RESUMO
Targeted protein degradation with proteolysis targeting chimeras (PROTACs) is a powerful therapeutic modality for eliminating disease-causing proteins through targeted ubiquitination and proteasome-mediated degradation. Most PROTACs have exploited substrate receptors of Cullin-RING E3 ubiquitin ligases such as cereblon and VHL. Whether core, shared, and essential components of the Cullin-RING E3 ubiquitin ligase complex can be used for PROTAC applications remains less explored. Here, we discovered a cysteine-reactive covalent recruiter EN884 against the SKP1 adapter protein of the SKP1-CUL1-F-box containing the SCF complex. We further showed that this recruiter can be used in PROTAC applications to degrade neo-substrate proteins such as BRD4 and the androgen receptor in a SKP1- and proteasome-dependent manner. Our studies demonstrate that core and essential adapter proteins within the Cullin-RING E3 ubiquitin ligase complex can be exploited for targeted protein degradation applications and that covalent chemoproteomic strategies can enable recruiter discovery against these targets.
Assuntos
Proteínas Culina , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Culina/metabolismo , Proteólise , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Targeted protein degradation with Proteolysis Targeting Chimeras (PROTACs) is a powerful therapeutic modality for eliminating disease-causing proteins through targeted ubiquitination and proteasome-mediated degradation. Most PROTACs have exploited substrate receptors of Cullin-RING E3 ubiquitin ligases such as cereblon and VHL. Whether core, shared, and essential components of the Cullin-RING E3 ubiquitin ligase complex can be used for PROTAC applications remains less explored. Here, we discovered a cysteine-reactive covalent recruiter EN884 against the SKP1 adapter protein of the SKP1-CUL1-F-box containing SCF complex. We further showed that this recruiter can be used in PROTAC applications to degrade neo-substrate proteins such as BRD4 and the androgen receptor in a SKP1- and proteasome-dependent manner. Our studies demonstrate that core and essential adapter proteins within the Cullin-RING E3 ubiquitin ligase complex can be exploited for targeted protein degradation applications and that covalent chemoproteomic strategies can enable recruiter discovery against these targets.
RESUMO
Crosslinked helix dimers (CHDs) are synthetic tertiary helical structure motifs designed to modulate interactions of proteins with binding partners. Helix dimers serve as mimics of coiled coils, which are known to be implicated in a multitude of protein complexes. Coiled coils are typically stable in long peptides (>21-28 residues), because sufficient intra- and interstrand contacts are not available in short peptides to coax strand assembly. To engineer conformationally stable CHDs in short sequences, we introduced a covalent linkage in place of an interhelical salt bridge and sculpted the helical interface with optimal hydrophobic packing. CHDs have shown efficacy for the disruption of targeted protein-protein interactions in biochemical, cellular, and animal models. This article describes our optimized approach to design and synthesize parallel and antiparallel helical tertiary structure mimics. Synthesis of CHDs involves conjugation of individual peptide segments, purification of the mono-conjugated strand, and alkylation of the two independent strands to yield crosslinked dimers. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Protocol for bis-triazole CHDs Basic Protocol 2: Protocol for dibenzyl ether CHDs.
Assuntos
Peptídeos , Proteínas , Sequência de Aminoácidos , Ligação Proteica , Domínios ProteicosRESUMO
Lung carcinoma is the main reason for cancer-associated deaths in the world. In a previous study, FCH domain only 1 (FCHo1) which is managed by protein kinase B (AKT), was shown to be activated in lung cancer. FCHo1 knockdown has previously been shown to cause cell death in lung cancer. However, the specific roles of FCHo1 in lung carcinoma remain elusive. Herein, we propose that FCHo1's intracellular mechanism targets the G1 to S phase transition, following the M phase. We demonstrated that F-BAR and mu homology domains exist separately in human lung tissues and that one truncated form is not detected in patients with lung cancer. Furthermore, quantitative global proteome analysis of FCHo1 indicated that the inhibition of G1/S phase transition and FCHo1 RNAi led to the death of cells in the G1/S phase. Noninvasive viral aerosol-mediated delivery of FCHo1 shRNA suppressed cancer progression in mice with non-small-cell lung cancer (NSCLC), suggesting that the delivery of FCHo1 shRNA could be a meaningful therapeutic strategy in lung cancer. Additional studies are needed to make clear the detailed mechanism of action of FCHo1.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Carcinoma , Neoplasias Pulmonares , Proteínas de Membrana , Animais , Biomarcadores , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/genética , Camundongos , RNA Interferente Pequeno/genéticaRESUMO
Aberrant Ras signaling is linked to a wide spectrum of hyperproliferative diseases, and components of the signaling pathway, including Ras, have been the subject of intense and ongoing drug discovery efforts. The cellular activity of Ras is modulated by its association with the guanine nucleotide exchange factor Son of sevenless (Sos), and the high-resolution crystal structure of the Ras-Sos complex provides a basis for the rational design of orthosteric Ras ligands. We constructed a synthetic Sos protein mimic that engages the wild-type and oncogenic forms of nucleotide-bound Ras and modulates downstream kinase signaling. The Sos mimic was designed to capture the conformation of the Sos helix-loop-helix motif that makes critical contacts with Ras in its switch region. Chemoproteomic studies illustrate that the proteomimetic engages Ras and other cellular GTPases. The synthetic proteomimetic resists proteolytic degradation and enters cells through macropinocytosis. As such, it is selectively toxic to cancer cells with up-regulated macropinocytosis, including those that feature oncogenic Ras mutations.
