Cellular thiol status-dependent inhibition of tumor cell growth via modulation of p27(kip1) translocation and retinoblastoma protein phosphorylation by 1'-acetoxychavicol acetate.

1'-Acetoxychavicol acetate (ACA) has been shown to inhibit tumor cell growth, but there is limited information on its effects on cell signaling and the cell cycle control pathway. In this study, we sought to determine how ACA alters cell cycle and its related control factors in its growth inhibitory effect in Ehrlich ascites tumor cells (EATC). ACA caused an accumulation of cells in the G1 phase and an inhibition of DNA synthesis, which were reversed by supplementation with N-acetylcysteine (NAC) or glutathione ethyl ester (GEE). Furthermore, ACA decreased hyperphosphorylated Rb levels and increased hypophosphorylated Rb levels. NAC and GEE also abolished the decease in Rb phosphorylation by ACA. As Rb phosphorylation is regulated by G1 cyclin dependent kinase and CDK inhibitor p27(kip1), which is an important regulator of the mammalian cell cycle, we estimated the amount of p27(kip1) levels by western blotting. Treatment with ACA had virtually no effect on the amount of p27(kip1) levels, but caused a decrease in phosphorylated p27(kip1) and an increase in unphosphorylated p27(kip1) as well as an increase in the nuclear localization of p27(kip1). These events were abolished in the presence of NAC or GEE. These results suggest that in EATC, cell growth inhibition elicited by ACA involves decreases in Rb and p27(kip1) phosphorylation and an increase in nuclear localization of p27(kip1), and these events are dependent on the cellular thiol status.

Cancer induction by an organic arsenic compound, dimethylarsinic acid (cacodylic acid), in F344/DuCrj rats after pretreatment with five carcinogens.

Arsenic (As) is environmentally ubiquitous and an epidemiologically significant chemical related to certain human cancers. Dimethylarsinic acid (cacodylic acid; DMA) is one of the major methylated metabolites of ingested arsenicals in most mammals. To evaluate the effects of DMA on chemical carcinogenesis, we conducted a multiorgan bioassay in rats given various doses of DMA. One-hundred twenty-four male F344/DuCrj rats were divided randomly into 7 groups (20 rats each for groups 1-5; 12 rats each for groups 6 and 7). To initiate multiple organs and tissues, animals in groups 1-5 were treated sequentially with diethylnitrosamine (100 mg/kg body weight, i.p., single dose at the commencement) and N-methyl-N-nitrosourea (20 mg/kg body weight, i.p., 4 times, on days 5, 8, 11, and 14). Thereafter, rats received 1,2-dimethylhydrazine (40 mg/kg body weight, s.c., 4 times, on days 18, 22, 26, and 30). During the same period, the animals were sequentially administered N-butyl-N-(4-hydroxybutyl)nitrosamine (0.05% in the drinking water, during weeks 1 and 2) and N-bis(2-hydroxypropyl)nitrosamine (0.1% in the drinking water, during weeks 3 and 4; DMBDD treatment). After a 2-week interval, groups 2-5 were given 50, 100, 200, or 400 ppm DMA, respectively, in the drinking water. Groups 6 and 7, which were not given DMBDD treatment, received 100 and 400 ppm DMA during weeks 6-30. All rats were killed at the end of week 30. In the initiated groups (groups 1-5), DMA significantly enhanced the tumor induction in the urinary bladder, kidney, liver, and thyroid gland, with respective incidences in group 5 (400 ppm DMA) being 80, 65, 65, and 45%. Induction of preneoplastic lesions (glutathione S-transferase placental form-positive foci in the liver and atypical tubules in the kidney) was also significantly increased in DMA-treated groups. Ornithine decarboxylase activity in the kidneys of rats treated with 100 ppm DMA was significantly increased compared with control values (P < 0.001). In conclusion, DMA is acting as a promoter of urinary bladder, kidney, liver, and thyroid gland carcinogenesis in rats, and we speculate that this may be related to cancer induction by As in humans.

Control by hyperthermia of ornithine decarboxylase in Ehrlich ascites tumor cells.

