CG2 were observed in the IL 1b and TNF treated MCF-7 JAK Signaling Pathway cells. Therefore, it seems that the regulation of the expression of inflammatory cytokines ABCG2 per cell-specific and tissue specific. To resolve this problem, we have two cell models of other cancers for which the existence of inflammatory micro-environment has been demonstrated confinement Lich stomach cancer and Geb Rmutterhalskrebs. We investigated the short-term effects of IL 1b, IL-6 and TNF mRNA, protein expression and function of cervical cancer HeLa cells ABCG2 online, and gastro-line cell carcinoma, 257th EPG85 To evaluate the potential of proinflammatory cytokines such as chemosensitizers of tumor cells resistant to the same experiments were performed on derevatives resistant cell lines overexpressing ABCG2, Including Evaluated by its rating and HeLaRDB EPG85 257RNV cells. explained utern that similarities and differences between the rules of ABCG2 of proinflammatory cytokines in various cell models k can improve our amplifier ndnisses RAF Signaling Pathway the underlying molecular mechanisms of drug resistance.
Materials and Methods of cell lines Four cell culture were used in this study, including normal line of Geb Rmutterhalskrebs, HeLa and ABCG2-overexpressing, daunorubicin Vascular Disrupting Agent resistant derivative, HeLaRDB, the gastric cell line, EPG85 257, and overexpressing ABCG2, mitoxantrone resistant derivative EPG85 257RNV. All cell lines were cultured in Leibovitz L 15 medium without L-glutamine, 10% FCS cultured at 37 with 5% CO 2. To the MDR Ph To obtain genotype, daunorubicin and mitoxantrone have regularly Pure and HeLaRDB EPG85 added 257RNV cells. These cells were cultured in a medium free daunorubicin or MX cultured for at least 1 week before each experiment. Real-time reverse transcription in each cell Not polymerase were seeded at a density of 2.02.5 9105 cells / well in a six-well plate t and at 37 overnight. Then they were incubated for 1272 h with various concentrations of IL 1b, IL-6 or TNF. Total RNA from subconfluent cells using a RNeasy MINIKIT treated isolated according to the manufacturer S instructions. First strand cDNA synthesis with oligo-dT primer was on 3 lg of total RNA using the MDV3100 Superscript II kit performed synthesis. In order to avoid DNA contamination, the RNA was treated with RNase-free DNase.
To evaluate the expression of ABCG2 mRNA in cancer cell lines after treatment with proinflammatory cytokines, were crossing the comparative methods on samples of cDNA using LightCycler 480 SYBR Green I Master Mix performed and analyzed on a LightCycler 480 System. Primer sequences and the optimum conditions of thermal cycles are listed in Table 1. Is therefore at the end of the PCR cycles were PCR products allm by using an analysis of the melting curve Hliches Erh Hen the temperature of 65 to 97C, a single PCR product was best Authorization by a dye SYBR Green detected. The software of the LightCycler 480 calculates the relative amount of target and reference genes on the PC and the basis of the results were defined as the ratio Ratio target / reference of the treated sample by the ratio Ratio target / reference split terms of the standard. The analysis of ABCG2 protein expression by flow cytometry To assess the effect of cytokines on E study.