Expression Levels Of N-cadherin Research Articles

Impact of Prospero Homeobox-1 (PROX-1) οn the Oncogenic Phenotypes of Hepatocellular Carcinoma Cells.

Transcriptional factor prospero homeobox-1 (PROX-1) is crucial for the embryonic development of various organs and cell fate specification. It exhibits either an oncogenic or tumor suppressive activity depending on cancer types. However, the relationship between PROX-1 and hepatocellular carcinoma (HCC) remains obscure. This study was conducted to investigate the effect of PROX-1 on the invasive and oncogenic phenotypes of human HCC cells. The effect of PROX-1 on tumor cell behavior was investigated by using a pcDNA-myc vector and a small interfering RNA in HepG2 and Huh7 human HCC cell lines. Flow cytometry, migration, invasion, proliferation, and tube formation assays were performed. PROX-1 expression in human HCC cells was explored by western blotting. PROX-1 overexpression enhanced tumor cell proliferation and inhibited apoptosis and cell cycle arrest by modulating the activities of caspase-3, PARP, and cyclin-dependent kinase inhibitors, including p21, p27, and p57 in HCC cells. After PROX-1 overexpression, the number of migrating and invading HCC cells significantly increased, and the expression levels of N-cadherin and Snail increased in HCC cells. PROX-1 overexpression enhanced angiogenesis through increased VEGF-A and VEGF-C expression and decreased angiostatin expression. PROX-1 overexpression also increased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and forkhead box O1 (FOXO1) in HCC cells. After PROX-1 knockdown, their phosphorylation was reversed. PROX-1 overexpression is associated with the invasive and oncogenic phenotypes of human HCC cells via GSK-3β and FOXO1 phosphorylation.

A study on the significance of serine hydroxymethyl transferase expression and its role in bladder cancer

Bladder cancer (BLCA) is a common malignant tumor in urinary system all over the world. However, due to its high recurrence rate and complex causes, clinicians often have limited options for surgical and drug treatments. Recent researchs on the molecular mechanism of BLCA have reveals its biological progress and potential for early diagnosis. Serine hydroxymethyltransferase 1/2 (SHMT1/2) is a crucial enzyme in the one-carbon metabolism of tumor cells, and the expression levels of these isozymes have been found to be associated with the biological progression of various malignant tumors. However, the impact of SHMT1/2 on the biological progression of bladder cancer and its molecular regulation mechanism remain unclear. In this research utilizes BLCA clinical sample data, the TCGA database, and in vitro cell experiments to predict the expression levels of SHMT1/2 in BLCA. The findings indicate that SHMT1 remained unchanged, while SHMT2 expression is increased in BLCA, which was related to poor prognosis. Additionally, SHMT2 affects the growth, migration, and apoptosis of bladder cancer cells in vitro. It also influences the expression levels of E-cadherin and N-cadherin, ultimately impacting the malignant biological progression of bladder tumors. These results establish a correlation between SHMT2 and the malignant biological progression of BLCA, providing a theoretical basis for the early diagnosis and treatment of bladder cancer.

Epithelial-Mesenchymal Transformation Promotes the Progression of Hepatocellular Carcinoma through NF-κB/MMP9 Axis.

Primary liver cancer (PHC) stands as one of the most prevalent malignant diseases in clinical settings. Studies have indicated that transcatheter arterial chemoembolization (TACE) treatment exhibits superior clinical outcomes, potentially increasing the complete necrosis rate in patients with PHC. A correlation exists between the clinical outcomes of TACE surgery and the process of epithelial-mesenchymal transition (EMT), yet the underlying mechanism remains a mystery. Hence, it is crucial to investigate the impact and mechanism of EMT on hepatocellular carcinoma (HCC). Retrospectively, patients with advanced liver cancer who underwent TACE were selected and categorized into two groups based on the assessment of clinical efficacy: the effective group and the ineffective group. The expression levels of nuclear factor-kappa B (NF-κB), matrix metalloproteinase 9 (MMP9), Ki-67, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), Vimentin, E-cadherin, and N-cadherin in tumor tissues were evaluated using reverse transcription-polymerase chain reaction (RT-PCR). In vitro, Huh7 cells were cultured, and lentivirus infections were utilized to inhibit the overexpression of NF-κB and MMP9. The determination of EMT and cell viability was conducted through Cell Counting Kit-8 (CCK-8) assays, RT-PCR, and Western blot. Sixty patients diagnosed with advanced liver cancer were selected for the study. Based on their clinical outcomes, 30 patients with advanced hepatocellular carcinoma were categorized into the effective group, while the remaining 30 patients were categorized into the ineffective group. The results of the Western blot analysis indicated that, in comparison to the effective group, the expression levels of NF-κB, MMP9, Ki-67, Bcl-2, Vimentin, and N-cadherin were significantly higher in the tumor tissues of the ineffective group. Conversely, the expression of Bax and E-cadherin was notably lower in the effective group. Following the individual knockdown of NF-κB and MMP9, the cell experiments revealed a remarkable decrease in the expression levels of Ki-67, Bcl-2, Vimentin, and N-cadherin, whereas the expression of Bax and E-cadherin showed significant elevation (p < 0.05). Furthermore, there was a significant increase in cell viability and a decrease in cell apoptosis after the knockdown of NF-κB and MMP9. The NF-κB/MMP9 signaling axis serves as a pivotal regulator that fosters proliferation and impedes apoptosis in Huh7 cells by modulating the process of EMT.

