RESUMO
Seven new peptaibols named tolypocladamides A-G have been isolated from an extract of the fungus Tolypocladium inflatum, which inhibits the interaction between Raf and oncogenic Ras in a cell-based high-throughput screening assay. Each peptaibol contains 11 amino acid residues, an octanoyl or decanoyl fatty acid chain at the N-terminus, and a leucinol moiety at the C-terminus. The peptaibol sequences were elucidated on the basis of 2D NMR and mass spectral fragmentation analyses. Amino acid configurations were determined by advanced Marfey's analyses. Tolypocladamides A-G caused significant inhibition of Ras/Raf interactions with IC50 values ranging from 0.5 to 5.0 µM in a nanobioluminescence resonance energy transfer (NanoBRET) assay; however, no interactions were observed in a surface plasmon resonance assay for binding of the compounds to wild type or G12D mutant Ras constructs or to the Ras binding domain of Raf. NCI 60 cell line testing was also conducted, and little panel selectivity was observed.
Assuntos
Antineoplásicos , Hypocreales , Aminoácidos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Hypocreales/química , Peptaibols/farmacologiaRESUMO
RAF kinases are essential effectors of RAS, but how RAS binding initiates the conformational changes needed for autoinhibited RAF monomers to form active dimers has remained unclear. Here, we present cryo-electron microscopy structures of full-length BRAF complexes derived from mammalian cells: autoinhibited, monomeric BRAF:14-3-32:MEK and BRAF:14-3-32 complexes, and an inhibitor-bound, dimeric BRAF2:14-3-32 complex, at 3.7, 4.1, and 3.9 Å resolution, respectively. In both autoinhibited, monomeric structures, the RAS binding domain (RBD) of BRAF is resolved, revealing that the RBD forms an extensive contact interface with the 14-3-3 protomer bound to the BRAF C-terminal site and that key basic residues required for RBD-RAS binding are exposed. Moreover, through structure-guided mutational studies, our findings indicate that RAS-RAF binding is a dynamic process and that RBD residues at the center of the RBD:14-3-3 interface have a dual function, first contributing to RAF autoinhibition and then to the full spectrum of RAS-RBD interactions.
Assuntos
Microscopia Crioeletrônica/métodos , Mutação , Neoplasias/patologia , Multimerização Proteica , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Conformação Proteica , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/químicaRESUMO
Activating mutations in RAS are found in approximately 30% of human cancers, resulting in the delivery of a persistent signal to critical downstream effectors that drive tumorigenesis. RAS-driven malignancies respond poorly to conventional cancer treatments and inhibitors that target RAS directly are limited; therefore, the identification of new strategies and/or drugs to disrupt RAS signaling in tumor cells remains a pressing therapeutic need. Taking advantage of the live-cell bioluminescence resonance energy transfer (BRET) methodology, we describe the development of a NanoBRET screening platform to identify compounds that modulate binding between activated KRAS and the CRAF kinase, an essential effector of RAS that initiates ERK cascade signaling. Using this strategy, libraries containing synthetic compounds, targeted inhibitors, purified natural products, and natural product extracts were evaluated. These efforts resulted in the identification of compounds that inhibit RAS/RAF binding and in turn suppress RAS-driven ERK activation, but also compounds that have the deleterious effect of enhancing the interaction to upregulate pathway signaling. Among the inhibitor hits identified, the majority were compounds derived from natural products, including ones reported to alter KRAS nanoclustering (ophiobolin A), to impact RAF function (HSP90 inhibitors and ROS inducers) as well as some with unknown targets and activities. These findings demonstrate the potential for this screening platform in natural product drug discovery and in the development of new therapeutic agents to target dysregulated RAS signaling in human disease states such as cancer.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Fibroblastos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Proteínas ras/agonistas , Proteínas ras/antagonistas & inibidores , Animais , Fibroblastos/metabolismo , Humanos , Ligantes , Nanotecnologia/métodos , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas ras/metabolismoRESUMO
Chronic lower back pain is one of the most common medical conditions leading to a significant decrease in quality of life. This study retrospectively analyzed whether the AxioBionics Wearable Therapy Pain Management (WTPM) System, a customized and wearable electrical stimulation device, alleviated chronic lower back pain, and improved muscular function. This study assessed self-reported pain levels using the visual analog scale before and during the use of the AxioBionics WTPM System when performing normal activities such as sitting, standing, and walking (n = 69). Results showed that both at-rest and activity-related pain were significantly reduced during treatment with the AxioBionics WTPM System (% reduction in pain: 64% and 60%, respectively; P < .05). Thus, this study suggests that the AxioBionics WTPM System is efficacious in treating chronic lower back pain even when other therapies have failed to sufficiently decrease reported pain levels.
