Ornithine Decarboxylase Protein Research Articles

Target of Rapamycin Mediated Ornithine Decarboxylase Antizyme Modulate Intracellular Putrescine and Ganoderic Acid Content in Ganoderma lucidum.

ABSTRACTPutrescine (Put) has been shown to play an important regulatory role in cell growth in organisms. As the primary center regulating the homeostasis of polyamine (PA) content, ornithine decarboxylase antizyme (AZ) can regulate PA content through feedback. Nevertheless, the regulatory mechanism of Put is poorly understood in fungi. Here, our analysis showed that GlAZ had a modulate effect on intracellular Put content by interacting with ornithine decarboxylase (ODC) proteins and reducing its intracellular protein levels. In addition, GlAZ upregulated the metabolic pathway of ganoderic acid (GA) biosynthesis in Ganoderma lucidum by modulating the intracellular Put content. However, a target of rapamycin (TOR) was found to promote the accumulation of intracellular Put after the GlTOR inhibitor Rap was added exogenously, and unbiased analyses demonstrated that GlTOR may promote Put production through its inhibitory effect on the level of GlAZ protein in GlTOR-GlAZ-cosilenced strains. The effect of TOR on fungal secondary metabolism was further explored, and the content of GA in the GlTOR-silenced strain after the exogenous addition of the inhibitor Rap was significantly increased compared with that in the untreated wild-type (WT) strain. Silencing of TOR in the GlTOR-silenced strains caused an increase in GA content, which returned to the WT state after replenishing Put. Moreover, the content of GA in GlTOR-GlAZ-cosilenced strains was also not different from that in the WT strain. Consequently, these results strongly indicate that GlTOR affects G. lucidum GA biosynthesis via GlAZ.IMPORTANCE Research on antizyme (AZ) in fungi has focused on the mechanism by which AZ inhibits ornithine decarboxylase (ODC). Moreover, there are existing reports on the regulation of AZ protein translation by TOR. However, little is known about the mechanisms that influence AZ in fungal secondary metabolism. Here, both intracellular Put content and GA biosynthesis in G. lucidum were shown to be regulated through protein interactions between GlAZ and GlODC. Furthermore, exploration of upstream regulators of GlAZ suggested that GlAZ was regulated by the upstream protein GlTOR, which affected intracellular Put levels and ganoderic acid (GA) biosynthesis. The results of our work contribute to the understanding of the upstream regulation of Put and provide new insights into PA regulatory systems and secondary metabolism in fungi.

Regulation of polyamine homeostasis through an antizyme citrullination pathway

This study reveals an uncovered mechanism for the regulation of polyamine homeostasis through protein arginyl citrullination of antizyme (AZ), a natural inhibitor of ornithine decarboxylase (ODC). ODC is critical for the cellular production of polyamines. AZ binds to ODC dimers and promotes the degradation of ODC via the 26S proteasome. This study demonstrates the protein citrullination of AZ catalyzed by peptidylarginine deiminase type 4 (PAD4) both in vitro and in cells. Upon PAD4 activation, the AZ protein was citrullinated and accumulated, leading to higher levels of ODC proteins in the cell. In the PAD4-overexpressing and activating cells, the levels of ODC enzyme activity and the product putrescine increased with the level of citrullinated AZ proteins and PAD4 activity. Suppressing cellular PAD4 activity reduces the cellular levels of ODC and downregulates cellular polyamines. Furthermore, citrullination of AZ in the C-terminus attenuates AZ function in the inhibition, binding, and degradation of ODC. This paper provides evidence to illustrate that PAD4-mediated AZ citrullination upregulates cellular ODC and polyamines by retarding ODC degradation, thus interfering with the homeostasis of cellular polyamines, which may be an important pathway regulating AZ functions that is relevant to cancer biology.

