Natural Extracts of Alnus japonica Induce BAK-Dependent Autophagy to Inhibit Liver Cancer Stem Cell Tumorigenesis

Background: Cancer stem cells (CSCs) contribute to hepatocellular carcinoma (HCC) progression and therapeutic resistance. Natural products with antioxidant and bioactive properties may offer novel strategies to suppress CSC-driven tumorigenesis. Methods: We investigated the effects of unfermented and fermented Alnus japonica bark extracts on CSC-like rG2-DC-1C cells. Cell proliferation, invasion, and xenograft tumor formation were assessed, and autophagy/apoptosis markers were analyzed. Results: Bark extracts reduced OCT4 expression, suppressed CSC proliferation and invasion, and inhibited xenograft tumor formation. Mechanistically, extracts activated BAK-dependent autophagy, evidenced by LC3B accumulation and p62 modulation, whereas diarylheptanoids Hirsutenone (Hir) and Oregonin (Ore) primarily induced apoptosis via Caspase-3 cleavage. Blocking autophagy with chloroquine or BAK knockdown reversed the anti-invasive effects of bark extracts, confirming BAK's role in CSC suppression. Component analysis suggests quercetin contributes to autophagy induction, though synergistic effects of other constituents remain possible. Conclusions: Together, these findings indicate that Alnus japonica bark extracts suppress CSC-driven liver tumorigenesis through autophagy, while Hir and Ore act via apoptosis, highlighting complementary mechanisms that broaden the therapeutic potential of this traditional medicinal plant and support further preclinical validation.