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

Theranostics. 2018 Feb 15;8(7):2044-2060. doi: 10.7150/thno.23304. eCollection 2018.

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; apoptosis; autophagy; cardiac glycosides; heat shock protein..

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Agents / administration & dosage
  • Antineoplastic Agents / pharmacology*
  • Autophagy / drug effects*
  • Blotting, Western
  • Cardenolides / administration & dosage
  • Cardenolides / pharmacology*
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Cell Survival / drug effects
  • Disease Models, Animal
  • Flow Cytometry
  • Fluorescent Antibody Technique
  • Formazans / analysis
  • Gene Expression Profiling
  • HSP90 Heat-Shock Proteins / metabolism
  • Humans
  • Lung Neoplasms / drug therapy*
  • Mice, Inbred BALB C
  • Mice, Nude
  • Microscopy, Electron, Transmission
  • Neoplasm Transplantation
  • Oncogene Protein v-akt / metabolism
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction / drug effects
  • Staining and Labeling
  • TOR Serine-Threonine Kinases / metabolism
  • Tetrazolium Salts / analysis
  • Treatment Outcome

Substances

  • Antineoplastic Agents
  • Cardenolides
  • Formazans
  • HSP90 Heat-Shock Proteins
  • Tetrazolium Salts
  • MTT formazan
  • MTOR protein, human
  • TOR Serine-Threonine Kinases
  • Oncogene Protein v-akt