Emetine dihydrochloride inhibits invasiveness and motility of hepatocellular carcinoma cells by blocking the MAPK pathway and inducing destabilization of Twist1

Emetine dihydrochloride inhibits invasiveness and motility of hepatocellular carcinoma cells by blocking the MAPK pathway and inducing destabilization of Twist1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Emetine dihydrochloride inhibits invasiveness and motility of hepatocellular carcinoma cells by blocking the MAPK pathway and inducing destabilization of Twist1 Haelim Yoon, Sayeon Cho This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4805487/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Hepatocellular carcinoma (HCC) is a malignant tumor that causes both extrahepatic and intrahepatic metastases. Epithelial to mesenchymal transition (EMT) is a crucial step in the development and metastasis of cancer. Emetine dihydrochloride (EDH) has been previously used as an anti-emetic and is now proposed as a replication inhibitor of SARS-CoV-2, but its effect against cancer metastasis has not been evaluated. Therefore, this study sought to investigate the regulatory mechanisms of cell migration from an EMT perspective using EDH in HCC cell lines. Methods HCC cell lines (Huh7, Hep3B, SNU449, SNU886, and PLC-PRF-5) were used to measure cell viability against EDH. The effect of EDH on migration was verified by wound healing analysis and migration analysis using Transwell. The effect of EDH on invasion was determined using an invasion assay in Matrigel-coated Transwell chambers. Spheroid invasion and soft agar colony formation assays were performed to verify the effect of EDH on anchorage-independent growth. Gelatin zymography was used to determine the activities of matrix metalloproteinase − 2 and − 9. The protein expression levels of Twist1, downstream target genes, and the mitogen-activated protein kinases (MAPKs) and AKT signaling pathways were determined through immunoblotting. The RNA expression levels of each gene were analyzed through RT-PCR and quantitative RT-PCR. Results EDH inhibited the motility of various HCC cell lines at non-toxic concentrations. The inhibitory effect of EDH on cancer cell motility resulted from a decrease in protein levels of Twist1, a key transcription factor involved in EMT. Depletion of Twist1 due to EDH treatment suppressed the expression of mesenchymal markers (N-cadherin and Vimentin) while increasing the expression of epithelial markers (E-cadherin). The regulatory pathway for the destabilization of Twist1 by EDH was mediated through the inactivation of the MAPK pathway. EDH specifically inactivated JNK and p38, thereby destabilizing the Twist1 protein, which is dependent on the S68 phosphorylation of Twist1. Conclusion EDH induces MAPK inactivation, which decreases Twist1 protein levels and ultimately suppresses mesenchymal properties. These results provide the first report on EDH from an EMT perspective and suggest its potential as an anticancer agent for HCC. Emetine anti-migration anti-invasion cancer cell motility EMT Twist1 HCC mesenchymal markers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Liver cancer is the third leading cause of cancer mortality worldwide [ 1 ]. Although surgical resection is the primary and most effective treatment for liver cancer patients, the prognosis for this disease remains poor due to its high 5-year recurrence rate [ 2 ]. Sorafenib, approved by the Food and Drug Administration in 2007, is currently the most widely used treatment for advanced hepatocellular carcinoma (HCC) [ 3 ]. However, side effects and limitations such as drug resistance [ 4 ] and toxicity [ 5 ] have been reported. Therefore, several studies have sought to enhance the anticancer activity of sorafenib by combining it with other chemical such as doxorubicin [ 6 ] and celecoxib [ 7 , 8 ]. Most cancer-related deaths are caused by metastasis rather than the primary cancer [ 9 ]. Metastasis begins when cancer cells move to other organs through the circulatory system, with epithelial-mesenchymal transition (EMT) being a crucial process [ 10 ]. During EMT, tumor cells lose epithelial properties such as cell polarity and cell-cell adhesion and acquire mesenchymal properties such as migration, invasion, and anti-apoptosis [ 10 – 12 ]. Key EMT-inducing transcription factors (EMT-TFs) such as Twist1/2, Snail1/2, and ZEB1/2 are intricately regulated by a network of signaling pathways, including TGF-β, Wnt-β-catenin, and Notch [ 12 ]. The most studied intracellular signaling pathways involved in EMT-TF expression are signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa B (NF-κB) [ 12 ]. AKT and mitogen-activated protein kinases (MAPKs) (extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38) pathways are known to regulate the activity of EMT-TFs [ 13 ]. The basic helix-loop-helix transcription factor Twist1 is a key regulator of EMT and highly expressed in various malignancies [ 14 ]. In various cancer types, Twist1 has been reported to contribute to cancer invasion and metastasis, increasing intravascular migration and extravasation through blood vessel walls [ 14 ]. Twist1 has been proven to upregulate N-cadherin, a mesenchymal marker, and downregulate E-cadherin, an epithelial marker, at the mRNA level through the E-box element located in the promoter of the target gene [ 14 ]. Additionally, Twist1 is associated with HCC metastasis, stimulating invasiveness through the expression of matrix metalloproteinase (MMP)-2 and − 9 [ 15 ]. Therefore, understanding the regulatory mechanism of Twist1 in the EMT process would provide valuable insights into the broader context of EMT and its impact on cancer metastasis. Emetine, an active ingredient isolated from ipecac species, is a protein synthesis inhibitor known to inhibit both intracellular ribosomal and mitochondrial protein synthesis and interfere with DNA and RNA synthesis [ 16 ]. Emetine has been traditionally used as an oral emetic and expectorant and is currently used as an antiprotozoal agent [ 17 ]. Interestingly, emetine has recently been studied as an antiviral agent, considered a potential drug with anti-coronavirus effects by inhibiting the replication of SARS-CoV-2 [ 17 – 19 ]. Emetine is formulated as a hydrobromide and hydrochloride salt when used as a drug, which increases the solubility of insoluble amines and facilitates absorption from the gastrointestinal tract [ 18 ]. In the 1970s, emetine dihydrochloride was used in clinical trials at the National Cancer Institute for its antitumor activity but was no longer developed due to severe toxicity [ 20 ]. Despite this, the apoptotic effect of emetine has been reported in various human cancer cells [ 21 , 22 ]. A recent study reported that emetine induces apoptosis not only by inhibiting of protein synthesis but also by regulating anti-apoptotic genes such as Bcl-x [ 21 ]. This apoptosis-inducing effect makes emetine a potential anticancer drug candidate for combination therapy, with some observed effects in lung cancer patients [ 23 ]. While emetine has been studied primarily for its apoptosis-inducing effects in various human cancer cells, this study aims to explore new functions of emetine by investigating its potential to inhibit cancer metastasis. In this report, we verified the effect of EDH on cell migration and invasion in HCC cell lines and investigated the mechanisms regulating cell motility from an EMT perspective. Methods Reagents and antibodies Emetine dihydrochloride (cat. No. 324693) used in the experiment was purchased as a powder from EMD Millipore Crop (Billerica, MA, USA) and dissolved in pure water. JNK inhibitor VIII (JNK inhibitor, HY-107598) and SB203580 (p38 inhibitor, HY-10256) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). MG132 (474790), Sorafenib (SML2653), and FLAG antibodies (F3165) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Twist1 (sc-81417), JNK (sc-7345), p38 (sc-7972), Akt1/2/3 (sc-81434), Vimentin (sc-32322), and β-actin (sc-47778) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). ERK1/2 (#9102), p-ERK1/2 (#9106), p-p38 (Thr180/Tyr182, #9211), p-JNK (Thr183/Tyr185, #9251), p-AKT (Ser473, #4060), p-NF-κB (Ser536, #3033), and p-STAT3 (Tyr705, #9145) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). E-cadherin (610182) and N-cadherin (610720) antibodies were obtained from BD Biosciences (San Jose, CA, USA). Polyclonal anti-mouse IgG Fc-tagged antibodies (LF-SA8001) and polyclonal anti-rabbit IgG-HRP (LF-SA8002) were purchased from AbFrontier (Young In Frontier Co., Ltd., Seoul, Korea). Cell culture Huh7 and Hep3B cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium or Dulbecco’s modified Eagle’s medium (DMEM) (WELGENE, Seoul, Korea) containing 1% penicillin-streptomycin (GIBCO, Grand Island, NY, USA) and 10% fetal bovine serum (FBS; WELGENE, Seoul, Korea). All cells were incubated at 37°C in a 5% CO 2 incubator. Cell viability assay Huh7 cells (4 × 10 4 cells/well) were seeded in 96-well plates and incubated overnight in a 5% CO 2 humidified air atmosphere. The cells were then treated with a mixture of EDH (50, 100, 200, and 400 nM) in a medium containing either 1% or 10% FBS. Cell viability was tested 24 and 48 hours after exposure to EDH using EZ-Cytox (DOGEN, Seoul, Korea) following the manufacturer’s instructions. Wound healing assay The day after seeding the Huh7 cells into 24-well plates (at 90% confluency), wounds were created using a pipette white tip. The culture medium was then aspirated and replaced with fresh medium containing 1% FBS and various concentrations of emetine dihydrochloride (50, 100, and 200 nM). The scratched cells were incubated for 24 and 48 hours, and cells that migrated to the wound surface were observed using a JuLI-Stage Real-Time Cell History Recorder (NanoEnTek Inc., Seoul, Korea). The change in wound closure is expressed as a percentage of wound healing in the treated groups compared to the control group. Each experiment was repeated three times, and the results were averaged. Transwell migration and invasion assay Cell migration was analyzed using a 24-Transwell plate (pore size; 8 µm, SPL, Korea) containing a polycarbonate membrane. Huh7 cells and Hep3B cells (5 × 10 4 cells/well) were seeded in the upper chamber. The cells were then treated with 250 µl of medium containing 1% FBS, with or without EDH. The lower chamber was filled with 500 µl of medium containing 10% FBS. Huh7 cells were incubated for 6 hours and Hep3B cells for 12 hours to allow migration through the membrane. The migrated cells on the membrane were then washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and stained with 0.5% crystal violet. Cells attached to the upper surface of the membrane were removed using cotton wool. The migrated cells were visualized by photographing the membrane under a microscope at 100× magnification. Three fields were photographed, and the most representative image was selected for the figure. To perform the Matrigel invasion assay, 30 µl of diluted Matrigel (0.5 mg/ml; BD Biosciences, Franklin Lakes, NJ, USA) was coated in the upper chamber for 3 hours. This procedure was similar to the migration assay, except that the Huh7 cells were incubated for 18 hours and the Hep3B cells were incubated for 24 hours. Analysis of soft agar colony formation The bottom of a 12-well plate was coated with 2× cell culture medium containing 0.5% agarose (Sigma-Aldrich, St. Louis, MO, USA). Huh7 cells (4 × 10 3 cells/well) were added to the coated upper layer by mixing with cell culture medium containing 0.3% agarose. Both the top and bottom layers were incubated at 4°C for 30 minutes each to make them semi-solid. The uppermost layer was then supplemented with cell culture medium with or without EDH. Fresh medium was replaced every 3 days, and the cells were incubated for 14 days at 37°C in a 5% CO 2 incubator. Colonies were stained with 0.05% crystal violet solution for 10 minutes at room temperature and decolorized using PBS. Each stained well was photographed using a digital camera. Gelatin zymography Huh7 cells (2 × 10 6 cells/well) were inoculated into a 12-well culture plate and cultured for one day. After washing with PBS, EDH was mixed in serum-free medium at each concentration (50, 100, and 200 nM), and 500 µl of each mixture was added to each well. After culturing for 24 hours, the cultured medium was collected and mixed with sample buffer [2.5 mM Tris-Cl (pH 6.8), 5% sodium dodecyl sulfate (SDS), 50% glycerol, 0.05% bromophenol blue]. The samples were then subjected to electrophoresis on 10% SDS-polyacrylamide gel containing 0.1% gelatin (G1393, Sigma-Aldrich, St. Louis, MO, USA). The separated gel was washed with a wash buffer [50 mM Tris-HCl (pH 7.5), 0.2 M NaCl, 5 mM CaCl 2 , 0.1 µM ZnCl and 2.5% Triton X-100] at room temperature for 30 minutes. Substrate buffer (0.016% NaN 3 and 1% Triton X-100) was added to the wash buffer, and the gel was incubated on a shaker at 37°C for 48 hours. After fixing for 30 minutes with fixing solution (40% methanol and 10% acetic acid), the gel was stained with 0.25% Coomassie Brilliant Blue for 1 hour. For decolorization, a decolorization solution (5% methanol and 8% acetic acid) was used. MMP activity assay Huh7 cells (2 × 10 6 cells/well) were seeded into 12-well culture plates and incubated for 24 hours in serum-free medium with or without EDH. MMP activity was analyzed using the collected medium with the Gelatinase/Collagenase Assay Kit (E12055, Thermo Fisher Scientific, USA). Experiments were performed according to the manufacturer’s instructions. Three-dimensional (3D) Spheroid Invasion analysis Huh7 cells (2 × 10 3 cells/well) were inoculated into a low-attachment 96-well plate (34896, SPL, Korea) and cultured for 72 hours. After removing the medium, 3 mg/ml of Matrigel (356230, BD Biosciences, USA) was added, and the cells were further cultured for 24 hours. The resulting spheroids were then treated with EDH in 10% FBS medium. Spheroids were recorded at 24-hour intervals using a JuLI-Stage Real-Time Cell History Recorder (NanoEnTek Inc., Seoul, Korea). Immunoblotting analysis After Huh7 cells were treated with EDH, cells were lysed in a lysis buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% Triton X-100, 0.5% IGEPAL CA-630, 1 mM EDTA, 1% glycerol; with 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, and 1 mM Na 3 VO 4 ]. EDH-treated and untreated protein samples were mixed with equal volumes of 5× SDS sample buffer [12 mM Tris-HCl (pH 6.8), 5% glycerol, 0.4% SDS, 0.02% bromophenol blue, and 1% β-mercaptoethanol]. After mixing, the samples were boiled at 100°C for 5 minutes. The samples were then electrophoresed on 12% SDS-PAGE and transferred to a nitrocellulose membrane. The protein-containing membranes were blocked with 5% nonfat dry skim milk and then incubated with specific primary antibodies (all antibodies diluted at 1:1000) in 3.3% BSA overnight at 4°C. The membranes were washed several times with 1× Tris-buffered saline with Tween 20 and then incubated with a secondary antibody for 2 hours at room temperature. Protein bands were detected using an enhanced chemiluminescent immunoblotting detection reagent (Pierce; Thermo Fisher Scientific, Inc.). All experiments were performed at least in triplicate, and the band intensity of each protein was analyzed with ImageJ. Reverse transcription-polymerase chain reaction (RT-PCR) analysis and quantitative RT-PCR (qRT-PCR) analysis Huh7 cells were inoculated in a 12-well plate and treated with EDH for 24 hours to extract total RNA (Lbozol, COSMO Genetech, Seoul, Korea). Using the extracted RNA (1 µg), complementary DNA (cDNA) was synthesized with the TOPscript cDNA synthesis kit (Enzynomics, Daejeon, Korea). RT-PCR and qRT-PCR were performed using the synthesized cDNA. Fluorescence values of PCR amplicons were obtained by performing qRT-PCR with a CFXConnect real-time thermocycler (Bio-Rad, Hercules, CA, USA). Gene expression levels were normalized to the Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) gene. According to the 2 −ΔΔCq method, gene expression levels were expressed compared to the control group. The RT-PCR and qRT-PCR primers are listed in Supplement table 1. Transfection Huh7 cells were inoculated to 70% confluency in a 6-well plate and incubated overnight. For transfection, the plasmid and linear polyethyleneimine (PolySciences, Warrington, USA) were mixed at a 1:3 ratio and left at room temperature for 30 min. Then, the mixture was added to each well and further cultured for 48 hours. Statistical analysis Data were analyzed using the Prism 3.0 statistical analysis program (GraphPad Software, San Diego, CA, USA). All data represent the mean value obtained from three or more independent experiments, and the error is presented as the mean ± standard deviation (SD). Post hoc analysis was conducted using Holm-Šídák’s method, and statistical analysis was performed using one-way ANOVA. Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. Non-significant differences are marked with "N.S." in the graph. Results EDH regulates the motility and invasiveness of HCC cell lines Using HCC Huh7 cells, wound healing assays were conducted as an initial screen of the drug reposition library provided by the Korea Chemical Bank. EDH (Fig. 1A) was identified as a potential inhibitor of Huh7 cell migration. Given that cancer cells possess high mobility and invasiveness leading to cancer metastasis, several HCC cell lines (Huh7, Hep3B, SNU449, SNU886, and PLC-PRF-5) were treated with EDH to evaluate its wound healing effects. When HCC cells were treated with different concentrations of EDH, wound closure was inhibited in a dose-dependent manner, compared to untreated cells (Fig. 1B and Supplement Fig. 1A). In addition, cell migration and invasion were evaluated using Transwell assays. Figure 1C shows that cell migration decreased in Huh7 and Hep3B cells treated with EDH in a concentration-dependent manner. Furthermore, cell invasion assays using Transwells with Matrigel-coated insert membranes indicated that EDH inhibited invasiveness in HCC cell lines (Fig. 1D and Supplement Fig. 1B). To confirm that the inhibitory effect of EDH on cancer cell migration in HCC cell lines was not due to cell death, the cytotoxicity of EDH on Huh7 and Hep3B cell lines was evaluated both under cell growth conditions (10% FBS in the media) and cell growth inhibition conditions (1% FBS in the media) (Supplement Fig. 2A). EDH did not cause cytotoxicity in HCC cells up to a concentration of 200 nM. Furthermore, when other HCC cell lines (SNU449, SNU886, and PLC-PRF-5) were treated with EDH, at least 80% of the cells survived at 200 nM EDH (Supplement Fig. 2B). Therefore, in subsequent experiments, HCC cell lines were treated with EDH concentrations of 200 nM or less to investigate the mechanisms by which EDH regulates cancer cell migration and invasion. Normal cells require extracellular matrix contact to grow and divide [24]. In contrast, metastatic cancer cells can grow in an anchorage-independent manner in soft agar [24]. Soft agar colony formation assays were performed to determine whether EDH inhibits the anchorage-independent colony formation of cancer cells. When Huh7 cells were treated with EDH, colony formation was inhibited in a dose-dependent manner compared to the negative control group (Fig. 1E). Additionally, the invasiveness of multicellular tumor spheroids was evaluated through a 3D spheroid invasion assay mimicking the conditions occurring during cancer metastasis in vivo [25]. When spheroids derived from Huh7 cells were exposed to EDH for a duration of up to 96 hours, a notable suppression in invasiveness was observed, even at a concentration of 50 nM EDH (Fig. 1F). Furthermore, when Huh7 cells were cultured under conventional two-dimensional cell culture conditions, EDH treatment markedly inhibited cell proliferation, which was particularly noticeable at concentrations above 100 nM (Supplement Fig. 3). These findings suggest that EDH is a promising suppressor of the anchorage-independent growth and invasion of Huh7 cells. EDH downregulates MMP activity in Huh7 cells The progression of cancer promotes the degradation of the extracellular matrix at the primary tumor site, thereby accelerating invasion into adjacent tissues [26]. Given the pivotal role of MMP-2 and -9 in extracellular matrix breakdown and subsequent cancer metastasis, the impact of EDH on MMP activity was explored [27]. The activities of secreted MMP-2 and -9 from Huh7 cells treated with EDH were assessed using gelatin zymography assays. As the concentration of EDH increased, the activities of both MMP-2 and -9 decreased notably (Fig. 2A). Furthermore, MMP enzymatic activity assays indicated that EDH downregulated MMP collagenase activity in a dose-dependent manner (Fig. 2B). Given that MMP overexpression increases the aggressiveness of cancer and is associated with poor prognosis, the expression levels of MMP2 and 9 in response to EDH treatment were validated using quantitative qRT-PCR. EDH decreased the gene expression levels of MMP2 and 9 in a dose-dependent manner (Fig. 2C). These results suggest that EDH regulates cell invasiveness by suppressing MMP2 and 9 mRNA expression. EDH downregulates protein levels of Twist1 in Huh7 cells To investigate the regulatory mechanism of EDH, the expression level of the Twist1 protein was evaluated. Twist1 plays a pivotal role as a transcription factor in orchestrating EMT and facilitating cancer invasion by modulating the expression of critical genes like E-cadherin and N-cadherin [12]. EDH dose-dependently decreased the protein level of Twist1 in Huh7 cells, whereas the levels of other EMT-TFs, Snail1 and ZEB1, were not significantly decreased by EDH (Fig. 3A and Supplement Fig. 4). At the highest concentration (200 nM), EDH treatment remarkably decreased Twist1 protein expression, not only in Huh7 cells but also in other HCC cell lines (Supplement Fig. 5). EDH did not inhibit the transcriptional expression of TWIST1 , indicating that its regulatory effect on Twist1 protein occurs post-transcriptionally (Fig. 3B). Moreover, when MG132, a potent proteasome inhibitor, was introduced to EDH-treated Huh7 cells, Twist1 protein levels did not decline (Fig. 3C). These results suggest that the inhibition of wound closure by EDH is due to a decrease in Twist1 levels. EDH regulates target genes of Twist1 Given the inhibitory effect of EDH on inhibits Twist1 protein expression in Huh7 cells, both mRNA and protein levels of Twist1 target genes were investigated. EDH-treated Huh7 cells exhibited increased mRNA expression of the epithelial marker CDH1 (E-cadherin protein coding gene), whereas the mRNA levels of mesenchymal markers CDH2 (N-cadherin protein coding gene) and VIM (Vimentin protein coding gene) decreased (Fig. 4A). In