| Purpose: To determine whether caffeine enhanced radiosensitization in an orthotopic transplant of LM3 human hepatocellular cancer in nude mice and to explore its possible mechanism.Methods and Materials: We infected LM3 hepatocellular carcinoma, a highly metastatic potential cell line, with the red fluorescent protein (RFP)-expressing plasmid DNA via lentiviral vector system to establish stable RFP-expressing LM3 hepatocellular cancer cell. About 1×107 RFP-expressing LM3 hepatocellular cancer cell were subcutaneously injected flank region of mice after conventional culture in vitro, when the tumor reached 1 cm in diameter, it was removed, cut into 2×2×2-mm3-sized pieces, and implanted into the liver, the mice were maintained under pathogen-free conditions after surgery. When the intrahepatic tumor fluorescent area reached approximately 0.5 cm in diameter, the mice were randomly divided into four groups: normal saline (NS); caffeine (Caff) alone; irradiation (IR) alone; and caffeine plus IR (Caff+IR) group. The intrahepatic tumors in both IR groups were exposed to IR of 5 Gy per fraction for three fractions, every other day, for a total of 15 Gy, caffeine in solution at a dose of 100 mg/kg was injected into the stomachs of mice after IR immediately. The red fluorescent areas were imaged before IR and every 3 days after IR and the resulting tumor growth curves were described. All animals were sacrificed on 30 days after IR, tumor volume was calculated, and a tumor volume histogram was described. The mice were sacrificed and tumor tissues were obtained at 12 hours after completion of IR. Cyclin-dependent kinase CDC2-Tyr15, cyclin CyclinB1 and apoptosis-related proteins caspase-3 were detected, and tumor apoptosis were analysed by using of TUNEL staining. Results: Caffeine enhanced radiosensitivity of LM3 hepatocellular cancer in vivo as described on resulting tumor growth curves: tumor growth speed in Caff + IR group was significantly lower than the other three groups. Tumor growth delay time in the Caff+IR group was 14.3 days compared with the NS group, 14.1 days compared with the Caff alone group, and 7.2 days compared with the IR alone group. The tumor volume histogram at the end of experiment also showed that tumor volume in caff + IR group was significantly lower than the other three groups. Immunohistochemical analysis of phospho-CDC2-Tyr15 (CDC2-Tyr15-P) protein: in the IR alone group, the proportion of CDC2-Tyr15-P positive cells with 5-Gy IR was 38.8±9.2%, with 10-Gy IR, 53.0±9.2%, and with 15-Gy IR, 68.4±10.6%; in the Caff+IR group, the proportions of CDC2-Tyr15-P positive cells were significantly lower than in the corresponding IR alone group, with 5-Gy IR, the proportion of CDC2-Tyr15-P positive cells was 24.2±7.3%, with 10-Gy IR, 26.8±8.5%, and with 15-Gy IR, 26.0±8.9%. Immunohistochemical analysis of CyclinB1 protein: in the IR alone group, the proportion of cyclinB1 positive cells with 5-Gy IR was 19.0±8.2%, with 10-Gy IR, 12.4±4.6%, and with 15-Gy IR, 7.0±3.7%; in the Caff+IR group, the proportions of cyclinB1 positive cells were significantly higher than in the corresponding IR alone group, with 5-Gy IR, the proportion of cyclinB1 positive cells was 33.8±9.5%, with 10-Gy IR, 33.4±9.3%, and with 15-Gy IR, 30.4±8.7%. TUNEL staining results: in the IR alone group, the proportion of TUNEL positive nuclei was 18.6±8.2% with 5-Gy IR, 21.2±6.1% with 10-Gy IR, and 24.2±7.2% with 15-Gy IR; in contrast, In the Caff+IR group, the proportions of TUNEL positive nuclei were significantly higher than in the corresponding IR alone group, with 5-Gy IR, the proportion of TUNEL positive nuclei was 31.0±8.8%, with 10-Gy IR, 43.2±10.9%, and with 15-Gy IR, 59.2±9.5%. caspase-3 protein expression was consistent with the TUNEL staining results.conclusion : The study proved caffeine might abrogate the radiation-induced G2 phase arrest of orthotopic transplant of LM3 human hepatocellular cancer by inhibiting phospho-CDC2-Tyr15 proteine expression levels and increasing cyclinB1 proteine expression level after IR, finally resulting in apoptosis of LM3 human hepatocellular cancer in vivo, enhancement of radiosensitivity might be due to decrease of DNA repair time during G2 arrest. Caffeine enhance radiosensitivity of orthotopic transplant of LM3 human hepatocellular cancer, and provide the feasibility of further clinical application.
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