RESUMO
Lysine-specific demethylase 1 (LSD1 or KDM1A ) has emerged as a critical mediator of tumor progression in metastatic castration-resistant prostate cancer (mCRPC). Among mCRPC subtypes, neuroendocrine prostate cancer (NEPC) is an exceptionally aggressive variant driven by lineage plasticity, an adaptive resistance mechanism to androgen receptor axis-targeted therapies. Our study shows that LSD1 expression is elevated in NEPC and associated with unfavorable clinical outcomes. Using genetic approaches, we validated the on-target effects of LSD1 inhibition across various models. We investigated the therapeutic potential of bomedemstat, an orally bioavailable, irreversible LSD1 inhibitor with low nanomolar potency. Our findings demonstrate potent antitumor activity against CRPC models, including tumor regressions in NEPC patient-derived xenografts. Mechanistically, our study uncovers that LSD1 inhibition suppresses the neuronal transcriptional program by downregulating ASCL1 through disrupting LSD1:INSM1 interactions and de-repressing YAP1 silencing. Our data support the clinical development of LSD1 inhibitors for treating CRPC - especially the aggressive NE phenotype. Statement of Significance: Neuroendocrine prostate cancer presents a clinical challenge due to the lack of effective treatments. Our research demonstrates that bomedemstat, a potent and selective LSD1 inhibitor, effectively combats neuroendocrine prostate cancer by downregulating the ASCL1- dependent NE transcriptional program and re-expressing YAP1.
RESUMO
Homeostasis of the biogenic polyamines spermine (Spm) and spermidine (Spd), present in µM-mM concentrations in all eukaryotic cells, is precisely regulated by coordinated activities of the enzymes of polyamine synthesis, degradation, and transport, in order to sustain normal cell growth and viability. Spermine oxidase (SMOX) is the key and most recently discovered enzyme of polyamine metabolism that plays an essential role in regulating polyamine homeostasis by catalyzing the back-conversion of Spm to Spd. The development of many types of epithelial cancer is associated with inflammation, and disease-related inflammatory stimuli induce SMOX. MDL72527 is widely used in vitro and in vivo as an irreversible inhibitor of SMOX, but it is also potent towards N1-acetylpolyamine oxidase. Although SMOX has high substrate specificity, Spm analogues have not been systematically studied as enzyme inhibitors. Here we demonstrate that 1,12-diamino-2,11-bis(methylidene)-4,9-diazadodecane (2,11-Met2-Spm) has, under standard assay conditions, an IC50 value of 169 µM towards SMOX and is an interesting instrument and lead compound for studying polyamine catabolism.
RESUMO
BACKGROUND: Airway epithelial injury is a crucial component of acute and severe asthma pathogenesis and a promising target for treatment of refractory asthma. However, the underlying mechanism of epithelial injury remains poorly explored. Although high levels of polyamines, mainly spermine, have been found in asthma and comorbidity, their role in airway epithelial injury and the cause of their altered levels in asthma have not been explored. METHODS: We measured key polyamine metabolic enzymes in lung samples from normal and asthmatic subjects and in mice with OVA-induced allergic airway inflammation (AAI). Polyamine metabolism was modulated using pharmacologic/genetic modulators. Epithelial stress and apoptosis were measured by TSLP levels and TUNEL assay, respectively. RESULTS: We found loss of the polyamine catabolic enzymes spermidine/spermine-N (1)-acetyltransferase-1 (SAT1) and spermine oxidase (SMOX) predominantly in bronchial epithelial cells (BECs) of human asthmatic lung samples and mice with AAI. In naïve mice, SAT1 or SMOX knockdown led to airway hyper-responsiveness, remodeling, and BEC apoptosis. Conversely, in mice with AAI, overexpression of either SAT1 or SMOX alleviated asthmatic features and reduced TSLP levels and BEC apoptosis. Similarly, while pharmacological induction of SAT1 and SMOX using the polyamine analogue bis(ethyl)norspermine (BENSPM) alleviated asthmatic features with reduced TSLP levels and BEC apoptosis, pharmacological inhibition of these enzymes using BERENIL or MDL72527, respectively, worsened them. Spermine accumulation in lungs correlated with BEC apoptosis, and spermine treatment caused apoptosis of human BEAS-2B cells in vitro. CONCLUSIONS: Spermine induces BEC injury. Induction of polyamine catabolism may represent a novel therapeutic approach for asthma via reversing BEC stress.