an EDH dose-dependency test, the protein levels of E-cadherin exhibited a dose-dependent increase upon EDH treatment, whereas those of N-cadherin and Vimentin decreased (Fig. 4B). These results suggest that EDH decre ases the Twist1 protein levels, thereby regulating its target genes. EDH inhibits MAPK signaling in Huh7 cells Multiple studies have provided insights into the molecular mechanisms involved in the EMT-mediated metastasis process [10, 12], with the MAPK signaling pathway playing a key role in regulating the Twist1 protein levels [12]. Given that EDH treatment decreases Twist1 protein levels, the phosphorylation on the activation loop of MAPKs (ERK, JNK, and p38) was examined by immunoblot analysis in EDH-treated Huh7 cells (Fig. 5A). EDH decreased the activation loop phosphorylation of MAPKs, and EDH more significantly inhibited the phosphorylation of JNK and p38 than that of ERK. Meanwhile, EDH showed no effect on the phosphorylation of AKT, a regulator of the post-translational process of Twist1 (Fig. 5B). STAT3 and NF-κB, which induce gene expression of Twist1, are not regulated by EDH (Supplement Fig. 6). When cells were treated with PD169316, a specific kinase inhibitor of p38, and JNK inhibitor VIII, each inhibitor decreased the Twist1 protein level (Fig. 5C). However, it is worth noting that the reduction of the Twist1 protein level by these inhibitors was much lower than that by EDH. Since MAPKs stabilize Twist1 by phosphorylating serine 68 (S68) of Twist1 [28], an inactive mutant of Twist1 substituted from serine to alanine (S68A) was overexpressed in Hep3B cells, after which the cells were treated with EDH. While EDH treatment decreased the protein level of wild-type Twist1, the protein level of Twist1 S68A remained unaffected (Fig. 5D). Collectively, these results suggest that EDH destabilizes Twist1 by inhibiting the phosphorylation of MAPKs. Considering that EDH regulates JNK and p38 and sorafenib regulates ERK [29], it could be proposed as a potential complement to address the limitations of sorafenib. When sorafenib and EDH were administered together, cell mobility and invasiveness were further decreased (Supplement Fig. 7A and B). Additionally, co-treatment of these drugs further decreased the expression of Twist1 in Huh7 cells without affecting cytotoxicity (Supplement Fig. 7C and D). These results suggest that the combined treatment of sorafenib and EDH effectively regulates the migration of Huh7 cells through inhibition of Twist1 and can serve as a basis for overcoming limitations through combined treatment with previously reported drugs. Discussion The high mortality rate of HCC has been attributed to early metastasis, including local or portal vein invasion as well as distal metastasis, and high resistance to drug therapy [ 11 ]. Multiple studies have validated the cell growth inhibition and apoptosis-inducing effects of EDH. However, the mechanisms through which EDH regulates EMT remain largely unexplored [ 18 ]. Our study confirmed the anti-migration and anti-invasion effects of EDH, and the regulatory mechanism of cancer metastasis inhibition was evaluated at the cellular level. The relationship between cancer cell metastasis and EMT-TFs has been reported in many previous studies [ 10 – 12 ]. Although the effects of EDH on HCC cells were mostly investigated with Huh7 cells in this study, EDH might show a similar effect on other HCC cells (Hep3B, SNU449, SNU886, and PLC-PRF-5), as EDH treatment significantly decreased Twist1 levels in the tested HCC cell lines. Considering that EDH did not regulate the mRNA expression level of Twist1 and that the protein level of Twist1 was not regulated by EDH in the presence of MG132, these results suggest that EDH regulates Twist1 post-translationally. AKT and MAPKs phosphorylate S42 and S68 of Twist1, respectively, thereby increasing protein stability [ 28 ]. Since EDH regulates MAPKs (especially JNK and p38) but not AKT, this result implicates that EDH down-regulates Twist1 through MAPK inhibition. The cancellation of EDH regulation following inactivation of the S68 residue of Twist1 and the reduction of Twist1 following inactivation of JNK and p38 through treatment with their respective inhibitors support a mechanism for MAPK-mediated reduction of Twist1 by EDH. Interestingly, EDH decreased Twist1 expression more effectively at a lower concentration (200 nM) than JNK and p38 inhibitors (10 and 5 µM, respectively). These findings suggest that EDH may have strategic advantages as an effective inhibitor of Twist1 compared with other MAPK inhibitors. Considering that sorafenib is an anti-HCC drug that regulates ERK, it was expected that the combined treatment of EDH and sorafenib would result in an additional effect on Twist1 regulation. Additionally, overexpression of Twist1 in Huh7 cells has been reported to increase resistance to sorafenib [ 4 ], suggesting that EDH co-administration could promote the therapeutic efficacy of sorafenib. However, our study only examined the inhibitory effect of EDH and sorafenib co-treatment at the cellular level, highlighting the need to further explore the mechanisms through which these two compounds jointly regulate Twist1 expression. Overexpression of Twist1 in HCC cell lines resulted in CDH2 and VIM upregulation, whereas CDH1 was downregulated [ 14 ]. Matsuo et al . reported that Twist1 overexpression in HCC patient tissues is associated with HCC metastasis and is inversely correlated with E-cadherin expression [ 30 ]. EDH regulates the expression of EMT markers at the mRNA level by lowering Twist1 protein levels. This suggests that EDH inhibits the mobility of HCC cell lines by upregulating E-cadherin and downregulating N-cadherin and Vimentin. Additionally, EDH affects the activity and expression of MMPs, which may further control the motility of HCC cells. Cancer cells use more than 20 MMPs to degrade the ECM [ 27 ], with MMP-2 and − 9 being key factors and biomarkers for cancer metastasis [ 31 ]. A report by Khales et al . found that Twist1 upregulates MMP expression and increases its activity in esophageal squamous-cell carcinoma cells [ 32 ]. Collectively, our findings suggest that the reduction of MMP activity by EDH, particularly the inhibition of MMP-2 and − 9, may contribute to regulating cell motility by targeting Twist1 degradation. Given that our studies were exclusively conducted in vitro , further in vivo studies on HCC metastasis inhibition are needed to validate our findings. Moreover, to confirm the role of EDH role in the molecular regulation of metastasis-related pathways, the mechanism through which EDH inhibits Twist1 should be explored in various cancer types. The direct mechanism of action of EDH on Twist1 regulation remains to be studied. Conclusions Our findings revealed the potential of EDH as a drug candidate to inhibit the metastasis of HCC by regulating the protein level of Twist1 through the MAPK pathway. Although previous studies have demonstrated that emetine inhibits cancer invasion by downregulating MAPK in lung cancer cells [ 33 ], its relationship with EMT-transcription factors had not been reported until now. This is the first study to investigate the anticancer effect of EDH by focusing on the EMT regulation mechanism. Given its inhibitory effect on cancer migration through Twist1 regulation, EDH shows potential as a candidate for HCC treatment. However, the regulatory mechanism of EDH on cancer metastasis should be further explored in other cancer types, and its combined effect with other drugs should be studied further. Abbreviations HCC Hepatocellular carcinoma EMT Epithelial to mesenchymal transition EDH Emetine dihydrochloride AMPK Mitogen-activated protein kinase EMT-TFs EMT-inducing transcription factors STAT3 Signal transducer and activator of transcription 3 NF-κB Nuclear factor kappa B ERK Extracellular signal-regulated kinase JNK c-Jun N-terminal Kinase MMP Matrix metallopeptidase HRP Horseradish peroxidase DMEM Dulbecco’s modified Eagle’s medium FBS Fetal bovine serum PBS Phosphate-buffered saline 3D three-dimensional SDS Sodium dodecyl sulfate RT-PCR Reverse transcription-polymerase chain reaction RT-qPCR Quantitative reverse transcription polymerase chain reaction GAPDH Glyceraldehyde 3-phosphate dehydrogenase SD Standard deviation Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The original gel images supporting the conclusions of this article are included within its additional file. Competing interests The authors declare that there are no conflicts of interest. Funding This research was supported by National Research Foundation of Korea (NRF) grants, funded by the Korea government (MSIT) (2021R1A2C1011196 and 2021M3A9G8024747) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1A6A1A03044296). Contributions Haelim Yoon conceptualized the study, wrote the original draft, and verified and visualized the data. Sayeon Cho conceptualized the study, supervised, reviewed and edited the manuscript, and obtained funding. Acknowledgments The drug reposition library used in the initial screening was kindly provided by Korea Chemical Bank (www.chembank.org) of Korea Research Institute of Chemical Technology. Author information Authors and affiliations Laboratory of Molecular and Pharmacological Cell Biology, College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea Haelim Yoon & Sayeon Cho Corresponding author Correspondence to Sayeon Cho. Additional information Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018, 68(6):394-424. Uchino K, Tateishi R, Shiina S, Kanda M, Masuzaki R, Kondo Y, Goto T, Omata M, Yoshida H, Koike K: Hepatocellular carcinoma with extrahepatic metastasis: clinical features and prognostic factors. Cancer 2011, 117(19):4475-4483. 