RESUMEN
BACKGROUND: Platelet hyperactivity induced by inflammation is a known risk factor for atherosclerosis and thrombosis, but its underlying mechanisms remain poorly understood. METHODS AND RESULTS: The signal transducer and activator of transcription 3 (STAT3) was activated in collagen-stimulated platelets. Activated STAT3 served as a protein scaffold to facilitate the catalytic interaction between the kinase Syk (spleen tyrosine kinase) and the substrate PLCγ2 to enhance collagen-induced calcium mobilization and platelet activation. The same interaction of STAT3 with Syk and PLCγ2 was detected in HEK293 cells transfected with cDNAs for Syk and PLCγ2 and stimulated with interleukin-6. Pharmacological inhibition of STAT3 blocked ≈50% of collagen- and a collagen-related peptide-induced but not thrombin receptor-activating peptide- or ADP-induced aggregation and ≈80% of thrombus formation of human platelets on a collagen matrix. This in vitro phenotype was reproduced in mice infused with STAT3 inhibitors and mice with platelet-specific STAT3 deficiency. By forming a complex with its soluble receptor, the proinflammatory cytokine interleukin-6 enhanced the collagen-induced STAT3 activation in human platelets. CONCLUSIONS: These data demonstrate a nontranscriptional activity of STAT3 that facilitates a crosstalk between proinflammatory cytokine and hemostasis/thrombosis signals in platelets. This crosstalk may be responsible for the platelet hyperactivity found in conditions of inflammation.
Asunto(s)
Agregación Plaquetaria/fisiología , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/fisiología , Vasculitis/metabolismo , Animales , Aterosclerosis/metabolismo , Plaquetas/citología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Colágeno/metabolismo , Colágeno/farmacología , Células HEK293 , Humanos , Interleucina-6/metabolismo , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfolipasa C gamma/metabolismo , Fosforilación/fisiología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT3/genética , Quinasa Syk , Trombosis/metabolismoRESUMEN
It is becoming increasingly evident that improving the cure rate of many cancers will require treatment regimens hit more than one validated tumor targets. Developing an anti-cancer agent that targets two oncoproteins simultaneously is a promising strategy for accomplishing this goal. It would be expected to promote drug efficacy, reduce therapy-resistant without introducing additional toxic side effects. HIF-1α is a key regulator of the cellular response to hypoxia and is involved in tumor angiogenesis and cancer cell survival, glucose metabolism, and invasion. Stat3 has several oncogenic functions, including suppression of anti-tumor immune responses and promotion of inflammation. Recently, we have identified the perylene derivative, TEL03, as a dual inhibitor that targets both HIF-1α and Stat3. TEL03 blocks the expression of both HIF-1α and Stat3, regulated oncogenes (e.g., Bcl-2, VEGF, Glut1, and others) in cancer cells, and induces cancer cell apoptosis. The results demonstrated that: (i) TEL03 blocks Stat3 phosphorylation, and inhibits Stat3 transcriptional activity; and (ii) interferes the binding of HIF-1α to p300/CBP inducing its degradation by proteasomes under hypoxic conditions. Our in vivo tests showed that as a dual inhibitor, TEL03 dramatically inhibited tumor growth, and provided the evidence that targeting both HIF-1α and Stat3 simultaneously could be a promising strategy for breast and pancreatic cancer therapies.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Imidas/química , Perileno/análogos & derivados , Perileno/química , Factor de Transcripción STAT3/metabolismo , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Expresión Génica/efectos de los fármacos , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Imidas/farmacología , Imidas/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Simulación del Acoplamiento Molecular , Perileno/farmacología , Perileno/uso terapéutico , Fosforilación/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/genética , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: The Stat3 pathway and the hypoxia-sensing pathway are both up-regulated in prostate cancer. Stat3 is a specific regulator of pro-carcinogenic inflammation and represents a promising therapeutic target. Hypoxia-inducible factor-1 (HIF-1)α, which mediates the cellular response to hypoxia, has been demonstrated to be over-expressed in many human cancers and is associated with poor prognosis and treatment failure in