Dietary arginine affects energy metabolism through polyamine turnover in juvenile Atlantic salmon (Salmo salar).

In the present study, quadruplicate groups of juvenile Atlantic salmon (Salmo salar) were fed plant protein-based diets with increasing arginine inclusions (range 28·8-37·4 g/kg DM) to investigate whether arginine supplementation affects growth and lipid accumulation through an elevated polyamine turnover. Dietary lysine was held at a constant concentration, just below the requirement. All other amino acids were balanced and equal in the diets. Arginine supplementation increased protein and fat accretion, without affecting the hepatosomatic or visceralsomatic indices. Dietary arginine correlated with putrescine in the liver (R 0·78, P= 0·01) and with ornithine in the muscle, liver and plasma (P= 0·0002, 0·003 and 0·0002, respectively). The mRNA of ornithine decarboxylase, the enzyme producing putrescine, was up-regulated in the white adipose tissue of fish fed the high-arginine inclusion compared with those fed the low-arginine diet. Concomitantly, spermidine/spermine-(N1)-acetyltransferase, the rate-limiting enzyme for polyamine turnover that consumes acetyl-CoA, showed an increased activity in the liver of fish fed the arginine-supplemented diets. In addition, lower acetyl-CoA concentrations were observed in the liver of fish fed the high-arginine diet, while ATP, which is used in the process of synthesising spermidine and spermine, did not show a similar trend. Gene expression of the rate-limiting enzyme for ß-oxidation of long-chain fatty acids, carnitine palmitoyl transferase-1, was up-regulated in the liver of fish fed the high-arginine diet. Taken together, the data support that increased dietary arginine activates polyamine turnover and ß-oxidation in the liver of juvenile Atlantic salmon and may act to improve the metabolic status of the fish.

E6201, a novel kinase inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1 and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase-1: in vivo effects on cutaneous inflammatory responses by topical administration.

E6201 [(3S,4R,5Z,8S,9S,11E)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,10-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione)] is a novel anti-inflammatory agent that has potent inhibitory effects on the production of proinflammatory cytokines from leukocytes and antiproliferative activity on keratinocytes. To characterize the in vivo pharmacological activity of E6201, topically administered E6201 was evaluated in several different animal models of dermatitis. E6201 formulated as an ointment or cream showed dose-dependent inhibition of croton oil-induced acute edema formation and neutrophil infiltration into mouse skin. In addition, E6201 cream inhibited the 1-fluoro-2,4-dinitrobenzene-induced contact hypersensitivity reaction mediated by T cells in mice. In this model, E6201 cream also suppressed the migration of neutrophils and lymphocytes into the inflammatory site. Pretreatment with E6201 cream attenuated phorbol-12 myristate 13-acetate-induced ornithine decarboxylase activity, a marker of proliferation in epidermis. Furthermore, E6201 ointment showed inhibitory effects on both mezerein-induced and interleukin (IL)-23-induced epidermal hyperplasia. E6201 also suppressed T cell receptor-stimulated IL-17 production from human T cells. These results indicate that topically administered E6201 may be a useful agent for the prevention and treatment of cutaneous inflammatory and hyperproliferative diseases such as psoriasis.

Anticancer activities of cranberry phytochemicals: an update.

Studies employing mainly in vitro tumor models show that extracts and compounds isolated from cranberry fruit (Vaccinium macrocarpon) inhibit the growth and proliferation of several types of tumor including breast, colon, prostate, and lung. Proanthocyanidin oligomers, flavonol and anthocyanin glycosides and triterpenoids are all likely contributors to the observed anticancer properties and may act in a complementary fashion to limit carcinogenesis. Possible chemopreventive mechanisms of action by cranberry phytochemicals include induction of apoptosis in tumor cells, reduced ornithine decarboxylase activity, decreased expression of matrix metalloproteinases associated with prostate tumor metastasis, and anti-inflammatory activities including inhibition of cyclooxygenases. A review of recent studies suggests a potential role for cranberry as a dietary chemopreventive and provides direction for future research.

Protective role of arginase in a mouse model of colitis.

