Induction Of Ornithine Decarboxylase Research Articles

Abstract 5905: Enterotoxigenic Bacteroides fragilis, a colon microbe, dysregulates polyamine catabolism to promote breast cancer progression

Abstract Background: With more than 2.3 million cases worldwide, breast cancer persists as a major health burden and warrants a greater understanding of the molecular triggers underlying this disease. Focusing on microbial dysbiosis, our lab discovered the pivotal role of the enterotoxigenic Bacteroides fragilis (ETBF) in promoting breast tumorigenesis through activation of key oncogenic pathways. A gap in knowledge exists regarding how microbes/microbial toxins modulate the oncogenic pathways in cancer cells. Polyamines are imperative for various physiological processes, including cell growth, survival, differentiation and apoptosis and are strongly associated with breast cancer. We questioned the involvement of polyamine pathway in mediating the oncogenic effects of microbial dysbiosis on breast cancer. Methods: Human Her2 expressing spontaneous breast cancer model, MMTV.f.HuHer2 mice, were infected with ETBF and tumor incidence and progression were monitored for 18 months. We used qRT-PCR, western blotting, immunocytochemistry and immunohistochemistry to examine expression of key nodes of polyamine pathway. Various growth, migration and invasion assays were performed to assess carcinogenic properties in breast cancer cells. We employed a colorimetric-based assay to analyze enzymatic activities of key polyamine metabolic members. ROS generation was examined via DCFA staining. Results: Our in vivo data showed that ETBF colonization in mice led to a considerable increase in the level of spermine oxidase (SMOX) in breast tumors, hinting at a correlation between ETBF and polyamine metabolism. Moreover, a marked rise in expression of γH2AX foci was observed in these tumors and in breast cancer cells, suggesting a critical role of ETBF in SMOX-related DNA damage. In agreement, we found SMOX upregulation in breast cancer cells. Our findings displayed a substantial increase in SMOX enzymatic activity in the presence of ETBF toxin, BFT, in breast tumor cells. BFT caused a marked rise in ROS generation of breast tumor cells, suggesting a plausible association of ETBF in SMOX-mediated oxidative stress. Interestingly, we noted a significant induction of ornithine decarboxylase (ODC) expression and activity in response to ETBF or BFT in breast cancer cells. Treatment of BFT-exposed breast cancer cells with SMOX inhibitors led to a sharp inhibition of neoplastic transformations, like cellular migration and γH2AX induction. Conclusion: Our collated evidences suggest the existence of an important cross-talk between key polyamine metabolic enzymes and ETBF that aids in exacerbating breast tumorigenesis. Of importance, therapeutic inhibition of SMOX dramatically impeded BFT-related breast tumorigenesis. Collectively, our results demonstrated the attractive potential of intervening the polyamine catabolic pathway for the management of breast cancer. Citation Format: Deeptashree Nandi, Sheetal Parida, Sowjanya Thatikonda, Jackson Foley, Tracy Stewart, Cynthia L. Sears, Robert A. Casero, Dipali Sharma. Enterotoxigenic Bacteroides fragilis, a colon microbe, dysregulates polyamine catabolism to promote breast cancer progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5905.

Inhibition of ornithine decarboxylase restores hypoxic pulmonary vasoconstriction in endotoxemic mice.

