Modulation of Autophagy by Ursolic and Betulinic Acids: Distinct Cytotoxic and Membrane‐Disruption in Malignant and Nonmalignant Cells

Autophagy is a critical adaptive mechanism in tumor cells that promotes survival under stress, but when dysregulated, it may trigger programmed cell death. The pentacyclic triterpenoids betulinic acid (BA) and ursolic acid (UA) are structurally related compounds that modulate autophagy; however, comparative insights into their effects on nonmalignant and malignant cells, as well as model membranes, remain limited. Here, we investigated the distinct cellular outcomes induced by UA and BA in nonmalignant keratinocytes (HaCaT) and malignant cell lines (A549, HeLa, MCF7, MES‐SA, PC3, SKMEL‐25/28), as well as their interactions with mitochondrial membrane mimetics. At 20 μM, BA reduced HaCaT proliferation by 70%, while UA achieved only 30% inhibition. BA induced pronounced mitochondrial dysfunction (i.e., 60%), mitophagy activation, and autophagy‐associated cell death linked to a lysosomal–mitochondrial stress axis. In contrast, UA induced lysosomal membrane permeabilization and the release of cathepsin B, resulting in ~50% cell death. In malignant cell lines, BA reduced viability to ~40%, whereas UA showed selective toxicity (53%–73% survival). Cotreatment with chloroquine enhanced UA's cytotoxicity by simulating BA‐like lysosomal accumulation. Biophysical assays revealed differential membrane disruption profiles: BA permeabilized cardiolipin‐rich membranes, while UA exerted milder surface‐level effects. These findings illustrate how structurally similar triterpenoids exert divergent effects on cellular membranes, autophagic flux, and cell fate, offering a foundation for designing selective anticancer agents that target the lysosomal–mitochondrial axis.