3′-Hydroxypterostilbene Inhibits 7,12-Dimethylbenz[a]anthracene (DMBA)/12-O-Tetradecanoylphorbol-13-Acetate (TPA)-Induced Mouse Skin Carcinogenesis

BackgroundA natural pterostilbene analogue isolated from the herb Sphaerophysa salsula, 3′-hydroxypterostilbene (HPSB), exhibits antiproliferative activity in several cancer cell lines; however, the inhibitory effects of HPSB on skin carcinogenesis remains unclear. PurposeThe aim of this study was to evaluate the inhibitory effects of HPSB on two-stage skin carcinogenesis in mice and its potential mechanism. Study Design and MethodsThis study investigated the anti-inflammatory and anti-tumor effects of HPSB in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated acute skin inflammation and 7,12-dimethylbenz[a]anthracene (DMBA)/TPA-induced two-stage skin carcinogenesis model. In addition, the effects of HPSB on the modulation of the phase I and phase II metabolizing enzymes in the DMBA-induced HaCaT cell model were investigated. ResultsThe results provide evidence that topical treatment with HPSB significantly inhibits TPA-induced epidermal hyperplasia and leukocyte infiltration through the down-regulation of cyclooxygenase-2 (COX-2), matrix metalloprotein-9 (MMP-9), and ornithine decarboxylase (ODC) protein expression in mouse skin. Furthermore, HPSB suppresses DMBA/TPA-induced skin tumor incidence and multiplicity via the inhibition of proliferating cell nuclear antigen (PCNA), Cyclin B1 and cyclin-dependent kinase 1 (CDK1) expression in the two-stage skin carcinogenesis model. In addition, pretreatment with HPSB markedly reduces DMBA-induced cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1 (CYP1B1) gene expression in human keratinocytes; however, HPSB does not significantly affect the gene expression of the phase II enzymes. ConclusionThis is the first study to show that topical treatment with HPSB prevents mouse skin tumorigenesis. Overall, our study suggests that natural HPSB may serve as a novel chemopreventive agent capable of preventing carcinogen activation and inflammation-associated tumorigenesis.

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).

Regulatory role of l-proline in fetal pig growth and intestinal epithelial cell proliferation.

l-proline (Pro) is a precursor of ornithine, which is converted into polyamines via ornithine decarboxylase (ODC). Polyamines plays a key role in the proliferation of intestinal epithelial cells. The study investigated the effect of Pro on polyamine metabolism and cell proliferation on porcine enterocytes in vivo and in vitro. Twenty-four Huanjiang mini-pigs were randomly assigned into 1 of 3 groups and fed a basal diet that contained 0.77% alanine (Ala, iso-nitrogenous control), 1% Pro or 1% Pro + 0.0167% α-difluoromethylornithine (DFMO) from d 15 to 70 of gestation. The fetal body weight and number of fetuses per litter were determined, and the small and large intestines were obtained on d 70 ± 1.78 of gestation. The in vitro study was performed in intestinal porcine epithelial (IPEC-J2) cells cultured in Dulbecco's modified Eagle medium-high glucose (DMEM-H) containing 0 μmol/L Pro, 400 μmol/L Pro, or 400 μmol/L Pro + 10 mmol/L DFMO for 4 d. The results showed that maternal dietary supplementation with 1% Pro increased fetal weight; the protein and DNA concentrations of the fetal small intestine; and mRNA levels for potassium voltage-gated channel, shaker-related subfamily, member 1 (Kv1.1) in the fetal small and large intestines (P < 0.05). Supplementing Pro to either gilts or IPEC-J2 cells increased ODC protein abundances and polyamine concentrations in the fetal intestines and IPEC-J2 cells (P < 0.05). In comparison with the Pro group, the combined administration of Pro and DFMO reduced the expression of ODC protein and spermine concentration in the fetal intestine, as well as the concentrations of putrescine, spermidine and spermine in IPEC-J2 cells (P < 0.05). Meanwhile, the percentage of cells in the S-phase and the mRNA levels of proto-oncogenes c-fos and c-myc were increased in response to Pro supplementation, whereas depletion of cellular polyamines with DFMO increased tumor protein p53 (p53) mRNA levels (P < 0.05). Taken together, dietary supplementation with Pro improved fetal pig growth and intestinal epithelial cell proliferation via enhancing polyamine synthesis.