Abstract 1819: Esculetin modulates SLC31A1 and ATP7B to influence copper homeostasis and trigger cell death in colorectal cancer

Abstract Background: Esculetin is a naturally occurring dihydroxycoumarin with anti-inflammatory, antioxidant, and anti-tumor activities. Copper ion transport proteins, notably SLC31A1 and ATP7B, are essential in maintaining intracellular copper homeostasis, averting disruptions that might lead to copper-induced cell death under normal physiological conditions. However, prior research has not explored Esculetin's role in inducing copper-induced cell death. Hence, this study aims to investigate the biological process of Esculetin-induced cell death and the molecular mechanism underlying dysregulation of intracellular copper ion homeostasis. Methods: Initially, the CCK-8 assay was used to determine the IC50 values of Esculetin on colorectal cancer cell lines (HCT116, HT29, SW480) and normal colonic epithelial cell lines (NCM460). In terms of biological behaviors, the effects of Esculetin on the proliferation of colorectal cancer cell lines were assessed using CCK-8, colony formation assay, and EdU staining. Flow cytometry was used to examine the impact of Esculetin on apoptosis and cell cycle distribution in colorectal cancer cells. A subcutaneous xenograft tumor model in nude mice was employed to evaluate the in vivo anti-tumor effects of Esculetin, while IHC staining was conducted to detect Ki-67 levels, and Western blotting was performed to assess EMT-related protein expression. To investigate copper ion homeostasis, qPCR was used to measure the expression levels of copper ion transporters SLC31A1 and ATP7B before and after Esculetin treatment. Results: The CCK-8 results revealed that the IC50 of Esculetin for normal colonic epithelial cell lines was significantly higher than that for colorectal cancer cell lines. Meanwhile, a notable inhibitory effect of Esculetin on the proliferation of colorectal cancer cells was observed. Additionally, Esculetin treatment suppressed colony formation and reduced the proportion of EdU-positive cells in colorectal cancer cells. Flow cytometry demonstrated an increase in apoptosis and S-phase arrest following Esculetin treatment. The subcutaneous xenograft tumor model showed significant inhibition of tumor growth by Esculetin, as evidenced by reduced Ki-67-positive cells in the Esculetin-treated group. Western blot analysis revealed a significant decrease in the expression levels of Vimentin and N-Cadherin and an increase in E-Cadherin expression. Furthermore, qPCR analysis post-Esculetin treatment showed an elevation in the inward copper transporter SLC31A1 levels, while the outward transporter ATP7B expression decreased. Conclusion: Esculetin induces dysregulation of intracellular copper ion homeostasis by upregulating SLC31A1 expression and downregulating ATP7B expression, leading to intracellular copper accumulation and subsequent disruption of copper homeostasis, thereby inducing copper-induced cell death. Citation Format: Fangyue Guo, Mengying Liu, Kaihe Zhu, Jiapeng Ru, Pei Li, Shanfeng Zhang. Esculetin modulates SLC31A1 and ATP7B to influence copper homeostasis and trigger cell death in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1819.

Plumbagin Regulates Snail to Inhibit Hepatocellular Carcinoma Epithelial-Mesenchymal Transition in vivo and in vitro.

Plumbagin (PL) has been shown to effe ctively inhibit autophagy, suppressing invasion and migration of hepatocellular carcinoma (HCC) cells. However, the specific mechanism remains unclear. This study aimed to investigate the effect of PL on tumor growth factor (TGF)-β-induced epithelial-mesenchymal transition (EMT) in HCC. Huh-7 cells were cultured, and in vivo models of EMT and HCC-associated lung metastasis were developed through tail vein and in situ injections of tumor cells. In vivo imaging and hematoxylin and eosin staining were used to evaluate HCC modeling and lung metastasis. After PL intervention, the expression levels of Snail, vimentin, E-cadherin, and N-cadherin in the liver were evaluated through immunohistochemistry and Western blot. An in vitro TGF-β-induced cell EMT model was used to detect Snail, vimentin, E-cadherin, and N-cadherin mRNA levels through a polymerase chain reaction. Their protein levels were detected by immunofluorescence staining and Western blot. In vivo experiments demonstrated that PL significantly reduced the expression of Snail, vimentin, and N-cadherin, while increasing the expression of E-cadherin at the protein levels, effectively inhibiting HCC and lung metastasis. In vitro experiments confirmed that PL up-regulated epithelial cell markers, down-regulated mesenchymal cell markers, and inhibited EMT levels in HCC cells. PL inhibits Snail expression, up-regulates E-cadherin expression, and down-regulates N-cadherin and vimentin expression, preventing EMT in HCC cells and reducing lung metastasis.

