Induction Of Ornithine Decarboxylase Activity Research Articles

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 > 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 >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<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<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<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

Elevated ornithine decarboxylase activity promotes skin tumorigenesis by stimulating the recruitment of bulge stem cells but not via toxic polyamine catabolic metabolites

Elevated expression of ornithine decarboxylase (ODC), the regulatory enzyme in polyamine biosynthesis, targeted to the epidermis is sufficient to promote skin tumor development following a single subthreshold dose of dimethylbenz(a)anthracene (DMBA). Since skin tumor promotion involves recruitment of hair follicle bulge stem cells harboring genetic lesions, we assessed the effect of increased epidermal ODC on recruitment of bulge stem cells in ODC-ER transgenic mice in which ODC activity is induced de novo in adult skin with 4-hydroxytamoxifen (4OHT). Bromodeoxyuridine-pulse labeling and use of K15.CrePR1;R26R;ODC-ER triple transgenic mice demonstrated that induction of ODC activity is sufficient to recruit bulge stem cells in quiescent skin. Because increased ODC activity not only stimulates proliferation but also increases reactive oxygen species (ROS) generation via subsequent induction of polyamine catabolic oxidases, we used an inhibitor of polyamine catabolic oxidase activity, MDL72527, to investigate whether ROS generation by polyamine catabolic oxidases contributes to skin tumorigenesis in DMBA-initiated ODC-ER transgenic skin. Newborn ODC-ER transgenic mice and their normal littermates were initiated with a single topical dose of DMBA. To assess tumor development originating from dormant bulge stem cells that possess DMBA-initiated mutations, epidermal ODC activity was induced in ODC-ER mice with 4OHT 5weeks after DMBA initiation followed by MDL72527 treatment. MDL72527 treatment resulted in a shorter tumor latency time, increased tumor burden, increased conversion to carcinomas, and lower tumor levels of p53. Thus, elevated epidermal ODC activity promotes tumorigenesis by stimulating the recruitment of bulge stem cells but not via ROS generation by polyamine catabolic oxidases.

Environmental pesticide exposure modulates cytokines, arginase and ornithine decarboxylase expression in human placenta

To evaluate the cytokine balance and enzymatic alterations induced by environmental pesticide exposure during pregnancy, this transversal study explored placentas derived from non-exposed women (control group-CG), and from women living in a rural area (rural group-RG), collected during intensive organophosphate (OP) pesticide spraying season (RG-SS) and during non-spraying season (RG-NSS). The exposure biomarkers blood cholinesterase and placental carboxylesterase (CaE) were significantly decreased in RG-SS. Among the cytokines studied IL-8, IL-6, TNFα, IL-10, TGFβ and IL-13, the expression frequency of IL-13 increased in RG-SS. Arginase and ornithine decarboxylase (ODC) enzymes were induced in syncytiotrophoblast and endothelial cells. Interestingly, the decrease in CaE activity was associated with arginase and ODC activity induction. These findings suggest that environmental pesticide exposure impacts the placenta by increasing the expression frequency of the anti-inflammatory cytokine IL-13, which may be related to the up-regulation of enzymes implicated in tissue repair.

Evidence that AMP-activated protein kinase can negatively modulate Ornithine decarboxylase activity in cardiac myoblasts

The responses of AMP-activated protein kinase (AMPK) and Ornithine decarboxylase (ODC) to isoproterenol have been examined in H9c2 cardiomyoblasts, AMPK represents the link between cell growth and energy availability whereas ODC, the key enzyme in polyamine biosynthesis, is essential for all growth processes and it is thought to have a role in the development of cardiac hypertrophy. Isoproterenol rapidly induced ODC activity in H9c2 cardiomyoblasts by promoting the synthesis of the enzyme protein and this effect was counteracted by inhibitors of the PI3K/Akt pathway. The increase in enzyme activity became significant between 15 and 30min after the treatment. At the same time, isoproterenol stimulated the phosphorylation of AMPKα catalytic subunits (Thr172), that was associated to an increase in acetyl coenzyme A carboxylase (Ser72) phosphorylation. Downregulation of both α1 and α2 isoforms of the AMPK catalytic subunit by siRNA to knockdown AMPK enzymatic activity, led to superinduction of ODC in isoproterenol-treated cardiomyoblasts. Downregulation of AMPKα increased ODC activity even in cells treated with other adrenergic agonists and in control cells. Analogue results were obtained in SH-SY5Y neuroblastoma cells transfected with a shRNA construct against AMPKα. In conclusion, isoproterenol quickly activates in H9c2 cardiomyoblasts two events that seem to contrast one another. The first one, an increase in ODC activity, is linked to cell growth, whereas the second, AMPK activation, is a homeostatic mechanism that negatively modulates the first. The modulation of ODC activity by AMPK represents a mechanism that may contribute to control cell growth processes.

