Fancy Romone M, Kim Harrison, Zhou Tong, Zinn Kurt R, Buchsbaum Donald J, Song Yuhua
Department of Biomedical Engineering, The University of Alabama at Birmingham, Birmingham 35294, Alabama.
Department of Radiology, The University of Alabama at Birmingham, Birmingham 35294, Alabama.
J Cell Biochem. 2017 Aug;118(8):2285-2294. doi: 10.1002/jcb.25882. Epub 2017 Apr 12.
Activation of death receptor-5 (DR5) leads to the formation of death-inducing signaling complex (DISC) for apoptotic signaling. TRA-8, a DR5 specific agonistic antibody, has demonstrated significant cytotoxic activity in vitro and in vivo without inducing hepatotoxicity. Calmodulin (CaM) that is overexpressed in breast cancer plays a critical role in regulating DR5-mediated apoptosis. However, the mechanism of CaM in regulating DR5-mediated apoptotic signaling remains unknown. In this study, we characterized CaM binding to DR5-mediated DISC for apoptosis in TRA-8 sensitive breast cancer cell lines using co-immunoprecipitation, fluorescence microscopic imaging, caspase signaling analysis, and cell viability assay. Results show that upon DR5 activation, CaM was recruited into DR5-mediated DISC in a calcium dependent manner. CaM antagonist, trifluoperazine (TFP), inhibited CaM recruitment into the DISC and attenuated DISC formation. DR5 oligomerization is critical for DISC formation for apoptosis. TFP decreased TRA-8 activated DR5 oligomerization, which was consistent with TFP's effect on DR5-mediated DISC formation. TFP and Ca chelator, EGTA, impeded TRA-8-activated caspase-dependent apoptotic signaling, and TFP decreased TRA-8-induced cell cytotoxicity. These results demonstrated CaM binding to DR5-mediated DISC in a calcium dependent manner and may identify CaM as a key regulator of DR5-mediated DISC formation for apoptosis in breast cancer. J. Cell. Biochem. 118: 2285-2294, 2017. © 2017 Wiley Periodicals, Inc.
死亡受体-5（DR5）的激活会导致凋亡信号转导的死亡诱导信号复合物（DISC）的形成。TRA-8是一种DR5特异性激动抗体，已在体外和体内显示出显著的细胞毒活性，且不会诱导肝毒性。在乳腺癌中过表达的钙调蛋白（CaM）在调节DR5介导的凋亡中起关键作用。然而，CaM调节DR5介导的凋亡信号转导的机制仍不清楚。在本研究中，我们使用免疫共沉淀、荧光显微镜成像、半胱天冬酶信号分析和细胞活力测定，对CaM与TRA-8敏感的乳腺癌细胞系中DR5介导的凋亡DISC的结合进行了表征。结果表明，在DR5激活后，CaM以钙依赖的方式被募集到DR5介导的DISC中。CaM拮抗剂三氟拉嗪（TFP）抑制CaM募集到DISC中并减弱DISC的形成。DR5寡聚化对于凋亡的DISC形成至关重要。TFP降低了TRA-8激活的DR5寡聚化，这与TFP对DR5介导的DISC形成的影响一致。TFP和钙螯合剂乙二醇双四乙酸（EGTA）阻碍了TRA-8激活的半胱天冬酶依赖性凋亡信号转导，并且TFP降低了TRA-8诱导的细胞毒性。这些结果表明CaM以钙依赖的方式与DR5介导的DISC结合，并可能确定CaM是乳腺癌中DR5介导的凋亡DISC形成的关键调节因子。《细胞生物化学杂志》118：2285 - 2294，2017年。©2017威利期刊公司
