E-cadherin Expression In Cells Research Articles

Silibinin upregulates E-cadherin expression in MKN-45 human gastric cancer cells

Background and objectives: Gastric cancer is currently known as one of the most important causes of cancer-driven death all over the world. In patients with gastric cancer, a significant proportion of death occurs due to metastasis. On the other hand, down modulated E-cadherin level has been reported as an important contributor to tumor cell invasion and metastasis. In this regard, the present work was aimed to evaluate the impact of silibinin, a flavonolignan with established anti-tumor efficacy, on cell viability and E-cadherin expression in the gastric cancer cell line MKN-45. Methods: To determine cell viability, MTT assay was performed 48 h after silibinin treatment (at concentrations of 100, 200 and 400 μM). In addition, quantitative real-time PCR was done following total RNA extraction and cDNA synthesis, to assess E-cadherin level in cells treated with silibinin. Results: The MTT results showed concentration-dependent reducing effect of silibinin on viability of MKN-45 cells. The findings of quantitative real-time PCR analysis demonstrated upregulated E-cadherin expression in cells treated with silibinin (significantly (p≤ 0.05) at concentration of 200 μM) compared to the control cells. Conclusions: The current study suggested that silibinin may exert anti-migratory/invasive effects on gastric cancer cells by enhancing E-cadherin expression, which needs to be further investigated.

Changes in cell junctions induced by inhibition of epidermal growth factor receptor in oral squamous cell carcinoma cells.

The benefits of epidermal growth factor receptor (EGFR) targeting in the treatment of head and neck cancer, have been documented. However, a minority of patients with head and neck cancer are unresponsive to EGFR targeting therapies. The present study evaluated the effects and limitations of an EGFR inhibitor on oral squamous cell carcinoma cells, particularly on cell-cell junctions mediated by epithelial (E)-cadherin. HSC-3 oral squamous cell carcinoma cells were treated with the EGFR inhibitor, AG1478 (0, 0.5, 2, 10 and 50 µM), and the effects of EGFR inhibition in HSC-3 cells were evaluated by wound healing assays, E-cadherin immunostaining and measurement of transepithelial electrical resistance in vitro. It was observed that treatment of oral squamous cell carcinoma cells with AG1478 suppressed cell motility, altered cell morphology and increased the number of cell-cell junctions compared with untreated control cells. Knockdown of EGFR induced a similar phenotype to that observed by the inhibition of EGFR. Furthermore, in oral squamous cell carcinoma cells treated with high-dose EGFR inhibitor (50 µM), the small number of cells that survived formed cell-cell junctions that were positive for E-cadherin expression. In cells treated with low concentrations of EGFR inhibitor (2 µM), recovery of epithelial properties was observed. The retention of E-cadherin expression in cells that survived high-dose EGFR inhibitor treatment may be a survival mechanism of cancer cells.

Cdc6 expression is induced by HPV16 E6 and E7 oncogenes and represses E-cadherin expression.

Cervical cancer is one of the most common cancers in women worldwide, and its development is related to two viral oncoproteins E6 and E7 from high-risk human papillomaviruses. Aberrant expression of E-cadherin is associated with epithelial-to-mesenchymal transition (EMT), and it is frequently seen in cervical cancer. However, the underlying mechanisms involved in E-cadherin suppression in cervical cancer are not clear. We studied the effects of human papillomavirus 16 (HPV16) E6 and E7 on E-cadherin and Cdc6 (cell division cycle 6) expression in the HCT-116 cell line. We also assessed the relationship between Cdc6 and E-cadherin expression in cells expressing HPV16 E6 and E7 proteins. The results showed that HPV16 E6 and E7 proteins reduce E-cadherin expression, and HPV16 E6-expressing cells undergo a more profound suppression of E-cadherin compared with cells expressing HPV16 E7. Our results also revealed that HPV16 E6 and E7 oncoproteins induce Cdc6 expression, whereas suppression of Cdc6 protein by short hairpin RNA restores E-cadherin expression. Induction of Cdc6 expression in HCT-116 cells was greater with E6 than with E7, a finding that was consistent with the corresponding changes in E-cadherin expression. These observations suggest that Cdc6 overexpression is an important factor for E-cadherin reduction in cells expressing HPV16 E6 and E7 proteins and may have an important role in the metastasis of HPV-associated cancers.Cancer Gene Therapy advance online publication, 11 November 2016; doi:10.1038/cgt.2016.51.

