Effects and mechanism of silent information regulator of transcription 1 in the drug-resistance of colonic cancer / 中华消化外科杂志

ABSTRACT

Objective To investigate the effects of mechanism of silent information regulator of transcription 1 (SIRT1) in the drug-resistance of colonic cancer.Methods The clinical data of 25 colonic cancer patients with 5-Fu-resistance and 30 colonic cancer patients with chemosensitivity who were admitted to the Henan Tumor Hospital from December 2012 to December 2013 were retrospectively analyzed.The specimens of colonic cancer were collected for study.(1) The protein expression of SIRT1 in patients with drug-resistance or chemotherapeutic sensitivity was tested by immunohistochemical staining.The protein expression of SIRT1 in the HCT116 and HCT1 16/5-FU cells was detected by Western blot.(2)HCT116/5-FU cells were interfered by siRNA and divided into the blank control group (cells untreated),the empty vector group (cells treated by siRNA) and the SIRT1 silence group (cells treated by SIRT1 siRNA).The protein expression of the HCT116/5-FU cells were inhibited by the c-Jun N-terminal kinase (JNK) and then divided into the SP600125 group [cells were treated by JNK signaling pathway inhibitor SP60012 (concentration30 μmol/L)for 12 hours],the DMSO group [cells were treated by DMSO (cells were treated by 0.1％ DMSO for 12 hours] and the control group (cells were treated by cell culture media).(3) Serine in the SIRT1 ser47 was mutated to alanine or aspartic acid,and mutations S47A (S47A group,serine to alanine) and S47D (S47D group,serine to aspartic acid) ; Untransfected HCT116/5-FU cells were in the S47 wild type group,and apCMV-3Tag-3 cells transfected by empty vector were served as negative control; all the HCT116/5-FU cells were interfered by 5-FU (concentration8 μmol/L) for 12 hours.HTC116 cells and HTC116/5-FU cells were treated by SIRT1 inhibitor resveratrol at concentrations of 0,1,10,50,100 nmol/L and SIRT1 activator niacinamide at concentrations of 0,1,2,3,4,5 ng/L.Cell proliferation was detected by MTF.(4) Cell apoptosis was detected by flow cytometry.(5) The expressions of related genes were detected by real-time PCR.(6)The expressions of related proteins were detected by western blot.The count data were analyzed using the chi-square test.The measurement data with normal distribution were presented as (x) ± s.The comparison among groups were analyzed using the one-way analysis of variance and LSD-t test.The pairwise comparisons were analyzed using the t text.Results (1) The results of immunohistochemical staining were as follows.The positive expressions of SIRT1 in patients with chemotherapeutic sensitivity and drug-resistance were 16.7％ (5/30) and 92.0％ (23/25),respectively,with significant difference (x2 =30.965,P ＜ 0.05).The relative mRNA and protein expressions of SIRT1 in HCT116/5-FU cells with drug-resistance were 1.870 ± 0.100 and 1.660 ± 0.109,which were significantly higher than 1.000 ± 0.070 and 1.000 ± 0.050 in HCT116/5-FU cells without drug-resistance (t =11.721,8.963,P ＜ 0.05).(2) The results of MTT were as follows.The proliferation rates of HCT116/5-FU cells treated by resveratrol at concentrations of 0,1,10,50 nmol/L were 100％ ±12％,105％± 14％,129％ ± 10％ and 144％ ± 17％,which were significantly higher than 41％ ± 10％,49％ ±11％,74％ ± 16％ and 105％ ± 17％ of HCT116 cells which were treated by reseratrol at the same contrations (t =8.226,-7.236,6.673,3.510,P ＜0.05).The proliferation rates of HCT116/5-FU cell treated by niacinamide at concentrations of 0,1,2 ng/L were 87％ ± 12％,78％ ± 12％,69％ ± 11％,which were significantly higher than 36％ ± 6％,32％± 5％,30％± 6％ of HCT116 cells which were treated by niacinamide at the same concentrations (t =-8.593,-8.006,-7.000,P ＜ 0.05).The proliferation rates of HCT116/5-FU cells in the blank control group,the empty vector group and the SIRT1 silence group were 100％± 8％,99％ ±9％,37％ ± 6％,with significant differences among the 3 groups (F =66.597,P ＜ 0.05),and the proliferation rate of HCT116/5-FU cells in the SIRT1 silence group was significantly lower than that in the blank control group (t =10.113,P ＜0.05).(3) The results of flow cytometry were as follows.The apoptotic rates of HCT116/5-FU cells in the SIRT1 silence group,the empty vector group and the blank control group were 60％ ± 5％,36％ ± 4％,35％ ±4％,with significant differences among the 3 groups (F =36.549,P ＜ 0.05),and the apoptotic rates of HCT1 16/5-FU cells in the SIRT1 silence group were significantly higher than that in the blank control group and the empty vector group (t =-7.215,-7.084,P ＜0.05).(4)The results of RT-PCR were as follows.The relative expression rates of P-gp mRNA in the HCT116/5-FU cells in the SIRT1 silence group,the empty vector group and the blank control group were 0.320 ± 0.030,0.990 ± 0.060,1.000 ± 0.090,with significant differences among the 3 groups (F =10.107,P ＜ 0.05),and the relative expression rate of P-gp mRNA in the SIRT1 silence group was significantly lower than that in the blank control group (t =11.463,P ＜ 0.05).The relative expression rates of P-gp mRNA in the HCT116/5-FU cells in the SP600125 group,the DMSO group and the control group were 0.240 ±.0.040,0.990 ± 0.100,1.000 ± 0.070,with significant difference among the 3 groups (F =19.002,P＜0.05),and the relative expression rates of P-gp mRNA in the SP600125 group was significantly lower than that in the control group (t =7.301,P ＜0.05).(5) The results of western blot were as follows.The relative expression rates of p-JNK protein in the HCT116/5-FU cells in the blank control group,the empty vector group and the SIRT1 silence group were 1.000 ± 0.090,1.090 ± 0.020,0.080 ± 0.010,with significant difference among the 3 groups (F =12.130,P ＜ 0.05).The ratios of p-SIRT1-S27/T-SIRT1,p-SIRT1-T530/T-SIRT1,p-SIRT1-S47/T-SIRT1 were 1.158 ±0.140,1.209 ±0.150,3.760 ±0.150 in HCT116 cells treated by 5-FU,and 1.120 ±0.109,1.130 ±0.100,2.160 ±0.110 in HCT116 cells treated by DMSO,with significant differences (F =9.763,10.261,P ＜0.05).The ratios of p-SIRT1-S47/T-SIRT1 in HCT116 cells treated by 5-FU and DMSO were 3.760 ± 0.150 and 2.160 ± 0.110,which were significantly higher than 0.940 ± 0.040 and 1.121 ± 0.110 in HCT116/5-FU cells (t =14.721,21.335,P ＜ 0.05).(6) The proliferation rates of HCT116/ 5-FU cells in the S47 wild type group,the negative control group,the S47A group and the S47D group were 41％± 31％,39％ ± 4％,64％ ± 2％ and 26％ ± 5％,with significant differences among the 4 groups (F =6.371,P ＜ 0.05).Conclusions SIRT1 promotes the proliferation of drug-resistant colonic cancer cells and increases the expression of P-gp via JNK signaling pathway,there by enhances cellular drug resistance.SIRT1 S47 is the critical site for 5-FU-resistance in HCT116/5-FU cells.