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• W4366085004 abstract "•Sulconazole has a broad spectrum of anticancer effects.•Sulconazole induces PANoptosis of esophageal cancer cells.•Sulconazole triggers mitochondrial oxidative stress and inhibits glycolysis.•Sulconazole increase the radiosensitivity of esophageal cancer cells. 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. 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. Esophageal cancer ranks seventh in incidence and sixth in mortality worldwide and is mainly comprised of esophageal squamous cell carcinoma and esophageal adenocarcinoma (1Lagergren J. Smyth E. Cunningham D. Lagergren P. Oesophageal cancer.Lancet. 2017; 390: 2383-2396Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 2Sung H. Ferlay J. Siegel R.L. Laversanne M. Soerjomataram I. Jemal A. et al.Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J. Clin. 2021; 71: 209-249Crossref PubMed Scopus (37605) Google Scholar). There are multiple choices for esophageal cancer treatment, including endoscopic management, surgery, chemotherapy, radiotherapy, and immunotherapy, depending on the tumor node metastasis stage combined with patient characteristics (3Kelly S. Harris K.M. Berry E. Hutton J. Roderick P. Cullingworth J. et al.A systematic review of the staging performance of endoscopic ultrasound in gastro-oesophageal carcinoma.Gut. 2001; 49: 534-539Crossref PubMed Scopus (315) Google Scholar). Early-stage esophageal cancers are adaptable to endoscopic resection, whereas locally advanced and nonmetastatic tumors are suitable for surgical management. Neoadjuvant, perioperative chemotherapy, radiotherapy, or chemoradiotherapy may be more suitable for patients with ≥ T2 tumors; immunotherapy trials are still on going. However, the 5-year survival rate of esophageal cancer patients is less than 20% (4Smyth E.C. Lagergren J. Fitzgerald R.C. Lordick F. Shah M.A. Lagergren P. et al.Oesophageal cancer.Nat. Rev. Dis. Primers. 2017; 317048Crossref PubMed Scopus (341) Google Scholar). The low survival rate can be attributed to the complex biological behavior of tumor cells, which show great resistance to tumor treatment. This is because the high levels of growth factors, cytokines, and hormones in esophageal cancer cells can effectively promote cell proliferation, differentiation, metabolism, and autophagy (5Vander Heiden M.G. Targeting cancer metabolism: a therapeutic window opens.Nat. Rev. Drug Discov. 2011; 10: 671-684Crossref PubMed Scopus (1135) Google Scholar, 6Nishihira T. Hashimoto Y. Katayama M. Mori S. Kuroki T. Molecular and cellular features of esophageal cancer cells.J. Cancer Res. Clin. Oncol. 1993; 119: 441-449Crossref PubMed Scopus (219) Google Scholar, 7Sohda M. Kuwano H. Current status and future prospects for esophageal cancer treatment.Ann. Thorac. Cardiovasc. Surg. 2017; 23: 1-11Crossref PubMed Scopus (150) Google Scholar). In addition, highly activated signal pathways mediated by growth factors and transmembrane receptors in tumor cells also greatly promote tumor growth and metastasis (8Adjei A.A. Hidalgo M. Intracellular signal transduction pathway proteins as targets for cancer therapy.J. Clin. Oncol. 2005; 23: 5386-5403Crossref PubMed Scopus (168) Google Scholar). In addition, based on a large sample size analysis of the genome, transcriptome and proteome studies have shown selective splicing and mutant genes; highly activated cell cycle–related pathways and receptor tyrosine signaling pathways play important roles in esophageal cancer (9Song Y. Li L. Ou Y. Gao Z. Li E. Li X. et al.Identification of genomic alterations in oesophageal squamous cell cancer.Nature. 2014; 509: 91-95Crossref PubMed Scopus (795) Google Scholar, 10Liu W. Xie L. He Y.H. Wu Z.Y. Liu L.X. Bai X.F. et al.Large-scale and high-resolution mass spectrometry-based proteomics profiling defines molecular subtypes of esophageal cancer for therapeutic targeting.Nat. Commun. 