Preventing Cancer by Inhibiting Ornithine Decarboxylase: A Comparative Perspective on Synthetic vs. Natural Drugs.

This perspective delves into the investigation of synthetic and naturally occurring inhibitors, their patterns of inhibition, and the effectiveness of newly utilized natural compounds as inhibitors targeting the Ornithine decarboxylase enzyme. This enzyme is known to target the MYC oncogene, thereby establishing a connection between polyamine metabolism and oncogenesis in both normal and cancerous cells. ODC activation and heightened polyamine activity are associated with tumor development in numerous cancers and fluctuations in ODC protein levels exert a profound influence on cellular activity for inhibition or suppressing tumor cells. This perspective outlines efforts to develop novel drugs, evaluate natural compounds, and identify promising inhibitors to address gaps in cancer prevention, highlighting the potential of newly designed synthetic moieties and natural flavonoids as alternatives. It also discusses natural compounds with potential as enhanced inhibitors.

Probenecid increases renal retention and antitumor activity of DFMO in neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial solid tumor in children. Interference with the polyamine biosynthesis pathway by inhibition of MYCN-activated ornithine decarboxylase (ODC) is a validated approach. The ODC inhibitor α-difluoromethylornithine (DFMO, or Eflornithine) has been FDA-approved for the treatment of trypanosomiasis and hirsutism and has advanced to clinical cancer trials including NB as well as cancer-unrelated human diseases. One key challenge of DFMO is its rapid renal clearance and the need for high and frequent drug dosing during treatment. METHODS: We performed in vivo pharmacokinetic (PK), antitumorigenic, and molecular studies with DFMO/probenecid using NB patient-derived xenografts (PDX) in mice. We used LC-MS/MS, HPLC, and immunoblotting to analyze blood, brain tissue, and PDX tumor tissue samples collected from mice. RESULTS: The organic anion transport 1/3 (OAT 1/3) inhibitor probenecid reduces the renal clearance of DFMO and significantly increases the antitumor activity of DFMO in PDX of NB (P < 0.02). Excised tumors revealed that DFMO/probenecid treatment decreases polyamines putrescine and spermidine, reduces MYCN protein levels and dephosphorylates retinoblastoma (Rb) protein (p-RbSer795), suggesting DFMO/probenecid-induced cell cycle arrest. CONCLUSION: Addition of probenecid as an adjuvant to DFMO therapy may be suitable to decrease overall dose and improve drug efficacy in vivo.

Characterising the Response of Human Breast Cancer Cells to Polyamine Modulation.

Breast cancer is a complex heterogeneous disease with multiple underlying causes. The polyamines putrescine, spermidine, and spermine are polycationic molecules essential for cell proliferation. Their biosynthesis is upregulated in breast cancer and they contribute to disease progression. While elevated polyamines are linked to breast cancer cell proliferation, there is little evidence to suggest breast cancer cells of different hormone receptor status are equally dependent on polyamines. In this study, we characterized the responses of two breast cancer cells, ER+ (oestrogen receptor positive) MCF-7 and ER- MDA-MB-231 cell lines, to polyamine modulation and determined the requirement of each polyamine for cancer cell growth. The cells were exposed to DFMO (a polyamine pathway inhibitor) at various concentrations under different conditions, after which several growth parameters were determined. Exposure of both cell lines to DFMO induced differential growth responses, MCF-7 cells showed greater sensitivity to polyamine pathway inhibition at various DFMO concentrations than the MDA-MB-231 cells. Analysis of intracellular DFMO after withdrawal from growth medium showed residual DFMO in the cells with concomitant decreases in polyamine content, ODC protein level, and cell growth. Addition of exogenous polyamines reversed the cell growth inhibition, and this growth recovery appears to be partly dependent on the spermidine content of the cell. Similarly, DFMO exposure inhibits the global translation state of the cells, with spermidine addition reversing the inhibition of translation in the breast cancer cells. Taken together, these data suggest that breast cancer cells are differentially sensitive to the antitumour effects of polyamine depletion, thus, targeting polyamine metabolism might be therapeutically beneficial in breast cancer management based on their subtype.

α-Difluoromethylornithine-Induced Cytostasis is Reversed by Exogenous Polyamines, Not by Thymidine Supplementation.

