Inhibition Of Epithelial-mesenchymal Transition Research Articles

Investigation of in-vitro Anti-Cancer and Apoptotic Potential of Garlic-Derived Nanovesicles against Prostate and Cervical Cancer Cell Lines.

Investigate the anti-cancerous potential of garlic-derived nanovesicles (GDNVs), exploring their cytotoxic effects on HeLa and PC-3 cell lines, and elucidate the underlying mechanisms, including apoptosis induction and inhibition of epithelial-mesenchymal transition (EMT). GDNVs were isolated using differential centrifugation and ultracentrifugation. Characterization was performed through dynamic light scattering (DLS), field-emission scanning electron microscopy (FESEM), and Fourier-transform infrared spectroscopy (FTIR). Cytotoxicity assessments on HeLa and PC-3 cell lines using MTT assay. Apoptosis induction was evaluated through nuclear morphology changes and quantification of apoptotic cells using DAPI and PI/annexin V analysis. Western blot of apoptosis-related proteins (bcl-2, bax, caspase-3) was analysed. Anti-metastatic potential was assessed using wound healing assay and EMT transition inhibition. Garlic-derived nanovesicles (GDNVs), characterized by a size of 134.2 nm, demonstrated a substantial and dose- as well as time-dependent anti-proliferative impact on HeLa and PC-3 cell lines. The induction of apoptosis was unequivocally established through discernible modifications in nuclear morphology. The apoptotic cell count in HeLa and PC-3 cells increased by 42.4 ± 4.2% and 38.2 ± 3.2%, respectively. Comprehensive Western blot demonstrated alterations in the expression of key apoptotic regulators, namely bcl-2, bax, and caspase-3, providing robust evidence for the initiation of apoptosis. Furthermore, GDNVs exerted a significant inhibitory effect (p < 0.001) on the migratory potential of both HeLa and PC-3 cells. Moreover, there was a discernible association between GDNVs and the suppression of Epithelial-Mesenchymal Transition (EMT), emphasizing their role in impeding the metastatic potential of these cancer cell lines. This study establishes, for the first time, the anti-cancerous potential of GDNVs. The observed dose- and time-dependent anti-proliferative effects, selective cytotoxicity, apoptosis induction, and anti-migratory potential highlight GDNVs as a promising candidate for cancer treatment.

P121 Characterisation of the miRNAome in Crohn’s Disease patients reveals transcriptomic changes associated with tissue composition and drug treatment

Abstract Background Gene regulation plays a major role in Crohn's Disease (CD) susceptibility and drug response. MicroRNA (miRNA)-mediated mechanisms tune gene expression at the post-transcriptional level. MiRNAs are 20-to-25-nt long non-coding RNAs that repress protein expression by direct inhibition or cleavage of the targets. Evaluating the miRNAome is emerging as a promising tool to clarify pathogenesis and identify promising biomarkers. We characterised the regulatory miRNAome across disease subtypes, gut tissue locations and infliximab treatment in CD. Methods We generated 120 transcriptomic profiles from terminal ileum and left colon biopsies from Hospital Universitario de la Princesa (Madrid). This cohort is composed of 10 patients with active endoscopy, 10 quiescent CD cases, and 10 controls (30 in total). In a balanced setting, half of the samples were incubated with infliximab for 18 hours at 37ºC and the rest with basal medium (Table 1). We carried out an exploratory analysis to gain insights into the determinants of miRNAome variability, including variance partition analyses and linear mixed modelling to detect significantly altered miRNAs. We also harnessed results from several publications including GSE16879 dataset to perform a comparative study with infliximab response signatures. Results Tissue location is the primary determinant of miRNA expression variability. The Variance Partition model identified 30 top miRNAs whose variability is maximised across regions. However, patient-specific effects also play a relevant role. We also identified 96 differentially expressed miRNAs between terminal ileum and left colon, with an overrepresentation of molecules involved in stress and developmental pathways (Fig.1). Among these, we find miR200s and miR429, that are associated with suppression of epithelial-mesenchymal transition and are up-regulated in colon more than in ileum. Down-regulation of miR196 is also shown in terminal ileum. Regarding the effects of infliximab treatment, we detected nine deregulated miRNAs which are associated with cellular response pathways. These include miR200s and miR192, which targets NOD2 and down-regulates NF-κB. Our results show an up-regulation of miR192 upon infliximab incubation (log2FC=2.37), which is associated with a reduction in TNFα expression and, consequently, with induced overexpression of miR192. We conclude that this miRNA is a potential target for therapy development. Conclusion In short, tissue location, and to a lesser extent disease subtype, are the main determinants of miRNAome in CD. A few treatment-responsive miRNAs like miR192 and miR200s are identified, in spite of detection challenges, emphasizing the need to improve our comprehension of the miRNA dynamics associated with drug response.