Endotoxemia impairs hypoxic pulmonary vasoconstriction which leads to systemic hypoxemia. This derogation is attributable to increased activity of nitric oxide synthase 2 and arginase metabolism. Gene expression analysis has shown increased expression of ornithine decarboxylase in lungs of endotoxemic mice, a downstream enzyme of arginase metabolism. The aim of this study was to investigate whether inhibition of ornithine decarboxylase increases hypoxic pulmonary vasoconstriction in lungs of endotoxemic mice. Mice received lipopolysaccharides or saline intraperitoneal, and hypoxic pulmonary vasoconstriction was measured using an isolated perfused mouse lung model. Additional mice with and without endotoxemia were pretreated with the ornithine decarboxylase-inhibitor difluoromethylornithine before examination of hypoxic pulmonary vasoconstriction. Hypoxic pulmonary vasoconstriction was defined as the difference of pulmonary arterial pressure between normoxic and hypoxic ventilation. In addition, lung tissue was analyzed using real-time quantitative polymerase chain reaction, Western blot and immunohistochemistry. Lipopolysaccharides caused an up-regulation of ornithine decarboxylase mRNA level (2.73 ± 0.19-fold increase, p < 0.05) as well as ornithine decarboxylase protein level (4.05 ± 0.37-fold increase, p < 0.05). Endotoxemia attenuated hypoxic pulmonary vasoconstriction when compared with untreated control mice (26.3 ± 9.7% vs. 67.0 ± 17.5%). Difluoromethylornithine (20, 100, 500 mg kg−1 body weight intraperitoneal) restored hypoxic pulmonary vasoconstriction in lungs of endotoxemic mice in a dose-dependent way (25.8 ± 9.9%, 57.3 ± 17.2%, 62.3 ± 12.4%) and decreased hypoxic pulmonary vasoconstriction in control mice (53.6 ± 13.6%, 40.0 ± 14.9%, 35.9 ± 12.4%). These results show that endotoxemia induces ornithine decarboxylase expression and suggest that ornithine decarboxylase contributes to the endotoxemia-induced impairment of hypoxic pulmonary vasoconstriction. Inhibition of ornithine decarboxylase might be a target in the therapy of diseases with inflammation impaired hypoxic pulmonary vasoconstriction, like the sepsis-associated acute respiratory distress syndrome (ARDS).

Knocking down raptor in human keratinocytes affects ornithine decarboxylase in a post-transcriptional Manner following ultraviolet B exposure.

Non-melanoma skin cancer (NMSC) is the most common form of cancer. Ultraviolet-B (UVB) radiation has been shown to be a complete carcinogen in the development of NMSC. The mammalian target of rapamycin complex 1 (mTORC1) is upregulated by UVB. Ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway, is also upregulated in response to UVB. However, the interplay between these two pathways after UVB exposure remains unclear. The studies described here compare mRNA stability between normal human keratinocytes (HaCaT cells) and HaCaT cells with low levels of raptor to investigate whether the induction of ODC by UVB is dependent on mTORC1. We show that the knockdown of mTORC1 activity led to decreased levels of ODC protein both before and after exposure to 20mJ/cm2 UVB. ODC mRNA was less stable in cells with decreased mTORC1 activity. Polysome profiles revealed that the initiation of ODC mRNA translation did not change in UVB-treated cells. We have shown that the ODC transcript is stabilized by the RNA-binding protein human antigen R (HuR). To expand these studies, we investigated whether HuR functions to regulate ODC mRNA stability in human keratinocytes exposed to UVB. We show an increased cytoplasmic localization of HuR after UVB exposure in wild-type cells. The ablation of HuR via CRISPR/Cas9 did not alter the stability of the ODC message, suggesting the involvement of other trans-acting factors. These data suggest that in human keratinocytes, ODC mRNA stability is regulated, in part, by an mTORC1-dependent mechanism after UVB exposure.

MRNA dynamics and alternative conformations adopted under low and high arginine concentrations control polyamine biosynthesis in Salmonella.

Putrescine belongs to the large group of polyamines, an essential class of metabolites that exists throughout all kingdoms of life. The Salmonella speF gene encodes an inducible ornithine decarboxylase that produces putrescine from ornithine. Putrescine can be also synthesized from arginine in a parallel metabolic pathway. Here, we show that speF expression is controlled at multiple levels through regulatory elements contained in a long leader sequence. At the heart of this regulation is a short open reading frame, orf34, which is required for speF production. Translation of orf34 interferes with Rho-dependent transcription termination and helps to unfold an inhibitory RNA structure sequestering speF ribosome-binding site. Two consecutive arginine codons in the conserved domain of orf34 provide a third level of speF regulation. Uninterrupted translation of orf34 under conditions of high arginine allows the formation of a speF mRNA structure that is degraded by RNase G, whereas ribosome pausing at the consecutive arginine codons in the absence of arginine enables the formation of an alternative structure that is resistant to RNase G. Thus, the rate of ribosome progression during translation of the upstream ORF influences the dynamics of speF mRNA folding and putrescine production. The identification of orf34 and its regulatory functions provides evidence for the evolutionary conservation of ornithine decarboxylase regulatory elements and putrescine production.