Polyamine spermidine is essential for the proliferation of eukaryotic cells. Administration of polyamine biosynthesis inhibitor α-difluoromethylornithine (DFMO) induces cytostasis that occurs in two phases; the early phase which can be reversed by spermidine, spermine, and some of their analogs, and the late phase which is characterized by practically complete depletion of cellular spermidine pool. The growth of cells at the late phase can be reversed by spermidine and by very few of its analogs, including (S)-1-methylspermidine. It was reported previously (Witherspoon et al. Cancer Discovery 3(9); 1072-81, 2013) that DFMO treatment leads to depletion of cellular thymidine pools, and that exogenous thymidine supplementation partially prevents DFMO-induced cytostasis without affecting intracellular polyamine pools in HT-29, SW480, and LoVo colorectal cancer cells. Here we show that thymidine did not prevent DFMO-induced cytostasis in DU145, LNCaP, MCF7, CaCo2, BT4C, SV40MES13, HepG2, HEK293, NIH3T3, ARPE19 or HT-29 cell lines, whereas administration of functionally active mimetic of spermidine, (S)-1-methylspermidine, did. Thus, the effect of thymidine seems to be specific only for certain cell lines. We conclude that decreased polyamine levels and possibly also distorted pools of folate-dependent metabolites mediate the anti-proliferative actions of DFMO. However, polyamines are necessary and sufficient to overcome DFMO-induced cytostasis, while thymidine is generally not.

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.

Allicin, a Potent New Ornithine Decarboxylase Inhibitor in Neuroblastoma Cells.

The natural product allicin is a reactive sulfur species (RSS) from garlic (Allium sativum L.). Neuroblastoma (NB) is an early childhood cancer arising from the developing peripheral nervous system. Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the biosynthesis of polyamines, which are oncometabolites that contribute to cell proliferation in NB and other c-MYC/MYCN-driven cancers. Both c-MYC and MYCN directly transactivate the E-box gene ODC1, a validated anticancer drug target. We identified allicin as a potent ODC inhibitor in a specific radioactive in vitro assay using purified human ODC. Allicin was ∼23 000-fold more potent (IC50 = 11 nM) than DFMO (IC50 = 252 µM), under identical in vitro assay conditions. ODC is a homodimer with 12 cysteines per monomer, and allicin reversibly S-thioallylates cysteines. In actively proliferating human NB cells allicin inhibited ODC enzyme activity, reduced cellular polyamine levels, inhibited cell proliferation (IC50 9-19 µM), and induced apoptosis. The natural product allicin is a new ODC inhibitor and could be developed for use in conjunction with other anticancer treatments, the latter perhaps at a lower than usual dosage, to achieve drug synergism with good prognosis and reduced adverse effects.

The rational discovery of multipurpose inhibitors of the ornithine decarboxylase.

Metabolic reprograming is a hallmark of cancer, and the polyamine metabolic network is dysregulated in many cancers. Ornithine decarboxylase (ODC) is a rate-limiting enzyme for polyamine synthesis in the polyamine metabolic network. In many cancer cells, ODC is over-expressed, so this enzyme has been an attracting anti-cancer drug target. In the catalysis axis (pathway), ODC converts ornithine to putrescine. Meanwhile, ODC's activity is regulated by protein-protein interactions (PPIs), including the ODC-OAZ1-AZIN1 PPI axis and its monomer-dimer equilibrium. Previous studies showed that when ODC's activity is inhibited, the PPIs might counteract the inhibition efficiency. Therefore, we proposed that multipurpose inhibitors that can simultaneously inhibit ODC's activity and perturb the PPIs would be very valuable as drug candidates and molecular tools. To discover multipurpose ODC inhibitors, we established a computational pipeline by combining positive screening and negative screening. We used this pipeline for the forward screening of multipurpose ligands that might inhibit ODC's activity, block ODC-OAZ1 interaction and enhance ODC non-functional dimerization. With a combination of different experimental assays, we identified three multipurpose ODC inhibitors. At last, we showed that one of these inhibitors is a promising drug candidate. This work demonstrated that our computational pipeline is useful for discovering multipurpose ODC inhibitors, and multipurpose inhibitors would be very valuable. Similar with ODC, there are a lot of proteins in human proteome that act as both enzymes and PPI components. Therefore, this work is not only presenting new molecular tools for polyamine study, but also providing potential insights and protocols for discovering multipurpose inhibitors to target more important protein targets.