MFGE8 regulates TGF-β-induced epithelial mesenchymal transition in endometrial epithelial cells in vitro.

This study investigated the role of milk fat globule-epidermal growth factor-factor 8 (MFGE8) in TGF-β-induced epithelial-mesenchymal transition (EMT) of endometrial epithelial cells. These were in vitro studies using human endometrial epithelial cells and mouse blastocysts. We investigated the ability of TGF-β to induce EMT in endometrial epithelial cells (HEC-1A) by assessment of cytological phenotype (by light and atomic force microscopy), changes in expression of the markers of cell adhesion/differentiation E- and N-cadherin, and of the transcription factor Snail (by immunofluorescence and immunoblotting), and competence to support embryo attachment in a mouse blastocyst outgrowth assay. We also studied the effects of E-cadherin expression in cells transfected by retroviral shRNA vectors specifically silencing MFGE8. Results demonstrated that TGF-β induced EMT as demonstrated by phenotypic cell changes, by a switch of cadherin expression as well as by upregulation of the expression of the mesenchymal markers Snail and Vimentin. Upon MFGE8 knockdown, these processes were interfered with, suggesting that MFGE8 and TGF-β together may participate in regulation of EMT. This study demonstrated for the first time that endometrial MFGE8 modulates TGF-β-induced EMT in human endometrium cells.

δEF1 associates with DNMT1 and maintains DNA methylation of the E-cadherin promoter in breast cancer cells.

Abnormal DNA methylation at the C-5 position of cytosine (5mC) of CpG dinucleotides is a well-known epigenetic feature of cancer. Levels of E-cadherin, which is regularly expressed in epithelial tissues, are frequently reduced in epithelial tumors due to transcriptional repression, sometimes accompanied by hypermethylation of the promoter region. δEF1 family proteins (δEF1/ZEB1 and SIP1/ZEB2), key regulators of the epithelial-mesenchymal transition (EMT), suppress E-cadherin expression at the transcriptional level. We recently showed that levels of mRNAs encoding δEF1 proteins are regulated reciprocally with E-cadherin level in breast cancer cells. Here, we examined the mechanism underlying downregulation of E-cadherin expression in three basal-type breast cancer cells in which the E-cadherin promoter region is hypermethylated (Hs578T) or moderately methylated (BT549 and MDA-MB-231). Regardless of methylation status, treatment with 5-aza-2′-deoxycytidine (5-aza), which inhibits DNA methyltransferases, had no effect on E-cadherin expression. Knockdown of δEF1 and SIP1 resulted in recovery of E-cadherin expression in cells lacking hypermethylation, whereas combined treatment with 5-aza synergistically restored E-cadherin expression, especially when the E-cadherin promoter was hypermethylated. Moreover, δEF1 interacted with DNA methyltransferase 1 (DNMT1) through the Smad-binding domain. Sustained knockdown of δEF1 family proteins reduced the number of 5mC sites in the E-cadherin promoter region, suggesting that these proteins maintain 5mC through interaction with DNMT1 in breast cancer cells. Thus, δEF1 family proteins appear to repress expression of E-cadherin during cancer progression, both directly at the transcriptional level and indirectly at the epigenetic level.

Abstract C28: MET receptor downregulation decreases oncogenic properties of cervical carcinoma cells in vitro and in vivo through E-cadherin re-expression and Slug downregulation

