Exosomes isolated from metabolically unhealthy normal weight and overweight phenotypes deteriorate the ER/PR positive breast cancer behavior.

Purpose: Obesity has been linked to a higher risk of postmenopausal breast cancer Yet, research indicates an opposite correlation between obesity and premenopausal breast cancer risk. Various obesity phenotypes based on metabolic health could play a significant part. This study aims to assess how plasma exosomes taken from women with varying obesity phenotypes impact MCF-7 cell migration, matrix metalloproteinase-2 activity, and apoptosis. Methods: The characterization of isolated exosomes and their internalization into MCF-7 cells was evaluated. The treatment of MCF-7 cells with exosomes isolated from different groups was done. Migration, the activity of MMP-2, mRNA expression of Bax and Bcl-2, protein expression of p-53 and Thr55 p-p53, and apoptosis were assessed. Results: Isolated exosomes from unhealthy obese individuals increase MCF-7 cell migration. Regarding MMP activities, unhealthy normal weight and overweight and healthy obese groups isolated exosomes increase the MMP-2 activity than the treated group with exosomes isolated from counterpart groups. Furthermore, unhealthy normal weight and overweight and healthy obese obtained exosomes decrease apoptosis compared to counterpart groups. Conclusion: Altogether, plasma exosomes derived from both unhealthy individuals with normal weight and overweight status, as well as those with unhealthy obesity, negatively impacted the behavior of estrogen/progesterone receptor-positive breast cancer cells. Supplementary Information: The online version contains supplementary material available at 10.1007/s40200-023-01295-1.

The obeticholic acid can positively regulate the cancerous behavior of MCF7 breast cancer cell line.

BACKGROUND: The diagnosis and treatment processes of cancer are among the main challenges of medical science in recent decades. The use of different therapeutic agents is one of the most common methods frequently utilized for cancer treatment. Accumulating evidence points to a potential effect of Obeticholic acid (OCA), a specific ligand for farnesoid X receptor, on the regulation of cancer-associated pathways. In spite of tremendous efforts to introduce OCA into the clinical setting, there is a great deal of uncertainty about its impact on breast cancer treatment. This study was performed to evaluate the effects of OCA on breast cancer. METHODS AND RESULTS: In this experiment, the MCF-7 (Michigan Cancer Foundation-7) cell line was treated with 0.1 µM OCA, and cancerous characteristics of the MCF-7 cell line was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide) assay, gelatin zymography, western blot, Real-time PCR, flow cytometry, and ELISA techniques. The results indicated that OCA increased the rate of apoptosis and the expression levels of PPARα (Peroxisome proliferator-activated receptor alpha) and TIMP-1 (tissue inhibitor of metalloproteinase-1) genes in this cell line, while it reduced the mRNA levels of MMP7 (matrix metalloproteinase 7) and Bcl-2 (B-cell lymphoma 2) genes, as well as the protein levels of the active form of AKT (protein kinase B), Erk1/2 (extracellular signal-regulated kinase 1/2) and STAT3 (Signal transducers and activators of transcription-3). Also, OCA decreased the activity of MMP9, while it increased the secretion of VEGF-A (vascular endothelial growth factor-A). CONCLUSIONS: It seems that OCA can exert anti-cancer effects on the MCF-7 cells by reducing growth, proliferation, migration, invasion, and regulation of the expression of genes involved in cancer-associated pathways. However, it should be noted that further studies are warranted to establish this concept, especially the increase of VEGF-A can be considered a challenge for the results of this study.

Metabolically healthy/unhealthy obesity and breast cancer: A possible role of plasma-derived extracellular vesicles on the cancerous behavior of triple-negative breast cancer.

PURPOSE: Obesity has known detrimental effects on breast cancer (BC) development and progression. However, it's essential to consider the obesity phenotype based on metabolic health. This study aims to evaluate the impact of circulating extracellular vesicles (EVs) from women with metabolically healthy or unhealthy normal weight, overweight, and obesity on MDA-MB-231 cell migration, invasion, and apoptosis. METHODS: Plasma EVs were isolated from different obesity phenotypes in women. EVs were characterized and EVs uptake by MDA-MB-231 cells was assessed. MDA-MB-231 cell lines were treated with EVs obtained from various studied groups, and migration, invasion, MMP-2 and MMP-9 activity, Bax and Bcl-2 mRNA expression, p-53 and Thr55 p-p53 protein expression, and apoptosis were assessed. RESULTS: EVs from obese individuals, regardless of phenotype, increased invasion and MMP-2 activity compared to healthy normal-weight EVs. Normal-weight EVs led to higher invasion under unhealthy conditions. BC cell migration was enhanced by EVs from healthy obese individuals compared to healthy normal-weight EVs. EVs from unhealthy obese women exhibited significantly lower p53/p-p53 levels and reduced apoptosis compared to healthy obese groups. CONCLUSION: It appears that EVs from both normal-weight women with unhealthy conditions and those with obesity or overweight, irrespective of metabolic status, worsened the cancerous behavior of TNBC cells. Therefore, considering metabolic health, in addition to BMI, is crucial for understanding obesity-related disorders.

