RESUMO
Chronic obstructive pulmonary disease (COPD) is the third leading cause of mortality globally and the risk of developing lung cancer is six times greater in individuals with COPD who smoke compared to those who do not smoke. Matrix metalloproteinases (MMPs) play a crucial role in the pathophysiology of respiratory diseases by promoting inflammation and tissue degradation. Furthermore, MMPs are involved in key processes like epithelial-to-mesenchymal transition (EMT), metastasis, and invasion in lung cancer. While EMT has traditionally been associated with the progression of lung cancer, recent research highlights its active involvement in individuals with COPD. Current evidence underscores its role in orchestrating airway remodeling, fostering airway fibrosis, and contributing to the potential for malignant transformation in the complex pathophysiology of COPD. The precise regulatory roles of diverse MMPs in steering EMT during COPD progression needs to be elucidated. Additionally, the less-understood aspect involves how these MMPs bi-directionally activate or regulate various EMT-associated signaling cascades during COPD progression. This review article explores recent advancements in understanding MMPs' role in EMT during COPD progression and various pharmacological approaches to target MMPs. It also delves into the limitations of current MMP inhibitors and explores novel, advanced strategies for inhibiting MMPs, potentially offering new avenues for treating respiratory diseases.
Assuntos
Progressão da Doença , Transição Epitelial-Mesenquimal , Metaloproteinases da Matriz , Doença Pulmonar Obstrutiva Crônica , Humanos , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Metaloproteinases da Matriz/metabolismo , Animais , Inibidores de Metaloproteinases de Matriz/farmacologia , Inibidores de Metaloproteinases de Matriz/uso terapêuticoRESUMO
This study presents an innovative approach for targeted drug delivery through the development of Glycyrrhizic acid-loaded zein nanoparticles (GA-LNPs) as a proficient carrier system. The juxtaposition of zein, a hydrophobic biological macromolecule as a protein carrier, and Glycyrrhizic acid (GA), a hydrophilic therapeutic compound, exemplifies the adaptability of hydrocolloids within cutting-edge drug delivery systems. The characterization and functional traits of research encompass multifaceted analyses of natural macromolecules, which elucidate the homogeneous and spherical morphology of GA-LNPs with an average size of 170.49 nm. The controlled drug release profile of GA, orchestrated under simulated gastrointestinal conditions, adheres to diffusion-based Higuchi kinetics, reflecting the controlled release of the natural macromolecules. The intermolecular interactions among Zein, GA, and cross-linker EDC, facilitated through molecular dynamics simulations, fortify the structural integrity of the encapsulation matrix. In Vitro studies revealed enhanced cellular uptake of GA-LNPs in MCF-7 breast cancer cells. This cellular internalization was further confirmed through cytotoxicity assessments using MTT and apoptosis assays (fluorescence microscopy), which demonstrated the prominent anticancer effects of GA-LNPs on MCF-7 in time/dose-dependent manner. The successful formulation of GA-LNPs, coupled with their sustained release and potent anticancer properties, makes them a potential platform for advanced targeted therapeutic strategies in biomedical applications.
Assuntos
Neoplasias da Mama , Portadores de Fármacos , Ácido Glicirrízico , Nanopartículas , Zeína , Ácido Glicirrízico/química , Ácido Glicirrízico/farmacologia , Zeína/química , Humanos , Nanopartículas/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Células MCF-7 , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Apoptose/efeitos dos fármacos , Simulação de Dinâmica Molecular , Feminino , Antineoplásicos/farmacologia , Antineoplásicos/química , Simulação por Computador , Sobrevivência Celular/efeitos dos fármacosRESUMO
Breast cancer is a major cause of death in women worldwide leading to requirement of new therapeutic strategies. Silymarin demonstrated the anti-cancer activity however, due to low bioavailability its use is restricted. This study aimed to improve the solubility of silymarin by developing a silymarin loaded zein nanoparticles (SLNPs) which was stabilized by beta cyclodextrin. Comprehensive physiochemical characterization studies based on DLS, FTIR, UV-Vis Spectroscopy, FE-SEM, TEM, XRD, DSC, NMR and TGA confirmed the successful synthesis of SLNPs via an anti-solvent precipitation method. FE-SEM and TEM images demonstrated the uniform size and spherical shape of nanoparticles with encapsulation and loading efficiencies of 84.32 ± 1.9 % and 15.25 ± 2.4 % respectively. The zein protein interaction with silymarin, and ß-cyclodextrin was shown to be beneficial via the use of molecular simulations and binding energy calculations. Cellular studies demonstrated dose and time dependent cytotoxicity of SLNPs on MCF-7 breast cancer cell. FACS, qRT-PCR and Western blotting showed Bax (pro-apoptotic) upregulation while Bcl-2 (anti-apoptotic) downregulation. Our findings suggest that these loaded nanoparticles are more efficient than pure drug, enhancing its bioavailability and paving the path for developing it as a promising nutraceutical to treat breast cancer.
