Cisplatin (DDP)-based adjuvant chemotherapy is widely used for the treatment of esophageal cancer. However, DDP resistance has become more common and thus new approaches are required to be explored. Cisplatin was used to induce autophagy in the human esophageal cancer cell line, EC9706 cells, and the effect of autophagy on the survival of EC9706 cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by CCK8 assay. Apoptosis and cell cycle were detected by flow cytometry. Monodansylcadaverine (MDC) was used to detect autophagy. Western blotting assay was used to investigate the molecular changes that occurred in the course of treatment. DDP inhibited cell proliferation, induced cell death and cell cycle arrest at S phage. Moreover, autophagy was activated through class III PI3K pathway. The expression of autophagy-related Beclin1 and LC3-I was up-regulated and part of LC3-I was converted into LC3-II. However, after the combination treatment of 3-MA and DDP, the cell inhibitory rate increased; the apoptosis rate and the numbers of cells in S phase also increased. Furthermore, the accumulation of autophagic vacuoles was decreased; the expression of Beclin1 and LC3 was significantly down-regulated and the release of cytochrome c was decreased. DDP-induced apoptosis in EC9706 cells can be enhanced by the inhibitor of autophagy, 3-MA. Autophagy might play a role as a self-protective mechanism in DDP-treated esophageal cancer cells, and its inhibition could be a novel strategy for the adjuvant chemotherapy of esophageal cancer.

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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.