Radiosensitization By Low Dose Gemcitabine On Human Hepatoma Cell Lines SMMC-7721

OBJECTIVE To explore the the radiosensitivity of low-dose gemcitabine (GEM) on human hepatocellular carcinoma cell line SMMC-7721 and its possible mechanism in vitro.MATERIALS AND METHODSCell linesThe human hepatocellular carcinoma cell lines SMMC-7721 were used in this study. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum in a humidified atmosphere containing 5% CO₂. Reagents and Radiation methodsPut the cells into four groups, (1) control group; (2) Radiation with 2Gy, 4Gy, 6Gy, 8Gy, 10Gy without Gemcitabine; (3) incubated with Gemcitabine at 0, 0. 001mg/ml, 0.01⁰.05 mg/ml and 0.1mg/ml without irradiation; (4) both Radiation and Gemcitabine (with different dose and different concentration). The cells were irradiated with a VARIAN (2100C) accelerator that was operated at 6MV X-ray and source-axis distance (SAD) of 100cm and field of 10cm* 10cm at a dose rate of 400cGy/min.Irridiation was performed in air at room temperature. Thenwe measured the cells surviving fraction by the mean of MTT; and made cells survival curve by colony forming method, and determined cell cycle with flow cytometry, and observed the morph changes of every group cells by optical microscope, transmission electron microscope and fluorescence microscope. DetectionThen we measured the cells surviving fraction by the mean of MTT; and made cells survival curve by colony forming method, and determined cell cycle with flow cytometry, and observed the morph changes of every group cells by optical microscope, transmission electron microscope and fluorescence microscope. Statistical analysisEach date point was based on three independent experiments. Statistical analysis was performed by ANOVA test with the software SPSS10.0. RESULTS CytotoxicityAfter 6 days of incubated with Gemcitabine, the surviving fraction of the cells of the other three groups was lower than that of the control group. The difference of surviving fraction of each two groups is significant (P<0.001). The inhibiting effect on surviving fraction of Gemcitabine was increased with increasing of concentration of the Gemcitabine in medium. The inhibiting effect on surviving fraction of Gemcitabine was best at 0.01-0.05mg/ml. Clonogenic AssaysGemcitabine markedly decreased the colony forming of the irridiated SMMC-7721 cells, the sensitization enhancement ratios (SER) was 1.92 ±0. 18. Flow CytometryGemcitabine enhanced S-phase blocking in SMMC-7721 cells. The ratio of the cells which was in S-phase was 43.5% in the group with 4Gy irradiation, and that was 56.4% in the group with 4Gy irradiation combined with Gemcitabine at 0.05mg/ml (P<0.05). ApoptosisMicrodosage GEM can increase apoptosis of SMMC-7721 induced by radiation significantly. It is clear that the apoptosis of radiation group (18.7%) is higher than that of radiation + GEM group (5.8%). CONCLUSIONSLow dose Gemcitabine has an enhancing effect on radiosensitivity in SMMC-7721 cell lines in vitro. The mechanism of radio potentiation by Gemcitabine probably enhanced S-phase blocking in SMMC-7721 cells to increase the ratio of radiosensitive cells by redistributing within the cell cycle. In addition, Gemcitabine had the ability to reduce the repair of radiation damage. Effect on of gemcitabine was increased with increasing of concentration of the gemcitabine in medium.