Assuntos
Asma/metabolismo , Epitélio/lesões , Poliaminas/metabolismo , Sistema Respiratório/patologia , Espermina/metabolismo , Animais , Apoptose , Asma/etiologia , Células Epiteliais/química , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Humanos , Pulmão/enzimologia , Camundongos , Espermina/efeitos adversosRESUMO
Chronic inflammation contributes to the development of various forms of cancer. The polyamine catabolic enzyme spermine oxidase (SMOX) is induced in chronic inflammatory conditions, including Helicobacter pylori-associated gastritis, where its production of hydrogen peroxide contributes to DNA damage and subsequent tumorigenesis. MicroRNA expression levels are also altered in inflammatory conditions; specifically, the tumor suppressor miR-124 becomes silenced by DNA methylation. We sought to determine if this repression of miR-124 is associated with elevated SMOX activity and concluded that miR-124 is indeed a negative regulator of SMOX. In gastric adenocarcinoma cells harboring highly methylated and silenced mir-124 gene loci, 5-azacytidine treatment allowed miR-124 re-expression and decreased SMOX expression. Overexpression of an exogenous miR-124-3p mimic repressed SMOX mRNA and protein expression as well as H2O2 production by >50% within 24 h. Reporter assays indicated that direct interaction of miR-124 with the 3'-untranslated region of SMOX mRNA contributes to this negative regulation. Importantly, overexpression of miR-124 before infection with H. pylori prevented the induction of SMOX believed to contribute to inflammation-associated tumorigenesis. Compelling human in vivo data from H. pylori-positive gastritis tissues indicated that the mir-124 gene loci are more heavily methylated in a Colombian population characterized by elevated SMOX expression and a high risk for gastric cancer. Furthermore, the degree of mir-124 methylation significantly correlated with SMOX expression throughout the population. These results indicate a protective role for miR-124 through the inhibition of SMOX-mediated DNA damage in the etiology of H. pylori-associated gastric cancer.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica , Inativação Gênica , MicroRNAs/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Regiões 3' não Traduzidas , Biópsia , Metilação de DNA , Regulação para Baixo , Gastrite/etiologia , Gastrite/patologia , Infecções por Helicobacter/complicações , Helicobacter pylori , Humanos , Neoplasias Gástricas/etiologia , Neoplasias Gástricas/patologia , Poliamina OxidaseRESUMO
Epigenetic silencing and inappropriate activation of gene expression are frequent events during the initiation and progression of cancer. These events involve a complex interplay between the hypermethylation of CpG dinucleotides within gene promoter and enhancer regions, the recruitment of transcriptional corepressors and the deacetylation and/or methylation of histone tails. These epigenetic regulators act in concert to block transcription or interfere with the maintenance of chromatin boundary regions. However, DNA/histone methylation and histone acetylation states are reversible, enzyme-mediated processes and as such, have emerged as promising targets for cancer therapy. This review will focus on the potential benefits and synergistic/additive effects of combining DNA-demethylating agents and histone deacetylase inhibitors or lysine-specific demethylase inhibitors together in epigenetic therapy for solid tumors and will highlight what is known regarding the mechanisms of action that contribute to the antitumor response.