Assuntos
Neoplasias da Mama , Nanopartículas , Silimarina , Zeína , Feminino , Humanos , Silimarina/farmacologia , Silimarina/química , Zeína/química , Simulação de Acoplamento Molecular , Neoplasias da Mama/tratamento farmacológico , Nanopartículas/química , Tamanho da PartículaRESUMO
In the cutting-edge era of developing precision therapeutics, nanoparticles have emerged as a potent drug delivery system. Altering the size of poorly water-soluble drugs to nanoscale could confer change in their physical properties, including enhanced water solubility and bioavailability. Evodiamine (EVO), a natural indolequinone alkaloid extract from Evodia rutaecarpa, has shown several important pharmacological applications, anti-cancer being one of them. Protein-based nano-drug delivery systems have gained the interest of researchers due to their better biocompatibility, biodegradability, non-immunogenicity and non-toxicity. In the present study, EVO encapsulated BSA nanoparticles (ENPs) were synthesized and characterized, which were nanoscale-sized (~ 150 nm), monodispersed, spherical shaped, and showed high entrapment efficiency (~ 86%) and controlled drug release. The in-vitro anti-cancer activity of ENPs on human breast cancer cells was dose- and time-dependent. The apoptotic molecular mechanism investigated using FACS, qRT-PCR, and western blotting analysis, revealed increased expression of p53 and Bax and decreased expression of Bcl-2. Biological studies demonstrated comparatively more efficient and targeted delivery of ENPs than pure EVO. The comprehensive physiochemical characterization and in-vitro validation collectively pinpoint ENPs as a promising avenue for harnessing the therapeutic potential of the natural anti-cancer compound EVO. The findings indicate improved cytotoxicity, positioning ENPs as a propitious strategy for advancing breast cancer treatment.
Assuntos
Neoplasias da Mama , Nanopartículas , Quinazolinas , Humanos , Feminino , Linhagem Celular Tumoral , Neoplasias da Mama/tratamento farmacológico , Apoptose , Nanopartículas/química , ÁguaRESUMO
Galectin-3 (Gal-3), a multifunctional carbohydrate-binding lectin, has emerged as a key player in various biological processes including inflammation, cancer, cardiovascular diseases and fibrotic disorders, however it remains unclear if Gal-3 is a bystander or drives lung tissue remodeling (LTR). Persistent exposure to cigarette smoke (CS) is the leading cause of oxidative and inflammatory damage to the lung tissues. CS-induced pathological increase in Gal-3 expression has been implicated in the pathogenesis of various respiratory conditions, such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and lung cancer. We and others have reported that CS induces Gal-3 synthesis and secretion, which modulates the pathological signaling pathways in lung epithelial cells implicating Gal-3 as a novel diagnostic marker and a factor driving LTR in CS-exposed lungs. Therefore, pharmacological interventions targeting Gal-3 and its upstream and downstream signaling pathways can help combat CS-induced LTR. Excitingly, preclinical models have demonstrated the efficacy of interventions such as Gal-3 expression inhibition, Gal-3 receptor blockade, and signaling pathways modulation open up promising avenues for future therapeutic interventions. Furthermore, targeting extracellular vesicles-mediated Gal-3 release and the potential of microRNA-based therapy are emerging as novel therapeutic approaches in CS-induced LTR and have been discussed in this article.
