Oxocrebanine, an aporphine alkaloid from Stephania hainanensis Lo et Y. Tsoong, inhibits hepatocellular carcinoma via coordinated induction of autophagy and mitotic arrest and dual inhibition of topoisomerase I/IIα

BackgroundLiver cancer is one of the malignant tumors that severely threaten human life and health worldwide. The development of novel drugs for the effective treatment of hepatocellular carcinoma (HCC) has thus become a critical research focus.PurposeTo explore the mechanism by which oxocrebanine induces autophagy and mediates M-phase cell cycle arrest in HCC Hep3B2.1-7 cells, clarify its inhibitory effect on topoisomerase and DNA damage-inducing ability, and analyze the regulatory mechanism of this drug on key signaling pathways of cell proliferation.MethodsPDK1 overexpression transfection assay was used to reversely verify the mechanism by which oxocrebanine induces autophagy in Hep3B2.1-7 cells. Flow cytometry combined with PI single staining was employed to clarify the effect of cell cycle arrest. Western Blot was used to detect the protein expression levels of p-H3, Cyclin B1, Aurora A, PLK1, p53, p21, Cdc2, and Cdc25C. Fluorescence microscopy was used to observe the expression of Cyclin B1 protein, and specific inhibitors were applied for reverse verification. Cell proliferation was detected by the CCK-8 assay, and DNA damage was evaluated by the comet assay. Western Blot was used to determine the expression levels of Topo I, Topo IIα, γ-H2AX, as well as key proteins of the Wnt/β-Catenin, PI3K/AKT/mTOR, MAPK/ERK, and JAK/STAT pathways. Pathway activators were added to further verify the changes in the expression of the above-mentioned proteins.ResultsFollowing transfection of PDK1 gene in Hep3B2.1-7 cells, oxocrebanine extremely significantly upregulated the LC3-II/LC3-I ratio and the protein expression levels of AKT and p-AKT (p<0.01). Oxocrebanine induced G2/M phase arrest in Hep3B2.1-7 cells, markedly elevated the protein expression levels of p-H3 and Cyclin B1 (p<0.01), caused Cyclin B1 to translocate from a uniform distribution to the cell membrane, and reduced the expression levels of Aurora A and PLK1 (p<0.01). Meanwhile, it upregulated the expression of p53, p21 and p-Cdc2, and downregulated that of Cdc2 and Cdc25C (p<0.01). After combined treatment with inhibitors, the upregulation of p53, p21 and Cyclin B1 was attenuated, and the inhibitory effect on Cdc2 was further enhanced. Oxocrebanine treatment inhibited the proliferation of Hep3B2.1-7 cells and partially reversed the increased cell proliferation induced by TOP1 and TOP2A overexpression. Oxocrebanine also downregulated the protein expression of Topo I and Topo IIα in Hep3B2.1-7 cells. The protein expression level of γ-H2AX was increased in a dose-dependent manner after oxocrebanine treatment, with obvious "comet" tailing observed, indicating that oxocrebanine could induce DNA damage. In addition, oxocrebanine downregulated the expression of key proteins in the Wnt/β-Catenin, PI3K/AKT/mTOR, MAPK/ERK and JAK/STAT pathways. Pathway activators elevated the expression of the above proteins and partially reversed the inhibitory effects of oxocrebaninecrebanine. Conclusion: oxycarbanine promote AKT phosphorylation, accelerate the conversion of LC3-I to LC3-II, and induce autophagy in HCC cells. By regulating Cyclin B1, Aurora A, PLK1, and the p53/p21/Cdc25C pathway, it induces M-phase cell cycle arrest in HCC cells. Oxocrebanine can inhibit the expression of topoisomerase, interfere with DNA replication and chromosome structure homeostasis, induce DNA damage, and thereby inhibit the proliferation of HCC cells. Meanwhile, it can inhibit the Wnt/β-catenin, PI3K/AKT/mTOR, MA