The role of alternative polyadenylation in epithelial-mesenchymal transition of non-small cell lung cancer.

The metastatic non-small cell lung cancer (NSCLC) is one of the cancers with high incidence, poor survival, and limited treatment. Epithelial-mesenchymal transition (EMT) is the first step by which an early tumor converts to an invasive one. Studying the underlying mechanisms of EMT can help the understanding of cancer metastasis and improve the treatment. In this study, 1013 NSCLC patients and 123 NSCLC cell lines are deeply analyzed for the potential roles of alternative polyadenylation (APA) in the EMT process. A trend of shorter 3'-UTRs (three prime untranslated region) is discovered in the mesenchymal samples. The identification of EMT-related APA events highlights the proximal poly(A) selection of CARM1. It is a pathological biomarker of mesenchymal tumor and cancer metastasis through losing miRNA binding to upregulate the EMT inducer of CARM1 and releasing miRNAs to downregulate the EMT inhibitor of RBM47. The crucial role of this APA event in EMT also guides its effect on drug responses. The patients with shorter 3'-UTR of CARM1 are more benefit from chemotherapy drugs, especially cisplatin. A stratification of NSCLC patients based on this APA event is useful for chemotherapy design in future clinics.

Oleoylethanolamide attenuates acute-to-chronic kidney injury: in vivo and in vitro evidence of PPAR-α involvement

Chronic kidney disease (CKD) development after acute kidney injury (AKI) involves multiple mechanisms, including inflammation, epithelial-mesenchymal transition (EMT), and extracellular matrix deposition, leading to progressive tubulointerstitial fibrosis. Recently, a central role for peroxisome-proliferator activated receptor (PPAR)-α has been addressed in preserving kidney function during AKI. Among endogenous lipid mediators, oleoylethanolamide (OEA), a PPAR-α agonist, has been studied for its metabolic and anti-inflammatory effects. Here, we have investigated OEA effects on folic acid (FA)-induced kidney injury in mice and the underlying mechanisms. OEA improved kidney function, normalized urine output, and reduced serum BUN, creatinine, and albuminuria. Moreover, OEA attenuated tubular epithelial injury, as shown by histological analysis, and decreased expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1. Gene expression analysis of kidney tissue indicated that OEA limited immune cell infiltration and inflammation. Moreover, OEA significantly inhibited Wnt7b and Catnb1 gene transcription and α-smooth muscle actin expression, indicating suppression of EMT. Accordingly, OEA exhibited an anti-fibrotic effect, as shown by Masson staining and the reduced levels of transforming growth factor (TGF)-β1, fibronectin, and collagen IV. Mechanistically, the nephroprotective effect of OEA was related to PPAR-α activation since OEA failed to exert its beneficial activity in FA-insulted PPAR-α-/- mice. PPAR-α involvement was also confirmed in HK2 cells where GW6471, a PPAR-α antagonist, blunted OEA activity on the TGF-β1 signalling pathway and associated pro-inflammatory and fibrotic patterns. Our findings revealed that OEA counteracts kidney injury by controlling inflammation and fibrosis, making it an effective therapeutic tool for limiting AKI to CKD progression.

Unraveling the molecular mechanisms of cell migration impairment and apoptosis associated with steroid sulfatase deficiency: Implications for X-linked ichthyosis

Steroid sulfatase (STS) deficiency is responsible for X-linked ichthyosis (XLI), a genetic disorder characterized by rough and dry skin caused by excessive keratinization. The impaired keratinization process leads to reduced cell mobility and increased apoptosis, which can cause an excessive buildup of the stratum corneum. In this study, we investigated the mechanisms underlying XLI and found that STS deficiency reduces cell mobility and increases apoptosis in human keratinocyte HaCaT cells. To explore these mechanisms further, RNA-sequencing was conducted on skin tissues from STS transgenic and knockout mice. Our RNA-seq results revealed that STS deficiency plays a critical role in regulating multiple signaling pathways associated with cell mobility and apoptosis, such as Wnt/β signaling and the Hippo signaling pathway. Knockdown of the STS gene using shRNA in HaCaT cells led to an upregulation of E-cadherin expression and suppression of key factors involved in epithelial-mesenchymal transition (EMT), such as N-cadherin and vimentin. Inhibition of EMT involved the Hippo signaling pathway and reduction of HIF-1α. Interestingly, inhibiting STS with shRNA increased mitochondrial respiration levels, as demonstrated by the extracellular flux oxygen consumption rate. Additionally, we observed a significant increase in ROS production in partial STS knockout cells compared to control cells. Our study demonstrated that the excessive generation of ROS caused by STS deficiency induces the expression of Bax and Bak, leading to the release of cytochrome c and subsequent cell death. Consequently, STS deficiency impairs cell mobility and promotes apoptosis, offering insights into the pathophysiological processes and potential therapeutic targets for XLI.