2021; 12: 4961Crossref PubMed Scopus (50) Google Scholar, 11Lin D.C. Hao J.J. Nagata Y. Xu L. Shang L. Meng X. et al.Genomic and molecular characterization of esophageal squamous cell carcinoma.Nat. Genet. 2014; 46: 467-473Crossref PubMed Scopus (460) Google Scholar, 12Liu W. Cui Y. Liu W. Liu Z. Xu L. Li E. Deep proteome profiling promotes whole proteome characterization and drug discovery for esophageal squamous cell carcinoma.Cancer Biol. Med. 2022; 19: 273-277Crossref PubMed Scopus (2) Google Scholar). These characteristics are important factors for determining the progression and therapeutic effects of esophageal cancer. In order to cure esophageal cancer, the Food and Drug Administration (FDA) has approved several drugs for esophageal cancer treatment in the past decades (13Bolger J.C. Donohoe C.L. Lowery M. Reynolds J.V. Advances in the curative management of oesophageal cancer.Br. J. Cancer. 2021; 126: 706-717Crossref PubMed Scopus (23) Google Scholar, 14Myint Z.W. Goel G. Role of modern immunotherapy in gastrointestinal malignancies: a review of current clinical progress.J. Hematol. Oncol. 2017; 10: 86Crossref PubMed Scopus (60) Google Scholar). However, drug discovery and development requires many stages, including discovery and development, preclinical research, clinical research, and FDA review, as well as FDA postmarket safety monitoring, which requires a large amount of money and time (15Parvathaneni V. Kulkarni N.S. Muth A. Gupta V. Drug repurposing: a promising tool to accelerate the drug discovery process.Drug Discov. Today. 2019; 24: 2076-2085Crossref PubMed Scopus (181) Google Scholar). Drug repositioning, the new use of old clinically approved drugs, can save time and cost with low risk (15Parvathaneni V. Kulkarni N.S. Muth A. Gupta V. Drug repurposing: a promising tool to accelerate the drug discovery process.Drug Discov. Today. 2019; 24: 2076-2085Crossref PubMed Scopus (181) Google Scholar, 16Aggarwal S. Verma S.S. Aggarwal S. Gupta S.C. Drug repurposing for breast cancer therapy: old weapon for new battle.Semin. Cancer Biol. 2021; 68: 8-20Crossref PubMed Scopus (66) Google Scholar). Recent years have witnessed that several alternative drugs have been successfully applied in clinical trials, such as statins and metformin (17Zhou C. Zhong X. Gao P. Wu Z. Shi J. Guo Z. et al.Statin use and its potential therapeutic role in esophageal cancer: a systematic review and meta-analysis.Cancer Manag. Res. 2019; 11: 5655-5663Crossref PubMed Scopus (7) Google Scholar, 18Wang S. Lin Y. Xiong X. Wang L. Guo Y. Chen Y. et al.Low-dose metformin reprograms the tumor immune microenvironment in human esophageal cancer: results of a phase II clinical trial.Clin. Cancer Res. 2020; 26: 4921-4932Crossref PubMed Scopus (75) Google Scholar). Sulconazole is listed as a broad-spectrum imidazole antifungal drug and has mainly been used to treat common skin fungal diseases in the UK since 1985 (19Benfield P. Clissold S.P. Sulconazole. A review of its antimicrobial activity and therapeutic use in superficial dermatomycoses.Drugs. 1988; 35: 143-153Crossref PubMed Scopus (38) Google Scholar). Our previous study discovered that sulconazole was effective in inhibiting the growth of esophageal cancer cells both in vitro and in vivo (10Liu W. Xie L. He Y.H. Wu Z.Y. Liu L.X. Bai X.F. et al.Large-scale and high-resolution mass spectrometry-based proteomics profiling defines molecular subtypes of esophageal cancer for therapeutic targeting.Nat. Commun. 