Assuntos
Complexos Multiproteicos/ultraestrutura , Conformação Proteica , Proteína Son Of Sevenless de Drosófila/ultraestrutura , Proteínas ras/ultraestrutura , Animais , Biomimética , Cristalografia por Raios X , Descoberta de Drogas , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/ultraestrutura , Células HCT116 , Sequências Hélice-Alça-Hélice/genética , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteoma/genética , Transdução de Sinais/genética , Proteína Son Of Sevenless de Drosófila/química , Proteína Son Of Sevenless de Drosófila/genética , Proteínas ras/química , Proteínas ras/genéticaRESUMO
Protein-protein interactions featuring intricate binding epitopes remain challenging targets for synthetic inhibitors. Interactions of NEMO, a scaffolding protein central to NF-κB signaling, exemplify this challenge. Various regulators are known to interact with different coiled coil regions of NEMO, but the topological complexity of this protein has limited inhibitor design. We undertook a comprehensive effort to block the interaction between vFLIP, a Kaposi's sarcoma herpesviral oncoprotein, and NEMO using small molecule screening and rational design. Our efforts reveal that a tertiary protein structure mimic of NEMO is necessary for potent inhibition. The rationally designed mimic engages vFLIP directly causing complex disruption, protein degradation and suppression of NF-κB signaling in primary effusion lymphoma (PEL). NEMO mimic treatment induces cell death and delays tumor growth in a PEL xenograft model. Our studies with this inhibitor reveal the critical nexus of signaling complex stability in the regulation of NF-κB by a viral oncoprotein.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfoma de Efusão Primária/metabolismo , NF-kappa B/metabolismo , Animais , Linhagem Celular , Dicroísmo Circular , Herpesvirus Humano 8/metabolismo , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Linfoma de Efusão Primária/genética , Masculino , Camundongos , Microscopia Confocal , Modelos Biológicos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Helical secondary and tertiary motifs are commonly observed as binding epitopes in natural and engineered protein scaffolds. While several strategies have been described to constrain α-helices or reproduce their binding attributes in synthetic mimics, general strategies to mimic tertiary helical motifs remain in their infancy. We recently described a synthetic strategy to develop helical dimers ( J. Am. Chem. Soc. 2015, 137, 11618-11621). We found that replacement of an interhelical salt bridge with a covalent bond can stabilize antiparallel motifs in short sequences. Here we show that the approach can be generalized to obtain antiparallel and parallel dimers as well as trimer motifs. Helical stabilization requires judiciously designed cross-linkers as well as optimal interhelical hydrophobic packing. We anticipate that these mimics would afford new classes of modulators of biological function.
Assuntos
Peptídeos/química , Biologia Computacional , Reagentes de Ligações Cruzadas/química , Peptídeos/síntese química , Conformação Proteica em alfa-Hélice , Estrutura Terciária de ProteínaRESUMO
A thiol-thioester exchange system has been used to measure the propensities of diverse ß-amino acid residues to participate in an α-helix-like conformation. These measurements depend on formation of a parallel coiled-coil tertiary structure when two peptide segments become linked by thioester formation. One peptide segment contains a "guest" site that accommodates diverse ß residues and is distal to the coiled-coil interface. We find that helix propensity is influenced by side chain placement within the ß residue [ß3 (side chain adjacent to nitrogen) slightly favored relative to ß2 (side chain adjacent to carbonyl)]. The previously recognized helix stabilization resulting from five-membered ring incorporation is quantified. These results are significant because so few quantitative thermodynamic measurements have been reported for α/ß-peptide folding.
