Centre of Experimental Medicine, Institute of Experimental Pharmacology and Toxicology Slovak Academy of Sciences, Dubravska cesta 9, 841 04 Bratislava, Slovak Republic.
Institute of Pharmacognosy, Interdisciplinary Excellence Centre, University of Szeged, 6720 Szeged, Hungary.
Life Sci. 2020 Nov 1;260:118419. doi: 10.1016/j.lfs.2020.118419. Epub 2020 Sep 12.
In our study, the anticancer effects of a semisynthetic p-quinol, protoapigenone 1'-O-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'-O-butyl ether moiety can be associated with improved senescence-inducing effect and, thus, enhanced chemotherapeutic potency of PABut compared to the unmodified natural protoflavone.
在本研究中，我们测试了半合成 p-苯醌，原儿茶素 1'-O-丁醚（PABut）对人黑色素瘤 A375 细胞的抗癌作用，并与天然同系物原儿茶素（PA）进行了比较。
用 MTT 法测定 PABut 和 PA 的细胞毒性。采用流式细胞术分析评估了这些化合物对 A375 细胞中 ROS 生成和细胞周期分布的影响。此外，通过 AO/EB 双重染色和流式细胞术评估了细胞凋亡。用分光光度法和 Western blot 分析定量测定衰老标志物。
PABut 和 PA 在亚微米浓度下对黑色素瘤 A375 细胞均表现出显著的细胞毒性。两种原儿茶素都能使细胞周期阻滞在 G2/M 期。然而，PABut 处理后细胞内 ROS 水平升高更为明显。经两种原儿茶素处理 48 小时后，细胞凋亡增加。两种受试原儿茶素均可显著上调 A375 细胞中 p21 蛋白水平。与 PA 不同，PABut 可显著降低 NAD 依赖性去乙酰化酶 SirT1 和 β-肌动蛋白的蛋白水平，同时轻度显著上调线粒体 SOD2 和衰老标志物 p16 蛋白和 SA-β-Gal 活性。然而，仅在 PA 处理后发现 p53 显著上调。
这些结果表明，PABut 和 PA 在黑色素瘤细胞中具有较高的化疗潜力，适合进一步测试。此外，与未修饰的天然原儿茶素相比，用 1'-O-丁醚修饰原儿茶素可增强诱导衰老的作用，从而提高 PABut 的化疗效力。