clinic. To develop a potent strategy to increase therapeutic efficacy and reduce drug resistance in prostate cancer therapy, we combined two anti-cancer agents: T40214 (a p-Stat3 inhibitor) and JG244 (a HIF-1α inhibitor) together to treat nude mice bearing human prostate tumor (DU145) and immunocompetent mice (C57BL/6) bearing murine prostate tumor (TRAMP-C2). METHODS: We employed in vitro and in vivo assays, including Western blots, cell cycle analysis, immunohistochemistry, TUNEL and xenograft models to determine the drug efficacy and mechanism of combination treatment of T40214 and JG244. RESULTS: We found that compared to treatment by T40214 or JG244 alone, the combination treatment using T40214 and JG244 together significantly suppressed growth of human or murine prostate tumors. Also, compared with apoptotic cells induced by T40214 or JG244 alone, the combined treatment greatly increased apoptosis in DU145 (P < 0.006) and TRAMP-C2 tumors (P < 0.008). CONCLUSIONS: Our results suggested that combination treatment including a HIF-1α/2α inhibitor not only has therapeutic efficacy in targeting HIF-1α/2α, but also could reduce the hypoxia-induced drug resistance to other therapies (e.g., T40214) and enhance drug efficacy. This approach could make prostate cancer treatments more effective.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Oligodesoxirribonucleótidos/farmacología , Oligonucleótidos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Factor de Transcripción STAT3/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Neoplasias de la Próstata/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hypoxia-inducible factor-1 (HIF-1) plays crucial roles in tumor promotion by upregulating its target genes, which are involved in energy metabolism, angiogenesis, cell survival, invasion, metastasis, and drug resistance. The HIF-1alpha subunit, which is regulated by O2-dependent hydroxylation, ubiquitination, and degradation, has been identified as an important molecular target for cancer therapy. We have rationally designed G-rich oligodeoxynucleotides (ODNs) as inhibitors of HIF-1alpha for human cancer therapy. The lead compounds, JG243 and JG244, which form an intramolecular parallel G-quartet structure, selectively target HIF-1alpha and decreased levels of both HIF-1alpha and HIF-2alpha (IC50 < 2 micromol/l) and also inhibited the expression of HIF-1-regulated proteins [vascular endothelial growth factor (VEGF), Bcl-2, and Bcl-XL], but did not disrupt the expression of p300, Stat3, or p53. JG-ODNs induced proteasomal degradation of HIF-1alpha and HIF-2alpha that was dependent on the hydroxylase activity of prolyl-4-hydroxylase-2. JG243 and JG244 dramatically suppressed the growth of prostate, breast, and pancreatic tumor xenografts. Western blots from tumor tissues showed that JG-ODNs significantly decreased HIF-1alpha and HIF-2alpha levels and blocked the expression of VEGF. The JG-ODNs are novel anticancer agents that suppress tumor growth by inhibiting HIF-1.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Oligonucleótidos/uso terapéutico , Animales , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/tratamiento farmacológico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Platelets arrest bleeding by adhering to and aggregating on the subendothelium exposed at the site of vessel injury. This process is initiated by the interaction between the subendothelium von Willebrand factor (VWF) and the glycoprotein (GP) Ib-IX-V complex on platelets. However, the same interaction also results in thrombosis at the site of a ruptured atherosclerotic plaque. Reagents regulating the GP Ib-VWF interaction will therefore have direct impact on haemostasis and thrombosis. We have characterised an oligonucleotide G-quartet (T30923) that specifically blocks VWF binding to GP Ibalpha, the VWF-binding subunit of the GP Ib-IX-V complex. We evaluated the potential interactions of T30923 with GP Ibalpha and VWF A1 domain by computer simulated molecular dockings, which identified four T30923 docking sites in the beta-sheets of the N-terminal region of GP Ibalpha (E14-D18, S39, D63-S64, and D83-S85). Experimentally, T30923 bound GP Ibalpha and dose-dependently blocked platelet aggregation induced by ristocetin and thrombin, but not by botrocetin, collagen, TRAP, and ADP. It also blocked shear-induced platelet aggregation and thrombus formation on immobilised VWF under arterial shear stress. These results demonstrate that T30923 may have therapeutic potentials to regulate the GP Ibalpha-VWF interaction.