Assuntos
Aminoácidos/química , Peptídeos/química , Sequência de Aminoácidos , Aminoácidos/síntese química , Estrutura Molecular , Peptídeos/síntese química , Conformação Proteica em alfa-Hélice , TermodinâmicaRESUMO
Honokiol (2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol) and magnolol (4-Allyl-2-(5-allyl-2-hydroxy-phenyl)phenol) are the major active polyphenol constituents of Magnolia officinalis (Magnoliaceae) bark, which has been widely used in traditional Chinese medicine (Houpu Tang) for the treatment of various diseases, including anxiety, stress, gastrointestinal disorders, infection, and asthma. The aim of this study was to investigate the direct effects of honokiol and magnolol on hepatic CYP1A and 2C-mediated metabolism in vitro using rat liver microsomes and in vivo using the Sprague-Dawley rat model. Honokiol and magnolol inhibited in vitro CYP1A activity (probe substrate: phenacetin) more potently than CYP2C activity (probe substrate: diclofenac): The mean IC50 values of honokiol for the metabolism of phenacetin and diclofenac were 8.59 µM and 44.7 µM, while those of magnolol were 19.0 µM and 47.3 µM, respectively. Notably, the systemic exposure (AUC and Cmax) of phenacetin, but not of diclofenac, was markedly enhanced by the concurrent administration of intravenous honokiol or magnolol. The differential effects of the two phytochemicals on phenacetin and diclofenac in vivo pharmacokinetics could at least be partly attributed to their lower IC50 values for the inhibition of phenacetin metabolism than for diclofenac metabolism. In addition, the systemic exposure, CL, and Vss of honokiol and magnolol tended to be similar between the rat groups receiving phenacetin and diclofenac. These findings improve our understanding of CYP-mediated drug interactions with M. officinalis and its active constituents.
Assuntos
Compostos de Bifenilo/administração & dosagem , Citocromo P-450 CYP1A1/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Diclofenaco/farmacocinética , Lignanas/administração & dosagem , Fígado/enzimologia , Fenacetina/farmacocinética , Administração Intravenosa , Animais , Compostos de Bifenilo/farmacologia , Cromatografia Líquida de Alta Pressão , Interações Medicamentosas , Regulação da Expressão Gênica/efeitos dos fármacos , Lignanas/farmacologia , Fígado/citologia , Microssomos Hepáticos/enzimologia , Estrutura Molecular , Ratos , Ratos Sprague-DawleyRESUMO
A large amount of fructose intake along with smoking is associated with increased incidence of diseases linked to metabolic syndrome. More research is necessary to understand the complex mechanism that ultimately results in metabolic syndrome and the effect, if any, of high fructose dietary intake and smoking on individual health. In this study, we investigated changes in ER-Golgi network and disturbance to secretion of adipokines induced by cigarette smoking (CS) and excess fructose intake and their contribution to the disruption of metabolic homeostasis. We used high fructose-induced metabolic disorder mice model by feeding them with high fructose diet for 8 weeks. For CS exposure experiment, these mice were exposed to CS for 28 days according to OECD guideline 412. Our results clearly showed that the immune system was suppressed and ER stress was induced in mice with exposure to CS and fed with high fructose. Furthermore, their concentrations of adipokines including leptin and adiponectin were aberrant. Such alteration in secretion of adipokines could cause insulin resistance which may lead to the development of type 2 diabetes.
Assuntos
Adipocinas/imunologia , Adipocinas/metabolismo , Apoptose/efeitos dos fármacos , Fumar Cigarros/efeitos adversos , Resistência à Insulina/imunologia , Doenças Metabólicas/imunologia , Animais , Açúcares da Dieta , Frutose , Masculino , Doenças Metabólicas/induzido quimicamente , Camundongos , Camundongos Endogâmicos C57BL , Poluição por Fumaça de Tabaco/efeitos adversosRESUMO
Background/Aim: Lung cancer shows the highest estimated deaths in both males and females in the Unites States. Importin 7 is overexpressed in lung adenocarcinoma tissues. In this study, we aimed to demonstrate the anticancer effect of importin 7 down-regulation, especially in lung cancer. Materials and Methods: Glycerol propoxylate triacrylate spermine (GPT-SPE) is a biocompatible carrier used for aerosol gene delivery. Repeated aerosol delivery of GPT-SPE/shImportin 7 complexes was performed to 10-week-old male K-ras LA1 mice (a murine lung cancer model) twice a week for 4 weeks (8 times) in a nose-only exposure chamber. Results: Aerosol delivery of GPT-SPE/shImportin 7 inhibits lung cancer in K-ras LA1 mice compared to control and scramble control groups. Moreover, importin 7-down-regulated stable cell-line demonstrates suppression of proliferation through Akt inhibition and apoptosis. Conclusion: Down-regulation of importin 7 significantly suppresses lung cancer in vitro and in vivo.
