Nigericin inhibits epithelial ovarian cancer metastasis by suppressing the cell cycle and epithelial−mesenchymal transition

Epithelial ovarian cancer (EOC) has the highest mortality among various types of gynecological malignancies. Most patients die of metastasis and recurrence due to cisplatin resistance. Thus, it is urgent to develop novel therapies to cure this disease. CCK-8 assay showed that nigericin exhibited strong cytotoxicity on A2780 and SKOV3 cell lines. Flow cytometry indicated that nigericin could induce cell cycle arrest at G0/G1 phase and promote cell apoptosis. Boyden chamber assay revealed that nigericin could inhibit migration and invasion in a dose-dependent manner by suppressing epithelial–mesenchymal transition (EMT) in EOC cells. These effects were mediated, at least partly, by the Wnt/β-catenin signaling pathway. Our results demonstrated that nigericin could inhibit EMT during cell invasion and metastasis through the canonical Wnt/β-catenin signaling pathway. Nigericin may prove to be a novel therapeutic strategy that is effective in patients with metastatic EOC.     

Chidamide alleviates TGF-β-induced epithelial–mesenchymal transition in lung cancer cell lines

Transforming growth factor-β (TGF-β)-induced epithelial–mesenchymal transition is a critical process in the initiation of metastasis of various types of cancer. Chidamide is a class I histone deacetylase inhibitor with anti-tumor activity. This study investigated the effects of chidamide on TGF-β-mediated suppression of E-cadherin expression in adenocarcinomic lung epithelial cells and the molecular mechanisms involved in these effects. Western blot analysis, confocal microscopy, Quantitative methyl-specific PCR and bisulfite sequencing were used to evaluate the effects of different treatments on chidamide ameliorating TGF-β induced-E-cadherin loss. H3 acetylation binding to the promoter of E-cadherin was detected by chromatin immunoprecipitations (CHIP). We found that chidamide reduced the level of lung cancer cell migration observed using a Boyden chamber assay (as an indicator of metastatic potential). Chidamide inhibited TGF-β-induced SMAD2 phosphorylation and attenuated TGF-β-induced loss of E-cadherin expression in lung cancer cells by Western blotting and confocal microscopy, respectively. Quantitative methyl-specific PCR and bisulfite sequencing revealed that TGF-β-enhanced E-cadherin promoter methylation was ameliorated in cells treated with chidamide. We demonstrated that histone H3 deacetylation within the E-cadherin promoter was required for TGF-β-induced E-cadherin loss; cell treatment with chidamide increased the H3 acetylation detected by CHIP. Taken together, our results demonstrate that TGF-β suppressed E-cadherin expression by regulating promoter methylation and histone H3 acetylation. Chidamide significantly enhanced E-cadherin expression in TGF-β-treated cells and inhibited lung cancer cell migration. These findings indicate that chidamide has a potential therapeutic use due to its capacity to prevent cancer cell metastasis.     

Genes suppressed by DNA methylation in non-small cell lung cancer reveal the epigenetics of epithelial–mesenchymal transition

Background

DNA methylation is associated with aberrant gene expression in cancer, and has been shown to correlate with therapeutic response and disease prognosis in some types of cancer. We sought to investigate the biological significance of DNA methylation in lung cancer.

Results

We integrated the gene expression profiles and data of gene promoter methylation for a large panel of non-small cell lung cancer cell lines, and identified 578 candidate genes with expression levels that were inversely correlated to the degree of DNA methylation. We found these candidate genes to be differentially methylated in normal lung tissue versus non-small cell lung cancer tumors, and segregated by histologic and tumor subtypes. We used gene set enrichment analysis of the genes ranked by the degree of correlation between gene expression and DNA methylation to identify gene sets involved in cellular migration and metastasis. Our unsupervised hierarchical clustering of the candidate genes segregated cell lines according to the epithelial-to-mesenchymal transition phenotype. Genes related to the epithelial-to-mesenchymal transition, such as AXL, ESRP1, HoxB4, and SPINT1/2, were among the nearly 20% of the candidate genes that were differentially methylated between epithelial and mesenchymal cells. Greater numbers of genes were methylated in the mesenchymal cells and their expressions were upregulated by 5-azacytidine treatment. Methylation of the candidate genes was associated with erlotinib resistance in wild-type EGFR cell lines. The expression profiles of the candidate genes were associated with 8-week disease control in patients with wild-type EGFR who had unresectable non-small cell lung cancer treated with erlotinib, but not in patients treated with sorafenib.

