Epithelial-mesenchymal Transition Protein Expression Research Articles

Alterations of endometrial epithelial-mesenchymal transition and MAPK signalling components in women with PCOS are partially modulated by metformin in vitro.

Growing evidence suggests that epithelial-mesenchymal transition (EMT) and its regulator mitogen-activated protein kinase (MAPK) contribute to endometria-related reproductive disorders. However, the regulation of EMT and MAPK signalling components in the endometrium from polycystic ovary syndrome (PCOS) patients has not been systematically investigated and remains elusive. In humans, how metformin induces molecular alterations in the endometrial tissues under PCOS conditions is not completely clear. Here, we recruited 7 non-PCOS patients during the proliferative phase (nPCOS), 7 non-PCOS patients with endometrial hyperplasia (nPCOSEH), 14 PCOS patients during the proliferative phase (PCOS) and 3 PCOS patients with endometrial hyperplasia (PCOSEH). Our studies demonstrated that compared with nPCOS, PCOS patients showed decreased Claudin 1 and increased Vimentin and Slug proteins. Similar to increased Slug protein, nPCOSEH and PCOSEH patients showed increased N-cadherin protein. Western blot and immunostaining revealed increased epithelial phosphorylated Cytokeratin 8 (p-CK 8) expression and an increased p-CK 8:CK 8 ratio in PCOS, nPCOSEH and PCOSEH patients compared to nPCOS patients. Although nPCOSEH and PCOSEH patients showed increased p-ERK1/2 and/or p38 protein levels, the significant increase in p-ERK1/2 expression and p-ERK1/2:ERK1/2 ratio was only found in PCOS patients compared to nPCOS patients. A significant induction of the membrane ERβ immunostaining was observed in the epithelial cells of PCOS and PCOSEH patients compared to nPCOS and nPCOSEH patients. While in vitro treatment with metformin alone increased Snail and decreased Claudin 1, N-cadherin and α-SMA proteins, concomitant treatment with metformin and E2 increased the expression of CK 8 and Snail proteins and decreased the expression of Claudin 1, ZO-1, Slug and α-SMA proteins. Our findings suggest that the EMT contributes to the switch from a healthy state to a PCOS state in the endometrium, which might subsequently drive endometrial injury and dysfunction. We also provide evidence that metformin differentially modulates EMT protein expression in PCOS patients depending on oestrogenic stimulation.

Apoptotic induction and anti-metastatic activity of eugenol encapsulated chitosan nanopolymer on rat glioma C6 cells via alleviating the MMP signaling pathway

Glioma is the prime cause of cancer allied mortality in adolescent people and it accounts about 80% of all malignant tumours. Eugenol is a major bioactive constituent present in the essential oils with numerous pharmacological benefits including nueroprotective activity. The major drawback of eugenol is its extreme volatile property and oxygen sensitivity therefore we increased the efficacy of drug; eugenol by encapsulating with chitosan polymer. Eugenol loaded chitosan polymer (EuCs) was characterized using FTIR, XRD, SEM, HR-TEM analysis and the encapsulation, drug release efficacy was assessed at in vitro condition. The induction of autophagy and anticancer efficacy of EuCs on glioma cells was evaluated with rat C6 glioma cells using MTT assay, acridine orange staining, immunocytochemical analysis of NFκβ protein expression and FLOW cytometric analysis. The anti-metastatic property of Eu-CS was assessed by immunoblotting and RT-PCR analysis of epithelial mesenchymal transition protein expression in EuCs treated rat C6 glioma cells. Our characterization analysis proves that EuCs possess essential physical and functional properties of copolymer to be utilized as a drug. Further the MTT analysis and AO staining confirms even in the presence of oncogenic inducer and autophagic inhibitors, EuCs exhibits apoptotic potency on rat C6 glioma cells. The result of immunocytochemical studies depicts the inhibition of NFκβ protein expression and flow cytometry studies confirm apoptosis induction by EuCs. The inhibition of metastasis by EuCs was proven by the decrease in epithelial mesenchymal transition protein expression inEu-Cs treated rat C6 glioma cells. Over all our results authentically confirms eugenol loaded chitosan nanopolymer persuasively induces apoptosis and inhibits metastasis in rat C6 glioma cells.

The Association Between Inflammation, Epithelial Mesenchymal Transition and Stemness in Colorectal Carcinoma.

