Downregulation Of Vimentin Research Articles

Hepatic stellate cells express thymosin Beta 4 in chronically damaged liver.

Although the various biological roles of thymosin β4 (Tβ4) have been studied widely, the effect of Tβ4 and Tβ4-expressing cells in the liver remains unclear. Therefore, we investigated the expression and function of Tβ4 in chronically damaged livers. CCl4 was injected into male mice to induce a model of chronic liver disease. Mice were sacrificed at 6 and 10 weeks after CCl4 treatment, and the livers were collected for biochemical analysis. The activated LX-2, human hepatic stellate cell (HSC) line, were transfected with Tβ4-specific siRNA and activation markers of HSCs were examined. Compared to HepG2, higher expression of Tβ4 in RNA and protein levels was detected in the activated LX-2. In addition, Tβ4 was up-regulated in human liver with advanced liver fibrosis. The expression of Tβ4 increased during mouse HSC activation. Tβ4 was also up-regulated and Tβ4-positive cells were co-localized with α-smooth muscle actin (α-SMA) in the livers of CCl4-treated mice, whereas such cells were rarely detected in the livers of corn-oil treated mice. The suppression of Tβ4 in LX-2 cells by siRNA induced the down-regulation of HSC activation-related genes, tgf-β, α-sma, collagen, and vimentin, and up-regulation of HSC inactivation markers, ppar-γ and gfap. Immunofluorescent staining detected rare co-expressing cells with Tβ4 and α-SMA in Tβ4 siRNA-transfected cells. In addition, cytoplasmic lipid droplets were observed in Tβ4 siRNA-treated cells. These results demonstrate that activated HSCs expressed Tβ4 in chronically damaged livers, and this endogenous expression of Tβ4 influenced HSC activation, indicating that Tβ4 might contribute to liver fibrosis by regulating HSC activation.

Identification of potential targets for diallyl disulfide in human gastric cancer MGC-803 cells using proteomics approaches.

Diallyl disulfide (DADS) is characterized as an effective agent for the prevention and therapy of cancer, however, mechanisms regarding its anticancer effects are not fully clarified. In the present study, we compared the protein expression profile of gastric cancer MGC-803 cells subjected to DADS treatment with that of untreated control cells to explore potential molecules regulated by DADS. Using proteomic approaches, we identified 23 proteins showing statistically significant differences in expression, including 9 upregulated and 14 downregulated proteins. RT-PCR and western blot analysis confirmed that retinoid-related orphan nuclear receptor α (RORα) and nM23 were increased by DADS, whereas LIM kinase-1 (LIMK1), urokinase-type plasminogen activator receptor (uPAR) and cyclin-dependent kinase-1 (CDK1) were decreased. DADS treatment and knockdown of uPAR caused suppression of ERK/Fra-1 pathway, downregulation of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-9 (MMP-9) and vimentin, and upregulation of tissue inhibitor of metalloproteinase-3 (TIMP-3) and E-cadherin, concomitant with inhibition of cell migration and invasion. Moreover, knockdown of uPAR potentiated the effects of DADS on MGC-803 cells. These data demonstrate that downregulation of uPAR may partially be responsible for DADS-induced inhibition of ERK/Fra-1 pathway, as well as cell migration and invasion. Thus, the discovery of DADS-induced differential expression proteins is conducive to reveal unknown mechanisms of DADS anti-gastric cancer.

Pterostilbene inhibits triple-negative breast cancer metastasis via inducing microRNA-205 expression and negatively modulates epithelial-to-mesenchymal transition

Breast cancer is the leading cause of cancer-related deaths among females in economically developing countries. Greater than 95% of breast malignancies are of epithelial origin; the induction of epithelial-to-mesenchymal transition (EMT) has been shown to initiate the metastatic process in breast carcinoma and remains the key target for drug development. Here, we examine the anti-metastatic potential of pterostilbene in modulating EMT process in breast cancer cells both in vitro and in vivo. The differential invasive ability among MCF7, Hs578t and MDA-MB-231 breast cancer cell lines were closely correlated with the expression of EMT markers, determined by Western blots and Matrigel-coated transwells assay. Pterostilbene inhibited the migratory and invasive potential of triple-negative MDA-MB-231 and Hs578t cells, accompanied by the up-regulation of E-cadherin and down-regulation of Snail, Slug, vimentin and ZEB1. Mechanistic investigations revealed a significant up-regulation of miR-205, which resulted in the reduction of Src expression in pterostilbene-treated breast cancer cells. Importantly, pterostilbene suppressed tumor growth and metastasis in MDA-MB-231-bearing NOD/SCID mice by reducing Src/Fak signaling; this observation was consistent with the negative correlations between miR-205 and Src expression in both normal and malignant breast tissues. Our findings provide supports for the usage of pterostilbene as an inhibitor of EMT process and potential candidate for adjuvant therapy.

