In our study, the anticancer effects of a semisynthetic -quinol, protoapigenone 1′--butyl ether (PABut), were tested in human melanoma A375 cells also in comparison with natural congener, protoapigenone (PA). The cytotoxic effect of PABut and PA was determined using MTT assay. Flow cytometry analysis was used to evaluate the influence of the compounds tested on ROS generation and cell cycle distribution in A375 cells. Moreover, apoptosis was evaluated by AO/EB dual staining as well as by flow cytometry. Markers of senescence were quantified by spectrofluorimetry and by Western blot analysis. Both PABut and PA showed significant cytotoxicity against melanoma A375 cells at sub-micromolar concentrations. Both protoflavones induced comparable cell cycle arrest in G2/M phase. However, a more profound upregulation of intracellular ROS levels was found following PABut treatment. An increased apoptosis in the cells following 48 h treatment with both protoflavones tested was also confirmed. Both compounds tested remarkably upregulated p21 protein levels in A375 cells. Unlike PA, PABut significantly decreased protein levels of NAD-dependent deacetylase SirT1 and β-actin accompanied by mild significant upregulation of mitochondrial SOD2 and senescence markers, p16 protein and SA-β-Gal activity. However, a significant upregulation of p53 only following PA treatment was found. These results suggest that PABut and PA confer high chemotherapeutic potential in melanoma cells and are suitable for further testing. Furthermore, modification of protoapigenone with 1′--butyl ether moiety can be associated with improved senescence-inducing effect and, thus, enhanced chemotherapeutic potency of PABut compared to the unmodified natural protoflavone.

中文翻译：

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原黄酮在黑色素瘤治疗中的作用：原芹酮及其合成烷基衍生物在 A375 细胞中的促氧化和促衰老作用


在我们的研究中，在人黑色素瘤 A375 细胞中测试了半合成对苯二酚原芹菜酮 1'-丁基醚 (PABut) 的抗癌作用，并与天然同类物原芹菜酮 (PA) 进行了比较。使用MTT法测定PABut和PA的细胞毒性作用。使用流式细胞术分析评估测试化合物对 A375 细胞中 ROS 生成和细胞周期分布的影响。此外，通过AO/EB双染色以及流式细胞术评估细胞凋亡。通过荧光分光光度法和蛋白质印迹分析对衰老标志物进行定量。 PABut 和 PA 在亚微摩尔浓度下均对黑色素瘤 A375 细胞表现出显着的细胞毒性。两种原黄酮均诱导类似的细胞周期停滞在 G2/M 期。然而，PABut 治疗后发现细胞内 ROS 水平出现更深刻的上调。还证实用两种测试的原黄酮处理 48 小时后细胞凋亡增加。经测试，这两种化合物均显着上调 A375 细胞中的 p21 蛋白水平。与 PA 不同，PABut 显着降低 NAD 依赖性脱乙酰酶 SirT1 和 β-肌动蛋白的蛋白质水平，同时轻微显着上调线粒体 SOD2 和衰老标记物、p16 蛋白和 SA-β-Gal 活性。然而，仅在 PA 处理后发现 p53 显着上调。这些结果表明 PABut 和 PA 对黑色素瘤细胞具有较高的化疗潜力，适合进一步测试。此外，与未修饰的天然原黄酮相比，用 1'-丁基醚部分修饰原芹酮可改善衰老诱导作用，从而增强 PABut 的化疗效力。