Miyahara Kana, Kazama Hiromi, Kokuba Hiroko, Komatsu Seiichiro, Hirota Ayako, Takemura Jun, Hirasawa Kazuhiro, Moriya Shota, Abe Akihisa, Hiramoto Masaki, Ishikawa Takashi, Miyazawa Keisuke
Department of Breast Surgery, Tokyo Medical University, Tokyo, Japan.
Department of Biochemistry, Tokyo Medical University, Tokyo, Japan.
Int J Oncol. 2016 Nov;49(5):1848-1858. doi: 10.3892/ijo.2016.3673. Epub 2016 Aug 29.
The ubiquitin-proteasome and autophagy-lysosome pathways are two major self-digestive systems for cellular proteins. Ubiquitinated misfolded proteins are degraded mostly by proteasome. However, when ubiquitinated proteins accumulate beyond the capacity of proteasome clearance, they are transported to the microtubule-organizing center (MTOC) along the microtubules to form aggresomes, and subsequently some of them are degraded by the autophagy-lysosome system. We previously reported that macrolide antibiotics such as azithromycin and clarithromycin block autophagy flux, and that concomitant treatment with the proteasome inhibitor bortezomib (BZ) and macrolide enhances endoplasmic reticulum (ER) stress-mediated apoptosis in breast cancer cells. As ubiquitinated proteins are concentrated at the aggresome upon proteasome failure, we focused on the microtubule as the scaffold of this transport pathway for aggresome formation. Treatment of metastatic breast cancer cell lines (e.g., MDA-MB‑231 cells) with BZ resulted in induction of aggresomes, which immunocytochemistry detected as a distinctive eyeball-shaped vimentin-positive inclusion body that formed in a perinuclear lesion, and that electron microscopy detected as a sphere of fibrous structure with some dense amorphous deposit. Vinorelbine (VNR), which inhibits microtubule polymerization, more effectively suppressed BZ-induced aggresome formation than paclitaxel (PTX), which stabilizes microtubules. Combined treatment using BZ and VNR, but not PTX, enhanced the cytotoxic effect and apoptosis induction along with pronounced ER stress loading such as upregulation of GRP78 and CHOP/GADD153. The addition of azithromycin to block autophagy flux in the BZ plus VNR-containing cell culture further enhanced the cytotoxicity. These data suggest that suppression of BZ-induced aggresome formation using an inhibitory drug such as VNR for microtubule polymerization is a novel strategy for metastatic breast cancer therapy.
泛素-蛋白酶体和自噬-溶酶体途径是细胞蛋白质的两个主要自我消化系统。泛素化的错误折叠蛋白大多由蛋白酶体降解。然而，当泛素化蛋白积累超过蛋白酶体清除能力时，它们会沿着微管被转运到微管组织中心（MTOC）形成聚集体，随后其中一些会被自噬-溶酶体系统降解。我们之前报道过，阿奇霉素和克拉霉素等大环内酯类抗生素会阻断自噬流，并且蛋白酶体抑制剂硼替佐米（BZ）与大环内酯类药物联合治疗会增强内质网（ER）应激介导的乳腺癌细胞凋亡。由于蛋白酶体功能衰竭时泛素化蛋白会聚集在聚集体中，我们将重点放在作为这种聚集体形成转运途径支架的微管上。用BZ处理转移性乳腺癌细胞系（如MDA-MB-231细胞）会诱导聚集体形成，免疫细胞化学检测显示其为在核周病变中形成的独特眼球状波形蛋白阳性包涵体，电子显微镜检测显示其为具有一些致密无定形沉积物的纤维结构球体。抑制微管聚合的长春瑞滨（VNR）比稳定微管的紫杉醇（PTX）更有效地抑制BZ诱导的聚集体形成。使用BZ和VNR联合治疗，但不使用PTX，会增强细胞毒性作用和凋亡诱导，同时伴有明显的ER应激负荷，如GRP78和CHOP/GADD153上调。在含BZ加VNR的细胞培养物中添加阿奇霉素以阻断自噬流会进一步增强细胞毒性。这些数据表明使用如VNR这种抑制微管聚合的药物抑制BZ诱导的聚集体形成是转移性乳腺癌治疗的一种新策略。
