doi:10.1016/j.bbrc.2005.06.113 
Copyright © 2005 Elsevier Inc. All rights reserved.
α-Tocopherol-mediated caspase-3 up-regulation enhances susceptibility to apoptotic stimuli
Noriyuki Miyoshia, b, Kisa Naniwaa, Takeshi Kumagaia, c, Koji Uchidaa, Toshihiko Osawaa and Yoshimasa Nakamuraa, d, 
, 
aLaboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
bLaboratory of Biochemistry, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8012, USA
cLaboratory of Hygienic Chemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan
dDepartment of Biofunctional Chemistry, Division of Bioscience, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan
Received 16 June 2005.
Available online 1 July 2005.
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Abstract
Although α-tocopherol is known as an essential micronutrient involved in various oxidative stress-related processes, its non-antioxidant activities have only been characterized in recent years. In this study, we reveal that (+)-α-tocopherol [RRR-α-tocopherol] enhances cellular susceptibility to both oxidative and non-oxidative apoptosis-inducing stimuli through up-regulation of caspase-3/CPP32 expression in several human cell lines. Exposure of (+)-α-tocopherol pretreated cells to known apoptosis-inducing stimuli, such as Fas, H2O2, or etoposide, resulted in an increase in cellular apoptotsis. In addition, (+)-α-tocopherol also elevated the pro-caspase-3 protein level and mRNA expression in a time- and dose-dependent manner, while other tocopherol analogues showed no effect. Experiments using a GC-specific DNA binding agent, mithramycin A, and an electrophoretic mobility shift assay demonstrated that Sp1 might mediate the enhanced expression of caspase-3. Our results also confirmed that (+)-α-tocopherol promotes the expression, but not the activation, of caspase-3 in various human cell lines. These findings provide biological evidence showing that (+)-α-tocopherol can amplify the apoptotic response by up-regulating the expression of pro-caspase-3.
Keywords: α-Tocopherol; Caspase-3; Apoptosis; Sp 1; Etoposide
Fig. 1. (+)-α-Tocopherol pretreatment enhances apoptosis induced by the various apoptotic stimuli. Jurkat or HL-60 cells were pretreated with 100 μM (+)-α-tocopherol (filled bars) or ethanol (open bars) for 12 h, washed three times, and then these treated cells were exposed to either Fas (500 ng/ml), H2O2 (50 μM), or etoposide (50 μM) for 9 h. Apoptosis was detected by TUNEL assay. Data are means of three experiments. *P < 0.05 versus ethanol pretreatment group.
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Fig. 2. (+)-α-Tocopherol up-regulates caspase-3 protein and mRNA. (A) Jurkat cells were treated with or without 100 μM (+)-α-tocopherol (VE) for different time periods as indicated (h). Immunoblot analyses were performed using an anti-caspase-3 antibody. In the absence of added VE, no change in caspase-3 level was observed as indicated by lane 0 and 24c. (B) Caspase-3 expressions were quantified by densitometric analysis. The data, expressed as the ratio of caspase-3/actin, represent the means of three experiments. *P < 0.05 versus 0 h control. (C) Jurkat cells were treated with (+)-α-tocopherol (VE) at each indicated concentration for 12 h (lanes 1–5) or hydrogen peroxide (H2O2) (lane 6) for 12 h. Cells were harvested and an immunoblot analysis was performed using anti-caspase-3, PARP, and actin antibodies. Closed triangles show full-length PARP (116 kDa), and open triangles show the cleaved ones (89 kDa). (D) The caspase-3 expressions were measured by densitometric analysis of an immunoblot. Data are means of two experiments. *P < 0.05 versus control. (E) Jurkat cells were treated with or without 100 μM (+)-α-tocopherol (VE) for different time periods as indicated (h). RT-PCR analysis was performed using a specific primer. (F) Expressions of caspase-3 mRNA were quantified by densitometric analysis of the results obtained with RT-PCR. Data are means of three experiments. *P < 0.05 versus 0 h control. (G) Effect of α-, β-, δ-, and γ-tocopherol on caspase-3 expression. Jurkat cells were treated with 100 μM tocopherol derivatives for 12 h. Immunoblot analysis was performed using anti-caspase-3 and anti-PARP monoclonal antibodies. (H) Caspase-3 expressions were quantified by densitometric analysis of an immunoblot. Significant difference is observed in a, versus control, P < 0.05; b, versus α-tocopherol, P < 0.05.
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Fig. 3. Sp1 regulates human caspase-3 promoter activity induced by (+)-α-tocopherol. (A) Effect of mithramycin A on caspase-3 expression up-regulated by (+)-α-tocopherol. Jurkat cells pretreated with mithramycin A (MMA) at each indicated concentration for 30 min were exposed to 100 μM (+)-α-tocopherol (VE) for 12 h. Immunoblot analyses were performed using anti-caspase-3 and anti-PARP antibodies. (B) Caspase-3 expressions were quantified by densitometric analysis of immunoblot data expressed as percent change with control set as 100%. Data are means of three experiments. Significance is expressed as a, versus control, P < 0.05; b, versus (+)-α-tocopherol (VE), P < 0.05. (C) Analysis of Sp1 binding to the caspase-3 promoter region. Nuclear extracts were prepared from Jurkat cells treated with 100 μM (+)-α-tocopherol (VE) for the indicated time. The mixture with or without nuclear extract and anti-Sp1 antibody was incubated with each labeled DNA probe (sequences are described under Materials and methods section). Samples were analyzed by polyacrylamide gel electrophoresis. The filled triangle indicates Sp1-specific complexes and the open triangle indicates their supershift bands.
Fig. 4. Up-regulation of caspase-3 by (+)-α-tocopherol affects its proteolytic activity. Jurkat cells were preincubated with or without 100 μM (+)-α-tocopherol for 12 h and stimulated with either Fas at 500 ng/ml, etoposide (ETP) at 50 μM, or H2O2 at 50 μM for 6 h. Inhibitory experiments were performed using caspase-3 inhibitor, Ac-Asp-Glu-Val-Asp-H (aldehyde) (Ac-DEVD-CHO) at 100 μM during apoptotic stimulation or mithramycin A at 100 nM for 30 min before (+)-α-tocopherol stimulation. Y-axis shows percent caspase-3-like activity relative to apoptotic stimulus-only control, which is set at 100%. Data are means of three experiments. Significance is expressed as *P < 0.05.
Fig. 5. (+)-α-Tocopherol up-regulates caspase-3 in various human cell lines. Five human culture cell lines (Jurkat, HL-60, HeLa, WiDr, and SH-SY5Y) were examined for effect of (+)-α-tocopherol on caspase-3 expression. Cells were treated with (+)-α-tocopherol at each indicated concentration for 12 h (lanes 1–5) or etoposide (ETP) (lane 6) for 12 h. Cells were harvested and an immunoblot analysis was performed using anti-caspase-3 and anti-PARP antibodies. Filled triangles show full-length PARP (116 kDa) and open triangles show the truncated PARP (89 kDa).
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