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
Hemin is an erythropoietic inductor capable of inducing autophagy in erythroid-like cell lines. Low-density lipoprotein receptor-related protein 1 (LRP1) is a transmembrane receptor involved in a wide range of cellular processes, such as proliferation, differentiation, and metabolism. Our aim was to evaluate whether LRP1 is responsible for hemin activity in K562 cells, with the results demonstrating a three-fold increase in LRP1 gene expression levels (P-values <0.001) when assessed by quantitative real-time RT-PCR (qRT-PCR). Moreover, a 70% higher protein amount was observed compared with control condition (P-values <0.01) by Western blot (WB). Time kinetic assays demonstrated a peak in light chain 3 (LC3) II (LC3II) levels after 8 h of hemin stimulation and the localization of LRP1 in the autophagosome structures. Silencing LRP1 by siRNA decreased drastically the hemin-induced autophagy activity by almost 80% compared with control cells (P-values <0.01). Confocal localization and biochemical analysis indicated a significant redistribution of LRP1 from early endosomes and recycling compartments to late endosomes and autophagolysosomes, where the receptor is degraded. We conclude that LRP1 is responsible for hemin-induced autophagy activity in the erythroblastic cell line and that hemin-LRP1 complex activation promotes a self-regulation of the receptor. Our results suggest that hemin, via the LRP1 receptor, favors erythroid maturation by inducing an autophagic response, making it a possible therapeutic candidate to help in the treatment of hematological disorders.
Assuntos
Autofagossomos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica , Hemina/farmacologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Autofagossomos/metabolismo , Autofagia/genética , Células HeLa , Humanos , Células K562 , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
The use of natural products in therapeutics has been growing over the years. Lignans are compounds with large pharmaceutical use, which has aroused interest in the search for new drugs to treat diseases. The present study evaluated the cytotoxicity of (-)-trachelogenin, a dibenzylbutyrolactone type lignan isolated from Combretum fruticosum, against several tumor and non-tumor cell lines using the MTT assay and its possible mechanism of action. (-)-Trachelogenin showed IC50 values ranging of 0.8-32.4µM in SF-295 and HL-60 cell lines, respectively and IC50 values >64µM in non-tumor cell lines. (-)-trachelogenin persistently induced autophagic cell death, with cytoplasmic vacuolization and formation of autophagosomes mediated by increasing LC3 activation and altering the expression levels of Beclin-1.
Assuntos
4-Butirolactona/análogos & derivados , Antineoplásicos Fitogênicos/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Combretum/química , Descoberta de Drogas , Caules de Planta/química , 4-Butirolactona/efeitos adversos , 4-Butirolactona/química , 4-Butirolactona/isolamento & purificação , 4-Butirolactona/farmacologia , Antineoplásicos Fitogênicos/efeitos adversos , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Autofagossomos/efeitos dos fármacos , Autofagossomos/patologia , Proteína Beclina-1/agonistas , Proteína Beclina-1/metabolismo , Brasil , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Combretum/crescimento & desenvolvimento , Etnofarmacologia , Células HCT116 , Humanos , Concentração Inibidora 50 , Medicina Tradicional , Proteínas Associadas aos Microtúbulos/agonistas , Proteínas Associadas aos Microtúbulos/metabolismo , Estrutura Molecular , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/metabolismo , Caules de Planta/crescimento & desenvolvimento , Vacúolos/efeitos dos fármacos , Vacúolos/patologiaRESUMO
Collapse of the mitochondrial membrane potential (MMP) is often considered the initiation of regulated cell death (RCD). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) is an uncoupler of the electron transport chain (ETC) that facilitates the translocation of protons into the mitochondrial matrix leading to the collapse of the MMP. Several cell stress responses such as mitophagy, mitochondrial biogenesis and the ubiquitin proteasome system may differentially contribute to restrain the initiation of RCD depending on the extent of mitochondrial damage. We induced graded mitochondrial damage after collapse of MMP with the mitochondrial uncoupler CCCP in Burkitt's lymphoma cells, and we evaluated the effect of several drugs targeting cell stress responses over RCD at 72 hr, using a multiparametric flow cytometry approach. CCCP caused collapse of MMP after 30 min., massive mitochondrial fission, oxidative stress and increased mitophagy within the 5-15 µM low-dose range (LDR) of CCCP. Within the 20-50 µM high-dose range (HDR), CCCP caused lysosomal destabilization and rupture, thus precluding mitophagy and autophagy. Cell death after 72 hr was below 20%, with increased mitochondrial mass (MM). The inhibitors of mitophagy 3-(2,4-dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(1H)-quinazolinone (Mdivi-1) and vincristine (VCR) increased cell death from CCCP within the LDR, while valproic acid (an inducer of mitochondrial biogenesis) also increased MM and cell death within the LDR. The proteasome inhibitor, MG132, increased cell death only in the HDR. Doxycycline, an antibiotic that disrupts mitochondrial biogenesis, had no effect on cell survival, while iodoacetamide, an inhibitor of glycolysis, increased cell death at the HDR. We conclude that mitophagy influenced RCD of lymphoma cells after MMP collapse by CCCP only within the LDR, while proteasome activity and glycolysis contributed to survival in the HDR under extensive mitochondria and lysosome damage.
