RESUMO
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and a member of the bHLH/PAS (basic Helix-Loop-Helix/Per-Arnt-Sim) family of proteins. The AhR was cloned and characterized for its role in mediating the toxicity of dioxins. Subsequent research has identified the role of AhR in suppression of cancer cell growth. We hypothesized that the AhR is a molecular target for therapeutic intervention in cancer, and that activation of the AhR by unique AhR ligands in cancer cells could have anti-cancer effects including induction of cell death. This study describes the discovery and characterization of a new class of anti-cancer agents targeting the AhR, that we designate as Select Modulators of AhR-regulated Transcription (SMAhRTs). We employed two independent small molecule screening approaches to identify potential SMAhRTs. We report the identification of CGS-15943 that activates AhR signaling and induces apoptosis in an AhR-dependent manner in liver and breast cancer cells. Investigation of the downstream signaling pathway of this newly identified SMAhRT revealed upregulation of Fas-ligand (FasL), which is required for AhR-mediated apoptosis. Our results provide a basis for further development of a new class of anti-cancer therapeutics targeting an underappreciated molecular target, the AhR.
Assuntos
Antineoplásicos/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Ativação Transcricional/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Linhagem Celular Tumoral , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Humanos , Ligantes , Camundongos , Quinazolinas/farmacologia , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Triazóis/farmacologiaRESUMO
Our previous study found that zinc finger protein 71 (ZNF71) mRNA expression was associated with chemosensitivity and its protein expression was prognostic of non-small-cell lung cancer (NSCLC). The Krüppel associated box (KRAB) transcriptional repression domain is commonly present in human zinc finger proteins, which are linked to imprinting, silencing of repetitive elements, proliferation, apoptosis, and cancer. This study revealed that ZNF71 KRAB had a significantly higher expression than the ZNF71 KRAB-less isoform in NSCLC tumors (n = 197) and cell lines (n = 117). Patients with higher ZNF71 KRAB expression had a significantly worse survival outcome than patients with lower ZNF71 KRAB expression (log-rank p = 0.04; hazard ratio (HR): 1.686 [1.026, 2.771]), whereas ZNF71 overall and KRAB-less expression levels were not prognostic in the same patient cohort. ZNF71 KRAB expression was associated with epithelial-to-mesenchymal transition (EMT) in both patient tumors and cell lines. ZNF71 KRAB was overexpressed in NSCLC cell lines resistant to docetaxel and paclitaxel treatment compared to chemo-sensitive cell lines, consistent with its association with poor prognosis in patients. Therefore, ZNF71 KRAB isoform is a more effective prognostic factor than ZNF71 overall and KRAB-less expression for NSCLC. Functional analysis using CRISPR-Cas9 and RNA interference (RNAi) screening data indicated that a knockdown/knockout of ZNF71 did not significantly affect NSCLC cell proliferation in vitro.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/biossíntese , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/biossíntese , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Intervalo Livre de Doença , Docetaxel/farmacologia , Feminino , Humanos , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Neoplasias/genética , Paclitaxel/farmacologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Taxa de SobrevidaRESUMO
Although new cancer therapeutics are discovered at a rapid pace, lack of effective means of delivery and cancer chemoresistance thwart many of the promising therapeutics. We demonstrate a method that confronts both of these issues with the light-activated delivery of a Bcl-2 functional converting peptide, NuBCP-9, using hollow gold nanoshells. This approach has shown not only to increase the efficacy of the peptide 30-fold in vitro but also has shown to reduce paclitaxel resistant H460 lung xenograft tumor growth by 56.4%.
Assuntos
Antineoplásicos/química , Sistemas de Liberação de Medicamentos , Ouro/química , Nanoconchas/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Liberação Controlada de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Terapia a Laser , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/fisiologiaRESUMO
The aryl hydrocarbon receptor (AhR) was initially identified as the receptor that binds and mediates the toxic effects induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and structurally related halogenated aromatics. Other toxic compounds including some polynuclear aromatic hydrocarbons act through the AhR; however, during the last 25 years, it has become apparent that the AhR plays an essential role in maintaining cellular homeostasis. Moreover, the scope of ligands that bind the AhR includes endogenous compounds such as multiple tryptophan metabolites, other endogenous biochemicals, pharmaceuticals and health-promoting phytochemicals including flavonoids, indole-3-carbinol and its metabolites. It has also been shown that like other receptors, the AhR is a drug target for multiple diseases including cancer, where both AhR agonists and antagonists effectively block many of the critical hallmarks of cancer in multiple tumor types. This review describes the anti-cancer activities of AhR ligands and demonstrates that it is time to separate the AhR from TCDD and exploit the potential of the AhR as a novel target for cancer chemotherapy.
