Briefly, cells were seeded in 96-well plates (4×103 for parental cells and 5.5×103 for resistant cells according to their doubling time) in 100 L of culture medium and incubated for 24 H to allow cell attachment and to reach a 50% confluence. this drug-resistance phenomenon. The aim of this study was to characterize new models of CDDP-resistant GC cell lines (AGS R-CDDP and MKN-28 R-CDDP) obtained through a stepwise increasing drug doses method, in order to understand the molecular mechanisms underlying chemoresistance as well as identify new therapeutic targets for the treatment of GC. Cell viability assays, cell death assays and the expression of resistance molecular markers confirmed that AGS R-CDDP and MKN-28 R-CDDP are reliable CDDP-resistant models. RNA-seq and bioinformatics analyses identified a total of 189 DEGs, including 178 up-regulated genes and 11 down-regulated genes, associated mainly to molecular functions involved in CDDP-resistance. DEGs were enriched in 23 metabolic pathways, among which c-Fms-IN-10 the most enriched was the and models of acquired or induced drug resistance is a Rabbit Polyclonal to HSP90B (phospho-Ser254) useful approach to better understand the mechanisms that trigger clinical resistance to chemotherapeutics. In addition, models can clarify the cellular and molecular mechanisms of novel anticancer agents, enabling comparisons with parental cells and intrinsically resistant cells [13]. The aim of this study was to characterize functionally models of CDDP-resistant gastric cancer based on two gastric cancer cell lines (AGS and MKN-28), which were developed through administering stepwise increases in drug dose. Materials and methods Ethics statements This study was approved by Ethical Committee of Universidad de La Frontera (Approval certificate N83/2015). Drugs Cisplatin (CDDP) was purchased from Selleck Chemicals (SelleckChem, USA). CDDP was reconstituted at a concentration of 3.3 mM diluted in 0.9% (p/v) NaCl and aliquots of stock solution were stored at ?80C. Cell lines and culture conditions AGS and MKN-28 cell lines were generously provided by Dr. Richard Peek (Vanderbilt University, Nashville, USA). AGS was established from a gastric adenocarcinoma obtained from a 54-year-old female [14] and MKN-28 from a moderately differentiated gastric tubular adenocarcinoma obtained from a 70-year-old female [15]. AGS and MKN-28 were cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (Thermofischer, USA) and 1% (v/v) penicillin and streptomycin (Thermofischer, USA). Cells were maintained at 37C in a 95% humidified atmosphere and 5% CO2 conditions. Cells were subcultured at 80% confluence and harvested after treatment with 0.25% trypsin and 0.02% EDTA (Corning, USA). Development of CDDP-resistant cell lines Induced drug-resistant cell lines, CDDP-resistant AGS cells (AGS R-CDDP) and CDDP-resistant MKN-28 cells (MKN-28 R-CDDP) were developed following Coleys protocol [16]. Briefly, the drug sensitivity of the parental cells was tested by establishing the starting dose of treatment at 20% of the EC50 concentration. Cells were seeded according to doubling time, and the starting dose of the drug was incorporated into the cells when they presented 20% confluence. The increase in drug doses was made every two subcultures, by doubling each earlier concentration. The cycle was repeated c-Fms-IN-10 30 occasions. Once cells acquired cisplatin resistance they were produced in drug-free medium for one month, freezing in liquid nitrogen and then awakened in medium comprising CDDP to confirm the level of drug resistance. The time for the development of this drug-resistant model was 12 months. Drug level of sensitivity assay Drug level of sensitivity analyses were performed using a standard viability assay (MTT assay). Briefly, cells were seeded in 96-well plates (4×103 for parental cells and 5.5×103 for resistant cells relating to their doubling time) in 100 L of culture medium and incubated for 24 H to allow cell attachment and to reach a 50% confluence. Next, cells were revealed for 72 H at different concentrations of CDDP, ranging from 0.01 M to 1000 M. Cells without CDDP were used as settings. After 72 H of incubation the medium was eliminated, and cells were washed with 100 L c-Fms-IN-10 of DPBS/Modified (Thermofischer, USA). Then, 0.5 mg/mL of MTT was added to each well, followed by 2 H incubation. As only practical mitochondrial dehydrogenase enzymes from viable cells can reduce MTT to form formazan, 100 L of propanol was used to fully dissolve this c-Fms-IN-10 purple precipitate. Absorbance was measured at 570 nm using the Infinite? NanoQuant spectrophotometer (TECAN, Switzerland). The EC50 ideals (drug concentration that inhibited cell growth at 50%) were estimated through the dose-response curve after 72 H of incubation under different drug concentrations. In this case, the percentage of viable cells was plotted according to the related drug concentrations, obtaining the ideals of half maximal effective concentration (EC50) by non-linear regression. The resistance index (RI) ideals were determined by dividing the EC50 ideals of c-Fms-IN-10 resistant cell lines from the EC50 ideals of parental cell lines, defining arbitrarily the chemoresistance as RI 2. Cell death assay A.