Protein Expression Levels Of E-cadherin Research Articles

Abstract 1396: Pathway-directed DNA demethylation as an epithelial to mesenchymal targeting strategy

Abstract It has been proposed that by silencing tumor suppressor genes, DNA methylation plays an important function in cancer initiation and progression. DNA hypomethylating agents are being developed as anti-cancer agents. However, this approach causes unrestricted hypomethylation, which could potentially activate tumor-promoting mechanisms. Therefore a strategy capable of inducing hypomethylation of selected genes is needed. Towards this goal, we designed a strategy to induce demethylation of a coordinated gene program by using Ten-eleven translocation (TET1) dioxygenase, which converts 5 -methylcytosine to 5-hydroxymethylcytosine. We constructed a fusion gene TET1-DBD containing the catalytic domain of TET1 and the DNA binding domain of Snail, the transcription factor that regulates epithelial-to-mesenchymal transition (EMT). We hypothesized that this fusion gene will bind to promoters targeted by Snail, induce hypomethylation, reactivate gene expression, and block EMT. To test this hypothesis, we used TET1-DBD and measured induction of EMT in an ovarian (SKOV3) and breast cancer (MCF10A) model driven by TGF-β. Genome-wide DNA methylation was assessed by Reduced Representation Bisulfite Sequencing (RRBS) to measure the downstream targets of TET1-DBD. TET1-DBD increased mRNA and protein expression levels of E-cadherin in transfected cells and prevented TGF-β induced expression of fibronectin and vimentin in MCF10A and SKOV3 cells. RRBS analysis demonstrated hypomethylation of genes targeted by Snail-DBD. Our results suggest that TET1-DBD selectively binds to and hypomethylates promoters of genes targeted by Snail, resulting in inhibition of the EMT program. We propose a novel approach to induce selective promoter hypomethylation and to block key oncogenic processes by using TET1 fused to a transcription factor “carrier”. Citation Format: HAO HUANG, Qing Yu, Maooj Kandpal, Ramana V. Davuluri, Daniela Matei. Pathway-directed DNA demethylation as an epithelial to mesenchymal targeting strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1396.

MicroRNA-206 regulates the epithelial-mesenchymal transition and inhibits the invasion and metastasis of prostate cancer cells by targeting Annexin A2.

The present study investigated the molecular mechanism by which microRNA-206 (miR-206) targets Annexin A2 (ANXA2) expression and inhibits the invasion and metastasis of prostatic cancer cells through regulation of the epithelial-mesenchymal transition (EMT). Using bioinformatics analysis, miR-206 was identified as the most promising candidate miRNA that targeted ANXA2. Prostate tissue specimens from 60 patients with prostate cancer, 30 patients with metastatic prostate cancer and 20 patients with benign prostatic hyperplasia (BPH) were examined for ANXA2 protein expression by immunohistochemistry and western blotting and for miR-206 expression by reverse transcription-quantitative polymerase chain reaction. Additionally, human prostate cancer PC-3 cells were transfected with miR-206 mimics, miR-206 inhibitors or a negative control sequence, and expression of ANXA2, E-cadherin and N-cadherin was detected by western blotting. Transwell assays were performed to determine the effect of altered miR-206 expression on the invasive behavior of PC-3 cells. Bioinformatics analysis predicted complementary binding between miR-206 and ANXA2 mRNA. ANXA2 protein expression was detected in a significantly higher proportion of BPH tissues (95%, 19/20) when compared with prostate cancer tissues (51.7%, 31/60; P<0.05). Similarly, ANXA2 was expressed in a significantly higher proportion of metastatic prostate cancer samples than that of prostate cancer samples (P<0.05). Expression of miR-206 was higher than that of ANXA2 in prostate cancer samples, but lower in BPH samples. Inhibition of miR-206 expression in PC-3 cells upregulated ANXA2 and E-cadherin protein expression levels, downregulated N-cadherin and vimentin, and promoted cell invasion in vitro. These data suggested that binding between miRNA-206 and ANXA2 mRNA may regulate EMT signaling, thereby suppressing the invasion and metastasis of prostatic cancer cells.

LncRNA AB073614 induces epithelial- mesenchymal transition of colorectal cancer cells via regulating the JAK/STAT3 pathway.

LncRNAs are involved in the metastasis and recurrence of human tumors, including colorectal cancer (CRC). We previously reported that lncRNA AB073614 promotes tumor proliferation and metastasis and predicted a poor clinical outcome of CRC patients. Herein, we investigated the underlying mechanism of lncRNA AB073614-related metastasis in CRC. The expression of lncRNA AB073614 in CRC tissues were evaluated by quantitative real-time PCR (qRT-PCR). Transwell assay was performed to detect the effects of lncRNA AB073614 on cell migration and invasion. Epithelial-mesenchymal transition (EMT) molecular markers and Janus kinase/signal transducer and activator of transcription (JAK/STAT3) pathway proteins expression levels were detected by Western blot and Immunofluorescence. We confirmed that lncRNA AB073614 was highly expressed in the colorectal cancer tissues. LncRNA AB073614 knockdown in SW480 and HCT116 cells significantly promoted the protein expression levels of E-cadherin and Occludin, and decreased the expressions of N-cadherin and Vimentin, then further decreased the cell migration and invasion ability. Interestingly, the expression of phosphorylated STAT3 was also down-regulated. Furthermore, SW480 and HCT116 cells were transfected with lncRNA AB073614 vector and treated with a JAK inhibitor, AT9283. The results showed that lncRNA AB073614 regulated EMT through JAK-STAT3 signaling pathway. All these results indicate that lncRNA AB073614 can induce the expression of EMT cell markers and regulate the process of EMT of CRC cells through regulating the JAK/STAT3 pathway activation.