Abstract 1788: Targeting ornithine decarboxylase down-regulates multiple pathways involved in precancerous lesion formation of esophageal squamous cell cancer

Abstract Precancerous lesion of esophageal squamous cell cancer (ESCC) owns the characteristic of bidirectional instability, which can develop to cancer or reverse to normal epithelium by appropriate interference. Therefore, the feature of precancerous lesion provides possibility for ESCC chemoprevention. In the current study, we found that ornithine decarboxylase (ODC) protein level is higher in esophageal precancerous lesions compared with normal esophageal epithelium. Knocking down ODC suppresses cell proliferation and clone formation of ESCC. Moreover, difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, decreases N-nitrosomethylbenzylamine (NMBA)-induced rats’ esophageal precancerous lesions. Further molecular study indicated that ODC down-regulates p38, ERK in the MAP kinase pathway and AKT/mTOR/p70S6K pathway, which are involved in the carcinogenesis process. Overall, our results indicate that ODC may be a potential target for ESCC chemoprevention. Citation Format: Yifei Xie, Qiong Wu, Jimin Zhao, Kangdong Liu. Targeting ornithine decarboxylase down-regulates multiple pathways involved in precancerous lesion formation of esophageal squamous cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1788.

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.

Aβ stimulates microglial activation through antizyme-dependent downregulation of ornithine decarboxylase.

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative disorders. Its pathology is associated with the deposition of amyloid β (Aβ), an abnormal extracellular peptide. Moreover, its pathological progression is closely accompanied by neuroinflammation. Specifically, Aβ-associated microglial overactivation may have the central role in AD pathogenesis. Interestingly, arginine metabolism may contribute to the equilibrium between M1 and M2 microglia. However, little is known about the involvement of arginine metabolism in Aβ-induced microglial neuroinflammation and neurotoxicity. Moreover, the underlying mechanism by which Aβ induces the transition of microglia to the M1 phenotype remains unclear. In this study, we investigated the role of Aβ in mediating microglial activation and polarization both in vitro and in vivo. Our results demonstrated that under the Aβ treatment, ornithine decarboxylase (ODC), a rate-limiting enzyme in the regulation of arginine catabolism, regulates microglial activation by altering the antizyme (AZ) + 1 ribosomal frameshift. Furthermore, the restoration of ODC protein expression levels has profound effects on inhibition of Aβ-induced M1 markers and thus attenuates microglial-mediated cytotoxicity. Altogether, our findings suggested that Aβ may contribute to M1-like activation by disrupting the balance between ODC and AZ in microglia.

Knockout of Raptor destabilizes ornithine decarboxylase mRNA and decreases binding of HuR to the ODC transcript in cells exposed to ultraviolet-B irradiation

Non-melanoma skin cancer (NMSC) is the most commonly diagnosed cancer in the United States. Ultraviolet-B (UVB) irradiation is the primary carcinogen responsible for stimulating NMSC development. Ornithine Decarboxylase (ODC), the first rate-limiting enzyme in the synthesis of polyamines, is upregulated in response to a variety of proliferation stimuli, including UVB exposure. Our previous studies have demonstrated regulation of ODC synthesis by the mammalian target of rapamycin complex 1 (mTORC1) in cells transformed by oncogenic Ras. The goal of these studies was to better understand the link between mTORC1 and ODC in nontransformed cells treated with UVB. We show that the ablation of mTORC1 activity by conditional knockout of its essential component Raptor led to decreased levels of ODC protein both before and after exposure to 10 mJ/cm2 UVB. Moreover, ODC mRNA was destabilized in the absence of Raptor, suggesting post-transcriptional regulation. We have previously shown that the ODC transcript is stabilized by the RNA binding protein (RBP) human antigen R (HuR), and the intracellular localization of HuR responds to changes in mTORC1 activity. To expand these studies, we investigated whether HuR functions to regulate ODC mRNA stability after UVB exposure. Our results show an increased localization of HuR to the cytoplasm after UVB exposure in wild-type cells compared to Raptor knockout cells, and this is accompanied by greater association of HuR with the ODC transcript. These data suggest that the localization of HuR in response to UVB is influenced, at least in part, by mTORC1 and that HuR can bind to and stabilize ODC mRNA after UVB exposure in an mTORC1-dependent manner.