The effect of hyperthermia on the activity and the messenger RNA levels of ornithine decarboxylase (ODC), which has a rapid rate of turnover in cultured cells, was studied in Ehrlich ascites tumor cells. When the cells were incubated at 42 degrees C, elevation of ODC activity by a change of the medium was prevented. Total RNA was isolated from cells treated at 37 degrees C or 42 degrees C, and the relative abundance of the ODC mRNA was measured by Northern blot analysis. These levels in heat-treated cells were comparable to those in control cells. Inhibition by hyperthermia was reversible. The recovery was suppressed by cycloheximide but not by actinomycin D. In hyperthermic-treated cells, the biological half-life of ODC was 14 min, which was the same time as for cells cultured at 37 degrees C. These results suggest that hyperthermic treatment of Ehrlich ascites tumor cells suppressed ODC induction during translation, not during transcription or after translation.

Posttranslational regulation of spermidine/spermine N1-acetyltransferase with stress.

The effect of stress on the activity and level of mRNA of spermidine/spermine N1-acetyltransferase (SAT), a polyamine degradation rate-limiting enzyme, was studied in Ehrlich ascites tumor cells. When the cells were treated with sodium arsenite or ethanol for 1 h at 37 degrees C, the activity of SAT increased time- and dose-dependently. Total RNA was isolated from cells treated with stress, and the relative abundance of the SAT mRNA was measured by Northern blot analysis. The amount was comparable to those in control cells. In stress-treated cells, the biological half-life of the enzyme was 48-55 min, but 27-30 min in control cells. When cells were treated with arsenite in the presence of cycloheximide, enzyme activity did not increase. In those cells, half-life of the enzyme was shorter than in the cells treated with arsenite alone. This suggests that stress-treatment of cells enhanced SAT activity posttranslationally and that some factor(s) which was synthesized de novo during the treatment of arsenite is involved in the stabilization of the enzyme.

Polyamine metabolism in the rat liver after orthotopic liver transplantation.

We examined the polyamine metabolism in liver transplanted after cold ischemia and effects of putrescine administration on liver injury, liver regeneration, and survival rate after orthotopic liver transplantation in the rat. Male Wistar rats were used as donors and recipients. Grafts were stored in Euro-Collins solution for 6 h at 4 degrees C. Orthotopic liver transplantation was performed by the three cuff technique. The activities of aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase elevated and peaked 4 h after liver transplantation. Hepatic ornithine decarboxylase and spermidine/spermine N1-acetyltransferase activities were also elevated and peaked 8 h after the operation. In agreement with the increases in ornithine decarboxylase and spermidine/spermine N1-acetyltransferase activities, the putrescine content increased and spermidine content decreased in the transplanted liver. Putrescine administrated intraperitoneally improved the survival rate, decreased serum transaminase level and increased the [3H]thymidine incorporation into the liver DNA. These findings suggest that both biosynthetic and biodegradative pathways are stimulated in liver transplantation, resulting in the increase in the formation of putrescine from ornithine and from spermidine, and that putrescine administration improve the survival rate by protecting the damaged graft after cold ischemia and reperfusion and by stimulating liver regeneration.

Effect of sucrose monostearate, an emulsifier, on polyamine metabolism and phosphatidylinositol turnover in Ehrlich ascites tumor cells.

Sucrose esters of fatty acids have antitumor activity. We studied the effect of sucrose monostearate (SS), an emulsifier, on polyamine metabolism and phosphatidylinositol turnover in Ehrlich ascites tumor cells. The activity of ornithine decarboxylase (ODC) was increased in the cells by changing the medium. This increase in the activity was inhibited by adding sucrose stearate, but not sucrose or stearate to the medium. The activity of spermidine/spermine N1-acetyltransferase (SAT), a rate-limiting enzyme of polyamine biodegradation, was enhanced with the addition of SS in a time- and dose-dependent manner. The elevation of SAT activity was completely prevented when cycloheximide was added to the culture simultaneously. In in vitro studies, SS at various concentrations up to 1 mM hardly affected the activities of ODC or SAT. The incorporation of [3H]inositol into both fractions of inositolphospholipid and inositol phosphates was inhibited by SS. These results suggest that the perturbation of polyamine metabolism and phosphatidylinositol turnover is involved in the mechanism of antitumor activity of SS in Ehrlich ascites tumor cells.

