Dual targeting of polyamine synthesis and uptake in diffuse intrinsic pontine gliomas.

Diffuse intrinsic pontine glioma (DIPG) is an incurable malignant childhood brain tumor, with no active systemic therapies and a 5-year survival of less than 1%. Polyamines are small organic polycations that are essential for DNA replication, translation and cell proliferation. Ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme in polyamine synthesis, is irreversibly inhibited by difluoromethylornithine (DFMO). Herein we show that polyamine synthesis is upregulated in DIPG, leading to sensitivity to DFMO. DIPG cells compensate for ODC1 inhibition by upregulation of the polyamine transporter SLC3A2. Treatment with the polyamine transporter inhibitor AMXT 1501 reduces uptake of polyamines in DIPG cells, and co-administration of AMXT 1501 and DFMO leads to potent in vitro activity, and significant extension of survival in three aggressive DIPG orthotopic animal models. Collectively, these results demonstrate the potential of dual targeting of polyamine synthesis and uptake as a therapeutic strategy for incurable DIPG.

Baicalein, 7,8-Dihydroxyflavone and Myricetin as Potent Inhibitors of Human Ornithine Decarboxylase.

BACKGROUND: Human ornithine decarboxylase (ODC) is a well-known oncogene, and the discovery of ODC enzyme inhibitors is a beneficial strategy for cancer therapy and prevention. METHODS: We examined the inhibitory effects of a variety of flavone and flavonol derivatives on ODC enzymatic activity, and performed in silico molecular docking of baicalein, 7,8-dihydroxyflavone and myricetin to the whole dimer of human ODC to investigate the possible binding site of these compounds on ODC. We also examined the cytotoxic effects of these compounds with cell-based studies. RESULTS: Baicalein, 7,8-dihydroxyflavone and myricetin exhibited significant ODC suppression activity with IC50 values of 0.88 µM, 2.54 µM, and 7.3 µM, respectively, which were much lower than that of the active-site irreversible inhibitor α-DL-difluoromethylornithine (IC50, the half maximal inhibitory concentration, of approximately 100 µM). Kinetic studies and molecular docking simulations suggested that baicalein, and 7,8-dihydroxyflavone act as noncompetitive inhibitors that are hydrogen-bonded to the region near the active site pocket in the dimer interface of the enzyme. Baicalein and myricetin suppress cell growth and induce cellular apoptosis, and both of these compounds suppress the ODC-evoked anti-apoptosis of cells. CONCLUSIONS: Therefore, we suggest that the flavone or flavonol derivatives baicalein, 7,8-dihydroxyflavone, and myricetin are potent chemopreventive and chemotherapeutic agents that target ODC.

Volatile Composition and Biological Activities of the Leaf Essential Oil from Zanthoxylum limoncello Grown in Oaxaca, México.

Zanthoxylum limoncello is a native plant from southern Mexico which is used as a timber source, condiment and as a traditional medicine. Herein, we report on the volatile content of the leaf essential oil and its biological activities. The annual essential oils (2015-2018) contained volatile organic compounds which exhibited a moderate growth inhibitory activity against H. pylori ATCC 53504 (MIC 121.4-139.7 µg mL-1 ), 26695 (MIC 85.5-94.9 µg mL-1 ) and J99 (MIC 94.7-110.4 µg mL-1 ). These hydrodistillates contained 2-undecanone (31.6-36.8 %; MIC 185.3-199.2 µg mL-1 ) and 2-undecenal (25.1-35.7 %; MIC 144.8-111.3 µg mL-1 ) as the most abundant compounds which were partially involved in the anti-H. pylori activity. The human ornithine decarboxylase enzyme (ODC1), which shows increased activity in several cancer types, was non-competitively inhibited (Vmax 2.7>0.8 Kcat s-1 ) by the essential oil of Z. limoncello as well as by 2-undecanone and 2-undecenal in accordance to in vitro kinetic studies. In silico calculations strongly suggest that the carbonyl group of these oxygenated hydrocarbons interacts with both Asn319 and Ala39 at the subunit A of ODC1. Considering that Ala39 is located close to Asn44, a crucial amino acid of the ODC's allosteric site, the non-competitive inhibition of the enzyme by 2-undecanone and 2-undecenal is endorsed. Finally, the essential oil of Z. limoncello and its main volatiles showed a significant (p<0.01) and prolonged repellent effect against Aedes aegypti.

