Sayeon Cho

Epithelial-mesenchymal transition (EMT) is a crucial step in the development and metastasis of cancer. Emetine was previously proposed as an antiemetic and a protein synthesis inhibitor, but its effect on cancer metastasis has not been evaluated. Therefore, this study sought to investigate the mechanisms regulating cell migration in the context of EMT using emetine dihydrochloride (EDH) in various HCC cell lines (Huh7, Hep3B, SNU449, SNU886, and PLC-PRF-5). EDH inhibited the migration and invasion of HCC cell lines at non-toxic concentrations. EDH inhibited cancer cell motility by reducing Twist1 protein levels, a key transcription factor involved in EMT. This reduction in Twist1 suppressed the expression of mesenchymal markers (N-cadherin and vimentin) while increasing the expression of epithelial marker E-cadherin. MG132 rescue and cycloheximide chase assays suggested that EDH may reduce Twist1 protein stability. EDH also suppressed JNK and p38 MAPK signaling, pathways previously implicated in the regulation of Twist1 phosphorylation at Serine 68 (S68). Consistently, EDH-induced reduction in Twist1 stability was attenuated in cells expressing the phosphorylation-deficient Twist1 S68A mutant, supporting a possible contribution of S68-associated MAPK signaling to EDH-mediated Twist1 regulation. Collectively, these findings suggest that EDH attenuates mesenchymal characteristics and motility in HCC cells, potentially through EMT-related mechanisms associated with altered Twist1 stability. Although further in vivo and mechanistic validation is required, our results suggest that EDH may have potential as an anti-metastatic candidate for HCC.

Background: Pogostemon stellatus (Lour.) Kuntze is a plant native to South and East Asia belonging to the Lamiaceae family, whose members are widely recognized for their anti-inflammatory and antioxidant properties. However, these properties have not been extensively studied in P. stellatus (Lour.) Kuntze. This study aimed to evaluate the anti-inflammatory and antioxidant effects of methanol extract of P. stellatus (Lour.) Kuntze (MPSK) in LPS-stimulated RAW 264.7 macrophages.Methods: RAW 264.7 macrophages were stimulated with LPS and treated with MPSK. Anti-inflammatory effects were assessed by measuring nitric oxide (NO) and prostaglandin E₂ (PGE₂) levels and the expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) at both mRNA and protein levels. Nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathway involvement was examined. Antioxidant capacity was determined using a DPPH radical scavenging assay, and nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream targets were evaluated.Results: MPSK markedly attenuated the production of inflammatory mediators and cytokines in LPS-induced RAW 264.7 macrophages. NO production was suppressed by 98%, compared with the LPS-treated group. PGE₂ production was also inhibited by 64%. Consistent with these findings, MPSK significantly reduced the protein and mRNA expression levels of iNOS and COX-2. Furthermore, MPSK suppressed the production and expression of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Notably, when MPSK was administered at 100 μg/mL, IL-6 and IL-1β production were reduced by 21% and 18%, respectively, compared with the LPS-treated group. These anti-inflammatory effects were associated with the inhibition of NF-κB and MAPK signaling pathways. Immunoblot analysis demonstrated that NF-κB protein expression was reduced by 72% upon treatment with MPSK, while the phosphorylation of ERK, JNK, and p38 was suppressed by 38%, 56%, and 35%, respectively. MPSK suppressed ROS levels in RAW 264.7 cells by 62%, demonstrating its potent antioxidant capacity. This antioxidant activity of MPSK was demonstrated by the upregulation of Nrf2 and its downstream antioxidant target genes.Conclusion: MPSK possesses both anti-inflammatory and antioxidant activities through regulation of NF-κB, MAPK, and Nrf2 pathways, suggesting its potential as a therapeutic agent against inflammation and oxidative stress.

Targeting RNA-dependent RNA polymerase (RdRp), a highly conserved enzyme essential for SARS coronavirus 2 (SARS-CoV-2) replication and transcription, represents a promising antiviral strategy due to its lower mutation rate than structural proteins such as Spike. This study introduces a cell-based assay system for screening potential SARS-CoV-2 RdRp inhibitors, contributing to ongoing efforts to identify effective antiviral agents. The assay utilizes a reporter vector containing the 3′ untranslated region (UTR), luciferase reporter gene, and 5’ UTR gene, sequentially arranged in reverse under the control of the cytomegalovirus promoter in the pcDNA3.1 vector. Co-transfection with SARS-CoV-2 RdRp resulted an increase in luminescence-based quantification of RdRp activity, achieving a Z-factor of 0.605, indicative of high reproducibility and reliability for high-throughput screening. Established RdRp inhibitors, including remdesivir, molnupiravir, tenofovir, and sofosbuvir, significantly reduced reporter activity, with remdesivir exhibiting the strongest inhibition. A newly identified RdRp inhibitor was further validated through primer extension polymerase and NMPylation assays, along with virus-based experiments, confirming its inhibitory mechanism. These results highlight the utility of this screening system in identifying effective RdRp-targeting antivirals, reinforcing the strategic importance of RdRp inhibition in combating SARS-CoV-2 and emerging variants.

