Inhibition Of Vimentin Expression Research Articles

Metformin is a Novel Suppressor for Vimentin in Human Gastric Cancer Cell Line.

Vimentin, an intermediate filament of mesenchymal cells, is upregulated in epithelial-mesenchymal transition (EMT) and has a main role in cancer metastasis. As a new strategy to control metastatic outgrowth, EMT markers are generally inhibited using some drugs or specific siRNA. In this study, AGS gastric cancer cells were treated with metformin and vimentin-specific siRNA (vim-siRNA) for 48 h. The impact of metformin and vim-siRNA on vimentin downregulation in AGS cells were analyzed by quantitative PCR and Western blot. Following treatment with metformin and vim-siRNA, cell motility, migration and invasion abilities of AGS cells were also analyzed. The results showed that inhibition of vimentin due to metformin was comparable with the vim-siRNA. Furthermore, wound-healing and invasion assays showed a significant decrease in migration and invasion of AGS cells following metformin and vim-siRNA treatment. Our finding for the first time indicated that metformin can be an alternative to specific siRNA for inhibition of vimentin expression and migration of AGS cell line. Taken together, our data indicates that the use of metformin might have a priority to siRNA for inhibition of gastric cancer cell behaviors siRNA is more unstable and expensive than metformin, and needs special vehicles and delivery strategies for efficient transfection of cells. Further in vivo studies can reveal metformin's potential in inhibition of EMT and metastasis of cancer cells.

MicroRNA-146a suppresses tumor malignancy via targeting vimentin in esophageal squamous cell carcinoma cells with lower fibronectin membrane assembly

BackgroundEsophageal squamous cell carcinoma (ESCC) is widely prevalent in Taiwan, and high metastatic spread of ESCC leads to poor survival rate. Fibronectin (FN) assembly on the cell membrane may induce ESCC mobility. MicroRNAs (MiRNAs) are abundant in and participate in tumorigenesis in many cancers. However, the role of MiRNA in FN assembly-related ESCC mobility remains unexplored.MethodsWe divided ESCC CE81T cells into high-FN assembly (CE81FN+) and low-FN assembly (CE81FN−) groups by flow cytometry. MiRNA microarray analysis identified miR-146a expression as the most down-regulated miRNA in comparison of CE81FN+ and CE81FN− cells.ResultsCell proliferation and migration were decreased when CE81FN+ cells overexpressed transgenic miR-146a compared to the parental cells, indicating an inverse correlation between low miR-146a expression and high proliferation as well as motility of FN assembly ESCC cells. Furthermore, vimentin is the target gene of miR-146a involved in ESCC tumorigenesis. MiR-146a suppressed cell proliferation, migration and invasion of CE81FN+ cells through the inhibition of vimentin expression, as confirmed by real-time PCR, Western blotting and Transwell™ assay. Analysis of one hundred and thirty-six paired ESCC patient specimens revealed that low miR-146a and high vimentin levels were frequently detected in tumor, and that the former was associated with late tumor stages (III and IV). Notably, either low miR-146a expression or high vimentin level was significantly associated with poor overall survival rate among ESCC patients.ConclusionsThis is the first report to link FN assembly in the cell membrane with miR-146a, vimentin and ESCC tumorigenesis both in vitro and in ESCC patients.

Parthenolide reduces metastasis by inhibition of vimentin expression and induces apoptosis by suppression elongation factor α − 1 expression

BackgroundUse of pharmaceutical agent for breast cancer chemotherapy is an interesting method that induces cells death by different way, such as apoptosis. Parthenolide is the main compound in feverfew that has been used to cure migraine and rheumatoid arthritis for long time. Parthenolide has been predominately investigated as inducer of apoptosis in human cancer cells. Purpose: We examined the expression of vimentin and Elongation factor α − 1 as breast cancer biomarkers in MCF7 cells exposure to Parthenolide. MethodIn this study, we investigated the antitumor mechanism of Parthenolide on the human breast cancer cell line MCF7, using SEM, flow cytometry and proteomics techniques. ResultComparative proteome analyses are shown Elongation factor1-α and vimentin was suppressed in response to Parthenolide treatment.

