Modifying effect of tuna orbital oil rich in docosahexaenoic acid and vitamin D3 on azoxymethane-induced colonic aberrant crypt foci in rats.

The modifying effect of dietary tuna (Thunnus thynnus orientalis) orbital oil rich in docosahexaenoic acid (DHA) and vitamin D3 (VD3) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. Animals were given three weekly subcutaneous injections of AOM (15 mg/kg body weight) to induce ACF. The rats were fed the experimental diet containing 5% tuna orbital oil (low fish oil), 23.5% tuna orbital oil (high fish oil), 5% corn oil (low corn oil) or 23.5% corn oil (high corn oil) for 5 weeks, starting 1 week before the first dose of AOM. Animals were sacrificed 2 weeks after the last AOM injection to count colonic ACF and assay the expression of cyclooxygenase (COX)-1 and -2. High corn oil diet significantly increased the development of ACF, when compared with low corn oil diet (P<0.005). High fish oil diet also increased ACF formation compared with low fish oil diet (P<0.01), but the increase was smaller than high corn oil diet. The frequency of ACF was significantly lower in the rats fed high fish oil diet than high corn oil diet (P<0.02). Moreover, frequency of ACF consisted of 4 or more crypts in rats fed the high fish oil diet was significantly lower than that of rats given high corn oil diet. COX-1 and COX-2 expression did not significantly differ among the groups. These results suggest that fish oil derived from tuna, which contains high amounts of DHA and VD3, suppresses the formation and growth of ACF without affecting COX-1 and COX-2 expression, and may have a preventive effect on colon carcinogenesis.

Cell membrane changes of structure and function in protein kinase inhibitor-induced polyploid cells.

Exogenous cyclic AMP has been thought to be a chemical without marked pharmacological effect until now, as it is not capable of penetrating the cell membrane in most eucaryotic cells. The present study obtained results consistent with those of most previous studies, showing that exogenous cyclic AMP itself did not interfere with the cell cycle even at the high dose of 100 microM. However, it was found that K252a, a potent inhibitor of protein kinases including protein kinase C, induced DNA re-replication, i.e. DNA synthesis at a elevated DNA ploidy in cells that had not undergone cytokinesis (leading to polyploidization), and that exogenous cyclic AMP markedly potentiated the K252a-induced polyploidization at a very low dose similar to the effective dose of membrane-permeable cyclic AMP analogue dibutyryl cyclic AMP. These findings suggested that the cell membrane changed during the formation of polyploid cells. This supposition was confirmed by scanning electron microscopy to observe structural changes and by determination of cellular attachment to investigate functional changes.

Citrus auraptene exerts dose-dependent chemopreventive activity in rat large bowel tumorigenesis: the inhibition correlates with suppression of cell proliferation and lipid peroxidation and with induction of phase II drug-metabolizing enzymes.

In our previous short-term experiment, Citrus auraptene inhibited the development of azoxymethane (AOM)-induced aberrant crypt foci, which are precursor lesions for colorectal carcinoma. In the present study, the possible inhibitory effect of dietary administration of auraptene was investigated using an animal colon carcinogenesis model with a colon carcinogen AOM. Male F344 rats were given s.c. injections of AOM (15 mg/kg body weight) once a week for 3 weeks to induce colon neoplasms. They also received diets containing 100 or 500 ppm auraptene for 4 weeks in groups of "initiation" feeding, starting 1 week before the first dosing of AOM. The diets containing auraptene were also given to rats for 38 weeks in groups of "postinitiation" feeding. At the termination of the study (38 weeks), dietary administration of auraptene caused dose-dependent inhibition in AOM-induced large bowel carcinogenesis. Auraptene feeding during the initiation phase reduced the incidence of colon adenocarcinoma by 49% at 100 ppm (P = 0.099) and 65% at 500 ppm (P = 0.0075). Auraptene administration during the postinitiation phase inhibited the incidence of colon adenocarcinoma by 58% at 100 ppm (P = 0.021) and 65% at 500 ppm (P = 0.0075). Also, the multiplicity of colon carcinoma was significantly reduced by initiation feeding at a dose level of 500 ppm (P < 0.01) and postinitiation feeding at a level of 100 and 500 ppm (P < 0.05 and P < 0.01, respectively). Feeding of auraptene suppressed the expression of cell proliferation biomarkers (ornithine decarboxylase activity and polyamine content) in the colonic mucosa and reduced the production of aldehydic lipid peroxidation [malondialdehyde and 4-hydroxy-2(E)-nonenal]. In addition, auraptene increased the activities of Phase II drug-metabolizing enzymes (glutathione S-transferase and quinone reductase) in the liver and colon. These findings suggest that the inhibitory effects of auraptene on AOM-induced colon tumorigenesis at the initiation level might be associated, in part, with increased activity of Phase II enzymes, and those at the postinitiation stage might be related to suppression of cell proliferation and lipid peroxidation in the colonic mucosa.

