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-arginine (L-Arg) and 5-fluorouracil (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.

1. Introduction

Liver cancer is one of the most prevalent and lethal malignant tumors and is the second leading cause of cancer-related death worldwide [1]. Among primary hepatic tumors, hepatocellular carcinoma (HCC) accounts for approximately 90% of cases. Standard interventions for early-stage liver cancer include surgical resection, localized ablation procedures, and liver transplantation. However, many patients are diagnosed at an advanced stage of the disease. The principal treatment modalities available encompass immunotherapy, radiotherapy, and chemotherapy. First-line chemotherapeutic agents, including anti-metabolites such as 5-fluorouracil (5-FU), anti-angiogenic agents such as sorafenib, and programmed death inhibitors such as atezolizumab, primarily target deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) [2].

Repairing DNA damage represents one of the body’s most common physiological processes, occurring predominantly through homologous recombination (HR) and non-homologous end joining (NHEJ) [3]. Mammalian cells have evolved sophisticated DNA repair mechanisms to address various forms of DNA damage—including mismatch repair, base excision repair, and nucleotide excision repair [4]. The key components involved in these processes include ataxia-telangiectasia mutated protein (ATM), ATM- and Rad3-related protein (ATR), and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). These proteins are integral to the PI3K-associated kinase family and play crucial roles in the HR and NHEJ pathways, which are essential for effective DNA damage repair [5]. Inhibition of DNA-PKcs or activation of ATM/ATR via phosphorylation impedes cellular proliferation while influencing DNA damage correction mechanisms. Phosphorylation events involving these proteins constitute critical steps within the DNA damage response pathway (DDR), a comprehensive signaling framework that cells have developed to endure adverse conditions while ensuring accurate transmission of genetic information to subsequent generations [6]. When DNA-PKcs is inhibited, it may result in a blockade within the NHEJ pathway—one of the primary mechanisms by which cells manage double-strand breaks (DSBs) in their genomic material [7]. Additionally, ATM and ATR activation play critical roles in the homologous recombination (HR) pathway, initiating a cascade of complex phosphorylation events that can arrest the cell cycle at the G1/S or G2/M transitions [8,9]. Consequently, the DNA-PKcs/ATM/ATR pathway represents a promising therapeutic target for cancer treatment. Upon DNA damage, the p53 protein undergoes phosphorylation, stabilizing it and promoting its nuclear accumulation. This activation induces the expression of genes associated with cell cycle arrest and apoptosis [10]. Simultaneously, DNA damage triggers the release of cytochrome c from mitochondria, activating caspase-9. Caspase-9 subsequently activates caspase-3, a key effector enzyme.