Polyamine metabolism in primary human colon adenocarcinoma cells (SW480) and their lymph node metastatic derivatives (SW620).

The natural polyamines are multifunctional constituents of all eucaryotic cells. The objective of this work was to compare aspects of polyamine metabolism in two related cell lines with the idea to investigate whether metabolic differences can be attributed to functional differences of the cells. The human colon carcinoma-derived cell lines SW480 and SW620 were chosen as models. SW480 cells were isolated from the primary tumour, SW620 cells from a lymph node of the same patient. SW620 cells grow faster, and the key regulatory enzymes of polyamine biosynthesis (ODC and AdoMetDC) are more active in the metastatic cells. Moreover, their ability to accumulate polyamines from the environment is more important than of SW480 cells. Likewise polyamine concentrations were markedly higher in SW620 cells, although they are much smaller than SW480 cells, and have a particularly small cytoplasmic space. Both cell lines show a striking diminution of ODC and AdoMetDC activities and changes in the polyamine patterns at the transition from exponential to non-exponential growth--most probably as a consequence of high cell density. Depletion of putrescine and spermidine due to inactivation of ODC by DFMO causes accumulation of cells in G1, and a proportional decrease of S-phase cells in both cell lines. Based on morphologic and other criteria SW480 and SW620 cells were typified as poorly differentiated. In agreement with their low grade of differentiation they exhibit a low alkaline phosphatase activity. However, the time-dependent decrease of alkaline phosphatase is not typical of differentiation patterns of other adenocarcinoma-derived cell lines or of normal enterocytes. The high capacity of de novo polyamine biosynthesis and of polyamine uptake is presumably a prerequisite for the rapid growth and invasiveness. The fact that these properties were more accentuated in the case of SW620 cells and paralleled enhanced metastatic properties indicate relationships between basic parameters of polyamine metabolism and malignancy.

Cytotoxic effects of the polyamine oxidase inactivator MDL 72527 to two human colon carcinoma cell lines SW480 and SW620.

N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) was considered to be a selective inactivator of FAD-dependent tissue polyamine oxidase. Recently MDL 72527 was reported to induce apoptosis in transformed hematopoietic cells through lysosomotropic effects. Since it is the only useful inhibitor of polyamine oxidase available at present, the re-evaluation of its properties seemed important. Human colon carcinoma-derived SW480 cells and their lymph node metastatic derivatives (SW620) were chosen for our study because they differ in various aspects of polyamine metabolism but have similar polyamine oxidase activities. MDL 72527 inhibited cell growth in a concentration-dependent manner, depleted intracellular polyamine pools, and caused the accumulation of N1-acetyl derivatives of spermidine and spermine. SW620 cells were more sensitive to the drug than were SW480 cells. At 150 micromol/L MDL 72527, SW620 cells accumulated in S-phase of the cell cycle, showed decreased polyamine transport rate, and showed no increase of polyamine N1-acetyltransferase activity. In contrast, SW480 cells were not arrested in a particular phase of the cell cycle, showed enhanced polyamine uptake, and showed a mild induction of acetyltransferase. The results suggest that MDL 72527 retains its value as a selective tool in short-term experiments only at concentrations not exceeding those necessary for the inactivation of polyamine oxidase. At concentrations above 50 micromol/L and at exposure times longer than 24 h, it may derange cell functions nonspecifically, and thus blur the results of studies intended to elucidate polyamine oxidase functions.

Resveratrol inhibits intestinal tumorigenesis and modulates host-defense-related gene expression in an animal model of human familial adenomatous polyposis.

We studied the effect of oral administration of resveratrol, a natural constituent of grapes, on tumorigenesis in Min mice. Min mice are congenic mice genetically predisposed to develop intestinal tumors as a result of a mutation of the Apc gene. Resveratrol (0.01% in the drinking water containing 0.4% ethanol) was administered for seven weeks to Min mice starting at five weeks of age. The control group was fed the same diet and received water containing 0.4% ethanol. Resveratrol prevented the formation of colon tumors and reduced the formation of small intestinal tumors by 70%. Comparison of the expression of 588 genes in the small intestinal mucosa showed that resveratrol downregulated genes that are directly involved in cell cycle progression or cell proliferation (cyclins D1 and D2, DP-1 transcription factor, and Y-box binding protein). In addition, resveratrol upregulated several genes that are involved in the recruitment and activation of immune cells (cytotoxic T lymphocyte Ag-4, leukemia inhibitory factor receptor, and monocyte chemotactic protein 3) and in the inhibition of the carcinogenic process and tumor expansion (tumor susceptibility protein TSG101, transforming growth factor-beta, inhibin-beta A subunit, and desmocollin 2). Our data highlight the complexity of the events associated with intestinal tumorigenesis and the multiplicity of the molecular targets of resveratrol. The high potency and efficacy of resveratrol support its use as a chemopreventive agent in the management of intestinal carcinogenesis.

