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1.
Macroautophagy is a catabolic process that delivers cytoplasmic components via the autophagosome to lysosomes for degradation. Measuring autophagic activity is critical to dissect molecular mechanisms and functions of autophagy but remains challenging due to the lack of a definitive method. We have recently developed a new fluorescent probe, GFP-LC3-RFP-LC3ΔG, to assess autophagic flux. Upon intracellular expression, the probe is cleaved by ATG4 family proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. The former is degraded by autophagy while the latter persists as an internal control in the cytosol. Autophagic flux can thus be quantified by obtaining the ratio of GFP:RFP signals. Using this method, we have identified several autophagy-modulating drugs by screening an approved drug library. We have also demonstrated that induced and basal autophagic flux can be monitored in zebrafish and mice. 相似文献
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We detail here a protocol using tandem-tagged mCherry-EGFP-LC3 (C-G-LC3) to quantify autophagic flux in single cells by ratiometric flow cytometry and to isolate subpopulations of cells based on their relative levels of autophagic flux. This robust and sensitive method measures autophagic flux rather than autophagosome number and is an important addition to the autophagy researcher’s array of tools for measuring autophagy. Two crucial steps in this protocol are i) generate cells constitutively expressing C-G-LC3 with low to medium fluorescence and low fluorescence variability, and ii) correctly set up gates and voltage/gain on a properly equipped flow cytometer. We have used this method to measure autophagic flux in a variety of cell types and experimental systems using many different autophagy stimuli. On a sorting flow cytometer, this technique can be used to isolate cells with different levels of basal autophagic flux, or cells with variable induction of flux in response to a given stimulus for further analysis or experimentation. We have also combined quantification of autophagic flux with methods to measure apoptosis and cell surface proteins, demonstrating the usefulness of this protocol in combination with other flow cytometry labels and markers. 相似文献
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Guillon-Munos A van Bemmelen MX Clarke PG 《Apoptosis : an international journal on programmed cell death》2005,10(5):1031-1041
The death of serum-deprived undifferentiated PC12 cells shows both autophagic and apoptotic features. Since it is still controversial whether the autophagy is instrumental in the cell death or a mere epiphenomenon, we tested the effects of inhibiting the autophagy by a variety of phosphoinositide 3-kinase inhibitors, and provided evidence that the autophagy, or a related trafficking event, is indeed instrumental in the cell death. Furthermore, by comparing the effects of PI3-K inhibition and caspase-inhibition on autophagic and apoptotic cellular events, we showed that in this case the autophagic and apoptotic mechanisms mediate cell death by parallel pathways and do not act in series.Financial support: grants 31-50598.97 and 31-61736.00 from the Swiss National Science Foundation 相似文献
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A. N. Luchnik 《Russian Journal of Developmental Biology》2000,31(3):186-189
A general principle of the maintenance of malignant growth in all types of tumors has been formulated. According to this principle,
stochastic but continuous death of some tumor cells due to the inherent genetic instability of their genome (fragility of
chromosomes) is the main event stimulating tumor growth. The dead cells trigger a complex multicomponent process of wound
healing expressed as further proliferation of living tumor cells, angiogenesis, stimulation of cell migration, and other events.
Stimulation of the proliferation of living cells leads to further death of cells and, as a result, to further stimulation
of the system of wound healing, etc. Thus, the tumor sacrifices a small amount of the dying cells to stimulate the proliferation
of all of its other cells. It is proposed that the nature of the genetic instability of malignant cells is related to the
appearance of an uninemic structure in some regions of chromosomes, in whole chromosomes, or in whole genomes. The author
bases his statements on the binemic structure of chromosomes, which has already been experimentally and theoretically substantiated.