Assuntos
Antineoplásicos/uso terapêutico , Metilação de DNA/genética , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/uso terapêutico , Histonas/metabolismo , Neoplasias/tratamento farmacológico , Sinergismo Farmacológico , Epigênese Genética , Humanos , Regiões Promotoras Genéticas , RNA/genética , RNA/metabolismo , Transcrição GênicaRESUMO
Helicobacter pylori infection causes gastric cancer, the third leading cause of cancer death worldwide. More than half of the world's population is infected, making universal eradication impractical. Clinical trials suggest that antibiotic treatment only reduces gastric cancer risk in patients with non-atrophic gastritis (NAG), and is ineffective once preneoplastic lesions of multifocal atrophic gastritis (MAG) and intestinal metaplasia (IM) have occurred. Therefore, additional strategies for risk stratification and chemoprevention of gastric cancer are needed. We have implicated polyamines, generated by the rate-limiting enzyme ornithine decarboxylase (ODC), in gastric carcinogenesis. During H. pylori infection, the enzyme spermine oxidase (SMOX) is induced, which generates hydrogen peroxide from the catabolism of the polyamine spermine. Herein, we assessed the role of SMOX in the increased gastric cancer risk in Colombia associated with the Andean mountain region when compared with the low-risk region on the Pacific coast. When cocultured with gastric epithelial cells, clinical strains of H. pylori from the high-risk region induced more SMOX expression and oxidative DNA damage, and less apoptosis than low-risk strains. These findings were not attributable to differences in the cytotoxin-associated gene A oncoprotein. Gastric tissues from subjects from the high-risk region exhibited greater levels of SMOX and oxidative DNA damage by immunohistochemistry and flow cytometry, and this occurred in NAG, MAG and IM. In Mongolian gerbils, a prototype colonizing strain from the high-risk region induced more SMOX, DNA damage, dysplasia and adenocarcinoma than a colonizing strain from the low-risk region. Treatment of gerbils with either α-difluoromethylornithine, an inhibitor of ODC, or MDL 72527 (N(1),N(4)-Di(buta-2,3-dien-1-yl)butane-1,4-diamine dihydrochloride), an inhibitor of SMOX, reduced gastric dysplasia and carcinoma, as well as apoptosis-resistant cells with DNA damage. These data indicate that aberrant activation of polyamine-driven oxidative stress is a marker of gastric cancer risk and a target for chemoprevention.
Assuntos
Adenocarcinoma , Infecções por Helicobacter/complicações , Helicobacter pylori/fisiologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/fisiologia , Neoplasias Gástricas , Adenocarcinoma/epidemiologia , Adenocarcinoma/genética , Adenocarcinoma/microbiologia , Adulto , Animais , Células Cultivadas , Colômbia/epidemiologia , Dano ao DNA/genética , Indução Enzimática , Gerbillinae , Infecções por Helicobacter/genética , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/genética , Fatores de Risco , Neoplasias Gástricas/epidemiologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologia , Poliamina OxidaseRESUMO
Very limited molecular knowledge exists about the identity and protein components of the ubiquitous polyamine transporters found in animal cells. However, a number of reports have been published over the last 5 years on potential candidates for metazoan polyamine permeases. We review the available evidence on these putative polyamine permeases, as well as establish a useful "identikit picture" of the general polyamine transport system, based on its properties as found in a wide spectrum of mammalian cells. Any molecular candidate encoding a putative "general" polyamine permease should fit that provided portrait. The current models proposed for the mechanism of polyamine internalization in mammalian cells are also briefly reviewed.
Assuntos
Poliaminas Biogênicas/metabolismo , Animais , Especificidade por SubstratoRESUMO
The polyamines spermidine and spermine have been hypothesized to possess different functions in the protection of DNA from reactive oxygen species. The growth and survival of mouse fibroblasts unable to synthesize spermine were compared to their normal counterparts in their native and polyamine-depleted states in response to oxidative stress. The results of these studies suggest that when present at normal or supraphysiological concentrations, either spermidine or spermine can protect cells from reactive oxygen species. However, when polyamine pools are pharmacologically manipulated to produce cells with low levels of predominately spermine or spermidine, spermine appears to be more effective. Importantly, when cells are depleted of both glutathione and endogenous polyamines, they exhibit increased sensitivity to hydrogen peroxide as compared to glutathione depletion alone, suggesting that polyamines not only play a role in protecting cells from oxidative stress but this role is distinct from that played by glutathione.