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.

Effect of the polyamine oxidase inactivator MDL 72527 on N(1)-(n-octanesulfonyl)spermine toxicity.

N(1)-(n-octanesulfonyl)spermine (N(1)OSSpm) is a potent calmodulin antagonist. In the present work, its toxicity to DHD/K12/TRb and CaCo-2 cells, two colon carcinoma-derived cell lines, was studied with the aim to identify those properties of the cells, which determine their sensitivity to N(1)OSSpm and related structures. Exposure of the cells to MDL 72527, a compound considered to be a selective inactivator of polyamine oxidase (PAO) increased the cytotoxicity of N(1)OSSpm to both cell lines. In contrast, toxicity of trifluoperazine, a calmodulin antagonist with a polyamine-unrelated structure, was not enhanced by MDL 72527. Combined exposure of cells to 2-(difluoromethyl)ornithine (DFMO) (a selective inactivator of ornithine decarboxylase), MDL 72527 and N(1)OSSpm produced a synergistic cytotoxic effect. Neither the intrinsic PAO activity of the cells (as determined with N(1), N(12)-diacetylspermine as substrate), nor their ability to accumulate the drug was a determinant of the cytotoxic effect of N(1)OSSpm. These data suggest that MDL 72527 has a target unrelated to PAO, which is responsible for the enhancement of N(1)OSSpm (and spermine) toxicity. Identification of this target may be of use if the therapeutic potentials of MDL 72527 are to be exploited.

Spermine cytotoxicity to human colon carcinoma-derived cells (CaCo-2).

Spermine is a constituent of all vertebrate cells. Nevertheless, it exerts toxic effects if it accumulates in cells. Spermine is a natural substrate of the FAD-dependent polyamine oxidase, a constitutive enzyme of many cell types. It has been reported that the toxicity of spermine was enhanced if polyamine oxidase was inhibited. We were interested to examine spermine toxicity to human colon carcinoma-derived CaCo-2 cells because, in contrast to most tumor cell lines, CaCo-2 cells undergo differentiation, which is paralleled by changes in polyamine metabolism. CaCo-2 cells were remarkably resistant to spermine accumulation, presumably because spermine is degraded by polyamine oxidase at a rate sufficient to provide spermidine for the maintenance of growth. Inactivation of polyamine oxidase increased the sensitivity to spermine. A major reason for the enhanced spermine cytotoxicity at low polyamine oxidase activity is presumably the profound depletion of spermidine, and the consequent occupation of spermidine binding sites by spermine. Hydrogen peroxide and the aldehydes 3-aminopropanal and 3-acetamidopropanal, the products of polyamine oxidase-catalyzed splitting of spermine and N1-acetylspermine, contribute little to spermine cytotoxicity. Activation of caspase by spermine was insignificant, and the formation of DNA ladders, another indicator of apoptotic cell death, could not be observed. Thus it appears that cell death due to excessive accumulation of spermine in CaCo-2 cells was mainly nonapoptotic. The content of brush border membranes did not change between days 6 and 8 after seeding, and it was not affected by exposure of the cells to spermine. However, the activities of alkaline phosphatase, sucrase, and aminopeptidase in nontreated cells were considerably enhanced during this period, but remained low if cells were exposed to spermine. These changes appear to indicate that differentiation is prevented by intoxication with spermine, although other explanations cannot be excluded.

Anti-proliferative effect of resveratrol, a natural component of grapes and wine, on human colonic cancer cells.

Resveratrol, a natural polyphenolic phytoalexine present in grapes and wines, has been reported to exert a variety of important pharmacological effects. We investigated the effects of resveratrol on the growth and polyamine metabolism of CaCo-2 human colon cancer cells. Treatment of the CaCo-2 cells with 25 microM resveratrol caused a 70% growth inhibition. The cells accumulated at the S/G2 phase transition of the cell cycle. No signs of cytotoxicity or apoptosis were detected. Resveratrol caused a significant decrease of ornithine decarboxylase (ODC) activity, a key enzyme of polyamine biosynthesis, which is enhanced in cancer growth. ODC inhibition resulted in the reduction of the intracellular putrescine content, indicating that polyamines might represent one of several targets involved in the anti-proliferative effects of resveratrol.

Inhibition of polyamine oxidase enhances the cytotoxicity of polyamine oxidase substrates. A model study with N1-(n-octanesulfonyl)spermine and human colon cancer cells.

