【摘要】 目的:探讨鸦胆子油(Brucea javanica oil,BJO)和卡培他滨(Capecitabine,Cap)合用对体外培养的人结直肠癌LOVO和HT29细胞周期和增殖及对Wnt/β-catenin信号通路的影响。方法:以CCK-8比色法,检测不同浓度的鸦胆子油(0.0625、0.125、0.25、0.50和1.00mg/ml)、卡培他滨(0.025、0.050、0.100、0.200、0.400μM)作用不同时间(2h、12h、24h和48h)以及单独、联合应用对LOVO和HT29细胞增殖能力影响;PI染色分析鸦胆子油、卡培他滨以及联合应用对细胞周期的影响;免疫印迹法检测结直肠癌细胞系(SW620、LOVO、LS174T、HT29和SW116)和人正常结直肠粘膜细胞系,以及鸦胆子油、卡培他滨单用及合用对细胞β-catenin和Wnt3a蛋白的表达影响;实时荧光定量PCR法检测结直肠癌细胞系(SW620、LOVO、LS174T、HT29和SW116)和人正常结直肠粘膜细胞系,鸦胆子油、卡培他滨单用及合用对细胞Wnt3a、β-catenin及其下游Cyclin D1和c-Myc mRNA含量的影响。结果:结直肠癌细胞系(SW620、LOVO、LS174T、HT29和SW116)中β-catenin蛋白和mRNA含量均明显高于正常结直肠上皮细胞系;0.25、0.50和1.00 mg/ml鸦胆子油作用12h(分别为:93.1%,90.8%和90.5%),24h(分别为:76.7%,71.1%和72.7%)可明显抑制LOVO细胞生长,且呈现时间和剂量依赖性的变化,同时抑制HT29细胞,卡培他滨与鸦胆子油作用相同;0.125、0.25、0.50和1.00mg/ml的鸦胆子油联合0.4μM卡培他滨比单独卡培他滨作用结直肠癌细胞系LOVO和HT29的细胞增殖率明显较低,且具有剂量依赖性,分别为:对LOVO分别为70.9%、67.0%、61.3%和55.7%;对HT29分别为70.1%、58.1%、48.3%和40.4%;1.00 mg/ml鸦胆子油和0.4μM卡培他滨合用于LOVO和HT29细胞48 h的增殖抑制率为55.7%和40.4%,明显低于单独使用0.4μM卡培他滨(72.5%和55.1%);DAPI染色证实,合用1.00mg/ml的鸦胆子油联合0.4μM卡培他滨比单独鸦胆子油和卡培他滨作用结直肠癌细胞系LOVO和HT29增殖抑制作用更强;联合使用明显增加两种细胞G1/G0期细胞数量,同时增加G2/M细胞数量;再者,联合使用明显抑制细胞Wnt3a、β-catenin蛋白和mRNA及其下游Cyclin D1和c-Myc mRNA含量表达。结论:鸦胆子油和卡培他滨合用可抑制结直肠癌细胞增殖,促进凋亡,该作用可能是通过抑制Wnt/β-catenin信号通路和细胞周期阻滞实现的。更多还原

【Abstract】 Objective: To explore the effect of Brucea javanica oil(BJO) in combination with Capecitabine(Cap) on apoptosis and Wnt/beta-catenin signaling pathway in human colorectal cancer(CRC) cell LOVO and HT29. Methods: Inhibitory effects of BJO and Cap on growths of LOVO and HT29 cells in the different time and different concentrations were determined by CCK-8 colorimetry assay,the cycle of cells treated with BJO and Cap was analyzed by flow cytometry with PI staining. Protein and mRNA levels of β-catenin and Wnt3 a in human normal rectal epithelial cell line FHC and CRC cell lines(SW620,LOVO,LS174 T,HT29 and SW116) were examined by western blot and semiquantitative RT-PCR(q-PCR). Results: Levels of β-catenin protein and mRNA in CRC cell lines(SW620,LOVO,LS174 T,HT29 and SW116) were significantly higher than those in FHC(P < 0. 05). 0. 25,0. 50 and 1. 00 mg/ml BJO significantly inhibited the growth of LOVO for 12 h(93. 075%,90. 823% and 90. 466% respectively) and 24 h(76. 672%,71. 059% and 72. 671% respectively)(P < 0. 05) in a dose and time dependent manner,also inhibited the growth of HT29 cells and had the same effect with that of Cap. 0. 125,0. 25,0. 50 and1. 00 mg/ml BJO combined with 0. 4μM Cap were significantly downregulated cell proliferation rates(70. 892%,67. 001%,61. 315% and55. 741% in LOVO,and 70. 095%,58. 128%,48. 328% and 40. 423% in HT29,respectively) in a dose-dependent manner(P < 0. 05),compared with Cap group. 1 mg/ml BJO in combination of 0. 4μM Cap inhibited proliferations of LOVO(55. 741%) and HT29(40. 423%)for 48 h,with significantly lower proliferation inhibition rates than those of BJO(72. 546%,55. 099%) alone(P < 0. 05). 1. 00 mg/ml BJO combined with 0. 4μM Cap induced lower proliferation inhibition rates of LOVO and HT29 than those of either Cap or BJO alone. Combined use of Cap and BJO promoted G1/G0 phase arrest and increased the number of cells in G2/M phase. BJO in combination with Cap significantly inhibited mRNA and protein expressions of β-catenin and Wnt3 a,and decreased mRNA expressions of cyclin D1 and c-Myc in LOVO and HT29 cells. Conclusion: BJO in combination with Cap can inhibit the proliferation of colorectal cancer cells,which may be mediated by activating Wnt/β-catenin signaling pathway and cell cycle arrest.更多还原