RESUMO
Lung cancer causes more than 140,000 deaths annually in the United States alone, and the prognosis for non-small cell lung cancer (NSCLC) is particularly poor. Therapies using small molecules that preferentially kill lung tumor cells by inducing cellular suicide (apoptosis) would therefore be highly desirable. Retinoids have shown promise as cancer preventive and cancer therapeutic agents. Retinoid signals are mediated by two classes of nuclear receptors: the retinoic acid receptors (RAR alpha, beta, and gamma) and the retinoid X receptors (RXR alpha, beta and gamma). These receptors usually bind as heterodimers to specific DNA sequences and/or interact with other transcriptional regulators, such as AP-1 (ref. 10) to regulate gene transcription. Synthetic retinoids can be made that activate only specific portions of the complex retinoid response network and activate selective biological programs. To identify retinoids with novel biological activities, we used a high-throughput "biological activity fingerprint" screen on a large library of retinoids and retinoid-related molecules (RRMs). We identified new structures that are highly effective against lung cancer cells in vitro, inducing apoptosis. We show here for one of these compounds that it is very effective against a human NSCLC in vivo in an animal model. These new molecules show a distinct pattern of receptor signaling.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Retinoides/uso terapêutico , Animais , Contagem de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Receptores do Ácido Retinoico/metabolismo , Retinoides/metabolismo , Retinoides/farmacologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Retinoids have a broad spectrum of biological activities and are useful therapeutic agents. Their physiological activities are mediated by two types of receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). RARs, as well as several related receptors, require heterodimerization with RXRs for effective DNA binding and function. However, in the presence of 9-cis-retinoic acid, a ligand for both RARs and RXRs, RXRs can also form homodimers. A series of retinoids is reported that selectively activates RXR homodimers but does not affect RAR-RXR heterodimers and thus demonstrates that both retinoid response pathways can be independently activated.
Assuntos
Receptores de Superfície Celular/metabolismo , Receptores do Ácido Retinoico , Retinoides/metabolismo , Fatores de Transcrição , Animais , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Cinética , Substâncias Macromoleculares , Estrutura Molecular , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptores X de Retinoides , Retinoides/química , Retinoides/farmacologia , Relação Estrutura-Atividade , Transcrição Gênica , Transfecção , Tretinoína/metabolismo , Tretinoína/farmacologiaRESUMO
Retinoids are known to inhibit the growth of a wide variety of cancer cells, including breast cancer cells. Advances made in recent years in the understanding of the molecular mechanisms of retinoid action have allowed the design of retinoids with selective activities. Such selective retinoids are of particular interest, because they may reduce the number of undesirable side effects observed with natural compounds. Here, we have compared the growth-inhibitory activities of natural retinoids with various selective retinoids, including anti-activator protein (AP)-1 selective compounds on estrogen receptor-positive and -negative breast cancer cell lines. In addition, we have investigated cooperativity between selective retinoids and IFNs and have begun to analyze the pathways that these two different growth inhibitors use for antagonizing breast cancer cell proliferation. We observe that several selective retinoids can inhibit breast cancer cells as efficiently as the natural compounds. Anti-AP-1-selective retinoids are as effective as retinoic acid receptor (RAR)-beta/gamma-selective compounds. This lets us conclude that retinoid-induced inhibition of breast cancer cell growth does not require retinoid receptor transactivation. Several synthetic retinoids including anti-AP-1-selective compounds show synergism with IFNs. However, true synergism between the two different types of growth regulators was seen only when both classes of molecules were used at low concentrations. RAR-beta/gamma and anti-AP-1-selective retinoids, but not RAR-alpha-selective compounds, induced increased RAR-gamma mRNA levels. Interestingly, IFNs at elevated concentrations (100 units/ml and higher) also induced increased RAR-gamma expression. Thus, when used at high concentrations, IFNs may activate growth-inhibitory pathways overlapping with those activated by retinoids. Because increased RAR-gamma expression is induced by the two different classes of breast cancer cell inhibitors, it is likely to have an important role in controlling the growth fo these cancer cells.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Interferons/farmacologia , Receptores do Ácido Retinoico/fisiologia , Retinoides/farmacologia , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Humanos , RNA Mensageiro/análise , Receptores do Ácido Retinoico/genética , Células Tumorais CultivadasRESUMO