Curcumin suppresses metastasis of triple-negative breast cancer cells by modulating EMT signaling pathways: An integrated study of bioinformatics analysis.

This study aimed to use bioinformatics approaches for predicting the anticancer mechanisms of curcumin on triple-negative breast cancer (TNBC) and to verify these predictions through in vitro experiments. Initially, the Cell Counting Kit-8 (CCK8) assay was employed to rigorously investigate the influence of curcumin on the proliferative capacity of TNBC cells. Subsequently, flow cytometry was employed to meticulously assess the impact of curcumin on cellular apoptosis and the cell cycle regulation. Transwell assays were employed to meticulously evaluate the effect of curcumin on the motility of TNBC cells. RNA sequencing was conducted, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of differentially expressed genes, aiming to elucidate the potential anticancer mechanisms underlying curcumin's effects. To thoroughly elucidate the interactions among multiple proteins, we constructed a protein-protein interaction (PPI) network. Finally, the expression levels of several key proteins, including fibronectin, mTOR, β-Catenin, p-Akt, Akt, N-Cadherin, p-S6, and S6, were assessed using the western blot. The CCK8 assay results showed that curcumin significantly inhibited the proliferation of Hs578T and MDA-MB-231 cells. Flow cytometry results showed that curcumin induced apoptosis in these cells and arrested the cell cycle at the G2/M phase. Additionally, Transwell assay results showed that curcumin effectively reduced the motility of Hs578T and MDA-MB-231 cells. Enrichment analysis of RNA sequencing data showed that the mechanism of action of curcumin was significantly associated with signaling pathways such as pathways in cancer, focal adhesion, and PI3K-Akt signaling pathways. Subsequently, we constructed a protein-protein interaction network to elucidate the interactions among multiple proteins. Finally, Western blotting analysis showed that curcumin significantly decreased the expression levels of key proteins including Fibronectin, mTOR, β-Catenin, p-Akt, Akt, N-Cadherin, p-S6, and S6. Curcumin exhibits its therapeutic potential in TNBC by modulating multiple signaling pathways. It may inhibit the epithelial-mesenchymal transition process by downregulating the expression of proteins involved in the mTOR and PI3K-Akt signaling pathways, thereby suppressing the motility of TNBC cells. These findings provide experimental evidence for considering curcumin as a potential therapeutic strategy in the treatment of TNBC.

JAM3: A prognostic biomarker for bladder cancer via epithelial–mesenchymal transition regulation

Understanding the intricate relationship between prognosis, immune function, and molecular markers in bladder cancer (BC) demands sophisticated analytical methods. To identify novel biomarkers for predicting prognosis and immune function in BC patients, we combined weighted gene co-expression network analysis (WGCNA) and least absolute shrinkage and selection operator (LASSO) regression analysis. This was conducted using data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Ultimately, we screened the junctional adhesion molecule 3 (JAM3) as an independent risk factor in BC. High levels of JAM3 were linked to adverse clinical parameters, such as higher T and N stages. Additionally, a JAM3-based nomogram model accurately predicted 1-, 3- and 5-year survival rates of BC patients, indicating potential clinical utility. Functional enrichment analysis revealed that high JAM3 expression activated the calcium signaling pathway, the extracellular matrix (ECM)-receptor interaction, and the PI3K-Akt signaling pathway, and was positively correlated with genes associated with epithelial–mesenchymal transition (EMT). Subsequently, we found that overexpression of JAM3 promoted the migration and invasion abilities in BC cells, regulating the expression levels of N-Cadherin, matrix metallopeptidase 2 (MMP2), and Claudin-1 thereby promoting EMT levels. Additionally, we showed that JAM3 was negatively correlated with anti-tumor immune cells such as CD8+T cells, while positively correlated with pro-tumor immune cells such as M2 macrophages, suggesting its involvement in immune cell infiltration. The immune checkpoint CD200 also showed a positive correlation with JAM3. Our findings revealed that elevated JAM3 levels are predictive of poor prognosis and immune cell infiltration in BC patients by regulating the EMT process.