2021; 12: 4961Crossref PubMed Scopus (50) Google Scholar). Here, we further demonstrate that sulconazole has significant inhibitory effects on a variety of tumor cells. It can not only inhibit the proliferation but also the migration of esophageal cancer cells. Mechanistically, sulconazole promoted PANoptosis by triggering oxidative stress and inhibiting glycolysis to increase radiosensitivity in esophageal cancer. Human cell lines (esophageal carcinoma cells KYSE30, KYSE150, and TE3; and esophageal epithelial cell line SHEE; liver carcinoma cells HepG2, Huh7, and normal Chang liver cells; gastric carcinoma cells SGC7901 and HGC27; lung carcinoma cells A549; breast carcinoma cells MDA-MB-453 and MCF7) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 mg/ml), and streptomycin (100 mg/ml) and kept at 37 °C in a humidified atmosphere containing 5% CO2. All cell lines were tested for mycoplasma contamination. 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%. Cells were inoculated into 96-well plates at an initial density of 1 × 104 cells per well. After cells became adherent, cells were refed with medium containing different concentrations of drugs and incubated for 24 h. Then, 20 μl MTS (G3518, Promega) was added to each well for 2 h. Finally, absorbance was measured at 492 nm using a Multiskan MK3 (Thermo Fisher Scientific). Cell viability curves were calculated and plotted with GraphPad Prism 8 software. KYSE30, KYSE150, and TE3 cells were inoculated into 12-well plates at 500 cells per well and cultured for 12 h until the cells adhered to the dish. Cells were treated with different concentrations of sulconazole for 24 h or different doses of ionizing radiation (IR) and then refed with fresh medium and cultured for 1 to 2 weeks. When there were at least 50 visible cell colonies in the culture plate, the cells were fixed with fixative (methanol:glacial acetic acid = 3:1), stained with 0.5% crystal violet, and then photographed with a Bio-Rad ChemiDoc MP. The number of clones was calculated with ImageJ (https://imagej.nih.gov/ij/) software and plotted with GraphPad Prism 8 (https://www.graphpad-prism.cn/) software. KYSE30 and KYSE150 cells were starved in serum-free medium for 12 h, then resuspended in serum-free medium, then 5 × 104 cells were inoculated into the upper chamber of a transwell, and 500 μl medium containing 10% FBS was added to the lower chamber until cell had adhered. Cells were treated with serum-free medium containing DMSO or 20 μM sulconazole for 24 h. The cells that had migrated to the lower part of the chamber were stained with 0.5% crystal violet. After taking photos, ImageJ software was used to calculate the number of cells passing through the chamber, which was plotted with GraphPad Prism 8 software. KYSE30 and KYSE150 cells were inoculated into 6-well plates. When the cells reached confluence, a sterile tip was used to scratch the middle of each well. Cells were then washed with PBS and cultured in medium containing 2% FBS with DMSO or 20 μM sulconazole for 24 h. To ensure that measurements were made at the same locations, the locations were recorded using a calibration scale on the ix73 inverted microscope (Olympus). The same area was photographed at 0 and 24 h, and the wound healing distance was analyzed by ImageJ software and plotted using GraphPad Prism 8 software. KYSE30 and KYSE150 cells were treated with DMSO or sulconazole (50 μM). Total RNA was extracted with TRIzol (Life Technologies). BGI constructed the RNA library and analyzed the RNA sequences with a BGISEQ-500 system. Data were aligned to genomes using STAR (version 2.7.6a). The differentially expressed mRNAs were identified by DESeq2 (version 1.16.1). The mRNA differences with fold change >1.5 or <0.67 and p-value <0.05 were considered significantly upregulated or downregulated. These differential genes were entered into the Metascape database for KEGG and HALLMARK enrichment analysis (https://metascape.org/). The enriched signal pathways were mapped by R language and GraphPad Prism 8 software. Experiments were designed to investigate the mechanism by which sulconazole inhibits the progression of esophageal cancer cells and leads to cell death. The esophageal cancer cell line KYSE30 was treated with either DMSO (control group) or sulconazole (50 μM) (experimental group) in three biological replicates, then processed according to the filter aided sample preparation method. The TMT global proteomic sequencing method has been previously described by us (10Liu W. Xie L. He Y.H. Wu Z.Y. Liu L.X. Bai X.F. et al.Large-scale and high-resolution mass spectrometry-based proteomics profiling defines molecular subtypes of esophageal cancer for therapeutic targeting.Nat. Commun. 