Assuntos
Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espermidina/fisiologia , Espermina/fisiologia , Animais , Apoptose , Células Cultivadas , Dano ao DNA , Eflornitina/farmacologia , Glutationa/farmacologia , Guanidinas/farmacologia , Marcação In Situ das Extremidades Cortadas , CamundongosRESUMO
The induction of polyamine catabolism by specific anti-tumour polyamine analogues has increased interest in the roles polyamine catabolism play in cell growth, death and response to various anti-tumour agents. The relatively recent finding of an inducible mammalian spermine oxidase (SMO/PAOh1), in addition to the two-step spermidine/spermine N(1)-acetyltransferanse (SSAT)/N(1)-acetylpolyamine oxidase (APAO) catabolic pathway, underscores the complexities of the regulation of polyamine catabolism by various stimuli. Furthermore, recent data indicate that infectious agents and mediators of inflammation can also up-regulate polyamine catabolism. Induction of SSAT by these agents can reduce intracellular polyamine concentrations and cell growth rate, thus providing a beneficial mechanism by which cells may adapt to inflammatory stress. However, increased polyamine catabolism can also result in substantial increases in intracellular reactive oxygen species (ROS) through the production of H(2)O(2) as a by-product of either APAO or SMO/PAOh1 activity. This increased generation of ROS can have different results, depending on the mechanism of induction and cell types involved. Targeted killing of tumour cells by agents that stimulate SSAT/APAO and/or SMO/PAOh1 is obviously a 'good' effect. However, induction of SMO/PAOh1 by inflammation or infectious agents has the potential to produce sufficient ROS in normal, non-tumour cells to lead to DNA damage, mutation and, potentially, carcinogenic transformation ('bad'). The variation in the induction of these polyamine catabolic enzymes, as well as the level and timing of this induction will dictate the cellular outcome in the presence of both desirable and undesirable effects ('ugly'). Here we discuss the relative role of each of the steps in polyamine catabolism in response to inflammatory stress.
Assuntos
Inflamação/fisiopatologia , Poliaminas/metabolismo , Anti-Inflamatórios não Esteroides/uso terapêutico , Transformação Celular Neoplásica , Homeostase , Humanos , Inflamação/tratamento farmacológico , Modelos Biológicos , NF-kappa B/metabolismo , Neoplasias/fisiopatologia , Transdução de SinaisRESUMO
The polyamines putrescine, spermidine and spermine are ubiquitous polycationic compounds that are found in nearly every cell type, and are required to support a wide variety of cellular functions. The existence of multiple cellular effector sites for naturally occurring polyamines implies that there are numerous targets for polyamine-based therapeutic agents. Through a programme aimed at the synthesis and evaluation of biologically active polyamine analogues, our laboratory has identified three distinct structural classes of polyamine derivatives that exhibit promising biological activity in vitro. We have synthesized more than 200 symmetrically and unsymmetrically substituted alkylpolyamines that possess potent antitumour or antiparasitic activity, depending on their backbone architecture and terminal alkyl substituents. Along similar lines, we have developed novel polyamino(bis)guanidines and polyaminobiguanides that are promising antitrypanosomal agents and that interfere with biofilm formation in the pathogenic bacterium Yersinia pestis. Finally, we recently reported a series of PAHAs (polyaminohydroxamic acids) and PABAs (polyaminobenzamides) that inhibit HDACs (histone deacetylases), and in some cases are selective for individual HDAC isoforms. These studies support the hypothesis that polyamine-based small molecules can be developed for use as biochemical probes and as potential therapies for multiple diseases.
Assuntos
Antineoplásicos/síntese química , Poliaminas/farmacologia , Poliaminas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Humanos , Putrescina/farmacologia , Espermidina/farmacologia , Espermina/farmacologia , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/farmacologia , Yersinia pestis/efeitos dos fármacosRESUMO
1,12-Diamino-3,6,9-triazadodecane, a new isosteric and charge-deficient analogue of spermine, is synthesized. Unlike spermine, the new analogue is an excellent chelator of Cu2+ ions. Possible applications of this compound for studying enzymes of polyamine metabolism and cellular functions of spermine are discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.