Ornithine decarboxylase as a therapeutic target for endometrial cancer.

Ornithine Decarboxylase (ODC) a key enzyme in polyamine biosynthesis is often overexpressed in cancers and contributes to polyamine-induced cell proliferation. We noted ubiquitous expression of ODC1 in our published endometrial cancer gene array data and confirmed this in the cancer genome atlas (TCGA) with highest expression in non-endometrioid, high grade, and copy number high cancers, which have the worst clinical outcomes. ODC1 expression was associated with worse overall survival and increased recurrence in three endometrial cancer gene expression datasets. Importantly, we confirmed these findings using quantitative real-time polymerase chain reaction (qRT-PCR) in a validation cohort of 60 endometrial cancers and found that endometrial cancers with elevated ODC1 had significantly shorter recurrence-free intervals (KM log-rank p = 0.0312, Wald test p = 5.59e-05). Difluoromethylornithine (DFMO) a specific inhibitor of ODC significantly reduced cell proliferation, cell viability, and colony formation in cell line models derived from undifferentiated, endometrioid, serous, carcinosarcoma (mixed mesodermal tumor; MMT) and clear cell endometrial cancers. DFMO also significantly reduced human endometrial cancer ACI-98 tumor burden in mice compared to controls (p = 0.0023). ODC-regulated polyamines (putrescine [Put] and/or spermidine [Spd]) known activators of cell proliferation were strongly decreased in response to DFMO, in both tumor tissue ([Put] (p = 0.0006), [Spd] (p<0.0001)) and blood plasma ([Put] (p<0.0001), [Spd] (p = 0.0049)) of treated mice. Our study indicates that some endometrial cancers appear particularly sensitive to DFMO and that the polyamine pathway in endometrial cancers in general and specifically those most likely to suffer adverse clinical outcomes could be targeted for effective treatment, chemoprevention or chemoprevention of recurrence.

Ornithine uptake and the modulation of drug sensitivity in Trypanosoma brucei.

Trypanosoma brucei, protozoan parasites that cause human African trypanosomiasis (HAT), depend on ornithine uptake and metabolism by ornithine decarboxylase (ODC) for survival. Indeed, ODC is the target of the WHO "essential medicine" eflornithine, which is antagonistic to another anti-HAT drug, suramin. Thus, ornithine uptake has important consequences in T. brucei, but the transporters have not been identified. We describe these amino acid transporters (AATs). In a heterologous expression system, TbAAT10-1 is selective for ornithine, whereas TbAAT2-4 transports both ornithine and histidine. These AATs are also necessary to maintain intracellular ornithine and polyamine levels in T. brucei, thereby decreasing sensitivity to eflornithine and increasing sensitivity to suramin. Consistent with competition for histidine, high extracellular concentrations of this amino acid phenocopied a TbAAT2-4 genetic defect. Our findings established TbAAT10-1 and TbAAT2-4 as the parasite ornithine transporters, one of which can be modulated by histidine, but both of which affect sensitivity to important anti-HAT drugs.-Macedo, J. P., Currier, R. B., Wirdnam, C., Horn, D., Alsford, S., Rentsch, D. Ornithine uptake and the modulation of drug sensitivity in Trypanosoma brucei.

Evaluation of a diospyrin derivative as antileishmanial agent and potential modulator of ornithine decarboxylase of Leishmania donovani.