Assuntos
Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Antineoplásicos/farmacologia , Humanos , Neoplasias/epidemiologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Dibenzodioxinas Policloradas/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/genéticaAssuntos
Apoptose , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Oligopeptídeos/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/fisiologia , Sobrevivência Celular/genética , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Inibidoras de Apoptose/fisiologia , Neoplasias/metabolismo , Neoplasias/patologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/química , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Carga Tumoral/efeitos dos fármacosRESUMO
Resistance to chemotherapy is a major cause of treatment failure and poor overall survival in patients with lung cancer. Identification of molecular targets present in resistant cancer cells is essential for addressing therapeutic resistance and prolonging lung cancer patient survival. Members of the B-cell lymphoma 2 (Bcl-2) family of proteins are associated with chemotherapeutic resistance. In this study, we found that pro-survival protein Bcl-2 is upregulated in paclitaxel resistant cells, potentially contributing to chemotherapy resistance. To exploit the increase in Bcl-2 expression for targeting therapy resistance, we investigated the effects of a peptide derived from the nuclear receptor Nur77 that converts Bcl-2 from an anti-apoptotic protein to a pro-apoptotic protein. The Nur77 derived peptide preferentially induced apoptosis in paclitaxel-resistant cancer cells with high expression of Bcl-2. This peptide also induced apoptosis of multidrug resistant H69AR lung cancer cells that express Bcl-2 and inhibited their growth in 3D spheroids. The Nur77 peptide strongly suppressed the growth of paclitaxel-resistant lung cancer cells in a zebrafish xenograft tumor model. Taken together, our data supports a new strategy for treating lung cancers that acquire resistance to chemotherapy through overexpression of Bcl-2.
RESUMO
Retinoid X receptor (RXR) plays a central role in the regulation of intracellular receptor signaling pathways by acting as a ubiquitous heterodimerization partner of many nuclear receptors, including the orphan receptor Nur77 (also known as TR3 [corrected] or NGFI-B), which translocates from the nucleus to mitochondria, where it interacts with Bcl-2 to induce apoptosis. Here, we report that RXRalpha is required for nuclear export and mitochondrial targeting of Nur77 through their unique heterodimerization that is mediated by dimerization interfaces located in their DNA-binding domain. The effects of RXRalpha are attributed to a putative nuclear export sequence (NES) present in its carboxyl-terminal region. RXRalpha ligands suppress NES activity by inducing RXRalpha homodimerization or altering RXRalpha/Nur77 heterodimerization. The RXRalpha NES is also silenced by RXRalpha heterodimerization with retinoic acid receptor or vitamin D receptor. Consistently, we were able to show that the mitochondrial targeting of the RXRalpha/Nur77 heterodimer and its induction of apoptosis are potently inhibited by RXR ligands. Together, our results reveal a novel nongenotropic function of RXRalpha and its involvement in the regulation of the Nur77-dependent apoptotic pathway [corrected]
Assuntos
Apoptose/fisiologia , Proteínas de Ligação a DNA/metabolismo , Receptor X Retinoide alfa/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Linhagem Celular , DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Humanos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Mutação , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides , Receptores dos Hormônios Tireóideos/química , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptor X Retinoide alfa/química , Receptor X Retinoide alfa/genética , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
TR3, also known as NGFI-B or nur77, is an immediate-early response gene and an orphan member of the steroid/thyroid/retinoid receptor superfamily. We previously reported that TR3 expression was induced by apoptotic stimuli and was required for their apoptotic effect in lung cancer cells. Here, we present evidence that TR3 was also induced by epidermal growth factor (EGF) and serum and was required for their mitogenic effect in lung cancer cells. Ectopic expression of TR3 in both H460 and Calu-6 lung cancer cell lines promoted their cell cycle progression and BrdU incorporation, while inhibition of TR3 expression by the small interfering RNA approach suppressed the mitogenic effect of EGF and serum. Analysis of TR3 mutants showed that both TR3 DNA binding and transactivation were required for its mitogenic effect. In contrast, they were dispensable for its apoptotic activity. Furthermore, confocal microscopy analysis demonstrated that TR3 functioned in the nucleus to induce cell proliferation, whereas it acted on mitochondria to induce apoptosis. In examining the signaling that regulates the mitogenic function of TR3, we observed that coexpression of constitutive-active MEKK1 inhibited TR3 transcriptional activity and TR3-induced proliferation. The inhibitory effect of MEKK1 was mediated through activation of Jun N-terminal kinase, which efficiently phosphorylated TR3, resulting in loss of its DNA binding. Together, our results demonstrate that TR3 is capable of inducing both proliferation and apoptosis in the same cells depending on the stimuli and its cellular localization.
