Ultrastructural and Immunocytochemical Characterization of Autophagic Vacuoles in Isolated Hepatocytes: Effects of Vinblastine and Asparagine on Vacuole Distributions

Fengsrud, Monica; Roos, Norbert; Berg, Trond; Liou, Willisa; Slot, Jan W.; Seglen, Per Ottar

doi:10.1006/excr.1995.1402

Cited by 116 publications

(110 citation statements)

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“…The dose-response curves of the cell survival after 24, 48 and 72 h of treatment revealed that both sensitive and resistant osteosarcoma cell lines showed a percent viability of about 50% after treatment VOA, given alone to both wild-type and MDR osteosarcoma cells, might induce death outcome alternative to apoptosis, as revealed for other natural alkaloid compounds, 29,30 and to study in depth its mechanism of action.…”

Section: Discussionmentioning

confidence: 93%

The plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells

Meschini¹,

Condello²,

Calcabrini³

et al. 2008

Autophagy

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In our previous studies, the bisindolic alkaloid voacamine (VOA), isolated from the plant Peschiera fuchsiaefolia, proved to exert a chemosensitizing effect on cultured multidrug resistant (MDR) osteosarcoma cells exposed to doxorubicin (DOX). In particular, VOA was capable of inhibiting P-glycoprotein action in a competitive way, thus explaining the enhancement of the cytotoxic effect induced by DOX on MDR cells. Afterwards, preliminary observations suggested that such an enhancement did not involve the apoptotic process but was due instead to the induction of autophagic cell death. The results of the present investigation demonstrate that the plant alkaloid VOA is an autophagy inducer able to exert apoptosis-independent cytotoxic effect on both wild-type and MDR tumor cells. In fact, under treatment condition causing about 50 percent of cell death, no evidence of apoptosis could be revealed by microscopical observations, Annexin V-FITC labeling and analysis of PARP cleavage, whereas the same cells underwent apoptosis when treated with apoptosis inducers, such as doxorubicin and staurosporine. Conversely, VOA-induced autophagy was clearly evidentiated by electron microscopy observations, monodansylcadaverine staining, LC3 expression, and conversion. These results were confirmed by the analysis of the modulating effects of the pretreatment with autophagy inhibitors prior to VOA administration. In addition, transfection of osteosarcoma cells with siRNA against ATG genes reduced VOA cytotoxicity. In conclusion, considering the very debated dual role of autophagy in cancer cells (protective or lethal, pro-or anti-apoptotic) our findings seem to demonstrate, at least in vitro, that a natural product able to induce autophagy can be effective against drug resistant tumors, either used alone or in association with conventional chemotherapeutics.

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Section: Discussionmentioning

confidence: 93%

The plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells

Meschini¹,

Condello²,

Calcabrini³

et al. 2008

Autophagy

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“…Transmission electron microscope examination [25] After treatment with CrTX, the cells were fixed in ice-cold 2.5% glutaraldehyde in 0.1 mol/L PBS and preserved at 4 °C for further processing. When processing resumed, the cells were post fixed in 1% osmium tetroxide in the same buffer, dehydrated in graded alcohols, embedded in Epon 812, sectioned with ultramicrotome, stained with uranyl acetate and lead citrate, followed by an examination with a transmission electron microscope (Philips Tecnai 10, Eindhoven, the Netherlands).…”

Section: Drug Preparation Crtx Was Supplied By Celtic Biotechmentioning

confidence: 99%

Autophagy is involved in cytotoxic effects of crotoxin in human breast cancer cell line MCF-7 cells

Yan

Yang

Qin

et al. 2007

Acta Pharmacologica Sinica

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Aim: To investigate the role of crotoxin (CrTX)‐induced autophagy in the death of MCF‐7 cells, a caspase‐3‐deficient, human breast cancer cell line. Methods: Cultured MCF‐7 cells were treated with various doses of CrTX, a phospholipase A2 (PLA2) isolated from the venom of the South American rattlesnake, Crotalus durissus terrificus. The cytotoxicity of CrTX in the presence and absence of caspase inhibitors was measured with methyl thiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) leakage assays. The activation of autophagy was determined with transmission electron microscope and monodansylcadaverin (MDC) labeling. The upregulation of lysosomal enzymes, the release of cyto‐chrome c (cyto‐c), and the nuclear translocation of the apoptosis inducing factor (AIF) were examined by immunoblotting and immunofluorescence. Results: CrTX inhibited the viability of MCF‐7 cells in a dose‐ and time‐dependent manner. CrTX‐activated autophagy was revealed by punctuate MDC labeling, and an increase in the formation of autophagosomes as well as apoptosis, as evidenced by nuclear condensation and fragmentation. The activation of cathepsin B, D, and L, in addition to the release of cytochrome c and the relocation of AIF into nuclei, were observed after CrTX treatment. Autophagy inhibitors 3‐methyladenine (3‐MA), NH4Cl, and the pan‐caspase inhibitor, Z‐Val‐Ala‐Asp‐fluoromethylketone (Z‐Vad‐fmk), attenuated CrTX‐induced cell death. Conclusion: An autophagic mechanism contributes to the apoptosis of MCF‐7 cells induced by CrTX.

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“…Autophagy starts with the formation of a multilayer membrane-bound autophagosome that sequesters fractions of the cytoplasm (4,5). In mammalian cells, most of autophagosomes receive inputs from endocytic compartments before fusing with lysosomes where the degradation of the sequestered material is completed (6,7).…”

mentioning

confidence: 99%

Ceramide-mediated Macroautophagy Involves Inhibition of Protein Kinase B and Up-regulation of Beclin 1

Scarlatti

Bauvy

Ventruti

et al. 2004

Journal of Biological Chemistry

415

376

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The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C 2 -ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B 1 . Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C 2 -ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C 2 -ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifendependent up-regulation. Tamoxifen and C 2 -ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.Macroautophagy or autophagy is an evolutionary conserved lysosomal pathway involved in the turnover of long lived proteins and organelles (1-3). Autophagy starts with the formation of a multilayer membrane-bound autophagosome that sequesters fractions of the cytoplasm (4, 5). In mammalian cells, most of autophagosomes receive inputs from endocytic compartments before fusing with lysosomes where the degradation of the sequestered material is completed (6, 7).The physiological importance of autophagy during starvation has been primarily highlighted in rat liver (8) and then in different cell types (reviewed in Refs. 9 and 10). At the same time, the term autophagic cell death or type II programmed cell death (PCD II) has been introduced (11) to describe a cell death different from apoptosis or type I programmed cell death (PCD I) (reviewed in Refs. 12 and 13). The recent progress made in characterization of the molecular mechanism controlling autophagy has brought a renewal of interest for this process (14). There is now evidence for the role of autophagy during development (15-17), in the life span extension (15,18), and in disease such as cancer (19,20), neurodegenerative disease (21, 22), and myopathies (23,24).A family of autophagy-related genes discovered in yeast and almost integrally conserved in all eucaryotic phyla controls the formation of the autophagosome (25). Two conjugation systems (Atg5p-Atg12p and Atg8p lipidation) are involved in...

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Ultrastructural and Immunocytochemical Characterization of Autophagic Vacuoles in Isolated Hepatocytes: Effects of Vinblastine and Asparagine on Vacuole Distributions

Cited by 116 publications

References 0 publications

The plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells

The plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells

Autophagy is involved in cytotoxic effects of crotoxin in human breast cancer cell line MCF-7 cells

Ceramide-mediated Macroautophagy Involves Inhibition of Protein Kinase B and Up-regulation of Beclin 1

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