TGF-β2-induced alterations of m6A methylation in hTERT RPE-1 cells

N6-methyladenosine (m6A) is a major type of RNA modification implicated in various pathophysiological processes. Transforming growth factor β2 (TGF-β2) induces epithelial-mesenchymal transition (EMT) in retinal pigmental epithelial (RPE) cells and promotes the progression of proliferative vitreoretinopathy (PVR). However, the role of m6A methylation in the EMT of human telomerase reverse transcriptase (hTERT) retinal pigmental epithelium (RPE)-1 cells has not been clarified. Here, we extracted RNA from RPE cells subjected to 0 or 20 ng/mL TGF-β2 for 72 h and identified differentially methylated genes (DMGs) by m6A-Seq and differentially expressed genes (DEGs) by RNA-Seq. We selected the genes related to EMT by conjoint m6A-Seq/RNA-Seq analysis and verified them by qRT-PCR. We then confirmed the function of m6A methylation in the EMT of RPE cells by knocking down the methyltransferase METTL3 and the m6A reading protein YTHDF1. Sequencing yielded 5814 DMGs and 1607 DEGs. Conjoint analysis selected 467 genes altered at the m6A and RNA levels that are closely associated with the EMT-related TGF-β, AGE-RAGE, PI3K-Akt, P53, and Wnt signaling pathways. We also identified ten core EMT genes ACTG2, BMP6, CDH2, LOXL2, SNAIL1, SPARC, BMP4, EMP3, FOXM1, and MYC. Their RNA levels were evaluated by qRT-PCR and were consistent with the sequencing results. We observed that METTL3 knockdown enhanced RPE cell migration and significantly upregulated the EMT markers N-cadherin (encoded by CDH2), fibronectin (FN), Snail family transcription repressor (SLUG), and vimentin. However, YTHDF1 knockdown had the opposite effects and decreased both cell migration and the N-cadherin, FN, and SLUG expression levels. The present study clarified TGF-β2-induced m6A- and RNA-level differences in RPE cells, indicated that m6A methylation might regulate EMT marker expression, and showed that m6A could regulate TGF-β2-induced EMT.

MiR-128-3p inhibits the proliferation, migration and invasion of human nasopharyngeal carcinoma cells via EMT

Purpose: To investigate the effect of overexpressing miR-128-3p on the migration, invasion, and proliferation of human nasopharyngeal cancer cells, and associated pathways.Methods: Cell counting kit-8 (CCK-8) and clone creation tests were used to investigate the effect of miR-128-3p inhibitor on the growth of C666-1 cells. The inhibitor control and miR-128-3p inhibitor were transfected into human nasopharyngeal cancer cells (C666-1) and allowed to incubate for 48 h. Cell migration and invasion assays were conducted using Transwell and Scratch assays. Cell cycle was assessed using propidium iodide (PI) single-staining method, while expression of miR-128-3p was evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Expression levels of vimentin, E-cadherin, and N-cadherin were determined by Western blot.Results: MiR-128-3p inhibitor transfection in C666-1 cells significantly increased cell proliferation, cell viability, and clonal proliferation, and also significantly reduced miR-128-3p expression levels, and enhanced cell migration and invasion. In C666-1 cells, inhibition of miR-128-3p induced epithelial mesenchymal transition (EMT).Conclusion: MiR-128-3p inhibits nasopharyngeal cancer cells from proliferating, migrating, and invading by regulating epithelial mesenchymal transition. MiR-128-3p may therefore be a viable therapeutic target for nasopharyngeal cancer. Future studies are required to yield pertinent data in support of this hypothesis.

Systematic analysis of the role of LDHs subtype in pan-cancer demonstrates the importance of LDHD in the prognosis of hepatocellular carcinoma patients

BackgroundLactate dehydrogenase (LDHs) is an enzyme involved in anaerobic glycolysis, including LDHA, LDHB, LDHC and LDHD. Given the regulatory role in the biological progression of certain tumors, we analyzed the role of LDHs in pan-cancers.MethodsCox regression, Kaplan–Meier curves, Receiver Operating Characteristic (ROC) curves, and correlation of clinical indicators in tumor patients were used to assess the prognostic significance of LDHs in pan-cancer. The TCGA, HPA, TIMER, UALCAN, TISIDB, and Cellminer databases were used to investigate the correlation between the expression of LDHs and immune subtypes, immune checkpoint genes, methylation levels, tumor mutational load, microsatellite instability, tumor-infiltrating immune cells and drug sensitivity. The cBioPortal database was also used to identify genomic abnormalities of LDHs in pan-cancer. A comprehensive assessment of the biological functions of LDHs was performed using GSEA. In vitro, HepG2 and Huh7 cells were transfected with LDHD siRNA and GFP-LDHD, the proliferation capacity of cells was examined using CCK-8, EdU, and colony formation assays; the migration and invasion of cells was detected by wound healing and transwell assays; western blotting was used to detect the levels of MMP-2, MMP-9, E-cadherin, N-cadherin and Akt phosphorylation.ResultsLDHs were differentially expressed in a variety of human tumor tissues. LDHs subtypes can act as pro-oncogenes or anti-oncogenes in different types of cancer and have an impact on the prognosis of patients with tumors by influencing their clinicopathological characteristics. LDHs were differentially expressed in tumor immune subtypes and molecular subtypes. In addition, LDHs expression correlated with immune checkpoint genes, tumor mutational load, and microsatellite instability. LDHD was identified to play an important role in the prognosis of HCC patients, according to a comprehensive analysis of LDHs in pan-cancer. In HepG2 and Huh7 cells, knockdown of LDHD promoted cell proliferation, migration, and invasion, promoted the protein expression levels of MMP-2, MMP-9, N-cadherin, and Akt phosphorylation, but inhibited the protein expression level of E-cadherin. In addition, LDHD overexpression showed the opposite changes.ConclusionLDHs subtypes can be used as potential prognostic markers for certain cancers. Prognostic and immunotherapeutic analysis indicated that LDHD plays an important role in the prognosis of HCC patients. In vitro experiments revealed that LDHD can affect HCC proliferation, migration, and invasion by regulating MMPs expression and EMT via Akt signaling pathway, which provides a new perspective on the anti-cancer molecular mechanism of LDHD in HCC.