Mechanism of action of ginsenoside Rh2: uptake and metabolism of ginsenoside Rh2 by cultured B16 melanoma cells.

The uptake and metabolism of ginsenoside Rh2 (Rh2) by B16 melanoma cells were studied. In a medium containing 2% fetal calf serum, the uptake of Rh2 reached a maximum of 3 nmol/10(6) cells at 3-6 h after Rh2 (12.5 microM) was added, but gradually decreased to 0.8 nmol/10(6) cells. In these cells, protopanaxadiol (PPD), which is an aglycon of Rh2, increased inversely with the decrease in Rh2 as a result of deglycosylation by the cells. When PPD (8 microM) was added to the medium, the uptake reached a plateau of 2.4 nmol/10(6) cells, within 0.5 h. The association constant of Rh2 (1.74 +/- 1.08 x 10(6) M-1) for bovine serum albumin (BSA) was significantly higher than that of PPD (9.90 +/- 1.10 x 10(4) M-1). In a serum-free medium, both Rh2 and PPD were incorporated within 1.5 h. The uptake rate constant of Rh2 (1.20 +/- 0.20 h-1) was not significantly different from that of PPD (1.02 +/- 0.15 h-1), but the release rate constant of PPD (2.12 +/- 0.38 h-1) was significantly lower than that of Rh2 (3.03 +/- 0.57 h-1). These differences in affinity for BSA and the release rate constants were thought to be the cause of the difference in uptake kinetics between these drugs. The effects of Rh2 and PPD on the cells were identical, and there was no difference in the lag periods before the appearance of their effects, despite their differing rates of uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

[Induction of phenotypic reverse transformation by plant glycosides in cultured cancer cells].

Crude ginsenosides extracted from the root of Panax ginseng C.A. Meyer inhibited the growth and colony forming ability of Morris hepatoma cells in soft agar suspension culture, and stimulate the serum protein synthesis of these cells, thus converting the cell characteristics both functionally and morphologically to those resembling original normal liver cells. We have called such a phenomenon "reverse transformation" or "redifferentiation" which can be regarded as decarcinogenesis. In this report, the results of our recent investigations are presented with particular reference to reverse transformation of B16 melanoma cells induced by ginsenoside Rh2 isolated from the methanol extract of crude ginseng saponin fraction and action mechanisms of ginsenoside Rh2 are also discussed.

An investigation of the immune suppression induced by some saikosaponin derivatives.

The relationship between lymphocyte activation by purified plant glycoside and the suppression of antibodies against sheep erythrocytes was investigated by an adoptive cell transfer system. Glycosides are one of the main components of Shôsaikotô--famous hematopoietic remedies of oriental medicine. In earlier reports, BALB/c mice treated with these drugs, suppressed the plaque-forming cell (PFC) response to T-dependent antigens. In contrast, the PFC response was enhanced for T-independent antigens. In this communication, an experiment was designed to determine the nature of the suppressive factor(s) produced in treated mice. Mice were orally or intraperitoneally administered 1 mg/kg of five types of purified SS derivatives every other day on five consecutive occasions. Spleen cells and serum were prepared from SS-treated mice and were then passively transferred to recipient mice. On the spleen cells (and not any of the sera) from drug-treated hosts effectively suppressed the PFC in recipients. The spleen cells of treated mice were further divided into adherent and non-adherent cells; each cell type was transferred into recipient animals. The observed suppressive activity was clearly detected in the non-adherent cell population. A subsequent analysis of the lymphocyte subpopulation revealed that Lyt.1.1 negative and Lyt.2.2 positive cells were responsible for this suppression. It was concluded that some SS derivatives elevate the activities of the major immunocyte population, with suppressor cell activity predominating.