Hepatitis C Virus Alters Metabolism of Biogenic Polyamines by a ROS-dependent Induction of Key Enzymes of Their Metabolism

Hepatitis C virus (HCV) infection often sets a chronic liver disease leading to fibrosis and hepatocellular carcinoma. These and other pathologies are induces by the virus by several mechanisms including pronounced oxidative stress and metabolic alterations. Though biogenic polyamines spermine and spermidine play pivotal role in liver functioning and even scavenging ROS, interplay between their metabolism and HCV infection has not been studied so far. Metabolism of spermine and spermidine is regulated mostly by three enzymes: ornithine decarboxylase (ODC) involved in their biosynthesis and by catabolic enzymes spermidine/spermine-N1-acetyl transferase (SSAT) and spermine oxidase (SMO). We have shown that chemically-induces oxidative stress triggers induction of ODC and SSAT via Nrf2 transcription factor. In lines with this, transient expression of two viral proteins, capsid and NS5A triggers a transient induction of both ODC and SSAT. In addition, both HCV core and NS5A induce sustained expression of spermine oxidase (SMO) in a ROS-independent fashion. Human hepatocarcinoma Huh7 cells harboring a full-length HCV replicon exhibited suppressed ODC and SSAT levels and elevated levels of SMO as well as depleted intracellular levels of spermine and spermidine. Noteworthy, SMO is a H2O2-producing enzyme implicated in development of gastric and colon cancer. Thus, role of HCV-induced SMO expression and of other alterations of polyamine metabolism in HCV replication and development of virus-associated liver pathologies should be explored in future.

Abstract 2006: AMX-513 polyamine depletion therapy inhibits tumor growth and reverses immunosuppression in cancers including MYC-driven neuroblastoma and pancreatic cancer

Abstract Tumorigenesis is associated with increased polyamine levels and involves the induction of ornithine decarboxylase (ODC), the initial rate-limiting enzyme in polyamine biosynthesis, and increased uptake of polyamines from the blood and diet. As well as contributing to proliferation, polyamines are reported to exert an immunosuppressive effect. Amplification of the MYC/MYCN oncogenes has been shown to directly induce ODC activity and inhibition of this enzyme by α-difluoromethylornithine (DFMO) markedly delays tumor development. Aminex Therapeutics is developing a polyamine depletion approach targeting both biosynthesis and transport of polyamines with AMX-513, a combination of the approved ODC inhibitor, DFMO, together with AMXT 1501, an alkylated polyamine mimetic which blocks polyamine uptake. In the syngeneic CT26.CL25 mouse model of colorectal cancer, AMX-513, dosed daily for four weeks, reduced tumor growth &amp;gt; 75% compared to vehicle-treated control in immunocompetent Balb/C mice. There was no effect in athymic nude mice indicating that tumor growth inhibition by AMX-513 is T-cell-dependent. In the induced transgenic K6/ODC squamous tumor mouse model, stable regression was sustained 10 weeks after treatment ended and was accompanied by tumor infiltrate increases in IFNγ and in CD3+ and CD8+ T-cells. Tumor infiltrates from AMX-513-treated KPC pancreatic cancer transgenic mice with tumor regressions showed &amp;gt;90% reductions in myeloid-derived suppressor cells (MDSCs; CD11b+ Gr-1+) but no changes in mature myeloid cells (CD11+Gr-1neg) by FACS analysis. AMX-513 treatment did not impact the percentage or number of CD4+CD25+FoxP3+ Tregs, but did significantly increase the percentage of activated CD8+ T cells in tumors. Neuroblastoma is an aggressive childhood cancer frequently associated with MYCN and ODC deregulation. In neuroblastoma cell lines, the AMX-513 combination was highly synergistic (CI&amp;lt;0.5). Prophylactic treatment of neuroblastoma-prone TH-MYCN transgenic mice with AMX-513 significantly extended survival compared to either agent alone (median survival time = 81.0±11.8 days versus DFMO alone = 57.1±7.1 days; P&amp;lt;0.0001). Treatment of mice with small palpable tumors with AMX-513 in combination with cyclophosphamide/topotecan significantly improved survival compared with either AMX-513 or cyclo/topo alone (5/9 long term survivors compared to 0/10 and 0/9 for cyclo/topo and AMT-513, respectively; P&amp;lt;0.001 in each case). Polyamine levels were significantly decreased in mice undergoing AMX-513 treatment compared to DFMO or AMXT 1501 alone. In conclusion, AMX-513 treatment alone or in combination with other cancer therapies results in significant tumor growth reduction in multiple cancer models and demonstrates novel immunotherapeutic potential. Clinical evaluation of AMX-513 is planned in 2017. Citation Format: Mark R. Burns, Kathy Fosnaugh, Michael G. Palfreyman, Laura Gamble, Jayne Murray, Sophie Allan, Georgina Eden, Sara Sarraf, Murray Norris, David Ziegler, Michelle Haber. AMX-513 polyamine depletion therapy inhibits tumor growth and reverses immunosuppression in cancers including MYC-driven neuroblastoma and pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2006. doi:10.1158/1538-7445.AM2017-2006

