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

Abstract

Environmental stress, exhaustive industrialization and the use of chemicals in our daily lives contribute to increasing incidence of cancer and other pathologies. Although the cancer treatment has revolutionized in last 2-3 decades, shortcomings such as (i) extremely high cost of treatment, (ii) poor availability of drugs, (iii) severe side effects and (iv) emergence of drug resistance have prioritized the need of developing alternate natural, economic and welfare (NEW) therapeutics reagents. Identification and characterization of such anti-stress NEW drugs that not only limit the growth of cancer cells but also reprogram them to perform their specific functions are highly desired. We recruited rat glioma- and human neuroblastoma-based assays to explore such activities of resveratrol, a naturally occurring stilbenoid. We demonstrate that nontoxic doses of resveratrol protect cells against a variety of stresses that are largely involved in age-related brain pathologies. These included oxidative, DNA damage, metal toxicity, heat, hypoxia, and protein aggregation stresses. Furthermore, it caused differentiation of cells to functional astrocytes and neurons as characterized by the upregulation of their specific protein markers. These findings endorse multiple bioactivities of resveratrol and encourage them to be tested for their benefits in animal models and humans.

Keywords: DNA damage; Oxidative stress; differentiation; hypoxia; old age; protection; protein aggregation; resveratrol.

Figure 1

Anti-stress screening of resveratrol in C6 cells. (A). Dose-dependent toxicity of chemical stresses listed in (B) and identification of their IC₂₀ doses using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cell viability assay. (C). Stress protection screening assay showing the anti-stress potential of resveratrol in C6 cells in pretreatment (PREP-defined by 24 h resveratrol pre-treatment followed by stress and recovery in resveratrol supplemented medium) and recovery (RECO-defined by stress followed by recovery in resveratrol supplemented medium) modes. Small but significant protection against all stresses was observed in PREP mode. RECO mode revealed protection against H2O2, UV and Benzopyrene stresses.

Figure 2

Anti-oxidative stress and DNA damage stress-protective potential of resveratrol. (A). Quantitative measure of H₂O₂ induced reactive oxygen species (ROS) formation and its suppression in resveratrol treated cells is shown. (B). Quantification of UV-C radiations induced DNA strand breaks as determined by Comet assay in control and resveratrol treated cells is shown. A total of 500 μM H₂O₂ and 10 mJ/cm² UV were taken as positive controls, respectively. Resveratrol protected the cells against H₂O₂ and UV stress both in the PREP and RECO modes of treatment.

Figure 3

Luciferase reporter assay on control, cobalt chloride (A) and heat-stressed (B) treated cells in PREP and RECO modes. Relative units of luminescence (luciferase activity (%)) from three independent experiments were plotted. Cells exposed to metal (200 µM cobalt chloride) and heat (42 °C) stress showed inactivation of luciferase activity that was revived in resveratrol-treated cells. (C). Benzopyrene (30 µM) and epinephrine (60 µM) suppressed catalase (innate antioxidant enzyme) that was restored in resveratrol-treated cells. (D). Sodium (meta) arsenite (20 µM) caused aggregation of GFP (shown by yellow arrowheads) that was de-aggregated in resveratrol-treated cells. A total of 250 µM benzopyrene and 300 µM epinephrine, and 150 µM sodium (meta)arsenite were taken as positive controls in (C) and (D), respectively.

Figure 4

Cell differentiation in glioma upon chronic resveratrol treatment. Flowchart (A) and images (B) summarizing morphological features of C6 cells, before and after induction of stress, throughout 30 days treatment with resveratrol (Res) with/without the prior stressed state. (C). Histogram showing the extent of differentiation (percent differentiation) as evaluated from three independent experiments. The number of cells showing differentiated morphology out of 100 cells from three random fields was plotted on a histogram, where control was taken as 100%. (D). Histogram showing loss of viability of C6 cells in response to stress followed by recovery with resveratrol treatment.

Figure 5

Molecular changes in control and resveratrol-treated stressed cells. (A). Western blot showing induction of DNA damage signaling and growth arrest with various chemical stresses listed in (B). Resveratrol-treated cells showed considerable reversal of the molecular changes. (C). Immunostaining showing induction of DNA damage (γH2AX) and senescence (HP1γ) in stressed cells. Resveratrol-treated cells showed considerable recovery. The decrease in mortalin in cells stressed with some stresses was recovered with resveratrol treatment.

Figure 6

Molecular basis for biphasic differentiation induced by resveratrol (Res) in C6 glioma: Immunostaining and Western blotting (quantification shown in histograms) for proteins specifically expressed in glial and neuronal cells. C6 cells treated with resveratrol for 6 weeks (Glial phenotype, A and B) and 10 weeks (Neuron phenotype, C and D) were used.

Figure 7

Resveratrol caused differentiation in human neuroblastoma cells. (A). Images summarizing cell morphology in the control and resveratrol (res)-treated cells for 34 days. The number of cells showing differentiated morphology out of 100 cells from three random fields were plotted on a histogram, where control was taken as 100%. Western blotting (B) and immunostaining (C) results showing induction of neuronal-type differentiation in resveratrol-treated human neuroblastoma cells along with the suppression of glial-related proteins.

Figure 8

Cell migration analyses of C6 cells upon treatment with resveratrol. Wound scratch migration assay with quantification (A) and radial migration assay using tumor spheroids (B) showing promigratory response in glioma cells with low dose resveratrol treatment. Western blotting (C) and immunostaining (D) showing upregulation of canonical markers for migration in cells treated with low dose of resveratrol; the high doses showed downregulation.