[An approach to prolongation of survival rate in tumor bearing mice using 5-fluorouracil in combination with various kinds of herb medicine].

The effect of 5-fluorouracil (5-FU) in combination with herb medicine on the prolongation of survival rate in tumor bearing mice was studied. 5-FU directly inhibited the growth of cancer cells in vitro. However, the herb medicines used in this experiment showed neither an inhibitory effect on the growth of cancer cells nor prevention of inhibitory effect of 5-FU on the growth of cancer cells. 5-FU in combination with various kinds of herb medicine prolonged the survival time of tumor bearing mice. 5-FU in combination with shosaiko-toh was most effective; its increase of life span (ILS) was by 56%. However, mitomycin C in combination with shosaiko-toh was not associated with the prolongation of the survival rate of tumor bearing mice. The long-term administration of 5-FU to rats resulted in decrease in body weight, food intake, and white blood cell count, and these symptoms were improved when 5-FU was used in combination with shosaiko-toh. However, the increase in A/G ratio due to a decrease in the serum globulin fraction and marked atrophy of the thymus were not improved even when shosaiko-toh was combined with 5-FU.

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

The 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.

An organ culture method for glandular stomach of new born rat.

An organ culture system has been developed for he long-term maintenance of the glandular stomach of new born Wistar rats. The explants from glandular stomach were cultured on Millipore filter in an organ culture dish with various media consisting of sera, buffers and additives under different gas phases of oxygen, carbon dioxide and nitrogen at 37 or 24 degrees C. The culture condition was evaluated by histological examination of explants and was divided into Grade (+++), (++), (+) and (-) depending upon the morphology and growth of the mucosal epithelium. D-MEM plus fetal calf serum (20%) supplemented with hydrocortisone (10 microgram/ml) or dexamethasone (5 microgram/ml) buffered with sodium bicarbonate and Hepes under a gas phase of O2 (95%)+CO2(5%) or (2(45%)+CO2(5%)+N2(50%) at 37 degrees C showed the best result. Other supplements tested, such as insulin, ascorbic acid and ferrous sulfate were ineffective. Addition of hydrocortisone or dexamethasone was similarly effective in serum-free media. Isologous rat serum had no advantage over fetal calf serum. Transplantation and autoradiography revealed the viability of the explants up to 35 days in vitro.

Ultrastructural studies of Morris hepatoma cells reversely transformed by ginsenosides.

Ginsenosides, which were extracted from Panax ginseng C.A. Meyer, induced well the development of subcellular organelles in cultured Morris hepatoma cells (MH1C1).

Antitumor effect of pyridine N-oxides having 1-(2-chloroethyl)-1-nitrosoureidoalkyl and 1-methyl-1-nitrosoureidoalkyl groups.

Pyridine N-oxides having 1-(2-chloroethyl)-1-nitrosoureidoalkyl or 1-methyl-1-nitrosoureidoalkyl groups were evaluated for their antitumor activity against AH13 hepatoma and L1210 leukemia. Among them, 1-(2-chloroethyl)-1-nitroso-3-(2-pyridylmethyl)urea N-oxide (1), its tosylate (2), 1-(2-chloroethyl)-1-nitroso-3-(2-pyridylethyl)urea N-oxide (4), and 1-(2-chloroethyl)-1-nitroso-3-(3-pyridylmethyl)urea N-oxide (6) were highly active against both tumors in ip-ip system. These compounds were also active in ip-iv and ip-po systems of L1210. On the other hand, pyridine N-oxides having 1-methyl-1-nitrosoureidoalkyl group were all inactive against AH13 and weakly active against L1210. Effect on blood cells in Donryu rats bearing EDEN-5 erythroblastic leukemia cells was tested with these 1-(2-chloroethyl)-1-nitrosoureidoalkylureas. These compounds caused leucopenia and compound (4) was only slightly effective against EDEN-5.