The cytotoxicity of gamma-secretase inhibitor I to breast cancer cells is mediated by proteasome inhibition, not by gamma-secretase inhibition.
The cytotoxicity of gamma-secretase inhibitor I to breast cancer cells is mediated by proteasome inhibition, not by gamma-secretase inhibition.
复制标题
DOI：
10.1186/bcr2347
复制
发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Allalunis-Turner J
中科院分区：
文献类型：
--
作者：
Han J;Ma I;Hendzel MJ;Allalunis-Turner J
Notch 是跨膜蛋白受体家族，其激活需要 γ 分泌酶的蛋白水解切割。由于异常的 Notch 信号传导可在转基因小鼠中诱导乳腺癌，并且 Notch 受体和配体的高表达水平与总体不良的临床结果相关，因此用小分子抑制 γ-分泌酶可能是治疗乳腺癌的一种有前途的方法。与这一假设一致，最近的两篇论文报道，γ-分泌酶抑制剂 I (GSI I)，Z-LLNle-CHO，在体外和体内对乳腺癌细胞都具有毒性。在本研究中，我们将 Z-LLNle-CHO 的活性和细胞毒性与两种高度特异性的 GSI（DAPT 和 L-685,458）以及三种结构上不相关的蛋白酶体抑制剂（MG132、lactacystin 和硼替佐米）的活性和细胞毒性进行了比较，以研究 Z-LLNle-CHO 在乳腺癌细胞中细胞毒性的机制。本研究使用了三种雌激素受体 (ER) 阳性细胞系 MCF-7、BT474 和 T47D，以及三种 ER 阴性细胞系 SKBR3、MDA-MB-231 和 MDA-MB-468。 SKBR3 和 BT474 细胞也过度表达 HER2/neu。通过使用 MTS 细胞活力/增殖测定来测量细胞毒性。通过免疫印迹和免疫荧光显微镜测量γ-分泌酶活性的抑制，以检测活性Notch1胞内结构域。通过使用基于细胞的蛋白酶体活性测定试剂盒，通过免疫印迹检测多泛素化蛋白的积累，并通过免疫荧光显微镜检测细胞泛素的重新分布来测定蛋白酶体抑制。我们发现，通过 DAPT 和 L-685,458 阻断 γ-分泌酶活性对一组六种乳腺癌细胞系的存活和增殖没有影响，而 Z-LLNle-CHO 即使在抑制 γ-分泌酶活性效率较低的浓度下也可能导致细胞死亡。此外，我们观察到 Z-LLNle-CHO 可以抑制蛋白酶体活性，并且这六种乳腺癌细胞系对 Z-LLNle-CHO 的相对细胞敏感性与三种蛋白酶体抑制剂观察到的相同。最后，我们发现 Z-LLNle-CHO 的细胞杀伤作用可以通过恢复蛋白酶体活性的化学物质来逆转。我们得出结论，Z-LLNle-CHO 在乳腺癌细胞中的细胞毒性是通过蛋白酶体抑制介导的，而不是通过 γ-分泌酶抑制介导的。
Notch is a family of transmembrane protein receptors whose activation requires proteolytic cleavage by γ-secretase. Since aberrant Notch signaling can induce mammary carcinomas in transgenic mice and high expression levels of Notch receptors and ligands correlates with overall poor clinical outcomes, inhibiting γ-secretase with small molecules may be a promising approach for breast cancer treatment. Consistent with this hypothesis, two recent papers reported that γ-secretase inhibitor I (GSI I), Z-LLNle-CHO, is toxic to breast cancer cells both in vitro and in vivo. In this study, we compared the activity and cytotoxicity of Z-LLNle-CHO to that of two highly specific GSIs, DAPT and L-685,458 and three structurally unrelated proteasome inhibitors, MG132, lactacystin, and bortezomib in order to study the mechanism underlying the cytotoxicity of Z-LLNle-CHO in breast cancer cells. Three estrogen receptor (ER) positive cell lines, MCF-7, BT474, and T47D, and three ER negative cell lines, SKBR3, MDA-MB-231, and MDA-MB-468, were used in this study. Both SKBR3 and BT474 cells also overexpress HER2/neu. Cytotoxicity was measured by using an MTS cell viability/proliferation assay. Inhibition of γ-secretase activity was measured by both immunoblotting and immunofluorescent microscopy in order to detect active Notch1 intracellular domain. Proteasome inhibition was determined by using a cell-based proteasome activity assay kit, by immunoblotting to detect accumulation of polyubiquitylated protein, and by immunofluorescent microscopy to detect redistribution of cellular ubiquitin. We found that blocking γ-secretase activity by DAPT and L-685,458 had no effect on the survival and proliferation of a panel of six breast cancer cell lines while Z-LLNle-CHO could cause cell death even at concentrations that inhibited γ-secretase activity less efficiently. Furthermore, we observed that Z-LLNle-CHO could inhibit proteasome activity and the relative cellular sensitivity of these six breast cancer cell lines to Z-LLNle-CHO was the same as observed for three proteasome inhibitors. Finally, we found that the cell killing effect of Z-LLNle-CHO could be reversed by a chemical that restored the proteasome activity. We conclude that the cytotoxicity of Z-LLNle-CHO in breast cancer cells is mediated by proteasome inhibition, not by γ-secretase inhibition.
登录
查看更多内容
影响因子：
4.6
作者：
Denlinger, CE;Rundall, BK;Jones, DR
通讯作者：
Jones, DR
影响因子：
5.3
作者：
Rand, MD;Grimm, LM;Aster, JC
通讯作者：
Aster, JC
影响因子：
11.2
作者：
Ban J;Bennani-Baiti IM;Kauer M;Schaefer KL;Poremba C;Jug G;Schwentner R;Smrzka O;Muehlbacher K;Aryee DN;Kovar H
通讯作者：
Kovar H
影响因子：
11.2
作者：
Rizzo P;Miao H;D'Souza G;Osipo C;Song LL;Yun J;Zhao H;Mascarenhas J;Wyatt D;Antico G;Hao L;Yao K;Rajan P;Hicks C;Siziopikou K;Selvaggi S;Bashir A;Bhandari D;Marchese A;Lendahl U;Qin JZ;Tonetti DA;Albain K;Nickoloff BJ;Miele L
通讯作者：
Miele L
影响因子：
2.5
作者：
Callahan, R;Raafat, A
通讯作者：
Raafat, A