Arginase is the endogenous inhibitor of inducible NO synthase (iNOS), because both enzymes use the same substrate, l-arginine (Arg). Importantly, arginase synthesizes ornithine, which is metabolized by the enzyme ornithine decarboxylase (ODC) to produce polyamines. We investigated the role of these enzymes in the Citrobacter rodentium model of colitis. Arginase I, iNOS, and ODC were induced in the colon during the infection, while arginase II was not up-regulated. l-Arg supplementation of wild-type mice or iNOS deletion significantly improved colitis, and l-Arg treatment of iNOS(-/-) mice led to an additive improvement. There was a significant induction of IFN-gamma, IL-1, and TNF-alpha mRNA expression in colitis tissues that was markedly attenuated with l-Arg treatment or iNOS deletion. Treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine worsened colitis in both wild-type and iNOS(-/-) mice. Polyamine levels were increased in colitis tissues, and were further increased by l-Arg. In addition, in vivo inhibition of ODC with alpha-difluoromethylornithine also exacerbated the colitis. Taken together, these data indicate that arginase is protective in C. rodentium colitis by enhancing the generation of polyamines in addition to competitive inhibition of iNOS. Modulation of the balance of iNOS and arginase, and of the arginase-ODC metabolic pathway may represent a new strategy for regulating intestinal inflammation.

Salix caprea inhibits skin carcinogenesis in murine skin: inhibition of oxidative stress, ornithine decarboxylase activity and DNA synthesis.

Chemoprevention of free radical-mediated diseases including cancer by natural products is an emerging discipline due to its wider applicability and acceptance. The present study deals with the chemopreventive effect of Salix caprea against phorbol ester-induced oxidative stress and tumor promotion in murine skin. In the present investigation, it was observed that a single application of 12-O-tetradecanoyl-13-phorbol acetate (TPA) (20 nmol/0.2 ml acetone/animal) caused a significant (P < 0.05) depletion of cutaneous antioxidants viz., glutathione, glutathione reductase, glutathione peroxidase, catalase and phase II drug metabolizing enzymes viz., glutathione-S-transferase, quinone reductase. An increase in the hydrogen peroxide generation and protein oxidation (measured in terms of protein carbonyl content) was also observed with a single application of TPA. However, the pretreatment of animals with different doses of Salix caprea (0.5, 1.0 and 1.5 mg/kg/0.2 ml acetone) caused a significant recovery in the TPA-mediated depletion in antioxidant levels. The pretreatment of animals with Salix caprea was observed to inhibit the TPA-mediated depletion in phase II enzymes. It was also observed that Salix caprea reversed the TPA-mediated depletion in the activity of phase II enzymes that is an important characteristic of cancer chemopreventive agents. Phorbol esters are known to induce the tumor promotion by increasing rate of DNA synthesis, ornithine decarboxylase activity (ODC), and xanthine oxidase activity. In the present investigation, it was observed that the pretreatment of animals with Salix caprea caused a significant (P < 0.05) depletion in the TPA-induced DNA synthesis, ODC and xanthine oxidase activity in mice skin. Salix caprea significantly reduced the tumor promotion in mice skin when tested in two-stage chemical carcinogenesis model. It was observed to inhibit significantly P < 0.05) the 7,12-dimethyl benz[a] anthracene (DMBA)-initiated phorbol ester promoted skin carcinogenesis. It was concluded from the results that Salix caprea is an effective antioxidant and chemopreventive agent against phorbol ester-induced tumor promotion.

Dietary lipids alter the effect of steroids on transport of glucose after intestinal resection: Part II. Signalling of the response.

BACKGROUND/PURPOSE: Glucocorticosteroids alter the function of the intestine. Budesonide (Bud) increases the jejunal D-glucose uptake, and this effect is prevented through a polyunsaturated fatty acid (PUFA) diet. This study was undertaken to assess the possible signalling effect of budesonide, prednisone (Pred), or dexamethasone (Dex) in animals with a 50% intestinal resection and fed chow or a diet enriched with saturated (SFA) or polyunsaturated fatty acids. METHODS: Northern blots were performed. RESULTS: Steroids reduced the jejunal but not the ileal expression of proglucagon. Ornithine decarboxylase (ODC) expression was reduced in the jejunum. CONCLUSIONS: c-jun, ODC, and proglucagon may be involved in the adaptive response that occurs with steroids and variations in dietary lipids after intestinal resection.

Effect of green tea extract on the induction of ornithine decarboxylase and the activation of extracellular signal-regulated kinase in bladder carcinoma ECV304 cells.