Decrease of ZEB1 expression inhibits the B16F10 cancer stem-like properties.

Increasing evidence supports that cancer stem cells (CSCs) are responsible for driving tumor initiation and maintenance. Zinc-finger E-box binding homeobox 1 (ZEB1) is a transcription factor for regulating tumor progression, and contributes to maintenance of CSC-like properties. The goal of the present study is to investigate the effect of decreasing ZEB1 expression on the B16F10 CSC-like properties. The recombinant shRNA targeting ZEB1 were transfected into melanoma B16F10 cells, and shZEB1-CD133(+)CD44(+) CSCs were isolated from the stable transfected cells using the magnetic-associated cell sorting method. The shZEB1-CD133(+)CD44(+) CSC-like properties were systematically analyzed. The results show the B16F10 shZEB1-CD133(+)CD44(+) CSCs significantly decreased the ability of clonogenicity, cellular proliferation, migration, and invasion. Importantly, tumorigenicity and tumor lung metastasis was significantly inhibited in B16F10 shZEB1-CD133(+)CD44(+) CSCs compared with B16F10 scramble-CD133(+)CD44(+) CSCs. The decrease of ZEB1 expression markedly resulted in down-regulation of vimentin and N-cadherin expression as well as up-regulation of E-cadherin expression in tumor tissues from the mice injected with B16F10 shZEB1-CD44(+)CD133(+) CSCs. These findings contribute to understanding the maintenance of B16F10 CD133(+)CD44(+) CSC-like properties that was closely associated with ZEB1 expression. ZEB1 may serve as a new therapeutic target for treatment of malignant melanoma.

Trichostatin A, a histone deacetylase inhibitor, reverses epithelial–mesenchymal transition in colorectal cancer SW480 and prostate cancer PC3 cells

Trichostatin A (TSA) is a kind of classical histone deacetylase (HDAC) inhibitor. In this study, we reported the reversal effects of TSA on EMT and investigated the possible involved molecular mechanisms in SW480 and PC3 cells. Firstly, we observed that TSA induced the reversal process of epithelial–mesenchymal transition (EMT) in SW480 and PC3 cells, resulting in attenuated cell invasion and migration abilities. TSA-induced EMT reversal was characterized by up-regulation of E-cadherin and down-regulation of Vimentin. Then, treatment with TSA also decreased the expression of transcription factor Slug. Furthermore, over-expression of Slug significantly caused down-regulation of E-cadherin and up-regulation of Vimentin. Meanwhile, TSA treatment in Slug-expressing cells could prevent these changes. These findings suggested that Slug played a crucial role in TSA-induced EMT reversal. Additionally, the study showed that TSA could induce the increase of HDAC1 and HDAC2 on the Slug gene promoter, which might be responsible for the suppression of Slug. Overall, TSA could reverse EMT in SW480 and PC3 cells and TSA-mediated down-regulation of Slug was involved in the reversal process.

Overexpression of miR-200a suppresses epithelial-mesenchymal transition of liver cancer stem cells.

Due to high incidence of invasion and intrahepatic metastasis, hepatocellular carcinoma (HCC) is one of the most aggressive tumors in the world, which is also associated with the acquisition of epithelial-mesenchymal transition (EMT). Increasing evidence suggests that cancer cells with EMT traits share many biological characteristics with cancer stem cells. And miR-200a has been known as a powerful regulator of EMT. Here, we sought to investigate the role of miR-200a in regulation of EMT phenotype of liver cancer stem cells (LCSCs). We used side population (SP) sorting to obtain cancer stem-like cells from HCC cell lines and identified that the SP fraction could be enriched with LCSCs. Then, we detected the expression of miR-200a and EMT makers in SP and non-SP cells. Our results suggested that miR-200a was down-regulated in SP cells, along with relatively low epithelial marker and high mesenchymal marker. In order to find the role of miR-200a in the manipulation of EMT, we transfected miR-200a mimic into LCSCs and found that overexpression of miR-200a resulted in down-regulation of N-cadherin, ZEB2, and vimentin, but up-regulation of E-cadherin. Moreover, overexpression of miR-200a resulted in decreased migration and invasion ability in LCSCs. In conclusion, our study revealed that miR-200a played an important role in linking the characteristics of cancer stem cells with EMT phenotype in HCC, and targeting miR-200a might be an effective strategy to weaken the invasive behavior of LCSCs.

