Charlie Norwood VA Medical Center, Augusta, GA, USA.
Department of Physiology, Medical College of Georgia at Georgia Regents University, Augusta, GA, USA.
Int J Oncol. 2014 May;44(5):1767-73. doi: 10.3892/ijo.2014.2343. Epub 2014 Mar 13.
Mitochondria constantly divide (mitochondrial fission) and fuse (mitochondrial fusion) in a normal cell. Disturbances in the balance between these two physiological processes may lead to cell dysfunction or to cell death. Induction of cell death is the prime goal of prostate cancer chemotherapy. Our previous study demonstrated that androgens increase the expression of a mitochondrial protein involved in fission and facilitate an apoptotic response to CGP37157 (CGP), an inhibitor of mitochondrial calcium efflux, in prostate cancer cells. However, the regulation and role of mitochondrial fusion proteins in the death of these cells have not been examined. Therefore, our objective was to investigate the effect of CGP on a key mitochondrial fusion protein, mitofusin 1 (Mfn1), and the role of Mfn1 in prostate cancer cell apoptosis. We used various prostate cancer cell lines and western blot analysis, qRT-PCR, siRNA, M30 apoptosis assay and immunoprecipitation techniques to determine mechanisms regulating Mfn1. Treatment of prostate cancer cells with CGP resulted in selective degradation of Mfn1. Mfn1 ubiquitination was detected following immunoprecipitation of overexpressed Myc-tagged Mfn1 protein from CGP-treated cells, and treatment with the proteasomal inhibitor lactacystin, as well as siRNA-mediated knockdown of the E3 ubiquitin ligase March5, protected Mfn1 from CGP-induced degradation. These data indicate the involvement of the ubiquitin-proteasome pathway in CGP-induced degradation of Mfn1. We also demonstrated that downregulation of Mfn1 by siRNA enhanced the apoptotic response of LNCaP cells to CGP, suggesting a likely pro-survival role for Mfn1 in these cells. Our results suggest that manipulation of mitofusins may provide a novel therapeutic advantage in treating prostate cancer.
线粒体在正常细胞中不断分裂（线粒体裂变）和融合（线粒体融合）。这两个生理过程之间的平衡失调可能导致细胞功能障碍或细胞死亡。诱导细胞死亡是前列腺癌化疗的首要目标。我们之前的研究表明，雄激素增加了参与裂变的线粒体蛋白的表达，并促进了前列腺癌细胞对线粒体钙外排抑制剂 CGP37157（CGP）的凋亡反应。然而，线粒体融合蛋白在这些细胞死亡中的调节和作用尚未被研究。因此，我们的目的是研究 CGP 对关键线粒体融合蛋白融合素 1（Mfn1）的影响，以及 Mfn1 在前列腺癌细胞凋亡中的作用。我们使用各种前列腺癌细胞系和 Western blot 分析、qRT-PCR、siRNA、M30 凋亡测定和免疫沉淀技术来确定调节 Mfn1 的机制。CGP 处理前列腺癌细胞导致 Mfn1 的选择性降解。在 CGP 处理细胞中免疫沉淀过表达的 Myc 标记的 Mfn1 蛋白后，检测到 Mfn1 的泛素化，并且用蛋白酶体抑制剂 lactacystin 以及 siRNA 介导的 E3 泛素连接酶 March5 的敲低处理，可保护 Mfn1 免受 CGP 诱导的降解。这些数据表明泛素-蛋白酶体途径参与了 CGP 诱导的 Mfn1 降解。我们还表明，siRNA 下调 Mfn1 增强了 LNCaP 细胞对 CGP 的凋亡反应，表明 Mfn1 在这些细胞中可能发挥了促生存作用。我们的研究结果表明，操纵融合蛋白可能为治疗前列腺癌提供新的治疗优势。