Assuntos
Neoplasias Pulmonares/metabolismo , MAP Quinase Quinase Quinase 1 , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Esteroides/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Apoptose , Sequência de Bases , Linhagem Celular Tumoral , Meios de Cultura , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitose/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Frações Subcelulares/metabolismo , Ativação TranscricionalRESUMO
The aryl hydrocarbon receptor (AhR) is a potential clinical target for cancer and autoimmune dysfunction. Identifying selective AhR modulators that produce desirable clinical outcomes represents an opportunity for developing new anti-cancer agents. Repurposing clinically-used drugs with established safety profiles that activate the AhR represents a good starting place to pursue this goal. In this study, we characterized the AhR-dependent effects of SU5416 (Semaxanib) following its identification in a small-molecule library screen. SU5416 potently activated AhR-dependent reporter genes, induced AhR nuclear localization, facilitated AhR-DNA binding, and increased, expression of its endogenous target genes. SU5416 significantly inhibited proliferation of Hepa1 hepatoma cells in an AhR-dependent manner, but did not induce apoptosis. SU5416 also inhibited the growth of human HepG2 liver cancer cells. The effects of SU5416 correlated with an increased G1 population and increased expression of cell cycle inhibitor p21cip1/waf1 at both the mRNA and protein level. Increased expression of p21cip1/waf1 by SU5416 required expression of both AhR and Arnt. In addition, evidence for long-term activation of the AhR in vivo by a single dose of SU5416 was identified by analyzing published microarray data. Our results provide support for continued investigation of the AhR as therapeutic for cancers such as hepatocellular carcinoma. In addition, our findings raise the possibility that some of the previously observed anti-proliferative effects of SU5416 may be due to activation of the AhR.
Assuntos
Antineoplásicos/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Indóis/farmacologia , Neoplasias Hepáticas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
We previously reported that raloxifene, an estrogen receptor modulator, is also a ligand for the aryl hydrocarbon receptor (AhR). Raloxifene induces apoptosis in estrogen receptor-negative human cancer cells through the AhR. We performed structure-activity studies with seven raloxifene analogs to better understand the structural requirements of raloxifene for induction of AhR-mediated transcriptional activity and apoptosis. We identified Y134 as a raloxifene analog that activates AhR-mediated transcriptional activity and induces apoptosis in MDA-MB-231 human triple negative breast cancer cells. Suppression of AhR expression strongly reduced apoptosis induced by Y134, indicating the requirement of AhR for Y134-induced apoptosis. Y134 also induced apoptosis in hepatoma cells without having an effect on cell cycle regulation. Toxicity testing on zebrafish embryos revealed that Y134 has a significantly better safety profile than raloxifene. Our studies also identified an analog of raloxifene that acts as a partial antagonist of the AhR, and is capable of inhibiting AhR agonist-induced transcriptional activity. We conclude that Y134 is a promising raloxifene analog for further optimization as an anti-cancer agent targeting the AhR.
