Role of pelitinib in the regulation of migration and invasion of hepatocellular carcinoma cells via inhibition of Twist1.

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.

Anti-migration and anti-invasion effects of LY-290181 on breast cancer cell lines through the inhibition of Twist1.

Breast cancer has become the most common cancer among women worldwide. Among breast cancers, metastatic breast cancer is associated with the highest mortality rate. Twist1, one of the epithelial-mesenchymal transition-regulating transcription factors, is known to promote the intravasation of breast cancer cells into metastatic sites. Therefore, targeting Twist1 to develop anti-cancer drugs might be a valuable strategy. In this study, LY-290181 dose-dependently inhibited migration, invasion, and multicellular tumor spheroid invasion in breast cancer cell lines. These anti-cancer effects of LY-290181 were mediated through the down-regulation of Twist1 protein levels. LY-290181 inhibited extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways. Therefore, our findings suggest that LY-290181 may serve as a basis for future research and development of an anti-cancer agent targeting metastatic cancers. [BMB Reports 2023; 56(7): 410-415].

Antioxidant and Anti-Inflammatory Effects of Bischofia javanica (Blume) Leaf Methanol Extracts through the Regulation of Nrf2 and TAK1.

Bischofia javanica (Blume) has been traditionally used to treat inflammatory diseases such as tonsillitis and ulcers throughout Asia, including China, Indonesia, and the Philippines: however, the molecular mechanisms by which B. javanica exerts its antioxidant and anti-inflammatory properties remain largely unknown. In this study, we analyzed the antioxidant and anti-inflammatory mechanisms of methanol extracts of B. javanica leaves (MBJ) in vitro and in vivo. MBJ decreased nitric oxide (NO) production and the expression of pro-inflammatory cytokines, including interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α, in lipopolysaccharide (LPS)-treated RAW 264.7 cells. The observed suppression of inflammatory responses by MBJ was correlated with an inhibition of the nuclear factor-κB (NF-κB) and the mitogen-activated protein kinase (MAPK) pathways. Additionally, MBJ induced nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that upregulates the expression of anti-inflammatory and antioxidant genes. Furthermore, MBJ exhibited antioxidant and anti-inflammatory effects in an acute hepatitis mouse model. In conclusion, our results confirm the medicinal properties of B. javanica, and therefore MBJ could be applied to improve inflammatory and redox imbalances in different types of pathologies.

Identification of a novel inhibitor of liver cancer cell invasion and proliferation through regulation of Akt and Twist1.

When primary cancer faces limited oxygen and nutrient supply, it undergoes an epithelial-mesenchymal transition, which increases cancer cell motility and invasiveness. The migratory and invasive cancer cells often exert aggressive cancer development or even cancer metastasis. In this study, we investigated a novel compound, 3-acetyl-5,8-dichloro-2-((2,4-dichlorophenyl)amino)quinolin-4(1H)-one (ADQ), that showed significant suppression of wound healing and cellular invasion. This compound also inhibited anchorage-independent cell growth, multicellular tumor spheroid survival/invasion, and metalloprotease activities. The anti-proliferative effects of ADQ were mediated by inhibition of the Akt pathway. In addition, ADQ reduced the expression of mesenchymal markers of cancer cells, which was associated with the suppressed expression of Twist1. In conclusion, ADQ successfully suppressed carcinogenic activity by inhibiting the Akt signaling pathway and Twist1, which suggests that ADQ may be an efficient candidate for cancer drug development.

Anti-Cancer Effects of Glaucarubinone in the Hepatocellular Carcinoma Cell Line Huh7 via Regulation of the Epithelial-To-Mesenchymal Transition-Associated Transcription Factor Twist1.

Hepatocellular carcinoma (HCC), the most common type of liver cancer, is a leading cause of cancer-related deaths. As HCC has a high mortality rate and its incidence is increasing worldwide, understanding and treating HCC are crucial for resolving major public health concerns. In the present study, wound healing screening assays were performed using natural product libraries to identify natural chemicals that can inhibit cancer cell migration. Glaucarubinone (GCB) showed a high potential for inhibiting cell migration. The anti-cancer effects of GCB were evaluated using the HCC cell line, Huh7. GCB showed anti-cancer effects, as verified by wound healing, cell migration, invasion, colony formation, and three-dimensional spheroid invasion assays. In addition, cells treated with GCB showed suppressed matrix metalloproteinase activities. Immunoblotting analyses of intracellular signaling pathways revealed that GCB regulated the levels of Twist1, a crucial transcription factor associated with epithelial-to-mesenchymal transition, and mitogen-activated protein kinase. The invasive ability of cancer cells was found to be decreased by the regulation of Twist1 protein levels. Furthermore, GCB downregulated phosphorylation of extracellular signal-regulated kinase. These results indicate that GCB exhibits anti-metastatic properties in Huh7 cells, suggesting that it could be used to treat HCC.

