Experimental Study Of Radiosensitivity Mechanism Of Gefitinib On Human Hepatocellular Carcinoma Bel7402

The liver was once considered as a radiation resistant organ, but in mo-dern basical and clinical research it is considered that the liver cells are late responding tissues. Theα/βof liver cells is greater than 10Gy. It indicate that the liver is an adiation-sensitive tissue, similar to poorly differentiated squamous cell carcinoma. EGFR overexpress in heaptocellular carcinoma, and it's closed with invasive tumor growth characteristics. In EGFR overe-xpressed tumor cells, the therapeutic dose of radiation can increase EGFR expression without corresponding ligand. It's a protective effect that the ra-diation activeted EGFR induced cell proliferation and increased DNA repair capacity. To enhance radiosensitivity, EGFR could be a therapeutic target.In our experiment, it was observed that the radiosensitivity of small molecule EGFR tyrosine kinase inhibitor Gefitinib on human hepatocellular carcinoma Be17402 cell line the in vitro activity.and tested that Gefitinib involved in the control DNA damage repair protein Mre11 expression or not. Further study involved in the mechanism of radiosensitization and provide theoretical basis to use Gefitinib as radiosensitizing agent.Objective:To explore whether Gefitinib can enhance the Radiosensiti-zation to human hepatocellular carcinoma Be17402 cell line in vitro and its potential mechanism.Methods:1. The inhibitory effect of Gefitinib on cell proliferation was measured by MTT assay, and to learn the value of IC50 and the degree of inhibition by different concentration of drug.2. Clonogenic assay:By choosing the different concentrations of Gefitinib combined with different doses of radiation therapy acting on cells to observe the number of cell cloning to calculate cell survival curve. Through the comparison to the relevant parameters of curve and the sensiti-zation enhancement ratio to observe the drugs on human heaptocellular carcinoma Be17402 cell line sensitizing effect of radiation.3. The effects of Gefitinib in combination with radiation on cell cycle distribution were analyzed by flow cytometry.4. The effects of Gefitinib in combination with radiation on the expres-sion of Mre11 protein was determined by western blot.Results:1. MTT experimental results showed that Gefitinib used alone has sign-ificantly inhibited effect on the human hepatocellular carcinoma Be17402 cell line in vitro. The efficiency of inhibitions is related the concentrations of the drug. The 50% inhibition concentrations (IC50) of Gefitinib were 7.26μmol/L for Be17402 cells. Form the cell survival curves, its effect of inhib-ition, likely linear relationship with its concentrations. The higher the conc-entration, the stronger the inhibition.2.Be17402 cells were cultured to Gefitinib in the concentrations (0.1×IC50, 0.2×IC50) and 6Gy irradiation, then were determined the number of cell clone and survival rate and the radiosensitivity parameters. Com-pared to the control group, there was significant difference between 0.1×IC50Gefitinib group and 0.2×IC50 Gefitinib group. (P<0.05). The radiosen-sitization enhancement ratios (SER) of Be17402 were 1.237 and 1.3456. Form the cell survival curves, the mean lethal dose(Do) was 3.1475Gy, the quasi-threshold dose(Dq) was 3.5055Gy, irradiation 2Gy survival rates (SF2) 0.8995 higher than two Gefitinib concentrations(0.1×IC50 and 0.2×IC50) combination with radiation treatment group. The effect of radiotherapy, likely linear is relationship with its concentrations. The higher the concent-ration is, the stronger the radiosensitivity is. The effect of synergism of ra-diotherapy and Gefitinib was clear and show some dose dependent in the human heaptocellular carcinoma Be17402 cell line in vitro.3. After Gefitinib monotherapy, G0/G1 phase cells of Be17402 was increased, G2/M phase cells increased slightly, while S phase cells decr-eesed. After irradiation alone, G2/M phase cells increased significantly. while the proportion of the S phase cells was reduced. When Gefitinib combination with radiotherapy, beside G2/M phase cells was significantly increased, G0/G1 phase and S phase cells were decreased and S phase was significantly lower than Gefitinib monotherapy or radiotherapy.4. The expression of Mre11 protein with the Gefitinib monotherapy than the control group no significant change in human hepatocellular carci-noma Be17402 cell line. Radiotherapy alone or combined with 0.2×IC50 Gef-itinib could reduced the expression of Mre11 protein of Be17402.Conclusion:1. Gefitinib could significantly inhibit the proliferation of Be17402 cells. IC50 is 7.26μmol/L, proliferation inhibition rate increased along with increased concentrion of Gefitinib.2. The radiosensitivity effcet of Be17402 cells can be enhanced by Gefitinib concentrations in the low cytotoxicity.The effect of radiosensit-ization increased gradually with the increment of concentrion. Survival curves and cell biololgical parameters of radiation showed that enhanced their radiosensitivity.3. Gefitinib synergy with radiation in human hepatocellular carcinoma Be17402 cell line radiation-sensitive G2/M phase arrest increased and radio-tolerance in S phase were decreased.4. The expression of Mre11 protein with the Gefitinib monotherapy than the control group no significant change in human hepatocellular carci-noma Be17402 cell line. Radiotherapy alone or combined with 0.2×IC50 Gefitinib could reduced the expression of Mre11 protein of Be17402. The radiosensitivity effcet of Gefitinib is probably associated with down regul-ating expression of Mre11 protein.