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Cervello M, Bachvarov D, Lampiasi N, Cusimano A, Azzolina A, McCubrey JA, Montalto G: Novel combination of sorafenib and celecoxib provides synergistic anti-proliferative and pro-apoptotic effects in human liver cancer cells. PLoS One 2013, 8(6):e65569. Webb T, Carter J, Roberts JL, Poklepovic A, McGuire WP, Booth L, Dent P: Celecoxib enhances [sorafenib + sildenafil] lethality in cancer cells and reverts platinum chemotherapy resistance. Cancer Biol Ther 2015, 16(11):1660-1670. Gao M, Deng C, Dang F: Synergistic antitumor effect of resveratrol and sorafenib on hepatocellular carcinoma through PKA/AMPK/eEF2K pathway. Food Nutr Res 2021, 65. Heerboth S, Housman G, Leary M, Longacre M, Byler S, Lapinska K, Willbanks A, Sarkar S: EMT and tumor metastasis. Clin Transl Med 2015, 4:6. Giannelli G, Koudelkova P, Dituri F, Mikulits W: Role of epithelial to mesenchymal transition in hepatocellular carcinoma. J Hepatol 2016, 65(4):798-808. Gonzalez DM, Medici D: Signaling mechanisms of the epithelial-mesenchymal transition. Sci Signal 2014, 7(344):re8. Kang E, Seo J, Yoon H, Cho S: The Post-Translational Regulation of Epithelial-Mesenchymal Transition-Inducing Transcription Factors in Cancer Metastasis. Int J Mol Sci 2021, 22(7). Zhao Z, Rahman MA, Chen ZG, Shin DM: Multiple biological functions of Twist1 in various cancers. Oncotarget 2017, 8(12):20380-20393. Sun T, Zhao N, Zhao XL, Gu Q, Zhang SW, Che N, Wang XH, Du J, Liu YX, Sun BC: Expression and functional significance of Twist1 in hepatocellular carcinoma: its role in vasculogenic mimicry. Hepatology 2010, 51(2):545-556. Emmanuel S. Akinboye OB: Biological activities of emetine. The Open Natural Products Journal 2011(4):8-15. Bleasel MD, Peterson GM: Emetine, Ipecac, Ipecac Alkaloids and Analogues as Potential Antiviral Agents for Coronaviruses. Pharmaceuticals (Basel) 2020, 13(3). Foreman KE, Jesse JN, 3rd, Kuo PC, Gupta GN: Emetine dihydrochloride: a novel therapy for bladder cancer. J Urol 2014, 191(2):502-509. Kumar R, Afsar M, Khandelwal N, Chander Y, Riyesh T, Dedar RK, Gulati BR, Pal Y, Barua S, Tripathi BN et al : Emetine suppresses SARS-CoV-2 replication by inhibiting interaction of viral mRNA with eIF4E. Antiviral Res 2021, 189:105056. Panettiere F, Coltman CA, Jr.: Experience with emetine hydrochloride (NSC 33669) as an antitumor agent. Cancer 1971, 27(4):835-841. Sun Q, Yogosawa S, Iizumi Y, Sakai T, Sowa Y: The alkaloid emetine sensitizes ovarian carcinoma cells to cisplatin through downregulation of bcl-xL. Int J Oncol 2015, 46(1):389-394. Uzor PF: Recent developments on potential new applications of emetine as anti-cancer agent. EXCLI J 2016, 15:323-328. Street EW: Cyclophosphamide plus emetine in lung cancer. Lancet 1972, 2(7773):381-382. Mori S, Chang JT, Andrechek ER, Matsumura N, Baba T, Yao G, Kim JW, Gatza M, Murphy S, Nevins JR: Anchorage-independent cell growth signature identifies tumors with metastatic potential. Oncogene 2009, 28(31):2796-2805. Ho WY, Yeap SK, Ho CL, Rahim RA, Alitheen NB: Development of multicellular tumor spheroid (MCTS) culture from breast cancer cell and a high throughput screening method using the MTT assay. PLoS One 2012, 7(9):e44640. Yilmaz M, Christofori G: EMT, the cytoskeleton, and cancer cell invasion. Cancer Metastasis Rev 2009, 28(1-2):15-33. Huang H: Matrix Metalloproteinase-9 (MMP-9) as a Cancer Biomarker and MMP-9 Biosensors: Recent Advances. Sensors (Basel) 2018, 18(10). Hong J, Zhou J, Fu J, He T, Qin J, Wang L, Liao L, Xu J: Phosphorylation of serine 68 of Twist1 by MAPKs stabilizes Twist1 protein and promotes breast cancer cell invasiveness. Cancer Res 2011, 71(11):3980-3990. Adnane L, Trail PA, Taylor I, Wilhelm SM: Sorafenib (BAY 43-9006, Nexavar), a dual-action inhibitor that targets RAF/MEK/ERK pathway in tumor cells and tyrosine kinases VEGFR/PDGFR in tumor vasculature. Methods Enzymol 2006, 407:597-612. Matsuo N, Shiraha H, Fujikawa T, Takaoka N, Ueda N, Tanaka S, Nishina S, Nakanishi Y, Uemura M, Takaki A et al : Twist expression promotes migration and invasion in hepatocellular carcinoma. BMC Cancer 2009, 9:240. Song Z, Wang J, Su Q, Luan M, Chen X, Xu X: The role of MMP-2 and MMP-9 in the metastasis and development of hypopharyngeal carcinoma. Braz J Otorhinolaryngol 2021, 87(5):521-528. Ardalan Khales S, Abbaszadegan MR, Majd A, Forghanifard MM: TWIST1 upregulates matrix metalloproteinase (MMP) genes family in esophageal squamous carcinoma cells. Gene Expr Patterns 2020, 37:119127. Kim JH, Cho EB, Lee J, Jung O, Ryu BJ, Kim SH, Cho JY, Ryou C, Lee SY: Emetine inhibits migration and invasion of human non-small-cell lung cancer cells via regulation of ERK and p38 signaling pathways. Chem Biol Interact 2015, 242:25-33. Additional Declarations No competing interests reported. Supplementary Files Originaluncroppedgelimages.pptx Supplementtableandfigure.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4805487","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":333922069,"identity":"b36db4a9-20cd-4452-9f2a-bc4defd1cc54","order_by":0,"name":"Haelim Yoon","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"Haelim","middleName":"","lastName":"Yoon","suffix":""},{"id":333922070,"identity":"496a888f-f383-4f8c-a98f-1e4b698055fe","order_by":1,"name":"Sayeon Cho","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYHACNgaGChj7ANFazpCshbGNFC3yM5KfPfg4z07e4ADzww8MZ+4R1mJwI83ccOa2ZMMNB9iMJRhuFBOhRSKHTZp32wHGDQcYzBgYPiQQ4zCQljkH7DccYP9GnBaGGyAtDQcSNxzgAdpygwgtBmeemUnOOJacPPMwT7FEwhliHNae/EziQ42dbd/x9o0fPhwjxmECMEXMQEyMBgYG/gNEKRsFo2AUjIKRDADngDkJ/pDG0AAAAABJRU5ErkJggg==","orcid":"","institution":"Chung-Ang University","correspondingAuthor":true,"prefix":"","firstName":"Sayeon","middleName":"","lastName":"Cho","suffix":""}],"badges":[],"createdAt":"2024-07-26 05:27:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4805487/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4805487/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63024773,"identity":"1df44666-d5e8-4938-99d7-87eabf00335c","added_by":"auto","created_at":"2024-08-22 08:14:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":672038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmetine dihydrochloride (EDH) suppresses the mobility of HCC cell lines. \u003c/strong\u003e\u0026nbsp;(A) Structure of EDH. (B) Wound healing assays were conducted by scratching a wound onto cell cultures using a white pipette tip, after which the cells were treated with EDH in 1% FBS medium. Representative images were taken at 48 hours using the JuLI-Stage Real-Time Cell History Recorder (scale bars, 0.5 mm). The wound closure value was measured as a percentage of the wound size at 48 hours point compared to the 0-hour point of each sample. Data were quantified from three independent experiments and presented as bar graphs. (C) For the migration assay, the upper chamber of the Transwell plate, which was seeded with Huh7 and Hep3B cells was treated with EDH (1% FBS medium). The bottom chamber was filled with 10% FBS medium and incubated for 6 hours. (D) To perform invasion assays, Huh7 and Hep3B cells were seeded on Matrigel-coated upper chambers and incubated for 18 and 24 hours, respectively. Cells were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet, and imaged using a microscope. Data are representative of three independent experiments. (E) Huh7 cells were mixed in 2×culture medium containing agarose. The agarose layer was treated with medium containing EDH at the indicated concentration, and cultured for 2 weeks, with the medium replaced every 3 days. Agarose colonies containing Huh7 cells were stained with crystal violet. (F) Huh7 cells formed into 3D spheroids were incubated with EDH for 96 hours. Images were taken at 24 hours intervals using JuLI-Stage Real-Time Cell History Recorder. The contour of the spheroid measured at 0 hours is indicated by a yellow line. The area of the spheroids was graphed by measuring the spheroid area using ImageJ. Data were collected from three independent experiments and values ​​are expressed as mean ± Standard deviation (SD). Statistical analysis was performed based on the control group at each time point. Data were analyzed by one-way ANOVA post hoc test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001 compared to the EDH-untreated group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4805487/v1/29c1ae9afaaa3348bb4140fe.png"},{"id":63022356,"identity":"e30ca6da-de2d-4c10-a4d5-cdebf45b196d","added_by":"auto","created_at":"2024-08-22 07:50:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":125809,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEDH regulates cell motility by inhibiting MMP activity and transcription. \u003c/strong\u003e(A) Huh7 cells were incubated with serum-free medium treated with different concentrations of EDH. After 24 hours, the collected culture medium was analyzed by zymography using SDS-PAGE gel containing gelatin. MMP-2 and -9 were measured using ImageJ and presented as bar graphs. (B) Gelatinase/collagenase activity assays were performed using culture medium from Huh7 cells treated with EDH. (C) mRNA expression levels of \u003cem\u003eMMP2 \u003c/em\u003eand \u003cem\u003e9\u003c/em\u003e were measured by qRT-PCR. The \u003cem\u003eGAPDH\u003c/em\u003e gene was used as a reference. All experiments were repeated independently three times and values ​​are expressed as mean ± SD. Data were analyzed by one-way ANOVA post hoc test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared to the EDH-untreated group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4805487/v1/2298d1dc02059f22672e6df3.png"},{"id":63024125,"identity":"99aa3aa0-4a3d-4003-b6d8-7ce2643aa2cc","added_by":"auto","created_at":"2024-08-22 08:06:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":172430,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEDH decreases the protein stability of Twist1 and inhibits the expression of Twist1 target genes. \u003c/strong\u003e(A) Huh7 cells were treated with EDH (50, 100, and 200 nM) for 24 hours, after which immunoblotting was performed using whole cell lysates to determine protein levels of Twist1. (B) Transcript levels of \u003cem\u003eTWIST1\u003c/em\u003e and \u003cem\u003eGAPDH\u003c/em\u003e were assessed by RT-PCR using mRNA harvested from Huh7 cells treated with EDH for 24 hours in 10% FBS medium. (C) Huh7 cells were pretreated with MG132 for 3 hours and then treated with EDH in 10% FBS culture medium for 3 hours.\u003cstrong\u003e \u003c/strong\u003eAll experiments were repeated independently three times and values ​​are expressed as mean ± SD. Data were analyzed by one-way ANOVA post hoc test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared to the EDH-untreated group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4805487/v1/6ceb2e682c3b3b15b335b8ad.png"},{"id":63024126,"identity":"8881d962-6b9d-453f-90b8-39547b1d7d61","added_by":"auto","created_at":"2024-08-22 08:06:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":185800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEDH represses target genes of Twist1, which regulates EMT properties. \u003c/strong\u003eTotal cell lysates of Huh7 cells treated with EDH for 24 hours were collected and subjected to (A) qRT-PCR to observe the mRNA expression levels of \u003cem\u003eCDH1\u003c/em\u003e, \u003cem\u003eCDH2\u003c/em\u003e, and \u003cem\u003eVIM \u003c/em\u003eas target genes of Twist1. (B) To evaluate protein expression, E-cadherin, N-cadherin, Vimentin, and β-actin were analyzed using immunoblotting. All data are representative of three experiments and expressed as the means ± SD. Data were analyzed by one-way ANOVA post hoc test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared to the EDH-untreated group.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4805487/v1/1c461c8ed1674ad1f5d7653a.png"},{"id":63022363,"identity":"f895f2d5-238a-40de-a897-1cefc39ea8d7","added_by":"auto","created_at":"2024-08-22 07:50:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":285280,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEDH regulates the MAPK signaling pathway, decreasing the expression of the Twist1. \u003c/strong\u003e(A and B) Huh7 cells were treated with EDH and incubated for 24 hours. Total cell lysates were used to confirm the protein levels of MAPK or AKT by western blotting. (C) Huh7 cells were treated with the indicated concentrations of p38 and JNK inhibitors for 6 hours and EDH for 3 hours. (D) Hep3B cells were overexpressed with FLAG-tagged Twist1 and mutant FLAG-tagged Twist1 S68A. The cells were then treated with EDH (200 nM) for 24 hours. The protein expression level of Twist was confirmed using the FLAG antibody. Data are representative of three experiments and expressed as means ± SD. Data were analyzed by one-way ANOVA post hoc test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared to the EDH-untreated group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4805487/v1/443a880e077777d9fd774db7.png"},{"id":87367315,"identity":"98188156-ef70-44df-b7ee-3c9a67ddebf4","added_by":"auto","created_at":"2025-07-23 06:47:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2374722,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4805487/v1/9f13ab9b-f2c6-43da-b79c-6506a0c34e1c.pdf"},{"id":63023264,"identity":"ddb30222-4ba1-4b13-b36a-3382b839895c","added_by":"auto","created_at":"2024-08-22 07:58:27","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":792867,"visible":true,"origin":"","legend":"","description":"","filename":"Originaluncroppedgelimages.