MiRNA-205 Is a Candidate Tumor Suppressor that Targets ZEB2 in Renal Cell Carcinoma

SummaryBackground: This study aims to characterize the function of downregulated MicroRNA miR-205 in renal cell carcinoma (RCC), and show how the downstream zinc finger E-box-binding homeobox 2 (ZEB2) is negatively regulated by miR-205. Materials and Methods: The expression of miR-205 was detected in RCC and adjacent non-tumor tissues using real-time polymerase chain reaction (PCR). The expression of miR-205 and ZEB2 was detected in RCC cell lines using real-time PCR. The luciferase reporter assay was used to assess ZEB2 as a target of miR-205. Protein levels of ZEB2, E-cadherin, and vimentin were measured by western blot after overexpression of miR-205 in ACHN cells. In vivo functions of miR-205 in ACHN cells were measured by MTT assays, migration and invasion assays, and flow cytometry. Results: MiR-205 was significantly downregulated in RCC samples and cell lines compared with matched non-tumor tissues and HK-2 cells, respectively. No significant difference was found in miR-205 expression between well differentiated and poorly to moderately differentiated groups or between phase I and phase II-III. ZEB2 was upregulated in RCC cell lines compared with expression in HK-2 cells. Upregulation of miR-205 expression caused the downregulation of ZEB2 and vimentin, and the upregulation of E-cadherin in ACHN cells. miR-205 also inhibited proliferation, migration, and invasion, and induced apoptosis of ACHN cells. Conclusion: miR-205 is a candidate tumor suppressor that targets ZEB2 in RCC.

Abstract 423: Epigenetic reprogramming of epithelial-mesenchymal transition in triple-negative breast cancer cells with DNA methyltransferase and histone deacetylase inhibitors

Abstract Incidence and mortality of triple negative breast cancer (TNBC) are increasing in women under the age of 40 who develop metastases in response to the epithelial to mesenchymal transition (EMT). The purpose of this study is to determine if the process of EMT can be abrogated by the use of DNA methytransferase (DNMT) and histone deacetylase (HDAC) inhibitors. For this purpose, we used two triple negative breast epithelial cell lines which represent a basal breast cancer progression model [Cancer Res 67 11147-11157, 2007]: 1) the trMCF cell line, which was transformed from MCF10F cells by treatment with 70 nM 17-β-estradiol, and 2) the bsMCF cell line, which was derived by Boyden chamber selection of invaded trMCF cells. trMCF cells express E-cadherin, form ducts and masses in 3D collagen matrix, and colonies in agar methocel, but are not tumorigenic when injected in SCID mice. bsMCF cells express high levels of vimentin, low levels of E-cadherin, are highly invasive, have lost ductulogenic capacity in 3D collagen matrix, show high colony formation in agar methocel, and are tumorigenic and metastatic when injected into the tail vein of SCID mice. We tested two DNMT inhibitors: Decitabine (DAC) and SGI-110 (SGI), and two HDAC inhibitors: Vorinostat (SAHA) and JNJ-26481585 (JNJ) for four days on the trMCF and bsMCF cell lines. The growth IC50 was calculated after four days of treatment using the MTT assay. Then, cells treated with the IC50 dose were used for wound healing, Matrigel invasion, 3D culture in collagen, and colony formation assay in agar methocel. Changes of EMT markers were evaluated both by immunocytochemical staining and Western blot analysis.The growth IC50 calculated by MTT assay were as follows: 300 nM for DAC in both cell lines; 1000 nM for SGI in trMCF, 500 nM for SGI in bsMCF; 300 nM for SAHA in trMCF, 700 nM for SAHA in bsMCF; 15 nM for JNJ in trMCF, and 25 nM for JNJ in bsMCF. Our results revealed that DNMT inhibitor SGI-110 was the best drug to reverse EMT of TNBC by inhibiting cell motility, invasion, and colony formation in agar methocel, as well as promoting cell differentiation by increasing the expression of E-cadherin, and down regulation of vimentin, and SLUG. These results suggest that this new generation of DNMT inhibitor is superior to previous DNMT and the HDAC inhibitors tested. (This work was supported by The Pennsylvania Cancer Cure Grant 6914101 and by NIH core grant CA06927 to Fox Chase Cancer Center. The compound SGI-110 was provided by Astex Pharmaceutical, Inc. Dublin CA). Citation Format: Yanrong Su, Nathan R. Hopfinger, Thomas J. Pogash, Theresa D. Nguyen, Julia Santucci-Pereira, Jose Russo. Epigenetic reprogramming of epithelial-mesenchymal transition in triple-negative breast cancer cells with DNA methyltransferase and histone deacetylase inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 423. doi:10.1158/1538-7445.AM2014-423