Asunto(s)
Glicoproteínas/química , Oligonucleótidos/química , Complejo GPIb-IX de Glicoproteína Plaquetaria/química , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Conformación Molecular , Oligonucleótidos/farmacología , Adhesividad Plaquetaria , Agregación Plaquetaria , Unión Proteica , Estructura Terciaria de Proteína , Estrés Mecánico , Factor de von Willebrand/químicaRESUMEN
BACKGROUND: Prostate cancer (PC) is the most common cancer among men in American and the second leading cause of cancer death. The treatment options employed for patients with advanced and metastatic PC are limited. As a critical mediator of oncogenic signaling, STAT3 is active in 82% of patients with PC. STAT3 has become a very important molecular target for PC therapy since it upregulates the oncogenes encoding apoptosis inhibitors, cell cycle regulators, and inducers of angiogenesis. However, no anti-tumor drug whose primary mode of action is to target STAT3 has yet reached the clinic. To this end, we have laid the initial groundwork to develop the STAT3-inhibiting G-quartet oligodeoxynucleotide (GQ-ODN), T40214, for treatment of PCs. METHODS: We employed in vitro and in vivo assays, including Western blots, EMSA, cell cycle analysis, TUNEL and xenograft models, to determine the drug efficacy and mechanism of T40214/PEI complex. RESULTS: The results demonstrated that (i) T40214 significantly inhibited STAT3 activation and induced apoptosis in both androgen-dependent and androgen-independent PC cells; (ii) T40214 delivered by ployethylenimine (PEI) significantly suppressed prostate tumor growth in tumor-bearing nude mice due to that T40214 inhibited STAT3 activation and then greatly promoted apoptosis, reduced angiogenesis and cell proliferation in prostate tumors. CONCLUSION: Our studies suggested that STAT3 is a critical oncogenic signal, which strongly influences the progression of PCs and that T40214/PEI complex is a promising candidate for treatment of patients with prostate tumors and represents a novel strategy for PC therapy.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Oligodesoxirribonucleótidos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Adenocarcinoma/patología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Desnudos , Neovascularización Patológica/tratamiento farmacológico , Oligodesoxirribonucleótidos/farmacología , Polietileneimina , Neoplasias de la Próstata/patología , Factor de Transcripción STAT3/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lung cancer is the leading cause of cancer mortality in the United States. Despite advances made over the past decades, the overall survival of patients with lung cancer remains dismal. Here we report novel G-quartet oligodeoxynucleotides (GQ-ODN) that were designed to selectively target signal transducer and activator of transcription 3 (Stat3), in the treatment of human non-small cell lung cancer (NSCLC). The objective of this study was to evaluate the effects of two novel GQ-ODN STAT3 inhibitors, T40214 and T40231, on NSCLC bearing nude mice. NSCLC bearing nude mice were assigned to 5 groups, which were treated by vehicle, control ODN, T40214, T40231, and Paclitaxel, respectively. Tumors were measured, isolated and analyzed using Western blotting, immuno-histochemistry, RPA and TUNEL. Results show that GQ-ODN T40214 and T40231 significantly suppress the growth of NSCLC tumors in nude mice by selectively inhibiting the activation of Stat3 and its downstream proteins Bcl-2, Bcl-xL, Mcl-1, survivin, VEGF, Cyclin D1 and c-myc; thereby, promoting apoptosis and reducing angiogenesis and cell proliferation. These findings validate Stat3 as an important molecular target for NSCLC therapy and demonstrate the efficacy of GQ-ODN in inhibiting Stat3 phosphorylation.