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

ABSTRACT

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 48 h, 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 microg/ml, or with oxaliplatin (L-OHP) of the concentration of 0.5 and 1.0 microg/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 microg/ml Rh123, was used to measure the mitochondrial membrane potential (DeltaPsim) 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 microg/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% to 46.5% +/- 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 microg/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 CaCl(2) of the LOVO cells were 40.0% +/- 3.5% and 37.4% +/- 2.3% respectively (P = 0.348). After treated with 100 microml/L 3-BrPA for 1, 3, and 5 h, the DeltaPsim 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.