Shikonin induces ferroptosis in osteosarcomas through the mitochondrial ROS-regulated HIF-1α/HO-1 axis

The most common malignant bone tumour is osteosarcoma, which has an unsatisfactory prognosis and unsatisfactory treatment. Ferroptosis shows promise as an effective OS therapy. A substance extracted from the Lithospermum erythrorhizon, Shikonin (SHK), inhibits a number of tumours, including ovarian, gastric, and lung cancers. However, whether SHK induces OS ferroptosis and its mechanisms are not clear.

Our study is aimed at investigating whether SHK causes ferroptosis and elucidating its molecular mechanism.

Cell counting Kit-8, cell cycle and cell apoptosis assay were utilised to assess therapeutic effect of SHK against OS. Normal cells, including H9C2, AML-12 and HK-2, haematoxylin and eosin staining and mice serum biomarker tests were used to assess SHK biosafety. Malondialdehyde (MDA) levels, the glutathione (GSH)/oxidized glutathione (GSSG) ratio, reactive oxygen species (ROS), lipid peroxide (LPO), and intracellular Fe²⁺ detection, quantitative reverse transcription PCR (qRT-PCR), Western blotting (WB) and rescue experiments were employed to confirm whether SHK induced ferroptosis in OS cells. Molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay were used to evaluate the direct binding between SHK and hypoxia-inducible factor-1α (HIF-1α). Protein stability and degradation analysis, small RNA interference, flow cytometry, qRT-PCR, and WB were used to investigate the molecular mechanism of ferroptosis. In vivo xenografts of nude mouse was used to study the anti-OS effect of SHK.

SHK significantly reduced OS cell viability and induced apoptosis and G2/M arrest. SHK increased intracellular levels of MDA, ROS, LPO, and Fe²⁺ while simultaneously reducing the GSH/GSSG ratio and GPX4 and SLC7A11 expression. CETSA and DARTS results demonstrated that SHK did not bind targetly to HIF-1α. Instead, mitochondrial ROS (MitoROS) promoted HIF-1α expression, resulting in HO-1 overexpression, excess Fe²⁺ production, ROS accumulation and GPX depletion, and ferroptosis. Furthermore, inhibition of MitoROS using Mito-TEMPO downregulated HIF-1α/HO-1 axis and mitigated the SHK-induced ferroptosis. In vivo, SHK effectively suppressed OS growth with favourable biosafety, confirming the molecular mechanism underlying SHK-induced ferroptosis in OS.

We observe that HIF-1α/HO-1 axis is the crucial factor in SHK-induced OS ferroptosis. Importantly, we demonstrate that HIF-1α is indirectly regulated by MitoROS rather than SHK bound directly to HIF-1α. Our research suggest that SHK is a potential drug candidate for OS treatment and may help in identifying novel therapeutic targets for OS.