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-Tocopherol inhibits human cancer cell cycle progression and cell proliferation by down-regulation of cyclins¹

Institute of Biochemistry and Molecular Biology, University of Bern, Switzerland

²Correspondence: University of Bern, Institute of Biochemistry and Molecular Biology, Bühlstrasse 28, 3012 Bern, Switzerland. E-mail: theresa.visarius{at}mci.unibe.ch

SPECIFIC AIMS

The aim of the present study was to compare the effect of - and -tocopherol on proliferation of human prostate carcinoma, colon adenocarcinoma, and osteosarcoma cells. Since epidemiological studies have suggested an anticancer activity of vitamin E and -tocopherol is the major form of vitamin E in the U.S. diet while -tocopherol is the predominant form in human plasma, we investigated the action of -tocopherol on the cell cycle, cell proliferation, and DNA synthesis and compared it with that of -tocopherol. We further analyzed the correlation between -tocopherol-mediated inhibition of cancer cell proliferation and protein levels of G₁-S transition-specific proteins, namely, cyclin D1, cyclin E, and cdk inhibitors p21^CIP1, p27^Kip1, and p16^INK4a.

PRINCIPAL FINDINGS

1. -Tocopherol inhibits cell proliferation more significantly than -tocopherol
Comparing the effects of -, ß-, and -tocopherol on cell growth, we observed that all three forms of vitamin E significantly inhibited cell proliferation whereas in the prostate cancer cell lines tested (DU-145 and LNCaP), -tocopherol induced a significantly stronger growth inhibition than either - or ß-tocopherol (P<0.05). Treated with 25 µM -tocopherol, androgen-independent prostate cancer cells (DU-145) grew only 14% relative to control cells whereas -tocopherol-treated cells grew 50% and ß-tocopherol-treated cells 59% in 24 h (Table 1 ). -Tocopherol-treated, androgen-dependent prostate cancer cells (LNCaP) grew only 26% relative to controls vs. 52% growth observed from -tocopherol-treated cells. Colon adenocarcinoma (CaCo-2) cells grew 36% when treated with -tocopherol and 50% when treated with -tocopherol (Table 1) . Thus, inhibition of cell proliferation in DU-145, LNCaP, and CaCo-2 cells was consistently inhibited more significantly by - than by - or ß-tocopherol (P<0.05). Human osteosarcoma cell proliferation, on the other hand, was only weakly inhibited by -tocopherol whereas proliferation of -tocopherol-treated cells was not distinguishable from that of controls (Table 1) .

2. -Tocopherol inhibits cell cycle progression and DNA synthesis
To assess the effect of -tocopherol on cell cycle progression and DNA synthesis we employed flow cytometry analysis and 5-bromo-2'-deoxy-uridine (BrdU) incorporation. -Tocopherol (25 µM) -treated DU-145 cells presented a higher G₁-phase population and a decreased S-phase population vs. control cells whereas 6 h after serum restimulation, the S-phase population of -tocopherol-treated cells (21.8%±6.3) was significantly lower than that of control cells (30%±1.8). Inversely, G₁-phase population was lower in control cells (52%±6) than in -tocopherol-treated cells (61.4%±3). No sub-G₁ population and no apoptotic cells were observed. In accordance with the G₁-S transition delay, the BrdU incorporation assay revealed decreased activity of DNA synthesis in the tested DU-145 and CaCo-2 cells.

Equimolar concentrations of - and -tocopherol (25 µM) inhibited DNA synthesis by 55% ± 5 and 32% ± 9, respectively, in DU-145 cells. In CaCo-2 cells, - and -tocopherol similarly inhibited DNA synthesis by 48% ± 22 and 25% ± 11, respectively. Thus, from the BrdU incorporation assay as from the proliferation assay, we conclude -tocopherol is more potent than -tocopherol in the cell lines investigated.

3. -Tocopherol down-regulates cyclin D1 and cyclin E levels
In correlation with the G₁-phase delay, -tocopherol (25 µM) inhibited the serum-stimulated increase of cyclin D1 and cyclin E. D-cyclins and cyclin E are key regulators of the G₁-S transition. Serum stimulation led to a nearly twofold increase of cyclin D1 within 8 h in DU-145 and LNCaP control cells, and this effect was significantly inhibited by -tocopherol (Fig. 1 ). Serum restimulation led to a constant increase of cyclin E levels in control cells and -tocopherol delayed this increase (Fig. 1) .

View larger version (50K): [in this window] [in a new window]   Figure 1. Effects of -tocopherol (25 µM) on cyclin D1 and cyclin E protein levels. Whole cell lysates were immunoblotted with antibodies against cyclin D1 and cyclin E at the times indicated. A) Cyclin D1: values are ß-actin corrected means ± SD of 4 independent experiments. *P < 0.05 vs. control at corresponding time. Western blot B) cyclin D1, C) cyclin E. Representative blots from 1 of 3 experiments.

The effect of -tocopherol (50 µM) on cyclin D1 and cyclin E levels was further assessed in LNCaP cells, where the pattern of down-regulation observed for the cyclins by -tocopherol was repeated.

4. Effect of -tocopherol on cdk inhibitors p21^CIP1, p27^Kip1, and p16^INK4a
The effect of -tocopherol on differences in protein levels from representatives of both cdk inhibitor (CKI) families was examined. Serum stimulation led to increased protein levels of all three CKIs tested, varying with time. From a baseline level observed at the end of serum deprivation, p27^Kip1 protein increased in control cells by greater than twofold within 2 h of serum stimulation and returned to baseline level over the next 11 h. Control cells further exhibited measurable p21^Cip1 protein levels, which peaked after 5 h and decreased again within 11 h. p16^INK4a, a CKI representative of the INK4 family, was nearly absent in the first 5 h but appeared 8 h after serum restimulation and decreased thereafter. -Tocopherol down-regulated protein levels of all three CKIs investigated, whereas p27^Kip1 and p21^Cip1 increased only minimally over the respective starting amounts present and p16^INK4a remained nearly undetectable over the 11 h period examined.

