Effect of atractylenolide Ⅲ on zearalenone-induced Snail1-mediated epithelial–mesenchymal transition in porcine intestinal epithelium

Background The intestinal epithelium performs essential physiological functions,such as nutrient absorption,and acts as a barrier to prevent the entry of harmful substances.Mycotoxins are prevalent contaminants found in ani-mal feed that exert harmful effects on the health of livestock.Zearalenone(ZEA)is produced by the Fusarium genus and induces gastrointestinal dysfunction and disrupts the health and immune system of animals.Here,we evaluated the molecular mechanisms that regulate the effects of ZEA on the porcine intestinal epithelium.Results Treatment of IPEC-J2 cells with ZEA decreased the expression of E-cadherin and increased the expression of Snai1 and Vimentin,which induced Snail1-mediated epithelial-to-mesenchymal transition(EMT).In addition,ZEA induces Snail-mediated EMT through the activation of TGF-βsignaling.The treatment of IPEC-J2 cells with atractyle-nolideⅢ,which were exposed to ZEA,alleviated EMT.Conclusions Our findings provide insights into the molecular mechanisms of ZEA toxicity in porcine intestinal epi-thelial cells and ways to mitigate it.

Eriocitrin inhibits proliferation and migration of lung adenocarcinoma cells by regulating epithelial mesenchymal transition

Objective:To investigate the effects of Eriocitrin on the proliferation and migration of Lung adenocarcinoma(LUAD)cells A549 and H1299,and the mechanism of Epithelial-Mesenchymal Transition(EMT).Methods:The effects of different Eriocitrin on the proliferation of LUAD cells A549 and H1299 were examined by CCK8 method.EMT-associated epithelial calmodulin(E-cadherin and N-cadherin),vimentin,ferroptosis-associated protein SLC7A11,GPX4,FTH were detected by Western Blot and expression of mRNA of EMT marker molecules E-cadherin,N-cadherin,Snail were detected by qRT-PCR.Effects of saccharomyces cerevisiae suberin on ferroptosis in LUAD cells as observed by lipid reactive oxygen species(ROS)assay.Results:Eriocitrin could significantly inhibit the proliferative behavior of LUAD cells A549 and H1299 and showed a certain dose-and time-dependence.Compared with the control group,different concentrations of Eriocitrin could significantly reduce the scratch healing rate after 24 and 48 h of action,and the difference was statistically significant(P<0.01).The expression of ROS is increased,EMT-related protein E-cadherin was increased in LUAD cells A549 and H1299 compared with the control group after the intervention with Eriocitrin.N-cadherin and Vimentin expression was decreased.E-cadherin mRNA expression was increased,and N-cadherin,Snail mRNA expression was decreased,expression of ferroptosis-associated protein SLC7A11,GPX4,FTH was decreased,the difference was statistically significant(P<0.05).Conclusion:Eriocitrin may inhibit the proliferation and migration of LUAD cells by regulating the EMT pathway and has potential application in LUAD prevention and adjuvant chemotherapy.

Analysis of Differential Gene Expression and Core Canonical Pathways Involved in the Epithelial to Mesenchymal Transition of Triple Negative Breast Cancer Cells by Ingenuity Pathway Analysis

Triple Negative Breast Cancer (TNBC) is a malignant form of cancer with very high mortality and morbidity. Epithelial to Mesenchymal Transition (EMT) is the most common pathophysiological change observed in cancer cells of epithelial origin that promotes metastasis, drug resistance and cancer stem cell formation. Since the information regarding differential gene expression in TNBC cells and cell signaling events leading to EMT is limited, this investigation was done by comparing transcriptomic data generated by RNA isolation and sequencing of a EMT model TNBC cell line in comparison to regular TNBC cells. RNA sequencing and Ingenuity Pathway Software Analysis (IPA) of the transcriptomic data revealed several upregulated and downregulated gene expressions along with novel core canonical pathways including Sirtuin signaling, Oxidative Phosphorylation and Mitochondrial dysfunction events involved in EMT changes of the TNBC cells.

