Simple SummaryThe role of autophagy and senescence in cancer resistance to ionizing radiation as a response to genotoxic stress is still only partially explored. The flavonoids quercetin and fisetin have previously been shown to sensitize cancer cells resistant to radiotherapy by targeting p16INK4and p21Kip1. Here, we examined their ability to modulate autophagy and senescence-associated inflammatory markers after irradiation in radioresistant cells. Quercetin or fisetin, in association with ionizing radiation, significantly activated AMPK and decreased ERK kinase activity, which was linked to autophagic stress response and apoptosis induction. In simple words, on one side, the combined treatment favored the induction of autophagy and senescence by activating AMPK; on the other side, it lowered the threshold for cell death and induced lethal autophagy and apoptosis by inhibiting the ERK pathway.AbstractResistance to cancer therapies remains a clinical challenge and an unsolved problem. In a previous study, we characterized a new colon cancer cell line, namely HT500, derived from human HT29 cells and resistant to clinically relevant levels of ionizing radiation (IR). Here, we explored the effects of two natural flavonoids, quercetin (Q) and fisetin (F), well-known senolytic agents that inhibit genotoxic stress by selectively removing senescent cells. We hypothesized that the biochemical mechanisms responsible for the radiosensitising effects of these natural senolytics could intercept multiple biochemical pathways of signal transduction correlated to cell death resistance. Radioresistant HT500 cells modulate autophagic flux differently than HT29 cells and secrete pro-inflammatory cytokines (IL-8), commonly associated with senescence-related secretory phenotypes (SASP). Q and F inhibit PI3K/AKT and ERK pathways, which promote p16INK4stability and resistance to apoptosis, but they also activate AMPK and ULK kinases in response to autophagic stress at an early stage. In summary, the combination of natural senolytics and IR activates two forms of cell death: apoptosis correlated to the inhibition of ERKs and lethal autophagy dependent on AMPK kinase. Our study confirms that senescence and autophagy partially overlap, share common modulatory pathways, and reveal how senolytic flavonoids can play an important role in these processes.