含氮黄芩素衍生物抗肿瘤活性及作用机理
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摘要
黄芩素具有低毒高效,资源丰富,药材来源方便,价格低廉等优点,近年来研究表明其具有抗氧化、抗病毒、抗菌、抗炎和镇静等功效,并有广泛的抗肿瘤活性,因此具有良好的开发前景。但是黄芩素溶解性差、生物利用度低,限制了其临床应用。近年来,随着对肿瘤分子机制更加深刻的研究,细胞周期调控理论取得了令人瞩目的成就,CDK抑制剂也成为研究的热点,但目前还没有一个CDK抑制剂真正应用于临床。大连理工大学精细化工国家重点实验室以黄芩素为底物,通过曼尼西反应合成系列含氮黄芩素衍生物,并通过CDK1/Cyclin B的抑制作用进行了筛选,得到九种活性较高的衍生物。
本论文在此基础上,以MCF-7, Hela, SMMC-7721, SGC-7901, BGC-823, HL-60和PC-3为体外肿瘤细胞系模型,对九种含氮黄芩素衍生物的抗肿瘤活性进行了进一步筛选,考察了药物对细胞增殖的抑制率,包括半数抑制浓度(IC50)、半数生长抑制浓度(GI50)、完全抑制浓度(TGI)、半数致死浓度(LC50),其中溶解度相对较高的8-N-甲基哌嗪亚甲基黄芩素(BA-f)、8-N-羟乙基哌嗪黄芩素(BA-i)和8-N-羟基哌啶黄芩素(BA-j)显示出了相对较高的抑制肿瘤增殖活性,其TGI值分别为25.4±2.67,26.5±2.47,28.1±2.85μM。以MCF-7为体外肿瘤细胞系模型代表,通过Hochest染色、PI单染、AnnexinV-PI双染、罗丹明123染色、caspase-3和caspase-8活性检测、Bcl-2和Bax蛋白检测等发现含氮黄芩素衍生物主要是通过将肿瘤细胞的细胞周期阻滞于G1、G2期,并使S期的细胞数目减少,表明其不仅是CDK1/cyclin B的抑制剂,还可能是CDK2/cyclinE和CDK9/cyclin T1的抑制剂。能够通过线粒体膜电位的降低,caspase-3和caspase-8活性的增加以及Bcl-2蛋白表达活性的降低,诱导肿瘤细胞凋亡而发挥抗增殖活性。琼脂糖凝胶电泳检测证明BA-f, BA-i, BA-j不直接作用于DNA,对拓扑异构酶Ⅰ没有抑制作用。
综上,合成的含氮黄芩素衍生物是一类新的CDK抑制剂,具有抗增殖活性,同时能够通过线粒体通路和死亡受体通路诱导细胞凋亡；由于不直接作用于DNA,对拓扑异构酶Ⅰ没有抑制作用,是一类毒副作用较小的抗肿瘤化合物,将成为高效抗肿瘤作用的创新药物。
Baicalein (5,6,7-trihydorxyflavone) is known to have a variety of attractive activities such as lipoxygenase inhibitory, antiviral, antibacterial, anti-inflammatory, hypotensive, and anticancer activities. For the reasons mentioned above, as well as its low toxicity and high efficacy, abundant resources and the advantage of low cost, baicalein has attracted more and more attention. However, the shortcomings such as its poor solubility and low bioavailability have limited its clinical application to some extents. In recent years, with profound development of the molecular mechanism of cancer, the theory of cell cycle regulation has obtained remarkable achievements, and CDK inhibitors are also being a hot topic. However, none of CDK inhibitors are in real clinical application. Thus, the design and synthesis of 8-aminomethylated baicalein analogues by Mannich reaction and assay for their CDK1/Cyclin B inhibitory activities by FRET have previously been done by State Key Laboratary of Fine Chemical of Dalian University of Technology, and nine of these compounds exhibited potent inhibitory activities against CDK1/Cyclin B.
In this paper, nine BAs were screened for their antiproliferative activities in various human cancer cell lines, and three parameters of dose-response activities (GI50, TGI and LC50) were evaluated. Among these, BA-f, BA-i and BA-j, which were much more soluble in aqueous solution than the rest, showed more effective antiproliferative activities and yielded mean TGI values of 25.4±2.67,26.5±2.47,28.1±2.85μM, respectively, in Hela, SMMC-7721, SGC-7901, BGC-823, HL-60 and PC-3 cancer cell lines. In addition, G1 and G2 growth arrests and decrease of S phase cell number demonstrated that BAs not only acted as CDK1/Cyclin B inhibitors, but could also inhibit CDK2/Cyclin E and CDK9/Cyclin T1. Moreover, increased caspase-8 and caspase-3 expressions, down-regulation of Bcl-2 expression and mitochondrial membrane potential (ΔΨm), which could induce apoptosis, were found to be elements that mainly contributed to the antiproliferative activities of BA-f, i and j. However, BA-f, i and j were not inhibitory toward DNA Topoisomerase I, and could not have direct effect on DNA.
