背景:显示许多植物次生代谢产物(PSM)嵌入DNA螺旋或与DNA凹槽相互作用。这可能会影响组蛋白-DNA相互作用,从而改变染色质结构和基因组功能。
方法:用15PSM处理后,在HeLa细胞中研究了核小体稳定性和接头组蛋白H1.2,H1.4和H1.5的定位,根据公开的数据,它们是DNA结合剂并具有抗癌活性。染色质重塑剂CBL0137用作对照。使用荧光显微镜研究了PSM的影响,流式细胞术,定量逆转录酶-聚合酶链反应(RT-qPCR),西方印迹。
结果:我们表明,用CBL0137处理1小时强烈抑制DNA合成,并导致强烈的接头组蛋白消耗,与核小体不稳定一致。没有PSM引起核小体不稳定,而大多数对接头组蛋白定位有显著影响。特别是,用11个无毒浓度的PSM进行细胞处理可诱导组蛋白H1.5明显易位到核仁,大多数PSM导致染色质部分中组蛋白H1.2和H1.4的消耗。姜黄素,白藜芦醇,小檗碱,柚皮苷,槲皮素引起所研究的接头组蛋白的所有三种变体的显着再分布,表明PSM对接头组蛋白DNA结合的影响有些重叠。我们证明了PSM,这诱导了组蛋白H1.5(小檗碱,姜黄素和柚皮素),影响细胞合成DNA的比例,表达或不表达细胞周期蛋白B并影响细胞周期分布。小檗碱诱导H1.5易位到核仁显示独立于细胞周期阶段(未分析中期)。
结论:我们首次揭示了PSM对细胞核中接头组蛋白位置的影响,这为PSM作为抗癌药物的研究开辟了新的方向。
BACKGROUND: Many plant secondary metabolites (PSMs) were shown to intercalate into DNA helix or interact with DNA grooves. This may influence histone-DNA interactions changeing chromatin structure and genome functioning.
METHODS: Nucleosome stability and linker histone H1.2, H1.4 and H1.5 localizations were studied in HeLa cells after the treatment with 15 PSMs, which are DNA-binders and possess anticancer activity according to published data. Chromatin remodeler CBL0137 was used as a control. Effects of PSMs were studied using fluorescent microscopy, flowcytometry, quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), western-blotting.
RESULTS: We showed that 1-hour treatment with CBL0137 strongly inhibited DNA synthesis and caused intensive linker histone depletion consistent with nucleosome destabilization. None of PSMs caused nucleosome destabilization, while most of them demonstrated significant influence on linker histone localizations. In particular, cell treatment with 11 PSMs at non-toxic concentrations induced significant translocation of the histone H1.5 to nucleoli and most of PSMs caused depletion of the histones H1.2 and H1.4 from chromatin fraction. Curcumin, resveratrol, berberine, naringenin, and quercetin caused significant redistribution of all three variants of the studied linker histones showing some overlap of PSM effects on linker histone DNA-binding. We demonstrated that PSMs, which induced the most significant redistribution of the histone H1.5 (berberine, curcumin and naringenin), influence the proportion of cells synthesizing DNA, expressing or non-expressing cyclin B and influence cell cycle distribution. Berberine induction of H1.5 translocations to nucleoli was shown to occur independently on the phases of cell cycle (metaphase was not analyzed).
CONCLUSIONS: For the first time we revealed PSM influence on linker histone location in cell nuclei that opens a new direction of PSM research as anticancer agents.