Establishment Of5-Fu Drug Resistant Colon Cancer Cell Line And The Exploration Of Its Biological Characteristics And The Related Drug-resistant Mechanisms

Objective：To establish a human5-Fluorouracil(5-Fu) resistant colon cancer cellline(DLD-1/5-FU) and investigate the differences of biological characteristics inmorphological changes, doubling time, drug resistance and cell cycle distribution. Todetect the proportion of cancer stem cells, the expression level of multidrug resistantprotein P-gp as well as the expression level of O(6)-Methylguanine-DNAMethyltransferase(MGMT) to determine whether they correlate with the drugresistance.Methods：Drug-resistance cell line（DLD-1/5-Fu）was induced by stepwise selectionon exposure to increasing doses of5-Fu in vitro. The shapes of the cells wereobserved by a high-powered microscope and the pictures were taken by a camera.Cell growth curve and the IC50of both parental and drug-resistant cell lines weredetected by CCK-8. Cell cycle distribution was measured by flow cytometry and theproportion of CD133+cells was also measured by it. The expression levels of P-gpand MGMT protein were both detected by western blotting. And the expression levelof mRNA of MGMT was determined by real time PCR.Results：After8months, the5-Fu resistant cell line DLD-1/5-Fu was successfullyinduced which could keep growing in the culture medium with the concentration of 5-Fu up to200μM. Compared with parental cells, the morphology of thedrug-resistant cells were irregular and branch-shaped. The growth rate of DLD-1/5-Fuwas slower than DLD-1and their doubling time were50.35±2.88h and42.61±2.17hrespectively (P＜0.05). The IC50of DLD-1was7.65±3.22μM，whereas DLD-1/5-Fuwas985.43±163.64μM， which showed that the drug-resistant cell line wascompletely insensitive to5-Fu and its resistance index(RI) was128.81(P＜0.01).Flow cytometry demonstrated that the percentage of DLD-1/5-Fu cells in phaseG0/G1increased（P＜0.05） and both S（P＞0.05） and G2/M（P＜0.05） phasedecreased. The proportion of cancer stem cells in drug resistant cell line was lowerthan the parental cells(P＜0.05).Western blot showed that the expression level of P-gpand MGMT protein were both significantly about twofold higher in DLD-1/5-Fu thanin DLD-1(P＜0.01). Real Time PCR demonstrated that the mRNA of MGMTexpressed in DLD-1/5-Fu was higher than that in DLD-1(P＜0.05),but was not asremarkable as its protein level.Conclusion：The5-Fu resistant cell line DLD-1/5-Fu was successfully establishedand its biological characteristics were quite different from that of the parental cellline. Meanwhile, cancer stem cells, P-gp and MGMT may contribute to the drugresistance to5-Fu in human colon cancer cells.