Generation of Scalable Hepatic Micro-Tissues as a Platform for Toxicological Studies.

BACKGROUND: Currently, there is an urgent need for scalable and reliable in vitro models to assess the effects of therapeutic entities on the human liver. Hepatoma cell lines, including Huh-7, show weakly resemblance to human hepatocytes, limiting their significance in toxicity studies. Co-culture of hepatic cells with non-parenchymal cells, and the presence of extracellular matrix have been shown to influence the biological behavior of hepatocytes. The aim of this study was to generate the scalable and functional hepatic micro-tissues (HMTs). METHODS: The size-controllable HMTs were generated through co-culturing of Huh-7 cells by mesenchymal stem cells and human umbilical vein endothelial cells in a composite hydrogel of liver-derived extracellular matrix and alginate, using an air-driven droplet generator. RESULTS: The generated HMTs were functional throughout a culture period of 28 days, as assessed by monitoring glycogen storage, uptake of low-density lipoprotein and indocyanine green. The HMTs also showed increased secretion levels of albumin, alpha-1-antitrypsin, and fibrinogen, and production of urea. Evaluating the expression of genes involved in hepatic-specific and drug metabolism functions indicated a significant improvement in HMTs compared to two-dimensional (2D) culture of Huh-7 cells. Moreover, in drug testing assessments, HMTs showed higher sensitivity to hepatotoxins compared to 2D cultured Huh-7 cells. Furthermore, induction and inhibition potency of cytochrome P450 enzymes confirmed that the HMTs can be used for in vitro drug screening. CONCLUSION: Overall, we developed a simple and scalable method for generation of liver micro-tissues, using Huh-7, with improved hepatic-specific functionality, which may represent a biologically relevant platform for drug studies.

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.

miR-27a and miR-214 exert opposite regulatory roles in Th17 differentiation via mediating different signaling pathways in peripheral blood CD4+ T lymphocytes of patients with relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) is one of the most prevalent autoimmune diseases, which involves the central nervous system. In this illness, Treg/Th17 cell imbalance causes the defect. Several studies revealed that T helper 17 (Th17) cells play a crucial role in pathogenesis, inflammation, and autoimmunity of several autoimmune diseases such as MS. In the present study, we assessed transcript levels of miR-27a and miR-214, in purified CD4+ T cells of MS patients, during relapsing and remitting phases in inducing differentiation of T naïve cells to Th17 cells. Forty RR-MS patient samples including those in relapsing (n=20) and remitting (n=20) phases were participated in this study. In addition, transcript levels of IL-17A, RORγt, IL-23R, Foxp3, and TGF-ß in purified CD4+ T cells of patients in relapsing and remitting phases of RRMS patients were compared to healthy controls. Expression levels of miR-27a and miR-214 were measured by RT-qPCR and compared to healthy control group (n=10). Data indicated upregulation of miR27a in relapsing phase of multiple sclerosis compared to remitting phase and healthy volunteers while miR-214 downregulated in relapsing phase of MS compared to remitting phase and healthy volunteers. In silico studies demonstrated pathways which miR-27a and miR-214 could effect on CD4+ T cell lineage fate including TGF-ß and mTOR signaling, respectively. Our data suggest that miR-27a may probably inhibit negative regulators of Th17 cell differentiation, thus promoting its differentiation while miR-214 has an adverse effect.

Thymoquinone and its therapeutic potentials.

Herbal medicine has attracted great attention in the recent years and is increasingly used as alternatives to chemical drugs. Several lines of evidence support the positive impact of medicinal plants in the prevention and cure of a wide range of diseases. Thymoquinone (TQ) is the most abundant constituent of the volatile oil of Nigella sativa seeds and most properties of N sativa are mainly attributed to TQ. A number of pharmacological actions of TQ have been investigated including anti-oxidant, anti-inflammatory, immunomodulatory, anti-histaminic, anti-microbial and anti-tumor effects. It has also gastroprotective, hepatoprotective, nephroprotective and neuroprotective activities. In addition, positive effects of TQ in cardiovascular disorders, diabetes, reproductive disorders and respiratory ailments, as well as in the treatment of bone complications as well as fibrosis have been shown. In addition, a large body of data shows that TQ has very low adverse effects and no serious toxicity. More recently, a great deal of attention has been given to this dietary phytochemical with an increasing interest to investigate it in pre-clinical and clinical researches for assessing its health benefits. Here we report on and analyze numerous properties of the active ingredient of N. sativa seeds, TQ, in the context of its therapeutic potentials for a wide range of illnesses. We also summarize the drug's possible mechanisms of action. The evidence reported sugests that TQ should be developed as a novel drug in clinical trials.