RESUMO
AIMS: Hydrogen sulfide (H2S) protects against ischemic and inflammatory injury following myocardial ischemia via induction of microRNA (miR)-21. We sought to determine whether H2S attenuates ischemic heart failure with reduced ejection fraction (HFrEF) and interrogate the role of cofilin-2, a target of miR-21, in this protective process. METHODS AND RESULTS: Adult male mice underwent myocardial infarction (MI) by coronary artery ligation after baseline echocardiography. Following MI, mice were treated with Na2S (100 µg/kg/day; intraperitoneal [IP]) or saline up to 28 days. End-diastolic pressure, measured by Millar catheter, was significantly increased (P < .05 vs sham) at 3 days post-MI in the saline group, which was attenuated with Na2S. Left ventricular (LV) fractional shortening decreased significantly at 28 days post-MI in the saline group but was preserved with Na2S and LV infarct scar size was smaller in Na2S group as compared to control. Apoptotic signaling, measured by Bcl-2/Bax ratio, was significantly increased in the saline group but was mitigated with Na2S. Survival rate was 2-fold higher in Na2S group compared to saline control (P < .05). Proteomic analysis and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (TOF)/TOF tandem mass spectrometry identified significant changes in proapoptotic cofilin-2 expression, a specific target of miR-21, between saline- and sodium sulfide -treated mice at 28 days post-MI. Western blot analysis confirmed a significant increase in cofilin-2 after MI, which was suppressed with Na2S treatment. Chronic Na2S treatment also attenuated inflammasome formation and activation leading to reduction of maladaptive signaling. CONCLUSION: Na2S treatment after MI preserves LV function and improves survival through attenuation of inflammasome-mediated adverse remodeling. We propose H2S donors as promising therapeutic tools for ischemic HFrEF.
Assuntos
Cofilina 2/metabolismo , Insuficiência Cardíaca/prevenção & controle , Sulfeto de Hidrogênio/farmacologia , Infarto do Miocárdio/tratamento farmacológico , Miocárdio/metabolismo , Sulfetos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Inflamassomos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
Two new cyclic depsipeptides named swinhopeptolides A (1) and B (2) have been isolated from the marine sponge Theonella swinhoei cf. verrucosa, collected from Papua New Guinea. They each contain 11 diverse amino acid residues and 13-carbon polyketide moieties attached at the N-terminus. Compounds 1 and 2 each exist as two conformers in DMSO-d6 due to cis/trans isomerism of the proline residue, and their structures were successfully assigned by extensive NMR analyses complemented by chemical degradation and derivatization studies. Swinhopeptolide B (2) contains a previously undescribed 2,6,8-trimethyldeca-(2E,4E,6E)-trienoic acid moiety N-linked to a terminal serine residue. Swinhopeptolides A (1) and B (2) showed significant inhibition of the Ras/Raf signaling pathway with IC50 values of 5.8 and 8.5 µM, respectively.
Assuntos
Depsipeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Theonella/química , Proteínas ras/antagonistas & inibidores , Aminoácidos/química , Animais , Depsipeptídeos/química , Depsipeptídeos/isolamento & purificação , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Papua Nova Guiné , Poríferos/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/metabolismoRESUMO
BACKGROUND: Multi-drug resistance (MDR) develops because cancer cells evade toxicity of several structurally unrelated drugs. Besides other mechanisms, MDR is linked to the overexpression of ATP Binding Cassette (ABC), transporters, among which ABCB1 is the best characterized one. Since overactivation of PI3K/Akt/mTOR plays a pivotal role in the growth of human cancers, we hypothesized whether dual PI3K and mTOR inhibitor, BEZ235 (BEZ, dactolisib) reverses resistance to doxorubicin (DOX). METHODS: Ovarian (A2780) and pancreatic (MiaPaca2) cancer cells were used to generate DOX-resistant clones by overexpressing ABCB1 or stepwise treatment of DOX. Intracellular accumulation of DOX was measured by flow cytometry after treatment with BEZ. RESULTS: BEZ treatment caused an increase in intracellular levels of DOX which was almost identical to the naïve parental cell lines. BEZ was found to be a weak substrate for ABCB1 as demonstrated by minimal increase in ATPase activity. BEZ treatment caused a dose-dependent decrease in cell viability in combination with DOX, which was associated with an increase in cleaved PARP expression in the drug resistant clones. CONCLUSIONS: These results suggest that BEZ is a non-substrate inhibitor of ABCB1 and is able to effectively re-sensitize cells overexpressing ABCB1 to the effects of DOX. GENERAL SIGNIFICANCE: Dual PI3 Kinase/mTOR inhibitor, BEZ, has the potential to reverse MDR in cancer patients.
