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. 2011;6(9):e24703.
doi: 10.1371/journal.pone.0024703. Epub 2011 Sep 12.

Resveratrol suppresses constitutive activation of AKT via generation of ROS and induces apoptosis in diffuse large B cell lymphoma cell lines

Affiliations

Resveratrol suppresses constitutive activation of AKT via generation of ROS and induces apoptosis in diffuse large B cell lymphoma cell lines

Azhar R Hussain et al. PLoS One. 2011.

Abstract

Background: We have recently shown that deregulation PI3-kinase/AKT survival pathway plays an important role in pathogenesis of diffuse large B cell lymphoma (DLBCL). In an attempt to identify newer therapeutic agents, we investigated the role of Resveratrol (trans-3,4', 5-trihydroxystilbene), a naturally occurring polyphenolic compound on a panel of diffuse large B-cell lymphoma (DLBCL) cells in causing inhibition of cell viability and inducing apoptosis.

Methodology/principal findings: We investigated the action of Resveratrol on DLBCL cells and found that Resveratrol inhibited cell viability and induced apoptosis by inhibition of constitutively activated AKT and its downstream targets via generation of reactive oxygen species (ROS). Simultaneously, Resveratrol treatment of DLBCL cell lines also caused ROS dependent upregulation of DR5; and interestingly, co-treatment of DLBCL with sub-toxic doses of TRAIL and Resveratrol synergistically induced apoptosis via utilizing DR5, on the other hand, gene silencing of DR5 abolished this effect.

