Department of Breast Oncology, Tokyo Medical University, Tokyo 160-8402, Japan.
Int J Oncol. 2012 Apr;40(4):1029-39. doi: 10.3892/ijo.2011.1317. Epub 2011 Dec 23.
The specific 26S proteasome inhibitor, bortezomib (BZ) potently induces apoptosis as well as autophagy in metastatic breast cancer cell lines such as MDA-MB-231 and MDA-MB-468. The combined treatment of clarithromycin (CAM) and BZ significantly enhances cytotoxicity in these cell lines. Although treatment with up to 100 µg/ml CAM alone had little effect on cell growth inhibition, the accumulation of autophagosomes and p62 was observed after treatment with 25 µg/ml CAM. This result indicated that CAM blocked autophagy flux. However, the combined treatment of BZ and CAM resulted in more pronounced autophagy induction, as assessed by increased expression ratios of LC3B-II to LC3B-I and clearance of intracellular p62, than treatment with BZ alone. This combination further enhanced induction of the pro-apoptotic transcription factor CHOP (CADD153) and the chaperone protein GRP78. Knockdown of CHOP by siRNA attenuated the death-promoting effect of BZ in MDA-MB-231 cells. A wild-type murine embryonic fibroblast (MEF) cell line also exhibited enhanced BZ-induced cytotoxicity with the addition of CAM, whereas a Chop knockout MEF cell line completely abolished this enhancement and exhibited resistance to BZ treatment. These data suggest that endoplasmic reticulum (ER)-stress mediated CHOP induction is involved in pronounced cytotoxicity by combining these reagents. Simultaneously targeting two major intracellular protein degradation pathways such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome pathway by CAM may improve the therapeutic outcome in breast cancer patients via ER-stress mediated apoptosis.
特定的 26S 蛋白酶体抑制剂硼替佐米（BZ）能有效诱导转移性乳腺癌细胞系（如 MDA-MB-231 和 MDA-MB-468）凋亡和自噬。克拉霉素（CAM）和 BZ 的联合治疗显著增强了这些细胞系的细胞毒性。尽管单独使用高达 100µg/ml 的 CAM 处理对细胞生长抑制几乎没有影响，但在用 25µg/ml 的 CAM 处理后，观察到自噬体和 p62 的积累。这一结果表明 CAM 阻断了自噬流。然而，BZ 和 CAM 的联合治疗导致更明显的自噬诱导，如 LC3B-II 与 LC3B-I 的表达比值增加以及细胞内 p62 的清除，比单独使用 BZ 更为明显。这种组合进一步增强了促凋亡转录因子 CHOP（CADD153）和伴侣蛋白 GRP78 的诱导。用 siRNA 敲低 CHOP 可减弱 BZ 在 MDA-MB-231 细胞中的促死亡作用。野生型小鼠胚胎成纤维细胞（MEF）细胞系在添加 CAM 后也表现出增强的 BZ 诱导细胞毒性，而 Chop 敲除 MEF 细胞系完全消除了这种增强作用并对 BZ 治疗产生抗性。这些数据表明，内质网（ER）应激介导的 CHOP 诱导参与了这些试剂联合使用时产生的显著细胞毒性。通过 BZ 靶向泛素-蛋白酶体系统和 CAM 靶向自噬-溶酶体途径这两个主要的细胞内蛋白降解途径，可能通过 ER 应激介导的凋亡改善乳腺癌患者的治疗效果。