Abstract 88: Epithelial-mesenchymal transition occurs in preneoplastic and neoplastic lesions of canine prostate

Abstract Background: The epithelial-mesenchymal transition (EMT) is an essential process in the tumor progression and metastasis. In human prostate carcinoma (PCa), the upregulation of cytokeratin and E-cadherin and down-regulation of vimentin have been associated with aggressive phenotype and poor prognosis. Due to the importance of canine cancer model it was evaluated the immunoexpression of AE1/AE3, E-cadherin and vimentin in canine prostatic lesions. Patients and Methods: A total of 75 prostatic tissues formalin-fixed paraffin embedded from dogs was selected: 10 normal prostatic tissues, 20 benign prostatic hyperplasia (BPH), 25 proliferative inflammatory atrophy (PIA) and 20 PCa. AE1/AE3 was detected with a monoclonal antibody (Invitrogen, 180132) at a 1:300 dilution, applied for 45 min at room temperature (RT). The antibody against Vimentin (V9, Invitrogen) and E-cadherin (NCH-38, Dako cytomatiomn) were monoclonal mouse antibodies, used at a 1:300 and 1:200, respectively, for 45 min at RT. The immunolabelling was performed by a polymer method (Histofine, Nichirei Biosciences,). A negative control was performed for all antibodies by omitting the primary antibody and substituting with Tris-buffered saline. The percentage of C-MYC, E-cadherin, and p63- positive cells per lesion was evaluated according to Prowatke et al. (2007). The samples were scored separately according to staining intensity and graded semi-quantitatively as negative, weakly positive, moderately positive, and strongly positive. The score was done in one 400 magnification field, considering only the lesion, since this was done in a TMA core of 1 mm. For statistical analyses, the immunostaining classifications were reduced to two categories: negative and positive. The negative category included negative and weakly positive staining. Chi-square or Fisher exact test was used to determine the association between the categorical variables. Results: All prostatic normal and BPH tissue were positive for cytokeratin, E-cadherin and negative for vimentin. Similarly, all PIA samples were positive for AE1/AE3. From those samples, 48% (12/25) were also positive for vimentin. 55% of PCa (11/25) was positive for vimentin and among these samples 75% (6/11) was also positive for AE1/AE3 and 45% (5/11) was negative for AE1/AE3. PIA and PCa presented a higher number of vimentin positive cells when compared with normal tissue (p=0.032). E-cadherin expression had no statistical difference among diagnosis groups, but we found a higher number of positive cases, with more than 51% of positive immunostaining in BPH and PIA (81.25% and 78.60% of the cases, respectively) than in PCa (55.55%). Conclusion: The carcinogenesis process regarding prostatic epithelial cells in dogs showed higher vimentin protein expression associated with concomitant loss of the cytokeratin and E-cadherin, similar in humans. Financial Support: Sao Paulo State Foundation (FAPESP) and CNPq. Citation Format: Carlos Eduardo Fonseca-Alves, Igor Simoes Tiagua Vicente, Luis Gabriel Rivera Calderon, Andre Augusto Justo, Silvia Regina Rogatto, Renée Laufer-Amorim. Epithelial-mesenchymal transition occurs in preneoplastic and neoplastic lesions of canine prostate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 88. doi:10.1158/1538-7445.AM2014-88

Cadherin and vimentin immunoexpression in the testis of normal and induced infertility models of albino rats.