Cancer Chemopreventive Retinoids: Validation and Analysis of &amp;lt;i&amp;gt;in Vivo&amp;lt;/i&amp;gt; and &amp;lt;i&amp;gt;in Vitro&amp;lt;/i&amp;gt; Bioassay Results

Several natural and synthetic retinoids (vitamin-A derived analogies) were examined for their potential anti-cancer activity in both in vivo animal models and a novel in vitro human keratinocyte clonal growth bioassay system. The natural retinoids included all-trans-retinoic (RA), 13-cis-retinoic acid, 4-oxoretinoic acid, and retinol. Among the synthetic retinoids tested were all trans N-(4-hydroxy(phenyl)retinamide, 3-substituted oxoretinoic acids, and 13 cis-N-ethylretinamide. The animal models employed were: 1) vitamin A-deficient hamster tracheal organ assay (HTOC); 2) the benzo(α)pyrene-induced squamous metaplasia in a hamster tracheal organ system (BP-HTOC); 3) the mouse skin tumor promoter (TPA)-induced ornithine decarboxylase enzyme assay(ODC); 4) the mouse skin papilloma (MPA) assay; and 5) a novel retinoid bioassay in which retinoids display IC50 values to inhibit clonal growth of NHK. All-trans-RA, 4-oxoretinoic acid and retinol were consistently more active than any of the synthetic derivatives in all bioassays tested. A statistical model was developed and significant positive correlations were found between: 1) ED50 values in the HTOC system and reduction in TPA-induced ODC enzyme activity; 2) tumors per animal in the MPA bioassay and suppression of TPA-induced ODC activity; and 3) a positive correlation between suppression of tumors per animal in the MPA assay, and retinoid inhibition of keratinocyte clonal growth. Test retinoids, were tested for their capacity to inhibit the clonal growth of a squamous carcinoma cell line (SCC-25), which were found to be 2 - 3 logs less sensitive for each tested retinoid than the corresponding activity against NHK cells. Antineoplastic retinoid drugs were reviewed.

The relevance of piroxicam for the prevention and treatment of nonmelanoma skin cancer and its precursors.

Piroxicam (PXM), a nonsteroidal anti-inflammatory drug, is an enolic benzothiazine and a potent member of the oxicam series. The drug suppresses the synthesis of proinflammatory enzymes, such as cyclo-oxygenases-1 and -2 (COX-1 and 2), downregulates the production of prostaglandins (PGs) and tromboxanes, and inhibits polyamines production by blocking ornithine decarboxylase induction involved in nonmelanoma skin carcinogenesis. In addition, PXM is able to induce tumor cell apoptosis and suppresses metalloproteinase 2 activities. Skin carcinogenesis is a multistep process in which the accumulation of genetic events leads to a gradually dysplastic cellular expression, deregulation of cell growth, and carcinomatous progression. COX-1 upregulation plays a significant role in PG and vascular epidermal growth factor production supporting tumor growth. Increased level of PGs in premalignant and/or malignant cutaneous tumors is also favored by upregulation of COX-2 and downregulation of the tumor suppressor gene 15-hydroxy-prostaglandin dehydrogenase. Chemoprevention can be a hopeful approach to inhibit carcinoma occurrence before an invasive tumor develops. The chemopreventive effect of nonsteroidal anti-inflammatory drugs on nonmelanoma skin cancers has been established. In this study, we highlighted the different modalities of action of PXM on the pathogenesis of nonmelanoma skin cancer, analyzing and evaluating binding modes and energies between COX-1 or COX-2 and PXM by protein–ligand molecular docking. Our clinical experience about the local use of PXM on actinic keratoses and field cancerization is also reported, confirming its efficacy as target therapy.