Multi-channel promotion of lung cancer progress by bone marrow derived mesenchymal stem cells in tumor microenvironment

Objective: To observe the growth and metastasis of lung cancer promoted by bone marrow derived mesenchymal stem cells (BMSCs) in tumor microenvironment and investigate the underlined mechanisms. Methods: Specific chemotaxis of BMSCs towards lung cancer was observed, and the tumor growth and metastasis were assessed in vivo. Furthermore, CD34 expression determined by immunohistochemistry was used to assess the microvessel density (MVD), and the expressions of GFP and α-SMA determined by immunofluorescence were used to detect the BMSCs derived mesenchymal cells. We investigated the effect of BMSCs on migration, invasion of lung cancer cells including A549 and H446 cells by using scratch assays and Transwell Assay in vitro. We also explored the effect of BMSCs on epithelial mesenchymal transition of A549 and H446 cells by observing the phenotype transition and E-Cadherin protein expression detected by Western blot. At last, we screened the potentially key soluble factors by enzyme linked immunosorbent assay (ELISA). Results: In NOD mice, labeled BMSCs injected via tail vein were special chemotaxis to tumor cells, and promoted the tumor growth [the time of tumor formation in A549+ BMSCs and A549 alone was (5.0±1.5) days and (10.0±3.6) days, respectively, P<0.05; the time of tumor formation in H446+ BMSCs and H446 alone was (5.2±1.5) days and (12.0±2.0) days, respectively, P<0.05]. The tumor incidence of A549+ BMSCs was 100%, significantly higher than 66.7% of A549 alone (P<0.05), while the tumor incidence of H446+ BMSCs was 83.0%, significantly higher than 50.0% of H446 alone (P<0.05). The BMSCs also increased the tumor volume [the tumor volume of A549+ BMSCs and A549 alone was (193.0±42.3) mm(3) and (97.8±42.9) mm(3,) respectively, P<0.05; the tumor volume of H446+ BMSCs and H446 alone was (78.6±34.8) mm(3) and (25.3±12.7) mm(3,) respectively, P<0.05] and facilitated the tumor metastasis (the tumor metastatic incidence of A549+ BMSCs and A549 alone was 100.0% and 16.7%, respectively, P<0.05; the tumor metastatic incidence of H446+ BMSCs and H446 alone was 100.0% and 0.0%, respectively, P<0.05). Furthermore, BMSCs increased tumor vessel formation (the MVD of A549+ BMSCs and A549 alone was 53.2±11.4 and 25.3±11.5, respectively, P<0.05; the MVD of H446+ BMSCs and H446 alone was 56.8±12.5 and 24.8±10.0, respectively, P<0.05). BMSCs were able to differentiate to fibroblasts in the lung squamous cell carcinoma and promoted the migration and invasion of lung cancer cells (the A of cells in the migrated lower chambers of A549+ BMSCs and A549 alone was 1.9±0.2 and 1.1±0.1, respectively, P<0.05; the A of cells in the migrated lower chambers of H446+ BMSCs and H446 alone was 1.9±0.3 and 0.9±0.2, respectively, P<0.05). The cell shape was longer and sharper, the intercellular junctions were reduced and the relative expression level of E-Cadherin protein in A549 co-cultured with BMDCs was 0.36, significantly down-regulated when compared to 0.55 of A549 alone (P<0.05), and the relative expression level of E-Cadherin protein in H446 co-cultured with BMDCs was 0.28, significantly down-regulated when compared to 0.46 of H446 cells alone (P<0.05). The concentration of IL-6 in the conditional medium of BMSCs, A549 co-cultured with BMSCs and H446 co-cultured with BMSCs was 910.5, 957.2, and 963.8, respectively, significantly up-regulated when compared to 18.8 of control group (P<0.05). The expression level of PGE2 in A549 co-cultured with BMSCs and H446 co-cultured with BMSCs was 130.5 and 87.2, significantly up-regulated when compared to 13.8 of control group and 23.8 of BMSCs group (P<0.05). Conclusions: Our results suggest that BMSCs contribute to the tumor growth through facilitating angiogenesis, and promote tumor metastasis through paracrine manner and down-regulation of E-Cadherin protein. IL-6 and PGE2 produced by BMDCs might be the potentially important cytokines.