According to several studies, green tea and individual catechins can inhibit the induction of ornithine decarboxylase (ODC), the key enzyme in the biosynthesis of polyamines. It has been suggested that the inhibition of ODC induction may offer an explanation to the anticancer and chemopreventive activities of green tea. In the present study, however, treatment of bladder carcinoma ECV304 cells with green tea extract (GTE) was not able to reduce the induction of ODC by fetal calf serum. Actually, in the absence of serum, GTE provoked a dose-dependent and remarkable induction of ODC activity. The induction of ODC, which could be elicited also by (-)-epigallocatechin 3-gallate, a major green tea component, required an early activation of extracellular signal-regulated kinase 1 and 2 (ERK), and both events appeared to be dependent on an alteration of the status of cellular thiol groups. Pretreatment with specific ERK or ODC inhibitors was able to prevent a late caspase activation but hardly affected the loss of cell viability provoked by GTE. In conclusion, to our knowledge, this is the first study showing that GTE can promote ODC induction in a tumor cell line.

[Effect of Astragalus injection in promoting IEC-6 cell differentiation through activating ornithine decarboxylase].

OBJECTIVE: To explore the mechanism of Astragalus injection (AI) in repairing mucous membrane by observing its effects on the proliferation, differentiation, migration as well as on intracellular content of ornithine decarboxylase (ODC) and polyamine in rat's small intestinal crypt-like cell line (IEC-6) in vitro. METHODS: AI was added in the IEC-6 cells after they had been cultured for 24 hours. Twelve hours after adding AI, the cells were collected to test the ODC mRNA level, ODC protein, ODC activity and the intracellular content of putrescine. Twenty-four hours after adding AI, the condition of cell proliferation and differentiation was observed. Another group of IEC-6 cells were injured after being cultured for 72 hours, and AI was added, the cell migration was observed at 24 hours, 48 hours and 72 hours after medication. RESULTS: AI could inhibit IEC-6 cell proliferation, promote the cell differentiation, but with no apparent effect on injured IEC-6 cell migratin. AI of 62.5-250 micrograms/ml concentration could increase the ODC mRNA level, as compared with control, the difference was significant (P < 0.05-0.01). ODC activity and intracellular content of putrescine could be gradually increased by AI in a dose-dependant manner. No effect of AI was shown on ODC protein in various dosage of AI groups. CONCLUSION: AI could promote IEC-6 cell differentiation by means of inducing the ODC activity and biosynthesis of polyamine, but without significant effect on cell migration.

Geraniol, a component of plant essential oils, inhibits growth and polyamine biosynthesis in human colon cancer cells.

Geraniol and other monoterpenes found in essential oils of fruits and herbs have been suggested to represent a new class of agents for cancer chemoprevention. As a first step in clarifying the mode of action of geraniol on colon carcinogenesis, we studied its effects on the growth of a human colon cancer cell line (Caco-2). Geraniol (400 microM) caused a 70% inhibition of cell growth, with cells accumulating in the S transition phase of the cell cycle, and concomitant inhibition of DNA synthesis. No signs of cytotoxicity or apoptosis were detected. Geraniol caused a 50% decrease of ornithine decarboxylase activity, a key enzyme of polyamine biosynthesis, which is enhanced in cancer growth. This led to a 40% reduction of the intracellular pool of putrescine. Geraniol also activated the intracellular catabolism of polyamines, indicated by enhanced polyamine acetylation. These observations indicate that polyamine metabolism is presumably a target in the antiproliferative properties of geraniol.

Asafoetida inhibits early events of carcinogenesis: a chemopreventive study.

Ferula (a genus of many species) commonly known as asafoetida is used as a flavoring agent in food and is used as a traditional medicine for many diseases in many parts of world. In the current investigation, we report the antioxidant and anticarcinogenic potential of asafoetida (Ferula narthex) in swiss albino mice. A single dose of TPA (20 nmol/0.2 ml acetone/animal), a known tumor promoter decreased the cellular antioxidant level significantly (p<0.01) when applied topically to mice skin. It also induced the ODC activity, rate of DNA synthesis, hydrogen peroxide level, xanthine oxidase activity and protein carbonyl content in mice skin significantly (p<0.01). These events are early biomarkers of carcinogenesis. However, the pretreatment of animals with asafoetida (300, 400 and 500 microg/200 microl acetone/animal) caused the reversal of all events significantly (p<0.01). The pretreament of animals with asafoetida recovered the antioxidant level and reversed the induced ODC activity and DNA synthesis significantly (p<0.01). We conclude that asafoetida is a potent antioxidant and can afford protection against free radical mediated diseases such as carcinogenesis.