Sertoli cells are important in determining the fate of spermatogenic cells by providing nutrition and structural support via cell junctions. Adhesion between Sertoli and germ cells is important for spermatogenesis. Cadherin are transmembrane proteins that mediate cell-cell adhesion, while, vimentin, the cytoskeletal intermediate filament plays an important role in spermatogenesis. The aim of the present study was to investigate cadherin and vimentin immunoexpression in the normal testis and in two types of altered spermatogenic states: the cyclophosphamide (CP) treatment and the cryptorchidism (Cx) models. Twenty four male albino rats were divided into control group: 6 rats receiving saline orally and the other 6 were sham-operated. CP group (n = 6): given 6 mg/kg/day of CP orally for 4 weeks. Cx group (n = 6): the left testis was surgically freed from the scrotum and fixed in the abdomen. Animals were sacrificed and the left testis dissected and prepared to be stained with haematoxylin and eosin stain and immunohistochemical stain against cadherin and vimentin. Morphometric measurements and statistical analysis were done. In CP-treated group there was degeneration of spermatocytes, vacuolations of Sertoli cells and absence of spermatozoa. These changes were more prominent in Cx group, in addition to interstitial hypercellularity. There was alsoa significant decrease in cadherin and vimentin immunostaining in CP-treated group that was more marked in the cryptorchidism group. A downregulation of both cadherin and vimentin was associated with both models of impaired spermatogenesis. This impairment could be attributed to disruption of the junctions between Sertoli and germ cells.

Steroid Sulfatase Promotes Invasion and is a Useful Marker for Predicting the Progression of Bladder Cancer

ABSTRACT Objectives: To assess the clinical and functional significance of steroid sulfatase (STS) in bladder cancer. STS is steroid sulfates activation enzyme and is considered one of the key enzymes in androgen signaling pathway. Androgen signal is recently suggested to be involved in the growth of bladder cancer. However, the role of STS in bladder cancer has not been elucidated. Methods: STS expression was measured in 170 bladder cancer tissues by immunohistochemistry. The effect of STS knockdown on cell proliferation, migration and invasion capacity was evaluated using bladder cancer cell line (T24). Results: The incidences of STS positive cancers were 21.3% and 41.2% in non-muscle invasive and muscle invasive bladder cancers, respectively (p = 0.0262). STS positive cancers showed shorter recurrence-free survival and cancer specific survival (CSS) (p = 0.0083, 0.0014, respectively). By multivariate analysis, STS expression level was identified as an independent prognostic factor for CSS (p = 0.043). Furthermore, in vitro knockdown of STS significantly reduced cell migration and invasion capacities of bladder cancer cells accompanied by up-regulation of E-cadherin and down-regulation of vimentin. However, the expression of androgen receptor (AR) was not correlated with that of STS, pathological stage or survival of patients with bladder cancer, suggesting that AR is not likely to play an important role in the progression of bladder cancer. Conclusions: The present study demonstrates that STS promotes the invasion capability of bladder cancer and is a useful marker for predicting the progression of bladder cancers. Disclosure: All authors have declared no conflicts of interest.

Wogonin suppresses human alveolar adenocarcinoma cell A549 migration in inflammatory microenvironment by modulating the IL-6/STAT3 signaling pathway.

Increasing evidence from various clinical and experimental studies has demonstrated that the inflammatory microenvironment facilitates tumor metastasis. Clinically, it will be a promising choice to suppress tumor metastasis by targeting inflammatory microenvironment. Our previous studies have demonstrated that wogonin (a bioflavonoid isolated from the traditional Chinese medicine of Huang-Qin) possesses the anti-metastatic and anti-inflammatory activity, but we have little idea about its efficacy on inflammatory-induced tumor metastasis and the mechanism underlying it. In this study, we focused on epithelial mesenchymal transition (EMT), the first step of tumor metastasis, to evaluate the effects of wogonin on tumor metastasis in inflammatory microenvironment. We found that wogonin inhibited THP-1 conditioned-medium- (CM-) and IL-6-induced EMT by inactivating STAT3 signal. And in wogonin-treated A549 cells which pretreated with THP-1 CM or IL-6, the expression level of E-cadherin, an EMT negative biomarker, increased while that of N-cadherin, Vimentin, and EMT-related transcription factors including Snail and Twist decreased. Moreover, wogonin inhibited IL-6-induced phosphorylation of STAT3, prevented p-STAT3 dimer translocation into the nucleus, and suppressed the DNA-binding activity of p-STAT3. Interestingly, similar results were obtained in the tumor xenografts mice, including downregulation of p-STAT3, N-cadherin, and Vimentin while up-regulation of E-cadherin. Wogonin also inhibit the metastasis of A549 cells in vivo. Taken all data together, we concluded that wogonin suppresses tumor cells migration in inflammatory microenvironment by inactivating STAT3 signal.

