Effects of dopamine on LC3-II activation as a marker of autophagy in a neuroblastoma cell model

Giménez-Xavier, Pol; Francisco, Roser; Santidrián, Antonio F.; Gil, Joan; Ambrosio, Santiago

doi:10.1016/j.neuro.2009.04.007

Cited by 44 publications

(32 citation statements)

References 47 publications

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“…In this system, we quantified autophagy by assessing the LC3-II/ LC3-I ratio, as we observed that, in SH-SY5Y cells, autophagy activation was variably associated with LC3-II increase and/or LC3-I decrease. 31 Under both nutrient-rich and starvation conditions, PINK1-FL cells showed a significant increase of the LC3-II/LC3-I ratio compared with control cells. Activation of autophagy was further confirmed by showing reduction of P62 levels in cells expressing PINK1 versus controls (Figure 5c).…”

Section: Resultsmentioning

confidence: 88%

The Parkinson-associated protein PINK1 interacts with Beclin1 and promotes autophagy

Michiorri

Gelmetti

Giarda³

et al. 2010

Cell Death Differ

229

176

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Mutations in the PINK1 gene cause autosomal recessive Parkinson's disease. The PINK1 gene encodes a protein kinase that is mitochondrially cleaved to generate two mature isoforms. In addition to its protective role against mitochondrial dysfunction and apoptosis, PINK1 is also known to regulate mitochondrial dynamics acting upstream of the PD-related protein Parkin. Recent data showed that mitochondrial Parkin promotes the autophagic degradation of dysfunctional mitochondria, and that stable PINK1 silencing may have an indirect role in mitophagy activation. Here we report a new interaction between PINK1 and Beclin1, a key pro-autophagic protein already implicated in the pathogenesis of Alzheimer's and Huntington's diseases. Both PINK1 N-and C-terminal are required for the interaction, suggesting that full-length PINK1, and not its cleaved isoforms, interacts with Beclin1. We also demonstrate that PINK1 significantly enhances basal and starvation-induced autophagy, which is reduced by knocking down Beclin1 expression or by inhibiting the Beclin1 partner Vps34. A mutant, PINK1 W437X , interaction of which with Beclin1 is largely impaired, lacks the ability to enhance autophagy, whereas this is not observed for PINK1 G309D , a mutant with defective kinase activity but unaltered ability to bind Beclin1. These findings identify a new function of PINK1 and further strengthen the link between autophagy and proteins implicated in the neurodegenerative process. Parkinson's disease (PD) is a frequent neurodegenerative disorder resulting from massive degeneration of the dopaminergic neurons in the substantia nigra. Although most cases are sporadic, several genes are known to cause familial PD, especially with early onset. 1 Mutations in the PINK1 gene are the second most frequent cause of autosomal recessive PD after those in the Parkin gene. 2,3 The PINK1 gene encodes a serine-threonine kinase with an N-terminal mitochondrial import sequence, first characterized as a protein aimed at maintaining mitochondrial integrity and preventing apoptosis in response to cellular stressors. 2,[4][5][6][7][8] This neuroprotective role is partly exerted through phosphorylation of the mitochondrial chaperon, TRAP1, although cytoplasm-restricted PINK1 was also shown to protect against MPTP damage. 9,10 The full-length PINK1 (PINK1-FL) is processed within mitochondria to generate two mature proteins; 4,11 all three isoforms localize both to the mitochondria and cytosol, their relative ratio being regulated by several factors. [10][11][12][13] Increasing data have demonstrated that absence of functional PINK1 induces abnormalities of mitochondrial morphology. 6,14,15 In several studies (mostly in Drosophila), PINK1 was shown to promote fission acting upstream of the Fis1-Drp1 machinery, and the mitochondrial phenotype observed in PINK1 knockout flies or silenced cells was associated to reduced fission. 16,17 Subsequent studies in mammalian cell systems contradicted these results, demonstrating that mutant or silenced PINK1 resulted in incre...

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Section: Resultsmentioning

confidence: 88%

The Parkinson-associated protein PINK1 interacts with Beclin1 and promotes autophagy

Michiorri

Gelmetti

Giarda³

et al. 2010

Cell Death Differ

229

176

View full text Add to dashboard Cite

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“…In the majority of the above-mentioned experimental conditions there were no significant changes in Beclin-1 levels, and in thyroid cancer cells a decrease of Beclin-1 protein level has been reported [78]. An increase of LC3 and LC3-II has been reported also after treatment with NPI-0052, ALLN (N-acetyl-leucyl-leucyl-norleucinal), epoxomicin and lactacystin of human prostate cancer cells [84], HCT116 cells [75], thyroid cancer cells [78], human dopaminergic neuroblastoma SH-SY5Y cells and dopaminergic neurons obtained from UPS-impaired mouse model of Parkinson's disease (PD) [85,86]. Differently than in the thyroid cancer cells [78], both in SH-SY5Y cells and in the midbrain of the UPS-impaired mouse model of PD, treatment with lactacystin significantly increased the level of not only LC3-I/II but also Beclin-1 protein, and reduced the levels of p-mTOR and mTOR [85].…”

Section: Effects Of Different Classes Of Proteasome Inhibitors On Autmentioning

confidence: 99%

Crosstalk between autophagy and proteasome protein degradation systems: possible implications for cancer therapy

Wójcik

2014

Folia Histochem Cytobiol.

