【作者】 陈晶；

【导师】 胡汛；

【作者基本信息】 浙江大学 ， 肿瘤学， 2012， 博士

【摘要】 2007年至2008,本研究小组报道了一系列萘醌类化合物——紫草素及其衍生物绕过耐药肿瘤细胞的抗凋亡——apoptosis和外排药物的机制,诱导肿瘤细胞发生程序性坏死——necroptosis,达到高效杀伤耐药肿瘤细胞的目的,为研发新的抗肿瘤药物提供了新的思路,即以紫草素及其衍生物的分子结构为模板来开发对肿瘤细胞的杀伤效果更好的药物,但是由于这些萘醌类化合物诱导肿瘤细胞程序性坏死的分子机制不确定,为新药开发方向带来一定的盲目性。本研究以发掘紫草素在肿瘤细胞中的靶蛋白为切入点,深入探讨紫草素及其衍生物,以及与紫草素分子结构类似的维生素K等萘醌类化合物参与肿瘤细胞中哪些分子机制,打乱了肿瘤细胞哪方面的生理平衡。首先通过solid-phase实验,我们发现紫草素与肿瘤细胞裂解液中M2型丙酮酸激酶特异结合,M2型丙酮酸激酶可能是紫草素的重要的靶蛋白。通过酶学实验,我们发现紫草素及其衍生物能够有效抑制M2型丙酮酸激酶的活性,其抑制效率远高于之前报道的M2型丙酮酸激酶小分子抑制剂一一compound3,并且紫草素对丙酮酸激酶不同的同工酶的抑制效果不同,其中对M2型丙酮酸激酶抑制效果最好。已知M2型丙酮酸激酶在肿瘤细胞里特异表达,是肿瘤细胞糖酵解代谢的最后一个限速酶,抑制了M2型丙酮酸激酶可能会对肿瘤细胞糖酵解代谢的有一定的抑制作用,药物处理后检测细胞培养液中葡萄糖和乳酸的含量,我们发现葡萄糖的消耗显著减少,乳酸的产生也显著降低,结果证实紫草素抑制了肿瘤细胞糖酵解的速率,并且对敏感肿瘤细胞株(MCF-7)和耐药细胞株(MCF-7/Adr、MCF-7/Bcl-2、MCF-7/Bcl-xL)的糖酵解代谢有类似的抑制效果。由于糖酵解调控肿瘤细胞能量和合成代谢基础物质的重要来源,抑制糖酵解很可能对肿瘤细胞造成致命打击,我们推测抑制M2型丙酮酸激酶的活性和糖酵解代谢可能是紫草素诱导肿瘤细胞产生程序性坏死的重要机制之一。通过细胞活性试验,我们发现M2型丙酮酸激酶mRNA被siRNA敲减以后,紫草素诱导产生程序性坏死的细胞比例变大,说明M2型丙酮酸激酶和紫草素诱导的程序性坏死有关。而将M1型丙酮酸激酶基因转染到肿瘤细胞中,导致细胞内丙酮酸激酶活性增加,细胞对紫草素的敏感度降低,再次证实细胞内M2型丙酮酸激酶是紫草素杀伤肿瘤细胞的重要的靶蛋白。通过相同的研究方法,我们发现维生素K3和K5对M2型丙酮酸激酶也有特异的抑制效果,它们也能够抑制肿瘤细胞糖酵解的速率,以及M2型丙酮酸激酶活性被抑制与维生素K3和K5单独杀伤肿瘤细胞的效果和作为辅助药物增加阿霉素毒性的效果相关。总之,本研究首次证实萘醌类化合物是M2型丙酮酸激酶的特异抑制剂,它们对肿瘤细胞的毒性和对M2型丙酮酸激酶活性的抑制相关,这为我们开发有前景的肿瘤药物提供了重要依据。更多还原

【Abstract】 From2007to2008, our group has reported a serial of naphthoqinones-shikonin and its derivatives were necroptosis inducers that could bypass cancer drug resistance, which provides a new direction to develop anti-cancer drugs. However, the mechanism was unclear, which brought blindness in developing new drugs. This study tries to uncover the target of shikonin in cancer cells, and explore the mechanism of necroptosis caused by shikonin and its derivatives. Vitamin Ks are another serial of naphthoquinones with similar chemical structure of shikonin, and they are discovered to be potential in cancer therapy. This study also tries to find out whether the mechanism of vitamin Ks in killing cancer cells is similar to shikonin, and which homeostasis they break in cancer cells. First, the result of solid-phase experiment showed that shikonin could bind to pyruvate kinase M2(PKM2) in cell lysate, which implies that PKM2would be an important target of shikonin in vivo. Through the enzyme activity assay, we found that shikonin and its derivatives could inhibit the activity of PKM2, and they were much more efficient than the reported small chemical inhibitor of PKM2compound3. Furthermore, we found that shikonin inhibited isozymes of pyruvate kinase in different extent, and PKM2among its isozymes was inhibited the most by shikonin. Since PKM2is specifically expressed in cancer cells, and the last rate-limiting enzyme of glycolysis, the inhibition of PKM2’s activity might induce the inhibition of glycolysis. Through quantifying glucose and lactate in culture medium after the treatment of cancer cells with shikonin, we proved that the consumption of glucose and the production of lactate were reduced, which demonstrated that the rate of glycolysis flux was attenuated by shikonin. And we also found that shikonin was efficient in inhibiting glycolysis of both sensitive cancer cell (MCF-7, MCF-7/Neo) and drug-resistant cancer cells (MCF-7/Adr, MCF-7/Bcl-2and MCF-7/Bcl-xL). Glycolysis is an important resource of energy and anabolism blocks in cancer cells. The inhibition of glycolysis might cause lethal damage to cancer cells. We hypothesized that shikonin might cause necroptosis partly by inhibiting PKM2and glycolysis. According to cell viability assay, we found that cancer cells were more sensitive to shikonin-induced necroptosis after the knock-down of PKM2by siRNA, which suggested that PKM2be related to shikonin-induced necroptosis. After the trasfection of PKM1into cancer cells, the activity of pyruvate kinase increased and cells were less sensitive to shikonin, which proved again that PKM2was an important target of shikonin in killing cancer cells. By similar experiments, we found that vitamin K3and K5inhibited PKM2specifically, as well as the rate of glycolysis. And we also discovered that PKM2was related to the toxicity caused by vitamin K3and K5alone or with doxorubicin. In conclusion, this study demonstrated for the first time that these naphthaquinones were specific inhibitors of PKM2, and the inhibition of PKM2was relevant to cancer cells’ sensitivity to naphthoquinones, which provides important clues in developing promising anti-cancer drugs.更多还原