Activation of ornithine decarboxylase protects against methylmercury toxicity by increasing putrescine

We previously reported significantly increased level of putrescine, a polyamine, in the brains of mice administered methylmercury. Moreover, addition of putrescine to culture medium reduced methylmercury toxicity in C17.2 mouse neural stem cells. In this study, the role of ornithine decarboxylase (ODC), an enzyme involved in putrescine synthesis, in response to methylmercury toxicity was investigated. Methylmercury increased ODC activity in mouse cerebrum and cerebellum, but this increase was hardly observed in the kidney and liver, where methylmercury accumulated at a high concentration. In the cerebrum and cerebellum, increased putrescine was observed with methylmercury administration. Methylmercury increased ODC activity in C17.2 cells, but this was almost completely abolished in the presence of an ODC inhibitor. Methylmercury also increased the level of ODC protein in mouse brain and C17.2 cells. In addition, C17.2 cells pretreated with ODC inhibitor showed higher methylmercury sensitivity than control cells. These results suggest that the increased ODC activity by methylmercury is involved in the increase in putrescine level, and ODC plays an important role in the reduction of methylmercury toxicity. This is the first study to provide evidence that increased ODC activity may be a protective response against methylmercury-induced neurotoxicity.

The ODC 3'-Untranslated Region and 5'-Untranslated Region Contain cis-Regulatory Elements: Implications for Carcinogenesis.

It has been hypothesized that both the 3′-untranslated region (3′UTR) and the 5′-untranslated region (5′UTR) of the ornithine decarboxylase (ODC) mRNA influence the expression of the ODC protein. Here, we use luciferase expression constructs to examine the influence of both UTRs in keratinocyte derived cell lines. The ODC 5′UTR or 3′UTR was cloned into the pGL3 control vector upstream or downstream of the luciferase reporter gene, respectively, and luciferase activity was measured in both non-tumorigenic and tumorigenic mouse keratinocyte cell lines. Further analysis of the influence of the 3′UTR on luciferase activity was accomplished through site-directed mutagenesis and distal deletion analysis within this region. Insertion of either the 5′UTR or 3′UTR into a luciferase vector resulted in a decrease in luciferase activity when compared to the control vector. Deletion analysis of the 3′UTR revealed a region between bases 1969 and 2141 that was inhibitory, and mutating residues within that region increased luciferase activity. These data suggest that both the 5′UTR and 3′UTR of ODC contain cis-acting regulatory elements that control intracellular ODC protein levels.

Insights on ornithine decarboxylase silencing as a potential strategy for targeting retinoblastoma

Ornithine Decarboxylase (ODC) is a key enzyme involved in polyamine synthesis and is reported to be up regulated in several cancers. However, the effect of ODC gene silencing in retinoblastoma is to be understood for utilization in therapeutic applications. Hence, in this study, a novel siRNA (small interference RNA) targeting ODC was designed and validated in Human Y79 retinoblastoma cells for its effects on intracellular polyamine levels, Matrix Metalloproteinase 2 & 9 activity and Cell cycle. The designed siRNA showed efficient silencing of ODC mRNA expression and protein levels in Y79 cells. It also showed significant reduction of intracellular polyamine levels and altered levels of oncogenic LIN28b expression. By this study, a regulatory loop is proposed, wherein, ODC silencing in Y79 cells to result in decreased polyamine levels, thereby, leading to altered protein levels of Lin28b, MMP-2 and MMP-9, which falls in line with earlier studies in neuroblastoma. Thus, by this study, we propose ODC silencing as a prospective strategy for targeting retinoblastoma.

