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Qaisiya M.,Italian Liver Foundation ONLUS | Brischetto C.,Italian Liver Foundation ONLUS | Jasprova J.,Charles University | Vitek L.,Charles University | And 3 more authors.
Archives of Toxicology | Year: 2016

Unconjugated bilirubin (UCB) in newborns may lead to bilirubin neurotoxicity. Few studies investigated the activation of endoplasmic reticulum stress (ER stress) by UCB. We performed an in vitro comparative study using undifferentiated SH-SY5Y, differentiated GI-ME-N neuronal cells and human U87 astrocytoma cells. ER stress and its contribution to inflammation and apoptosis induced by UCB were analyzed. Cytotoxicity, ER stress and inflammation were observed only in neuronal cells, despite intracellular UCB accumulation in all three cell types. UCB toxicity was enhanced in undifferentiated SH-SY5Y cells and correlated with a higher mRNA expression of pro-apoptotic CHOP. Mouse embryonic fibroblast knockout for CHOP and CHOP siRNA-silenced SH-SY5Y increased cells viability upon UCB exposure. In SH-SY5Y, ER stress inhibition by 4-phenylbutyric acid reduced UCB-induced apoptosis and decreased the cleaved forms of caspase-3 and PARP proteins. Reporter gene assay and PERK siRNA showed that IL-8 induction by UCB is transcriptionally regulated by NFкB and PERK signaling. These data suggest that ER stress has an important role in the UCB-induced inflammation and apoptosis, and that targeting ER stress may represent a potential therapeutic approach to decrease UCB-induced neurotoxicity. © 2016 Springer-Verlag Berlin Heidelberg


PubMed | Italian Liver Foundation ONLUS and Charles University
Type: | Journal: Archives of toxicology | Year: 2016

Unconjugated bilirubin (UCB) in newborns may lead to bilirubin neurotoxicity. Few studies investigated the activation of endoplasmic reticulum stress (ER stress) by UCB. We performed an in vitro comparative study using undifferentiated SH-SY5Y, differentiated GI-ME-N neuronal cells and human U87 astrocytoma cells. ER stress and its contribution to inflammation and apoptosis induced by UCB were analyzed. Cytotoxicity, ER stress and inflammation were observed only in neuronal cells, despite intracellular UCB accumulation in all three cell types. UCB toxicity was enhanced in undifferentiated SH-SY5Y cells and correlated with a higher mRNA expression of pro-apoptotic CHOP. Mouse embryonic fibroblast knockout for CHOP and CHOP siRNA-silenced SH-SY5Y increased cells viability upon UCB exposure. In SH-SY5Y, ER stress inhibition by 4-phenylbutyric acid reduced UCB-induced apoptosis and decreased the cleaved forms of caspase-3 and PARP proteins. Reporter gene assay and PERK siRNA showed that IL-8 induction by UCB is transcriptionally regulated by NFB and PERK signaling. These data suggest that ER stress has an important role in the UCB-induced inflammation and apoptosis, and that targeting ER stress may represent a potential therapeutic approach to decrease UCB-induced neurotoxicity.


Qaisiya M.,Italian Liver Foundation ONLUS | Coda Zabetta C.D.,Italian Liver Foundation ONLUS | Bellarosa C.,Italian Liver Foundation ONLUS | Tiribelli C.,Italian Liver Foundation ONLUS | Tiribelli C.,University of Trieste
Cellular Signalling | Year: 2014

Unconjugated bilirubin (UCB) is responsible for neonatal jaundice and high level of free bilirubin (Bf) can lead to kernicterus. Previous studies suggest that oxidative stress is a critical component of UCB-induced neurotoxicity. The Nrf2 pathway is a powerful sensor for cellular redox state and is activated directly by oxidative stress and/or indirectly by stress response protein kinases. Activated Nrf2 translocates to nucleus, binds to Antioxidant Response Element (ARE), and enhances the up-regulation of cytoprotective genes that mediate cell survival. The aim of the present study was to investigate the role of Nrf2 pathway in cell response to bilirubin mediated oxidative stress in the neuroblastoma SH-SY5Y cell line. Cells exposed to a toxic concentration of UCB (140. nM Bf) showed an increased intracellular ROS levels and enhanced nuclear accumulation of Nrf2 protein. UCB stimulated transcriptional induction of ARE-GFP reporter gene and induced mRNA expression of multiple antioxidant response genes as: xCT, Gly1, γGCL-m, γGCL-c, HO-1, NQO1, FTH, ME1, and ATF3. Nrf2 siRNA decreased UCB induced mRNA expression of HO1 (75%), NQO1 (54%), and FTH (40%). The Nrf2-related HO-1 induction was reduced to 60% in cells pre-treated with antioxidant (NAC) or specific signaling pathway inhibitors for PKC, P38α and MEK1/2 (80, 40 and 25%, respectively). In conclusion, we demonstrated that SH-SY5Y cells undergo an adaptive response against UCB-mediated oxidative stress by activation of multiple antioxidant response, in part through Nrf2 pathway. © 2013 Elsevier Inc.


PubMed | University of Trieste and Italian Liver Foundation ONLUS
Type: Journal Article | Journal: Cellular signalling | Year: 2014

Unconjugated bilirubin (UCB) is responsible for neonatal jaundice and high level of free bilirubin (Bf) can lead to kernicterus. Previous studies suggest that oxidative stress is a critical component of UCB-induced neurotoxicity. The Nrf2 pathway is a powerful sensor for cellular redox state and is activated directly by oxidative stress and/or indirectly by stress response protein kinases. Activated Nrf2 translocates to nucleus, binds to Antioxidant Response Element (ARE), and enhances the up-regulation of cytoprotective genes that mediate cell survival. The aim of the present study was to investigate the role of Nrf2 pathway in cell response to bilirubin mediated oxidative stress in the neuroblastoma SH-SY5Y cell line. Cells exposed to a toxic concentration of UCB (140 nM Bf) showed an increased intracellular ROS levels and enhanced nuclear accumulation of Nrf2 protein. UCB stimulated transcriptional induction of ARE-GFP reporter gene and induced mRNA expression of multiple antioxidant response genes as: xCT, Gly1, GCL-m, GCL-c, HO-1, NQO1, FTH, ME1, and ATF3. Nrf2 siRNA decreased UCB induced mRNA expression of HO1 (75%), NQO1 (54%), and FTH (40%). The Nrf2-related HO-1 induction was reduced to 60% in cells pre-treated with antioxidant (NAC) or specific signaling pathway inhibitors for PKC, P38 and MEK1/2 (80, 40 and 25%, respectively). In conclusion, we demonstrated that SH-SY5Y cells undergo an adaptive response against UCB-mediated oxidative stress by activation of multiple antioxidant response, in part through Nrf2 pathway.

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