Conclusions

Our results demonstrate that the underlying biology of genes regulated by DNA methylation may have predictive value in lung cancer that can be exploited therapeutically.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1079) contains supplementary material, which is available to authorized users.     

Cigarette smoke-induced alveolar epithelial–mesenchymal transition is mediated by Rac1 activation

Background

Epithelial–mesenchymal transition (EMT) is the major pathophysiological process in lung fibrosis observed in chronic obstructive pulmonary disease (COPD) and lung cancer. Smoking is a risk factor for developing EMT, yet the mechanism remains largely unknown. In this study, we investigated the role of Rac1 in cigarette smoke (CS) induced EMT.

Methods

EMT was induced in mice and pulmonary epithelial cells by exposure of CS and cigarette smoke extract (CSE) respectively.

Results

Treatment of pulmonary epithelial cells with CSE elevated Rac1 expression associated with increased TGF-β1 release. Blocking TGF-β pathway restrained CSE-induced changes in EMT-related markers. Pharmacological inhibition or knockdown of Rac1 decreased the CSE exposure induced TGF-β1 release and ameliorated CSE-induced EMT. In CS-exposed mice, pharmacological inhibition of Rac1 reduced TGF-β1 release and prevented aberrations in expression of EMT markers, suggesting that Rac1 is a critical signaling molecule for induction of CS-stimulated EMT. Furthermore, Rac1 inhibition or knockdown abrogated CSE-induced Smad2 and Akt (PKB, protein kinase B) activation in pulmonary epithelial cells. Inhibition of Smad2, PI3K (phosphatidylinositol 3-kinase) or Akt suppressed CSE-induced changes in epithelial and mesenchymal marker expression.

Conclusions and general significance

Altogether, these data suggest that CS initiates EMT through Rac1/Smad2 and Rac1/PI3K/Akt signaling pathway. Our data provide new insights into the fundamental basis of EMT and suggest a possible new course of therapy for COPD and lung cancer.     

Extracellular matrix proteins regulate epithelial–mesenchymal transition in mammary epithelial cells

Mouse mammary epithelial cells undergo transdifferentiation via epithelial–mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland.     

MiR-876-5p suppresses epithelial–mesenchymal transition of lung cancer by directly down-regulating bone morphogenetic protein 4

Lung cancer is the leading cause of cancer-related death throughout the world. We aimed to investigate the role of a novel microRNA-876-5p and its potential molecular target bone morphogenetic protein 4 (BMP-4), in the epithelial–mesenchymal transition (EMT) of lung cancer. Expressions of microRNA-876-5p and its potential target BMP-4 were analysed in lung cancer cells and patient tissues. Luciferase activity assay was conducted to verify direct targeting of microRNA-876-5p to the 3′-UTR of BMP-4 mRNA. Migration, invasion capacities of lung cancer cells expressing microRNA-876-5p were analysed, and characteristics of lung cancer EMT protein markers were also evaluated. A xenograft tumour mouse model was established to address the roles of microRNA-876-5p and BMP-4 in lung cancer EMT in vivo. MicroRNA-876-5p was decreased while BMP-4 was increased in lung cancer cells and tissues. MicroRNA-876-5p directly targeted 3′-UTR of BMP-4 mRNA to inhibit its expression. MicroRNA-876-5p expression significantly inhibited the migration, invasion and EMT of lung cancer cells in vitro, as well as metastasis in vivo, which required BMP-4 expression. MicroRNA-876-5p suppresses EMT of lung cancer by directly down-regulating BMP-4, both of which could serve as potential therapeutic targets in the treatment of lung cancer.     

