A Cell System-Assisted Strategy for Evaluating the Natural Antioxidant-Induced Double-Stranded DNA Break (DSB) Style

Abstract

Natural antioxidants derived from plants exert various physiological effects, including antitumor effects. However, the molecular mechanisms of each natural antioxidant have not yet been fully elucidated. Identifying the targets of natural antioxidants with antitumor properties in vitro is costly and time-consuming, and the results thus obtained may not reliably reflect in vivo conditions. Therefore, to enhance understanding regarding the antitumor effects of natural antioxidants, we focused on DNA, one of the targets of anticancer drugs, and evaluated whether antioxidants, e.g., sulforaphane, resveratrol, quercetin, kaempferol, and genistein, which exert antitumor effects, induce DNA damage using gene-knockout cell lines derived from human Nalm-6 and HeLa cells pretreated with the DNA-dependent protein kinase inhibitor NU7026. Our results suggested that sulforaphane induces single-strand breaks or DNA strand crosslinks and that quercetin induces double-strand breaks. In contrast, resveratrol showed the ability to exert cytotoxic effects other than DNA damage. Our results also suggested that kaempferol and genistein induce DNA damage via unknown mechanisms. Taken together, the use of this evaluation system facilitates the analysis of the cytotoxic mechanisms of natural antioxidants.

Arsenic affects homologous recombination and single‐strand annealing but not end‐joining pathways during DNA double‐strand break repair

“…1A). We previously reported that LIG4 À/À or RAD54 À/À cells exhibit greater sensitivity to DSBinducing agents than do wild-type cells [14,17]. Thus, our results suggest that the cytotoxicity associated with arsenic exposure is not caused by DSB induction per se.…”

“…Double-strand breaks are the most lethal type of DNA lesions, occurring as one-or two-ended [10][11][12][13]. Ionizing radiation and DNA topoisomerase II (Top2) inhibitors (such as etoposide) induce two-ended DSBs [14]. One-ended DSBs are unique in that they arise when replication forks collide with single-stranded breaks or DNA crosslinks [14].…”

“…Ionizing radiation and DNA topoisomerase II (Top2) inhibitors (such as etoposide) induce two-ended DSBs [14]. One-ended DSBs are unique in that they arise when replication forks collide with single-stranded breaks or DNA crosslinks [14]. In human cells, DSBs can be repaired by either of the four different pathways: homologous recombination (HR), non-homologous end joining (NHEJ), polymerase theta-mediated end joining (TMEJ), and single-strand annealing (SSA) [10][11][12][13].…”

“…SSA utilizes homologous sequences in the vicinity of DSBs to directly join the ends; thus, repair by SSA is accompanied by longer deletions relative to those introduced during NHEJ or TMEJ [13]. The repair of one-ended DSBs via NHEJ, TMEJ, and SSA can be particularly cytotoxic; for example, NHEJ-deficient cells exhibit resistance to one-ended DSB-inducing agents, such as camptothecin and poly(ADP-ribose) polymerase (PARP) inhibitors [14,16,17]. Therefore, HR is important for survival of cells treated with one-ended DSBinducing agents [17,18].…”