2021; 12: 4961Crossref PubMed Scopus (50) Google Scholar). In short, tryptic peptides were desalinated and lyophilized with StageTips and then labeled with TMT-11plex (Pierce) according to the manufacturer's instructions. Finally, TMT MS experiments were performed on a nanoscale EASY-nLC 1200UHPLC system or nanoU3000UHPLC system (Thermo Fisher Scientific). The samples of the experimental group and the control group were compared by unpaired t test. Protein differences with fold change >1.3 or <0.77 and p-value <0.05 were considered significantly upregulated or downregulated. These differential proteins were entered into the Metascape database for KEGG and HALLMARK enrichment analysis (https://metascape.org/). The enriched signal pathways were mapped by R language and GraphPad Prism 8 software. Data were collected using Xcalibur software (Thermo Fisher Scientific, version 3.0) (https://thermo.flexnetoperations.com/control/thmo/index). Raw data were processed using Proteome Discoverer (https://thermo.flexnetoperations.com/control/thmo/index) (version 2.2), and MS/MS spectra were searched against the reviewed SwissProt human proteome database (20,311 entries). The release/download date was 29th August, 2020. All searches were carried out with a precursor mass tolerance of 20 ppm, fragment mass tolerance of 0.02 Da, oxidation (Met) (+15.9949 Da), TMT6plex (Lys) (229.163 Da), and acetylation (protein N-terminus) (+42.0106 Da) as variable modifications, carbamidomethylation (Cys) (+57.0215 Da), TMT6plex (N-terminal) (229.163 Da) as fixed modification, and three trypsin missed cleavages allowed. Only peptides of at least six amino acids in length were considered. Peptide and protein identifications were filtered by Proteome Discoverer to control a false discovery rate < 1%. At least one unique peptide was required for protein identification. KYSE30 and KYSE150 cells were seeded at a density to achieve 70% confluence into 12-well plates. After cell adhesion, cells were treated with different concentrations of drugs for 12 h. To measure cell death, Annexin V/PI (C1062, Beyotime) was used for the detection of cell apoptosis or necroptosis. In addition, the cells were stained with 5 μg/ml propidium iodide (PI), and the percentage of the PI-positive cells was analyzed using a BD Accuri C6 flow cytometer. There were 1 × 104 cells counted per sample. PI-positive cells may have undergone apoptosis, pyroptosis, necroptosis, or ferroptosis. For live cell imaging, 100 nM SYTOX Green (S7020, Thermo Fisher Scientific) was added for 20 min, then photographed using a Lionheart FX (BioTek). The resulting images were analyzed by ImageJ software, and the percentage of green-positive cells in each image was calculated. Methods for measuring glycolytic metabolic alterations of cellular metabolism have been described in our previous study (20Zeng R.J. Zheng C.W. Gu J.E. Zhang H.X. Xie L. Xu L.Y. et al.RAC1 inhibition reverses cisplatin resistance in esophageal squamous cell carcinoma and induces downregulation of glycolytic enzymes.Mol. Oncol. 