Anti-GD2 induced allodynia in rats can be reduced by pretreatment with DFMO.

BACKGROUND: Anti-GD2 therapy with dinutuximab is effective in improving the survival of high-risk neuroblastoma patients in remission and after relapse. However, allodynia is the major dose-limiting side effect, hindering its use for neuroblastoma patients at higher doses and for other GD2-expressing malignancies. As polyamines can enhance neuronal sensitization, including development of allodynia and other forms of pathological pain, we hypothesized that polyamine depletion might prove an effective strategy for relief of anti-GD2 induced allodynia. METHOD: Sprague-Dawley rats were allowed to drink water containing various concentrations of difluoromethylornithine (DFMO) for several days prior to behavioral testing. Anti-GD2 (14G2a) was injected into the tail vein of lightly sedated animals and basal mechanical hindpaw withdrawal threshold assessed by von Frey filaments. Endpoint serum DFMO and polyamines, assessed 24h after 14G2a injection, were measured by HPLC and mass spectrometry. RESULTS: An i.v. injection of 14G2a causes increased paw sensitivity to light touch in this model, a response that closely mimics patient allodynia. Animals allowed to drink water containing 1% DFMO exhibited a significant reduction of 14G2a-induced pain sensitivity (allodynia). Increasing the dosage of the immunotherapeutic increased the magnitude (intensity and duration) of the pain behavior. Administration of DFMO attenuated the enhanced sensitivity. Consistent with the known actions of DFMO on ornithine decarboxylase (ODC), serum putrescene and spermidine levels were significantly reduced by DFMO, though the decrease in endpoint polyamine levels did not directly correlate with the behavioral changes. CONCLUSIONS: Our results demonstrate that DFMO is an effective agent for reducing anti-GD2 -induced allodynia. Using DFMO in conjunction with dinutuximab may allow for dose escalation in neuroblastoma patients. The reduction in pain may be sufficient to allow new patient populations to utilize this therapy given the more acceptable side effect profile. Thus, DFMO may be an important adjunct to anti-GD2 immunotherapy in addition to a role as a potential anti-cancer therapeutic.

Inhibition of the polyamine synthesis enzyme ornithine decarboxylase sensitizes triple-negative breast cancer cells to cytotoxic chemotherapy.

Treatment of patients with triple-negative breast cancer (TNBC) is limited by a lack of effective molecular therapies targeting this disease. Recent studies have identified metabolic alterations in cancer cells that can be targeted to improve responses to standard-of-care chemotherapy regimens. Using MDA-MB-468 and SUM-159PT TNBC cells, along with LC-MS/MS and HPLC metabolomics profiling, we found here that exposure of TNBC cells to the cytotoxic chemotherapy drugs cisplatin and doxorubicin alter arginine and polyamine metabolites. This alteration was because of a reduction in the levels and activity of a rate-limiting polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). Using gene silencing and inhibitor treatments, we determined that the reduction in ODC was mediated by its negative regulator antizyme, targeting ODC to the proteasome for degradation. Treatment with the ODC inhibitor difluoromethylornithine (DFMO) sensitized TNBC cells to chemotherapy, but this was not observed in receptor-positive breast cancer cells. Moreover, TNBC cell lines had greater sensitivity to single-agent DFMO, and ODC levels were elevated in TNBC patient samples. The alterations in polyamine metabolism in response to chemotherapy, as well as DFMO-induced preferential sensitization of TNBC cells to chemotherapy, reported here suggest that ODC may be a targetable metabolic vulnerability in TNBC.

Ornithine decarboxylase inhibition downregulates multiple pathways involved in the formation of precancerous lesions of esophageal squamous cell cancer.