Inhibition of Inflammatory Responses by Centella asiatica via Suppression of IRAK1-TAK1 in Mouse Macrophages.

Centella asiatica (L.) Urb. (C. asiatica) has been widely treated for inflammation-related diseases in China for thousands of years. While C. asiatica showed relevant effects as traditional medicine, the mechanism of C. asiatica suppressing inflammation has not been thoroughly investigated. Therefore, this study was conducted to reveal the anti-inflammatory mechanism of methanol fraction from C. asiatica (MCA) at the molecular level in murine macrophages. Levels of inflammation-related mediators were observed with treatment of MCA. MCA significantly suppressed nitric oxide production and iNOS expression in RAW 264.7 macrophages. Prostaglandin E2 production was alleviated by MCA via the downregulation of cyclooxygenase-2. MCA treatment also reduced pro-inflammatory tumor necrosis factor-[Formula: see text] and interleukin (IL)-6 levels. LPS/D-GalN-induced acute hepatitis in mouse was alleviated by MCA treatment. In addition, MCA decreased the phosphorylation of inhibitory [Formula: see text]B[Formula: see text] (I[Formula: see text]B[Formula: see text]) at Ser32/36 and thereby blocked I[Formula: see text]B[Formula: see text] degradation. TXY motif phosphorylation in the activation loops of mitogen-activated protein kinases (MAPKs) was also suppressed by MCA treatment. Further investigation revealed that MCA inhibited transforming growth factor-[Formula: see text]-activated kinase 1 (TAK1) phosphorylation and IL-1 receptor-associated kinase (IRAK1) degradation, the upstream kinases activating nuclear factor [Formula: see text]B and MAPKs. Taken together, MCA exhibited anti-inflammatory properties via the downregulation of IRAK1-TAK1 signaling pathways.

Anti-cancer effects of ethanol extract of Reynoutria japonica Houtt. radix in human hepatocellular carcinoma cells via inhibition of MAPK and PI3K/Akt signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Reynoutria japonica Houtt. has been used as a traditional medicine of cancer in East Asia for thousands of years. However, the mechanism of the anti-cancer effect of R. japonica has not been investigated at the molecular level. The regulation of intracellular signaling pathways by the extract of R. japonica radix needs to be evaluated for a deeper understanding and application of the anti-cancer effect of R. japonica radix. AIM OF THE STUDY: The purpose of this study was to evaluate the inhibitory effects of the ethanol extracts of R. japonica radix (ERJR) on cancer metastasis and the regulation mechanism of metastasis by ERJR in human hepatocellular carcinomas. MATERIALS AND METHODS: Suppression of cancer metastasis by ERJR in SK-Hep1 and Huh7 cells were investigated. Prior to experiments, the cytotoxic effect of ERJR was examined by cell viability assays. To evaluate the inhibitory effects of ERJR on cancer metastasis, wound-healing assays, invasion assays, zymography, and multicellular tumor spheroids (MCTS) assays were performed. Molecular mechanisms in the suppressive regulation of metastasis by ERJR were verified by measuring the expression levels of metastatic markers, and the phosphorylation and protein levels of cancer metastasis-related signaling pathways. RESULTS: In all experiments, ERJR was used at a maximum concentration of 20 µg/ml, which did not show cytotoxicity in SK-Hep1 and Huh7 cells. We examined the inhibitory effects of ERJR on cancer metastasis. In wound-healing and invasion assays, ERJR treatment effectively suppressed the wound-recovery of Huh7 cells and inhibited the invasion ability of SK-Hep1 cells. Also, ERJR treatment significantly decreased the enzymatic activity of matrix metalloproteinase-2 and -9 in SK-Hep1 cells. ERJR suppressed the growth of MCTS in SK-Hep1 cells in a dose-dependent manner. These results indicated that ERJR effectively inhibited the invasive and proliferative ability of SK-Hep1 and Huh7 cells. Moreover, ERJR treatment reduced the expression levels of Snail1, Twist1, N-cadherin, and Vimentin, which are metastatic markers, by inhibiting the activation of protein kinase B and mitogen-activated protein kinases in SK-Hep1 cells. CONCLUSIONS: These results verified the molecular mechanism of ERJR that has been used in traditional anti-cancer remedy and suggest that it can be developed as a promising therapy for cancer metastasis in the future.