Assuntos
Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Serina-Treonina Quinases TOR/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Linhagem Celular Tumoral , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Quinolinas/farmacologiaRESUMO
AIMS: Deregulation of mTOR (mammalian target of rapamycin) signalling occurs in diabetes, which exacerbates injury following myocardial infarction (MI). We therefore investigated the infarct-limiting effect of chronic treatment with rapamycin (RAPA, mTOR inhibitor) in diabetic mice following myocardial ischaemia/reperfusion (I/R) injury and delineated the potential protective mechanism. METHODS AND RESULTS: Adult male diabetic (db/db) or wild-type (WT) (C57) mice were treated with RAPA (0.25 mg/kg/day, intraperitoneal) or vehicle (5% DMSO) for 28 days. The hearts from treated mice were subjected to global I/R in Langendorff mode. Cardiomyocytes, isolated from treated mice, were subjected to simulated ischaemia/reoxygenation (SI/RO) to assess necrosis and apoptosis. Myocardial infarct size was increased in diabetic heart following I/R as compared to WT. Likewise, enhanced necrosis and apoptosis were observed in isolated cardiomyocytes of diabetic mice following SI/RO. Treatment with RAPA reduced infarct size as well as cardiomyocyte necrosis and apoptosis of diabetes and WT mice. RAPA increased STAT3 phosphorylation and miRNA-17/20a expression in diabetic hearts. In addition, RAPA restored AKT phosphorylation (target of mTORC2) but suppressed S6 phosphorylation (target of mTORC1) following I/R injury. RAPA-induced cardioprotection against I/R injury as well as the induction of miR-17/20a and AKT phosphorylation were abolished in cardiac-specific STAT3-deficient diabetic mice, without alteration of S6 phosphorylation. The infarct-limiting effect of RAPA was obliterated in cardiac-specific miRNA-17-92-deficient diabetic mice. The post-I/R restoration of phosphorylation of STAT3 and AKT with RAPA were also abolished in miRNA-17-92-deficient diabetic mice. Additionally, RAPA suppressed the pro-apoptotic prolyl hydroxylase (Egln3/PHD3), a target of miRNA-17/20a in diabetic hearts, which was abrogated in miRNA-17-92-deficient diabetic mice. CONCLUSION: Induction of STAT3-miRNA-17-92 signalling axis plays a critical role in attenuating MI in RAPA-treated diabetic mice. Our study indicates that chronic treatment with RAPA might be a promising pharmacological intervention for attenuating MI and improving prognosis in diabetic patients.
Assuntos
Diabetes Mellitus/tratamento farmacológico , MicroRNAs/metabolismo , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Modelos Animais de Doenças , Preparação de Coração Isolado , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Necrose , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
The Ras GTPases are frequently mutated in human cancer, and, although the Raf kinases are essential effectors of Ras signaling, the tumorigenic properties of specific Ras-Raf complexes are not well characterized. Here, we examine the ability of individual Ras and Raf proteins to interact in live cells using bioluminescence resonance energy transfer (BRET) technology. We find that C-Raf binds all mutant Ras proteins with high affinity, whereas B-Raf exhibits a striking preference for mutant K-Ras. This selectivity is mediated by the acidic, N-terminal segment of B-Raf and requires the K-Ras polybasic region for high-affinity binding. In addition, we find that C-Raf is critical for mutant H-Ras-driven signaling and that events stabilizing B-Raf/C-Raf dimerization, such as Raf inhibitor treatment or certain B-Raf mutations, can allow mutant H-Ras to engage B-Raf with increased affinity to promote tumorigenesis, thus revealing a previously unappreciated role for C-Raf in potentiating B-Raf function.