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Oligodesoxirribonucleótidos/uso terapéutico , Factor de Transcripción STAT3/antagonistas & inhibidores , Animales , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/efectos de los fármacos , Ciclina D1/análisis , Ciclina D1/antagonistas & inhibidores , Ciclina D1/metabolismo , Inmunohistoquímica , Proteínas Inhibidoras de la Apoptosis , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica/metabolismo , Oligodesoxirribonucleótidos/farmacología , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Fosforilación , Conformación Proteica , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-myc/análisis , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/metabolismo , Survivin , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína bcl-X/análisis , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/metabolismoRESUMEN
Signal transducer and activator of transcription 3 (Stat3) is a critical mediator of oncogenic signaling activated frequently in many types of human cancer where it contributes to tumor cell growth and resistance to apoptosis. Stat3 has been proposed as a promising target for anticancer drug discovery. Recently, we developed a series of G-quartet oligodeoxynucleotides (GQ-ODN) as novel and potent Stat3 inhibitors, which significantly suppressed the growth of prostate and breast tumors in nude mice. In the present study, we showed that GQ-ODN specifically inhibited DNA-binding activity of Stat3 as opposed to Stat1. Computer-based docking analysis revealed that GQ-ODN predominantly interacts with the SH2 domains of Stat3 homodimers to destabilize dimer formation and disrupt DNA-binding activity. We employed five regimens in the treatment of nude mice with tumors of head and neck squamous cell carcinoma (HNSCC): placebo, paclitaxel, GQ-ODN T40214, GQ-ODN T40231, and T40214 plus paclitaxel. The mean size of HNSCC tumors over 21 days only increased by 1.7-fold in T40214-treated mice and actually decreased by 35% in T40214 plus paclitaxel-treated mice whereas the mean size of HNSCC tumors increased 9.4-fold in placebo-treated mice in the same period. These findings show that GQ-ODN has potent activity against HNSCC tumor xenografts alone and in combination with paclitaxel.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Oligodesoxirribonucleótidos/uso terapéutico , Factor de Transcripción STAT3/antagonistas & inhibidores , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis , ADN de Neoplasias/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Guanina/química , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Ratones , Ratones Desnudos , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/química , Paclitaxel/uso terapéutico , Factor de Transcripción STAT3/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We report here a novel mutation in the cytochrome b5 reductase gene resulting in type I methemoglobinemia. A single T->C transition in exon 8 at position 25985 was identified, changing codon 217 from Leu to Pro (L217P). The mutation is located in the NADH binding domain at the base of alpha-helix Nalpha3, a region of sequence highly conserved from yeast to man. A quantitative assessment of the thermodynamic cost of this mutation at 37 degrees C revealed a ten-fold drop in the free energy of stability. Alterations in hydrogen bonding and solvent accessibility surrounding residue 217 were predicted based on computer modeling.
