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

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 (p70S6K) 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/p70S6K-induced actin reorganization and MMPs expression.

www.nature.com/scientificreports/ effect. Therefore, concentrations of ETD lower than 50 µM were employed in subsequent experiments to eliminate interference from the cytotoxic and proliferative effects of the compound on cell migration and invasion.

ETD inhibits cancer metastatic behaviors in non-small-cell lung cancer cells.
Since-cell migration and invasion are prerequisite steps in the metastasis process; to evaluate whether ETD can suppress metastatic cells, a transwell migration assay was performed. The results showed that ETD significantly reduced the number of migrated cells in a dose-dependent manner ( Fig. 2A). Consistently, a wound healing assay was also conducted to evaluate the effect of ETD on collective cell migration in both cell types. As seen in Fig. 2B, migration was clearly decreased in the ETD-treated group, particularly with 50 µM ETD, compared with the control.
To evaluate the anti-invasive effects of ETD, a transwell invasion assay was performed. The results demonstrated that 25 and 50 µM ETD extensively inhibited lung cancer cell invasion, with an inhibition rate of approximately 40-80% (Fig. 2C). Since cancer cells acquire survival mechanisms to overcome cell detachment-induced apoptosis during systemic circulation 18 , colony formation assays in soft agar were performed to explore the effect of ETD on anchorage-independent growth. The results showed that the colonies formed on soft agar were markedly smaller for cells treated with ETD than control cells, indicating that this compound was able to suppress anchorage-independent growth (Fig. 2D). These data suggest that ETD is an effective compound suppressing the metastatic behaviors of lung cancer cells.

ETD suppresses cell migration and invasion via actin stress fiber reorganization and MMP inhibition.
Actin stress fibers, contractile actin bundles, are required for cell motility during metastasis 19 . Our results demonstrated that actin stress fibers were clearly present in nontreated cells, but they were markedly reduced after treatment with 50 µM ETD (Fig. 3A,B). We also investigated whether ETD-attenuated cell motility is involved in the alteration of lamellipodia formation. Immunofluorescence assay revealed that ETD effectively disrupted lamellipodial assembly (Fig. 3A). Quantitative analysis of lamellipodia demonstrated that the areas of lamellipodia formation in A549 and H460 cells were notably decreased in the presence of ETD (Fig. 3C). Since the Rac1 protein plays an important role in actin reorganization 20 , the active state of GTP-Rac1 was then examined. Western blot analysis showed that ETD strongly suppressed Rac1 activity ( Supplementary Fig. S1), suggesting that ETD inhibited these actin dynamics in a Rac1-dependent manner. Furthermore, the mRNA levels of MMP-2 and MMP-9, extracellular matrix-degrading enzymes required for the cancer invasive process, were significantly downregulated by ETD in a dose-dependent manner (Fig. 3D). These data suggested that ETD suppresses lung cancer cell migration and invasion via alteration of actin organization and reduction of MMPs expression.
ETD attenuates Akt/mTOR/p70 S6K -mediated actin reorganization. The Akt signaling pathway has been reported to control necessary cancer behaviors during metastasis, including cell motility and invasion 11 . To explore whether the Akt pathway is involved in ETD-mediated suppression of cell migration and invasion, Western blot analysis was performed. As seen in Fig. 4A, the level of Ser473-phosphorylated Akt (p-Akt), an active state of Akt, was gradually decreased in A549 and H460 cells treated with ETD, whereas total Akt was unchanged. Western blot analysis also revealed that ETD was able to downregulate the expression levels of p-mTOR (Ser2448) and p-p70 S6K (Thr389), members of a downstream signaling axis of Akt that regulates cell motility and invasion 21,22 , in A549 and H460 cells, suggesting that Akt/mTOR/p70 S6K signaling is a possible target by which ETD inhibits non-small-cell lung cancer cell migration and invasion.