RESUMO
The aryl hydrocarbon receptor (AhR) has a fundamental role during postnatal liver development and is essential for mediating dioxin toxicity. However, the genetic programs mediating, both, the toxic and physiological effects downstream of the transcription factor AhR are in major parts unknown. We have identified the proto-oncogene c-jun as a novel target gene of AhR. Induction of c-jun depends on activation of p38-mitogen-activated protein kinase (MAPK) by an AhR-dependent mechanism. None of the kinases that are known to phosphorylate p38-MAPK is activated by AhR. Neither the dephosphorylation rate of p38-MAPK is reduced. Furthermore, increased p38-MAPK phosphorylation in response to dioxins does not require ongoing transcription. These findings establish activating 'cross-talk' with MAPK signaling as a novel principle of AhR action, which is apparently independent of the AhR's function as a DNA-binding transcriptional activator.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Dibenzodioxinas Policloradas/farmacologia , Proteínas Proto-Oncogênicas c-jun/genética , Receptores de Hidrocarboneto Arílico/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Genes Reporter , Humanos , Proto-Oncogene Mas , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
The peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor family member that can form a heterodimeric complex with retinoid X receptor (RXR) and initiate transcription of target genes. In this study, we have examined the effects of the PPAR gamma ligand ciglitazone and the RXR ligand SR11237 on growth and induction of retinoic acid receptor (RAR) beta expression in breast and lung cancer cells. Our results demonstrated that ciglitazone and SR11237 cooperatively inhibited the growth of ZR-75-1 and T-47D breast cancer and Calu-6 lung cancer cells. Gel shift analysis indicated that PPAR gamma, in the presence of RXR, formed a strong complex with a retinoic acid response element (beta retinoic acid response element) in the RAR beta promoter. In reporter gene assays, RXR ligands and ciglitazone, but not the PPAR gamma ligand 15d-PGJ(2), cooperatively promoted the transcriptional activity of the beta retinoic acid response element. Ciglitazone, but not 15d-PGJ(2), strongly induced RAR beta expression in human breast and lung cancer cell lines when used together with SR11237. The induction of RAR beta expression by the ciglitazone and SR11237 combination was diminished by a PPAR gamma-selective antagonist, bisphenol A diglycidyl ether. All-trans-retinoic acid or the combination of ciglitazone and SR11237 was able to induce RAR beta in all-trans-retinoic acid-resistant MDA-MB-231 breast cancer cells only when the orphan receptor chick ovalbumin upstream promoter transcription factor was expressed, or in the presence of the histone deacetylase inhibitor trichostatin A. These studies indicate the existence of a novel RAR beta-mediated signaling pathway of PPAR gamma action, which may provide a molecular basis for developing novel therapies involving RXR and PPAR gamma ligands in potentiating antitumor responses.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores do Ácido Retinoico/genética , Tiazolidinedionas , Fatores de Transcrição/fisiologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzoatos/farmacologia , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Neoplasias Pulmonares/genética , Receptores Citoplasmáticos e Nucleares/genética , Retinoides/farmacologia , Tiazóis/farmacologia , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that plays multiple roles in regulation of immune and inflammatory responses. The ability of certain AhR ligands to induce regulatory T cells (Tregs) has generated interest in developing AhR ligands for therapeutic treatment of immune-mediated diseases. To this end, we designed a screen for novel Treg-inducing compounds based on our understanding of the mechanisms of Treg induction by the well-characterized immunosuppressive AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We screened a ChemBridge small molecule library and identified 10-chloro-7H-benzimidazo[2,1-a]benzo[de]Iso-quinolin-7-one (10-Cl-BBQ) as a potent AhR ligand that was rapidly metabolized and not cytotoxic to proliferating T cells. Like TCDD,10-Cl-BBQ altered donor CD4(+) T cell differentiation during the early stages of a graft versus host (GVH) response resulting in expression of high levels of CD25, CTLA-4 and ICOS, as well as several genes associated with Treg function. The Treg phenotype required AhR expression in the donor CD4(+) T cells. Foxp3 was not expressed in the AhR-induced Tregs implicating AhR as an independent transcription factor for Treg induction. Structure-activity studies showed that unsubstituted BBQ as well as 4, 11-dichloro-BBQ were capable of inducing AhR-Tregs. Other substitutions reduced activation of AhR. Daily treatment with 10-Cl-BBQ during the GVH response prevented development of GVH disease in an AhR-dependent manner with no overt toxicity. Together, our data provide strong support for development of select BBQs that activate the AhR to induce Tregs for treatment of immune-mediated diseases.