Apoptotic effects of human amniotic fluid mesenchymal stem cells conditioned medium on human MCF-7 breast cancer cell line.

Introduction: Breast cancer, as the most common malignancy among women, is shown to have a high mortality rate and resistance to chemotherapy. Research has shown the possible inhibitory role of Mesenchymal stem cells in curing cancer. Thus, the present work used human amniotic fluid mesenchymal stem cell-conditioned medium (hAFMSCs-CM) as an apoptotic reagent on the human MCF-7 breast cancer cell line. Methods: Conditioned medium (CM) was prepared from hAFMSCs. After treating MCF-7 cells with CM, a number of analytical procedures (MTT, real-time PCR, western blot, and flow cytometry) were recruited to evaluate the cell viability, Bax and Bcl-2 gene expression, P53 protein expression, and apoptosis, respectively. Human fibroblast cells (Hu02) were used as the negative control. In addition, an integrated approach to meta-analysis was performed. Results: The MCF-7 cells' viability was decreased significantly after 24 hours (P < 0.0001) and 72 hours (P < 0.05) of treatment. Compared with the control cells, Bax gene's mRNA expression increased and Bcl-2's mRNA expression decreased considerably after treating for 24 hours with 80% hAFMSCs-CM (P = 0.0012, P < 0.0001, respectively); an increasing pattern in P53 protein expression could also be observed. The flow cytometry analysis indicated apoptosis. Results from literature mining and the integrated meta-analysis showed that hAFMSCs-CM is able to activate a molecular network where Bcl2 downregulation stands in harmony with the upregulation of P53, EIF5A, DDB2, and Bax, leading to the activation of apoptosis. Conclusion: Our finding demonstrated that hAFMSCs-CM presents apoptotic effect on MCF-7 cells; therefore, the application of hAFMSCs-CM, as a therapeutic reagent, can suppress breast cancer cells' viabilities and induce apoptosis.

The Ability of Antibody against Truncated Matrix Metalloproteinase 9 Peptide to Evaluate the Native Protein on Tear Drops of Dry Eye Disease Patients by a Point-of-Care Diagnostic System.

Purpose: To obtain a reactive and specific antibody against truncated matrix metalloproteinase 9 (MMP-9), that has reactivity toward the native protein. Precision, accuracy, specificity, and sensitivity were evaluated using a point-of-care test. Methods: An in silico study was used to confirm the anti peptide truncated MMP-9 is against native MMP-9. After an antibody titer assessment, purification, and characterization, the anti MMP-9 was assessed. The cut-off value was determined using the purified gelatinases of the supernatant HCT 116 cell line. The supernatant was purified by preparative native-polyacrylamide gel electrophoresis based on charge and size of the proteins. Furthermore, quality control (QC) of the results were performed following standard densitometry methods. Results: A truncated MMP-9 is the major epitope peptide that can trigger the immune system to scavenge for a specific and reactive antibody against the native MMP-9. The MMP-9 native protein is purified from the supernatant of the HCT 116 cell line and the commercially available, full-length MMP-9. The cut-off value was estimated at 30 µg/mL. QC results indicated that the specificity was 80%, sensitivity was 96.7%, accuracy was 91%, and precision was 91.66%. The area under curve was 0.827 (P < 0.001). The positive predictive value was 83%, and the negative predictive value was 96%. Conclusions: The antibody against the synthetic epitope peptide can detect the native MMP-9. Native MMP-9 is considered the main biomarker in an immunoassay POCT and is used to diagnose dry eye disease (DED). In accordance with QC results, MMP-9 point of care test can be utilized for screening patients suffering from DED.

A theoretical strategy for acceleration of human immune response against SARS-CoV-2: a fusion protein harboring virus-binding and pre-exposed antigen domains.