Anticancer effects of methanol extract of Myrmecodia platytyrea Becc. leaves against human hepatocellular carcinoma cells via inhibition of ERK and STAT3 signaling pathways.

Myrmecodia platytyrea Becc., a member of the Rubiaceae family, is found throughout Southeast Asia and has been traditionally used to treat cancer. However, there is limited pharmacological information on this plant. We investigated the anticancer effects of the methanol extract of Myrmecodia platytyrea Becc. leaves (MMPL) and determined the molecular mechanisms underlying the effects of MMPL on metastasis in human hepatocellular carcinoma (HCC) cells. MMPL dose-dependently inhibited cell migration and invasion in SK­Hep1 and Huh7 cells. In addition, MMPL strongly suppressed the enzymatic activity of matrix metalloproteinases (MMP­2 and MMP­9). Diminished telomerase activity by MMPL resulted in the suppression of both telomerase activity and telomerase-associated gene expression. The levels of urokinase-type plasminogen activator receptor (uPAR) expression as well as the phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) were also attenuated by MMPL. The above results collectively suggest that MMPL has anticancer effects in HCC and that MMPL can serve as an effective therapeutic agent for treating human liver cancer.

Dual-specificity phosphatase 5 acts as an anti-inflammatory regulator by inhibiting the ERK and NF-κB signaling pathways.

Although dual-specificity phosphatase 5 (DUSP5), which inactivates extracellular signal-regulated kinase (ERK), suppresses tumors in several types of cancer, its functional roles remain largely unknown. Here, we show that DUSP5 is induced during lipopolysaccharide (LPS)-mediated inflammation and inhibits nuclear factor-κB (NF-κB) activity. DUSP5 mRNA and protein expression increased transiently in LPS-stimulated RAW 264.7 cells and then returned to basal levels. DUSP5 overexpression in RAW 264.7 cells suppressed the production of pro-inflammatory tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), whereas knockdown of DUSP5 increased their expression. Investigation of two major inflammatory signaling pathways, mitogen-activated protein kinase (MAPK) and NF-κB, using activator protein-1 (AP-1) and NF-κB reporter plasmids, respectively, showed that NF-κB transcription activity was downregulated by DUSP5 in a phosphatase activity-independent manner whereas AP-1 activity was inhibited by DUSP5 phosphatase activity towards ERK,. Further investigation showed that DUSP5 directly interacts with transforming growth factor beta-activated kinase 1 (TAK1) and inhibitor of κB (IκB) kinases (IKKs) but not with IκBα. DUSP5 binding to IKKs interfered with the association of TAK1 with IKKs, suggesting that DUSP5 might act as a competitive inhibitor of TAK1-IKKs association. Therefore, we propose that DUSP5 negatively regulates ERK and NF-κB in a phosphatase activity-dependent and -independent manner, respectively.

Suppression of inflammation by the rhizome of Anemarrhena asphodeloides via regulation of nuclear factor-κB and p38 signal transduction pathways in macrophages.

The rhizome of Anemarrhena asphodeloides Bunge (A. asphodeloides) has been used as a traditional East Asian medicine for the treatment of various types of inflammatory disease. However, to the best of our knowledge, there have been no systemic studies regarding the molecular mechanisms of action of the A. asphodeloides rhizome anti-inflammatory effects. The aim of the present study was to elucidate the anti-inflammatory effects and underlying mechanism of action of ethanol extracts of the rhizome of A. asphodeloides (EAA) in murine macrophages. Non-cytotoxic concentrations of EAA (10-100 µg/ml) significantly decreased the production of NO and interleukin (IL)-6 in lipopolysaccharide (LPS)-stimulated macrophages, while the production of tumor necrosis factor-α was not regulated by EAA. EAA-mediated reduction of nitric oxide (NO) was due to reduced expression levels of inducible NO synthase (iNOS). Furthermore, protein expression levels of LPS-induced cyclooxygenase-2, another inflammatory enzyme, were alleviated in the presence of EAA. EAA-mediated reduction of those proinflammatory mediators was due to inhibition of nuclear factor-κB (NF-κB) and activator protein 1 transcriptional activities followed by the stabilization of inhibitor of κ Bα and inhibition of p38, respectively. These results indicate that EAA suppresses LPS-induced inflammatory responses by negatively regulating p38 and NF-κB, indicating that EAA is a candidate treatment for alleviating inflammation.