Epithelial-mesenchymal transition transcription factors and mi RNAs: “Plastic surgeons” of breast cancer

Growing evidence suggests that breast cancer cell plasticity arises due to a partial reactivation of epithelialmesenchymal transition(EMT) programs in order to give cells pluripotency, leading to a stemness-like phenotype. A complete EMT would be a dead end program that would render cells unable to fully metastasize to distant organs. Evoking the EMT-mesenchymal-toepithelial transition(MET) cascade promotes successful colonization of distal target tissues. It is unlikely that direct reprogramming or trans-differentiation without passing through a pluripotent stage would be thepreferred mechanism during tumor progression. This review focuses on key EMT transcriptional regulators, EMT-transcription factors involved in EMT(TFs) and the mi RNA pathway, which are deregulated in breast cancer, and discusses their implications in cancer cell plasticity. Cross-regulation between EMT-TFs and mi RNAs, where mi RNAs act as co-repressors or co-activators, appears to be a pivotal mechanism for breast cancer cells to acquire a stem cell-like state, which is implicated both in breast metastases and tumor recurrence. As a master regulator of mi RNA biogenesis, the ribonuclease type Ⅲ endonuclease Dicer plays a central role in EMTTFs/mi RNAs regulating networks. All these EMT-MET key regulators represent valuable new prognostic and predictive markers for breast cancer as well as promising new targets for drug-resistant breast cancers.

Role of epithelial-mesenchymal transition in gastric cancer initiation and progression

Gastric cancer is one of the most common malignant tumors worldwide.Due to its intricate initiation and progression mechanisms,early detection and effective treatment of gastric cancer are difficult to achieve.The epithelial-mesenchymal transition(EMT)is characterized as a fundamental process that is critical for embryonic development,wound healing and fibrotic disease.Recent evidence has established that aberrant EMT activation in the human stomach is closely associated with gastric carcinogenesis and tumor progression.EMT activation endows gastric epithelial cells with increased characteristics of mesenchymal cells and reduces their epithelial features.Moreover,mesenchymal cells tend to dedifferentiate and acquire stem cell or tumorigenic phenotypes such as invasion,metastasis and apoptosis resistance as well as drug resistance during EMT progression.There are a number of molecules that indicate the stage of EMT(e.g.,E-cadherin,an epithelial cell biomarker);therefore,certain transcriptional proteins,especially E-cadherin transcriptional repressors,may participate in the regulation of EMT.In addition,EMT regulation may be associated with certain epigenetic mechanisms.The aforementioned molecules can be used as early diagnostic markers for gastric cancer,and EMT regulation can provide potential targets for gastric cancer therapy.Here,we review the role of these aspects of EMT in gastric cancer initiation and development.

Effect of h GC-MSCs from human gastric cancer tissue on cell proliferation, invasion and epithelial-mesenchymal transition in tumor tissue of gastric cancer tumor-bearing mice

Objective: To study the effect of h GC-MSCs from human gastric cancer tissue on cell proliferation, invasion and epithelial-mesenchymal transition in tumor tissue of gastric cancer tumor-bearing mice. Methods: BABL/c nude mice were selected as experimental animals and gastric cancer tumor-bearing mice model were established by subcutaneous injection of gastric cancer cells, randomly divided into different intervention groups. h GC-MSCs group was given different amounts of gastric cancer cells for subcutaneous injection, PBS group was given equal volume of PBS for subcutaneous injection. Then tumor tissue volume were determined, tumor-bearing mice were killed and tumor tissues were collected, m RNA expression of proliferation, invasion, EMT related molecules were determined. Results: 4, 8, 12, 16, 20 d after intervention, tumor tissue volume of h GC-MSCs group were significantly higher than those of PBS group and the more the number of h GC-MSCs, the higher the tumor tissue volume; m RNA contents of Ki-67, PCNA, Bcl-2, MMP-2, MMP-7, MMP-9, MMP-14, N-cadherin, vimentin, Snail and Twist in tumor tissue of h GC-MSCs group were higher than those of PBS group, and m RNA contents of Bax, TIMP1, TIMP2 and E-cadherin were lower than those of PBS group. Conclusions: h GC-MSCs from human gastric cancer tissue can promote the tumor growth in gastric cancer tumor-bearing mice, and the molecular mechanism includes promoting cell proliferation, invasion and epithelial-mesenchymal transition.

