Involvement of ornithine decarboxylase in the control of proliferation of the HT29 human colon cancer cell line. effect of vasoactive intestinal peptide on enzyme activity

Butyrate is a short chain fatty acid (SCFA) produced by bacterial fermentation of dietary fibers in the colon lumen which severely affects the proliferation of colon cancer cells in in vitro experiments. Although butyrate is able to interfere with numerous cellular targets including cell cycle regulator expression, little is known about butyrate metabolism and its possible involvement in its effect upon colon carcinoma cell growth. In this study, we found that HT-29 Glc 2/1 cells strongly accumulated and oxidized sodium butyrate without producing ketone bodies, nor modifying oxygen consumption nor mitochondrial ATP synthesis. HT-29 cells accumulated and oxidized sodium acetate at a higher level than butyrate. However, sodium butyrate, but not sodium acetate, reduced cell growth and increased the expression of the cell cycle effector cyclin D3 and the inhibitor of the G1/S cdk-cyclin complexes p21/WAF1/Cip1, demonstrating that butyrate metabolism downstream of acetyl-CoA synthesis is not required for the growth-restraining effect of this SCFA. Furthermore, HT-29 cells modestly incorporated the 14 C-labelled carbon from sodium butyrate into cellular triacylglycerols and phospholipids. This incorporation was greatly increased when d-glucose was present in the incubation medium, corresponding to the capacity of hexose to circulate in the pentose phosphate pathway allowing NADPH synthesis required for lipogenesis. Interestingly, when HT-29 cells were cultured in the presence of sodium butyrate, their capacity to incorporate 14 C-labelled sodium butyrate into triacylglycerols and phospholipids was increased more than twofold. In such experimental conditions, HT-29 cells when observed under an electronic microscope, were found to be characterized by an accumulation of lipid droplets in the cytosol. Our data strongly suggest that butyrate acts upon colon carcinoma cells upstream of acetyl-CoA synthesis. In contrast, the metabolism downstream of acetyl-CoA [i.e. oxidation in the tricarboxylic acid (TCA) cycle and lipid synthesis] likely acts as a regulator of butyrate intracellular concentration.Keywords: butyrate; metabolism; growth; colon carcinoma cells.Epidemiological studies generally suggest a protective role of dietary fibers in colon carcinogenesis [1]. Dietary fibers contained in vegetables, fruit and cereals are heterogeneous group of compounds with different physicochemical properties [2]. They escape digestion and are recovered in the colon lumen. The different proposed mechanisms of action in which fibers may protect against colorectal tumours development include the inactivation of carcinogenic substances, an increase in faecal bulk with shortening of the bowel transit time and a decreased contact time [3]. In the large intestine, fibers can undergo anaerobic fermentation by intestinal flora leading to the production of various metabolites including short chain fatty acids (SCFA, mainly acetate, propionate and butyrate). Among these three, butyrate has received much attention due to its ability to severely ...

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“…ODC is considered as a protooncogene product important for cell growth [35,36] and malignant transformation [37].…”

Section: Discussionmentioning

confidence: 99%

Butyrate metabolism upstream and downstream acetyl‐CoA synthesis and growth control of human colon carcinoma cells

Leschelle¹,

Delpal²,

Goubern

et al. 2000

European Journal of Biochemistry

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“…culture medium does not reverse the SNP effect suggests that although inhibition of putrescine synthesis by NO could play a role in the inhibitory effect on growth after 2 days of culture, this would not be the primary target. Hence, when an ODC inhibitor, namely difluoromethylornithine, was added to the culture medium of HT-29 cells, it inhibited cell growth, an effect which could be reversed by adding putrescine [8].…”

Section: Discussionmentioning

confidence: 99%

“…L-Ornithine is the precursor of putrescine, spermidine and spermine by the action of ornithine decarboxylase, spermidine synthase and spermine synthase respectively. In HT-29 cells, the inhibition of cellular synthesis of polyamines leads to the almost complete arrest of cellular proliferation [8]. In HT-29 cells, a small part of L-arginine taken up is degraded into nitric oxide and L-citrulline [7].…”

Section: Introductionmentioning

confidence: 99%

Sodium nitroprusside inhibits proliferation and putrescine synthesis in human colon carcinoma cells

Blachier¹,

Robert²,

Selamnia³

et al. 1996

FEBS Letters

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In human colon carcinoma HT-29 Glc t+ cells, L-arginine is the common precursor of polyamines which are absolutely necessary for cellular profiferation and nitric oxide (NO) with reported anti-proliferative activity. The aim of the present work was to test the effect of the NO donor sodium nitroprusside (SNP) on polyamine synthesis and cellular growth in HT-29 cells. SNP in the micromolar range inhibits cellular putrescine synthesis and this effect is greatly reversed by haemoglobin, supporting the view that the effect of SNP is related to the generation of NO. This corresponds to the inhibition by SNP of ornithine decarboxylase activity. Furthermore, SNP inhibits cellular proliferation. The effect of SNP is reversed by haemoglobin after 2 days of treatment but not after 4 days. Although no acute toxic effect of SNP was detected after 90 min incubation, it greatly enhanced the cellular death rate after several days in culture as estimated by the LDH leakage test. In conclusion, our data raise the possibility of an inhibitory interrelationship between NO and polyamine metabolic pathways. NO induced inhibition of putrescine synthesis and growth in HT-29 cells is discussed from a causal perspective.