2019; 13: 2010-2030Crossref PubMed Scopus (36) Google Scholar). In short, 1 × 104 KYSE30 and KYSE150 cells were inoculated into 96-well plates and treated with sulconazole (30 μM and 50 μM) for 24 h. Then, glucose uptake and lactate production were measured with a Glucose Uptake-Glo Assay kit (Promega). KYSE30 cells were treated with DMSO and sulconazole (30 μM), and KYSE150 cells were treated with DMSO and sulconazole (50 μM) for 24 h. Total RNA was extracted with TRIzol (15596018, Life Technologies). Then, reverse transcription to complementary DNA was performed using HiScript III RT SuperMix for qPCR (+gDNA wiper) (R323-01, Vazyme). Quantitative RT-PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme) and an Applied Biosystems 7500/7500 Fast Real-Time PCR System (Thermo Fisher Scientific). The primers are listed in the supplemental Table S1. Protein extraction and Western blotting were performed as described previously (21Zheng Z.Y. Yang P.L. Luo W. Yu S.X. Xu H.Y. Huang Y. et al.STAT3beta enhances sensitivity to concurrent chemoradiotherapy by inducing cellular necroptosis in esophageal squamous cell carcinoma.Cancers (Basel). 2021; 13: 901Crossref PubMed Scopus (14) Google Scholar). Briefly, extracted proteins were separated by 8% - 15% SDS-PAGE. After the proteins were electrophoretically transferred to polyvinylidene fluoride (IPVH00010, Millipore) membrane, nonspecific binding was blocked by incubation with 5% skim milk, then membranes were incubated with the following primary antibodies: anti-PARP (CST, 9542S, 1:1000), anti-BCL2 (Proteintech, 12789-1-AP, 1:1000), anti-BAX (Proteintech, 50599-2-Ig, 1:1000), anti-caspase3 (CST, 14220, 1:1000), anti-cleaved caspase3 (CST, 9661S, 1:1000), anti-GSDME (Abcam, ab215191, 1:1000), anti-GSDMD (Proteintech, 20770-1-AP, 1:1000), anti-MLKL (Proteintech, 21066-1-AP, 1:1000), anti-pMLKL (Abcam, ab196436, 1:1000), anti-RIPK1 (CST, 3493T, 1:1000), anti-HK Ⅰ (CST, 8337T, 1:1000), anti-HK Ⅱ (CST, 8337T, 1:1000), anti-PFKP (CST, 8337T, 1:1000), anti-PKM1/2 (CST, 8337T, 1:1000), anti-PKM2 (CST, 8337T, 1:1000), anti-LDHA (CST, 8337T, 1:1000), anti-PDH (CST, 8337T, 1:1000), anti-AKT (CST, 4685S, 1:1000), anti-pAKT (CST, 4060S, 1:1000), anti-MEK1 (SB, 101351-T38, 1:1000), anti-pMEK (CST, 9121S, 1:1000), anti-ERK (CST, 4695S, 1:1000), anti-pERK (CST, 4370T, 1:1000), anti-STAT3 (CST, 9139S, 1:1000), and anti-pSTAT3 (CST, 9145S, 1:1000). Membranes were then washed and incubated with the appropriate horseradish peroxidase–conjugated secondary antibodies: anti-rabbit IgG (CST, 7074S, 1:2000) and m-IgGκ BP-HRP (Santa Cruz, sc-516102, 1:4000). A ChemiDoc MP Imaging System was used to visualize the proteins (Bio-Rad). Cells were seeded in 96-well plates at 1 × 104 cells per well. After 12 h, different concentrations of drugs were added for an additional 12 h. Then, the supernatants of the cultures were transferred to new wells, and lactate dehydrogenase (LDH) was measured with a CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (G1780, Promega). Finally, absorbance was measured at 492 nm with a Thermo Multiskan MK3 (Thermo Fisher Scientific). The LDH release ratio was calculated by the following equation: (LDH sample - LDH background - LDH untreated)/(LDH maximum - LDH background) × 100%. KYSE30 and KYSE150 cells were inoculated into 12-well plates. After cell attachment to the dish, cells were treated with DMSO and sulconazole (50 μM) for 24 h to measure reactive oxygen species (ROS) production and mitochondrial membrane potential or 12 h to measure lipid peroxidation. Then, cells were harvested and washed with Hank’s Balanced Salt Solution and incubated cells with 5 μM MitoSOX (M36008, Thermo Fisher Scientific), 100 nM TMRE (HY-D0985A, MedChemExpress), and 5 μM BODIPY 581/591 C11 (D3861, Thermo Fisher Scientific) for 30 min at 37 °C, protected from light. Then, cells were washed twice with Hank’s Balanced Salt Solution and analyzed using a BD Accuri C6 flow cytometer. Transmission electron microscopy (TEM) analysis was performed with a JEM-F200 transmission microscope (JEOL). Samples were fixed with a solution containing 2.5% glutaraldehyde for more than 2 h at 4 °C, then washed in PBS buffer three times at 37 °C for 40s each time. Samples were treated with 0.1% Millipore-filtered cacodylate-buffered tannic acid, postfixed with 1% buffered osmium, and stained en bloc with 1% Millipore-filtered uranyl acetate. Samples were