Assuntos
Fumar Cigarros , Doença Pulmonar Obstrutiva Crônica , Humanos , Biomarcadores/metabolismo , Galectina 3/metabolismo , Pulmão/metabolismo , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Produtos do TabacoRESUMO
Cordycepin gets rapidly metabolized in the body into inactive form due to its structural similarity to adenosine, thus inhibiting its development as a medicinal agent. This study was aimed to improve the solubility and stability of cordycepin, a potential drug with known antiproliferative activity, by encapsulating it in bovine serum albumin: ß-cyclodextrin nanoparticles. Cordycepin-loaded nanoparticles (CLNPs) were synthesized using the antisolvent method and characterized thoroughly using various techniques. Our dynamic light scattering measurement showed a particle size and zeta potential of 160 ± 2.75 nm and -20.21 ± 2.1 mV, respectively, for CLNPs. Transmission electron microscopy studies revealed that particles were spherical in morphology. These CLNPs showed sustained release of cordycepin with encapsulation and loading efficiency of 81.62 ± 1.5 and 27.02 ± 2.0%, respectively, based on high-performance liquid chromatography and UV-vis studies. Based on differential scanning calorimetry and zeta potential studies, CLNPs improve cordycepin stability and solubility. Our molecular simulations and binding energy calculation also showed favorable protein interaction between cordycepin, bovine serum albumin, and ß-cyclodextrin, further supporting the notion of improved stability. In vitro cytotoxicity, apoptosis, and cellular uptake studies on breast cancer cells showed that the synthesized nanoparticles had greater cytotoxicity as compared to free cordycepin.
Assuntos
Nanopartículas , Soroalbumina Bovina , Soroalbumina Bovina/química , Portadores de Fármacos/química , Desoxiadenosinas/farmacologia , Nanopartículas/química , Tamanho da PartículaRESUMO
Breast cancer (BC) incidence and associated mortality have increased in tandem with the growth in obesity among the females worldwide. An adipokine, visfatin, has been shown to potentially impact glucose, lipid, and protein metabolism, and promote cancer growth however, the mechanism underlying the effect of visfatin on lipid metabolism dysregulation contributing to BC cell survival, proliferation, and metastasis has not been elucidated. Herein, we have investigated the role of visfatin on the induction of Sterol regulatory element binding protein (SREBP-1) and its upstream and downstream mediators in MCF-7 breast cancer cells. The survival and proliferation was investigated using MTT and Trypan blue assays, cytosolic lipid accumulation was observed using Nile red staining, mRNA and protein expressions were examined using RT-qPCR and western blotting, respectively, and cell cycle analysis was performed using fluorescence-activated cell sorting. Our results indicate that visfatin increased the survival and proliferation of MCF-7 cells in a time- and dose-dependent manner and augmented lipid buildup via activation of SREBP-1 and its associated downstream lipid synthesizing enzymes, at both mRNA and protein levels in MCF-7 cells. Inhibiting SREBP-1 using fatostatin or silencing with siRNA abrogated excessive lipid deposition by suppressing the expression of genes related to lipid synthesis pathway. Further, in-silico study showed high affinity binding of visfatin with epidermal growth factor receptor (EGFR), which was confirmed in an in-vitro study where visfatin increased the phosphorylation of EGFR at tyrosine residue and activated its downstream proteins via phosphorylation of AKT and GSK3ß in MCF-7 cells. Inhibition of GSK3ß by phosphorylation led to increased activity of SREBP-1 and associated downstream proteins. In summary, SREBP-1 may be a critical player in visfatin-induced lipid synthesis and accumulation in BC cells via activation of EGFR/AKT/GSK3ß pathway leading to increased cell survival and proliferation of BC cells.
