Sulconazole Induces PANoptosis by Triggering Oxidative Stress and Inhibiting Glycolysis to Increase Radiosensitivity in Esophageal Cancer

Abstract

Esophageal cancer is the seventh most common cancer in the world. Although traditional treatment methods such as radiotherapy and chemotherapy have good effects, their side effects and drug resistance remain problematic. The repositioning of drug function provides new ideas for the research and development of anticancer drugs. We previously showed that the Food and Drug Administration-approved drug sulconazole can effectively inhibit the growth of esophageal cancer cells, but its molecular mechanism is not clear. Here, our study demonstrated that sulconazole had a broad spectrum of anticancer effects. It can not only inhibit the proliferation but also inhibit the migration of esophageal cancer cells. Both transcriptomic sequencing and proteomic sequencing showed that sulconazole could promote various types of programmed cell death and inhibit glycolysis and its related pathways. Experimentally, we found that sulconazole induced apoptosis, pyroptosis, necroptosis, and ferroptosis. Mechanistically, sulconazole triggered mitochondrial oxidative stress and inhibited glycolysis. Finally, we showed that low-dose sulconazole can increase radiosensitivity of esophageal cancer cells. Taken together, these new findings provide strong laboratory evidence for the clinical application of sulconazole in esophageal cancer.

Keywords: PANoptosis; esophageal cancer; glycolysis; oxidative stress; radiosensitivity; sulconazole.

Graphical abstract

Fig. 1

Sulconazole inhibits the viability of various cancer cell lines.A, molecular structure of sulconazole. B–D, viability of esophageal cancer cell lines (KYSE30 and KYSE150) and an esophageal epithelial cell line (SHEE) (B), liver cancer cell lines (HepG2 and Huh7) and a normal liver cell line (Chang liver) (C), gastric cancer cell lines (SGC7901 and HGC27), lung cancer cell line (A549), and breast cancer cell lines (MDA-MB-543 and MCF7) (D) were assessed after treatment with sulconazole at concentrations as indicated for 24 h. The data are representative of three independent experiments and presented as the mean ± SD.

Fig. 2

Sulconazole inhibits the proliferation and migration of esophageal cancer cell lines.A, cell clonogenic assay was used to verify the inhibition of proliferation of KYSE30 (left panel) and KYSE150 (right panel) cells after sulconazole treatment for 24 h. B, transwell assay was used to verify the inhibitory effect of sulconazole on migration of KYSE30 (left panel) and KYSE150 (right panel) cells after sulconazole treatment for 24 h. The number of cells was counted in random fields. C and D, migration of KYSE30 and KYSE150 cells was detected by wound healing assay after sulconazole treatment for 24 h. Diagrams (left panel) were used for quantitative analyses of migration distance. All data are representative of three independent experiments. p-values were calculated by unpaired two-sided Student’s t tests. ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 3

Dysregulated proteins and pathways in esophageal cancer cells treated with sulconazole were analyzed by transcriptomics and proteomics.A, flowchart of the experimental process of transcriptomic sequencing and proteomic sequencing. B, Venn diagram of 1.5-fold change upregulated and downregulated genes from transcriptomic sequencing. C, HALLMARK enrichment analysis of dysregulated genes. D, KEGG enrichment analysis of dysregulated genes. E, volcano plot of 1.3-fold change upregulated and downregulated proteins from proteomic sequencing. F, KEGG enrichment analysis of dysregulated proteins. G, HALLMARK enrichment analysis of dysregulated proteins.

