Shizuo Odashima

Effects of glycyrrhizin and glycyrrhetinic acid on growth and melanogenesis in cultured B16 melanoma cells.

The effects of glycyrrhizin (GL) and its aglycone, glycyrrhetinic acid (GA), on the growth and differentiation of mouse melanoma (B16) cells in culture were studied. GA inhibits the growth of B16 melanoma cells, causes morphological alterations and stimulates melanogenesis. GL also resulted in the same changes but only when the concentration was about 20 times more than that needed for GA. When GA was removed after 84 h of treatment, the growth rate recovered slightly, but the doubling time was about twice that of the control. Cytofluorometric analysis showed that the growth inhibition of GA is the result of inhibition of the transfer from G1 to S phase.

Induction of phenotypic reverse transformation by ginsenosides in cultured Morris hepatoma cells

Abstract Ginsenosides (oligoglycosides) extracted from the root of Panax Ginseng, C. A. Meyer, are found to induce the reverse transformation of Morris hepatoma cells (MH 1 C 1 ) in vitro . After 24 subcultures of growth medium containing ginsenoside, MH 1 C 1 cells were morphologically changed. These cells were relatively large and polygonal in shape with abundant fine granular cytoplasm and clear intercellular space, and resembled normal hepatocyte-like cells. These cells reversely transformed, by ginsenosides, showed much less ability to grow in a 0.33% soft agar suspension culture in comparison to control MH 1 C 1 cells, but the doubling time of these cells was 30 hr which is similar to that of original Morris hepatoma cells. In these reversely transformed cells, a remarkable increase in uptake of l- 3 H -ornithine in an arginine deficient medium, an increase of activity of succinate-cytochrome c reductase and a decrease in 5′ -nucleotidase activity were observed.

Protective effect of saikosaponin-d isolated from Bupleurum falcatum L. on CCl4-induced liver injury in the rat

SummaryThe effects of saikosaponin-d extracted from the roots of Bupleurum falcatum L. on carbon tetrachloride-induced hepatic injury were studied in rats. Pretreatment with saikosaponin-d produced a remarkable inhibitory action on acute hepatic injury by CCl4. A significant inhibition of lipid peroxidation induced by an acute dose of CCl4 in the liver of rats pre-treated with saikosaponin-d was also noted.Continuous injection of CCl4 caused liver cirrhosis in rats but the severity of cirrhosis was reduced in rats treated simultaneously with CCl4 and saikosaponin-d.

Different metastatic potentials of ras- and src-transformed BALB/c 3T3 A31 variant cells.

The metastatic phenotype of tumor cells is thought to be induced by an aberrant signaling cascade or cascades that are different from those required for tumorigenicity. Oncogene‐transfected cells with different tumorigenicities and metastatic potentials have been used to identify such pathways and responsible molecules. However, oncogenes that can induce tumorigenicity in recipient cells also frequently induce the metastatic phenotype at the same time. The difficulty in obtaining cell lines that are tumorigenic but not metastatic has hampered such studies. In this report, we transfected the activated c‐Ha‐ras oncogene into BALB/c 3T3 A31 variant cells and found that the transfectants were tumorigenic but they did not form metastatic lung nodules in the experimental metastasis assay. The phenotype was very stable and was maintained during cultivation. On the other hand, the metastatic potentials of either the transfected cells or the original variant cells could be induced by transfection of the v‐src oncogene. The src transfectants formed extensive nodules in lung when injected into the tail veins of congeneric mice. The cell motility of the metastatic src transfectants on Matrigel‐coated dishes was greater than that of the ras transfectants. The src transfectants were also invasive in Matrigel when analyzed on a filter. These variant cells transformed by the ras and src oncogenes will be a useful system for identifying the signaling cascades responsible for the metastatic potential of tumors.

Isolation of a highly metastatic cell line to lymph node in human oral squamous cell carcinoma by orthotopic implantation in nude mice

To isolate a variant cell line with increased lymph node metastasising potential from human oral squamous cell carcinoma (SCC) we performed in vivo selection using orthotopic implantation in nude mice. Human oral SCC cells (HSC-3) were injected orthotopically into the tongue of nude mice. After 3 weeks their cervical lymph nodes were excised and the tumour cells metastasised to the lymph nodes were isolated and injected into the tongue of nude mice again, this procedure was performed three times. The resultant cells, designated as HSC-3-M3, metastasised to cervical lymph nodes in 90% of mice, while parental HSC-3 cells metastasised in only 30% of mice after injection into the tongue. HSC-3 and HSC-3-M3 cell lines which have the same origin but different lymphatic metastatic capacities could be a useful model system for studying mechanisms involved in lymph node metastasis of human oral SCC.