To confirm whether the Akt pathway and its downstream effectors are required for the inhibitory effects of ETD on cell migration and invasion, we knocked down Akt in A549 and H460 cells by specific small interfering RNA (siAkt). The results demonstrated that the level of Akt, p-Akt and its downstream kinase (p-p70 S6K ) and p-mTOR were declined in response to siAkt transfection (Fig. 4B). The migrating cells and the mRNA levels of MMP-2 and MMP-9 were reduced accordingly ( Fig. 4C,D). In addition, the reduction in p-Akt, p-p70 S6K and p-mTOR became more obvious after treatment with ETD in Akt knockdown cells. Together with, the suppression of cell migration and MMPs expression were potentiated by ETD treatment (Fig. 4C,D). These data supported our hypothesis that the Akt/mTOR/p70 S6K pathway participates in the ETD-induced attenuation of cell migration and invasion.
Epithelial-to-mesenchymal transition (EMT) has been reported to potentiate cancer cell movement 23 , and we tested whether ETD impedes the EMT process. Western blot analysis demonstrated that mesenchymal markers, including Snail, Slug and N-cadherin, were not significantly altered ( Supplementary Fig. S2A). However, transforming growth factor β (TGF-β)-induced cell migration and anchorage-independent growth was extensively attenuated by ETD ( Supplementary Fig. S2B-D). These data suggest that this inhibitory effect of ETD might function, in part, through suppression of Akt activity, since Akt participates in the noncanonical TGF-β pathway 24 .
ETD directly binds to Akt via the protein kinase domain. We further investigated whether Akt activity might be a result of direct interaction between ETD and Akt. A molecular docking study demonstrated that ETD binds to the ATP binding site in the protein kinase domain of Akt (Fig. 5A). The key interactions stabilizing the complex are hydrogen bonding and van der Waals interactions (Fig. 5B). The methoxy and phenol groups of ETD can form hydrogen bonds with the amide backbone of Ala230 (1.82 Å), which is a backbone amide in the kinase hinge, and the carboxylic acid side chain of Asp292 (1.93 Å), respectively. Other amino acids forming van der Waals interactions are Leu156, Val164, Ala177, Lys179, Thr211, Met227, Glu228, Tyr229, Met281, Thr291, and Phe438 (Supplementary Table S3). The methoxy group is oriented towards the gatekeeper Met227. Based on these interactions, the free binding energy between ETD and Akt is − 8.85 kcal/mol, and the ligand efficiency www.nature.com/scientificreports/ (LE) value of ETD is − 0.44 kcal/mol per heavy atom. These data indicate that ETD has a potential interaction with Akt and may interfere with Akt phosphorylation.

ETD inhibits in vivo lung cancer metastasis.
To confirm the role of ETD in lung tumor metastasis, A549-luciferase cells pretreated with 50 µM ETD were injected into the tail veins of mice. Three days after cell inoculation, the extent of lung metastasis was quantified by bioluminescence imaging (Fig. 6A). Pretreatment of cells with ETD demonstrated a significant reduction in metastatic foci (Fig. 6B). Quantitative analysis also revealed that compared with the control group, the group pretreated with ETD had remarkably suppressed lung cell metastasis (Fig. 6C), supporting the potent antimetastatic activity of ETD in lung cancer cells.