World health organization has called for academic research and development of new chemotherapeutic strategies to overcome the emerging resistance and side effects exhibited by the drugs currently used against leishmaniasis. Diospyrin, a bis-naphthoquinone isolated from Diospyros montana Roxb., and its semi-synthetic derivatives, were reported for inhibitory activity against protozoan parasites including Leishmania. Presently, we have investigated the antileishmanial effect of a di-epoxide derivative of diospyrin (D17), both in vitro and in vivo. Further, the safety profile of D17 was established by testing its toxicity against normal macrophage cells (IC50∼20.7 µM), and also against normal BALB/c mice in vivo. The compound showed enhanced activity (IC50∼7.2 µM) as compared to diospyrin (IC50∼12.6 µM) against Leishmania donovani promastigotes. Again, D17 was tested on L. donovani BHU1216 isolated from a sodium stibogluconate-unresponsive patient, and exhibited selective inhibition of the intracellular amastigotes (IC50∼0.18 µM). Also, treatment of infected BALB/c mice with D17 at 2mg/kg/day reduced the hepatic parasite load by about 38%. Subsequently, computational docking studies were undertaken on selected enzymes of trypanothione metabolism, viz. trypanothione reductase (TryR) and ornithine decarboxylase (ODC), followed by the enzyme kinetics, where D17 demonstrated non-competitive inhibition of the L. donovani ODC, but could not inhibit TryR.

PLP-dependent enzymes as potential drug targets for protozoan diseases.

The chemical properties of the B(6) vitamers are uniquely suited for wide use as cofactors in essential reactions, such as decarboxylations and transaminations. This review addresses current efforts to explore vitamin B(6) dependent enzymatic reactions as drug targets. Several current targets are described that are found amongst these enzymes. The focus is set on diseases caused by protozoan parasites. Comparison across a range of these organisms allows insight into the distribution of potential targets, many of which may be of interest in the development of broad range anti-protozoan drugs. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

Effects of psoralen from Psoralea corylifolia on quinone reductase, ornithine decarboxylase, and JB6 cells transformation promotion.

The cancer chemopreventive effect of psoralen isolated from the seeds of Psoralea corylifolia was investigated in the induction of quinone reductase (QR) activity, intracellular detoxification enzyme, inhibition of 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity, a key regulatory enzyme for polyamine metabolism, and tumor promotion in mouse epidermal JB6 cells, sensitive to tumor promoters (clone 415a P+ cells), which are related to suppress multistage carcinogenesis including initiation and promotion. Psoralen was isolated and identified from the ethyl acetate-soluble fraction of the methanolic extract from the seeds. Psoralen was active in induction of QR activity, the concentration of psoralen required to induce 1.5 fold QR activity was 14.8 µg/mL. Also, this pure compound inhibited TPA-induced ODC activity by 50% (designated IC(50)) at the concentration 15.6 µg/mL and exhibited inhibition of TPA-induced tumor promotion in mouse epidermal JB6 cells with an IC(50) value of 17.1 µg/mL. Therefore, it is extrapolated that psoralen has the potential capable of inhibiting the initiation and/or promotion stage of carcinogenesis by induction of QR activity, inhibition of TPA-induced ODC activity and mouse epidermal JB6 cells tumor promotion.

Quercetin inhibits human DLD-1 colon cancer cell growth and polyamine biosynthesis.

BACKGROUND/AIM: Polyamines and ornithine decarboxylase are involved in cell growth and differentiation. The polyphenol quercetin may exert anti-tumour properties by influencing proliferation, differentiation, and apoptosis. The aim of the study was to investigate the effects of increasing concentrations of quercetin (from 0.1 to 100 µM) on polyamine biosynthesis, cell proliferation, and apoptosis in the DLD-1 cells. MATERIALS AND METHODS: Polyamine levels and ornithine decarboxylase activity were evaluated by HPLC and radiometric technique, respectively. The proliferative response was estimated by 3-(4,5 dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) test and [(3)H]-thymidine incorporation in cell DNA. Apoptosis was investigated by DNA fragmentation. RESULTS: At concentrations ≥50 µM, quercetin significantly reduced ornithine decarboxylase activity, putrescine and spermidine levels compared to controls and cells treated with 0.1 µM concentration. Quercetin concentrations ≥70 µM caused a significant reduction in the conversion of MTT tetrazolium salt and [(3)H]-thymidine incorporation. The same concentrations were needed to induce the apoptosis. CONCLUSION: The present study demonstrates that quercetin can affect growth of DLD-1 cells by both decreasing polyamine biosynthesis and inducing apoptosis. Due to the extensive dietary consumption of polyphenols, such as quercetin, the biological activity of these compounds deserves further investigation.