Background/Aim: Hepatocellular carcinoma (HCC) is a main type of liver cancer with high metastatic potential, and its incidence is steadily increasing worldwide. However, the development of new drugs for the treatment of HCC is still insufficient. This study aimed to determine the anticancer effect of NSC-38270, a natural product, on HCC. Materials and Methods: After treating HCC Huh7 cells with NSC-38270, cell growth, wound healing, migration, and invasion assays were conducted. We investigated the effects of NSC-38270 on Twist1, a crucial epithelial–mesenchymal transition (EMT)-related transcription factor. In addition, apoptosis, histone H2A.X activation, and cell morphology assays were performed in Huh7 and immortalized normal liver cells following treatment with NSC-38270. Results: NSC-38270 reduced the migration and invasion ability of Huh7 cells, accompanied by a decrease in Twist1. Furthermore, NSC-38270 induced apoptosis in Huh7 cells, whereas apoptosis was not observed in immortalized normal liver cells (THLE-2 cells and Chang liver cells). Conclusion: NSC-38270 exhibited significant inhibitory effects on the migration and invasion of Huh7 cells by repressing Twist1. Importantly, it induced cancer cell-specific apoptotic effects. These findings suggest that NSC-38270 holds promising potential as a therapeutic candidate for cancer treatment.

Background/Aim: Hepatocellular carcinoma (HCC) is a prevalent type of cancer worldwide. Although sorafenib is the only chemotherapy agent used for HCC, there is a need to discover a more potent anticancer agent with reduced side-effects. The compound, (S)-3-(3-fluoro-4-methoxybenzyl)-5,6,7-trimethoxychroman-4-one (FMTC), was designed to inhibit tubulin assembly but its specific mechanisms of action have not been previously investigated. Herein, we investigated the regulation mechanisms by which FMTC affects the proliferation of the HCC cell line, Huh7. Materials and Methods: The effects of FMTC on cell viability and growth were analyzed in the HCC cell line, Huh7. Cell cycle and apoptosis regulated by FMTC were analyzed using flow cytometry. To verify the regulation of mRNA and protein expression of cell proliferation-related factors by FMTC in Huh7 cells, RT-qPCR and western blot analyses were employed. Results: FMTC suppressed cell division dose-dependently by triggering cell cycle arrest at the G₂/M phase via p21 up-regulation. The increased phosphorylation of histone H3 on Ser-10 and the condensation of chromatin in FMTC-treated cells indicated mitotic arrest. Prolonged FMTC-induced cell cycle arrest triggered apoptosis. Conclusion: FMTC inhibits the proliferation of human liver cancer cells by up-regulating p21, thereby inducing cell cycle arrest at the G₂/M phase. These findings highlight FMTC as a novel agent for HCC treatment.

Background: Overexpression of Twist1, one of the epithelial-mesenchymal transition-transcription factors (EMT-TFs), is associated with hepatocellular carcinoma (HCC) metastasis. Pelitinib is known to be an irreversible epidermal growth factor receptor tyrosine kinase inhibitor that is used in clinical trials for colorectal and lung cancers, but the role of pelitinib in cancer metastasis has not been studied. This study aimed to investigate the anti-migration and anti-invasion activities of pelitinib in HCC cell lines.Methods: Using three HCC cell lines (Huh7, Hep3B, and SNU449 cells), the effects of pelitinib on cell cytotoxicity, invasion, and migration were determined by cell viability, wound healing, transwell invasion, and spheroid invasion assays. The activities of MMP-2 and -9 were examined through gelatin zymography. Through immunoblotting analyses, the expression levels of EMT-TFs (Snail1, Twist1, and ZEB1) and EMT-related signaling pathways such as mitogen-activated protein kinases (MAPKs) and Akt signaling pathways were measured. The activity and expression levels of target genes were analyzed by reporter assay, RT-PCR, quantitative RT-PCR, and immunoblotting analysis. Statistical analysis was performed using one-way ANOVA with Dunnett's Multiple comparison tests in Prism 3.0 to assess differences between experimental conditions.Results: In this study, pelitinib treatment significantly inhibited wound closure in various HCC cell lines, including Huh7, Hep3B, and SNU449. Additionally, pelitinib was found to inhibit multicellular cancer spheroid invasion and metalloprotease activities in Huh7 cells. Further investigation revealed that pelitinib treatment inhibited the migration and invasion of Huh7 cells by inducing Twist1 degradation through the inhibition of MAPK and Akt signaling pathways. We also confirmed that the inhibition of cell motility by Twist1 siRNA was similar to that observed in pelitinib-treated group. Furthermore, pelitinib treatment regulated the expression of target genes associated with EMT, as demonstrated by the upregulation of E-cadherin and downregulation of N-cadherin.Conclusion: Based on our novel finding of pelitinib from the perspective of EMT, pelitinib has the ability to inhibit EMT activity of HCC cells via inhibition of Twist1, and this may be the potential mechanism of pelitinib on the suppression of migration and invasion of HCC cells. Therefore, pelitinib could be developed as a potential anti-cancer drug for HCC.