Assuntos
Neoplasias da Mama , Proteínas Proto-Oncogênicas c-akt , Feminino , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias da Mama/patologia , Lipogênese , Regulação para Cima , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Nicotinamida Fosforribosiltransferase , Glicogênio Sintase Quinase 3 beta/metabolismo , Receptores ErbB/metabolismo , RNA Mensageiro/metabolismo , LipídeosRESUMO
AIMS: An elevated level of galectin-3, a carbohydrate-binding lectin implicated in tumorigenesis, metastasis, and epithelial-mesenchymal transition (EMT), has been found in cigarette smokers. However, the regulation of its expression and role in the pathogenesis of CS-induced EMT and lung cancer metastasis is unclear. Here, we have investigated the mechanism of CS-induced and galectin-3-mediated EMT in airway epithelial cells (AECs). MAIN METHODS: A549 adenocarcinoma cells and primary small airway epithelial cells cultured on an air-liquid interface (ALI) were exposed to cigarette smoke extract (CSE), and MTT, trypan blue, migration, invasion, tumor spheroid and colony formation assays were performed to assess EMT phenotype. Immunoblotting was performed to assess EMT and stemness markers and other regulatory proteins. KEY FINDINGS: CSE exposure affected cell survival and morphology, migration, invasion, and clonogenicity of AECs, which were concomitant with an increase in the expression of EMT markers, galectin-3, and runt-related transcription factor-2 (RUNX-2), an osteogenic transcription factor and upstream regulator of galectin-3. Chemical inhibition or silencing of RUNX-2 downregulated galectin-3 and modulated EMT marker expression, migration, invasion, and clonogenicity in CSE-exposed AECs. Recombinant human galectin-3 also induced EMT and stemness-associated changes in the AECs, and GB1107, a galectin-3 inhibitor, ameliorated these changes. Further, CSE-induced intracellular ROS enabled an increase in RUNX-2 and galectin-3 expression, which were reversed by n-acetyl-cysteine. SIGNIFICANCE: These results provide a novel mechanistic insight into CSE-induced EMT via RUNX-2/galectin-3 axis mediated through ROS, which promoted EMT-associated changes, including invasion, migration, and stemness in AECs, which could be implicated in CS-induced lung cancer progression.
Assuntos
Adenocarcinoma de Pulmão , Fumar Cigarros , Neoplasias Pulmonares , Humanos , Transição Epitelial-Mesenquimal , Galectina 3 , Espécies Reativas de Oxigênio , Neoplasias Pulmonares/patologia , Fatores de TranscriçãoRESUMO
Obesity has reached a pandemic proportion and is responsible for the augmentation of multimorbidity including certain cancers. With the rise in obesity amongst the female population globally, a concomitant increase in breast cancer (BC) incidence and related mortality has been observed. In the present review, we have elucidated the cellular and molecular insight into the visfatin-mediated cellular plasticity programs such as Epithelial to mesenchymal transition (EMT) and Endothelial to mesenchymal transition (EndoMT), and stemness-associated changes in BC cells. EMT and EndoMT are responsible for inducing metastasis in cancer cells and conferring chemotherapy resistance, immune escape, and infinite growth potential. Visfatin, an obesity-associated adipokine implicated in metabolic syndrome, has emerged as a central player in BC pathogenesis. Several studies have indicated the presence of visfatin in the tumor microenvironment (TME) where it augments EMT and EndoMT of BC cells. Further, Visfatin also modulates the TME by acting on the tumor stroma cells such as adipocytes, infiltrated immune cells, and adipose-associated stem cells that secrete factors such as cytokines, and extracellular vesicles responsible for augmenting cellular plasticity program. Visfatin induced altered metabolism of the cancer cells and molecular determinants such as non-coding RNAs involved in EMT and EndoMT have been discussed. We have also highlighted specific therapeutic targets that can be exploited for the development of effective BC treatment. Taken together, these advanced understandings of cellular and molecular insight into the visfatin-mediated cellular plasticity programs may stimulate the development of better approaches for the prevention and therapy of BC, especially in obese patients.
Assuntos
Neoplasias da Mama , Nicotinamida Fosforribosiltransferase , Neoplasias da Mama/patologia , Citocinas/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Humanos , Nicotinamida Fosforribosiltransferase/metabolismo , Obesidade/metabolismo , Microambiente TumoralRESUMO
Smoking tobacco is a major risk factor for the development of lung cancer, COPD, and other lung pathologies in smokers. Cigarette smoke (CS), which is comprised of several toxic components, is known to cause oxidative stress and inflammation-induced lung damage. Since airway epithelial cells act as the primary barrier, they protect the lung tissues from environmental insults, including CS. Upon exposure to these insults, airway epithelial cells act as the initial site of injury and orchestrate the pathophysiology of lung cancer. Scientists have been using cigarette smoke extract (CSE) in the preclinical model of in vitro cell culture to understand the effect of CS on the cellular, biochemical, and molecular changes in the lung epithelial cells. However, the standard procedure to prepare the CSE in the laboratory with a low-cost assembly and obtaining a reproducible quality of CSE in different batches is a challenge. Here, in this chapter, we delineate the method for the preparation of CSE using a discontinuous puff-based system which is an economical and reproducible method to prepare CSE in the laboratory. This method is suitable for studying CSE-induced molecular changes in lung diseases, including lung cancer, using in vitro models of lung adenocarcinoma cells.
