Jan 20, 2012 autophagy inhibition using hydrochloroquine in breast cancer patients the safety and scientific validity of this study is the responsibility of the study sponsor and investigators. Advanced fluorescent probes for imaging distinct processes in the autophagy pathway see a list of the products featured in this article. Chloroquine phosphate the information contained here is subject to changes as i experiment and learn more about chloroquine phosphate what it treats marine ich cryptocaryon irritans, marine velvet disease amyloodinium ocellatum, brooklynella hostilis and uronema marinum. Autophagy protects against palmitateinduced apoptosis in.
Product specialist, sigma life science autophagy is a highly regulated process by which longlived proteins, organelles, and protein aggregates are captured within autophagosomes, which are then fused with lysosomes for. The zebrafish danio rerio is an attractive model for studying the circadian clock and autophagy. During the autophagy process, lc3i is converted to lc3ii by lipidation, resulting in lc3ii migrating differently on sdspage. Does chloroquine, an antimalarial drug, affect autophagy in. The response of gics to radiation was associated with autophagy and the late autophagy inhibitor chloroquine in combination with radiation caused g0g1 phase arrest, promoted gics apoptosis and impaired the repair of radiationinduced dna damage in gics, and decreased the gics tumorsphere numbers and diameters. Lc3ii is used as a quantitative marker of autophagy since it is required for the formation of the autophagosome and its level is proportional to the amount of autophagosomes in the cell. Autophagy is a membrane trafficking process delivering cytoplasmic material to the lysosome for degradation. The core autophagy related atg complexes in mammals are ulk1 protein kinases, atg9wipi1 and vps34beclin1 class iii pi3kinase complexes, and the atg12 and lc3 conjugation systems.
Tumor cells require autophagy to remove misfolded proteins or damaged cellular organelles. The different steps of canonical autophagy have been well characterized and involve the assembly of at least 36 autophagy related atg proteins into distinct complexes. To determine concentrations of bafilomycin and chloroquine which result in. While it has been suggested that autophagy may elicit cardioprotective and prosurvival modulating functions, excessive activation of autophagy can also be detrimental. Zebrafish as a model to understand autophagy and its role. Autophagy is an evolutionarily conserved process that plays a key role in the maintenance of overall cellular health. Here we show that in human gallbladder carcinoma gbc cells lines, sgc996 and gbcsd, autophagy is induced by the dna damaging agent 5fluorouracil 5fu. Rapamycin upregulation of autophagy reduces infarct size and.
A toxicological study using zebrafish danio rerio as a model. Left a549 cells were treated with chloroquine and stained with invitrogen hoechst 33342, invitrogen hcs cellmask deep red, and an antilc3b with invitrogen alexa fluor 488 goat antirabbit antibody. Historically known for its antimalarial activity, chloroquine is a widely used biological research tool for studying autophagy inhibition. Mitochondrial maintenance involves mitophagy, a selective autophagy process that removes abnormal mitochondria. Centrifugation is not needed during the rinse procedure. We administered chloroquine orally to idsko mice from 4 to 25 weeks of age. As expected, p62 was absent from p62 knockout cells fig. Chloroquine diphosphate was originally developed as a treatment against malaria. While in combination with the pretreatment of chloroquine cq, a inhibitor of autophagy, the. Suppression of autophagy by chloroquine sensitizes 5. A novel transgenic zebrafish line allows for in vivo quantification of. To investigate whether chloroquine, which inhibits autophagy, would preserve cell integrity, we used the automatic acquisition feature of our largescale sem system and examined whether chloroquine treatment led to a decrease in the number of autophagy like vacuoles. Lysosomal inhibitor chloroquine can be used to block autophagosome turnover leading to accumulation of lc3bii, rendering it a useful tool compound when assessing the autophagy activities of cells following various treatments.
Chloroquine also inhibits autophagy by specifically blocking acidification of the lysosomes which fuse with autophagosomes. Jun 21, 2014 autophagy, a cell survival process has recently been recognized as being involved in human strokes 1, 2. Mitophagy regulated by the pink1parkin pathway intechopen. Effect of short and longterm highfat feeding on autophagy. Autophagy activation in zebrafish heart regeneration. We further assessed the autophagy activities in these models. The drug was added to 1h starved cells at different ph values because cq affects ph dependently. It is the ability of chloroquine to inhibit autophagy in. Primary rat cortical neurons at div7 were used for experiments. This inhibits lysosomal hydrolases and prevents autophagosomal fusion and degradation, which can result in apoptotic or necrotic cell death 14. Hydroxychloroquine inhibits autophagy to potentiate.