Coronavirus disease 2019 (COVID-19) is a respiratory disease that outbreaks since December 2019 and spread globally. Various methods have been used to treat SARS-CoV-2 that is generally based on the information obtained from the therapeutic approaches used for SARS-COV and MERS patients. In this article, we introduce a theoretical strategy in which a two-domain fusion protein presents the virus to the immune system. This fusion protein contains a viral-binding domain such as the ACE2 domain and a domain such as the hepatitis B antigen that has previously been exposed to the immune system. This two-domain fusion protein, could be called "virus-presenting fusion protein", would attach to the virus spike protein via the ACE2 domain while the hepatitis B antigen would be bound by anti-hepatitis B antibodies facilitating the opsonization and presentation of the virus to the immune system. We believe that this virus-presenting fusion protein will accelerate the immune response to the SARS-CoV-2 virus.

Paper based analytical devices for blood grouping: a comprehensive review.

The clinical importance of blood group (BG) antigens is related to their ability to induce immune antibodies that can cause hemolysis. Yet, ABO and D (Rh) are still considered to be the key antigens for healthy blood transfusion and secondary antigens are the next priority. Serological typing is the most widely used typing method. Rapid and accurate blood grouping plays an important role in some clinical conditions, rather than conventional techniques. Hence, developing a simple and economical model for rapid blood grouping would facilitate these tests. In recent decades, paper-based microfluidics such as µPADs has gained much interest in wide application areas such as point-of-care diagnostic. In this study, we evaluated µPADs that are performed for blood grouping and its recent progress. A comprehensive literature search was performed using databases including PUBMED, SCOPUS, Web of Science and Google Scholar. Keywords were blood grouping or typing, paper analytical device, rapid test, etc. After investigation of search results, 16 papers from 2010 to 2020 were included. Further information in detail was classified in Table 1. Generally, two principles for blood typing µPADs are introduced. The lateral chromatographic flow method and the vertical flow-through method that detects BG in a visual-based manner. To detect results with acceptable clarity many factors and challenges like paper, blood sample, buffer, Ab and RBC interaction and also µPADs stability need to be considered, which are discussed. In conclusion, the simplicity, stability, cheapness, portability and biocompatibility of µPADs for blood grouping confirming its utility and also they have the capability to robust, universal blood-grouping platform. Table 1 Summary of blood grouping tests using paper-based analytical devices Antigens Type of diagnosis Validation method Sample No Accuracy Action time Paper type Stability Sample dilution Buffer Ref A, B, Rh Forward volunteers records 5 - - Whatman No. 4 - 1/2 PBS* (Khan et al. 2010) A, B, Rh Forward gel assay test and conventional slide test 100 100% 1 min Whatman No. 4 and Kleeenex paper towel 7 Days in 4 °C 1/1 NSS (Al-Tamimi et al. 2012) A, B, Rh Forward gel card assay 99 100% 20 Sec + Washing Kleeenex paper towel - 1/1 NSS (Li et al. 2012) A, B, Rh Forward - - - - Kleeenex paper towel - 45/100 PSS (Li et al. 2013) A, B, Rh Forward gel card assay 98 100% 1.5 min Kleeenex paper towel - 85/100 PBS (Guan et al. 2014b) C, E, c, e, K, Jka, Jkb, M, N, S, P1, and Lea Forward gel card assay 266 100% - Kleeenex paper towel - 1/1 NSS (Li et al. 2014b) A, B, Rh Forward and Reverse conventional slide test 96 ≈ 91% 10 min Whatman No. 1 21 Days in 4 °C 1/2 NSS (Noiphung et al. 2015) C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S and s, P1, Lea and Leb Forward - 478 - - Kleeenex paper towel - 1/1 NSS, PBS (Then et al. 2015) A, B Forward and Reverse conventional slide test 76 100% 5-8 min Whatman No. 4 38 Days in 4 °C 1/4, 1/1 NSS (Songjaroen and Laiwattanapaisal 2016) D, K Forward volunteers records 210 - 7.5 min Kleenex paper towel - 1/1 NSS (Yeow et al. 2016) A, B, c, e, D, C, E, M, N, S, s, P1, Jka, Jkb, Lea, Leb, Fya, and Fyb Forward and Reverse gel card assay 3550 ≈100% 30 s Fiber glass and cotton linter 180 Days in 25 °C 45/100, 1/1 PBS (Zhang et al. 2017) A, B Forward conventional slide test 598 100% 3 min Whatman No. 113 14 Day in 4 °C 1/1 NSS (Songjaroen et al. 2018) A, B, Rh Forward conventional slide test - - 30 Sec + Washing Unrefined sisal paper - 1/2 NSS (Casals-Terré et al. 2019) A, B, Rh Forward - - - - Whatman No.1 - 1/1 NSS (Ansari et al. 2020) ABO & Rh Forward and Reverse conventional slide test - 100% Unrefined Eucalyptus papers - 1/2 NSS, PBS (Casals-Terré et al. 2020) A, B, Rh Forward - - - 30 Sec + Washing Whatman No. 4 modified with chitosan ≥ 100 days in 25 °C 1/1 NSS (Parween et al. 2020) *phosphate buffer saline, normal saline solution.