Discussion
Lung cancer is one of the most serious malignancies worldwide due to its rapid metastasis 1 . Cell migration and invasion are recognized as critical steps in cancer metastasis, and the inhibition of these aggressive behaviors is of interest as a promising therapeutic approach. Previous studies have reported that phenolic compounds from Dendrobium spp. of Thai orchids exhibit antimetastatic activity via different molecular mechanisms 14,25,26 . In the present study, we first demonstrated the potent effect of ETD, a phenanthrene derivative isolated from Thai orchids, on suppressing lung cancer metastasis in both in vivo and in vitro studies. Furthermore, the underlying mechanism involved with the regulation of actin cytoskeleton rearrangement and MMP expression via the Akt/ mTOR/p70 S6K signaling pathway. The migration and invasion of cancer cells are hallmarks of malignancy, enabling cancer cell dissemination to distant organs 3 . It has been reported that reorganization of actin filaments is required for cancer cell migration and invasion 5,19 . Dynamic changes in the actin cytoskeleton promote the formation of discrete structures in cancer cells, including lamellipodia and stress fibers, which are essential for directional movement 19,27 . Several studies have demonstrated that disruption of actin structures is able to attenuate migration and invasion abilities in various cancer cell lines [28][29][30] , which is in agreement with our finding that the formation of stress fibers and lamellipodia was obviously disrupted in ETD-treated lung cancer cells and consequently resulted in decreased cell motility and invasion. Accumulating studies have revealed that Rac1, a member of the Rho family of small GTPases, participates in the organization of actin filaments and remodeling of the plasma membrane 20 . The GTP binding protein Rac1, in its active form, activates the Arp2/3 complex by binding with the SCAR/WAVE regulatory complex, which promotes the elongation of actin at the leading edge of motile cells 31 . Rac1 also functions as a direct regulator of actin stress fiber formation 32 . Overactivation of Rac1 has been found in various human cancers, including non-small-cell lung cancer 33 . The downregulation of Rac1 was shown to reduce the number of stress fibers 34 and attenuate cancer cell migration and metastasis 35 . In agreement with our findings, the disruption of actin-based structures, including stress fibers and lamellipodia, is known to be related to a decrease in the active form of Rac1 in response to ETD treatment.
It is well known that PI3K/Akt signaling plays a dominant role in governing cancer cell migration and invasion. The activation of Akt participates in the reorganization of the actin cytoskeleton and mediates contraction of the cellular body through several downstream signaling pathways 36 . mTOR1, a downstream serine threonine kinase effector, was actively phosphorylated at Ser2448 by PI3K/Akt 37 . Loss of mTORC1 activity as a consequence of Akt inhibition contributed to a disruption of F-actin organization, including in lamellipodia and filopodia formation, at the leading edge of cancer cells 38 . In addition, p70 S6K is reported to be a downstream target of the PI3K/Akt/mTORC1 axis 39 . p70 S6K phosphorylated at Thr389 potently induces Rac1-mediated lamellipodia formation 9,36,40 . Inhibition of Akt/mTORC1/p70 S6K signaling resulted in an alteration of actin reorganization in favor of impeding cell motility 38 , suggesting an intriguing approach for attenuating cancer metastasis. Our findings also demonstrate that ETD significantly decreased Akt phosphorylation and activation of its downstream molecules mTOR and p70 S6K , leading to the suppression of lung cancer cell migration. Furthermore, several studies have documented that activation of the PI3K/Akt/mTOR/p70 S6K signaling pathway triggers the expression of proteolytic enzymes facilitating cancer invasion, including MMP-2 and MMP-9 10,41 , and in particular, p70 S6K is an important transcription factor responsible for MMP-9 synthesis 41 . Based on this evidence and our finding, the reduction in MMP-2 and MMP-9 expressions induced by ETD in lung cancer cells is a consequence of inactivation of Akt and its downstream effectors. 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 54 . Each dot plot represents a single colony. All data are presented as the mean ± SEM (n = 3). *p < 0.05 vs untreated control group.  www.nature.com/scientificreports/ By considering to the molecular structure of ETD, we further revealed how ETD has an inhibitory effect on Akt and whether there is an interaction among them. Akt consists of pleckstrin homology (PH), catalytic kinase, and regulatory domains, and its activity is regulated by phosphorylation and dephosphorylation processes in an Akt conformation-dependent manner. A recent study indicated that the quinone analog phenanthrene acts as a potent Akt inhibitor via direct interaction with Cys296 and Cys310 in a catalytic domain and induces Akt dephosphorylation 42 . Our study also demonstrated that ETD binds to the catalytic domain but at different sites. However, the interacting residues overlapped with those of the Akt inhibitors CID-20759629 43 and A-674563 44 (Supplementary Table S3), and the LE values were comparable to that in our study.