However, the functional role of autophagy in bacterial cancer therapy has been little investigated. The role of autophagy and apoptosis during embryo development. Chloroquine, the treatment for rheumatoid arthritis, sle, hiv and malaria for half a century, is one of the only autophagy inhibitors, whose effectiveness and safety have been proven in. A guide for the laboratory use of zebrafish danio brachydanio rerio. Jci autophagy inhibition enhances therapyinduced apoptosis. Research studies demonstrate that chloroquine accumulates in acidic lysosomes and increases the lysosomal ph. Lycorine downregulates hmgb1 to inhibit autophagy and. Role of autophagy in glycogen breakdown and its relevance to.
This line is useful for indirectly monitoring autophagic activity in neurons in vivo and screening for macroautophagyautophagymodulating compounds. Under oxidative stress, ros produced in melanocytes could induce autophagy and activate the nrf2 antioxidant pathway, remove toxic molecules, and maintain the redox homeostasis of melanocytes, while continuous stimulation of ros might eventually lead to inactivation of autophagy and excessive activation of nrf2 antioxidant pathway and break the redox homeostasis of melanocytes, resulting in. It has since been implicated in cancer research as it inhibits cell growth and induces cell death in a number of cancer cell lines. Parkinsons disease pd is a movement disorder caused by the specific loss of dopaminergic neurons in the substantia nigra of the midbrain. Chloroquine covid19 research cell signaling technology. Chloroquine, an autophagy inhibitor, potentiates the. Fertilization triggers cell remodeling from each gamete to a totipotent zygote. A toxicological study using zebrafish danio rerio as a model 12 introduction there has been an increase in the demand for new, robust, and costeffective ways to assess chemicals for their effect on human health, particularly early development.
We use zebrafish larvae to investigate the role of bacterial autophagy. Autophagy inhibition using hydrochloroquine in breast cancer. Zebrafish has become a popular model system for the study of vertebrate development. Jul 14, 2016 bacteriabased living anticancer agents have emerged as promising therapeutics. Multiple roles of the cytoskeleton in bacterial autophagy plos. The zebrafish book a guide for the laboratory use of.
Autophagy is a lysosomedependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation. Autophagy is a highly regulated cell death process which cells use to recycle cellular contents for survival. Inhibition of autophagy with bafilomycin and chloroquine. However, little is known about autophagy in dietinduced nafld in zebrafish. This model has potential for exploring how autophagy affects disease pathogenesis. Mridul roy 1, long liang 1, xiaojuan xiao 1, yuanliang peng 1, yuhao luo 1, weihua zhou 1, ji zhang 1, lugui qiu 2, shuaishuai zhang 2, feng liu 3, mao ye 4, wen zhou 5, jing liu 1.
Autophagy, a cellular degradative pathway, is an important selfdefense mechanism in response to various stresses. Listing a study does not mean it has been evaluated by the u. Cahova1 1department of metabolism and diabetes, institute for clinical and experimental medicine, prague, czech republic received march 19, 2012 accepted may 24, 2012 summary. Involvement of endoplasmic reticulum stress, autophagy and. Autophagy is a protective mechanism for human melanoma cells. In mice, autophagy increases in most organs under starvation conditions, with muscles showing a particularly clear response. We have determined that autophagy regulates pancreatic beta cell survival in response to pdx1 deficiency and nutrient deprivation. Sep 26, 2016 involvement of endoplasmic reticulum stress, autophagy and apoptosis in advanced glycation end productsinduced glomerular mesangial cell injury skip to main content thank you for visiting. Two genes implicated in pd, pink1 and parkin, regulate mitophagy in cultured cells. Genetic inhibition of autophagy promotes p53 lossofheterozygosity. Continue to the next level to keep filling your fish tank with interesting and diverse zebra fish.
Chloroquine, a widely used antimalaria drug, is currently under investigation in clinical trials on cancer patients. Autophagy promotes pancreatic beta cell death in response to. The effect of chloroquine cq on autophagy was studied in starved tetrahymena pyriformis. Effect of short and longterm highfat feeding on autophagy flux and lysosomal activity in rat liver z. However, autophagy is a survival pathway for tumor cells to overcome metabolic stress and to resist cell death in response to radiation and chemotherapeutic agents 32, 35. This is important because of the increase in the amount of chemicals produced and released into the. In mammalian cells, two major catabolic processesthe ubiquitinproteasome system ups and the autophagy pathwaymaintain cell health by degrading proteins and organelles that have either reached the end of their life or become compromised. Analyzing autophagy in zebrafish pubmed central pmc. The zebrafish as a new model for the in vivo study of shigella plos. We here give an overview of different methods used to analyse autophagy in zebrafisha relatively new model for studying autophagy and briefly discuss what has been done so. Autophagy1 is a complex of adaptive cellular response that enhances cancer cell survival in the face of cellular stresses such as chemothery. Chloroquine, an antimalarial drug, inhibits autophagy by preventing degradation of autolysosomes. A major role of autophagy is therefore to liberate amino acids, fatty acids, and glucose that can be used to maintain cellular functions during stress and starvation.