dehydrated in 50%, 70%, and 90% ethanol, respectively, and finally washed with anhydrous ethanol three times, then infiltrated and embedded in LX-112 medium. Medium was allowed to polymerize at 40 °C for 30 min, 60 °C for 10 min, 70 °C for 10 min, and 80 °C for 20 min, then samples were cut into 60 to 80 nm thick sections on an ultrathin sectioning machine, stained with uranyl acetate for 30 min and lead citrate for 5 min, and then examined with a JEM-F200 transmission electron microscope at an accelerating voltage of 80 kV. Digital images were obtained using an AMT Imaging System (Advanced Microscopy Techniques Corp). Statistical analyses were performed using GraphPad Prism 8. All experimental results are described by means ± SD. Independent groups of samples were evaluated using an unpaired two-sided Student’s t test. For comparisons between multiple groups, a two-way ANOVA was used. All specific statistical details are displayed in the figure captions and source data. All statistically relevant experiments were performed with at least three biological replicates. We first evaluated the anticancer effect of sulconazole through cell function experiments in vitro. Cell viability assays showed that the IC50 (half maximal inhibitory concentration) of sulconazole on the esophageal cancer KYSE30 and KYSE150 cell lines was 43.68 μM and 49.15 μM, respectively, and on the SHEE esophageal epithelial cell line was 87.66 μM (Fig. 1B). The IC50 for HepG2 and Huh7 liver cancer cells was 31.81 μM and 19.50 μM, respectively, and 53.93 μM for the normal Chang liver cell line (Fig. 1C). In addition, it also had significant inhibitory effect on gastric cancer (SGC7901, IC50 = 35.31 μM and HGC27, IC50 = 37.24 μM), lung cancer (A549, IC50 = 53.59 μM), and breast cancer (MDA-MB-453, IC50 = 38.73 μM and MCE7, IC50 = 39.04 μM) cell lines (Fig. 1D). These results suggest that sulconazole has a broad-spectrum anticancer effect. The cell clonogenic assay results showed that 20 μM sulconazole could effectively inhibit colony formation of esophageal cancer cells (Fig. 2A). Transwell assays showed that sulconazole markedly reduced the number of cells invading through the chamber (Fig. 2B). In a wound healing assay, sulconazole inhibited the wound healing rate of KYSE30 and KYSE150 cells (Fig. 2, C and D). In conclusion, our results suggest that sulconazole effectively inhibits the progression of esophageal cancer cells in vitro. To investigate the underlying molecular mechanism(s) of sulconazole-mediated inhibition of esophageal cancer cells, RNA and protein of esophageal cancer cells treated with sulconazole were extracted and the transcriptomes (KYSE30 and KYSE150) and proteome (KYSE30) were sequenced (Fig. 3A). After sulconazole treatment, a large number of genes were significantly upregulated and downregulated (FC > 1.5 or <0.67, p < 0.05). There were 2235 genes upregulated, and 2262 genes downregulated in both KYSE30 and KYSE150 cell lines (Fig. 2B). KEGG and HALLMARK analysis of differentially expressed genes showed that the upregulated genes resulting from sulconazole treatment were mostly enriched in apoptosis, ferroptosis, autophagy, mitophagy, and related signal pathways. Downregulated genes were focused on DNA replication/repair, cell cycle, glycolysis, and oxidative phosphorylation (Fig. 2, C and D). Proteomic sequencing showed 419 proteins were upregulated and 434 proteins were downregulated (FC > 1.3 or <0.77, p < 0.05) (Fig. 2E). Similarly, proteome results were also enriched in autophagy, apoptosis, and ferroptosis, in addition to downregulation of glycolysis and oxidative phosphorylation (Fig. 2, F and G). These results indicate that sulconazole suppresses esophageal cancer progression by inhibiting glycolysis and promoting multiple types of programmed cell death (PCD). PANoptosis usually considered regulated by PANoptosome complex is an inflammatory PCD. In addition, the most important point is that PANoptosis cannot be characterized by apoptosis, pyroptosis, necroptosis, and ferroptosis alone (22Malireddi R.K.S. Gurung P. Kesavardhana S. Samir P. Burton A. Mummareddy H. et al.Innate immune priming in the absence of TAK1 drives RIPK1 kinase activity-independent pyroptosis, apoptosis, necroptosis, and inflammatory disease.J. Exp. Med. 2020; 217https://doi.org/10.1084/jem.20191644Crossref PubMed Google Scholar, 23Lee S. Karki R. Wang Y. Nguyen L.N. Kalathur R.C. Kanneganti T.D. AIM2 forms a complex with pyrin and ZBP1 to drive PANoptosis and host defence.Nature. 