent spindle form in all bone marrow samples from young cases while the cell shape became more round by aging. Notably, the number of colonies was reduced in aged samples as compared to parallel young subject samples (P < 0.05). We also detected that the expression of endothelial related markers diminished by aging. Conclusion: The results of this study suggest that the age-related vascular abnormalities could be presumably related to the decline in stemness capacity of EPCs.

Taq1B Polymorphism of Cholesteryl Ester Transfer Protein (CETP) and Its Effects on the Serum Lipid Levels in Metabolic Syndrome Patients.

The metabolic syndrome (MetS) is one of the most important risk factors for type 2 diabetes and cardiovascular disease. This syndrome is characterized by abdominal obesity, hypertension, insulin resistance, and dyslipidemia. The plasma origin of Cholesteryl ester transfer protein (CETP) is responsible for transferring cholesterol esters from high-density lipoprotein particles to apolipoprotein B containing lipoproteins compartment. We conducted this study to investigate the association between CETP gene Taq1B (rs708272) polymorphism in the metabolic syndrome among Iranian subjects. A sample size of 200 patients diagnosed with MetS together with 200 healthy donors as control were enrolled in this study. The investigation of polymorphism was performed by the use of polymerase chain reaction and restriction fragment length polymorphism analysis. To determine the relationship between polymorphism and lipid profile, we measured lipids and CETP concentration in metabolic syndrome and control subjects. Genotype distribution and allelic frequencies of polymorphism were determined and compared in both groups. Our findings showed that all clinical and biochemical characteristics in patients differed from the control group. The results showed that genotype and allele frequency of the Taq1B polymorphism was not significantly different between two groups. Instinctively, CETP was significantly higher in metabolic syndrome (1.64 ± 0.32 µg/ml) than in control (1.53 ± 0.34 µg/ml). A low level of CETP was found in blood of B2B2 typified genotype. In spite of Taq1B polymorphism on ester transfer protein concentration, no direct correlation was found between this polymorphism and metabolic syndrome.

Effects of combination of melatonin and laser irradiation on ovarian cancer cells and endothelial lineage viability.

The main goal of anti-cancer therapeutic approaches is to induce apoptosis in tumor masses but not in the normal tissues. Nevertheless, the combination of photodynamic irradiation with complementary oncostatic agents contributes to better therapeutic performance. Here, we applied two different cell lines; SKOV3 ovarian carcinoma cells and HUVECs umbilical cord cells as in vitro models to pinpoint whether pharmacological concentration of melatonin in combination with photodynamic therapy induces cell cytotoxicity. The cells were separately treated with various concentrations of melatonin (0 to 10 mM) and photodynamic irradiation alone or in combination. Cells were preliminary exposed to increasing concentrations of melatonin for 24 h and subsequently underwent laser irradiation for 60 s with an output power of 80 mW in continuous mode at 675 nm wavelength and a total light dose of 13.22 J/cm2. Cell viability, apoptosis/necrosis rates, and reactive oxygen species levels as well as heat shock protein 70 expression were monitored after single and combined treatments. A statistical analysis was performed by applying one-way analysis of variance (ANOVA) and post hoc Tukey's test. Combination treatment of both cell lines caused a marked increase in apoptosis/necrosis rate, reactive oxygen species generation, and heat shock protein 70 expression compared to incubation of the cells with each agent alone (p < 0.05). SKOV3 cancer cells expressed higher level of heat shock protein 70 under experimental procedure as compared to HUVECs (p < 0.05). Our results introduce melatonin as a potent stimulus for enhancing the efficacy of laser on induction of apoptosis in tumor cells.

Potent anti-angiogenic and cytotoxic effect of conferone on human colorectal adenocarcinoma HT-29 cells.

BACKGROUND: Cancer is one of the leading causes of death worldwide, both in developed and developing countries. Of note, colorectal adenoma encompasses a high rate of gastrointestinal-associated cancer death in human being. Today, different strategies, including surgery approaches, photodynamic therapy, radiation and particularly natural compounds have been extensively used to manage tumor behavior in human body. METHODS: The objective of the present study was to elucidate the multilateral effects of conferone on HT-29 cell lines. In addition to cell cytotoxicity, the extent of lipid peroxidation, MDA formation, catalase, superoxide dismutase and intracellular ROS levels, as markers of oxidative stress, were also studied. P-glycoprotein-mediated cellular efflux effectiveness, anti-angiogenic and finally anti-migratory capacities of conferone-exposed HT-29 cells were monitored over a course of 72 h. RESULTS: It was found that, conferone mediated cell proliferation arrest and induced cell death through both apoptosis and necrosis phenomena. HT-29 cells, exposed to 20 µM conferone, under gone oxidative stress and total content of reactive oxygen species was increased in a time-dependent manner. Intracellular accumulation of rhodamine 123 and cell's swelling under iso- and hypo-osmotic conditions could be related to P-glycoprotein incorrect performance in the presence of conferone. A significant reduction in CD31 positive cells population and in vitro tubulogenesis of endothelial cells was also observed after incubation with conditioned medium collected from 72 h conferone-treated HT-29 cells. Conferone also precluded angiogenesis capability of treated HT-29 cells through an altered secretome profile, including vascular endothelial growth factor, Angiopoietin-1 and -2 factors. In addition to anti-angiogenic properties of conferone, a profound decrease in migration capability of HT-29 cells was also evident.