Epithelial-mesenchymal transition in breast cancer progression and metastasis

Breast cancer is the most common cancer in women,and approximately 90% of breast cancer deaths are caused by local invasion and distant metastasis of tumor cells.Epithelial-mesenchymal transition(EMT) is a vital process for large-scale cell movement during morphogenesis at the time of embryonic development.Tumor cells usurp this developmental program to execute the multi-step process of tumorigenesis and metastasis.Several transcription factors and signals are involved in these events.In this review,we summarize recent advances in breast cancer researches that have provided new insights in the molecular mechanisms underlying EMT regulation during breast cancer progression and metastasis.We especially focus on the molecular pathways that control EMT.

Attenuated LKB1-SIK1 signaling promotes epithelial-mesenchymal transition and radioresistance of non–small cell lung cancer cells

Background: Radiotherapy is one of the main therapeutic approaches for non–small cell lung cancer(NSCLC). However, radioresistant cancer cells can eventually cause tumor relapse and even fatal metastasis. It is thought that radioresistance and metastasis could be potentially linked by epithelial?mesenchymal transition(EMT). In this study, we established radioresistant NSCLC cells to investigate the potential relationship among radioresistance, EMT, and enhanced metastatic potential and the underlying mechanism involving liver kinase B1(LKB1)?Salt?inducible kinase 1(SIK1) signaling.Methods: The radioresistant cell lines A549 R and H1299 R were generated by dose?gradient irradiation of the paren?tal A549 and H1299 cells. The radioresistance/sensitivity was evaluated by Cell Counting Kit?8 assay, apoptosis analysis, and/or clonogenic cell survival assay. The EMT phenotype and the signaling change were assessed by Western blot?ting. The abilities of invasion and migration were evaluated by transwell assays and wound healing assays.Results: The radioresistant cell lines A549 R and H1299 R displayed mesenchymal features with enhanced invasion and migration. Mechanistically, A549 R and H1299 R cells had attenuated LKB1?SIK1 signaling, which leaded to the up?regulation of Zinc?finger E?box?binding homeobox factor 1(ZEB1)—a transcription factor that drives EMT. Re?expression of LKB1 in A549 R cells reversed the EMT phenotype, whereas knockdown of LKB1 in H1299 R cells further promoted the EMT phenotype. Moreover, re?expression of LKB1 in A549 cells increased the radiosensitivity, whereas knockdown of LKB1 in H1299 cells decreased the radiosensitivity.Conclusions: Our findings suggest that attenuated LKB1?SIK1 signaling promotes EMT and radioresistance of NSCLC cells, which subsequently contributes to the enhanced metastatic potential. Targeting the LKB1?SIK1?ZEB1 pathway to suppress EMT might provide therapeutic benefits.

Systematic review of the old and new concepts in the epithelial-mesenchymal transition of colorectal cancer

Epithelial-to-mesenchymal transition(EMT) is defined as the transformation of an epithelial cell into a spindle cell with the loss of membrane E-cadherin expression and the gain of mesenchymal markers positivity. In the field of colorectal cancer(CRC), first data about EMT was published in 1995 and more than 400 papers had been written up to March 2016. Most of them are focused on the molecular pathways and experimentally-proved chemoresistance. In the present article, an update in the field of EMT in CRC based on the review of the literature and personal experience of the authors is presented. The information about the molecular and immunohistochemical(IHC) particularities of these processes and their possible role in the prognosis of CRC were also up-dated. This article focuses on the IHC quantification of the EMT, the immunoprofile of tumor buds and on the relation between EMT, angiogenesis, and stem cells activation. The EMT-induced chemoresistance vs chemotherapyor radiotherapy-induced EMT and cellular senescence was also synthesized for both conventional and targeted therapy. As a future perspective, the EMTangiogenesis-stemness link could be used as a possible valuable parameter for clinical follow-up and targeted therapeutic oncologic management of patients with CRC. Association of dexamethasone and angiotensin converting enzyme inhibitors combined with conventional chemotherapies could have clinical benefits in patients with CRC. The main conclusion is that, although many studies have been published, the EMT features are still incompletely elucidated and newly discovered EMT markers provide confusing data in understanding this complicated process, which might have significant clinical impact.