Close to 180,000 women will be diagnosed with breast cancer this year in the United States and more than 43,000 will die from this disease. Antiestrogens have shown promise, but they can only be effective against estrogen-dependent stages of the disease. We identify here a retinoid antagonist, MX781, that is effective against estrogen receptor-positive and -negative breast cancer cells. Although classical retinoids show limited efficacy and significant side effects, this novel compound kills breast cancer cells by inducing apoptosis and is effective against estrogen receptor-negative human breast cancer tumors in vivo. Remarkably, MX781 is well tolerated and does not seem to have significant toxicity. This novel retinoid antagonist, therefore, represents a promising new candidate for the treatment of breast cancer.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Éteres/farmacologia , Receptores de Estrogênio/análise , Retinoides/antagonistas & inibidores , Retinoides/farmacologia , Animais , Neoplasias da Mama/química , Neoplasias da Mama/patologia , Caspases/fisiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células Tumorais CultivadasRESUMO
Overexpression of the multifunctional growth factor transforming growth factor-beta 1 (TGF beta 1) has been connected to numerous diseases in human. TGF beta 1 expression is largely governed by three AP-1 binding sites located in two different promoters of this gene. We have examined the ability of retinoid receptors to inhibit the activity of the two promoters (especially the promoter 1) by cotransfection assays in the hepatocellular carcinoma cell line HepG2. When the TGF beta 1 promoter activity is induced by 12-O-tetradecanoyl phorbol13-acetate (an activator of AP-1-controlled gene transcription), this activity can be strongly repressed by retinoic acid receptor-alpha (RAR alpha), RAR beta, or retinoid X receptor-alpha (RXR alpha) as well as other members of the nuclear receptor family. Repression was hormone dependent and a function of receptor concentration. Heterodimerization of RAR alpha or RAR beta with RXR alpha did not modify the inhibition activities of these receptors, indicating that heterodimer formation is not required for antagonizing of AP-1 activity. On further examining the anti-AP-1 activity of RXR alpha we observed that three different AP-1-controlled promoters (TGF beta 1, collagenase, and cFos) can be inhibited. Using gel shift assays, we demonstrated that RXR alpha inhibits Jun and Fos DNA binding and that 9-cis RA enhances this inhibition, suggesting that a mechanism involving direct protein-protein interaction between RXR and AP-1 components mediates the inhibitory effect observed in vivo. Transfection analyses with RXR alpha point mutations revealed that residues L422, C432, and, to a lesser extent, residues L418 and L430, are involved in ligand-induced anti-AP1 activity of RXR alpha in vivo. Thus both types of retinoid receptors can inhibit AP-1-activated promoters, including the TGF beta 1 gene promoter, via a mechanism that involves protein-protein interaction.
Assuntos
Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores do Ácido Retinoico/fisiologia , Fatores de Transcrição , Fator de Crescimento Transformador beta/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Carcinoma Hepatocelular , Colagenases/genética , Fibrossarcoma , Genes fos , Humanos , Neoplasias Hepáticas , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores do Ácido Retinoico/genética , Receptores X de Retinoides , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
Glucocorticoid excess increases fat mass, preferentially within omental depots; yet circulating cortisol concentrations are normal in most patients with metabolic syndrome (MS). At a pre-receptor level, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) activates cortisol from cortisone locally within adipose tissue, and inhibition of 11beta-HSD1 in liver and adipose tissue has been proposed as a novel therapy to treat MS by reducing hepatic glucose output and adiposity. Using a transformed human subcutaneous preadipocyte cell line (Chub-S7) and human primary preadipocytes, we have defined the role of glucocorticoids and 11beta-HSD1 in regulating adipose tissue differentiation. Human cells were differentiated with 1.0 microM cortisol (F), or cortisone (E) with or without 100 nM of a highly selective 11beta-HSD1 inhibitor PF-877423. 11beta-HSD1 mRNA expression increased across adipocyte differentiation (P<0.001, n=4), which was paralleled by an increase in 11beta-HSD1 oxo-reductase activity (from nil on day 0 to 5.9+/-1.9 pmol/mg per h on day 16, P<0.01, n=7). Cortisone enhanced adipocyte differentiation; fatty acid-binding protein 4 expression increased 312-fold (P<0.001) and glycerol-3-phosphate dehydrogenase 47-fold (P<0.001) versus controls. This was abolished by co-incubation with PF-877423. In addition, cellular lipid content decreased significantly. These findings were confirmed in the primary cultures of human subcutaneous preadipocytes. The increase in 11beta-HSD1 mRNA expression and activity is essential for the induction of human adipogenesis. Blocking adipogenesis with a novel and specific 11beta-HSD1 inhibitor may represent a novel approach to treat obesity in patients with MS.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/fisiologia , Adipogenia/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a Ácido Graxo/genética , Glucocorticoides/metabolismo , Humanos , Síndrome Metabólica/etiologia , PPAR gama/genética , RNA Mensageiro/análiseRESUMO
Cytosolic 3,3',5-tri-iodo-L-thyronine-binding proteins (CTBP I, II and IV species) from human red blood cells undergo rapid loss of activity at low temperatures. Cold treatment of CTBPs was accompanied by dissociation of the polymeric protein to the 60 kDa inactive monomer. Re-activation of the cold-inactivated CTBP IV by warming resulted in association of the monomer to the active polymeric form. A similar association-dissociation phenomenon was also obtained isothermically, though pH changes. We conclude that CTBP I and CTBP II are polymeric forms of CTBP IV.