The inhibition of YAP Signaling Prevents Chronic Biliary Fibrosis in the Abcb4-/- Model by Modulation of Hepatic Stellate Cell and Bile Duct Epithelium Cell Pathophysiology.

Primary sclerosing cholangitis (PSC) represents a chronic liver disease characterized by poor prognosis and lacking causal treatment options. Yes-associated protein (YAP) functions as a critical mediator of fibrogenesis; however, its therapeutic potential in chronic biliary diseases such as PSC remains unestablished. The objective of this study is to elucidate the possible significance of YAP inhibition in biliary fibrosis by examining the pathophysiology of hepatic stellate cells (HSC) and biliary epithelial cells (BEC). Human liver tissue samples from PSC patients were analyzed to assess the expression of YAP/connective tissue growth factor (CTGF) relative to non-fibrotic control samples. The pathophysiological relevance of YAP/CTGF in HSC and BEC was investigated in primary human HSC (phHSC), LX-2, H69, and TFK-1 cell lines through siRNA or pharmacological inhibition utilizing verteporfin (VP) and metformin (MF). The Abcb4-/- mouse model was employed to evaluate the protective effects of pharmacological YAP inhibition. Hanging droplet and 3D matrigel culture techniques were utilized to investigate YAP expression and activation status of phHSC under various physical conditions. YAP/CTGF upregulation was observed in PSC patients. Silencing YAP/CTGF led to inhibition of phHSC activation and reduced contractility of LX-2 cells, as well as suppression of epithelial-mesenchymal transition (EMT) in H69 cells and proliferation of TFK-1 cells. Pharmacological inhibition of YAP mitigated chronic liver fibrosis in vivo and diminished ductular reaction and EMT. YAP expression in phHSC was effectively modulated by altering extracellular stiffness, highlighting YAP's role as a mechanotransducer. In conclusion, YAP regulates the activation of HSC and EMT in BEC, thereby functioning as a checkpoint of fibrogenesis in chronic cholestasis. Both VP and MF demonstrate effectiveness as YAP inhibitors, capable of inhibiting biliary fibrosis. These findings suggest that VP and MF warrant further investigation as potential therapeutic options for the treatment of PSC.

A disulfiram derivative against lung cancer via the Notch signaling pathway without neurotoxicity and hepatotoxicity.

The exploration of novel anti-lung cancer small-molecule drugs is important for drug resistance and adverse effects of chemotherapeutic drugs in current clinics. Disulfiram (DSF), as an antidote, has been proven to have excellent antitumor effects in combination with copper (Cu). However, the risk for potential neurotoxicity and hepatotoxicity in clinical use, as well as its poor water solubility, limits its use. In this study, we identified a DSF derivative, S-(N,N-diethyldithiocarbamoyl)-N-acetyl-L-cysteine, which could greatly increase the water solubility by converting it to a calcium salt (DS-NAC). The anti-lung cancer pharmacodynamic studies in vitro of DS-NAC were evaluated and a mouse model of lung cancer in situ was established to explore the therapeutic effects of DS-NAC compared with DSF and oxaliplatin (OXA). The results demonstrated that DS-NAC combined with Cu had superior cytotoxicity to DSF and OXA in the CCK8 assay against lung cancer cells, and exhibited potent anti-metastatic, epithelial-mesenchymal transition inhibition. In addition, DS-NAC showed better antitumor effects than DSF and comparable effects to OXA in lung cancer in situ model. In terms of the antitumor mechanism, we discovered that DS-NAC in combination with Cu exerted a greater inhibitory effect on the Notch pathway than DSF, which may account for its excellent antitumor effects. Finally, we verified the safety of DS-NAC in vivo, showing lower hepatotoxicity and neurotoxicity compared with DSF and OXA. DS-NAC is a promising anti-lung cancer drug with a favorable safety profile.

