Involved Mechanism Of The Acquired Resistance To 5-FU In Human Colon Cancer

[ Background]: Colorectal cancer is one of the most common malignant tumors. It is the second place on the list of mortality caused by cancers in in North America and other developed countries, and is 4^th to 6^th in our country with an increasing incidence. In the past two decades, substantial advances have been made in the treatment of colorectal cancer. For example, 5-fluorouracil (5-FU)-based adjuvant chemotherapy has been efficacious in reducing mortality for lymph node-positive colon cancer and has become the standard of care. However, recurrence and metastasis due to drug resistance remain major obstacles. New strategies to overcome resistance to 5-FU and an improved curative rate for colorectal cancer are urgently needed. An antimetabolite chemotherapeutic drug, 5-FU causes cell injury by inhibiting thymidylate synthase or by incorporating itself into DNA or RNA. High-level expression of thymidylate synthase, increased activity of deoxyuridine triphosphatase, methylation of the MLH1 gene, and overexpression of Bcl-2, Bcl-XL, and Mcl-1 proteins have all been reported to lead to resistance to 5-FU, which suggests that multiple factors might contribute to 5-FU resistance. However, the precise mechanisms of this resistance remain unclear. In this study, 5-FU resistant cell lines were established by repeat application of 5-FU on human colon cancer cell lines DLD1 and DLD-TRAIL/R. Then the characteristics of 5-FU resistant cells were analyzed for clarifying the mechanisms of resistance to 5-FU.[Methods]: Cell viability assay was performed by counting viable cells microscopically with a hemocytometer;western blot analysis was used for determining the expression of various proteins;flow cytometry assay was done for cell cycle analysis;the software of Curve Expert was used for IC50 of four cell lines after treatment with agents;differences between treatment groups were assessed using paired Student's t test.[Results]: 1). Selection of 5-Fluorouracil-Resistant cells and cell viability: 5-FU resistant cells were derived from DLD1 and DLD1-TRAIL/R cells with increasing (IQuM—400,uM ) concentration of 5-FU. Subsequent study revealed that the 5-FU concentration at which growth was inhibited in half of the cells (IC50) was 2.5 ,Mmol/L for parental cells and 210.6 /anol/L for 5-FU/R cells (an 84-fold difference) and 4.4 ^mol/L for TRAIL/R cells and 105.7 ,amol/L for TF/R cells (a 24-fold difference). Furthermore, both 5-FU resistance cell lines remain sensitive to Paclitaxol, Cisplatin, Mitomycin C and TRAIL protein. Thus, resistance of these cells seemed to be specific to 5-FU. 2). Expression of Bcl-2 Family Members in 5-Fluorouracil-Resistant Cells and S-Phase Arrest in 5-Fluorouracil-Resistant Cells: No obvious changes of thymidylate synthase> Bax> Bak> Bcl-2^ Bcl-w> XIAP> survivin and ILP-2 were detected in 5-FU-resistant cells with or without 5-FU treatment. Mdr protein expression was more pronounced in TF/R cells than in other cells, and Bik, Bcl-Xs, and Bcl-XL protein expression was higher in 5-FU/R cells than in parental cells and higher in TF/R cells than in TRAIL/R cells. In other words, the expression of these three Bcl-2 family proteins was higher in 5-FU-resistant cells than in 5-FU-sensitive cells. Among these three proteins, Bcl-XL was the only one that is antiapoptotic. Thus, Bcl-XL protein may play an important actor in 5-FU resistance. We evaluated cell cycle profiles after treatment with arious concentrations of 5-FU for 3 days. We found that 5-FU induced S-phase arrest in 5-FU-sensitive cell lines but not in 5-FU-resistant cell lines. The expression level of the phosphorylated histone H2A.X, a hallmark of DNA damage, was high in 5-FU-sensitive cell lines within 24 hours of treatment with 200 jumol/L 5-FU butremained low in 5-FU-resistant cell lines even after 3 days of treatment. Our findings suggested that 5-Fumediated resistance related with DNA damage. 3). Effect of Over-expression of Bcl-XL Protein on Cell Sensitivity to 5-Fluorouracil: We stably transfected with Bcl-XL plasmid into DLD1 cells and derived DLDl/Bcl-XL cells which overexpressed Bcl-XL protein. Dose-effect analysis showed that the IC50 of 5-FU was 4.5 ftmolfL in DLDl/Bcl-XL cells and 2.5 ^mol/L in parental cells (a 1.8-fold difference). Furthermore, Western blot and cell cycle analysis showed over-expression of Bcl-XL protein had certain protective effect on the apoptosis induced by 5-FU. However, 5-FU still could induce cell cycle arrest and phosphorylated H2A.X in DLDl/Bcl-XL cells.[Conclusion]: 1). Over-expression of Bcl-XL as well as 5-Fu-induced DNA damage and cell cycle arrest existed in the acquired 5-FU-resistant cells. 2). Over-expression of Bcl-XL protein had certain effects on the apoptosis but not on the DNA damage and cell cycle arrest induced by 5-FU in colon cells. 3). Over-expression of Bcl-XL protein is not the major reason for the colon cell lines resistant to 5-FU, other unknown mechanisms might be involved.