The high incidence and mortality of esophageal squamous cell cancer (ESCC) is a major health problem worldwide. Precancerous lesions of ESCC may either progress to cancer or revert to normal epithelium with appropriate interventions; the bidirectional instability of the precancerous lesions of ESCC provides opportunities for intervention. Reports suggest that the upregulation of ornithine decarboxylase (ODC) is closely related to carcinogenesis. In this study, we investigated whether ODC may act as a target for chemoprevention in ESCC. Immunohistochemistry (IHC) assays indicate that ODC expression is higher in esophageal precancerous lesions compared with normal tissue controls. Its overexpression promotes cell proliferation and transformation of normal esophageal epithelial cells, and its activity is increased after N-nitrosomethylbenzylamine (NMBA) induction in Shantou human embryonic esophageal cell line (SHEE) and human immortalized cells (Het1A) cells. In addition, p38 α, extracellular regulated kinase (ERK1/2) in the mitogen-activated protein kinase pathway and protein kinase B (AKT)/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (p70S6K) pathways are activated in response to NMBA treatment. Difluoromethylornithine (DFMO) is an ODC inhibitor, which inhibits NMBA-induced activation of p38 α, ERK1/2 and AKT/mTOR/p70S6K pathways; this has been verified by Western blotting. DFMO was also found to suppress the development of esophageal precancerous lesions in an NMBA-induced rat model; IHC demonstrated p38 α, ERK1/2, and AKT/mTOR/p70S6K pathways to be downregulated in these rats. These findings indicate the mechanisms by which ODC inhibition suppresses the development of esophageal precancerous lesions by downregulating p38 α, ERK1/2, and AKT/mTOR/p70S6k signaling pathways, ODC may be a potential target for chemoprevention in ESCC.

Unforeseen Possibilities To Investigate the Regulation of Polyamine Metabolism Revealed by Novel C-Methylated Spermine Derivatives.

The biogenic polyamines, spermine (Spm) and spermidine, are organic polycations present in millimolar concentrations in all eukaryotic cells participating in the regulation of vital cellular functions including proliferation and differentiation. The design and biochemical evaluation of polyamine analogues are cornerstones of polyamine research. Here we synthesized and studied novel C-methylated Spm analogues: 2,11-dimethylspermine (2,11-Me2Spm), 3,10-dimethylspermine (3,10-Me2Spm), 2-methylspermine, and 2,2-dimethylspermine. The tested analogues overcame growth arrest induced by a 72 h treatment with α-difluoromethylornithine, an ornithine decarboxylase (ODC) inhibitor, and entered into DU145 cells via the polyamine transporter. 3,10-Me2Spm was a poor substrate of spermine oxidase and spermidine/spermine-N1-acetyltransferase (SSAT) when compared with 2,11-Me2Spm, thus resembling 1,12-dimethylspermine, which lacks the substrate properties required for the SSAT reaction. The antizyme (OAZ1)-mediated downregulation of ODC and inhibition of polyamine transport are crucial in the maintenance of polyamine homeostasis. Interestingly, 3,10-Me2Spm was found to be the first Spm analogue that did not induce OAZ1 and, consequently, was a weak downregulator of ODC activity in DU145 cells.

Purification and Characterization of Ornithine Decarboxylase from Aspergillus terreus; Kinetics of Inhibition by Various Inhibitors.

l-Ornithine decarboxylase (ODC) is the rate-limiting enzyme of de novo polyamine synthesis in humans and fungi. Elevated levels of polyamine by over-induction of ODC activity in response to tumor-promoting factors has been frequently reported. Since ODC from fungi and human have the same molecular properties and regulatory mechanisms, thus, fungal ODC has been used as model enzyme in the preliminary studies. Thus, the aim of this work was to purify ODC from fungi, and assess its kinetics of inhibition towards various compounds. Forty fungal isolates were screened for ODC production, twenty fungal isolates have the higher potency to grow on L-ornithine as sole nitrogen source. Aspergillus terreus was the most potent ODC producer (2.1 µmol/mg/min), followed by Penicillium crustosum and Fusarium fujikuori. These isolates were molecularly identified based on their ITS sequences, which have been deposited in the NCBI database under accession numbers MH156195, MH155304 and MH152411, respectively. ODC was purified and characterized from A. terreus using SDS-PAGE, showing a whole molecule mass of ~110 kDa and a 50 kDa subunit structure revealing its homodimeric identity. The enzyme had a maximum activity at 37 °C, pH 7.4-7.8 and thermal stability for 20 h at 37 °C, and 90 days storage stability at 4 °C. A. terreus ODC had a maximum affinity (Km) for l-ornithine, l-lysine and l-arginine (0.95, 1.34 and 1.4 mM) and catalytic efficiency (kcat/Km) (4.6, 2.83, 2.46 × 10-5 mM-1·s-1). The enzyme activity was strongly inhibited by DFMO (0.02 µg/mL), curcumin (IC50 0.04 µg/mL), propargylglycine (20.9 µg/mL) and hydroxylamine (32.9 µg/mL). These results emphasize the strong inhibitory effect of curcumin on ODC activity and subsequent polyamine synthesis. Further molecular dynamic studies to elucidate the mechanistics of ODC inhibition by curcumin are ongoing.