ZNF692 promotes cell proliferation, invasion and migration of human prostate cancer cells by targeting the EMT signaling pathway

BackgroundProstate cancer poses a considerable threat to human health. At present, the mechanism of tumor progression remains unclear. ZNF692 is overexpressed in many tumors, and the high expression of ZNF692 is correlated with tumor aggressiveness and tumor phenotype of prostate cancer, suggesting that ZNF692 may play an important role in tumor biology of prostate cancer. This paper aims to elucidate the relationship between them.MethodsThe expression level of ZNF692 was verified in normal prostate cells (RWPE-1) and prostate cancer cells (LNCaP, PC3, DU145). PC3 cells were selected to construct the ZNF692 knockout prostate cancer cell line. The changes of cell proliferation, apoptosis, invasion and metastasis were detected by CCK8, Edu staining, Transwell assay and scratch assay. The expression levels of related proteins were detected by Western blot.ResultsAt the cellular level, ZNF692 was overexpressed to varying degrees in prostate cancer cell lines, with the highest expression in PC3 cell lines. CCK8 and Edu results showed that the proliferation of prostate cancer PC3 cells that knocked down ZNF692 was slowed. Transwell assay and scratch assay showed reduced invasion and migration of prostate cancer PC3 cells that knocked out ZNF692. Flow cytometry showed that the apoptosis rate of prostate cancer PC3 cells after ZNF692 knockout was increased. In addition, after ZNF692 silencing, the expression level of epithelial phenotype E-cadherin increased in PC3 cells, while the expression level of interstitial phenotype N-cadherin, Vimentin, c-Myc, and CyclinA1 decreased. The state of prostate cancer PC3 cells that overexpressed ZNF692 was reversed from the state after ZNF692 was knocked down.ConclusionZNF692 can be used as a new prognostic marker and a potential biologic therapeutic target for PCa. By inhibiting the expression of c-myc and cyclinA1, the EMT signaling pathway is regulated to provide evidence for its potential molecular mechanism.

Platelet-derived Growth Factor Activates Pericytes in the Microvessels of Chronic Subdural Hematoma Outer Membranes.

Angiogenesis is one of the growth mechanisms of chronic subdural hematoma (CSDH). Pericytes have been implicated in the capillary sprouting during angiogenesis and are involved in brain ischemia and diabetic retinopathy. This study examined the pericyte expressions in CSDH outer membranes obtained during trepanation surgery. Eight samples of CSDH outer membranes and 35 samples of CSDH fluid were included. NG2, N-cadherin, VE-cadherin, Tie-2, endothelial nitric oxide synthase (eNOS), platelet-derived growth factor (PDGF) receptor-β (PDGFR-β), a well-known marker of pericytes, phosphorylated PDGFR-β at Tyr751, and β-actin expressions, were examined using western blot analysis. PDGFR-β, N-cadherin, and Tie-2 expression levels were also examined using immunohistochemistry. The concentrations of PDGF-BB in CSDH fluid samples were measured using enzyme-linked immunosorbent assay kits. NG2, N-cadherin, VE-cadherin, Tie-2, eNOS, PDGFR-β, and eNOS expressions in CSDH outer membranes were confirmed in all cases. Furthermore, phosphorylated PDGFR-β at Tyr751 was also detected. In addition, PDGFR-β, N-cadherin, and Tie-2 expressions were localized to the endothelial cells of the vessels within CSDH outer membranes by immunohistochemistry. The concentration of PDGF-BB in CSDH fluids was significantly higher than that in cerebrospinal fluid. These findings indicate that PDGF activates pericytes in the microvessels of CSDH outer membranes and suggest that pericytes are crucial in CSDH angiogenesis through the PDGF/PDGFR-β signaling pathway.