N(1)-(n-octanesulfonyl)spermine (N(1) OSSpm) is a substrate of polyamine oxidase. It shares several properties with spermine, such as antagonism of NMDA-type glutamate receptors, calmodulin antagonism, and cytotoxicity, but it is more potent by orders of magnitude in these regards than spermine. The human colon carcinoma-derived cell line CaCo-2 was used as a model to study the toxicity of N(1) OSSpm as a function of polyamine oxidase (PAO) activity and differentiation. If the formation of hydrogen peroxide and aminoaldehyde by the PAO-catalysed reactions was prevented by selective inactivation of the enzyme with MDL 72527, cytotoxicity of N(1)OSSpm was not diminished, but on the contrary, enhanced. Exponentially growing CaCo-2 cells were considerably more sensitive to N(1)OSSpm than differentiating cells. The results suggest that cytotoxic substrates of PAO exhibit enhanced cytotoxicity in cells, if PAO activity is inhibited. Since tumour cells are known to have lower polyamine oxidase activities than their normal counterparts, it will be interesting to explore whether cytotoxic substrates of polyamine oxidase, for which N(1)OSSpm is an example, are suited to preferentially kill tumour cells.

Promotion of intestinal carcinogenesis by Streptococcus bovis.

The involvement of Streptococcus bovis, an member of the human gut flora, in colorectal neoplastic diseases is an object of controversy. The aim of this study was to determine the effects of S.bovis and of antigens extracted from the bacterial cell wall on early preneoplastic changes in the intestinal tract. Adult rats received i. p. injections of azoxymethane (15 mg/kg body weight) once per week for 2 weeks. Fifteen days (week 4) after the last injection of the carcinogen, the rats received, by gavage twice per week during 5 weeks, either S.bovis (10(10) bacteria) or wall-extracted antigens (100 microg). One week after the last gavage (week 10), we found that administration of either S.bovis or of antigens from this bacterium promoted the progression of preneoplastic lesions through the increased formation of hyperproliferative aberrant colonic crypts, enhanced the expression of proliferation markers and increased the production of IL-8 in the colonic mucosa. Our study suggests that S.bovis acts as a promoter of early preneoplastic lesions in the colon of rats. The fact that bacterial wall proteins are more potent inducers of neoplastic transformation than the intact bacteria may have important implications in colon cancer prevention.

Blood polyamine levels after oral ornithine load, a diagnostic marker of hyperproliferative premalignant and malignant stages in a model of colon carcinogenesis.

This study was performed to determine whether a single oral dose of ornithine (Orn), the substrate of ornithine decarboxylase (ODC), increases blood concentrations of polyamines premalignant stage, and whether blood polyamine levels could be used as predictive markers of cancer development. Male Wistar rats were divided into two groups, control and 1,2-dimethylhydrazine (DMH)-treated rats. DMH (20 mg/kg body weight) was injected intraperitoneally once weekly for 10 weeks. Five, 7, and 10 weeks after the last injection when premalignant aberrant crypt foci have developed in the colon, blood levels of putrescine (PUT), spermidine (SPD), and spermine (SPM) were estimated before and after an oral load of ORN. The results showed that after a single oral load of Orn, blood PUT, but not SPD and SPM, concentrations were significantly higher in DMH-treated rats compared with control rats, indicating enhancement of ODC activity. These results support the view that the increased blood concentration of PUT after administration of Orn may be a useful marker to detect hyperproliferative premalignant and malignant stages of cancer development.

Nitric oxide synthase inhibition promotes carcinogen-induced preneoplastic changes in the colon of rats.

l-Arginine is metabolized either to polyamines through arginase and ornithine decarboxylase (ODC) activities or to citrulline and nitric oxide (NO, nitrogen monoxide) through the NO synthase (NOS) pathway. Polyamine levels and ODC activity are high in tumor cells. The aim of this study was to test whether N(G)-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NOS, modulates colon carcinogenesis. Adult male Wistar rats were treated with azoxymethane (AOM, 15 mg/kg ip), a chemical carcinogen, once a week for 2 weeks. One week after the second injection the rats were randomly divided into two groups. One group (n = 8) received l-NAME (10 mg/kg body wt/day) in drinking water. The control group (n = 8) received tap water. After 5 weeks, the rats receiving l-NAME showed enhanced mean basal arterial blood pressure, decreased heart rate, and a significant decrease of the cGMP content in the colonic mucosa. In both groups, AOM induced the formation of colonic aberrant crypt foci (ACF). In l-NAME-treated rats, the number of ACF was higher than in controls by 47%. ODC activity was enhanced by 11-fold. S-Adenosyl-methionine-decarboxylase activity and putrescine concentration were significantly increased in the colonic mucosa of l-NAME-treated rats. The data suggest that l-NAME promotes carcinogen-induced preneoplastic changes in the colon by inhibiting NOS activity and by stimulating polyamine biosynthesis.

