RESUMO
Caveolin-1 (Cav-1) is an integral membrane protein present in all organelles, responsible for regulating and integrating multiple signals as a platform. Mitochondria are extremely adaptable to external cues in chronic liver diseases, and expression of Cav-1 may affect mitochondrial flexibility in hepatic stellate cells (HSCs) activation. We previously demonstrated that exogenous expression of Cav-1 was sufficient to increase some classical markers of activation in HSCs. Here, we aimed to evaluate the influence of exogenous expression and knockdown of Cav-1 on regulating the mitochondrial plasticity, metabolism, endoplasmic reticulum (ER)-mitochondria distance, and lysosomal activity in HSCs. To characterize the mitochondrial, lysosomal morphology, and ER-mitochondria distance, we perform transmission electron microscope analysis. We accessed mitochondria and lysosomal networks and functions through a confocal microscope and flow cytometry. The expression of mitochondrial machinery fusion/fission genes was examined by real-time polymerase chain reaction. Total and mitochondrial cholesterol content was measured using Amplex Red. To define energy metabolism, we used the Oroboros system in the cells. We report that GRX cells with exogenous expression or knockdown of Cav-1 changed mitochondrial morphometric parameters, OXPHOS metabolism, ER-mitochondria distance, lysosomal activity, and may change the activation state of HSC. This study highlights that Cav-1 may modulate mitochondrial function and structural reorganization in HSC activation, being a potential candidate marker for chronic liver diseases and a molecular target for therapeutic intervention.
Assuntos
Caveolina 1 , Células Estreladas do Fígado , Caveolina 1/genética , Caveolina 1/metabolismo , Colesterol/metabolismo , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/patologia , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismoRESUMO
Cryptococcus neoformans and Cryptococcus gattii are the etiological agents of cryptococcosis, a high mortality disease. The development of such disease depends on the interaction of fungal cells with macrophages, in which they can reside and replicate. In order to dissect the molecular mechanisms by which cryptococcal cells modulate the activity of macrophages, a genome-scale comparative analysis of transcriptional changes in macrophages exposed to Cryptococcus spp. was conducted. Altered expression of nearly 40 genes was detected in macrophages exposed to cryptococcal cells. The major processes were associated with the mTOR pathway, whose associated genes exhibited decreased expression in macrophages incubated with cryptococcal cells. Phosphorylation of p70S6K and GSK-3ß was also decreased in macrophages incubated with fungal cells. In this way, Cryptococci presence could drive the modulation of mTOR pathway in macrophages possibly to increase the survival of the pathogen.
RESUMO
Autophagy is a cellular bulk degradation process used as an alternative source of energy and metabolites and implicated in various diseases. Inefficient autophagy in nutrient-deprived cancer cells would be beneficial for cancer therapy making its modulation valuable as a therapeutic strategy for cancer treatment, especially in combination with chemotherapy. Dipyridamole (DIP) is a vasodilator and antithrombotic drug. Its major effects involve the block of nucleoside uptake and phosphodiestesase inhibition, leading to increased levels of intracellular cAMP. Here we report that DIP increases autophagic markers due to autophagic flux blockage, resembling autophagosome maturation and/or closure impairment. Treatment with DIP results in an increased number of autophagosomes and autolysosomes and impairs degradation of SQSTM1/p62. As blockage of autophagic flux decreases the recycling of cellular components, DIP reduced the intracellular ATP levels in cancer cells. Autophagic flux blockage was neither through inhibition of lysosome function nor blockage of nucleoside uptake, but could be prevented by treatment with a PKA inhibitor, suggesting that autophagic flux failure mediated by DIP results from increased intracellular levels of cAMP. Treatment with DIP presented antiproliferative effects in vitro alone and in combination with chemotherapy drugs. Collectively, these data demonstrate that DIP can impair autophagic degradation, by preventing the normal autophagosome maturation, and might be useful in combination anticancer therapy.
Assuntos
Adenocarcinoma/patologia , Autofagia/efeitos dos fármacos , Dipiridamol/farmacologia , Neoplasias da Próstata/patologia , Trifosfato de Adenosina/metabolismo , Antineoplásicos/farmacologia , Autofagossomos/efeitos dos fármacos , Autofagossomos/ultraestrutura , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Masculino , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/genética , Ensaio Tumoral de Célula-TroncoRESUMO
Glioblastoma multiforme is the most aggressive type of primary brain tumor associated with few therapeutic opportunities and poor prognosis. In this study, we evaluated the efficacy of combining temozolomide (TMZ) with suberoylanilide hydroxamic acid (SAHA) - a specific histone deacetylases inhibitor - in glioma models in vitro and in vivo. In glioma cell lines, combined TMZ/SAHA promoted more cytotoxicity, G2/M arrest and apoptosis than either drugs alone. G2/M arrest was detected as soon as 24â¯h post drug exposure and preceded apoptosis, which occurred from 72â¯h treatment. TMZ and SAHA, alone or combined, also stimulated autophagy as evaluated by means of acridine orange staining and immunodetection of LC3I-II conversion and p62/SQSTM1 degradation. Time-course of autophagy accompanied G2/M arrest and preceded apoptosis, and blockage of late steps of autophagy with chloroquine (CQ) augmented SAHA/TMZ toxicity leading to apoptosis. In orthotopic gliomas in vivo, combined SAHA/TMZ showed better antitumor efficacy than either drugs alone, and adding CQ to the regimen improved antiglioma effects of SAHA and TMZ monotherapies without further benefit on combined SAHA/TMZ. In summary, the herein presented data suggest that autophagy acts as a protective response that impairs efficacy of SAHA and TMZ. Inhibiting autophagy termination with CQ may offer means to improve antitumor effects of SAHA and TMZ in gliomas and possibly other cancers.
Assuntos
Cloroquina/uso terapêutico , Temozolomida/uso terapêutico , Vorinostat/uso terapêutico , Animais , Antimaláricos/administração & dosagem , Antimaláricos/uso terapêutico , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Astrócitos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Beclina-1/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cloroquina/administração & dosagem , Quimioterapia Combinada , Técnicas de Silenciamento de Genes , Humanos , Ratos , Ratos Wistar , Temozolomida/administração & dosagem , Vorinostat/administração & dosagemRESUMO
Acridine Orange is a cell-permeable green fluorophore that can be protonated and trapped in acidic vesicular organelles (AVOs). Its metachromatic shift to red fluorescence is concentration-dependent and, therefore, Acridine Orange fluoresces red in AVOs, such as autolysosomes. This makes Acridine Orange staining a quick, accessible and reliable method to assess the volume of AVOs, which increases upon autophagy induction. Here, we describe a ratiometric analysis of autophagy using Acridine Orange, considering the red-to-green fluorescence intensity ratio (R/GFIR) to quantify flow cytometry and fluorescence microscopy data of Acridine-Orange-stained cells. This method measured with accuracy the increase in autophagy induced by starvation or rapamycin, and the reduction in autophagy produced by bafilomycin A1 or the knockdown of Beclin1 or ATG7. Results obtained with Acridine Orange, considering R/GFIR, correlated with the conversion of the unlipidated form of LC3 (LC3-I) into the lipidated form (LC3-II), SQSTM1 degradation and GFP-LC3 puncta formation, thus validating this assay to be used as an initial and quantitative method for evaluating the late step of autophagy in individual cells, complementing other methods.