Curcumin inhibits the invasion and migration of pancreatic cancer cells by upregulating TFPI-2 to regulate ERK- and JNK-mediated epithelial-mesenchymal transition.

Pancreatic cancer (PC) is one of the most deadly human malignancies. Curcumin is a natural polyphenolic compound with wide-ranging pharmacological effects. Growing evidence suggests that curcumin has anticancer activity against PC, but the mechanism remains incompletely elucidated. This study aimed to investigate the effects and mechanisms of curcumin on the invasion and migration of PC cells. Effect of curcumin on tissue factor pathway inhibitor (TFPI)-2 mRNA expression in PC cells was initially identified using qRT-PCR. Cytotoxicity of curcumin was assessed with MTT assays and IC50 was calculated. Involvement of ERK and JNK pathways, as well as protein expression of TFPI-2 and epithelial-mesenchymal transition (EMT)-related markers, were detected using immunoblotting. Invasion and migration of PC cells were examined using Transwell assays. TFPI-2 expression was manipulated by transfection with siRNA and shRNA. Rescue assays were used to validate the effect of curcumin on cell invasion and migration via TFPI-2. Curcumin increased the expression of TFPI-2 mRNA and protein in PC cells and attenuated cell invasion and migration. Curcumin also inhibited ERK and JNK pathways and EMT in PC cells. Knockdown of TFPI-2 partially reversed the inhibition of ERK and JNK pathways and EMT by curcumin. Mechanistically, curcumin upregulated TFPI-2, thereby inhibiting the ERK and JNK pathways, leading to the inhibition of EMT in PC cells. Collectively, curcumin inhibits ERK- and JNK-mediated EMT through upregulating TFPI-2, which in turn suppresses the migration and invasion of PC cells. These findings provide new insights into the antitumor mechanism of curcumin.

The Nexus of Inflammation-Induced Epithelial-Mesenchymal Transition and Lung Cancer Progression: A Roadmap to Pentacyclic Triterpenoid-Based Therapies.

Lung cancer is the leading cause of cancer-related death worldwide. Its high mortality is partly due to chronic inflammation that accompanies the disease and stimulates cancer progression. In this review, we analyzed recent studies and highlighted the role of the epithelial-mesenchymal transition (EMT) as a link between inflammation and lung cancer. In the inflammatory tumor microenvironment (iTME), fibroblasts, macrophages, granulocytes, and lymphocytes produce inflammatory mediators, some of which can induce EMT. This leads to increased invasiveness of tumor cells and self-renewal of cancer stem cells (CSCs), which are associated with metastasis and tumor recurrence, respectively. Based on published data, we propose that inflammation-induced EMT may be a potential therapeutic target for the treatment of lung cancer. This prospect is partially realized in the development of EMT inhibitors based on pentacyclic triterpenoids (PTs), described in the second part of our study. PTs reduce the metastatic potential and stemness of tumor cells, making PTs promising candidates for lung cancer therapy. We emphasize that the high diversity of molecular mechanisms underlying inflammation-induced EMT far exceeds those that have been implicated in drug development. Therefore, analysis of information on the relationship between the iTME and EMT is of great interest and may provide ideas for novel treatment approaches for lung cancer.

Activation of the TGF-β1/EMT signaling pathway by claudin-1 overexpression reduces doxorubicin sensitivity in small cell lung cancer SBC-3 cells

Small-cell lung cancer (SCLC), which accounts for about 15 % of all lung cancers, progresses more rapidly than other histologic types and is rarely detected at an operable early stage. Therefore, chemotherapy, radiation therapy, or their combination are the primary treatments for this type of lung cancer. However, the tendency to acquire resistance to anticancer drugs is a severe problem. Recently, we found that an intercellular adhesion molecule, claudin (CLDN) 1, known to be involved in the migration and invasion of lung cancer cells, is involved in the acquisition of anticancer drug resistance. In the present study, we investigated the effect of CLDN1 on the anticancer-drug sensitivity of SCLC SBC-3 cells. Since epithelial-mesenchymal transition (EMT), which is involved in cancer cell migration and invasion, is well known for its involvement in anticancer-drug sensitivity via inhibition of apoptosis, we also examined EMT involvement in decreased anticancer-drug sensitivity by CLDN1. Sensitivity to doxorubicin (DOX) in SBC-3 cells was significantly decreased by CLDN1 overexpression. CLDN1 overexpression resulted in increased TGF-β1 levels, enhanced EMT induction, and increased migratory potency of SBC-3 cells. The decreased sensitivity of SBC-3 cells to anticancer drugs upon TGF-β1 treatment suggested that activation of the TGF-β1/EMT signaling pathway by CLDN1 causes the decreased sensitivity to anticancer drugs and increased migratory potency. Furthermore, treatments with antiallergic agents tranilast and zoledronic acid, known EMT inhibitors, significantly mitigated the decreased sensitivity of CLDN1-overexpressing SBC-3 cells to DOX. These results suggest that EMT inhibitors might effectively overcome reduced sensitivity to anticancer drugs in CLDN1-overexpressing SCLC cells.