Autophagy or autophagocytosis from the ancient greek autophagos, meaning selfdevouring and kytos, meaning hollow is the natural, regulated mechanism of the cell that removes unnecessary or dysfunctional components. Ad western blot analyses of lc3i and lc3ii in lysates in neurons exposed to increasing concentrations of bafilomycin baf or chloroquine cq for 24 h. While it is closely linked with apoptosis, autophagy is primarily characterized as a catabolic mechanism by which cellular energy homeostasis is maintained, and by which cellular organelles and proteins are degraded. There are numerous methods of inducing autophagy including serum starvation and rapamycin which both inhibit mtor signalling. Autophagy and apoptosis are crucial processes during embryo development of invertebrates and vertebrates organisms, as they are necessary for the formation of a new organism, starting from a fertilized egg. Autophagy cellular selfeating is a highly regulated degradation process of the eukaryotic cell during which parts of the cytoplasm are delivered into, and broken down within, the lysosomal. It is the ability of chloroquine to inhibit autophagy in tumor cells that has. Sbi0206965 was shown to suppress autophagy induced by mtor inhibition. When a proliferating tetrahymena culture is transferred to a starvation medium, autophagy commences although cells most advanced in the cell cycle will divide. To study autophagy in zebrafish, we identified two zebrafish atg8 homologs, lc3 and gabarap, and generated two transgenic zebrafish lines expressing gfptagged versions of.
Palmitate painduced liver injury is considered a risk factor for the development of nonalcoholic fatty liver disease. Chloroquine cq is a lysosomotropic agent with an extensive range of biological effects 1. We further tested the protein levels of keap1 and p62 in autophagy deficient cells. Autophagy plays a major role in metabolic adaptation in rastransformed cells 36, 37. The major form of autophagy, macroautophagy referred to as autophagy herein, is a cellular process that normally recycles old, or damaged, organelles and proteins by vesicular seclusion followed by lysosomal degradation. Keap1 facilitates p62mediated ubiquitin aggregate clearance. Autophagy is a cellular degradative pathway that delivers cytoplasmic cargo to the lysosome. This is in part due to the lack of adequate transgenic lines, that could be used to monitor andor alter autophagy in zebrafish. Programmed cell death pcd and cell survival are two sides of the same coin. Whether autophagy observed in treated cancer cells represents a mechanism that allows tumor cells to survive therapy or a mechanism for initiating a nonapoptotic form of programmed cell death remains controversial.
Autophagy is a key pathway in the development of endocrine resistance in breast cancer, and targeting autophagy can reverse antiestrogen resistance. Effects of bafilomycin and chloroquine on autophagy and cell survival. We describe activation of autophagy early in the regenerative response to muscle injury, while blocking autophagy using chloroquine or atg5 and becn1 knockdown reduced the rate of. There is a growing literature on the use of zebrafish to model human disease. The best studied of these processes is macroautophagy, which will be referred to as autophagy hereafter. Recent studies have made some progress in investigating the induction of autophagy in fish cells, the. Tem is the classical method to detect autophagic structures within eukaryotic cells fig.
It has been successfully used in zebrafish too, detecting such structures in diverse tissues. Autophagy is a process by which a cell breaks down macromolecules in response to starvation or stress signals. Analyzing autophagy in zebrafish congcong he and daniel j. The macroautophagy hereafter referred to as autophagy pathway has received little attention as a potential cause of beta cell death. Chloroquine interestingly induces the formation of autophagosomes but blocks the formation of autophagolysosomes and hence ultimately is an inhibitor of autopha.
Chloroquine phosphate reef2reef saltwater and reef aquarium. Ijms free fulltext autophagy in the central nervous. To determine if autophagy is important for tumor suppression, we inhibited autophagy in transgenic zebrafish via stable, tissuespecific. Cloning of zebrafish lc3 and generation of a transgenic gfplc3 fish line provide excellent tools to monitor autophagy in this organism. Thus, autophagy has a fundamental and driving role in developing tumors and. There are many forms of autophagy including macroautophagy, microautophagy and chaperonemediated autophagy. Accordingly, lc3bii levels can serve as a measure of autophagy activity. Zebrafish danio rerio is a fresh water fish that inhabits rivers in india.
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