Abstract Introduction: Cervical carcinoma, despite the development of vaccine, is the second cause of death among women suffering from cancer, especially in low and middle incomes countries. There is currently no effective therapy for advanced cervical cancer and it is important to determine molecular markers, which can help to develop new therapeutic strategies. MET receptor plays an important role during embryonic development, wound healing as well as cancer development and progression. MET is overexpressed in a variety of human cancers increasing their aggressiveness by stimulating tumor cells proliferation, survival and motility. In cervical carcinoma, MET receptor has been shown i) to promote scattering and morphogenesis of tumor cells, ii) to regulate migration and cytoskeleton changes, iii) to translocate of B-catenin transcription factor into the nucleus and iv) to up-regulate cyclin D1 expression. Objectives: In this study we asked whether downregulation of MET receptor changes cell phenotype into more benign and inhibits tumorigenesis of cervical cancer cells. Materials and Methods: Cervical carcinoma cell line, HTB-35, was transduced with MET and LacZ shRNA expressing virus. The level of various genes expression was estimated by real-time RT-PCR. Protein expression was evaluated by Western blot. Immunofluorescence and immunohistochemistry staining was used to study the expression of E-cadherin in cells under culture condition and in paraffin tumor section. In in vivo study, cells were injected subcutaneously into NOD-SCID mice (5x106/mouse) and after 4 weeks, tumors were isolated, measured and weighted. All experiments were repeated at least 3 times. For the statistical analysis, P values were assessed with two-tailed Student's t test. Results: In our study, we showed that silencing of MET receptor decreases proliferation rate by downregulation of cyclin D1 and c-myc. The growth rate between control and MET deficient cells differed significantly for each time point and, after 96 hours of culture, was three times higher for control cells. In MET deficient cells, the expression of cyclin D1 and c-myc was significantly lower in comparison to control cells at mRNA and protein level. Downregulation of MET receptor significantly decreases the expression of epithelial to mesenchymal transition regulating factor, Slug, and increases the expression of E-cadherin, a hallmark of epithelial phenotype in the in vitro study. We observed that downregulation of MET expression dramatically decreases tumor growth in NOD-SCID mice. HTB-35 control cells formed tumors of approximate weight of 1.7 g whereas MET deficient cells formed very small tumors with the mean weight of 0.08 g, or no tumors at all. Moreover, tumors formed by cells with MET receptor downregulation showed the expression of E-cadherin. Summary: We show that MET receptor downregulation decreases oncogenic properties of cervical carcinoma cells in vitro and in vivo and changes cell phenotype into more epithelial and less aggressive. Thus, inhibition of HGF-MET receptor axis might help in the future to develop new treatment strategies to block cervical carcinoma development and progression. Acknowledgement: This study was supported by research grants to K.M. from the Polish Ministry of Science and Higher Education NN 401142339, NN 401054839. Citation Format: Katarzyna Miekus, Marta Pawlowska, Grazyna Drabik, Marcin Majka. MET receptor downregulation decreases oncogenic properties of cervical carcinoma cells in vitro and in vivo through E-cadherin re-expression and Slug downregulation. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C28.

Mir-373 Affects Human Lung Cancer Cells' Growth and its E-Cadherin Expression

The aims of this study was to elucidate whether the expression of E-cadherin can be affected by the recombinant has-mir-373 eukaryotic expression plasmid vector through tests in vitro, and to analyze the relationship between the expression of E-cadherin and tumor growth. According to the has-mir-373 sequence in miRBase database, two template DNA sequences were designed. The has-mir-373 sequence and a control sequence were synthesized and cloned into pGenesil-1 eukaryotic expression plasmid vector. The recombinant plasmids were transfected into human lung cancer A549 cells by liposome-mediated method. The mir-373 expression in A549 cells was detected by using real-time quantitative polymerase chain reaction (real-time PCR). MTT (methyl thiazolyl tetrazolium) was used to analyze the growth of cancer cell cycle. RT-PCR and Western blotting were used to evaluate the levels of E-cadherin mRNA and protein expression, respectively. The expression of E-cadherin in cells was determined by immunocytochemistry. The mobility capability of transfected cells were evaluated by using wound healing assay in vitro. The fluorescent light was observed under fluorescent microscope. RT-PCR indicated that the mRNA of E-cadherin increased, and the Western blotting results also displayed that mir-373 promoted the expression of the E-cadherin protein. Compared with the control groups, MTT method and wound healing assay demonstrated that both the growth rate and migration of A549 cells transfected with the recombinant has-mir-373 eukaryotic expression plasmid was also decreased significantly (p < 0.001). The differences between the other two control groups were not significant (p > 0.05). The immunocytochemistry demonstrated a significant increase of E-cadherin protein levels in the cells transfected with mir-373, but not in the cells of the control group. Mir-373 could increase the expression levels of the E-cadherin and decrease the migration ability of human lung cancer A549 cells in vitro.