Effects of an aqueous extract of processed bidi tobacco on the growth of hamster tracheal epithelial cells.

Inhalation of tobacco dust is responsible for elevated genotoxicity and pulmonary ailments in workers engaged in processing tobacco for the manufacture of bidis, the Indian version of cigarettes. Tracheal tissue being the major site of interaction with tobacco dust, the effects of different concentrations of an aqueous extract of bidi tobacco (ATE) on the growth of a hamster tracheal epithelial cell line (HTE) were investigated. Colony forming efficiency assay revealed that ATE was cytotoxic only at the highest concentration of 5.0 mg/ml. In cultures treated with 1.25 mg/ml ATE, the cell doubling time and growth rate were similar to that of the controls, while a significant increase in cell doubling time (29.4+/-0.3 h vs 14.0+/-3.75 h, P<0.001) was observed at 2.5 mg/ml ATE concentration. Exposure of HTE cells to the non-toxic ATE concentration of 2.5 mg/ml was found to stimulate ornithine decarboxylase (ODC) activity, incorporation of [3H] methyl thymidine into DNA and increase in the S phase fraction was seen by flow cytometry. However, a 56% reduction in the growth rate of cultures treated with 2.5 mg/ml ATE was related to the prolongation of the traverse of cells through S phase. ATE-induced growth suppression was reversed when cultures were grown in ATE-free medium or upon repeated exposure to ATE. The findings suggest that increased tracheal cell proliferation induced by chronic inhalation of tobacco dust may contribute to the development of pulmonary disorders and possibly neoplasia in exposed individuals.

The effect of high doses of folic acid on the overexpression of ornithine decarboxylase and S-adenosylmethionine content in human colon adenomatous polyps.

It was shown that the 3-month supplementation of patients having colon polyps with folic acid (5 mg/day) led to a 35% decrease in abnormally high ornithine decarboxylase activity in polyps that was accompanied by a 43% increase of S-adenosylmethionine content in polyps.

Increased translation efficiency and antizyme-dependent stabilization of ornithine decarboxylase in amino acid-supplemented human colon adenocarcinoma cells, Caco-2.

The mechanisms of the response of ornithine decarboxylase(ODC), the rate-limiting enzyme in polyamine biosynthesis, to amino acid supplementation were studied in the human colon adenocarcinoma cell line, Caco-2. Supplementation of serum-deprived, subconfluent Caco-2 cells with any one of a series of amino acids (10 mM) resultedin increased ODC activity, reaching a maximum of approx. 12.5-fold after approx. 4 h, over control cells either not supplemented or supplemented with iso-osmolar D-mannitol. Glycine, L-asparagine and L-serine, as well as their D-enantiomers, were the strongest effectors and acted in a concentration-dependent manner; millimolar concentrations of most of these amino acids being sufficient to significantly increase ODC activity. In contrast, supplementation with D-methionine, L-lysine, L-aspartate or L-glutamate had little or no effect on ODC activity, whereas supplemental L-methionine, L-arginine, L-ornithine or L-cysteine was inhibitory. Polyamine assays showed that the putrescine content of cells varied in accordance with the changes in ODC activity. Western-blot and Northern-blot analyses revealed specifically increased levels of ODC protein but not mRNA,respectively, in response to supplementation with an ODC-inducing amino acid. Suppression of the increase in cycloheximide-treated cellsconfirmed a requirement for protein synthesis. Pulse-labelling of cellswith [(35)S]methionine showed a 3-fold increase in thesynthesis of ODC protein after 4 h of supplementation with glycineor L-serine. Supplemental glycine also augmented, reversibly, the half-life of ODC by almost 4-fold and simultaneously decreased the activity of putrescine-induced free antizyme. These results suggest that translational, but not transcriptional, regulation of ODC takes part in ODC induction by amino acids in Caco-2 cells. However, it also appears to occur in concert with decreased enzyme in activation and/or degradation.

Prostate cancer chemoprevention by green tea: in vitro and in vivo inhibition of testosterone-mediated induction of ornithine decarboxylase.