Altered production of the active oxygen species is involved in enhanced cytotoxic action of acylated derivatives of ascorbate to tumor cells.

Our previous study shows that 6-O-acyl derivatives of L-ascorbic acid inhibits more markedly cell growth of mouse Ehrlich carcinoma than ascorbic acid. The present study shows that 6-O-palmitoyl ascorbic acid but not ascorbic acid prolongs the lifespan of mice into which tumors such as Meth A fibrosarcoma, MM46 mammary carcinoma, Ehrlich carcinoma and sarcoma 180 are implanted. The potentiated cytotoxicity of 6-O-palmitoyl ascorbic acid is not due to an increase in duration time of the cytotoxic action, because 6-O-palmitoyl ascorbic acid is gradually inactivated during contact with tumor cells and exhibits a similar action time curve to that of ascorbic acid as shown by clonal growth assay. Cytotoxicity of 6-O-palmitoyl ascorbic acid is markedly diminished by combined addition of catalase and superoxide dismutase (SOD), as shown by dye exclusion assay, whereas the cytotoxicity was slightly reduced by either enzyme alone but not by the specifically inactivated or heat-denatured enzymes. In contrast, cytotoxicity of ascorbic acid is abolished by catalyse but not SOD. Autooxidation of 6-O-palmitoyl ascorbic acid was not inhibited by catalase plus SOD. The results indicate that cytotoxicity of 6-O-palmitoyl ascorbic acid is attributed at least partly to both hydrogen peroxide (H2O2) and superoxide (O2-.) generated at the early stage. Cytotoxicity of 6-O-palmitoyl ascorbic acid is also appreciably attenuated by singlet oxygen (1O2) scavengers such as hydroquinone, 1,4-diazobicyclo-2,2,2-octane or sodium azide, but not by hydroxyl radical scavengers including butylated hydroxytoluene, D-mannitol, benzoic acid and ethanol. Thus, in contrast to cytotoxicity of ascorbic acid mediated entirely by H2O2 initially generated, acylated ascorbic acid produces a diversity of active oxygen species including H2O2, O2-. and other species secondarily generated via disproportion, which may be additively involved in the enhanced cytotoxic action.

Effects of butylated hydroxyanisole on ornithine decarboxylase activity and its gene expression induced by phorbol ester tumor promoter.

Butylated hydroxyanisole (BHA) is a phenolic antioxidant that has been found to suppress the activity of skin tumor promoters. In this study, we investigated the effect of BHA on the activity of ornithine decarboxylase (ODC, an indicator of tumor promotion) and its gene expression induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in mouse skin. TPA-induced ODC activity was markedly inhibited by the topical application of 55 mumol of BHA (the inhibition rate at 6 h was about 80%). In Northern and dot-blot analysis, the TPA-induced increase in ODC mRNA was shown to be markedly reduced by the same dose of BHA (the inhibition rate at 4 h was about 60%). These results suggest the involvement of a decrease in ODC gene expression in the mechanism of the inhibition of ODC activity by BHA.

Differential effects of cyclosporine A on ornithine decarboxylase activity induced by ultraviolet-B and PUVA in mouse skin.

The inhibitory effect of cyclosporine A (CsA) on the activity of ornithine decarboxylase (ODC) induced by phorbol ester tumor promoter has been reported. In the present study, the effects of CsA on ODC activity induced by ultraviolet-B (UV-B) and PUVA in the skin of the SKH/hr 1 hairless mouse were investigated. Topical application of CsA (1.7 mumol) to the dorsal skin irradiated with 50 mJ/cm2 UV-B did not remarkably change ODC activity. On the other hand, if CsA was applied simultaneously with 3 J/cm2 UV-A 1 h after treatment with 0.3% 8-methoxypsoralen, PUVA-induced ODC activity was suppressed by about 60% at 12 and 24 h after UV-A irradiation. In the dot blotting analysis of ODC-specific mRNA level, a significant but slight decrease in ODC mRNA level (about 20% inhibition) was observed in the PUVA-treated group compared with the control group (vehicle and UV-A). The inhibition of PUVA-induced ODC activity by CsA may have been caused in part by the decrease in ODC mRNA level and in part by a post-transcriptional regulation mechanism.

