AIM: To elucidate the detailed mechanisms underlying the appreciable effects of JG3, a novel marine-derived oligosaccharide, on cell migration using a Chinese hamster ovary (CHO) cell line stably over-expressing heparanase. METHODS: A retrovirus infection system was used to establish a CHO-K1 cell line stably transfected with heparanase. Immunocytochemistry was used to assess cell morphology. Flow cytometry was selected to analyze the activation of beta1-integrin, and Western blotting was used to analyze the downstream effects on the cell adhesion pathway. An affinity precipitation assay was used to determine activation of the small GTPases, Rac1, and RhoA. RESULTS: JG3 abolished heparanase-driven formation of focal adhesions and cell spreading. Although JG3 failed to block the heparanase-triggered activation of beta1-integrin or the phosphorylation of Src, the oligosaccharide caused a significant dephosphorylation of FAK and subsequent inactivation of Erk. Furthermore, JG3 was found to arrest the activation of Rac1. CONCLUSION: All these findings help form an alternative view to understand the mechanisms underlying the inhibitory effects of JG3 on cell motility.Acta Pharmacologica Sinica (2009) 30: 1033-1038; doi: 10.1038/aps.2009.97; published online 22 June 2009.
AIM: To elucidate the detailed mechanisms underlying the appreciable effects of JG3, a novel marine-derived oligosaccharide, on cell migration using a Chinese hamster ovary (CHO) cell line stably over-expressing heparanase. METHODS: A retrovirus infection system was used to establish a CHO-K1 cell line stably transfected with heparanase. Immunocytochemistry was used to assess cell morphology. Flow cytometry was selected to analyze the activation of beta1-integrin, and Western blotting was used to analyze the downstream effects on the cell adhesion pathway. An affinity precipitation assay was used to determine activation of the small GTPases, Rac1, and RhoA. RESULTS: JG3 abolished heparanase-driven formation of focal adhesions and cell spreading. Although JG3 failed to block the heparanase-triggered activation of beta1-integrin or the phosphorylation of Src, the oligosaccharide caused a significant dephosphorylation of FAK and subsequent inactivation of Erk. Furthermore, JG3 was found to arrest the activation of Rac1. CONCLUSION: All these findings help form an alternative view to understand the mechanisms underlying the inhibitory effects of JG3 on cell motility.Acta Pharmacologica Sinica (2009) 30: 1033-1038; doi: 10.1038/aps.2009.97; published online 22 June 2009.