鸦胆子苦醇抑制结直肠癌细胞增殖与Nrf2通路相关性研究

Inhibitory effects of Brusatol on proliferation and Nrf2 pathway in colorectal cancer cells

目的鸦胆子苦醇(brusatol,BRU)系苦木科植物鸦胆子果实中提取的一种苦木内酯类化合物,近年来被发现能有效抑制肺癌细胞的Nrf2通路,在抗肿瘤治疗中的应用潜力受到关注。本研究着重探讨BRU对结直肠癌细胞增殖和Nrf2通路的影响。方法采用不同浓度BRU处理HCT116和HT29细胞,MTT法检测细胞活力,台盼蓝染色观察细胞增殖,Hoechst 33342单染和AnnexinⅤ/PI双染检测凋亡,PI单染检测细胞周期,BrdU免疫荧光法研究DNA的合成,试剂盒法检测细胞内还原性辅酶Ⅱ(nicotinamide adenine dinucleotide phosphate,NADPH)和GSH水平。蛋白质印迹法检测BRU对HCT116细胞Nrf2蛋白表达的影响,QRT-PCR法检测BRU对Nrf2靶基因6-磷酸葡萄糖脱氢酶(glucose-6-phosphate dehydrogenase,G6PD)、6-磷酸葡萄糖酸脱氢酶(6-phospho gluconat dehydrogenase,6GPD)、异柠檬酸脱氢酶同工酶1(isocitrate dehydrogenase 1,IDH1)和苹果酸酶1(malic enzyme 1,ME1)转录水平的影响。结果MTT结果显示,15~240nmol/L的BRU处理可降低HCT116细胞和HT29细胞的活力。用60和240nmol/L的BRU分别处理HCT116和HT29细胞48h,能显著抑制细胞增殖。BrdU免疫荧光结果显示,BRU处理24h能显著抑制2种细胞的DNA合成,抑制率分别达到(76.64±6.78)%和(62.87±8.62)%,t值分别为8.615和5.852,P值分别为0.002和0.005。凋亡检测结果显示,BRU在60nmol/L条件下未明显诱导HCT116细胞凋亡。用60nmol/L的BRU处理HCT116细胞,能使Nrf2蛋白水平在2和4h分别减少(48.67±3.21)%和(43.67±11.85)%,t值分别为10.382和2.133,P值分别为0.001和0.025。另外,60nmol/L的BRU处理8h还能显著降低结肠癌细胞NADPH和GSH的水平。QRT-PCR法检测结果显示,BRU能在8h内时间依赖性的抑制HCT116细胞Nrf2靶基因G6PD、6GPD、IDH1和ME1的转录。结论 BRU可以快速而短暂的抑制结肠癌细胞Nrf2信号通路,在不明显诱导细胞凋亡的条件下有效抑制细胞增殖。BRU对结肠癌细胞增殖的抑制作用可能与Nrf2信号通路有关。

OBJECTIVE Brusatol（BRU）,one of ester compounds extracted from fruit of Brucea javanica,has been confirmed as an Nrf2 inhibitor in lung cancer cells and has attracted more and more attention in tumor therapy.Here the effects of BRU on the Nrf2 pathway and proliferation in colon cancer cells were studied.METHODS HCT116 cells and HT29 cells were treated with various concentrations of BRU.Cell viabilities were detected by MTT assay.Cell proliferation rates were evaluated by trypan blue staining.Apoptosis was detected by Hoechst 33342 staining and Annexin Ⅴ/PI staining.Cell cycle was studied using flow cytometry with PI staining.DNA synthesis was determined by use of BrdU incorporation.The intracellular NADPH and GSH levels were detected using commercial assay kit.The Nrf2 protein level was detected by Western blot.The mRNA levels of G6 PD,6 GPD,IDH1 and ME1 were determined by QRT-PCR.RESULTS MTT assay showed that 15-240 nmol/L BRU reduced the cell viability in both HCT116 cells and HT29 cells.The cell proliferation was significantly inhibited when HCT116 cells and HT29 cells were treated with 60 and 240 nmol/L BRU for 48 h,respectively.Using BrdU incorporation,we found DNA synthesis in two types of colorectal cancer cells were repressed when treated with BRU for 24 h.The inhibition rates of DNA synthesis in HCT116 cells and HT29 cells were（76.64±6.78）%（t=8.615,P=0.002）and（62.87±8.62）%（t=5.852,P=0.005）,respectively.No obvious apoptosis was found when HCT116 cells were treated with 60 nmol/L BRU.But under the same conditions,BRU induced rapid decrease in Nrf2 protein level.The reduction of Nrf2 protein were reached（48.67±3.21）%（t=10.382,P=0.001）and（43.67±11.85）%（t=2.133,P=0.025）when HCT116 cells were treated with 60 nmol/L BRU for 2 hand 4 h,respectively.In addition,the intracellular NADPH and GSH levels were also significantly reduced by treatment with BRU for 8 hin colorectal cancer cells.QRT-PCR analysis indicated that the mRNA levels of G6 PD,6 GPD,IDH1 and ME1,the Nrf2-target genes,were reduced by BRU time-dependently in HCT116 cells.CONCLUSIONS BRU induced rapid and transient inhibition in Nrf2 pathway in colon cancer cells.BRU significantly inhibited the proliferation of colon cancer cells without inducing apoptosis and the mechanism of action might be related to the inhibition of the Nrf2 pathway.