Objective:To elucidate the effect of bepridil, an Na +/Ca 2+ exchanger (NCX) inhibitor, on the proliferation, migration and apoptosis of melanoma cells, and to explore their potential underlying mechanisms. Methods:Six paraffin-embedded tissue specimens were collected from 3 patients histopathologically diagnosed with melanocytic nevi and 3 patients histopathologically diagnosed with melanoma in the Hospital for Skin Diseases, Chinese Academy of Medical Sciences from January to December 2023. NCX1 expression in tissues was determined by immunohistochemical staining, and Western blot analysis was performed to verify the expression of NCX1 in primary melanocytes and melanoma cell lines A375, A875, SKMEL-28, M14, MV3, and SK-MEL-5. Cell counting kit 8 (CCK8) assay was conducted to evaluate the effect of bepridil at different concentrations on the viability of melanoma cells, and the proliferation curve was drawn to calculate the half inhibitory concentration (IC50) of bepridil. Some melanoma cells were then treated with bepridil at IC50 (bepridil groups), and cells treated with dimethyl sulfoxide-containing media served as control groups. Intracellular Ca 2+ levels were assessed in A375 and SK-MEL-28 cells using the Fluo-4 calcium assay kit, the migration and apoptosis of A375, SK-MEL-28, and A2058 cells were estimated by Transwell assay and flow cytometry respectively. The effects of bepridil treatment on gene expression and pathways in A375 cells were evaluated by transcriptome sequencing, Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The content of reactive oxygen species (ROS) in melanoma cells after the bepridil treatment was detected by flow cytometry, and the expression of endoplasmic reticulum stress- and mitochondrial apoptotic pathway-related molecules was determined by Western blot analysis. Comparisons between two groups were performed by t test. Results:Immunohistochemical assay showed that the expression of NCX1 was significantly higher in the melanoma tissues (0.320 ± 0.020) than in the melanocytic nevus tissues (0.235 ± 0.008, t = 4.04, P = 0.016) ; Western blot analysis showed that the NCX1 protein bands were darker in color in the melanoma cell lines than in the primary melanocytes. CCK8 assay showed a gradual decrease in melanoma cell viability with increasing concentrations of bepridil. In the A375 and SK-MEL-28 cells, the fluorescence intensity of calcium was higher in the bepridil groups after the treatment with bepridil at IC50 (25 μmol/L) (64.82 ± 2.98, 75.84 ± 2.07, respectively) than in the corresponding control groups (37.10 ± 2.33, 66.54 ± 1.47, respectively, both P < 0.05) ; in the A375, SK-MEL-28, and A2058 cells, the migration ability was lower in the bepridil groups (103.00 ± 9.07, 67.33 ± 7.22, 61.33 ± 1.76, respectively) than in the corresponding control groups (400.00 ± 25.17, 276.70 ± 14.63, 116.00 ± 10.69, respectively, all P < 0.05), while their apoptosis rates were higher in the bepridil groups (5.72% ± 0.06%, 13.58% ± 0.86%, 25.76% ± 1.95%, respectively) than in the corresponding control groups (3.99% ± 0.50%, 6.47% ± 0.88%, 8.01% ± 0.36%, respectively, all P < 0.05). Transcriptome sequencing revealed 119 up-regulated genes and 164 down-regulated genes in bepridil-treated A375 cells compared with control cells, and the differentially expressed genes were mainly associated with metabolic pathways, endoplasmic reticulum stress, and tumor-related pathways. The ROS levels were higher in bepridil-treated A375, SK-MEL-28, and A2058 cells (1 907 ± 33, 7 607 ± 535, 3 380 ± 300, respectively) than in the corresponding control groups (1 646 ± 16, 4 386 ± 163, 2 110 ± 66, respectively, all P < 0.05). Western blot analysis showed that the expression of C/EBP homologous protein and activating transcription factor 4 was higher in bepridil-treated A375 and SK-MEL-28 cells than in the corresponding control groups, but was lower in the A375 and SK-MEL-28 cells treated with bepridil and BAPTA (a calcium chelator) than in the corresponding cells treated with bepridil alone. Conclusion:The NCX inhibitor bepridil could increase intracellular Ca 2+ levels, suppress the proliferation and migration, and promote the apoptosis of melanoma cells, which may be closely related to biological processes such as endoplasmic reticulum stress.

Objective:To investigate the expression of epigenetic inhibitor polycomb group proteins such as enhancer of zeste homolog 1/2 (EZH1/EZH2), embryonic ectoderm development protein (EED) and suppressor of zeste 12 (SUZ12) in common cutaneous T-cell lymphomas and lymphoproliferative disorders (CTCL/LPD) .Methods:Totally, 93 paraffin-embedded skin samples of CTCL/LPD and 8 of lichen planus were collected from Hospital for Skin Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College between 2012 and 2019, and subjected to immunohistochemical staining to determine the protein expression of EZH2, EED, SUZ12 and EZH1. Statistical analysis was carried out with SPSS 25.0 software by using chi-square test and Spearman correlation analysis.Results:The 93 cases of CTCL/LPD included 44 cases of mycosis fungoides (MF), 17 natural killer/T cell lymphoma (NK/TCL), 8 primary cutaneous anaplastic large cell lymphoma (PC-ALCL), 8 lymphomatoid papulosis (LyP), 8 hydroa vacciniforme-like lymphoproliferative disorder (HV-like LPD) and 8 cases of subcutaneous panniculitis-like T cell lymphoma (SPTCL). Among the 93 CTCL/LPD cases, 83 (89.2%) were positive for EZH2, 81 (87.1%) for EED, 78 (83.9%) for SUZ12 and 37 (39.8%) for EZH1; among the 8 cases of lichen planus, 1 was positive for EZH2, all were positive for EZH1, and all were negative for EED and SUZ12. The expression of EZH2, EED, SUZ12 and EZH1 in lichen planus samples significantly differed from all the CTCL/LPD samples ( χ2 = 41.75, 39.74, 39.36, 32.83, respectively, all P < 0.001), and from MF, NK/TCL, PC-ALCL, LyP, HV-like LPD and SPTCL samples separately (α = 0.008 3, all P < 0.001). Meanwhile, the score of EZH2 expression was negatively correlated with that of EZH1 expression in the MF, NK/TCL, PC-ALCL, LyP, HV-like LPD and SPTCL tissues ( rs = -0.60, -0.68, -0.89, -0.74, -0.93, -0.80, respectively, all P < 0.05) . Conclusion:Polycomb group proteins EZH2, EED, SUZ12 and EZH1 are abnormally expressed in CTCL/LPD lesions.