Increase In Expression Of Snail Research Articles

Role of heat shock protein 90 in hypoxia-induced angiogenesis and epithelial-mesenchymal transition for esophageal squamous cancer cells.

395 Background: Hypoxia is known as an important trigger for the development and progression of human cancers. Heat shock proteins (Hsps) may be up-regulated in response to cellular stressors including hypoxia. To date, the functional role of Hsps within hypoxic tumor microenvironment for esophageal squamous cell cancer (ESCC) remains poorly defined. Methods: CoCl2 was used to induce hypoxia that mimics a hypoxic tumor microenvironment in cultured ESCC cells. Successful induction of hypoxia was confirmed by 2’,7’ –dichlorofluorescein diacetate (DCFDA) assay by detection of cellular reactive oxygen species (ROS). 7-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a selective Hsp90 inhibitor, was used to treat 2 ESCC cell lines, KYSE-170 and -510 cells pretreated with or without CoCl2, an agent to induce a hypoxic tumor microenvironment. Cytotoxicity (MTT) and migration assays were conducted in KYSE-17 and -510 ESCC cells in response to different concentrations of CoCl2, followed by protein expression of Hsp 90, vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1a (HIF-1a), mTOR as well as markers related epithelial-mesenchymal transition (EMT) such as snail or E-cadherin, by immunoblot or ELISA. In parallel, cell proliferation and migration assays of ESCC cells were analyzed. Results: CoCl2 induced hypoxia was supported by an induction of reactive oxygen species (ROS). CoCl2 (200 mM) significantly suppressed cell viability and proliferation with a concomitant up-regulation of VEGF and HIF-1a in a dose-dependent fashion. In contrast, cell migration was significantly increased in response to CoCl2, while down-regulating E-cadherin with concomitant increase in Snail expression. 17-DMAG decreased expression of VEGF and HIF-1a. In addition, it inhibited cell migration and invasion of ESCC cells were analyzed. Conclusions: Our data have shown that CoCl2 induced hypoxia promotes EMT and angiogenesis, which are inhibited by 17-DMAG, suggesting that hypoxia induced EMT and angiogenesis is Hsp 90 dependent in ESCC.

The Role of ROR1 in Chemoresistance and EMT in Endometrial Cancer Cells.

Background and Objectives: Receptor tyrosine kinase-like orphan receptor type 1 (ROR1) plays a critical role in embryogenesis and is overexpressed in many malignant cells. These characteristics allow ROR1 to be a potential new target for cancer treatment. The aim of this study was to investigate the role of ROR1 through in vitro experiments in endometrial cancer cell lines. Materials and Methods: ROR1 expression was identified in endometrial cancer cell lines using Western blot and RT-qPCR. The effects of ROR1 on cell proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT) markers were analyzed in two endometrial cancer cell lines (HEC-1 and SNU-539) using either ROR1 silencing or overexpression. Additionally, chemoresistance was examined by identifying MDR1 expression and IC50 level of paclitaxel. Results: The ROR1 protein and mRNA were highly expressed in SNU-539 and HEC-1 cells. High ROR1 expression resulted in a significant increase in cell proliferation, migration, and invasion. It also resulted in a change of EMT markers expression, a decrease in E-cadherin expression, and an increase in Snail expression. Moreover, cells with ROR1 overexpression had a higher IC50 of paclitaxel and significantly increased MDR1 expression. Conclusions: These in vitro experiments showed that ROR1 is responsible for EMT and chemoresistance in endometrial cancer cell lines. Targeting ROR1 can inhibit cancer metastasis and may be a potential treatment method for patients with endometrial cancer who exhibit chemoresistance.

547 THE ROLE OF HEAT SHOCK PROTEIN 90 IN HYPOXIA INDUCED ANGIOGENESIS AND EPITHELIAL-MESENCHYMAL TRANSITION OF ESOPHAGEAL SQUAMOUS CELL CANCER

Abstract Hypoxia is known as an important trigger for the development of metastases in human cancers. Heat shock proteins (Hsps) are up-regulated by cellular stressors including hypoxia. To date, the functional role of Hsps within the hypoxic tumor microenvironment for esophageal squamous cell cancer (ESCC) remains poorly defined. Methods CoCl₂ was used to induce hypoxia in cultured ESCC cells which was confirmed by 2′,7′ –dichlorofluorescin diacetate (DCFDA) assay. 7-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a selective Hsp90 inhibitor, was used to treat 2 ESCC cell lines, KYSE-170 and -510 cells pretreated with or without CoCl₂₂₂₂₂ in different concentrations, followed by cytotoxicity (MTT) and migration assays. In parallel, expression of Hsp90, vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1α (HIF-1α), and markers related to epithelial-mesenchymal transition (EMT) such as snail/E-cadherin, by immunoblot or ELISA while analyzing cell proliferation and migration of treated ESCC cells. Results CoCl₂ induced hypoxia was supported by induction of reactive oxygen species (ROS). CoCl₂ (200 μM) significantly suppressed cell viability and proliferation with a concomitant up-regulation of VEGF and HIF-1α in a dose-dependent fashion. In contrast, cell migration was significantly increased in response to CoCl₂ while down-regulating E-cadherin with a concomitant increase in Snail expression. 17-DMAG decreased expression of VEGF and HIF-1α while inhibiting cell migration and invasion. Conclusion Our data demonstrate that CoCl2 induced hypoxia promotes EMT and angiogensis, which are inhibited by 17-DMAG. These results suggest that hypoxia induced EMT and angiogensis is Hsp90 dependent in ESCC.