Down-regulated expression of vimentin induced by mechanical stress in fibroblasts derived from patients with ossification of the posterior longitudinal ligament.

The aim of this study was to investigate the potential role of vimentin in the signal transduction pathways initiated by mechanical stimulation that contribute to ossification of the posterior longitudinal ligament of the spine (OPLL). We investigated the effects of in vitro cyclic stretch on cultured spinal ligament cells derived from OPLL (OPLL cells) and non-OPLL (non-OPLL cells) patients. The expression levels of the osteoblast-specific genes encoding osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen (COL I) were assessed by semi-quantitative reverse transcription-polymerase chain reaction. Vimentin protein expression in OPLL cells was detected by Western blotting. Small interfering RNA (siRNA) interference targeting vimentin was performed in OPLL cells induced by mechanical stress, and the expression levels of OCN, ALP and COL I were assessed. In response to mechanical stretch, the expression levels of OCN, ALP, and COL I were increased in OPLL cells, whereas no change was observed in non-OPLL cells. Furthermore, knockdown of vimentin protein expression by siRNA resulted in an increase in the mRNA expression levels of OCN, ALP, and COL I. The down-regulation of vimentin induced by mechanical stress plays an important role in the progression of OPLL through the induction of osteogenic differentiation in OPLL cells.

Vimentin regulates neuroplasticity in transected spinal cord rats associated with micRNA138.

Spinal cord injury (SCI) often results in motor disability concomitant with limit neuroplasticity; the underlying mechanism, however, is still unclear. This study established spinal cord transection rats model (T10), then performed cDNA microarray analysis and found that vimentin located in astrocytes was increased significantly in scar tissues after transection. To understand the role of vimentin and it's mechanism of regulation, RNA interference and luciferase assay were used. Vimentin knockdown in the scar tissues showed a significant improvement on locomotor function in hindlimbs, while vimentin overexpression exhibited an opposite effect. In vitro, vimentin downregulation or overexpression can effectively inhibit or increase astrogliosis, respectively. Moreover, by using biological informatics technology, we predicted that vimentin may be as the target of micRNA138 (miR-138), and confirmed that miR-138 could regulate vimentin by luciferase activity assay. The present results not only validated the exact role of vimentin in transected spinal cord, but also exhibited a novel regulation mechanism, in which miR-138 may regulate vimentin to promote neuroplasticity. It, therefore, provides a novel target for gene drug discovery based on miRNA-138 or vimentin for the treatment of SCI in the future clinic trial.

Blocking PI3K/Akt signaling attenuates metastasis of nasopharyngeal carcinoma cells through induction of mesenchymal-epithelial reverting transition.

In the present study, we evaluated the role of phosphatidylinositol-3 OH kinase/protein kinase B (PI3K/Akt) signaling on changes to epithelial-to-mesenchymal reverting transition (EMrT) in nasopharyngeal carcinoma (NPC). Protein expression levels of p-Akt (Ser473), and the epithelial‑to-mesenchymal transition (EMT) markers E-cadherin, vimentin, α smooth muscle actin (α-SMA), were examined in clinical samples from 130 cases of undifferentiated non-keratinizing NPC, and 20 cases of benign nasopharyngitis. The relationship between protein expression levels and the statue of NPC lymph node metastasis was analyzed. The poorly‑differentiated NPC cell line CNE2Z was treated with various concentrations of the PI3K inhibitor, LY294002, and western blotting and quantitative polymerase chain reaction assays were used to analyze the activation of PI3K/Akt signaling and expression of E-cadherin, vimentin and α-SMA. The ability of cellular migration and invasion was assessed using Transwell assays. The in vivo effects of LY294002 on metastasis and expression of EMT markers in CNE2Z cells was evaluated using tumor xenograft experiments. The expression levels of p-Akt (Ser473) in NPC samples were higher than those in nasopharyngitis. There were reduced levels of membrane E-cadherin protein expression, and increased cytosol vimentin and α-SMA expression levels in NPC samples compared with those in nasopharyngitis samples. High expression levels of p-Akt (Ser473), vimentin, and α-SMA, and low expression levels of E-cadherin were positively associated with lymph node metastasis of NPC cells. Treating CNE2Z cells with LY294002 inhibited p-Akt (Ser473), vimentin and α-SMA expression but upregulated E-cadherin expression, leading to significantly attenuated cell invasion and migration. Administration of mice with LY294002 resulted in upregulation of membrane E-cadherin, and downregulation of vimentin and α-SMA in CNE2Z xenografts, with reduced pulmonary metastasis. Our findings suggest that inhibiting the PI3K/Akt pathway using LY294002 attenuated NPC metastasis via induction of EMrT.