Assuntos
Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Neoplasias/enzimologia , Quinases raf/metabolismo , Proteínas ras/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Mutação , Células NIH 3T3 , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/genética , Esferoides Celulares , Quinases raf/genética , Proteínas ras/genéticaRESUMO
The Raf protein kinases are key intermediates in cellular signal transduction, functioning as direct effectors of the Ras GTPases and as the initiating kinases in the ERK cascade. In human cancer, Raf activity is frequently dysregulated due to mutations in the Raf family member B-Raf or to alterations in upstream Raf regulators, including Ras and receptor tyrosine kinases. First-generation Raf inhibitors, such as vemurafenib and dabrafenib, have yielded dramatic responses in malignant melanomas containing B-Raf mutations; however, their overall usefulness has been limited by both intrinsic and acquired drug resistance. In particular, issues related to the dimerisation of the Raf kinases can impact the efficacy of these compounds and are a primary cause of drug resistance. Here, we will review the importance of Raf dimerisation in cell signalling as well as its effects on Raf inhibitor therapy, and we will present the new strategies that are being pursued to overcome the 'Raf Dimer Dilemma'.
Assuntos
Neoplasias/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Quinases raf/química , Resistencia a Medicamentos Antineoplásicos , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Modelos Moleculares , Terapia de Alvo Molecular , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Oximas/farmacologia , Oximas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Multimerização Proteica , Transdução de Sinais , Vemurafenib/farmacologia , Vemurafenib/uso terapêutico , Quinases raf/antagonistas & inibidores , Quinases raf/genéticaRESUMO
BACKGROUND AND PURPOSE: Enhanced mammalian target of rapamycin (mTOR) signalling contributes to the pathogenesis of diabetes and plays a critical role in myocardial ischaemia/reperfusion (I/R) injury. Rapatar is a novel nanoformulated micellar of rapamycin, a putative inhibitor of mTOR that has been rationally designed to increase water solubility of rapamycin to facilitate p.o. administration and enhance bioavailability. We examined the effect of Rapatar on the metabolic status and protection against myocardial I/R injury in type 2 diabetic mice. EXPERIMENTAL APPROACH: Adult male db/db mice were treated daily for 10 weeks with Rapatar (0.75 mg·kg-1 ·day-1 , p.o.) or vehicle. Isolated hearts were connected to a Langendorff perfusion system and subjected to global ischaemia (30 min) and reperfusion (1 h). KEY RESULTS: Rapatar reduced fasting plasma glucose and triglyceride levels, prevented the gain in body weight and also improved glucose tolerance and insulin sensitivity in db/db mice compared with control. Cardiac function was improved following Rapatar treatment in db/db mice. Myocardial infarct size was reduced in Rapatar-treated mice with improved post-ischaemic rate-force product. Western blot analyses demonstrated a significant inhibition of phosphorylation of ribosomal protein S6 (downstream target of mTORC1), but not Akt (Ser473 , target of mTORC2) following chronic treatment with Rapatar. Rapatar also induced phosphorylation of AMPK, STAT3, ERK1/2 and glycogen synthase kinase 3ß, without interfering with phosphorylation of p38. CONCLUSION AND IMPLICATIONS: Our studies indicate that chronic treatment with Rapatar improves metabolic status and cardiac function with a reduction of infarct size following myocardial I/R injury in diabetic mice.
Assuntos
Diabetes Mellitus Experimental/prevenção & controle , Cardiomiopatias Diabéticas/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Nanoestruturas/química , Substâncias Protetoras/uso terapêutico , Sirolimo/uso terapêutico , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Cardiomiopatias Diabéticas/tratamento farmacológico , Masculino , Camundongos , Camundongos Obesos , Micelas , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Substâncias Protetoras/química , Sirolimo/químicaRESUMO
The chemotherapeutic use of doxorubicin (Dox) is hindered due to the development of irreversible cardiotoxicity. Specifically, childhood cancer survivors are at greater risk of Dox-induced cardiovascular complications. Because of the potent cardioprotective effect of phosphodiesterase 5 (PDE5) inhibitors, we examined the effect of long-acting PDE5 inhibitor tadalafil (Tada) against Dox cardiotoxicity in juvenile mice. C57BL/6J mice (6 weeks old) were treated with Dox (20 mg/kg, i.v.) and (or) Tada (10 mg/kg daily for 14 days, p.o.). Cardiac function was assessed by echocardiography following 5 and 10 weeks after Dox treatment. The expression of cardiac proteins was examined by Western blot analysis. Dox treatment caused diastolic dysfunction in juvenile mice indicated by increasing the E/E' (early diastolic myocardial velocity to early tissue Doppler velocity) ratio as compared with control at both 5 and 10 weeks after Dox treatment. Co-treatment of Tada and Dox preserved left ventricular diastolic function with reduction of E/E'. Dox treatment decreased the expression of SERCA2 and desmin in the left ventricle; however, only desmin loss was prevented with Tada. Also, Dox treatment increased the expression of myosin heavy chain (MHCß), which was reduced by Tada. We propose that Tada could be a promising new therapy for improving cardiac function in survivors of childhood cancer.