CONCLUSION AND SIGNIFICANCE

The present study demonstrates that -tocopherol is more potent than - or ß-tocopherol in inhibiting proliferation of DU-145, LNCaP, and CaCo-2 cells and that -tocopherol prevents cell cycle progression via reduction of cyclin D1 and cyclin E levels. Parallel to the inhibition of proliferation, DNA synthesis is inhibited more significantly by - than by -tocopherol. To our knowledge, this study demonstrates for the first time down-regulation of cancer cell growth by -tocopherol on DNA synthesis, G₁-S transition delay, and protein levels for proteins that are important in G₁-S transition, resulting in a diminution of cell proliferation without apparent apoptosis or necrosis. For - and -tocopherol, several non-antioxidant functions have been described. Since -tocopherol has a weaker antioxidant capacity than -tocopherol but DU-145, LNCaP, and CaCo-2 cell growth was inhibited more significantly by - than by -tocopherol, we suggest a novel non-antioxidant function to be at the basis of this -tocopherol control of cell proliferation.

-Tocopherol possesses unique features that distinguish it from -tocopherol. For example, -tocopherol was shown to be superior to -tocopherol in inhibiting neoplastic transformation of embryonic fibroblasts, and cyclooxygenase activity in macrophages and epithelial cells is known to be inhibited by -tocopherol but not by -tocopherol. Similarly, -tocopherol has specific non-antioxidant properties not shared by other tocopherols. In rat A7r5 smooth muscle cells, control of cell proliferation is mediated by inhibition of PKC- activity, a function not shared by - or -tocopherol. Although a specific target for - and -tocopherol has not been identified, our results indicate a more potent growth inhibition effect of - than of -tocopherol in the prostate and colon cancer cell lines examined.

A possible explanation for the down-regulation of cyclin D1 and p21^CIP1 we observed may be found by comparison with established experiments conducted with the phosphatidylinositol 3'-kinase (PI3K) inhibitors Ly294002 and wortmannin. Stimulation of the PI3K pathway by serum leads to activation of AKT, inhibition of GSK-3ß, and increased cyclin D1 and p21^CIP1 protein levels. Ly294002 and wortmannin in some cell lines are known to reduce cyclin D1 and p21^CIP1 protein abundance by allowing uninhibiting GSK-3ß activity. In LNCaP cells, the PI3K pathway was shown to be a dominant growth factor-activated cell survival pathway, whereas LNCaP and DU-145 cells are known to have a mutation in the PTEN gene. PTEN is a phosphatase that deposphorylates 3-phosphorylated inositol phospholipids, triggering substrates of the PI3K pathway. Serum stimulation activates the PI3K pathway and leads to increased cyclin D1 and p21^CIP1 concentrations. In LNCaP cells, treatment with the PI3K inhibitors Ly294002 and wortmannin induced apoptosis. Since no differences in apoptosis or necrosis between control and tocopherol-treated cells was observed in our experimental system (<3% total), we speculate that -tocopherol, rather than blocking the PI3K pathway, interacts by a homeostatic mechanism in inhibiting this pathway (Fig. 2 ).

View larger version (35K): [in this window] [in a new window]   Figure 2. Schematic diagram of differences observed between tocopherol-treated and control cells. In prostate and colon cancer cells, serum stimulation led to increased cell proliferation as well as increased DNA synthesis and protein levels for proteins important in G1-S transition; -tocopherol diminished these effects.

- and -tocopherol can have different functions in cells; accordingly, their uptake and metabolism by the human body are regulated differently and depend on both the diet and the activity of cytochrome P450 3A. In muscle, adipose and brain tissue -tocopherol levels depend more directly on diet and can reach the same levels as -tocopherol. In plasma, -tocopherol concentrations are lower than -tocopherol, and -tocopherol supplementation suppresses -tocopherol levels. Conversely, -tocopherol supplementation increases -tocopherol plasma concentration without significantly lowering -tocopherol levels. Diet can vary in total amount of vitamin E as well as in properties of -, -, and other tocopherols. For example, oil from corn, soybean, sesame, nuts, walnuts, pecans, or peanuts are rich sources of -tocopherol; a typical U.S. dies contains 70% -tocopherol in total vitamin E consumption. A comparison of the frequency of different tumors in relationship with the amount of -tocopherol consumed in the diet may cast some light on the significance of the in vitro findings relative to an in vivo protection.

Our finding that -tocopherol inhibits proliferation of prostate and colon cancer cells more potently than -tocopherol provides a cellular mechanism supporting the concept emerging from epidemiological studies that a greater magnitude of risk reduction for prostate and colon cancer may occur if both total vitamin E consumption and the amount of -tocopherol in the diet or in supplementation are increased. Further studies will focus on the regulation of -tocopherol on factors upstream of cyclin D1. Regulation of the PI3K pathway by -tocopherol and its effects on the turnover of cyclin D1 are ongoing studies. Molecular characterization of the -tocopherol induced cell cycle control demonstrated in this study will be further developed by addressing these issues.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0362fje; to cite this article, use FASEB J. (October 4, 2002) 10.1096/fj.02-0362fje

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