The role of curcumae rhizoma-sparganii rhizoma medicated serum in epithelial-mesenchymal transition in the triple negative breast cancer: Pharmacological role of CR-SR in the TBNC

The aim of this study was to investigate the effect of Curcumae Rhizoma-Sparganii Rhizoma (CR-SR) medicated serum on the changes of epithelial-mesenchymal transition (EMT) in the triple-negative breast cancer (TNBC). The EMT model was developed by using the TNBC MDA-MB-468 cells, which were treated with TGF-β1. The migration and invasion abilities of TGF-β1-treated MDA-MB-468 cells were detected by wound healing assay and transwell assay. Protein expression levels of E-cadherin and vimentin were determined by western blot. CR-SR medicated serum repressed the phenotypic transition in the TGF-β1-induced MDA-MB-468 cells. Moreover, CR-SR medicated serum inhibited TGF-β1-induced cell proliferation, migration and invasion. Besides, CR-SR medicated serum could reduce TGF-β1-induced up-regulation of the phosphorylation levels of Smad3, and reduce the expression of several transcription factors (Snail1, Snail2 and Twist1). CR-SR medicated serum might suppress TGF-β1-induced EMT in TNBC by decreasing the phosphorylated Smad3 (p-Smad3) pathway in vitro.

PIAS1 inhibited the metastasis of gastric cancer cell by epithelial-mesenchymal transition regulation within the inflammatory microenvironment.

Protein inhibitor of activated signal transducer and activator of transcription-1 (PIAS1) is an important regulator of the inflammatory signaling network, the expression of which was decreased in gastric cancer and implicated in the development of cancer. However, its mechanism has not been elucidated. The aim of the present study was to investigate the effect of PIAS1 on epithelial-mesenchymal transition (EMT) of gastric cancer cells within the inflammatory microenvironment. Recombinant adenovirus Ad5/F35-PIASl and Ad5/F35-null plasmids were constructed to transfect SGC7901 cells. Subsequently, these plasmids were confirmed by reverse transcription polymerase chain reaction and western blotting. The cells were treated with IL-6 or Ad5/F35-PIASl+IL-6, and the control cells were treated with Ad5/F35-null+IL-6. The morphological changes to the cells were observed using inverted microscopy. The effect of PIAS1 on cell migration and invasion was evaluated by scratch wound healing and Transwell chamber assays, and the protein expression of EMT markers and phosphatidylinositol 3-kinase (PI3K)/serine/threonine-protein kinase (Akt)/matrix metalloproteinase (MMP)-9 signaling pathway was examined by western blotting. Transfection with Ad5/F35-PIASl markedly increased the PIAS1 expression in SGC7901 cells. The cells acquired the more typical spindle-shape phenotype of mesenchymal cells following co-culture with IL-6; the cells co-cultured with IL-6 and Ad5/F35-PIASl acquired changes concordant with an epithelial phenotype. The overexpression of PIAS1 significantly decreased the migratory and invasive capacities of the SGC7901 cells (P<0.01). Western blotting indicated that the expression levels of E-cadherin protein in the cells treated with Ad5/F35-PIASl+IL-6 were increased significantly and the expression levels of zinc finger protein SNAI, Twist-related protein 1, vimentin and MMP-9, and the activation of PI3K/Akt proteins were decreased when compared with IL-6- or Ad5/F35-null+IL-6-treated cells (both P<0.01). PIAS1 may inhibit EMT in gastric cancer cells within the inflammatory microenvironment via the regulation of PI3K/Akt pathway activation, and may serve an important role in the inhibition of tumor invasion and metastasis with in this microenvironment.

Downregulation of SNHG1 suppresses cell proliferation and invasion by regulating Notch signaling pathway in esophageal squamous cell cancer.

Long non-coding RNAs (lncRNAs) exert important functions involved in tumorigenesis and cancer progression including esophageal squamous cell cancer (ESCC), however, the clinical role and underlying biological function of Small Nucleolar RNA Host Gene 1 (SNHG1) in ESCC is not well known. Quantitative Real-time polymerase chain reaction (QRT-PCR) was used to detect the SNHG1 expression levels in ESCC tissues and adjacent non-cancerous tissues. Chi-square test was used to evaluate the association between clinicopathological features and SNHG1 expression in ESCC patients, Kaplan-Meier curve and log rank test was performed to analyze the association between overall survival and SNHG1 expression. Cell proliferation and invasion was assessed by MTT assay, colony formation, and transwell cell invasion assays. Western blot were also performed to examine protein expression levels of E-cadherin, Vimentin and N-cadherin, Notch 1 and Hes-1. We demonstrated that lncRNA SNHG1 was significantly up-regulated in ESCC tissues compared with adjacent non-cancerous tissues in ESCC patients. Meanwhile, increased lncRNA SNHG1 expression levels markedly correlated with lymph node metastasis, depth of invasion, TNM stage and reduced over survival time in ESCC patients. Furthermore, MTT assay, colony formation, transwell cell invasion, qRT-PCR and Western-blot assays demonstrated that knockdown of lncRNA SNHG1 could inhibit cell proliferation and cell invasion capacity and cell Epithelial-Mesenchymal Transition (EMT) phenomenon by up-regulation E-cadherin and down-regulating Vimentin and N-cadherin in ESCC cells. Besides, we demonstrated that knockdown of SNHG1 suppressed the Notch signaling pathway by reducing the Notch1 and Hes-1 expression levels in ESCC cells. These results indicated that lncRNA SNHG1 may be a potential predictor of prognosis in ESCC patients and a novel target for ESCC treatment.