Assuntos
Fumar Cigarros , Neoplasias Pulmonares , Doença Pulmonar Obstrutiva Crônica , Células Epiteliais/patologia , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Nicotiana/efeitos adversos , Nicotiana/químicaRESUMO
Airway epithelial cells arrayed in the inner lining of the airways of the lung are believed to be the major source for the development of malignancy of the lung. The advent of in vitro cell culture model made it easy to understand the molecular mechanism of carcinogenesis at a cellular level, where the airway epithelial cells are cultured on a 2D surface submerged in the culture media. However, this method of culturing airway epithelial cells does not reflect their true nature, and thus results obtained have their limitations. Further, they exhibit dissimilar morphology, transcriptome, and secretome when compared to the cells in vivo. Thus, the experimental data obtained from 2D culture models are inconclusive and, in most cases, could not be validated further in in vivo settings. These limitations can be addressed by culturing the airway epithelial cells on air-liquid interface (ALI), where they develop ciliated morphology similar to that of the lung. Experiments performed with this 3D model provide reliable data that are more realistic, and, in many cases, could replace the requirement of further in vivo validation. Here, we provide the detailed protocol of a 3D culture system called ALI culture for growing human-derived primary small airway epithelial cells to study the cellular and molecular changes associated with lung cancer.
Assuntos
Células Epiteliais , Neoplasias Pulmonares , Técnicas de Cultura de Células/métodos , Células Cultivadas , Humanos , Pulmão , Neoplasias Pulmonares/patologiaRESUMO
Cigarette smoke exposure leads to upregulation of cyclooxygenase-2 (COX-2), an inducible enzyme that synthesizes prostaglandin E2 (PGE2) and promotes airway inflammation. COX-2 overexpression is frequently implicated in inflammation, invasion, metastasis, and epithelial-mesenchymal transition (EMT). However, its detailed molecular mechanism in cigarette smoke induced EMT is not clear. Further, fisetin, a bioflavonoid, exhibits antioxidant and anti-inflammatory properties, but its effect in modulating COX-2-mediated inflammation and downstream sequelae remains unexplored. Therefore, we have investigated the mechanism of cigarette smoke-induced COX-2-mediated EMT in airway epithelial cells and examined the role of fisetin in controlling this aberration. MTT, trypan blue staining, gelatin zymography, Western blotting, invasion, wound healing, and tumor sphere formation assays in cigarette smoke extract (CSE) and/or fisetin treated airway epithelial cells, and in-silico molecular docking studies were performed. Results revealed that CSE exposure increased the expression and activity of COX-2, MMP-2/9, and ß-catenin and also enhanced expression of EMT markers leading to higher migration and invasion potential of airway epithelial cells. A specific COX-2 inhibitor NS-398 as well as fisetin treatment reversed the expression of EMT biomarkers, reduced the activity of MMP-2/9, and blocked the migration and invasion potential induced by CSE. Further, PGE2 also increased MMPs activity, invasion, and migration potential similar to CSE, which were significantly reversed by fisetin. In-silico studies showed a high binding affinity of fisetin to key EMT associated proteins, validating its anti-EMT potential. Thus, our study firstly unearths the mechanism of CSE-induced EMT in airway epithelial cells via COX-2/MMP/ß-catenin pathway, and secondly, it reveals that fisetin could significantly reverse CSE-induced EMT by inhibiting COX-2, indicating that fisetin could be an effective drug candidate for cigarette smoke-induced lung dysfunction.
Assuntos
Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Flavonóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Poluição por Fumaça de Tabaco/efeitos adversos , Células A549 , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Flavonóis/metabolismo , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Simulação de Acoplamento Molecular , Nitrobenzenos/farmacologia , Ligação Proteica , Sulfonamidas/farmacologia , Nicotiana/química , beta Catenina/metabolismoRESUMO
Black berry (Syzygium cumini) fruit is useful in curing diabetic complications; however, its role in diabetes-induced cardiomyopathy is not yet known. In this study, we investigated the regulation of gelatinase-B (MMP-9) by S. cumini methanol seed extract (MSE) in diabetic cardiomyopathy using real-time PCR, RT-PCR, immunocytochemistry, gel diffusion assay, and substrate zymography. The regulatory effects of MSE on NF-κB, TNF-α, and IL-6 were also examined. Identification and estimation of polyphenol constituents present in S. cumini extract were carried out using reverse-phase HPLC. Further, in silico docking studies of identified polyphenols with gelatinase-B were performed to elucidate molecular level interaction in the active site of gelatinase-B. Docking studies showed strong interaction of S. cumini polyphenols with gelatinase-B. Our findings indicate that MSE significantly suppresses gelatinase-B expression and activity in high-glucose- (HG-) stimulated cardiomyopathy. Further, HG-induced activation of NF-κB, TNF-α, and IL-6 was also remarkably reduced by MSE. Our results suggest that S. cumini MSE may be useful as an effective functional food and dietary supplement to regulate HG-induced cardiac stress through gelatinase.