Uninemic regions have an exceedingly high frequency of spontaneous chromosome aberrations, due to blockage of the mechanism
of underlying repair of the DNA double break in the absence of a second DNA copy. Possible approaches to a search for more
efficient methods of therapy are discussed. 相似文献
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Zhi Gen Leng Shao Jian Lin Ze Rui Wu Yu Hang Guo Lin Cai Han Bing Shang 《Autophagy》2017,13(8):1404-1419
Dopamine agonists such as bromocriptine and cabergoline have been successfully used in the treatment of pituitary prolactinomas and other neuroendocrine tumors. However, their therapeutic mechanisms are not fully understood. In this study we demonstrated that DRD5 (dopamine receptor D5) agonists were potent inhibitors of pituitary tumor growth. We further found that DRD5 activation increased production of reactive oxygen species (ROS), inhibited the MTOR pathway, induced macroautophagy/autophagy, and led to autophagic cell death (ACD) in vitro and in vivo. In addition, DRD5 protein was highly expressed in the majority of human pituitary adenomas, and treatment of different human pituitary tumor cell cultures with the DRD5 agonist SKF83959 resulted in growth suppression, and the efficacy was correlated with the expression levels of DRD5 in the tumors. Furthermore, we found that DRD5 was expressed in other human cancer cells such as glioblastomas, colon cancer, and gastric cancer. DRD5 activation in these cell lines suppressed their growth, inhibited MTOR activity, and induced autophagy. Finally, in vivo SKF83959 also inhibited human gastric cancer cell growth in nude mice. Our studies revealed novel mechanisms for the tumor suppressive effects of DRD5 agonists, and suggested a potential use of DRD5 agonists as a novel therapeutic approach in the treatment of different human tumors and cancers. 相似文献
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Carlo Follo Dario Barbone William G. Richards Raphael Bueno V. Courtney Broaddus 《Autophagy》2016,12(7):1180-1194
Understanding the role of autophagy in cancer has been limited by the inability to measure this dynamic process in formalin-fixed tissue. We considered that 3-dimensional models including ex vivo tumor, such as we have developed for studying mesothelioma, would provide valuable insights. Using these models, in which we could use lysosomal inhibitors to measure the autophagic flux, we sought a marker of autophagy that would be valid in formalin-fixed tumor and be used to assess the role of autophagy in patient outcome. Autophagy was studied in mesothelioma cell lines, as 2-dimensional (2D) monolayers and 3-dimensional (3D) multicellular spheroids (MCS), and in tumor from 25 chemonaive patients, both as ex vivo 3D tumor fragment spheroids (TFS) and as formalin-fixed tissue. Autophagy was evaluated as autophagic flux by detection of the accumulation of LC3 after lysosomal inhibition and as autophagy initiation by detection of ATG13 puncta. We found that autophagic flux in 3D, but not in 2D, correlated with ATG13 positivity. In each TFS, ATG13 positivity was similar to that of the original tumor. When tested in tissue microarrays of 109 chemonaive patients, higher ATG13 positivity correlated with better prognosis and provided information independent of known prognostic factors. Our results show that ATG13 is a static marker of the autophagic flux in 3D models of mesothelioma and may also reflect autophagy levels in formalin-fixed tumor. If confirmed, this marker would represent a novel prognostic factor for mesothelioma, supporting the notion that autophagy plays an important role in this cancer. 相似文献
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Dandan Song Hongsheng Liang Bo Qu Yijing Li Jingjing Liu Yanan Zhang Lu Li Li Hu Xiangtong Zhang Aili Gao 《Journal of cellular biochemistry》2019,120(1):622-633
Glioma, the most predominant primary malignant brain tumor, remains uncured due to the absence of effective treatments. Hence, it is imperative to develop successful therapeutic agents. This study aimed to explore the antitumor effects and mechanisms of ivermectin (IVM) in glioma cells in vitro and in vivo. The effects of IVM on cell viability, cell cycle arrest, apoptosis rate, and morphological characteristics were determined respectively by MTT assay/colony formation assay, flow cytometry, and transmission electron microscope. In addition, the expression levels of cycle-related and apoptosis-associated proteins were individually examined by Western blot analysis. Moreover, cell proliferation and apoptosis analyses were carried out by TUNEL, Ki-67, cleaved caspase-3, and cleaved caspase-9 immunostaining assay. Our results demonstrated that IVM has a potential dosage-dependent inhibition effect on the apoptosis rate of glioma cells. Meanwhile, the results also revealed that IVM induced apoptosis by increasing caspase-3 and caspase-9 activity, upregulating the expressions of p53 and Bax, downregulating Bcl-2, activating cleaved caspase-3 and cleaved caspase-9, and blocking cell cycle in G0/G1 phase by downregulating levels of CDK2, CDK4, CDK6, cyclin D1, and cyclin E. These findings suggest that IVM has an inhibition effect on the proliferation of glioma cells by triggering cell cycle arrest and inducing cell apoptosis in vitro and in vivo, and probably represents promising agent for treating glioma. 相似文献