Selective cyclooxygenase-2 inhibitor NS-398 attenuates myocardial fibrosis in mice after myocardial infarction via Snail signaling pathway.

The role of NS-398 in Snail pathway of myocardial cells in mice after myocardial infarction and its effect on myocardial fibrosis were investigated in this study. C57BL/6 mice were selected to establish mouse models of myocardial infarction with permanent ligation of anterior descending branch and sham-operation models without ligation. After successful establishment of models, 30 mice were randomly divided into sham-operation group, myocardial infarction group and drug intervention group. The drug intervention group was treated with intraperitoneal injection of NS-398 (5 mg/kg) at 1 week after modeling for 3 weeks. The survival status of mice after operation was monitored, the cardiac function was detected via echocardiography, the collagen levels in heart tissue pathological sections were detected via Masson staining and Sirius red staining. Moreover, the expressions of Snail and type I collagen levels were detected via immunohistochemistry, and the Snail protein expression level and the activity and expression level of E-cadherin protein were detected via Western blotting. At 4 weeks after establishment of myocardial infarction model, the fibrosis reaction was obvious, and the cardiac function was decreased, accompanied with Snail activation. The administration of NS-398 for 3 weeks inhibited the Snail activity expression and significantly improved the fibrosis degree after infarction. However, it did not improve the cardiac function. Inhibiting Snail improved the fibrosis reaction after infarction, in which Snail/E-cadherin signaling pathway was involved. NS-398 improves the myocardial fibrosis in mice after myocardial infarction through inhibiting the Snail signaling pathway.

The role of aplysia ras homolog I in colon cancer cell invasion and adhesion.

Aplysia ras homolog I (ARHI) acts as a tumor suppressor in certain cancer cells. However, the role of ARHI in colon cancer development has not previously been reported. The present study aimed to investigate the functional role of ARHI in colon cancer focusing on the aspect of metastasis. Furthermore, the molecular mechanism underlying its function was explored. The present study detected the expression of ARHI in a human colon epithelial cell line and colon cancer cell lines using reverse transcription-quantitative polymerase chain reaction and western blotting analysis. It was demonstrated that ARHI expression was significantly downregulated in colon cancer cell lines compared with the normal colon epithelial cell line (P<0.05). An ARHI-pcDNA3.1 plasmid was transfected into HCT116 cells to overexpress ARHI. The number of invaded cells and the adhesive ability were significantly decreased in the ARHI overexpression group compared with the control group, as determined by cell invasion and adhesion assays (P<0.05). Furthermore, ARHI overexpression led to increased mRNA and protein expression levels of E-cadherin, and decreased mRNA and protein expression levels of N-cadherin and vimentin. Wnt/β-catenin signaling was suppressed in HCT116 cells overexpressing ARHI. Lithium chloride, a wnt/β-catenin signaling activator, was able to attenuate the effect of ARHI on HCT116 cell invasion and adhesion. In addition, the effect of ARHI on epithelial-mesenchymal transition (EMT) in HCT116 cells was reversed by the activation of wnt/β-catenin signaling. In conclusion, the present study provided novel evidence that ARHI could inhibit colon cancer cell invasion and adhesion through suppressing EMT, and these effects were achieved, at least partially, via the suppression of the wnt/β-catenin signaling pathway. The present findings may help in developing novel therapeutic approaches for colon cancer.

Bcl-2 associated athanogene 3 affects the epithelial-mesenchymal transition in human cervical cancer