Evaluation of epithelial-mesenchymal transitioned circulating tumor cells in patients with resectable gastric cancer: Relevance to therapy response

AIM: To evaluate the epithelial-to-mesenchymal transition(EMT) of circulating tumor cells(CTCs) in gastric cancer patients.METHODS: We detected tumor cells for expression of four epithelial(E^+) transcripts(keratins 8, 18, and 19 and epithelial cell adhesion molecule) and two mesenchymal(M^+) transcripts(Vimentin and Twist) by a quantifiable, dual-colorimetric RNA-in situ hybridization assay. Between July 2014 and October 2014, 44 patients with gastric cancer were recruited for CTC evaluation. Blood samples were obtained from selected patients during the treatment course [before surgery, after surgery and at the 6^(th) cycle of XELOX based chemotherapy(about 6 mo postoperatively)].RESULTS: We found the EMT phenomenon in which there were a few biphenotypic E^+/M^+ cells in primary human gastric cancer specimens. Of the 44 patients, the presence of CTCs was reported in 35(79.5%) patients at baseline. Five types of cells including from exclusively E^+ CTCs to intermediate CTCs and exclusively M^+ CTCs were identified(4 patients with M^+ CTCs and 10 patients with M^+ or M^+ > E^+ CTCs). Further, a chemotherapy patient having progressive disease showed a proportional increase of mesenchymal CTCs in the post-treatment blood specimens. We used NCI-N87 cells to analyze the linearity and sensitivity of Can Patrol^(TM) system and the correlation coefficient(R^2) was 0.999.CONCLUSION: The findings suggest that the EMT phenomenon was both in a few cells of primary tumors and abundantly in CTCs from the blood of gastric cancer patients, which might be used to monitor therapy response.

Epithelial-mesenchymal transition- activating transcription factors- multifunctional regulators in cancer

The process of epithelial to mesenchymal transition(EMT), first noted during embryogenesis, has also been reported in tumor formation and leads to the development of metastatic growth. It is a naturally occurring process that drives the transformation of adhesive,non-mobile epithelial like cells into mobile cells with a mesenchymal phenotype that have ability to migrate to distant anatomical sites. Activating complex network of embryonic signaling pathways, including Wnt, Notch,hedgehog and transforming growth factor-β pathways,lead to the upregulation of EMT activating transcription factors, crucial for normal tissue development and maintenance. However, deregulation of tightly regulated pathways affecting the process of EMT has been recently investigated in various human cancers. Given the critical role of EMT in metastatic tumor formation,better understanding of the mechanistic regulation provides new opportunities for the development of potential therapeutic targets of clinical importance.

Inflammatory microenvironment contributes to epithelial-mesenchymal transition in gastric cancer

Gastric cancer(GC) is the fifth most common malignancy in the world. The major cause of GC is chronic infection with Helicobacter pylori(H. pylori). Infection with H. pylori leads to an active inflammatory microenvironment that is maintained by immune cells such as T cells, macrophages, natural killer cells, among other cells. Immune cell dysfunction allows the initiation and accumulation of mutations in GC cells, inducing aberrant proliferation and protection from apoptosis. Meanwhile, immune cells can secrete certain signals, including cytokines, and chemokines, to alter intracellular signaling pathways in GC cells. Thus, GC cells obtain the ability to metastasize to lymph nodes by undergoing the epithelial-mesenchymal transition(EMT), whereby epithelial cells lose their epithelial attributes and acquire a mesenchymal cell phenotype. Metastasis is a leading cause of death for GC patients, and the involved mechanisms are still under investigation. In this review, we summarize the current research on how the inflammatory environment affects GC initiation and metastasis via EMT.

Mechanisms of fibrogenesis in liver cirrhosis:The molecular aspects of epithelial-mesenchymal transition

Liver injuries are repaired by fibrosis and regeneration.The cause of fibrosis and diminished regeneration,especially in liver cirrhosis,is still unknown.Epithelialmesenchymal transition(EMT) has been found to be associated with liver fibrosis.The possibility that EMT could contribute to hepatic fibrogenesis reinforced the concept that activated hepatic stellate cells are not the only key players in the hepatic fibrogenic process and that other cell types,either hepatic or bone marrow-derived cells could contribute to this process.Following an initial enthusiasm for the discovery of this novel pathway in fibrogenesis,more recent research has started to cast serious doubts upon the real relevance of this phenomenon in human fibrogenetic disorders.The debate on the authenticity of EMT or on its contribution to the fibrogenic process has become very animated.The overall result is a general confusion on the meaning and on the definition of several key aspects.The aim of this article is to describe how EMT participates to hepatic fibrosis and discuss the evidence of supporting this possibility in order to reach reasonable and useful conclusions.