Assuntos
Cardiotônicos/farmacologia , Proteínas do Citoesqueleto/metabolismo , Doxorrubicina , Miocárdio/enzimologia , Inibidores da Fosfodiesterase 5/farmacologia , Tadalafila/farmacologia , Disfunção Ventricular Esquerda/prevenção & controle , Função Ventricular Esquerda/efeitos dos fármacos , Fatores Etários , Animais , Western Blotting , Cardiotoxicidade , Desmina/metabolismo , Modelos Animais de Doenças , Ecocardiografia Doppler , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Cadeias Pesadas de Miosina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Disfunção Ventricular Esquerda/induzido quimicamente , Disfunção Ventricular Esquerda/enzimologia , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
Ras pathway signaling plays a critical role in cell growth control and is often upregulated in human cancer. The Raf kinases selectively interact with GTP-bound Ras and are important effectors of Ras signaling, functioning as the initiating kinases in the ERK cascade. Here, we identify a route for the phospho-inhibition of Ras/Raf/MEK/ERK pathway signaling that is mediated by the stress-activated JNK cascade. We find that key Ras pathway components, the RasGEF Sos1 and the Rafs, are phosphorylated on multiple S/TP sites in response to JNK activation and that the hyperphosphorylation of these sites renders the Rafs and Sos1 unresponsive to upstream signals. This phospho-regulatory circuit is engaged by cancer therapeutics, such as rigosertib and paclitaxel/Taxol, that activate JNK through mitotic and oxidative stress as well as by physiological regulators of the JNK cascade and may function as a signaling checkpoint to suppress the Ras pathway during conditions of cellular stress.
Assuntos
Glicina/análogos & derivados , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Paclitaxel , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Sulfonas , Ativação Enzimática/efeitos dos fármacos , Glicina/farmacocinética , Glicina/farmacologia , Células HeLa , Humanos , Estresse Oxidativo , Paclitaxel/farmacocinética , Paclitaxel/farmacologia , Fosforilação , Sulfonas/farmacocinética , Sulfonas/farmacologia , Proteínas ras/metabolismoRESUMO
We previously reported that Sildenafil enhances apoptosis and antitumor efficacy of doxorubicin (DOX) while attenuating its cardiotoxic effect in prostate cancer. In the present study, we investigated the mechanism by which sildenafil sensitizes DOX in killing of prostate cancer (PCa) cells, DU145. The death receptor Fas (APO-1 or CD95) induces apoptosis in many carcinoma cells, which is negatively regulated by anti-apoptotic molecules such as FLIP (Fas-associated death domain (FADD) interleukin-1-converting enzyme (FLICE)-like inhibitory protein). Co-treatment of PCa cells with sildenafil and DOX for 48 hours showed reduced expression of both long and short forms of FLIP (FLIP-L and -S) as compared to individual drug treatment. Over-expression of FLIP-s with an adenoviral vector attentuated the enhanced cell-killing effect of DOX and sildenafil. Colony formation assays also confirmed that FLIP-S over-expression inhibited the DOX and sildenafil-induced synergistic killing effect as compared to the cells infected with an empty vector. Moreover, siRNA knock-down of CD95 abolished the effect of sildenafil in enhancing DOX lethality in cells, but had no effect on cell killing after treatment with a single agent. Sildenafil co-treatment with DOX inhibited DOX-induced NF-κB activity by reducing phosphorylation of IκB and nuclear translocation of the p65 subunit, in addition to down regulation of FAP-1 (Fas associated phosphatase-1, a known inhibitor of CD95-mediated apoptosis) expression. This data provides evidence that the CD95 is a key regulator of sildenafil and DOX mediated enhanced cell death in prostate cancer.
