Visnagin选择性保护doxorubicin诱发的细胞死亡的代谢机制

Doxorubicin and other anthracycline family members are the most widely used chemotherapy drugs. Clinically their usages are restricted because of severe cardiotoxicity. Even with suboptimal dosage, many patients still developed heart disease later in their lives. Therefore it would be valuable to develop selective cardioprotectant, which protects heart but does not interferes with cancer treatment. We previously discovered visnagin as such reagent and determined MDH2 as its binding target. This made us speculate that visnagin acts by modulating metabolic pathway, which comparts its selectivity and efficacy. Our preliminary data showed that similar to visnagin manipulation of glucose catabolism with MDH2 knockdown, TCA cycle intermediates or oxidative phosphorylation inhibitors selectively protect against doxorubicin. In addition, inhibition of glycolysis with 2-deoxyglucose (in presence of pyruvate) abrogated visnagin’s protective effect. Of interest, blocking glycolysis pathway only reduced ATP production in those cells that can be protected by visnagin, suggesting that there exist different metabolic fluxes along glycolysis in visnagin sensitive and insensitive cells. These results implicate that selective protection of visnagin may be attributed to glycolysis and/or associated branches. Furthermore, recent studies suggest that doxorubicin induced cell death via histone eviction, which is regulated by histone methylation. Histone methylation is in turn controlled by metabolic state. Taken together, we hypothesize that (1) visnagin enhances glycolysis/serine biosynthesis flux in visnagin sensitive cells, which promotes s-adenosyl-methionine (DNA/histone methylation substrate) production through one carbon metabolism/methionine cycle, and (2) upregulation of histone methylation prevents doxorubicin induced histone eviction. At last, we will also develop more potent visnagin derivatives based on of mechanistic study that visnagin binds to mitochondrial enzyme MDH2.

作为应用最广的一类抗癌化疗药，doxorubicin及其家族的其他成员因为强烈的心脏毒性，不得不牺牲抗癌效果而使用较低剂量；即便如此，也有相当一部分癌症病人在治疗结束后会患上doxorubicin诱发的心脏病。因而研发可以保护心脏但不影响癌症治疗的保护剂有重要的临床应用意义。我们之前发现visnagin在多种体内体外模型中选择性保护心肌细胞而不干扰doxorubicin杀死癌细胞，并且还发现visnagin的靶点是三羧酸循环的酶MDH2， 暗示visnagin也许是通过改变细胞的代谢状态起作用。另外，已知doxorubicin可以通过从染色质排挤组蛋白引起细胞死亡，而细胞代谢可以通过调节组蛋白甲基化阻止组蛋白的排挤作用。本项目试图验证visnagin通过影响细胞特异的糖酵解/分支途径提高组蛋白的甲基化从而抑制doxorubicin引起的细胞死亡。

阿霉素是临床上应用最广的抗癌化疗药，但其应用在很大程度上因其心脏毒性被限制，因而发现可以有效缓解阿霉素心脏副作用而不影响癌症治疗的佐剂有重要的临床意义。我们之前筛选出一类以visnagin为代表的小分子可以在细胞，斑马鱼和小鼠模型中选择性拮抗阿霉素诱导的细胞死亡，本研究的目的旨在明确visnagin的选择性保护机制。基于前期结果visnagin可以和葡萄糖代谢三羧酸循环中的酶MDH2结合，并且一些三羧酸循环的代谢中间产物及抑制三羧酸循环下游的电子传递链可以提高阿霉素处理的细胞的存活率，我们提出假说：visnagin通过影响葡萄糖代谢途径拮抗阿霉素诱导的细胞死亡。因为阿霉素可以通过多种途径诱导多种形式的细胞死亡，我们还需要明确visnagin拮抗哪种细胞死亡途径。此外，因为visnagin的潜在靶点MDH2特异存在于线粒体，开发可以靶向线粒体的visnagin类化合物或许有更好的保护作用。围绕这些问题特别是visnagin选择保护性机制，我们的研究发现：<1> visnagin拮抗依赖于拓扑异构酶2 （topoisomerase 2，TOP2）的DNA损伤/细胞凋亡途径 <2>虽然所有细胞都表达TOP2，但阿霉素只能在部分细胞中诱导拓扑异构酶2和DNA形成共价复合物(TOP2-DNAcc)---产生DNA损伤的必要中间产物，<3>visnagin可以降低阿霉素诱导的TOP2-DNAcc的水平，并且visnagin只能选择性保护可以诱导形成TOP2-DNAcc的细胞（TOP2-DNAcc+ cells）, <4>visnagin不能通过降低TOP2的蛋白水平或直接TOP2酶活性减低阿霉素诱导的TOP2-DNAcc水平。之后我们将进一步研究visnagin如何通过影响翻译后修饰或TOP2与染色质的结合拮抗阿霉素诱导的TOP2-DNAcc形成，另外我们还将探究在不同细胞中阿霉素选择性诱导形成TOP2-DNAcc的机制。因为敲低MDH2不能在所有被visnagin保护的细胞中拮抗阿霉素诱导的细胞死亡及visnagin在缺失线粒体的酵母细胞中还有保护作用，我们怀疑visnagin通过三羧酸之外的途径起作用。虽然三羧酸循环的代谢中间产物如琥珀酸，α-酮戊二酸和电子传递链抑制剂还可以拮抗阿霉素诱导的细胞死亡，但极有可能通过其他机制起保护作用，这也是我们下一步的研究内容。