Gliadin activates arginase pathway in RAW264.7 cells and in human monocytes

Celiac disease (CD) is an autoimmune enteropathy triggered in susceptible individuals by the ingestion of gliadin-containing grains. Recent studies have demonstrated that macrophages play a key role in the pathogenesis of CD through the release of inflammatory mediators such as cytokines and nitric oxide (NO). Since arginine is the obliged substrate of iNOS (inducible nitric oxide synthase), the enzyme that produces large amount of NO, the aim of this work is to investigate arginine metabolic pathways in RAW264.7 murine macrophages after treatment with PT-gliadin (PTG) in the absence and in the presence of IFNγ. Our results demonstrate that, besides strengthening the IFNγ-dependent activation of iNOS, gliadin is also an inducer of arginase, the enzyme that transforms arginine into ornithine and urea. Gliadin treatment increases, indeed, the expression and the activity of arginase, leading to the production of polyamines through the subsequent induction of ornithine decarboxylase. This effect is strengthened by IFNγ. The activation of these pathways takes advantage of the increased availability of arginine due to a decreased system y+l-mediated efflux, likely ascribable to a reduced expression of Slc7a6 transporter. A significant induction of arginase expression is also observed in human monocytes from healthy subject upon treatment with gliadin, thus demonstrating that gluten components trigger changes in arginine metabolism in monocyte/macrophage cells.

P38δ Regulates p53 to Control p21Cip1 Expression in Human Epidermal Keratinocytes

PKCδ suppresses keratinocyte proliferation via a mechanism that involves increased expression of p21(Cip1). However, the signaling mechanism that mediates this regulation is not well understood. Our present studies suggest that PKCδ activates p38δ leading to increased p21(Cip1) promoter activity and p21(Cip1) mRNA/protein expression. We further show that exogenously expressed p38δ increases p21(Cip1) mRNA and protein and that p38δ knockdown or expression of dominant-negative p38 attenuates this increase. Moreover, p53 is an intermediary in this regulation, as p38δ expression increases p53 mRNA, protein, and promoter activity, and p53 knockdown attenuates the activation. We demonstrate a direct interaction of p38δ with PKCδ and MEK3 and show that exogenous agents that suppress keratinocyte proliferation activate this pathway. We confirm the importance of this regulation using a stratified epidermal equivalent model, which mimics in vivo-like keratinocyte differentiation. In this model, PKCδ or p38δ knockdown results in reduced p53 and p21(Cip1) levels and enhanced cell proliferation. We propose that PKCδ activates a MEKK1/MEK3/p38δ MAPK cascade to increase p53 levels and p53 drives p21(Cip1) gene expression.

Inhibitory effects of Momordica grosvenori Swingle extracts on 12-O-tetradecanoylphorbol 13-acetate-induced skin inflammation and tumor promotion in mouse skin

Our previous data showed that the Momordica grosvenori Swingle extract (MSE) exhibited the anti-inflammatory effect through markedly suppressed LPS-induced up-regulation of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and ODC (ornithine decarboxylase) gene expression in RAW 264.7 cells. Regarding the link between inflammation and carcinogenesis, we further investigated the bio-molecular mechanisms of both anti-inflammatory and anti-tumor activities in vivo using a TPA (12-O-tetradecanoyl phorbol 13-acetate)-stimulated mouse skin model. Pretreatment with MSE in mouse skin has led to the reduction of TPA-induced nuclear translocation of the nuclear factor-κB (NFκB) subunits as well as phosphorylation of IκBα and p65 subsequent reduction of IκBα degradation. In addition, the MSE inhibitory effect on upstream of NFκB was found to involve the transcriptional effects of MAPK signaling as indicated by strong suppression on TPA-induced activation of extracellular signal regulate kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK)1/2, phosphatidylinositol 3-kinase (PI3K) and Akt. Moreover, MSE significantly inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-TPA-induced skin tumor formation in mice measured by the tumor multiplicity of papillomas at 20 weeks. The results suggested that MSE contained promising functional ingredients capable of preventing inflammation-associated tumorigenesis.

