Kiyoko Shinkai

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.

Multiple forms of human adenosine deaminase: I. Purification and characterization of two molecular species

Abstract 1. 1. Gel filtration of human tissue extracts with Sephadex G-200 revealed the existence of two molecular species of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) differing markedly in their molecular sizes. 2. 2. Tissue-specific distributions of these species were also revealed. 3. 3. The larger and smaller enzymes were purified more than 1000-fold, nearly to homogeneity, from the lung and the stomach, respectively. 4. 4. Determination of molecular weights of the enzymes using a calibrated Sephadex G-200 column resulted in the values of 230 000 and 47 000, which were in reasonable accordance with the values obtained by sucrose density gradient centrifugation. 5. 5. No significant differences were observed enzymically or immunologically between the properties of the two enzymes except for their heat-stabilities and molecular activities. 6. 6. A reversible conversion of the larger deaminase to the smaller was demonstrated when the former was treated with guanidine. 7. 7. The smaller enzyme was apparently converted to the larger after incubating the former enzyme sample with crude lung supernatant. 8. 8. The possible structural relationship between the two species of human adenosine deaminase is discussed.

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.

Inhibition by ginsenoside Rg3 of bombesin-enhanced peritoneal metastasis of intestinal adenocarcinomas induced by azoxymethane in Wistar rats

The effects of concomitant use of bombesin and ginsenoside Rg3 on the incidence of peritoneal metastasis of intestinal adenocarcinomas induced by azoxymethane were investigated in male inbred Wistar rats. From the start of the experiment, rats were given weekly s.c. injections of azoxymethane (7.4mg/kg body weight) for 10 weeks and s.c. injection of bombesin (40μg/kg body weight) every other day, and from week 20, s.c. injections of ginsenoside Rg3 (2.5 or 5.0mg/kg body weight) every other day until the end of the experiment in week 45. Bombesin significantly increased the incidence of intestinal tumors and cancer metastasis to the peritoneum in week 45. It also significantly increased the labeling index of intestinal cancers. Although administration of a higher dose of ginsenoside Rg3 with bombesin had little or no effect on the enhancement of intestinal carcinogenesis by bombesin, the location, histologic type, depth of involvement, infiltrating growth pattern, labeling and apoptotic indices and tumor vascularity of intestinal cancers, it significantly decreased the incidence of cancer metastasis. These findings indicate that ginsenoside Rg3 inhibits cancer metastasis through activities that do not affect the growth or vascularity of intestinal cancers.

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.

Multiple forms of human adenosine deaminase. II. Isolation and properties of a conversion factor from human lung

Abstract 1. 1. A conversion factor, which can convert the small form ( E S ; 47 000 mol. wt) of human adenosine deaminase (EC 3.5.4.4) to the large form ( E L ; 230 000 mol. wt), was purified about 600-fold from normal human lung by (NH4)2SO4 fractionation, Sephadex and DEAE-cellulose chromatography, and preparative acrylamide electrophoresis. 2. 2. The purified preparation, heat labile and free from the deaminase activity, exhibited upon disc electrophoresis a single protein band associated with the converting activity and also showed a maximum absorption at 280 nm. Its molecular weight was found to be 139 000 when judged by gel filtration with Sephadex G-200. 3. 3. The factor did not require sulfhydryl compounds nor bivalent metal ions for exerting its activity. 4. 4. Results of quantitative analysis of the rate of formation of E L from known amounts of conversion factor and E S , immunochemical characterizations of the two forms of the enzyme in relation to conversion factor by using antiserum against the factor, and polyacrylamide electrophoresis of E L and the factor in the presence of sodium dodecyl sulfate strongly suggest that E L is a complex of E S and conversion factor. 5. 5. No detectable activity of conversion factor was demonstrated in an extract of lung cancer tissue.

INVOLVEMENT OF SMALL GTPASES RHO AND RAC IN THE INVASION OF RAT ASCITES HEPATOMA CELLS

Lysophosphatidic acid (LPA) triggers the invasion of a mesothelial cell monolayer by rat ascites hepatoma (MM1) cells. LPA also induces rapid morphological changes of MM1 cells, cell surface blebbing and pseudopodia formation. Pseudopodia formation is tightly correlated with cellular invasiveness. Clostridium Botulinum C3 exoenzyme and genistein abrogated the formation of blebs and pseudopodia together with the inhibition of invasion, indicating that GTPase Rho and certain tyrosine kinases are involved in both processes. MM1 cells expressing constitutively active Rho exhibited the invasion and the formation of blebs and pseudopodia in the absence of LPA. In contrast, MM1 cells expressing constitutively active Rac were not invasive in the absence of LPA, but were invasive in the presence of LPA. Their morphological response to LPA was almost the same as that of parental MM1 cells. Expression of dominant negative Rac suppressed the invasiveness to approximately 3% of that of parental MM1 cells, together with the inhibition of pseudopodia formation. Thus, Rho and Rac are cooperatively involved in both the invasion and the related morphological changes of MM1 cells. Rho activation is sufficient both for the induction of invasion and the morphological changes leading to the invasion, whereas Rac activation is necessary but not sufficient by itself. We propose that Rho activation is not mediated by Rac but the cooperation of both GTPases is essential to trigger the invasive behavior of MM1 cells.

Ng-nitro-L-arginine methyl ester inhibits bone metastasis after modified intracardiac injection of human breast cancer cells in a nude mouse model

We investigated the effects of NG‐nitro‐L‐arginine‐methyI ester (L‐NAME), a nitric oxide synthase (NOS) inhibitor, on hone metastasis of human breast cancer, MDA‐231 cells. Tumor cells (2 × 105 cells in 0.2 ml of phosphate‐buffered saline; PBS) were injected through the diaphragm into the left ventricle of the heart of laparotomized nude mice (male 5‐week‐old ICR‐nu/nu). L‐NAME (2 mg/ mouse/injection in 0.1 ml of PBS) was given intraperitoneally to mice 6 h and 3 h before and immediately, 3 h, 6 h, 18 h and 21 h after the intraeardlac injection of tumor cells. As a control, 0.1 ml of PBS was injected instead of L‐NAME. The effect of NG‐nitro‐D‐arginine‐methyl ester CD‐NAME; 2 mg/mouse/injection), an inactive analogue of L‐NAME, was also investigated to evaluate the specificity of L‐NAME action. Radiographical examination 31 days after the tumor‐cell injection showed that the incidence and number of osteolytic bone metastases and the number of bones with metastasis in L‐NAME‐treated mice were significantly reduced compared with those in PBS‐treated mice (P<0,05). The differences between PBS‐treated and D‐NAME‐treated mice were not significant. Our findings suggest that specific and appropriate NOS inhibitors may represent a new pharmacological approach to therapy for cancer patients at risk of developing osteolytic bone metastases.

In vitro Invasion of Endothelial Cell Monolayer by Rat Ascites Hepatoma Cells

To study the tissue preference of invasion, we developed an assay system for the invasion of endothelial cells as a modification of the previously established assay of tumor cell invasion of meso‐thelial cells. Rat ascites hepatoma cells (AH 130) that had been seeded on a monolayer of cultured endothelial cells penetrated and formed tumor cell colonies under the monolayer. The penetration was time‐dependent and the number of penetrated tumor cells and colonies was proportional to the number of tumor cells seeded. Comparison of the in vitro tumor cell invasion of endothelial cell monolayer with that of cultured mesothelial cell layer showed that a clone from the tumor cells (CI‐30) which was highly penetrative into the mesothelial cell layer had only a limited ability to penetrate the endothelial cell layer.