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The impact of inflammatory cells in malignant ascites on small intestinal ICCs' morphology and function 下载免费PDF全文
Yan He Xiuli Wang Lei Gao Jiade Li Meisi Yan Duanyang Liu Yufu Wang Lei Zhang Xiaoming Jin 《Journal of cellular and molecular medicine》2015,19(9):2118-2127
Malignant ascites is one of the common complication at the late stage of abdominal cancers, which may deteriorate the environment of abdominal cavity and lead to potential damage of functional cells. Interstitial cells of Cajal (ICCs) are mesoderm‐derived mesenchymal cells that function normal gastrointestinal motility. The pathological changes of ICCs or the reduced number may lead to the motility disorders of gastrointestinal tract. In this study, through analysis of malignant ascites which were obtained from cancer patients, we found that inflammatory cells, including tumour‐infiltrating lymphocytes, accounted for 17.26 ± 1.31% and tumour‐associated macrophages, occupied 19.06 ± 2.27% of total cells in the ascites, suggesting these inflammatory cells, in addition to tumour cells, may exert important influence on the tumour environment of abdominal cavity. We further demonstrated that the number of mice ICCs were significant decreased, as well as morphological and functional damage when ICCs were in the simulated tumour microenvironment in vitro. Additionally, we illustrated intestinal myoelectrical activity reduced and irregular with morphological changes of ICCs using the mice model of malignant ascites. In conclusion, our data suggested that inflammatory cells in malignant ascites may damage ICCs of the small intestine and lead to intestinal motility disorders. 相似文献
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Ladislav Tamás Marta Šimonovi ová Jana Huttová Igor Mistrík 《Acta Physiologiae Plantarum》2004,26(1):85-93
In order to characterise the possible mechanisms involved in Al toxicity some functional characteristics were analysed in
young barley (Hordeum vulgare L.) seedlings cultivated between moistened filter paper. Transfer of germinated barley seeds into hydroponic culture system
caused significant stress, which was manifested by root-growth inhibition and elevated Evans blue uptake of root tips. Hydroponics
caused stress unabled the analysis of Al-induced stress in the young barley roots during the first day of cultivation. Several
(3–4) days are required for adaptation of barley seedlings to hydroponics in spite of strong aeration of the medium. Using
filter paper compared to cultivation in solution application of much higher Al concentrations were required to inhibit root
growth. Al-induced root growth inhibition, Al uptake, damage of plasma-membrane (PM) permeability of root cells, as well as
elevated oxalate oxidase - OxO (EC 1.2.3.4) activity were significantly correlated. While 1 mM Al concentration had no effect
on barley roots growing on filter paper, 5 to 100 mM Al concentration inhibited root growth, enhanced cell death and induced
oxalate oxidase activity with increasing intensity. The time course analysis of OxO gene expression and OxO activity showed
that 10 mM Al increased OxO activity as soon as 3 h after exposure of roots to Al reaching its maximum at about 18 h after
Al application. These results indicate that expression of OxO is activated very early after exposure of barley to Al, suggesting
its role in oxidative stress and subsequent cell death caused by Al toxicity in plants. 相似文献
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Wen Yue Ahmed Hama? Giovanni Tonelli Chantal Bauvy Valérie Nicolas Hugo Tharinger Patrice Codogno Maryam Mehrpour 《Autophagy》2013,9(5):714-729