Assuntos
Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , Doença Enxerto-Hospedeiro/prevenção & controle , Isoquinolinas/metabolismo , Isoquinolinas/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Animais , Benzimidazóis/química , Benzimidazóis/farmacocinética , Doença Enxerto-Hospedeiro/imunologia , Imunoterapia , Isoquinolinas/química , Isoquinolinas/farmacocinética , Cinética , Ligantes , Camundongos , Relação Estrutura-AtividadeRESUMO
A novel role of the dihydroorotatedehydrogenase (DHODH) inhibitor leflunomide as a potential anti-melanoma therapy was recently reported (Nature 471:518-22, 2011). We previously reported that leflunomide strongly activates the transcriptional activity of the Aryl Hydrocarbon Receptor (AhR). We therefore tested whether the AhR regulates the anti-proliferative effects of leflunomide in melanoma. We first evaluated the expression of AhR in melanoma cells and found that AhR is highly expressed in A375 melanoma as well as in several other cancer cell types. To evaluate whether AhR plays a role in regulating the growth inhibitory effects of leflunomide in A375 cells, we generated a stable cell line from parental A375 cells expressing a doxycycline (DOX) inducible AhR shRNA. Using these cells in the absence or presence of DOX (normal AhR levels or AhR-knockdown, respectively) we found that the anti-proliferative effects of leflunomide, but not its metabolite A771726, were strongly dependent upon AhR expression. It has been well established that supplementation of cells with exogenous uridine completely rescues the anti-proliferative effects due to DHODH inhibition. Thus, we performed uridine rescue experiments in A375 cells to determine whether the anti-proliferative effects of leflunomide are solely due to DHODH inhibition as previously reported. Interestingly, saturating levels of uridine only modestly rescued A375 cells from the anti-proliferative effects of both leflunomide and A771726, indicating additional mechanism(s), apart from DHODH inhibition are responsible for the anti-proliferative effects of leflunomide in melanoma cells. Uridine also did not rescue MDA-MB-435S melanoma cell proliferation after leflunomide treatment. Our results reveal that the AhR is a molecular target of leflunomide and support the feasibility of the clinical application of leflunomide for treating melanoma. Furthermore, analysis of expression data from 967 cancer cell lines revealed that AhR is expressed in multiple different cancer types supporting the intriguing possibility of targeting the AhR for therapy in a number of cancers.
Assuntos
Isoxazóis/farmacologia , Melanoma/metabolismo , Melanoma/patologia , Receptores de Hidrocarboneto Arílico/metabolismo , Compostos de Anilina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Crotonatos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Di-Hidro-Orotato Desidrogenase , Fluoresceínas/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Hidroxibutiratos/farmacologia , Leflunomida , Nitrilas , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais/efeitos dos fármacos , Succinimidas/metabolismo , Toluidinas , Uridina/farmacologiaRESUMO
BACKGROUND: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity and biological activity of dioxins and related chemicals. The AhR influences a variety of processes involved in cellular growth and differentiation, and recent studies have suggested that the AhR is a potential target for immune-mediated diseases. METHODOLOGY/PRINCIPAL FINDINGS: During a screen for molecules that activate the AhR, leflunomide, an immunomodulatory drug presently used in the clinic for the treatment of rheumatoid arthritis, was identified as an AhR agonist. We aimed to determine whether any biological activity of leflunomide could be attributed to a previously unappreciated interaction with the AhR. The currently established mechanism of action of leflunomide involves its metabolism to A771726, possibly by cytochrome P450 enzymes, followed by inhibition of de novo pyrimidine biosynthesis by A771726. Our results demonstrate that leflunomide, but not its metabolite A771726, caused nuclear translocation of AhR into the nucleus and increased expression of AhR-responsive reporter genes and endogenous AhR target genes in an AhR-dependent manner. In silico Molecular Docking studies employing AhR ligand binding domain revealed favorable binding energy for leflunomide, but not for A771726. Further, leflunomide, but not A771726, inhibited in vivo epimorphic regeneration in a zebrafish model of tissue regeneration in an AhR-dependent manner. However, suppression of lymphocyte proliferation by leflunomide or A771726 was not dependent on AhR. CONCLUSIONS: These data reveal that leflunomide, an anti-inflammatory drug, is an agonist of the AhR. Our findings link AhR activation by leflunomide to inhibition of fin regeneration in zebrafish. Identification of alternative AhR agonists is a critical step in evaluating the AhR as a therapeutic target for the treatment of immune disorders.
