Anticancer Effects of 1,3-Dihydroxy-2-Methylanthraquinone and the Ethyl Acetate Fraction of Hedyotis Diffusa Willd against HepG2 Carcinoma Cells Mediated via Apoptosis.
Anticancer Effects of 1,3-Dihydroxy-2-Methylanthraquinone and the Ethyl Acetate Fraction of Hedyotis Diffusa Willd against HepG2 Carcinoma Cells Mediated via Apoptosis.
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DOI：
10.1371/journal.pone.0151502
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Li QS
中科院分区：
文献类型：
--
作者：
Li YL;Zhang J;Min D;Hongyan Z;Lin N;Li QS
白花蛇舌草是我国传统医药中常用的一种药物，具有疗效温和、毒性低的特点，可用于治疗多种疾病，包括癌症。本研究的目的是鉴定白花蛇舌草中的主要抗癌成分，并探讨其作用机制。用乙酸乙酯对白花蛇舌草进行提取和分级，得到H-乙酸乙酯级分，其显示出比用磺酰罗丹明B测定获得的其他级分对HepG 2细胞更高的抗癌活性。乙酸乙酯部位的活性成分为1，3-二羟基-2-甲基蒽醌（DMQ），在125 μmol/L时，抑制率高达48.9 ± 3.3%，选择性高达9.4 ± 4.5倍（p<0.01）。用级分和DMQ处理的HepG 2细胞使用光学和荧光显微镜在形态上可视化。Annexin V-异硫氰酸荧光素/碘化丙啶染色流式细胞术、DNA梯状条带和细胞周期分布测定。机制研究显示半胱天冬酶-3、半胱天冬酶-8和半胱天冬酶-9蛋白酶活性的上调（p<0.001），表明线粒体凋亡和死亡受体途径的参与。进一步的研究表明，与对照组相比，DMQ和组分处理的HepG 2细胞中的活性氧增加（p<0.01），而线粒体膜电位显著降低（p<0.001）。Western blot结果显示，Bax、p53、Fas、FasL、p21和细胞色素C呈剂量依赖性上调（p<0.01），Bcl-2、线粒体细胞色素C、cyclin E和CDK 2呈剂量依赖性下调（p<0.01）。逆转录-聚合酶链反应（RT-PCR）结果显示，p53、Bax mRNA表达增加（P<0.001），Bcl-2 mRNA表达减少（P<0.001）。caspase-8抑制剂Z-IETD-FMK或caspase-9抑制剂Z-LEHD-FMK预处理可减弱DMQ及其组分对HepG 2细胞的生长抑制和凋亡诱导作用。这些结果表明，DMQ和H-乙酸乙酯部位的白花蛇舌草具有潜在的抗肿瘤作用。此外，作用机制可能涉及线粒体凋亡和死亡受体途径。
Hedyotis Diffusa Willd, used in Traditional Chinese Medicine, is a treatment for various diseases including cancer, owing to its mild effectiveness and low toxicity. The aim of this study was to identify the main anticancer components in Hedyotis Diffusa Willd, and explore mechanisms underlying their activity. Hedyotis Diffusa Willd was extracted and fractionated using ethyl acetate to obtain the H-Ethyl acetate fraction, which showed higher anticancer activity than the other fractions obtained against HepG2 cells with sulforhodamine B assays. The active component of the H-Ethyl acetate fraction was identified to be 1,3-dihydroxy-2-methylanthraquinone (DMQ) with much high inhibitory rate up to 48.9 ± 3.3% and selectivity rate up to 9.4 ± 4.5 folds (p<0.01) at 125 μmol/L. HepG2 cells treated with the fraction and DMQ visualized morphologically using light and fluorescence microscopy. Annexin V—fluorescein isothiocyanate / propidium iodide staining flow cytometry, DNA ladder and cell cycle distribution assays. Mechanistic studies showed up-regulation of caspase-3, -8, and -9 proteases activities (p<0.001), indicating involvement of mitochondrial apoptotic and death receptor pathways. Further studies revealed that reactive oxygen species in DMQ and the fraction treated HepG2 cells increased (p<0.01) while mitochondrial membrane potential reduced significantly (p<0.001) compared to the control by flow cytometry assays. Western blot analysis showed that Bax, p53, Fas, FasL, p21 and cytoplasmic cytochrome C were up-regulated (p<0.01), while Bcl-2, mitochondrial cytochrome C, cyclin E and CDK 2 were down-regulated dose-dependently (p<0.01). The reverse transcriptase-polymerase chain reaction showed that mRNA expressions of p53 and Bax increased (p<0.001) while that of Bcl-2 decreased (p<0.001). Pre-treatment with caspase-8 inhibitor Z-IETD-FMK, or caspase-9 inhibitor Z-LEHD-FMK, attenuated the growth-inhibitory and apoptosis-inducing effects of DMQ and the fraction on HepG2 cells. These results suggested that DMQ and the H-Ethyl acetate fraction of Hedyotis Diffusa Willd showed potential anticancer effects. Furthermore, the mechanisms of action may involve mitochondrial apoptotic and death receptor pathways.