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4805487/v1/fcaea1e066e34110613e0da7.pptx"},{"id":63022357,"identity":"eee90bf5-7e88-495a-b2a8-ad8102990dcf","added_by":"auto","created_at":"2024-08-22 07:50:27","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1642200,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementtableandfigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-4805487/v1/3761d23171f8dcfb36815e80.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Emetine dihydrochloride inhibits invasiveness and motility of hepatocellular carcinoma cells by blocking the MAPK pathway and inducing destabilization of Twist1","fulltext":[{"header":"Background","content":"\u003cp\u003eLiver cancer is the third leading cause of cancer mortality worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although surgical resection is the primary and most effective treatment for liver cancer patients, the prognosis for this disease remains poor due to its high 5-year recurrence rate [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Sorafenib, approved by the Food and Drug Administration in 2007, is currently the most widely used treatment for advanced hepatocellular carcinoma (HCC) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, side effects and limitations such as drug resistance [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and toxicity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] have been reported. Therefore, several studies have sought to enhance the anticancer activity of sorafenib by combining it with other chemical such as doxorubicin [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and celecoxib [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMost cancer-related deaths are caused by metastasis rather than the primary cancer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Metastasis begins when cancer cells move to other organs through the circulatory system, with epithelial-mesenchymal transition (EMT) being a crucial process [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. During EMT, tumor cells lose epithelial properties such as cell polarity and cell-cell adhesion and acquire mesenchymal properties such as migration, invasion, and anti-apoptosis [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Key EMT-inducing transcription factors (EMT-TFs) such as Twist1/2, Snail1/2, and ZEB1/2 are intricately regulated by a network of signaling pathways, including TGF-β, Wnt-β-catenin, and Notch [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The most studied intracellular signaling pathways involved in EMT-TF expression are signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa B (NF-κB) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. AKT and mitogen-activated protein kinases (MAPKs) (extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38) pathways are known to regulate the activity of EMT-TFs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The basic helix-loop-helix transcription factor Twist1 is a key regulator of EMT and highly expressed in various malignancies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In various cancer types, Twist1 has been reported to contribute to cancer invasion and metastasis, increasing intravascular migration and extravasation through blood vessel walls [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Twist1 has been proven to upregulate N-cadherin, a mesenchymal marker, and downregulate E-cadherin, an epithelial marker, at the mRNA level through the E-box element located in the promoter of the target gene [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Additionally, Twist1 is associated with HCC metastasis, stimulating invasiveness through the expression of matrix metalloproteinase (MMP)-2 and \u0026minus;\u0026thinsp;9 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, understanding the regulatory mechanism of Twist1 in the EMT process would provide valuable insights into the broader context of EMT and its impact on cancer metastasis.\u003c/p\u003e \u003cp\u003eEmetine, an active ingredient isolated from ipecac species, is a protein synthesis inhibitor known to inhibit both intracellular ribosomal and mitochondrial protein synthesis and interfere with DNA and RNA synthesis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Emetine has been traditionally used as an oral emetic and expectorant and is currently used as an antiprotozoal agent [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Interestingly, emetine has recently been studied as an antiviral agent, considered a potential drug with anti-coronavirus effects by inhibiting the replication of SARS-CoV-2 [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Emetine is formulated as a hydrobromide and hydrochloride salt when used as a drug, which increases the solubility of insoluble amines and facilitates absorption from the gastrointestinal tract [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the 1970s, emetine dihydrochloride was used in clinical trials at the National Cancer Institute for its antitumor activity but was no longer developed due to severe toxicity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Despite this, the apoptotic effect of emetine has been reported in various human cancer cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A recent study reported that emetine induces apoptosis not only by inhibiting of protein synthesis but also by regulating anti-apoptotic genes such as \u003cem\u003eBcl-x\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This apoptosis-inducing effect makes emetine a potential anticancer drug candidate for combination therapy, with some observed effects in lung cancer patients [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. While emetine has been studied primarily for its apoptosis-inducing effects in various human cancer cells, this study aims to explore new functions of emetine by investigating its potential to inhibit cancer metastasis. In this report, we verified the effect of EDH on cell migration and invasion in HCC cell lines and investigated the mechanisms regulating cell motility from an EMT perspective.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eReagents and antibodies\u003c/h2\u003e\n \u003cp\u003eEmetine dihydrochloride (cat. No. 324693) used in the experiment was purchased as a powder from EMD Millipore Crop (Billerica, MA, USA) and dissolved in pure water. JNK inhibitor VIII (JNK inhibitor, HY-107598) and SB203580 (p38 inhibitor, HY-10256) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). MG132 (474790), Sorafenib (SML2653), and FLAG antibodies (F3165) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Twist1 (sc-81417), JNK (sc-7345), p38 (sc-7972), Akt1/2/3 (sc-81434), Vimentin (sc-32322), and \u0026beta;-actin (sc-47778) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). ERK1/2 (#9102), p-ERK1/2 (#9106), p-p38 (Thr180/Tyr182, #9211), p-JNK (Thr183/Tyr185, #9251), p-AKT (Ser473, #4060), p-NF-\u0026kappa;B (Ser536, #3033), and p-STAT3 (Tyr705, #9145) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). E-cadherin (610182) and N-cadherin (610720) antibodies were obtained from BD Biosciences (San Jose, CA, USA). Polyclonal anti-mouse IgG Fc-tagged antibodies (LF-SA8001) and polyclonal anti-rabbit IgG-HRP (LF-SA8002) were purchased from AbFrontier (Young In Frontier Co., Ltd., Seoul, Korea).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eCell culture\u003c/h2\u003e\n \u003cp\u003eHuh7 and Hep3B cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium or Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (WELGENE, Seoul, Korea) containing 1% penicillin-streptomycin (GIBCO, Grand Island, NY, USA) and 10% fetal bovine serum (FBS; WELGENE, Seoul, Korea). All cells were incubated at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eCell viability assay\u003c/h2\u003e\n \u003cp\u003eHuh7 cells (4 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) were seeded in 96-well plates and incubated overnight in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified air atmosphere. The cells were then treated with a mixture of EDH (50, 100, 200, and 400 nM) in a medium containing either 1% or 10% FBS. Cell viability was tested 24 and 48 hours after exposure to EDH using EZ-Cytox (DOGEN, Seoul, Korea) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eWound healing assay\u003c/h2\u003e\n \u003cp\u003eThe day after seeding the Huh7 cells into 24-well plates (at 90% confluency), wounds were created using a pipette white tip. The culture medium was then aspirated and replaced with fresh medium containing 1% FBS and various concentrations of emetine dihydrochloride (50, 100, and 200 nM). The scratched cells were incubated for 24 and 48 hours, and cells that migrated to the wound surface were observed using a JuLI-Stage Real-Time Cell History Recorder (NanoEnTek Inc., Seoul, Korea). The change in wound closure is expressed as a percentage of wound healing in the treated groups compared to the control group. Each experiment was repeated three times, and the results were averaged.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eTranswell migration and invasion assay\u003c/h2\u003e\n \u003cp\u003eCell migration was analyzed using a 24-Transwell plate (pore size; 8 \u0026micro;m, SPL, Korea) containing a polycarbonate membrane. Huh7 cells and Hep3B cells (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) were seeded in the upper chamber. The cells were then treated with 250 \u0026micro;l of medium containing 1% FBS, with or without EDH. The lower chamber was filled with 500 \u0026micro;l of medium containing 10% FBS. Huh7 cells were incubated for 6 hours and Hep3B cells for 12 hours to allow migration through the membrane. The migrated cells on the membrane were then washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and stained with 0.5% crystal violet. Cells attached to the upper surface of the membrane were removed using cotton wool. The migrated cells were visualized by photographing the membrane under a microscope at 100\u0026times; magnification. Three fields were photographed, and the most representative image was selected for the figure. To perform the Matrigel invasion assay, 30 \u0026micro;l of diluted Matrigel (0.5 mg/ml; BD Biosciences, Franklin Lakes, NJ, USA) was coated in the upper chamber for 3 hours. This procedure was similar to the migration assay, except that the Huh7 cells were incubated for 18 hours and the Hep3B cells were incubated for 24 hours.