Abstract 4774: Polyamines are rich and induces epigenetic change by inhibition of LSD1 in cancer stem-like cells

Abstract Cancer stem cells (CSC) are highly tumorigenic and resistant to chemotherapy and radiotherapy. We have previously reported that a small fraction of cancer cells with low proteasome activity had CSC-like properties. Ornithine decarboxylase (ODC) is an enzyme which converts ornithine to polyamines including putrescine, spermidine, and spermine. Proteasomal degradation of ODC is known to be ubiquitin-independent. The aim of this study was to explain how low proteasome activity gave CSC-like properties. Western blot showed high ODC protein level in CSC-like cells in cervical cancer and osteosarcoma cell lines, although ODC mRNA level is not high. These findings suggest that ODC protein level is high in CSC-like cells because ODC protein degradation is inactive. To investigate polyamine metabolism in CSC-like cells, we performed GC-MS analysis and found that putrescine, spermidine, and spermine were enriched in CSC-like cells. Furthermore, flux analysis showed conversion from ornithine to polyamines was more rapid in CSC-like cells. We performed a computational structure analysis and found that polyamines bind and inhibit demethylase site of LSD1 which is known to be a demethylase of histone H3K4 and a nuclear homolog of amine oxidases. Spermine was the strongest binding energy. An enzyme assay showed that LSD1 activity was dose-dependently inhibited by polyamines and spermine was the strong inhibitor. Finally, we analyzed performed ChIP sequencing analysis by using HeLa CSC-like cells and found that more global enrichment of H3K4me3 and less global enrichment of H3K4me1 than non-CSC-like cells. In conclusion, polyamines are rich in CSC, and be able to inhibit LSD1 activty and induce epigenetic alterations especially in H3K4 methylation in CSC. Citation Format: Keisuke Tamari, Hideshi Ishii, Masamitsu Konno, Naohiro Nishida, Jun Koseki, Koichi Kawamoto, Fumiaki Isohashi, Yuichiro Doki, Masaki Mori, Kazuhiko Ogawa. Polyamines are rich and induces epigenetic change by inhibition of LSD1 in cancer stem-like cells [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 4774. doi:10.1158/1538-7445.AM2017-4774

Targeting ornithine decarboxylase (ODC) inhibits esophageal squamous cell carcinoma progression

To explore the function of ornithine decarboxylase in esophageal squamous cell carcinoma progression and test the effectiveness of anti-ornithine decarboxylase therapy for esophageal squamous cell carcinoma. In this study, we examined the expression pattern of ornithine decarboxylase in esophageal squamous cell carcinoma cell lines and tissues using immunohistochemistry and Western blot analysis. Then we investigated the function of ornithine decarboxylase in ESCC cells by using shRNA and an irreversible inhibitor of ornithine decarboxylase, difluoromethylornithine. To gather more supporting pre-clinical data, a human esophageal squamous cell carcinoma patient-derived xenograft mouse model (C.B-17 severe combined immunodeficient mice) was used to determine the antitumor effects of difluoromethylornithine in vivo. Our data showed that the expression of the ornithine decarboxylase protein is increased in esophageal squamous cell carcinoma tissues compared with esophagitis or normal adjacent tissues. Polyamine depletion by ODC shRNA not only arrests esophageal squamous cell carcinoma cells in the G2/M phase, but also induces apoptosis, which further suppresses esophageal squamous cell carcinoma cell tumorigenesis. Difluoromethylornithine treatment decreases proliferation and also induces apoptosis of esophageal squamous cell carcinoma cells and implanted tumors, resulting in significant reduction in the size and weight of tumors. The results of this study indicate that ornithine decarboxylase is a promising target for esophageal squamous cell carcinoma therapy and difluoromethylornithine warrants further study in clinical trials to test its effectiveness against esophageal squamous cell carcinoma.