Enhanced antitumor effect via combination of triptolide with 5-fluorouracil in pancreatic cancer

Background: Pancreatic cancer is one of the most aggressive types of cancer, and lack of effective treatment results with a very low 6-month survival rate. This study explores the enhancement of therapeutic effect on human pancreatic cancer cell line (AsPC-1) via combination of triptolide (TPL) and 5-fluorouracil (5-FU). Methods: Cell proliferation was measured by sulforhodamine B (SRB), apoptotic cells were assessed by flow cytometry and western blot for cleaved caspase-3 and vimentin to explore the role of vimentin protein in pancreatic cancer cell line. In vivo , AsPC-1 xenografts were treated by TPL, 5-FU and combination respectively and tumor samples were tested by western blot. Results: The results showed that TPL and 5-FU had the effects on AsPC-1 cell line with the dose dependence individually. The combination of TPL and 5-FU enhanced the cytotoxicity significantly compared to 5-FU or TPL alone. Combination index (CI) indicated the effect of TPL combined with 5-FU was highly synergistic. Furthermore, pancreatic cancer cells treated with TPL plus 5-FU exhibited increased apoptosis and higher levels of pro-apoptotic proteins including caspase-3 activity compared to cells treated with TPL or 5-FU alone. In the mechanism study, high vimentin expression was involved in the enhanced apoptosis induced by TPL plus 5-FU. It indicated that the vimentin expression was inhibited by TPL plus 5-FU significantly. In vivo , the tumor growth of AsPC-1 xenografts was much slower in the group treated with TPL plus 5-FU compared to the group treated with TPL or 5-FU alone. Conclusions: (I) TPL has a potent therapeutic effect on pancreatic cancer cell lines; (II) the combination of TPL with 5-FU enhances the therapeutic effect in vitro and in vivo TPL showed high synergistic effect with 5-FU; (III) the inhibitory effect of TPL plus 5-FU on pancreatic cancer is mediated by the induction of apoptosis and TPL enhanced the apoptosis via inhibition of vimentin expression.

Epithelial-to-mesenchymal transition correlates with gefitinib resistance in NSCLC cells and the liver X receptor ligand GW3965 reverses gefitinib resistance through inhibition of vimentin.

The role of epithelial-to-mesenchymal transition in cancer drug resistance is increasingly acknowledged. We examined whether epithelial-to-mesenchymal transition affects gefitinib resistance in non-small cell lung cancer (NSCLC) cells. Cell viability was detected by CCK-8 assay, VIM expression levels were determined by quantitative real-time polymerase chain reaction. Western blot and immunocytochemistry were performed to determine the protein expression level of vimentin. We observed morphologic differences between gefitinib-sensitive and -insensitive cells. Compared with the sensitive parental cell line, HCC827, vimentin expression levels were increased in HCC827 cells with acquired gefitinib resistance. Vimentin expression was also markedly upregulated in cells with intrinsic gefitinib resistance, and upregulated vimentin expression was correlated with gefitinib sensitivity. Our previous study demonstrated that coadministration of gefitinib and GW3965 resulted in decreased cell proliferation and induced apoptosis. Therefore, we investigated the relationship among GW3965, vimentin, and gefitinib resistance in NSCLC cells by analysis of the expression of vimentin in cells treated with a combination of gefitinib and GW3965. Gefitinib treatment led to increased levels of intracellular vimentin, while combined treatment with gefitinib and GW3965 resulted in decreased vimentin expression levels through reduction of gefitinib drug resistance in NSCLC cells. Overall, these findings suggest that vimentin expression is associated with sensitivity to gefitinib, and our study highlights the potential usefulness of the drug, GW3965, for reversal of gefitinib resistance through inhibition of vimentin expression.

Cardioprotective effects of monocyte locomotion inhibitory factor on myocardial ischemic injury by targeting vimentin

Monocyte locomotion inhibitory factor (MLIF), a heat-stable pentapeptide produced by Entamoeba histolytica, has anti-inflammatory function and protective effect on ischemic stroke. In this study, we evaluated the effect of MLIF on myocardial ischemia. Mice were subjected to ischemia/reperfusion by occlusion of the left anterior descending artery (LAD). After sacrifice, the serum concentrations of cardiac troponin I (cTnI), creatine kinase (CK), lactate dehydrogenase (LDH) as well as the heart infarct size were measured. HE and TUNEL staining were used to observe the pathological damage and the apoptotic cells. For in vitro study, the oxygen-glucose deprivation(OGD) model was established in H9c2 cells. MTT assay and flow cytometry assay were performed to evaluate cell viability and apoptosis. The expression of JNK and caspase 3 was assessed by western blot analysis. Pull-down assay was used to detect the specific binding protein of MLIF in myocardial cells. MLIF significantly reduced the infarct size, and the cTnI, CK and LDH levels, amelioratived pathological damage and reduced the apopotosis compared with the myocardial I/R model group. MLIF improved cell survival and inhibited apoptosis and necrosis by inhibiting the p-JNK and cleaved caspase3 expression. Furthermore, the binding protein of MLIF in myocardial cells was vimentin. Inhibition of vimentin expression by withaferin A or vimentin siRNA repressed the protective effects of MLIF in OGD-provoked H9c2 cells. Taken together, our results demonstrate that the cardioprotective effects of MLIF on myocardial ischemia injury are related to reductions in the inflammatory response and apoptosis by targeting vimentin.

