Chelerythrine inhibits esophageal squamous cell carcinoma progression via PINK1-Parkin-mediated mitophagy

Abstract

Background: Chelerythrine (CHE) exhibits notable anti-inflammatory and antitumor properties, while its impact on esophageal squamous cell carcinoma (ESCC), especially the underlying mechanisms remain unclear. In this study, we aim to investigate the roles and mechanism of CHE in the ESCC treatment.

Methods: Human ESCC cell lines and organoids were used for in vitro cell experiments, BALB/c nude mice were used for in vivo animal experiments. To investigate the underlying mechanism of CHE treatment, drug library screen, RNA sequencing analysis, TMT-based quantitative proteomic analysis, western blotting analysis, immunofluorescence, immunohistochemistry, quantitative real-time polymerase chain reaction, mitochondrial membrane potential assay, apoptosis assay, detection of mitochondrial reactive oxygen species (mtROS), autophagic flux monitoring, transmission electron microscopy, and seahorse XF-96 metabolic flux analysis were used to assess the effect of CHE and relevant mechanism.

Results: CHE dose-dependently inhibited the proliferation, migration, and invasion of ESCC cells. CHE also induced cell apoptosis and triggered PTEN-induced kinase 1 (PINK1)-Parkin-mediated mitophagy-mediated cell death by elevating the production of reactive oxygen species in mitochondria and diminishing mitochondrial membrane potential (MMP). However, the production of autophagosomes and autolysosomes induced by CHE altered when used in combination with the autophagy inhibitors 3-methyladenine (3-MA) or bafilomycin A1 (BafA1), indicating that it induced complete autophagic flux in the cells. Mechanistically, CHE affected multiple signaling pathways associated with ubiquitin-mediated proteolysis, mitophagy, and mitochondrial energy metabolism, indicating its close involvement in mitophagy occurrence. In addition, CHE treatment significantly reduced tumor size and weight in nude mice bearing KYSE150 tumors and retarded the growth of organoids derived from patients, it also reduced the ratio of M2 macrophage in tumor microenvironment and cell metabolism.

Conclusions: CHE activates PINK1-Parkin-mediated mitophagy and disrupts mitochondrial homeostasis, and it also affects the tumor environment and cell metabolism, ultimately leading to cell death, supporting the potential of CHE for ESCC therapy.

Keywords: Chelerythrine; Esophageal squamous cell carcinoma; Mitophagy; PINK1-Parkin pathway.

Fig. 1

CHE significantly attenuates the viability and proliferation of ESCC cells following drug screening. A Workflow schematic for CHE screening in a compound screening library. B The structure of CHE molecules. C Effects of CHE on the viability of KYSE30, KYSE150, and KYSE450 cells. D Determination of the IC50 values for CHE in ESCC cells. E The viability of HET-1 A, KYSE30 and KYSE150 cells upon treatments with CHE for 6 h (n = 6). F 2.5 µM CHE significantly suppressed cell proliferation in KYSE30 and KYSE150 cells. G The viability of KYSE30 and KYSE150 cells decreased more dramatically after combinational use of CHE and DDP than use alone for 24 h (n = 6). Data are mean ± SD. **p < 0.01, *** p < 0.001, and **** p < 0.0001 represents the comparison with the control group

Fig. 2

CHE treatment suppresses multiple malignant behaviors of ESCC cells. A Colony formation by KYSE30 and KYSE150 cells was reduced following treatment with varying concentrations of CHE. B Statistical analysis of colony formation in KYSE30 and KYSE150 cells following CHE treatment. C, D Reduced wound healing capability in KYSE30 cells after exposure to 0–5 µM CHE. Scale bar = 500 μm. E, F Impaired wound healing in KYSE150 cells following exposure to 0–5 µM CHE. Scale bar = 500 μm. G, H Decreased migration and invasion capabilities in KYSE30 cells upon treatments with varying concentrations of CHE for 48 h. Scale bar = 100 μm. I, J Attenuated migration and invasion capabilities of KYSE150 cells upon treatments with varying concentrations of CHE for 48 h. Scale bar = 100 μm. K Promotion of apoptosis in KYSE30 and KYSE150 cells by different concentrations of CHE for 48 h. L Statistical analysis of apoptosis in KYSE30 and KYSE150 cells following CHE treatment. M–P Western blot analysis of apoptosis-related protein expression in KYSE30 and KYSE150 cells after treatment with 5 µM CHE for 12 h. GAPDH served as an internal control. Data are mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and “ns” represents not significant