Polyamines in 1 alpha, 25-dihydroxycholecalciferol-induced differentiation of human promyelocytic leukemia cells, HL-60.

The human promyelocytic leukemia cell line, HL-60, differentiated into macrophage/monocytes in the presence of 1 alpha,25-dihydroxycholecalciferol [1 alpha,25(OH)2D3], as assessed by the percentage of morphologically mature cells and their ability to reduce nitroblue tetrazolium. In this study of the mechanism involved, the activities of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase (SAT), the rate-limiting enzymes of polyamine metabolism, as well as the cellular levels of polyamine were measured. ODC activity reached a peak 24 h after the addition of 1 alpha,25(OH)2D3 and then decreased, while SAT activity gradually increased as differentiation commenced. An increase in putrescine and decreases in spermidine and spermine were also observed. Addition of alpha-difluoromethylornithine, an irreversible inhibitor of ODC, with or without methylglyoxalbis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, caused no effect on 1 alpha,25(OH)2D3-induced cell differentiation, although the cellular levels of putrescine and spermidine decreased markedly. Addition of alpha-difluoromethylornithine markedly suppressed cell proliferation; this effect was reversed by the addition of exogenous putrescine. Addition of exogenous spermidine or spermine to overcome activation of SAT also had no effect on 1 alpha,25(OH)2D3-induced cell differentiation. These results suggest both that polyamine metabolism is not important in 1 alpha,25(OH)2D3-induced differentiation of HL-60 cells, but that it is intimately involved in the proliferation of these cells.

Calcium ionophore A23187 induction of spermidine/spermine N1-acetyltransferase activity in bovine lymphocytes.

The divalent cation ionophore A23187 increased the activity in bovine lymphocytes of spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme in polyamine biodegradation. The enzyme was induced in a dose- and time-dependent manner. Induction was suppressed by indomethacin, but not by trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) or palmitoylcarnitine. These results suggest that the activation of phospholipase A2 involves the induction of spermidine/spermine N1-acetyltransferase. Ornithine decarboxylase, a rate-limiting enzyme in polyamine biosynthesis, was not suppressed by indomethacin but was by TFP and W-7. The molecular mechanism of the induction of spermidine/spermine N1-acetyltransferase and ornithine decarboxylase may be different.

Phorbol esters stimulate spermidine/spermine N1-acetyltransferase activity in mitogen-stimulated bovine lymphocytes.

Phorbol 12-myristate-13-acetate (PMA) is shown to induce spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme of polyamine biodegradation, in bovine lymphocytes. When PMA and phytohemagglutinin (PHA) were added simultaneously, the enzyme activity was stimulated synergistically. The ability of phorbol esters to stimulate the enzyme activity was consistent with their tumor-promoting ability. Phorbol, which is not a tumor promotor, was incapable of stimulating the enzyme activity. Phorbol diacetate weakly stimulated the activity of the acetylase. Phorbol dibutyrate had a similar stimulatory effect to PMA. These results suggest that the spermidine/spermine N1-acetyltransferase may play an important role in changes in polyamine levels in phorbol ester-treated cells and that the increase in the enzyme activity may have some relationship to the control of cell growth and differentiation by phorbol esters.

Inhibition by interferon (alpha + beta) of mouse liver regeneration and its reversal by putrescine.

Mouse interferon (alpha + beta) given to mice by intraperitoneal injection suppressed both the accumulation of putrescine and stimulation of DNA synthesis in liver caused by partial hepatectomy. The suppression of DNA synthesis was completely reversed by exogenous putrescine. The same results were obtained when core 2',5'-oligoadenylate instead of interferon was given to partially hepatectomized mice. These results suggest that interferon inhibits putrescine formation through elevating the 2',5'-oligoadenylate level and thus inhibits DNA synthesis in the regenerating liver.

