Mutsuko Mukai

Inhibition of in vitro Tumor Cell Invasion by Ginsenoside Rg3

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.

Y-27632, an inhibitor of Rho-associated protein kinase, suppresses tumor cell invasion via regulation of focal adhesion and focal adhesion kinase

Migration of rat ascites hepatoma (MM1) cells, invasion and phagokinetic movement were induced by the combination of lysophosphatidic acid (LPA) and fibronectin (FN). Induction of migratory activity was tightly correlated with morphological change of MM1 cells from spherical or polygonal‐shaped cells to fusiform‐shaped ones with pseudopodia. MM1 cells were mobile in a fusiform shape, whereas those of a spherical or polygonal shape were not. A small GTPase Rho and one of its downstream effectors ROCK (Rho‐associated coiled‐coil forming protein kinase), play essential roles in these processes, as evidenced by suppression of migration and morphological change of MM1 cells by Clostridium botulinum C3 exoenzyme, an inhibitor of Rho, or by Y‐27632, an inhibitor of ROCK. Y‐27632 also suppressed the formation of fusiform‐shaped pseudopodia‐carrying MM1 cells that was induced by stimulation with the combination of LPA and FN. LPA and FN also evoked the formation of focal adhesions and actin bundles, and tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. The inhibitory effect of Y‐27632 on LPA‐induced migration and morphological change of MM1 cells was considered to be mediated, at least in part, by impaired formation of focal adhesions and actin bundles. Y‐27632 suppressed LPA‐induced tyrosine phosphorylation of FAK and paxillin, suggesting that ROCK regulates these molecules and Y‐27632 inhibits cellular migration and morphological change, at least in part, through this regulation.

Intravenous anesthetic, propofol inhibits invasion of cancer cells.

Intravenous anesthetic, propofol (2,6-diisopropylphenol), is extensively used for general anesthesia without knowing the effects on cancer. We found here that clinically relevant concentrations of propofol (1-5 microg/ml) decreased the invasion ability of human cancer cells (HeLa, HT1080, HOS and RPMI-7951). In the HeLa cells treated with propofol, formation of actin stress fibers as well as focal adhesion were inhibited, and propofol had little effect on the invasion ability of the HeLa cells with active Rho A (Val(14)-Rho A). In addition, continuous infusion of propofol inhibited pulmonary metastasis of murine osteosarcoma (LM 8) cells in mice. These results suggest that propofol inhibits the invasion ability of cancer cells by modulating Rho A and this agent might be an ideal anesthetic for cancer surgery.

Inhibition of tumor invasion and metastasis by a novel lysophosphatidic acid (cyclic LPA).

Fetal calf serum (FCS) and 1‐oleoyl lysophosphatidic acid (LPA) were previously found to be potent inducers of invasion (transcellular migration) in an in vitro system. A novel LPA, composed of cyclic phosphate and cyclopropane‐containing hexadecanoic acid (PHYLPA), first isolated from myxoamoebae of Physarum polycephalum, and its synthetic derivatives (cLPA) were tested for their ability to inhibit tumor cell invasion and metastasis. Amoung these, Pal‐cLPA, which has a palmitoyl moiety, was most potent in inhibiting invasion, with 93.8% inhibition at the concentration of 25 μM. Invasion in vitro by mouse melanoma cells (B16), human pancreatic adenocarcinoma cells (PSN‐1), human lung cancer cells (OC‐10) and human fibrosarcoma cells (HT‐1080) was also inhibited by Pal‐cLPA. The stimulation of MM1 cells with LPA triggered F‐actin formation, which was impaired by the addition of Pal‐cLPA at invasion‐inhibitory concentration. Pal‐cLPA induced a rapid increase in adenosine 3′,5′‐cyclic monophosphate (cAMP) concentration in MM1 cells. The addition of dibutyryl cAMP significantly abrogated LPA‐induced invasion by MM1 cells and actin polymerization in the cells. The inhibition of MM1 cell invasion by Pal‐cLPA may be ascribed to an increased level of cAMP. Pal‐cLPA also suppressed invasion in vitro by MM1 cells induced by FCS dose dependently, without affecting proliferation. It also suppressed the pulmonary metastasis of B16 mouse melanoma cells injected into the tail vein of C57BL/6 mice. Thus, Pal‐cLPA is effective in inhibiting invasion and metastasis of a variety of tumor cells. Int. J. Cancer 81:918–922, 1999.

rho-Mediated protein tyrosine phosphorylation in lysophosphatidic-acid-induced tumor-cell invasion.

Rat ascites hepatoma cells (MMI) invade a mesothelial cell monolayer in vitro in assay medium containing serum, but not in serum‐free medium. Serum could be completely replaced by I‐oleoyl lysophosphatidic acid (LPA) in inducing invasion. LPA‐induced 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 110‐ to 130‐kDa proteins in MMI cells but not in mesothelial cells. These concentrations of LPA were over 10 times higher (10 to 25 μM) 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 p21. Invasion of MCL by MMI cells induced by serum and that by B16FE7 cells induced by LPA were inhibited by genistein or C3 as well. By immunoprecipitation, we detected p125 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.

