[Effects of 5-Aza-dC on 5-Fu chemosensitivity by modulating TIP30 gene expression in human colorectal cancer cells].

Objective To investigate the effect of 5-Aza-2'-deoxycytidine (5-Aza-dC) on TIP30 gene expression and the relationship between TIP30 expression and the sensitivity to 5-fluouracil (5-Fu) in colorectal cancer cells.Methods The methylation profile of TIP30 gene in HCT116 colorectal cancer cells was determined by methylation-specific PCR.The levels of TIP30 mRNA and protein were determined by RT-PCR and Western blot after the 5-Aza-dC treatment.MTT assay was used to detect the chemosensitivity of HCT116 cells to 5-Fu.Results TIP30 gene displayed complete DNA methylation in the HCT116 cells without 5-Aza-dC pretreatment.After the 5-Aza-dC treatment for 3 days,only demethylating PCR amplification product was detected and TIP30 gene showed DNA demethylation.With the prolongation of the time of removal of 5-Aza-dC treatment,methylated and demethylated PCR amplification products were observed and TIP30 gene displayed both DNA methylation and DNA demethylation in the colorectal cancer cells.At the day 10 after removal of 5-Aza-dC,methylating PCR amplification product appeared and TIP30 gene showed DNA methylation.No expressions of TIP30 mRNA and protein were detected in the HCT116 cells untreated with 5-Aza-dC.After the treatment of 5-Aza-dC for 3 d and then removed the 5-Aza-dC,the expressions of TIP30 mRNA and protein were increased obviously.With the prolonged time after 5-Aza-dC removal,the expressions of TIP30 mRNA and protein decreased and reached the lowest level on day 10.The IC50 values of 5-Fu were 41.62,33.17 and 4.96 μg/ml in the HCT116 cells pretreated with 5-Aza-dC,d0 and d10 with the drug removal after drug treatment for 3 d,respectively.Conclusions The results of this study show that the expression of TIP30 gene may be associated with its DNA methylation status and may affect the sensitivity of colorectal cancer cells to 5-Fu. Key words: Colorectal neoplasms;  5-Aza-2'-deoxycytidine;  DNA methylation; Fluorouracil;  Chemosensitivity