After washing, 5104 cells were added to each well for the times indicated in the determine legends

After washing, 5104 cells were added to each well for the times indicated in the determine legends. (or a nucleolin-like protein) is usually a signaling receptor for P-selectin on Colo-320 cells and suggest a mechanism for linkage of nucleolin to P-selectin-induced signal transduction pathways that regulate the adhesion and the spreading of Colo-320 on VPC 23019 fibronectin substrates. strong class=”kwd-title” Keywords: P-Selectin, cell-surface nucleolin, cell adhesion, signal transduction Introduction Metastasis is usually a multi-step cascade of cellular events by which cancer cells leave the original tumor mass and establish new colonies at distant sites in the body [1, 2]. In the hematogenous phase of metastasis, cancer cells that reach the blood stream must eventually gain the ability to attach to the blood or lymphatic vessel endothelium prior to successful tumor cell transendothelial migration and colonization of host organs [3, 4]. Both integrins and selectins are thought to be involved in the process of tumor cell metastasis. Selectins are vascular adhesion molecules involved in leukocyte trafficking, inflammation, thrombosis, autoimmunity, and cancer. Three major members of the selectin family have been identified: L-selectin, E-selectin and P-selectin. L-Selectin is usually constitutively expressed on leukocytes. P-and E-selectins are expressed on activated endothelial cells. P-selectin is also expressed on thrombin-activated platelets. All three members of the selectin family can bind to human tumor cells and cancer-derived cell lines [5-11]. Both E-selectin and P-selectin can support the tethering of tumor cells to endothelium in flow conditions [6, 12, 13], and P-selectin can induce interactions between cancer cells and platelets to form emboli which facilitate the arrest of cancer cells [7, 13-17]. Moreover, P- and/or L-selectin-deficient mice reveal a pronounced inhibition of metastasis compared with wild-type mice [18, 19]. The absence of P-selectin also reduces primary tumor growth compared to P-selectin wild-type controls [20]. Integrins are transmembrane glycoproteins that function as cell adhesion VPC 23019 receptors for cell-substrate and cell-cell interactions. Each integrin consists of VPC 23019 an and a subunit. Integrin-mediated cell adhesion is usually subject to multiple signaling pathways that can control either the affinity of integrins for their ligands or the clustering of integrins around the cell surface [21-23]. It is believed that dysregulation of integrin-mediated cell adhesion can contribute to the promotion of the metastatic process [24-26]. Recently, we have shown that P-selectin binding to Colo-320 human colon carcinoma cells specifically activates the 51 integrin, which results in the increase of cell attachment and VPC 23019 cell spreading on fibronectin substrates. This selectin-mediated integrin activation results from at least two distinct intracellular signaling pathwaysthe p38 MAPK and the PI 3-K pathwaysthat are linked by a p38 MAPK-PI 3-K signaling complex [27]. These results suggest that P-selectin binding to specific receptors on cancer cells can activate signals that regulate tumor cell adhesion and possibly proliferation. Here, we have identified nucleolin (or a nucleolin-like protein) as a Colo-320 cell P-selectin receptor by using affinity chromatography, mass-spectrometry (MS), immunoassays, and RNAi knock-down. We show that nucleolin is usually expressed around the cell-surface. An anti-nucleolin mAb (D3) significantly blocks Colo-320 cell adhesion to immobilized P-selectin. Moreover, nucleolin becomes clustered around the plasma membrane when bound P-selectin-IgG-Fc chimeric protein is usually cross-linked. We also found that P-selectin binding to Colo-320 cells induces tyrosine phosphorylation specifically of cell-surface nucleolin, but not of nucleolin ELF3 expressed in cytoplasm or nucleus, and formation of a nucleolin/p38 MAPK/PI 3-K signaling complex. Using siRNA to inhibit specifically nucleolin expression in Colo-320 cells, we have exhibited that both the P-selectin binding to Colo-320 cells and formation of the P-selectin-mediated p38 MAPK/PI 3-K signaling complex require nucleolin expression. These results show that nucleolin is usually a signaling P-selectin receptor on Colo-320 human colon VPC 23019 carcinoma cells and suggest a mechanism.