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

doi:10.21203/rs.3.rs-4805487/v1

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

https://doi.org/10.21203/rs.3.rs-4805487/v1

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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

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.

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% CO₂ incubator.

Cell viability assay

Huh7 cells (4 × 10⁴ cells/well) were seeded in 96-well plates and incubated overnight in a 5% CO₂ 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 × 10⁴ 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 × 10³ 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% CO₂ 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 × 10⁶ 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 CaCl₂, 0.1 µM ZnCl and 2.5% Triton X-100] at room temperature for 30 minutes. Substrate buffer (0.016% NaN₃ 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 × 10⁶ 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 × 10³ 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 Na₃VO₄]. 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.

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.

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.

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.

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

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

Standard deviation

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.

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

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Abstract

Figures

Background

Methods

Reagents and antibodies

Cell culture

Cell viability assay

Wound healing assay

Transwell migration and invasion assay

Analysis of soft agar colony formation

Gelatin zymography

MMP activity assay

Three-dimensional (3D) Spheroid Invasion analysis

Immunoblotting analysis

Reverse transcription-polymerase chain reaction (RT-PCR) analysis and quantitative RT-PCR (qRT-PCR) analysis

Transfection

Statistical analysis

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1