The inhibition of macrophage foam cell formation by tetrahydroxystilbene glucoside is driven by suppressing vimentin cytoskeleton.

Macrophage foam cell formation triggered by oxLDL is an important event that occurs during the development of atherosclerosis. 2,3,5,4'-Tetrahydroxystilbene-2-O-β-d-glucoside (TSG) exhibits significant anti-atherosclerotic activity. Herein we used U937 cells induced by PMA and oxLDL in vitro to investigate the inhibitory effects of TSG on U937 differentiation and macrophage foam cell formation. TSG pretreatment markedly inhibited cell differentiation induced by PMA, macrophage apoptosis and foam cell formation induced by oxLDL. The inhibition of vimentin expression and cleavage was involved in these inhibitory effects of TSG. The suppression of vimentin by siRNA in U937 significantly inhibited cell differentiation, apoptosis and foam cell formation. Using inhibitors for TGFβR1 and PI3K, we found that vimentin production in U937 cells is regulated by TGFβ/Smad signaling, but not by PI3K-Akt-mTOR signaling. Meanwhile, TSG pretreatment inhibited both the expression of TGFβ1 and the phosphorylation of Smad2 and Smad3, and TSG suppressed the nuclear translocation of Smad4 induced by PMA and oxLDL. Furthermore, TSG attenuated the induced caspase-3 activation and adhesion molecules levels by PMA and oxLDL. PMA and oxLDL increased the co-localization of vimentin with ICAM-1, which was attenuated by pretreatment with TSG. These results suggest that TSG inhibits macrophage foam cell formation through suppressing vimentin expression and cleavage, adhesion molecules expression and vimentin-ICAM-1 co-localization. The interruption of TGFβ/Smad pathway and caspase-3 activation is responsible for the downregulation of TSG on vimentin expression and degradation, respectively.

Abstract 4102: The involvement of EMT in heparanase promoted bone-metastasis in multiple myeloma

Abstract Multiple myeloma (MM), a hematologic malignancy, preferentially grows in bone marrow and frequently metastasizes between bones. Our previous studies have demonstrated that heparanase (HPSE), an enzyme overexpressed in MM cells, promotes MM bone-metastasis and progression. However, the mechanism of HPSE action is unclear. Epithelial-mesenchymal-transition (EMT) is a process giving epithelial cells the features of mesenchymal cells which has been shown to be critical in the metastatic process of epithelial-origin tumors. Although MM is not an epithelial-derived malignancy, MM cells indeed express epithelial marker E-cadherin and mesenchymal marker Vimentin, and have the ability to adhere to and interact with the extracellular matrix (ECM). In the present study, we determined the role of EMT in HPSE-induced MM bone metastasis via: (1) Using Western blot to evaluate the expression of EMT markers E-cadherin, Vimentin and Fibronectin in MM cells that were transfected with empty vector (HPSE-low) or HPSE cDNA (HPSE-high), as well as MM cells treated with recombinant human HPSE (rHPSE) for 48hrs; (2) staining HPSE and EMT markers on tumors established by HPSE-low or HPSE-high cells in SCID mice (n = 10), and on bone marrow biopsies of 35 MM patients; (3) knocking down Vimentin in HPSE-high-luc MM cells by Vimentin shRNA (VIM k/d-luc cells) and testing bone homing of VIM k/d-luc cells in SCID mice by i.v. and s.c. injection of these cells. Tumor growth and bone-metastasis was determined by measuring human kappa levels (a soluble marker of MM cells) in mouse sera and weekly bioluminescent imaging. Western blots demonstrated HPSE-high and rHPSE treated MM cells expressed significantly higher levels of the mesenchymal markers Vimentin and Fibronectin, and lower levels of the epithelial marker E-cadherin, compared to HPSE-low or PBS treated MM cells. Significantly increased expression of Vimentin and decreased expression of E-cadherin was revealed in tumors formed by HPSE-high cells compared with the tumors formed by HPSE-low cells. Immunostaining of bone marrow biopsies of MM patients indicated HPSE and Vimentin expression in MM cells have a significant positive correlation (rs = 0.414, p = 0.014). Finally, both SCID-i.v. injection and SCID-s.c. injection models showed that knocking down mesenchymal marker Vimentin in HPSE-high MM cells successfully blocked HPSE-induced bone metastasis and inhibited tumor growth, compared with mice bearing shRNA control HPSE-high MM cells. In conclusion, our studies demonstrated (1) EMT-like features are a novel biomarker of poor prognosis in MM; (2) HPSE promotes MM metastasis to bone and disease progression, at least in part, through inducing EMT-like features in MM cells; (3) Inhibition of Vimentin expression in MM cells impairs HPSE-induced tumor growth and bone metastasis. Note: This abstract was not presented at the meeting. Citation Format: Qianying Pan, Juan Li, Patrick D. Rowan, Timothy N. Trotter, Yang Yang. The involvement of EMT in heparanase promoted bone-metastasis in multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4102. doi:10.1158/1538-7445.AM2015-4102

