Expression Of Vimentin In Cells Research Articles

Effects of obatoclax combined with gemcitabine on breast cancer cells under hypoxia condition

Objective: To explore the effects of obatoclax(OBX) combined with gemcitabine(GEM) on breast cancer cells MCF-7 and BT-20 cell activity, migration, invasion and apoptosis under hypoxia condition.Methods: Breast cancer cells MCF-7 and BT-20 were divided into normal group, hypoxia group, GEM group, OBX+GEM group. Normal group: Cells were cultured at 37℃, 5% CO2 for 24 h and 48 h; Hypoxia group: Cells were cultured at 37℃, 1% O2, 5% CO2, 94% N2 for 24 h and 48 h; GEM group: Cells were cultured at 37℃, 1% O2, 5% CO2, 94% N2, adding 10 μmol/L GEM for 24 h and 48 h; OBX + GEM group: Cells were cultured at 37℃, 1% O2, 5% CO2, 94% N2, adding 10 μmol/L GEM and 50 nmol/L OBX for 24 h and 48 h. Western blot method was used to detect the expressions of HIF-1α in MCF-7 and BT-20 cells under normal oxygen and hypoxia condition. CCK-8 method was used to detect cancer cell activity, each group was provided with 15 compound holes. Scratch experiment was used to detect cells migration ability, each group was provided with 6 compound holes. Western blot method was used to detect the expressions of vimentin, E-Cadherin and p53 protein in cells of each group. Results: Under hypoxia condition, the expression of HIF-1α in MCF-7 and BT-20 cells was much higher than that under normal oxygen(P＜0.05). Compared with hypoxia group, GEM could reduce MCF-7 and BT-20 cells migration ability(P＜0.01)and cell activity(P＜0.05), while decrease the expression of vimentin protein(P＜0.01)and promote the expressions of E-Cadherin (P＜0.01)and p53 protein(P＜0.01) in tumor cells under hypoxia condition. In OBX combined with GEM group, the cell activity and the migration ability of MCF-7 and BT-20 were reduced significantly(P＜0.01). The expression of vimentin in cells was further reduced(P＜0.01). The expressions of E-Cadherin(P＜0.01)and p53(P＜0.01) protein were increased significantly compared with GEM group. Conclusion: Under hypoxia condition, OBX combined with a low-dose of GEM can significantly inhibit the growth, migration and invasion of breast cancer cells, and enhance the pro-apoptotic effect of GEM, but the specific mechanism needs further study.

1,25(OH)D vitamin D promotes NOS2 expression in response to bacterial and viral PAMPs in primary bovine salivary gland fibroblasts.

The faecal-oral route is a predominant mode of infectious disease transmission and yet the immunology of the bovine oral cavity is poorly understood. The objectives of this study were to develop an in vitro cell model of bovine salivary gland cells and to characterize the role of vitamin D on the expression of innate immune genes induced by stimulation with bacterial and viral pathogen-associated molecular patterns (PAMPs). Submandibular glandular tissue was excised post-mortem, processed, cells isolated and cultured until confluency after which cells were incubated with the active form of vitamin D (1,25(OH)D) for 18h before stimulation with lipopolysaccharide (LPSμg/ml), lipoteichoic acid (LTAμg/ml) or polyinosinic:polycytidylic acid (poly I:C-20μg/ml) PAMPs for 6h and immune gene expression was assessed by Quantitative Real-Time PCR (RT-qPCR). RT-qPCR analysis of vimentin expression in cells derived from the bovine submandibular gland shows that cultured cells were fibroblast in origin. These cells significantly induce the pro-inflammatory cytokine IL1B, β-defensin and cathelicidin genes but these were not significantly altered in response to 1,25(OH)D. In contrast, 1,25(OH)D significantly up-regulates the expression of the NOS2 gene encoding iNOS in bovine submandibular stromal cells compared to EtOH (vehicle) control and this is a maintained response to all three bacterial and viral ligands. We have developed a new in vitro model to allow detailed investigations of mechanisms to enhance oral immunity in cattle. We show that these cells are fibroblast in nature, immunologically competent and vitamin D responsive. Their vitamin D-mediated enhancement of NOS2 expression warrants further investigation in saliva as a potential mechanism to boost oral immunity against infectious agents.

