Sanguinarine exerts anti-hepatocellular carcinoma activity by targeting FDX1 to induce FDX1/LIAS/DLAT/HSP70 axis-dependent cuproptosis

Abstract

Hepatocellular carcinoma (HCC), the predominant type of primary liver cancer, represents an extremely aggressive malignancy. The induction of cuproptosis has developed into a favorable therapeutic direction for HCC, considering its strong association with HCC. Sanguinarine (San), a benzophenanthridine alkaloid derived from traditional herbs such as Chelidonium majus L., demonstrates broad-spectrum anticancer activities against various cancer cell types. However, the precise molecular mechanisms underlying its effects in the treatment of HCC remain largely undefined. This investigation seeks to examine the anti-HCC effects of San and to explore the mechanisms underlying these effects through the induction of cuproptosis. In vitro experiments demonstrate that San markedly inhibits the proliferation, movement, and epithelial-mesenchymal transition of HCC cells while enhancing their apoptosis. In vivo, San notably impedes tumor growth and upregulates the cuproptosis signature markers ferredoxin 1 (FDX1), oligomeric dihydrolipoamide S-acetyltransferase (DLAT), and heat shock protein 70 (HSP70) in HCC xenograft tumor models. Mechanistically, San induces proteotoxic stress and cuproptosis in HCC cells by increasing copper concentration, upregulating the expression of FDX1, lipoic acid synthetase (LIAS), HSP70, and lipoylated DLAT aggregation, and simultaneously reducing mitochondrial membrane potential and intracellular glutathione and pyruvate levels. Moreover, the combination of San with copper ionophores (Elesclomol-CuCl2) exhibits synergistic effects in promoting cuproptosis. FDX1 silencing markedly diminishes San-induced suppression of cell proliferation and FDX1 and HSP70 levels in HCC cells. Additionally, molecular docking analysis predicts that San exhibits the highest potential for binding with FDX1. Surface plasmon resonance experiments and cellular thermal shift assay confirm that San strongly interacts with FDX1 and markedly enhances the thermostability of FDX1. In conclusion, our findings indicate that San substantially inhibits the progression of HCC by targeting FDX1/LIAS/DLAT/HSP70 axis-dependent cuproptosis.

1. Introduction

Hepatocellular carcinoma (HCC) represents the sixth most prevalent malignant tumor and the third principal source of cancer-associated deaths globally [1,2], following lung and colorectal cancers [3]. The existing therapeutic approaches for HCC, including surgery [3,4], chemotherapy, radiotherapy, targeted therapy, and immunotherapy [5,6], fail to markedly improve prognosis or extend survival among individuals with advanced HCC [7], leading to the median survival time in HCC patients being limited to 6–20 months [7,8]. Furthermore, the substantial expenses, antimedicinal resistance, adverse effects, and toxicities associated with these therapeutic approaches substantially limit the anticancer efficacy of tumor chemotherapy drugs and the first- and second-line therapies for HCC [1,7,9]. Consequently, an urgent requirement exists to uncover new molecular pathways and druggable targets for HCC while also developing more effective agents with reduced toxicity for its treatment.

In 2022, Tsvetkov et al. [10] coined the term “cuproptosis” to describe a novel copper-lipoylation-induced regulatory cell death. This distinctive death exhibits multiple characteristic features, including elevated copper ion levels within cells, fatal aggregation of lipoylated dihydrolipoamide S-acetyltransferase (DLAT) in mitochondrial tricarboxylic acid cycle processes and enhanced heat shock protein 70 (HSP70) expression [11–13]. Mechanistically, cuproptosis operates through the essential regulator ferredoxin 1 (FDX1), which encodes a mitochondrial matrix reductase responsible for converting Cu2+ into the more toxic Cu+ form [14,15]. FDX1 further mediates the lipoylation modification of DLAT in cooperation with lipoic acid synthetase (LIAS), leading to lipoylated DLAT oligomerization facilitated by excess mitochondrial copper ions, along with mitochondrial damage and increases in HSP70 levels [11–17]. This cascade ultimately leads to cuproptosis through the resultant mitochondrial protein toxicity. Current research has emphasized the essential function of cuproptosis in cancer development and outcomes [15,18–20]. Many studies have revealed that FDX1, LIAS, and DLAT influence HCC development, survival outcomes, immune cell presence, and treatment efficacy [14,17,19,21–25]. Reduced FDX1 levels are linked to unfavorable outcomes and reduced survival in HCC, whereas higher FDX1 expression is linked to improved survival in HCC [19,25]. Additionally, FDX1 overexpression [19] or increased oligomeric DLAT [26] inhibits the growth of HCC cell lines and HCC progression in vivo. Therefore, targeting cuproptosis signaling molecules opens up new possibilities for precise clinical diagnosis and treatment in HCC.