Detoxified pneumolysin derivative ΔA146Ply inhibits triple- negative breast cancer metastasis mainly via mannose receptor-mediated autophagy inhibition

ABSTRACT The detoxified pneumolysin derivative ΔA146Ply has been proven to have a direct anti-triple negative breast cancer effect by our group, but its work model remains unclear. In this study, we focused on its ability to inhibit triple-negative breast cancer metastasis. We found that ΔA146Ply suppressed the migration and invasion of triple-negative breast cancer cells by activating mannose receptor and toll-like receptor 4. Their activation triggers the activation of the mammalian target of rapamycin signalling, sequentially leading to autophagy, transforming growth factor-β1, and epithelial-mesenchymal transition inhibition. Furthermore, the combination of doxorubicin and ΔA146Ply significantly inhibited triple-negative breast cancer progression and prolonged survival in tumour-bearing mice. Taken together, our study provides an alternative microbiome-based mannose receptor-targeted therapy for triple-negative breast cancer and a novel theoretical and experimental basis for the downstream signalling pathway of the mannose receptor.

Silibinin Prevents TGFβ-Induced EMT of RPE in Proliferative Vitreoretinopathy by Inhibiting Stat3 and Smad3 Phosphorylation.

The purpose of this study was to investigate the effects of silibinin on epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) and proliferative vitreoretinopathy (PVR) formation, as well as its underlying molecular mechanism. Cellular morphological change and EMT molecular markers were evaluated by using phase contrast imaging, qPCR, and Western blot (WB) to investigate the impact of silibinin on the EMT of ARPE-19 cells. Scratch assay and transwell assay were used to study the effect of silibinin on cell migration. An intravitreally injected RPE-induced rat PVR model was used to assess the effect of silibinin on PVR in vivo. RNA-seq was applied to study the molecular mechanism of silibinin-mediated PVR prevention. Silibinin inhibited TGFβ1-induced EMT and migration of RPE in a dose-dependent manner in vitro. Moreover, silibinin prevented proliferative membrane formation in an intravitreal injected RPE-induced rat PVR model. In line with these findings, RNA-seq revealed a global suppression of TGFβ1-induced EMT and migration-related genes by silibinin in RPEs. Mechanistically, silibinin reduced TGFβ1-induced phosphorylation levels of Smad3 and Stat3, and Smad3 nuclear translocation in RPE. Silibinin inhibits the EMT of RPE cells in vitro and prevents the formation of PVR membranes in vivo. Mechanistically, silibinin inhibits Smad3 phosphorylation and suppresses Smad3 nuclear translocation through the inhibition of Stat3 phosphorylation. These findings suggest that silibinin may serve as a potential treatment for PVR.

GK921, a transglutaminase inhibitor, strengthens the antitumor effect of cisplatin on pancreatic cancer cells by inhibiting epithelial-to-mesenchymal transition

Pancreatic adenocarcinoma (PAAD), a common digestive malignant tumor, presents high mortality rates and limited treatment methods. Currently, chemotherapy remains the main therapy method for patients with PAAD. As a classical chemotherapy drug, cisplatin (DDP) is limited by dose-related toxicity in patients with PAAD. In this study, we demonstrated that TGM2 may be a treatment and prognosis marker in pancreatic cancer patients. Co-treatment of low dose of DDP and GK921, a transglutaminase (TGM2) inhibitor, is capable of synergistically inhibiting the PAAD cell viability and proliferation in vitro and in vivo. Based on in vitro study, GK921 inhibited the epithelial-to-mesenchymal transition (EMT) induced by TGM2 as well as aggravated cell cycle arrest and apoptosis resulted from DDP, making pancreatic cancer cells more sensible to DDP. Our results showed that GK921 increased the protein levels regarding E-cadherin as well as decreased the protein level regarding Snail2, N-cadherin, which indicated that GK921 inhibited EMT in pancreatic cancer cells. Snail2 overexpression inhibited GK921/DDP-induced cell apoptosis, as well as mitigated the GK921/DDP-caused cell death and the EMT inhibition. In vivo studies also found GK921/DDP combination can further inhibit the growth of PAAD without significantly side effects. To sum up, we showed that GK921 increased PAAD cells sensitivity to DDP via inhibiting EMT. As revealed, DDP/GK921 co-treatment could promisingly serve for treating PAAD patients.