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eAnalysis of soft agar colony formation\u003c/h2\u003e\n \u003cp\u003eThe bottom of a 12-well plate was coated with 2\u0026times; cell culture medium containing 0.5% agarose (Sigma-Aldrich, St. Louis, MO, USA). Huh7 cells (4 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well) were added to the coated upper layer by mixing with cell culture medium containing 0.3% agarose. Both the top and bottom layers were incubated at 4\u0026deg;C for 30 minutes each to make them semi-solid. The uppermost layer was then supplemented with cell culture medium with or without EDH. Fresh medium was replaced every 3 days, and the cells were incubated for 14 days at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. Colonies were stained with 0.05% crystal violet solution for 10 minutes at room temperature and decolorized using PBS. Each stained well was photographed using a digital camera.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eGelatin zymography\u003c/h2\u003e\n \u003cp\u003eHuh7 cells (2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well) were inoculated into a 12-well culture plate and cultured for one day. After washing with PBS, EDH was mixed in serum-free medium at each concentration (50, 100, and 200 nM), and 500 \u0026micro;l of each mixture was added to each well. After culturing for 24 hours, the cultured medium was collected and mixed with sample buffer [2.5 mM Tris-Cl (pH 6.8), 5% sodium dodecyl sulfate (SDS), 50% glycerol, 0.05% bromophenol blue]. The samples were then subjected to electrophoresis on 10% SDS-polyacrylamide gel containing 0.1% gelatin (G1393, Sigma-Aldrich, St. Louis, MO, USA). The separated gel was washed with a wash buffer [50 mM Tris-HCl (pH 7.5), 0.2 M NaCl, 5 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.1 \u0026micro;M ZnCl and 2.5% Triton X-100] at room temperature for 30 minutes. Substrate buffer (0.016% NaN\u003csub\u003e3\u003c/sub\u003e and 1% Triton X-100) was added to the wash buffer, and the gel was incubated on a shaker at 37\u0026deg;C for 48 hours. After fixing for 30 minutes with fixing solution (40% methanol and 10% acetic acid), the gel was stained with 0.25% Coomassie Brilliant Blue for 1 hour. For decolorization, a decolorization solution (5% methanol and 8% acetic acid) was used.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eMMP activity assay\u003c/h2\u003e\n \u003cp\u003eHuh7 cells (2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well) were seeded into 12-well culture plates and incubated for 24 hours in serum-free medium with or without EDH. MMP activity was analyzed using the collected medium with the Gelatinase/Collagenase Assay Kit (E12055, Thermo Fisher Scientific, USA). Experiments were performed according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eThree-dimensional (3D) Spheroid Invasion analysis\u003c/h2\u003e\n \u003cp\u003eHuh7 cells (2 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well) were inoculated into a low-attachment 96-well plate (34896, SPL, Korea) and cultured for 72 hours. After removing the medium, 3 mg/ml of Matrigel (356230, BD Biosciences, USA) was added, and the cells were further cultured for 24 hours. The resulting spheroids were then treated with EDH in 10% FBS medium. Spheroids were recorded at 24-hour intervals using a JuLI-Stage Real-Time Cell History Recorder (NanoEnTek Inc., Seoul, Korea).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunoblotting analysis\u003c/h2\u003e\n \u003cp\u003eAfter Huh7 cells were treated with EDH, cells were lysed in a lysis buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% Triton X-100, 0.5% IGEPAL CA-630, 1 mM EDTA, 1% glycerol; with 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, and 1 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e]. EDH-treated and untreated protein samples were mixed with equal volumes of 5\u0026times; SDS sample buffer [12 mM Tris-HCl (pH 6.8), 5% glycerol, 0.4% SDS, 0.02% bromophenol blue, and 1% \u0026beta;-mercaptoethanol]. After mixing, the samples were boiled at 100\u0026deg;C for 5 minutes. The samples were then electrophoresed on 12% SDS-PAGE and transferred to a nitrocellulose membrane. The protein-containing membranes were blocked with 5% nonfat dry skim milk and then incubated with specific primary antibodies (all antibodies diluted at 1:1000) in 3.3% BSA overnight at 4\u0026deg;C. The membranes were washed several times with 1\u0026times; Tris-buffered saline with Tween 20 and then incubated with a secondary antibody for 2 hours at room temperature. Protein bands were detected using an enhanced chemiluminescent immunoblotting detection reagent (Pierce; Thermo Fisher Scientific, Inc.). All experiments were performed at least in triplicate, and the band intensity of each protein was analyzed with ImageJ.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eReverse transcription-polymerase chain reaction (RT-PCR) analysis and quantitative RT-PCR (qRT-PCR) analysis\u003c/h2\u003e\n \u003cp\u003eHuh7 cells were inoculated in a 12-well plate and treated with EDH for 24 hours to extract total RNA (Lbozol, COSMO Genetech, Seoul, Korea). Using the extracted RNA (1 \u0026micro;g), complementary DNA (cDNA) was synthesized with the TOPscript cDNA synthesis kit (Enzynomics, Daejeon, Korea). RT-PCR and qRT-PCR were performed using the synthesized cDNA. Fluorescence values of PCR amplicons were obtained by performing qRT-PCR with a CFXConnect real-time thermocycler (Bio-Rad, Hercules, CA, USA). Gene expression levels were normalized to the Glyceraldehyde 3-phosphate dehydrogenase (\u003cem\u003eGAPDH\u003c/em\u003e) gene. According to the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Cq\u003c/sup\u003e method, gene expression levels were expressed compared to the control group. The RT-PCR and qRT-PCR primers are listed in Supplement table 1.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eTransfection\u003c/h2\u003e\n \u003cp\u003eHuh7 cells were inoculated to 70% confluency in a 6-well plate and incubated overnight. For transfection, the plasmid and linear polyethyleneimine (PolySciences, Warrington, USA) were mixed at a 1:3 ratio and left at room temperature for 30 min. Then, the mixture was added to each well and further cultured for 48 hours.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eData were analyzed using the Prism 3.0 statistical analysis program (GraphPad Software, San Diego, CA, USA). All data represent the mean value obtained from three or more independent experiments, and the error is presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). \u003cem\u003ePost hoc\u003c/em\u003e analysis was conducted using Holm-\u0026Scaron;\u0026iacute;d\u0026aacute;k\u0026rsquo;s method, and statistical analysis was performed using one-way ANOVA. Statistical significance is indicated as follows: *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Non-significant differences are marked with \u0026quot;N.S.\u0026quot; in the graph.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEDH regulates the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emotility\u003c/strong\u003e \u003cstrong\u003eand invasiveness of HCC cell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing HCC Huh7 cells, wound healing assays were conducted as an initial screen of the drug reposition library provided by the Korea Chemical Bank. \u0026nbsp; EDH (Fig. 1A) was identified as a potential inhibitor of Huh7 cell migration. Given that cancer cells possess high mobility and invasiveness leading to cancer metastasis, several HCC cell lines (Huh7, Hep3B, SNU449, SNU886, and PLC-PRF-5) were treated with EDH to evaluate its wound healing effects. When HCC cells were treated with different concentrations of EDH, wound closure was inhibited in a dose-dependent manner, compared to untreated cells (Fig. 1B and Supplement Fig. 1A). In addition, cell migration and invasion were evaluated using Transwell assays. Figure 1C shows that cell migration decreased in Huh7 and Hep3B cells treated with EDH in a concentration-dependent manner. Furthermore, cell invasion assays using Transwells with Matrigel-coated insert membranes indicated that EDH inhibited invasiveness in HCC cell lines (Fig. 1D and Supplement Fig. 1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo confirm that the inhibitory effect of EDH on cancer cell migration in HCC cell lines was not due to cell death, the cytotoxicity of EDH on Huh7 and Hep3B cell lines was evaluated both under cell growth conditions (10% FBS in the media) and cell growth inhibition conditions (1% FBS in the media) (Supplement Fig. 2A). EDH did not cause cytotoxicity in HCC cells up to a concentration of 200 nM. Furthermore, when other HCC cell lines (SNU449, SNU886, and PLC-PRF-5) were treated with EDH, at least 80% of the cells survived at 200 nM EDH (Supplement Fig. 2B). Therefore, in subsequent experiments, HCC cell lines were treated with EDH concentrations of 200 nM or less to investigate the mechanisms by which EDH regulates cancer cell migration and invasion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNormal cells require extracellular matrix contact to grow and divide [24]. In contrast, metastatic cancer cells can grow in an anchorage-independent manner in soft agar [24]. Soft agar colony formation assays were performed to determine whether EDH inhibits the anchorage-independent colony formation of cancer cells. When Huh7 cells were treated with EDH, colony formation was inhibited in a dose-dependent manner compared to the negative control group (Fig. 1E). Additionally, the invasiveness of multicellular tumor spheroids was evaluated through a 3D spheroid invasion assay mimicking the conditions occurring during cancer metastasis \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003e[25]. When spheroids derived from Huh7 cells were exposed to EDH for a duration of up to 96 hours, a notable suppression in invasiveness was observed, even at a concentration of 50 nM EDH (Fig. 1F). Furthermore, when Huh7 cells were cultured under conventional two-dimensional cell culture conditions, EDH treatment markedly inhibited cell proliferation, which was particularly noticeable at concentrations above 100 nM (Supplement Fig. 3). These findings suggest that EDH is a promising suppressor of the anchorage-independent growth and invasion of Huh7 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEDH downregulates MMP activity in Huh7 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe progression of cancer promotes the degradation of the extracellular matrix at the primary tumor site, thereby accelerating invasion into adjacent tissues [26]. Given the pivotal role of MMP-2 and -9 in extracellular matrix breakdown and subsequent cancer metastasis, the impact of EDH on MMP activity was explored [27]. The activities of secreted MMP-2 and -9 from Huh7 cells treated with EDH were assessed using gelatin zymography assays. As the concentration of EDH increased, the activities of both MMP-2 and -9 decreased notably (Fig. 2A). Furthermore, MMP enzymatic activity assays indicated that EDH downregulated MMP collagenase activity in a dose-dependent manner (Fig. 2B). Given that MMP overexpression increases the aggressiveness of cancer and is associated with poor prognosis, the expression levels of \u003cem\u003eMMP2\u003c/em\u003e and \u003cem\u003e9\u003c/em\u003e in response to EDH treatment were validated using quantitative qRT-PCR. EDH decreased the gene expression levels of \u003cem\u003eMMP2\u003c/em\u003e and \u003cem\u003e9\u003c/em\u003e in a dose-dependent manner (Fig. 2C). These results suggest that EDH regulates cell invasiveness by suppressing \u003cem\u003eMMP2\u0026nbsp;\u003c/em\u003eand \u003cem\u003e9\u003c/em\u003e mRNA expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEDH downregulates protein levels of Twist1 in Huh7 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the regulatory mechanism of EDH, the expression level of the Twist1 protein was evaluated. Twist1 plays a pivotal role as a transcription factor in orchestrating EMT and facilitating cancer invasion by modulating the expression of critical genes like E-cadherin and N-cadherin [12]. EDH dose-dependently decreased the protein level of Twist1 in Huh7 cells, whereas the levels of other EMT-TFs, Snail1 and ZEB1, were not significantly decreased by EDH (Fig. 3A and Supplement Fig. 4). At the highest concentration (200 nM), EDH treatment remarkably decreased Twist1 protein expression, not only in Huh7 cells but also in other HCC cell lines (Supplement Fig. 5). EDH did not inhibit the transcriptional expression of \u003cem\u003eTWIST1\u003c/em\u003e, indicating that its regulatory effect on Twist1 protein occurs post-transcriptionally (Fig. 3B). Moreover, when MG132, a potent proteasome inhibitor, was introduced to EDH-treated Huh7 cells, Twist1 protein levels did not decline (Fig. 3C). These results suggest that the inhibition of wound closure by EDH is due to a decrease in Twist1 levels.