Conclusion/significance: Altogether, these data suggest that Resveratrol acts as a suppressor of AKT/PKB pathway leading to apoptosis via generation of ROS and at the same time primes DLBCL cells via up-regulation of DR5 to TRAIL-mediated apoptosis. These data raise the possibility that Resveratrol may have a future therapeutic role in DLBCL and possibly other malignancies with constitutive activation of the AKT/PKB pathway.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Resveratrol suppresses growth and induces apoptosis in DLBCL cells.
(A) DLBCL cell lines were incubated with 0–100 µM Resveratrol for 24 hours. Cell viability was measured by MTT assays as described in Materials and Methods. The graph displays the mean +/− SD (standard deviation) of three independent experiments, * p<0.05, statistically significant (Students t-test). (B) SUDHL4, HBL-1 and OCILY3 cells were treated with 25 and 50 µM Resveratrol for 24 hours and apoptosis was measured by Live/Dead Assay. (C) DLBCL cells were treated with 25 and 50 µM Resveratrol for 24 hours. Thereafter, the cells were washed, fixed and stained with propidium iodide, and analyzed for DNA content by flow cytometry as described in “Materials and Methods”. (D) DLBCL cells were treated with 25 and 50 µM Resveratrol (as indicated) for 24 hours and cells were subsequently stained with flourescein-conjugated annexin-V and propidium iodide (PI) and analyzed by flow cytometry.
Figure 2
Figure 2. Resveratrol inhibits constitutive active AKT and its downstream effectors in DLBCL cells.
(A) SUDHL4 and HBL-1 cells were treated with various doses of Resveratrol. Equal amount of protein from each sample was immunoblotted with Phospho-Akt-Ser473, total AKT Phospho-FOXO1, total FOXO1, p-GSK3, Phospho-Bad and Beta-actin. (B) SUDHL4 cells were treated with 25 and 50 µM Resveratrol for 24 hours. Cells were lysed and immunoprecipitated with either AKT antibody or IgG along with protein A beads overnight. After incubation, beads were washed and incubated with kinase buffer and 2 µl GSK-3 Protein/ATP mixture at 30°C for 4 hours. Beads were boiled and equal amount of protein was immunoblotted with antibody against p-GSK3. (C) SUDHL4 and HBL-1 cells were loaded with 10 µM H2DCFDA for 45 minutes and then were incubated with 25 µM Resveratrol for indicated time periods. Cells were re-suspended in PBS and analyzed for intracellular accumulation of H2DCFDA by flow cytometry (D) SUDHL4 and HBL-1 cell lines were loaded with 10 µM H2DCFDA for 45 minutes and then were pre-treated with 10mM NAC for 2 hours followed by treatment with 50 µM Resveratrol for various time periods. Cells were re-suspended in PBS and analyzed using flow cytometry. Bar graph displays the mean +/− SD (standard deviation) of three independent experiments, * p<0.05, statistically significant (Students t-test). (E) SUDHL4 and HBL-1 cells were pretreated with 10mM NAC for 2 hours followed by treatment with 50 µM Resveratrol for 24 hours. After cell lysis, equal amounts of proteins were immunoblotted with antibodies against p-AKT and beta actin as indicated.
Figure 3
Figure 3. Resveratrol-induced mitochondrial signaling pathway in DLBCL cells.
(A) After treating with 50 µM Resveratrol for indicated time periods, HBL-1 and SUDHL4 cells were lysed and immuno-precipitated with anti-Bax 6A7 antibody for detection of conformationally changed Bax protein. In addition, the total cell lysates were immuno-blotted with specific anti-Bax polyclonal antibody. (B) HBL-1 and SUDHL4 cells were pre-treated with either, 10mM NAC and 80 µM z-VAD/fmk for 2 hours and subsequently treated with 50 µM Resveratrol for 8 hours. Cells were lysed and immunoprecipitated with anti-Bax 6A7 antibody and proteins were immunoblotted with Bax rabbit polyclonal antibody. (C) DLBCL cells were treated with and without 25 and 50 µM Resveratrol for 24 hours. Live cells with intact mitochondrial membrane potential and dead cells with lost mitochondrial membrane potential was measured by JC-1 staining and analyzed by flow cytometry as described in Materials and Methods. (D) SUDHL4 and HBL-1 cells were treated with 25 and 50 µM Resveratrol for 24 hours. Mitochondrial free cytosolic fractions were isolated and immunoblotted with antibody against cytochrome c and Beta-actin.
Figure 4
Figure 4. Activation of caspases -9, -3 and cleavage of PARP induced by Resveratrol treatment in DLBCL cells.
(A) SUDHL4 and HBL-1 cells were treated with and without 25 and 50 µM Resveratrol for 24 hours. Cells were lysed and equal amounts of proteins were immunoblotted with antibodies against caspase-9, caspase-3, cleaved caspase-3, PARP and Beta-actin. SUDHL4 and HBL-1 cells were pretreated with either 80 µM z-VAD (B) or 10mM NAC (C) for 2 hours and subsequently treated with 50 µM Resveratrol for 24 hours. Cells were lysed and equal amounts of proteins were immunoblotted with antibodies against caspase-9, caspase-3 cleaved caspase-3 and beta-actin. (D) SUDHL4 and HBL-1 cells were pretreated with either 80 µM of z-VAD-fmk or 10mM NAC for 2 hours and subsequently treated with 50 µM Resveratrol for 24 hours. Following treatment, cells were stained with fluorescein conjugated annexinV/PI and apoptosis was measured by flow cytometry.
Figure 5
Figure 5. Reservatrol-mediated upregulation of DR5.
(A) SUDHL4 cells were treated with 50 µM Resveratrol for indicated time periods. After cell lysis, equal amounts of proteins were immuno-blotted with antibodies against DR5 and beta actin. (B) SUDHL4 and HBL-1 cells were pre-treated with either 80 µM of z-VAD (upper panel) or 10mM NAC (lower panel) for 2 hours and subsequently treated with 50 µM Resveratrol for 24 hours. Cells were lysed and equal amounts of proteins were immunoblotted with antibodies against DR5 and beta-actin. (C) SUDHL4 cells were either transfected with 50 and 100nM siRNA, specific against DR5 or scrambled siRNA for 48 hours. Cells were then treated with 25 and 50 µM Resveratrol for 24 hours, following which cells were stained with fluorescent-conjugated Annexin V/PI and analyzed by flow cytometry. (D) SUDHL4 cells were either transfected with 100nM siRNA, specific against DR5 or scrambled siRNA for 48 hours and treated with 25 and 50 µM Resveratrol for 24 hours. Cells were lysed and equal amounts of proteins were immuno-blotted with antibodies against DR5, caspase-3, cleaved caspase-3, PARP and beta-actin.
Figure 6
Figure 6. Resveratrol treatment potentiates TRAIL mediated apoptosis in DLBCL cells.
(A) SUDHL4 and HBL-1 cells were treated with either 10 µM Resveratrol in the presence and absence of 1 and 5ng TRAIL for 24 hours. Following treatment, cells were stained with fluorescent-conjugated Annexin V/PI and analyzed by flow cytometry. (B) SUDHL4 cells were treated with either 10 µM Resveratrol in the presence and absence of 1 and 5ng TRAIL for 24 hours. Following treatment, cells were lysed and equal amounts of proteins were immuno-blotted with antibodies against caspase-8, caspase-3, PARP, and beta- actin. (C) SUDHL4 and HBL-1 cells were transfected with either scrambled siRNA (100nM) or DR5 specific siRNA (100nM) for 48 hours and then cells were treated with 10 µM Resveratrol in the presence of either 1 or 5ng TRAIL for 24 hours following which cells were stained with fluorescent-conjugated Annexin V/PI and analyzed by flow cytometry, * denotes statistical significance (p<0.05) or (D) cells were stained with 50 µM calcein AM and 8 µM ethidium homodimer and visualized under an Olympus fluorescent microscope using a long-pass filter. (E) SUDHL4 and HBL-1 cells were transfected with either scrambled siRNA (100nM) or DR5 specific siRNA (100nM) for 48 hours and then cells were treated with 0 µM Resveratrol in the presence of either 1 or 5ng TRAIL for 24 hours following which cells were lysed and immunoblotted with antibodies against DR5 and Betaactin.
Figure 7
Figure 7. Schematic representation of Resveratrol-induced apoptosis in DLBCL.

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