Investigation On The Establish Of SACC/DDP And The Mechanism Of Its Multidrug Resistance

Objective:1. To establish a human oral Salivary Adenoid Cystic Carcinoma cell line (SACC/DDP) by exposing parent cell line periodically to DDP of .steady concentration. And changes in cellular morphology was observed.2. To determine the sensitivity of SACC/DDP cells to 4 anticancer agents.3. To detect the expressions of Multidrug Resistance-associated Protein (MRP),so as to study the mechanism of the Multidrug Resistance(MDR) of human SACC/DDP cell line. Methods: Our study included three parts.1. SACC cell line, which purchased from Hua-xi medical center of Sichuan University, was exposed to DDP with concentration of lug/ml for 48 hours every month. 6 months later, the cell line of SACC/DDP was established. Changes in celluar morphology were observed by invert microscope and recorded by taking photo. Change in population doubling time between SACC and SACC/DDP was detected, and the cell growth curves werre also determined. 6 and!2 month later, the multidrug-resistance was detected by MTT method.2. MTT method, Flow cytometry and AO fluorescent stain were adopted to observe killing effect, cell circle and apoptosis of SACC cell and SACC/DDP cell with various chemotherapeutic agents (MTX, PYM, VCR, MMC ) at different concentrations. The expression of Bax protein was also determined.3. Immunohistochemical SP, Western-blot and RT-PCR were taken to test the expressions of MRP at protein and mRNA levels. The changes in the drugs' concentration inside cells were detected with HPLC method.Results:1. DDP induced Multidrug Resistance in Human Salivary Adenoid Cystic Carcinoma Cell Line.1.1 SACC/DDP cell line was established with stable growth level after exposing to DDP of lug/ml for 6 months, and the morphology of cell was changed.1.2 The population doubling time of SACC/DDP was 40.2 hours, which is apparently different from that of its parental cell-61.9 hours (p<0.05).1.3 The drug resistant index of SACC/DDP was 15.08, which indicates that the drug resistance of SACC/DDP had been increased. And the stability of drug-resistance was 82% and 68% 6 and 12 months later.2. Evaluation of the Multidrug Resistance of SACC/DDP2.1 All of the four chemotherapeutic agents-VCR, MTX, PYM, MMC had strong killing effects on SACC cell line. While their effects on SACC/DDP cell line reduced.2.2 FCM results manifested the obvious changes in cell circle between SACC/DDP and SACC cell line. The number ratio of cells in GO stage to that in Gi stage (GO/G1) clearly declined in SACC/DDP cell line, and so did Gz/M. These changes were related to the concentration of chemotherapeutic agents.2.3 The apoptotic proportion of SACC cell line was rather low at the nature state, and increased dose-dependently with pure chemotherapy. Nevertheless, the apoptotic proportion of SACC/DDP cell line was reduced obviously compared to SACC cell line. The positive expression rate of Bax protein was 20.7% in SACC/DDP, obviously lower than that in SACC cell line (P<0.01).3. The expression of MRP in SACC/DDP3.1 The expression of MRP in SACC/DDP was 84.2%, which was apparently higher than that in SACC.3.2 The expression of the protein of MRP in SACC/DDP was 74.70?7.56, which was higher than that in SACC.3.3 The expression of mRNA of MRP in SACC/DDP was 0.907?.330, while that in SACC was 0.212?.101.There was obvious difference between SACC and SACC/DDP cell line.3.4 The concentration of the chemotherapeutic agents in SACC/DDP was lower than that in SACC.And the concentrations increased after being cultured with Staurosprine for 2 hours.Conclusion:1 The multidrug resistant human Salivary Adenoid Cystic Carcinoma cell line (SACC/DDP) could be estabished by exposing parent cell line periodically to DDP. Theinmorphology and population doubling time of cell line have been changed, and the new cell line has a stable drug-resistance.2 SACC/DDP was resistant to chemotherapeutic agents with different structure.And it was proved that SACC/DDP had the ability to produce MDR. More over, th...