sirolimus and monodansylcadaverine

Changes in the two main intracellular degradation systems, the Ubiquitin-Proteasome System and the Autophagy-Lysosome pathway (ALP) are widely discussed as a hallmark of the aging process. To follow the age-related behavior of both degradation systems we examined their impact on ferritin, known to be degradable by both. Ferritin H was analyzed in young and senescent human fibroblasts, revealing a higher steady-state level in the senescent cells. By blocking both proteolytic systems, we confirmed that particularly the ALP plays a crucial role in ferritin H turnover. However, an unexpected increase in lysosomal activity in the senescent cells, suggests a dysregulation in the autophagy pathway. To further investigate the impaired ferritin H turnover, confocal microscopic colocalization studies of ferritin H with lysosomal-associated membrane protein 2a (Lamp2a) and monodansylcadaverine (MDC) were performed and clearly revealed the degradation of ferritin by macroautophagy. By induction of autophagy via inhibition of mTOR using rapamycin an increase of ferritin H turnover was obtained in senescent cells, demonstrating a mTOR dependent reduction of autophagy in senescent human fibroblasts.

Topics: Apoferritins; Autophagy; Cadaverine; Cellular Senescence; Deferoxamine; Enzyme Inhibitors; Fibroblasts; Foreskin; Gene Expression; Hemin; Humans; Kinetics; Lysosomal-Associated Membrane Protein 2; Lysosomes; Male; Primary Cell Culture; Proteasome Endopeptidase Complex; Proteolysis; Sirolimus; TOR Serine-Threonine Kinases

N-n-butyl haloperidol iodide (F2), a novel compound derived from haloperidol, protects against the damaging effects of ischemia/reperfusion (I/R) injury in vitro and in vivo. In this study, we hypothesized the myocardial protection of F2 on cardiomyocyte hypoxia/reoxygenation (H/R) injury is mediated by inhibiting autophagy in H9c2 cells. The degree of autophagy by treatment with F2 exposed to H/R in H9c2 cell was characterized by monodansylcadaverine, transmission electron microscopy, and expression of autophagy marker protein LC3. Our results indicated that treatment with F2 inhibited autophagy in H9c2 cells exposed to H/R. 3-methyladenine, an inhibitor of autophagy, suppressed H/R-induced autophagy, and decreased apoptosis, whereas rapamycin, a classical autophagy sensitizer, increased autophagy and apoptosis. Mechanistically, macrophage migration inhibitory factor (MIF) was inhibited by F2 treatment after H/R. Accordingly, small interfering RNA (siRNA)-mediated MIF knockdown decreased H/R-induced autophagy. In summary, F2 protects cardiomyocytes during H/R injury through suppressing autophagy activation. Our results provide a new mechanistic insight into a functional role of F2 against H/R-induced cardiomyocyte injury and death.

Topics: Adenine; Animals; Apoptosis; Autophagy; Cadaverine; Cell Line; Cytoprotection; Dose-Response Relationship, Drug; Haloperidol; Intramolecular Oxidoreductases; Macrophage Migration-Inhibitory Factors; Microtubule-Associated Proteins; Myocardial Reperfusion Injury; Myocytes, Cardiac; Protective Agents; Rats; RNA Interference; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection

Rapamycin (Rapa), an inhibitor of mammalian target of Rapamycin (mTOR), is an immunosuppressive agent that has anti-proliferative effects on some tumors. This study aims to investigate the effects of Rapa suppressing proliferation of pancreatic carcinoma PC-2 cells in vitro and its molecular mechanism involved in antitumor activities. MTT assays showed that the inhibition of proliferation of PC-2 cells in vitro was in a time- and dose-dependent manner. By using transmission electron microscopy, apoptosis bodies and formation of abundant autophagic vacuoles were observed in PC-2 cells after Rapa treatment. Flow cytometry assays also showed Rapa had a positive effect on apoptosis. MDC staining showed that the fluorescent density was higher and the number of MDC-labeled particles in PC-2 cells was greater in the Rapa treatment group than in the control group. RT-PCR revealed that the expression levels of p53, Bax and Beclin 1 were up-regulated in a dose-dependent manner, indicating that Beclin 1 was involved in Rapa induced autophagy and Rapa induced apoptosis as well as p53 up-regulation in PC-2 cells. The results demonstrated that Rapa could effectively inhibit proliferation and induce apoptosis and autophagy in PC-2 cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; bcl-2-Associated X Protein; Beclin-1; Cadaverine; Cell Line, Tumor; Cell Proliferation; Cell Shape; Flow Cytometry; Humans; Membrane Proteins; Mice; NIH 3T3 Cells; Pancreatic Neoplasms; RNA, Messenger; Sirolimus; TOR Serine-Threonine Kinases; Vacuoles

Despite of similarities between glioma stem/progenitor cells (GSPCs) and neural stem/progenitor cells (NSPCs), inhibition of differentiation is a distinct characteristic of GSPCs. In this study, we investigated the effects of autophagy impairment on inhibition of differentiation of GSPC, and its molecular mechanism. GSPCs were kept by our laboratory; NSPCs were isolated from human fetal brain tissue. We found that the autophagic activity in GSPCs was significantly lower than that in NSPCs. However, the autophagic activity markedly increased after GSPCs were induced to differentiate by fetal calf serum (FCS). The autophagy inhibitors 3-methyladenine and Bafilomycin A1 (BFA) inhibited the FSC-induced differentiation of GSPCs. And autophagy activator Rapamycin could promote differentiation of GSPCs. In order to disclose whether the loss of PTEN in GSPC is related to the deficiency of autophagic activity in GSPCs (for PTEN being lost in the GSPCs studied by us), we introduced the wild type gene of PTEN into GSPCs, and found that the autophagic activity was restored significantly after the gene transduction. The low autophagic activity in GSPCs leads to the inhibition of differentiation of GSPCs, and the loss of PTEN in GSPCs probably is an underlying mechanism for the low autophagic activity in GSPCs. These results suggest that bust autophagic activity target at PTEN might be a potential therapy target for glioma therapy.

Topics: Adenine; Autophagy; Blotting, Western; Cadaverine; Cell Differentiation; Cells, Cultured; Central Nervous System Agents; Glioma; Humans; Immunohistochemistry; Macrolides; Microscopy, Electron, Transmission; Neurons; PTEN Phosphohydrolase; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus; Stem Cells; Transduction, Genetic

The induction of the autophagy machinery, a process for the catabolism of cytosolic proteins and organelles, constitutes a crucial mechanism in innate immunity. However, the involvement of autophagy in human neutrophils and the possible inducers of this process have not been completely elucidated. In this study, the induction of autophagy was examined in human neutrophils treated with various activators and detected by the formation of acidified autophagosomes through monodansylcadaverine staining and via LC-3B conversion screened by immunoblotting and immunofluorescence confocal microscopy. In addition, the expression of the ATG genes was assessed by real-time RT-PCR. We provide evidence that autophagy is implicated in human neutrophils in both a phagocytosis-independent (rapamycin, TLR agonists, PMA) and phagocytosis (Escherichia coli)-dependent initiation manner. ROS activation is a positive mechanism for autophagy induction in the case of PMA, TLR activation and phagocytosis. Furthermore, LC3B gene expression was uniformly upregulated, indicating a transcriptional level of regulation for the autophagic machinery. This study provides a stepping stone toward further investigation of autophagy in neutrophil-driven inflammatory disorders.