The effect of hepatic ischemia-reperfusion on polyamine metabolism in some organs of the rat.

Hepatic ischemia was produced by clamping the portal venous and hepatic arterial blood supply to the left lateral and median lobes of the rat liver. Hepatic ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SAT) activities in ischemic and nonischemic regions were increased and, respectively, peaked by 6 hr and 3 hr after 1 hr of hepatic ischemia. Hepatic putrescine contents in ischemic and nonischemic regions were increased and peaked by 6 hr. However, hepatic spermidine and spermine were not increased. an increase in ODC activity was also observed in the spleen and the kidney after 1 hr of hepatic ischemia. [3H]thymidine incorporation into DNA was observed in the liver and the spleen--however it was not observed in the kidney--after hepatic ischemia.

Growth inhibitory effect of green tea extract in Ehrlich ascites tumor cells involves cytochrome c release and caspase activation.

We reported previously that the mechanism by which Green tea extract (GTE) elicited growth-inhibitory effects in Ehrlich ascites tumor cells involved a decrease in ornithine decarboxylase (ODC) activity and in cell viability. Decrease in ODC activity has been associated with apoptotic cell death and we therefore studied changes in cytochrome c release and caspase activation, which characterize apoptosis. GTE caused a dose- and time-dependent increase in caspase-3-like protease activation, preceded by a release of cytochrome c from the mitochondria. Inhibiting the activation of caspase-3 with acetyl-Asp-Glu-Val-Asp-alpha-aldehyde (caspase inhibitor) caused a reversal in the effect on cell viability.

Control by treatment with lithium chloride of ornithine decarboxylase in Ehrlich ascites tumor cells.

The activity of ornithine decarboxylase (ODC) was increased in Ehrlich ascites tumor cells by a change of the medium. This increase in the activity was inhibited by the addition of LiCl to the medium. Na+ and Mg2+ did not affect the enzyme activity. The inhibition of the enzyme activity with LiCl was not reversed by the addition of inositol or dibutyryl cyclic AMP. Total RNA was isolated from cells treated with LiCl and the relative abundance of the ODC mRNA was measured by Northern blot analysis. These levels in cells treated with LiCl were comparable to those in control cells. In the treated cells, the biological half-life of ODC was 14 min, which was the same as for the control cells. The inhibition by LiCl of ODC activity was not due to the nonspecific toxicity of LiCl. These results suggest that treatment of Ehrlich ascites tumor cells with LiCl suppressed ODC induction during translation, not during transcription or after translation.

Promotive action of acylated ascorbate on cellular DNA synthesis and growth at low doses in contrast to inhibitory action at high doses or upon combination with hyperthermia.

Effects of 6-O-palmitoyl ascorbate (ascorbate) developed to increase the antitumour activity of ascorbic acid on DNA synthesis and proliferation of Ehrlich ascites tumour cells were investigated. Treatment of the cells with the acylated ascorbate at 25-50 microM for 1 h resulted in no effect on DNA synthesis, assayed by pulse incorporation of [3H]thymidine after a culture period of 20 h, but led to 49%-87% enhanced DNA synthesis after 4 days, suggesting that long-term culture is required for promotion by ascorbate to occur. At a dose as high as 75 microM acylated ascorbate, however, cellular DNA synthesis was 64% inhibited after 20 h and 99% after 4 days. The results suggest that acylated ascorbate exhibits a dual action on DNA synthesis: promotion at low doses and inhibition at high doses, both of which are potentiated in a time-dependent manner. In contrast to the above-mentioned results at 37 degrees C, acylated ascorbate at 25-75 microM inhibited but did not promote DNA synthesis at 42 degrees C whatever the culture period. Similar results were exhibited when proliferation of cells cultured for a long period was investigated. At 37 degrees C, 50 microM acylated ascorbate increased the number of the cells to 3.6 times the control values after 8 days and to 1.9 times after 11 days; in contrast, a 75-microM dose decreased the cell number considerably. Combination with hyperthermia (42 degrees C) suppressed the increase and cell growth was completely inhibited at 75 microM.