Apigenin Inhibits Human SW620 Cell Growth by Targeting Polyamine Catabolism.

Apigenin is a nonmutagenic flavonoid that has antitumor properties. Polyamines are ubiquitous cellular polycations, which play an important role in the proliferation and differentiation of cancer cells. Highly regulated pathways control the biosynthesis and degradation of polyamines. Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the metabolism, and spermidine/spermine-N1-Acetyl transferase (SSAT) is the rate-limiting enzyme in the catabolism of polyamines. In the current study, the effect of increasing concentrations of apigenin on polyamine levels, ODC and SSAT protein expression, mRNA expression, cell proliferation and apoptosis, and the production of reactive oxygen species (ROS) was investigated in SW620 colon cancer cells. The results showed that apigenin significantly reduced cell proliferation, decreased the levels of spermidine and spermine, and increased previously downregulated putrescine contents. Apigenin also enhanced SSAT protein and mRNA levels and the production of reactive oxygen species in SW620 cells, though it had no significant effect on the levels of ODC protein or mRNA. Apigenin appears to decrease the proliferation rate of human SW620 cells by facilitating SSAT expression to induce polyamine catabolism and increasing ROS levels to induce cell apoptosis.

In-silico Identification of Candidate Inhibitory Ligands against Ornithine Decarboxylase Enzyme for Human Sleeping Sickness Causing Trypanosoma brucei

Ornithine Decarboxylase (ODC) catalyzes the decarboxylation of ornithine to putrescine. This is known to be a crucial step in the biosynthesis of polyamines. These polyamines are necessary for microbial cell growth and proliferation. Hence, ODC enzyme is the best target to treat African sleeping sickness disease-causing protozoan parasite, Trypanosoma brucei. ODC is a 5'-pyridoxal phosphate (PLP) dependent, an obligate homodimer enzyme with two identical active sites at the dimer interface, comprising the beta or alpha barrel domain from one subunit and beta-sheet domain from the other subunit. The catalytic residues are contributed to the active site from both monomers. An X-ray crystallography study on ODC in the wild T. brucei has revealed two structural changes upon ligand binding; an amino acid residue specifically Lys-69 is displaced by putrescine forming a new interaction and a side chain of Cys-360 moves to the active site. Mutation of the Cys residue to Ala or Ser amino acid reduces the Kcat energy of the decarboxylation reaction drastically. Interestingly, ligand ZINC01703953 shown interaction with ODC protein, Lys-69 functional amino acid with docked score of -8.28 out of 35 ligands tested based on Virtual Screening (VS) with AutoDock suite in the current study. Another, top scoring (-9.69) ligand, ZINC67855534 is found to interact with amino acid residues, involved in the active site formation of the ODC enzyme from our current VS experiment. Hence, the ligands ZINC01703953 and ZINC67855534 could possibly consider as potential candidates against T. brucei upon further in-vitro experimental validations.

Mitogen-Activated Protein Kinase and Intracellular Polyamine Signaling Is Involved in TRPV1 Activation-Induced Cardiac Hypertrophy.

