G2013 modulates TLR4 signaling pathway in IRAK-1 and TARF-6 dependent and miR-146a independent manner.

Inflammation is inseparable part of different diseases especially cancer and autoimmunity. During inflammation process toll like receptor 4(TLR4) responds to lipopolysaccharide (LPS), one of the bacterial components, and TLR4 signaling leads to interleukine-1 receptor associated kinase-1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor6 (TRAF6) activation which ultimately results in nuclear factor- ĸB (NF-ĸB) activation as the main transcription factor of inflammatory cytokines. Conversely, NF-ĸB over activation induces miR-146a in innate immune cells which can consequently reduce TRAF6, IRAK1, and NF-ĸB activation in a negative feedback. G2013 is a novel designed non-steroidal anti-inflammatory drug (NSAID) which was recently shown to be effective in experimental autoimmune encephalomyelitis (EAE) mouse model. The aim of this study was to evaluate G2013 effects on inflammatory (IRAK1 and TRAF6) and anti-inflammatory (miR-146a) factors of TLR4 signaling pathway. For this purpose, cytotoxicity of G2013 has been evaluated by MTT assay. Expression level of miR-146a in PBMCs and IRAK1 along with TRAF6 in HEK-293 TLR4 cells have been determined using real time PCR. Our results showed that IC50 of G2013 was 25µg/ml, thus 5 and 25 µg/ml concentrations used for further treatments as low dose and high dose concentrations. Our results showed that IRAK1 expression reduced between 5 to 8 fold after treatment by G2013 in a dose dependent manner (p<0.001). In parallel TRAF6 expression declined between 3 to 10 fold dose dependently (p<0.05). However, miR-146a expression was not affected after treatment with low dose and high dose of G2013. In conclusion our data showed that G2013 can regulate TLR4 signaling pathway during inflammation by reducing downstream signaling molecules, IRAK1 and TRAF6 without altering miR-146a expression.

siRNA-mediated silencing of MDR1 reverses the resistance to oxaliplatin in SW480/OxR colon cancer cells.

One of the most challenging aspects of colon cancer therapy is rapid acquisition of multidrug resistant phenotype. The multidrug resistance gene 1 (MDR1) product, p—glycoprotein (P—gp), pump out a variety of anticancer agents from the cell, giving rise to a general drug resistance against chemotherapeutic agents. The aim of this study was to investigate the effect of a specific MDR1 small interference RNA (siRNA) on sensitivity of oxaliplatin—resistant SW480 human colon cancer cell line (SW480/OxR) to the chemotherapeutic drug oxaliplatin. SW480 cells were made resistant by continuous incubation with stepwise serially increased concentrations of oxaliplatin over a 6—months period. Resistance cell were subsequently transfected with specific MDR1 siRNA. Relative MDR1 mRNA expression was measured by Quantitative real—time PCR. Western blot analysis was performed to determine the protein levels of P—gp. The cytotoxic effects of oxaliplatin and MDR1 siRNA, alone and in combination were assessed using MTT and the number of apoptotic cells was determined with the TUNEL assay. MDR1 siRNA effectively reduced MDR1 expression in both mRNA and protein levels. MDR1 down—regulation synergistically increased the cytotoxic effects of oxaliplatin and spontaneous apoptosis SW480/OxR. Our data demonstrates that RNA interference could down regulate MDR1 gene expression and reduce the P—gp level, and partially reverse the drug resistance in SW480/OxR cells in vitro. Therefore, the results could suggest that MDR1 silencing may be a potent adjuvant in human colon chemotherapy.

Human leukocyte antigen and esophageal cancer in the northwestern region of Iran.

Esophageal squamous cell carcinoma is the 6th most commonly occurring cancer worldwide. A relationship between HLA A1 and B40 and esophageal cancer was described in patients examined in China. The aim of this study was to investigate the relation of HLA class 1 and esophageal carcinoma in the northwestern region of Iran. Using specific monoclonal antibodies, different human leukocyte antigens (HLA) were quantified in 100 patients suffering esophageal carcinoma in Tabriz, a major city located in the Northwestern region of Iran. These data were compared to those of 100 healthy matched individuals as a control group from the same region. HLA B14 and A24 were increased and showed statistically significant correlation in squamous cell carcinoma. These findings may also indicate the association between genetic factors and esophageal carcinoma. Further studies are suggested for detecting correlation of HLA and esophageal carcinoma in other regions.

Production and characterization of monoclonal antibodies against human IgG in Balb/c mouse.

Immunoglobulin G (IgG) is the main immunoglobulin in natural human serum. It constitutes 70 to 75 percent of all immunoglobulins. Monoclonal antibodies have many applications in diagnosis, treatment and purification. The conjugated monoclonal antibodies against human IgG are used in most diagnostic kits. For production of monoclonal antibodies against human IgG, spleen cells of the most immune mouse were fused with SP2/0 (myeloma cells) using Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. The suitable clones were selected for limiting dilution (L.D). Then, the supernatant of suitable monoclones were assessed for cross reactivity with IgM & IgA by ELISA and confirmed by immunobloting. The subclasses of the selected monoclonal antibodies were determined and the clones were frozen and kept in liquid nitrogen. Finally, suitable monoclone was injected into the mouse, intraperitoneally, that has been primed with Pristane. In this study, 127 clones were obtained of which 15 clones had absorbance more than 1 which two of them with absorbance about 1.5 were selected for limiting dilution. The yield of limiting dilution was 6 clones with absorbance about 1.8 which did not show cross reactivity with IgM & IgA. Among these clones, G2 monoclone with IgG1 subclass was selected as suitable one and it was reproduced in FCS free RPMI 1640. For large scale production in invivo, the suitable clone was implanted in the peritoneum of the Balb/c mouse and its titer was measured, which showed 1/100,000 dilution for ascitic fluid, having no cross reactivity with IgM & IgA. Monoclonal antibody was purified by chromatography, confirmed by SDS- PAGE, conjugated with enzyme and applied for diagnostic kits.

Development of hybridoma cells producing monoclonal antibody against human epsilon (epsilon) chain.

Epsilon (epsilon) chain is one of the five classes of immunoglobulins that plays an important role in allergic diseases. Production of monoclonal antibodies by a single clonotype against different epitopes of epsilon chain have high priority in development of diagnostic kits. In this study, an attempt was made to produce monoclonal antibodies against human epsilon chain. Balb/c mice were immunized with semipurified epsilon chain and spleen cells were fused with Sp2/0 mouse myeloma cell line in the presence of poly ethylene glycol. Supernatant of hybridoma cells were screened for detection of antibody by Enzyme-linked Immuno Sorbent Assay (ELISA) method. Cloning of selective high absorbance wells were done with limiting dilution method. The suitable clones (monoclones) were selected by ELISA and confirmed by immunoblot. The subclasses of the chosen monoclonal antibodies were determined and the clones freezed and kept in liquid nitrogen. During this study three successful fusions were carried out, which resulted in over than 100 clones with high production of anti- epsilon chain. Twelve clones with the highest titers were selected for cloning. After limiting dilution more than 50 monoclonal antibodies were produced and the unsuitable one (C1F2) displayed higher absorbance in reaction with purified IgE, relatively high cross-reactivity with IgM, and the highest cross-reactivity with IgG. In Immunoblotting, presence of relatively high-density band in reaction with IgE was confirmed. The unsuitable monoclonal antibody was shown to be IgG1 subclass with kappa light chain. It seems that, this monoclonal antibody could not be successfully useful in diagnostic kits.