己糖激酶-Ⅱ基因在人结肠癌细胞中的表达及其治疗意义

Expression of hexokinase-Ⅱ gene in human colon cancer cells and the therapeutic significance of inhibition thereof

目的检测己糖激酶-Ⅱ基因(HK-Ⅱ)在人结肠癌细胞中的表达,探讨抑制 HK-Ⅱ对结肠癌的治疗效应及机制。方法应用逆转录-聚合酶链反应(RT-PCR)和 Western 印迹检测4种人结肠癌细胞 HK-Ⅱ基因的表达情况。在 HK-Ⅱ抑制剂(3-BrPA)诱导下,通过 MTF 法观察结肠癌细胞增殖和化疗敏感性变化;用琼脂糖凝胶电泳分析细胞凋亡 DNA 片段;底物比色法测定半胱氨酸蛋白水解酶(caspase-3)活性;流式细胞术和紫外分光光度仪分别检测线粒体膜电位(ΔΨm)、线粒体膜通透转换孔(PTP)开放的变化。结果 HK-Ⅱ基因在4种人结肠癌细胞中明显表达。抑制 HK-Ⅱ(3-BrPA)能明显控制结肠癌细胞增殖并诱导细胞凋亡。300μmol/L 3-BrPA 刺激 LOVO、HCT-116、HT-29、SW480细胞48 h 后,细胞增殖抑制率分别为83.7%±2.0%、83.4%±4.1%、89.7%±3.7%和80.6%±0.9%。与小剂量3-BrPA(25 μmol/L)联合作用 LOVO 细胞48 h,低剂量表阿霉素(0.05、0.1μg/ml)的细胞增殖抑制率由5.1%±1.3%和10.5%±2.0%分别增至46.5%±3.2%和57.9%±3.3%(P<0.01);而低剂量 L-OHP(0.5、1.0μg/ml)的细胞增殖抑制率则由19.2%±6.1%和32.2%±2.2%分别提高到48.4%±3.2%和60.5%±4.6%(P<0.01)。50、100、150 μmol/L 3-BrPA 诱导 LOVO 细胞24 h 后,caspase-3活性分别为1.13±0.11、1.60±0.20和3.03±0.11(P=0.000)。3-BrPA 作用 LOVO 细胞线粒体20 min 后,PTP 开放程度为40.0%±3.5%,而对照组(CaCl_2)为37.4%±2.3%,两者相比,差异无统计学意义(P=0.348)。100 μmol/L 3-BrPA 刺激LOVO 细胞1、3、5 h 后,ΔΨm下降率分别为12.7%、15.4%、26.8%。结论 HK-Ⅱ基因可望成为结肠癌的有效治疗靶点。

Objective To investigate the expression of hexokinase （HK）-Ⅱ gene in human colon cancer cells and the therapeutic significance of inhibition of HK-Ⅱ gene. Methods Human colon cancer cells of the lines HCT-116, LOVO, HT-19, and SW480 were cultured. The mRNA expression and protein expression of HK-Ⅱ in these cells were detected by RT-PCR and Western blotting respectively. 3- Bromopyruvic acid （3-BrPA） , a HK-Ⅱ specific inhibitor, of different concentrations was added into the culture fluid of the colon cancer cells for 48h, then MTT method was used to examine the proliferation of the cells. 3-BrPA combined with adriamycin （ ADM ） of the concentrations of 0.05 and 0. 1 μg/ml, or with oxaliplastin （L-OHP） of the concentration of 0. 5 and 1.0 μg/ml was added into culture fluid for 48 h to observe the change of the cell proliferation rate. 3-BrPA of different concentrations was co-cultivated with LOVO cells for 24 h, and then enzyme labeling instrument was used to measure the activity of caspase-3, a cell apoptosis signal. 3-BrPA and CaCl2 were added into the culture fluid of LOVO cells and then ultra-violet spectrum was used to detect the mitochondrial permeability transition pore （PTP） opening degree. Flow cytometry （FCM）, with addition of 10 μg/ml Rh123, was used to measure the mitochondrial membrane potential （△Ψm） of LOVO cells. Results Both HK-Ⅱ mRNA and HK-Ⅱ protein were expressed in the HCT-116, LOVO, HT-19, and SW480 cells. Treated by 3-BrPA combined with ADM of the concentrations of 0. 05 and 0. 1 μg/ml for 48 h, the cell proliferation inhibiting rates in the 4 lines were increased from 5.1%±1.3% and 10.5%±2.0% to46.5% ±3.2% and 57.9% ± 3.3% respectively （all P〈0.01）. Treated by 3-BrPA combined with L-OHP of the concentration of 0. 5 and 1.0 μg/ml for 48 h, the cell proliferation inhibiting rates were increased from 19. 2% ± 6. 1% and 32.2% ± 2.2% to 48.4% ±3.2% and 60. 5% ± 4. 6% respectively （ all P 〈 0. 01 ） . 24 h after the co-cultivation of 3-BrPA of different concentrations, the caspase-3 activity of the LOVO cells was increased along with the increase of the concentration of 3-BrPA （P = 0. 000）. The PTP opening degrees induced by 3-BrPA and CaCl2 of the LOVO cells were 40. 0% ±3.5% and 37. 4% ±2. 3% respectively （P=0. 348）. After treated with 100 μml/L 3- BrPA for 1, 3, and 5 h, the △Ψm decrease rates of the LOVO cells were 12. 7% , 15.4% , and 26. 8% respectively. Conclusion HK-Ⅱ gene may be an effective therapeutic target for gene may be an effective therapeutic target for gene may be an effective therapeutic target for colon cancer.