Department of Pharmacology and Biochemistry, School of Pharmacy, Fudan University, Shanghai 201203, China.
Chemical Biology, Roche Pharmaceutical Research and Early Development, Roche Innovation Center Shanghai, Shanghai 201203, China.
Acta Pharmacol Sin. 2017 Oct;38(10):1381-1393. doi: 10.1038/aps.2017.9. Epub 2017 May 1.
The flavonoid quercetin exhibits significant anticancer activities with few side effects. In the current study, we characterized TL-2-8, a quercetin derivative, as a novel anticancer agent in vitro and in vivo. Cell proliferation and viability were assessed using Cell Counting Kit-8 and CellTiter-Blue assay, respectively. Cell death was examined using PI staining or a TUNEL assay. Mitophagy was determined by measuring autophagic flux and by confocal imaging. Protein expression was examined by Western blotting. We found that TL-2-8 selectively inhibited the proliferation and decreased the viability of various cancer cells (the anti-proliferation IC50 values in MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cells at 72 h were 8.28, 8.56, and 9.58 μmol/L, respectively), and it displayed only slight cytotoxicity against normal MCF-10A and HEK-293 cells. In MDA-MB-231 and MDA-MB-468 breast cancer cells, TL-2-8 treatment induced the degradation of multiple Hsp90 client proteins without inducing Hsp70. TL-2-8 (3, 6, 12 μmol/L) dose-dependently inhibited the expression of AHA1, an activator of Hsp90 ATPase, and decreased Hsp90-AHA1 complex formation, leading to decreased Hsp90 chaperone function and reduced polo-like kinase 1 (PLK1) signaling. Consequently, impaired mitophagy was induced via the downregulation of lysosomal-associated membrane protein 2 (LAMP2). The in vivo anticancer effects of TL-2-8 were evaluated in an MDA-MB-231 breast cancer xenograft model, which was treated with TL-2-8 (25, 50, 100 mg·kg·d, po). Administration of TL-2-8 resulted in tumor growth inhibition rates of 37.9%, 58.9% and 70.9%, respectively, whereas quercetin treatment (100 mg·kg·d, po) produced only a lower tumor growth inhibition rate (49.5%). Furthermore, TL-2-8 treatment significantly extended the lifespan of mice bearing MDA-MB-231 breast cancer cell xenografts. Our results demonstrate that TL-2-8 induces significant cell death and immature mitophagy in breast cancer cells in vitro and in vivo via AHA1 abrogation.
类黄酮槲皮素具有显著的抗癌活性，副作用较少。在本研究中，我们将槲皮素衍生物 TL-2-8 鉴定为一种新型的体外和体内抗癌药物。使用细胞计数试剂盒-8 和 CellTiter-Blue 检测分别评估细胞增殖和活力。通过 PI 染色或 TUNEL 检测来检查细胞死亡。通过测量自噬通量和共聚焦成像来确定线粒体自噬。通过 Western blotting 检测蛋白表达。我们发现 TL-2-8 选择性抑制增殖并降低各种癌细胞的活力（72 小时 MDA-MB-231、MDA-MB-468 和 MCF-7 乳腺癌细胞的抗增殖 IC50 值分别为 8.28、8.56 和 9.58 μmol/L），并且对正常 MCF-10A 和 HEK-293 细胞仅有轻微的细胞毒性。在 MDA-MB-231 和 MDA-MB-468 乳腺癌细胞中，TL-2-8 处理诱导多种 Hsp90 客户蛋白的降解，而不诱导 Hsp70。TL-2-8（3、6、12 μmol/L）剂量依赖性地抑制 AHA1 的表达，AHA1 是 Hsp90 ATPase 的激活剂，并降低 Hsp90-AHA1 复合物的形成，导致 Hsp90 伴侣功能降低和 polo 样激酶 1（PLK1）信号减少。因此，通过下调溶酶体相关膜蛋白 2（LAMP2）诱导受损的线粒体自噬。在 MDA-MB-231 乳腺癌异种移植模型中评估 TL-2-8 的体内抗癌作用，该模型用 TL-2-8（25、50、100 mg·kg·d，po）治疗。TL-2-8 给药分别导致肿瘤生长抑制率为 37.9%、58.9%和 70.9%，而槲皮素治疗（100 mg·kg·d，po）仅产生较低的肿瘤生长抑制率（49.5%）。此外，TL-2-8 治疗显著延长了携带 MDA-MB-231 乳腺癌细胞异种移植物的小鼠的寿命。我们的结果表明，TL-2-8 通过 AHA1 阻断在体外和体内诱导乳腺癌细胞中显著的细胞死亡和不成熟的线粒体自噬。