Notch mediated epithelial to mesenchymal transformation is associated with increased expression of the Snail transcription factor

Notch signalling pathway has been implicated as an important contributor to epithelial to myofibroblast transformation (EMT) in tumourigenesis. However, its role in kidney tubular cells undergoing EMT is not defined. This study assessed Notch signalling and the downstream effects on Snail in cultured proximal tubular epithelial cells. EMT was induced by exposure to transforming growth factor beta-1 (TGFβ 1) and angiotensin II (AngII). The expressions of Notch1, Snail, E-cadherin and α-smooth muscle actin (α-SMA) were determined by Western blot. Matrix Metalloproteinase (MMP)-2 and -9 production were determined by zymography. The specific roles of Notch1-ICD and Snail were determined by gene expression or siRNA technique respectively. TGFβ 1 and AngII resulted in EMT as characterized by the expected decrease in E-cadherin expression, an increase in α-SMA, MMP-2 and MMP-9 expression and associated increase of Notch1 and Snail. Over-expression of Notch1-ICD similarly resulted in increased Snail expression, loss of E-cadherin and increasedα-SMA. Inhibiting Snail degradation by pre-treatment with lithium chloride (LiCl) led to a further decrease in E-cadherin expression in cells concurrently exposed to TGFβ 1 + AngII, confirming that Snail is a repressor of E-cadherin. Silencing of Snail blocked TGFβ 1 + AngII induced EMT. Inhibition of Notch activation, by concurrent exposure to DAPT during the induction of EMT attenuated the decrease in E-cadherin expression, limited the increase in α-SMA and MMP-2 and -9 expression and decreased Snail expression. These results suggest a direct role for Notch signalling via the Snail pathway in the development of EMT and renal fibrosis.

The role of E‐cadherin in nevogenesis: an experimental study using epidermal reconstructs

In the development of congenital nevi, how nevus cells migrate in the dermis remains unclear. As shown in an earlier study designed to investigate Unna's Abtropfung hypothesis, dermal invasion does not occur when nevus cells are seeded on epidermal reconstructs. In melanoma, the decrease of E-cadherin expression is associated with the dermal invasion of melanoma cells. To study the expression of E-cadherin in dermal-cultured nevus cells from congenital nevi and its relevance to explain the absence of dermal invasion noted in epidermis reconstructed with cultured nevus cells. Comparison of the immunohistochemical expression pattern of E-cadherin in congenital nevi in vivo and after culture in monolayers and in a three-dimensional system. E-cadherin was not expressed in vivo by dermal nevus cells, either isolated or in nests. However, in monolayer cultures, dermal nevus cells expressed E-cadherin. When these cells were used in reconstructed epidermis, nevus cells did not invade the dermis and they expressed E-cadherin when isolated and just weakly or not when grouped in junctional nests. The absence of dermal invasion of nevus cells could be due to the expression of E-cadherin in these cells in reconstructed epidermis. Our experiments suggest, a restoration of the control of keratinocytes, that nevus cells escape in the dermal compartment.

Expression of epithelial cadherin in the developing and adult pig ovary.

Epithelial cadherin (E-cadherin) is one member of a family of intracellular calcium (Ca(2+))-dependent adhesion molecules that mediates selective cell-cell adhesion in a variety of species. Since changes in adhesive function accompany ovarian tissue development and remodeling, we were interested in studying the expression of E-cadherin during ovarian maturation in the pig. The objectives of this study were 1) to investigate the pattern of E-cadherin mRNA and protein expression during ovarian ontogeny in the pig, 2) to identify specific cells expressing E-cadherin in the mature porcine ovary, and 3) to compare E-cadherin expression in cells of morphologically healthy and atretic follicles. The results showed the presence of a 120-kDa protein, corresponding to E-cadherin, which was highest in fetal and neonatal ovaries and declined markedly (3- to 8-fold, p < 0.05) with maturity (16 wk to adult). In the adult ovary, E-cadherin was highest in ovarian surface epithelium (OSE) whereas in healthy follicles, granulosa cells had the highest levels. A significant decline (p < 0.05) in E-cadherin expression was evident in granulosa and theca cells from atretic follicles when compared with E-cadherin expression in cells of healthy follicles. A 4.2-kb E-cadherin transcript was detected in ovaries from 15-day-old pigs, and expression was markedly reduced (p < 0.05) by 16 wk of age. In the ovary of pregnancy, there was a faint E-cadherin mRNA signal, whereas in follicles the signal was stronger and uniform across follicle size. This is the first report that E-cadherin is expressed by the porcine ovary during ontogeny and that follicular and OSE cells of the adult ovary express the protein. Although the role of E-cadherin in the porcine ovary is unknown, the decline in E-cadherin expression in atretic follicular cells suggests that E-cadherin has a role in maintaining the structural integrity of the ovarian follicle during growth and development.