Different forms of vitamin E revisited

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Hi All, In the below PDF-available paper at

apater.mun.ca is two versions. First come the

Medline citation. Then comes the express

format of the paper. Third comes the full paper.

Which do you like better?

The paper is on the important issue of the different

forms of vitamin E. That the ones having the best

effect did not correlate with their anti-oxidant ability

seemed to me be striking.

Cheers, Al.

Gysin R, Azzi A, Visarius T.

Gamma-tocopherol inhibits human

cancer cell cycle progression and cell

proliferation by down-regulation of cyclins.

FASEB J. 2002 Dec;16(14):1952-4.

PMID: 12368234 [PubMed - indexed for MEDLINE]

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 alpha-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 p21CIP1, p27Kip1, and

p16INK4a.

PRINCIPAL FINDINGS

1. -Tocopherol inhibits cell proliferation

more significantly than -tocopherol

Comparing the effects of -, -, 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-tocopherol (P0.05).

Treated with 25 M -tocopherol, androgen-indepen-dent

prostate cancer cells (DU-145) grew only 14%

relative to control cells whereas -tocopherol-treated

cells grew 50% and -tocopherol-treated cells 59% in

24 h (Table 1). -Tocopherol-treated, androgen-depen-dent

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 -tocoph-erol

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-tocopherol

(P0.05). Human osteosarcoma cell proliferation, on

the other hand, was only weakly inhibited by -tocoph-

erol 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 cy-tometry

analysis and 5-bromo-2-deoxy-uridine (BrdU)

incorporation. -Tocopherol (25 M) -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 signifi-cantly

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 tran-sition

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

M) 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 -tocoph-erol

in the cell lines investigated.

3. -Tocopherol down-regulates cyclin D1 and cyclin

E levels

In correlation with the G1 -phase delay, -tocopherol

(25 M) 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) .

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.

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).

The effect of -tocopherol (50 M) 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 in-creased

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 ofserum

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 represen-tative

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 re-mained

nearly undetectable over the 11 h period

examined.

CONCLUSION AND SIGNIFICANCE

The present study demonstrates that -tocopherol is

more potent than -or-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 inhib-

ited more significantly by - than by -tocopherol. To

our knowledge, this study demonstrates for the first

time down-regulation of cancer cell growth by -to-copherol

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 prolifera-tion

without apparent apoptosis or necrosis. For - and

-tocopherol, several non-antioxidant functions have

been described. Since -tocopherol has a weaker anti-oxidant

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 distin-guish

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 toco-pherols.

In rat A7r5 smooth muscle cells, control of cell

proliferation is mediated by inhibition of PKC- activ-ity,

a function not shared by -or-tocopherol. Al-though

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) inhibi-tors

Ly294002 and wortmannin. Stimulation of the

PI3K pathway by serum leads to activation of AKT,

inhibition of GSK-3, 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-3 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 ob-served

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).

- 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 concen-trations

are lower than -tocopherol, and -tocopherol

supplementation suppresses -tocopherol levels. Con-versely,

-tocopherol supplementation increases -to-copherol

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 con-sumed

in the diet may cast some light on the signifi-cance

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 character-ization

of the -tocopherol induced cell cycle control

demonstrated in this study will be further developed by

addressing these issues.

--------------------------------------------

Gysin R, Azzi A, Visarius T.

Gamma-tocopherol inhibits human

cancer cell cycle progression and cell

proliferation by down-regulation of cyclins.

FASEB J. 2002 Dec;16(14):1952-4.

PMID: 12368234 [PubMed - indexed for MEDLINE]

Effects of gamma-tocopherol on the cell

cycle and proliferation were examined in

human prostate carcinoma, colorectal

adenocarcinoma, and osteosarcoma cells.

Many epidemiological studies have

suggested an anticancer activity of vitamin E,

yet mechanistic studies are sparse to date.

Vitamin E consists of four tocopherols

(alpha-, beta-, gamma-, delta-)

and the corresponding tocotrienols.

Because gamma-tocopherol is the

predominant form of tocopherol found in

the U.S. diet, while alpha-tocopherol is the form

of vitamin E most readily found in

dietary supplements, we compared

physiologically relevant concentrations of

these tocopherols and found a more

significant growth inhibition effect for

gamma- than for alpha-tocopherol.

Flow cytometry analysis of gamma-tocopherol

treated prostate carcinoma DU-145 cells

showed decreased progression into the

S-phase. This effect was associated with

reduced DNA synthesis as measured by

5-bromo-2'-deoxy-uridine incorporation.

Furthermore, Western-blot analysis of

gamma-tocopherol treated cells showed

decreased levels of cyclin D1 and cyclin

E. Taken together, the results indicate

that gamma-tocopherol inhibits cell

cycle progression via reduction of cyclin

D1 and cyclin E levels. Because

gamma-tocopherol has a weaker antioxidant

capacity than a-tocopherol and

gamma-tocopherol more significantly

inhibited cell proliferation as well as DNA

synthesis than alpha-tocopherol, we

suggest a non-antioxidant mechanism to be at

the basis of this effect.