Evaluating the effect of a mixture of two main conjugated linoleic acid isomers on hepatic steatosis in HepG2 cellular model.

Hepatic steatosis is an early form of non-alcoholic fatty liver disease (NAFLD), caused by abnormal fat deposition in the hepatocytes. Conjugated linoleic acid (CLA) is a group of positional and geometric dienoic isomers of linoleic acid that attract significant attention because of its beneficial effects on chronic diseases such as cancer, obesity, and metabolic syndrome. This study examined the influence of a mixture of two main CLA isomers (CLA-mix) on lipid accumulation and lipid metabolism-related genes using HepG2 cells treated with palmitic acid (PA) as an in vitro model for hepatic steatosis. Methods and Results: HepG2 cells were treated for 24 h: control (BSA), model (BSA + PA), and treated groups (BSA-PA + non-toxic concentrations of CLA-mix). Intracellular lipid deposition, triglyceride (TG), total cholesterol (TC) and gene expression were measured by Oil-Red O staining, colorimetric assay kits and real-time PCR, respectively. CLA-mix at high concentrations had significantly decreased intracellular total lipid and TG deposition compared to the model group. However, none of the CLA-mix concentrations had a significant effect on the intracellular TC level. CLA-mix significantly increased the expression of some genes mainly regulated by PPARα but did not alter the expression of lipogenesis-related genes. Conclusions: These results demonstrate that high concentrations of CLA-mix protect against hepatic steatosis and play a role in regulating fatty acid oxidation and bile excretion through the PPARα pathway. It is suggested that the effect of different ratios of two main CLA isomers on the amount and ratio of bile compounds be investigated in future studies.

Positive Correlation between Circulating Fetuin-A and Severity of Coronary Artery Disease in Men.

OBJECTIVE: Fetuin-A serves a dual function; its high levels are associated with metabolic syndrome, type 2 diabetes, obesity, insulin resistance, and nonalcoholic fatty liver disease, and on the other hand, it serves as a potent inhibitor of ectopic vascular calcification. Due to the opposing findings, the aim of the current study was to investigate serum fetuin-A levels in men with coronary artery disease (CAD). METHODS: In the case-control study, anthropometric and biochemical parameters were determined in 83 men (43 CAD patients and 40 control subjects). At last, the serum fetuin-A levels were measured using the fetuin-A human enzyme-linked immunosorbent assay (ELISA) kit. RESULTS: A significant difference was detected among the two groups for triglyceride and cholesterol levels (P=0.003 and P=0.002, respectively). The mean fetuin-A levels were determined 230.57 ± 63.76 and 286.35 ± 64.07 µg/ml for the control group and the CAD patients, respectively (P<0.001). Fetuin- A was significantly correlated to the severity of CAD (r 0.393, P<0.001) and associated with the risk of CAD in subjects (OR [CI] = 1. 144 [1.060-1. 235]; p = 0.001). A cut-off value of 237.4 µg/ml had good sensitivity (76.7%) and specificity (65.0%) for differentiating between two groups [area under curve (AUC) = 0.732 (CI=0.621-0.842); p < 0.001]. CONCLUSION: Our results indicated that fetuin-A levels were positively correlated to the severity of CAD. The findings suggest that there is a possible link between pathogenic mechanisms of atherosclerosis and fetuin-A; however, more investigations are needed in this regard.

Anti-Inflammatory Effects of Plasma Circulating Exosomes Obtained from Normal-Weight and Obese Subjects on Hepatocytes.

