• The combination of L-arginine and 5-fluorouracil synergistically inhibits HCC cell proliferation by inducing oxidative stress, DNA damage, and apoptosis.
• Mechanistically, the treatment downregulates DNA-PKcs and PI3K/AKT signaling while activating ATM/ATR and downstream checkpoint kinases, leading to G2/M arrest.
• Pharmacological inhibition of DNA-PKcs or PI3K enhances the therapeutic effect, whereas ATM/ATR inhibition or ROS scavenging reverses it, confirming the pathway's role.
• In vivo validation in a DEN-induced rat liver cancer model supports the translational potential of this combination strategy for HCC therapy.