Effect of saikosaponin derivatives upon the immune response against T-dependent and T-independent antigens in mice

Effect of water soluble extracts and purified derivatives from the root of the plant Bupletum falcatum L. upon the immune response of BALB/c mice was investigated using heterologous erythrocytes and bacterial lipopolysaccharide. This Chinese Medicine is famous for its reputed anti-inflammatory, anti-tumor and other biological activities, and has been widely used alone or in a complexed form since ancient times in Oriental Countries. The effect on immune responsiveness of this drug was judged by the measurement of antibody secreting cells causing localized hemolysis in agar gel. The purified derivatives, Saikosaponin (SS) a and d, suppressed anti-SRBC plaque-forming cells (PFC), but the other derivatives, b1 and b2 and c, did not. Moreover, derivatives a and d also enhanced anti-LPS PFC. From experiments establishing minimum effective doses for anti-SRBC PFC in mice, one mg/kg was optimum for BALB/c mice. The effect of the drug on antibody formation against heterologous erythrocytes was nonspecific because treated mice had greatly depressed PFC responses against both sheep and rabbit erythrocytes. The phenotypic effect of this drug as judged by PFC against T-dependent or T-independent antigen was not identified. However, it was suggested that the purified derivatives SSa and d stimulated both T- and B-cells. The possible mechanism of effect against immunocompotent cells is discussed.

Effects of saikosaponin-d on aminonucleoside nephrosis in rats

The effects of saikosaponin-d extracted from the roots of Bupleurum falcatum L. on aminonucleoside nephrosis were studied in rats. Urine protein excretion in rats receiving aminonucleoside alone was significantly elevated on the 2nd day after the last injection of aminonucleoside and reached a peak on the 11th day. Urinary protein on the 11th day was reduced by 48 and 46%, respectively in animals treated with saikosaponin-d from the 2nd and 8th day after the last injection of aminonucleoside. Electron microscopically, the degree of abnormality, e.g. loss or fusion of foot processes, in the glomerular epitherial cells was significantly lower in the rats treated with saikosaponin-d after aminonucleoside injection than in the rats treated with aminonucleoside alone. It is concluded that saikosaponin-d prevents the development of proteinuria induced by aminonucleoside in the rat.

G1 phase-specific suppression of the Cdk2 activity by ginsenoside Rh2 in cultured murine cells.

Ginsenoside Rh2, a plant glycoside with a dammarane skeleton resembling a steroid skeleton as an aglycone, has anticancer potentials in vitro or in vivo. To elucidate the molecular mechanisms of the effects of Rh2, we have examined the Cyclin-dependent kinase-2 (Cdk2) activity in G1 arrested B16 melanoma cells and in S phase-arrested Meth-A sarcoma cells, that have been treated with Rh2. The kinase activity was suppressed in B16 cells but not in Meth-A cells. In addition, Rh2 was found to induce G1 arrest and concomitantly suppress the Cdk2 activity in carcinogen-susceptible BALB/c 3T3 A31-1-1 and A31-1-13 cell lines. Thus, Rh2 has a G1 phase-specific suppressive effect on the Cdk2 activity, supporting further evaluation of Rh2 and its related compounds in cancer chemoprevention studies.

Effects of saikosaponin‐d on enhanced CCl4‐hepatotoxicity by phenobarbitone

The effects of saikosaponin‐d extracted from the roots of Bupleurum falcatum L. on increased toxicity of CCl4 and increased activities of microsomal enzymes induced by phenobarbitone have been examined. Saikosaponin‐d showed protection against the CCl4‐hepatotoxicity enhanced by phenobarbitone. It also inhibited increases in the content of cytochrome P450 and NADPH‐cytochrome c reductase activity, which are induced by the phenobarbitone treatment, but the spectral characteristics of P450 were not altered. The rate of microsomal lipid peroxidation by NADPH and CCl4 was significantly lowered in‐vitro in rats pretreated with phenobarbitone and saikosaponin‐d compared with those pretreated with phenobarbitone alone.

Isolation and characterization of a low metastatic variant from EL-4 mouse T-lymphoma

A variant cell line (EL-4ad) which adhered to a tissue culture dish was isolated from highly metastatic EL-4 murine T-lymphoma. The experimental and spontaneous metastatic ability of EL-4ad was lower than that of the EL-4 parent cell line. The cell surface phenotypes of both cell lines were CD2+3+4−8− 45+TCRαβ+TCRγδ−, but the level of CD2 expression of EL-4ad was much lower than that of EL-4. Furthermore, EL-4ad had higher binding ability to fibronectin and expressed more PNA receptors on the cell surface than EL-4. These differences indicated that either the maturation stage of the less metastatic variant was lower than that of the parent cell line or the activation state of the two cell lines differed. EL-4ad showed higher in vitro invasiveness and adhesiveness to liver cells, and these characters were not consistent with the reduced metastatic ability of this variant. Neuraminidase-releasable cell surface sialic acid levels did not differ significantly between the cell lines. Neither cell line was adhesive to laminin, type IV collagen or reconstituted basement membrane. These metastasis-related properties could not explain the decreased metastatic ability of EL-4ad. On the other hand, EL-4ad was more sensitive to NK activity than EL-4 in vivo, and this was thought to be a major cause of its decreased metastasic ability. The molecules or mechanisms involved in the differentiation or activation of T-cells may be responsible for the sensitivity of tumor cells to NK activity.