Cytoplasmic Accumulation of the RNA-binding Protein HuR Stabilizes the Ornithine Decarboxylase Transcript in a Murine Nonmelanoma Skin Cancer Model

Ornithine decarboxylase (ODC) is the first and usually rate-limiting enzyme in the polyamine biosynthetic pathway. Under normal physiological conditions, polyamine content and ODC enzyme activity are highly regulated. However, the induction of ODC activity is an early step in neoplastic transformation. The studies described here use normal mouse keratinocytes (C5N cells), and spindle carcinoma cells (A5 cells) to explore the regulation of ODC in nonmelanoma skin cancer development. Previous results have shown that induction of ODC activity is both necessary and sufficient for the promotion of skin tumors. We see a marked increase in ODC enzyme activity in A5 cells compared with C5N keratinocytes, which correlates with a 4-fold stabilization of ODC mRNA. These data suggest that ODC is post-transcriptionally regulated in skin tumor development. Thus, we sought to investigate whether the ODC transcript interacts with the RNA-binding protein HuR, which is known to bind to and stabilize its target mRNAs. We show that HuR is able to bind to the ODC 3'-UTR in A5 cells but not in C5N cells. Immunofluorescence results reveal that HuR is present in both the nucleus and cytoplasm of A5 cells, whereas C5N cells exhibit strictly nuclear localization of HuR. Knockdown experiments in A5 cells showed that when HuR is depleted, ODC RNA becomes less stable, and ODC enzyme activity decreases. Together, these data support the hypothesis that HuR plays a causative role in ODC up-regulation during nonmelanoma skin cancer development by binding to and stabilizing the ODC transcript.

Amino acids regulate expression of Antizyme‐1 and modulate Ornithine Decarboxylase Activation

In a glucose‐salt solution (EBSS), asparagine (ASN) stimulates ornithine decarboxylase (ODC) activity in a dose‐dependent manner. We determined the mechanism by which ASN induces ODC activity. In the absence of amino acids (AA), an antizyme 20‐kDa isoform (AZ‐1‐20), which inhibits ODC activity, increased in a time‐dependent fashion. ASN prevented the accumulation of AZ‐1‐20 and rapidly induced ODC activity, as did glutamine and α‐amino iso butyric acid. In contrast, lysine and valine induced AZ‐1‐20 expression and decreased ASN‐induced ODC activity. ODC induction by ASN was lower in DMEM containing AA as compared to that in DMEM without AA, suggesting that AZ‐1‐20‐inducing AA may partially neutralize the effect of ASN. Incubation in EBSS for 6 hrs increased AZ‐1‐20 protein levels, and addition of ASN decreased accumulated AZ‐1‐20. Cycloheximide similarly caused elimination of accumulated AZ‐1‐20 but did not alter the rate of decrease due to ASN. Actinomycin‐D failed to prevent AZ‐1‐20 expression in EBSS. These results suggest that ASN modulates AZ‐1‐20 levels by regulating synthesis. ASN prevented the phosphorylation and caused accumulation of eukaryotic initiation factor binding protein (4EBP1). Thus, by preventing the phosphorylation of 4EBP1, ASN causes it to accumulate and bind to eIF4E resulting in the inhibition of the synthesis of AZ‐1‐20, allowing the activation of ODC.