Assuntos
Anti-Inflamatórios/farmacologia , Hiperglicemia/patologia , Metaloproteinase 9 da Matriz/metabolismo , Miócitos Cardíacos/patologia , Estresse Oxidativo , Extratos Vegetais/farmacologia , Sementes/química , Syzygium/química , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucose , Hiperglicemia/genética , Inflamação/patologia , Interleucina-6/metabolismo , Metaloproteinase 9 da Matriz/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenóis/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos , Especificidade por Substrato/efeitos dos fármacos , Termodinâmica , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: Cysteinyl leukotrienes (CysLTs), a group of inflammatory lipid mediators, are found elevated in obese-asthmatic patients. Leukotriene D4 (LTD4), a representative CysLT, is implicated in promoting lung inflammation and remodelling in allergic asthma, but its role in non-allergic asthma, especially in obese-asthmatic patients, is not known. Here, using primary human small airway epithelial cells (SAECs) we have investigated the mechanism of LTD4-induced inflammation and remodelling and assessed high proneness of obese mice to develop asthma upon challenge with allergen ovalbumin (OVA). METHODS: Primary human small airway epithelial cells (SAECs) were stimulated with different concentrations of LTD4 for different time intervals and various inflammatory markers were measured through cytokine array, membrane-based ELISA and Western blotting. An air-liquid interface (ALI) model of SAECs was used to study the effects of LTD4-induced remodelling in SAECs using Western blotting, H&E staining and PAS staining. Further, OVA-based murine model was used to examine the propensity of high-fat diet (HFD)-fed obese mice to develop asthma symptoms by studying the infiltration of inflammatory cells (assessed by bronchioalveolar lavage (BAL) cytology) and airway remodelling (assessed by histopathology) upon allergen exposure. RESULTS: The human primary small airway epithelial cells (SAECs) treated with LTD4 showed significant alterations in the levels of inflammatory markers such as GM-CSF, TNF-α, IL-1ß, EGF and eotaxin in dose- and time-dependent manner. Further, LTD4 enhanced the activation of inflammasomes as evidenced by increased levels of NALP3, cleaved caspase-1 and IL-1ß. LTD4 also enhanced inflammation by increasing the expression of COX-2 in SAECs. The airway remodelling markers Vimentin and Muc5AC were found elevated in ALI culture of SAECs when stimulated with LTD4, as it also increased TGF-ß levels and activation of Smad2/3 phosphorylation in SAECs. Last, sensitization and challenge of HFD-fed obese mice with OVA showed increased infiltration of inflammatory cells in BAL and enhanced levels of remodeling phenotypes like loss of cilia, mucus cell metaplasia and collagen deposition in mice lung tissues. CONCLUSION: The results suggest that LTD4 could induce inflammatory response in human airway epithelial cell by activating NALP3 inflammasome. LTD4 could further promote airway epithelial cells' remodelling through TGF-ß/smad2/3-mediated pathway. Our in vivo results suggested that obesity predisposed the OVA challenged mice to develop lung inflammation and remodelling akin to asthma-like phenotypes during obesity.
