Resveratrol Delays Age-Related Deterioration and Mimics Transcriptional Aspects of Dietary Restriction without Extending Life Span

22 páginas, 4 figuras.A small molecule that safely mimics the ability of dietary restriction (DR) to delay age-related diseases in laboratory animals is greatly sought after. We and others have shown that resveratrol mimics effects of DR in lower organisms. In mice, we find that resveratrol induces gene expression patterns in multiple tissues that parallel those induced by DR and every-other-day feeding. Moreover, resveratrol-fed elderly mice show a marked reduction in signs of aging, including reduced albuminuria, decreased inflammation, and apoptosis in the vascular endothelium, increased aortic elasticity, greater motor coordination, reduced cataract formation, and preserved bone mineral density. However, mice fed a standard diet did not live longer when treated with resveratrol beginning at 12 months of age. Our findings indicate that resveratrol treatment has a range of beneficial effects in mice but does not increase the longevity of ad libitum-fed animals when started midlife.This work was supported by grants from the American Heart Association (0425834T to J.A.B. and 0435140N to A.C.) and from the NIH (RO1GM068072, AG19972, and AG19719 to D.A.S.), (HL077256 to Z.U.), (HD034089 to L.W), (2RO1 EY011733 to N.S.W.), Spanish grant (BFU2005-03017 to P.N.), and by the generous support of Mr. Paul F. Glenn and The Paul F. Glenn Laboratories for the Biological Mechanisms of Aging.Peer reviewe

Vorinostat Enhances Cytotoxicity of SN-38 and Temozolomide in Ewing Sarcoma Cells and Activates STAT3/AKT/MAPK Pathways.

Histone deacetylase inhibitors (HDACi) have been evaluated in patients with Ewing sarcoma (EWS) but demonstrated limited activity. To better understand the potential for HDACi in EWS, we evaluated the combination of the HDACi vorinostat, with DNA damaging agents SN-38 (the active metabolite of irinotecan and topoisomerase 1 inhibitor) plus the alkylating agent temozolomide (ST). Drugs were evaluated in sequential and simultaneous combinations in two EWS cell lines. Results demonstrate that cell viability, DNA damage and reactive oxygen species (ROS) production are dependent on the sequence of drug administration. Enhanced cytotoxicity is exhibited in vitro in EWS cell lines treated with ST administered before vorinostat, which was modestly higher than concomitant treatment and superior to vorinostat administered before ST. Drug combinations downregulate cyclin D1 to induce G0/G1 arrest and promote apoptosis by cleavage of caspase-3 and PARP. When ST is administered before or concomitantly with vorinostat there is activation of STAT3, MAPK and the p53 pathway. In contrast, when vorinostat is administered before ST, there is DNA repair, increased AKT phosphorylation and reduced H2B acetylation. Inhibition of AKT using the small molecule inhibitor MK-2206 did not restore H2B acetylation. Combining ST with the dual ALK and IGF-1R inhibitor, AZD3463 simultaneously inhibited STAT3 and AKT to enhance the cytotoxic effects of ST and further reduce cell growth suggesting that STAT3 and AKT activation were in part mediated by ALK and IGF-1R signaling. In summary, potent antiproliferative and proapoptotic activity were demonstrated for ST induced DNA damage before or simultaneous with HDAC inhibition and cell death was mediated through the p53 pathway. These observations may aid in designing new protocols for treating pediatric patients with high-risk EWS

Vorinostat Enhances Cytotoxicity of SN-38 and Temozolomide in Ewing Sarcoma Cells and Activates STAT3/AKT/MAPK Pathways

<div><p>Histone deacetylase inhibitors (HDACi) have been evaluated in patients with Ewing sarcoma (EWS) but demonstrated limited activity. To better understand the potential for HDACi in EWS, we evaluated the combination of the HDACi vorinostat, with DNA damaging agents SN-38 (the active metabolite of irinotecan and topoisomerase 1 inhibitor) plus the alkylating agent temozolomide (ST). Drugs were evaluated in sequential and simultaneous combinations in two EWS cell lines. Results demonstrate that cell viability, DNA damage and reactive oxygen species (ROS) production are dependent on the sequence of drug administration. Enhanced cytotoxicity is exhibited <i>in vitro</i> in EWS cell lines treated with ST administered before vorinostat, which was modestly higher than concomitant treatment and superior to vorinostat administered before ST. Drug combinations downregulate cyclin D1 to induce G0/G1 arrest and promote apoptosis by cleavage of caspase-3 and PARP. When ST is administered before or concomitantly with vorinostat there is activation of STAT3, MAPK and the p53 pathway. In contrast, when vorinostat is administered before ST, there is DNA repair, increased AKT phosphorylation and reduced H2B acetylation. Inhibition of AKT using the small molecule inhibitor MK-2206 did not restore H2B acetylation. Combining ST with the dual ALK and IGF-1R inhibitor, AZD3463 simultaneously inhibited STAT3 and AKT to enhance the cytotoxic effects of ST and further reduce cell growth suggesting that STAT3 and AKT activation were in part mediated by ALK and IGF-1R signaling. In summary, potent antiproliferative and proapoptotic activity were demonstrated for ST induced DNA damage before or simultaneous with HDAC inhibition and cell death was mediated through the p53 pathway. These observations may aid in designing new protocols for treating pediatric patients with high-risk EWS.</p></div

STAT3, AKT and MAPK activation in EWS cell lines following exposure to ST and vorinostat.