Breast cancer tissue contains a small population of cells that have the ability to self-renew; these cells are known as cancer stem-like cells (CSCs). We have recently shown that autophagy is essential for the tumorigenicity of these CSCs. Salinomycin (Sal), a K+/H+ ionophore, has recently been shown to be at least 100 times more effective than paclitaxel in reducing the proportion of breast CSCs. However, its mechanisms of action are still unclear. We show here that Sal blocked both autophagy flux and lysosomal proteolytic activity in both CSCs and non-CSCs derived from breast cancer cells. GFP-LC3 staining combined with fluorescent dextran uptake and LysoTracker-Red staining showed that autophagosome/lysosome fusion was not altered by Sal treatment. Acridine orange staining provided evidence that lysosomes display the characteristics of acidic compartments in Sal-treated cells. However, tandem mCherry-GFP-LC3 assay indicated that the degradation of mCherry-GFP-LC3 is blocked by Sal. Furthermore, the protein degradation activity of lysosomes was inhibited, as demonstrated by the rate of long-lived protein degradation, DQ-BSA assay and measurement of cathepsin activity. Our data indicated that Sal has a relatively greater suppressant effect on autophagic flux in the ALDH+ population in HMLER cells than in the ALDH− population; moreover, this differential effect on autophagic flux correlated with an increase in apoptosis in the ALDH+ population. ATG7 depletion accelerated the proapoptotic capacity of Sal in the ALDH+ population. Our findings provide new insights into how the autophagy-lysosomal pathway contributes to the ability of Sal to target CSCs in vitro. 相似文献
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Cursino L Smajs D Smarda J Nardi RM Nicoli JR Chartone-Souza E Nascimento AM 《Journal of applied microbiology》2006,100(4):821-829
AIMS: The antagonistic activity of the Escherichia coli strain H22 against enteric bacteria was studied both in vitro and in vivo. METHODS AND RESULTS: In vitro, bacterial strains belonging to seven of nine genera of the family Enterobacteriaceae (Enterobacter, Escherichia, Klebsiella, Morganella, Salmonella, Shigella and Yersinia) were inhibited by the strain H22. Six days after simultaneous oral inoculation in germ-free mice, E. coli strain H22 reduced the faecal population of Shigella flexneri 4 to undetectable levels (P < 0.05). In ex vivo assay, inhibitory zones against Sh. flexneri 4 were observed around faecal samples from mice inoculated with E. coli strain H22. The in vitro inhibition of Sh. flexneri 4 was shown to be mediated by microcin C7. In addition to microcin C7, strain H22 was shown to produce aerobactin, new variants of colicins E1 and Ib, and bacteriophage particles with morphology similar to the phages of the family Myoviridae. CONCLUSIONS: Altogether, the properties of E. coli H22, observed both under in vitro and in vivo conditions, suggest its potential use as a probiotic strain for livestock and humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The strain H22 was shown to produce several antimicrobial compounds with inhibitory capabilities against pathogenic or potentially pathogenic enterobacteria. 相似文献
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Christopher W. Stackpole Suraj S. Kalbag Laura Groszek 《In vitro cellular & developmental biology. Animal》1995,31(3):244-251
Summary Four mouse B16 melanoma subclones representing distinct stages in the benign-to-malignant progression of that tumor (G3.15,
G3.5, G3.12, and G3.26), and three phenotype conversion variants with enhanced malignancy (G3.15*, G3.5*, and G3.12*), were
comparatively examined for exogenous mitogen and growth factor requirements and for responsiveness to exogenous and endogenous
growth modulators in monolayer culture. Growth behavior in serum-free medium with or without mitogen or growth factor supplements,
and in supplemented quiescent serum-containing medium, confirmed previous indications that the G3.5 and G3.15* phenotypes
were identical, as were the G3.26 and G3.12* phenotypes. However, G3.12 differed from the closest conversion equivalent, G3.5*,
and probably represents an aberrant phenotype within this sequence. There was a direct relationship between degree of malignancy
(G3.15 → G3.5 → G3.5* → G3.26), growth capacity in serum-free medium, and responsiveness to transferrin. Only G3.5*, G3.26,
and G3.12* cells were growth-autonomous in serum-free medium and also highly responsive to mitogens. The polypeptide growth
factors epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor, transforming growth factor-α, and insulinlike growth factor-1 and -2 were generally stimulatory in quiescent medium, but the degree of growth promotion
was unrelated to malignancy level. Transforming growth factor-β1 was inhibitory to the more benign populations (G3.15, G3.5, and G3.15*) but stimulated proliferation of other cells. All
populations produced autocrine fibronectin, and G3.12, G3.5*, G3.26, and G3.12* cells also produced autocrine transferrin.