Assuntos
Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias da Próstata/patologia , Citrato de Sildenafila/farmacologia , Agentes Urológicos/farmacologia , Receptor fas/metabolismo , Antibióticos Antineoplásicos/farmacologia , Apoptose , Western Blotting , Proliferação de Células , Sinergismo Farmacológico , Imunofluorescência , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Pancreatic cancer has the lowest 5-year survival rate of all major cancers despite decades of effort to design and implement novel, more effective treatment options. In this study, we tested whether the dual phosphoinositide 3-kinase/mechanistic target of rapamycin inhibitor BEZ235 (BEZ) potentiates the antitumor effects of doxorubicin (DOX) against pancreatic cancer. Cotreatment of BEZ235 with DOX resulted in dose-dependent inhibition of the phosphoinositide 3-kinase/mechanistic target of rapamycin survival pathway, which corresponded with an increase in poly ADP ribose polymerase cleavage. Moreover, BEZ cotreatment significantly improved the effects of DOX toward both cell viability and cell death in part through reduced Bcl-2 expression and increased expression of the shorter, more cytotoxic forms of BIM. BEZ also facilitated intracellular accumulation of DOX, which led to enhanced DNA damage and reactive oxygen species generation. Furthermore, BEZ in combination with gemcitabine reduced MiaPaca2 cell proliferation but failed to increase reactive oxygen species generation or BIM expression, resulting in reduced necrosis and apoptosis. Treatment with BEZ and DOX in mice bearing tumor xenographs significantly repressed tumor growth as compared with BEZ, DOX, or gemcitabine. Additionally, in contrast to the enhanced expression seen in MiaPaca2 cells, BEZ and DOX cotreatment reduced BIM expression in H9C2 cardiomyocytes. Also, the Bcl-2/Bax ratio was increased, which was associated with a reduction in cell death. In vivo echocardiography showed decreased cardiac function with DOX treatment, which was not improved by combination treatment with BEZ. Thus, we propose that combining BEZ with DOX would be a better option for patients than current standard of care by providing a more effective tumor response without the associated increase in toxicity.
Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Doxorrubicina/farmacologia , Imidazóis/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Antibióticos Antineoplásicos/farmacologia , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Cardiotoxicidade , Sobrevivência Celular , Doxorrubicina/efeitos adversos , Sinergismo Farmacológico , Feminino , Células HCT116 , Humanos , Imidazóis/uso terapêutico , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Miócitos Cardíacos/efeitos dos fármacos , Neoplasias Pancreáticas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Quinolinas/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Diabetic patients suffer augmented severity of myocardial infarction. Excessive activation of the mammalian target of rapamycin (mTOR) and decreased activation of STAT3 are implicated in diabetic complications. Considering the potent cardioprotective effect of mTOR inhibitor, rapamycin, we hypothesized that reperfusion therapy with rapamycin would reduce infarct size in the diabetic hearts through STAT3 signaling. Hearts from adult male db/db or wild type (WT) C57 mice were isolated and subjected to 30 min of normothermic global ischemia and 60 min of reperfusion in Langendorff mode. Rapamycin (100 nM) was infused at the onset of reperfusion. Myocardial infarct size (IS) was significantly reduced in rapamycin-treated mice (13.3 ± 2.4 %) compared to DMSO vehicle control (35.9 ± 0.9 %) or WT mice (27.7 ± 1.1 %). Rapamycin treatment restored phosphorylation of STAT3 and enhanced AKT phosphorylation (target of mTORC2), but significantly reduced ribosomal protein S6 phosphorylation (target of mTORC1) in the diabetic heart. To determine the cause and effect relationship of STAT3 in cardioprotection, inducible cardiac-specific STAT3-deficient (MCM TG:STAT3(flox/flox)) and WT mice (MCM TG:STAT3(flox/flox)) were made diabetic by feeding high fat diet (HFD). Rapamycin given at reperfusion reduced IS in WT mice but not in STAT3-deficient mice following I/R. Moreover, cardiomyocytes isolated from HFD-fed WT mice showed resistance against necrosis (trypan blue staining) and apoptosis (TUNEL assay) when treated with rapamycin during reoxygenation following simulated ischemia. Such protection was absent in cardiomyocytes from HFD-fed STAT3-deficient mice. STAT3 signaling plays critical role in reducing IS and attenuates cardiomyocyte death following reperfusion therapy with rapamycin in diabetic heart.