BackgroundInflammation plays an important albeit dual role in carcinogenesis. Survival studies have highlighted the prognostic significance of peritumorous inflammation. Currently, the theoretical background allows inflammation, epithelial mesenchymal transition (EMT) and the closely associated stem cell differentiation in colorectal carcinoma (CRC) to be linked. However, there is scarce direct morphological evidence.Purpose and methodsThe aim of our study was to investigate the role of inflammation in cancer growth and invasion by analyzing the association between inflammation and known morphological prognostic features of colorectal cancer, EMT, stemness and mismatch repair (MMR) protein expression. The study was designed as a retrospective morphological and immunohistochemical assessment of 553 consecutive cases of surgically treated primary CRC.ResultsThere were statistically significant associations between high-grade inflammation and lower pT (p = 0.002), absence of lymph node metastases (p < 0.001) and less frequent lymphatic (p = 0.003), venous (p = 0.017), arterial (p = 0.012), perineural (p = 0.001) and intraneural (p = 0.01) invasion. In contrast, Crohn’s like reaction (CLR) by density of lymphoid follicles in the invasive front lacked significant differences in regard to pT, pN, tumor invasion into surrounding structures (blood or lymphatic vessels, nerves), grade or necrosis (all p > 0.05). The expression of E-cadherin, CD44 and MMR proteins yielded no statistically significant associations with peritumorous inflammation by Klintrup-Mäkinen score or the density of lymphoid follicles. Nevertheless, E-cadherin levels were significantly associated with the density of eosinophils (p = 0.007).ConclusionHigh-grade peritumorous inflammation is associated with beneficial morphologic CRC features, including less frequent manifestations of invasion, and is not secondary to tissue damage and necrosis. CLR is not associated with cancer spread by pTN; this finding indirectly suggests an independent role of CLR in carcinogenesis. Further, inflammation by Klintrup-Mäkinen grade and CLR is not dependent on epithelial-mesenchymal transition and stem cell differentiation. Our study highlights the complex associations between inflammation, tumor morphology, EMT, stemness and MMR protein expression in human CRC tissues.

The potential influence of long non-coding RNA PRKG1-AS1 on oral squamous cell carcinoma: A comprehensive study based on bioinformatics and in vitro validation.

Oral squamous cell carcinoma (OSCC) is one of the most frequent malignancies in oral cancer. Herein, we aimed to investigate the influence of lncRNA protein kinase cGMP-dependent type I-Antisense RNA 1 (PRKG1-AS1) in OSCC progression. Basing on the data acquired from TCGA database, the expression and prognostic value of PRKG1-AS1 in OSCC patients were assessed. The expression of PRKG1-AS1 in OSCC cells was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by Cell Counting Kit-8 (CCK8) and colony-forming assays. Transwell assay was employed to test cell invasion and migration. The protein expression of epithelial-mesenchymal transition (EMT)-related markers was detected by Western blotting. The consequences displayed that PRKG1-AS1 was highly expressed in OSCC tissues and high expression of PRKG1-AS1 predicted poor outcomes. The expression of PRKG1-AS1 was higher in CAL27, SCC-9, and SCC-4 than that in normal human oral keratinocytes (NHOK). The results of biological experiments showed that deficiency of PRKG1-AS1 suppressed cell growth, invasion, and migration in CAL27 cells, and over-expression of PRKG1-AS1 accelerated cell growth, invasion, and migration in SCC-4 cells. Finally, silencing of PRKG1-AS1 obviously facilitated the protein expression levels of E-cadherin and reduced levels of N-cadherin, Vimentin, and Snail in CAL27 cells whereas over-expression of PRKG1-AS1 led to opposite results in SCC-4 cells. These outcomes indicated that PRKG1-AS1 functioned as a facilitator in OSCC cell growth, migration, and invasion, which all might be achieved by regulating EMT.

