Screeing And Identification Of Novel Proteins And Genes Related To Multidrug Resistance In Human Gastric Cancer Cell Line SGC7901

Multi-drug resistance (MDR) of tumor cells is the main obstacle of cancer chemotherapy. Previous studies have shown that the mechanisms of MDR involve Pgp, MRP, LRP, BCRP, GSH/GST, PKC, Topo DNA plerosis, genes related to apoptosis and changes of cellular environment (such as pH, hypoxia and temperature). Very little is known for MDR of gastric carcinoma cells. Preliminary studies have shown specific features in MDR of gastric carcinoma cells, which cannot be completely explained by any known mechanisms of MDR.Aim of the study: This research project aimed to investigate the expression and regulation of MDR-related proteins and genes in gastric cancer SGC7901 cells and Vincristine-resistant SGC7901 (SGC7901/VCR) cells for significance in the development of MDR in cancer cells.Methods: Two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) using O'Farrell system and immobilized pH gradients (IPG) were applied to compare the differential expression of MDR proteins in gastric cancer SGC7901 and SGC7901/VCRcells. The differentially expressed proteins were identified by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). A modified differential-display polymerase chain reaction (DD-PCR) was used to examine the expression of mRNA in SGC7901/VCR cells versus SGC7901 controls. The differentially expressed mRNA were cloned and the cDNA fragments were confirmed by reverse-Northern blot hybridization, sequencing analysis and Northern bolt analysis. One of the cDNAs was further analyzed by3'-RACE and multiple tissue expression (MTE) array.Results: This study has optimized the 2-D PAGE methods to demonstrate the differentially expressed proteins in SGC7901 and SGC7901/VCR gastric cancer cells. Thirty proteins were found significantly different in their expression levels with 6 higher and 19 lower in SGC7901/VCR cells. Five proteins were found to be unique to SGC7901 or SGC7901/VCR cell (3 in SGC7901/VCR cells, 2 in 7901 cells). One of the differentially expressed proteins was sequenced by MALDI-MS. Identified by DD-PCR, 20 cDNAs were found with higher expression in SGC7901/VCR cells. Sequencing analysis revealed that seven of the mRNAs were encoded by known genes which may be related to MDR. Thirteen of the cDNA were products of unknown genes. The size and abundance of 4 of the novel gene products were confirmed by Northern blot analysis (GRP-2, 1.9kb; GRP-19, 2.0kb; GRP-28, 1.6kb; GRP-32, 1.7kb, respectively). Results confirmed their high expression level in SGC7901/VCR cells. GRP-2 was further characterized by MTE array hybridization to show its high expression in normal lung and weak expression in other histocytes, suggesting that it may be an important gene in MDR of gastric carcinoma.Summary: Novel proteins with regulated expression were found in gastric cancer SGC7901/VCR cell line. Twenty cDNAs were found with up-regulated expression ingastric cancer SGC7901/VCR cells. Cloning and sequencing revealed that seven of them encode known proteins which may be related to MDR. Thirteen of the cDNAs are products of unknown genes with GRP-2 showing up-regulated expression in SGC7901/VCR cell. These differentially expressed proteins and genes provide materials for further investigation on the molecular mechanism of MDR.