ZEB and Snail expression indicates epithelial-mesenchymal transition in canine melanoma

Melanoma progression is associated with the epithelial-mesenchymal transition (EMT) when tumor cells reduce E-cadherin and increase N-cadherin expression resulting in an escape from the microenvironment via loss of cellular adhesion and gain of motility. Transcription factor proteins Snail and ZEB trigger EMT by repression of epithelial markers and activation of mesenchymal properties. This study evaluated E-cadherin, N-cadherin, Snail, ZEB1 and ZEB2 expression by IHC and investigated their relationship with morphological characteristics in cutaneous and oral canine melanoma. Results from melanoma cases demonstrated E-cadherin expression in 45% (9/20) of oral and 58% (22/38) of cutaneous tumors, while N-cadherin expression was observed in 95% (18/19) of oral and 92% (34/37) of cutaneous melanoma. Cytoplasmic and nuclear N-cadherin expression was positively correlated with ZEB1 expression, while the cell membrane N-cadherin expression was positively correlated with ZEB2. In addition, an increase in nuclear N-cadherin expression was associated with reduced Snail expression in cutaneous melanoma and an increase in Snail expression in oral melanoma, indicating that the correlation between N-cadherin and Snail expression is coincident with tumor location. Our data suggest that ZEB family protein is associated with N-cadherin translocation from cell membrane to the cytoplasm and nuclei, and may act as important transcription factors of EMT regulation in canine melanoma.

ATP Synthase Subunit Epsilon Overexpression Promotes Metastasis by Modulating AMPK Signaling to Induce Epithelial-to-Mesenchymal Transition and Is a Poor Prognostic Marker in Colorectal Cancer Patients.

Metastasis remains the major cause of death from colon cancer. We intend to identify differentially expressed genes that are associated with the metastatic process and prognosis in colon cancer. ATP synthase epsilon subunit (ATP5E) gene was found to encode the mitochondrial F0F1 ATP synthase subunit epsilon that was overexpressed in tumor cells compared to their normal counterparts, while other genes encoding the ATP synthase subunit were repressed in public microarray datasets. CRC cells in which ATP5E was silenced showed markedly reduced invasive and migratory abilities. ATP5E inhibition significantly reduced the incidence of distant metastasis in a mouse xenograft model. Mechanistically, increased ATP5E expression resulted in a prominent reduction in E-cadherin and an increase in Snail expression. Our data also showed that an elevated ATP5E level in metastatic colon cancer samples was significantly associated with the AMPK-AKT-hypoxia-inducible factor-1α (HIF1α) signaling axis; silencing ATP5E led to the degradation of HIF1α under hypoxia through AMPK-AKT signaling. Our findings suggest that elevated ATP5E expression could serve as a marker of distant metastasis and a poor prognosis in colon cancer, and ATP5E functions via modulating AMPK-AKT-HIF1α signaling.

Notch1 increases Snail expression under high reactive oxygen species conditions in hepatocellular carcinoma cells

Notch1 and reactive oxygen species (ROS) modulate important pathways associated with tumor development and progression. Notably, Notch1 expression is upregulated in 41.8% of hepatocellular carcinoma (HCC) patients and ROS levels increases as HCC progresses from Grade I to Grade III. It has been established that Notch1 and ROS modulate Snail expression in malignant tumors; however, the mechanism regulating Snail protein expression is not yet known. In this study, we observed that Notch1 and ROS cooperatively increase the levels of Snail protein in Huh7 (hepatoma) cells. On its own, signaling through Notch1 increases transcription of Snail without changing protein levels. In contrast, the combined activation of the Notch1 and ROS-induced phosphoinositide 3-kinase/Akt (PI3K/Akt) signaling pathways resulted in the high expression of Snail protein. This increase in Snail expression was associated with increased Huh7 cells invasiveness. Furthermore, we observed that correlation between Snail and Notch1 expression was the strongest in advanced grade HCC tissue. In conclusion, Notch1 and ROS-induced PI3K/Akt signals cooperatively increase Snail expression and may induce malignancy in HCC.