Targeting cancer-associated adipocyte-derived CXCL8 inhibits triple-negative breast cancer progression and enhances the efficacy of anti-PD-1 immunotherapy

Cancer-associated adipocytes (CAAs), one of the primary stromal components, exhibit intimate crosstalk and release multiple cell factors mediating local and systemic biological effects. However, the role of CAAs in the regulation of systemic immune responses and their potential value in the clinical treatment of triple-negative breast cancer (TNBC) are not well described. Transcriptome sequencing was performed on CAA and normal adipocyte (NA) tissues isolated from surgically resected samples from TNBC patients and healthy controls. Cytokines, including C-X-C motif chemokine ligand 8 (CXCL8, also known as IL-8), secreted from NAs and CAAs were compared by transcriptome sequencing and enzyme-linked immunosorbent assay (ELISA). Proliferation, migration and invasion assays were employed to analyze the role of CAAs and CAA-derived CXCL8 (macrophage inflammatory protein-2 (MIP2) as a functional surrogate in mice). TNBC syngraft models were established to evaluate the curative effect of targeting CXCL8 in combination with anti-PD-1 therapies. Real-time quantitative polymerase chain reaction (RT-qPCR), western blotting (WB), polymerase chain reaction (PCR) array, flow cytometry, immunohistochemistry (IHC), and immunofluorescence (IF) were applied to analyze immune cell infiltration and epithelial–mesenchymal transition (EMT) markers. Specifically, we demonstrated that CAAs and CAA-derived CXCL8 played important roles in tumor growth, EMT, metastasis and tumor immunity suppression. CAA-derived CXCL8 remodeled the tumor immune microenvironment not only by suppressing CD4+ T and CD8+ T immune cell infiltration but also by upregulating CD274 expression in TNBC. The combination of targeting the CXCL8 pathway and blocking the PD-1 pathway synergistically increased the tumor immune response and inhibited tumor progression. Thus, our results highlight the molecular mechanisms and translational significance of CAAs in tumor progression and immune ecosystem regulatory effects and provide a better understanding of the potential clinical benefit of targeting CAA-derived CXCL8 in antitumor immunity and as a new therapeutic moiety in TNBC.

Abnormal activation of the Wnt3a/β-catenin signaling pathway promotes the expression of T-box transcription factor 3(TBX3) and the epithelial-mesenchymal transition pathway to mediate the occurrence of adenomyosis.

T-box transcription factor 3(TBX3) is a transcription factor that can regulate cell proliferation, apoptosis, invasion, and migration in different tumor cells; however, its role in adenomyosis (ADM) has not been previously studied. Some of ADM's pathophysiological characteristics are similar to those of malignant tumors (e.g., abnormal proliferation, migration, and invasion). We hypothesized that TBX3 might have a role in ADM. We used tamoxifen-induced Institute of Cancer research (ICR) mice to establish ADM disease model. The study procedure included western blotting and immunohistochemistry to analyze protein levels; additionally, we used intraperitoneal injection of Wnt/β-catenin pathway inhibitor XAV-939 to study the relationship between TBX3 and Wnt/β-catenin pathway as well as Anti-proliferation cell nuclear antigen( PCNA) and TUNEL to detect cell proliferation and apoptosis, respectively. TBX3 overexpression and epithelial-to-mesenchymal transition (EMT) in ADM mice was found to be associated with activation of the Wnt3a/β-catenin pathway. Treatment with XAV-939 in ADM mice led to the inhibition of both TBX3 and EMT; moreover, abnormal cell proliferation was suppressed, the depth of invasion of endometrium cells was limited. Thus, the use of XAV-939 effectively inhibited further invasion of endometrial cells. These findings suggest that TBX3 may play an important role in the development of ADM. The expression of TBX3 in ADM was regulated by the Wnt3a/β-catenin pathway. The activation of the Wnt3a/β-catenin pathway in ADM promoted TBX3 expression and induced the occurrence of EMT, thus promoting cell proliferation and inhibiting apoptosis, ultimately accelerating the development of ADM. The study provides a reference for the diagnosis of ADM.

