αKG-induced oxidative stress and mTOR inhibition as a therapeutic strategy for liver cancer

Abstract

Despite the availability of targeted therapies, liver cancer remains a severe health burden. The need for adjuvant therapy to improve treatment efficacy and prevent recurrence is emerging. Alpha-ketoglutarate (αKG) is an intermediate in the tricarboxylic acid cycle and a cofactor for various oxygenases. A critical role of this multifunctional metabolite has started to be revealed in physiological and pathological conditions. We found that αKG exerts various anti-tumor effects in liver cancer cells. Our kinetic transcriptome study suggested that increasing reactive oxygen species and inhibiting mTORC1 signaling underlies. Indeed, αKG treatment elevated oxidative stress and induced DNA damage, presumably caused by early downregulation of the antioxidant gene SLC7A11. Further, we validated impaired mTOR signaling and decreased cellular energy production. This unique mechanism underscores αKG's potential as a liver cancer therapy by harnessing oxidative stress and disrupting metabolic signaling. These findings could provide valuable insights into further exploration of αKG as a promising therapeutic agent in liver cancer.

Keywords: ATP; Alpha-Ketoglutarate; Anti-tumor; Liver cancer; ROS; mTOR.

Fig. 1

αKG exhibits anti-tumor effects. A Dose-dependent effect of DM-αKG on the proliferation of HepG2 and Huh7 cells (mean ± SEM, ***p < 0.001, n = 3). B Time-dependent effect of DM-αKG on the proliferation of HepG2 and Huh7 cells (mean ± SEM, ***p < 0.001, n = 3). C Protein levels of Caspase-3, Cleaved Caspase-3, and Beta actin in HepG2 and Huh7 cells following DM-αKG treatment. D Time-dependent induction of apoptosis in HepG2 and Huh7 cells treated with DM-αKG. E Time-dependent effect of DM-αKG on wound healing in HepG2 and Huh7 cells (mean ± SEM, **p < 0.01, ***p < 0.001, n = 3) (Scale bar = 500 μm)

Fig. 2

DM-αKG alters transcriptome patterns in liver cancer cells. A Protein levels of HIF-1α, p53, p21, LC3B-I, LC3B-II, and Beta actin in HepG2 and Huh7 cells following DM-αKG treatment. B Schematic representation of the experimental design for DM-αKG treatment in liver cancer cells. C MDS plot showing transcriptome clustering of Control and DM-αKG-treated samples at different time points (3, 6, 9, 12, 24, and 48 h). D Expression changes in the ferroptosis-related gene set at early, intermediate, and late time points following DM-αKG treatment. Fold changes were calculated by normalizing each time point to the control condition. E GSEA analysis following DM-αKG treatment. F Gene expression changes in HepG2 cells after DM-αKG treatment. Time points (early, intermediate, and late) are categorized into upregulated and downregulated genes relative to the control (0 h), represented by eight groups. Fold changes were calculated by normalizing each time point to the control condition. U indicates upregulation, while D indicates downregulation. G Hallmark pathway analysis of genes consistently upregulated (UUU) across early, intermediate, and late time points following DM-αKG treatment in HepG2 cells. H Hallmark pathway analysis of genes consistently downregulated (DDD) across early, intermediate, and late time points following DM-αKG treatment in HepG2 cells

Fig. 3

αKG treatment increases ROS levels. A ROS levels measured by DCF fluorescence intensity in HepG2 and Huh7 cells following DM-αKG treatment (mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, n = 3). B Representative images of comet assay illustrating the degree of DNA damage and quantification of comet tail moments in HepG2 and Huh7 cells after DM-αKG treatment (mean ± SEM, *p < 0.05, ***p < 0.001, n = 3) (Scale bar = 200 μm). C Fold changes in ROS regulator expression based on microarray analysis following DM-αKG treatment. D Relative SLC7A11 expression in HepG2 and Huh7 cells following DM-αKG treatment (mean ± SEM, ***p < 0.001, n = 3). E Protein levels of H3K4me3, H3K9me3, and H3 in HepG2 and Huh7 cells after DM-αKG treatment

Fig. 4

αKG suppresses mTOR signaling in liver cancer cells. A GSEA analysis following DM-αKG treatment. B Protein levels of p-S6, S6, p-4EBP1, 4EBP1, and Beta actin in HepG2 and Huh7 cells following DM-αKG treatment. C Gene expression changes observed at early time points post-DM-αKG treatment. The corresponding gene list is provided in the supplementary Table 2–5. D Schematic representation of the experimental design and ATP levels in HepG2 and Huh7 cells following DM-αKG treatment and under starvation conditions. E Summary diagram illustrating the overall effects of αKG treatment in liver cancer cells