Asunto(s)
Citocromo-B(5) Reductasa/genética , Metahemoglobinemia/enzimología , Metahemoglobinemia/genética , Mutación , Adulto , Citocromo-B(5) Reductasa/química , Femenino , Genotipo , Humanos , India , Masculino , TermodinámicaRESUMEN
Approximately half of all medical illnesses can be attributed to insufficient or excessive apoptosis. Apoptosis resistance is a cardinal feature of cancer, mediated in many instances, by signal transducer and activator of transcription (STAT) 3. We identified G-quartet oligodeoxynucleotides (GQ-ODNs) as potent and selective inhibitors of Stat3 DNA binding activity in vitro. We report here that GQ-ODNs are capable of inhibiting the growth of nude mouse xenografts of breast and prostate tumors. We developed a rat model of severe hemorrhagic shock (HS) to assess the benefits of promoting Stat3 activity in diseases marked by excessive apoptosis. Administration of the Stat3-activating cytokine IL-6 at the initiation of resuscitation from HS activated intra-cardiac Stat3, reversed cardiac apoptosis, left ventricular dysfunction and hypovolemic circulatory collapse (HCC) and resulted in a 5-fold reduction in mortality; pre-treatment of rats with GQ-ODN prevented the reversal of cardiac apoptosis and HCC by IL-6. Thus, targeting of Stat3 may be a useful for treatment of multiple cancers; agents that activate Stat3 may be beneficial in acute insults that cause apoptosis in organs critical for survival.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Neoplasias Experimentales/tratamiento farmacológico , Oligodesoxirribonucleótidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Femenino , Humanos , Interleucina-6/farmacología , Masculino , Ratones , Ratones Desnudos , Modelos Biológicos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Trasplante de Neoplasias , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Choque/prevención & control , Transducción de Señal/efectos de los fármacos , Trasplante HeterólogoRESUMEN
Stat3 is a signaling molecular and oncogene activated frequently in many human malignancies including the majority of prostate, breast, and head and neck cancers; yet, no current chemotherapeutic approach has been implemented clinically that specifically targets Stat3. We recently developed G-rich oligodeoxynucleotides, which form intramolecular G-quartet structures (GQ-ODN), as a new class of Stat3 inhibitor. GQ-ODN targeted Stat3 protein directly inhibiting its ability to bind DNA. When delivered into cells using polyethyleneimine as vehicle, GQ-ODN blocked ligand-induced Stat3 activation and Stat3-mediated transcription of antiapoptotic genes. To establish the effectiveness of GQ-ODN as a potential new chemotherapeutic agent, we systemically administered GQ-ODN (T40214 or T40231) plus polyethyleneimine or polyethyleneimine alone (placebo) by tail-vein injection into nude mice with prostate and breast tumor xenografts. Whereas the mean volume of breast tumor xenografts in placebo-treated mice increased >7-fold over 18 days, xenografts in the GQ-ODN-treated mice remained unchanged. Similarly, whereas the mean volume of prostate tumor xenografts in placebo-treated mice increased 9-fold over 10 days, xenografts in GQ-ODN-treated mice increased by only 2-fold. Biochemical examination of tumors from GQ-ODN-treated mice demonstrated a significant reduction in Stat3 activation, levels of the antiapoptotic proteins Bcl-2 and Bcl-xL, and an 8-fold increase in the number of apoptotic cells compared with the tumors of placebo-treated mice. Thus, GQ-ODN targeting Stat3 induces tumor cell apoptosis when delivered into tumor xenografts and represents a novel class of chemotherapeutic agents that holds promise for the systemic treatment of many forms of metastatic cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de Unión al ADN/antagonistas & inhibidores , Oligonucleótidos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Transactivadores/antagonistas & inhibidores , Animales , Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , División Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Guanosina/química , Guanosina/genética , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Moleculares , Oligonucleótidos/química , Oligonucleótidos/genética , Fosforilación , Polietileneimina/farmacología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Factor de Transcripción STAT3 , Relación Estructura-Actividad , Especificidad por Sustrato , Transactivadores/genética , Transactivadores/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The ability of certain DNA sequences to form G-quartet structures has been exploited recently to develop novel anti-cancer agents including small molecules that promote