Chondroitin sulfate protects SH-SY5Y cells from oxidative stress by inducing heme oxygenase-1 via phosphatidylinositol 3-kinase/Akt.
We investigated the mechanism of the neuroprotective properties of chondroitin sulfate (CS), an endogenous perineuronal net glycosaminoglycan, in human neuroblastoma SH-SY5Y cells subjected to oxidative stress. Preincubation with CS for 24 h afforded concentration-dependent protection against H2O2-induced toxicity (50 microM for 24 h) measured as lactic dehydrogenase released to the incubation media; cell death was prevented at the concentrations of 600 and 1000 microM. Cell death caused by a combination of 10 microM rotenone plus 1 microM oligomycin-A (Rot/oligo) was also reduced by CS at concentrations ranging from 0.3 to 100 microM; in this toxicity model, maximum protection was achieved at 3 microM (48%). No significant protection was observed in a cell death model of Ca2+ overload (70 mM K+, for 24 h). H2O2 and Rot/oligo generated reactive oxygen species (ROS) measured as an increase in the fluorescence of dichlorofluorescein diacetate-loaded cells. CS drastically reduced ROS generation induced by both H2O2 (extracellular ROS) and Rot/oligo (intracellular ROS). CS also increased the expression of phosphorylated Akt and heme oxygenase-1 by 2-fold. The protective effects of CS were prevented by chelerythrine, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), cycloheximide, and Sn(IV)-protoporphyrin IX. Taken together, these results show that CS can protect SH-SY5Y cells under oxidative stress conditions by activating protein kinase C, which phosphorylates Akt that, via the phosphatidylinositol 3-kinase/Akt pathway, induces the synthesis of the antioxidant protein heme oxygenase-1.
Cañas
Noelia
N
Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo 4, E-28029 Madrid, Spain.
Valero
Teresa
T
Villarroya
Mercedes
M
Montell
Eulàlia
E
Vergés
Josep
J
García
Antonio G
AG
López
Manuela G
MG
D017209:Apoptosis / Q000187:drug effects; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002809:Chondroitin Sulfates / Q000494:pharmacology*; D004790:Enzyme Induction; D005434:Flow Cytometry; D051547:Heme Oxygenase-1 / Q000096:biosynthesis*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D015151:Immunoblotting; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009840:Oligomycins / Q000633:toxicity; D016877:Oxidants / Q000633:toxicity; D018384:Oxidative Stress / Q000187:drug effects*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D012402:Rotenone / Q000633:toxicity
D018696:Neuroprotective Agents; D009840:Oligomycins; D016877:Oxidants; D017382:Reactive Oxygen Species; D012402:Rotenone; C031004:oligomycin A; D002809:Chondroitin Sulfates; D006861:Hydrogen Peroxide; D007770:L-Lactate Dehydrogenase; D051547:Heme Oxygenase-1; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt