Apple Flavonoids Suppress Carcinogen-Induced DNA Damage
Apple Flavonoids Suppress Carcinogen-Induced DNA Damage in Human Bronchial Epithelial Cells
The work Apple Flavonoids Suppress Carcinogen-Induced DNA Damage in Normal Human Bronchial Epithelial Cells explores the potential of flavonoids found in apple skins to reduce DNA damage in normal human bronchial epithelial cells induced by carcinogenic chemicals. Genomic instability is a key factor in cancer initiation and progression, so the selection of natural compounds that are able to improve DNA repair or decrease DNA damage is a relevant approach in chemoprevention. The authors characterize an apple flavonoid fraction (AF4), previously reported as exerting antioxidant, anti-inflammatory and neuroprotective effects, and test the capacity of AF4 to counteract genotoxic stress in normal human bronchial epithelial cells (BEAS-2B).
This study starts by describing how mammalian DNA is constantly under attack from harmful agents such as nitrosamines, chemotherapeutic drugs and environmental toxins. These agents result in the formation of reactive oxygen species (ROS) that can cause DNA strand breakage and double-strand breaks (DSBs). When DSBs occur, cells trigger the DNA damage response (DDR), a signaling pathway driven by ATM, ATR, CHK1, CHK2, p53 and γ-H2AX. In cases of severe damage, cells die by apoptosis; in cases of successful repair, genomic stability is restored. The authors hypothesize that AF4 may decrease ROS levels and prevent DNA fragmentation, inhibit activation of the DDR, and promote expression of DNA repair proteins such as DNA-PKcs and Ku80.
To examine this hypothesis, BEAS-2B cells were pretreated with 50 (μg/mL) AF4 and then exposed to four carcinogens: NNK, NNK-acetate (NNK-Ae), methotrexate (MTX), and cisplatin. The MTS assay was used to assess cytotoxicity. The level of ROS was determined by measuring DCFH-DA fluorescence. Western blotting was used to measure antioxidant enzymes (SOD1, catalase and GPX1). The evaluation of DNA damage was performed by γ-H2AX immunofluorescence and comet assay. ELISA was used to measure DNA fragmentation. The activation of the DDR was evaluated by Western blotting of phosphorylated ATM, ATR, CHK1, CHK2, p53 and γ-H2AX. The activation of the DNA repair machinery was evaluated using Ku80 expression levels and DNA-PKcs phosphorylation.
AF4 was found to be effective in significantly reducing the cytotoxic effects of NNK-Ae, MTX and NNK. However, cisplatin remained quite toxic even after AF4 pretreatment, suggesting that AF4 is not effective against all types of DNA damage. ROS analysis indicated that oxidative stress caused by carcinogens almost doubled, while AF4 pretreatment restored ROS levels close to the control. Additionally, AF4 resulted in higher intracellular antioxidant levels, including induction of SOD1, implying improved resistance to oxidative stress. The DNA damage assays showed that NNK-Ae, MTX and cisplatin generated significant γ-H2AX formation, characteristic of DSBs. For NNK-Ae and MTX, pretreatment with AF4 was protective at the histone level, with a significant decrease in γ-H2AX foci. These results were confirmed by comet assay: AF4 decreased DNA tail damage from 97% (NNK-Ae) and 68% (MTX) to much lower levels. AF4 by itself was not found to be a DNA-damaging agent, showing that it is not toxic to normal cells.
Using DNA fragmentation analysis, it was found that carcinogen-induced apoptotic DNA cleavage was prevented by AF4. This points to the role of AF4 in preserving DNA integrity and preventing the triggering of early apoptotic pathways by genotoxic stress.
DDR signaling analysis demonstrated that NNK-Ae and MTX induced activation of ATR, CHK1, CHK2, p53 and γ-H2AX. ATR phosphorylation and downstream checkpoint activation were inhibited by AF4 pretreatment, suggesting less DNA damage. Interestingly, phosphorylation of a key protein in the non-homologous end joining (NHEJ) repair pathway was increased by AF4. AF4 was unable to activate DNA-PKcs when NU7026 was used as an inhibitor, showing that AF4 stimulates repair pathways rather than simply decreasing damage.
In conclusion, the study indicates that apple flavonoids have an anti-carcinogenic effect on normal lung epithelial cells through their ability to inhibit the production of free radicals. AF4 inhibits ROS formation and prevents DNA fragmentation, inhibits DDR activation and promotes DNA repair through phosphorylation of DNA-PKcs. The results indicate that apple flavonoids may have the potential to serve as natural chemopreventive agents that keep the genome stable when exposed to carcinogenic stresses.
Critical Analysis: There was strong evidence from this study that apple flavonoids protect normal cells against genotoxic agents. Multiple assays (cytotoxicity, ROS measurement, γ-H2AX staining, comet assay, DDR protein analysis and activation of DNA-PKcs) provide a comprehensive picture of AF4’s protective mechanisms. The mechanistic understanding gained from both ATR suppression and activation of DNA-PKcs is of particular value, as it demonstrates that AF4 can not only prevent damage but also promote repair. However, the study is only in vitro, and the bioavailability of AF4 in humans is not known. Flavonoids are frequently extensively metabolized into conjugates that may have different activity in vivo. Further, the resistance of cisplatin to AF4 protection indicates that flavonoids do not protect against all genotoxic agents. AF4 should be studied in animal models, in long-term dietary intake studies, and to identify the specific flavonoids that most contribute to activation of DNA repair. Nevertheless, this study demonstrates that apple flavonoids have a potential role in chemoprevention by lowering oxidative stress, preventing DNA damage and facilitating repair.