G-quartet formation within the c-MYC promoter thereby repressing c-MYC transcription and introducing G-quartet-forming oligodeoxynucleotides (GQ-ODN) into cancer cells resulting in p53-dependent cell cycle arrest and inhibition of DNA replication. GQ-ODNs also have been developed as potent inhibitors of signal transducer and activator of transcription (STAT) 3, a critical mediator of oncogenic signaling in many cancers. This review summarizes the rational design of G-quartet forming DNA drugs as Stat3 inhibitors. Topics that are reviewed include the strategy of structure-based drug design, establishment of a structure-activity relationship, development of a novel intracellular delivery system for G-quartet-forming DNA agents and in vivo drug testing to assess the anti-cancer effects of DNA drugs in tumor xenografts. Results to date with GQ-ODN targeting Stat3 are encouraging, and it is hoped that continued pursuit of the methodology outlined here may lead to development of an effective agent for treatment of metastatic cancers, such as prostate and breast, in which Stat3 is constitutively activated.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Conformación de Ácido Nucleico , Oligonucleótidos/síntesis química , Oligonucleótidos/farmacología , Animales , Proteínas de Unión al ADN/antagonistas & inhibidores , Diseño de Fármacos , Humanos , Relación Estructura-Actividad Cuantitativa , Factor de Transcripción STAT3 , Transactivadores/antagonistas & inhibidoresRESUMEN
Stat3 is an oncogene that is activated in many human cancer cells. Genetic approaches that disrupt Stat3 activity result in inhibition of cancer cell growth and enhanced cell apoptosis supporting the development of novel drugs targeting Stat3 for cancer therapy. G-quartet oligodeoxynucleotides (ODNs) were demonstrated to be potent inhibitors of Stat3 DNA binding activity in vitro with the G-quartet ODN, T40214, having an IC(50) of 7 microM. Computer-simulated docking studies indicated that G-quartet ODNs mainly interacted with the SH2 domain of Stat3 and were capable of inserting between the SH2 domains of Stat3 dimers bound to DNA. We demonstrated that the G-rich ODN T40214, which forms a G-quartet structure at intracellular but not extracellular K+ ion concentrations, is delivered efficiently into the cytoplasm and nucleus of cancer cells where it inhibited IL-6-stimulated Stat3 activation and suppressed Stat3-mediated upregulation of bcl-x and mcl-1 gene expression. Thus, G-quartet represents a new class of drug for targeting of Stat3 within cancer cells.
Asunto(s)
Proteínas de Unión al ADN/genética , Marcación de Gen/métodos , Oligodesoxirribonucleótidos/farmacología , Transactivadores/genética , Secuencia de Bases , Línea Celular Tumoral , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Dimerización , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Modelos Moleculares , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Factor de Transcripción STAT3 , Supresión Genética , Transactivadores/química , Transactivadores/metabolismoAsunto(s)
Proteínas HMGA/genética , Leucemia de Células T/genética , Leucemia de Células T/patología , Nanopartículas , Oligodesoxirribonucleótidos/administración & dosificación , Factor de Transcripción STAT3/genética , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Leucemia de Células T/metabolismo , Leucemia de Células T/terapia , Masculino , Ratones , Ratones Transgénicos , Factor de Transcripción STAT3/metabolismo , Carga TumoralRESUMEN
The mounting evidences have shown that signal transducer and activator of transcription 3 (Stat3) is a critical target for cancer therapy. Recently, we developed a G-quartet oligonucleotide T40214 as a novel and potent Stat3 inhibitor. T40214 specifically inhibited DNA-binding activity of Stat3 and significantly suppressed the growth of many tumor xenografts in nude mice. To determine the mechanism of GQ-ODNs selectively targeting Stat3, we established a 3D model of complex T40214/p-Stat3 dimer based on experimental evidences. The binding site of T40214 within Stat3 dimer was determined by statistical docking analysis. The results indicated that T40214 strongly interacted within the region from residue E638 through E652 of Stat3 dimer. The binding model refined by Hex docking disclosed that T40214 binds to SH2 domain of Stat3 and forms H-bonds with residues Q643, Q644, N646, and N647, which are critical for the binding interaction. The 3D models also suggested that T40214 inhibits Stat3 activity through disrupting the binding interaction between Stat3 dimer and DNA duplex for transcription. Our computational studies provided a platform for future structure-based drug design of novel Stat3 inhibitors.