INTRODUCTION: Obesity is a disorder with low-grade chronic inflammation that plays a key role in hepatic inflammation and steatosis. Moreover, there are studies to support the role of exosomes in cellular communications, the regulation of metabolic homeostasis and immunomodulatory activity. Accordingly, we aimed to evaluate the influence of plasma circulating exosomes derived from females with normal-weight and obesity on the secretion of inflammatory cytokines in human liver cells. METHODS: Plasma circulating exosomes were isolated from four normal (N-Exo) and four obese (OExo) women. The exosomes were characterized and approved for CD63 expression (common exosomal protein marker) and morphology/size using the western blot and TEM methods, respectively. The exosomes were used for the stimulation of HepG2 cells in vitro. After 24 h of incubation, the protein levels of TNF-α, IL-6, and IL-1ß were measured in the culture supernatant of HepG2 cells using the ELISA kit. RESULTS: The protein levels of IL-6 and TNF-α in the cells treated with O-Exo and N-Exo reduced significantly in comparison with the control group (P=0.039 and P<0.001 respectively), while significant differences were not found between normal and obese groups (P=0.808, and P=0.978 respectively). However, no significant differences were found among the three groups in terms of IL-1ß levels (P=0.069). Based on the correlation analysis, the protein levels of IL-6 were positively correlated with TNF-α (r 0.978, P<0.001). CONCLUSION: These findings suggest that plasma circulating exosomes have probably antiinflammatory properties independent of body mass index and may decrease the secretion of inflammatory cytokines in the liver. However, further in vitro and in vivo investigations are needed to address the anti-inflammatory function of N-Exo and O-Exo in human liver cells and/or other cells.

The possible effect of silver nanoparticles on glyceraldehyde-3-phosphate dehydrogenase activity and formation of amyloid-like aggregates in MCF-7 cell line.

Silver nanoparticles (AgNPs) are widely used in medicine, however, the underlying mechanisms of their action on cellular signaling have not been completely determined, and fundamental studies are required to clarify them. We aimed to investigate AgNPs effects on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as both the internal control gene and the redox-sensitive enzyme involved in apoptosis-related pathways and the formation of amyloid aggregates. To achieve this purpose, MCF-7 cells were treated with different concentrations (0, 3, 22, and 200 µg/ml) of AgNPs and then cell viability, generation of reactive oxygen species (ROS), induction of apoptosis, expression of GAPDH gene, the formation of amyloid aggregates, and GAPDH activity were assessed. The results indicated that treatment with AgNPs significantly reduced cell viability and increased apoptosis in a dose-dependent manner. The ROS levels increased at lower concentrations of AgNPs (up to 22 µg/ml) and during short-term exposure (30 min). The level of GAPDH gene expression was significantly upregulated by 1.22, 1.47, and 1.56 fold, in the concentrations of 3, 22, and 200 µg/ml, respectively. The amount of amyloid aggregates was significantly increased in a dose-dependent manner. The results of enzyme activity showed that AgNPs were affected on the activity of GAPDH protein, however, it has fluctuated that could not be interpreted by our limited data. In conclusion, our results suggested that AgNPs could affect the GAPDH gene expression and enzyme activity, therefore the selection of GAPDH as a gene and protein internal control in the (AgNPs)-related studies requires careful consideration. Additionally, AgNPs may cause apoptosis due to the increase in the production of amyloid aggregates.

Specific cellular internalization and pH-responsive behavior of doxorubicin loaded PLGA-PEG nanoparticles targeted with anti EGFRvIII antibody.

AIM: Antibody-conjugated nanoparticles have attracted much attention in the field of cancer treatment due to the enhancement of the tumor cell response to anticancer drugs as well as reducing the side effects of chemotherapeutic agents on healthy tissues. However, most studies in this field generally mentioned the specific cellular uptake of conjugated nanoparticles. In this study, we loaded doxorubicin (DXR: as an effective antineoplastic agent) in PLGA-PEG (D,L-lactic-co-glycolic acid)-(polyethylene glycol) biocompatible polymeric nanoparticles (NPs) and then conjugated with anti-EGFRvIII antibody. The resulting nanoparticles had remarkable sensitivity to pH decrease and were capable of targeting specific cells. MATERIALS AND METHODS: To this aim, PLGA-PEG-COOH was used for the synthesis of nanoparticles and stabilized by polyvinyl alcohol (PVA) according to the nanoprecipitation method. The carboxylic groups on the surface of PLGA-PEG NPs were activated by EDC/NHS and covalently conjugated to amino groups of the monoclonal antibody. The prepared NPs were characterized by Zetasizer and transmission electron microscopy (TEM). The resulting NPs were evaluated in terms of entrapment efficiency (EE), drug loading efficiency (DLE), drug-release profile, and cell internalization. Intrinsic cytotoxicity was assessed by the MTT, apoptosis (Annexin V-PI) and cell cycle assays. KEY FINDINGS: The in vitro drug release assessment of conjugated particles (MAb-DXR-PLGA NPs) showed a slow sustained DXR release in physiological pH (7.4) values, while the initial drug release was markedly higher (the 1.9 fold) in acidic pH (6.5) ranges. The selectivity for cellular internalization of MAb-DXR-PLGA NPs into U87MG vIII cells (overexpressing EGFRvIII) in comparison with U87MG cells (lacking EGFRvIII expression) was also confirmed. The MTT assay demonstrated that the cytotoxicity of MAb-DXR-PLGA NPs against U87MG vIII cells was more pronounced when compared with BSA-DXR-PLGA NPs. The results of the MTT assay were also confirmed by apoptosis and cell cycle assays. SIGNIFICANCE: Our findings suggest that the designed anti-EGFRvIII MAb-DXR-PLGA NPs could be considered as a proper option for targeted drug delivery systems due to pH sensitivity and specific cellular internalization.