Recently, we have shown that ornithine decarboxylase (ODC), a rate-controlling enzyme in the polyamine biosynthetic pathway, is overexpressed in prostate cancer (PCA) and prostatic fluid in humans (R. R. Mohan et al., Clin. Cancer Res., 5: 143-147, 1999). ODC is also characterized as an androgen-responsive gene, and the androgenic stimulation regulates the development and growth of both normal and tumorigenic prostate cells. Thus, chemopreventive approaches aimed toward the modulation of ODC could be effective against PCA. Green tea polyphenols (GTPs) possess strong chemopreventive properties against a variety of animal tumor models and in some human epidemiological studies. At least two epidemiological studies have suggested that people who consume tea regularly may have a decreased risk of PCA. In this study, we investigated the effect of GTPs against testosterone-mediated induction of ODC in human prostate carcinoma cells, LNCaP as an in vitro model, and in Cpb:WU rats and C57BL/6 mice as in vivo models. Treatment of LNCaP cells with testosterone resulted in induction of ODC activity in a dose-dependent manner. Pretreatment of the cells with GTPs resulted in a significant inhibition of testosterone-caused induction of ODC activity in a dose-dependent manner. Similar effects of GTPs were observed in anchorage-independent growth assay of LNCaP cells where pretreatment of the cells with GTP was found to result in dose-dependent inhibition of colony formation. Testosterone treatment of the cells resulted in a significant increase in the level of ODC mRNA, and this increase was almost completely abolished by prior treatment of the cells with GTPs. The administration of testosterone (10 mg/kg body weight, i.p.) to sham-operated and castrated Cpb:WU rats resulted in 2- and 38-fold increases in ODC activity, respectively, in the ventral prostate. Oral feeding of 0.2% GTPs in drinking water for 7 days before testosterone administration resulted in 20 and 54% decreases in testosterone-caused induction of ODC activity in sham-operated and castrated rats, respectively. Similar results were obtained with C57BL/6 mice, where testosterone treatment at similar dosage resulted in a 2-fold increase in ODC activity in the ventral prostate and prior oral feeding with 0.2% GTPs resulted in 40% inhibition in this induction.

New bioactive flavonoids and stilbenes in cubé resin insecticide.

Fractionation of cubé resin from Lonchocarpus utilus and L. urucu roots led to the isolation and identification of 11 minor flavonoids and stilbenes containing the gem-dimethylpyran moiety or a dihydrodiol derivative thereof. The eight new compounds were as follows: the isoflavonoid cis-4'',5''-dihydro-4'',5' '-dihydroxylonchocarpusone (2); four (2S)-6-(gamma, gamma-dimethylallyl)-6'',6''-dimethylpyran[2'',3'':7, 8]flavanones with substituents of 5-hydroxy-3',4'-dimethoxy (3), 5, 3'-dihydroxy-4'-methoxy (4), 5,4'-dihydroxy-3'-methoxy (5), and 3', 4'-dimethoxy (6); and three 6'',6''-dimethylpyran[2'',3'':3', 4']stilbenes with 4-hydroxy-5'-methoxy (9), 3,5'-dimethoxy-4-hydroxy (10) and 3,4,5-trimethoxy (11) substitution patterns. Structure-activity relationships for inhibition of NADH:ubiquinone oxidoreductase activity (bovine heart electron transport particles) and phorbol ester-induced ornithine decarboxylase activity (cultured MCF-7 cells) generally parallel those for cytotoxicity (MCF-7 and Hepa 1clc7 cells).

Short-term biomarkers of tumor promotion in mouse skin treated with petroleum middle distillates.

Topical application of certain petroleum middle distillates (PMD) to mice produces skin tumors after long latency, and initiation/promotion protocols indicate that this effect is associated with their tumor promoting activity. Since induction of sustained, potentiated epidermal hyperplasia is predictive of promoting activity, five compositionally distinct PMD [hydrodesulfurized kerosene (API 81-07); hydrodesulfurized PMD (API 81-10); odorless light petroleum hydrocarbons; severely hydrotreated light vacuum distillate (LVD); and lightly refined paraffinic oil (LRPO)] were assessed for their effects on epidermal hyperplasia. PMD were administered (2 x/week for 2 weeks) to skin of CD-1 mice. Four quantitative biomarkers of epidermal hyperplasia were evaluated: epidermal thickness, number of nucleated epidermal cells per unit length of basement membrane, labeling (BrdUrd) index of epidermal cells, and induction of epidermal ornithine decarboxylase (ODC) activity. As positive controls, 12-O-tetradecanoylphorbol-13-acetate (TPA) and n-dodecane were utilized. PMD-induced skin irritation was evaluated visually and/or histopathologically. All five PMD produced dose-dependent, skin irritation and epidermal hyperplasia. On a weight basis the magnitude of the maximal PMD-induced effects was similar to that produced by n-dodecane, but > 1000-fold less than that produced by TPA. Epidermal hyperplasia and subacute skin irritancy produced by the five PMD were similar. Of the four short-term markers of tumor promotion assessed, labeling index and epidermal ODC activity were predictive of the relative promoting activities of those PMD for which tumorigenicity bioassay data are available, i.e., API 81-07 > API 81-10 > LRPO. An apparent discrepancy to the predictability of epidermal ODC activity occurred with LRPO:toluene [1:1 (v/v)]. This mixture is nontumorigenic, yet significantly induced epidermal ODC activity. This mixture, however, produced severe epidermal toxicity that precluded any meaningful analysis of short-term biomarkers in relationship to biological activity.

