No abstract

The effect of plant glycosides on tumor cell invasion was examined. Among the glycosides tested, ginsenoside Rgs was found to be a potent inhibitor of invasion by rat ascites hepatoma cells (MM1), B16FE7 melanoma cells, human small cell lung carcinoma (OC10), and human pancreatic adenocarcinoma (PSN‐1) cells, when examined in a cell monolayer invasion model. Structurally analogous ginsenosides, Rb2, 20(R)‐ginsenoside Rg2 and 20(S)‐ginsenoside Rg3 (a stereoisomer of Rg3), showed little inhibitory activity. Neither Rh1, Rh2, 20(R)‐ginsenosides Rh1 Rb1, Rc nor Re had any effect. The effective ginsenoside, Rg3, tended to inhibit experimental pulmonary metastasis by highly metastatic mouse melanoma B16FE7 cells as well. Taking account of our previous finding that 1‐oleoyl‐lysophosphatidic acid (LPA) induced invasion by MM1 cells in the monolayer invasion model, the effect of Rg3 on molecular events associated with the invasion induced by LPA was analyzed in order to understand the mechanism of the inhibition. Rg3, which suppressed the invasion induced by LPA, dose‐dependently inhibited the LPA‐triggered rise of intracellular Ca2+. Protein tyrosine phosphorylation triggered by LPA was not inhibited by Rg3.

No abstract

We demonstrated previously that rat ascites hepatoma MM1 cells require both lysophosphatidic acid (LPA) and fibronectin (FN) for phagokinetic motility and transcellular migration and that these events are regulated through the RhoA-ROCK pathway. It remains to be elucidated, however, how the signals from both LPA and FN are integrated into cell migration. To examine this, total cellular lysates after stimulation with LPA or FN were subjected to time-course immunoblot analysis with anti-phosphotyrosine antibodies (Abs). Consequently, tyrosine-phosphorylation of paxillin was obviously persistent after stimulation with FN ؉ LPA as compared to after stimulation with either alone. Cell migration plays an essential role in various physiological and pathological phenomena such as embryogenesis, wound repair, inflammation and invasion and metastasis of cancer cells. 1,2 Cell migration is primarily mediated by integrin binding to extracellular matrices (ECM) and regulated by organization of the actin cytoskeleton and the formation of focal adhesion complexes, which are induced through activation of the Rho-family small GTP-binding proteins. [3][4][5] Integrins not only act as a scaffold but also generate important signals leading to cell migration. Integrinmediated signaling events that regulate cytoskeletal organization require Rho-family GTPases and conversely integrin-mediated adhesion itself regulates Rho-family GTPases. 6 Cell motility is closely related to morphology, growth, 7 differentiation and survival, in which different signals from other cell surface receptors are also implicated. 8 It remains to be elucidated, however, how the integrin and growth-factor signaling pathways are integrated into cell migration.Paxillin (p68-kD) is one of the integrin assembly proteins and can interact directly with several integrin assembly proteins, including vinculin, talin, ␤1 integrin, focal adhesion kinase (FAK), c-Src and Csk. 9,10 Three human isoforms of paxillin (␣, ␤ and ␥) and 2 homologous murine isoforms (␣ and ␤) have been identified, 11,12 and the ␣ form (original paxillin) appears to play a more dominant role. 13 Integrin-mediated tyrosine phosphorylation also enables paxillin to interact with various signaling molecules: tyrosine residues (Y) 31 and 118 being especially predominant targets of phosphorylation by kinases and creating binding sites for the SH2 domain of adaptor protein Crk. 14 -16 Thus, paxillin plays a pivotal role in cell adhesion, migration and further oncogenic transformation. 9,10 Because several cytokines and growth factors also induce tyrosine phosphorylation of paxillin, 9,10 it is suggested that the signals from both cytokine-and growth-factor receptors and those from integrins converge on paxillin.In efforts to evaluate the invasive capacity of cancer cells and to understand the mechanisms of cancer-host interactions including transcellular (transmonolayer) migration of cancer cells, we have developed an in vitro system: rat ascites hepatoma MM1 cells are allowed to invade a mesothelial cel...

Rat ascites hepatoma cells (MM I) invade a mesothelial cell monolayer in vitro inassay medium containing serum, but not in serum-free medium. Serum could be completely replaced by I -oleoyl lysophosphatidic acid (LPA) in inducing invasion. LPAinduced invasion was inhibited by genistein, a tyrosine-kinase inhibitor. Protein tyrosine phosphorylation in response to LPA was thus analyzed in order to determine the molecular mechanism of invasion. LPA of invasion-inducible concentrations evoked a transient increase in tyrosine phosphorylation, mainly of I IOto 130-kDa proteins in MMI cells but not in mesothelial cells. These concentrations of LPA were over I0 times higher (I 0 to 25 pM) than those necessary to produce a variety of biological actions, such as tyrosine phosphorylation in fibroblasts, neurite retraction and platelet aggregation. Protein tyrosine phosphorylation and invasion by MMI cells induced by LPA are largely regulated by rho p21, because both were inhibited by Clostridium botulinum C3 exo-enzyme, which is known to specifically inactivate rho p2 I. Invasion of MCL by MM I cells induced by serum and that by B l6FE7 cells induced by LPA were inhibited by genistein or C3 as well. By immunoprecipitation, we detected pl25 focal adhesion kinase (FAK) as a major protein of 110-to 130-kDa tyrosine phosphorylated in response to LPA. Tyrosine phosphorylation of paxillin by LPA was also detected. 0 1996 Wiley-Liss, Inc.