EMT is one of the crucial processes driving cancer metastasis. It involves genotypic and phenotypic changes of cells from an epithelial-like morphology to cells with loose cell-cell adhesion and a mesenchymal-like morphology 45 . Epithelial cells undergoing EMT decrease the expression of cell adhesion molecules, elevate the expression of mesenchymal markers and rearrange their cytoskeletons 45,46 . TGF-β, a multifunctional cytokine involved in many tumor cell functions, is a key modulator of the EMT mechanism 47 . The binding of TGF-β to its receptor initiates SMAD phosphorylation and activates downstream cascades in the canonical pathway 48 . TGF-β mediates EMT-associated transcription factors (TFs), including those of the Snail and the Slug families, and repressors of the E-cadherin promotor through SMAD signaling, which suppresses the expression of cell adhesion molecules 49 . In this study, we found that ETD was able to suppress TGF-β-induced metastatic phenotypes in A549 cells; however, ETD had no effect on Snail and Slug, a direct transcriptional repressor of E-cadherin, and N-cadherin expression (Supplementary Fig. S2). Since TGF-β-mediated EMT occurs through canonical and noncanonical pathways 24,50 , these data suggest that ETD diminishes TGF-β-induced metastatic phenotypes independent of canonical mechanisms. In addition, PI3K/Akt and the Rho GTPase family were reported to participate in a noncanonical pathway contributing to TGF-β-induced EMT 24,51 , suggesting that the inhibitory effect of ETD on TGF-β-enhanced cell migration is caused by ETD-induced suppression of Akt signaling and Rac1.
In conclusion, this study demonstrated that ETD attenuates lung cancer cell metastasis in an in vivo and an in vitro studies. ETD exhibits an inhibitory effect on lung cancer cell migration and invasion via inhibition of Akt/mTOR signaling, and thereby modulates actin reorganization and downregulates MMP expressions (Fig. 7). This study suggests that the novel pharmacological activity of ETD warrants further research and development of this compound for ultimate use against non-small-cell lung cancer metastasis.

Materials and methods
Cell culture. Human non-small-cell lung A549 and H460 cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). A549 and H460 cells were grown in DMEM and RPMI, respectively. Both of medium were supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin-streptomycin and 2 mM L-glutamine. Cells were maintained in a 37 °C humidified incubator with 5% CO 2 . All media and supplements were purchased from GIBCO (Grand Island, NY, USA). Erianthridin preparation. ETD (Fig. 1A) was extracted from the whole plant of Dendrobium formosum Roxb. ex Lindl. as previously reported 52 . ETD was dissolved in dimethyl sulfoxide (DMSO) to make stock solution. The desired concentrations of ETD used were prepared by dilution with culture media. The final concentration of DMSO in all experiments was less than 0.1% which shows no observable toxic effect to cells.
Cell viability assay. A549 and H460 cells (10 4 cells/well) were seeded onto 96-well plates and incubated at 37 °C with 5% CO 2 overnight. Then, cells were treated with various concentrations of ETD (0-500 µM) for 24, 48 and 72 h. Cell viability was examined by MTT assay as previously described 14 . The mean optical density in the indicated group was used to calculate the percentage of cell viability.
Cell proliferation assay. A549 and H460 cells (2 × 10 3 cells/well) were seeded onto 96-well plates and treated with non-toxic concentrations of ETD (0-50 µM) for 24, 48 and 72 h. At the end of incubation period, 10 μL of MTT solution (5 mg/mL) was added and incubated at 37 °C for 4 h. The medium was removed and replaced with 100 μL of DMSO to dissolve formazan crystals after incubation. The intensity of solution was measured at 570 nm by microplate reader. The absorbance in indicated group was calculated and represented as relative cell growth compared to control group.
Wound healing assay. A549 and H460 cells (2 × 10 4 cells/well) were seeded onto 96-well and incubated overnight. Cell migration was examined by Wound scratching assay as described 53 . The wound spaces were photographed under a phase contrast microscopy. The space area was quantified using ImageJ software (NIH) 54 and represented as relative cell migration to the control group.