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.

Combination therapy with local radiofrequency ablation and YC-1 inhibits the proliferation and metastasis of hepatocellular carcinoma through activating β-catenin signaling.

This study was aimed to investigate the possible role of combination therapy of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) and radiofrequency ablation (RFA) in the treatment of hepatocellular carcinoma (HCC). The two kinds of HCC HCCLM3 and HepG2 cells were intervened by the RFA and YC-1 respectively to construct the model cell. Then cell proliferation, apoptosis, migration and invasion were analyzed using the MTT, flow cytometry and transwell assay respectively. Moreover, the cell signal-related protein expression was also analyzed using western blotting. Our results showed that the combination therapy of RFA and YC-1 significantly inhibited the proliferation and induced the apoptosis of the two kinds of cells (P < 0.05). Besides, cell migration and invasion were inhibited by the combination of RFA and YC-1, followed with the EMT symbols of E-cadherin, N-cadherin and vimentin expression in cells (P < 0.05). Moreover, the β-catenin signal was activated by the combination treatment. Taken together, the data presented in this study reveals that the novel method about combination of RFA and YC-1 may opposite an improve effect on the treatment of HCC than the RFA single treatment. Also, the combination therapy of RFA and YC-1 may well inhibit the development and metastasis of HCC via preventing the EMT through activating the β-catenin signal.

Inositol induces mesenchymal-epithelial reversion in breast cancer cells through cytoskeleton rearrangement

Inositol displays multi-targeted effects on many biochemical pathways involved in epithelial-mesenchymal transition (EMT). As Akt activation is inhibited by inositol, we investigated if such effect could hamper EMT in MDA-MB-231 breast cancer cells. In cancer cells treated with pharmacological doses of inositol E-cadherin was increased, β-catenin was redistributed behind cell membrane, and metalloproteinase-9 was significantly reduced, while motility and invading capacity were severely inhibited. Those changes were associated with a significant down-regulation of PI3K/Akt activity, leading to a decrease in downstream signaling effectors: NF-kB, COX-2, and SNAI1. Inositol-mediated inhibition of PS1 leads to lowered Notch 1 release, thus contributing in decreasing SNAI1 levels. Overall, these data indicated that inositol inhibits the principal molecular pathway supporting EMT. Similar results were obtained in ZR-75, a highly metastatic breast cancer line. These findings are coupled with significant changes on cytoskeleton. Inositol slowed-down vimentin expression in cells placed behind the wound-healing edge and stabilized cortical F-actin. Moreover, lamellipodia and filopodia, two specific membrane extensions enabling cell migration and invasiveness, were no longer detectable after inositol addiction. Additionally, fascin and cofilin, two mandatory required components for F-actin assembling within cell protrusions, were highly reduced. These data suggest that inositol may induce an EMT reversion in breast cancer cells, suppressing motility and invasiveness through cytoskeleton modifications.

RUNX3 regulates vimentin expression via miR‐30a during epithelial–mesenchymal transition in gastric cancer cells

Runt-related transcription factor 3 (RUNX3) is a putative tumour suppressor via regulating the expression of a series of target genes. Clinical studies demonstrated that loss of RUNX3 expression is associated with gastric cancer progression and poor prognosis, but the underlying mechanism is not entirely clear. Accumulating evidence shows that the epithelial–mesenchymal transition (EMT) plays an important role in cancer relapse and metastasis. Therefore, we addressed whether RUNX3 has a role in the EMT in gastric cancer. Knockdown of RUNX3 promoted cell invasion and increased the protein expression of the mesenchymal marker vimentin in human gastric cancer cells. Overexpression of RUNX3 suppressed cell invasion and decreased the protein expression of vimentin in the cells and inhibited gastric cancer cells colonization in nude mice. Furthermore, overexpression of RUNX3 increased the expression of microRNA-30a (miR-30a), and miR-30a directly targeted the 3′ untranslated region of vimentin and decreased its protein level. miR-30a inhibitor abrogated RUNX3-mediated inhibition of cell invasion and downregulation of vimentin. Thus, RUNX3 suppressed gastric cancer cell invasion and vimentin expression by activating miR-30a. In gastric cancer patients, levels of RUNX3 were positively correlated with miR-30a and negatively associated with the levels of vimentin. Collectively, our data suggest a novel molecular mechanism for the tumour suppressor activity of RUNX3. Effective therapy targeting the RUNX3 pathway may help control gastric cancer cell invasion and metastasis by inhibiting the EMT.

