Studies On Expression Of MDR Related Proteins,signal Transduction Pathway And Ion Channels Of Gastric Cancer Cells

Multidrug resistance (MDR) is the main obstacle of chemotherapy for malignant tumor, especially for gastric cancer. Several molecules have been found to confer MDR phenotype of tumor cells, such as Pgp, MRP, LRP, BCRP, GSH/GSH, TopoII, apoptosis associated proteins and anti-apoptosis proteins. However, these molecules could not interpret completely how tumor cells develop MDR. It is a hot point in tumor area to find novel MDR related molecules and new means to reverse MDR. We have prepared a multidrug resistant gastric cancer cell line SGC7901/VCR, which derived from human gastric cancer cell line SGC7901 by stepwise selection using vincristine as inducing reagent. SGC7901/VCR cells could survive 0.2ug/ml of vincristine. It was confirmed that SGC7901/VCR cells overexpress Pgp and MGrl Ag, and that they are cross-resistant to sveral anti-cancer drugs, which suggests SGC7901/VCR is a good cell model for study on gastric cancer MDR. The present study will use SGC7901/VCR as foundation to explore the relationships between ion channel, signal transduction and gastric cancer MDR by means of whole cell patch clamp, laser scanning confocal microscopy (LSCM), flow cytometry (FCM) and other advanced methods. The results will be helpful to uncover novel mechnisms of multidrug resistance and make a good themry foundation for new MDR reversal means.AIM To prepare multidrug resistant gastric cancer sublines with differentresistant index by stepwise selection of SGC7901/VCR cells and use them as cell models: (1) To determine the correlation between protein kinase C (PKC), protein kinase A (PKA) and MDR of gastric cancer cells. (2) To detect alterations of ion channels on multidrug resistant gastric cancer cells and its regulation. (3) To observe the effect of multidrug-resistance associated monoclonal antibody MGrl on PKC activity of multidrug resistant gastric cancer cells. (4) To observe the differential expression of MG7Ag on drug-resistant and drug-sensitive gastric cancer cells and its effects on intracellular drug accumulation of gastric cancer cells.METHODS (1) Multidrug resistant gastric cancer sublines were prepared by stepwise selection in vitro and identified by using immunohistochemistry and flow cytometry. (2) The expression of PKC was determined by using immunohistochemistry and Western blotting, and the activities of PKC and PKA were detected by using competitive protein binding method. (3) The effects of PKC activator and inhibitor on drug sensitivity were determined by MTT assay, and the effects of PKC activator and inhibitor on drug accumulation were determined by FCM. (4) To use double-labelled immunofluorescence and LSCM to detect the co-expression of PKC and MGrlAg. (5) The alterations of ion channels and its regulations were detected by using whole cell patch clamp. (6) The alterations of subcellular location of PKC isoenzymes in sweeling-activated SGC7901/VCR cells were detected by immunofluorescence. (7) The expression of potassium channels Kvl.5 and Kv2.1 were determined by immunohistochemstry. (8) The effect of potassium channel blocker on drug accumulation was determined by FCM. (9) The differential expression of MG7Ag on drug-resistant and drug-sensitive gastric cancer cells was detected by immunohistochemistry and FCM. (10) The effects of monoclonal antibody MG7 on drug sensitivity and drug accumulation of drug-resistant gastric cancer cells were determined by using MTT assay and FCM, respectively.RESULTS(1) Multidrug resistant gastric cancer cell sublines with different resistantindex, SGC7901/VCR (0.3ug/ml), SGC7901/VCR (0.7ug/ml) and SGC7901/VCR (l.Oug/ml), were successfully established. These cells exhibited increased expression of MDR related protein Pgp and MGrl Ag. And they also showed significant morphological alterations such as increased cell volum, polygonal shape, much more and longer villi, increased synapse-like connections, much more mitochondrions and part-confluence of some cells.(2) All of multidrug resistant gastric cancer cell sublines an...