Asunto(s)
G-Cuádruplex , Oligonucleótidos/química , Oligonucleótidos/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/química , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Simulación por Computador , Dimerización , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Estructura Cuaternaria de Proteína , Factor de Transcripción STAT3/genética , Homología de Secuencia de AminoácidoRESUMEN
G-CSFR cytoplasmic tyrosine (Y) residues (Y704, Y729, Y744, and Y764) become phosphorylated upon ligand binding and recruit specific Src homology 2 domain-containing proteins that link to distinct yet overlapping programs for myeloid cell survival, differentiation, proliferation, and activation. The structural basis for recruitment specificity is poorly understood but could be exploited to selectively target deleterious G-CSFR-mediated signaling events such as aberrant Stat3 activation demonstrated in a subset of acute myeloid leukemia patients with poor prognosis. Recombinant Stat3 bound to G-CSFR phosphotyrosine peptide ligands pY704VLQ and pY744LRC with similar kinetics. Testing of three models for Stat3 Src homology 2-pY ligand binding in vitro and in vivo revealed unique determinants for Stat3 recruitment and activation by the G-CSFR, the side chain of Stat3 R609, which interacts with the pY ligand phosphate group, and the peptide amide hydrogen of E638, which bonds with oxygen/sulfur within the + 3 Q/C side chain of the pY ligand when it assumes a beta turn. Thus, our findings identify for the first time the structural basis for recruitment and activation of Stat3 by the G-CSFR and reveal unique features of this interaction that can be exploited to target Stat3 activation for the treatment of a subset of acute myeloid leukemia patients.
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Fosfopéptidos/fisiología , Fosfotirosina/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocito/fisiología , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/metabolismo , Arginina/genética , Arginina/metabolismo , Línea Celular , Biología Computacional , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Ligandos , Lisina/genética , Lisina/metabolismo , Modelos Moleculares , Fosfotirosina/fisiología , Unión Proteica/genética , Factor de Transcripción STAT3/genéticaRESUMEN
Stat3 is constitutively activated in many human cancers where it functions as a critical mediator of oncogenic signaling through transcriptional activation of genes encoding apoptosis inhibitors (e.g. Bcl-x(L), Mcl-1 and survivin), cell-cycle regulators (e.g. cyclin D1 and c-Myc) and inducers of angiogenesis (e.g. vascular endothelial growth factor). This article reviews several approaches that have been pursued for targeting Stat3 in cancer therapy including antisense strategies, tyrosine kinase inhibition, decoy phosphopeptides, decoy duplex oligonucleotides and G-quartet oligodeoxynucleotides (GQ-ODN). The GQ-ODN strategy is reviewed in somewhat greater detail than the others because it includes a novel system that effectively delivers drug into cells and tissues, addresses successfully the issue of specificity of targeting Stat3 versus Stat1, and has demonstrated efficacy in vivo.
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Anticarcinógenos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias/metabolismo , Factor de Transcripción STAT3/metabolismo , Inductores de la Angiogénesis/farmacología , Animales , Apoptosis/fisiología , Proteínas de Ciclo Celular/farmacología , Movimiento Celular/fisiología , Proliferación Celular , Humanos , Proteínas Inhibidoras de la Apoptosis/farmacología , Ligandos , Neoplasias/tratamiento farmacológico , Oligonucleótidos/farmacología , Oligonucleótidos Antisentido/farmacología , Fosfopéptidos/farmacología , Plásmidos/farmacología , Conformación Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas , Factor de Transcripción STAT3/genética , Transducción de Señal/genética , Activación Transcripcional/genética , Triterpenos/farmacología , Tirfostinos/farmacología , Dominios Homologos src/fisiologíaRESUMEN
Human nuclear DNA topoisomerase I (top1) plays a crucial role in DNA replication, transcription, and chromosome condensation. In this study, we show that intra- and intermolecular guanosine quartets (G-quartets) can inhibit top1-mediated DNA cleavage at a high affinity site. Top1-mediated DNA cleavage was also inhibited by a 16-mer single-stranded oligodeoxynucleotide (ODN) containing a G-rich sequence (G(2)T(2)G(5)TG(2)TG(3)) and by its RNA equivalent, neither of which form G-quartet structures. A comparison of various single-stranded ODN for their ability to inhibit top1-mediated DNA cleavage indicated that G-rich sequences containing repeats of 2 or 3 consecutive guanines interspaced with thymines specifically inhibited top1. We also found that both single-stranded and G-quartet-forming ODNs bind to top1 without being cleaved by the enzyme. These results demonstrate that either DNA or RNA G-rich single-stranded and G-quartet-forming oligonucleotides can bind to top1 and prevent cleavage of duplex DNA.