Liver Function in Rats Acclimatized to a Simulated Altitude of 5500 m

We examined the functional and morphological characteristics of the liver in rats acclimatized to a simulated altitude of 5500 m. We examined the metabolic activity and cytoplasmic distribution of liver mitochondria and the capacity of the liver to regenerate after partial hepatectomy. Mitochondrial respiration, oxidative phosphorylation, the respiratory control ratio (RCR), and the morphological characteristics of mitochondria in liver sections were studied after 3 months acclimatization to high altitude (HA). Partial hepatectomy was performed in a subset of animals after 30 days acclimatization to 5500 m. The rate of hepatic regeneration, induction of ornithine decarboxylase and uridine diphosphate glucuronyltransferase (UGT1a1), and plasma bilirubin were measured 24, 48, 72, and 96 hours after hepatectomy. Acclimatization to 5500 m did not affect the mitochondrial respiratory capacity or oxidative phosphorylation. The RCR decreased and acid phosphatase activity increased, which suggests that there were subtle changes in mitochondrial integrity. In addition, mitochondria were distributed more homogeneously in hepatocytes. Hepatic regeneration, which was associated with 25-fold induction of the ornithine decarboxylase, did not differ between controls and the altitude-exposed animals. Plasma bilirubin levels rose markedly 24 hours after hepatectomy, but returned to control levels 48 hours after the operation in the altitude-exposed animals. Thus, the remarkable functional capacity of the liver was retained at simulated HA. Redistribution of hepatic mitochondria seems to play an important role in maintaining hepatic function despite severe cellular hypoxia.

Polyamines Are Critical for the Induction of the Glutamate Decarboxylase-dependent Acid Resistance System in Escherichia coli

As part of our studies on the biological functions of polyamines, we have used a mutant of Escherichia coli that lacks all the genes for polyamine biosynthesis for a global transcriptional analysis on the effect of added polyamines. The most striking early response to the polyamine addition is the increased expression of the genes for the glutamate-dependent acid resistance system (GDAR) that is important for the survival of the bacteria when passing through the acid environment of the stomach. Not only were the two genes for glutamate decarboxylases (gadA and gadB) and the gene for glutamate-γ-aminobutyrate antiporter (gadC) induced by the polyamine addition, but the various genes involved in the regulation of this system were also induced. We confirmed the importance of polyamines for the induction of the GDAR system by direct measurement of glutamate decarboxylase activity and acid survival. The effect of deletions of the regulatory genes on the GDAR system and the effects of overproduction of two of these genes were also studied. Strikingly, overproduction of the alternative σ factor rpoS and of the regulatory gene gadE resulted in very high levels of glutamate decarboxylase and almost complete protection against acid stress even in the absence of any polyamines. Thus, these data show that a major function of polyamines in E. coli is protection against acid stress by increasing the synthesis of glutamate decarboxylase, presumably by increasing the levels of the rpoS and gadE regulators.

The induction of cardiac ornithine decarboxylase by β2‐adrenergic agents is associated with calcium channels and phosphorylation of ERK1/2

The role that the induction of cardiac ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, by beta-adrenergic agents may have in heart hypertrophy is a controversial issue. Besides, the signaling pathways related to cardiac ODC regulation have not been fully elucidated. Here we show that in Balb C mice the stimulation of cardiac ODC activity by adrenergic agents was mainly mediated by β2 -adrenergic receptors, and that this induction was lower in the hypertrophic heart. Interestingly, this stimulation was abolished by the L-calcium channel antagonists verapamil and nifedipine. In addition, whereas the treatment with β2 -adrenergic agents was associated to both the increases in ODC, ODC-antizyme inhibitor 1 (AZIN1), c-fos and c-myc mRNA levels and the phosphorylation of CREB and MAP kinases ERK1 and ERK2 (ERK1/2), the co-treatment with L-calcium channel blockers differentially prevented most of these changes. These results suggest that the stimulation of cardiac ODC by β2 -adrenergic agents is associated with the activation of MAP kinases through the participation of L-calcium channels, and that by itself p-CREB does not appear to be sufficient for the transcriptional activation of ODC. In addition, post-translational mechanisms related with the induction of AZIN1 appear to be related to the increase of cardiac ODC activity.