Objective: To investigate the expression of Bcl-2 associated athanogene 3 (BAG3) in cervical cancer tissues and cells and its role in epithelial mesenchymal transition (EMT) of cervical cancer. Methods: (1) Cervical cancer samples were collected from September 2015 to March 2017 in the Qilu Hospital of Shandong University and Shangdong Provincial Hospital. While, 50 normal tissues were collected from August 2015 to March 2017 in the Dezhou Municiple Hospital, which were obtained from patients with uterine myoma underwent hysterectomy and patients with cervical biopsy. Reverse transcription (RT)-PCR and western blot were used to detect the expression of BAG3 mRNA and protein, and their clinical significances were analyzed. (2) The expression of BAG3 mRNA and protein was detected using RT-PCR and western blot method in HeLa and SiHa cell lines and normal cervical epithelial cells. The experiment was divided into two groups, BAG3 small interfering RNA transfected group (si-BAG3) and the control group transfected with small interfering RNA (siRNA). Cell counting kit 8 (CCK-8) analysis was used to detect cell proliferation of two groups. Wound-healing and transwell assay were used to detect the migration and invasion ability of HeLa and SiHa cells. The xenograft model of cervical cancer in nude mice was used to observe the effect of BAG3 on tumor xenografts and the tumor-related biomarkers were tested by western blot. Results: (1) The expression levels of BAG3 mRNA and protein in cervical carcinoma tissues were 1.20±0.15 and 1.10±0.16, which were significantly higher than that in normal cervical tissue, 0.23±0.04 and 0.29±0.03 (both P<0.01). The results showed that the expression levels of BAG3 mRNA and protein were significantly correlated with cervical carcinoma staging and lymph node metastasis (P<0.05).However, its expression was not correlated with the patient's age, pathological grade, and diameter of tumor (all P>0.05). (2) Compared with normal cervical epithelial cells, the expression of BAG3 mRNA and protein levels in HeLa and SiHa cells were significantly increased (P<0.01), the expression levels of BAG3 mRNA and protein in HeLa and SiHa cells transfected with si-BAG3 were significantly lower than that in control group (all P<0.01). After post-transfected 72 hours, A value of HeLa and SiHa with transfection were significantly lower than those in control group [(0.88±0.08) vs (1.22±0.13), (0.92±0.09) vs (1.35±0.12); both P<0.01]. After post-transfected 24 hours, the migration level of HeLa and SiHa cells with transfection were significantly lower than those in the control group [(20.1±2.1)% vs (58.6±5.6)%, and (21.1±2.1)% vs (61.7±5.4)%; both P<0.01]. The transmembrane cell number in HeLa and SiHa cells with transfection were 76±11 and 71±8, which were significantly less than those in control group (131±12 and 129±14; both P<0.01). After the inoculation into nude mice, tumor formation time of HeLa and SiHa cells with transfection were (9.5±0.5) and (10.5±1.3) days, respectively, which were significantly longer than those in control group [(4.5±0.5) and (5.2±1.1) days; both P<0.05]. Compared with those in the control group, the expression level of Slug, N-cadherin and matrix metalloproteinase-2 (MMP-2) protein in HeLa and SiHa cells with transfected in tumor tissues were significantly decreased (all P<0.01), while the expression level of E-cadherin protein was significantly increased (P<0.01). Conclusion: BAG3 could be involved in the proliferation, migration and invasion of cervical cancer cells by affecting cervical cancer EMT, and BAG3 may be an effective target for the treatment of cervical cancer.

Correlation analysis between the parameters of contrast-enhanced ultrasonography in evaluating cervical cancer metastasis and expression of E-cadherin.

The study aims to investigate the correlation between the parameters of contrast-enhanced ultrasonography in evaluating cervical cancer metastasis and expression of E-cadherin. All 120 patients with cervical cancer underwent contrast-enhanced ultrasonography. According to the results of postoperative pathological examination, patients were divided into distant metastasis group (group A), lymph node metastasis without distant metastasis group (group B) and no metastasis group (group C). Expression of E-cadherin in cervical cancer tissues was detected by enzyme-linked immunosorbent assay (ELISA). Correlations between the parameters of contrast-enhanced ultrasonography in evaluating cervical cancer metastasis and expression of E-cadherin were analyzed by Pearsons correlation analysis. Comparison of parameters of contrast-enhanced ultrasonography showed that, the baseline intensity of group A was 11.9±2.2 dB, which was significantly lower than that of group B and C. Baseline intensity of group B was significantly lower than that of group C (13.0±2.4 vs. 15.3±3.6 dB), significant differences were found among three groups (P<0.05). The sensitivity and specificity of the use of enhanced intensity ~83.7 dB in evaluating tumor metastasis of patients with cervical cancer were 82.42 and 79.32%, respectively. Expression level of E-cadherin protein in group A was 0.030±0.003 ng/ml, which was significantly lower than that in group B and C (P<0.05), expression level of E-cadherin protein in group A was significantly lower than that in group C (0.037±0.007 vs. 0.045±0.012 ng/ml), significant differences in the expression level of E-cadherin protein were found among the three groups of cervical cancer patients (P<0.05). Pearsons correlation analysis showed that there was a positive correlation between the baseline intensity of contrast-enhanced ultrasonography and the expression level of E-cadherin (P<0.05), while there was a negative correlation between the enhanced intensity of contrast-enhanced ultrasonography and the expression level of E-cadherin (P<0.05). Contrast-enhanced ultrasonography can be used to determine the tumor metastasis of cervical cancer patients, in addition, the combined use of contrast-enhanced ultrasonography and E-cadherin protein expression can significantly improve the diagnosis and treatment of cervical cancer.

The mechanism of epithelial-mesenchymal transition induced by TGF-β1 in neuroblastoma cells.

Neuroblastoma is the second most common extracranial malignant solid tumor that occurs in childhood, and metastasis is one of the major causes of death in neuroblastoma patients. The epithelial-mesenchymal transition (EMT) is an important mechanism for both the initiation of tumor invasion and subsequent metastasis. Therefore, this study investigated the mechanism by which transforming growth factor (TGF)-β1 induces EMT in human neuroblastoma cells. Using quantitative RT-qPCR and western blot analyses, we found that the mRNA and protein expression levels of E-cadherin were significantly decreased, whereas that of α-SMA was significantly increased after neuroblastoma cells were treated with different concentrations of TGF-β1. A scratch test and Transwell migration assay revealed that cell migration significantly and directly correlated with the concentration of TGF-β1 indicating that TGF-β1 induced EMT in neuroblastoma cells and led to their migration. Inhibiting Smad2/3 expression did not affect the expression of the key molecules involved in EMT. Further investigation found that the expression of the glioblastoma transcription factor (Gli) significantly increased in TGF-β1-stimulated neuroblastoma cells undergoing EMT, accordingly, interfering with Gli1/2 expression inhibited TGF-β1-induced EMT in neuroblastoma cells. GANT61, which is a targeted inhibitor of Gli1 and Gli2, decreased cell viability and promoted cell apoptosis. Thus, TGF-β1 induced EMT in neuroblastoma cells to increase their migration. Specifically, EMT induced by TGF-β1 in neuroblastoma cells did not depend on the Smad signaling pathway, and the transcription factor Gli participated in TGF-β1-induced EMT independent of Smad signaling.