Assuntos
Quelantes/química , Cobre/química , Espermina/análogos & derivados , Espermina/química , Quelantes/síntese química , Espermina/síntese químicaRESUMO
Interest in polyamine catabolism has increased since it has been directly associated with the cytotoxic response of multiple tumour types to exposure to specific anti-tumour polyamine analogues. Human polyamine catabolism was considered to be a two-step pathway regulated by the rate-limiting enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) that provides substrate for an acetylpolyamine oxidase (APAO). Further, the super-induction of SSAT by several anti-tumour polyamine analogues has been implicated in the cytotoxic response of specific solid-tumour phenotypes to these agents. This high induction of SSAT has been correlated with cellular response to the anti-tumour polyamine analogues in several systems and considerable progress has been made in understanding the molecular mechanisms that regulate the analogue-induced expression of SSAT. A polyamine response element has been identified and the transacting transcription factors that bind and stimulate transcription of SSAT have been cloned and characterized. The link between SSAT activity and cellular toxicity is thought to be based on the production of H(2)O(2) by the activity of the constitutive APAO that uses the SSAT-produced acetylated polyamines. The high induction of SSAT and the subsequent activity of APAO are linked to the cytotoxic response of some tumour cell types to specific polyamine analogues. However, we have recently cloned a variably spliced human polyamine oxidase (PAOh1) that is inducible by specific polyamine analogues, efficiently uses unacetylated spermine as a substrate, and also produces toxic H(2)O(2) as a product. The results of studies with PAOh1 suggest that it is an additional enzyme in polyamine catabolism that has the potential to significantly contribute to polyamine homoeostasis and drug response. Most importantly, PAOh1 is induced by specific polyamine analogues in a tumour-phenotype-specific manner in cell lines representative of the major forms of solid tumours, including lung, breast, colon and prostate. The sensitivity to these anti-tumour polyamine analogues can be significantly reduced if the tumour cells are co-treated with 250 microM of the polyamine oxidase inhibitor N (1), N (4)-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527), suggesting that the H(2)O(2) produced by PAOh1 does in fact play a direct role in the observed cytotoxicity. These results strongly implicate PAOh1 as a new target that, in combination with SSAT, may be exploited for therapeutic advantage. The current understanding of the role and regulation of these two important polyamine catabolic enzymes are discussed.
Assuntos
Antineoplásicos/farmacologia , Poliaminas Biogênicas/metabolismo , Acetiltransferases/biossíntese , Desenho de Fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/biossíntese , Poliamina OxidaseRESUMO
The natural polyamines and their analogues have been implicated in transcriptional regulation of specific genes. Human polyamine-modulated factor-1 (hPMF-1) was the first polyamine-responsive transcription factor identified. Here the mouse homologue of the hPMF-1 gene is described. Interestingly, the mouse gene (mPMF-1) codes for two alternatively spliced mRNAs. Both of the mouse splice variants, mPMF-1S and mPMF-1L, possess C-terminal coiled-coil domains nearly identical to that found in hPMF-1 and are highly homologous with the human protein. The C-terminal coiled-coil structure is necessary for transcriptional activation. However, the shorter protein, mPMF-1S, does not contain an N-terminal coiled-coil region as do both hPMF-1 and the longer mPMF-1L. mPMF-1L mRNA codes for a protein of 202 amino acids, 37 amino acids longer than the human protein. By contrast, mPMF-1S codes for only 133 amino acids, as a result of two exons being omitted compared with mPMF-1L. Both mouse transcription factors can interact with Nrf-2 (nuclear factor-E2-related factor 2), the normal partner of hPMF-1, substantiating the importance of the C-terminal coiled-coil region responsible for this interaction. Finally, the expression of mPMF-1 is induced when mouse M1 myeloid leukaemia cells are exposed to polyamine analogues, suggesting control similar to that observed for the hPMF-1.
