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The tubulin inhibitor MG-2477 induces autophagy-regulated cell death, ROS accumulation and activation of FOXO3 in neuroblastoma.

ABSTRACT: Neuroblastoma is the most frequent extra-cranial solid tumor in children with still high mortality in stage M. Here we studied the tubulin-inhibitor MG-2477 as a possible therapeutic agent for neuroblastoma therapy and uncovered that MG-2477 induces death in neuroblastoma cells independent of PKB-activation status and stage. MG-2477 triggers within 30 minutes extensive autophagosome-formation that finally leads to cell death associated with mitotic catastrophe. Autophagy is critical for MG-2477-induced death and is regulated by the BH3-only protein PMAIP1/NOXA which sequesters the anti-apoptotic BCL2-protein BCLXL and thereby displaces and activates the autophagy-regulator BECN1/beclin1. Knockdown of NOXA or overexpression of its pro-survival binding partners MCL1 and BCLXL counteracts MG-2477-induced cell death. MG-2477 also rapidly induces the repression of the anti-apoptotic protein Survivin, which promotes autophagy and cell death. We further observed the accumulation of reactive oxygen species (ROS) that triggers autophagy induction suggesting a change of the PI3 kinase-III/BECN1 complex and activates the transcription factor FOXO3, which contributes to final cell death induction. The combined data suggest that MG-2477 induces a sequential process of ROS-accumulation, autophagy and FOXO3-activation that leads to cell death in neuroblastoma cells.

SUBMITTER: Hagenbuchner J

PROVIDER: S-EPMC5458265 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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The tubulin inhibitor MG-2477 induces autophagy-regulated cell death, ROS accumulation and activation of FOXO3 in neuroblastoma.

Hagenbuchner Judith J Lungkofler Lorena L Kiechl-Kohlendorfer Ursula U Viola Giampietro G Ferlin Maria Grazia MG Ausserlechner Michael J MJ Obexer Petra P

Oncotarget 20170501 19

Neuroblastoma is the most frequent extra-cranial solid tumor in children with still high mortality in stage M. Here we studied the tubulin-inhibitor MG-2477 as a possible therapeutic agent for neuroblastoma therapy and uncovered that MG-2477 induces death in neuroblastoma cells independent of PKB-activation status and stage. MG-2477 triggers within 30 minutes extensive autophagosome-formation that finally leads to cell death associated with mitotic catastrophe. Autophagy is critical for MG-2477- ...[more]

PMID: 28415610

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Project description:BackgroundNeuroblastoma is the most common solid tumor in childhood and develops from undifferentiated progenitor cells of the sympathetic nervous system. In neuronal tumor cells DNA-damaging chemotherapeutic agents activate the transcription factor FOXO3 which regulates the formation of reactive oxygen species (ROS) and cell death as well as a longevity program associated with therapy resistance. We demonstrated before that C10ORF10/DEPP, a transcriptional target of FOXO3, localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification. In the present study, we investigated the impact of FOXO3 and DEPP on the regulation of autophagy. Autophagy serves to reduce oxidative damage as it triggers a self-degradative process for the removal of aggregated or misfolded proteins and damaged organelles.MethodsThe effect of FOXO3 and DEPP on autophagy induction was analyzed using live cell fluorescence microscopy and immunoblot analyses of SH-EP cells transfected with a plasmid for EYFP-LC3 and with siRNAs specific for LC3, respectively. ROS steady-state levels were measured with reduced MitoTrackerRed CM-H2XROS. Cellular apoptosis was analyzed by flow cytometry and the caspase 3/7 assay.ResultsWe report for the first time that DEPP induces ROS accumulation and thereby mediates the formation of autophagosomes as inhibition of ROS formation by N-acetyl-cysteine completely blocks autophagy. We further demonstrate that H2O2-treatment triggers autophagy-induction by FOXO3-mediated DEPP expression. Importantly, knockdown of DEPP was sufficient to efficiently inhibit autophagy-induction under different stress conditions such as serum starvation and genotoxic stress, suggesting that DEPP expression is critical for the initiation of autophagy in neuroblastoma. FOXO3-triggered autophagy partially protects neuroblastoma cells from cell death. Consistent with this concept, we demonstrate that inhibition of autophagy by LC3-knockdown significantly increased etoposide- and doxorubicin-induced apoptosis. These results were also confirmed by the use of the autophagy-inhibitor chloroquine that significantly enhanced the chemotherapeutic effect of etoposide and doxorubicin in neuronal tumor cells.ConclusionTargeting FOXO3/DEPP-triggered autophagy is a promising strategy to sensitize neuroblastoma cells to chemotherapy.