2021; 597: 415-419Crossref PubMed Scopus (126) Google Scholar). In addition, studies have shown that chemotherapeutic drugs can also induce noninflammasome-dependent PANoptosis (24Lin J.F. Hu P.S. Wang Y.Y. Tan Y.T. Yu K. Liao K. et al.Phosphorylated NFS1 weakens oxaliplatin-based chemosensitivity of colorectal cancer by preventing PANoptosis.Signal. Transduct. Target Ther. 2022; 7: 54Crossref PubMed Scopus (36) Google Scholar). Based on the results of the above analysis, we explored whether sulconazole could induce PANoptosis. First, we found that esophageal cancer cells treated with sulconazole resulted in time-dependent and dose-dependent cell death, including PI-positive cells that are indicative of cellular apoptosis, necroptosis, pyroptosis, or ferroptosis (Figs. 4B and S1, A and C). Meanwhile, we also collected cell lysates for Western blot detection and found that poly ADP-ribose polymerase (PARP) and cleaved PARP also changed accordingly (Figs. 4A and S1, B and D), which indicates apoptosis. Next, in order to explore how sulconazole triggers apoptosis, we treated esophageal cancer cells in a dose-dependent manner at the same time (12 h) and used Western blotting to detect apoptosis-related upstream signal pathways. We found that BCL2 decreased and cleaved BAX and cleaved caspase3 increased after sulconazole treatment (Fig. 4A). It has been reported that during chemotherapy, pyroptosis can occur with apoptosis and depends on the activity of caspase3 (25Wang Y. Gao W. Shi X. Ding J. Liu W. He H. et al.Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin.Nature. 2017; 547: 99-103Crossref PubMed Scopus (1434) Google Scholar), so we tested the pyroptosis effector proteins GSDME and GSDMD and found that the cleaved GSDME, but not cleaved GSDMD, was increased. A simultaneous LDH assay showed that the release of LDH also increased (Fig. 4, A and C). Further, after combination of sulconazole and caspase inhibitor z-VAD (Z-VAD-FMK), cleavage of both PARP and GSDME was obviously inhibited and the percentage of dead cells was also reduced (supplemental Fig. S1, E and F), The above results combined show that sulconazole can induce apoptosis and pyroptosis via a BCL2–Bax–caspase3 axis. Flow cytometry analysis after treatment of cells with sulconazole and staining with Annexin V/PI showed that the percentages of live cells in Q4 (Annexin V-/PI-) decreased, whereas early apoptotic cells in Q3 (Annexin-V+/PI-) and late apoptotic cells and necrotic cells in Q2 (Annexin V+/PI+) increased (Fig. 4, D–F), suggesting that cell necroptosis also occurs. We found that following sulconazole treatment, cells also showed robust phosphorylation of pseudokinase mixed lineage kinase-like domain (MLKL), an inducer of necroptosis (26Karki R. Sharma B.R. Tuladhar S. Williams E.P. Zald" @default.
• W4366085004 created "2023-04-19" @default.
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• W4366085004 date "2023-06-01" @default.
• W4366085004 modified "2023-10-16" @default.
• W4366085004 title "Sulconazole Induces PANoptosis by Triggering Oxidative Stress and Inhibiting Glycolysis to Increase Radiosensitivity in Esophageal Cancer" @default.
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• W4366085004 doi "https://doi.org/10.1016/j.mcpro.2023.100551" @default.
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