Tetrahydroxystilbene glucoside improves TNF-α-induced endothelial dysfunction: involvement of TGFβ/Smad pathway and inhibition of vimentin expression.

Endothelial dysfunction plays an important role in the pathogenesis of atherogenesis. 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), an active component of the rhizome extract from Polygonum multiflorum (PM), exhibits significant anti-atherosclerotic activity. Here, we used human umbilical vein endothelial cells (HUVECs) induced by tumor necrosis factor-α (TNF-α) in vitro to investigate the cytoprotective effects of TSG on TNF-α-induced endothelial injury and the related mechanisms. Pretreatment with 50 and 100 μM TSG markedly attenuated TNF-α-induced loss of cell viability and release of lactate dehydrogenase (LDH) and inhibited TNF-α-induced cell apoptosis. The inhibition of vimentin expression was involved in the cytoprotection afforded by TSG. Using inhibitors for PI3K and TGFβ or siRNA for Akt and Smad2, we found that vimentin production in HUVECs is regulated by TGFβ/Smad signaling, but not by PI3K-Akt-mTOR signaling. Meanwhile, TSG inhibited both the expression of TGFβ1 and the phosphorylation of Smad2 and Smad3, and TSG suppressed the nuclear translocation of Smad4 induced by TNF-α. These results suggest that TSG protects HUVECs against TNF-α-induced cell damage by inhibiting vimentin expression via the interruption of the TGFβ/Smad signaling pathway.

Requirement of Vimentin Filament Assembly for β3-Adrenergic Receptor Activation of ERK MAP Kinase and Lipolysis

Catecholamine stimulation of beta-adrenergic receptors (betaAR) in adipocytes activates the cAMP-dependent protein kinase to promote liberation of fatty acids as a fuel source. The adipocyte beta3AR also activates extracellular signal-regulated kinases (ERK)-1 and -2 through direct recruitment and activation of Src kinase. This pathway together with cAMP-dependent protein kinase contributes to maximal beta3AR-stimulated lipolysis. In a search for other molecules that might associate with beta3AR upon agonist stimulation, we identified vimentin using a proteomics approach. Immunoprecipitation of beta3AR from adipocytes in the absence or presence of the beta3AR agonist CL316,243, followed by Western blotting for vimentin confirmed this specific interaction. Since vimentin has also been identified on lipid droplets, the functional consequences of blocking the expression or structural integrity of vimentin intermediate filaments on beta3AR regulation of ERK activation and lipolysis was assessed. Following disruption of intermediate filaments with beta,beta'-iminodipropionitrile, as confirmed by confocal microscopy, beta3AR-stimulated ERK activation was blocked, and lipolysis was reduced by more than 40%. Independently, depletion of vimentin by small hairpin RNA (shRNA) completely inhibited beta3AR-mediated ERK activation and significantly reduced lipolysis. By contrast, disruption of actin-containing microfilaments by cytochalasin D or microtubules by nocodazole had no effect on either lipolysis or ERK activation. These results indicate that vimentin plays an essential role in the signal transduction pathway from beta3AR to the activation ERK and its contribution to lipolysis.