Ascorbic acid inhibits ferric nitrilotriacetate induction of ornithine decarboxylase, DNA synthesis, oxidative stress, and hepatotoxicity in rats

Ascorbic acid (AA) is a naturally occurring phenolic compound with antioxidant properties used in food, cosmetics, and pharmaceutical products. In this study, the effect of AA on ferric nitrilotriacetate (Fe-NTA)-induced hepatotoxicity in rats has been examined. Fe-NTA alone enhances ornithine decarboxylase activity to 4.5-fold and tritiated thymidine incorporation in DNA to 3.6-fold in livers compared with the corresponding saline-treated controls. The enhanced ornithine decarboxylase activity and DNA synthesis showed a reduction to 3.02- and 1.88-fold, respectively, at a higher dose of 2 mg AA per day per animal, compared with the Fe-NTA-treated groups. Fe-NTA treatment also enhanced the hepatic microsomal lipid peroxidation to 1.7-fold compared to saline-treated controls. These changes were reversed significantly in animals receiving pretreatment of AA. The present data shows that AA can reciprocate the toxic effects of Fe-NTA and can serve as a potent chemopreventive agent to suppress oxidant-induced tissue injury and hepatotoxicity in rats.

Ornithine: The Overlooked Molecule in the Regulation of Polyamine Metabolism3

We overexpressed a mouse ornithine decarboxylase gene under the control of a constitutive and an estradiol-inducible promoter in Arabidopsis thaliana to increase our understanding of the regulation of polyamine metabolism. Of particular interest was the role of the substrate ornithine not only in the regulation of polyamine biosynthesis, but also in the accumulation of related amino acids in response to short-term induction of this enzyme. We hypothesized that the inducible expression of the transgene would mimic the natural responses of plants to changing conditions, e.g. under stress conditions and during rapid growth. Our results reveal that ornithine, even though present in relatively small quantities (compared with other amino acids of the glutamate-arginine-proline pathway), may not only be the key regulator of polyamine biosynthesis in Arabidopsis, but it may also regulate the entire subset of pathways for glutamate to arginine and to proline. Indirectly, it could also regulate putrescine catabolism, therefore contributing to the γ-aminobutyric acid content of the cells. Furthermore, the induction of mouse ornithine decarboxylase resulted in up- and down-regulation of several amino acids in the transgenic plants. It was learned that the turnover of putrescine in both the wild type and the transgenic plants occurs rapidly, with a half-life of 6-8 h.

Helicobacter pyloriCagA induces ornithine decarboxylase upregulation via Src/MEK/ERK/c-Myc pathway: implication for progression of gastric diseases

Helicobacter pylori (H. pylori) dysregulates the expression of various genes resulting in gastric precursor lesions and cancer. Meanwhile, ornithine decarboxylase (ODC) is a key enzyme that catalyzes the formation of polyamines which are critical for cell growth. So far, the possible regulation of ODC by H. pylori and its virulence factors, and the associated mechanism in gastric epithelial cells remains undefined. In the present study, we found that cellular ODC protein was upregulated by wild-type H. pylori infection and ectopic expression of a cytotoxin-associated gene A (CagA). As a negative control, there was no such effect by cagA-mutant H. pylori infection. Results of signal protein inhibitor treatment demonstrated that the Src, MEK (mitogen-activated protein kinase kinase) and ERK (extracellular signal-regulated kinase) pathway was involved. Moreover, when c-Myc was inhibited, the stimulatory effect of CagA on ODC expression was abolished. Clinically, a positive correlation between c-Myc and ODC expression was observed in patient-derived abnormal gastric tissues. These results implied that the Src/MEK/ERK/c-Myc pathway was required for CagA-mediated ODC induction. Finally, inhibition of ODC expression led to decreased foci formation of gastric epithelial cells before and after H. pylori infection, and ODC protein was over-expressed in precancerous gastric lesions and primary gastric cancer. Collectively, our findings provide new insights into the mechanism behind H. pylori-infection-associated gastric diseases.