REG3A promotes the proliferation, migration, and invasion of gastric cancer cells.

The mechanism underlying the metastasis of gastric cancer (GC) cells remains elusive. REG3A is considered an oncogene in various cancers, but in GC its role is unclear. Here, we report that the expression of REG3A was significantly increased in the tumor tissues of patients with GC compared with the matched normal tissues. Knockdown of REG3A induced by specific small interfering RNA (siRNA) significantly repressed the proliferation of GC cells for 24 h or 48 h. Moreover, knockdown of REG3A significantly suppressed the migration, invasion, and adhesion of GC cells in vitro. Furthermore, knockdown of REG3A reduced the phosphorylation of JAK2 and STAT3, and altered the messenger RNA (mRNA) and protein expression levels of E-cadherin, Snail, RhoC, MTA1, MMP-2, and MMP-9. Taken together, REG3A is overexpressed in GC and promotes the proliferation, migration, invasion, and adhesion of GC cells by regulating the JAK2/STAT3 signal pathway. REG3A may be a potential therapeutic target for GC.

Interaction of macrophages and endometrial cells induces epithelial-mesenchymal transition-like processes in adenomyosis.

Adenomyosis is a nonneoplastic condition characterized by the benign invasion of ectopic endometrium into the myometrium. Macrophages play significant roles in epithelial-mesenchymal transition (EMT) and adenomyosis. An EMT associated with adenomyosis has been extensively studied. This study investigated the process by which the interaction of macrophages with endometrial cells induces EMT in Ishikawa cells and epithelial cells of adenomyosis. Specimens were collected after hysterectomy or resection of adenomyosis lesions from women with adenomyosis and curettage from women without adenomyosis or endometriosis. Immunohistochemistry and immunofluorescent staining demonstrated that CD68-positive macrophages aggregated in adenomyosis lesions, along with the increased protein expressions of n-cadherin, vimentin, and S100A4. By contrast, the protein expressions of e-cadherin and CK7 were decreased. After the primary endometrium cells were cocultured with THP-1-derived macrophages, the protein expression levels of n-cadherin, vimentin, and S100A4 of endometrium cells were increased, whereas the protein expression levels of e-cadherin and CK7 were decreased. The proportion of alternatively activated (M2) macrophages derived from THP-1 macrophages was also increased. The M2 macrophages elicited a bidirectional effect on Ishikawa cells by inducing EMT-like or mesenchymal-epithelial transition-like processes. The apoptotic rate of the Ishikawa cells cocultured with macrophages was increased, whereas their cell proliferation rate was decreased. Transmission electron microscopy indicated that the number of intercellular junctions of the cocultured Ishikawa cells was reduced. Microarray-based gene expression analysis revealed that transforming growth factor-β1/Smad3 and interleukin-6/JAK2/STAT3 signaling pathways were upregulated. Therefore, macrophages can induce EMT-like processes in adenomyosis and undergo polarization to M2.

Prometastatic mechanisms of CAF-mediated EMT regulation in pancreatic cancer cells.

Tumor metastasis are accompanied by the EMT (epithelial-mesenchymal transition)-MET (mesenchymal-epithelial transition) two-step process. In this study, we investigated the importance of cancer associated fibroblasts (CAF) in the process. First, the primary cultures of isolated pancreatic CAF, fibroblasts of normal pancreatic tissues(NF), and normal hepatic stellate cells (HSF) were identified and verified via the expression of α-SMA and vimentin. Using an indirect three-dimensional co-culture model, the morphological changes were observed by light microscopy and laser scanning confocal microscopy. The invasive and migration capacity of pancreatic cancer cells was determined by Transwell chamber migration assay or scratch assay. The mRNA and protein expression levels of E-cadherin, vimentin, and Gli1 were determined by RT-PCR and western blotting. Primary cultures of isolated CAF, NF, HSF showed satisfactory growth with active proliferation. Indirect co-culture with CAF, BxPc-3 and Panc-1 cells showed significant partial EMT, reduced E-cadherin expression, and enhanced vimentin expression as compared with the single culture and NF/HSF co-culture groups, with corresponding increases in migratory and invasive capacities. PCR and western blotting results showed that mRNA and protein expression levels of Gli1 in CAF and Snail in cancer cells were increased. This process could be reversed by inhibition of hedgehog (HH) signaling in CAF. In the tumor microenvironment, activation of CAF is the key event in mediating partial EMT, and its mechanism may be associated with paracrine action after activation of HH signaling in CAF.