Inhibition of ornithine decarboxylase 1 facilitates pegylated arginase treatment in lung adenocarcinoma xenograft models.

Arginine depletion has shown anticancer effects among arginine auxotrophic cancers. An anti­proliferative effect of pegylated arginase (BCT­100) has been shown in acute myeloid leukaemia, hepatocellular carcinoma and mesothelioma. The aim of the present study was to evaluate the effect of BCT­100 in lung adenocarcinoma. A panel of lung adenocarcinoma cell lines and xenograft models were used to investigate the effect of BCT­100. Protein expression, arginine level, putrescine level, spermidine level and apoptosis were analyzed by western blotting, ELISA, high performance liquid chromatography, dot blot and TUNEL assay, respectively. BCT­100 converts arginine to ornithine. BCT­100 reduced in vitro cell viability across different lung adenocarcinoma cell lines and suppressed tumour growth in an HCC4006 xenograft, while paradoxical growth stimulation was observed in H358, HCC827, H1650 and H1975 xenografts. Upon BCT­100 treatment, ornithine decarboxylase 1 (ODC1) was induced in two solid tumour xenografts (H1650 and H1975). It was postulated that the accumulated ornithine could be channeled via ODC1 to produce polyamines that promoted tumour growth. The action of an ODC1 inhibitor (α­difluoromethylornithine, DFMO) was studied in the restoration of the anticancer effects of BCT­100 in lung adenocarcinoma. In both H1650 and H1975 xenografts, a combination of DFMO and BCT­100 significantly suppressed tumour growth, resulting in doubled median survival compared with the control. Putrescine was decreased in almost all treatment arms in the H1650, H1975 and HCC4006 xenografts. Nonetheless spermidine was reduced only following DFMO/BCT­100 treatment in the H1650 and H1975 xenografts. Apoptosis was enhanced in the combined treatment arm in both H1650 and H1975 xenografts. In the HCC4006 xenograft, addition of DFMO did not alter the tumour suppressive effect of BCT­100. In conclusion, inhibition of ODC1 by DFMO was crucial in facilitating BCT­100 treatment in lung adenocarcinoma that was partially mediated by depleting arginine and polyamines with consequent apoptosis.

Urtica dioica inhibits cell growth and induces apoptosis by targeting Ornithine decarboxylase and Adenosine deaminase as key regulatory enzymes in adenosine and polyamines homeostasis in human breast cancer cell lines.

Breast cancer is a heterogeneous and multifactorial disease with variable disease progression risk, and treatment response. Urtica dioica is a traditional herb used as an adjuvant therapeutic agent in cancer. In the present study, we have evaluated the effects of the aqueous extract of Urtica dioica on Adenosine deaminase (ADA) and Ornithine decarboxylase (ODC1) gene expression in MCF-7, MDA-MB-231, two breast cancer cell lines being estrogen receptor positive and estrogen receptor negative, respectively.  Cell lines were cultured in suitable media. After 24 h, different concentrations of the extract were added and after 72 h, ADA and ODC1 gene expression as well as BCL2 and BAX apoptotic genes were assessed by Taqman real time PCR assay. Cells viability was assessed by MTT assay, and apoptosis was also evaluated at cellular level. The intra and extracellular levels of ODC1 and ADA enzymes were evaluated by ELISA. Results showed differential expression of ADA and ODC1 genes in cancer cell lines. In MCF-7 cell line, the expression level of ADA was upregulated in a dose-dependent manner but its expression did not change in MDA-MB cell line. ODC1 expression was increased in both examined cell lines. Also, increased level of the apoptotic BAX/BCL-2 ratio was detected in MCF-7 cells. These results demonstrated that Urtica dioica induces apoptosis in breast cancer cells by influencing ODC1 and ADA genes expression, and estrogen receptors. The different responses observed with these cell lines could be due to the interaction of Urtica dioica as a phytoestrogen with the estrogen receptor.

