16 research outputs found
Heme modulates intestinal epithelial cell activation: involvement of NADPHox-derived ROS signaling
Insulin/IGF-I Signaling Pathways Enhances Tumor Cell Invasion through Bisecting GlcNAc N-glycans Modulation. An Interplay with E-Cadherin
<div><p>Changes in glycosylation are considered a hallmark of cancer, and one of the key targets of glycosylation modifications is E-cadherin. We and others have previously demonstrated that E-cadherin has a role in the regulation of bisecting GlcNAc <i>N</i>-glycans expression, remaining to be determined the E-cadherin-dependent signaling pathway involved in this <i>N</i>-glycans expression regulation. In this study, we analysed the impact of E-cadherin expression in the activation profile of receptor tyrosine kinases such as insulin receptor (IR) and IGF-I receptor (IGF-IR). We demonstrated that exogenous E-cadherin expression inhibits IR, IGF-IR and ERK 1/2 phosphorylation. Stimulation with insulin and IGF-I in MDA-MD-435 cancer cells overexpressing E-cadherin induces a decrease of bisecting GlcNAc <i>N</i>-glycans that was accompanied with alterations on E-cadherin cellular localization. Concomitantly, IR/IGF-IR signaling activation induced a mesenchymal-like phenotype of cancer cells together with an increased tumor cell invasion capability. Altogether, these results demonstrate an interplay between E-cadherin and IR/IGF-IR signaling as major networking players in the regulation of bisecting <i>N</i>-glycans expression, with important effects in the modulation of epithelial characteristics and tumor cell invasion. Here we provide new insights into the role that Insulin/IGF-I signaling play during cancer progression through glycosylation modifications.</p> </div
Effects of exogenous E-cadherin on the cell morfology and expression of epithelial and mesenchymal markers in MDA-MDA-435 cells.
<p>(A) Total cell lysates from MDA-MB-435+mock and MDA-MB-435+E-cad were obtained and analyzed by Western blot for E-cadherin. MDA-MB-435+mock cells do not show detectable levels of E-cadherin. Actin was used as a loading control. (B) The same amount of cells were seeded, and at the same time of culture phase contrast images show the cell morphology of MDA-MB-435+mock, which exhibit a mesenchymal-like phenotype, and MDA-MB-435+E-cad exhibit an epithelial-like phenotype. The inserts represent higher magnifications of the figure. Scale bar = 20 µm. (C) The bar graph shows the relative amount of E-cadherin, occludin, β-catenin, vimentin, fibonectin and N-cadherin mRNA levels by qRT-PCR. MDA-MB-435+E-cad cells exhibit a significant decreased expression of the mesenchymal markers fibronectin and N-cadherin. Values were normalized to the amount of mRNA in MDA-MB-435+mock. Error bars indicate the means + S.E.M. (n = 3). ** = P < 0.01, *** =P < 0.001, Student's t-test. </p
Effects of stimulation with insulin and IGF-I on cell invasion.
<p>Representative images of cell invasion through Matrigel using 8 mm pore of a polycarbonate membrane. Nuclei were stained with DAPI. We observed a significant increase of tumor cell invasion of MDA-MB-435+Ecad after stimulation with insulin or IGF-I. The bar graph shows the amount of cells/field. Error bars indicate the means + S.E.M. (n = 3). ** = P < 0.01 ANOVA test.</p
Effects of exogenous E-cadherin expression on the phosphoproteome profile of tyrosine kinase receptors and downstream proteins.
