Resveratrol prevents antibody-induced apoptotic death of retinal cells through upregulation of Sirt1 and Ku70
© Anekonda et al; licensee BioMed Central Ltd. 2008
Received: 16 October 2008
Accepted: 01 December 2008
Published: 01 December 2008
To determine whether resveratrol, a natural plant-derived drug, has protective effects against antibody-induced apoptosis of retinal cells in vitro and to provide insights on the mechanism of resveratrol protection.
E1A.NR3 retinal cells pretreated with 40 μM resveratrol were grown in the presence of anti-recoverin (Rec-1), anti-enolase (Enol-1) antibodies, and normal purified immunoglobulins. When the cells were exposed to resveratrol before treatment with Enol-1 or Rec-1 antibodies, 30–55% more cells survived compared to the resveratrol-untreated cells. Western blotting showed a reduction in proapoptotic protein Bax in the cytoplasm and mitochondria of resveratrol-treated cells. Resveratrol-pretreated cells also showed a significant decrease in intracellular calcium and an inhibition of caspase-3 activity as compared to the untreated cells. Sirt1 expression was greatly reduced in the cells grown in the presence of Rec-1 and Enol-1, but it increased about five times in the resveratrol-pretreated cells. Immunocytochemistry revealed that Sirt1 expression in the cytoplasm and nucleus was colocalized with Ku70 expression in resveratrol-treated cells, suggesting possible interaction with each other in the cell. The pattern of the Ku70 cellular localization also overlapped with the Bax cellular localization in treated and untreated cells.
In vitro protection of retinal cells from apoptosis by resveratrol occurred through multiple early molecular events, such as reduction of intracellular calcium levels, down-regulation of Bax, up-regulation of Sirt1 and Ku70 activities, and inhibition of caspase-3 activity. These findings will help designing future in vivo and pre-clinical treatments for autoimmune retinopathies.
Patients with autoimmune retinopathies (AR), including cancer-associated retinopathy (CAR), suffer from retinal degeneration and progressively lose their vision. Currently available corticosteroid and immunomodulation therapies have limited roles in modifying the progression of AR or CAR . Therefore, a safe and reliable treatment is urgently needed for these patients. Furthermore, age is the strongest risk factor for the incidence of retinal degeneration in adult Americans . The prevalence of vision impairments and blindness increases after the age of 40 and is particularly rapid after age 75 . We believe that designing an effective therapy for the treatment of autoimmune retinopathies involves both understanding the disease mechanism and utilizing anti-aging mechanisms in therapeutics.
CAR and AR are associated with circulating autoantibodies [4, 5]. The most common autoantibodies found in association with vision loss are against recoverin and α-enolase . In both cases, an increased intracellular calcium ([Ca+2]i) caused by antibody triggered the apoptotic pathway, and in patients, it can lead to degeneration of photoreceptors in the retina [6–9]. In this study, we evaluated the effect of resveratrol, a polyphenolic phytoalexin, on levels of [Ca+2]i and on protection of retinal cells from antibody-induced apoptotic death in vitro.
Resveratrol has strong anti-aging properties and has been shown to play a neuroprotective role in several neurological disorders [10–14] by protecting brain cells from death. Recent studies also directly link the beneficial effects of resveratrol to prevention of vision loss [15–18]. These studies strongly suggest that resveratrol could be useful for treating vision and neurological disorders associated with diverse pathologies. The protective effects of resveratrol on the retinal cells were examined in the in vitro study using undifferentiated, immortalized rat retinal E1A.NR3 cells, which express markers specific for photoreceptors, bipolar cells, ganglion cells, and retinal glial cells .
The molecular mechanism of resveratrol in cellular protection is not fully understood. Resveratrol acts by inducing the anti-aging protein Sirt1 in organisms ranging from yeasts to mammals [20, 21]. Sirt1 exhibits anti-apoptotic properties by deacetylating Ku70 protein in HEK293T kidney cells . Ku70, a DNA repair protein present in the nucleus in its native deacetylated form, sequesters Bax in the cytoplasm, and thereby performs a protective role in the cell . In our recent study on antibody-induced apoptosis in retinal cells, the upregulated Bax translocated to mitochondria and triggered mitochondria-mediated caspase-3-mediated apoptosis and ultimately caused retinal cell death [6, 9]. We hypothesize that resveratrol upregulates Sirt1 and Ku70 in retinal cells and suppresses Bax in the cytoplasm, therefore protecting cells from apoptotic death induced by anti-retinal antibody.
