Caffeine enhances radiosensitization to orthotopic transplant LM3 hepatocellular carcinoma in vivo

Abstract

The aim of this study was to determine whether caffeine enhanced radiosensitization in an orthotopic transplant of LM3 human hepatocellular cancer in nude mice. LM3 hepatocellular carcinoma cells were infected with red fluorescent protein and irradiated, and cell cycle distribution and survival fraction were detected. A nude mouse model of orthotopic transplant of red fluorescent protein-expressing LM3 hepatocellular cancer was established. Nude mice were divided into four groups: control (NS); caffeine (Caff) alone; irradiation (IR) alone; and caffeine + IR (Caff + IR). Tumor growth curves were described. Expression of cyclin and apoptosis were evaluated by analysis of phosphorylated cyclin dependent kinase 1 (CDC2) Tyr15 (CDC2-Tyr15-P), cyclinB1, TUNEL staining, and caspase-3. Caffeine abrogated IR-induced G(2) phase arrest and decreased survival of irradiated LM3 cells. Caffeine enhanced radiosensitivity of LM3 hepatocellular cancer in vivo. 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. At 15 Gy, expression of CDC2-Tyr15-P in the Caff + IR group (26.0 +/- 8.9%) was significantly lower than in the IR alone group (68.4 +/- 10.6%), expression of cyclinB1 and proportion of TUNEL-positive cells in the Caff + IR group (30.4 +/- 8.7% and 59.2 +/- 9.5%, respectively) was significantly higher than in the IR alone group (7.0 +/- 3.7% and 24.2 +/- 7.2%, respectively), expression of caspase-3 was consistent with the TUNEL staining results. This study suggested that caffeine might enhance the radiosensitivity of LM3 hepatocellular cancer in vivo, and may be feasible for further clinical applications.

Figure 1

Caffeine abolished radiation‐induced G₂ phase arrest in LM3 hepatocellular carcinoma cells. (A) The proportion of cells in G₁ phase arrest and G₂ phase arrest as a function of dose 16 h after irradiation (IR). (B) The proportion of cells in G₂ phase arrest as a function of time after 12 Gy IR with or without caffeine (Caff). Data are expressed as the mean ± SD for five independent experiments. *P < 0.05 compared with the IR alone group; **P < 0.01 compared with the IR alone group.

Figure 2

Clonogenic surviving fraction of LM3 hepatocellular carcinoma cells following treatment with irradiation (IR) alone or IR with 2.5 mM caffeine (Caff). The surviving fraction of cells was normalized by the occurrence of cell death in the group treated with 0 Gy IR. Data are expressed as the mean ± SD for five independent experiments.

Figure 3

Caffeine (Caff) sensitized hepatocellular tumor tissues to ionizing irradiation (IR). (A) Tumor fluorescence images before IR and at day 30 after IR. (B) Tumor growth curves. (C) Tumor volume histogram at the end of the experiment. Data are expressed as the mean ± SD (n = 10 per group). NS, normal saline. *P < 0.05 compared with the IR alone group; **P < 0.01 compared with the IR alone group.

Figure 4

Phosphorylated cyclin dependent kinase 1 (CDC2) Tyr15 (CDC2‐Tyr15‐P) protein expression in LM3 hepatocellular carcinoma cells and the effect of irradiation (IR) with or without caffeine (Caff). (A) Western blot analysis of CDC2‐Tyr15‐P expression at 0 Gy, 12 h and 36 h after 5‐Gy IR, and 12 h after 10‐Gy and 15‐Gy IR. (B) Immunohistochemical analysis of CDC2‐Tyr15‐P positive cells at 0 Gy, 12 h after 5‐Gy, 10‐Gy, and 15‐Gy IR. The number of positive cells in 10 randomly selected areas was counted under the microscope (original magnification, ×200). Data are expressed as the mean ± SD (n = 5 per group). NS, normal saline. *P < 0.05 compared with the IR alone group; **P < 0.01 compared with the IR alone group.

Figure 5

CyclinB1 protein expression in LM3 hepato‐cellular carcinoma cells and the effect of irradiation (IR) with or without caffeine (Caff). (A) Western blot analysis of cyclinB1 expression at 0 Gy, 12 h and 36 h after 5‐Gy IR, and 12 h after 10‐Gy and 15‐Gy IR. (B) Immunohistochemical analysis of cyclinB1‐positive cells at 0 Gy, 12 h after 5‐Gy, 10‐Gy, and 15‐Gy IR. The number of positive cells in 10 randomly selected areas were counted under the microscope (original magnification, ×200). Arrows indicate cyclinB1‐positive cells. Data are expressed as the mean ± SD (n = 5 per group). NS, normal saline. *P < 0.05 compared with the IR alone group; **P < 0.01 compared with the IR alone group.

Figure 6

Caffeine (Caff) enhanced apoptosis to ionizing irradiation (IR) in LM3 hepatocellular carcinoma cells. (A) Results of TUNEL assay at 0 Gy, 12 h after 5‐Gy, 10‐Gy, and 15‐Gy IR with or without caffeine. The number of positive cells in 10 randomly selected areas was counted under the microscope (original magnification, ×200). (B) Western blot analysis of caspase‐3 expression at 0 Gy, 12 h after 5‐Gy, 10‐Gy, and 15‐Gy IR with or without caffeine. Data are expressed as the mean ± SD (n = 5 per group). NS, normal saline. *P < 0.05 compared with the IR alone group; **P < 0.01 compared with the IR alone group.