L-arginine synergistic with 5-fluorouracil intervenes in DNA damage repair via the DNA-PKcs/ATM/ATR pathway in hepatocellular carcinoma cells.

Abstract

DNA damage repair is a critical physiological process. The combined treatment of L-Arg and 5-FU significantly inhibits cell proliferation, enhances nitric oxide (NO) production via inducible nitric oxide synthase (iNOS), and promotes the accumulation of reactive oxygen species (ROS). This heightened oxidative stress triggers DNA damage and apoptosis, as evidenced by a substantial increase in the Bax/Bcl-2 ratio; the activation of caspase-9, caspase-3, and PARP cleavage; and increased level of phosphorylated p53. Moreover, the combination treatment induces G2/M phase arrest, with a significant increase in p-H2AX (Ser 139) (known as γ-H2AX) expression, indicating extensive DNA damage. Mechanistically, the combined treatment modulates DNA damage response pathways by downregulating DNA-PKcs. Concurrently, it enhances the phosphorylation of ATM, ATR, CHK1, CHK2, and BRCA1. Additionally, the L-Arg and 5-FU combination downregulates PI3K/AKT signaling. AZD-7648 (a DNA-PKcs inhibitor) and LY294002 (a PI3K inhibitor) enhance p-ATM and p-ATR activation, resulting in elevated apoptosis and increased γ-H2AX expression. In contrast, the inhibition of ATM/ATR by CGK733 suppresses this response, reducing apoptosis and DNA damage signaling. Additionally, the ROS scavengers NAC and iNOS, when applied separately, restore p-AKT and DNA-PKcs expression; suppress the upregulation of p-ATM, p-ATR, and γ-H2AX; and ultimately reduce apoptosis. These findings are validated in a DEN-induced rat liver cancer model. In summary, 5-FU and L-Arg synergistically increase iNOS/NO-driven ROS accumulation, inducing γ-H2AX-marked DNA damage through dual modulation of repair pathways (inhibiting PI3K/AKT/DNA-PKcs while activating ATM/ATR), ultimately triggering p53-mediated G2/M arrest and apoptosis in hepatocellular carcinoma cells.