Fig. 3

CHE treatment affects the expression pattern and pathways in ESCC cells at the mRNA level. A Cluster analysis plot illustrating differential gene expression at the mRNA level in KYSE150 cells after treatment with CHE. B Scatter plot displaying pooled upregulated and downregulated genes. C Detailed information for the top 10 upregulated and downregulated genes following CHE treatment. D Cellular components, biological processes, and molecular functions clustering analysis of differentially expressed genes after CHE treatment. E The top 20 KEGG pathway maps that were significantly enriched in differentially expressed genes following CHE treatment, as determined via KEGG pathway cluster analysis. F The top five upregulated genes and the differentially spliced genes in the mitophagy pathway following CHE treatment. G Verification of the top five differentially expressed genes in the mitophagy pathway through qRT-PCR. Data are mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and “ns” indicates not significant

Fig. 4

CHE affects various proteins associated with mitochondrial metabolism and signaling pathways in ESCC cells via proteomic assay. A Heat map illustrating differential protein expression in KYSE150 cells treated with CHE. B The number of altered proteins in KYSE150 cells following CHE treatment. C A volcano map depicting differential protein expression in KYSE150 cells, with arrows indicating the top 5 upregulated and downregulated proteins after CHE treatment. D Rose plot categorizing differential proteins by subcellular localization, with the number in brackets representing the proportion of differential proteins in each subcellular location. E Detailed information for the top 10 differentially expressed proteins that exhibited the highest upregulation and downregulation following CHE treatment. F The top 20 most significant functional results of KEGG pathway enrichment analysis in KYSE150 cells treated with CHE. G A bar chart represents KEGG pathway significant enrichment, with longer bars representing more significant enrichment. H The top 20 most significant functional results of Reactome pathway enrichment analysis in KYSE150 cells treated with CHE. I A bar chart representing Reactom pathway significant enrichment, with longer bars representing more significant enrichment

Fig. 5

Enhanced mtROS production induces apoptosis in ESCC cells following CHE treatment. A Confocal images depicting KYSE30 cells stained with MitoSOX Red following pretreatment with 5 mM NAC for 2 h, followed by CHE treatment for 4 h. Scale bar = 10 μm. B The average fluorescence intensity values of MitoSOX Red in KYSE30 cells (n = 12). C Cell viability of KYSE30 cells treated with CHE after NAC pretreatment for 2 h (n = 5). D Confocal micrographs illustrating KYSE150 cells stained with MitoSOX Red after pretreatment with 5 mM NAC for 2 h, followed by CHE treatment for 4 h. Scale bar = 10 μm. E The average fluorescence intensity values of MitoSOX Red in KYSE150 cells (n = 12). F Cell viability of KYSE150 cells treated with CHE after NAC pretreatment for 2 h (n = 5). G Fluorescence intensity analysis of MitoSOX Red values for KYSE30 cells via flow cytometry after CHE treatment at varying concentrations for 12 h. H Statistical analysis of the fluorescence intensity values of MitoSOX Red in KYSE30 cells. I Fluorescence intensity analysis of MitoSOX Red values in KYSE150 cells through flow cytometry following CHE treatment at varying concentrations for 12 h. J Statistical analysis of fluorescence intensity values of MitoSOX Red in KYSE150 cells. K Apoptosis analysis in KYSE30 cells following pretreatment with NAC (5 mM) for 2 h and treatment with CHE (5 µM) for 48 h with Annexin-V/PI staining and flow cytometry. L Statistical analysis of the apoptosis rate in KYSE30 cells following various treatments. M Apoptosis analysis in KYSE150 cells following pretreatment with NAC (5 mM) for 2 h and treatment with CHE (5 µM) for 48 h using Annexin-V/PI staining and flow cytometry. N Statistical analysis of the apoptosis rate in KYSE150 cells following various treatments. All data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and “ns” denotes not significant