Suppression of 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity by resveratrol derivatives

AbstractAs demonstrated previously, resveratrol (3,4',5-trihydroxy-trans-stilbene) inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC), the key rate limiting enzyme in mammalian polyamine synthesis. Using human bladder epithelial carcinoma HTB-24 cells in culture where resveratrol inhibits induction with an IC50 of 8.8 µM, we now report potential metabolites demonstrate greater activity [tetrabutylammonium (E)-4-(3,5-dihydroxystyryl)phenyl sulfate (IC50 1.2 µM), resveratrol tripotassium 3,5,4'-trisulfate (IC50 1.8 µM), resveratrol tripotassium 3,4'-disulfate (IC50 1.8 µM), and resveratrol tripotassium 3,5-disulfate (IC50 2.3 µM)]. Based on RT-PCR studies, ODC inhibition occurs at the transcriptional level, but this was not due to direct inhibition of protein kinase C (e.g., resveratrol IC50, 79 µM; resveratrol tripotassium 3,5-disulfate IC50, 49 µM). Additional work is underway to more fully investigate this potentially important observation. [This work was supported by program project P01 CA48112 awarded by the National Cancer Institute. SL acknowledges Indo-US Science and Technology Forum (IUSSTF), New Delhi for a Research Fellowship].

Suppression of 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity by resveratrol derivatives

As demonstrated previously, resveratrol (3,4',5-trihydroxy-trans-stilbene) inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC), the key rate limiting enzyme in mammalian polyamine synthesis. Using human bladder epithelial carcinoma HTB-24 cells in culture where resveratrol inhibits induction with an IC50 of 8.8 µM, we now report potential metabolites demonstrate greater activity [tetrabutylammonium (E)-4-(3,5-dihydroxystyryl)phenyl sulfate (IC50 1.2 µM), resveratrol tripotassium 3,5,4'-trisulfate (IC50 1.8 µM), resveratrol tripotassium 3,4'-disulfate (IC50 1.8 µM), and resveratrol tripotassium 3,5-disulfate (IC50 2.3 µM)]. Based on RT-PCR studies, ODC inhibition occurs at the transcriptional level, but this was not due to direct inhibition of protein kinase C (e.g., resveratrol IC50, 79 µM; resveratrol tripotassium 3,5-disulfate IC50, 49 µM). Additional work is underway to more fully investigate this potentially important observation. [This work was supported by program project P01 CA48112 awarded by the National Cancer Institute. SL acknowledges Indo-US Science and Technology Forum (IUSSTF), New Delhi for a Research Fellowship].

The Enzymatic Activities of the Escherichia coli Basic Aliphatic Amino Acid Decarboxylases Exhibit a pH Zone of Inhibition

The stringent response regulator ppGpp has recently been shown by our group to inhibit the Escherichia coli inducible lysine decarboxylase, LdcI. As a follow-up to this observation, we examined the mechanisms that regulate the activities of the other four E. coli enzymes paralogous to LdcI: the constitutive lysine decarboxylase LdcC, the inducible arginine decarboxylase AdiA, the inducible ornithine decarboxylase SpeF, and the constitutive ornithine decarboxylase SpeC. LdcC and SpeC are involved in cellular polyamine biosynthesis, while LdcI, AdiA, and SpeF are involved in the acid stress response. Multiple mechanisms of regulation were found for these enzymes. In addition to LdcI, LdcC and SpeC were found to be inhibited by ppGpp; AdiA activity was found to be regulated by changes in oligomerization, while SpeF and SpeC activities were regulated by GTP. These findings indicate the presence of multiple mechanisms regulating the activity of this important family of decarboxylases. When the enzyme inhibition profiles are analyzed in parallel, a "zone of inhibition" between pH 6 and pH 8 is observed. Hence, the data suggest that E. coli utilizes multiple mechanisms to ensure that these decarboxylases remain inactive around neutral pH possibly to reduce the consumption of amino acids at this pH.