Given the important role of Rac1 activation

Given the important role of Rac1 activation selleck compound in cell migration, we next examined the effect of Notch1 over expression on cell migration using a Boyden chamber system. Notch1 knockdown inactivated Rac1 and inhibited ICC cell migration In reciprocal experiments, we examined whether knocking down endogenous Notch1 would inhibit Rac1 activity and cell migration using Notch1 siRNA. The efficiency of the Notch1 siRNA was examined by Western blot. Compared with scramble siRNA transfected cells, the GTP Rac1 level was dramatically decreased in Notch1 siRNA transfected cells. Similarly, suppression of Notch1 expression inhibited cell migration compared with scramble siRNA transfected cells. Knock down of endogenous Notch1 inhibits Rac1 activity and cell migration. ICC 9810 cells were transfected with Notch1 siRNA or scramble siRNA.

Notch1 expression did not stimulate ICC cancer cell proliferation To exclude the possibility that the increased cell migra tion was due to Rac1 activation, not cell proliferation, we over expressed and knocked down Notch1 in ICC 9810 cells, which express moderate levels of Notch1. As shown in Figure 5, BrdU incorporation analysis indicated that Notch1 expression did not affect cell proliferation. Notch1 over expression induced an EMT phenotype in ICC cancer cells To demonstrate that the ICN is sensitive to gamma secretase inhibitor, we performed western blot analysis to evaluate the ICN expression in ICC 9810 cells treated with 1 umol L GSI. This analysis confirmed that ICN was induced at the protein level within 24 hours of ex posure to the GSI but was not induced by the DMSO control.

Notch activation has been shown to induce an epithelial to mesenchymal transition in breast cancer Given that Notch1 expression was associated with ICC metasta sis, we further investigated whether a link exists between Notch1 expression and the EMT phenomenon in ICC. Ex ogenous Notch1 was transfected into ICC 9810 cells that express moderate levels of Notch1. Increased Notch1 ex pression was accompanied by enhanced expression of SMA and Vimentin and loss of E cadherin expression, which are hallmarks of EMT. An examination of cell morphology and immunofluorescence indicated that Notch1 over expression resulted in morphological changes and cytoskeletal reorganization in ICC 9810 cells. Discussion Notch genes encode large transmembrane proteins that act as receptors for the Delta, Serrate, Lag 2 first family of ligands. Four different Notch proteins and the following five known ligands exist in mam mals, Delta like 1, Delta like 3, Delta like 4, Jagged 1 and Jagged 2. Notch signaling plays multiple roles in development and tissue homeostasis, and these roles can be subverted during oncogenic transform ation.

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