Transwell migration and invasion assay. Transwell migration and invasion were determined using transwell chambers with and without Matrigel-coating. A549 and H460 cells (5 × 10 4 cells/well) were seeded onto the upper chamber of 24-well transwell plates containing serum free media, and 600 μL media containing 10% FBS, a chemo-attractant, was added in the lower chamber. Cells were incubated for 18-20 h to allow cells movement into the underneath of membrane. Cells at the upper chamber were removed using cotton-swab, and www.nature.com/scientificreports/ migrating or invading cells at the lower surface of membrane were fixed with methanol and stained with DAPI. Cells from at least five random fields were imaged using fluorescence microscope (Nikon Inverted Microscope Eclipse Ti-U Ti-U/B, NY, USA) and presented as a relative value to the number of migrating or invading cells in the control group.
Anchorage-independent growth assay. Anchorage-dependent growth was performed by maintaining the cells in soft agar as previously described 14 . Briefly, cells were treated with various non-toxic concentrations of ETD in 500 μL complete media by addition onto the upper layer of soft agar. At the end of incubation time, the colonies were stained with 0.01% crystal violet for 30 min at room temperature and washed with deionized water. All colonies per well were imaged under a phase contrast microscope. The size (μm) of the colonies was measured by ImageJ software (NIH) 54 .
Immunofluorescence assay. A549 and H460 cells were plated at a density of 2 × 10 3 cells onto the coverslip, and treated with non-toxic doses of ETD for 48 h. Immunostaining for actin was performed as described 14 .
Cells were imaged using a fluorescence microscope (model IX81, Olympus, Japan). The number of actin stress fibers per cell and the extension of lamellipodia were analyzed by ImageJ software (NIH) 54 in comparison to control group.
Quantitative real-time PCR (qRT-PCR). A549 and H460 cells were treated with non-toxic doses of ETD for 48 h. Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Valencia, CA, USA) following the manufacture's instruction. The qualitative real-time PCR was performed for MMP-2 and MMP-9 expressions using One step TB Green PrimeScript PLUS RT-PCR Kit (Takara, Japan). The primers used were listed in Supplementary Table S2. The expression levels of the target genes were calculated using (2 −ΔΔCt ) method.
Western blot analysis. A549 and H460 cells were treated with non-toxic doses of ETD and incubated for 24 h. At the end of incubation, cells were lysed in TMEM lysis buffer as described 53 . An equal protein content was dissolved by SDS-polyacrylamide gels and electrotransferred onto polyvinyl difluoride (PVDF) membranes. The membranes were blocked, incubated with specific primary and secondary antibodies as described in Supplementary Table S1. The blots were visualized by enhanced chemiluminescence system using Immobilon Western chemiluminescent HRP substrate (Millipore, MA, USA). GAPDH was used as a loading control. Quantification of the band intensity of protein expression was performed using ImageJ software (NIH) 54 .
In vivo tail vein metastasis assay. Five to six-week old CB17-Prkdc scid mice were obtained from the CLEA Japan, Inc. (Tokyo, Japan) and maintained under specific pathogen-free conditions throughout the study. The mice were randomly separated into six mice in each group. A number of 10 6 A549-luc cells, that were incubated with or without 50 μM ETD for 24 h, were injected into tail vein of mice. The mice were sacrificed after injection 3 d, and lung metastasis was evaluated by IVIS Lumina II System (Caliper Life Science, MA, USA).  Statistical analysis. Data are presented as mean ± S.E.M at least three-independent experiments, and all data were analyzed using Prism 8 (GraphPad Software, Inc., San Diego, CA, USA). The student's t-test was used to analyze statistical differences between two groups. The One-way ANOVA with Tukey's Multiple Comparison Test was applied for determination the statistical significance between control and treatment groups. P-values less than 0.05 were considered statistically significance.
Ethic statement. All protocols were performed in accordance with relevant guideline. Animal experiments were approved by the Animal Experiment Ethics Committee of the University of Toyama (A2019INM-5), and were carried in accordance with the ARRIVE guidelines.