Metabolism of polyamines in transgenic cells of carrot expressing a mouse ornithine decarboxylase cDNA.

The metabolisms of arginine (Arg), ornithine (Orn), and putrescine were compared in a nontransgenic and a transgenic cell line of carrot (Daucus carota L.) expressing a mouse Orn decarboxylase cDNA. [14C]Arg, [14C]Orn, and [14C]putrescine were fed to cells and their rates of decarboxylation, uptake, metabolism into polyamines, and incorporation into acid-insoluble material were determined. Transgenic cells showed higher decarboxylation rates for labeled Orn than the nontransgenic cells. This was correlated positively with higher amounts of labeled putrescine production from labeled Orn. With labeled Arg, both the transgenic and the nontransgenic cells exhibited similar rates of decarboxylation and conversion into labeled putrescine. When [14C]putrescine was fed, higher rates of degradation were observed in transgenic cells as compared with the nontransgenic cells. It is concluded that (a) increased production of putrescine via the Orn decarboxylase pathway has no compensatory effects on the Arg decarboxylase pathway, and (b) higher rates of putrescine production in the transgenic cells are accompanied by higher rates of putrescine conversion into spermidine and spermine as well as the catabolism of putrescine.

Protective effects of silymarin against photocarcinogenesis in a mouse skin model.

BACKGROUND: Nonmelanoma skin cancer is the most common cancer among humans; solar UV is its major cause. Therefore, it is important to identify agents that can offer protection against this cancer. PURPOSE: We evaluated the protective effects of silymarin, a flavonoid compound isolated from the milk thistle plant, against UVB radiation-induced nonmelanoma skin cancer in mice and delineated the mechanism(s) of its action. METHODS: For long-term studies, three different protocols of treatment were employed, each evaluating protection by silymarin at a different stage of carcinogenesis. Female SKH-1 hairless mice were subjected to 1) UVB-induced tumor initiation followed by phorbol ester-mediated tumor promotion, 2) 7,12-dimethylbenz[a]anthracene-induced tumor initiation followed by UVB-mediated tumor promotion, and 3) UVB-induced complete carcinogenesis. Forty mice were used in each protocol and were divided into control and treatment groups. Silymarin was applied topically at a dose of 9 mg per application before UVB exposure, and its effects on tumor incidence (% of mice with tumors), tumor multiplicity (number of tumors per mouse), and average tumor volume per mouse were evaluated. In short-term studies, the following parameters were measured: formation of sunburn and apoptotic cells, skin edema, epidermal catalase and cyclooxygenase (COX) activities, and enzymatic activity and messenger RNA (mRNA) expression for ornithine decarboxylase (ODC), a frequently observed marker at tumor promotion stage. Fisher's exact test was used to evaluate differences in tumor incidence, two-sample Wilcoxon rank sum test was used for tumor multiplicity and tumor volume, and Student's t test was used for all other measurements. All statistical tests were two-sided. RESULTS: In the protocol with UVB-induced tumor initiation, silymarin treatment reduced tumor incidence from 40% to 20% (P = .30), tumor multiplicity by 67% (P = .10), and tumor volume per mouse by 66% (P = .14). In the protocol with UVB-induced tumor promotion, silymarin treatment reduced tumor incidence from 100% to 60% (P<.003), tumor multiplicity by 78% (P<.0001), and tumor volume per mouse by 90% (P<.003). The effect of silymarin was much more profound in the protocol with UVB-induced complete carcinogenesis, where tumor incidence was reduced from 100% to 25% (P<.0001), tumor multiplicity by 92% (P<.0001), and tumor volume per mouse by 97% (P<.0001). In short-term experiments, silymarin application resulted in statistically significant inhibition in UVB-caused sunburn and apoptotic cell formation, skin edema, depletion of catalase activity, and induction of COX and ODC activities and ODC mRNA expression. CONCLUSIONS AND IMPLICATION: Silymarin can provide substantial protection against different stages of UVB-induced carcinogenesis, possibly via its strong antioxidant properties. Clinical testing of its usefulness is warranted.