Assuntos
Adenocarcinoma/metabolismo , Carioferinas/genética , Neoplasias Pulmonares/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Apoptose , Carcinogênese , Linhagem Celular Tumoral , Regulação para Baixo , Técnicas de Silenciamento de Genes , Técnicas de Transferência de Genes , Humanos , Carioferinas/metabolismo , Pulmão/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Interferente Pequeno/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus is elevated in cancer cells. Therefore, proteins of the ER-Golgi intermediate compartment (ERGIC) attract significant attention as targets for cancer treatment. Enhanced cancer cell growth and epithelial-mesenchymal transition by ERGICs correlates with poor-prognosis of lung cancer. This prompted us to assess whether knockdown of ERGIC3 may decrease lung cancer growth. To test the hypothesis, the effects of ERGIC3 short hairpin RNA (shERGIC3) on ER stress-induced cell death and lung tumorigenesis were investigated both in vitro and in vivo. Knockdown of ERGIC3 led to ER stress-induced autophagic cell death and suppression of proliferation in the A549 human lung cancer cell-line. Moreover, non-invasive aerosol-delivery of shERGIC3 using the biocompatible carrier glycerol propoxylate triacrylate and spermine (GPT-SPE) inhibited lung tumorigenesis in the K-rasLA1 murine model of lung cancer. Our data suggest that suppression of ERGIC3 could provide a framework for the development of effective lung cancer therapies.
Assuntos
Adenocarcinoma/metabolismo , Estresse do Retículo Endoplasmático , Neoplasias Pulmonares/metabolismo , Pulmão/metabolismo , Proteínas de Membrana/metabolismo , Células A549 , Adenocarcinoma/patologia , Animais , Autofagia/genética , Carcinogênese , Processos de Crescimento Celular , Estresse do Retículo Endoplasmático/genética , Transição Epitelial-Mesenquimal , Genes ras , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
Inorganic phosphate (Pi) is required by all living organisms for the development of organs such as bone, muscle, brain, and lungs, regulating the expression of several critical genes as well as signal transduction. However, little is known about the effects of prolonged dietary Pi consumption on lung cancer progression. This study investigated the effects of a high-phosphate diet (HPD) in a mouse model of adenocarcinoma. K-rasLA1 mice were fed a normal diet (0.3% Pi) or an HPD (1% Pi) for 1, 2, or 4 months. Mice were then sacrificed and subjected to inductively coupled plasma mass/optical emission spectrometry and laser ablation inductively coupled plasma mass-spectrometry analyses, western blot analysis, histopathological, immunohistochemical, and immunocytochemical analyses to evaluate tumor formation and progression (including cell proliferation, angiogenesis, and apoptosis), changes in ion levels and metabolism, autophagy, epithelial-to-mesenchymal transition, and protein translation in the lungs. An HPD accelerated tumorigenesis, as evidenced by increased adenoma and adenocarcinoma rates as well as tumor size. However, after 4 months of the HPD, cell proliferation was arrested, and marked increases in liver and lung ion levels and in energy production via the tricarboxylic acid cycle in the liver were observed, which were accompanied by increased autophagy and decreased angiogenesis and apoptosis. These results indicate that an HPD initially promotes but later inhibits lung cancer progression because of metabolic adaptation leading to tumor cell quiescence. Moreover, the results suggest that carefully regulated Pi consumption are effective in lung cancer prevention.