Promotion of intestinal carcinogenesis by dietary methionine.

The metabolism of the polyamines spermidine and spermine is known to be enhanced in rapidly proliferating cells. Methionine is a precursor of the aminopropyl moieties of these amines. Therefore, it was of interest to study the effects of a methionine supplemented diet on polyamine metabolism and preneoplastic changes occurring in the intestinal tract of rats treated with the chemical carcinogen azoxymethane (AOM). Adult Wistar rats received 15 mg AOM/kg body wt (i.p.) once each week for 2 weeks. Thereafter, the rats were randomly divided into two groups and received controlled isoenergetic diets containing the same amount of folate, choline and vitamin B12 during 12 weeks: one group was kept on a standard diet; the other was fed the same diet, except that 1% L-methionine was added at the expense of carbohydrates. After 12 weeks, the administration of the methionine-supplemented diet stimulated the turnover rate of ileal epithelial cells, indicating enhanced crypt cell proliferation. Furthermore, in this group, a 2-fold increase in the number of aberrant hyperproliferative crypts and the appearance of tumors was observed in the colon. These effects were accompanied by the increased formation of spermidine and spermine due to the enhancement of S-adenosylmethionine decarboxylase activity and by the upregulation of Cdx-1, a homeobox gene with oncogenic potentials. The experimental data do not support the view of a chemopreventive effect of dietary methionine supplementation on intestinal carcinogenesis in rats, even at an early phase of preneoplastic development, but rather suggest that methionine promotes intestinal carcinogenesis.

Concomitant changes in polyamine pools and DNA methylation during growth inhibition of human colonic cancer cells.

The effects of CGP 48664 and DFMO, selective inhibitors of the key enzymes of polyamine biosynthesis, namely, of S-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), were investigated on growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. Both inhibitors caused growth inhibition and affected similarly the initial expression of the differentiation marker sucrase. In the presence of the AdoMetDC inhibitor, ODC activity and the intracellular pool of putrescine were enhanced, whereas the spermidine and spermine pools were decreased. In the presence of the ODC inhibitor, the AdoMetDC activity was enhanced and the intracellular pools of putrescine and spermidine were decreased. With both compounds, the degree of global DNA methylation was increased. Spermine and spermidine (but not putrescine) selectively inhibited cytosine-DNA methyltransferase activity. Our observations suggest that spermidine (and to a lesser extent spermine) controls DNA methylation and may represent a crucial step in the regulation of Caco-2 cell growth and differentiation.

Premature stimulation of rat sucrase-isomaltase (SI) by exogenous insulin and the analog B-Asp10 is regulated by a receptor-mediated signal triggering SI gene transcription.

The mechanism(s) by which insulin enhance prematurely the activity of brush border membrane (BBM) hydrolases in rat immature intestine is unknown. Therefore, we have compared the responses of four BBM enzymes [sucrase-isomaltase (SI), maltase, lactase-phloridzine hydrolase (LPH), and aminopeptidase] with exogenous insulin, the analog B-Asp10, IGF-I, and antireceptor MAb [insulin-receptor (IR) MAb] given to preweaning pups. Low doses of insulin caused a precocious induction of SI and of SI mRNA and stimulated maltase activity without effect on LPH nor on aminopeptidase activities. IGF-I given at the same dose as that of insulin had no detectable effect on these enzymes. Administration to sucklings of IR MAb prevented the effect of endogenous insulin by inhibiting the expression of SI and maltase without effect on LPH activity. B-Asp10, an insulin analogue that exhibits in vitro a 3.5-fold increase in receptor affinity with sustained signaling of the receptor tyrosine kinase, caused an overexpression of SI by 3.5-fold and of maltase by 1.5-fold compared with equivalent doses of normal insulin. The premature increases in SI activity, SI mRNA, and maltase activity in response to insulin were dose-dependent and were associated with dose-dependent increases in intracellular spermine and spermidine concentrations. In conclusion, these data suggest that the premature induction of SI by insulin is mediated by a dose-dependent signal initiated by binding of the hormone to its intestinal receptor, which after transduction into the cell indirectly triggers the transcription of the SI gene, possibly by changes in intracellular polyamine concentrations.

Preventive administration of ornithine alpha-ketoglutarate improves intestinal mucosal repair after transient ischemia in rats.

