SK (SK), a secondary plant metabolite from Lithospermum erythrorhizon, is an inducer of oxidative stress and a DNA Topoisomerase inhibitor with promising anticancer properties. However, the underlying mechanisms, especially the involvement of autophagy in cancer cell death, are poorly understood. Here, we report a novel mechanism of action that activates a noncanonical, Beclin1-independent but ATG5-dependent autophagy pathway triggered by oxidative stress in two distinct subtypes of triple-negative breast cancer (TNBC) cell lines: mesenchymal stem cell-like MDA-MB-231 and basal-like-1 MDA-MB-468. We observed that this noncanonical autophagy pathway specifically targets and degrades nuclear material by nucleophagy. Electron microscopy analysis of both cell lines revealed distinct nuclear alterations, including envelope-limited chromatin sheets (ELCS), nuclear buds, and micronuclei after SK treatment. Furthermore, numerous autophagosomes and lysosomes were found in close proximity to the nuclear membrane, suggesting the occurrence of nucleophagy. The localization of γ-H2AX in nuclear buds and micronuclei observed by confocal microscopy indicated cytosolic leakage of damaged DNA. Additionally, Western blot analysis confirmed the role of the cGAS-STING pathway, which is essential for detecting damaged DNA in the cytosol. Inner nuclear membrane protein Lamin B1 was found to interact with LC3II and was subsequently degraded through the nucleophagy pathway. Knockout of ATG5 using CRISPR-Cas9 reduced autophagy, while Beclin1 knockdown did not reduce LC3II conversion, indicating that the process follows a noncanonical autophagy pathway that is dependent on ATG5 and independent of Beclin1. SK induces oxidative stress, leading to mitochondrial depolarization and DNA damage accumulation, which subsequently triggers autophagy and ultimately causes apoptotic cell death. Treatment with the ROS scavenger N-acetylcysteine (NAC) reduced nuclear stress, mitochondrial dysfunction, autophagy, and cell death, emphasizing the role of oxidative stress in SK-induced cell death. MDA-MB-468 cells exhibited greater sensitivity to SK-induced nuclear stress and cell death compared to MDA-MB-231 cells. Taken together, we demonstrate that SK exerts its anticancer effects in TNBC cells through the generation of oxidative stress and noncanonical autophagy, thus highlighting SK's potential for targeted anticancer therapeutics.