Transforming growth factor-β1 induces bronchial epithelial cells to mesenchymal transition by activating the Snail pathway and promotes airway remodeling in asthma

Airway remodeling is characterized by airway wall thickening, subepithelial fibrosis, increased smooth muscle mass, angiogenesis and an increase in mucous glands, which may lead to a chronic and obstinate asthma with pulmonary function depression. In the present study, we observed substantially thickened lung tissue with extensive fibrosis in ovalbumin-sensitized mice, which was interrelated with transforming growth factor-β1 (TGF-β1) expression in bronchoalveolar lavage fluid. In vitro experiments further demonstrated that TGF-β1 resulted in epithelial-mesenchymal transition (EMT) in bronchial epithelial cells, which was characterized by the expected decrease in E-cadherin expression and the increase in vimentin and α-smooth muscle actin expression, as well as the associated increase in Snail expression at mRNA and protein levels. Furthermore, the downregulation of Snail by small interfering RNA (siRNA) attenuated the TGF-β1‑induced EMT-like phenotype. Of note, a significantly increased synthesis of fibronectin was observed following TGF-β1 treatment, which further supported the hypothesis that EMT is a pivotal factor in peribronchial fibrosis. In combination, the results indicated that myofibroblasts deriving from bronchial epithelial cells via EMT may contribute to peribronchial fibrosis and that Snail may be an important factor in this phenomenon.

Overexpression of integrin-linked kinase (ILK) promotes glioma cell invasion and migration and down-regulates E-cadherin via the NF-κB pathway

Integrin-linked kinase (ILK) is a ubiquitously expressed serine/threonine protein kinase that has been implicated in cancer development, progression and metastasis. The aim of the present study was to characterize the role of ILK in glioma cell invasion and migration. We generated a recombinant eukaryotic expression vector containing the human ILK gene and transfected it into human glioma SHG-44 cells. Real-time PCR and western blot analysis were used to identify the stable transformants. The wound healing and Transwell invasion assays showed that ectopic overexpression of ILK in SHG-44 cells significantly promoted their migration and invasion capabilities in culture. This was accompanied by a decrease in expression of E-cadherin and an increase in expression of Snail and Slug. Moreover, the decrease in E-cadherin expression induced by ILK overexpression was greatly restored by the nuclear factor-κB (NF-κB) inhibitor BAY 11-7028 or small interfering RNA targeting NF-κB p65, indicating an involvement of NF-κB in ILK-induced down-regulation of E-cadherin. In conclusion, our data underscore a novel role for ILK in glioma invasion and metastasis processes, implicating potential for therapeutic interference.

Snail Regulates Type I Collagen Expression in Cardiac Fibroblast and Myofibroblast

Snail is a transcription factor that binds to the E‐box sequence to regulate the promoter activity of target genes and is involved in cell signaling, wound healing, cell migration, apoptosis, and EMT. We evaluated the gene expression in Sprague‐Dawely rat myocardial infarction (MI) models. Snail expression was found up regulated in MI heart tissue relative to non‐MI. We then investigated the regulatory role of Snail in angiotensin II (AngII) induced collagen expression. Primary cultures of cardiac fibroblast and myofibroblasts (isolated from site of MI) treated with 10−7 M AngII, a strong stimulator of collagen I expression, resulted in significant increase in Snail expression, confirmed by RT‐PCR. Fibroblast and myofibroblast treated with losartan, an AngII type 1 receptor (AT1) blocker, resulted in reduced Snail expression. Snail siRNA transfection confirmed its regulatory role in AngII induced collagen I expression, which resulted in significant down regulation of collagen I expression (*p= <0.001). These data demonstrate AngII induced collagen expression is regulated by Snail, which is under the control of AT1 receptor mediated signaling pathways in cardiac fibroblast and myofibroblasts.

Mesenchymal transition in kidney collecting duct epithelial cells

Progressive organ damage due to tissue scarring and fibrosis is a paradigm shared by numerous human diseases including chronic kidney disease. The purpose of this study was to confirm the hypothesis that collecting duct (CD) epithelial cells can undergo mesenchymal transition (EMT) in vitro. The mechanism by which CDs undergo EMT is complex and involves both early and late cellular events. Early events include rapid insulin-like growth factor (IGF)-induced Akt and GSK-3beta phosphorylation, associated with early disruption of E-cadherin-beta-catenin membrane colocalization, with translocation of E-cadherin to endosomes, with translocation of beta-catenin to the nucleus, and with an increase in Snail expression. Transforming growth factor-beta1, on the other hand, induced early activation of Smad3 and its translocation to the nucleus, Erk1/2 phosphorylation, and early disruption of membrane E-cadherin localization. The late consequences of these events included a phenotypic transformation of the cells to a mesenchymal morphology with associated increase in vimentin and alpha-smooth muscle actin protein expression and a decrease in total cellular E-cadherin expression, detectable as early as 24 h after stimulation.