Zaarur Nava, Gabai Vladimir L, Porco John A, Calderwood Stuart, Sherman Michael Y
Department of Biochemistry, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA.
Cancer Res. 2006 Feb 1;66(3):1783-91. doi: 10.1158/0008-5472.CAN-05-3692.
Novel classes of anticancer drugs, including proteasome inhibitors and Hsp90 inhibitors, potently induce heat shock proteins (Hsps). Because Hsps show antiapoptotic activities, we suggested that suppression of such induction may sensitize cancer cells to these drugs. Here, we knocked out the major heat shock transcription factor HSF-1 in several cancer cell lines using small interfering RNA and showed that such cells, which can no longer induce Hsps in response to proteasome and Hsp90 inhibitors, become more sensitive to these drugs. Furthermore, we developed a high-throughput screen for small molecules that inhibit induction of Hsps. The first step was a cell-based screen for inhibitors of Hsps-mediated luciferase refolding followed by a counterscreen for toxicity. The second step was a direct testing for inhibition of Hsp induction by immunoblotting with anti-Hsp72 antibody. After screening of 20,000 compounds from several diversity libraries, we focused on a compound we called NZ28, which potently inhibited induction of Hsps by heat shock, proteasome, and Hsp90 inhibitors in a variety of cell lines, and showed no significant toxicity. After testing of a set of analogues of NZ28, we identified a structural element that was critical for the activity. We also identified another inhibitor of the Hsp induction that was practically nontoxic. This compound, which we called emunin, strongly sensitized myeloma cells to proteasome and Hsp90 inhibitors and prostate carcinoma cells to proteasome inhibitors. This work indicates that targeting the heat shock response may facilitate use of proteasome and Hsp90 inhibitors for cancer treatment.
新型抗癌药物类别，包括蛋白酶体抑制剂和热休克蛋白90（Hsp90）抑制剂，能有效诱导热休克蛋白（Hsps）。由于热休克蛋白具有抗凋亡活性，我们推测抑制这种诱导作用可能会使癌细胞对这些药物更敏感。在此，我们使用小干扰RNA在几种癌细胞系中敲除了主要的热休克转录因子HSF-1，结果显示这些细胞在受到蛋白酶体和Hsp90抑制剂刺激时不再能诱导热休克蛋白，并且对这些药物变得更加敏感。此外，我们开发了一种针对抑制热休克蛋白诱导的小分子高通量筛选方法。第一步是基于细胞的筛选，寻找能抑制热休克蛋白介导的荧光素酶重折叠的抑制剂，随后进行毒性反筛选。第二步是通过用抗Hsp72抗体进行免疫印迹直接检测热休克蛋白诱导的抑制情况。从几个多样化文库中筛选了20000种化合物后，我们聚焦于一种名为NZ28的化合物，它能有效抑制多种细胞系中热休克、蛋白酶体和Hsp90抑制剂诱导的热休克蛋白，且无明显毒性。在测试了一组NZ28类似物后，我们确定了一个对活性至关重要的结构元件，并鉴定出另一种几乎无毒性的热休克蛋白诱导抑制剂。这种我们称为emunin的化合物，能使骨髓瘤细胞对蛋白酶体和Hsp90抑制剂以及前列腺癌细胞对蛋白酶体抑制剂高度敏感。这项研究表明，针对热休克反应可能有助于蛋白酶体和Hsp90抑制剂用于癌症治疗。