Influence of plasma circulating exosomes obtained from obese women on tumorigenesis and tamoxifen resistance in MCF-7 cells.

Obesity is associated with breast cancer aggressiveness and drug resistance. Although the underlying mechanisms are unknown, recent studies indicated that exosomes have a principal contributory role in obesity-associated metabolic complications. Hence, we investigated whether obesity can mediate breast cancer progression and resistance to tamoxifen by plasma-derived-exosomes from obese women or not. Plasma exosomes isolated from five normal-weight (N-Exo) and five obese women (O-Exo) were characterized for size, zeta potential, and CD63 expression. After the treatment of MCF-7 cells with N-Exo and O-Exo, cell proliferation, migration, invasion as well as levels of MMP-9 and MMP-2 were evaluated by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, wound healing, transwell, and zymography methods, respectively. For evaluating resistance to tamoxifen, the cell viability, apoptosis, and the p53 protein were evaluated using the MTT assay, flow cytometry, and western blot methods, respectively. Cell proliferation, migration, and invasion were significantly increased in the cells treated with O-Exo than untreated cells (p = .001, p = .018, p = .034, respectively). Levels of MMP-2 and MMP-9 were remarkably increased in the cells treated with O-Exo in comparison with ones treated with N-Exo (p = .040, p = .043, respectively). As for resistance to tamoxifen, O-Exo had significantly the greater anti-apoptotic effects in comparison with the N-Exo group (p = .013). Besides, p53 levels were significantly decreased in the cells treated with O-Exo than ones treated with N-Exo (p = .045). The cell viability was significantly more in cells treated with O-Exo in comparison with the cells only treated with tamoxifen (p = .040). Our findings demonstrated that circulating exosomes derived from obese women could lead to tumorigenesis and tamoxifen resistance in breast cancer cells. However, more studies are needed to establish this notion.

Effect of circulating exosomes derived from normal-weight and obese women on gluconeogenesis, glycogenesis, lipogenesis and secretion of FGF21 and fetuin A in HepG2 cells.

BACKGROUND: It is generally accepted that obesity can lead to metabolic disorders such as NAFLD and insulin resistance. However, the underlying mechanism has been poorly understood. Moreover, there is evidence to support the possible role of exosomes in the metabolic homeostasis regulation. Accordingly, we aimed to determine the effect of plasma circulating exosomes derived from obese and normal-weight women on insulin signaling and the secretion of hepatokines in human liver cells. METHODS: Plasma exosomes isolated from four obese (O-Exo) women and four normal-weight (N-Exo) female candidates were characterized for size, zeta potential, and CD63 protein expression and were used for stimulation of HepG2 cells. Then, cell viability, as well as levels of glycogen and triglyceride (TG), were evaluated. Levels of fetuin-A and FGF21 were measured using the ELISA kit. Expression of glucose 6-phosphatase (G6pase) and phosphoenolpyruvate carboxykinase (PEPCK) genes were determined using qRT-PCR. Western blot analysis was carried out to evaluating the phosphorylation of GSK3ß. RESULTS: The TG levels increased significantly in the cells treated with O-Exo than the control (vehicle) group (P = 0.005) and normal-weight group (P = 0.018). Levels of p-GSK3ß and glycogen were significantly reduced by O-Exo in comparison with control (P = 0.002, P = 0.018, respectively). The mRNA expression of G6pase and PEPCK enzymes increased in the cells treated with O-Exo in comparison with the vehicle group (P = 0.017, P = 0.010, respectively). The levels of FGF21 in the supernatant of cells treated with O-Exo and N-Exo were significantly lower than the control group (P = 0.007). CONCLUSION: It appears that obesity-related circulating exosomes can impair insulin signaling pathways and associated components, increase intracellular TG content, and decrease FGF21 secretion in the hepatocytes.

