Mimnaugh Edward G, Xu Wanping, Vos Michele, Yuan Xitong, Isaacs Jennifer S, Bisht Kheem S, Gius David, Neckers Len
Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Rockville, Maryland 20850, USA.
Mol Cancer Ther. 2004 May;3(5):551-66.
The ansamycin antibiotic, geldanamycin, targets the hsp 90 protein chaperone and promotes ubiquitin-dependent proteasomal degradation of its numerous client proteins. Bortezomib is a specific and potent proteasome inhibitor. Both bortezomib and the geldanamycin analogue, 17-N-allylamino-17-demethoxy geldanamycin, are in separate clinical trials as new anticancer drugs. We hypothesized that destabilization of hsp 90 client proteins with geldanamycin, while blocking their degradation with bortezomib, would promote the accumulation of aggregated, ubiquitinated, and potentially cytotoxic proteins. Indeed, geldanamycin plus bortezomib inhibited MCF-7 tumor cell proliferation significantly more than either drug alone. Importantly, while control cells were unaffected, human papillomavirus E6 and E7 transformed fibroblasts were selectively sensitive to geldanamycin plus bortezomib. Geldanamycin alone slightly increased protein ubiquitination, but when geldanamycin was combined with bortezomib, protein ubiquitination was massively increased, beyond the amount stabilized by bortezomib alone. In geldanamycin plus bortezomib-treated cells, ubiquitinated proteins were mostly detergent insoluble, indicating that they were aggregated. Individually, both geldanamycin and bortezomib induced hsp 90, hsp 70, and GRP78 stress proteins, but the drug combination superinduced these chaperones and caused them to become detergent insoluble. Geldanamycin plus bortezomib also induced the formation of abundant, perinuclear vacuoles, which were neither lysosomes nor autophagosomes and did not contain engulfed cytosolic ubiquitin or hsp 70. Fluorescence marker experiments indicated that these vacuoles were endoplasmic reticulum derived and that their formation was prevented by cycloheximide, suggesting a role for protein synthesis in their genesis. These observations support a mechanism whereby the geldanamycin plus bortezomib combination simultaneously disrupts hsp 90 and proteasome function, promotes the accumulation of aggregated, ubiquitinated proteins, and results in enhanced antitumor activity.
安莎霉素类抗生素格尔德霉素作用于热休克蛋白90（hsp 90）这一蛋白质伴侣，并促进其众多客户蛋白的泛素依赖性蛋白酶体降解。硼替佐米是一种特异性强效蛋白酶体抑制剂。硼替佐米和格尔德霉素类似物17-N-烯丙基氨基-17-去甲氧基格尔德霉素都作为新型抗癌药物正在进行单独的临床试验。我们推测，用格尔德霉素使hsp 90客户蛋白不稳定，同时用硼替佐米阻断其降解，会促进聚集的、泛素化的且可能具有细胞毒性的蛋白的积累。事实上，格尔德霉素加硼替佐米对MCF-7肿瘤细胞增殖的抑制作用明显强于单独使用这两种药物中的任何一种。重要的是，虽然对照细胞未受影响，但人乳头瘤病毒E6和E7转化的成纤维细胞对格尔德霉素加硼替佐米具有选择性敏感性。单独使用格尔德霉素会使蛋白泛素化略有增加，但当格尔德霉素与硼替佐米联合使用时，蛋白泛素化会大量增加，超过单独使用硼替佐米时稳定的量。在经格尔德霉素加硼替佐米处理的细胞中，泛素化蛋白大多不溶于去污剂，表明它们发生了聚集。单独来看，格尔德霉素和硼替佐米都会诱导hsp 90、hsp 70和葡萄糖调节蛋白78（GRP78）应激蛋白，但药物组合会超诱导这些伴侣蛋白并使其变得不溶于去污剂。格尔德霉素加硼替佐米还会诱导大量核周空泡的形成，这些空泡既不是溶酶体也不是自噬体，且不含有被吞噬的胞质泛素或hsp 70。荧光标记实验表明这些空泡源自内质网，且其形成可被环己酰亚胺阻止，这表明蛋白质合成在其形成过程中发挥作用。这些观察结果支持了一种机制，即格尔德霉素加硼替佐米的组合同时破坏hsp 90和蛋白酶体功能，促进聚集的、泛素化蛋白的积累，并导致增强的抗肿瘤活性。