KAI1 reverses the epithelial-mesenchymal transition in human pancreatic cancer cells

Background: Epithelial-mesenchymal transition (EMT) plays an important role in pancreatic cancer (PC). In the present study, we investigated the effects of KAI1 gene overexpression on the EMT of human PC cell lines, MIA PaCa-2 and PACN-1. Methods: Plasmids overexpressing KAI1 and pCMV were transfected into MIA PaCa-2 and PACN-1 cells, respectively. After selection of differently transfected cells by G418, KAI1 protein levels were examined by Western blotting, and transfected cells were renamed as MIA PaCa-2-K, MIA PaCa-2-p, PACN-1-K and PACN-1-p. Wound healing and Transwell migration assays were then performed comparing the two groups of cells. EMT-related markers were analyzed by Western blotting. Results: The percentage of wound closure significantly decreased in MIA PaCa-2-K cells compared with MIA PaCa-2-p and MIA PaCa-2 cells after 24, 48 and 72 h ( P < 0.05). In PACN-1-K cells, the percentage of wound closure significantly decreased as well ( P < 0.05). Numbers of invading MIA PaCa-2, MIA PaCa-2-p and MIA PaCa-2-K cells were determined as 48.0 ±15.4, 50.0 ±12.4, and 12.0 ±3.8, respectively. The corresponding numbers of invading PACN-1, PACN-1-p and PACN-1-K cells were 29.0 ±10.6, 31.0 ±11.4, and 8.0 ±4.2, respectively. KAI1 overexpression induced a significant upregulation of E-cadherin and also significant downregulation of Snail, vimentin, matrix metalloproteinase 2 (MMP2) and MMP9 (all P < 0.05) in PC cells. Conclusions: KAI1 reversed EMT-related marker expression and inhibited migration and invasion of PC cells. Thus, KAI1 might represent a novel potential therapeutic target for PC.

Qinggan Huoxue Recipe suppresses epithelial-tomesenchymal transition in alcoholic liver fibrosis through TGF-β1/Smad signaling pathway

