Mimnaugh Edward G, Xu Wanping, Vos Michele, Yuan Xitong, Neckers Len
Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, 9000 Rockville Pike, Building 10, Room 1-5940, Bethesda, MD 20892-1107, USA.
Mol Cancer Res. 2006 Sep;4(9):667-81. doi: 10.1158/1541-7786.MCR-06-0019.
Geldanamycin and Velcade, new anticancer drugs with novel mechanisms of action, are currently undergoing extensive clinical trials. Geldanamycin interrupts Hsp90 chaperone activity and causes down-regulation of its many client proteins by the ubiquitin-proteasome pathway; Velcade is a specific proteasome inhibitor. Misfolded Hsp90 clients within the endoplasmic reticulum (ER) lumen are cleared by ER--associated protein degradation, a sequential process requiring valosin-containing protein (VCP)-dependent retrotranslocation followed by ubiquitination and proteasomal proteolysis. Cotreatment of cells with geldanamycin and Velcade prevents destruction of destabilized, ubiquitinated Hsp90 client proteins, causing them to accumulate. Here, we report that misfolded protein accumulation within the ER resulting from geldanamycin and Velcade exposure overwhelms the ability of the VCP--centered machine to maintain the ER secretory pathway, causing the ER to distend into conspicuous vacuoles. Overexpression of dominant-negative VCP or the "small VCP--interacting protein" exactly recapitulated the vacuolated phenotype provoked by the drugs, associating loss of VCP function with ER vacuolization. In cells transfected with a VCP--enhanced yellow fluorescent protein fluorescent construct, geldanamycin plus Velcade treatment redistributed VCP--enhanced yellow fluorescent protein from the cytoplasm and ER into perinuclear aggresomes. In further support of the view that compromise of VCP function is responsible for ER vacuolization, small interfering RNA interference of VCP expression induced ER vacuolization that was markedly increased by Velcade. VCP knockdown by small interfering RNA eventually deconstructed both the ER and Golgi and interdicted protein trafficking through the secretory pathway to the plasma membrane. Thus, simultaneous geldanamycin and Velcade treatment has far-reaching secondary cytotoxic consequences that likely contribute to the cytotoxic activity of this anticancer drug combination.
格尔德霉素和万珂是具有全新作用机制的新型抗癌药物，目前正在进行广泛的临床试验。格尔德霉素会干扰热休克蛋白90（Hsp90）的伴侣活性，并通过泛素-蛋白酶体途径导致其许多客户蛋白下调；万珂是一种特异性蛋白酶体抑制剂。内质网（ER）腔内错误折叠的Hsp90客户蛋白通过内质网相关蛋白降解被清除，这是一个连续的过程，需要含缬酪肽蛋白（VCP）依赖的逆向转运，随后进行泛素化和蛋白酶体蛋白水解。用格尔德霉素和万珂共同处理细胞可防止不稳定的、泛素化的Hsp90客户蛋白被破坏，导致它们积累。在此，我们报告，格尔德霉素和万珂暴露导致内质网内错误折叠蛋白积累，超过了以VCP为中心的机制维持内质网分泌途径的能力，导致内质网膨胀形成明显的液泡。显性负性VCP或“小VCP相互作用蛋白”的过表达完全重现了药物引发的空泡化表型，将VCP功能丧失与内质网空泡化联系起来。在用VCP增强型黄色荧光蛋白荧光构建体转染的细胞中，格尔德霉素加万珂处理使VCP增强型黄色荧光蛋白从细胞质和内质网重新分布到核周聚集体中。为进一步支持VCP功能受损导致内质网空泡化这一观点，VCP表达的小干扰RNA干扰诱导了内质网空泡化，而万珂使其明显增加。小干扰RNA介导的VCP敲低最终破坏了内质网和高尔基体，并阻断了通过分泌途径向质膜的蛋白质转运。因此，同时使用格尔德霉素和万珂治疗会产生深远的继发性细胞毒性后果，这可能促成了这种抗癌药物组合的细胞毒性活性。
