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Regulation of apoptotic processes in neurodegeneration and cancer: In vitro studies on human neuroblastoma cells
Stockholm University, Faculty of Science, Department of Neurochemistry.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Apoptosis is a highly controlled process of cell death, which is vital for maintenance of all multicellular organisms. Aberrant regulation of apoptosis can give rise to pathological conditions such as neurodegenerative diseases and cancer. Here, we used different human neuroblastoma cell lines to study mechanisms that may be involved in either neurodegeneration or resistance to cancer treatment. First, we have designed and developed tau-anchored FRET sensors (tAFSs) for live cell imaging of local caspase activation. Using these sensors we showed that the Alzheimer’s disease related neurotoxic peptide, amyloid-β, induced a global activation of caspase-3 and -6, but not -9, in neuronally differentiated SH-SY5Y cells. We also investigated the possible role of NF-κB in the resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in different human neuroblastoma cell lines. While N-type SH-SY5Y and IMR32 cells were unaffected, S-type SK-N-AS cells were clearly sensitized to TRAIL by different NF-κB inhibitory agents. However, no correlation between NF-κB inhibition and sensitization to TRAIL could be observed. Instead, induction of reactive oxygen species (ROS) seemed to play a more important role. Furthermore, using tAFSs we also showed that TRAIL resistance in SK-N-AS cells is mainly due to incomplete activation of caspase-3, and could be reversed by different PKC inhibitors.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2012. , 71 p.
National Category
Natural Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-79157ISBN: 978-91-7447-553-1 (print)OAI: oai:DiVA.org:su-79157DiVA: diva2:547743
Public defence
2012-10-19, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2012-09-27 Created: 2012-08-28 Last updated: 2012-09-05Bibliographically approved
List of papers
1. Anchored FRET sensors detect local caspase activation prior to neuronal degeneration
Open this publication in new window or tab >>Anchored FRET sensors detect local caspase activation prior to neuronal degeneration
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2011 (English)In: Molecular Neurodegeneration, ISSN 1750-1326, E-ISSN 1750-1326, Vol. 6, 35- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Recent studies indicate local caspase activation in dendrites or axons during development and in neurodegenerative disorders such as Alzheimer's disease (AD). Emerging evidences point to soluble oligomeric amyloid-beta peptide as a causative agent in AD.

RESULTS: Here we describe the design of fluorescence resonance energy transfer (FRET)-based caspase sensors, fused to the microtubule associated protein tau. Specific caspase sensors preferentially cleaved by caspase-3, -6 or -9 were expressed in differentiated human neuroblastoma SH-SY5Y cells. The anchoring of the sensors resulted in high FRET signals both in extended neurites and soma and made analysis of spatiotemporal signal propagation possible. Caspase activation was detected as loss of FRET after exposure to different stimuli. Interestingly, after staurosporine treatment caspase-6 activation was significantly delayed in neurites compared to cell bodies. In addition, we show that exposure to oligomer-enriched amyloid-beta peptide resulted in loss of FRET in cells expressing sensors for caspase-3 and -6, but not -9, in both soma and neurites before neurite degeneration was observed.

CONCLUSIONS: Taken together, the results show that by using anchored FRET sensors it is possible to detect stimuli-dependent differential activation of caspases and to distinguish local from global caspase activation in live neuronal cells. Furthermore, in these cells oligomer-enriched amyloid-beta peptide induces a global, rather than local activation of caspase-3 and -6, which subsequently leads to neuronal cell death.