Assuntos
Remodelação das Vias Aéreas/imunologia , Asma/imunologia , Células Epiteliais/imunologia , Inflamação/imunologia , Leucotrieno D4/imunologia , Obesidade/imunologia , Alérgenos/imunologia , Animais , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Células Cultivadas , Citocinas/imunologia , Humanos , Inflamassomos/imunologia , Inflamação/patologia , Contagem de Leucócitos , Masculino , Camundongos Endogâmicos BALB C , Mucina-5AC/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Obesidade/patologia , Ovalbumina/imunologia , Proteína Smad2/imunologia , Proteína Smad3/imunologia , Vimentina/imunologiaRESUMO
Carvacrol (CV), a monoterpene possesses wide range of biological activities but has limited application due to low aqueous solubility and poor bioavailability. To address this issue and enhance bioavailability and efficacy of carvacrol, lecithin stabilized zein nanoparticles were investigated. Precipitation method was used for synthesis of nanoparticles and characterized using various techniques. CV entrapped under optimized parameters has size around 250 nm with -15 mV zeta potential. SEM studies showed nanoparticles with spherical morphology and size in accordance with DLS studies. FTIR, NMR and DSC were used to determine the molecular interaction between CV and lecithin stabilized zein nanoparticles. Molecular docking studies were performed to understand the interaction between protein and drug at molecular level. Our results demonstrated the presence of two active sites within zein, showing strong binding interactions with carvacrol. The encapsulation efficiency of 78% with loading efficiency of 13% was obtained as per HPLC and UV-Vis studies. Cytotoxicity assay indicated that the CV loaded nanoparticles induce cytotoxicity against colon cancer (SW480) cells further confirmed by acridine orange and ethidium bromide dual staining assay. Fluorescent tagged nanoparticles revealed significant cellular uptake of drug. Our results suggest that CV can be conveniently delivered via oral route after incorporating into lecithin stabilized zein nanoparticles and may prove effective for colon cancer treatment.
Assuntos
Nanopartículas , Zeína , Cimenos , Portadores de Fármacos , Simulação de Acoplamento Molecular , Tamanho da PartículaRESUMO
In the past decade, naturally occurring phytoconstituents have emerged as potential therapeutic agents and alternative to synthetic drugs. However, efficient delivery of hydrophobic phytoconstituents into the body with desired therapeutic efficacy is a key challenge for the pharmaceutical industries due to their insolubility in water and low oral bioavailability. Nanosuspension formulations have shown promises to improve the delivery of the hydrophobic molecules with simultaneously avoiding the drawbacks like carrier toxicity and scale-up issues of other nanotechnology-based drug delivery systems. In this study, we have used morin hydrate (MH), a flavonol, and developed MH nanosuspension formulation (MHNS) to improve its poor physiochemical properties and low oral bioavailability. Different stabilizers with varying concentrations were investigated for preparing nanosuspension. MHNS was characterized by DLS, TEM, FTIR, DSC, powder XRD and was evaluated for its solubility, dissolution, partition coefficient, in-vitro anticancer activity and pharmacokinetics in rats. The optimized nanosuspension formulation, with a size of <100 nm, is capable of increasing aqueous solubility, dissolution rate, and oral bioavailability of MH. Moreover, the therapeutic efficacy, in terms of cytotoxicity to human lung cancer cells, of MH was also increased after formulating into nanosuspension form.
Assuntos
Flavonoides , Nanopartículas , Administração Oral , Animais , Disponibilidade Biológica , Ratos , Solubilidade , SuspensõesRESUMO
Bone regeneration is a multi-step, overlapping process, in which angiogenesis and osteogenesis are the key players. Several attempts have been made to promote angiogenesis-coupled osteogenesis using scaffolding technology. However, the recreation of functional vasculature during bone regeneration is an unparalleled challenge. In this study, a dual drug-delivering polycaprolactone-collagen fibrous scaffold is reported to promote early osteogenesis and angiogenesis. Simvastatin as a pro-angiogenic and dexamethasone as an osteoinductive drug were encapsulated to functionalize the electrospun fibers. The optically transparent fibrous mat represented the sustained and sequential release of drugs for 28 days. The fibrous mesh increased cell proliferation and enhanced the osteogenic differentiation up to 21 days. The alkaline phosphatase activity and mineral deposition were comparatively higher on dual drug-releasing fibers when compared to control fibers. The dual drug-releasing osteoconductive fibers demonstrated osteogenesis as early as 7 days with a 3.7 and 1.5 fold increase in the expression of osteogenic differentiation markers (RUNX2 and osteocalcin), respectively. In vitro angiogenesis using primary human umbilical vein endothelial cells (pHUVECs) showed no significant difference in cell proliferation among control fibers and dual drug-releasing fibers. However, the angioinductive nature of simvastatin released from the fibers demonstrated tube formation and 2 fold higher angiogenic score. The mRNA and protein expression study of angiogenic markers (VEGFR2 and eNOS) by polymerase chain reaction and western blotting depicted the angioinducing potential of dual drug-releasing fibers. VEGFR2 and eNOS mRNA expressions increased by 1.1 and 1.6 fold, respectively, whereas their protein expression increased by 3.2 and 1.7 fold, respectively. The overall results demonstrate the synergistic effect of osteoconductive substrate and osteoinductive dual drugs to promote early osteogenesis, and release of the pro-angiogenic drug promotes angiogenesis.