Fig. 6

CHE depolarizes MMP and induces mitophagy in ESCC cells. A MMP was assessed in KYSE30 cells treated with 5 µM CHE or 10 µM CCCP for 6 h, and mitochondrial fluorescence transitions were observed using a fluorescence microscope. JC-1 monomers (green, indicative of decreased MMP), JC-1 aggregates (red, representing normal MMP), and the merged image indicated the intracellular localization of JC-1 monomers and aggregates. Scale bar = 50 µm. B Fluorescence intensity values of JC-1 monomers in KYSE30 cells treated with 5 µM CHE or 10 µM CCCP for 6 h were analyzed through flow cytometry. C MMP was evaluated in KYSE150 cells treated with 5 µM CHE or 10 µM CCCP for 6 h, and mitochondrial fluorescence transitions were observed with a fluorescence microscope. D Fluorescence intensity values of JC-1 monomers in KYSE150 cells treated with 5 µM CHE or 10 µM CCCP for 6 h were analyzed through flow cytometry. E, F Representative images of KYSE30 and KYSE150 cells labeled with fluorescent probes for mitochondria (MitoBright Red) and autophagy biomarkers (LC3B). Scale bar = 5 µm. G TEM images of KYSE150 cells before and after CHE treatment. Yellow arrows represent mitochondria, red arrows represent lysosomes, and green arrows represent autophagosomes. Scale bar = 5 µm, 1 µm. H Confocal images of KYSE150 cells expressing mRFP-GFP-LC3 were pretreated with 2 mM 3-MA for 2 h and then CHE for 4 h. Scale bar = 5 µm, 2 µm. White arrows indicate autophagosomes (right) and autolysosomes (left). I Quantification of autophagosomes and autolysosomes in KYSE150 cells in panel H (n = 5). J, K The viability of KYSE30 and KYSE150 cells treated with CHE after 3-MA pretreatment for 2 h (n = 5). L Confocal images of KYSE150 cells expressing mRFP-GFP-LC3 after pretreatment with 50 nM BafA1 for 2 h and treatment with CHE for 4 h. Scale bar = 5 µm, 2 µm. White arrows indicate autophagosomes (right) and autolysosomes (left). M Quantification of autophagosomes and autolysosomes in KYSE150 cells in panel L (n = 5). N, O The viability of KYSE30 and KYSE150 cells treated with CHE after BafA1 pretreatment for 2 h (n = 5). All data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and “ns” indicates not significant

Fig. 7

PINK1-Parkin pathway mediates mitophagy induced by CHE treatment. A Western blot analysis of changes in mitophagy-related proteins in KYSE30 cells treated with 5 µM CHE and 10 µM CCCP for 12 h. B Quantitave and statistical analysis of the intensities of altered proteins in KYSE30 cells. C Western blot analysis of alterations in mitophagy-related proteins in KYSE150 cells treated with 5 µM CHE and 10 µM CCCP for 12 h. D Quantitative and statistical analysis of the intensities of changed proteins in KYSE150 cells. E, F Representative images of PINK1 and Parkin proteins and mitochondria labeled with the fluorescent probe MitoBright Red in KYSE30 cells. Scale bar = 5 μm. G, H Representative images of PINK1 and Parkin proteins and mitochondria labeled with the fluorescent probe MitoBright Red in KYSE150 cells. Scale bar = 5 μm. I, J Western blot and statistical analysis of the alterations in Ub levels in KYSE30 cells treated with 5 µM CHE and 10 µM CCCP for 12 h. K, L Western blot and statistical analysis of the alterations in Ub levels in KYSE150 cells treated with 5 µM CHE and 10 µM CCCP for 12 h. All images represent three independent experiments. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 8

CHE retards the growth of KYSE150 in vivo via PINK1-Prakin-mediated mitophagy. A Diagrammatic representation of the animal experiment involving mice treated with 5 mg/kg CHE and control mice. B Representative images of mice and dissected tumors from tumor-bearing mice treated with or without CHE. C Tumor weight significantly decreased in CHE-treated mice in comparison with control mice (n = 5, p < 0.01). D Tumor volume was significantly reduced in CHE-treated mice in comparison with control mice (n = 5, p < 0.05). E Changes in body weight of CHE-treated and control mice (n = 5, p < 0.05). F, G The protein levels of PINK1, Parkin, and SQSTM1/p62 significantly increased in CHE-treated xenograft tumors compared to control (p < 0.05 for PINK1 and SQSTM1/p62 proteins, p < 0.001 for Parkin protein). H H&E staining of tumor tissue from CHE-treated mice and control mice (Scale bar = 100 μm, 20 μm). I, J Increased levels of PINK1 protein in CHE-treated xenograft tumors validated with immunohistochemistry (Scale bar = 100 μm, 50 μm, p < 0.0001). K, L Increased levels of Parkin protein in CHE-treated xenograft tumors validated with immunohistochemistry (Scale bar = 100 μm, 50 μm, p < 0.0001). M Decreased levels of CD206 protein in CHE-treated xenograft tumors validated with immunohistochemistry (Scale bar = 100 μm, 50 μm). N The viability of ESCC-derived organoids decreased along with the increased levels of CHE. O The determination of IC50 in ESCC organoids using CHE. P CHE treatment reduced the viability of organoids derived from patients with ESCC (n = 6, p < 0.01). Q Schematic representation of the mechanism by which CHE treatment triggers mitophagy in ESCC cells. All data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001