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. 2020 Jul 13;11(7):524.
doi: 10.1038/s41419-020-2730-7.

Echinatin suppresses esophageal cancer tumor growth and invasion through inducing AKT/mTOR-dependent autophagy and apoptosis

Pan Hong #  1 , Qin-Wen Liu #  2 , Yao Xie  2 , Qi-Hua Zhang  1 , Long Liao  1 , Qing-Yu He  3 , Bin Li  4 , Wen Wen Xu  5
Affiliations

Echinatin suppresses esophageal cancer tumor growth and invasion through inducing AKT/mTOR-dependent autophagy and apoptosis

Pan Hong et al. Cell Death Dis. .

Abstract

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors with poor survival. It is urgent to search for new efficient drugs with good stability and safety for clinical therapy. This study aims to identify potential anticancer drugs from a compound library consisting of 429 natural products. Echinatin, a compound isolated from the Chinese herb Glycyrrhiza uralensis Fisch, was found to markedly induce apoptosis and inhibit proliferation and colony-formation ability in ESCC. Confocal fluorescence microscopy data showed that echinatin significantly induced autophagy in ESCC cells, and autophagy inhibitor bafilomycinA1 attenuated the suppressive effects of echinatin on cell viability and apoptosis. Mechanistically, RNA sequencing coupled with bioinformatics analysis and a series of functional assays revealed that echinatin induced apoptosis and autophagy through inactivation of AKT/mTOR signaling pathway, whereas constitutive activation of AKT significantly abrogated these effects. Furthermore, we demonstrated that echinatin had a significant antitumor effect in the tumor xenograft model and markedly suppressed cell migration and invasion abilities of ESCC cells in a dose-dependent manner. Our findings provide the first evidence that echinatin could be a novel therapeutic strategy for treating ESCC.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Echinatin inhibits ESCC cell proliferation.
a Chemical structure of echinatin. b KYSE30 and KYSE270 cells were treated with echinatin at different concentrations (for up to 40 μM), and cell viability determined by CCK-8 assay. c The colony formation of KYSE30 and KYSE270 cells was inhibited upon exposure to echinatin. d The detection of apoptosis in ESCC cells treated with different concentrations of echinatin for 48 h by an Annexin V-FITC/PI double staining assay. e Western blot analysis was performed to detect the expression of cleaved caspase-3, caspase-3, and cleaved PARP in the ESCC cells treated with indicated concentrations of echinatin for 48 h. Bars, SD; **P < 0. 01, ***P < 0.001.
Fig. 2
Fig. 2. Echinatin induces autophagy in ESCC cells.
The expression of LC3 in the KYSE30 and KYSE270 cells exposed to echinatin was compared by immunofluorescence (a) and Western blot (c), b The number of LC3 puncta per cell and the percentage of the cells with LC3 puncta were counted under florescence microscope. KYSE30 and KYSE270 cells were treated with different concentrations of echinatin, with or without pretreated with BafA1 (0.5 nM, 12 h), and then the viability and colony-formatoin ability determined by CCK-8 assay (d) and colony-formation assay (e), respectively. Comparison of apoptosis and expression of apoptotic markers in ESCC cell treated with different concentrations of echinatin with or without BafA1 pretreatment (0.5 nM, 12 h) by an Annexin V-FITC/PI double staining assay (f) and western blot (g). Bar = 10 μm. Bars, SD; *P < 0.05, **P < 0. 01, ***P < 0.001.
Fig. 3
Fig. 3. The AKT/mTOR signaling pathway mediates the effect of echinatin on autophagy and apoptosis in ESCC cells.
a Ingenuity pathway analysis (IPA) suggested a dysregulation of AKT pathway in echinatin-treated KYSE30 cells. b KYSE30 and KYSE270 cells were exposed to different concentrations of echinatin for 48 h, and western blot analysis was performed to detect the expression levels of AKT, p-AKT, mTOR, and p-mTOR. c–f The KYSE30 and KYSE270 cells transfected with AKT (T308D/S473D)-expressing plasmid or vector control were treated with echinatin at the indicated concentrations for 48 h, and then compared for p-mTOR expression (c), cell viability (d), apoptosis (e), and expression levels of AKT, p-AKT, mTOR, p-mTOR, LC3, cleaved caspase-3 and cleaved PARP (f). Bars, SD; *P < 0.05, **P < 0. 01.
Fig. 4
Fig. 4. Echinatin enhances the sensitivity of ESCC cells to 5-FU.
a, b The viability and colony-formation ability of the KYSE30 and KYSE270 cells treated with 5-FU (1.25 or 0.3 μM), echinatin (20 μM) alone, or the combination of 5-FU and echinatin for up to 5d was determined by CCK-8 assay (a) and colony-formation assay (b), respectively. c Echinatin increased apoptosis in the 5-FU-treated ESCC cells, indicated by higher expression levels of cleaved caspase-3 and cleaved PARP. Bars, SD; *P < 0.05, **P < 0. 01, ***P < 0.001.
Fig. 5
Fig. 5. Echinatin inhibits migration and invasion in ESCC cells.
The migration (a) and invasion (b) abilities of KYSE30 and KYSE270 cells treated with echinatin at different concentrations for 24 h were determined by chamber migration and invasion assays. c Western blot analysis of expressions of vimentin, β-catenin and E-cadherin in the KYSE30 and KYSE270 cells treated with echinatin at the indicated concentrations for 24 h. d, e The KYSE30 and KYSE270 cells transfected with AKT (T308D/S473D)-expressing plasmid or vector control were treated with echinatin at the indicated concentrations for 24 h, and then compared for migration ability (d), invasion ability (e). Bars, SD; *P < 0.05, **P < 0. 01, ***P < 0.001.
Fig. 6
Fig. 6. Echinatin suppresses the growth of ESCC tumor xenograft in nude mice.
The nude mice bearing KYSE270-derived tumor xenografts were orally administrated with echinatin (20 mg/kg or 50 mg/kg) every 2 days (n = 6 per group), whereas the control group received the vehicle only. a Tumor curves showed that echinatin significantly suppressed the growth of tumor xenografts. b Expression levels of AKT, p-AKT, mTOR, p-mTOR and LC3 in the tumors from mice treated with echinatin or vehicle were detected by Western blot. c Body weight of nude mice during the experimental period. d Hematoxylin and eosin (H&E) staining of lung, liver, and kidney specimens collected from mice of the treatment and control groups. e Comparison of serum ALT and AST level between echinatin-treated and control groups. Bars, SD; ***P < 0.001.
Fig. 7
Fig. 7. Schematic diagram summarizing the action mechanism of echinatin in cancer cells.
Activation of AKT/mTOR signaling inhibits autophagy to regulate the balance between proliferation and apoptosis in ESCC cells, while echinatin induces cell autophagy and apoptosis via inhibiting AKT/mTOR pathway, leading to the suppression of cell growth and tumorigensis.
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