Complex Behavior of ALDH1A1 and IGFBP1 in Liver Metastasis from a Colorectal Cancer.

Using our data set (GSE50760) previously established by RNA sequencing, the present study aimed to identify upregulated genes associated with colorectal cancer (CRC) liver metastasis (CLM) and verify their biological behavior. The potential roles of candidate genes in tumors were assessed using cell proliferation and invasion assays. Tissue samples were collected from 18 CRC patients with synchronous CLM and two CRC cell lines (SW480 and SW620) were used for transfection and cloning. The roles of the genes identified in CLM were verified using immunohistochemistry in 48 nude mice after intrasplenic transplantation of CRC cells. mRNA and protein expression was determined by quantitative real-time reverse transcription polymerase chain reaction and western blot, respectively. Nine genes were initially selected according to the relevance of their molecular function and biological process and, finally, ALDH1A1 and IGFBP1 were chosen based on differential mRNA expression and a positive correlation with protein expression. The overexpression of ALDH1A1 and IGFBP1 significantly and time-dependently decreased cell proliferation (p ≤ 0.001–0.003) and suppressed invasiveness by ≥3-fold over control cells (p < 0.001) in the SW480 cell line, whereas they had a slight effect on reducing SW620 cell proliferation. The protein expression levels of E-cadherin, N-cadherin, claudin-1, and vimentin were significantly higher in CLM than in primary tumor tissues (p < 0.05). However, the cadherin switch, namely, N-cadherin overexpression with reduced E-cadherin expression, was not observed in CLM tissues and transfected CRC cells. Irrespective of reduced proliferation and invasion found on in vitro cell assays, persistent overexpression of β-catenin, vimentin, and ZO-1 in IGFBP1-overexpressing SW480 cells possibly contributed to CLM development in mice implanted with IGFBP1-overexpressing SW480 cells (CLM occurrences: SW480/IGFBP1-transfected mice vs. SW480/vector- and SW480/ALDH1A1-transfected mice, 4/8 vs. 0/10, p = 0.023). In conclusion, ALDH1A1 and IGFBP1 are differentially overexpressed in CLM and may play a dual role, functioning as both tumor suppressors and metastasis promoters in CRC.

Experimental research of IKKε through E-cadherin regulation of the human brain glioma migration and invasion ability

Objective To investigate the effect of IKKe in glioma cells on the regulation of E-cadherin expression and cell migration and invasion ability. Methods The IKKe overexpression vectors and shRNA-IKKe lentivirus were constructed. The TP483 glioma cell lines were transfected by the IKKe overexpression plasmid. The expression level of IKKe in glioma cells was upregulated. The U251 and U138 glioma cell lines were transfected by the shRNA-IKKe lentivirus, IKKe expression level in glioma cells was downregulated. The expression levels of IKKe and E-cadherin in cells were detected by RT-PCR and Western blot. The expression changes of IKKe and E-cadherin after transfecting were observed by using the immunofluorescence assay. The cell migration and invasion abilities were detected by using the scratch test and Transwell assay. Results Compared with the non-transfected group (TP483 control group), the expression level of IKKe protein increased (P=0.000), and the expression level of E-cadherin protein decreased (P=0.000) in the transfected IKKe expression plasmid group (TP483 treaed group). In the glioma cells transfected IKKe shRNA(the U251 treated group and the U138 treated group) , the expression level of IKKe protein decreased significantly compared with the non-transfected group (the U251 treated group and the U138 treated group; all P<0.05). At the same time, the expression levels of E-cadherin protein in each treated group were all increased compared with the control group (P<0.05). The scratch repair rate of the TP483 treated group was higher than that of the control group (56.39±0.93% vs. 46.43±1.68%; P=0.035). After transfection of overexpression plasmid for 24 h, the number of TP483 cells passing through the filter membrane increased compared with the control group (55.8±6.0 vs. 24.6±2.3; P=0.000). The scratch repair rates of the U251 and U138 treated groups decreased compared with their respective control groups (the U251 treated group: 50.40±1.09%, the U251 control group: 87.29±11.11%, P=0.043; the U138 treated group: 48.20±1.34%, the U138 control group: 73.15±6.62%, P=0.035); After transfection for 24 h, the number of cells passing through the filter membrane decreased in the U251 and the U138 groups compared with the control group (the U251 treated group: 77.8±6.4, the control group: 124.0±13.4, P=0.000; the U138 treated group: 25.8±4.2, the control group: 54.8±7.1, P=0.000). Conclusions The low expression of IKKe can effectively inhibit the invasion and migration of glioma cells, and the high expression of IKKe can enhance their invasion and migration abilities. Its possible mechanism is to influence the invasion and migration abilities of glioma cells through regulating the expression level of E-cadherin. Key words: Glioma; Transcellular cell migration; Neoplasm invasiveness; Ⅰ-kappa B kinase; E-cadherin

Heterophyllin B inhibits the adhesion and invasion of ECA-109 human esophageal carcinoma cells by targeting PI3K/AKT/β-catenin signaling.