Platelets promote breast cancer cell MCF-7 metastasis by direct interaction: surface integrin \u03b12\u03b21-contacting-mediated activation of Wnt-\u03b2-catenin pathway

BackgroundIntegrin-mediated platelet-tumor cell contacting plays an important role in promoting epithelial-mesenchymal transition (EMT) transformation of tumor cells and cancer metastasis, but whether it occurs in breast cancer cells is not completely clear.ObjectiveThe purpose of this study was to investigate the role of integrin α2β1 in platelet contacting to human breast cancer cell line MCF-7 and its effect on the EMT and the invasion of MCF-7 cells.MethodsHuman platelets were activated by thrombin, and separated into pellets and releasates before the co-incubation with MCF-7 cells. Cell invasion was evaluated by transwell assay. The surface integrins on pellets and MCF-7 cells were inhibited by antibodies. The effect of integrin α2β1 on Wnt-β-catenin pathway was assessed by integrin α2β1-silencing and Wnt-β-catenin inhibitor XAV. The therapeutic effect of integrin α2β1-silencing was confirmed in the xenograft mouse model.ResultsPellets promote the invasion and EMT of MCF-7 cells via direct contacting of surface integrin α2β1. The integrin α2β1 contacting activates Wnt-β-catenin pathway and promotes the expression of EMT proteins in MCF-7 cells. The activated Wnt-β-catenin pathway also promotes the autocrine of TGF-β1 in MCF-7 cells. Both Wnt-β-catenin and TGF-β1/pSmad3 pathways promote the expression of EMT proteins. Integrin α2β1-silencing inhibits breast cancer metastasis in vivo.ConclusionsThe direct interaction between platelets and tumor cells exerts its pro-metastatic function via surface integrin α2β1 contacting and Wnt-β-catenin activation. Integrin α2β1-silencing has the potential effect of inhibiting breast cancer metastasis.

Antitumor Activity of Vanicoside B Isolated from Persicaria dissitiflora by Targeting CDK8 in Triple-Negative Breast Cancer Cells.

A flavonoid glycoside, quercitrin (1), and two phenylpropanoyl sucrose derivatives, vanicoside B (2) and lapathoside C (3), were isolated for the first time from the herb Persicaria dissitiflora. Vanicoside B (2) exhibited antiproliferative activity against a panel of cancer cell lines in triple-negative breast cancer (TNBC) MDA-MB-231 cells. The underlying mechanisms of the antitumor activity of 2 were investigated in TNBC cells. Upregulation of cyclin-dependent kinase 8 (CDK8) was observed in a claudin-low molecular subtype of TNBC cells. A molecular modeling study indicated that 2 showed a high affinity for CDK8. Further investigations revealed that 2 suppressed CDK8-mediated signaling pathways and the expression of epithelial-mesenchymal transition proteins and induced cell cycle arrest and apoptosis in MDA-MB-231 and HCC38 TNBC cells. Moreover, 2 inhibited tumor growth without overt toxicity in a nude mouse xenograft model implanted with MDA-MB-231 cells. Taken together, these findings demonstrate the significance of CDK8 activity in TNBC and suggest a potential use of 2 as a therapeutic candidate for the treatment of aggressive human triple-negative breast cancer.

Prognostic value of multiple epithelial mesenchymal transition‑associated proteins in intrahepatic cholangiocarcinoma

The aim of the present study was to investigate the expression of epithelial mesenchymal transition (EMT)-associated proteins and their prognostic value in intrahepatic cholangiocarcinoma (ICC). The expression of six EMT-associated proteins, including E-cadherin, N-cadherin, Vimentin, Snail family transcriptional repressor 1 (Snail), Snail family transcriptional repressor 2 (Slug) and S100 calcium binding protein A4 (S100A4) was determined by immunohistochemistry in 109 patients with ICC who had received surgery. Survival analysis showed that patients with low E-cadherin expression (P<0.001) or high S100A4 (P<0.001) or Snail (P<0.001) expression had a reduced survival time. Based on the numbers of alterations in the expression of EMT-associated proteins as determined by immunohistochemical analysis, the patients were categorized as low (score, 0-3; n=75) or high (score, ≥4; n=34) EMT expression groups. The high EMT expression group was significantly associated with positive lymph node metastasis (P=0.023) and late Tumor-Node-Metastasis (TNM) stage (P<0.001). Furthermore, patients in the high EMT expression group had a significantly poorer overall survival time than those in the low EMT expression group (P<0.001). Multivariate analysis indicated that EMT status was a significant independent predictor for overall survival time (P=0.004), and was linked to surgical margin (P=0.013) and TNM stage (P<0.001). In conclusion, the reduced expression of E-cadherin and high expression of Snail and S100A4 were significantly associated with the poor survival of patients with ICC after surgery. The EMT protein expression status was associated with ICC progression, and may be considered as an independent prognostic indicator for patients with ICC.

