To study the inhibitory effects on microphthalmia-associated transcription factor (MITF)-related biological aspects in malignant melanomas (MMs) in the presence or absence of the low-molecular MITF specific inhibitor ML329, cell viability, cellular metabolic functions, and three-dimensional (3D) spheroid formation efficacy were compared among MM cell lines including SK-mel-24, A375, dabrafenib- and trametinib-resistant A375 (A375DT), and WM266-4. Upon exposure to 2 or 10 μM of ML329, cell viability was significantly decreased in WM266-4, SK-mel-24, and A375DT cells, but not A375 cells, in a dose-dependent manner, and these toxic effects of ML329 were most evident in WM266-4 cells. Extracellular flux assays conducted using a Seahorse bioanalyzer revealed that treatment with ML329 increased basal respiration, ATP-linked respiration, proton leakage, and non-mitochondrial respiration in WM266-4 cells and decreased glycolytic function in SK-mel-24 cells, whereas there were no marked effects of ML329 on A375 and A375DT cells. A glycolytic stress assay under conditions of high glucose concentrations also demonstrated that the inhibitory effect of ML329 on the glycolytic function of WM266-4 cells was dose-dependent. In addition, ML329 significantly decreased 3D-spheroid-forming ability, though the effects of ML329 were variable among the MM cell lines. Furthermore, the mRNA expression levels of selected genes, includingSTAT3as a possible regulator of 3D spheroid formation,KRASandSOX2as oncogenic-signaling-related factors,PCG1aas the main regulator of mitochondrial biogenesis, andHIF1aas a major hypoxia transcriptional regulator, fluctuated among the MM cell lines, possibly supporting the diverse ML329 effects mentioned above. The findings of diverse ML329 effects on various MM cell lines suggest that MITF-associated biological activities are different among various types of MM.
为研究在存在或不存在低分子MITF特异性抑制剂ML329的情况下，对恶性黑色素瘤中MITF相关生物学特性的抑制作用，本研究比较了包括SK-mel-24、A375、达拉非尼和曲美替尼耐药A375（A375DT）以及WM266-4在内的黑色素瘤细胞系的细胞活力、细胞代谢功能和三维球体形成效能。当暴露于2或10 μM的ML329时，WM266-4、SK-mel-24和A375DT细胞的细胞活力显著降低，而A375细胞则未受影响，且这种效应呈剂量依赖性，其中ML329对WM266-4细胞的毒性作用最为明显。通过Seahorse生物分析仪进行的细胞外通量分析显示，ML329处理增加了WM266-4细胞的基础呼吸、ATP相关呼吸、质子泄漏和非线粒体呼吸，并降低了SK-mel-24细胞的糖酵解功能，而对A375和A375DT细胞则无明显影响。在高葡萄糖浓度条件下进行的糖酵解应激实验也表明，ML329对WM266-4细胞糖酵解功能的抑制作用呈剂量依赖性。此外，ML329显著降低了三维球体形成能力，但其效果在不同黑色素瘤细胞系中存在差异。同时，所选基因的mRNA表达水平在不同黑色素瘤细胞系中波动，这些基因包括作为三维球体形成可能调节因子的STAT3、作为致癌信号相关因子的KRAS和SOX2、作为线粒体生物合成主要调节因子的PCG1a以及作为主要缺氧转录调节因子的HIF1a，这可能支持了上述ML329的多样化效应。ML329对不同黑色素瘤细胞系的多样化效应表明，MITF相关的生物活性在不同类型的黑色素瘤中存在差异。
Significant and Various Effects of ML329-Induced MITF Suppression in the Melanoma Cell Line
肿瘤(癌症)患者之家