Treatment with phorbol esters leads to spermine depletion and inhibition of DNA synthesis in phytohemagglutinin-activated bovine lymphocytes.

Phorbol 12-myristate-13-acetate (PMA) inhibited an increase in [3H]thymidine incorporation induced by phytohemagglutinin (PHA) in cultured bovine lymphocytes. Cellular levels of putrescine increased in the presence of PHA and PMA but the levels of spermidine and spermine had decreased to the control levels by 40 h. In cells treated with PHA and PMA, the activity of spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme in polyamine biodegradation, was stimulated synergistically. Phorbol esters with tumor-promoting ability also stimulated the enzyme activity and a reciprocal correlation between the enzyme activity and DNA synthesis was observed. Addition of spermine reversed the PHA- and PMA-induced inhibition of DNA synthesis but putrescine and spermidine failed to restore it. These results suggest that the enhancement of spermidine/spermine N1-acetyltransferase activity results in the depletion of intracellular spermine and a concomitant decrease in DNA synthesis.

Effects of Ca2+ ionophore A23187, 1,2-dioctanoylglycerol, and dibutyryl cAMP on the activity and expression of ornithine decarboxylase in guinea pig lymphocytes.

The Ca2+ ionophore A23187 induced small increases in ornithine decarboxylase activity and ornithine decarboxylase mRNA in guinea pig lymphocytes. 1,2-Dioctanoylglycerol potentiated the A23187-induced ornithine decarboxylase activity and the accumulation of mRNA for this enzyme. Dibutyryl cAMP also potentiated the enzyme activity, but had little effect on the accumulation of mRNA. 1,2-Dioctanoylglycerol and 12-O-tetradecanoylphorbol-13-acetate potentiated ornithine decarboxylase activity that had been increased by treatment with both A23187 and dibutyryl cAMP with a consistent increase in the ornithine decarboxylase mRNA. However, dibutyryl cAMP augmented ornithine decarboxylase activity that had been increased by the combination of A23187 and 1,2-dioctanoylglycerol without affecting the ornithine decarboxylase mRNA level. These results suggest that the protein kinase C and cyclic AMP pathways are involved in the enhancement of ornithine decarboxylase activity in guinea pig lymphocytes, but that the mechanisms of the enhancement differ for each pathway, the former increasing the ornithine decarboxylase mRNA level, but not the latter.

Potentiation by retinoic acid of ornithine decarboxylase induction by phytohemagglutinin or phorbol 12-myristate 13-acetate in guinea pig lymphocytes.

Retinoic acid potentiated the increases in ornithine decarboxylase (L-ornithine carboxy-lyase [EC 4.1.1.17]) activity, [3H]difluoromethylornithine binding to ornithine decarboxylase, intracellular levels of polyamines and DNA synthesis in guinea pig lymphocytes stimulated with phytohemagglutinin. The stimulatory effect on the ornithine decarboxylase induction was dependent on the dose of retinoic acid and on the time of addition of the drug. Retinoic acid has to be added not later than 2 h after phytohemagglutinin to elicit the potentiation. Retinyl acetate also potentiated ornithine decarboxylase induction caused by phytohemagglutinin. Both of these retinoids augmented ornithine decarboxylase induction caused by phorbol 12-myristate 13-acetate. The half-life of ornithine decarboxylase activity estimated after addition of actinomycin D was longer in cells treated with phytohemagglutinin or phorbol 12-myristate 13-acetate together with retinoic acid than in cells treated with the mitogen alone. The half-life after addition of cycloheximide was not affected by retinoic acid. These results suggest that the retinoids are stimulators rather than inhibitors of ornithine decarboxylase induction caused by phytohemagglutinin or phorbol 12-myristate 13-acetate in guinea pig lymphocytes and that retinoic acid potentiates the enzyme activity at the transcriptional or posttranscriptional, but not at the post-translational stage.