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEDH regulates target genes of Twist1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the inhibitory effect of EDH on inhibits Twist1 protein expression in Huh7 cells, both mRNA and protein levels of Twist1 target genes were investigated. EDH-treated Huh7 cells exhibited increased mRNA expression of the epithelial marker \u003cem\u003eCDH1\u003c/em\u003e (E-cadherin protein coding gene), whereas the mRNA levels of mesenchymal markers \u003cem\u003eCDH2\u003c/em\u003e (N-cadherin protein coding gene) and \u003cem\u003eVIM\u0026nbsp;\u003c/em\u003e(Vimentin protein coding gene) decreased (Fig. 4A). In an EDH dose-dependency test, the protein levels of E-cadherin exhibited a dose-dependent increase upon EDH treatment, whereas those of N-cadherin and Vimentin decreased (Fig. 4B). These results suggest that EDH decre ases the Twist1 protein levels, thereby regulating its target genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEDH inhibits MAPK signaling in Huh7 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMultiple studies have provided insights into the molecular mechanisms involved in the \u0026nbsp;EMT-mediated metastasis process [10, 12], with the MAPK signaling pathway playing a key role in regulating the Twist1 protein levels [12]. Given that EDH treatment decreases Twist1 protein levels, the phosphorylation on the activation loop of MAPKs (ERK, JNK, and p38) was examined by immunoblot analysis in EDH-treated Huh7 cells (Fig. 5A). EDH decreased the activation loop phosphorylation of MAPKs, and EDH more significantly inhibited the phosphorylation of JNK and p38 than that of ERK. Meanwhile, EDH showed no effect on the phosphorylation of AKT, a regulator of the post-translational process of Twist1 (Fig. 5B). STAT3 and NF-\u0026kappa;B, which induce gene expression of Twist1, are not regulated by EDH (Supplement Fig. 6). When cells were treated with PD169316, a specific kinase inhibitor of p38, and JNK inhibitor VIII, each inhibitor decreased the Twist1 protein level (Fig. 5C). However, it is worth noting that the reduction of the Twist1 protein level by these inhibitors was much lower than that by EDH. Since MAPKs stabilize Twist1 by phosphorylating serine 68 (S68) of Twist1 [28], an inactive mutant of Twist1 substituted from serine to alanine (S68A) was overexpressed in Hep3B cells, after which the cells were treated with EDH. While EDH treatment decreased the protein level of wild-type Twist1, the protein level of Twist1 S68A remained unaffected (Fig. 5D). Collectively, these results suggest that EDH destabilizes Twist1 by inhibiting the phosphorylation of MAPKs. Considering that EDH regulates JNK and p38 and sorafenib regulates ERK [29], it could be proposed as a potential complement to address the limitations of sorafenib. When sorafenib and EDH were administered together, cell mobility and invasiveness were further decreased (Supplement Fig. 7A and B). Additionally, co-treatment of these drugs further decreased the expression of Twist1 in Huh7 cells without affecting cytotoxicity (Supplement Fig. 7C and D). These results suggest that the combined treatment of sorafenib and EDH effectively regulates the migration of Huh7 cells through inhibition of Twist1 and can serve as a basis for overcoming limitations through combined treatment with previously reported drugs.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe high mortality rate of HCC has been attributed to early metastasis, including local or portal vein invasion as well as distal metastasis, and high resistance to drug therapy [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Multiple studies have validated the cell growth inhibition and apoptosis-inducing effects of EDH. However, the mechanisms through which EDH regulates EMT remain largely unexplored [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our study confirmed the anti-migration and anti-invasion effects of EDH, and the regulatory mechanism of cancer metastasis inhibition was evaluated at the cellular level.\u003c/p\u003e \u003cp\u003eThe relationship between cancer cell metastasis and EMT-TFs has been reported in many previous studies [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Although the effects of EDH on HCC cells were mostly investigated with Huh7 cells in this study, EDH might show a similar effect on other HCC cells (Hep3B, SNU449, SNU886, and PLC-PRF-5), as EDH treatment significantly decreased Twist1 levels in the tested HCC cell lines. Considering that EDH did not regulate the mRNA expression level of Twist1 and that the protein level of Twist1 was not regulated by EDH in the presence of MG132, these results suggest that EDH regulates Twist1 post-translationally. AKT and MAPKs phosphorylate S42 and S68 of Twist1, respectively, thereby increasing protein stability [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Since EDH regulates MAPKs (especially JNK and p38) but not AKT, this result implicates that EDH down-regulates Twist1 through MAPK inhibition. The cancellation of EDH regulation following inactivation of the S68 residue of Twist1 and the reduction of Twist1 following inactivation of JNK and p38 through treatment with their respective inhibitors support a mechanism for MAPK-mediated reduction of Twist1 by EDH. Interestingly, EDH decreased Twist1 expression more effectively at a lower concentration (200 nM) than JNK and p38 inhibitors (10 and 5 \u0026micro;M, respectively). These findings suggest that EDH may have strategic advantages as an effective inhibitor of Twist1 compared with other MAPK inhibitors. Considering that sorafenib is an anti-HCC drug that regulates ERK, it was expected that the combined treatment of EDH and sorafenib would result in an additional effect on Twist1 regulation. Additionally, overexpression of Twist1 in Huh7 cells has been reported to increase resistance to sorafenib [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], suggesting that EDH co-administration could promote the therapeutic efficacy of sorafenib. However, our study only examined the inhibitory effect of EDH and sorafenib co-treatment at the cellular level, highlighting the need to further explore the mechanisms through which these two compounds jointly regulate Twist1 expression.\u003c/p\u003e \u003cp\u003eOverexpression of Twist1 in HCC cell lines resulted in \u003cem\u003eCDH2\u003c/em\u003e and \u003cem\u003eVIM\u003c/em\u003e upregulation, whereas \u003cem\u003eCDH1\u003c/em\u003e was downregulated [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Matsuo \u003cem\u003eet al\u003c/em\u003e. reported that Twist1 overexpression in HCC patient tissues is associated with HCC metastasis and is inversely correlated with E-cadherin expression [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. EDH regulates the expression of EMT markers at the mRNA level by lowering Twist1 protein levels. This suggests that EDH inhibits the mobility of HCC cell lines by upregulating E-cadherin and downregulating N-cadherin and Vimentin. Additionally, EDH affects the activity and expression of MMPs, which may further control the motility of HCC cells. Cancer cells use more than 20 MMPs to degrade the ECM [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], with MMP-2 and \u0026minus;\u0026thinsp;9 being key factors and biomarkers for cancer metastasis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. A report by Khales \u003cem\u003eet al\u003c/em\u003e. found that Twist1 upregulates MMP expression and increases its activity in esophageal squamous-cell carcinoma cells [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Collectively, our findings suggest that the reduction of MMP activity by EDH, particularly the inhibition of MMP-2 and \u0026minus;\u0026thinsp;9, may contribute to regulating cell motility by targeting Twist1 degradation.\u003c/p\u003e \u003cp\u003eGiven that our studies were exclusively conducted \u003cem\u003ein vitro\u003c/em\u003e, further \u003cem\u003ein vivo\u003c/em\u003e studies on HCC metastasis inhibition are needed to validate our findings. Moreover, to confirm the role of EDH role in the molecular regulation of metastasis-related pathways, the mechanism through which EDH inhibits Twist1 should be explored in various cancer types. The direct mechanism of action of EDH on Twist1 regulation remains to be studied.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur findings revealed the potential of EDH as a drug candidate to inhibit the metastasis of HCC by regulating the protein level of Twist1 through the MAPK pathway. Although previous studies have demonstrated that emetine inhibits cancer invasion by downregulating MAPK in lung cancer cells [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], its relationship with EMT-transcription factors had not been reported until now. This is the first study to investigate the anticancer effect of EDH by focusing on the EMT regulation mechanism. Given its inhibitory effect on cancer migration through Twist1 regulation, EDH shows potential as a candidate for HCC treatment. However, the regulatory mechanism of EDH on cancer metastasis should be further explored in other cancer types, and its combined effect with other drugs should be studied further.