Abstract: 【Abstract】 Objective To elucidate the effect of bepridil, an Na+/Ca2+ 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 Ca2+ 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 Ca2+ 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.

Key words: Melanoma, Cell proliferation, Transcriptome, Endoplasmic reticulum stress, Na /Ca2 exchanger inhibitors, Cell migration, Bepridil