Fig. 4

Sulconazole induces PANoptosis of esophageal cancer cells.A, Western blot analyses for the expression of BCL2, BAX, cleaved BAX, caspase3, cleaved caspase3, PARP, cleaved PARP, GSDME, GSDME-N, GSDMD, GSDMD-N, p-MLKL, MLKL, RIPK1, cleaved RIPK1, and β-Actin after sulconazole treatment for 12 h. B–D, flow cytometry (B) and quantification analysis (C and D) with Annexin V/PI staining evaluating the percentages of live cells in Q4 (Annexin V^-/PI^-), early apoptotic cells in Q3 (Annexin V⁺/PI^-), and late apoptotic cells and necrotic cells in Q2 (Annexin V⁺/PI⁺) among KYSE30 and KYSE150 treated with DMSO or sulconazole for 12 h. E, cell death in KYSE30 and KYSE150 cells treated with sulconazole for 12 h were detected by flow cytometry, and the PI-positive cells were calculated and shown in the diagrams. F, LDH release of KYSE30 and KYSE150 cells after sulconazole treatment for 12 h. G, heat map of ferroptosis-related genes that were differentially expressed in KYSE30 and KYSE150 cells with or without sulconazole treatment. H and I, the mRNA expression of ferroptosis-related genes by quantitative RT-PCR after sulconazole treatment for 24 h J–M, measurement of lipid peroxidation after sulconazole Ferr-1 (20 μM) and DFOM (100 μM) treatment for 12 h. Bar graph showing relative levels of lipid peroxidation by C11-BODIPY staining in KYSE30 and KYSE150 cells (L and M). N, representative images of cell death in KYSE30 and KYSE150 cells after sulconazole treatment for 12 h. O, diagrams were used for quantitative analyses of green dead cells (SYTOX Green-positive) in (N). The data in (B–O) are representative of three independent experiments and presented as the mean ± SD. p-values were calculated by two-way ANOVA. ∗∗p < 0.01, ∗∗∗p < 0.001. DFOM, deferoxamine mesylate; Ferr-1, ferrostatin-1; LDH, lactate dehydrogenase; MLKL, mixed lineage kinase-like domain; PI, propidium iodide.

Fig. 5

Sulconazole triggers mitochondrial oxidative stress and inhibits glycolysis.A, transmission electron microscopy (TEM) images of KYSE30 cells subjected to the indicated treatments for 24 h. White arrows indicate mitochondria. Scale bars represent left, 1 μm; right, 500 nm. B; PI, propidium iodideD, mitochondrial membrane potential analysis (B), ROS level analysis (C), and glucose uptake analysis (D) in KYSE30 and KYSE150 cells after sulconazole treatment for 24 h. E, heat map of 19 glycolysis-related enzymes that were differentially expressed in KYSE30 and KYSE150 cells with or without sulconazole treatment. F and G, the mRNA expression of glycolysis pathway enzymes by quantitative RT-PCR in KYSE30 (F) and KYSE150 (G) cells. H, Western blot analyses of glycolysis-related enzymes after sulconazole treatment for 24 h in KYSE30 (left panel) and KYSE150 (right panel) cells. Quantification of the blots is shown below. I, Western blot analyses for the expression of p-AKT, AKT, p-MEK, MEK, p-ERK, ERK, p-STAT3, and STAT3 after sulconazole treatment for 24h in KYSE30 (left panel) and KYSE150 (right panel) cells. The data in (B–D, F and G) are representative of three independent experiments and presented as the mean ± SD. p-values were calculated by two-way ANOVA. ∗∗p < 0.01, ∗∗∗p < 0.001. ROS, reactive oxygen species.

Fig. 6

Sulconazole increases radiosensitivity of esophageal cancer cells.A, flowchart of the clonogenic assay under sulconazole/IR chemoradiotherapy treatment. B, typical diagrams of radiation survival of KYSE30, KYSE150, and TE3 cells. C, D and E, statistical histogram of numbers of colonies formed. F, KYSE30 was treated with DMSO or sulconazole (20 μM) for 24 h, and γH2AX was detected at different time points after 4 Gy irradiation. G, ROS levels were detected after treatment with DMSO (control), sulconazole (20 μM), 4 Gy, or sulconazole (20 μM) combined with 4 Gy for 24 h in KYSE30 and KYSE150 cells. The data in (C–E, G and H) are representative of three independent experiments and presented as the mean ± SD. p-values were calculated by two-way ANOVA (C–E) and unpaired two-sided Student’s t test (G and H). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. IR, ionizing radiation; ROS, reactive oxygen species.