Gene expression analysis of renal cell carcinoma cell line A-498 after 8-hour treatment with englerin A.

Natural products possess enormous structural and chemical diversity and are a rich source of drugs or drug-like leads. Although the final products may not necessarily represent the active ingredients of the natural source, the majority of all drugs in the market have their origin in nature. Englerin A is guaiane sesquiterpene that was isolated from the Tanzanian plant Phyllanthus engleri and has attracted much attention because of its unique structure and cytotoxicity profile in cancer cells. Englerin A was documented to have potent cytotoxicity preferentially against renal cancer cells when tested against the NCI60 cell panel. Very recently, however, a study which screened englerin A against 524 cancer cell lines from the cancer cell line encyclopedia reported that englerin A was also cytotoxic to a small subset of cancer cells in addition to renal cancer cells.[19]. In addition to its unique structure and cytotoxicity profile, englerin A is distinctive because it can cause cell death by multiple death mechanisms including necrosis and apoptosis, with apoptosis occurring in the absence of caspase activation. The distinctive features of englerin A suggest that it has a unique mechanism of action and recent reports, though lacking consensus, support this notion. Englerin A has been shown to activate protein kinase C theta (PKCθ) and was proposed to affect cell viability by promoting glucose dependence while simultaneously starving cells of glucose. Englerin A has also been shown to increase cytosolic calcium levels which may play a role in the decreased phosphorylation and activity of the oncoprotein, EWS-FLI1, in Ewing’s sarcoma. Two other groups have reported that englerin A inhibits tumor cell growth by activating the transient receptor potential cation channel, subfamily C, member 4 (TRPC4) ion channel, while a third group reported that englerin A antagonized L-type calcium channels. However, in at least two of these studies, the concentration of englerin A that was required to modulate these calcium channels was much higher than that required to kill renal carcinoma cells and other cells sensitive to the cytotoxic effects of englerin A. For instance, the most recent study reported that englerin A displaced a radiolabeled tool compound bound to an L-type calcium channel in a concentration-dependent manner with a Ki of 5.7 M. In contrast, the EC50 of englerin A in reducing the viability or growth of most renal cancer cell lines in the NCI60 cell panel is less than 60 nM, suggesting that modulation of L-type or TRPC calcium channels is not required for englerin A to induce toxic effects in these cells. It is still not clear what mechanisms account for the cytotoxicity of englerin A at nanomolar levels in these cells. We anticipate that there may be other mechanisms and targets of englerin A as natural products generally have multiple targets. In the current study, we chose microarray as well as other technologies to explore the mechanism(s) of action of englerin A at a more global level. Gene expression analysis was performed on A-498 cells treated with 100 nM englerin A during 8 hours. Four replicates of vehicle control and englerin A treated A-498 cells were used in the experiment.

天然产物（natural products）具有极为丰富的结构与化学多样性，是药物或类药先导化合物的重要来源。尽管最终产物未必对应天然来源中的活性成分，但市面上绝大多数药物均源自天然产物。恩格列宁A（Englerin A）是一种愈创木烷倍半萜（guaiane sesquiterpene），从坦桑尼亚产的恩氏叶下珠（Phyllanthus engleri）中分离得到，其独特的结构及对癌细胞的细胞毒性谱使其备受关注。当在NCI60细胞谱（NCI60 cell panel）中进行测试时，恩格列宁A被证实对肾癌细胞具有强效的选择性细胞毒性。然而近期一项研究通过癌细胞系百科全书（cancer cell line encyclopedia）中的524株癌细胞系筛选恩格列宁A后发现，除肾癌细胞外，恩格列宁A对一小部分其他癌细胞系同样具有细胞毒性[19]。除独特的结构与细胞毒性特征外，恩格列宁A的另一显著特点是可通过包括坏死与凋亡在内的多种死亡机制诱导细胞死亡，且其介导的细胞凋亡不依赖半胱天冬酶激活。恩格列宁A的这些独特属性提示其作用机制具有唯一性，尽管现有研究尚未达成共识，但近期相关报道均支持这一观点。已有研究表明，恩格列宁A可激活蛋白激酶Cθ（protein kinase C theta, PKCθ），并被认为通过增强细胞对葡萄糖的依赖、同时剥夺细胞葡萄糖供应来影响细胞活力。另有研究显示，恩格列宁A可升高胞浆钙离子水平，这可能与尤因肉瘤（Ewing’s sarcoma）中癌蛋白EWS-FLI1的磷酸化水平及活性降低有关。另有两个研究团队报道，恩格列宁A通过激活瞬时受体电位阳离子通道C亚家族成员4（transient receptor potential cation channel, subfamily C, member 4, TRPC4）抑制肿瘤细胞生长，而第三个团队则发现恩格列宁A可拮抗L型钙通道（L-type calcium channels）。但在上述至少两项研究中，调节这些钙通道所需的恩格列宁A浓度，远高于杀伤肾癌细胞及其他对恩格列宁A细胞毒性敏感的细胞所需的浓度。例如，最新一项研究报道，恩格列宁A以浓度依赖性方式置换结合于L型钙通道的放射性标记工具化合物，其抑制常数（Ki）为5.7 μM。与之形成对比的是，在NCI60细胞谱中，多数肾癌细胞系的恩格列宁A半数有效浓度（EC50）低于60 nM，这提示调节L型或TRPC家族钙通道并非恩格列宁A诱导这些细胞产生毒性效应的必要条件。目前仍不清楚恩格列宁A在纳摩尔级别下对这些细胞产生细胞毒性的具体机制。我们推测，与多数天然产物一样，恩格列宁A可能存在其他作用靶点与机制。在本研究中，我们采用基因芯片（microarray）及其他技术，从更全局的层面探究恩格列宁A的作用机制。我们对经100 nM恩格列宁A处理8小时的A-498细胞开展了基因表达分析，实验中共设置四组溶剂对照（vehicle control）样本与四组恩格列宁A处理的A-498细胞样本。