Platelets Promote Breast Cancer Cell MCF-7 Metastasis by Direct Interaction: Surface Integrin α2β1 Contacting Mediated Activation of Wnt-β-Catenin Pathway

Background: Integrin-mediated platelet-tumor cell contacting plays an important role in promoting epithelial-mesenchymal transition (EMT) transformation of tumor cells and cancer metastasis, but whether it occurs in breast cancer cells is not completely clear. Objective: The purpose of this study was to investigate the role of integrin α2β1 in platelet contacting to human breast cancer cell line MCF-7 and its effect on the EMT and the invasion of MCF-7 cells. Methods: Human platelets were activated by thrombin, and separated into pellets and releasates before the co-incubation with MCF-7 cells. Cell invasion was evaluated by transwell assay. The surface integrins on pellets and MCF-7 cells were inhibited by antibodies. The effect of integrin α2β1 on Wnt-β-catenin pathway was assessed by integrin α2β1-silencing and Wnt-β-catenin inhibitor XAV. The therapeutic effect of integrin α2β1-silencing was confirmed in the xenograft mouse model. Results: Pellets promote the invasion and EMT of MCF-7 cells via direct contacting of surface integrin α2β1. The integrin α2β1 contacting activates Wnt-β-catenin pathway and promotes the expression of EMT proteins in MCF-7 cells. The activated Wnt-β-catenin pathway also promotes the autocrine of TGF-β1 in MCF-7 cells. Both Wnt-β-catenin and TGF-β1/pSmad3 pathways promote the expression of EMT proteins. Integrin α2β1-silencing inhibits breast cancer metastasis in vivo. Conclusions: The direct interaction between platelets and tumor cells exerts its pro-metastatic function via surface integrin α2β1 contacting and Wnt-β-catenin activation. Integrin α2β1-silencing has the potential effect of inhibiting breast cancer metastasis. Funding: This study was supported by the National Natural Science Foundation of China (Grant No. 81702595). Declaration of Interest: All authors declare that they have no competing interests. Ethical Approval: This study was approved by the Institute Research Medical Ethics Committee of the First Affiliated Hospital of Zhengzhou University. All the individuals were participating in this study with written informed consent. All animals-treatment operations were executed according to the Zhengzhou University Ethical Guidelines for Animal Experiment.

Upregulation of LASP2 inhibits pancreatic cancer cell migration and invasion through suppressing TGF-β-induced EMT.

LASP2 (LIM and SH3 protein 2), a member of the LIM-protein subfamily of the nebulin group, was first identified as a splice variant of the nebulin gene. In the past, investigators mainly focused on the impact of LASP2 on cardiac diseases because of its identification in the myocardium. Recently, several studies have reported that LASP2 is associated with the progression of various cancers. However, there have been no investigations on the expression and function of LASP2 in pancreatic cancer (PC). In this study, we performed the quantitative real-time polymerase chain reaction and Western blot analysis to detect the expression of LASP2 in PC tissues and cell lines. PC cells were transfected with LASP2 overexpression plasmid or the negative control in the presence or absence of tumor growth factor-β (TGF-β). The transwell assays were used to measure the effects of LASP2 on PC cell migration and invasion. The protein expression of epithelial-mesenchymal transition (EMT) markers was detected using Western blot assay. Our results demonstrated that LASP2 was downregulated in PC tissues and cell lines. In addition, upregulation of LASP2 inhibited the PC cell migration and invasion. We also found that LASP2 upregulation reversed TGF-β-induced EMT in PC cells. Taken together, we provided novel evidence supporting the tumor-suppressor role of LASP2 in PC and suggested it as a potential therapeutic target in PC treatment.

MicroRNA-95-3p promoted the development of prostatic cancer via regulating DKK3 and activating Wnt/β-catenin pathway.

Previous studies have shown that microRNA-95-3p (miR-95-3p) plays a crucial role in multiple human cancers except for prostatic cancer (PCa). Therefore, the function of miR-95-3p was investigated in PCa in the present work. The expression of miR-95-3p was measured by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) assay. Western blot assay was used to examine the protein expression of epithelial-mesenchymal transition (EMT) markers. In addition, the function of miR-95-3p was detected through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and transwell assays. Dual Luciferase assay was applied to confirm the relationship between miR-95-3p and dickkopf-3 (DKK3). The tumor growth was observed through xenograft tumor formation assay. The upregulation of miR-95-3p was detected in PCa tissues and cell lines, which predicted poor prognosis of PCa patients. Moreover, miR-95-3p promoted cell proliferation, migration and invasion in PCa by targeting DKK3 and activating the Wnt/β-catenin pathway. MiR-95-3p also promoted the tumor growth of PCa in vivo. Besides that, downregulation of DKK3 was identified in PCa and low DKK3 expression predicted poor prognosis of PCa patients. MiR-95-3p promoted the development of PCa via targeting DKK3 and activating the Wnt/β-catenin pathway.