Assuntos
Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/etiologia , Fosfatos/efeitos adversos , Fósforo na Dieta/efeitos adversos , Animais , Apoptose , Western Blotting , Proliferação de Células , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Fosfatos/administração & dosagem , Fósforo na Dieta/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Small interfering RNA (siRNA)-mediated gene silencing represents a promising strategy for treating diseases such as cancer; however, specific gene silencing requires an effective delivery system to overcome the instability and low transfection efficiency of siRNAs. To address this issue, a polysorbitol-based transporter (PSOT) was prepared by low molecular weight branched polyethylenimine (bPEI) crosslinked with sorbitol diacrylate (SDA). Osteopontin (OPN) gene, which is highly associated with non-small cell lung cancer (NSCLC) was targeted by siRNA therapy using siRNA targeting OPN (siOPN). Characterization study confirmed that PSOT formed compact complexes with siOPN and protected siOPN against degradation by RNase. PSOT/siOPN complexes demonstrated low cytotoxicity and enhanced transfection efficiency in vitro, suggesting that this carrier may be suitable for gene silencing. In the A549 and H460 lung cancer cell lines, PSOT/siOPN complexes demonstrated significant silencing efficiency at both RNA and protein levels. To study in vivo tumor growth suppression, two lung cancer cell-xenograft mouse models were prepared and PSOT/siOPN complexes were delivered into the mice through intravenous injection. The siOPN-treated groups demonstrated significantly reduced OPN expression at both the RNA and protein levels as well as suppression of tumor volume and weight. Taken together, siOPN delivery using PSOT may present an effective and novel therapeutic system for lung cancer treatment.
Assuntos
Portadores de Fármacos/química , Técnicas de Transferência de Genes , Neoplasias Pulmonares/terapia , Osteopontina/genética , Polietilenoimina/química , RNA Interferente Pequeno/genética , Sorbitol/química , Animais , Western Blotting , Linhagem Celular Tumoral , Expressão Gênica , Inativação Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Interferente Pequeno/administração & dosagem , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this study, we examined the mechanism underlying the effect of Saururus chinensis Baill (saururaceae) on hepatocellular carcinoma HepG2 cells. HepG2 cells and Chang cells were exposed to various concentrations of S. chinensis Baill extract (SC-E) for 24 h. SC-E affected more significantly HepG2 cells than Chang cells in terms of cell viability and ATP production. Therefore, current study examined detailed mechanism how SC-E affected HepG2 cell survival. We found that SC-E (75 and 150 µg/ml) induced apoptosis via oxidative stress. SC-E also caused CCAAT-enhancer-binding protein homologous protein (CHOP) activation by dissociating the binding immunoglobulin protein (BiP) from inositol-requiring 1α (IRE1α) in the endoplasmic reticulum (ER) and induced Bax, cytochrome c release to cytosol, caspase-3 activation, and poly ADP ribose polymerase (PARP) cleavage, resulting in HepG2 cell apoptosis. Furthermore, SC-E caused ER Ca(2+) leakage into the cytosol; ER dilation and mitochondrial membrane damage were observed in transmission electron microscopy (TEM). Taken together, our results demonstrated that SC-E induced cancer cell apoptosis specifically through ER stress.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Extratos Vegetais/farmacologia , Saururaceae/química , Antineoplásicos Fitogênicos/efeitos adversos , Sinalização do Cálcio/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/ultraestrutura , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Metabolismo Energético/efeitos dos fármacos , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/ultraestrutura , Medicina Tradicional do Leste Asiático , Microscopia Eletrônica de Transmissão , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/efeitos adversos , República da CoreiaRESUMO
BACKGROUND/AIM: Lung cancer has the highest mortality rate among cancers and current therapies are not efficient. Therefore, novel therapeutic methods are urgently needed. Here, we examined the effectiveness of simultaneous Akt1 inhibition and Pdcd4 over-expression using a dual expression system in suppressing tumorigenesis in K-ras(LA1) mice (a lung cancer model). MATERIALS AND METHODS: An shRNA targeting Akt1 (shAkt1) and cDNA of programmed cell death protein 4 (Pdcd4) were inserted into a dual expression vector (shAkt1+Pdcd4). A sorbitol diacrylate-polyethylenimine (SDA-PEI) carrier was used because of low toxicity and high transfection efficiency. Aerosolized SDA-PEI/shAkt1+Pdcd4 complex was delivered to the mice twice a week for 4 weeks using a nose-only exposure inhalation chamber. RESULTS: Simultaneous Akt1 inhibition and Pdcd4 over-expression synergistically induced potent antitumor effect. Analysis revealed significant reduction in lung tumor number. CONCLUSION: Dual expression of shAkt1 and Pdcd4 effectively suppresses lung tumorigenesis.