A Randomized, Double-Blind, Placebo-Controlled Phase 3 Skin Cancer Prevention Study of α-Difluoromethylornithine in Subjects with Previous History of Skin Cancer

AbstractPreclinical studies have shown that the inhibition of ornithine decarboxylase (ODC) by α-difluoromethylornithine (DFMO) and resultant decreases in tissue concentrations of polyamines (putrescine and spermidine) prevents neoplastic developments in many tissue types. Clinical studies of oral DFMO at 500 mg/m2/day revealed it to be safe and tolerable and resulted in significant inhibition of phorbol ester–induced skin ODC activity. Two hundred and ninety-one participants (mean age, 61 years; 60% male) with a history of prior nonmelanoma skin cancer (NMSC; mean, 4.5 skin cancers) were randomized to oral DFMO (500 mg/m2/day) or placebo for 4 to 5 years. There was a trend toward a history of more prior skin cancers in subjects randomized to placebo, but all other characteristics including sunscreen and nonsteroidal anti-inflammatory drug use were evenly distributed. Evaluation of 1,200 person-years of follow-up revealed a new NMSC rate of 0.5 events/person/year. The primary end point, new NMSCs, was not significantly different between subjects taking DFMO and placebo (260 versus 363 cancers, P = 0.069, two-sample t test). Evaluation of basal cell (BCC) and squamous cell cancers separately revealed very little difference in squamous cell cancer between treatment groups but a significant difference in new BCC (DFMO, 163 cancers; placebo, 243 cancers; expressed as event rate of 0.28 BCC/person/year versus 0.40 BCC/person/year, P = 0.03). Compliance with DFMO was >90% and it seemed to be well tolerated with evidence of mild ototoxicity as measured by serial audiometric examination when compared with placebo subjects. The analysis of normal skin biopsies revealed a significant (P < 0.05) decrease in 12-0-tetradecanoylphorbol-13-acetate–induced ODC activity (month 24, 36, and 48) and putrescine concentration (month 24 and 36 only) in DFMO subjects. Subjects with a history of skin cancer taking daily DFMO had an insignificant reduction (P = 0.069) in new NMSC that was predominantly due to a marked reduction in new BCC. Based on these data, the potential of DFMO, alone or in combination, to prevent skin cancers should be explored further. Cancer Prev Res; 3(1); 35–47

Patulin causes DNA damage leading to cell cycle arrest and apoptosis through modulation of Bax, p 53 and p 21/WAF1 proteins in skin of mice

Patulin (PAT), a mycotoxin found in apples, grapes, oranges, pear and peaches, is a potent genotoxic compound. WHO has highlighted the need for the study of cutaneous toxicity of PAT as manual labour is employed during pre and post harvest stages, thereby causing direct exposure to skin. In the present study cutaneous toxicity of PAT was evaluated following topical application to Swiss Albino mice. Dermal exposure of PAT, to mice for 4 h resulted in a dose (40–160 μg/animal) and time (up to 6 h) dependent enhancement of ornithine decarboxylase (ODC), a marker enzyme of cell proliferation. The ODC activity was found to be normal after 12 and 24 h treatment of patulin. Topical application of PAT (160 μg/100 μl acetone) for 24–72 h caused (a) DNA damage in skin cells showing significant increase (34–63%) in olive tail moment, a parameter of Comet assay (b) significant G 1 and S-phase arrest along with induction of apoptosis (2.8–10 folds) as shown by annexin V and PI staining assay through flow cytometer. Moreover PAT leads to over expression of p 21/WAF1 (3.6–3.9 fold), pro apoptotic protein Bax (1.3–2.6) and tumor suppressor wild type p 53 (2.8–3.9 fold) protein. It was also shown that PAT induced apoptosis was mediated through mitochondrial intrinsic pathway as revealed through the release of cytochrome C protein in cytosol leading to enhancement of caspase-3 activity in skin cells of mice. These results suggest that PAT has a potential to induce DNA damage leading to p 53 mediated cell cycle arrest along with intrinsic pathway mediated apoptosis that may also be correlated with enhanced polyamine production as evident by induction of ODC activity, which may have dermal toxicological implications.