Plasmodium immunotherapy combined with gemcitabine has a synergistic inhibitory effect on tumor growth and metastasis in murine Lewis lung cancer models.

Our previous studies have demonstrated that Plasmodium immunotherapy (infection) has antitumor effects in mice. However, as a new form of immunotherapy, this therapy has a weakness: its specific killing effect on tumor cells is relatively weak. Therefore, we tested whether Plasmodium immunotherapy combined with gemcitabine (Gem), a representative chemotherapy drug, has synergistic antitumor effects. We designed subcutaneously and intravenously implanted murine Lewis lung cancer (LLC) models to test the antitumor effect of Plasmodium chabaudi ASS (Pc) infection in combination with Gem treatment and explored its underlying mechanisms. We found that both Pc infection alone and Gem treatment alone significantly inhibited tumor growth in the subcutaneous model, and combination therapy was more effective than either monotherapy. Monotherapy only tended to prolong the survival of tumor-bearing mice, while the combination therapy significantly extended the survival of mice, indicating a significant synergistic effect of the combination. In the mechanistic experiments, we found that the combination therapy significantly upregulated E-cadherin and downregulated Snail protein expression levels, thus inhibiting epithelial-mesenchymal transition (EMT) of tumor cells, which may be due to the blockade of CXCR2/TGF-β-mediated PI3K/Akt/GSK-3β signaling pathway. The combination of Pc and Gem plays a synergistic role in inhibiting tumor growth and metastasis, and prolonging mice survival in murine lung cancer models. These effects are partially attributed to the inhibition of EMT of tumor cells, which is potentially due to the blockade of CXCR2/TGF-β-mediated PI3K/Akt/GSK-3β/Snail signaling pathway. The clinical transformation of Plasmodium immunotherapy combined with Gem for lung cancer is worthy of expectation.

2-Methoxyestradiol ameliorates paraquat-induced pulmonary fibrosis by inhibiting the TGF-β1/Smad2/3 signaling pathway

Paraquat (PQ) is a highly effective and highly toxic herbicide that is highly toxic to both humans and animals. Pulmonary fibrosis is the primary cause of fatality in patients with PQ poisoning, there is no effective drug treatment yet. 2-Methoxyestradiol (2ME) is a natural metabolite of estradiol with anti-tumor, anti-angiogenesis, and anti-proliferative effects. Whether 2ME has the potential to inhibit pulmonary fibrosis induced by PQ is unclear. This study aims to investigate the potential effects and mechanism of 2ME on PQ-induced pulmonary fibrosis. C57BL/6 mice and A549 cells were exposed to PQ to establish pulmonary fibrosis model. In vivo, Hematoxylin and eosin (H&E) staining was utilized to assess the pathological characteristics. Masson's trichrome staining was employed to evaluate the collagen deposition. Western blot and immunohistochemistry were conducted to determine the expressions of fibrosis markers. In vitro, the expressions of epithelial–mesenchymal transition (EMT) markers were detected using western blot and immunofluorescence to evaluated the potential inhibition of PQ-induced EMT by 2ME. And proteins associated with the TGF-β1/Smad2/3 signaling pathway were measured by western blot in vivo and in vitro. The result found that 2ME can ameliorated PQ-induced pulmonary fibrosis and inhibit the activation of TGF-β1/Smad2/3 signaling pathway. These findings suggest that 2ME may serve as a potential therapeutic agent for treating PQ-induced pulmonary fibrosis.

Armillaria ostoyae extracts inhibit EMT of cancer cell lines via TGF-β and Wnt/β-catenin signaling components

Armillaria ostoyae is an edible mushroom, appreciated in human nutrition, with several medicinal properties already proven. However, its phenolic composition and effects on cancer have never been investigated so far, especially regarding specific signaling pathways included in first steps of cancer metastatic process, epithelial-mesenchymal transition (EMT) and migration. We report phenolic profile of two Armillaria ostoyae extracts, ethanol and diethyl ether (E and DE) and their effects on colorectal carcinoma (HCT-116) and cervical adenocarcinoma (HeLa) cells. Phenolics were detected using HPLC, while effects on cancer cells were investigated using MTT test; expression of markers related to EMT on gene and protein level by flow cytometry, immunofluorescence and qRT-PCR method. Antimigratory potential was assessed on collective and individual type of migration by Wound healing assay and RTCA technique. Sinapic acid was detected as the main phenolic component in E, while rutin and naringin were dominant in DE type of extract. Extracts notably suppressed pro-EMT markers: vimentin, N-cadherin, β-catenin, Snail, Smad2 and MMP-9, and elevated anti-EMT marker E-cadherin. Consequently, collective and single cell migration were significantly inhibited via regulation of TGF-β and Wnt/β-catenin signaling components. DE extract was more efficient in suppression of EMT and migration of colorectal and cervical cancer cells.