Assuntos
Proteínas Reguladoras de Apoptose/biossíntese , Terapia Genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas de Ligação a RNA/biossíntese , Animais , Proteínas Reguladoras de Apoptose/imunologia , Carcinogênese/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Proteína Oncogênica p21(ras)/genética , Polietilenoimina/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/imunologiaRESUMO
Inorganic phosphate (Pi) plays crucial roles in several biological processes and signaling pathways. Pi uptake is regulated by sodium-dependent phosphate (Na/Pi) transporters (NPTs). Moreover, Pi is used as a food additive in food items such as sausages, crackers, dairy products, and beverages. However, the high serum concentration of phosphate (> 5.5 mg/dL) can cause adverse renal effects, cardiovascular effects including vascular or valvular calcification, and stimulate bone resorption. In addition, Pi can also alter vital cellular signaling, related to cell growth and cap-dependent protein translation. Moreover, intake of dietary Pi, whether high (1.0%) or low (0.1%), affects organs in developing mice, and is related to tumorigenesis in mice. The recommended dietary allowance (RDA) of Pi is the daily dietary intake required to maintain levels above the lower limit of the range of normal serum Pi concentration (2.7 mg/dL) for most individuals (97-98%). Thus, adequate intake of Pi (RDA; 700 mg/day) and maintenance of normal Pi concentration (2.7-4.5 mg/dL) are important for health and prevention of diseases caused by inadequate Pi intake.
Assuntos
Fenômenos Biológicos , Aditivos Alimentares , Fosfatos/efeitos adversos , Fosfatos/toxicidade , Transdução de Sinais , Animais , Fenômenos Biológicos/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Sistema Cardiovascular/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , Camundongos , Proteínas de Transporte de Fosfato/fisiologia , Fosfatos/metabolismo , Fosfatos/fisiologia , Recomendações Nutricionais , Transdução de Sinais/efeitos dos fármacos , Sódio/fisiologiaRESUMO
INTRODUCTION: Lung cancer has the highest mortality rate among all cancers in both men and women. Aerosol delivery is a noninvasive method for gene delivery to the lungs, although efficient and biocompatible vectors need to be developed for lung cancer therapy. AREAS COVERED: This review summarizes recent advances in airway gene delivery for lung cancer treatment in animal models using viral vectors or cationic polymers. Viral vectors including lentiviruses and adenoviruses have been used for airway gene delivery because of their high transfection efficiency. Cationic polymers have also been developed for aerosol gene therapy owing to their biocompatibility and ease of modification. EXPERT OPINION: Efficient delivery and specific promoters are needed for lung cancer therapy. Capsid engineering or PEGylation can lower immunogenicity. Moreover, immunotherapy and oncolytic viruses need to be tested with aerosol delivery for lung cancer therapy. Meanwhile, naturally existing cationic materials may allow the development of novel and biocompatible carriers. In combination with various technologies for aerosol delivery, novel and specific carriers could be developed for lung cancer therapy in the future. Finally, standardized protocols for quantifying and manufacturing viral vectors and cationic polymers need to be developed in order to ensure biosafety.
Assuntos
Terapia Genética/métodos , Vetores Genéticos/genética , Neoplasias Pulmonares/terapia , Aerossóis , Animais , Cátions , Feminino , Técnicas de Transferência de Genes , Humanos , Masculino , Polímeros/química , TransfecçãoRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0077121.].
RESUMO
The sodium-dependent phosphate co-transporter 2b (NPT2b) plays an important role in maintaining phosphate homeostasis. In previous studies, we have shown that high dietary inorganic phosphate (Pi) consumption in mice stimulated lung tumorigenesis and increased NPT2b expression. NPT2b has also been found to be highly expressed in human lung cancer tissues. The association of high expression of NPT2b in the lung with poor prognosis in oncogenic lung diseases prompted us to test whether knockdown of NPT2b may regulate lung cancer growth. To address this issue, aerosols that contained small interfering RNA (siRNA) directed against NPT2b (siNPT2b) were delivered into the lungs of K-ras (LA1) mice, which constitute a murine model reflecting human lung cancer. Our results clearly showed that repeated aerosol delivery of siNPT2b successfully suppressed lung cancer growth and decreased cancer cell proliferation and angiogenesis, while facilitating apoptosis. These results strongly suggest that NPT2b plays a role lung tumorigenesis and represents a novel target for lung cancer therapy.