Keyword
Amyloid-beta, Caspases, FRET, Live Cell Imaging, Neurite degeneration, Neurodegeneration, Spatiotemporal analysis
National Category
Biological Sciences Chemical Sciences
Research subject
Biochemistry; Neurochemistry and Neurotoxicology; Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-58646 (URN)10.1186/1750-1326-6-35 (DOI)000291991900001 ()21605370 (PubMedID)
Funder
Swedish Research Council, 2010-4481Swedish Research Council, 2010-4505
Available from: 2011-06-07 Created: 2011-06-07 Last updated: 2017-12-11Bibliographically approved
2. Increased spatiotemporal resolution of caspase activation by anchoring FRET-based sensors to cytoskeleton
Open this publication in new window or tab >>Increased spatiotemporal resolution of caspase activation by anchoring FRET-based sensors to cytoskeleton
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(English)Manuscript (preprint) (Other academic)
Keyword
AFS, Apoptosis, Caspases, FRET, Live Cell Imaging
National Category
Biological Sciences Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-79138 (URN)
Funder
Swedish Research Council
Available from: 2012-08-28 Created: 2012-08-28 Last updated: 2016-01-29Bibliographically approved
3. Sensitization to TRAIL-induced apoptosis in human neuroblastoma SK-N-AS cells by NF-kappa B inhibitors is dependent on reactive oxygen species (ROS)
Open this publication in new window or tab >>Sensitization to TRAIL-induced apoptosis in human neuroblastoma SK-N-AS cells by NF-kappa B inhibitors is dependent on reactive oxygen species (ROS)
2011 (English)In: Journal of Neuro-Oncology, ISSN 0167-594X, E-ISSN 1573-7373, Vol. 104, no 2, 459-472 p.Article in journal (Refereed) Published
Abstract [en]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in a variety of cancer cell lines with almost no toxicity toward normal cells. However, many neuroblastoma cells acquire resistance to TRAIL by mechanisms that are poorly understood. The objective of this study was to investigate involvement of the transcription factor NF-kappa B in the resistance of human neuroblastoma SK-N-AS cells to TRAIL-induced apoptosis. We used five compounds previously reported to inhibit NF-kappa B activity. SN50, curcumin, oridonin, and pyrrolidine dithiocarbamate (PDTC) all sensitized cells to TRAIL-induced apoptosis. In contrast, N-alpha-tosyl-l-phenylalanyl chloromethyl ketone (TPCK) did not affect sensitivity to TRAIL, although reporter gene assay clearly showed inhibition of NF-kappa B activity. In addition, neither curcumin nor oridonin had any inhibitory effect on NF-kappa B activity at concentrations at which sensitization to TRAIL was observed. Instead, the free radical scavenger N-acetyl-l-cysteine (NAC) completely blocked the effect on TRAIL-induced apoptosis caused by curcumin, oridonin, and PDTC. Furthermore, exposure of SK-N-AS cells to H(2)O(2) could mimic the TRAIL-sensitizing effect of other agents. In conclusion, our results suggest that sensitization of neuroblastoma SK-N-AS cells to TRAIL-induced apoptosis is correlated with induction of reactive oxygen species (ROS) rather than inhibition of NF-kappa B.

Keyword
Apoptosis, Neuroblastoma, NF-kappa B, ROS, TRAIL
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-68003 (URN)10.1007/s11060-010-0516-y (DOI)000294263800006 ()
Note

authorCount :2

Available from: 2012-01-03 Created: 2012-01-02 Last updated: 2017-12-08Bibliographically approved
4. TRAIL resistance in human neuroblastoma SK-N-AS cells is dependent on protein kinase C and involves inhibition of caspase-3 proteolytic processing
Open this publication in new window or tab >>TRAIL resistance in human neuroblastoma SK-N-AS cells is dependent on protein kinase C and involves inhibition of caspase-3 proteolytic processing
2012 (English)In: Journal of Neuro-Oncology, ISSN 0167-594X, E-ISSN 1573-7373, Vol. 109, no 3, 503-512 p.Article in journal (Refereed) Published
Abstract [en]

Neuroblastoma is the most common solid extracranial cancer form in childhood with an etiology that is mostly unknown. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been proposed as a promising future anticancer drug candidate, highly malignant neuroblastoma has been reported to acquire TRAIL resistance by mechanisms that are poorly understood. Here, we show by western blot analysis, and live cell imaging using anchored FRET sensors, that the resistance to TRAIL-induced apoptosis in human neuroblastoma SK-N-AS cells depends on an incomplete processing of procaspase-3, generating an immature and catalytically inactive 21 kDa fragment. We have previously shown that the naturally occurring compound curcumin can sensitize SK-N-AS cells to TRAIL. In the present study, we show that curcumin also has a similar effect on human neuroblastoma SHEP1 cells. Furthermore, we show that curcumin and TRAIL co-treatment induces complete maturation and activation of caspase-3 in both cell lines. The mechanisms behind this effect seem to be dependent on protein kinase C (PKC), since inhibition of PKC using bisindolylmaleimide XI, could also sensitize these cells to TRAIL through a similar effect on caspase-3 activation. Moreover, TRAIL co-treatment with bisindolylmaleimide XI or curcumin resulted in down-regulation of X-linked inhibitor of apoptosis protein. In conclusion, our study shows that PKC can be involved in TRAIL resistance in human neuroblastoma cells by preventing caspase-3 maturation.

Keyword
AFS, Caspases, Curcumin, Neuroblastoma, PKC, TRAIL
National Category
Natural Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-79146 (URN)10.1007/s11060-012-0932-2 (DOI)000308441700007 ()22798207 (PubMedID)
Funder
Swedish Research Council
Available from: 2012-08-28 Created: 2012-08-28 Last updated: 2017-12-07Bibliographically approved

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