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“…ODC inhibitors such as DFMO are known to cause cytostasis by depleting intracellular polyamines (11,(13)(14)(15). The cytostatic effect of DFMO is thus preventable by the addition of exogenous putrescine, the product of ODC, but not by ornithine, the substrate for ODC, as illustrated in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, the requirement of polyamines for maintenance of cell growth and function is clearly established (10-12). Direct evidence for this view has been provided by experiments in which polyamine synthesis was prevented in mammalian cells in culture by mutations of key enzymes (such as ODC) or by the application of enzyme inhibitors (11,(13)(14)(15).NO, like the enzyme-activated irreversible ODC inhibitor, ␣-dif luoromethylornithine (DFMO), decreases intracellular polyamine levels in RASMC (9). The cytostatic effects of NO and DFMO on RASMC can be reversed by the addition of exogenous polyamines but not ornithine, supporting the view that NO, like DFMO, inhibits ODC (9).…”

mentioning

confidence: 99%

Role of p42/p44 mitogen-activated-protein kinase and p21 ^waf1/cip1 in the regulation of vascular smooth muscle cell proliferation by nitric oxide

Bauer

Buga

Ignarro

2001

Proc. Natl. Acad. Sci. U.S.A.

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The purpose of this study was to determine the involvement of the p42͞p44 mitogen-activated protein kinase (MAPK) pathway and induction of p21 waf1/cip1 in the antiproliferative effects of nitric oxide (NO) on rat aortic smooth muscle cells (RASMC). NO, like ␣-difluoromethylornithine (DFMO), interferes with cell proliferation by inhibiting ornithine decarboxylase (ODC) and, therefore, polyamine synthesis. S-nitroso-N-acetylpenicillamine or (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate inhibited RASMC growth at concentrations as low as 3 M, and DFMO elicited effects at concentrations of 100 M or greater. The cytostatic effect of NO and DFMO was prevented by the MAPK kinase 1͞2 inhibitors PD 098,059 or U0126. This finding suggests that the p42͞p44 MAPK pathway is involved in the inhibition of RASMC proliferation by NO. Western blot analysis revealed that treatment of RASMC with NO or DFMO leads to activation of p42͞p44 MAPK and induction of p21 waf1/cip1 . This effect was prevented by MAPK kinase 1͞2 inhibitors, suggesting that induction of p21 waf1/cip1 depended on activation of p42͞p44. Moreover, activation of p42͞p44 and induction of p21 waf1/cip1 were prevented by exogenous putrescine but not ornithine, suggesting this effect was due to the inhibition of ODC by NO or DFMO. Finally, activation of p42͞p44 MAPK and induction of p21 waf1/cip1 were cGMPindependent. Neither 1H-(1,2,4)oxadiazolo[4,3-␣]quinoxalin-1-one nor zaprinast influenced the cytostatic effect of NO or DFMO or their ability to activate these signal transduction pathways. These observations suggest that inhibition of ODC and accompanying putrescine production are the underlying mechanisms by which NO and DFMO activate the MAPK pathway to promote induction of p21 waf1/cip1 and consequent inhibition of cell proliferation.polyamines ͉ ornithine decarboxylase ͉ atherosclerosis ͉ MAPK V ascular smooth muscle cells (VSMC) are normally quiescent in the adult, rarely exhibiting mitogenic activity in their contractile state (1). However, vascular injury stimulates VSMC growth by disrupting the physiological balance between inhibition and stimulation. Over-proliferation of VSMC contributes to many clinical vascular pathologies, the causes of which vary from mechanical (restenosis after balloon angioplasty), to immunological (transplant arteriosclerosis), to multifactorial (atherosclerosis). Increased VSMC proliferation has been attributed to dysfunction of or damage to the endothelial lining in the area of intimal thickening and, more specifically, to decreased nitric oxide (NO) production. This effect is made evident by several studies in which NO donor agents or NO synthase gene transfer were shown to inhibit neointimal formation after balloon angioplasty in animal models (2-4). The effect inverse has been shown in studies where animals were subjected to balloon angioplasty and then treated with inhibitors of NO synthase (4). In these studies, intimal formation was enhanced by the inhibition of NO production.The mechanism by whi...

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Involvement of ornithine decarboxylase in the control of proliferation of the HT29 human colon cancer cell line. effect of vasoactive intestinal peptide on enzyme activity

Cited by 40 publications

References 28 publications

Butyrate metabolism upstream and downstream acetyl‐CoA synthesis and growth control of human colon carcinoma cells

Butyrate metabolism upstream and downstream acetyl‐CoA synthesis and growth control of human colon carcinoma cells

Sodium nitroprusside inhibits proliferation and putrescine synthesis in human colon carcinoma cells

Role of p42/p44 mitogen-activated-protein kinase and p21 ^waf1/cip1 in the regulation of vascular smooth muscle cell proliferation by nitric oxide

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Involvement of ornithine decarboxylase in the control of proliferation of the HT29 human colon cancer cell line. effect of vasoactive intestinal peptide on enzyme activity

Cited by 40 publications

References 28 publications

Butyrate metabolism upstream and downstream acetyl‐CoA synthesis and growth control of human colon carcinoma cells

Butyrate metabolism upstream and downstream acetyl‐CoA synthesis and growth control of human colon carcinoma cells

Sodium nitroprusside inhibits proliferation and putrescine synthesis in human colon carcinoma cells

Role of p42/p44 mitogen-activated-protein kinase and p21 waf1/cip1 in the regulation of vascular smooth muscle cell proliferation by nitric oxide

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Role of p42/p44 mitogen-activated-protein kinase and p21 ^waf1/cip1 in the regulation of vascular smooth muscle cell proliferation by nitric oxide