E1A.NR3 cells  were grown in a 96-well microplate at a density of 2 × 104/well in 100 μl volume with 0–40 μM resveratrol for 16 hrs. 0.8 mg/ml of Rec-1 or Enol-1 were added to the culture for another 72 hrs. The cell viability was measured as described in a prior study .
Intracellular calcium assay
[Ca2+]i was measured using the Fluo-4 NW Calcium Assay (Molecular Probes) as previously described . Briefly, 2 × 104/well E1A.NR3 cells were grown in 96-well plates overnight. Then 100 μl Fluo-4 NW dye was added for 30 min at 37°C. After adding 50 μl/well of antibody (0.8 mg/ml of Enol-1, Rec-1, normal IgG) or thapsigargin (2 μM) alone, or after 15 min pre-treatment with 40 μM resveratrol, measurements were made using an FLx800 Microplate Fluorescence Reader (Bio-Tek Instruments, Inc.).
Cells (25 × 104) were pre-treated with 0–40 μM resveratrol for 16 hrs, followed by incubation with 0.8 mg/ml of Rec-1 or Enol-1 for 2 hrs. For total protein extraction, cells were harvested, lysed, and centrifuged for 30 min at 15,000 g. Cytosolic and mitochondrial fractions of cells treated with 0.8 mg/ml antibody for 45 min were obtained as described in . The protein content in each fraction was determined using a BCA Protein Assay (Pierce).
The blots containing 50 μg total, cytosolic or mitochondrial protein extracts were blocked in of 5% nonfat milk in 0.1% Tween 20/PBS for 1 hr and then HRE-conjugated antibodies were used as follows: 1:500 for mouse anti-Bax (Santa Cruz Biotech), 1:500 for rabbit anti-Sirt1 (Santa Cruz Biotech), 1:500 for goat anti-Ku70 (Santa Cruz Biotech), 1:1000 for goat anti-α-tubulin (Santa Cruz Biotech) and rabbit anti-Cox-IV (Cell Signaling) antibodies. Blots were developed using LuminiGOLD ECL Western Blotting Detection Kit (SgnaGen Lab) and exposed to an X-Omat film (Kodak) until dark bands appeared. Blots were analyzed by densitometry using Kodak Digital Science 1D Image Analysis software. α-tubulin was used as a control for cytoplasmic and total proteins, and Cox-IV for mitochondrial proteins.
Retinal cells were grown on a slide (2 × 104/well) overnight pretreated with 40 μM resveratrol for 16 hrs and then apoptosis was induced with 0.8 mg/ml Rec-1 or Enol-1 for 2 hrs. Next, cells were washed, fixed in 4% paraformaldehyde, and permeabilized with 0.3% Triton X-100. After 30 min blocking with 1% BSA in PBS, one of the following antibodies was added: mouse anti-Bax, 1:50; rabbit anti-Sirt1, 1:200; or goat anti-Ku70, 1:50 (Santa Cruz Biotech, Inc.) at 4°C overnight. Secondary antibodies conjugated to fluorochromes (Alexa Fluor 488, green and Alexa Fluor 594, red) (Invitrogen) were added for 1 hr. The images were photographed under a Leica DM5000B fluorescence microscope.
Determination of caspase-3 activity
5 × 105/well cells were grown overnight and 40 μM resveratrol was added 16 hrs before the induction of apoptosis with 0.8 mg/ml Rec-1 or Enol-1 for 8, 16, and 24 hr in triplicate. DMSO (0.5%) was used as a control. Following antibody treatments, cells were harvested, lysed, and centrifuged. A 50 μl supernatant was used for determining caspase-3 activity using an EnzChek caspase-3 fluorescent assay (Molecular Probes). Caspase-3 activity was determined from a standard curve and normalized to the total protein content in each sample.
Results are expressed as mean ± SE. The treatment differences were assessed by one-way ANOVAs and Mann-Whitney t-tests. A P value less than 0.05 was considered significant.
Our previously published studies have established specific timelines for antibody-induced cell death, including the rise of [Ca2+]i (3–30 min), expression of Bax (15–120 min), and activation of caspase-3 (16–24 hrs) in E1A-NR3 retinal cells following incubation with antibody [5, 6, 9, 24].
Discussion and conclusion
Dulbecco's modified Eagle's medium
Fetal Bovine Serum
Plasma membrane ATPase Ca2+ channel
Retinal pigment epithelium
Sarco-Endoplasmic Reticulum Ca2+-ATPase channel
We thank Dr. Gail Seigel for the E1A.NR3 retinal cells. This work is supported by grants from the Collins Medical Trust (TA) and the National Institute of Health EY13053 (GA), and by an unrestricted grant to the Casey Eye Institute from the foundation Research to Prevent Blindness, New York, NY.
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