Theranostics 2018; 8(7):2044-2060. doi:10.7150/thno.23304

Research Paper

3′-epi-12β-hydroxyfroside, a new cardenolide, induces cytoprotective autophagy via blocking the Hsp90/Akt/mTOR axis in lung cancer cells

Yan Sun1*, Yong-Hao Huang1*, Feng-Ying Huang1*, Wen-Li Mei2, Quan Liu3, Cai-Chun Wang1, Ying-Ying Lin1, Canhua Huang1,4, Yue-Nan Li1,5✉, Hao-Fu Dai2✉, Guang-Hong Tan1✉

1. 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
2. Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571199, China;
3. The Department of Medical Oncology, Affiliated Hospital of Jiangnan University and Fourth People's Hospital of Wuxi, Wuxi 214062, China;
4. State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, China;
5. The Pathology Department of the First Affiliated Hospital, Hainan Medical College, Haikou 570103, China.
*Equal contribution.

Abstract

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.

Keywords: 3′-epi-12β-hydroxyfroside, cardiac glycosides, autophagy, apoptosis, heat shock protein.

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How to cite this article:
Sun Y, Huang YH, Huang FY, Mei WL, Liu Q, Wang CC, Lin YY, Huang C, Li YN, Dai HF, Tan GH. 3′-epi-12β-hydroxyfroside, a new cardenolide, induces cytoprotective autophagy via blocking the Hsp90/Akt/mTOR axis in lung cancer cells. Theranostics 2018; 8(7):2044-2060. doi:10.7150/thno.23304. Available from http://www.thno.org/v08p2044.htm