DIMES, Department of Experimental Medicine, Human Anatomy, University of Genoa, Via Antonio de Toni 14, 16132, Genoa, Italy.
DiMI, Department of Internal Medicine and Medical Specialities, University of Genoa, Viale Benedetto XV 2, 16132, Genoa, Italy.
J Exp Clin Cancer Res. 2017 Nov 3;36(1):154. doi: 10.1186/s13046-017-0615-0.
ERBB2 is overexpressed in up to 20-30% of human breast cancers (BCs), and it is associated with aggressive disease. Trastuzumab (Tz), a humanized monoclonal antibody, improves the prognosis associated with ERBB2-amplified BCs. However, the development of resistance remains a significant challenge. Carnosic acid (CA) is a diterpene found in rosemary and sage, endowed with anticancer properties. In this in vitro study, we have investigated whether Tz and CA have cooperative effects on cell survival of ERBB2 overexpressing (ERBB2) cells and whether CA might restore Tz sensitivity in Tz-resistant cells.
We have studied BC cell migration and survival upon CA and Tz treatment. In particular, migration ability was assessed by transwell assay while cell survival was assessed by MTT assay. In addition, we have performed cell cycle and apoptosis analysis by high-resolution DNA flow cytometry and annexin-V, resazurin and sytox blue staining by flow cytometry, respectively. The expression of proteins involved in cell cycle progression, ERBB2 signaling pathway, and autophagy was evaluated by immunoblot and immunofluorescence analysis. Cellular structures relevant to the endosome/lysosome and autophagy pathways have been studied by immunofluorescence and transmission electron microscopy.
We report that, in ERBB2 BC cells, CA reversibly enhances Tz inhibition of cell survival, cooperatively inhibits cell migration and induces cell cycle arrest in G0/G1. These events are accompanied by ERBB2 down-regulation, deregulation of the PI3K/AKT/mTOR signaling pathway and up-regulation of both CDKN1A/p21 and CDKN1B/p27. Furthermore, we have demonstrated that CA impairs late autophagy and causes derangement of the lysosomal compartment as shown by up-regulation of SQSTM1/p62 and ultrastructural analysis. Accordingly, we have found that CA restores, at least in part, sensitivity to Tz in SKBR-3 Tz-resistant cell line.
Our data demonstrate the cooperation between CA and Tz in inhibiting cell migration and survival of ERBB2 BC cells that warrant further studies to establish if CA or CA derivatives may be useful in vivo in the treatment of ERBB2 cancers.
ERBB2 在多达 20-30%的人类乳腺癌（BC）中过表达，并且与侵袭性疾病相关。曲妥珠单抗（Tz），一种人源化单克隆抗体，改善了与 ERBB2 扩增的 BC 相关的预后。然而，耐药性的发展仍然是一个重大挑战。迷迭香酸（CA）是一种在迷迭香和鼠尾草中发现的二萜，具有抗癌特性。在这项体外研究中，我们研究了 Tz 和 CA 是否对 ERBB2 过表达（ERBB2）细胞的细胞存活有协同作用，以及 CA 是否可能恢复 Tz 耐药细胞对 Tz 的敏感性。
我们研究了 CA 和 Tz 处理后 BC 细胞的迁移和存活。特别是，通过 Transwell 测定评估迁移能力，而通过 MTT 测定评估细胞存活。此外，我们通过高分辨率 DNA 流式细胞术进行细胞周期和凋亡分析，通过流式细胞术分别用 Annexin-V、Resazurin 和 Sytox Blue 染色进行分析。通过免疫印迹和免疫荧光分析评估参与细胞周期进程、ERBB2 信号通路和自噬的蛋白质的表达。通过免疫荧光和透射电子显微镜研究与内体/溶酶体和自噬途径相关的细胞结构。
我们报告说，在 ERBB2 BC 细胞中，CA 可逆地增强了 Tz 对细胞存活的抑制作用，协同抑制细胞迁移并诱导细胞周期停滞在 G0/G1 期。这些事件伴随着 ERBB2 的下调、PI3K/AKT/mTOR 信号通路的失调以及 CDKN1A/p21 和 CDKN1B/p27 的上调。此外，我们已经证明 CA 会损害晚期自噬并导致溶酶体区室的紊乱，这表现为 SQSTM1/p62 的上调和超微结构分析。因此，我们发现 CA 至少部分恢复了 SKBR-3 Tz 耐药细胞系对 Tz 的敏感性。
我们的数据表明 CA 和 Tz 在抑制 ERBB2 BC 细胞的迁移和存活方面具有协同作用，这需要进一步的研究来确定 CA 或 CA 衍生物是否可用于体内治疗 ERBB2 癌症。