The Influence of Psychological Stress on the Initiation and Progression of Diabetes and Cancer.

CONTEXT: Psychological stress can be considered a risk factor for the initiation and progression of many pathological conditions, including type 1 and 2 diabetes mellitus and cancer. OBJECTIVES: The aim of this review article was to evaluate the molecular and cellular mechanisms linking psychological stress to the onset and progression of diabetes and cancer. EVIDENCE ACQUISITION: The current review was conducted to survey and analyze studies related to the effects of psychological stress on diabetes and cancer. RESULTS: Psychological stress may make individuals prone to the development of diabetes through the impairment of the hypothalamic-pituitary-adrenal (HPA) axis function, sympathetic nerves system (SNS), lipid profile, cytokines balance, renin-angiotensin system (RAS), and insulin signaling pathway. Additionally, psychological stress can contribute to the development of cancer through the perturbation in the HPA axis, SNS function, and cytokines balance. Psychological stress is also capable of decreasing the levels of oxytocin and dopamine, leading to an increased risk of cancer in susceptible individuals. CONCLUSIONS: It seems that psychological stress plays a significant role in the onset and progression of diabetes and cancer. The identification of the pathways triggered by psychological stress would open up a new avenue for the understanding of molecular mechanisms by which diabetes and cancer could be managed or even prevented.

A critical challenge in the development of antibody: Selecting the appropriate fragment of the target protein as an antigen based on various epitopes or similar structure.

The main challenge in the development of antibody is to select the appropriate antigen particularly when a truncated protein is used for immunization or as vaccine antigen. In previous studies, fragment selection was mainly based on epitopes and less often on the structure. Fewer studies have paid attention to the prediction of the truncated protein 3D structure and retained its similarity in the native and truncated proteins. Here we used in silico analysis to select two fragments of Pyruvate Kinase M2 (PKM2), as a tumor marker. One fragment, M-tPKM2, had a shorter sequence with one epitope although the predicted 3D structure was similar to the native PKM2. The other fragment, R-tPKM2, had a longer sequence and thus more epitopes, but had a different structure from the native PKM2. Recombinant truncated proteins were expressed in E. coli and purified via affinity chromatography. Secondary structure elements in purified proteins were determined by Circular Dichroism, then they were utilized to develop antibodies in mice. Both antigens could elicit high immune response against themselves (OD450 = 3.326 ± 0.562 for M-tPKM2; OD450 = 3.562 ± 0.110 for R-tPKM2). However, significantly higher response against PKM2 was observed among the mice immunized with M-tPKM2 (p < 0.0001 by One way ANOVA followed by Tukey's post hoc comparison). Also, the monoclonal antibody produced against the M-tPKM2 could recognize the native PKM2 in the MCF7 cells. Our finding suggested that for the purpose of designing an antigen with the ability to produce a potent antibody against the target protein, it is better to select sequences which have a similar structure in truncated and native proteins, even at the cost of having shorter sequences and fewer epitopes.

Production and Characterization of Monoclonal and Polyclonal Antibodies Against Truncated Recombinant Dickkopf-1 as a Candidate Biomarker.

Several studies have reported an increased serum level of Dickkopf (DKK-1) protein in a variety of cancers, including multiple myeloma, lung, colorectal, bone loss, and Alzheimer's disease. This protein has potential to be used as a biomarker for the diagnosis of some cancers, especially bone loss in multiple myeloma. In the present study, to measure the concentration level of DKK-1 protein, rabbit polyclonal antibody (pAb) and mouse monoclonal antibodies (mAbs) were produced against this protein. New Zealand white rabbits and BALB/c mice were immunized with the chimeric recombinant DKK-1 antigen. Immunized mouse spleen cells were fused with SP2/0 cells to generate anti-rDKK-1 antibody-producing hybridoma cells. Antibodies were purified by protein A affinity chromatography and assessed using sodium dodecyl sulfate polyacrylamide gel, western blotting and enzyme-linked immunosorbent assay. These results implied that the pAb and mAb were produced against the DKK-1 protein. The Kd value of 5 × 10-9 M was recorded for the mAb MR6F3 toward native DKK-1, and the Ig isotype was identified as IgG2b. No cross-reactivity was shown with DKK-2 by MR6F3. Collectively, our results revealed that the produced pAb and mAb could be used in the measurement of DKK-1 protein.