Assuntos
Regeneração Óssea , Colágeno/química , Sistemas de Liberação de Medicamentos , Neovascularização Fisiológica/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Poliésteres/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Células 3T3 , Animais , Varredura Diferencial de Calorimetria , Diferenciação Celular , Proliferação de Células , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Dexametasona/administração & dosagem , Eletroquímica , Perfilação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Óxido Nítrico Sintase Tipo III/metabolismo , Preparações Farmacêuticas , Sinvastatina/administração & dosagem , Propriedades de Superfície , Termogravimetria , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Acacetin, a bioflavanoid, contains anti-inflammatory and anti-cancer activities as shown in different experimental models. However, its anticancer potential and mechanism of action against colorectal cancer cells is largely unknown. Here, we have investigated the efficacy of acacetin using two colorectal adenocarcinoma SW480 and HCT-116 cell lines. Cell survival was examined by Trypan-blue exclusion and MTT assays, cell cycle analysis by FACS, apoptosis was assessed using Annexin V FITC assay and nuclear condensation by Hoechst staining, ROS level by DCFDA and Mitosox, and protein expression level by Western blotting. Acacetin reduced the cell survival and proliferation of both types of cells, and induced S- and G2-M phase arrest and also reduced the levels of ß-catenin and its downstream target c-myc. Further, acacetin induced apoptosis as examined by Annexin-V FITC and nuclear condensation. It increased intracellular ROS production, especially mitochondrial ROS. Acacetin increased mitochondrial membrane potential depolarization and Bax:Bcl-2 ratio. Although significant changes in caspases -8 and -9 and PARP level was not observed, acacetin could induce the truncation and subsequent translocation of activated AIF from mitochondria to cytosol, which could further induce chromosomal breakage leading to apoptosis. In conclusion, Acacetin induces mitochondrial ROS-mediated cell death in a caspase-independent manner in SW480 and HCT-116 colon carcinoma cells by inducing apoptosis inducing factor (AIF), which may potentiate its anticancer and chemotherapeutic prospects against colorectal carcinoma.
Assuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Flavonas/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Flavonas/metabolismo , Células HCT116 , Humanos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo , beta Catenina/metabolismoRESUMO
Matrix metalloproteinases (MMPs) have been implicated in EMT but their role in the regulation of cigarette smoke-induced EMT in airway epithelium is not clear. We have therefore investigated the potential role of MMP-2 and -9 in cigarette smoke extract (CSE) induced EMT using A549 lung epithelial cells and human small airway epithelial cells (SAEC). The cells were treated with different concentration of CSE, and MTT and trypan blue assays, acridine orange-ethidium bromide assay, gelatin zymography, Western blotting, immunofluorescence studies, Boyden-chamber assay, wound healing assay and air-liquid interface (ALI) culture were used to assess different cellular and molecular changes associated with EMT. The results depict that CSE increased the cytotoxicity along with a concurrent increase in the expression and activity of MMP-2 and -9. CSE further altered EMT markers like E-cadherin, N-cadherin, vimentin, and the molecular modulators of EMT such as ß-catenin and pGSK-3ß. Further, CSE also upregulated EGFR, AKT, and ERK1/2 in airway epithelial cells. SB-3CT, a known inhibitor of MMP-2 and -9, altered and reversed the expression of markers of EMT and kinases, validating the role of MMP-2 and -9 in CSE-induced EMT. Fisetin, a plant-derived bioflavonoid, also reversed the expression of EMT markers and molecular regulators in a similar fashion as SB-3CT. In summary, this study highlights the role of MMP-2 and -9 in CSE-induced EMT and curate its molecular cascade through EGFR/AKT/ERK/ß-catenin axis, which could be restored by MMP-2 and -9 inhibitor and fisetin. Fisetin is hitherto unknown to modulate CSE-induced MMPs activity in airway epithelial cells, and our study suggests its potential role as a therapeutic approach in CSE-induced EMT in lung epithelial cells.