AIM: To investigate the mechanism by which Qinggan Huoxue Recipe (QGHXR) inhibits epithelial-to-mesenchymal transition (EMT) in rats with alcoholic liver fibrosis (ALF).METHODS: A total of 75 male SD rats were used to induce ALF. Serum biochemical indicators, including alanine aminotransferase, aspartate aminotransferase, laminin and hyaluronidase, were measured. Liver histopathological changes were evaluated using hematoxylin-eosin and Sirius red staining. EMT was examined by analyzing the expression of the epithelial marker E-cadherin and the mesenchymal markers vimentin and fibronectin using RT-PCR and Western blot. The inhibitory effect of QGHXR on EMT markers, as well as its effect on molecules associated with the transforming growth factor (TGF)-&#x003b2;1/Smad signaling pathway, including TGF-&#x003b2;1, Smad3, snail, occludin, ZO-1 and claudin, was also examined.RESULTS: Compared with normal control rats, ALF rats exhibited a decrease in E-cadherin levels (mRNA: ALF 0.16 &#x000b1; 0.05 vs control 1.00 &#x000b1; 0.08; protein: ALF 0.09 &#x000b1; 0.05 vs control 0.70 &#x000b1; 0.17, P &#x0003c; 0.01) and an increase in vimentin and fibronectin levels (mRNA: 11.43 &#x000b1; 0.39 vs 1.00 &#x000b1; 0.19 and 9.91 &#x000b1; 0.34 vs 1.00 &#x000b1; 0.44, respectively, P &#x0003c; 0.01; protein: 1.13 &#x000b1; 0.42 vs 0.09 &#x000b1; 0.03 and 1.16 &#x000b1; 0.43 vs 0.09 &#x000b1; 0.00, respectively, P &#x0003c; 0.01). This indicates that EMT occurred in ALF rats. In addition, the TGF-&#x003b2;1/Smad signaling pathway was activated in ALF rats, as evidenced by the increase in TGF-&#x003b2;1 and snail levels (mRNA: 1.76 &#x000b1; 0.12 vs 1.00 &#x000b1; 0.05 and 6.98 &#x000b1; 0.41 vs 1.00 &#x000b1; 0.10, respectively, P &#x0003c; 0.01; protein: 1.43 &#x000b1; 0.05 vs 0.12 &#x000b1; 0.03 and 1.07 &#x000b1; 0.29 vs 0.07 &#x000b1; 0.02, respectively, P &#x0003c; 0.01) and the decrease in Smad3 levels (mRNA: 0.05 &#x000b1; 0.01 vs 1.00 &#x000b1; 0.12, P &#x0003c; 0.01; protein: 0.06 &#x000b1; 0.05 vs 0.89 &#x000b1; 0.12, P &#x0003c; 0.01). Furthermore, levels of the tight junction markers occludin, ZO-1 and claudin decreased in ALF rats compared with healthy control rats (mRNA: 0.60 &#x000b1; 0.09 vs 1.00 &#x000b1; 0.12, 0.11 &#x000b1; 0.00 vs 1.00 &#x000b1; 0.12 and 0.60 &#x000b1; 0.01 vs 1.00 &#x000b1; 0.08, respectively, P &#x0003c; 0.01; protein: 0.05 &#x000b1; 0.01 vs 0.87 &#x000b1; 0.40, 0.09 &#x000b1; 0.05 vs 0.89 &#x000b1; 0.18 and 0.04 &#x000b1; 0.03 vs 0.95 &#x000b1; 0.21, respectively, P &#x0003c; 0.01). In ALF rats treated with QGHXR, E-cadherin levels increased (mRNA: QGHXR 0.67 &#x000b1; 0.04 vs ALF model 0.16 &#x000b1; 0.05, P &#x0003c; 0.01; protein: QGHXR 0.66 &#x000b1; 0.21 vs ALF model 0.09 &#x000b1; 0.05, P &#x0003c; 0.01), and vimentin and fibronectin levels decreased (mRNA: 6.57 &#x000b1; 1.05 vs 11.43 &#x000b1; 0.39 and 1.45 &#x000b1; 1.51 vs 9.91 &#x000b1; 0.34, respectively, P &#x0003c; 0.01; protein: 0.09 &#x000b1; 0.03 vs 1.13 &#x000b1; 0.42 and 0.10 &#x000b1; 0.01 vs 1.16 &#x000b1; 0.43, respectively, P &#x0003c; 0.01). In addition, QGHXR inhibited the expression of TGF-&#x003b2;1 and increased the expression of Smad3 (mRNA: 1.03 &#x000b1; 0.11 vs 1.76 &#x000b1; 0.12, 0.70 &#x000b1; 0.10 vs 0.05 &#x000b1; 0.01, respectively, P &#x0003c; 0.05 and P &#x0003c; 0.01; protein: 0.12 &#x000b1; 0.03 vs 1.43 &#x000b1; 0.05 and 0.88 &#x000b1; 0.20 vs 0.06 &#x000b1; 0.05, respectively, P &#x0003c; 0.01). QGHXR treatment also reduced the levels of the EMT-inducing transcription factor snail (mRNA: 2.28 &#x000b1; 0.33 vs 6.98 &#x000b1; 0.41, P &#x0003c; 0.01; protein: 0.08 &#x000b1; 0.02 vs 1.07 &#x000b1; 0.29, P &#x0003c; 0.01) and increased the occludin, ZO-1 and claudin levels (mRNA: 0.73 &#x000b1; 0.05 vs 0.60 &#x000b1; 0.09, 0.57 &#x000b1; 0.04 vs 0.11 &#x000b1; 0.00 and 0.68 &#x000b1; 0.03 vs 0.60 &#x000b1; 0.01, respectively, P &#x0003c; 0.01, P &#x0003c; 0.01 and P &#x0003c; 0.05; protein: 0.92 &#x000b1; 0.50 vs 0.05 &#x000b1; 0.01, 0.94 &#x000b1; 0.22 vs 0.09 &#x000b1; 0.05 and 0.94 &#x000b1; 0.29 vs 0.04 &#x000b1; 0.03, respectively, P &#x0003c; 0.01). The effects of QGR and HXR on the TGF-&#x003b2;1/Smad signaling pathway were similar to that of QGHXR; however, the QGR- and HXR-induced changes in vimentin mRNA levels, the QGR-induced changes in fibronectin mRNA levels and the HXR-induced changes in snail and TGF-&#x003b2;1 mRNA levels were not significant.CONCLUSION: Qinggan Huoxue Recipe inhibits EMT in ALF rats by modulating the TGF-&#x003b2;1/Smad signaling pathway, suggesting that the mechanism underlying the amelioration of ALF induced by QGHXR is associated with this pathway.