Ornithine decarboxylase overexpression in mouse 10T1/2 fibroblasts: cellular transformation and invasion.

BACKGROUND: Ornithine decarboxylase (ODC) plays a pivotal role in the synthesis of polyamines, a group of chemical compounds that are essential for cell growth. Recent reports have shown that ODC overexpression may be involved in malignant transformation of immortalized NIH 3T3 cells. We have demonstrated that ODC-overproducing mouse breast cancer cells are more invasive in vitro than control cells. However, little information is available concerning the relationship between ODC overexpression, tumor invasion, and metastasis and the signal transduction pathways involved in ODC-induced transformation and invasion. PURPOSE: Our purpose was twofold: 1) to determine whether ODC overexpression is directly involved in tumor cell invasion and 2) to determine whether ODC overexpression induces mitogen-activated protein (MAP) kinase activities that are associated with cell growth and transformation. METHODS: We transfected C3H clone 8 mouse 10T1/2 fibroblasts with an expression vector that carries a complementary DNA encoding rat ODC. Neomycin-resistant cells that overproduced ODC (4-6.5 times the control levels) were isolated. The transformed phenotype of these cells was determined by assessing colony formation and anchorage-independent growth in soft agar. The invasiveness of the cells was studied by means of an invasion assay that used Matrigel-coated filters in Boyden chambers. The MAP kinase activity of the cells was assayed by an in-gel kinase assay, using myelin basic protein as the substrate. RESULTS: Overexpression of ODC induced not only cell transformation and anchorage-independent growth in soft agar but also invasiveness through a Matrigel-coated filter. The ODC-overproducing transfectants showed enhanced MAP kinase activity that paralleled the magnitude of cell invasiveness. CONCLUSIONS: ODC plays a pivotal role not only in cell transformation but also in cancer cell invasion. ODC overexpression enhanced MAP kinase activity. IMPLICATIONS: Our results demonstrate a connection between the polyamine/ODC and the MAP kinase signal transduction pathways and suggest that MAP kinase may play a pivotal role in ODC-induced cell transformation and invasion.

Ornithine decarboxylase over-expression stimulates mitogen-activated protein kinase and anchorage-independent growth of human breast epithelial cells.

In these experiments we tested the hypothesis that constitutive activation of polyamine(PA) biosynthesis may contribute to mammary carcinogenesis. Spontaneously immortalized normal human MCF-10A breast epithelial cells were infected with the retroviral vector pLOSN containing a cDNA which codes for a truncated and more stable ornithine decarboxylase (ODC), the rate-limiting enzyme in PA synthesis. Upon chronic selective pressure with alpha-difluoromethyl-ornithine (DFMO) (an irreversible inhibitor of ODC), infected MCF-10A cells exhibited an approximately 250-fold increase in ODC activity, which persisted despite discontinuation of DFMO. ODC-over-expressing MCF-10A cells showed a modest decrease in S-adenosylmethionine decarboxylase and an increase in spermidine/spermineN1-acetyltransferase. Analysis of cellular PA profile revealed a selective accumulation of putrescine without alterations in spermidine and spermine contents. Lesser degrees of increased ODC activity were obtained reproducibly by re-exposing the cells to incremental small doses of DFMO. We observed a bell-shaped dose-related positive effect of ODC activity on clonogenicity in soft agar of MCF-10A cells. Since anchorage-dependent growth was actually reduced, such positive influence on this feature of transformation was not a non-specific consequence of a growth advantage provided by ODC over-expression. In addition, we observed a close parallelism between the dose-dependent effects of ODC expression on clonogenicity and activity of the ERK-2 kinase, a central element of the MAPK cascade. Our data demonstrate an interaction between PA and the MAPK signalling pathway and suggest that the latter may be involved in ODC-induced transformation of mammary epithelial cells.