BackgroundThe transient receptor potential vanilloid type 1 (TRPV1) is expressed in the cardiovascular system, and increased TRPV1 expression has been associated with cardiac hypertrophy. Nevertheless, the role of TRPV1 in the pathogenesis of cardiac hypertrophy and the underlying molecular mechanisms remain unclear.Methods and ResultsIn cultured cardiomyocytes, activation of TRPV1 increased cell size and elevated expression of atrial natriuretic peptide mRNA and intracellular calcium level, which was reversed by TRPV1 antagonist capsazepine. Increased expression of phosphorylated calmodulin‐dependent protein kinase IIδ and mitogen‐activated protein kinases were found in TRPV1 agonist capsaicin‐treated cardiomyocytes. Selective inhibitor of calmodulin‐dependent protein kinase IIδ decreased phosphorylation of extracellular signal–regulated kinases and p38. Capsaicin induced an increase in expression of ornithine decarboxylase protein, which is the key enzyme in polyamine biosynthesis in cardiomyocytes. Nevertheless, there was no obvious change of ornithine decarboxylase expression in TRPV1 knockdown cells after capsaicin treatment, and specific inhibitors of calmodulin‐dependent protein kinase IIδ or p38 downregulated the capsaicin‐induced expression of ornithine decarboxylase. Capsazepine alleviated the increase in cross‐sectional area of cardiomyocytes and the ratio of heart weight to body weight and improved cardiac function, including left ventricular internal end‐diastolic and ‐systolic dimensions and ejection fraction and fractional shortening percentages, in mice treated with transverse aorta constriction. Capsazepine also reduced expression of ornithine decarboxylase and cardiac polyamine levels. Transverse aorta constriction induced increases in phosphorylated calmodulin‐dependent protein kinase IIδ and extracellular signal–regulated kinases, and p38 and Serca2a were attenuated by capsazepine treatment.ConclusionsThis study revealed that the mitogen‐activated protein kinase signaling pathway and intracellular polyamines are essential for TRPV1 activation–induced cardiac hypertrophy.

Modulation of L-Arginine-Arginase Metabolic Pathway Enzymes: Immunocytochemistry and mRNA Expression in Peripheral Blood and Tissue Levels in Head and Neck Squamous Cell Carcinomas in North East India.

Arginine may play important roles in tumor progression by providing ornithine for polyamine biosynthesis, required for cell growth. The aim of this work was to determine the expression of arginine metabolic pathway enzymes in head and neck squamous cell carcinoma (HNSCC) in northeast India. The expressions of arginase isoforms (ARG1 and ARG2), ornithine aminotransferase (OAT) and ornithine decarboxylase (ODC) were examined in fifty paired HNSCC and adjacent non-tumor tissues by immunohistochemistry. Immunocytochemistry, semiquantitative reverse transcription sq-PCR and quantitative real-time qPCR were used to assess protein and mRNA expressions in peripheral blood of fifty HNSCC patients and hundred controls. ARG1 and ODC protein and mRNA were strongly expressed in peripheral blood from HNSCC patients. No ARG2 expression was observed. In vivo, expression of ARG1, ARG2 and ODC was significantly higher in tumor than in non-tumor tissues. Most tumors expressed low levels of OAT, with no difference in tissues or blood, compared to controls. The absolute extent of maximal ARG1 upregulation with qPCR showed 6.23 fold increase in HNSCC. These findings strongly suggest that in HNSCCs, the ARG1 pathway is stimulated leading to the formation of polyamines as indicated by higher ODC expression, which promote tumor growth.

Ornithine Decarboxylase Activity Is Required for Prostatic Budding in the Developing Mouse Prostate.

The prostate is a male accessory sex gland that produces secretions in seminal fluid to facilitate fertilization. Prostate secretory function is dependent on androgens, although the mechanism by which androgens exert their effects is still unclear. Polyamines are small cationic molecules that play pivotal roles in DNA transcription, translation and gene regulation. The rate-limiting enzyme in polyamine biosynthesis is ornithine decarboxylase, which is encoded by the gene Odc1. Ornithine decarboxylase mRNA decreases in the prostate upon castration and increases upon administration of androgens. Furthermore, testosterone administered to castrated male mice restores prostate secretory activity, whereas administering testosterone and the ornithine decarboxylase inhibitor D,L-α-difluromethylornithine (DFMO) to castrated males does not restore prostate secretory activity, suggesting that polyamines are required for androgens to exert their effects. To date, no one has examined polyamines in prostate development, which is also androgen dependent. In this study, we showed that ornithine decarboxylase protein was expressed in the epithelium of the ventral, dorsolateral and anterior lobes of the adult mouse prostate. Ornithine decarboxylase protein was also expressed in the urogenital sinus (UGS) epithelium of the male and female embryo prior to prostate development, and expression continued in prostatic epithelial buds as they emerged from the UGS. Inhibiting ornithine decarboxylase using DFMO in UGS organ culture blocked the induction of prostatic buds by androgens, and significantly decreased expression of key prostate transcription factor, Nkx3.1, by androgens. DFMO also significantly decreased the expression of developmental regulatory gene Notch1. Other genes implicated in prostatic development including Sox9, Wif1 and Srd5a2 were unaffected by DFMO. Together these results indicate that Odc1 and polyamines are required for androgens to exert their effect in mediating prostatic bud induction, and are required for the expression of a subset of prostatic developmental regulatory genes including Notch1 and Nkx3.1.

