Promoting the Anti-Tumor Activity of Radiotherapy on Lung Cancer through a Modified Ketogenic Diet and the AMPK Signaling Pathway

Abstract

Taking advantage of the characteristics of high metabolic heterogeneity of tumor cells, the modified ketogenic diet (KD) combined with radiotherapy was used to investigate and analyze the radiosensitivity of a lung cancer model from the perspective of energy metabolism. Different concentrations of glucose and βhydroxybutyrate (βHB) were used at the cellular level to simulate the level of ketone bodies. A cell counting kit was used to detect the effect of different concentrations of glucose (2.78mM, 5.56mM, 12.5mM, and 25mM) and βHB (0mM, 5mM, and 10mM) combined with radiotherapy on the proliferation of LLC cells. Flow cytometry was used to detect tumor cell cycle and apoptosis. Immunofluorescence was used to detect the expression of γH2AX, a DNA damage marker, and western blot was used to detect the expression of AMPK and ρ-AMPK. At the animal level, C57BL/6J female mice were used to establish a transplanted tumor model of lung cancer, and fed with different fat ratio diets combined with radiotherapy. The volume, tumor size, blood glucose level, blood ketone level, survival time and safety of the mice were monitored and observed. The LLC cells were treated with different concentrations of glucose and βHB. The results showed that the survival rate of LLC cells decreased significantly with the increase of irradiation dose when the glucose concentration was 5.56mM and 2.78mM; However, the survival rate of cells in low glucose medium added with βHB was significantly lower than that of the control group, and the survival rate of LLC decreased significantly with the extension of culture time after irradiation (p < 0.001). After irradiation, LG (low glucose) group, LG+βHB 5mM group and LG+βHB 10mM group had a significantly higher proportion of G2 phase, and a significantly higher proportion of early and late phase than the control group. γH2AX foci were detected in LG group, LG +βHB 5mM group and LG +βHB 10mM group at 2h and 24h after radiotherapy, which were significantly higher than those in the control group (p < 0.05). The median survival time was 38 days in the PT group, 55 days in the PT+RT group, 41 days in the 45F group, and not reached in the 45F+RT group. HE staining showed no tumor metastasis and toxic side effects in liver and kidney. The expression of ρ-AMPK/AMPK in the combined treatment group was higher than that in the other groups. The expression of ρ-AMPK/AMPK in RT, 45F and combined treatment group was higher than that in PT group. The expression of ρ-AMPK/AMPK in RT group was higher than that in 45F group, and the difference was statistically significant (p < 0.01). Modified ketogenic diet can enhance the anti-tumor effect of radiotherapy in LLC tumor-bearing mice by reducing glucose and increasing the energy supply ratio from fat.