TGF-β1 regulates cell fate during epithelial–mesenchymal transition by upregulating survivin

Members of the transforming growth factor beta (TGF-β) superfamily are multifunctional cytokines that regulate several cellular processes, including cell cycle arrest, differentiation, morphogenesis, and apoptosis. TGF-β promotes extracellular matrix production and morphological change. Morphogenetic responses to TGF-β include cell migration and epithelial–mesenchymal transition (EMT), which are critical during embryogenesis, development of fibrotic diseases, and the spreading of advanced carcinomas. The purpose of this study was to clarify how TGF-β regulates the fate of retinal pigment epithelial (RPE) cells. TGF-β1 promoted cell cycle progression and phosphorylation of retinoblastoma protein (Rb) in ARPE-19 cells. TGF-β1 induced survivin expression, which in turn stabilized tubulin and Aurora B. RT-PCR and western blot analysis revealed that survivin expression increased in ARPE-19 cells following TGF-β1 treatment. When survivin was depleted, TGF-β1 induced cell cycle arrest and apoptosis and also reduced Rb phosphorylation. In conclusion, the present study shows that induction of EMT in human RPE cells upregulates survivin, leading to survivin-dependent inhibition of cell cycle arrest and apoptosis. Whether cells undergo EMT or apoptosis in response to TGF-β1 is dependent on their cell cycle state, and TGF-β1 regulates the cell cycle via survivin.     

Overexpression of LVRN impedes the invasion of trophoblasts by inhibiting epithelial–mesenchymal transition

Laeverin(LVRN)was first detected on the outer layer of the chorion laeve and migrating extravillous trophoblasts(EVTs).It is an enzyme that plays an important role in the placentation and pathophysiology of preeclampsia(PE).Previous studies have indicated that LVRN may be required for the invasion of human trophoblast cells.Paradoxically,LVRN was found to be highly expressed in the trophoblasts of PE patients with impaired invasive capacities.In this study,we detected the expression of LVRN in the placentas of PE patients(n=5)and normal term pregnancy women(n=5)as a control group by immunohistochemistry.LVRN was elevated in decidua(P=0.0083)and villi(P=0.0079)of PE patients.Next,LVRN was overexpressed via adeno-associated virus-mediated gene transfer in trophoblastic cell lines HTR8,Swan71,and JAR.Matrigel transwell assay and wound healing assay showed that overexpression of LVRN impeded the invasion of these three cell lines.Western blot analysis showed that LVRN overexpression caused downregulation of N-cadherin and vimentin and upregulation of E-cadherin,suggesting the inhibitory role of LVRN in epithelial–mesenchymal transition(EMT).Moreover,our data indicated that long noncoding RNA NONSTAT103348(lnc10-7)was elevated in PE patients.Silencing lnc10-7 led to decreased LVRN expression.Taken together,although the basal level of LVRN may be crucial for cell invasion,overexpression of LVRN may abrogate the cell invasiveness,suggesting a multifaceted role of LVRN in the pathogenesis of PE.     

The effect of Pokemon on bladder cancer epithelial–mesenchymal transition

Objective

This study aimed at detecting Pokemon expression in bladder cancer cell and investigating the relationship between Pokemon and epithelial–mesenchymal transition. Furthermore, we investigated the functions of Pokemon in the carcinogenesis and development of bladder cancer. This study was also designed to observe the inhibitory effects of siRNA expression vector on Pokemon in bladder cancer cell.

Methods

The siRNA expression vectors which were constructed to express a short hairpin RNA against Pokemon were transfected to the bladder cancer cells T24 with a liposome. Levels of Pokemon, E-cadherin and β-catenin mRNA and protein were examined by real-time quantitative-fluorescent PCR and Western blot analysis, respectively. The effects of Pokemon silencing on epithelial–mesenchymal transition of T24 cells were evaluated with wound-healing assay.

Results

Pokemon was strongly inhibited by siRNA treatment, especially siRNA3 treatment group, as it was reflected by Western blot and real-time PCR. The gene and protein of E-cadherin expression level showed increased markedly after Pokemon was inhibited by RNA interference. While there were no differences in the levels of gene and protein of β-catenin among five groups. The bladder cancer cell after Pokemon siRNA interference showed a significantly reduced wound-closing efficiency at 6, 12 and 24 h.

Conclusions

Our findings suggest Pokemon may inhibit the expression of E-cadherin. The low expression of E-cadherin lead to increasing the phenotype and apical-base polarity of epithelial cells. These changes of cells may result in the recurrence and progression of bladder cancer at last.     

