Rat Glioma Cell-Based Functional Characterization of Anti-Stress and Protein Deaggregation Activities in the Marine Carotenoids, Astaxanthin and Fucoxanthin

Abstract

Stress, protein aggregation, and loss of functional properties of cells have been shown to contribute to several deleterious pathologies including cancer and neurodegeneration. The incidence of these pathologies has also been shown to increase with age and are often presented as evidence to the cumulative effect of stress and protein aggregation. Prevention or delay of onset of these diseases may prove to be unprecedentedly beneficial. In this study, we explored the anti-stress and differentiation-inducing potential of two marine bioactive carotenoids (astaxanthin and fucoxanthin) using rat glioma cells as a model. We found that the low (nontoxic) doses of both protected cells against UV-induced DNA damage, heavy metal, and heat-induced protein misfolding and aggregation of proteins. Their long-term treatment in glioma cells caused the induction of physiological differentiation into astrocytes. These phenotypes were supported by upregulation of proteins that regulate cell proliferation, DNA damage repair mechanism, and glial differentiation, suggesting their potential for prevention and treatment of stress, protein aggregation, and age-related pathologies.

Anti-Stress, Glial- and Neuro-Differentiation Potential of Resveratrol: Characterization by Cellular, Biochemical and Imaging Assays

“…It has been established that the brain functions deteriorate with prolonged exposure to a variety of stresses [12]. Brain-derived cells in culture have been established as a reliable model to study the molecular mechanism of stress-induced damage and its prevention/recovery by natural and synthetic reagents [5,15,39]. In these premises, we used C6 cells in conjunction with a variety of stress models including oxidative stress, smoke, hypoxia, heat-stress, metal and emotional stress to investigate the anti-stress potential of resveratrol.…”

“…Heat stress has been shown to cause misfolding of proteins leading to the abnormalities in the cytoskeleton and cell functions. Heavy metal poisoning has been shown to cause aggregation and sedimentation of proteins called p-bodies or stress granules [5]. These granules are known to cause permanent proteinopathies, not limiting to amyloidosis and dementia.…”

“…Several molecules have been suggested for this purpose. We previously showed the potential of natural compounds like the marine carotenoids and cucurbitacin B-withanone cocktail to induce astrocytic morphology and mixed type biochemical differentiation in rat glioma cells [5,15]. Proteins with the most remarkable changes included GFAP (indicating astrocytic differentiation) and MAP2 (indicating neuronal differentiation).…”

“…Cells (10 × 10 4 /well) were plated in 6-well plates in two identical sets and allowed to settle overnight. The cells were transfected with pGL4-Luc plasmid expressing a full-length Luciferase driven by a constitutive promoter for 48 h, as described previously [5]. Cells were treated with/without 2 µM resveratrol for 4 h, followed by either heat-stress at 42 • C and 5% CO 2 or treatment with 200 µM cobalt chloride (hypoxia) for 2 h, and recovery at 37 • C either in the control or 2 µM resveratrol-supplemented medium for another 24 h. The first set of cells were taken for immunocytochemistry for Luciferase protein as described later and the second was lysed using passive lysis buffer for Luciferase expression estimation using the Luciferase Assay System (Promega, E1501) following the manufacturer's protocol.…”

“…Cells stably expressing GFP protein were prepared as previously described [5]. The 5 × 10 4 cells/well were plated in 6-well plates, allowed to settle overnight, and pretreated with 2 µM resveratrol.…”