SB431542 partially inhibits high glucose-induced EMT by restoring mitochondrial homeostasis in RPE cells

BackgroundThe epithelial–mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells participated in the development of retinal fibrosis. SB431542 is a small molecule inhibitor with inhibitory effects on the ALK4, ALK5 and ALK7. Our study aimed to explore the effect of SB431542 on the EMT of RPE cells and to provide new ideas for the treatment of retinal fibrosis.MethodsWe performed fundus fluorescein angiography, optical coherence tomography and hematoxylin–eosin staining in vivo to observe the effect of SB431542 on choroidal neovascularization (CNV)-induced retinopathy. The proliferation, migration, cytoskeleton, adhesion, reactive oxygen species (ROS), mitochondrial morphology and membrane potential of RPE cells were observed in vitro through fluorescein diacetate staining, Cell Counting Kit-8 experiment, wound healing assay, phalloidin staining, immunofluorescence, MitoSOX, DCFH-DA, MitoTracker and JC-10 staining. Western blot, reverse transcription quantitative and immunofluorescence were used to detect the expression of EMT–related markers, pERK1/2, pGSK3β and β-catenin.ResultsSB431542 significantly alleviated retinopathy in the CNV model. The proliferation, migration and adhesion in RPE cells decreased to a certain extent in SB431542 treatment. SB431542 partially normalized the structure of RPE cells. The expression levels of E-cadherin increased, while the expression levels of laminin and N-cadherin decreased with SB431542 treatment. SB431542 reduced the production of total ROS, mitochondrial SOX and recovered the mitochondrial membrane potential to a certain degree. In addition, our study showed that SB431542 downregulated the phosphorylation of ERK1/2, GSK3β and the expression of β-catenin.ConclusionSB431542 improved EMT in RPE cells by maintaining mitochondrial homeostasis via the ERK1/2 and GSK3β/β-catenin pathways.1HzEs7TmoETCuNYu8FcEmbVideo Graphical SB431542 inhibits EMT in RPE cells under high glucose conditions.

TGF-β1 induces epithelial-to-mesenchymal transition in chronic rhinosinusitis with nasal polyps through microRNA-182.

Epithelial-to-mesenchymal transition (EMT) in nasal epithelial cells is involved in tissue remodeling of chronic rhinosinusitis with nasal polyps (CRSwNP). Our study investigated the molecular mechanisms that microRNA-182 (miR-182) regulated EMT in eosinophilic (Eos) and non-eosinophilic (non-Eos) CRSwNP. To investigate the mechanism by which miR-182 regulates EMT in human nasal epithelial cells (hNEPCs). The expression of EMT markers (E-cadherin, N-cadherin and vimentin), transforming growth factor (TGF)-β1, and miR-182 were determined by western blotting and reverse transcription-quantitative PCR (RT-qPCR). Fluorescence in situ hybridization (FISH) was used to detect the miR-182 localization. Additionally, EMT markers expression and cell morphology changes were checked upon treatment with TGF-β1, or TGF-β1 with miR-182 inhibitor, or miR-182 mimics, or miR-182 inhibitor alone in hNEPCs. In both Eos CRSwNP and non-Eos CRSwNP, the expression levels of E-cadherin were downregulated while the expression levels of N-cadherin, vimentin, TGF-β1 and miR-182 were significantly upregulated compared with control nasal tissues. Additionally, more significant changes in these EMT markers were observed in the Eos-CRSwNP when compared with the non-Eos CRSwNP. Invitro hNEPCs model, TGF-β1 upregulated miR-182 expression and promoted EMT in hNEPCs, indicated by changes in cell morphology and EMT markers expression. Furthermore, these upregulations were reversed by miR-182 inhibitor. This study showed that miR-182-induced EMT in response to TGF-β1 might promote nasal polypogenesis in both Eos CRSwNP and non-Eos CRSwNP, thus providing potential targets for the future development of novel therapeutic approaches for the management of CRSwNP.

MiR-383-5p inhibits the proliferation and migration of lung adenocarcinoma cells by targeting SHMT2.

Purpose: To explore the effects of miR-383-5p and serine hydroxymethyltransferase 2 (SHMT2) on the proliferation and migration of lung adenocarcinoma cells. Methods: SHMT2 expression in lung adenocarcinoma and normal tissues was investigated using The Cancer Genome Atlas database. Immunohistochemical analysis was performed to confirm SHMT2 expression in lung adenocarcinoma and adjacent normal lung tissues. Bioinformatics analysis and luciferase reporter assays were used to analyze the relationship between miR-383-5p and SHMT2 expression. The protein expression levels of SHMT2, vimentin, N-cadherin, E-cadherin, Bcl-2, and cyclinD1 were analyzed using western blotting. The reverse transcription-quantitative polymerase chain reaction was used to detect SHMT2 knockdown efficiency, miR-383-5p overexpression, and inhibition efficiency. The proliferative ability of cells was detected using the Cell Counting Kit-8 assay. The Transwell assay was used to detect the migration ability of cells. Results: SHMT2 expression was significantly increased in patients with lung adenocarcinoma compared to that in control patients; the higher the SHMT2 expression the worse the outcomes were in patients with lung adenocarcinoma. SHMT2 knockdown inhibited the proliferation, migration, and epithelial-mesenchymal transition of lung adenocarcinoma A549 and H1299 cells. MiR-383-5p directly targeted and downregulated SHMT2 in A549 and H1299 cells. The effects of miRNA-383-5p on the proliferation and migration of these cells differed from those of SHMT2. Exogenous overexpression of SHMT2 reversed the miR-383-5p-induced proliferation and migration inhibition in A549 and H1299 cells. Conclusion: MiR-383-5p inhibits the proliferation and migration of lung adenocarcinoma cells by targeting and downregulating SHMT2.

Capsaicin and Cold exposure promote EMT-mediated premetastatic niche formation to facilitate colorectal cancer metastasis.

Colorectal cancer (CRC) is a common malignant tumor worldwide. Capsaicin and cold exposure were positively correlated with CRC metastasis. However, the mechanisms of action underlying capsaicin and cold exposure in 1,2-dimethylhyrazine (DMH)-induced CRC remain unknown. Multiple assays were utilized in the present study, including methylene blue, hematoxylin eosin (H&E) and immunohistochemistry (IHC) staining, western blotting and Duolink proximity ligation assay (PLA), in order to assess the influence of capsaicin and cold exposure on CRC rat models induced by DMH. The present study reported that capsaicin and cold exposure treatment significantly increased the size and number of colonic tumors, and the CRC metastasis rate in the capsaicin and cold exposure groups was higher than that in DMH model group.Moreover, it was observed that capsaicin and cold exposure increased mRNA and protein expression levels of LAMC2 and integrin-β1 induced by DMH. Duolink PLA results indicated that cold exposure and capsaicin significantly promoted interaction formation between LAMC2 and ITGB1 in CRC rats induced by DMH. Furthermore, western blot and IHC analysis confirmed that cold exposure and capsaicin inhibited DMH-induced decreases in the expression levels of E-cadherin, and increases in the expression levels of p-FAK, Snails, Fibronectin and N-cadherin. In addition, the serum levels of IL-1β and IL-6 in capsaicin and cold exposure group were higher than those of model group. In conclusion, our study suggests that both capsaicin and cold exposure may contribute to EMT-mediated the formation of premetastatic niche, which may lead to CRC metastasis by activating the early interaction between LAMC2 and integrin-β1.

GLUL stabilizes N-Cadherin by antagonizing β-Catenin to inhibit the progresses of gastric cancer

Glutamate-ammonia ligase (GLUL, also known as glutamine synthetase) is a crucial enzyme that catalyzes ammonium and glutamate into glutamine in the ATP-dependent condensation. Although GLUL plays a critical role in multiple cancers, the expression and function of GLUL in gastric cancer remain unclear. In the present study, we have found that the expression level of GLUL was significantly lower in gastric cancer tissues compared with adjacent normal tissues, and correlated with N stage and TNM stage, and low GLUL expression predicted poor survival for gastric cancer patients. Knockdown of GLUL promoted the growth, migration, invasion and metastasis of gastric cancer cells in vitro and in vivo, and vice versa, which was independent of its enzyme activity. Mechanistically, GLUL competed with β-Catenin to bind to N-Cadherin, increased the stability of N-Cadherin and decreased the stability of β-Catenin by alerting their ubiquitination. Furthermore, there were lower N-Cadherin and higher β-Catenin expression levels in gastric cancer tissues compared with adjacent normal tissues. GLUL protein expression was correlated with that of N-Cadherin, and could be the independent prognostic factor in gastric cancer. Our findings reveal that GLUL stabilizes N-Cadherin by antagonizing β-Catenin to inhibit the progress of gastric cancer.

Polyphyllin I attenuates the invasion and metastasis via downregulating GRP78 in drug-resistant hepatocellular carcinoma cells.

