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, 8 (1), 8009

Cypripedin Diminishes an Epithelial-To-Mesenchymal Transition in Non-Small Cell Lung Cancer Cells Through Suppression of Akt/GSK-3β Signalling

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Cypripedin Diminishes an Epithelial-To-Mesenchymal Transition in Non-Small Cell Lung Cancer Cells Through Suppression of Akt/GSK-3β Signalling

Surassawadee Treesuwan et al. Sci Rep.

Abstract

Lung cancer appears to have the highest rate of mortality among cancers due to its metastasis capability. To achieve metastasis, cancer cells acquire the ability to undergo a switch from epithelial to mesenchymal behaviour, termed the epithelial-to-mesenchymal transition (EMT), which is associated with poor clinical outcomes. Drug discovery attempts have been made to find potent compounds that will suppress EMT. Cypripedin, a phenanthrenequinone isolated from Thai orchid, Dendrobium densiflorum, exhibits diverse pharmacological activities. In this study, we found that cypripedin attenuated typical mesenchymal phenotypes, including migratory behaviour, of non-small cell lung cancer H460 cells, with a significant reduction of actin stress fibres and focal adhesion and with weakened anchorage-independent growth. Western blot analysis revealed that the negative activity of this compound on EMT was a result of the down-regulation of the EMT markers Slug, N-Cadherin and Vimentin, which was due to ATP-dependent tyrosine kinase (Akt) inactivation. As a consequence, the increase in the Slug degradation rate via a ubiquitin-proteasomal mechanism was encouraged. The observation in another lung cancer H23 cell line also supported this finding, indicating that cypripedin exhibits a promising pharmacological action on lung cancer metastasis that could provide scientific evidence for the further development of this compound.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Cytotoxicity of cypripedin on lung cancer H460 cells. (A) Chemical structure of cypripedin. (B) H460 cells were treated with various concentrations (0–100 µM) of cypripedin for 24, 48 and 72 h; cell viability was measured by MTT assay and is represented as a mean of the relative value. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with untreated control cells. (C) H460 cells were treated with various concentrations (0–100 µM) of cypripedin for 72 h; apoptotic cells were investigated by Hoechst 33342 nuclear staining dye. The percentage of apoptotic cells was calculated comparing to the control cells. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with control cells. (D) The cells with apoptotic nuclei are illustrated (scale bar is 10 µm). (E) H460 cells were treated with non-toxic concentrations (0–20 µM) of cypripedin for 24, 48 and 72 h; cell growth was examined by cell proliferation assay and is presented as a relative value. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with untreated control cells.
Figure 2
Figure 2
Cypripedin suppresses cell migration and an anchorage-independent growth. (A) H460 cells were pre-treated with non-toxic concentrations (0–20 µM) of cypripedin for 72 h, and the wound healing assay was performed. The wound space was captured and measured at 0, 24, 48 and 72 h. The wound area was calculated and presented as a relative value to the area at the initial time point. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with control cells. (B) The cells were treated similarly with cypripedin and were subjected to the transwell migration assay. After 18 h, the migrated cells were fixed with MeOH and were stained with DAPI. The migrated cells that were underneath of membrane were imaged by fluorescence microscopy (scale bar is 10 µm) and were calculated as the relative number of migrated cells of the cypripedin treated group over the untreated control group. The data are presented mean ± SEM (n = 4). *p < 0.05 compared with control cells. (C) H460 cells were seeded on cover slips and treated with non-toxic concentrations (0–20 µM) of cypripedin for 72 h. The actin stress fibres (red), focal adhesion protein paxillin (green) and nuclei staining DAPI (blue) were analysed by immunofluorescence assay and were imaged by a confocal fluorescence microscope (scale bar is 10 µm). The number of actin stress fiber (SFs) and paxllin-adhered stress fiber (FAs; arrow) were quantified. The data are presented as mean ± SEM from at least 50 cells. *p < 0.05 compared with control cells. (D) H460 cells were treated similarly with cypripedin (0–20 µM) for 72 h and were subjected to an anchorage-independent growth assay. After 14 d, the colonies were stained by crystal violet. The dot plot represents the value of a single colony. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with control cells.
Figure 3
Figure 3
Cypripedin attenuated in vitro tumourigenesis and spheroid-based cell migration. (A) H460 cells were mixed with 4% Matrigel and cultured onto Matrigel coated-cell culture plate in the presence or absence of cypripedin (20 µM). After 10 d, spheroid was immunostained for actin (red) and DNA (blue). The data are presented as a mean of spheroid diameter ± SEM (n = 25). *p < 0.05 compared with control cells. Scale bar is 20 µm. (B) Spheroids were generated under detached condition as described in Method, seeded onto cell culture plate and treated with or without cypripedin (20 µM). Images were captured at d0 and d3 with 20x and 40x magnification, and cell migration was analyzed from the migrating distance (between red line and black line). The data are presented as a mean of migrating distance ± SEM (n = 15). *p < 0.05 compared with control cells. Scale bar is 100 µm.