<p><b>(A)</b> Immunoblot analysis of lysates of A4573 and TC32 cells following exposure to single (ST and V) and combination (ST/V, V/ST and STV) drug treatments, using antibodies against STAT3, p-STAT3 (Y705), AKT, p-AKT (S473), MAPK, p-MAPK (p42/44), histone 2B (H2B), and ac-H2B (K5). GAPDH was loading control. <b>(B)</b> HDAC activity was measured in A4573, and <b>(C)</b> TC32 cell lysates using the HDAC Activity Assay Kit (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142704#sec002" target="_blank">Methods</a>). Data are presented as mean absorbance (±SE), n = 3. Asterisks denote significant differences for drug combinations, **p< 0.01.</p

Summary of EWS cellular response to ST and vorinostat drug combinations.

<p>The figure depicts EWS cellular response following DNA damage (DNA double-strand breaks and ROS production) and HDAC inhibition. ST promotes DNA damage and vorinostat inhibits HDACs leading to activation of p53, inhibition of CD1, cleavage of caspase-3 and induction of apoptosis. A secondary drug response involves activation of STAT3, mediated by Src, and activation of AKT and MAPK mediated in part through ALK and IGF-1R. V/ST combination facilitates greater cell proliferation (Survival) and ST/V and STV combinations facilitate greater cell death (Apoptosis). Abbreviations: HDAC—Histone deacetylase; ROS—reactive oxygen species, CD1 –cyclin D1, STAT3 –signal transducer and activator of transcription 3; MAPK—mitogen-activated protein kinase, ALK—anaplastic lymphoma kinase; IGF-1R –insulin-like growth factor 1 receptor. Red arrows represent drug inhibition; Solid gray arrows represent constitutive signaling pathways; Solid blunt lines represent inhibition of signals; Dotted gray lines represent activation of signaling pathways in response to drug treatments.</p

STAT3, AKT and MAPK inhibition in EWS cell lines following exposure to ST and vorinostat, MK-2206 and AZD3463.

<p><b>(A)</b> Immunoblot analysis of lysates of A4573 and TC32 cells following exposure to media only (Control, C); ST/V and V/ST with (+) or without (-) 5.0 μM MK-2206 using antibodies against AKT, p-AKT (S473), HDAC1, histone 2B (H2B) and ac-H2B (K5). GAPDH was loading control. <b>(B)</b> Immunoblot analysis of A4573 and TC32 cells following exposure to single (ST and V) and combination (ST/V, V/ST and STV) drug treatments, using an antibody against MGMT. <b>(C)</b> A4573 cells and <b>(D)</b> TC32 cells were incubated with media only (Control, C); ST/V and V/ST with (+) or without (-) 20 nM AZD3463 and viable cells were measured at 48 h by the MTT assay. Data represents mean absorbance (±SE), n = 6. Asterisks denote significant differences between (+) versus (-) 20 nM AZD3463, **p< 0.01 <b>(E)</b> Immunoblot analysis of lysates of A4573 and TC32 cells following exposure to media only (Control, C); ST/V and V/ST with (+) or without (-) 20 nM AZD3463 using antibodies against ALK, IGF-1R, STAT3 (Y705), p-STAT3, AKT, p-AKT (S473), MAPK, p-MAPK (p42/44).</p

DNA damage and intracellular ROS generation in EWS cells following exposure to ST and vorinostat.

<p><b>(A)</b> Immunoblot analysis of γ-H2AX in lysates of A4573 and TC32 subjected to single agents (ST and V) and drug combination (ST/V, V/ST and STV) treatments. GAPDH was loading control. <b>(B)</b> Comet images illustrating DNA fragmentation in TC32 cells treated with single agents and drug combinations. Comet assays were performed in triplicate. <b>(C)</b> The tail intensity relative to the total cell DNA intensity is represented as the percent tail DNA for each treatment group. Data shown represent mean ± SE for each experimental group (n = 50 cells). <b>(D)</b> The tail lengths of comets were measured in individual cells and expressed relative to the control group. Data shown represent mean ± SE for each experimental group (n = 50 cells). Asterisks denote significant differences for drug combinations, **p< 0.01. Asterisks denote significant differences in drug combinations, **p< 0.01 <b>(E)</b> Intracellular ROS production in TC32 cells following single and combination treatments was determined using dichlorofluorescein acetate (DCFA) dye. ROS fluorescence was quenched by N-acetyl-cysteine (NAC). Emitted fluorescence (Em/Ex 490/530 nm) in treated cells was compared with untreated control cells. Measurements were taken for 2 independent experiments, and the data are mean ± SE. Asterisks denote significant differences for drug combinations, **p< 0.01.</p

Effects of combining SN-38 plus Temozolomide (ST) and Vorinostat (V) on EWS cell proliferation.

<p><b>(A)</b> A4573 cells were treated with SN-38 plus temozolomide (ST) and vorinostat (V) as single agents; in sequence combinations of ST followed by vorinostat (ST/V), and vorinostat followed by ST (V/ST) and simultaneous combination (STV), detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142704#pone.0142704.t001" target="_blank">Table 1</a>. For single agents alone and STV combination, cells received drug-free media for 24 h, followed by respective drugs for 24 h. For sequence combinations, cells received a single agent alone for 24 h, followed by the other agent alone for 24 h. Viable cells were measured at 48 h using the MTT assay. Data are presented as mean absorbance ±SE of six replicates, n = 6. Asterisks denote statistically significant differences for drug combinations, **p< 0.01. <b>(B)</b> Percentage of viable TC32 cells following 48 h drug treatments. <b>(C)</b> After 48 h single agent and combination treatments, cells received drug-free media for an additional 24 h. Cell viability was measured at 72 h by the MTT assay. <b>(D)</b> Percentage of viable TC32 cells subjected to 48 h drug treatments followed by 24 h incubation with drug-free media. <b>(E)</b> Phase contrast images depicting morphology of TC32 control cells and ST/V and V/ST treatment groups. Scale bar-200 μm.</p