GSK3β inhibits epithelial-mesenchymal transition via the Wnt/β-catenin and PI3K/Akt pathways

AIM： To investigate the regulatory mechanism of glycogen synthase kinase 3β（GSK3β） in epithelialmesenchymal transition（EMT） process after proliferative vitreoretinopathy（PVR） induction. METHODS： Experimental PVR was induced by intravitreal injection of retinal pigment epithelium（RPE） cells in the eyes of rabbits. A PI3 K/Akt inhibitor（wortmannin） and a GSK3β inhibitor（Li Cl） were also injected at different time during PVR progress. Electroretinogram（ERG）, ocular fundus photographs, and B-scan ultrasonography were used to observe the PVR progress. Western blot test on the extracted retina were performed at 1, 2, 4 wk. The expression of the mesenchymal marker vimentin was determined by immunohistochemistry. Toxicity of wortmannin and Li Cl were evaluated by ERG and Td Tmediated d UTP nick-end labeling（TUNEL） assay. The vitreous was also collected for metabolomic analysis. RESULTS： Experimental PVR could significantly lead to EMT, along with the suppressed expression of GSK3β and the activation of Wnt/β-catenin and PI3 K/Akt pathways. It was verified that upregulating the expression of GSK3β could effectively inhibit EMT process by suppressing Wnt/β-catenin and PI3 K/Akt pathways. CONCLUSION： GSK3β effectively inhibits EMT via the Wnt/β-catenin and PI3 K/Akt pathways. GSK3β may be regarded as a promising target of experimental PVR inhibition.

Integrin-linked kinase overexpression promotes epithelial-mesenchymal transition via nuclear factor-κB signaling in colorectal cancer cells

AIM: To investigate the effect of integrin-linked kinase (ILK) on proliferation, metastasis, and invasion of the colorectal cancer cell line SW480.METHODS: In this study, the colorectal cancer cell line SW480 was stably transfected with ILK plasmids, and small interfering RNA (siRNA) was used to knockdown expression of nuclear factor (NF)-&#x003ba;B/p65. Methylthiazole tetrazolium (MTT) assay was performed to measure proliferation, and the wound healing migration assay and matrigel invasion assay were used to test the metastasis and invasion ability of SW480 cells. To explore the epithelial-mesenchymal transition (EMT) process, embryonic development, and the invasion and metastasis of tumors, the protein level of E-cadherin, vimentin, snail, and slug was detected by western blot. Immunofluorescence was also used to detect E-cadherin expression. Western blot was used to determine the level of phosphorylated-inhibitor of kappa B (I&#x003ba;B)a, inhibitor of gamma B (I&#x003b3;B)a, and nuclear factor kappa B (NF-&#x003ba;B) expressions and to explore the ILK signaling pathway.RESULTS: Western blot results revealed that ILK expression significantly increased when ILK was overexpressed in SW480 cells (P &#x0003c; 0.05). Proliferation, metastasis, and invasion ability were improved in the vector-ILK group compared to the vector group (P &#x0003c; 0.05). Immunofluorescence results revealed that E-cadherin fluorescence intensity decreased after ILK was overexpressed (P &#x0003c; 0.05). Western blot results revealed that the protein expression of E-cadherin was reduced, while vimentin, snail, and slug were upregulated when ILK was overexpressed in SW480 cells (P &#x0003c; 0.05). In order to determine the role of the NF-&#x003ba;B signaling pathway in ILK overexpression promoted EMT occurrence, we overexpressed ILK in SW480 cells and found that levels of NF-&#x003ba;B/p65 and cytoplasmic phosphorylated-I&#x003ba;Ba were increased and that cytoplasmic I&#x0043a;Ba levels were decreased compared to the control group (P &#x0003c; 0.05). Furthermore, NF-&#x003ba;B/p65 knockout revealed that E-cadherin was increased in the overexpressed ILK group.CONCLUSION: ILK overexpression improved the proliferation, metastasis, and invasion ability of SW480 cells, and this effect may be mediated by the NF-&#x003ba;B signaling pathway.