Drug target identification, validation, characterisation and exploitation for treatment of Acanthamoeba (species) infections.

New more efficacious antimicrobials as required for the treatment of Acanthamoeba infections as those currently available require arduous treatment regimes, are not always effective and are poorly active against the cystic stages. Herein, we review potential drug targets including tubulin, alternative oxidase, amino acid biosynthesis and myosin. In addition, we review the literature for current missing tools and resources for the identification, validation and development of new antimicrobials for this organism. Additional targets should come to light through a concerted genome sequencing effort.

Anti-inflammatory and antitumor promotional effects of a novel urinary metabolite, 3',4'-didemethylnobiletin, derived from nobiletin.

We reported previously that 3',4'-didemethylnobiletin (DDMN) is the major metabolite of nobiletin in mouse urine. In this study, we examined DDMN's molecular mechanism of action and its anti-inflammatory and antitumor properties. We demonstrated that topical application of DDMN effectively inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated transcription of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and ornithine decarboxylase (ODC) messenger RNA and protein expression in mouse skin. Pretreatment with DDMN has resulted in the reduction of TPA-induced nuclear translocation of the nuclear factor-kappa B (NF-kappaB) subunit. DDMN also reduced DNA binding by blocking phosphorylation of inhibitor kappaB (IkappaB) alpha and p65 and caused subsequent degradation of IkappaBalpha. DDMN inhibited TPA-induced phosphorylation and nuclear translocation of the signal transducer and activator of transcription 3. Moreover, DDMN suppressed TPA-induced activation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt and protein kinase C that are upstream of NF-kappaB and activator protien-1. We also found that DDMN significantly inhibited TPA-induced mouse skin inflammation by decreasing inflammatory parameters. Furthermore, DDMN significantly inhibited 7,12-dimethylbenz[a]anthracene/TPA-induced skin tumor formation measured by the tumor multiplicity of papillomas at 20 weeks. Presented data for the first time reveal that DDMN is an effective antitumor agent that functions by downregulating inflammatory iNOS, COX-2 and ODC gene expression in mouse skin. It is suggested that DDMN is a novel functional agent capable of preventing inflammation-associated tumorigenesis.

L-arginine reduces cell proliferation and ornithine decarboxylase activity in patients with colorectal adenoma and adenocarcinoma.

PURPOSE: Evidence suggests that the majority of colorectal carcinomas arise from adenomas, and L-arginine suppresses colorectal tumorigenesis. We suppose that L-arginine may inhibit the process of carcinogenesis from colorectal adenoma to adenocarcinoma. The aim of this study was to investigate the effects of L-arginine on the formation and development of colorectal tumors. EXPERIMENTAL DESIGN: We selected 60 patients with colorectal cancer and 60 patients with colorectal adenoma (CRA) and divided them into four groups of 30 patients each. We gave 30 g (120 mL) of L-arginine everyday for 3 days to the test groups, whereas L-arginine was substituted by 5% glucose in the control groups. The expression of the proliferating cell nuclear antigen, survivin, and nitric oxide synthase was examined immunohistochemically, and ornithine decarboxylase (ODC) activity was examined spectrophotometrically. Serum nitric oxide (NO) was detected by the Griess assay. RESULTS: In patients with CRA, the proliferating cell nuclear antigen and survivin labeling indexes and ODC activity of the tumor and paratumor mucosa in the L-arginine-treated group after L-arginine treatment were significantly lower as compared with the corresponding pretreatment values (P < 0.01). Moreover, inducible nitric oxide synthase expression in the tumor markedly increased after L-arginine treatment (P < 0.05). Serum NO levels in the patients with colorectal cancer were markedly higher than those in the patients with CRA, and L-arginine treatment was responsible for this increase (P < 0.05). CONCLUSIONS: Our results show that L-arginine can restrain crypt cell hyperproliferation and the expression of survivin, an inhibitor of apoptosis protein. This suggests that L-arginine can block the formation and development of colorectal tumors, and this effect might be related to the increased serum NO concentration and decreased ODC activity.

