Molecular Mechanisms Of Multidrug Resistance On Gastric Cancer Cells Mediated By MGr1-Ag

Gastric cancer is the one of the most prevalent malignant tumor with the highest mortality in China, mostly due to failure of chemotherapy. The major cause of chemotherapeutic failure is the multidrug resistance (MDR) of the cancer cells, inherited or acquired during drug treatment. MDR-related molecules identified so far (such as P-gp, MRP, LRP, BCRP, GST, PKC and TopoII) can n ot e xplain a 111 he M DR t ypes o f gastric c ancer, i ndicating t hat there are unknown MDR-related molecules and mechanisms in gastric cancer. MGrl-Ag, identified by our institute, is a novel MDR-related molecule found in vincristine-resistant gastric cancer cell line, SGC7901/VCR. Previous studies showed that MGrl antibody could partially reverse the MDR phenotype of gastric cancer. Investigating the functions of MGrl-Ag and its role in MDR will help to understand the development and modulation of cancer MDR phenotype.Objective To study the expression, distribution and significance of MGrl-Ag in normal and malignant tissues of stomach, observe the expression levels of MGrl-Ag in drug resistant and sensitive gastric cancer cell lines, establish stable cell sub lines with MGrl-Ag overexpressed or downregulatedand reveal the roles of MGrl-Ag in gastric cancer MDR.Methods 1) Tissue arrays of normal and malignant stomach tissues were stained with MGrl antibody by immunohistochemical (IHC) method to study the expression and distribution patterns of MGrl-Ag; 2) Expression of MGrl-Ag in drug resistant or sensitive gastric cancer cell lines was detected by Western blot; 3) cDNA of MGrl-Ag was amplified from SGC7901/VCR cells, cloned into pUCm-T vector, the recombinant vectors was confirmed by endonuclease digestion and sequencing; 4) Sense (pcDNA3.1/MGrl-Ag) and antisense (pcDNA3.1/antiMGrl-Ag) expression vectors were constructed by subcloning the MGrl-Ag gene into pcDNA3.1/V5-his vectors in a orientation-directed manner; 5)Using lipofectamine, pcDNA3.1/ MGrl-Ag plasmids were transfected into MGC803 cells, and the pcDNA3.1/ antiMGrl-Ag was transected into SGC7901/VCR cells. Empty pcDNA3.1 vectors were transfected the respective cells as controls; 6) Western blot was used to detect the changes of MGrl-Ag in the tansfected cells; 7) MTT assay was used to draw the growth curves of the transfected cells and the control cells; 8) Flow cytometry (FCM) was used to examine the cell cycle distribution of the transfected cells and their control counterparts and the proliferous index (PI) was calculated; 9) In vitro drug sensitivity of the transfected cells and their controls was tested by MTT assay. Their sensitivity to 8 kinds of chemotherapeutic drugs such as adriamycin was examined and the IC50 values were calculated; 10) FCM was used to study the accumulation and detention of adriamycin in the cells. Western blot was used to study the changes of P-gP and MRP expression in the transfected cells; 11) Annexin/PI staining was used to analyze the apoptosis of the transfected cells, and apoptosis index was calculated. Apoptosis-associated molecules, Bcl-2 and Bax were examined bywestern blot.Results 1) As shown by IHC, 92 of 144 gastric cancer samples were stained positively by MGr-1 antibody (63.89%). 52 of 72 normal stomach tissue samples were MGrl-Ag positive (80.56%), the difference in MGrl-Ag expression between normal and cancer tissues was of statistical significance (P<0.05); 68 of 72 tumor tissues were with confirmed pathological diagnosis of differentiation state. MGrl-Ag was positive in 83.33% well differentiated, 62.50% medium differentiated and 58.97% poorly differentiated carcinoma, there were significant difference between well and poorly differentiated carcinoma (PO.01) while no significant difference between medium and poorly differentiated carcinoma (P>0.05). In samples with whole layer tumor infiltration, 82.35% were MGrl-Ag positive. In serous membrane-infiltrated samples, muscular layer-infiltrated and submucosa-infiltrated, the positive rates of MGrl-Ag were 57.14%, 47.83% and 25.00% respectively. There were significan...