关键词: 黑色素瘤, 3胍基丙酸, 丝裂原活化蛋白激酶, 细胞增殖, 肿瘤迁移

Abstract: 【Abstract】 Objective To investigate the effects of the solute carrier family 6 member 8 （SLC6A8） inhibitor 3-guanidinopropionic acid （β-GPA） on the migration of melanoma cell lines A375 and SK-MEL-28 and to explore the underlying mechanisms. Methods Melanoma A375 and SK-MEL-28 cells were treated with β-GPA at concentrations of 0 （control group）, 1, 5, 10, 20, 40, 80, 100, and 200 mmol/L for 24 h. The inhibitory effects of β-GPA on melanoma cell proliferation were evaluated using the Cell Counting Kit-8 （CCK-8） assay. To investigate the effects of β-GPA on melanoma cell migration, A375 and SK-MEL-28 cells were treated with 0 （control group）, 10, 20, and 40 mmol/L β-GPA, and migration ability was assessed using Transwell migration assays and wound-healing assays. A375 cells were treated with 0 （control group） or 20 mmol/L β-GPA for 24 h, and transcriptome sequencing was performed to identify differentially expressed genes between the control and treatment groups. Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis was conducted, and Western blot analysis was further used to verify the regulatory effects of 0, 10, 20, and 40 mmol/L β-GPA on the c-Jun N-terminal kinase （JNK） signaling pathway. The C57BL/6 mouse melanoma lung metastasis model was established to evaluate the therapeutic effects of β-GPA on melanoma metastasis. The mice were divided into 4 groups: control group, 100 mg/kg β-GPA group, 500 mg/kg β-GPA group, and 30 mg/kg dabrafenib group （positive control）. Comparisons among multiple groups were performed using one-way analysis of variance followed by Dunnett′s post hoc test. Results CCK-8 assays showed that the half-maximal inhibitory concentration （IC50） values of β-GPA for A375 and SK-MEL-28 cells were 58.63 ± 3.74 mmol/L and 76.38 ± 1.35 mmol/L, respectively. β-GPA inhibited melanoma cell proliferation in a concentration-dependent manner （both P < 0.001）. Transwell migration assays demonstrated that, in A375 cells, the numbers of migrated cells in the 10, 20, and 40 mmol/L β-GPA treatment groups （47.00 ± 2.65, 38.33 ± 1.53, and 32.67 ± 0.58, respectively） were all significantly lower than that in the control group （86.67 ± 2.31, all P < 0.01）. In SK-MEL-28 cells, the numbers of migrated cells in the 10, 20, and 40 mmol/L β-GPA treatment groups （68.33 ± 3.51, 55.33 ± 4.16, and 49.67 ± 2.52, respectively） were also significantly lower than that in the control group （89.67 ± 2.52, all P < 0.01）. Wound-healing assays showed that, in A375 cells, migration distances at 12 h after treatment with 10, 20, and 40 mmol/L β-GPA （93.22 ± 7.64 μm, 66.38 ± 10.66 μm, and 64.27 ± 6.81 μm, respectively） were significantly shorter than that in the control group （132.10 ± 11.67 μm, all P < 0.01）. At 24 h, migration distances in the 10, 20, and 40 mmol/L β-GPA treatment groups （169.50 ± 8.48 μm, 141.20 ± 14.88 μm, and 88.98 ± 4.24 μm, respectively） were also significantly shorter than that in the control group （238.00 ± 7.44 μm, all P < 0.001）. In SK-MEL-28 cells, migration distances at 12 h after treatment with 10, 20, and 40 mmol/L β-GPA （115.80 ± 7.61 μm, 123.20 ± 5.47 μm, and 97.88 ± 9.12 μm, respectively） were significantly shorter than that in the control group （203.00 ± 21.40 μm, all P < 0.01）. At 24 h, migration distances in the 10, 20, and 40 mmol/L β-GPA treatment groups （212.90 ± 2.88 μm, 220.60 ± 8.90 μm, and 187.90 ± 21.74 μm, respectively） were significantly shorter than that in the control group （348.40 ± 15.71 μm, all P < 0.001）. Transcriptomic sequencing revealed that β-GPA significantly regulated signaling pathways including estrogen, phospholipase D, and mitogen-activated protein kinase （MAPK） pathways. Western blot analysis showed that, in A375 cells, the relative expression levels of phosphorylated JNK （p-JNK） protein in the 10, 20, and 40 mmol/L β-GPA treatment groups （0.88 ± 0.06, 0.85 ± 0.08, and 0.69 ± 0.07, respectively） were significantly lower than that in the control group （1.00 ± 0.07, all P < 0.05）. In SK-MEL-28 cells, the relative expression levels of p-JNK protein in the 10, 20, and 40 mmol/L β-GPA treatment groups （0.75 ± 0.09, 0.52 ± 0.08, and 0.50 ± 0.09, respectively） were also significantly lower than that in the control group （1.00 ± 0.06, all P < 0.05）. In vivo experiments using the C57BL/6 mouse melanoma lung metastasis model showed that the numbers of melanoma nodules in the 100 mg/kg β-GPA group, 500 mg/kg β-GPA group, and 30 mg/kg dabrafenib group （7.33 ± 0.76, 3.33 ± 1.02, and 5.17 ± 1.08, respectively） were all significantly lower than that in the control group （17.17 ± 1.07, all P < 0.001）. Conclusion β-GPA inhibits the migration of melanoma cells, and the underlying mechanism may be related to suppression of the JNK signaling pathway.

Key words: Melanoma, 3-Guanidinopropanoic acid, Mitogen-activated protein kinase, Cell proliferation, Tumor migration