Upregulated Interleukin 21 Receptor Enhances Proliferation and Epithelial-Mesenchymal Transition Process in Benign Prostatic Hyperplasia.

Background: Interleukins (ILs) and related chronic inflammation have been found to contribute to the development of benign prostatic hyperplasia (BPH) in recent decades. As a late member of the ILs family, IL-21 receptor (IL-21R) can modulate cell proliferation, however, IL-21R activity in the prostate has not been examined. The current study aimed to elucidate a potential role of IL-21R in the development of BPH.Material and Methods: Human prostate tissues, cell lines and rats were used. QRT-PCR, Western blot, and immunohistochemistry, along with hematoxylin and eosin, Masson's trichrome, and immunofluorescent staining were performed. BPH-1 cells with IL-21R silenced were cultured or co-cultured with macrophages (active THP-1, AcTHP-1). Apoptosis and cell cycle phases were determined via flow cytometry. Epithelial-mesenchymal transition (EMT) processes were also examined. In vivo, rat prostatitis was induced with intraprostatic injected lipopolysaccharide (LPS).Results: IL-21R was highly expressed in human as well as rat prostate, mainly in the epithelial compartment. BPH concomitant with prostatitis significantly upregulated the expression of IL-21R. Knockdown of IL-21R induced cell apoptosis and cycle arrest at G0/G1 phase, and blocked the EMT process in BPH-1 cells. When IL-21R silenced BPH-1 cells were co-cultured with AcTHP-1 cells, these aforementioned processes and IL-21R change were completely reversed. Prostatic hyperplasia was observed with IL-21R upregulated in LPS induced prostatitis rats. More specifically, the expression of apoptosis, cyclin, and EMT proteins in this rat model are altered in a manner consistent with that seen in the cell line model.Conclusions: Our novel data demonstrates the expression and functional activities of IL-21R in the mechanism for development of BPH. IL-21R mainly localized in prostate epithelium and it was upregulated in hyperplastic prostate tissues. IL-21R enhanced proliferation of BPH-1 cells, via inhibiting cell apoptosis, and modulating cell cycles, as well as the EMT process, in response to inflammatory stimuli.

Quercetin inhibits transforming growth factor β1-induced epithelial-mesenchymal transition in human retinal pigment epithelial cells via the Smad pathway.

PurposeThe purpose of this study was to evaluate the effect and mechanism of quercetin on TGF-β1-induced retinal pigment epithelial (RPE) cell proliferation, migration, and extracellular matrix secretion.Materials and methodsCell counting kit-8, transwell, wound-healing assays, and ELISA were used to assess viability, migration, and collagen I secretion, respectively. Western blot analysis and qPCR were employed to detect mRNA and protein expression levels, respectively.ResultsQuercetin suppressed TGF-β1-induced cell proliferation, migration, and collagen I secretion. The results also showed that mRNA and protein expression of epithelial–mesenchymal transition (EMT)-related markers such as alpha-smooth muscle actin and N-cadherin was downregulated by quercetin in TGF-β1-treated RPE cells; conversely, quercetin upregulated the expression of E-cadherin and tight junction protein 1 (ZO-1). In addition, quercetin could inhibit mRNA and protein expression of matrix metalloproteinases. Quercetin may reverse the progression of EMT via the Smad2/3 pathway.ConclusionOur results demonstrate the protective effects of quercetin on RPE cell EMT, revealing a potential therapeutic agent for proliferative vitreoretinopathy treatment.

Expression of Epithelial-Mesenchymal Transition Proteins in Pancreatic Anaplastic (Undifferentiated) Carcinoma.

