Erianthridin suppresses non-small-cell lung cancer cell metastasis through inhibition of Akt/mTOR/p70S6K signaling pathway

Abstract

Cancer metastasis is a major cause of the high mortality rate in lung cancer patients. The cytoskeletal rearrangement and degradation of extracellular matrix are required to facilitate cell migration and invasion and the suppression of these behaviors is an intriguing approach to minimize cancer metastasis. Even though Erianthridin (ETD), a phenolic compound isolated from the Thai orchid Dendrobium formosum exhibits various biological activities, the molecular mechanism of ETD for anti-cancer activity is unclear. In this study, we found that noncytotoxic concentrations of ETD (≤ 50 μM) were able to significantly inhibit cell migration and invasion via disruption of actin stress fibers and lamellipodia formation. The expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 was markedly downregulated in a dose-dependent manner after ETD treatment. Mechanistic studies revealed that protein kinase B (Akt) and its downstream effectors mammalian target of rapamycin (mTOR) and p70 S6 kinase (p70^S6K) were strongly attenuated. An in silico study further demonstrated that ETD binds to the protein kinase domain of Akt with both hydrogen bonding and van der Waals interactions. In addition, an in vivo tail vein injection metastasis study demonstrated a significant effect of ETD on the suppression of lung cancer cell metastasis. This study provides preclinical information regarding ETD, which exhibits promising antimetastatic activity against non-small-cell lung cancer through Akt/mTOR/p70^S6K-induced actin reorganization and MMPs expression.

Figure 1

Cytotoxicity of erianthridin (ETD) in A549 and H460 cells. (A) The chemical structure of ETD (3,4-dimethoxy-9,10-dihydrophenanthrene-2,7-diol) is shown. (B) A549 and H460 cells were treated with the indicated concentrations of ETD for 24, 48 and 72 h. Cell viability was analyzed using the MTT assay and is represented as a percentage. (C) A549 and H460 cells were treated with nontoxic concentrations of ETD for 24, 48 and 72 h. Cell proliferation was evaluated by the MTT assay. The rate of cell growth was calculated as a value relative to time 0 h. The data are presented as the mean ± SEM (n = 3). *p < 0.05 vs untreated control cells.

Figure 2

ETD inhibits metastatic behaviors of non-small-cell lung cancer cells. (A) A549 and H460 cells were seeded onto a transwell chamber and treated with nontoxic concentrations of ETD (0–50 µM). After 20 h, the migrated cells were stained with DAPI and imaged by fluorescence microscopy. The scale bar is 10 µm. (B) A monolayer of the cells was scratched with a pipette tip to generate a wound, and the cells were treated with 0–50 µM ETD. The wound area was photographed under a microscope at 0, 48 and 72 h. The wound area was quantified at each time point relative to the area at the initial time point. (C) A549 and H460 cells were seeded onto a transwell chamber coated with Martigel and treated with nontoxic concentrations of ETD (0–50 µM). The scale bar is 10 µm. (D) Anchorage-independent growth assays were conducted by seeding cells into 24-well plates coated with 0.5% agarose. Cells were incubated with ETD and allowed to grow for 10 d. The colony size was measured using ImageJ. Each dot plot represents a single colony. All data are presented as the mean ± SEM (n = 3). *p < 0.05 vs untreated control group.

Figure 3

ETD suppresses cell migration and invasion via alteration of actin stress fiber organization and inhibition of MMP expression. (A) A549 and H460 cells were cultured on cover slips and treated with 50 µM ETD for 48 h. Cells were stained with phalloidin (actin, red) and DAPI (blue) and observed by confocal microscopy at ×20 magnification. The images in the box are enlarged in the right panel. Arrows indicate actin stress fibers and lamellipodia. The scale bar is 20 µm. (B) The number of stress fibers per cell and (C) the area of lamellipodia per cell were analyzed relative to those of the control group using ImageJ. The data are presented as mean ± SEM from at least 50 cells. *p < 0.05 vs untreated control group. (D) Cells were treated with 0–50 µM ETD for 48 h, and MMP-2 and MMP-9 mRNA expressions were quantified by quantitative RT-PCR. The mRNA expression level of the treatment group was calculated relative to that in the control group. The data are presented as the mean ± SEM (n = 3). *p < 0.05 vs untreated control group.

Figure 4

ETD inhibits cell migration and invasion via an Akt/mTOR/p70^S6K-dependent mechanism. (A) A549 and H460 cells were treated with nontoxic concentrations of ETD for 24 h. The protein expression levels of p-Akt, Akt, p-mTOR, mTOR and p-p70^S6K were examined by Western blot analysis. The protein expression levels are displayed as the mean ± SEM (n = 3). *p < 0.05 vs untreated control group. (B) A549 and H460 cells were transfected with siRNA against Akt (siAkt) or si-mismatch control siRNA (siCtrl). After transfection for 18 h, cells were incubated with 50 µM ETD for 24 h and examined by Western blot analysis. The protein expression levels are shown as the mean ± SEM (n = 3). *p < 0.05 vs untreated cells, ^#p < 0.05 vs untreated siCtrl cells. (C) A549 and H460 cells were transfected with siRNA against Akt (siAkt) or si-mismatch control siRNA (siCtrl). After transfection for 48 h, cells were subjected to transwell migration assay in the presence or absence of 50 µM ETD. The scale bar is 10 µm. The data are shown as the mean ± SEM (n = 3). *p < 0.05 vs untreated cells, ^#p < 0.05 vs untreated siCtrl cells. (D) A549 and H460 cells were transfected with siRNA against Akt (siAkt) or si-mismatch control siRNA (siCtrl). After transfection for 18 h, cells were treated with 0–50 µM ETD for 48 h. MMP-2 and MMP-9 mRNA expressions were quantified by quantitative RT-PCR. The data are presented as the mean ± SEM (n = 3). *p < 0.05 vs untreated cells, ^#p < 0.05 vs untreated siCtrl cells.

Figure 5

ETD directly binds to Akt. (A) Molecular docking analysis of ETD and Akt was performed by AutoDock4.2 and PyMOL program. (B) A schematic diagram, created by BIOVIA Discovery Studio Visualizer 2017, of the interacting residues between ETD and Akt is shown. Hydrogen bonds are displayed in green. The van der Waals interactions are shown in light green. The anion-π and sulfur-π interactions are shown in orange and yellow, respectively.

Figure 6

ETD attenuates an in vivo lung cancer metastasis. (A) Scheme for an in vivo lung cancer metastasis experiment. A549 cells expressing luciferase were treated with 50 µM of ETD for 24 h and injected into the tail vein of mice. (B) After 3 d of injection, the lung tissues were collected, and metastatic cancer cells were detected by IVIS imaging system. (C) Quantified values of bioluminescence intensity in ETD and control group are shown as a total flux (p/s). The data are presented as mean ± SEM (n = 6). *p < 0.05 vs untreated control group.

Figure 7

A Scheme diagram of this study. ETD suppresses lung cancer cell migration and invasion through inhibition of Akt/mTOR/p-p70^S6K signaling pathway. This diagram was created with “Biorender.com”.