1,3-Bis(2-chloroethyl)-1-nitrosourea enhances the inhibitory effect of Resveratrol on 5-fluorouracil sensitive/resistant colon cancer cells

AIM:To study the mechanism of 5-fluorouracil(5-FU)resistance in colon cancer cells and to develop strategies for overcoming such resistance by combination treatment.METHODS:We established and characterized a 5-FU resistance(5-FU-R)cell line derived from continuous exposure(25μmol/L)to 5-FU for 20 wk in 5-FU sensitive HCT-116 cells.The proliferation and expression of different representative apoptosis and anti-apoptosis markers in 5-FU sensitive and 5-FU resistance cells were measured by the MTT assay and by Western blotting,respectively,after treatment with Resveratrol(Res)and/or 1,3-Bis(2-chloroethyl)-1-nitrosourea(BCNU).Apoptosis and cell cycle arrest was measured by 4',6'-diamidino-2-phenylindole hydrochloride staining and fluorescence-activated cell sorting analysis,respectively.The extent of DNA damage was measured by the Comet assay.We measured the visible changes in the DNA damage/repair cascade by Western blotting.RESULTS:The widely used chemotherapeutic agents BCNU and Res decreased the growth of 5-FU sensitive HCT-116 cells in a dose dependent manner.Combined application of BCNU and Res caused more apoptosis in5-FU sensitive cells in comparison to individual treatment.In addition,the combined application of BCNU and Res caused a significant decrease of major DNA base excision repair components in 5-FU sensitive cells.We established a 5-FU resistance cell line(5-FU-R)from 5-FU-sensitive HCT-116(mismatch repair deficient)cells that was not resistant to other chemotherapeutic agents(e.g.,BCNU,Res)except 5-FU.The 5-FU resistance of 5-FU-R cells was assessed by exposure to increasing concentrations of 5-FU followed by the MTT assay.There was no significant cell death noted in5-FU-R cells in comparison to 5-FU sensitive cells after5-FU treatment.This resistant cell line overexpressed anti-apoptotic[e.g.,AKT,nuclear factorκB,FLICE-like inhibitory protein),DNA repair(e.g.,DNA polymerase beta(POL-β),DNA polymerase eta(POLH),protein Flap endonuclease 1(FEN1),DNA damage-binding protein 2(DDB2)]and 5-FU-resistance proteins(thymidylate synthase)but under expressed pro-apoptotic proteins(e.g.,DAB2,CK1)in comparison to the parental cells.Increased genotoxicity and apoptosis were observed in resistant cells after combined application of BCNU and Res in comparison to untreated or parental cells.BCNU increased the sensitivity to Res of 5-FU resistant cells compared with parental cells.Fifty percent cell death were noted in parental cells when 18μmol/L of Res was associated with fixed concentration(20μmol/L)of BCNU,but a much lower concentration of Res(8μmol/L)was needed to achieve the same effect in 5-FU resistant cells.Interestingly,increased levels of adenomatous polyposis coli and decreased levels POL-β,POLH,FEN1 and DDB2 were noted after the same combined treatment in resistant cells.CONCLUSION:BCNU combined with Res exerts a synergistic effect that may prove useful for the treatment of colon cancer and to overcome drug resistance.

合成头孢泊肟酯中间体的气相色谱跟踪分析

目的 对合成头孢泊肟酯中间体 1-氯乙基氯甲酸酯、1-氯乙基异丙基碳酸酯 ,1-碘代乙基异丙基碳酸酯作定性、定量的跟踪分析 ,帮助确定最佳的合成工艺条件。方法 用 GC- MS、核磁共振、SE- 5 4(30 m×0 .5 3mm× 0 .2 5μm)毛细管柱气相色谱法进行定性、定量。结果 合成 1-氯乙基氯甲酸酯最高含量达 6 1.3%,1-氯乙基异丙基碳酸酯含量达 95 .4%,1-碘代乙基异丙基碳酸酯含量达 87.5 %。结论 实验表明本色谱方法分离度好 ,重现性好 ,对探索最佳合成工艺路线具有指导意义。