An efficient and reproducible method to culture Bergmann and cortical radial glia using textured PMMA.

Radial glia cells comprise the principal population of neural stem cells (NSC) during development. Attempts to develop reproducible radial glia and NSC culture methods have met with variable results, yielding non-adherent cultures or requiring the addition of growth factors. Recent studies demonstrated that a 2-μm patterned poly-methyl methacrylate (ln2 PMMA) grooved scaffold, by mimicking the biophysical and microtopographic properties of the embryonic NSC niche, induces the de-differentiation of glial cells into functional radial glia cells. Here we describe a method for obtaining cultures of adherent Bergmann radial glia (BRG) and cortical radial glia (CRG). The growth substrate is ln2 PMMA and the addition of growth factors is not required. Postnatal glia obtained from mouse cerebellum or cerebral cortex and grown on ln2 PMMA adopted a BRG/CRG phenotype characterized by a bipolar shape, the up-regulation of progenitor markers such as nestin and Sox2, and the down-regulation of vimentin and GFAP. Neurons cultured over the BRG/CRG aligned their processes with those of the glial shafts, thus mimicking the behavior of migrating neuronal cells. The ln2 PMMA culture method offers an ideal system for analyzing both the biochemical factors controlling the neurogenic potential of BRG/CRG and neuronal migration. The ln2 PMMA method is a reproducible system to obtain immature BRG/CRG preparations in vitro. It can be used to study the properties of CNS progenitor cells as well as the interactions between radial glia and neurons, and supports cultured progenitors for use in different applications.

Down-regulation of Gli-1 inhibits hepatocellular carcinoma cell migration and invasion.

Glioma-associated oncogene homolog-1 (Gli-1) is considered a marker of Hedgehog pathway activation and is associated with the progression of several cancers. We have previously reported that Gli-1 was correlated with invasion and metastasis in hepatocellular carcinoma (HCC). However, the exact roles and mechanisms of Gli-1 in HCC invasion are unclear. In this study, we found that small interfering RNA mediated down-regulation of Gli-1 expression significantly suppressed adhesion, motility, migration, and invasion of both SMMC-7721 and SK-Hep1 cells. Furthermore, down-regulation of Gli-1 significantly reduced expressions and activities of both matrix metalloproteinase (MMP)-2 and MMP-9. In addition, we found that down-regulation of Gli-1 resulted in up-regulation of E-cadherin and concomitant down-regulation of Snail and Vimentin, consistent with inhibition of epithelial-mesenchymal transition (EMT). Taken together, our results suggest that down-regulation of Gli-1 suppresses HCC cell migration and invasion likely through inhibiting expressions and activations of MMP-2, 9 and blocking EMT.

Serine protease inhibitor Kazal type 1 promotes epithelial-mesenchymal transition through EGFR signaling pathway in prostate cancer

Overexpression of serine protease inhibitor Kazal type 1 (SPINK1) defines an aggressive molecular subtype of ETS fusion-negative prostate cancer (PCa) patients in western countries. However, how SPINK1 contributes to PCa invasion and metastasis is largely unknown. Fluorescence in situ hybridization and immunohistochemistry were utilized to detect ERG rearrangement, SPINK1 expression, and EGFR aberrations in a cohort of 211 PCa patients with radical prostatectomy. Real-time quantitative PCR and Western blotting were used to study the transcript and protein expression levels. Cellular distribution of E-cadherin and vimentin were observed by immunofluorescence. Cellular function was evaluated by siRNA, transwell, and wound healing assay, respectively. SPINK1-induced Epithelial-mesenchymal transition (EMT) in benign prostate RWPE cells, manifested by acquisition of mesenchymal morphology, alternation of EMT markers as well as migration and invasion capabilities. Knockdown of SPINK1 in 22RV1 PCa cells results in up-regulation of E-cadherin and down-regulation of vimentin. SPINK1-induced EMT is mediated by EGFR, in which MAPK/MEK/ERK pathway is mainly involved. Connective tissue growth factor (CTGF) might be an important down-stream molecule of SPINK1-EGFR axis. Clinically, SPINK1 and EGFR were significantly co-overexpressed in a cohort of Chinese PCa patients (n > 200). SPINK1 is an unfavorable prognostic factor in Chinese PCas (P = 0.025). These findings suggest that SPINK1 promotes EMT through EGFR signaling pathway in PCa and SPINK1 could be a new prognostic marker in Chinese PCas.