Assuntos
Proteínas de Transporte/metabolismo , Eritrócitos/metabolismo , Proteínas de Membrana/metabolismo , Hormônios Tireóideos , Tri-Iodotironina/metabolismo , Células Cultivadas , Temperatura Baixa , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Ligação a Hormônio da TireoideRESUMO
Four cytosolic 3,5,3'-triiodo-L-thyronine-binding proteins (CTBP) were isolated from hemoglobin-free human erythrocyte on DEAE-cellulose column by linear gradient of NaCl (0-0.4 M). CTBP I, II, and IV underwent rapid loss of their activities at low temperatures, whereas CTBP III was cold-insensitive. Reactivation of cold-inactivated CTBPs by warming was obtained at 20 and 37 degrees C. CTBP I, II, and IV were not inhibited by thiol-blocking agents, whereas CTBP III was blocked. Scatchard analysis of L-3,5,3'-triodo-thyronine binding showed a high affinity site with Kd on the order of 10(-10) M for CTBP II and Kd values of about 10(-9) M for CTBP I and IV and of about 10(-8) M for CTBP III. The order of affinity of iodothyronine analogues to CTBPs was similar in CTBP I, II, and IV but different in CTBP III. Chromatography on Sephacryl S-200 HR showed the elution of a single peak for each CTBP. The apparent molecular weights were about 200,000, 200,000, 25,000, and 60,000 for CTBP I, II, III, and IV, respectively. The physiological relevance of these CTBPs is discussed.
Assuntos
Proteínas de Transporte/isolamento & purificação , Eritrócitos/química , Proteínas de Membrana/isolamento & purificação , Hormônios Tireóideos , Tri-Iodotironina/metabolismo , Sítios de Ligação , Ligação Competitiva , Proteínas de Transporte/metabolismo , Citosol/química , Humanos , Concentração de Íons de Hidrogênio , Cinética , Proteínas de Membrana/metabolismo , Peso Molecular , Compostos de Sulfidrila/farmacologia , Temperatura , Proteínas de Ligação a Hormônio da TireoideRESUMO
Similar cold-sensitive properties, values of dissociation constants (Kd = 1 x 10(-10) M), and regulatory effectors were found for the cold-sensitive cytosolic 3,5,3'-triiodo-L-thyronine (L-T3)-binding protein (CTBP) and pyruvate kinase from human erythrocyte. Various metabolites of the blood cell were assayed for their effects on CTBP activity after heat and cold preincubation treatments. Among these compounds, five- and six-carbon phosphorylated sugars were effective in protecting the CTBP activity against cold inactivation, whereas only ATP and dATP blocked activation by heat treatments. The effects of fructose 1,6-bisphosphate, fructose 2,6-bisphosphate, and ATP were obtained at physiological concentrations. Three-carbon phosphorylated intermediates of glycolysis, ADP, AMP, cAMP, and GTP had no effect on cold and heat treatments. The monomer-tetramer interconversion of the enzyme was also regulated by fructose 1,6-bisphosphate and ATP. The association is under the control of fructose 1,6-bisphosphate, whereas the dissociation is under ATP control. This regulation may have physiological relevance since the hormone binds to the tetrameric form of the enzyme at a site other than the active site.