OBJECTIVES: To investigate whether the preventive enteral administration of a polyamine precursor, such as ornithine alpha-ketoglutaiate (OKG), has a beneficial effect on the repair process of the intestinal mucosa after transient mesenteric vascular occlusion. DESIGN: A controlled laboratory study. SETTING: A research laboratory facility at the Research Institute for Digestive Cancer. SUBJECTS: Male Wistar rats, weighing 330 to 350 g, housed individually in plastic cages under standardized conditions (23 degrees C, 73 degrees F, 60% relative humidity, 12-hr light and 12-hr dark cycles). INTERVENTIONS: Intragastrically, 1.5 or 17 hrs before surgery, animals received either distilled water (water group), or an amino acid solution of either OKG (1 g/kg) or L-lysine (0.68 g/kg) in distilled water under isonitrogenous conditions. Intestinal ischemia was produced in anesthetized rats by occluding the superior mesenteric artery for 90 mins with a microbulldog clamp. At the end of the ischemic period, the clamp was removed, allowing reperfusion, and the abdomen was closed. MEASUREMENTS AND MAIN RESULTS: At 0, 4, and 24 hrs after ischemia/reperfusion, the midjejuno-ileum was resected. Intestinal morphology, polyamine content, and hydrolase activities were determined. In all groups at the end of the ischemic period, the villi were dismantled with preservation of the crypts. Rats treated with OKG exhibited a restoration of short villi by 4 hrs after ischemia/reperfusion. In other groups, the villi remained extensively denuded. By 24 hrs, only rats treated with OKG showed a complete recovery of normal mucosal architecture. The amount of the polyamines, putrescine, and spermidine were significantly enhanced by 4 hrs after ischemia/reperfusion in the mucosa of rats treated with OKG, as compared with the two other groups. At a functional level, sucrase and aminopeptidase activities remained significantly higher by 4 hrs of ischemia/reperfusion in rats treated with OKG as compared with rats receiving water or lysine. By 24 hrs, hydrolase activities were normalized in rats treated with OKG, whereas an important deficit in hydrolase activities remained in rats receiving either water or lysine. CONCLUSIONS: OKG administration to rats did not prevent ischemic damage of the intestinal mucosa, but it accelerated the repair of the mucosa during reperfusion. OKG favored the restitution of normal villus architecture and the functional recovery of the intestinal mucosa. We hypothesized that OKG-triggered metabolites might mediate the restitution process and contribute to the healing of the intestinal mucosa by minimizing cell injury and by promoting the replacement of injured cells.

Suppression of preneoplastic changes in the intestine of rats fed low levels of polyamines.

Administration for 7 days of an enteral diet that is naturally deficient in polyamines strikingly reduces the preneoplastic changes observed in the intestines of adult Wistar rats previously treated with the carcinogen 1,2-dimethylhydrazine. On the contrary, supplementing the enteral diet with spermidine favors preneoplastic development. The effects of the low-polyamine diet included a 40% decline in the putrescine content of the intestinal mucosa, a significant decrease in the turnover rate of the epithelial cells from the crypts to villus tip in the ileum, and a 2-fold reduction in the number of abnormal colonic crypts. The experimental data support the view that it might be of interest to control the dietary intake of polyamines in the clinical management of cancer patients.

[Spontaneous calcified coronary embolism and myocardial infarction. Apropos of a case]. / Embolie calcaire coronaire spontanée et infarctus du myocarde. A propos d'une observation.

The authors report a rare case of myocardial infarction due to calcific coronary embolisation in a patient with previously asymptomatic calcific aortic stenosis. The diagnosis was suggested by the finding of a lacunar image in the distal segment of the left anterior descending artery exactly corresponding to a punctiform mobile calcification visible before opacification of the coronary arteries. The clinical features of coronary embolism and in particular of calcific embolism are reviewed.

[Echography and scintigraphy using technetium 99m pyrophosphate in the diagnosis of cardiac amyloidosis]. / Contribution de l'échographie et de la scintigraphie au pyrophosphate de technetium 99m au diagnostic de l'amylose cardiaque.

The diagnosis of amyloid cardiomyopathy was only based, until the last few years, on the results of invasive techniques. It seems presently that the combined contribution of cardiac sonography and scintigraphy using technetium 99m pyrophosphate, makes, most of the time, this diagnosis possible without need for additional examinations. This notion is illustrated by a typical case-report and data from the literature. Demonstration on the cardiac sonogram of a thickening of the walls-while the context and especially the electrocardiogram are not in favor of a left ventricular hypertrophy--associated with a very particular "hyperechoing" aspect and an abnormal fixation on the scintigram, may be considered specific of this disease.