RESUMO
New approaches that are more efficient and able to specifically reach lung tumors are needed. We developed new hyaluronan-based nanoparticles targeting CD44 receptors of two different sizes and compared their lung cancer cells targeting efficacy in vitro and in vivo. The nanoparticles' cellular uptake was dose-dependent, and specific to hyaluronan receptors, particularly CD44. The binding and internalization differed according to nanoparticle size. In vivo biodistribution studies in two orthotopic lung tumor models showed that intrapulmonary nebulized nanoparticles accumulated in lungs, but not in the tumor nodules. In contrast, despite a significant liver capture, intravenous injection led to a better accumulation of the nanoparticles in the lung tumors compared with the surrounding healthy lung tissues. We demonstrated that the hyaluronan-based nanoparticles size plays significant role in cellular uptake and biodistribution. Small nanoparticles showed active targeting of CD44-overexpressing tumors, suggesting that they could be used as drug-delivery system. FROM THE CLINICAL EDITOR: Combating cancers remains an important goal in clinical medicine. In this study, the authors investigated the ability of two hyaluronan-based nanoparticles targeting CD44 receptors to home in on lung cancer cells in an in-vivo orthotropic model. The preferential uptake of smaller sized nanoparticles via intravenous route has further enhanced the existing knowledge of future drug designs.
Assuntos
Sistemas de Liberação de Medicamentos , Receptores de Hialuronatos/genética , Ácido Hialurônico/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas/administração & dosagem , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Linhagem Celular Tumoral , Portadores de Fármacos , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Neoplasias Pulmonares/patologia , Nanopartículas/química , Tamanho da Partícula , Polissacarídeos/administração & dosagem , Polissacarídeos/química , Distribuição Tecidual/efeitos dos fármacosRESUMO
To select the appropriate patients for treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), it is important to gain a better understanding of the intracellular pathways leading to EGFR-TKI resistance, which is a common problem in patients with lung cancer. We recently reported that mutant KRAS adenocarcinoma is resistant to gefitinib as a result of amphiregulin and insulin-like growth factor-1 receptor overexpression. This resistance leads to inhibition of Ku70 acetylation, thus enhancing the BAX/Ku70 interaction and preventing apoptosis. Here, we determined the intracellular pathways involved in gefitinib resistance in lung cancers and explored the impact of their inhibition. We analyzed the activation of the phosphatidyl inositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) pathway in lung tumors. The activation of AKT was associated with disease progression in tumors with wild-type EGFR from patients treated with gefitinib (phase II clinical trial IFCT0401). The administration of IGF1R-TKI or amphiregulin-directed shRNA decreased AKT signaling and restored gefitinib sensitivity in mutant KRAS cells. The combination of PI3K/AKT inhibition with gefitinib restored apoptosis via Ku70 downregulation and BAX release from Ku70. Deacetylase inhibitors, which decreased the BAX/Ku70 interaction, inhibited AKT signaling and induced gefitinib-dependent apoptosis. The PI3K/AKT pathway is thus a major pathway contributing to gefitinib resistance in lung tumors with KRAS mutation, through the regulation of the BAX/Ku70 interaction. This finding suggests that combined treatments could improve the outcomes for this subset of lung cancer patients, who have a poor prognosis.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/tratamento farmacológico , Mutação/genética , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas/genética , Quinazolinas/farmacologia , Proteínas ras/genética , Acetilação , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma Mucinoso/tratamento farmacológico , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Nucleares/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Gefitinibe , Histona Desacetilase 1/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Autoantígeno Ku , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Metástase Neoplásica , Fosforilação/efeitos dos fármacos , Prognóstico , Estudos Prospectivos , Proteínas Proto-Oncogênicas p21(ras) , Receptor IGF Tipo 1/metabolismo , Células Tumorais Cultivadas , Proteína X Associada a bcl-2/metabolismoRESUMO