Assuntos
Linfoma de Burkitt/tratamento farmacológico , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Desacopladores/farmacologia , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/patologia , Autofagia/efeitos dos fármacos , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Iodoacetamida/farmacologia , Leupeptinas/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Quinazolinonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Vincristina/farmacologiaRESUMO
Although graphene oxide (GO), a nanomaterial with hexagonal planar layer, has been widely studied due to its applications in neurobiology that include drug delivery and tissue engineering, additional studies to assess its potential toxic effects are still needed. Thus, this study evaluated the effects of GO exposure (at 5, 10, 50 or 100mg/L) during six consecutive days on mortality, hatching, spontaneous movement, heart rate, morphology, locomotion behavior, acetylcholinesterase (AChE) activity, dopamine levels and relative gene expression of developmental neurology-related genes using zebrafish larvae. In the 5mg/L dose, synapsin IIa expression up-regulation was seen concomitantly with down-regulation of dat expression, showing a potential compensatory mechanism. Moreover, the 10mg/L exposure caused an increase in heart rate, in absolute turn angle, brain cell damage and a decrease in dopamine levels. These alterations may be associated with autophagosome formation found in GO-exposed larval brain. No changes were observed on higher doses of GO exposure, probably due to nanomaterial agglomeration. Taken together, these results show that toxic effects of GO exposure are not dose-dependent, and are preeminent in lower concentrations. Additional studies are needed to deepen the specific mechanisms of GO neurotoxicity and are required to elucidate its potential biomedical use.
Assuntos
Grafite/química , Grafite/farmacologia , Larva/efeitos dos fármacos , Óxidos/química , Óxidos/farmacologia , Animais , Autofagossomos/efeitos dos fármacos , Nanotecnologia , Peixe-ZebraRESUMO
Activated hepatic stellate cells (HSC) are the major source of collagen I in liver fibrosis. Eugenia uniflora L. is a tree species that is widely distributed in South America. E. uniflora L. fruit-popularly known as pitanga-has been shown to exert beneficial properties. Autophagy contributes to the maintenance of cellular homeostasis and survival under stress situation, but it has also been suggested to be an alternative cell death pathway. Mitochondria play a pivotal role on signaling cell death. Mitophagy of damaged mitochondria is an important cell defense mechanism against organelle-mediated cell death signaling. We previously found that purple pitanga extract induced mitochondrial dysfunction, cell cycle arrest, and death by apoptosis and necrosis in GRX cells, a well-established activated HSC line. We evaluated the effects of 72-h treatment with crescent concentrations of purple pitanga extract (5 to 100 µg/mL) on triggering autophagy in GRX cells, as this is an important mechanism to cells under cytotoxic conditions. We found that all treated cells presented an increase in the mRNA expression of autophagy-related protein 7 (ATG7). Concomitantly, flow cytometry and ultrastructural analysis of treated cells revealed an increase of autophagosomes/autolysosomes that consequentially led to an increased mitophagy. As purple pitanga extract was previously found to be broadly cytotoxic to GRX cells, we postulated that autophagy contributes to this scenario, where cell death seems to be an inevitable fate. Altogether, the effectiveness on inducing activated HSC death can make purple pitanga extract a good candidate on treating liver fibrosis.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Eugenia/química , Células Estreladas do Fígado/patologia , Extratos Vegetais/farmacologia , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Linhagem Celular , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fitoterapia , Extratos Vegetais/uso terapêuticoRESUMO
Toxoplasma gondii is an obligate intracellular protozoan parasite, the causative agent of toxoplasmosis, one of the most widespread zoonoses in the world. During the host immune response, tissue cysts are formed, allowing the maintenance of the parasite within the host cell. Autophagy, a degradation process of cellular components, is critical for cellular homeostasis. Recently, it has been proposed that autophagy participates in host-pathogen interactions. Autophagic inducers (rapamycin or glucose plus serum deprivation) inhibited infection and parasite proliferation in a clinically relevant model of primary skeletal muscle cells (SkMC). The ultrastructural analysis showed in SkMC submitted to autophagic stimuli the presence of structures suggestive of autophagosomes close to the parasitophorous vacuole containing degraded parasites. Fluorescence microscopy results pointed out the increase in LC3 puncta in these cells after incubation with autophagic inducers. In the present study, SkMC autophagy controlled the proliferation of tachyzoites inside the cell, data reinforced by ultrastructural evidences and increased LC3 expression.