The aim of this study was to identify an association of pancreatic anaplastic carcinoma (APC) with the epithelial-mesenchymal transition (EMT). Resected APCs (n = 24) were examined to assess components of APCs, including carcinomatous, transitional, and sarcomatous regions. Analysis was performed based on the immunoreactivity of E-cadherin and 3 EMT-related proteins: Slug (zinc finger protein SNAI2), Twist (Twist-related protein 1), and Zeb1 (zinc finger E-box-binding homeobox 1). Expression score was determined based on staining intensity and stained area of the target cells. Finally, we performed a hierarchical clustering based on the expression pattern of E-cadherin and EMT-related proteins of the sarcomatous component. The expression score of E-cadherin decreased in the order of sarcomatous > transitional > carcinomatous components (P < 0.01). Although there were significant differences in the immunohistochemical scores of Slug, Twist, and Zeb1 between carcinomatous and transitional components (P < 0.01), the significant difference in immunohistochemical score of Zeb1 between transitional and sarcomatous components was found (P < 0.05). Furthermore, APCs were divided into 2 subgroups based on the expression patterns of E-cadherin and EMT-related proteins (hierarchical clustering analysis). Consequently, these subgroups were distinguished by Twist expression. Epithelial-mesenchymal transition plays an essential role in the pathogenesis of APC.

Chemo‐preventive and therapeutic effect of the dietary flavonoid kaempferol: A comprehensive review

Kaempferol, a natural flavonoid present in several plants, possesses a wide range of therapeutic properties such as antioxidant, anticancer, and anti-inflammatory. It has a significant role in reducing cancer and can act as a therapeutic agent in the treatment of diseases and ailments such as diabetes, obesity, cardiovascular diseases, oxidative stress, asthma, and microbial contamination disorders. Kaempferol acts through different mechanisms: It induces apoptosis (HeLa cervical cancer cells), decreases cell viability (G2/M phase), downregulates phosphoinositide 3-kinase (PI3K)/AKT (protein kinase B) and human T-cell leukemia/lymphoma virus-I (HTLV-I) signaling pathways, suppresses protein expression of epithelial-mesenchymal transition (EMT)-related markers including N-cadherin, E-cadherin, Slug, and Snail, and metastasis-related markers such as matrix metallopeptidase 2 (MMP-2). Accordingly, the aim of the present review is to collect information pertaining to the effective role of kaempferol against various degenerative disorders, summarize the antioxidant, anti-inflammatory, anticancer, antidiabetic, and antiaging effects of kaempferol and to review the progress of recent research and available data on kaempferol as a protective and chemotherapeutic agent against several ailments.

Serine protease modulation of Dependence Receptors and EMT protein expression

ABSTRACT Expression of the tumour suppressor Deleted in Colorectal Cancer (DCC) and the related protein neogenin is reduced by the mammalian serine protease chymotrypsin or the bacterial serine protease subtilisin, with increased cell migration. The present work examines whether these actions are associated with changes in the expression of cadherins, β-catenin and vimentin, established markers of the Epithelial-Mesenchymal Transition (EMT) which has been linked with cell migration and tumour metastasis. The results confirm the depletion of DCC and neogenin and show that chymotrypsin and subtilisin also reduce expression of β-catenin in acutely prepared tissue sections but not in human mammary adenocarcinoma MCF-7 or MDA-MB-231 cells cultured in normal media, or primary normal human breast cells. A loss of β-catenin was also seen in low serum media but transfecting cells with a dcc-containing plasmid induced resistance. E-cadherin was not consistently affected but vimentin was induced by low serum-containing media and was increased by serine proteases in MCF-7 and MDA-MB-231 cells in parallel with increased wound closure. Vimentin might contribute to the promotion of cell migration. The results suggest that changes in EMT proteins depend on the cells or tissues concerned and do not parallel the expression of DCC and neogenin. The increased cell migration induced by serine proteases is not consistently associated with the expression of the EMT proteins implying either that the increased migration may be independent of EMT or supporting the view that EMT is not itself consistently related to migration. (241).