Focused ultrasound and microbubble (FUS + MB)-mediated blood-brain barrier (BBB) permeability enhancement can facilitate targeted brain-drug delivery. While controlling the magnitude of BBB permeability enhancement is necessary to limit tissue damage, little work has attempted to decouple these concepts. This work investigated the relationship between BBB permeability enhancement and the relative transcription of inflammatory mediators 4 h following sonication. Three microbubble formulations, Definity, BG8774, and MSB4, were compared, with the dose of each formulation normalized to gas volume. While changes in the transcription of key proinflammatory mediators, such as Il1b, Ccl2, and Tnf, were correlated to the magnitude of BBB permeability enhancement, these correlations were not independent of microbubble formulation; microbubble size distribution may play an important role, as linear regression analyses of BBB permeability magnitude versus differential gene expression for these proinflammatory mediators revealed significantly greater slopes for MSB4, a monodisperse microbubble with mean diameter of 4 µm, compared to Definity or BG8774, both polydisperse microbubbles with mean diameters below 2 µm. Additionally, the function of an acoustic feedback control algorithm, based on the detection threshold of ultraharmonic emissions, was assessed. While this control strategy was effective in limiting both wideband emissions and red blood cell extravasation, microbubble formulation was found to influence the magnitude of BBB leakage and correlations to acoustic emissions. This work demonstrates that while the initial magnitude of FUS + MB-mediated BBB permeability enhancement has a clear influence on the subsequent inflammatory responses, microbubble characteristics influence these relationships and must also be considered.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Microbolhas/uso terapêutico , Terapia por Ultrassom/métodos , Animais , Transporte Biológico , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos , Masculino , Permeabilidade , Ratos , Ratos Sprague-Dawley , Sonicação/métodos , Ondas UltrassônicasRESUMO
Recent advances in the field of monodisperse microbubble synthesis by flow focusing allow for the production of foam-free, highly concentrated and monodisperse lipid-coated microbubble suspensions. It has been found that in vitro, such monodisperse ultrasound contrast agents (UCAs) improve the sensitivity of contrast-enhanced ultrasound imaging. Here, we present the first in vivo study in the left ventricle of rat and pig with this new monodisperse bubble agent. We systematically characterize the acoustic sensitivity and safety of the agent at an imaging frequency of 2.5 MHz as compared with three commercial polydisperse UCAs (SonoVue/Lumason, Definity/Luminity and Optison) and one research-grade polydisperse agent with the same shell composition as the monodisperse bubbles. The monodisperse microbubbles, which had a diameter of 4.2 µm, crossed the pulmonary vasculature, and their echo signal could be measured at least as long as that of the polydisperse UCAs, indicating that microfluidically formed monodisperse microbubbles are stable in vivo. Furthermore, it was found that the sensitivity of the monodisperse agent, expressed as the mean echo power per injected bubble, was at least 10 times higher than that of the polydisperse UCAs. Finally, the safety profile of the monodisperse microbubble suspension was evaluated by injecting 400 and 2000 times the imaging dose, and neither physiologic nor pathologic changes were found, which is a first indication that monodisperse lipid-coated microbubbles formed by flow focusing are safe for in vivo use. The more uniform acoustic response and corresponding increased imaging sensitivity of the monodisperse agent may boost emerging applications of microbubbles and ultrasound such as molecular imaging and therapy.