Effect of gallic acid on renal biochemical alterations in male Wistar rats induced by ferric nitriloacetic acid.

The present study is an effort to identify a potent chemopreventive agent against various diseases (including cancer) in which oxidative stress and cell proliferation plays an important causative role. This study was designed to investigate the effect of gallic acid against ferric nitrilotriacetic acid (Fe-NTA)-induced carcinogen/ drug metabolizing phase I and phase II enzymes, antioxidative parameters, kidney markers, tumour promotion markers and lipid peroxidation (LPO) in kidney of male Wistar rats. Fe-NTA (9 mg Fe/kg body weight, intraperitoneally) caused significant depletion in the detoxification and antioxidant enzyme armoury with concomitant elevation in renal LPO, serum creatinine, blood urea nitrogen, hydrogen peroxide generation, ornithine decarboxylase activity and [3H]thymidine incorporation into renal DNA. However, pretreatment of animals with gallic acid (10 and 20 mg/kg body weight) resulted in a significant decrease in the levels of the parameters measured (P <0.001). Renal glutathione content (P <0.001), glutathione metabolizing enzyme (P <0.001) and antioxidant enzyme levels were also recovered to a significant level (P <0.001). The enhanced reduced glutathione level and enzyme activities involved in xenobiotic metabolism and maintaining antioxidant status of cells are suggestive of a chemopreventive efficacy of gallic acid against Fe-NTA-mediated oxidative stress, toxicity and cell proliferative response in Wistar rats.

Effect of dietary apigenin on colonic ornithine decarboxylase activity, aberrant crypt foci formation, and tumorigenesis in different experimental models.

The efficacy of dietary apigenin, a dietary flavonoid, in colon cancer prevention was investigated by evaluating the inhibition of the ornithine decarboxylase (ODC) activity and the formation of aberrant crypt foci (ACF) and by studying the ability of apigenin to block colon carcinogenesis in two mouse models. First, the activity of ODC was measured in colon cancer cells (Caco-2) and in the colon epithelium of CF-1 mice. Apigenin at 10 and 30 muM significantly inhibited the ODC activity of Caco-2 cells by 26% and 57%, respectively. Colonic ODC activity in CF-1 mice was reduced with 0.1% dietary apigenin by 42% compared with the control, but this difference was not statistically significant. Second, ACF formation was evaluated in azoxymethane (AOM)-induced CF-1 mice. Female CF-1 mice at 6 wk of age were i.p. injected with 5 mg/kg body weight (BW) AOM once to induce ACF. ACF formation in CF-1 mice was reduced by 50% (P < 0.05) with 0.1% dietary apigenin fed for 6 wk when compared with the control. Dietary apigenin inhibited ACF only in the distal region of the CF-1 mouse colon. Finally, tumorigenesis studies were conducted using two different mouse models: AOM-induced CF-1 mice and Min mice with mutant adenomatous polyposis coli (APC) gene. Female CF-1 mice at 6 wk of age were i.p. injected with 10 mg/kg BW AOM weekly for 6 (AOM Study I) or 4 (AOM Study II) wk to induce tumors. CF-1 mice were fed diets containing 0.025% or 0.1% apigenin for 23-25 wk. Female Min mice were fed diets for 10 wk beginning at 5 wk of age. In two AOM-treated mouse colon tumor studies 0.025% and 0.1% dietary apigenin modestly reduced tumors in the group fed 0.025% apigenin (25% incidence in comparison with 65% in the controls) in a non-dose response manner. Apigenin failed to inhibit adenoma formation in the Min mouse study. These results suggest that dietary apigenin showed promise in cancer prevention by reducing the ODC activity and ACF formation, however, clear evidence of cancer prevention was not obtained in mouse tumor studies. Further investigation of the potential chemopreventive effect of apigenin in carcinogenesis is warranted.

