SENOLYTIC ACTIVITY OF FLAVONOIDS IN ENHANCING TYPE-I AND TYPE-II CELL DEATH IN HUMAN RADIORESISTANT CELL LINES

The role of autophagy and senescence as a cellular response to genotoxic stress in the context of cancer resistance to DNA-damaging agents such as ionizing radiation represents still a limited explored field. We recently characterized new cellular models resistant to clinically relevant doses of ? radiations and derived from human osteosarcoma SAOS and colon adenocarcinoma HT29. These new cell lines, namely SAOS400 and HT500, have been employed to study the regulation of autophagy, senescence and apoptosis following treatments with ionizing radiation [1]. We also provided data on the role of natural flavonoids, such as quercetin and fisetin, being well-known senolytic agents able to selectively eliminate senescent cells and bypass the resistance to genotoxic stress [1-3]. Here, we report that radioresistant HT500 cells were characterized by high levels of SA ?-gal (Senescence-Associated ?-galactosidase) activity, commonly associated with a specific type of senescence known as "therapy-induced senescence" (TIS)and that quercetin and fisetin can reduce the expression of this marker. Therefore, we hypothesized that the biochemical mechanisms responsible for the radiosensitising effects of senolytic agents could intercept changes in the intracellular ROS concentrations as a response to genotoxic stress. This event can induce forms of resistance to cell death such as senescence and protective autophagy through multiple biochemical pathways of signal transduction (e.g. ATP/AMPK/ULK1, PI3K/AKT, MAPK)targeted by natural flavonoids. We demonstrated by immunoblot analysis that 40 microM quercetin and fisetin after 10 and 20min of treatment inhibit the phosphorylation of PI3K/AKT and ERK1/2 kinases, responsible for apoptosis resistance and p16INK4stability, and, at the same time, they activate AMPK and ULK kinases confirming our hypothesis. Finally, we showed that in combination with clinically relevant doses of radiation, natural senolytics activate in HT500 cells two forms of cell death: apoptosis (type 1) measured with caspase-3 activity and nuclear Hoechst staining, and lethal autophagy (type2) assessed by selective autophagy inhibitor and fluorescent autophagosome dye. In conclusion, we confirmed that senescence and autophagy partially overlap, sharing common modulatory pathways, revealing important roles of senolytic flavonoids in these processes that deserve future investigations. [1] Russo M, Moccia S, Spagnuolo C, Tedesco I, Lauria F, Russo GL.Int. J. Mol. Sci. 2022, 23(1), 301. [2] Kirkland JL, Tchkonia T. J Intern Med. 2020 288(5):518-536 [3] Li W, Qin L, Feng R, Hu G, Sun H, He Y, Zhang R. Mech Ageing Dev. 2019 Jul;181:1-6.