Assuntos
Meios de Contraste , Microbolhas , Ultrassonografia/métodos , Animais , Ratos , Ratos Sprague-Dawley , SuínosRESUMO
Combinations of therapeutic agents could synergistically enhance the response of lung cancer cells. Co-delivery systems capable of transporting chemotherapeutics with different physicochemical properties and with the simultaneous release of drugs remain elusive. Here, we assess the ability of nanoparticles of 30-nm diameter obtained from the self-assembly of hyaluronan-based copolymer targeting CD44 receptors to encapsulate both gefitinib and vorinostat for effective combinational lung cancer treatment. Drug loading was performed by nanoprecipitation. Drug release experiments showed a slow release of both drugs after 5â¯days. Using two- and three-dimensional lung adenocarcinoma cell cultures, we observed that the nanoparticles were mostly found at the periphery of the CD44-expressing spheroids. These drug-loaded nanoparticles were as cytotoxic as free drugs in the two- and three-dimensional systems and toxicity was due to apoptosis induction. In mouse models, intravenous injection of hyaluronan-based nanoparticles showed a selective delivery to subcutaneous CD44-overexpressing tumors, despite a significant liver capture. In addition, the systemic toxicity of the free drugs was reduced by their co-delivery using the nanoparticles. Finally, intrapulmonary administration of drug-loaded nanoparticles, to avoid a possible hepatic toxicity due to their accumulation in the liver, showed a stronger inhibition of orthotopic lung tumor growth compared to free drugs. In conclusion, hyaluronan-based nanoparticles provide active targeting partially mediated by CD44, less-toxic drug release and improved antitumor efficiency.
Assuntos
Antineoplásicos/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Gefitinibe/administração & dosagem , Ácido Hialurônico/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas/administração & dosagem , Vorinostat/administração & dosagem , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Feminino , Gefitinibe/química , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/química , Neoplasias Pulmonares/metabolismo , Camundongos Nus , Nanopartículas/química , Vorinostat/químicaRESUMO
Development of drug resistance limits the efficacy of targeted therapies. Alternative approaches using different combinations of therapeutic agents to inhibit several pathways could be a more effective strategy for treating cancer. The effects of the approved epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (gefitinib) or a multi-targeted kinase inhibitor (sorafenib) in combination with a histone deacetylase inhibitor (vorinostat) on cell proliferation, cell cycle distribution, apoptosis, and signaling pathway activation in human lung adenocarcinoma and hepatocarcinoma cells with wild-type EGFR and mutant KRAS were investigated. The effects of the synergistic drug combinations were also studied in human lung adenocarcinoma and hepatocarcinoma cells in vivo. The combination of gefitinib and vorinostat synergistically reduced cell growth and strongly induced apoptosis through inhibition of the insulin-like growth factor-1 receptor/protein kinase B (IGF-1R/AKT)-dependent signaling pathway. Moreover, the gefitinib and vorinostat combination strongly inhibited tumor growth in mice with lung adenocarcinoma or hepatocarcinoma tumor xenografts. In contrast, the combination of sorafenib and vorinostat did not inhibit cell proliferation compared to a single treatment and induced G2/M cell cycle arrest without apoptosis. The sorafenib and vorinostat combination sustained the IGF-1R-, AKT-, and mitogen-activated protein kinase-dependent signaling pathways. These results showed that there was synergistic cytotoxicity when vorinostat was combined with gefitinib for both lung adenocarcinoma and hepatocarcinoma with mutant KRAS in vitro and in vivo but that the combination of vorinostat with sorafenib did not show any benefit. These findings highlight the important role of the IGF-1R/AKT pathway in the resistance to targeted therapies and support the use of histone deacetylase inhibitors in combination with EGFR-tyrosine kinase inhibitors, especially for treating patients with mutant KRAS resistant to other treatments.
RESUMO
Two new flavonoid glycosides, namely 3'-(2'',4''-dihydroxybenzyloxy)acanthophorin B (1b) and beta,2,3',4,4',6-hexahydroxy-alpha-(alpha-L-rhamnopyranosyl)dihydrochalcone (2) were isolated from the leaves of Psorospermum androsaemifolium together with quercetin (1), acanthophorin B (1a), alpha- (3) and beta-amyrine (3a), vismiaquinone (4), 12-hentriacontanol and hentriacontane. The structures of these secondary metabolites were established using detailed spectroscopic analysis and by comparison with published data. Compounds 1, 1a, 1b, 2, 3, 3a and 4 showed weak antifungal and antibacterial activities.