Butea monosperma and chemomodulation: protective role against thioacetamide-mediated hepatic alterations in Wistar rats.

The present study was carried out to study the effect of Butea monosperma, a known liver acting drug on the tumor promotion related events of carcinogenesis in rat liver. Thioacetamide (TAA) was used to induce tumor promotion response and oxidative stress and caused significant depletion in the detoxification and antioxidant enzyme armory with concomitant elevation in malondialdehyde (MDA) formation, hydrogen peroxide (H(2)O(2)) generation, ornithine decarboxylase (ODC) activity and unscheduled DNA synthesis. However, B. monosperma pretreatment at two different doses restored the levels of the above-said parameters (p < 0.001) in a dose-dependent manner. The alcoholic extract of B. monosperma used in the present study seems to offer dose-dependent protection and maintain the structural integrity of hepatic cells. This was evident from the significant reduction in TAA-induced serum GOT, GPT, Lactate dehydrogenase (LDH) and gamma-Glutamyl transpeptidase activity (GGT) activities (p < 0.001). These investigations validate the use of B. monosperma in liver disorders by Ayurvedic physicians. Overall results indicate that the methanolic extract of B. monosperma possesses hepatoprotective effects and also it might suppress the promotion stage via inhibition of oxidative stress and polyamine biosynthetic pathway.

Chemopreventive effects of various concentrations of alpha-santalol on skin cancer development in CD-1 mice.

Previous studies from this laboratory have indicated that alpha-santalol (5%) provides chemopreventive effects in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin cancer in CD-1 and SENCAR mice. Skin cancer development is associated with increased ornithine decarboxylase (ODC) activity, DNA synthesis and rapid proliferation of epidermal cells. The purpose of this investigation was to determine the effects of various concentrations (1.25% and 2.5%) of alpha-santalol on DMBA-initiated and TPA-promoted skin cancer development, TPA-induced ODC activity, and DNA synthesis in CD-1 mice. alpha-Santalol treatment at both concentrations (1.25% and 2.5%) prevented the skin cancer development. alpha-Santalol treatment (1.25% and 2.5%) resulted in a significant decrease in the TPA-induced ODC activity and incorporation of [3H]thymidine in DNA in the epidermis of CD-1 mice. There was no significant difference in the effects of 1.25% and 2.5% alpha-santalol on tumour incidence, multiplicity, epidermal TPA-induced ODC activity, or DNA synthesis in CD-1 mice.

Anticarcinogenic effect of Nymphaea alba against oxidative damage, hyperproliferative response and renal carcinogenesis in Wistar rats.