The present study aimed to measure the effect of heterophyllin B (HB) on the adhesion and invasion of ECA-109 human esophageal carcinoma cells, and examine the possible mechanism involved. A Cell Counting kit 8 assay was performed to determine the cell viability. Cell adhesion and invasion were determined following treatment of the ECA-109 cells with HB (0, 10, 25 and 50 µM) for 24 h. The levels of phosphorylated (p-)ATK and p-phosphoinositide 3-kinase (PI3K), and the protein levels of β-catenin were measured using western blot analysis. The mRNA and protein expression levels of E-cadherin, vimentin, snail, matrix metalloproteinase (MMP)2 and MMP9 were detected using reverse trancsription-quantitative polymerase chain reaction and western blot analyses, respectively. HB (10, 25 and 50 µM) significantly suppressed the adhesion and invasion of the ECA-109 human esophageal carcinoma cells in a dose-dependant manner. The expression levels of p-ATK, p-PI3K and β-catenin were markedly decreased. The expression of E-cadherin was promoted, whereas the expression levels of snail, vimentin, MMP 2 and MMP 9 were decreased significantly in the ECA-109 cells treated with HB. In addition, HB inhibited the adhesion and invasion induced by PI3K activating peptide in the ECA-109 cells, and the protein expression levels were also adjusted. These results suggested that HB effectively suppressed the adhesion and invasion of the human esophageal carcinoma cells by mediating the PI3K/AKT/β-catenin pathways and regulating the expression levels of adhesion- and invasion-associated genes.

E-cadherin expression in a rat model of acute pancreatitis.

A clinical requirement exists for early biomarkers that can predict the severity of acute pancreatitis (AP). In order to determine whether E-cadherin is associated with the severity of AP, a pancreatitic rat model was established and the expression levels of E-cadherin were detected. A study population of 24 Sprague Dawley rats was administered intraperitoneal injections of various concentrations of L-arginine in order to induce pancreatitis. Rats were assigned to the severe acute pancreatitis (SAP) or mild acute pancreatitis (MAP) group based on the results of histological evaluations and the serum levels of amylase. An additional 8 rats received intraperitoneal injections of NaCl solution, as a control group. For each group, the serum concentrations of soluble E-cadherin and the expression levels of E-cadherin protein in the pancreatic tissue were detected. The results indicated that the rat model of pancreatitis was successfully established. Rats in the high concentration L-arginine treatment group, which exhibited a higher pancreatitis pathology score and level of serum amylase, were assigned to the SAP group. Low concentration L-arginine group rats were assigned to the MAP group. The pathology scores and levels of serum amylase in the SAP and MAP group rats were higher compared with the control group rats. The levels of serum E-cadherin were the most elevated in the SAP group. Statistically significant differences were detected in the SAP and MAP groups compared with the control group, and in the SAP group compared with the MAP group (P<0.05). Furthermore, the levels of E-cadherin protein in the pancreatic tissue were elevated in the SAP group compared with the MAP and control groups. In conclusion, the present study demonstrated that E-cadherin was overexpressed in SAP rats, and the overexpression of E-cadherin may be associated with the severity of AP.

Promoting effect of connective tissue growth factor on expression of E-cadherin in human Tenon capsule fibroblasts

Background Scarring of filtration channel following glaucoma filtering surgery is a main cause of the failure of the surgery. The proliferation, epithelial-mesenchymal transition and extracellular matrix remodeling of fibroblasts are thought to be the primary pathological mechanism of scarring. Connective tissue growth factor (CTGF) plays a promoting role in the formation of scar. Whether CTGF participates in mesenchymal-epithelial transition of human Tenon capsule fibroblasts (HTFs) is not clear yet. Objective This study attempted to investigate the effect of CTGF on the mesenchymal-epithelial transition of HTFs in vitro. Methods HTFs were cultured and passaged in high glucose DMEM medium with 10% fetal bovine serum, and the cells of generation 3-6 were used in this experiment. The cells were divided into the blank control group and CTGF-treated group and were routinely cultured in the blank control group. CTGF was added in the medium in the CTGF-treated group, with the final concentration 50 ng/ml CTGF. Immunofluorescence staining was used to identify and locate the expression of E-cadherin protein in the cells, and Western blot assay was employed to quantitatively analyze the expression level of E-cadherin protein in 48 hours after culture. Results The HTFs grew well with the spindle-like shape and vortex-like arrangement. The red fluorescence (E-cadherin protein) in the cytoplasm and blue fluorescence in the cellular nucleus were seen in the CTGF-treated group, but only nucleus with blue fluorescence were obtained in the blank control group. Western blot assay showed that the E-cadherin protein expression was absent in the blank control group, however, the relative expression level of E-cadherin protein in the cells was 0. 63±0. 08. Conclusions E-cadherin protein is not expressed in fibroblast derived from mesenchymal tissue. However, CTGF can induce the expression of E-cadherin in HTFs. This study suggests that CTGF promotes the mesenchymal-epithelial transition of HTFs in vitro. Key words: Humans; Fibroblasts; Tenon capsule; Connective tissue growth factor; Transdifferentiation; Glaucoma/surgery; Bleb; Scarring