Figure 4
Figure 4
Cypripedin inhibits the epithelial to mesenchymal transition (EMT) in lung cancer H460 cells. (A) After H460 cells were treated with non-toxic concentration (0–20 µM) of cypripedin for 72 h, the protein expression levels of EMT markers Slug, Snail, Vimentin and N-Cadherin were analysed by Western blotting, and the intensity was qualified by densitometry. GAPDH was reprobed to confirm equal loading. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with control cells. Vimentin (B) and N-Cadherin (C) were visualized by immunofluorescence staining assay. The fluorescence intensity was analysed by ImageJ software. The diagram illustrates signal intensity along the dotted line, whereas each bar presented the relative mean intensity/cell from at least 50 cells. (D) The mRNA expression levels of the EMT markers were measured by quantitative RT-PCR and are presented as relative value to the untreated control cells. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with control cells.
Figure 5
Figure 5
Cypripedin down-regulates Slug via an Akt-GSK-3β-dependent mechanism. (A) After treatment with non-toxic concentrations (0–20 µM) of cypripedin for 72 h, the protein expression levels of p-Akt (Ser 473), Akt, p-GSK-3β (Ser 9), and GSK-3β were analysed by Western blotting, and the intensity was qualified by densitometry. GAPDH was reprobed to confirm equal loading. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with control cells. (B) H460 cells were treated with an Akt inhibitor LY294002 (0–10 µM) for 18 h; the protein expression levels of p-Akt (Ser 473), Akt, p-GSK-3β (Ser 9) and GSK-3β were analysed by Western blotting, and the intensity was qualified by densitometry. GAPDH was reprobed to confirm equal loading. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with control cells. (C) After the cells were treated similarly with an Akt inhibitor LY294002 (0–10 µM), cell migration was evaluated by the wound healing assay. Wound space was captured and measured at 0, 12 and 24 h. The wound area was calculated and presented as relative value to those at initial time point. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with control cells. (D) H460 cells were transfected with siAkt (100 and 200 nM) or si-mismatch control. After post-transfection for 72 h, the protein expression levels of p-Akt (Ser 473), Akt, p-GSK-3β (Ser 9), GSK-3β and Slug were analysed by Western blotting, and the intensity was qualified by densitometry. GAPDH was reprobed to confirm equal loading. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with untreated control cells.
Figure 6
Figure 6
Cypripedin mediates Slug degradation via ubiquitin-proteasomal pathway. (A) H460 cells were pre-treated with cycloheximide (CHX, 10 µg/mL) for 1 h before incubation with or without 20 µM of cypripedin for 0–4 h. (B) H460 cells were pre-treated with or without a proteasome inhibitor, MG132 (10 µM) for 1 h, before treatment with 20 µM of cypripedin for 0–4 h. Slug expression was evaluated by Western blot analysis, and the intensity was qualified by densitometry. GAPDH was reprobed to confirm equal loading. The relative Slug level over the experiment periods was presented, and the half-life was calculated. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with control cells. (C) H460 cells were pre-treated with MG132 10 µM for 1 h, followed by incubation with cypripedin (0–20 µM) for 3 h. The Slug ubiquitination was analysed by immunoprecipitation assay. The lysates were obtained, and then Slug was pulled down with anti-Slug antibody, and the Slug pull-down samples were collected and subjected to immunoblotting to confirm the equal levels of Slug substrate.
Figure 7
Figure 7
Cypripedin suppresses the epithelial to mesenchymal transition (EMT) in lung cancer H23 cells. (A) H23 cells were treated with various concentrations (0–200 µM) of cypripedin for 24, 48 and 72 h and cell viability was measured by MTT assay. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with control cells. (B) H23 cells were treated with non-toxic concentrations (0–50 µM) of cypripedin for 24, 48 and 72 h, and cell growth was examined by cell proliferation assay and presented as a relative value. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with control cells. (C) H23 cells were pre-treated with non-toxic concentrations (0–50 µM) of cypripedin for 72 h, and the wound healing assay was performed. Wound space was captured and measured at 0, 24, 48 and 72 h. The wound area was calculated and presented as a relative value to those at initial time points. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with control cells. (D) After H23 cells were treated with non-toxic concentrations (0–50 µM) of cypripedin for 72 h, the protein expression levels of EMT markers Slug, Snail and Vimentin were analysed by Western blotting, and the intensity was qualified by densitometry. GAPDH was reprobed to confirm equal loading. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with control cells. (E) The effect of cypripedin on EMT-regulated proteins was investigated using Western blot analysis. After treatment with non-toxic concentrations (0–50 µM) of cypripedin for 72 h, the protein expression levels of p-Akt (Ser 473), Akt, p-GSK-3β (Ser 9), GSK-3β and Slug were analysed by Western blotting, and the intensity was qualified by densitometry. GAPDH was reprobed to confirm equal loading. The data are presented as mean ± SEM (n = 4). *p < 0.05 compared with control cells.
Figure 8
Figure 8
A schematic diagram summarizes the underlying mechanism of cypripedin-suppressing EMT in lung cancer cells.

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