Abstract
Scope Human neoplastic transformation due to DNA damage poses an increasing global healthcare concern. Maintaining genomic integrity is crucial for avoiding tumor initiation and progression. The present study aimed to investigate the efficacy of an apple flavonoid fraction (AF4) against various carcinogen-induced toxicity in normal human bronchial epithelial cells and its mechanism of DNA damage response and repair processes. Methods and Results AF4-pretreated cells were exposed to nicotine-derived nitrosamine ketones (NNK), NNK acetate (NNK-Ae), methotrexate (MTX), and cisplatin to validate cytotoxicity, total reactive oxygen species, intracellular antioxidants, DNA fragmentation, and DNA tail damage. Furthermore, phosphorylated histone (γ-H2AX) and proteins involved in DNA damage (ATM/ATR, Chk1, Chk2, and p53) and repair (DNA-PKcs and Ku80) mechanisms were evaluated by immunofluorescence and western blotting, respectively. The results revealed that AF4-pretreated cells showed lower cytotoxicity, total ROS generation, and DNA fragmentation along with consequent inhibition of DNA tail moment. An increased level of γ-H2AX and DNA damage proteins was observed in carcinogen-treated cells and that was significantly (p ≤ 0.05) inhibited in AF4-pretreated cells, in an ATR-dependent manner. AF4 pretreatment also facilitated the phosphorylation of DNA-PKcs and thus initiation of repair mechanisms. Conclusion Apple flavonoids can protect in vitro oxidative DNA damage and facilitate repair mechanisms.
Highlights
Mammalian genomic DNA is susceptible to various environmental, cytotoxic, or genotoxic agents that sense DNA damage and activate signaling cascades for effective repair mechanisms
We investigated the effects of AF4 on normal human bronchial epithelial cells (BEAS-2B) challenged with known carcinogenic chemical agents such as 4-(methylnitrosamino)-1-(3-pyridyl-d4)-1-butanone (NNK), 4-[(acetoxymethyl) nitrosamino]-1-(3-pyridyl)-1-butanone (NNK acetate; nitrosamine ketones (NNK)-Ae), methotrexate (MTX), and cisplatin
AF4 pretreatment did not show any significant reduction in cytotoxicity for cisplatin-treated cells and found to be morphologically distinct with rounded-shape or detached cells
Summary
Mammalian genomic DNA is susceptible to various environmental, cytotoxic, or genotoxic agents that sense DNA damage and activate signaling cascades for effective repair mechanisms. Under a normal circumstance with a specific type of DNA lesion, DNA damage is commonly repaired through nonhomologous end joining (NHEJ)/homologous recombination (HR) mechanisms [1, 2]. Alkylating agents, platinum drugs, antimetabolites, topoisomerase inhibitors and ionizing radiations, nitrosoureas, aziridine compounds, alkyl sulphonates, and triazine compounds are some of the electrophiles that covalently transfer alkyl-groups onto the DNA bases, disrupting the DNA helix and induces DNA breaks [3]. DNA double-strand breaks (DSBs) are the most lethal lesions that can result in mutations, chromosomal aberrations, and cell death [4, 5]. Maintaining genomic integrity possess global healthcare challenge and should be well addressed
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