Topics: Adenine; Autophagy; Cadaverine; Chromones; Coloring Agents; Escherichia coli; Guanosine; Humans; Hydrogen-Ion Concentration; Inflammation; Microscopy, Confocal; Morpholines; Neutrophils; Phagosomes; Poly I-C; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sirolimus; Small Ubiquitin-Related Modifier Proteins; Tetradecanoylphorbol Acetate; Toll-Like Receptors; Transcription, Genetic; Vacuoles

Morphological and biochemical studies have shown that autophagosomes fuse with endosomes forming the so-called amphisomes, a prelysosomal hybrid organelle. In the present report, we have analyzed this process in K562 cells, an erythroleukemic cell line that generates multivesicular bodies (MVBs) and releases the internal vesicles known as exosomes into the extracellular medium. We have previously shown that in K562 cells, Rab11 decorates MVBs. Therefore, to study at the molecular level the interaction of MVBs with the autophagic pathway, we have examined by confocal microscopy the fate of MVBs in cells overexpressing green fluorescent protein (GFP)-Rab11 and the autophagosomal protein red fluorescent protein-light chain 3 (LC3). Autophagy inducers such as starvation or rapamycin caused an enlargement of the vacuoles decorated with GFP-Rab11 and a remarkable colocalization with LC3. This convergence was abrogated by a Rab11 dominant negative mutant, indicating that a functional Rab11 is involved in the interaction between MVBs and the autophagic pathway. Interestingly, we presented evidence that autophagy induction caused calcium accumulation in autophagic compartments. Furthermore, the convergence between the endosomal and the autophagic pathways was attenuated by the Ca2+ chelator acetoxymethyl ester (AM) of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), indicating that fusion of MVBs with the autophagosome compartment is a calcium-dependent event. In addition, autophagy induction or overexpression of LC3 inhibited exosome release, suggesting that under conditions that stimulates autophagy, MVBs are directed to the autophagic pathway with consequent inhibition in exosome release.

Topics: Amino Acids; Autophagy; Autophagy-Related Protein 12; Cadaverine; Calcium; Chelating Agents; Culture Media, Serum-Free; Cytoplasmic Vesicles; Egtazic Acid; Exocytosis; HSC70 Heat-Shock Proteins; Humans; K562 Cells; Membrane Fusion; Microtubule-Associated Proteins; Models, Biological; Monensin; Nocodazole; Proteins; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins; Recombinant Fusion Proteins; RNA, Small Interfering; Sirolimus; Small Ubiquitin-Related Modifier Proteins; Transfection; Vinblastine

Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic components and organelles in a vacuole called an autophagosome that finally fuses with the lysosome. Rab7 is a member of the Rab family involved in transport to late endosomes and in the biogenesis of the perinuclear lysosome compartment. To assess the role of Rab7 in autophagy we stably transfected CHO cells with wild-type pEGFP-Rab7, and the mutants T22N (GDP form) and Q67L (GTP form). Autophagy was induced by amino acid starvation and the autophagic vacuoles were labeled with monodansylcadaverine. By fluorescence microscopy we observed that Rab7wt and the active mutant Rab7Q67L were associated with ring-shaped vesicles labeled with monodansylcadaverine indicating that these Rab proteins associate with the membrane of autophagic vesicles. As expected, in cells transfected with the negative mutant Rab7T22N the protein was diffusely distributed in the cytosol. However, upon induction of autophagy by amino acid starvation or by rapamycin treatment this mutant clearly decorated the monodansylcadaverine-labeled vesicles. Furthermore, a marked increase in the size of the monodansylcadaverine-labeled vacuoles induced by starvation was observed by overexpression of the inactive mutant T22N. Similarly, there was an increase in the size of vesicles labeled with LC3, a protein that specifically localizes on the autophagosomal membrane. Taken together the results indicate that a functional Rab7 is important for the normal progression of autophagy.

Topics: Animals; Autophagy; Cadaverine; Cell Fractionation; CHO Cells; Cricetinae; Cricetulus; Cytosol; Fluorescent Dyes; Green Fluorescent Proteins; Lysosomes; Microscopy, Fluorescence; Mutation; Plasmids; rab GTP-Binding Proteins; Recombinant Fusion Proteins; Sirolimus; Vacuoles