Phase I, phase II 효소 및 ornithine decarboxylase에 미치는 해양심층수의 영향

동해에서 취수한 해양심층수의 암예방 효능을 얄아보고자 암발생 억제물질의 생화학적 표식자(biochemical markers)인 CYP 1A2 활성, phase II 효소인 QR과 GST의 활성, GSH 함량 및 ODC 활성에 미치는 영향을 살펴보았다. 해양심층수는 암의 개시단계(initiation)를 유발시키는 것으로 알려진 CYP 1A2 활성을 경도의존적으로 저해시켰다. 해양심층수를 Hepa 1clc7 세포에 경도별<TEX>$(100{\sim}1,000)$</TEX>로 처리하였을 때 phase II 생체 해독효소인 QR과 GST의 활성은 최대 20%의 증가를 나타내었고, 외부의 독성물질이나 대사산물로부터 세포를 보호하는 역할을 하는 GSH의 함량은 <TEX>$26{\sim}40%$</TEX>의 증가를 나타내었다. 그리고 발암과정의 촉진/진행단계에 관여하는 ODC의 활성은 해양심층수의 경도 800과 1,000에서 20%와 35%의 저해율을 나타내었으며, 경도 1,000을 처리한 군에서는 양성대조군인 DFMO와 같은 저해율을 나타내었다. 그러므로 본 연구 결과에 의하면 동해 해양심층수는 발암과정과 관련된 개시 및 촉진/진행단계를 저해시켜 암예방 효능을 나타낼 것으로 사료된다. Deep sea water was tested for cancer chemopreventive activity by measuring the activities of <TEX>${\beta}-$</TEX> naphthoflavone <TEX>$({\beta}-NF)-induced$</TEX> cytochrome P 450 1A2 (CYP 1A2), quinone reductase (QR) and glutathione-S-transferase (GST), glutathione (GSH) levels, and ornithine decarboxylase (ODC) activity. The in vitro incubation of rat liver microsome with deep sea water (a hardness range of <TEX>$100{\sim}1,000$</TEX>) showed a hardness-dependent inhibition of CYP 1A2 activity. QR and GST activities were induced about <TEX>$1.1{\sim}1.2$</TEX> fold with the treatment of deep sea water in murine hepatoma Hepa 1clc7 cells. In addition GSH levels were increased <TEX>$1.3{\sim}1.4$</TEX> fold in a hardness range of <TEX>$100{\sim}1,000$</TEX>. The deep sea water showed 20.3 and 35.0% inhibition of 12-O- tetradecanoylphorbol-13-a-cetate (TPA)-induced ODC activity at hardness 800 and 1,000, respectively. Therefore, deep sea water is worth further investigation with respect to cancer chemoprevention or therapy.

New transition state–based inhibitor for human ornithine decarboxylase inhibits growth of tumor cells

Pyridoxal 5'-phosphate (PLP)-dependent ornithine decarboxylase (ODC) is the key enzyme in polyamine synthesis. ODC is overexpressed in many tumor cells and thus a potential drug target. Here we show the design and synthesis of a coenzyme-substrate analogue as a novel precursor inhibitor of ODC. Structural analysis of the crystal structure of human ODC disclosed an additional hydrophobic pocket surrounding the epsilon-amino group of its substrate ornithine. Molecular modeling methods showed favorable interactions of the BOC-protected pyridoxyl-ornithine conjugate, termed POB, in the active site of human ODC. The synthesized and purified POB completely inhibited the activity of newly induced ODC activity at 100 micromol/L in glioma LN229 and COS7 cells. In correlation with the inhibition of ODC activity, a time-dependent inhibition of cell growth was observed in myeloma, glioma LN18 and LN229, Jurkat, COS7, and SW2 small-cell lung cancer cells if DNA synthesis and cell number were measured, but not in the nontumorigenic human aortic smooth muscle cells. POB strongly inhibited cell proliferation not only of low-grade glioma LN229 cells in a dose-dependent manner (IC(50) approximately 50 micromol/L) but also of high-grade glioblastoma multiforme cells. POB is much more efficient in inhibiting proliferation of several types of tumor cells than alpha-DL-difluoromethylornithine, the best known irreversible inhibitor of ODC.

Increasing ornithine decarboxylase activity is another way of prolactin preventing methotrexate-induced apoptosis: Crosstalk between ODC and BCL-2