Abstract 2886: Differential effects of soy isoflavones on Akt and vimentin expression in human prostate cancer cells and mouse xenograft tumors

Abstract Studies have reported that soy isoflavones inhibit growth in a number of carcinoma cell lines. The overexpression of Akt, a prosurvival pathway, and vimentin, a mesenchymal marker, in urological carcinomas has been associated with tumor progression and poor prognosis. Our study investigated the effects of a soy isoflavone concentrate (ISF) on the expression of Akt and vimentin in a) cultured human prostate cancer cells and b) xenograft tumors in mice. METHODS: Human prostate cancer PC-3 cells, which are androgen-insensitive and do not express p53 or PTEN, were grown in culture. Hypoxia exposure was at 1% oxygen vs room air for 48 hours. Akt, phosphorylated-Akt (phosAkt), carbonic anhydrase IX (CAIX) and vimentin expression in cells was measured by Western immunoblot analysis. Male nude mice on a soy-free control diet were injected with PC-3 cells into the hind flank. On day 5, half the mice were placed on a diet containing 0.5% soy isoflavone concentrate (ISF). Tumor size was monitored biweekly. Resected tumors were fixed in formalin and paraffin-embedded. Immunohistochemical (IHC) analysis of tumors used antibodies against PCNA, TUNEL, CAIX, Akt, phosAkt, and vimentin. RESULTS: In culture, PC-3 cells constitutively express high levels of Akt and vimentin proteins, which is not altered by hypoxia; in contrast, phosAkt and CAIX proteins are highly expressed only under hypoxic conditions. ISF caused dose-dependent inhibition of DNA synthesis and cell viability without evidence of apoptotic cell death. In culture, ISF decreased the constitutive expression of vimentin, as well as the hypoxia-mediated induction of pAkt and CAIX. In mice, the soy ISF diet significantly inhibited xenograft tumor growth. At 39 days, median tumor size was 862 mm3 in soy-free controls, and 520 mm3 in mice on the ISF diet (p=0.02). IHC analysis of tumors shows that ISF decreased the expression of nuclear PCNA, while TUNEL positive apoptotic cells was increased 4-fold. The ISF diet markedly suppressed levels of total Akt and activated phosAkt. In contrast, staining for CAIX protein was not different in control vs ISF-tumors. Vimentin was clearly expressed in PC-3 tumors and was increased by exposure to irradiation (IR, 2Gy). Interestingly, ISF suppressed IR-induced vimentin expression. Thus, soy ISF treatment inhibited PC-3 cell proliferation both in vitro and in vivo, but different mechanisms are involved in the regulation of expression of the dominant Akt prosurvival signaling pathway, as well as a major mesenchymal marker vimentin. CONCLUSIONS: Together, data indicate that a soy ISF diet strongly inhibits the growth of prostate xenograft tumors which is mediated by differential effects on hypoxia-regulated genes in the tumor microenvironment. (Supported by NIH CA102386 and the Maren Foundation) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2886.