Toxicity of silver nanoparticles on different tissues of Balb/C mice.

AIM: The main purpose of the current study was to evaluate the toxicity of silver nanoparticles (Ag NPs) on adult Balb/C mice. MAIN METHODS: Twenty five Balb/C mice purchased and divided into four groups of five. Group one serves as control and injected by normal saline; group's two to four were injected by Ag NPs at 0.25, 0.50 and 1 mg/kg, respectively. KEY FINDINGS: Overall, current results indicate that all concentration of Ag NPs have potential for induction of toxicity in different tissues. Ag NPs at concentration >0.25 mg/kg result in pathological changes in liver, spleen, brain, heart, lungs, kidneys, and testicles tissues; as well as it lead to significant change in sperm quality and quantity, and blood brain barrier (BBB) permeability. Ag NPs at the lowest concentration (0.25 mg/kg) significantly changed the oxidative stress levels in serum and liver tissue but did not change the level of liver enzymes and renal markers in serum. SIGNIFICANCE: The current research results support clearly the toxic effects of Ag NPs at very low concentration and suggest that further in vivo investigation are required to be able to confirm the safety of nanoparticle derived application to use in life.

Evaluation of targeted curcumin (CUR) loaded PLGA nanoparticles for in vitro photodynamic therapy on human glioblastoma cell line.

In this study, antibody-conjugated biodegradable polymeric nanoparticles were developed to enhance the photodaynamic efficiency of curcumin (CUR) on glioblastoma tumor cells. Poly (D, l-lactic-co-glycolic acid) nanoparticles (PLGA NPs) were synthesized and stabilized by polyvinyl alcohol (PVA). Poly(ethylene-alt-maleic anhydride) (PEMA) was used to provide carboxyl groups on the surface of NPs. The CUR or FITC (fluorescein isothiocyanate) was encapsulated in PLGA NPs using the nanoprecipitation method. The carboxylic groups on the surface of the PLGA NPs were covalently conjugated to the amino groups of a monoclonal antibody against EGFRvIII (A-EGFRvIII-f). The prepared NPs were fully characterized by Zetasizer, scanning electron microscope (SEM), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR), and then entrapment efficiency (EE), drug loading efficiency (DLE), CUR release, cell internalization, intrinsic cytotoxicity, and phototoxicity were evaluated. Furthermore, the effect of monoclonal antibody (MAb) on the tyrosine phosphorylation of EGFRvIII after photodynamic therapy (PDT) was assessed. The immunoreactivity of the antibody in MAb-PLGA NPs was preserved during the process of conjugation. The selective cellular internalization of MAb-PLGA NPs (FITC or CUR loaded) into the DKMG/EGFRvIII cells (EGFRvIII overexpressed human glioblastoma cell line) in comparison with DK-MGlow (human glioblastoma cell line with low level of EGFRvIII) was also confirmed. MAb-CUR-PLGA NPs were able to show more effective photodynamic toxicity (56% vs. 24%) on the DKMG/EGFRvIII cells compared to CUR-PLGA NPs. These results suggest that the anti-EGFRvIII MAb-CUR-PLGA NPs have potential of targeted drug delivery system for PDT in the overexpressed EGFRvIII tumor cells.

Expression of miRNAs Targeting mTOR and S6K1 Genes of mTOR Signaling Pathway Including miR-96, miR-557, and miR-3182 in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer. Aberrant expression of genes in mTOR pathway and their targeting miRNAs plays an important role in TNBC. The aim of this study was to determine the expression of mTOR and S6K1 and their targeting miRNAs in breast cancer cell lines and clinical samples. miRNAs targeting 3'-UTR of mTOR and S6K1 mRNAs were predicted using bioinformatic algorithms. MDA-MB-231, MCF-7, and MCF-10A as well as 20 TNBC samples were analyzed for gene and miRNA expression using quantitative real-time PCR (RT-qPCR). A receiver operating characteristic (ROC) curve analysis was performed for evaluation of candidate miRNAs as diagnostic biomarkers. miR-96 and miR-557 targeting mTOR and S6K1 mRNAs, respectively, were selected, and miR-3182 was selected as the miRNA targeting both genes. The miRNAs were down-regulated in cell lines, while their target mRNAs were up-regulated. Similar findings were observed in clinical samples. The ROC curve analysis revealed decline in expression of these miRNAs. We suggest that miR-96, miR-557, and miR-3182 can be used as inhibitory agents for mTOR and S6K1 in TNBC-targeted therapy.