Key Laboratory of Tropical Diseases and Translational Medicine of the Ministry of Education & Hainan Provincial Key Laboratory of Tropical Medicine, Hainan Medical College, Haikou 571199, China.
Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571199, China.
Theranostics. 2018 Feb 15;8(7):2044-2060. doi: 10.7150/thno.23304. eCollection 2018.
Cardenolides have potential as anticancer drugs. 3'-epi-12β-hydroxyfroside (HyFS) is a new cardenolide structure isolated by our research group, but its molecular mechanisms remain poorly understood. This study investigates the relationship between its antitumor activities and autophagy in lung cancer cells. Cell growth and proliferation were detected by MTT, lactate dehydrogenase (LDH) release, 5-ethynyl-20-deoxyuridine (EDU) and colony formation assays. Cell apoptosis was detected by flow cytometry. Autophagic and signal proteins were detected by Western blotting. Markers of autophagy and autophagy flux were also detected by immunofluorescence, transmission electron microscopy and acridine orange staining. Real time RT-PCR was used to analyze the gene expression of Hsp90. Hsp90 ubiquitination was detected by coimmunoprecipitation. The antitumore activities of HyFS were observed in nude mice. HyFS treatment inhibited cell proliferation and induced autophagy in A549 and H460 lung cancer cells, but stronger inhibition of cell proliferation and induction of cell apoptosis were shown when HyFS-mediated autophagy was blocked. The Hsp90/Akt/mTOR axis was found to be involved in the activation of HyFS-mediated autophagy. Evidence of direct interaction between Hsp90 and Akt was observed. HyFS treatment resulted in decreased levels of heat shock protein 90 (Hsp90) and phosphorylated Akt, overexpression of Hsp90 increased activation of autophagy, and inhibition of Hsp90 expression decreased autophagy. In addition, ubiquitin-mediated degradation of Hsp90 and subsequent dephosphorylation of its client protein Akt were also found in HyFS-treated lung cancer cells. Moreover, combination treatment with HyFS and chloroquine showed remarkably increased tumor inhibition in both A549- and H460-bearing mice. Our results demonstrate that HyFS induced cytoprotective autophagy through ubiquitin-mediated degradation of Hsp90, which further blocked the Akt/mTOR pathway in lung cancer cells. Thus, a combination of a HyFS-like cardenolide and an autophagic inhibitor is a potential alternative approach for the treatment of lung cancer.
卡烯内酯具有抗癌药物的潜力。3'-表-12β-羟基佛罗斯苷（HyFS）是本研究组分离的一种新的卡烯内酯结构，但对其分子机制知之甚少。本研究探讨了其在肺癌细胞中的抗肿瘤活性与自噬之间的关系。通过 MTT、乳酸脱氢酶（LDH）释放、5-乙炔基-20-脱氧尿苷（EDU）和集落形成实验检测细胞生长和增殖。通过流式细胞术检测细胞凋亡。通过 Western blot 检测自噬和信号蛋白。通过免疫荧光、透射电子显微镜和吖啶橙染色检测自噬和自噬流的标志物。通过实时 RT-PCR 分析 Hsp90 的基因表达。通过共免疫沉淀检测 Hsp90 的泛素化。在裸鼠中观察到 HyFS 的抗肿瘤活性。HyFS 处理抑制 A549 和 H460 肺癌细胞的增殖并诱导自噬，但当阻断 HyFS 介导的自噬时，对细胞增殖的抑制作用更强，诱导细胞凋亡。发现 Hsp90/Akt/mTOR 轴参与 HyFS 介导的自噬的激活。观察到 Hsp90 和 Akt 之间存在直接相互作用的证据。HyFS 处理导致热休克蛋白 90（Hsp90）和磷酸化 Akt 的水平降低，Hsp90 的过表达增加自噬的激活，抑制 Hsp90 的表达减少自噬。此外，还发现 HyFS 处理的肺癌细胞中存在 Hsp90 的泛素介导降解及其客户蛋白 Akt 的去磷酸化。此外，在 A549 和 H460 荷瘤小鼠中，HyFS 与氯喹联合治疗显示出明显增加的肿瘤抑制作用。我们的研究结果表明，HyFS 通过 Hsp90 的泛素介导降解诱导细胞保护性自噬，进而阻断肺癌细胞中的 Akt/mTOR 通路。因此，HyFS 样卡烯内酯与自噬抑制剂的联合使用可能是治疗肺癌的一种有潜力的替代方法。
