Ascorbate induces G2/M arrest and necrosis without generation of direct DNA double-strand breaks

Ascorbate is used for combination therapy with promising results in clinical trials. The proposed main ascorbate cytotoxic effect is DNA damage induction through excessive oxidative stress. However, the involvement of DNA double-strand breakage (DSB) formation and repair in ascorbate-induced DNA damage remains to be revealed. We here used wild-type cells and cells with knock-outs (KO) of the key non-homologous end-joining (NHEJ) repair proteins DNA-PKcs and XRCC4 and tested 2D and 3D cell viability, growth, DSB (DNA fragmentation and DSB surrogate markers 53BP1 and γH2AX), apoptosis, necrosis, and cell cycle in response to ascorbate. Ascorbate reduced cell survival and viability in a concentration-dependent manner, with a tendency toward XRCC4 KO cells more sensitivity. Unlike radiation, ascorbate did not produce prompt DSB. However, 24 h after exposure, there was a clear increase in 53BP1 foci in both NHEJ-proficient and KO cells and evident pan-nuclear γH2AX response, especially in XRCC4 KO cells, which may indicate nuclear degradation leading to DSB formation over time. Further, ascorbate induced G2/M arrest with a more pronounced effect in XRCC4 KO cells. For higher doses (>1 mM) we observed a rapid (24 h) necrotic response without activation of apoptosis. The cell line's different response seems to be related to their cell cycle regulation rather than the NHEJ status. These findings provide novel mechanistic insights into DNA damage formation and cellular response to ascorbate. Our data suggest that ascorbate does not generate prompt or direct DSBs. Instead, it induces delayed DSBs stemming from nuclear necrotic degradation, driven by cell cycle regulation rather than NHEJ status. • Ascorbate reduced survival with a tendency toward XRCC4 KO cells more sensitivity. • Unlike radiation, ascorbate did not produce prompt DSB. • 53BP1 foci and pan-nuclear γH2AX indicate necrotic nuclear degradation. • Ascorbate generates G2/M arrest with a more pronounced effect in XRCC4 KO cells. • Different cellular response seems to be related to cell cycle rather than NHEJ.