Polydatin attenuates reactive oxygen species-induced airway remodeling by promoting Nrf2-mediated antioxidant signaling in asthma mouse model

Reactive oxygen species (ROS) and epithelial–mesenchymal transition (EMT) play a critical role in transforming growth factor (TGF)-β1-mediated fibrotic airway remodeling in asthma. Polydatin (PD) is a small natural molecule in Chinese medicine; it is isolated from Polygonum cuspidatum and has antioxidative properties. In this study, we aimed to determine whether PD was protective against ROS-induced pulmonary fibrosis in asthma. Ovalbumin (OVA) was used to induce asthma in a mouse model that was treated with or without PD. We also created nuclear factor erythroid 2-related factor 2 (Nrf2) knockdown BEAS-2B cells and investigated whether PD reversed TGF-β1-induced pulmonary epithelial cell EMT by promotion of Nrf2-mediated antioxidation. Immunofluorescence showed that ROS and TGF-β1 expression was significantly increased in lung tissue from the OVA-induced asthma model. PD treatment inhibited activity of ROS and TGF-β1. Immunohistochemistry showed that PD treatment decreased OVA-induced lung ROS, TGF-β1 expression and fibroblasts. Western blotting showed that PD treatment reversed OVA-induced NADPH oxidase (NOX)1/4 expression by promoting Nrf2-mediated heme oxygenase-1 and NADPH dehydrogenase (quinone)-1 expression. PD treatment suppressed OVA-induced EMT and lung fibroblast protein expression in lung tissue. Nrf2 downregulation suppressed the protective effect of PD by promoting TGF-β1-induced ROS and EMT and accumulation of extracellular-matrix-related protein. All these data indicate that PD has potential therapeutic effects in asthma by promoting Nrf2-mediated antioxidation.

CAFs enhance paclitaxel resistance by inducing EMT through the IL‑6/JAK2/STAT3 pathway.

Carcinoma-associated fibroblasts (CAFs) are the major components of mesenchymal cells in the inflammatory tumor microenvironment. They are involved in epithelial-mesenchymal transition (EMT) and chemotherapy resistance by directly contacting with cancer cells or secretory cytokines. In the present study, we examined the role of CAFs in the induction of EMT in ovarian cancer. Primary ovarian cancer cells, CAFs and normal fibroblasts (NFs) were isolated from fresh cancer tissue and cultured for immunohistochemistry studies. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of IL-6 in the culture supernatants of these cells. The expression of IL-6 at the mRNA level was examined by RT-PCR. The expression of IL-6 at the protein level in ovarian cancer tissues was determined using an immunofluorescence assay in both tissue sections and cell lobes. OVCAR3 cells were treated with the culture supernatants collected from CAFs and NFs. IL-6 monoclonal antibody (mAb) was employed to neutralize IL-6. The expression of phosphorylated STAT3 was assessed. Changes in EMT, proliferation, invasion and proapoptotic protein expression were also examined. Flow cytometry was performed to detect the changes in apoptosis resistance of OVCAR3 cells. The JAK2/STAT3 pathway-specific inhibitor AG490 was used to block this pathway and the β-TGF inhibitor was used to inhibit EMT. The clinical data of patients treated in our hospital were collected between January 1st, 2009 and June 30th, 2013. The expression of interstitial IL-6 in paraffin-embedded tissues was detected by immunohistochemistry. The relationship between the expression of interstitial IL-6 and the treatment response was examined by linear regression and multiple linear regression analyses. We found that CAFs were the main source of IL-6 in ovarian cancer tissue. CAFs promoted the phosphorylation of STAT3 in ovarian cancer and enhanced the proliferation, invasion and EMT. Enhanced EMT may lead to apoptosis resistance, inhibitory expression of pro-apoptotic proteins and paclitaxel resistance. A total of 255 patients were enrolled in this retrospective study. Univariate and multivariate analyses revealed that age, CA125, interstitial IL-6 expression and cytoreduction satisfaction were closely related to the sensitivity of the TP (docetaxel plus cisplatin or carbopatin) regimen in ovarian cancer (P<0.05). These results demonstrated that CAFs highly secreted IL-6 and promoted β-TGF-mediated EMT in ovarian cancer via the JAK2/STAT3 pathway, leading to inhibited apoptosis and subsequent paclitaxel resistance. Therefore, CAFs may be a new therapeutic target for the treatment of ovarian cancer.