Drug resistance to chemotherapy agents presents a major obstacle to the effective treatment of hepatocellular carcinoma (HCC), a common type of liver cancer. Increasing evidence indicates a link between drug resistance and the recurrence of HCC. Polyphyllin I (PPI), a promising pharmaceutical candidate, has shown potential therapeutic advantages in the treatment of sorafenib-resistant hepatocellular carcinoma (SR-HCC cells). In this study, we sought to investigate the mechanism underlying the inhibitory effect of PPI on the invasion and metastasis of SR-HCC cells. Our in vitro studies included scratch wound-healing migration assays and transwell assays to examine PPI's effect on HCC cell migration and invasion. Flow cytometry was employed to analyze the accumulation or efflux of chemotherapy drugs. The results of these experiments demonstrated that PPI increased the susceptibility of HCC to sorafenib while inhibiting SR-HCC cell growth, migration, and invasion. Molecular docking analysis revealed that PPI exhibited a higher binding affinity with GRP78. Western blot analysis and immunofluorescence experiments showed that PPI reduced the expression of GRP78, E-cadherin, N-cadherin, Vimentin, and ABCG2 in SR-HCC cells. Interference with and overproduction of GRP78 in vitro impacted the proliferation, migration, invasion, and metastasis of HCC cells. Further examination revealed that PPI hindered the expression of GRP78 protein, resulting in a suppressive effect on SR-HCC cell migration and invasion. Histological examination of tumor tissue substantiated that administering PPI via gavage to HepG2/S xenograft nude mice inhibited tumor growth and significantly reduced tumor size, as evidenced by xenograft experiments involving nude mice. Hematoxylin and eosin (HE) staining of tumor tissue specimens, along with immunohistochemistry (IHC), were conducted to evaluate the expression levels of Ki67, GRP78, N-cadherin, Vimentin, and ABCG2. The results indicated that PPI administration decreased the levels of proteins associated with metastasis and markers of drug resistance in tumor tissues, impeding tumor growth and spread. Overall, our findings demonstrated that PPI effectively suppressed the viability, proliferation, invasion, and metastasis of SR-HCC cells both in vitro and in vivo by modulating GRP78 activity. These findings provide new insights into the mechanism of PPI inhibition of SR-HCC cell invasion and metastasis, highlighting PPI as a potential treatment option for sorafenib-resistant HCC.

Anticancer and anti-metastasis activity of 1,25 dihydroxycholecalciferols and genistein in MCF-7 and MDA-MB-231 breast cancer cell lines

A powerful steroid hormone precursor, 1,25 dihydroxycholecalciferols (1,25(OH)2D3), and dietary phytoestrogen (genistein) are essential compounds that act by binding to nuclear receptors and altering gene expression. They have many biological benefits, some of which have anticancer properties. We studied the impact of 1,25(OH)2D3 and genistein on the proliferation, progression, and metastasis of MCF-7 and MDA-MB-231 cells when they were used alone or in combination and investigated whether there was a synergistic effect between genistein and 1,25(OH)2D3. To achieve these goals, a variety of assays, including flow cytometry, cell invasion assays, cell adhesion assays, Western blotting, and RT‒PCR, were used.Our findings showed that genistein, 1,25(OH)2D3, and the two combined all effectively declined the growth of MCF-7 and MDA-MB-231 cells by arresting the cells in the G0/G1 phase and inducing an apoptotic pathway. Stimulation of apoptosis was achieved by upregulating the expression of BAX and CASP3 genes and downregulating the expression levels of BCL-2 gene. Furthermore, both compounds suppress metastasis by reducing cell adhesion and cell migration/invasion by elevating the expression level of E-cadherin and reducing the expression level of P-cadherin and N-cadherin. Additionally, both genistein and 1,25(OH)2D3 increased the expression level of ERK1 and reduced the expression levels of JNK, p38, Ras, and MEK proteins, which reduced metastasis, enhanced the response to cancer treatment, and improved overall survival. Thus, genistein and 1,25(OH)2D3 can both be considered key candidates in the search for new breast cancer treatments.

Extracellular Mechanical Stimuli Alters the Metastatic Progression of Prostate Cancer Cells within 3D Tissue Matrix.

This study aimed to understand extracellular mechanical stimuli's effect on prostate cancer cells' metastatic progression within a three-dimensional (3D) bone-like microenvironment. In this study, a mechanical loading platform, EQUicycler, has been employed to create physiologically relevant static and cyclic mechanical stimuli to a prostate cancer cell (PC-3)-embedded 3D tissue matrix. Three mechanical stimuli conditions were applied: control (no loading), cyclic (1% strain at 1 Hz), and static mechanical stimuli (1% strain). The changes in prostate cancer cells' cytoskeletal reorganization, polarity (elongation index), proliferation, expression level of N-Cadherin (metastasis-associated gene), and migratory potential within the 3D collagen structures were assessed upon mechanical stimuli. The results have shown that static mechanical stimuli increased the metastasis progression factors, including cell elongation (p < 0.001), cellular F-actin accumulation (p < 0.001), actin polymerization (p < 0.001), N-Cadherin gene expression, and invasion capacity of PC-3 cells within a bone-like microenvironment compared to its cyclic and control loading counterparts. This study established a novel system for studying metastatic cancer cells within bone and enables the creation of biomimetic in vitro models for cancer research and mechanobiology.