Analysis of epithelial–mesenchymal transition markers in psoriatic epidermal keratinocytes

Psoriasis is similar to endpoints of epithelial–mesenchymal transition (EMT), a process of epithelial cells transformed into fibroblast-like cells. The molecular epithelial and mesenchymal markers were analysed in psoriatic keratinocytes. No obvious alteration of epithelial markers E-cadherin (E-cad), keratin 10 (K10), K14 and K16 was detected in psoriatic keratinocytes. However, significantly increased expression of Vim, FN, plasminogen activator inhibitor 1 (PAI-1) and Slug was seen. IL-17A and IL-13 at 50 ng ml⁻¹ strongly decreased expression of K10, Vim and FN. TGF-β1 at 50 ng ml⁻¹ promoted the production of N-cad, Vim, FN and PAI-1. Slug was decreased by dexamethasone (Dex), but E-cad was upregulated by Dex. Silencing of ERK partially increased E-cad and K16, but remarkably inhibited K14, FN, Vim, β-catenin, Slug and α5 integrin. Moreover, inhibition of Rho and GSK3 by their inhibitors Y27632 and SB216763, respectively, strongly raised E-cad, β-catenin and Slug. Dex decreased Y27632-mediated increase of β-catenin. Dex at 2.0 µM inhibited SB216763-regulated E-cad, β-catenin and slug. In conclusion, EMT in psoriatic keratinocytes may be defined as an intermediate phenotype of type 2 EMT. ERK, Rho and GSK3 play active roles in the process of EMT in psoriatic keratinocytes.     

S1PR3 deficiency alleviates radiation-induced pulmonary fibrosis through the regulation of epithelial–mesenchymal transition by targeting miR-495-3p

Radiation-induced pulmonary fibrosis (RIPF) is a life-threatening complication of thoracic radiotherapy, which contributes to continued deterioration in pulmonary function. Sphingosine-1 phosphate receptor 3 (S1PR3) has been identified as a crucial molecule in fibrosis. Accumulating evidence indicated that the inhibition of the S1PRs ameliorates fibrogenesis. Thus, this study aims to explore whether S1PR3 participates in RIPF and elucidates the molecular mechanisms underlying S1PR3-modulated epithelial–mesenchymal transition (EMT) in transforming growth factor-β1-induced pulmonary epithelia. A recombinant adeno-associated viral-mediated S1PR3 (AAV-S1PR3) gene therapy analyzed the effect of S1PR3 gene deficiency on the altered histology structure and molecular mechanisms in the lung of mice with whole-lung irradiation. Compared with the AAV-negative control mice, AAV-mediated S1PR3 knockdown in the lung of mice attenuated pulmonary fibrosis induced by the radiation, as indicated by the alleviation of collagen accumulation, lessened histopathological alterations, and the suppression of inflammatory cells infiltration. S1PR3 deficiency reversed the RIPF concomitantly with abrogated EMT-related protein (α-smooth muscle actin). Consistently, S1PR3-deficient pulmonary epithelia inhibited the EMT process changes and fibrosis formation. Furthermore, S1PR3 was designated as one of the target genes for microRNA-495-3p (miR-495-3p). The inhibition of miR-495-3p promoted the expression of S1PR3 in pulmonary epithelia, whereas the overexpression of miR-495-3p inhibited the S1PR3/SMAD2/3 pathway and suppressed the EMT process. Collectively, miR-495-3p might be a negative regulator of the EMT process in fibrosis formation by inhibiting the targeted S1PR3 gene. These results established a link between the S1PR3 gene, the EMT process, and the fibrosis, suggesting the pharmacological blockage of S1PR3 as a potential therapeutic strategy for RIPF.     

Down-regulated miR-15a mediates the epithelial–mesenchymal transition in renal tubular epithelial cells promoted by high glucose

High glucose (HG) has been reported to be associated with renal dysfunction. And one potential mechanism underlining the dysfunction is the epithelial–mesenchymal transition (EMT) of renal tubular epithelial cells. Present study showed that EMT was induced in the HG-treated renal tubular epithelial cells by promoting the expression of mesenchymal phenotype molecules, such as α-SMA and collagen I, and down-regulating the expression of epithelial phenotype molecule E-cadherin. Moreover, we have identified the down-regulation of miR-15a which was accompanied with the HG-induced EMT. And the miR-15a overexpression inhibited the α-SMA, collagen I expression, and the promotion of E-cadherin expression by targeting and down-regulating AP4 which was also significantly promoted by the HG in the renal tubular epithelial cells. Thus, this study revealed that the weakening regulation on the AP4 expression by miR-15a might contribute to the HG-induced EMT in the renal tubular epithelial cells.