Derivatives of Dictyostelium Differentiation-Inducing Factor-3 Promote Autophagic Flux, Mitochondrial Dysfunction and Iron-Associated Oxidative Stress

Novel anticancer agents are needed to overcome therapeutic resistance, and autophagy represents a promising target. The chlorinated alkylphenones Dictyostelium differentiation-inducing factors 1 and 3 (DIF-1 and DIF-3) and their derivatives exert anti-proliferative and anti-metastatic effects; however, their molecular mechanisms remain incompletely understood. Using the GFP-LC3-RFP-LC3ΔG autophagic flux reporter, we identified Bu-DIF-3 and CP-DIF-3 as autophagy-promoting derivatives and characterized their actions principally in HeLa cells, with selected autophagy and AMP-activated protein kinase (AMPK) measurements in HAP1 cells. Both suppressed mTORC1 signaling, activated AMPK, and reduced mitochondrial membrane potential (MMP). Bu-DIF-3 additionally induced reactive oxygen species and redox stress response genes, including SLC7A11 and CHAC1. SLC7A11 induction may reflect a compensatory antioxidant response. Unlike CP-DIF-3, Bu-DIF-3 increased the FerroOrange-detectable labile Fe²⁺ signal, and deferoxamine partially restored the reduced MTT signal, supporting possible iron involvement in reduced metabolic activity. Thus, modification of the DIF-3 methoxy group modulates autophagy-promoting activity and associated stress responses. Bu-DIF-3 was associated with oxidative stress and altered labile Fe²⁺ homeostasis; whether a ferroptosis-related mechanism contributes remains unresolved. These findings provide a cellular basis for further evaluation of Bu-DIF-3 and related DIF derivatives.