Only G3.12 cells failed to utilize both of those factors. Reversible mitogen-stimulated G3.12 cell growth was accompanied
by partial and reversible responsiveness to both autocrine transferrin and fibronectin, whereas permanent stimulation by both
factors characterized all growth-autonomous populations. 相似文献
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Tengfang Ling Bo Zhang Weiti Cui Mingzhu Wu Jinshan Lin Wenting Zhou Jingjing Huang Wenbiao Shen 《Plant science》2009,177(4):331-340
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Santosh K. Dasari Eyal Schejter Shani Bialik Aya Shkedy Vered Levin-Salomon Smadar Levin-Zaidman 《Cell cycle (Georgetown, Tex.)》2017,16(21):2003-2010
Autophagy is critical for homeostasis and cell survival during stress, but can also lead to cell death, a little understood process that has been shown to contribute to developmental cell death in lower model organisms, and to human cancer cell death. We recently reported1 on our thorough molecular and morphologic characterization of an autophagic cell death system involving resveratrol treatment of lung carcinoma cells. To gain mechanistic insight into this death program, we performed a signalome-wide RNAi screen for genes whose functions are necessary for resveratrol-induced death. The screen identified GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase, as an important mediator of autophagic cell death. Here we further show the physiological relevance of GBA1 to developmental cell death in midgut regression during Drosophila metamorphosis. We observed a delay in midgut cell death in two independent Gba1a RNAi lines, indicating the critical importance of Gba1a for midgut development. Interestingly, loss-of-function GBA1 mutations lead to Gaucher Disease and are a significant risk factor for Parkinson Disease, which have been associated with defective autophagy. Thus GBA1 is a conserved element critical for maintaining proper levels of autophagy, with high levels leading to autophagic cell death. 相似文献
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Liang Chen Shuning Bi Qiuren Wei Zhijun Zhao Chaojie Wang Songqiang Xie 《Journal of cellular and molecular medicine》2020,24(9):5387-5401
Oesophageal squamous cell carcinoma (ESCC), the most common form of oesophageal malignancies in the Asia‐Pacific region, remains a major clinical challenge. In this study, we found that ivermectin, an effective antiparasitic drug that has been approved for patients to orally treat onchocerciasis for over 30 years, displayed potent antitumour activity against ESCC cells in vitro and in nude mice. We demonstrated that ivermectin significantly inhibited cell viability and colony formation, and induced apoptosis through a mitochondrial‐dependent manner in ESCC cells. Ivermectin also abrogated ESCC cell migration, invasion, as well as the protein levels of MMP‐2 and MMP‐9. Mechanistically, ivermectin strongly inhibited the expression of PAK1; by further gain‐ and loss‐of‐function experiments, we confirmed that PAK1 played a crucial role in ivermectin‐mediated inhibitory effects on ESCC cells. In addition, the data indicated that ivermectin promoted PAK1 degradation through the proteasome‐dependent pathway. Additionally, ivermectin synergized with chemotherapeutic drugs including cisplatin and 5‐fluorouracil to induce apoptosis of ESCC cells. Interestingly, the in vivo experiments also confirmed that ivermectin effectively suppressed tumour growth and lung metastasis of ESCC. Collectively, these results indicate that ivermectin exerts a potent antitumour activity against ESCC and is a promising therapeutic candidate drug for ESCC patients, even those carrying metastasis. 相似文献
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An earlier model in which uptake of essential nutrients for which the cell is auxotrophic, regulates cell division, is discussed
in the light of new experimental findings, specifically the purification of a new type of transport-inhibitory protein from
rat liver and the properties of the protein. The possible role of such proteins in malignant transformation is also discussed. 相似文献