Roles of Rho/Rock Signaling Pathway in Silica-induced Epithelial-mesenchymal Transition in Human Bronchial Epithelial Cells

Objective To investigate the roles of Rho/Rock signaling pathway in silica-induced Epithelial-mesenchymal transition （EMT） in human bronchial epithelial cells （BEC） in vitro. Methods Human BEC were incubated with silica with various concentrations for indicated times. Cell viability was assayed by MTT test. Morphologic Changes were observed by microscope. Mesenchymal marker a-smooth muscle actin （a-SMA）, vimentin （Vim）, and epithelial marker E-cadherin （E-cad） were analyzed by Western Blot. The pull-down assay was used to measure Rho activity. In the prevention experiments, the specific inhibitor for Rho effector ROCK （Y27632） was used to inhibit the activity of Rho. Results Human BEC stimulated with silica were converted from a ＂cobblestone＂ epithelial structure into an elongated fibroblast-like shape structure. Incubation of human BEC with silica induced de novo expression of a-SMA and Vim, and loss of E-cad. Also, silica treatment resulted in Rho activation in human BEC. Y27632 up-regulated the E-cad expression but attenuated a-SMA and Vim expression in silica-stimulated cells. Conclusion The activation of Rho/ROCK signaling pathways is most likely involved in Silica-induced EMT in human bronchial epithelial cells.

In vitro inhibition of proliferation,migration and epithelial-mesenchymal transition of human lens epithelial cells by fasudil

AIM： To study the potential role of fasudil as a treatment for posterior capsular opacification（PCO） of the human crystalline lens.METHODS： Human lens epithelial cells（HLECs; line SRA01/04） was exposed to transforming growth factor-β2（TGF-β2） to induce the process of epithelial-mesenchymal transition（EMT）. Fasudil was applied to the cell samples. Its effect on overall HLECs proliferation and migration was studied, as was its influence on EMT induction by TGF-β2 using cell migration assay, MTT colorimetric assay and Western blot assay.RESULTS： Fasudil inhibited the proliferation of SRA01/04. Its effect was time-and concentration-dependent. The migration of SRA01/04 cells was significantly reduced 24-72 h after fasudil treatment, and the half maximal inhibitory concentration（IC50） was 22.37 μmol/mL at 72 h. Reversal of the elongated, fibroblast-like shape changes induced by TGF-β2 in SRA01/04 cells was observed. Fasudil up-regulated the expression of Connexin43 protein and down-regulated the expression of α-SMA protein compared with the cells treated with TGF-β2. Furthermore, when exposed to fasudil, the phosphorylation of Rhoassociated protein kinase（Rock） and myosin light chain（MLC） could not be activated in the cell preparations.CONCLUSION： Fasudil suppresses the proliferation and migration of SRA01/04 cells, and inhibits the process of EMT induced by TGF-β2. These results suggest that fasudil may serve as a therapeutic agent for PCO.

Side Population Cells in Human Gallbladder Cancer Cell Line GBC-SD Regulated by TGF-β-induced Epithelial-mesenchymal Transition

Mounting evidence has shown that side population （SP） cells are enriched for cancer stem cells （CSCs） responsible for cancer malignancy. In this study, SP technology was used to isolate a small subpopulation of SP cells in human gallbladder cancer cell line GBC-SD, and SP cells which had superior potential for proliferation in vitro and tumorigenesis in vivo were identified. Importantly, the abundance of GBC-SD SP cells was increased by a transforming growth factor-β （TGF-β）-induced epithelial-mesenchymal transition （EMT）, and this effect was accompanied with a strong up-regulation of ABCG2 mRNA expression, and a decreased sensitivity to mitoxantrone. SP cells were restored upon the removal of TGF-β and the reversion of the cells to an epithelial phenotype, and smad3-specific siRNA reduced SP abundance in response to TGF-β. In conclusion, TGF-β-induced EMT by smad-dependent signaling pathway promotes cancer development and anti-cancer drug resistant phenotype by augmenting the abundance of GBC-SD SP cells, and a better understanding of mechanisms involved in TGF-β-induced EMT may provide a novel strategy for preventing cancer progression.