Assuntos
Cardiomiopatias Diabéticas/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Fator de Transcrição STAT3/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Western Blotting , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatias Diabéticas/patologia , Modelos Animais de Doenças , Imunossupressores/farmacologia , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Traumatismo por Reperfusão Miocárdica/etiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sirolimo/farmacologiaRESUMO
The phosphodiesterase 5 (PDE5) inhibitors, including sildenafil (Viagra™), vardenafil (Levitra™), and tadalafil (Cialis™) have been developed for treatment of erectile dysfunction. Moreover, sildenafil and tadalafil are used for the management of pulmonary arterial hypertension in patients. Since our first report showing the cardioprotective effect of sildenafil in 2002, there has been tremendous growth of preclinical and clinical studies on the use of PDE5 inhibitors for cardiovascular diseases and cancer. Numerous animal studies have demonstrated that PDE5 inhibitors have powerful protective effect against myocardial ischemia/reperfusion (I/R) injury, doxorubicin cardiotoxicity, ischemic and diabetic cardiomyopathy, cardiac hypertrophy, Duchenne muscular dystrophy and the improvement of stem cell efficacy for myocardial repair. Mechanistically, PDE5 inhibitors protect the heart against I/R injury through increased expression of nitric oxide synthases, activation of protein kinase G (PKG), PKG-dependent hydrogen sulfide generation, and phosphorylation of glycogen synthase kinase-3ß - a master switch immediately proximal to mitochondrial permeability transition pore and the end effector of cardioprotection. In addition, PDE5 inhibitors enhance the sensitivity of certain types of cancer to standard chemotherapeutic drugs, including doxorubicin. Many clinical trials with PDE5 inhibitors have focused on the potential cardiovascular and anti-cancer benefits. Despite mixed results of these clinical trials, there is a continuing strong interest by basic scientists and clinical investigators in exploring their new clinical uses. It is our hope that future new mechanistic investigations and carefully designed clinical trials would help in reaping additional benefits of PDE5 inhibitors for cardiovascular disease and cancer in patients.
Assuntos
Diabetes Mellitus/tratamento farmacológico , Cardiopatias/tratamento farmacológico , Neoplasias/tratamento farmacológico , Inibidores da Fosfodiesterase 5/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Diabetes Mellitus/enzimologia , Cardiopatias/enzimologia , Humanos , Neoplasias/enzimologia , Inibidores da Fosfodiesterase 5/farmacologiaRESUMO
BACKGROUND: Maintaining physiological levels of hydrogen sulfide during ischemia is necessary to limit injury to the heart. Because of the anti-inflammatory effects of hydrogen sulfide, we proposed that the hydrogen sulfide donor, sodium sulfide (Na2S), would attenuate myocardial injury through upregulation of protective microRNA-21 (miR-21) and suppression of the inflammasome, a macromolecular structure that amplifies inflammation and mediates further injury. METHODS AND RESULTS: Na2S-induced miR-21 expression was measured by quantitative polymerase chain reaction in adult primary rat cardiomyocytes and in the mouse heart. We measured inflammasome formation and activity in cardiomyocytes challenged with lipopolysaccharide and ATP or simulated ischemia/reoxygenation and in the heart after regional myocardial ischemia/reperfusion, in the presence or absence of Na2S. To assess the direct anti-inflammatory effects of hydrogen sulfide in vivo, we used a peritonitis model by way of intraperitoneal injection of zymosan A. Na2S attenuated inflammasome formation and activity, measured by counting cytoplasmic aggregates of the scaffold protein apoptosis speck-like protein containing a caspase-recruitment domain (-57%) and caspase-1 activity (-50%) in isolated cardiomyocytes and in the mouse heart (all P<0.05). Na2S also inhibited apoptosis (-38%) and necrosis (-43%) in cardiomyocytes in vitro and reduced myocardial infarct size (-63%) after ischemia/reperfusion injury in vivo (all P<0.05). These protective effects were absent in cells treated with the miR-21 eraser, antagomiR-21, and in miR-21 knockout mice. Na2S also limited the severity of inflammasome-dependent inflammation in the model of peritonitis (P<0.05) in wild-type but not in miR-21 knockout mice. CONCLUSIONS: Na2S induces cardioprotective effects through miR-21-dependent attenuation of ischemic and inflammatory injury in cardiomyocytes.