The biomarkers associated with epithelial-mesenchymal transition in human keloids

IntroductionA keloid is a type of benign fibrotic disease with similar features to malignancies, including anti-apoptosis, over-proliferation, and invasion. Epithelial-mesenchymal transition (EMT) is a crucial mechanism that regulates the metastatic behavior of tumors. Thus, identifying EMT biomarkers is paramount in comprehensively understanding keloid pathogenesis. MethodsTo identify the differentially expressed genes (DEGs) GSE92566 dataset, with 3 normal skin and 4 keloid tissues, was downloaded from GEO databases to identify the differentially expressed genes (DEGs). Further, EMT-related genes were downloaded from dbEMT 2.0 databases and intersected with GSE92566 DEGs to identify EMT-related-DEGs (ERDEGs). Subsequently, the ERDEGs were used for GO, KEGG, gene set enrichment analysis (GSEA), protein-protein interaction (PPI), and miRNAs-mRNAs network analysis. To predict small molecules for EMT inhibition, the ERDEGs were imported to cMAP databases, whereas hub genes were imported to DGidb databases. Finally, we carried out qRT-PCR and in vitro experiments to validate our findings. ResultsA total of 122 ERDEGs were identified, including 59 upregulated and 63 down-regulated genes. Moreover, enrichment analysis revealed that focal adhesion, AMPK signal pathway, Wnt signal pathway, and EMT biological process were significantly enriched. STRING databases and Cytoscape software were used to construct the PPI network and EMT-related hub genes. Further, 3 modules were explored from the PPI network using the Molecular Complex Detection (MCODE) plugin. In the Cytohubba plugin, 10 hub genes were explored, including FN1, EGF, SOX9, CDH2, PROM1, EPCAM, KRT19, ITGB1, CD24, and KRT18. These genes were then enriched for the focal adhesion pathway. We constructed a microRNA (miRNA)-mRNA network, which predicted hsa-miR-155–5p (8 edges), hsa-miR-124–3p (7 edges), hsa-miR-145–5p (5 edges), hsa-miR-20a-5p (5 edges) and hsa-let-7b-5p (4 edges) as the most connected miRNAs regulating EMT. Based on the ERDEGs and 10 hub genes mentioned above, ribavirin demonstrated high drug-targeting relevance. Subsequently, qRT-PCR confirmed that the expression of FN1, ITGB1, CDH2, and EPCAM corroborated with previous findings. qRT-PCR also showed that the expression levels of hsa-miR-124–3p and hsa-miR-145–5p were significantly lower in keloids and hsa-miR-155–5p was upregulated in keloids. Finally, by treating human keloid fibroblasts (HKFs) with ribavirin in vitro, we confirmed that ribavirin could inhibit HKFs proliferation and EMT. ConclusionIn summary, this work provides novel EMT biomarkers in keloids and predicts new small target molecules for keloid therapy. Our findings improve the understanding of keloid pathogenesis, providing new treatment options.

Nanosecond pulsed cold atmospheric plasma jet suppresses proliferation and migration of human glioblastoma cells via apoptosis promotion and EMT inhibition

Glioblastoma (GBM) is one of the most aggressive and challenging cancers to treat. Despite extensive research on dozens of cancer cells, including GBM, the effect of cold atmospheric plasma (CAP) on the invasive migration of GBM cells has received limited attention, and the underlying mechanisms remain poorly understood. This study aims to investigate the potential molecular mechanism of ns-CAPJ in inhibiting the invasive migration of human GBM cells. The findings indicate that ns-CAPJ significantly reduces GBM cell invasion and migration, and induces apoptosis in GBM cells. Further mechanistic studies demonstrate a direct correlation between the suppression of the epithelial-mesenchymal transition (EMT) signaling pathway and ns-CAPJ’s inhibitory effect on GBM cell invasion and migration. Additionally, combined with the N-acetyl cysteine (NAC, a ROS inhibitor) assay, we found that the ROS stimulated by the ns-CAPJ plays an important role in suppressing the EMT process. This work is expected to provide new insight into understanding the molecular mechanisms of how ns-CAPJ inhibits the proliferation and migration of human GBM cells.