AZ Street in Vasopressin Synthesis.

The antizyme (AZ) protein binds to and mediates the degradation of ornithine decarboxylase (ODC), the rate-limiting enzyme responsible for the conversion of Lornithine to putrescine. Biosynthetic polyamines (spermidine and spermine) formed from putrescine are present in all living cells. ODC degradation can be prevented by AZ inhibitors (Azin1 or Azin2), which bind to AZ to form a stable complex. Therefore, relationships between ODC, AZ, and Azin proteins are important in the regulation of polyamine synthesis. Polyamines are known to be multifunctional molecules that have also been implicated in the modulation of the gene expression (1). A recent study (2) has used a transcriptomic strategy and multiple other approaches including lentiviral vector techniques to show, for the first time, that polyamines are involved in the regulation of the arginine vasopressin (AVP) gene expression. Greenwood et al (2) demonstrated that Azin1, as well as AZ1, ODC, and polyamines (spermidine and spermine), are abundant in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) of the hypothalamus in rats. The hypothalamo-neurohypophyseal system is one of the most widely studied neuroendocrine model systems (3). The magnocellular neurosecretory cells of the SON and PVN project their axon terminals to the posterior pituitary from which they secrete their products, AVP and oxytocin (OT), into the systemic circulation. AVP, also called antidiuretic hormone, acts on AVP receptor 2 in the kidney to promote reabsorption of water via aquaporin 2. Greenwood et al (2) found that Azin1 mRNA levels were significantly increased in the SON and the PVN after dehydration and salt loading [drinking of 2% (wt/vol) NaCl solution], referred to as chronic hyperosmotic stressors. They also showed that acute osmotic challenge (ip administration of hypertonic saline) caused a rapid increase of Azin1 mRNA as well as AVP heteronuclear (hn) RNA levels in the SON and the PVN. AVP hnRNA levels reflect theactivationof AVPgene transcription in theSON and PVN after acute osmotic stimulation (4). These observations raised the possibility that polyamines may be involved in controlling AVP gene expression and hence body fluid balance in vivo. To test this hypothesis, Greenwood et al (2) designed in vivo experiments to study whether Azin1 overexpression or knockdown, using lentiviral vectors, could change the expression of the AVP gene in the SON and/or PVN. Thus, they showed that Azin1 overexpression in the SON caused a significant decrease in AVP mRNA levels. In contrast, short hairpin RNA-mediated knockdown of Azin1 significantly increased AVP mRNA levels in the SON and decreased plasma osmolality because of activated AVP synthesis and elevated secretion of AVP into the bloodstream. In an ex vivo study (organotypic hypothalamic culture), they clearly confirmed that ODC inhibition (blockade of putrescine synthesis from L-ornithine) caused a significant increase of AVP hnRNA but not OT hnRNA. It is very interesting that the effect of the ODC inhibitor on the AVP gene expression is so specific. This study used a transcriptomic strategy to identify significant genes from all of the expressed genes in the specific region of the brain after a physiological manipulation. This strategy is a very useful and effective way to find novel molecules that may regulate the target gene expression. The targetmoleculesdescribedbyMurphyandhis colleagues (2)are AVP and OT. Despite being cloned in early 1980s, it still is unclear which molecules regulate the expressions of these genes under physiological manipulations.