Journal: Molecular & Cellular Proteomics : MCP

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

doi: 10.1016/j.mcpro.2023.100551

Figure Lengend Snippet: 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.

Article Snippet: Sulconazole nitrate (HY-B1460A), Z-VAD-FMK (z-VAD) (HY-16658B), ferrostatin-1 (HY-100579), deferoxamine mesylate (HY-B0988), and necrostatin-1 (Nec) (HY-15760) were purchased from MedChemExpress and dissolved in dimethyl sulfoxide (DMSO) for storage, and the final concentration of DMSO used in all experiments did not exceed 0.1%.

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

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

doi: 10.1016/j.mcpro.2023.100551

Figure Lengend Snippet: 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.

Article Snippet: Sulconazole nitrate (HY-B1460A), Z-VAD-FMK (z-VAD) (HY-16658B), ferrostatin-1 (HY-100579), deferoxamine mesylate (HY-B0988), and necrostatin-1 (Nec) (HY-15760) were purchased from MedChemExpress and dissolved in dimethyl sulfoxide (DMSO) for storage, and the final concentration of DMSO used in all experiments did not exceed 0.1%.

Techniques: Migration, Clonogenic Assay, Inhibition, Transwell Assay, Wound Healing Assay

Journal: Molecular & Cellular Proteomics : MCP

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

doi: 10.1016/j.mcpro.2023.100551

Figure Lengend Snippet: 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.

Article Snippet: Sulconazole nitrate (HY-B1460A), Z-VAD-FMK (z-VAD) (HY-16658B), ferrostatin-1 (HY-100579), deferoxamine mesylate (HY-B0988), and necrostatin-1 (Nec) (HY-15760) were purchased from MedChemExpress and dissolved in dimethyl sulfoxide (DMSO) for storage, and the final concentration of DMSO used in all experiments did not exceed 0.1%.

Techniques: Sequencing

Journal: Molecular & Cellular Proteomics : MCP

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

doi: 10.1016/j.mcpro.2023.100551

Figure Lengend Snippet: 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.

Article Snippet: Sulconazole nitrate (HY-B1460A), Z-VAD-FMK (z-VAD) (HY-16658B), ferrostatin-1 (HY-100579), deferoxamine mesylate (HY-B0988), and necrostatin-1 (Nec) (HY-15760) were purchased from MedChemExpress and dissolved in dimethyl sulfoxide (DMSO) for storage, and the final concentration of DMSO used in all experiments did not exceed 0.1%.

Techniques: Western Blot, Expressing, Flow Cytometry, Staining, Quantitative RT-PCR

Journal: Molecular & Cellular Proteomics : MCP

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

doi: 10.1016/j.mcpro.2023.100551

Figure Lengend Snippet: 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 iodide D , 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.

Article Snippet: Sulconazole nitrate (HY-B1460A), Z-VAD-FMK (z-VAD) (HY-16658B), ferrostatin-1 (HY-100579), deferoxamine mesylate (HY-B0988), and necrostatin-1 (Nec) (HY-15760) were purchased from MedChemExpress and dissolved in dimethyl sulfoxide (DMSO) for storage, and the final concentration of DMSO used in all experiments did not exceed 0.1%.

Techniques: Transmission Assay, Electron Microscopy, Membrane, Expressing, Quantitative RT-PCR, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

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

doi: 10.1016/j.mcpro.2023.100551

Figure Lengend Snippet: 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.

Article Snippet: Sulconazole nitrate (HY-B1460A), Z-VAD-FMK (z-VAD) (HY-16658B), ferrostatin-1 (HY-100579), deferoxamine mesylate (HY-B0988), and necrostatin-1 (Nec) (HY-15760) were purchased from MedChemExpress and dissolved in dimethyl sulfoxide (DMSO) for storage, and the final concentration of DMSO used in all experiments did not exceed 0.1%.

Techniques: Clonogenic Assay, Irradiation, Control