Assuntos
Proteínas de Transporte/sangue , Eritrócitos/metabolismo , Glicólise , Proteínas de Membrana/sangue , Piruvato Quinase/sangue , Hormônios Tireóideos , Tri-Iodotironina/sangue , Trifosfato de Adenosina/farmacologia , Carboidratos/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Temperatura Baixa , Citosol/metabolismo , Frutosedifosfatos/farmacologia , Humanos , Cinética , Substâncias Macromoleculares , Proteínas de Membrana/efeitos dos fármacos , Ribonucleotídeos/farmacologia , Proteínas de Ligação a Hormônio da TireoideRESUMO
Retinoic acid (RA) and its natural and synthetic analogs, the retinoids, regulate many biological processes, including development, differentiation, cell growth, morphogenesis, metabolism and homeostasis. Retinoid effects are mediated by specific nuclear receptors, the RARs and RXRs. Because of their ability to control cell growth and induce differentiation, retinoids are being examined for the prevention and treatment of several cancers. The majority of retinoids so far analyzed and available inhibit primarily cell proliferation and tumor progression but cannot eliminate cancer cells. In addition, the beneficial effects of the natural retinoids are undermined by undesirable side effects, possibly due to indiscriminate activation of all retinoid receptor subtypes and response pathways. Here, we show that a synthetic retinoid, CD-271, that activates selectively the RAR gamma subtype in a given context, shows increased anti-proliferative activity against certain carcinoma cells over all-trans-retinoic acid (tRA). CD-271 exhibits enhanced activity against DU-145 prostate adenocarcinoma cells through apoptosis-inducing activity, while tRA does not. The selective anti-cancer cell action appears to be receptor-mediated as an RAR antagonist reverses the inhibition. This profile was not seen with other selective retinoids, such as RAR alpha-selective agonists, anti-AP-1 compounds and a non-apoptosis inducing RAR gamma agonist. Our data point to a specific role for RAR gamma in controlling the growth of the prostate, consistent with previous RAR gamma gene knockout data. The identified retinoid represents a new class of compounds with potential for the treatment of prostate cancer.
Assuntos
Androgênios/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias da Próstata/tratamento farmacológico , Tretinoína/uso terapêutico , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Neoplasias da Próstata/patologia , Elementos de Resposta/genética , Transcrição Gênica/efeitos dos fármacos , Tretinoína/análogos & derivados , Células Tumorais CultivadasRESUMO
Retinoids have shown promise as anti-cancer and cancer preventative agents. All-trans-N-(4-hydroxyphenyl)retinamide (4HPR) belongs to a new group of retinoids that not only inhibit the proliferation of cancer cells but also can induce apoptosis in certain cancer cells. Because of its increased efficacy against cancer cells and its low toxicity it has been entered into a number of clinical trials. However, its mechanism of action is not known, and it had been assumed that it is not a true retinoid. Here we analyze its ability to function as an activator of nuclear retinoid receptors (RARs and RXRs). We observe that, in transactivation assays, 4HPR is a potent transactivator with RARgamma and a moderate activator with RARbeta but is not an activator with RARalpha and RXRalpha. Furthermore, RARgamma-selective transactivation by 4HPR is enhanced on some response elements and reduced on others when compared to natural retinoids. In contrast to transactivation, 4HPR in transrepression assays functions mostly with RARalpha, RARbeta, and RXRalpha. Optimal receptor activation is seen at 4HPR concentrations at which it is a potent growth inhibitor and inducer of apoptosis. We conclude that 4HPR is a highly selective activator of retinoid receptors. We propose that this selective activation of the nuclear receptors is likely to be the basis for its specific biological activities and its favorable pharmaceutical properties.
Assuntos
Anticarcinógenos/farmacologia , Antineoplásicos/farmacologia , Fenretinida/farmacologia , Receptores do Ácido Retinoico/metabolismo , Apoptose/efeitos dos fármacos , Northern Blotting , Neoplasias da Mama , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , Receptores X de Retinoides , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Retinoids regulate many biological processes, including differentiation, morphogenesis and cell proliferation. They are also important therapeutic agents, but their clinical usefulness is limited because of side effects. Retinoid activities are mediated by specific nuclear receptors, the RARs and RXRs, which can induce transcriptional activation through specific DNA sites or by inhibiting the transcription factor AP-1 (refs 12-15), which usually mediates cell proliferation signals. Because the two types of receptor actions are mechanistically distinct, we investigated whether conformationally restricted retinoids, selective for each type of receptor action, could be identified. Here we describe a new class of retinoids that selectively inhibits AP-1 activity but does not activate transcription. These retinoids do not induce differentiation in F9 cells but inhibit effectively the proliferation of several tumour cell lines, and could thus serve as candidates for new retinoid therapeutic agents with reduced side effects.