The Culture of Cancer Cell Lines as Tumorspheres Does Not Systematically Result in Cancer Stem Cell Enrichment

Cancer stem cells (CSC) have raised great excitement during the last decade and are promising targets for an efficient treatment of tumors without relapses and metastases. Among the various methods that enable to enrich cancer cell lines in CSC, tumorspheres culture has been predominantly used. In this report, we attempted to generate tumorspheres from several murine and human cancer cell lines: B16-F10, HT-29, MCF-7 and MDA-MB-231 cells. Tumorspheres were obtained with variable efficiencies from all cell lines except from MDA-MB-231 cells. Then, we studied several CSC characteristics in both tumorspheres and adherent cultures of the B16-F10, HT-29 and MCF-7 cells. Unexpectedly, tumorspheres-forming cells were less clonogenic and, in the case of B16-F10, less proliferative than attached cells. In addition, we did not observe any enrichment in the population expressing CSC surface markers in tumorspheres from B16-F10 (CD133, CD44 and CD24 markers) or MCF-7 (CD44 and CD24 markers) cells. On the contrary, tumorspheres culture of HT-29 cells appeared to enrich in cells expressing colon CSC markers, i.e. CD133 and CD44 proteins. For the B16-F10 cell line, when 1 000 cells were injected in syngenic C57BL/6 mice, tumorspheres-forming cells displayed a significantly lower tumorigenic potential than adherent cells. Finally, tumorspheres culture of B16-F10 cells induced a down-regulation of vimentin which could explain, at least partially, the lower tumorigenicity of tumorspheres-forming cells. All these results, along with the literature, indicate that tumorspheres culture of cancer cell lines can induce an enrichment in CSC but in a cell line-dependent manner. In conclusion, extensive characterization of CSC properties in tumorspheres derived from any cancer cell line or cancer tissue must be performed in order to ensure that the generated tumorspheres are actually enriched in CSC.

MiR-200a inhibits epithelial-mesenchymal transition of pancreatic cancer stem cell

BackgroundPancreatic cancer is one of the most aggressive cancers, and the aggressiveness of pancreatic cancer is in part due to its intrinsic and extrinsic drug resistance characteristics, which are also associated with the acquisition of epithelial-to-mesenchymal transition (EMT). Increasing evidence suggests that EMT-type cells share many biological characteristics with cancer stem-like cells. And miR-200 has been identified as a powerful regulator of EMT.MethodsCancer Stem Cells (CSCs) of human pancreatic cancer cell line PANC-1 were processed for CD24, CD44 and ESA multi-colorstaining, and sorted out on a BD FACS Aria II machine. RT-qPCR was performed using the miScript PCR Kit to assay the expression of miR-200 family. In order to find the role of miR-200a in the process of EMT, miR-200a mimic was transfected to CSCs.ResultsPancreatic cancer cells with EMT phenotype displayed stem-like cell features characterized by the expression of cell surface markers CD24, CD44 and epithelial-specific antigen (ESA), which was associated with decreased expression of miR-200a. Moreover, overexpression of miR-200a was resulted in down-regulation of N-cadherin, ZEB1 and vimentin, but up-regulation of E-cadherin. In addition, miR-200a overexpression inhibited cell migration and invasion in CSCs.ConclusionIn our study, we found that miR-200a played an important role in linking the characteristics of cancer stem-like cells with EMT-like cell signatures in pancreatic cancer. Selective elimination of cancer stem-like cells by reversing the EMT phenotype to mesenchymal-to-epithelial transition (MET) phenotype using novel agents would be useful for prevention and/or treatment of pancreatic cancer.