Possible involvement of calcium channels and plasma membrane receptors on Staurosporine-induced neurite outgrowth.

Staurosporine as a protein kinases inhibitor induced cell death or neurite outgrowth in PC12 cells. We investigated the involvement of calcium channel and plasma membrane receptors on staurosporine inducing neurite outgrowth in PC12 cells. PC12 cells were preincubated with NMDA receptor inhibitors (1.8 mM ketamine and 1µM MK801, treatment 1) or L-Type Calcium channels (100 µM nifedipine and 100 µM flavoxate hydrochloride, treatment 2) or calcium-calmoduline kinasses (10 µM trifluoprazine, treatment 3) and nifedipine, MK801, flavoxate hydrochloride and ketamine (treatment4) or without pretreatments (control). Then, the cells were cultured in RPMI culture medium containing 214nM staurosporine for induction of neurite outgrowth. The percentage of Cell cytotoxicity and apoptotic index was assessed. Total neurite length (TNL) and fraction of cell differentiation were assessed. After 24h, the percentage of cell cytotoxicity were increased in treatments 1, 2 and 4 compared with control (p<0.05). After 6h, apoptotic index was similar between all treatments. After 12h, apoptotic index were increased in treatment 4 compared with control (p<0.05). After 24h, apoptotic index were increased in treatments 1, 2 and 4 compared with control (p<0.05). TNL were decreased in treatments 1, 2 and 4 compared with control in different times of assessment (6, 12 and 24 h) (p<0.05). The fraction of cell differentiation were decreased in treatments 1, 2 and 4 compared with control (p<0.05). It can be concluded that the possible involvement of L-type calcium channel and the N-methyl D-aspartate receptor on staurosporine-induced neurite outgrowth process in PC12 cells.

Evaluation of Methylation Status in the 5'UTR Promoter Region of the DBC2 Gene as a Biomarker in Sporadic Breast Cancer.

OBJECTIVE: Breast cancer is one of the most common malignancies in women worldwide. It is caused by a number of genetic and epigenetic factors. Aberrant hypermethylation of the promoter regions in specific genes is a key event in the formation and progression of breast cancers as well as the DBC2 gene, as a tumor suppressor gene. Different studies show that the DBC2 gene is inactivated through epigenetic mechanisms such as methylation in its promoter region. In this study, authors have tried to analyze methylation status in the promoter region of DBC2 gene in affected women and healthy controls. MATERIALS AND METHODS: In this experimental study, we evaluated the methylation status of DBC2 gene with nested methylation-specific PCR (MSPCR) using specific methylated and unmethylated primer sets, in three separate PCR reactions. We used 50 tissue and blood samples of patients with breast cancer, 5 normal tissues and also 30 normal blood samples. Results were evaluated by the Mann-Whitney test, SPSS 16.0 statistical software. RESULTS: Nested MSPCR results demonstrated that the frequency of the DBC2 promoter region methylation status in tumor and blood samples of the affected patients was significantly higher than that of the corresponding normal controls. CONCLUSION: DBC2 gene inactivation by methylation of CpG islands in the promoter region probably is a crucial step in the process of cell proliferation and susceptibility to different cancers, including breast cancer. Our study provides new evidence that aberrant methylation of the DBC2 gene is involved in the tumorigenesis of breast cancer. DNA methylation in this gene is proven to be a potential marker for tumor diagnosis and prognosis, as well as a novel therapeutic target.