Prolactin has more than 300 separate functions including affecting mammary growth, differentiation, secretion and anti-apoptosis. In the previous studies, prolactin induced Bcl-2 expression to prevent apoptosis and also provoked the activity of ornithine decarboxylase (ODC). Our previous data showed that ODC overexpression upregulates Bcl-2 and prevents tumor necrosis factor alpha (TNF-alpha)- and methotrexate (MTX)-induced apoptosis. Here, we further investigate whether prolactin prevents MTX-induced apoptosis through inducing ODC activity and the relationship between ODC and Bcl-2 upon prolactin stimulation. Prolactin prevented MTX-induced apoptosis in a dose-dependent manner in HL-60 cells. Following prolactin stimulation, ODC enzyme activity also shows an increase in a dose-dependent manner, expressing its maximum level at 3 h, and rapidly declining thereafter. Prolactin-induced ODC activity is completely blocked by a protein kinase C delta (PKCdelta) inhibitor, rottlerin. However, there are no changes in the expressions of ODC mRNA and protein level after prolactin stimulus. It indicates that prolactin may induce ODC activity through the PCKdelta pathway. Besides, Bcl-2 expresses within 1 h of prolactin treatment and this initiating effect of prolactin is not inhibited by alpha-difluoromethylornithine (DFMO). However, Bcl-2 is further enhanced following prolactin stimulation for 4 h and this enhancement is blocked by DFMO. Bcl-2 has no effect on ODC activity and protein levels, but ODC upregulates Bcl-2, which is inhibited by DFMO. Overall, there are two different forms of prolactin effect, it induces Bcl-2 primarily, and following this it stimulates ODC activity. Consequently induced ODC activity further enhances the expression of Bcl-2. The anti-apoptotic effect of prolactin is diminished by DFMO and recovered by putrescine. Obviously, ODC activity is one basis for the anti-apoptotic mechanisms of prolactin. A Bcl-2 inhibitor, HA14-1, together with DFMO, completely blocks the anti-apoptotic effects of prolactin. These results suggest that increasing ODC activity is another way of prolactin preventing MTX-induced apoptosis and that this induction of ODC activity enhances the expression of Bcl-2 strongly enough to bring about the anti-apoptotic function.

Chemopreventive effect of protein extract ofAsterina pectinifera in HT-29 human colon adenocarcinoma cells

We investigated the effect of protein extract of Asterina pectinifera on the activity of 4 enzymes that may play a role in adenocarcinoma of the colon: quinone reductase (QR), glutathione S-transferase (GST), ornithine decarboxylase (ODC), and cyclooxygenase (COX)-2. QR and GST activity increased in HT-29 human colon adenocarcinoma cells increased that had been exposed to 4 concentrations of the protein extract (80, 160, 200, and 240 microg/mL). Additionally, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ODC activity decreased significantly in cells exposed to the extract in concentrations of 160 microg/mL (p<0.05), 200 microg/mL (p<0.005), and 240 microg/mL (p<0.005). TPA-induced COX-2 activity also decreased in cells exposed to extract concentrations of 10, 20, 40, and 60 microg/mL. COX-2 expression was also inhibited in cells exposed to this extract. These results suggest that this protein extract of A pectinifera has chemopreventive activity in HT-29 human colon adenocarcinoma cells, and therefore, may have the potential to function as a chemopreventive agent in human colorectal cancer.

Efficacy of glutamine-enriched enteral nutrition in an experimental model of mucosal ulcerative colitis.

Intact intestinal epithelium and associated lymphatic tissue act as body defences against luminal toxins. This barrier may become threatened or compromised in inflammatory bowel disease, leading to an increase in mucosal permeability and subsequent translocation of endotoxins. The effect of oral glutamine on gut mucosal ornithine decarboxylase activity and on endotoxin levels in portal vein blood was studied in a guinea-pig model of carrageenan-induced colitis. Despite failure to show induction of ornithine decarboxylase activity by glutamine administration, the mean endotoxin level of portal vein blood in guinea-pigs fed a glutamine-enriched elemental diet was 25.3 pg/ml compared with 71.2 pg/ml in animals given a standard elemental diet (P < 0.01). A glutamine-enriched elemental diet may be therapeutically beneficial in patients with inflammatory bowel disease.

Ferulic Acid Stabilizes a Solution of Vitamins C and E and Doubles its Photoprotection of Skin

Ferulic acid is a potent ubiquitous plant antioxidant. Its incorporation into a topical solution of 15%l-ascorbic acid and 1%alpha-tocopherol improved chemical stability of the vitamins (C+E) and doubled photoprotection to solar-simulated irradiation of skin from 4-fold to approximately 8-fold as measured by both erythema and sunburn cell formation. Inhibition of apoptosis was associated with reduced induction of caspase-3 and caspase-7. This antioxidant formulation efficiently reduced thymine dimer formation. This combination of pure natural low molecular weight antioxidants provides meaningful synergistic protection against oxidative stress in skin and should be useful for protection against photoaging and skin cancer.

