Effects Of 5-Aza-CdR On The Apoptosis Induced By 5-FU In Human Colon Cancer Cell Line Caco-2

Objective:(1) To investigate methylation status of promoter of p16 in four different colon cancer cell lines.(2) To analyse the changes of the growth rate,sensitivity in response to the chemotherapy and apoptosis induced by 5-fluorouracil(5-FU) before and after the treatment with demethylation agent 5-aza-2'-deoxycytidine(5-Aza-CdR) on p16 methylated colon cancer cell line Caco-2.These results may provide insight information for understanding the colon carcinogenesis and be helpful in combined therapy in colon cancer.Methods:In the present study,we examined the expression of p16 methylation state in four different colon cancer cell lines by methyl ation specific polymerase chain reaction(MSP).colon cancer cell line Caco-2 was treated with 5-Aza-CdR in different concentrations(0.625μmol/L,1.25μmol/L,2.5μmol/L and 5μmol/L) and were investigated for the expression of p16 mRNA by reverse transcriptase-polymerase chain reaction(RT-PCR).MTT method was used to observe the change of inhibitory rate of Caco-2 to 5-Aza-CdR and 5-FU,RT-PCR and western-blotting were used to observe the change of the expression of apoptosis-related gene Bnip3/bax/ bcl-2,flow cytometry was used to observe the changes of the apoptosis rate of Caco-2 and ordinary Giemsa staining with light microscopy and Hoechst 33342 staining with fluorescence microscopy were used to observe specific and typical morphology of apoptosis before and after treatment.Results:(1)We examined the expression of p16 mRNA and its methylation status in four different colon cancer cell lines by MSP and found that the promoter region of p16 gene were completely methylation in two cell lines(Caco-2 and RKO) and hemimethylation in two cell lines(SW480 and HCT116).(2)The expression of p16 mRNA was detected in Caco-2 colon cancer cell lines after 72 hours treatment of 5-Aza-CdR(0.625μmol/L,1.25μmol/L,2.5μmol/L and 5μmol/L) in vitro.The promoter region of p16 gene showed partly methylation after the treatment of 5-Aza-CdR at 1.25μmol/L,2.5μmol/L and 5μmol/L.(3) Reduced cell proliferaton was detected in Caco-2 after the treatment of 5-Aza-CdR/5-FU in vitro.(4) Early apoptosis of Caco-2 treated with 5-FU combined with 5-Aza-CdR or not were enhanced signlficantly with the prolonging of administration times.The sensitivity to apoptosis induced by 5-FU and 5-Aza-CdR exceeded the simple additive effect of two drugs(P＜0.05).(5) Caco-2 cell line treated with 5-Aza-CdR and 5-FU showed specific and typical morphology of apoptosis.(6) RT-PCR and Western-blotting results indicated that 5-Aza-CdR up-regulated the expression of apoptosis-related gene bnip3 and bax,and down-regulated the gene bcl-2(P＜0.01) on Caco-2 cell line treated with 5-FU.Conclusions:(1) Inactivation of p16 in Caco-2 probably due to hypermethylation of its promoter region,p16 can be re-expressed after 5-Aza-CdR treatment.(2) Reduced cell proliferaton was detected in Caco-2 after the treatment of 5-Aza-CdR /5-FU in vitro.(3) The sensitivity to apoptosis induced by 5-FU and 5-Aza-CdR exceeded the simple additive effect of two drugs,and showed specific and typical morphology of apoptosis.(4) The apoptotic effect on Caco-2 induced by 5-FU was time-dependent.The combination of 5-Aza-CdR and 5-FU could more significantly promote apoptosis than 5-Aza-CdR or 5-FU alone,5-Aza-CdR could increase the sensitivity of Caco-2 to 5-FU through up-regulating the expression of apoptosis-related gene bnip3 and bax,and down-regulating the gene bcl-2.