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Núcleo Celular/enzimología , ADN-Topoisomerasas de Tipo I/metabolismo , ADN de Cadena Simple/metabolismo , Guanosina/química , Oligodesoxirribonucleótidos/metabolismo , Oligorribonucleótidos/metabolismo , Secuencia de Bases , Camptotecina/farmacología , ADN de Cadena Simple/química , Humanos , Oligodesoxirribonucleótidos/químicaRESUMEN
Several groups have demonstrated that G-rich oligonucleotides forming G-quartet structures display activity as potential drugs, such as potent HIV inhibitors. The delivery of G-quartet oligonucleotides to their intracellular targets is a key obstacle to overcome for their clinical success. Here we have developed a novel system to deliver G-rich oligonucleotides into the cell nucleus, e.g., the site of HIV integration. On the basis of the property of potassium-induced formation of G-quartet structure, we explored the difference of K(+) concentrations inside (140 mM) and outside (4 mM) cells to induce the G-rich oligonucleotides to form different structures inside and outside cells. The key steps of this delivery system include the following: (i) First, the G-quartet structure is denatured to form a lipid-DNA complex, so that the molecules can be well delivered into cells. (ii) Then the delivered molecules are induced to form G-quartet structures by potassium inside cells since the G-quartet structure is the primary requirement for inhibition of HIV-1 HIV integrase (IN) activity. The molecules of a novel G-quartet HIV inhibitor, T40214, with the sequence of (GGGC)(4) were successfully delivered into the nuclei of target cells, which significantly decreased HIV-1 replication and increased the probability to target HIV-1 IN in infected cells.
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Sistemas de Liberación de Medicamentos/métodos , Guanina/metabolismo , Inhibidores de Integrasa VIH/metabolismo , Líquido Intracelular/metabolismo , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/metabolismo , Potasio/química , Línea Celular , ADN/química , ADN/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Guanina/química , Guanina/farmacología , Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Cinética , Liposomas , Sustancias Macromoleculares , Desnaturalización de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/farmacología , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Integración Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Stat3 is an Src homology (SH)2-containing protein constitutively activated in a wide variety of human cancers following its recruitment to YXXQ-containing motifs, which results in resistance to apoptosis. Despite resolution of the crystal structure of Stat3 homodimer bound to DNA, the structural basis for the unique specificity of Stat3 SH2 for YXXQ-containing phosphopeptides remains unresolved. We tested three models of this interaction based on computational analysis of available structures and sequence alignments, two of which assumed an extended peptide configuration and one in which the peptide had a beta-turn. By using peptide immunoblot affinity assays and mirror resonance affinity analysis, we demonstrated that only phosphotyrosine (Tyr(P)) peptides containing +3 Gln (not Leu, Met, Glu, or Arg) bound to wild type Stat3. Examination of a series of wild type and mutant Stat3 proteins demonstrated loss of binding to pYXXQ-containing peptides only in Stat3 mutated at Lys-591 or Arg-609, whose side chains interact with the Tyr(P) residue, and Stat3 mutated at Glu-638, whose amide hydrogen bonds with oxygen within the +3 Gln side chain when the peptide ligand assumes a beta-turn. These findings support a model for Stat3 SH2 interactions that could form the basis for anticancer drugs that specifically target Stat3.