Project description:BACKGROUND:FOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma. In the present study we investigated the regulation of C10orf10/DEPP by the transcription factor FOXO3. As a physiological function of C10orf10/DEPP has not been described so far we analyzed its effects on cellular ROS detoxification and death sensitization in human neuroblastoma cells. METHODS:The effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS. The cellular localization of DEPP was determined by confocal microscopy of EYFP-tagged DEPP, fluorescent peroxisomal- and mitochondrial probes and co-immunoprecipitation of the PEX7 receptor. RESULTS:We report for the first time that DEPP regulates ROS detoxification and localizes to peroxisomes and mitochondria in neuroblastoma cells. FOXO3-mediated apoptosis involves a biphasic ROS accumulation. Knockdown of DEPP prevented the primary and secondary ROS wave during FOXO3 activation and attenuated FOXO3- and H2O2-induced apoptosis. Conditional overexpression of DEPP elevates cellular ROS levels and sensitizes to H2O2 and etoposide-induced cell death. In neuronal cells, cellular ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that allows binding to the PEX7 receptor and import into peroxisomes, we analyzed the effect of DEPP on cellular detoxification by measuring enzyme activity of catalase. Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells. DEPP directly interacts with the PEX7 receptor and localizes to the peroxisomal compartment. In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells. CONCLUSION:The combined data indicate that in neuroblastoma DEPP localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification, which sensitizes tumor cells to ROS-induced cell death.

Project description:The regulated turnover of synaptic vesicle (SV) proteins is thought to involve the ubiquitin-dependent tagging and degradation through endo-lysosomal and autophagy pathways. Yet, it remains unclear which of these pathways are used, when they become activated, and whether SVs are cleared en masse together with SV proteins or whether both are degraded selectively. Equally puzzling is how quickly these systems can be activated and whether they function in real-time to support synaptic health. To address these questions, we have developed an imaging-based system that simultaneously tags presynaptic proteins while monitoring autophagy. Moreover, by tagging SV proteins with a light-activated ROS generator, Supernova, it was possible to temporally control the damage to specific SV proteins and assess their consequence to autophagy-mediated clearance mechanisms and synaptic function. Our results show that, in mouse hippocampal neurons of either sex, presynaptic autophagy can be induced in as little as 5-10 min and eliminates primarily the damaged protein rather than the SV en masse. Importantly, we also find that autophagy is essential for synaptic function, as light-activated damage to, for example, Synaptophysin only compromises synaptic function when autophagy is simultaneously blocked. These data support the concept that presynaptic boutons have a robust highly regulated clearance system to maintain not only synapse integrity, but also synaptic function.SIGNIFICANCE STATEMENT The real-time surveillance and clearance of synaptic proteins are thought to be vital to the health, functionality, and integrity of vertebrate synapses and are compromised in neurodegenerative disorders, yet the fundamental mechanisms regulating these systems remain enigmatic. Our analysis reveals that presynaptic autophagy is a critical part of a real-time clearance system at synapses capable of responding to local damage of synaptic vesicle proteins within minutes and to be critical for the ongoing functionality of these synapses. These data indicate that synapse autophagy is not only locally regulated but also crucial for the health and functionality of vertebrate presynaptic boutons.

Project description:Acetylshikonin is a shikonin derivative originated from Lithospermum erythrorhizon roots that exhibits various biological activities, including granulation tissue formation, promotion of inflammatory effects, and inhibition of angiogenesis. The anticancer effect of acetylshikonin was also investigated in several cancer cells; however, the effect against renal cell carcinoma (RCC) have not yet been studied. In this study, we aimed to investigate the anticarcinogenic mechanism of acetylshikonin in A498 and ACHN, human RCC cell lines. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), cell counting, and colony forming assay showed that acetylshikonin induced cytotoxic and antiproliferative effects in a dose- and time-dependent manner. Cell cycle analysis and annexin V/propidium iodide (PI) double staining assay indicated the increase of subG1 phase and apoptotic rates. Also, DNA fragmentation was observed by using the TUNEL and comet assays. The intracellular ROS level in acetylshikonin-treated RCC was evaluated using DCF-DA. The ROS level was increased and cell viability was decreased in a dose- and time-dependent manner, while those were recovered when cotreated with NAC. Western blotting analysis showed that acetylshikonin treatment increased the expression of FOXO3, cleaved PARP, cleaved caspase-3, -6, -7, -8, -9, γH2AX, Bim, Bax, p21, and p27 while decreased the expressions of CYP2J2, peroxiredoxin, and thioredoxin-1, Bcl-2, and Bcl-xL. Simultaneously, nuclear translocation of FOXO3 and p27 was observed in cytoplasmic and nuclear fractionated western blot analysis. Acetylshikonin was formerly identified as a novel inhibitor of CYP2J2 protein in our previous study and it was evaluated that CYP2J2 was downregulated in acetylshikonin-treated RCC. CYP2J2 siRNA transfection augmented that apoptotic effect of acetylshikonin in A498 and ACHN via up-regulation of FOXO3 expression. In conclusion, we showed that the apoptotic potential of acetylshikonin against RCC is mediated via increase of intracellular ROS level, activation of FOXO3, and inhibition of CYP2J2 expressions. This study offers that acetylshikonin may be a considerable alternative therapeutic option for RCC treatment by targeting FOXO3 and CYP2J2.

Dataset Information

The tubulin inhibitor MG-2477 induces autophagy-regulated cell death, ROS accumulation and activation of FOXO3 in neuroblastoma.

Publications

The tubulin inhibitor MG-2477 induces autophagy-regulated cell death, ROS accumulation and activation of FOXO3 in neuroblastoma.

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