Difluoromethylornithine Combined with a Polyamine Transport Inhibitor Is Effective against Gemcitabine Resistant Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is highly chemo-resistant and has an extremely poor patient prognosis, with a survival rate at five years of <8%. There remains an urgent need for innovative treatments. Targeting polyamine biosynthesis through inhibition of ornithine decarboxylase with difluoromethylornithine (DFMO) has had mixed clinical success due to tumor escape via an undefined transport system, which imports exogenous polyamines and sustains intracellular polyamine pools. Here, we tested DFMO in combination with a polyamine transport inhibitor (PTI), Trimer44NMe, against Gemcitabine-resistant PDAC cells. DFMO alone and with Trimer44NMe significantly reduced PDAC cell viability by inducing apoptosis or diminishing proliferation. DFMO alone and with Trimer44NMe also inhibited in vivo orthotopic PDAC growth and resulted in decreased c-Myc expression, a readout of polyamine pathway dysfunction. Moreover, dual inhibition significantly prolonged survival of tumor-bearing mice. Collectively, these studies demonstrate that targeting polyamine biosynthesis and import pathways in PDAC can lead to increased survival in pancreatic cancer.

Ornithine Decarboxylase Inhibition: A Strategy to Combat Various Diseases.

Ornithine decarboxylase is the first enzyme in the polyamine biosynthetic pathway. It is the rate-limiting enzyme which is included during the change of ornithine to putrescine which is the first polyamine. Polyamines (putrescine, spermidine, spermine) are natural and synthetic compounds which contain two or more amino group. Polyamines are highly implicated in cellular functions such as cell growth & multiplication, DNA stabilization, gene transcription and translation, ion-channel activity, etc. Elevated levels of polyamines are found in highly proliferating tumor cells. Hence inhibition of this enzyme was found useful in cancer. α-DL-difluoromethylornithine(DFMO) (Eflornithine) an enzyme- activated irreversible inhibitor was the first of this type. However, its use as an anticancer agent did not continue for long due to various reasons. Polyamines have also been found to play an important role in other infectious microorganisms. Eflornithine is successfully used in diseases such as African sleeping sickness and is being researched against a number of tropical diseases. It is widely used against hirsutism in women. Various other product (putrescine) based analogs and transition state or PLP (cofactor) based analogs are being synthesized against diseases such as Leishmaniasis, Malaria and others discussed in the article.

Herbacetin suppresses cutaneous squamous cell carcinoma and melanoma cell growth by targeting AKT and ODC.

Herbacetin is a flavonol compound that is found in plants such as flaxseed and ramose scouring rush herb, it possesses a strong antioxidant capacity, and exerts anticancer effects on colon and breast cancer. However, the effect of herbacetin on skin cancer has not been investigated. Herein, we identified herbacetin as a dual V-akt murine thymoma viral oncogene homolog (AKT) and ornithine decarboxylase (ODC) inhibitor, and illustrated its anticancer effects in vitro and in vivo against cutaneous squamous cell carcinoma (SCC) and melanoma cell growth. To identify the direct target(s) of herbacetin, we screened several skin cancer-related protein kinases, and results indicated that herbacetin strongly suppresses both AKT and ODC activity. Results of cell-based assays showed that herbacetin binds to both AKT and ODC, inhibits TPA-induced neoplastic transformation of JB6 mouse epidermal cells, and suppresses anchorage-independent growth of cutaneous SCC and melanoma cells. The inhibitory activity of herbacetin was associated with markedly reduced NF-κB and AP1 reporter activity. Interestingly, herbacetin effectively attenuated TPA-induced skin cancer development and also exhibited therapeutic effects against solar-UV-induced skin cancer and melanoma growth in vivo. Our findings indicate that herbacetin is a potent AKT and ODC inhibitor that should be useful for preventing skin cancers.

[Low-molecular-weight regulators of biogenic polyamine metabolism affect cytokine production and expression of hepatitis С virus proteins in Huh7.5 human hepatocarcinoma cells].

