Icaritin induces paraptosis in hepatocellular carcinoma cells by targeting BHLHE40 via endoplasmic reticulum stress and mitochondrial dysfunction

Objective

Icaritin, a flavonoid derived from the traditional Chinese medicine Epimedium, exhibits diverse biological activities; however, the mechanisms underlying its effects against hepatocellular carcinoma (HCC) remain unclear. This study aimed to investigate the anticancer properties of icaritin and elucidate the mechanisms of icaritin-induced cell death.

Methods

The effects of icaritin-induced paraptosis were assessed using CellTiter-Glo, EdU, and colony formation assays, and phenotypic observations. Transcriptome analysis was performed to identify the dysregulated genes associated with icaritin-induced paraptosis. Western blotting and qRT-PCR were used to analyze icaritin-induced changes in protein and mRNA levels, respectively. Mito-GFP, ROS, and MMP assays were conducted to monitor the mitochondrial status. IPA, molecular docking, CETSA, shRNA and cell-derived xenografts confirmed the role of BHLHE40 in icaritin-induced paraptosis in vivo and in vitro

Results

Icaritin induced paraptosis in HCC cells, which was characterized by cytoplasmic vacuolation and caspase-independent. Transcriptomic analysis indicated that icaritin triggered ER stress and mitochondrial dysfunction, validated by molecular and biochemical assays. IPA, molecular docking, and CETSA analyses identified BHLHE40 as a crucial mediator of this process. BHLHE40 knockdown inhibited ER stress and mitochondrial dysfunction, significantly reducing icaritin-induced paraptosis in HCC cells. Animal experiments demonstrated that silencing of BHLHE40 diminished the inhibitory effects of icaritin on tumor growth in xenograft models.

Conclusion

These results highlight the potent anticancer effects of icaritin, particularly its ability to induce paraptosis by targeting BHLHE40. This study provides a comprehensive understanding of the anticancer mechanisms of icaritin and suggests that targeting BHLHE40 represents a novel therapeutic strategy for enhancing the efficacy of cancer treatment.

Abbreviations

HCC: Hepatocellular carcinoma; TCM: Traditional Chinese Medicine; ER: Endoplasmic Reticulum; MMP: Mitochondrial Membrane Potential; ROS: Reactive Oxygen Species; CTG: CellTiter-Glo; EdU: 5-ethynyl-2′-deoxyuridine; UPR: Unfolded Protein Response; BHLHE40: Basic helix-loop-helix family member E40; IPA: Ingenuity Pathway Analysis; CETSA: Cellular Thermal Shift Assay; PI: Propidium Iodide;PCR: Polymerase Chain Reaction; qRT-PCR: Quantitative Reverse Transcription PCR; GFP: Green Fluorescent Protein; JC-1: 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; ATF4: Activating Transcription Factor 4; PERK: Protein kinase RNA-like ER kinase; DDIT3: DNA Damage Inducible Transcript 3; LMNA: Lamin A/C; IC50: Half Maximal Inhibitory Concentration; PEG300: Polyethylene glycol 300; PBS: Phosphate Buffered Saline; BSA: Bovine Serum Albumin; HRP: Horseradish Peroxidase; SRA: Sequence Read Archive; PCD: Programmed Cell Death; COX8A: Cytochrome c oxidase subunit 8A; DCFH-DA: 2′,7′-Dichlorodihydrofluorescein diacetate; CHX: Cycloheximide; zVAD-FMK: Carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone; PARP: Poly (ADP-ribose) polymerase; DMSO: Dimethyl sulfoxide; SPF: Specific Pathogen-Free; IACUC: Institutional Animal Care and Use Committee