ABSTRACT: This experiment is designed to evaluate gene expression alteration following silencing SMAD2/3 and overexpressing CCT6A in A549 lung cancer cells. Total RNA were extraced from indicated stable cell lines treated with or with out TGF-Î²
Project description:This experiment is designed to evaluate gene expression alteration following silencing SMAD2/3 and overexpressing CCT6A in A549 lung cancer cells. Total RNA were extraced from indicated stable cell lines treated with or with out TGF-β
Project description:This experiment is designed to evaluate gene expression alteration following silencing SMAD2/3 and overexpressing CCT6A in A549 lung cancer cells. Overall design: Total RNA were extraced from indicated stable cell lines treated with or with out TGF-β
Project description:Paradoxically, during early tumor development in many cancer types, TGF-? acts as a tumor suppressor, whereas in the advanced stages of these cancers, increased TGF-? expression is linked to high metastasis and poor prognosis. These findings suggest that unidentified mechanisms may function to rewire TGF-? signaling toward its prometastatic role in cancer cells. Our current study using non-small-cell lung carcinoma (NSCLC) cell lines, animal models, and clinical specimens demonstrates that suppression of SMAD2, with SMAD3 function intact, switches TGF-?-induced transcriptional responses to a prometastatic state. Importantly, we identified chaperonin containing TCP1 subunit 6A (CCT6A) as an inhibitor and direct binding protein of SMAD2 and found that CCT6A suppresses SMAD2 function in NSCLC cells and promotes metastasis. Furthermore, selective inhibition of SMAD3 or CCT6A efficiently suppresses TGF-?-mediated metastasis. Our findings provide a mechanism that directs TGF-? signaling toward its prometastatic arm and may contribute to the development of therapeutic strategies targeting TGF-? for NSCLC.
Project description:The control of radioresistance and metastatic potential of surviving cancer cells is important for improving cancer eradication by radiotheraphy. The distal-less homeobox2 (DLX2) gene encodes for a homeobox transcription factor involved in morphogenesis and its deregulation was found in human solid tumors and hematologic malignancies. Here we investigated the role of DLX2 in association with radiation-induced epithelial to mesenchymal transition (EMT) and stem cell-like properties and its regulation by Smad2/3 signaling in irradiated A549 and MDA-MB-231 human cancer cell lines. In irradiated A549 and MDA-MB-231 cells, EMT was induced as demonstrated by EMT marker expression, phosphorylation of Smad2/3, and migratory and invasive ability. Also, irradiated A549 and MDA-MB-231 cells showed increased cancer stem cells (CSCs) marker. Interestingly, DLX2 was overexpressed upon irradiation. Therefore, we examined the role of DLX2 in radiation-induced EMT and radioresistance. The overexpression of DLX2 alone induced EMT, migration and invasion, and CSC marker expression. The reduced colony-forming ability in irradiated cells was partially restored by DLX2 overexpression. On the other hand, the depletion of DLX2 using si-RNA abolished radiation-induced EMT, CSC marker expression, and phosphorylation of Smad2/3 in irradiated A549 and MDA-MB-231 cells. Also, depletion of DLX2 increased the radiation sensitivity in both cell lines. Moreover, knockdown of Smad2/3, a key activator of TGF-?1 pathway, abrogated the radiation-induced DLX2 expression, indicating that radiation-induced DLX2 expression is dependent on Smad2/3 signaling. These results demonstrated that DLX2 plays a crucial role in radioresistance, radiation-induced EMT and CSC marker expression, and the expression of DLX2 is regulated by Smad2/3 signaling in A549 and MDA-MB-231 cell lines.
Project description:To find out genes regulated by TGF-Î² in A549 cells, we compared gene expression of cells treated with 1ng/ml TGF-Î² versus non-treated cells and find out that expression of one transmembrane protein, TM4SF20, is reduced by TGF-Î². A549 cells were treated with or without 1ng/ml TGF-Î² for 12h. RNA was extracted and hybridized on Affymetrix microarrays
Project description:Idiopathic pulmonary fibrosis (IPF) is a chronic and usually progressive lung disease and the epithelial-mesenchymal transition (EMT) may play an important role in the pathogenesis of pulmonary fibrosis. IL-17 is a proinflammatory cytokine which promotes EMT profiles in lung inflammatory diseases. In this study, we investigated the effect of IL-17 on EMT in alveolar epithelial cell line A549 and the role of TGFβ1-Smad and ERK signaling pathways in the process. Morphological observation on the cells was performed under inverted microscope. The mRNA and protein expressions of E-cad and α-SMA were detected by quantitative RT-PCR and western blotting. The mRNA and protein expressions of TGF-β1 were analyzed via quantitative RT-PCR and ELISA. Expressions of Smad2/3, p-Smad2/3, ERK1/2, p-ERK1/2 and p-JNK were examined by western blotting. The results indicated that IL-17 can induce A549 cells to undergo morphological changes and phenotypic markers changes, such as down-regulated E-cad expression and up-regulated α-SMA expression. Additionally, IL-17 enhanced TGF-β1 expression and stimulated Smad2/3 and ERK1/2 phosphorylation in A549 cells. However, there were no significant differences in the expression of phosphorylated JNK in A549 cells with or without IL-17 treatment. SB431542 or U0126 treated cells showed inhibited morphological changes and phenotypic markers expression, such as up-regulated E-cad expression and down-regulated α-SMA expression. In summary, our results suggest that IL-17 can induce A549 alveolar epithelial cells to undergo EMT via the TGF-β1 mediated Smad2/3 and ERK1/2 activation.