Superoxide radical potentiates invasive capacity of rat ascites hepatoma cells in vitro

Effect of superoxide radical (O2-) produced extracellularly by hypoxanthine (HX) and xanthine oxidase (XO) on invasive capacity of rat ascites hepatoma cells was studied. Invasive capacity was estimated in vitro by counting the number of tumor cell colonies penetrated underneath cultured mesothelial cell monolayer. When the tumor cells had been treated with non-toxic doses of HX and XO, the formation of penetrated colonies increased with increasing concentrations of XO. This increment was completely inhibited by scavengers of active oxygen radicals, superoxide dismutase (SOD) in combination with catalase (CAT) added simultaneously at the time of HX-XO treatment.

Biological functions of a novel lipid mediator, cyclic phosphatidic acid.

A novel bioactive lipid, cyclic phosphatidic acid (cPA), was isolated originally from myxoamoebae of a true slime mold, Physarum polycephalum, and has now been detected in a wide range of organisms from slime molds to humans. It has a cyclic phosphate at the sn-2 and sn-3 positions of the glycerol carbons, and this structure is absolutely necessary for its activities. This substance shows specific biological functions, including antimitogenic regulation of the cell cycle, regulation of actin stress fiber formation and rearrangement, inhibition of cancer cell invasion and metastasis, regulation of differentiation and viability of neuronal cells, and mobilization of intracellular calcium. Although the structure of cPA is similar to that of lysophosphatidic acid (LPA), its biological activities are apparently distinct from those of LPA. In the present review, we focus mainly on the enzymatic formation of cPA, the antimitogenic regulation of the cell cycle, the inhibition of cancer cell invasion and metastasis, and the neurotrophic effect of cPA.

Expression of S100A6 and S100A4 in Matched Samples of Human Colorectal Mucosa, Primary Colorectal Adenocarcinomas and Liver Metastases

Objective: S100A6 and S100A4, two of S100 protein family, have been suggested to be associated with cancer tumorigenesis and metastasis. The aim of this study was to evaluate the expression levels of S100A6 and S100A4 in matched samples of primary human colorectal adenocarcinomas (T), adjacent normal colorectal mucosa (N) and liver metastases (M). This gave us the advantage of directly comparing levels of S100A6 and S100A4 expression within the same genetic background. Methods: In matched samples of N, T and M from 10 colorectal adenocarcinoma patients, expressions of S100A6 and S100A4 were studied by Western blot and immunohistochemical analyses using specific antibodies against each protein. Results: The expression levels of S100A6 were significantly higher in T than in N (p < 0.05), while those of S100A4 showed no difference between T and N. There were no significant differences in the expression levels of S100A6 or S100A4 between M and T. Similar results were obtained by immunohistochemical analysis. Moreover, S100A6 was stained more intensely in invading fronts than in central portions of both T and M. Conclusions: The observed differential expression of S100A6 and S100A4 suggests that S100A6, rather than S100A4, is associated with human colorectal adenocarcinoma tumorigenesis and invasion/metastasis.

Hepatoma cell migration through a mesothelial cell monolayer is inhibited by cyclic AMP-elevating agents via a Rho-dependent pathway

1‐Oleoyl lysophosphatidic acid (LPA) induces transmonolayer migration (in vitro invasion) of rat ascites hepatoma MM1 cells and their morphological changes leading to the migration. We have previously shown that an LPA analog, palmitoyl cyclic phosphatidic acid (Pal‐cPA), suppresses transmonolayer migration of MM1 cells by rapidly increasing the intracellular cyclic AMP (cAMP) concentration. We report here that various cAMP‐elevating agents, including dibutyryl cAMP, forskolin, cholera toxin and 3‐isobutyl‐1‐methylxanthine, consistently inhibited LPA‐induced transmonolayer migration of MM1 cells. Moreover, pull‐down assays for GTP‐bound, active RhoA demonstrated that the blockage by cAMP‐elevating agents of morphological changes leading to the migration was probably mediated through inhibiting RhoA activation.

Suppression of the invasive capacity of rat ascites hepatoma cells by knockdown of Slingshot or LIM kinase.

Actin cytoskeletal reorganization is essential for tumor cell migration, adhesion, and invasion. Cofilin and actin-depolymerizing factor (ADF) act as key regulators of actin cytoskeletal dynamics by stimulating depolymerization and severing of actin filaments. Cofilin/ADF are inactivated by phosphorylation of Ser-3 by LIM kinase-1 (LIMK1) and reactivated by dephosphorylation by Slingshot-1 (SSH1) and -2 (SSH2) protein phosphatases. In this study, we examined the roles of cofilin/ADF, LIMK1, and SSH1/SSH2 in tumor cell invasion, using an in vitro transcellular migration assay. In this assay, rat ascites hepatoma (MM1) cells were overlaid on a primary-cultured rat mesothelial cell monolayer and the number of cell foci that transmigrated underneath the monolayer in the presence of lysophosphatidic acid (LPA) was counted. The knockdown of cofilin/ADF, LIMK1, or SSH1/SSH2 expression by small interfering RNAs (siRNAs) significantly decreased the LPA-induced transcellular migration of MM1 cells and their motility in two-dimensional culture. Knockdown of LIMK1 also suppressed fibronectin-mediated cell attachment and focal adhesion formation. Our results suggest that both LIMK1-mediated phosphorylation and SSH1/SSH2-mediated dephosphorylation of cofilin/ADF are critical for the migration and invasion of tumor cells and that LIMK1 is involved in the transcellular migration of tumor cells by enhancing both adhesion and motility of the cells.