Increased expression of annexin I and thioredoxin detected by two-dimensional gel electrophoresis of drug resistant human stomach cancer cells

The therapy of advanced cancer using chemotherapy alone or in combination w ith radiation or hyperthermia yields an overall response rate of about 20-5 0%. This success is often marred by the development of resistance to cytost atic drugs. Our aim was to study the global analysis of protein expression in the development of chemoresistance in vitro. We therefore used a cell cu lture model derived from the gastric carcinoma cell line EPG 85-257P. A cla ssical multidrug-resistant subline EPG85-257RDB selected to daunorubicin an d an atypical multidrug-resistant cell variant EPG85-257RNOV selected to mi toxantrone, were analysed using two-dimensional electrophoresis in immobili zed pH-gradients (pH 4.0-8.0) in the first dimension and linear polyacrylam ide gels (12%) in the second dimension. After staining with coomassie brill iant blue, image analysis was performed using the PDQuest system. Spots of interest were isolated using preparative two-dimensional electrophoresis an d subjected to microsequencing. A total of 241 spots from the EPG85-257RDB- standard and 289 spots from the EPG85-257RNOV-standard could be matched to the EPG85-257P-standard. Microsequencing after enzymatic hydrolysis in gel, mass spectrometric data and sequencing of the peptides after their fractio nation using microbore HPLC identified that two proteins annexin I and thio redoxin were overexpressed in chemoresistant cell lines. Annexin I was pres ent in both the classical and the atypical multidrug-resistant cells. Thior edoxin was found to be overexpressed only in the atypical multidrug-resista nt cell line. (C) 1998 Elsevier Science B.V. All rights reserved.