Wang Lei, Cai Wei, Han Bing, Zhang Jue, Yu Bing, Chen Ming
Burn and Plastic Surgery, Zhongda Hospital Affiliated Southeast University, Nanjing, 210009, People's Republic of China.
Burn and Plastic Surgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, 210011, People's Republic of China.
Onco Targets Ther. 2021 Feb 25;14:1261-1273. doi: 10.2147/OTT.S283548. eCollection 2021.
Malignant melanoma was characterized by insensitive chemotherapy, drug resistance, and high metastatic ability, which resulted in the main reason for the mortality among skin-related cancers. The current agents were not sufficient to improve the treatment status of melanoma patients, and it was still needed to develop new chemotherapeutic drugs for melanoma. Our study aimed to study the anticancer effects and potential mechanisms of ouabain on melanoma cells.
The inhibitory effects of ouabain were determined by CCK8 and colony formation assays, and the morphological changes of melanoma cells were observed by inverted microscope. The apoptosis induction and cell cycle distribution were detected by annexin V/PI double staining and PI staining, respectively. The expression of the biomarker proteins in apoptosis and G2/M phase were determined by Western blotting analysis. The effects of ouabain on the migration of melanoma cells were measured by transwell migration assay and wound closure analysis. The potential mechanisms of ouabain in melanoma cells were analyzed by transcriptome sequencing.
Our present study demonstrated that ouabain exhibited strong inhibitory effects on cell proliferation and triggered dramatical morphological changes of melanoma cells. Moreover, ouabain induced significant apoptosis in A375 rather than SK-Mel-28 cells via upregulation of bax expression and downregulation of bcl-2 expression. Consistently, ouabain treatment induced cell cycle arrest at G2/M phase in both A375 and SK-Mel-28 cells via upregulation of cyclin B1 and downregulation of cdc2 and cdc25c. Importantly, ouabain suppressed the migration of A375 and SK-Mel-28 cells. Furthermore, the transcriptome sequencing demonstrated that p53 and MAPK signaling pathway might play important roles in the inhibitory effects of ouabain.
Our study revealed that ouabain exhibited dramatical anticancer effects, which provided a novel application for cardiac glycoside drugs in the clinical treatment of melanoma.
恶性黑色素瘤具有化疗不敏感、耐药及高转移能力的特点，这是皮肤相关癌症患者死亡的主要原因。目前的药物不足以改善黑色素瘤患者的治疗状况，仍需要开发新的黑色素瘤化疗药物。本研究旨在探讨哇巴因对黑色素瘤细胞的抗癌作用及潜在机制。
采用CCK8法和集落形成实验测定哇巴因的抑制作用，通过倒置显微镜观察黑色素瘤细胞的形态变化。分别采用膜联蛋白V/碘化丙啶双染法和碘化丙啶染色法检测细胞凋亡诱导情况和细胞周期分布。通过蛋白质免疫印迹分析确定凋亡和G2/M期生物标志物蛋白的表达。采用Transwell迁移实验和伤口愈合分析检测哇巴因对黑色素瘤细胞迁移的影响。通过转录组测序分析哇巴因在黑色素瘤细胞中的潜在机制。
我们目前的研究表明，哇巴因对细胞增殖具有强烈的抑制作用，并引发黑色素瘤细胞显著的形态变化。此外，哇巴因通过上调bax表达和下调bcl-2表达，在A375细胞而非SK-Mel-28细胞中诱导显著凋亡。同样，哇巴因处理通过上调细胞周期蛋白B1和下调cdc2及cdc25c，使A375和SK-Mel-28细胞的细胞周期停滞在G2/M期。重要的是，哇巴因抑制A375和SK-Mel-28细胞的迁移。此外，转录组测序表明p53和丝裂原活化蛋白激酶信号通路可能在哇巴因的抑制作用中发挥重要作用。
我们的研究表明，哇巴因具有显著的抗癌作用，为强心苷类药物在黑色素瘤临床治疗中的应用提供了新途径。
