| At the first part,we added the 5-fluorouracil to MGC803 with gradually increased concentration to establish the resistant cell line MGC803/5-FU,and we also detected the differences between MGC803 and MGC803/5-FU.In this study,the resistant cell line MGC803/5-FU was established by exposingMGC803 to 5-fluorouracil with gradually increased concentration.The cell morphology was observed under the inverted microscope.The median inhibitory concentrations of 5-fluorouracil and other anticancer drugs on both MGC803 and MGC803/5-FU were achieved by the CCK8 assay.The drug resistant stability of MGC803/5-FU was tested during the six months since it was established.And then,the apoptosis ratewas detected by DAPI staining and flow cytometry and cell cycle distributions were also detected by flow cytometry after stained by PI.The difference of colony formation between them was tested and the multiplication rates were compared,too.The difference in protein expressions of P-gp,TS,β-catenin were detected by western blotting.Thedrug resistant cell line MGC803/5-FU was established after MGC803 was exposed to 5-FU eight months.And the resistance index on 5-FU was 12.75 and it can be kept above 9 during six months after established.MGC803/5-FU is not resistant to otheranticancer drugsremarkably.The apoptosis rate and coloning efficiency of MGC803/5-FU are lower remarkably compared with MGC803.The doubling time of MGC803/5-FUincreases obviously.The percent of MGC803/5-FU at the G0/G1 phase increases and the percent at the S phase decreases compared to MGC803.The protein expression of TS increases obviously while there is no difference at the protein expression of P-gp compared to MGC803.Besides,the proteins about the signal path of Wnt/β-catenin change.At the second part,the effect and mechanism on proliferation and apoptosis of novel compound ZYL-263 onbothMGC803 andMGC803/5-FU was researched.Firstly,the effect on proliferation on MGC803 and MGC803/5-FU of ZYL-263 was detected by CCK8 assay.The efficiency of colony formation was tested after the two cell lines were exposed at ZYL-263 in different concentrations.And then,the morphology of apoptosis was observed with DAPI staining after MGC803 and MGC803/5-FU was induced by ZYL-263.Also,their reactive oxide species and apoptosis rate were tested by flow cytometry after MGC803 and MGC803/5-FU were exposed at ZYL-263 in different concentrations.Finally,the protein expressions of Bcl-2、 Bax、Total-Caspase-7、Cleaved-Caspase-7、Total-PARP-1、Cleaved-PARP-1、TS were detected by western blotting after MGC803 and MGC803/5-FU were induced by ZYL-263.ZYL-263 can inhibit the proliferation of MGC803 and MGC803/5-FU and its effect on MGC803/5-FU was more obvious.ZYL-263 can inhibit the colony formation of MGC803 and MGC803/5-FU.ZLY-263 induces the apoptosis of MGC803 and MGC803/5-FU,and the apoptosis rate of MGC803/5-FU was higher.ZYL-263 can make MGC803 and MGC803/5-FU induce more reactive oxide species.ZYL-263 can reduce the protein expressions of Bcl-2,Total-Caspase-7,Total-PARP-1 whileup-regulatetheprotein expressions of Cleaved-Caspase-7,Cleaved-PARP-1,Baxof both MGC803 and MGC803/5-FU in concentration-dependent manner.Besides,ZYL-263 can reduce the protein expression of TS of MGC803/5-FU but not MGC803.In conclusion,we successfully established the human gastric resistant cell line MGC803/5-FU.And we researched the possible mechanisms and it can be used as a model to study the problem of drug resistance.Besides,the new compound ZYL-263 can induce apoptosis of MGC803 and MGC803/5-FU by up-regulating the expressions of Bax,Cleaved-Caspase-7,Cleaved-PARP-1 and down-regulating the expressions of Bcl-2,Total-Caspase-7,Total-PARP-1.Especially,ZYL-263 can down-regulate the expression of TS of MGGC803/5-FU.This research may provide a new way tofind new compounds to solve the drug resistance. |
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