Project description:Background:Epithelial-mesenchymal transition (EMT) plays an important role in fibrosis, chronic inflammation, tumor metastasis, etc. Glycyrrhizin, an active component extracted from licorice plant, has been reported to treat a variety of inflammatory reactions through inhibiting high-mobility group box1 (HMGB1), which has been suggested to be a significant mediator in EMT process. However, whether glycyrrhizin affects the EMT process or not remains unclear. Methods:Human alveolar epithelial cell line A549 and normal human bronchial epithelial cell line BEAS-2B were treated with extrinsic TGF-?1 to induce EMT. Elisa was used to detect HMGB1 concentrations in cell supernatant. RNA interference and lentivirus infection experiments were performed to investigate the involvement of HMGB1 in EMT process. Cell Counting Kit-8 (CCK-8) was used to detect the viability of A549 and BEAS-2B cells treated with glycyrrhizin. Finally, the effects of glycyrrhizin on EMT changes, as well as the underlying mechanisms, were evaluated via Western blot, immunofluorescence and transwell assays. Results:Our results showed that HMGB1 expression was increased by TGF-?1, and knockdown of HMGB1 expression reversed TGF-?1-induced EMT in A549 and BEAS-2B cells. Ectopic HMGB1 expression or TGF-?1 treatment caused a significant increase in HMGB1 release. Notably, we found that glycyrrhizin treatment effectively suppressed TGF-?1-induced EMT process by inhibiting HMGB1. Also, glycyrrhizin significantly inhibited the migration of both A549 and BEAS-2B cells promoted by TGF-?1. Mechanistically, HMGB1 overexpression could activate Smad2/3 signaling in A549 and BEAS-2B cells. Glycyrrhizin significantly blocked the phosphorylation of Smad2/3 stimulated either by TGF-?1 or by ectopic HMGB1 in A549 and BEAS-2B cells. Conclusions:HMGB1 is a vital mediator of EMT changes induced by TGF-?1 in lung epithelial cells. Importantly, glycyrrhizin can effectively block Smad2/3 signaling pathway through inhibiting HMGB1, thereby suppressing the EMT progress.
Project description:<h4>Rationale</h4>Airway hyperreactivity and remodeling are characteristic features of asthma. Interactions between the airway epithelium and environmental allergens are believed to be important in driving development of pathology, particularly because altered epithelial gene expression is common in individuals with asthma.<h4>Objectives</h4>To investigate the interactions between a modified airway epithelium and a common aeroallergen in vivo.<h4>Methods</h4>We used an adenoviral vector to generate mice overexpressing the transforming growth factor-beta signaling molecule, Smad2, in the airway epithelium and exposed them to house dust mite (HDM) extract intranasally.<h4>Measurements and main results</h4>Smad2 overexpression resulted in enhanced airway hyperreactivity after allergen challenge concomitant with changes in airway remodeling. Subepithelial collagen deposition was increased and smooth muscle hyperplasia was evident resulting in thickening of the airway smooth muscle layer. However, there was no increase in airway inflammation in mice given the Smad2 vector compared with the control vector. Enhanced airway hyperreactivity and remodeling did not correlate with elevated levels of Th2 cytokines, such as IL-13 or IL-4. However, mice overexpressing Smad2 in the airway epithelium showed significantly enhanced levels of IL-25 and activin A after HDM exposure. Blocking activin A with a neutralizing antibody prevented the increase in lung IL-25 and inhibited subsequent collagen deposition and also the enhanced airway hyperreactivity observed in the Smad2 overexpressing HDM-exposed mice.<h4>Conclusions</h4>Epithelial overexpression of Smad2 can specifically alter airway hyperreactivity and remodeling in response to an aeroallergen. Moreover, we have identified novel roles for IL-25 and activin A in driving airway hyperreactivity and remodeling.
Project description:Targeted therapies against cancer stem cells, which are enriched in side populations (SP), involves interruption of Wnt-signalling. Furthermore, EpCAM is a SP marker and modulator of Wnt-signalling. Therefore, the effects of an anti-EpCAM treatment on SP-cells and WNT/Î²-catenin signalling was studied. SP of the human lung adenocarcinoma cell line A549 was obtained by fluorescence activated cell sorting and whole genome scans helped to define their molecular phenotype after anti-EpCAM antibody treatment. Anti-EpCAM treated and untreated A549 cells were subjected to Hoechst 33342 dye exclusion assay and sorted to SP and non-SP fractions by FACS. Three biological replicates.
Project description:We have shown that water solubilized versions of a zinc ionophore increase intracellular concentrations of free zinc and have antiproliferative activity in exponential phase A549 lung cancer cultures. The gene expression profiles of A549 lung cancer cultures treated with the lead compound PCI-5002 reveal the activation of stress response pathways. Medium supplementation with zinc (25 Î¼M) led to activation of additional oxidative stress response as well as apoptotic pathways. We propose that the pharmacologic delivery of zinc to tumors using water solubilized ionophores is a potential approach to cancer therapy. Experiment Overall Design: A549 human lung cancer cells (1 x 105 cells per T-25 flask in 7 mL complete RPMI 1640 medium) were seeded 8 days prior to treatment of non-cycling plateau phase cultures with drug. At 4 hours prior to RNA isolation, sapphyrin PCI-5002 (10 Î¼M final concentration), ZnOAc2 (25 Î¼M final concentration), the combination, or control (5% mannitol) solution was added to the cultures. Each experiment was performed in triplicate. After incubation, all cultures were washed twice with HBSS supplemented with 0.5% BSA and total RNA was isolated and subjected to analysis on Human Genome U133 Plus 2.0 Arrays (Affymetrix, Santa Clara, CA), as described. ArrayAssist software (Stratagene/Affymetrix) and the RMA (Robust Microarray Analysis) algorithm were used to generate scaled gene expression values.