Acetylshikonin induces necroptosis via the RIPK1/RIPK3-dependent pathway in lung cancer

Abstract

Despite advances in therapeutic strategies, lung cancer remains the leading cause of cancer-related death worldwide. Acetylshikonin is a derivative of the traditional Chinese medicine Zicao and presents a variety of anticancer properties. However, the effects of acetylshikonin on lung cancer have not been fully understood yet. This study explored the mechanisms underlying acetylshikonin-induced cell death in non-small cell lung cancer (NSCLC). Treating NSCLC cells with acetylshikonin significantly reduced cell viability, as evidenced by chromatin condensation and the appearance of cell debris. Acetylshikonin has also been shown to increase cell membrane permeability and induce cell swelling, leading to an increase in the population of necrotic cells. When investigating the mechanisms underlying acetylshikonin-induced cell death, we discovered that acetylshikonin promoted oxidative stress, decreased mitochondrial membrane potential, and promoted G2/M phase arrest in lung cancer cells. The damage to NSCLC cells induced by acetylshikonin resembled results involving alterations in the cell membrane and mitochondrial morphology. Our analysis of oxidative stress revealed that acetylshikonin induced lipid oxidation and down-regulated the expression of glutathione peroxidase 4 (GPX4), which has been associated with necroptosis. We also determined that acetylshikonin induces the phosphorylation of receptor-interacting serine/threonine-protein kinase 1 (RIPK1)/RIPK3 and mixed lineage kinase domain-like kinase (MLKL). Treatment with RIPK1 inhibitors (necrostatin-1 or 7-Cl-O-Nec-1) significantly reversed acetylshikonin-induced MLKL phosphorylation and NSCLC cell death. These results indicate that acetylshikonin activated the RIPK1/RIPK3/MLKL cascade, leading to necroptosis in NSCLC cells. Our findings indicate that acetylshikonin reduces lung cancer cells by promoting G2/M phase arrest and necroptosis.

Keywords: ROS; acetylshikonin; human lung cancer; necroptosis.

Figure 1

Acetylshikonin decreased cancer cell viability and induced chromatin condensation and nuclear debris formation. (A) Chemical structure of acetylshikonin. (B) CCK-8 assay results of MRC-5 cells and H1299 and A549 cells following incubation with acetylshikonin (0.5–10 μM) for 24 h (n = 4). (C, D) Fluorescence microscope images showing DAPI staining results and cell morphology of H1299 and A549 cells following treatment with acetylshikonin (0.5–10 μM). Red arrows indicated nuclear debris. Scale bar = 50 μm. MRC-5 cells and untreated cells were used as controls. Results are shown as means ± SD. ^*p < 0.05 compared to untreated control.

Figure 2

Acetylshikonin promoted DNA fragmentation and cell cycle arrest in G2/M phase. (A, B) Flow cytometry image results indicating cell cycle progression in H1299 and A549 cells following treatment with acetylshikonin (0.5–10 μM) for 24 h and PI staining for 30 min (n = 4). (C, D) Western blot analysis showing CDK1 and cyclin B1 protein expression in H1299 and A549 cells treated with acetylshikonin (0.5–10 μM) for 6 h (n = 4). Untreated cells were used as controls. Results are shown as means ± SD. ^*p < 0.05 compared to untreated control.

Figure 3

Acetylshikonin increased the membrane permeability of NSCLC cells and the proportion of necrotic NSCLC cells. (A, B) Flow cytometry results for Annexin V/PI showing the incidence of cell death among H1299 and A549 cells following treatment with acetylshikonin (0.5–10 μM) for 24 h (n = 4). (C) Phase microscope images of NSCLC cells following incubation with acetylshikonin (0.5–10 μM) for 24 h. Red arrows indicate swollen blebs. Scale bar = 50 μm. (D, E) PI staining results of H1299 and A549 cells following treatment with acetylshikonin (0.5–10 μM) for 4 h. Scale bar = 200 μm. Untreated cells were used as controls. Results are shown as means ± SD. ^*p < 0.05 compared to untreated control.

Figure 4

Acetylshikonin induced intracellular ROS production and depolarization of mitochondrial membrane potential. (A, B) Flow cytometry results indicating ROS production in H1299 and A549 cells following incubation with acetylshikonin (0.5–10 μM) and H₂DCFDA for 30 min (n = 4). (C) Fluorescence microscope images used to analyze the MMP of NSCLC cells following incubation with acetylshikonin (0.5–10 μM) for 24 h and JC-1 staining for 30 min (n = 4). Scale bar = 50 μm. Untreated cells were used as controls. Results are shown as means ± SD. ^*p < 0.05 compared to untreated control.

Figure 5

Acetylshikonin induced necroptotic lipid peroxidation in NSCLC cells. (A) Transmission electron microscopy analysis showing impaired membrane integrity (blue arrow) after treating H1299 cells with acetylshikonin for 6 h. The red arrow indicates mitochondria, and the white arrow indicates lysosomes. 5,000×: Scale bar = 2 μm. 20,000×: Scale bar = 0.5 μm. (B–D) Fluorescence microscope images and flow cytometry results indicating lipid peroxidation in H1299 and A549 cells incubated with acetylshikonin (0.5–10 μM) and BODIPY™ 581/591 C11 for 30 min (n = 4). Scale bar = 200 μm. (E, F) Western blot analysis showing GPX4 protein expression in H1299 and A549 cells following treatment with acetylshikonin (0.5–10 μM) for 24 h (n = 4). Untreated cells were used as controls. Results are shown as means ± SD. ^*p < 0.05 compared to untreated control.

Figure 6

Acetylshikonin promoted cell death via necroptotic RIPK1, RIPK3, and MLKL signaling activation. (A) Fluorescence microscope images showing MLKL phosphorylation in H1299 and A549 cells following incubation with acetylshikonin (2.5 μM) for 0–4 h. Scale bar = 50 nm. (B, C) Western blot analysis showing RIPK1, RIPK3, and MLKL protein phosphorylation levels in NSCLC cells treated with acetylshikonin (2.5 μM) for 0–4 h (n = 4). (D) Fluorescence microscope images showing MLKL phosphorylation in H1299 and A549 cells preincubated with necrostatin-1 (20 nM) and 7-Cl-O-Nec-1 (30 nM) for 1 h and then incubated with acetylshikonin (2.5 μM) for a further 4 h. Scale bar = 50 nm. (E, F) CCK-8 assays indicating the viability of H1299 and A549 cells preincubated with necrostatin-1 (20 nM) and 7-Cl-O-Nec-1 (30 nM) for 1 h and then incubated with acetylshikonin (2.5 μM) for a further 24 h (n = 4). Untreated cells were used as controls. Results are shown as means ± SD. ^*p < 0.05 compared to untreated control. ^#p < 0.05 compared to acetylshikonin alone group.