Hepatitis C virus (HCV) induces the expression of the genes of proinflammatory cytokines, the excessive production of which may cause cell death, and contribute to development of liver fibrosis and hepatocarcinoma. The relationship between cytokine production and metabolic disorders in HCV-infected cells remains obscure. The levels of biogenic polyamines, spermine, spermidine, and their precursor putrescine, may be a potential regulator of these processes. The purpose of the present work was to study the effects of the compounds which modulate biogenic polyamines metabolism on cytokine production and HCV proteins expression. Human hepatocarcinoma Huh7.5 cells have been transfected with the plasmids that encode HCV proteins and further incubated with the following low-molecular compounds that affect different stages of polyamine metabolism: (1) difluoromethylornithine (DFMO), the inhibitor of ornithine decarboxylase, the enzyme that catalyzes the biosynthesis of polyamines; (2) N,N'-bis(2,3-butane dienyl)-1,4-diaminobutane (MDL72.527), the inhibitor of proteins involved in polyamine degradation; and (3) synthetic polyamine analog N^(I),N^(II)-diethylnorspermine (DENSpm), an inducer of polyamine degradation enzyme. The intracellular accumulation and secretion of cytokines (IL-6, IL-1ß, TNF-α, and TGF-ß) was assessed by immunocytochemistry and in the immunoenzyme assay, while the cytokine gene expression was studied using reverse transcription and PCR. The effects of the compounds under analysis on the expression of HCV proteins were analyzed using the indirect immunofluorescence with anti-HCV monoclonal antibodies. It has been demonstrated that, in cells transfected with HCV genes, DFMO reduces the production of three out of four tested cytokines, namely, TNF-α and TGF-ß in cells that express HCV core, Е1Е2, NS3, NS5A, and NS5B proteins, and IL-1ß in the cells that express HCV core, Е1Е2, and NS3 proteins. MDL72527 and DENSpm decreased cytokine production to a lesser extent. Incubation with DFMO led to a 28-32% decrease in the number of cells expressing NS5B or NS5A, both of which are key components of the HCV replication complex. The results obtained in the work indicate that a further detailed study of the antiviral activity of DFMO is required in order to assess its potential as an anti-hepatitis C therapeutic agent.

A Label-Free Continuous Fluorescence-Based Assay for Monitoring Ornithine Decarboxylase Activity with a Synthetic Putrescine Receptor.

Polyamines play an important role in cell growth, differentiation, and cancer development, and the biosynthetic pathway of polyamines is established as a drug target for the treatment of parasitic diseases, neoplasia, and cancer chemoprevention. The key enzyme in polyamine biosynthesis is ornithine decarboxylase (ODC). We report herein an analytical method for the continuous fluorescence monitoring of ODC activity based on the supramolecular receptor cucurbit[6]uril (CB6) and the fluorescent dye trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DSMI). CB6 has a significantly higher binding constant to the ODC product putrescine (>107 M-1) than to the substrate L-ornithine (340 M-1). This enables real-time monitoring of the enzymatic reaction through a continuous fluorescence change caused by dye displacement from the macrocycle by the formed product, which allowed a straightforward determination of enzyme kinetic parameters ( kcat = 0.12 s-1 and KM = 24 µM) and inhibition constants of the two ODC inhibitors α-difluoromethylornithine (DFMO) and epigallocatechin gallate (EGCG). The potential for high-throughput screening (HTS) was demonstrated by excellent Z' factors (>0.9) in a microplate reader format, and the sensitivity of the assay is comparable to or better than most established complementary methods, which invariably have the disadvantage of not being compatible with direct implementation and upscaling to HTS format in the drug discovery process.

Ornithine decarboxylase or gamma-glutamylcysteine synthetase overexpression protects Leishmania (Vianna) guyanensis against antimony.

Trypanosomatids present a unique mechanism for detoxification of peroxides that is dependent on trypanothione (bisglutathionylspermidine). Ornithine decarboxylase (ODC) and γ-glutamylcysteine synthetase (GSH1) produce molecules that are direct precursors of trypanothione. In this study, Leishmania guyanensis odc and gsh1 overexpressor cell lines were generated to investigate the contribution of these genes to the trivalent antimony (SbIII)-resistance phenotype. The ODC- or GSH1-overexpressors parasites presented an increase of two and four-fold in SbIII-resistance index, respectively, when compared with the wild-type line. Pharmacological inhibition of ODC and GSH1 with the specific inhibitors α-difluoromethylornithine (DFMO) and buthionine sulfoximine (BSO), respectively, increased the antileishmanial effect of SbIII in all cell lines. However, the ODC- and GSH1-overexpressor were still more resistant to SbIII than the parental cell line. Together, our data shows that modulation of ODC and GSH1 levels and activity is sufficient to affect L. guyanensis susceptibility to SbIII, and confirms a role of these genes in the SbIII-resistance phenotype.