The present study investigates the prophylactic effect of Nymphaea alba against ferric nitrilotriacetate (Fe-NTA)-induced renal oxidative stress, hyperproliferative response and renal carcinogenesis in Wistar rats. Treatment with Fe-NTA (9 mg Fe/kg body weight, intraperitoneally) enhanced iron-ascorbate-induced renal lipid peroxidation, xanthine oxidase, gamma-glutamyl transpeptidase and hydrogen peroxide (H2O2) generation with reduction in renal glutathione content, antioxidant enzymes, viz., glutathione peroxidase, glutathione reductase, catalase, glucose-6-phosphate dehydrogenase and phase-II metabolising enzymes such as glutathione-S-transferase and quinone reductase. It also elevated the levels of blood urea nitrogen, serum creatinine, ornithine decarboxylase (ODC) activity and thymidine [3H] incorporation into renal DNA. It also enhanced DEN-initiated renal carcinogenesis by increasing the percentage incidence of renal tumors. Treatment of rats orally with N. alba (100 and 200 mg/kg body weight) resulted in significant decrease in gamma-glutamyl transpeptidase, lipid peroxidation, xanthine oxidase, H2O2 generation, blood urea nitrogen, serum creatinine, renal ODC activity, DNA synthesis (p < 0.001) and incidence of tumors. Renal glutathione content (p < 0.01), glutathione metabolizing enzymes (p < 0.001) and antioxidant enzymes were also recovered to significant level (p < 0.001). Thus, our results show that N. alba is a potent chemopreventive agent and suppresses Fe-NTA-induced oxidative stress, hyperproliferative response and renal carcinogenesis in Wistar rats.

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

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

Study on prevention of two-stage skin carcinogenesis by Hibiscus rosa sinensis extract and the role of its chemical constituent, gentisic acid, in the inhibition of tumour promotion response and oxidative stress in mice.

We designed the present study to investigate the role of gentisic acid in the chemopreventive activity of Hibiscus rosa sinensis extract on 7,12-dimethyl benz(a)anthracene (DMBA)/croton oil-mediated carcinogenesis in mouse skin via 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced tumour promotion response and oxidative stress. Single topical application of DMBA followed by twice weekly applications of croton oil after one week for 20 weeks resulted in 100% incidence of tumours in animals in 15 weeks. However, application of H. rosa sinensis extract 30 minutes prior to the application of croton oil twice weekly for 20 weeks caused significant reduction in the number of tumours per mouse and the percentage of tumour-bearing mice. Also, the latency period for the appearance of the first tumour was delayed on H. rosa sinensis pretreatment. A single topical application of TPA caused significant depletion in reduced glutathione (GSH) content, activities of its metabolizing and antioxidant enzymes, while malondialdehyde (MDA) formation, H2O2 content, ornithine decarboxylase (ODC) activity and DNA synthesis were significantly increased. Interestingly, pretreatment of H. rosa sinensis extract (3.5 mg and 7 mg/kg body weight) and gentisic acid (2.0 microg and 4.0 microg/0.2 ml acetone per animal) restored the levels of GSH, and its metabolizing and antioxidant enzymes (P<0.05). There was also a statistically significant reduction in MDA formation and H2O2 content (P<0.05) at both doses. Although inhibition of ODC activity by gentisic acid was not dose-dependent, thymidine incorporation in DNA (P<0.05) was dose-dependently recovered by the plant extract and its chemical constituent. We therefore propose that gentisic acid has a role in the modulatory activity of H. rosa sinensis extract.

Proteasomes begin ornithine decarboxylase digestion at the C terminus.

Proteasomes denature folded protein substrates and thread them through a narrow pore that leads to the sequestered sites of proteolysis. Whether a protein substrate initiates insertion from its N or C terminus or in a random orientation has not been determined for any natural substrate. We used the labile enzyme ornithine decarboxylase (ODC), which is recognized by the proteasome via a 37-residue C-terminal tag, to answer this question. Three independent approaches were used to assess orientation as follows. 1) The 461-residue ODC protein chain was interrupted at position 305. The C-terminal fragment was degraded by purified proteasomes, but because processivity requires continuity of the polypeptide chain, the N-terminal fragment was spared. 2) A proteasome-inhibitory viral sequence prevented degradation when introduced near the C terminus but not when inserted elsewhere in ODC. 3) A bulky tightly folded protein obstructed in vivo degradation most effectively when positioned near the C terminus. These data demonstrate that the proteasome initiates degradation of this native substrate at the C terminus. The co-localization of entry site and degradation tag to the ODC C terminus suggests that recognition tags determine the site for initiating entry. Flexibility of a polypeptide terminus may promote the initiation of degradation.