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHepatocellular carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpithelial to mesenchymal transition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEmetine dihydrochloride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAMPK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitogen-activated protein kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMT-TFs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEMT-inducing transcription factors\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSTAT3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSignal transducer and activator of transcription 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNF-κB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNuclear factor kappa B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eERK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular signal-regulated kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJNK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ec-Jun N-terminal Kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMatrix metallopeptidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHRP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHorseradish peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e3D\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethree-dimensional\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSodium dodecyl sulfate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT-PCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReverse transcription-polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT-qPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eQuantitative reverse transcription polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGAPDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlyceraldehyde 3-phosphate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original gel images supporting the conclusions of this article are included within its additional file.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by National Research Foundation of Korea (NRF) grants, funded by the Korea government (MSIT) (2021R1A2C1011196 and\u0026nbsp;2021M3A9G8024747) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1A6A1A03044296).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaelim Yoon conceptualized the study, wrote the original draft, and verified and visualized the data. Sayeon Cho conceptualized the study, supervised, reviewed and edited the manuscript, and obtained funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe drug reposition library\u0026nbsp;used in the initial screening was kindly provided by Korea Chemical Bank (www.chembank.org) of Korea Research Institute of Chemical Technology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLaboratory of Molecular and Pharmacological Cell Biology, College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaelim Yoon \u0026amp; Sayeon Cho\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Sayeon Cho.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher’s Note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. \u003cem\u003eCA Cancer J Clin \u003c/em\u003e2018, 68(6):394-424.\u003c/li\u003e\n\u003cli\u003eUchino K, Tateishi R, Shiina S, Kanda M, Masuzaki R, Kondo Y, Goto T, Omata M, Yoshida H, Koike K: Hepatocellular carcinoma with extrahepatic metastasis: clinical features and prognostic factors. \u003cem\u003eCancer \u003c/em\u003e2011, 117(19):4475-4483.\u003c/li\u003e\n\u003cli\u003eCheng Y, Luo R, Zheng H, Wang 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Longacre M, Byler S, Lapinska K, Willbanks A, Sarkar S: EMT and tumor metastasis. \u003cem\u003eClin Transl Med \u003c/em\u003e2015, 4:6.\u003c/li\u003e\n\u003cli\u003eGiannelli G, Koudelkova P, Dituri F, Mikulits W: Role of epithelial to mesenchymal transition in hepatocellular carcinoma. \u003cem\u003eJ Hepatol \u003c/em\u003e2016, 65(4):798-808.\u003c/li\u003e\n\u003cli\u003eGonzalez DM, Medici D: Signaling mechanisms of the epithelial-mesenchymal transition. \u003cem\u003eSci Signal \u003c/em\u003e2014, 7(344):re8.\u003c/li\u003e\n\u003cli\u003eKang E, Seo J, Yoon H, Cho S: The Post-Translational Regulation of Epithelial-Mesenchymal Transition-Inducing Transcription Factors in Cancer Metastasis. \u003cem\u003eInt J Mol Sci \u003c/em\u003e2021, 22(7).\u003c/li\u003e\n\u003cli\u003eZhao Z, Rahman MA, Chen ZG, Shin DM: Multiple biological functions of Twist1 in various cancers. \u003cem\u003eOncotarget \u003c/em\u003e2017, 8(12):20380-20393.\u003c/li\u003e\n\u003cli\u003eSun T, Zhao 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Akinboye OB: Biological activities of emetine. \u003cem\u003eThe Open Natural Products Journal \u003c/em\u003e2011(4):8-15.\u003c/li\u003e\n\u003cli\u003eBleasel MD, Peterson GM: Emetine, Ipecac, Ipecac Alkaloids and Analogues as Potential Antiviral Agents for Coronaviruses. \u003cem\u003ePharmaceuticals (Basel) \u003c/em\u003e2020, 13(3).\u003c/li\u003e\n\u003cli\u003eForeman KE, Jesse JN, 3rd, Kuo PC, Gupta GN: Emetine dihydrochloride: a novel therapy for bladder cancer. \u003cem\u003eJ Urol \u003c/em\u003e2014, 191(2):502-509.\u003c/li\u003e\n\u003cli\u003eKumar R, Afsar M, Khandelwal N, Chander Y, Riyesh T, Dedar RK, Gulati BR, Pal Y, Barua S, Tripathi BN\u003cem\u003e et al\u003c/em\u003e: Emetine suppresses SARS-CoV-2 replication by inhibiting interaction of viral mRNA with eIF4E. \u003cem\u003eAntiviral Res \u003c/em\u003e2021, 189:105056.\u003c/li\u003e\n\u003cli\u003ePanettiere F, Coltman CA, Jr.: Experience with emetine hydrochloride (NSC 33669) as an antitumor agent. \u003cem\u003eCancer \u003c/em\u003e1971, 27(4):835-841.\u003c/li\u003e\n\u003cli\u003eSun Q, Yogosawa S, Iizumi Y, Sakai T, Sowa Y: The alkaloid emetine sensitizes ovarian carcinoma cells to cisplatin through downregulation of bcl-xL. \u003cem\u003eInt J Oncol \u003c/em\u003e2015, 46(1):389-394.\u003c/li\u003e\n\u003cli\u003eUzor PF: Recent developments on potential new applications of emetine as anti-cancer agent. \u003cem\u003eEXCLI J \u003c/em\u003e2016, 15:323-328.\u003c/li\u003e\n\u003cli\u003eStreet EW: Cyclophosphamide plus emetine in lung cancer. \u003cem\u003eLancet \u003c/em\u003e1972, 2(7773):381-382.\u003c/li\u003e\n\u003cli\u003eMori S, Chang JT, Andrechek ER, Matsumura N, Baba T, Yao G, Kim JW, Gatza M, Murphy S, Nevins JR: Anchorage-independent cell growth signature identifies tumors with metastatic potential. \u003cem\u003eOncogene \u003c/em\u003e2009, 28(31):2796-2805.\u003c/li\u003e\n\u003cli\u003eHo WY, Yeap SK, Ho CL, Rahim RA, Alitheen NB: Development of multicellular tumor spheroid (MCTS) culture from breast cancer cell and a high throughput screening method using the MTT assay. \u003cem\u003ePLoS One \u003c/em\u003e2012, 7(9):e44640.\u003c/li\u003e\n\u003cli\u003eYilmaz M, Christofori G: EMT, the cytoskeleton, and cancer cell invasion. \u003cem\u003eCancer Metastasis Rev \u003c/em\u003e2009, 28(1-2):15-33.\u003c/li\u003e\n\u003cli\u003eHuang H: Matrix Metalloproteinase-9 (MMP-9) as a Cancer Biomarker and MMP-9 Biosensors: Recent Advances. \u003cem\u003eSensors (Basel) \u003c/em\u003e2018, 18(10).\u003c/li\u003e\n\u003cli\u003eHong J, Zhou J, Fu J, He T, Qin J, Wang L, Liao L, Xu J: Phosphorylation of serine 68 of Twist1 by MAPKs stabilizes Twist1 protein and promotes breast cancer cell invasiveness. \u003cem\u003eCancer Res \u003c/em\u003e2011, 71(11):3980-3990.\u003c/li\u003e\n\u003cli\u003eAdnane L, Trail PA, Taylor I, Wilhelm SM: Sorafenib (BAY 43-9006, Nexavar), a dual-action inhibitor that targets RAF/MEK/ERK pathway in tumor cells and tyrosine kinases VEGFR/PDGFR in tumor vasculature. \u003cem\u003eMethods Enzymol \u003c/em\u003e2006, 407:597-612.\u003c/li\u003e\n\u003cli\u003eMatsuo N, Shiraha H, Fujikawa T, Takaoka N, Ueda N, Tanaka S, Nishina S, Nakanishi Y, Uemura M, Takaki A\u003cem\u003e et al\u003c/em\u003e: Twist expression promotes migration and invasion in hepatocellular carcinoma. \u003cem\u003eBMC Cancer \u003c/em\u003e2009, 9:240.\u003c/li\u003e\n\u003cli\u003eSong Z, Wang J, Su Q, Luan M, Chen X, Xu X: The role of MMP-2 and MMP-9 in the metastasis and development of hypopharyngeal carcinoma. \u003cem\u003eBraz J Otorhinolaryngol \u003c/em\u003e2021, 87(5):521-528.\u003c/li\u003e\n\u003cli\u003eArdalan Khales S, Abbaszadegan MR, Majd A, Forghanifard MM: TWIST1 upregulates matrix metalloproteinase (MMP) genes family in esophageal squamous carcinoma cells. \u003cem\u003eGene Expr Patterns \u003c/em\u003e2020, 37:119127.\u003c/li\u003e\n\u003cli\u003eKim JH, Cho EB, Lee J, Jung O, Ryu BJ, Kim SH, Cho JY, Ryou C, Lee SY: Emetine inhibits migration and invasion of human non-small-cell lung cancer cells via regulation of ERK and p38 signaling pathways. \u003cem\u003eChem Biol Interact \u003c/em\u003e2015, 242:25-33.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Emetine, anti-migration, anti-invasion, cancer cell motility, EMT, Twist1, HCC, mesenchymal markers","lastPublishedDoi":"10.21203/rs.3.rs-4805487/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4805487/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHepatocellular carcinoma (HCC) is a malignant tumor that causes both extrahepatic and intrahepatic metastases. Epithelial to mesenchymal transition (EMT) is a crucial step in the development and metastasis of cancer. Emetine dihydrochloride (EDH) has been previously used as an anti-emetic and is now proposed as a replication inhibitor of SARS-CoV-2, but its effect against cancer metastasis has not been evaluated. Therefore, this study sought to investigate the regulatory mechanisms of cell migration from an EMT perspective using EDH in HCC cell lines.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHCC cell lines (Huh7, Hep3B, SNU449, SNU886, and PLC-PRF-5) were used to measure cell viability against EDH. The effect of EDH on migration was verified by wound healing analysis and migration analysis using Transwell. The effect of EDH on invasion was determined using an invasion assay in Matrigel-coated Transwell chambers. Spheroid invasion and soft agar colony formation assays were performed to verify the effect of EDH on anchorage-independent growth. Gelatin zymography was used to determine the activities of matrix metalloproteinase \u0026minus;\u0026thinsp;2 and \u0026minus;\u0026thinsp;9. The protein expression levels of Twist1, downstream target genes, and the mitogen-activated protein kinases (MAPKs) and AKT signaling pathways were determined through immunoblotting. The RNA expression levels of each gene were analyzed through RT-PCR and quantitative RT-PCR.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEDH inhibited the motility of various HCC cell lines at non-toxic concentrations. The inhibitory effect of EDH on cancer cell motility resulted from a decrease in protein levels of Twist1, a key transcription factor involved in EMT. Depletion of Twist1 due to EDH treatment suppressed the expression of mesenchymal markers (N-cadherin and Vimentin) while increasing the expression of epithelial markers (E-cadherin). The regulatory pathway for the destabilization of Twist1 by EDH was mediated through the inactivation of the MAPK pathway. EDH specifically inactivated JNK and p38, thereby destabilizing the Twist1 protein, which is dependent on the S68 phosphorylation of Twist1.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEDH induces MAPK inactivation, which decreases Twist1 protein levels and ultimately suppresses mesenchymal properties. These results provide the first report on EDH from an EMT perspective and suggest its potential as an anticancer agent for HCC.\u003c/p\u003e","manuscriptTitle":"Emetine dihydrochloride inhibits invasiveness and motility of hepatocellular carcinoma cells by blocking the MAPK pathway and inducing destabilization of Twist1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-22 07:50:22","doi":"10.21203/rs.3.rs-4805487/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8ac6caad-dfc4-4b77-a508-76d8181732e6","owner":[],"postedDate":"August 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-23T06:39:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-22 07:50:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4805487","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4805487","identity":"rs-4805487","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}