Induction of Ornithine Decarboxylase Activity Is a Necessary Step for Mitogen-Activated Protein Kinase Kinase–Induced Skin Tumorigenesis

A transgenic mouse line overexpressing a constitutively active mutant of MEK1, a downstream effector of Ras, driven by the keratin 14 (K14) promoter, has been used to test the hypothesis that ornithine decarboxylase (ODC) induction during tumor promotion following a single initiating event [i.e., the activation of the Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway], is a necessary step in skin carcinogenesis. K14-MEK mice exhibit moderate hyperplasia, with spontaneous skin tumor development within 5 weeks of birth. Analysis of epidermis and dermis showed induction of MEK protein and ERK1/ERK2 phosphorylation, but no change in Akt-1, suggesting that the PI 3-kinase pathway, another pathway downstream of ras, is not activated. Examination of tumors revealed high levels of ODC protein and activity, indicating that activation of signaling cascades dependent on MEK activity is a sufficient stimulus for ODC induction. When K14-MEK mice were given alpha-difluoromethylornithine (DFMO), a suicide inactivator of ODC, in the drinking water from birth, there was a dramatic delay in the onset of tumor growth ( approximately 6 weeks), and only 25% of DFMO-treated mice developed tumors by 15 weeks of age. All untreated K14-MEK mice developed tumors by 6 weeks of age. Treatment of tumor-bearing mice with DFMO reduced both tumor size and tumor number within several weeks. Tumor regression was the result of both inhibition of proliferation and increased apoptosis in tumors. The results establish ODC activation as an important component of the Raf/MEK/ERK pathway, and identify K14-MEK mice as a valuable model with which to study the regulation of ODC in ras carcinogenesis.

Effect of Green Tea Extract on the Induction of Ornithine Decarboxylase and the Activation of Extracellular Signal-Regulated Kinase in Bladder Carcinoma ECV304 Cells

According to several studies, green tea and individual catechins can inhibit the induction of ornithine decarboxylase (ODC), the key enzyme in the biosynthesis of polyamines. It has been suggested that the inhibition of ODC induction may offer an explanation to the anticancer and chemopreventive activities of green tea. In the present study, however, treatment of bladder carcinoma ECV304 cells with green tea extract (GTE) was not able to reduce the induction of ODC by fetal calf serum. Actually, in the absence of serum, GTE provoked a dose-dependent and remarkable induction of ODC activity. The induction of ODC, which could be elicited also by (-)-epigallocatechin 3-gallate, a major green tea component, required an early activation of extracellular signal-regulated kinase 1 and 2 (ERK), and both events appeared to be dependent on an alteration of the status of cellular thiol groups. Pretreatment with specific ERK or ODC inhibitors was able to prevent a late caspase activation but hardly affected the loss of cell viability provoked by GTE. In conclusion, to our knowledge, this is the first study showing that GTE can promote ODC induction in a tumor cell line.

Glyceryl trinitrate, a nitric oxide donor, abrogates ferric nitrilotriacetate-induced oxidative stress and renal damage

Ferric nitrilotriacetate (Fe-NTA), a common water pollutant and a known renal carcinogen, acts through the generation of oxidative stress and hyperproliferative response. In the present study, we show that the nitric oxide (NO) generated by the administration of glyceryl trinitrate (GTN) affords protection against Fe-NTA-induced oxidative stress and proliferative response. Administration of Fe-NTA resulted in a significant ( P<0.001) depletion of renal glutathione (GSH) content with concomitant increase in lipid peroxidation and elevated tissue damage marker release in serum. Parallel to these changes, Fe-NTA also caused down-regulation of GSH metabolizing enzymes including glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione- S-transferase and several fold induction in ornithine decarboxylase (ODC) activity and rate of DNA synthesis. Subsequent exogenous administration of GTN at doses of 3 and 6 mg/kg body weight resulted in significant ( P<0.001) recovery of GSH metabolizing enzymes and amelioration of tissue GSH content, in a dose-dependent manner. GTN administration also inhibited malondialdehyde (MDA) formation, induction of ODC activity, enhanced rate of DNA synthesis, and pathological deterioration in a dose-dependent fashion. Further, administration of NO inhibitor, N G-nitro- l-arginine methyl ester ( l-NAME), exacerbated Fe-NTA-induced oxidative tissue injury, hyperproliferative response, and pathological damage. Overall, the study suggests that NO administration subsequent to Fe-NTA affords protection against ROS-mediated damage induced by Fe-NTA.