Ferrerol overcomes the invasiveness of lung squamous cell carcinoma cells by regulating the expression of inducers of Epithelial Mesenchymal Transition

In recent years Epithelial Mesenchymal Transition (EMT) has been proposed as a mechanism indispensable to acquisition of metastatic properties by tumor cells. In this study we tested the ability of Ferrerol, a Chinese herb-derived compound to ablate the EMT in human lung squamous cell carcinoma cells. Human lung squamous cell carcinoma cells, Calu-1 were treated with various concentrations of Ferrerol for 24 h to examine its effect on their viability by the MTT assay. Only those concentrations which showed least effect on the viability of Calu-1 cells were further used to evaluate the expression of epithelial and mesenchymal markers by western blotting. Furthermore the effect of such concentrations on the migration and invasion of Calu-1 cells was determined by wound healing and transwell invasion assays respectively. The results demonstrated that Ferrerol treatment led to the downregulation of Slug and Zeb-1, transcriptional regulators of EMT with the concomitant increase and decrease in the expression of E-cadherin and vimentin respectively. These data were further supported by migration and invasion assays which demonstrated that Ferrerol treatment caused inhibited the migration and invasion of Calu-1 lung squamous cell carcinoma cells. Taken together, our results indicate that Ferrerol suppresses lung squamous cell carcinoma cell metastatic potential by modulating the expression of EMT proteins.

Inhibitory effects of 3,3′-diindolylmethane on epithelial-mesenchymal transition induced by endocrine disrupting chemicals in cellular and xenograft mouse models of breast cancer

As a phytoestrogen, 3,3′-diindolylmethane (DIM) plays a chemopreventive role by inhibiting cancer progression. In this study, we examined the effects of 17β-estradiol (E2), two endocrine disrupting chemicals (EDCs), triclosan (TCS) and bisphenol A (BPA), and DIM on epithelial-mesenchymal transition (EMT) and metastatic behaviors of estrogen receptor (ER)-positive MCF-7 breast cancer cells. An in vitro assay revealed that E2 (10−9 M), TCS (10−5–10−7 M), and BPA (10−5–10−7 M) induced MCF-7 cell proliferation compared to a control through the ER pathway. In addition, E2, TCS, and BPA changed the cell morphology from the epithelial to the mesenchymal phenotype and increased the migration and invasion capacity of MCF-7 cells via ER; however, co-treatment with DIM (20 μM) effectively suppressed E2, TCS, and BPA-induced cell proliferation, EMT, migration, and invasion of MCF-7 cells. Western blot assay revealed that DIM regulated the protein expression of EMT- and metastasis-related genes toward the inhibition of these processes. Moreover, E2, TCS, and BPA increased the protein expression of CXCR4, which is a receptor of chemokine CXCL12 that is positively involved in breast cancer metastasis via an ER-dependent pathway. Conversely, DIM and a CXCR4 antagonist (AMD3100) decreased CXCR4 protein expression, which led to inhibition of the EMT process, indicating that DIM may suppress E2, TCS or BPA-induced EMT, migration, and invasion of MCF-7 breast cancer cells by suppressing CXCR4 protein expression. These in vitro effects of E2, TCS, BPA, and DIM were also identified in a xenografted mouse model transplanted with MCF-7 breast cancer cells. Taken together, DIM is a potent chemopreventive compound for preventing metastatic behaviors of breast cancer cells induced by EDCs with cancer-related toxicity.

Altered expression of epithelial mesenchymal transition and pluripotent associated markers by sex steroid hormones in human embryonic stem cells.

Embryonic stem (ES) cells are pluripotent stem cells derived from a developmental stage of pre‑implanted embryos. The present study investigated the effect of female sex steroid hormones on the characteristics of human ES cells by using a feeder‑free culture protocol. In a feeder‑free condition without sex hormones, human ES cells assumed the form of tightly packed cells that grow in a monolayer. The cells had clean and defined edges with no evidence of differentiation and expressed several markers specific for undifferentiated ES cells including POU class 5 homeobox 1 (POU5F1), sex determining region Y‑box 2 (SOX2) and NANOG homeobox (NANOG). It was then investigated if female sex steroid hormones including 17β‑estradiol (E2) and progesterone (P4) altered the protein expression of epithelial-mesenchymal transition (EMT) associated markers in addition to pluripotency markers including POU5F1, SOX2 and NANOG in human ES cells. The protein expression levels of N‑cadherin, Snail and Slug were increased while E‑cadherin expression was decreased by treatment of E2 or P4, and the expression levels of POU5F1, SOX2 and NANOG were decreased by the treatment of E2 or P4. When E2 and P4 were treated in combination with an estrogen receptor inhibitor (ICI 182,780) and progesterone receptor inhibitor (RU486) respectively, their effects on EMT and pluripotency of ES cells were restored to control levels. The results suggested that E2 and P4 may regulate EMT and pluripotency of human ES cells by mediating their receptors. The present study may aid in the understanding of the role of sex steroid hormones in the cellular biology of human ES cells.