山奈酚的细胞核内处置调控DNA甲基转移酶抑制结肠癌分子机制及对中药山奈药效的影响

Our group has proved that flavonoids in traditional Chinese medicines (TCMs) may subject to intestinal triple recycling, which resulted in their specific high concentration and long-term exposure in the intestine. Our previous studies found that Kaempferia galanga Linn.(KGL), one of TCMs used for gastrointestinal treatments, and its corresponding biological flavonoid kaempferol could both significantly suppress colon cancer with respective inhibition rate of 86.94% and 73.52%. Further mechanism study revealed that kaempferol in KGL directly bound with DNA methyltransferases (DNMTs) and decreased 42.8% of CpG methylation in DACT2 promoter, subsequently inhibited β-catenin translocating into nucleus, ultimately suppressed colon cancer initiation. On the other hand, our preliminary data also showed that kaempferol in the nucleus could be metabolized by glucuronidation enzymes, and inhibition of efflux transporters (ETs) contrarily increased the nuclear distribution of kaempferol. Therefore, we hypothesized that the nuclear disposition of kaempferol may directly regulate DNMTs to exert anti-tumor efficacy. While some other components in KGL may serve as ETs inhibitors to markedly enhance the cancer prevention efficacy of kaempferol itself by increasing the concentration and retention of kaempferol in the nucleus. Thus, in this proposal, molecular pharmacokinetics and biological methodologies were employed in nucleus to find out 1) the regulatory mechanisms between nuclear pharmacokinetics of kaempferol and DNMTs catalytic activity; 2) screening the promising ETs inhibitors from KGL; 3) determining the effect of kaempferol nuclear disposition on its anti-colon cancer efficacy. By studying PK-PD correlation of kaempferol and KGL in nucleus, our study may provide novel strategy and experimental evidence for understanding the specific anti-cancer efficacy of TCMs.

我们已证实黄酮山奈酚因肠道循环，可高浓度、长滞留于肠细胞。前期发现：中药山奈及山奈酚可显著抑制结肠癌(抑瘤率各为86.94%和73.52%)；山奈酚与核内DNA甲基转移酶(DNMTs)直接结合，使抑癌基因DACT2启动区42.8%CpG岛去甲基化，阻碍β-catenin入核，抑制肿瘤；预实验又发现，山奈酚在细胞核中被葡萄糖醛酸化酶代谢，抑制核膜外排转运蛋白(ETs)外排代谢物，可显著增加山奈酚核内分布与浓度。因此，推测山奈酚入核处置直接调控DNMTs；山奈中有成分可抑制ETs外排，增加山奈酚核内浓度与滞留，是山奈/山奈酚抑制结肠癌的关键机制。为此，拟采用分子药代与生物学方法，揭示山奈酚核处置对DNMTs的调控及机制；筛查山奈中抑制ETs外排的活性成分；研究山奈酚核内处置对山奈整体药效的影响。课题创新地从核内PK-PD及调控，阐明山奈/山奈酚作用方式与药效机制，是中药药效研究的新思路和新方法

中药山奈主要成分山奈酚与核内DNA甲基转移酶(DNMTs)直接结合，使抑癌基因DACT2启动区CpG岛去甲基化，阻碍β-catenin入核，抑制肿瘤，且其在细胞核中被葡萄糖醛酸化酶代谢，抑制核膜外排转运蛋白(ETs)外排代谢物，可显著增加山奈酚核内分布与浓度。因此，本项目主要围绕UGTs代谢酶及ETs外排转运蛋白的介导山奈酚核内处置行为对DNMTs调控，抑制甲基化发生，促进下游靶点的转录，从而抑制结直肠癌，为阐明山奈酚对结直肠癌的作用方式与药效机制提供新的思路和方法。首先，明确山奈酚对结肠癌的低毒无副作用及其潜在靶点入核行为的调控关系，我们发现靶点SMARCA4（蛋白为BRG1）是山奈酚重要的靶点。进一步通过敲除UGTs-ETs发现山奈酚受UGTs-ETs的核处置，山奈酚在核中以更丰富的原型形式存在，其中以ETs中的MRP2/PGP和UGTs中的UGT1A9/ UGT1A1影响效果较明显。接着，通过生物信息学分析挖掘发现DNMTs调控机制及山奈酚对其下游靶基因的作用，发现靶点TPR、GNAS、INHBB等主要参与染色体同源重组、DNA构象改变、损伤修复以及Ras、MAPK、趋化因子等信号传导途径调控。最后通过体内体外实验探究 “UGTs-ETs”对山奈酚-DNMTs互作及抑制结肠癌机制，发现 “UGTs-ETs” 抑制后可提高山奈酚对结肠癌肿瘤的抑制率，以及抑制DNMT下游靶基因甲基化，促进DNMT下游靶基因的转录激活，从而抑制结肠癌。此外，探究了山奈酚对肠息肉、胆汁酸的调控及肠道菌群影响及降低结肠癌中蛋白糖基化水平及潜在机制。本课题揭示了“UGTs-ETs”对山奈酚-DNMTs的调控关系，并阐明山奈酚的核内处置受到UGTs-ETs调控及与DNMTs的下游靶基因调控途径，为山奈酚的药用价值多样性开发提供数据支持。