Inhibitory effects of the ginsenoside Rg 3 on phorbol ester-induced cyclooxygenase-2 expression, NF-κB activation and tumor promotion

Our previous studies demonstrated the anti-oxidant and anti-tumor promotional properties of the methanol extract of heat-processed Panax ginseng C.A. Meyer [Cancer Lett. 150 (2000) 41]. In the present work, we have evaluated anti-inflammatory as well as anti-tumor promoting effects of Rg 3, a major ginsenoside derived from heat-processed ginseng. Pretreatment of dorsal skins of female ICR mice with Rg 3 significantly inhibited 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase activity and 7,12-dimethylbenz[ a]anthracene-initiated papilloma formation. In another experiment, Rg 3 pretreatment abrogated the expression of cyclooxygenase-2 in TPA-stimulated mouse skin. Rg 3 also inhibited the TPA-induced activation of the eukaryotic transcription factor, NF-κB in both mouse skin and cultured human pro-myelocytic leukemia (HL-60) cells. Moreover, Rg 3 exerted potent inhibitory effects on the activation of another transcription factor, activator protein-1 (AP-1) that is responsible for c- jun and c- fos oncogenic transactivation. Based on these findings, it is likely that the anti-tumor promoting activity of Rg 3 is mediated possibly through down-regulation of NF-κB and AP-1 transcription factors.

Inhibitory effects of the standardized extract (DA‐9601) of Artemisia asiatica Nakai on phorbol ester–induced ornithine decarboxylase activity, papilloma formation, cyclooxygenase‐2 expression, inducible nitric oxide synthase expression and nuclear transcription factor κB activation in mouse skin

Artemisia asiatica Nakai has been used in traditional Asian medicine for the treatment of inflammatory and other disorders. Previous studies have revealed that the formulated ethanol extract (DA-9601) of A. asiatica has pronounced antioxidative and antiinflammatory activities and exhibits cytoprotective effects against experimentally induced gastrointestinal, hepatic and pancreatic damage. In the present study, we assessed the inhibitory effect of DA-9601 on tumor promotion, which is closely linked to inflammatory tissue damage. As an initial approach to evaluating the possible antitumor-promoting potential of DA-9601, its effects on TPA-induced ear edema were examined in female ICR mice. Pretreatment of the inner surface of the mouse ear with DA-9601 30 min prior to topical application of TPA inhibited ear edema at 5 hr. TPA-stimulated expression of epidermal COX-2 and iNOS was also mitigated by topical application of the same extract. Moreover, DA-9601 abrogated the TPA-mediated activation of NF-kappa B/Rel and AP-1 in mouse epidermis. Suppression of epidermal NF-kappa B by DA-9601 appeared to be mediated in part through inhibition of I kappa B alpha degradation, thereby blocking the nuclear translocation of p65, the functional subunit of NF-kappa B. DA-9601 also significantly suppressed TPA-induced ODC activity and papilloma formation in mouse skin. Taken together, these findings suggest that DA-9601 derived from A. asiatica possesses potential chemopreventive activities.

Low iron state is associated with reduced tumor promotion in a two-stage mouse skin carcinogenesis model

The purpose of this study was to investigate the effect of low iron state in a two-stage mouse skin carcinogenesis model using 7,12-dimethylbenz[ a]anthracene (DMBA)-initiated and benzoyl peroxide (BPO)-promoted cutaneous tumorigenesis. All mice were treated with DMBA. Low iron state was achieved by injection with phenylhydrazine hydrochloride and feeding low iron diet. A low iron state resulted in a decrease in tumor incidence (papillomas and carcinomas) and number of tumors/mouse. Also, the conversion of papillomas to carcinomas was lower in mice on a low iron state. BPO treatment enhanced epidermal lipid peroxidation (LPO) and was accompanied by a depletion in the level of epidermal reduced glutathione (GSH) and decrease in the activities of antioxidant enzymes. BPO treatment also increased ornithine decarboxylase (ODC) activity and [ 3H]thymidine incorporation into cutaneous DNA. Mice in a low iron state were less susceptible to the effects of BPO treatment, as was apparent from a partial recovery of GSH levels and the activities of antioxidant enzymes, as well as a lower induction in ODC activity, [ 3H]thymidine incorporation into cutaneous DNA and lesser epidermal LPO. As expected, cutaneous iron levels were lower in mice on a low iron state. Thus, our data show that the tumor-promoting potential of BPO is reduced by low iron state in a two-stage mouse skin carcinogenesis model.