3'-epi-12β-hydroxyfroside, a new cardenolide, induces cytoprotective autophagy via blocking the Hsp90/Akt/mTOR axis in lung cancer cells.
3'-epi-12β-hydroxyfroside, a new cardenolide, induces cytoprotective autophagy via blocking the Hsp90/Akt/mTOR axis in lung cancer cells.
复制标题
3'-epi-12beta-Hydroxyfroside 是一种新的卡烯内酯，通过阻断肺癌细胞中的 Hsp90/Akt/mTOR 轴诱导细胞保护性自噬
作者：
Sun Y;Huang YH;Huang FY;Mei WL;Liu Q;Wang CC;Lin YY;Huang C;Li YN;Dai HF;Tan GH
理由：强心内酯具有作为抗癌药物的潜力。 3′-epi-12β-羟基froside（HyFS）是我们课题组分离出的一种新的强心内酯结构，但其分子机制仍知之甚少。本研究探讨其抗肿瘤活性与肺癌细胞自噬之间的关系。方法：通过MTT、乳酸脱氢酶（LDH）释放、5-乙炔基-20-脱氧尿苷（EDU）和集落形成试验检测细胞生长和增殖。通过流式细胞术检测细胞凋亡。通过蛋白质印迹法检测自噬和信号蛋白。还通过免疫荧光、透射电子显微镜和吖啶橙染色检测自噬和自噬通量的标志物。采用实时RT-PCR分析Hsp90的基因表达。通过免疫共沉淀检测 Hsp90 泛素化。在裸鼠体内观察HyFS的抗肿瘤活性。结果：HyFS处理抑制A549和H460肺癌细胞的细胞增殖并诱导自噬，但当HyFS介导的自噬被阻断时，表现出更强的细胞增殖抑制和诱导细胞凋亡。 Hsp90/Akt/mTOR 轴被发现参与 HyFS 介导的自噬的激活。观察到 Hsp90 和 Akt 之间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 水平降低，Hsp90 的过度表达增加自噬的激活，而抑制 Hsp90 表达则减少自噬。此外，在 HyFS 处理的肺癌细胞中还发现了泛素介导的 Hsp90 降解及其客户蛋白 Akt 的去磷酸化。此外，HyFS 和氯喹联合治疗显示 A549 和 H460 小鼠的肿瘤抑制显着增强。结论：我们的结果表明，HyFS 通过泛素介导的 Hsp90 降解诱导细胞保护性自噬，从而进一步阻断肺癌细胞中的 Akt/mTOR 通路。因此，HyFS 样卡烯内酯和自噬抑制剂的组合是治疗肺癌的潜在替代方法。
Rationale: 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. Methods: 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. Results: 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. Conclusion: 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.
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