Mitochondrial oxidative phosphorylation inhibition by 9α,11α-dihydroxy-kaurenoic acid promotes ROS-associated apoptosis and suppresses cancer stemness in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality worldwide, highlighting the urgent need for novel therapeutic strategies. In this study, we investigated the anticancer activity and molecular mechanism of ent-9α,11α-dihydroxy-15-oxo-kaur-16-en-19-oic acid (9α,11α-dihydroxy-kaurenoic Acid; 9α,11αOH-KA, also known as adenostemmoic acid B), a natural kaurane-type diterpenoid isolated from Adenostemma lavenia, against NSCLC. 9α,11αOH-KA exhibited potent and preferential cytotoxicity toward NSCLC cells compared with non-tumorigenic fibroblasts, accompanied by inhibition of cell migration and induction of apoptosis. Moreover, 9α,11αOH-KA significantly suppressed tumorsphere formation and reduced the expression of cancer stem cells (CSCs)-associated markers, including CD44, CD133, and Notch-1, indicating impairment of stemness-associated properties. Mechanistically, 9α,11αOH-KA induced mitochondrial dysfunction associated with impaired oxidative phosphorylation (OXPHOS), as evidenced by reduced basal and maximal oxygen consumption rate (OCR), depletion of intracellular ATP, and dissipation of mitochondrial membrane potential. These mitochondrial alterations were accompanied by pronounced reactive oxygen species (ROS) accumulation, which contributed to apoptotic cell death, as antioxidant pretreatment attenuated both ROS generation and apoptosis. Importantly, oral administration of 9α,11αOH-KA significantly suppressed A549 xenograft tumor growth without observable systemic toxicity. Collectively, 9α,11αOH-KA exhibits strong anti-NSCLC activity through dual suppression of mitochondrial bioenergetics and CSCs-associated properties. These findings highlight 9α,11αOH-KA as a promising phytochemical candidate for the development of mitochondria-targeting therapeutics for NSCLC.