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Abstract:The Ubiquitin-Proteasomes System (UPS) and autophagy, two main intracellular protein degradation pathways within the eukaryotic cells which were originally regarded as rather independent, seem to be very closely related. Proteasome inhibitors, including the multipathway inhibitor bortezomib, are drawing increased attention for their therapeutic potential in the treatment of chronic inflammation and cancer, especially tumours with a high degree of malignancy. The over-activation of autophagy induces cell death and may act as a powerful tumour-suppressing mechanism. However, autophagy, serving as an important mechanism to generate nutrients in time of cellular stresses, may directly contribute to the survival of cells treated with proteasome inhibitors, and in consequence, may decrease the effectiveness of therapy. Results of studies performed on several cancer cell lines demonstrated synergy between proteasome inhibitors and autophagy inhibitors. Those results became the base for ongoing clinical trials investigating autophagy inhibition in combination with anti-cancer therapies, including bortezomib. This review provides summary of the latest data on the functioning of the UPS and the mechanisms of autophagy. The new insights describing the main pathways of autophagy activation in response to UPS inhibition related to: (i) Unfolded Protein Response, (ii) PI3K/Akt/mTOR pathway, and (iii) formation of aggresomes, are discussed. It is concluded that concomitant inhibition of the two main cellular protein degradation systems may provide new therapeutic modalities for cancer treatment.

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“…In the PC-3 prostate cancer cell line, SFN causes redistribution of LC3 into autophagosomes and 3-methyladenine (3-MA), an inhibitor of autophagy, is able to restrain this process (8). The adenine analogue blocks class III PI3K complex activities and the mammalian target of rapamycin pathway, leading to the inhibition of autophagy (17). The present study used the BE(2)-C human neuroblastoma cell line containing the amplified MYCN gene (responsible for high levels of aggressiveness) and P53 mutation, to characterize the effects of synthetic SFN on cells in vitro.…”

Section: Inhibition Of Autophagy By 3-methyladenine Potentiates Sulfomentioning

confidence: 99%

Inhibition of autophagy by 3-methyladenine potentiates sulforaphane-induced cell death of BE(2)-C human neuroblastoma cells

Horwacik¹,

Gaik²,

Durbas³

et al. 2012

Molecular Medicine Reports

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Sulforaphane (SFN) is an isothiocyanate present in cruciferous vegetables, which has been shown to exert an anti-cancer effect when tested in vitro and in vivo. The anti-cancer effects of SFN encompass induction of cytoprotective autophagy; therefore, the present study aimed to determine whether the chemopreventive activity of SFN may be potentiated by inhibition of autophagy. The present study provided detailed insight into the susceptibility of human neuroblastoma cells to treatment with synthetic SFN, in combination with an inhibitor of autophagy, 3-methyladenine (3-MA). The present study confirmed the suppression of the viability of the human neuroblastoma cell line BE(2)-C by SFN and reported the inhibition of DNA synthesis, as determined by a decrease in tritiated thymidine incorporation. Furthermore, the results verified the effectiveness of SFN in inducing apoptosis in the BE(2)-C cell line as demonstrated by caspase activation, increased protein expression levels of B-cell lymphoma 2-associated X protein and loss of mitochondrial membrane potential. Combined treatment of the cells with SFN with 3-MA proved to be effective in decreasing cell viability, through a mechanism that may proceed via the early induction of autophagy by SFN, followed by induction of apoptosis, as well as inhibition of autophagy by 3-MA.

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Effects of dopamine on LC3-II activation as a marker of autophagy in a neuroblastoma cell model

Cited by 44 publications

References 47 publications

The Parkinson-associated protein PINK1 interacts with Beclin1 and promotes autophagy

The Parkinson-associated protein PINK1 interacts with Beclin1 and promotes autophagy

Crosstalk between autophagy and proteasome protein degradation systems: possible implications for cancer therapy

Inhibition of autophagy by 3-methyladenine potentiates sulforaphane-induced cell death of BE(2)-C human neuroblastoma cells

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