The Establishment Of The Paclitaxel-resistant Human Gastric Cancer Cell Line MGC803/PTX And Study On The Anti-drug-resistance Effect Of Novel β-lactams Compounds

Part One:The establishment of paclitaxel-resistant human gastric cancer cell line MGC803/PTX and the research on the mechanisms of resistanceThe drug resistant cell line MGC803/PTX was established by gradually increasing density based on paclitaxel, to investigate the differentia in MGC803 and MGC803/PTX cell line. Methods:The cell line MGC803/PTX was established by gradually increasing density based on paclitaxel over a period of 10 months. The morphological features were observed by invert microscope during cancer cell exposure to paclitaxel. The drug resistance index and stability of cell line was detected by CCK8 assay. The multidrug resistance of MGC803/PTX to anticancer agents was evaluated by CCK8 assay. The growth curve of parent and drug resistant cell lines are depicted with cytometry. Clone formation rate was detected by clone formation assay. Apoptosis was measured by Flow cytometry(FCM), and the cellular distribution is analyzed with the FCW too.The protein expressions of P-gp, Bcl-2, Bax, PARP,β-catenin and p-GSK-3β of drug resistant and parent cell line were detected by western blotting,to explore the possible mechanism of resistance drug in MGC803/PTX. Results:The drug resistant cell line MGC803/PTX was made up after induction of ten months,MGC803/PTX cells were of 9.3 fold resistance to PTX. The morphologic of MGC803 convert long shuttle to short polygon with smaller size during induction. MGC803/PTX cells were of 4.38 fold resistance to 5-FU,1.4 fold resistance to ADR. The proliferation rate of MGC803/PTX cells was significantly slower than that of the parental cells, the population doubling time of MGC803 and MGC803/PTXwere separately 22.5h and 24.55 h. The drug resistant cell clone formation ability obviously higher than that of parental cells;increased nucleus dyeing, generation of apoptosis body, nuclear chromatin condensed were observed in apoptosis cell, but drug-resistant cell apoptosis rate is obviously lower than that of parental cells; Flow cytometry testing results also show that drug-resistant cell line MGC803/PTX significantly below MGC803 cells, apoptosis rate were 1.8% and 24.2% respectively, MGC803 / PTX distribution of cell cycle in G0/G1 phase stage(71.95%), S(14.8%), G2(10.15%), MGC803 cells in G0/G1 phase 62.4%), S period(11.275%), G2(20. 47%), the drug resistant cells in G0/G1 phase increased obviously.P-gp, Bcl-2 and PARP protein expressions were efficiently higher in the MGC803/PTX than in the MGC803, but Bax protein expressions were lower in the MGC803/PTX than in the MGC803. Additional,β-catenin and p-GSK-3β protein expressions were down-regulation in the drug resistant cells. Conclusions: 1 The stable human gastric cancer drug resistant cell line MGC803/PTX was successfully established. 2 The biological characteristics of drug resistant cell line MGC803/PTX were significantly changed,such as cell morphology, cell proliferation rate, apoptosis rate,cell cycle distribution. 3 P-gp, Bcl-2 and PARP protein expressions were efficiently higher in the MGC803/PTX than in the MGC803, but Bax protein expressions was lower in the MGC803/PTX than in the MGC803. This may be part of the reason for its drug resistance. At the same time, the β-catenin and p-GSK-3β proteins expression compared with MGC803 cells also have differences, its resistance mechanism associated with Wnt/β-catenin related pathways. Part Two:Study the effect of compound 20 on MGC803/PTXTo explore the effect of new compound 20 on MGC803/PTX of drug resistant cell line and preliminary study on its mechanism. Methods:CCK8 assay was used to detect the antitumor activity of compound 20 to MGC803/PTX at different time of action. The effect of compound 20 on the proliferation of single cell was detected by clone formation assay. Hoechst 33258 staining was used to observe the morphological changes of MGC803/PTX cells after the action of compound 20. High content analysis technology combined with PI staining to detect the effect of compound 20 on cell cycle arrest. The expression of P-gp, Bcl-2, Bax, Pro-PARP1,Cleaved caspase-3 were detected by Western blot. Results:The results of CCK8 assay showed that compound 20 had a good activity on both MGC803 and MGC803/PTX cells.The obvious shrinkage chromatin,nuclear fragmentation were observed in MGC803/PTX after the action of compound 20,and the cell cycle distribution of MGC803/PTX was changed,G2/M increased significantly. Compound 20 can up regulate the expression of Bax and Cleaved caspase-3 proteins, and down regulate the expression of P-gp, Bcl-2 and Pro-PARP1 proteins in a concentration-dependent manner. Conclusions: 1 Compound 20 had a strong inhibitory effect on MGC803/PTX cells. 2 Compound 20 can block MGC803/PTX cells in G2/M phase. 3 The mechanism of compound 20 is related to the apoptosis pathway, which can down regulate the expression of P-gp, Bcl-2 and Pro-PARP1 proteins, up regulate of Bax and Cleaved caspase-3 protein, and ultimately lead to apoptosis of MGC803/PTX cells. 4 Novel β-lactams compound 20 has an obvious effect in the MGC803/PTX cells, it is considered to develop relevant preparations and to study of the mechanism.