Assuntos
Anti-Inflamatórios/farmacologia , Isoxazóis/farmacologia , Receptores de Hidrocarboneto Arílico/agonistas , Animais , Sequência de Bases , Linhagem Celular , Citocromo P-450 CYP1A2/metabolismo , Primers do DNA , Imunofluorescência , Genes Reporter , Leflunomida , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Regeneração , Peixe-Zebra/genética , Peixe-Zebra/fisiologiaRESUMO
Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their anticancer effects through cyclooxygenase-2 (COX-2)-dependent and independent mechanisms. Here, we report that Sulindac, an NSAID, induces apoptosis by binding to retinoid X receptor-alpha (RXRalpha). We identified an N-terminally truncated RXRalpha (tRXRalpha) in several cancer cell lines and primary tumors, which interacted with the p85alpha subunit of phosphatidylinositol-3-OH kinase (PI3K). Tumor necrosis factor-alpha (TNFalpha) promoted tRXRalpha interaction with the p85alpha, activating PI3K/AKT signaling. When combined with TNFalpha, Sulindac inhibited TNFalpha-induced tRXRalpha/p85alpha interaction, leading to activation of the death receptor-mediated apoptotic pathway. We designed and synthesized a Sulindac analog K-80003, which has increased affinity to RXRalpha but lacks COX inhibitory activity. K-80003 displayed enhanced efficacy in inhibiting tRXRalpha-dependent AKT activation and tRXRalpha tumor growth in animals.
Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor X Retinoide alfa/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sulindaco/análogos & derivados , Sulindaco/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citoplasma/metabolismo , Dinoprostona/metabolismo , Desenho de Fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Receptor X Retinoide alfa/genética , Receptor X Retinoide alfa/metabolismo , Deleção de Sequência/fisiologia , Transdução de Sinais/fisiologia , Sulindaco/metabolismo , Sulindaco/uso terapêutico , Ativação Transcricional/efeitos dos fármacos , Transfecção , Tretinoína/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor; the AhR Per-AhR/Arnt-Sim (PAS) domain binds ligands. We developed homology models of the AhR PAS domain to characterize previously observed intra- and interspecies differences in ligand binding using molecular docking. In silico structure-based virtual ligand screening using our model resulted in the identification of pinocembrin and 5-hydroxy-7-methoxyflavone, which promoted nuclear translocation and transcriptional activation of AhR and AhR-dependent induction of endogenous target genes.
Assuntos
Modelos Moleculares , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Receptores de Hidrocarboneto Arílico/genética , Ativação Transcricional/efeitos dos fármacos , Interface Usuário-ComputadorRESUMO
Bcl-2 can be converted into a proapoptotic molecule by nuclear receptor Nur77. However, the development of Bcl-2 converters as anticancer therapeutics has not been explored. Here we report the identification of a Nur77-derived Bcl-2-converting peptide with 9 amino acids (NuBCP-9) and its enantiomer, which induce apoptosis of cancer cells in vitro and in animals. The apoptotic effect of NuBCPs and their activation of Bax are not inhibited but rather potentiated by Bcl-2. NuBCP-9 and its enantiomer bind to the Bcl-2 loop, which shares the characteristics of structurally adaptable regions with many cancer-associated and signaling proteins. NuBCP-9s act as molecular switches to dislodge the Bcl-2 BH4 domain, exposing its BH3 domain, which in turn blocks the activity of antiapoptotic Bcl-X(L).
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Esteroides/metabolismo , Animais , Antineoplásicos/química , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta a Droga , Feminino , Células HeLa , Humanos , Células Jurkat , Camundongos , Camundongos Knockout , Camundongos SCID , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Oligopeptídeos/química , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptores de Esteroides/genética , Estereoisomerismo , Fatores de Tempo , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Defects in apoptosis mechanisms play important roles in malignancy and autoimmunity. Orphan nuclear receptor Nur77/TR3 has been demonstrated to bind antiapoptotic protein Bcl-2 and convert it from a cytoprotective to a cytodestructive protein, representing a phenotypic conversion mechanism. Of the 6 antiapoptotic human Bcl-2 family members, we found that Nur77/TR3 binds strongest to Bcl-B, showing selective reactivity with Bcl-B, Bcl-2, and Bfl-1 but not Bcl-X(L), Mcl-1, or Bcl-W. Nur77 converts the phenotype of Bcl-B from antiapoptotic to proapoptotic. Bcl-B is prominently expressed in plasma cells and multiple myeloma. Endogenous Bcl-B associates with endogenous Nur77 in RPMI 8226 myeloma cells, where RNA interference experiments demonstrated dependence on Bcl-B for Nur77-induced apoptosis. Furthermore, a Nur77-mimicking peptide killed RPMI 8226 myeloma cells through a Bcl-B-dependent mechanism. Because Bcl-B is abundantly expressed in plasma cells and some myelomas, these findings raise the possibility of exploiting the Nur77/Bcl-B mechanism for apoptosis for eradication of autoimmune plasma cells or myeloma.