<p>(A) Total cell lysates from MDA-MB-435+mock and MDA-MB-435+E-cad were obtained and analyzed by Phospho-RTK array using 300 µg of proteins. The phosphor-RTK coordinates are shown on the right side of figure illustrating the localization of the spots containing immobilized antibodies on the nitrocellulose membrane. B17 and B18 represent the IR spots, whereas B19 and B20 represent IGF-IR spots, both receptors show decreased phosphorylation levels upon E-cadherin expression. (B) Total cell lysates from MDA-MB-435+mock and MDA-MB-435+E-cad were obtained and analyzed by Western blot for phospho-IR(Tyr1150-51)/phospho-IGF-IR(Tyr1135-36), IR, IGFR, Akt, phospho-Akt (Ser 473), ERK 1/2, phospho-ERK 1/2 and β-catenin. MDA-MB-435+E-cad cells show significant decreased levels of phospho-IR/phospho-IGF-IR and phospho-ERK1/2, comparing with mock cells. Tubulin was used as a loading control. The bar graphs show the relative amount of proteins levels normalized to tubulin. Error bars indicate the means + S.E.M. (n = 3). ** = P < 0.01, Student's t-test.</p
Effects of stimulation with insulin and IGF-I on the phosphorylation of tyrosine kinase receptors and downstream proteins.
<p>(A,B) Total cell lysates from MDA-MB-435+mock, MDA-MB-435+E-cad and MDA-MB-435+E-cad stimulated (24h) with insulin or IGF-1 were obtained and analyzed by Western blot for phospho-IR(Tyr1150-51)/phospho-IGF-IR(Tyr1135-36), IR, IGFR, Akt, phospho-Akt (Ser 473), ERK 1/2, phospho-ERK 1/2, β-catenin and E-cadherin. Increased phosphorylation levels of IR, IGF-IR, and ERK 1/2 were observed after stimulation with insulin or IGF-I. Tubulin was used as a loading control. No changes were observed for the total expression levels of Akt and ERK1/2. (C) The bar graphs show the relative amount of proteins levels normalized to tubulin. Error bars indicate the means + S.E.M. (n = 3). ** = P < 0.01, ANOVA t-test.</p
Effects of insulin and IGF-I stimulation on the mRNA expression levels of epithelial and mesenchymal markers.
<p>(A) The bar graphs show the relative amount of E-cadherin, occludin, β-catenin, vimentin, fibronectin and N-cadherin mRNA levels by qRT-PCR. Significant down-regulation of the mRNA levels of epithelial markers (occludin and β-catenin) and an up-regulation of the mesenchymal marker fibronectin were observed after insulin and IGF-I stimulation. Values were normalized to the amount of mRNA in MDA-MB-435+E-cad. Error bars indicate the means + S.E.M. (n = 3). * = P < 0.05, ** = P < 0.01, ANOVA test. <b>Effects of stimulation with insulin and IGF-I on the fibronectin protein levels</b>. (B) Total cell lysates from MDA-MB-435+E-cad,MDA-MB-435+E-cad stimulated (24h) with insulin or IGF-1, and MDA-MB-435+mock were obtained and analyzed by Western blot for fibronectin. Increased fibronectin expression levels are observed after stimulation with insulin or IGF-I. The bar graphs show the relative amount of fibronectin levels normalized to tubulin.</p
Effects of insulin and IGF-I stimulation on the expression levels of bisecting GlcNAc <i>N</i>-glycans, in general and specifically on E-cadherin.
<p>(A) Total cell lysates from MDA-MB-435+mock, MDA-MB-435+E-cad and MDA-MB-435+E-cad stimulated (24h) with insulin or IGF-1 were obtained and analyzed by Lectin blot for E-PHA. The bar graphs show the relative amount of bisecting GlcNAc <i>N</i>-glycans levels in the whole protein lysate. MDA-MB-435+E-cad cells stimulated with insulin (100 ng/mL) and IGF-I (50 ng/mL) showed a significant decrease of the overall levels of bisecting GlcNAc N-glycans. The values were normalized to tubulin. Error bars indicate the means + S.E.M. (n = 3). ** = P < 0.01 ANOVA test. (B) Total cell lysates from MDA-MB-435+mock, MDA-MB-435+E-cad and MDA-MB-435+E-cad stimulated (24h) with insulin or IGF-1 were obtained and immunoprecipitated using E-cadherin antibody. The immunoprecipitates were analyzed by Western blot for E-cadherin and Lectin blot for E-PHA. The bar graphs show the relative amount of E-cadherin-linked bisecting GlcNAc <i>N</i>-glycans levels. Activation of insulin and IGF-I signaling pathway led to a decreased modification of E-cadherin with bisecting GlcNAc N-glycan structures.</p