Assuntos
Fatores de Transcrição/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Poliaminas Biogênicas/metabolismo , Poliaminas Biogênicas/farmacologia , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição de Proteínas de Ligação GA , Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Fígado/metabolismo , Camundongos , Dados de Sequência Molecular , Biossíntese de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/genética , Coelhos , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Mammalian polyamine catabolism is under the control of two enzymes, spermidine/spermine N1-acetyltransferase and the flavin adenine dinucleotide-dependent polyamine oxidase (PAO). In this study, the cloning and initial characterization of human PAO is reported. A 1894-bp cDNA with an open reading frame of 1668-bp codes for a protein of 555 amino acids. In vitro transcription/translation of this cDNA clone produces the expected M(r) 61,900 protein with PAO activity. The PAO activity of this clone is inhibited by MDL 72,527, a specific inhibitor of mammalian PAO. However, neither pargyline, a specific monoamine oxidase inhibitor, nor semicarbazide, a specific diamine oxidase inhibitor, inhibits the PAO activity of this clone. PAO has been referred to as being constitutively expressed. However, 24-h exposure of a non-small cell lung carcinoma cell line, NCI H157, to 10 microM of N1,N"-bis(ethyl)norspermine results in approximately 5-fold induction of PAO mRNA and a >3-fold induction of PAO activity. These results demonstrate that in at least one cell type, PAO is up-regulated in response to polyamine analogue exposure. The PAO clone described here should provide a useful tool, which will facilitate the dissection of the role of polyamine catabolism in normal growth and in response to the antitumor polyamine analogues.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Poliaminas/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes/genética , Humanos , Cinética , Dados de Sequência Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Poliaminas/química , Biossíntese de Proteínas , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Transcrição Gênica , Células Tumorais Cultivadas , Poliamina OxidaseRESUMO
A novel series of alkyl- or aralkyl-substituted polyamine analogues was synthesized containing a 3-7-3 polyamine backbone. These analogues were evaluated in vitro, and in one case in vivo, for activity as antitrypanosomal agents, and for activity against opportunistic infection caused by Microsporidia. Compound 21 inhibits trypanosomal growth with an IC(50) as low as 31nM, while compound 24 shows promising activity in vitro against trypanosomes, and against Microsporidia in vitro and in vivo.
Assuntos
Antiprotozoários/química , Poliaminas/química , Poliaminas/farmacologia , Tripanossomicidas/química , Animais , Antiprotozoários/farmacologia , Linhagem Celular , Encephalitozoon cuniculi/efeitos dos fármacos , Concentração Inibidora 50 , Camundongos , Camundongos Knockout , Microsporida/efeitos dos fármacos , Coelhos , Relação Estrutura-Atividade , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
Agmatine is a biogenic amine with the capacity to regulate a number of nonreceptor-mediated functions in mammalian cells, including intracellular polyamine content and nitric oxide generation. We observed avid incorporation of agmatine into several mammalian cell lines and herein characterize agmatine transport in mammalian cells. In transformed NIH/3T3 cells, agmatine uptake is energy dependent with a saturable component indicative of carrier-mediated transport. Transport displays an apparent Michaelis-Menten constant of 2.5 microM and a maximal velocity of 280 pmol x min(-1) x mg(-1) protein and requires a membrane potential across the plasma membrane for uptake. Competition with polyamines, but not cationic molecules that utilize the y+ system transporter, suppresses agmatine uptake. Altering polyamine transporter activity results in parallel changes in polyamine and agmatine uptake. Furthermore, agmatine uptake is abrogated in a polyamine transport-deficient human carcinoma cell line. These lines of evidence demonstrate that agmatine utilizes, and is dependent on, the polyamine transporter for cellular uptake. The fact that this transport system is associated with proliferation could be of consequence to the antiproliferative effects of agmatine.