Immunocytochemical studies on the vimentin distribution and cell proliferation of fibroblasts in patients with Friedreich's ataxia

Fibroblasts obtained from patients with Friedreich's ataxia and normal control subjects were studied by immunocytochemistry for intermediate filament vimentin and also for in vitro proliferation. Trypsinized cells were seeded on coverslips and incubated for 1.5 h and 24 h. The expression of vimentin in cells was investigated by immunofluorescence microscopy. Cell proliferation was studied with BrdU antibody technique. Cells from patients with Friedreich's ataxia showed a slower outgrowth of vimentin filaments in comparison to cells from normal controls. These cells also incorporated less 5-bromo-2'-deoxyuridine (BrdU) into their DNA. The observations may be relevant to the clinical manifestations of the disease which involves many organs in addition to brain and spinal cord.

Co-expression of GFAP and vimentin in astrocytes proliferating in response to injury in the mouse cerebral hemisphere. A combined autoradiographic and double immunocytochemical study

Changes in the distribution of proliferating astrocytes expressing glial fibrillary acidic protein (GFAP) and/or vimentin were examined in the injured cerebral hemisphere in adult mice. The injury was followed by [3H]thymidine injections at different time intervals. The brain sections were doubly immunostained for GFAP and vimentin and subjected to autoradiography. In that way three cell types were distinguished immunocytochemically: 1.(1) astrocytes co-expressing glial fibrillary acidic protein (GFAP) and vimentin2.(2) astrocytes expressing only GFAP3.(3) astrocyte-like cells expressing vimentin. Thereafter, numbers of immunopositive and autoradiographically labelled cells and their locations within the region of injury were recorded at each stage of the experiment. Two hours as well as 1 day after the injury proliferation of GFAP-positive astrocytes and of those co-expressing GFAP and vimentin could only be seen as statistically insignificant phenomena. On day 2 the reactive proliferation of each immunocytochemically defined cell type was already maximal, then gradually decreased and its last signs were recorded on day 8. On day 2, among all the proliferating GFAP-positive astrocytes, 67.2% were also vimentin-positive. Later, the proportion declined to 50.7% and 38.5% on days 4 and 8, respectively. The labelled astrocyte-like vimentin-positive cells were located closest to the lesion margins. In comparison, the astrocytes co-expressing GFAP and vimentin and those expressing exclusively GFAP, occupied regions progressively farther from the lesion site. At the initial stages of the response to injury, vimentin expression in cells starting their reactive proliferation did not precede the expression of GFAP. This was considered as an argument against a hypothesis that reactive astrocyte division induces a two-stage increase in the cytoskeletal protein level in which synthesis of vimentin precedes that of GFAP.

Cell cycle-dependent vimentin expression in elutriator-synchronized, TPA-treated MPC-11 mouse plasmacytoma cells

We correlated cell cycle progression and vimentin expression at the single cell level by multiparameter flow cytometry in populations of MPC-11 cells enriched in different cell cycle phases by centrifugal elutriation and subsequently treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Synchronized, untreated cultures showed a uniform, synchronous progression through the cell cycle during further cultivation. A 6-h TPA treatment of G1-phase-enriched cultures induced both a partial G1-phase arrest in the same cycle and a moderate fraction of cells to become vimentin positive. However, nearly all cells of the cultures enriched in S- or in G2/M-phase cells could be arrested by TPA treatment at the earliest in the G1 phase of the second cell cycle and displayed higher fractions of positive cells as well as higher average levels of vimentin. After 20 h of treatment, the G1-phase arrest was almost complete. In terms of fractions of vimentin-positive cells as well as of average cellular vimentin content, the differences between the cultures resembled, albeit on a higher level, those between the respective cultures treated with TPA for 6 h. These observations might explain the striking bimodal distribution of individual cellular vimentin content detectable in G1-phase fractions of asynchronous, TPA-treated cultures. The pattern of vimentin mRNA accumulation in synchronized cultures after short-term TPA treatment strongly suggests that the cell cycle-dependent pattern of vimentin expression is caused, at least in part, by different levels of vimentin mRNA accumulated in the cells. Since proteinaceous mediator(s) are obviously involved in TPA-induced vimentin expression in MPC-11 cells, cell cycle-dependent vimentin expression in these cells may be dependent on cell cycle-dependent regulation of the activity and/or concentration of such mediator(s).