Assuntos
Sulfeto de Hidrogênio/metabolismo , MicroRNAs/genética , Isquemia Miocárdica/genética , Animais , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Isquemia Miocárdica/imunologia , Isquemia Miocárdica/metabolismo , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/metabolismo , Sulfetos/metabolismo , Regulação para CimaRESUMO
Elevated mammalian target of rapamycin (mTOR) signaling contributes to the pathogenesis of diabetes, with increased morbidity and mortality, mainly because of cardiovascular complications. Because mTOR inhibition with rapamycin protects against ischemia/reperfusion injury, we hypothesized that rapamycin would prevent cardiac dysfunction associated with type 2 diabetes (T2D). We also investigated the possible mechanisms and novel protein targets involved in rapamycin-induced preservation of cardiac function in T2D mice. Adult male leptin receptor null, homozygous db/db, or wild type mice were treated daily for 28 days with vehicle (5% DMSO) or rapamycin (0.25 mg/kg, intraperitoneally). Cardiac function was monitored by echocardiography, and protein targets were identified by proteomics analysis. Rapamycin treatment significantly reduced body weight, heart weight, plasma glucose, triglyceride, and insulin levels in db/db mice. Fractional shortening was improved by rapamycin treatment in db/db mice. Oxidative stress as measured by glutathione levels and lipid peroxidation was significantly reduced in rapamycin-treated db/db hearts. Rapamycin blocked the enhanced phosphorylation of mTOR and S6, but not AKT in db/db hearts. Proteomic (by two-dimensional gel and mass spectrometry) and Western blot analyses identified significant changes in several cytoskeletal/contractile proteins (myosin light chain MLY2, myosin heavy chain 6, myosin-binding protein C), glucose metabolism proteins (pyruvate dehydrogenase E1, PYGB, Pgm2), and antioxidant proteins (peroxiredoxin 5, ferritin heavy chain 1) following rapamycin treatment in db/db heart. These results show that chronic rapamycin treatment prevents cardiac dysfunction in T2D mice, possibly through attenuation of oxidative stress and alteration of antioxidants and contractile as well as glucose metabolic protein expression.
Assuntos
Antibacterianos/farmacologia , Proteínas Contráteis/biossíntese , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Sirolimo/farmacocinética , Serina-Treonina Quinases TOR/metabolismo , Animais , Proteínas Contráteis/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Glucose/genética , Glucose/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Miocárdio/patologia , Proteômica , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genéticaRESUMO
The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with clinically relevant chemotherapies to kill gastrointestinal/genitourinary cancer cells. In bladder cancer cells, regardless of H-RAS mutational status, at clinically achievable doses, PDE5 inhibitors interacted in a greater than additive fashion with doxorubicin/mitomycin C/gemcitabine/cisplatin/paclitaxel to cause cell death. In pancreatic tumor cells expressing mutant active K-RAS, PDE5 inhibitors interacted in a greater than additive fashion with doxorubicin/gemcitabine/paclitaxel to cause cell death. The most potent PDE5 inhibitor was sildenafil. Knock down of PDE5 expression recapitulated the combination effects of PDE5 inhibitor drugs with chemotherapy drugs. Expression of cellular FLICE-like inhibitory protein-short did not significantly inhibit chemotherapy lethality but did significantly reduce enhanced killing in combination with sildenafil. Overexpression of B-cell lymphoma-extra large suppressed individual and combination drug toxicities. Knock down of CD95 or Fas-associated death domain protein suppressed drug combination toxicity. Combination toxicity was also abolished by necrostatin or receptor interacting protein 1 knock down. Treatment with PDE5 inhibitors and chemotherapy drugs promoted autophagy, which was maximal at â¼24 hour posttreatment, and 3-methyl adenine or knock down of Beclin1 suppressed drug combination lethality by â¼50%. PDE5 inhibitors enhanced and prolonged the induction of DNA damage as judged by Comet assays and γhistone 2AX (γH2AX) and checkpoint kinase 2 (CHK2) phosphorylation. Knock down of ataxia telangiectasia mutated suppressed γH2AX and CHK2 phosphorylation and enhanced drug combination lethality. Collectively our data demonstrate that the combination of PDE5 inhibitors with standard of care chemotherapy agents for gastrointestinal/genitourinary cancers represents a novel modality.