Assuntos
Agmatina/metabolismo , Poliaminas/metabolismo , Agmatina/química , Animais , Transporte Biológico , Linhagem Celular , Dinitrofenóis/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Iodoacetatos/farmacologia , Cinética , Camundongos , Estrutura Molecular , Putrescina/metabolismo , Desacopladores/farmacologiaRESUMO
Polyamines are essential for cell growth and differentiation. Structural polyamine analogues have been shown to have antitumor activity in experimental models including breast cancer. The ability of polyamine analogues to alter activity of cytotoxic chemotherapeutic agents in breast cancer models has not been evaluated. This study evaluates the ability of two polyamine analogues, N1-ethyl-N11-[(cyclopropyl)methyl]-4,8-diazaundecane (CPENSpm) and N1-ethyl-N11-[(cycloheptyl)methyl]-4,8-diazaundecane (CHENSpm) to synergize with cytotoxics in five human breast cancer cell lines. Antagonism, additivity, or synergy of the combinations was determined using the median effect/combination index model. The chemotherapeutic agents chosen, cis-diaminechloroplatinum(II), doxorubicin, 5-fluorouracil, fluorodeoxyuridine, 4-hydroperoxycyclophosphamide, paclitaxel, docetaxel, and vinorelbine, all have antitumor activity in breast cancer and represent a spectrum of mechanisms. Three treatment schedules of polyamine analogue and cytotoxic were tested in MCF-7 and MDA-MB-468 lines, demonstrating a schedule-dependence of synergistic growth inhibition. Cytotoxic agent alone for 24 h followed by polyamine analogue alone for 96 h resulted in the most synergistic combinations and the greatest synergy. This schedule was then tested in three additional breast cancer lines, and several synergistic combinations were again identified. Two cytotoxics, vinorelbine and the fluoropyrimidines, showed the most promise in combination with the polyamine analogues. They were able to synergize with one or both polyamine analogues in most of the breast cancer cell lines. CPENSpm was also able to synergize with virtually all of the cytotoxics in the estrogen receptor alpha-positive MCF-7 and T-47D lines. These preclinical data demonstrate a treatment schedule and combinations of polyamine analogues and cytotoxics that will be important to study mechanistically and clinically for breast cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Células Tumorais Cultivadas/efeitos dos fármacos , Vimblastina/análogos & derivados , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Sinergismo Farmacológico , Feminino , Fluoruracila/administração & dosagem , Humanos , Paclitaxel/administração & dosagem , Poliaminas/administração & dosagem , Poliaminas/metabolismo , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologia , Vimblastina/administração & dosagem , VinorelbinaRESUMO
Polyamines and polyamine analogues have been demonstrated to modulate the transcription of various genes. Spermidine/spermine N(1)-acetyltransferase (SSAT) is transcriptionally regulated through the interaction of at least two trans-acting transcription factors, NF-E2-related factor 2 (Nrf-2) and PMF-1 (polyamine modulated factor-1). Nrf-2 has previously been shown to regulate transcription of other genes through interactions between its C-terminal leucine zipper and the leucine-zipper region of other members of the small Maf protein family (the term "Maf" is derived from MusculoAponeurotic-Fibrosarcoma virus). Here it is demonstrated that the interaction between Nrf-2 and PMF-1 is mediated through the binding of the leucine-zipper region of Nrf-2 and a C-terminal coiled-coil region of PMF-1 that does not contain a leucine zipper. Mutations that interrupt either the leucine zipper of Nrf-2 or the coiled-coil region of PMF-1 are demonstrated to alter the ability of these factors to interact, thus their ability to regulate the transcription of the SSAT gene is lost.
Assuntos
Acetiltransferases/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Acetiltransferases/química , Sequência de Aminoácidos , Sequência de Bases , DNA Recombinante , Humanos , Zíper de Leucina , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fator 2 Relacionado a NF-E2 , Técnicas do Sistema de Duplo-HíbridoAssuntos
Antineoplásicos/síntese química , Antiparasitários/síntese química , Poliaminas/síntese química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antiparasitários/química , Antiparasitários/farmacologia , Apoptose , Transporte Biológico , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Infecções Oportunistas/tratamento farmacológico , Poliaminas/química , Poliaminas/farmacologia , Espécies Reativas de Oxigênio , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismoRESUMO
Reactive oxygen species have recently been demonstrated to play a role in numerous cellular signal transduction pathways. Here we investigate the involvement of H2O2 in Raf-1-mediated differentiation in the human medullary thyroid carcinoma (MTC) cell line TT:deltaRaf-1:ER. Catalase, but not Cu/Zn superoxide dismutase, completely inhibited Raf-1-induced differentiation of beta-estradiol-treated TT: deltaRaf-1:ER. In addition, catalase treatment down-regulated RET expression at both the mRNA and protein levels and induced apoptosis in the parental TT cell line and uninduced TT:deltaRaf-1:ER human MTC cells. These results implicate H2O2 as a downstream mediator of c-Raf-1-induced differentiation and as a survival factor in MTC cells.