In summary, the results suggested that 8-aminomethylated baicalein analogues is a novel class of CDK inhibitors which has high antiproliferative activity and could induce apoptosis, which is mediated by the mitochondrial mediated pathway and death receptor-related pathway. And because it could not inhibit DNA and DNA TopoⅠ, BAs is less toxic and may be explored as a possible novel drug with enhanced anticancer effect.
本论文在此基础上,以MCF-7, Hela, SMMC-7721, SGC-7901, BGC-823, HL-60和PC-3为体外肿瘤细胞系模型,对九种含氮黄芩素衍生物的抗肿瘤活性进行了进一步筛选,考察了药物对细胞增殖的抑制率,包括半数抑制浓度(IC50)、半数生长抑制浓度(GI50)、完全抑制浓度(TGI)、半数致死浓度(LC50),其中溶解度相对较高的8-N-甲基哌嗪亚甲基黄芩素(BA-f)、8-N-羟乙基哌嗪黄芩素(BA-i)和8-N-羟基哌啶黄芩素(BA-j)显示出了相对较高的抑制肿瘤增殖活性,其TGI值分别为25.4±2.67,26.5±2.47,28.1±2.85μM。以MCF-7为体外肿瘤细胞系模型代表,通过Hochest染色、PI单染、AnnexinV-PI双染、罗丹明123染色、caspase-3和caspase-8活性检测、Bcl-2和Bax蛋白检测等发现含氮黄芩素衍生物主要是通过将肿瘤细胞的细胞周期阻滞于G1、G2期,并使S期的细胞数目减少,表明其不仅是CDK1/cyclin B的抑制剂,还可能是CDK2/cyclinE和CDK9/cyclin T1的抑制剂。能够通过线粒体膜电位的降低,caspase-3和caspase-8活性的增加以及Bcl-2蛋白表达活性的降低,诱导肿瘤细胞凋亡而发挥抗增殖活性。琼脂糖凝胶电泳检测证明BA-f, BA-i, BA-j不直接作用于DNA,对拓扑异构酶Ⅰ没有抑制作用。
综上,合成的含氮黄芩素衍生物是一类新的CDK抑制剂,具有抗增殖活性,同时能够通过线粒体通路和死亡受体通路诱导细胞凋亡；由于不直接作用于DNA,对拓扑异构酶Ⅰ没有抑制作用,是一类毒副作用较小的抗肿瘤化合物,将成为高效抗肿瘤作用的创新药物。
Baicalein (5,6,7-trihydorxyflavone) is known to have a variety of attractive activities such as lipoxygenase inhibitory, antiviral, antibacterial, anti-inflammatory, hypotensive, and anticancer activities. For the reasons mentioned above, as well as its low toxicity and high efficacy, abundant resources and the advantage of low cost, baicalein has attracted more and more attention. However, the shortcomings such as its poor solubility and low bioavailability have limited its clinical application to some extents. In recent years, with profound development of the molecular mechanism of cancer, the theory of cell cycle regulation has obtained remarkable achievements, and CDK inhibitors are also being a hot topic. However, none of CDK inhibitors are in real clinical application. Thus, the design and synthesis of 8-aminomethylated baicalein analogues by Mannich reaction and assay for their CDK1/Cyclin B inhibitory activities by FRET have previously been done by State Key Laboratary of Fine Chemical of Dalian University of Technology, and nine of these compounds exhibited potent inhibitory activities against CDK1/Cyclin B.
In this paper, nine BAs were screened for their antiproliferative activities in various human cancer cell lines, and three parameters of dose-response activities (GI50, TGI and LC50) were evaluated. Among these, BA-f, BA-i and BA-j, which were much more soluble in aqueous solution than the rest, showed more effective antiproliferative activities and yielded mean TGI values of 25.4±2.67,26.5±2.47,28.1±2.85μM, respectively, in Hela, SMMC-7721, SGC-7901, BGC-823, HL-60 and PC-3 cancer cell lines. In addition, G1 and G2 growth arrests and decrease of S phase cell number demonstrated that BAs not only acted as CDK1/Cyclin B inhibitors, but could also inhibit CDK2/Cyclin E and CDK9/Cyclin T1. Moreover, increased caspase-8 and caspase-3 expressions, down-regulation of Bcl-2 expression and mitochondrial membrane potential (ΔΨm), which could induce apoptosis, were found to be elements that mainly contributed to the antiproliferative activities of BA-f, i and j. However, BA-f, i and j were not inhibitory toward DNA Topoisomerase I, and could not have direct effect on DNA.
In summary, the results suggested that 8-aminomethylated baicalein analogues is a novel class of CDK inhibitors which has high antiproliferative activity and could induce apoptosis, which is mediated by the mitochondrial mediated pathway and death receptor-related pathway. And because it could not inhibit DNA and DNA TopoⅠ, BAs is less toxic and may be explored as a possible novel drug with enhanced anticancer effect.
引文
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