Key words: vitamin E • G1-S transition • cyclin D1 • cyclin E

Prostate and colorectal cancers are leading causes of cancer-related deaths

among men in

Western nations (1). Although dietary intake of vitamin E is known from

epidemiological

studies to prevent cancer disease progression in vivo and to inhibit

proliferation of prostate

carcinoma cells in vitro, little is known about its mechanism of action. Vitamin

E consists of four

tocopherols (alpha-delta) and the corresponding tocotrienols. a -Tocopherol is

the predominant

form of vitamin E in human plasma and, until recently, the protective action of

vitamin E has

been attributed only to its antioxidant activity, which in vitro is in the order

alpha>>>delta (2). In

the past ten years a -tocopherol additionally was found to have non-antioxidant

functions, such

as, inhibition of cell proliferation (3), inhibition of platelet adhesion (4),

inhibition of protein

kinase C (PKC) (5), activation of protein phosphatase type 2A (PP2A) (6), or

down-regulation of

the scavenger receptor CD36 on its expression level (7). Newer studies have

indicated that also

? -tocopherol possesses unique features that distinguish it from other vitamin E

forms (8). ? -

Tocopherol is the most abundant form of vitamin E in the U.S. diet and the

second most common

form in human plasma (9). Interest in the use of vitamin E for the prevention of

prostate cancer

increased when a statistically significant 32% reduction in the incidence of

prostate cancer and a

41% reduction in mortality from this disease were shown in men consuming a

-tocopherol

supplements who took part in an a -tocopherol ß -carotene controlled trial (10).

Thereafter, a

nested case-control study of 578 prostate cancer patients demonstrated a

suggestive inverse

association for a -tocopherol and aggressive prostate cancer (11). Furthermore,

a small Swiss

study demonstrated that smokers with low prediagnostic levels of vitamin E had

an increased

risk of fatal prostate cancer (12). In summary, several epidemiological studies

have shown a

protective effect for vitamin E against prostate cancer.

Most studies relating vitamin E to cancer incidence focused only on a

-tocopherol. Recently,

however, a nested case-control study examined the association of ? -tocopherol

with the incidence

of prostate cancer and compared it with the effect of a -tocopherol. This study

indicated an

inverse association between tocopherol plasma concentrations and the incidence

of prostate

cancer much more significantly for ? -tocopherol than for a -tocopherol. Men in

the highest

quintile of plasma ? -tocopherol concentrations had a fivefold reduction in the

risk of prostate

cancer compared with those in the lowest quintile (13).

The influence of tocopherols on the prevention of colon cancer has also been a

subject of

interest. Incidence of colon cancer was reduced in several animal models where

animals were

supplemented with vitamin E (14). Epidemiological studies relating human colon

cancer to

vitamin E were, however, less consistent (15). The above-mentioned studies

notably focused on

a -tocopherol, although, in the etiology of colon cancer, distinct biologic

potencies,

pharmacokinetics, and different capacities to prevent neoplastic transformation

have been

suggested for both a - and ? -tocopherol (15). In humans with colon cancer,

levels of

prostaglandin E2 are higher in tumors than in adjacent normal mucosa (16), which

is

hypothesized to promote the growth of colon cancer by increasing the

proliferation rate of tumor

cells (17). In human macrophages and epithelial cells ? -tocopherol, but not a

-tocopherol, was

found to inhibit prostaglandin E2 production by inhibiting cyclooxygenase-2

(COX-2) activity

(18).

In human plasma and tissues a -tocopherol concentrations are significantly

higher than that of ? -

tocopherol. ? -Tocopherol is more rapidly metabolized than a -tocopherol (19)

and a -tocopherol

supplementation competitively decreases plasma and tissue ? -tocopherol

concentrations (20). A

recent vitamin E supplementation study, however, showed that human tissue ?

-tocopherol

concentrations, as percentage of a -tocopherol, were substantially greater than

had been

recognized before. ? -Tocopherol tissue concentrations were 1.4 to 4.6 times

greater in adipose

tissue, muscle, skin, and vein than in plasma (21). Taken together, several

studies suggested a

protective effect of vitamin E against prostate and colon cancer. a -Tocopherol

is more present in

human plasma and tissues than ? -tocopherol, but still little is known about the

potency of the

different tocopherols on prevention of cancer cell proliferation.

The aim of the present study was to compare effects of a - ß -, and ?

-tocopherol on the control of

cancer cell proliferation and to characterize the mechanism of action of ?

-tocopherol. We

included ß -tocopherol in this study to differentiate between the effects

produced by antioxidant

activity versus antioxidant-independent events. ß -Tocopherol is an isomer of ?

-tocopherol and,

because it possesses a similar antioxidant capacity as a -tocopherol, was used

to discriminate

between antioxidant and non-antioxidant functions. We analyzed the effect of the

three vitamin E

forms in the androgen-independent prostate carcinoma cell line DU-145, in the

androgen-

dependent prostate carcinoma cell line LNCaP, in the colon adenocarcinoma cell

line CaCo-2,

and in the osteosarcoma cell line SaOs-2. The effect of ? -tocopherol on cell

cycle progression

was determined by flow cytometry analysis and by 5-bromo-2™-deoxy-uridine (BrdU)

incorporation. The effect of ? -tocopherol on the G1-S transition specific

cyclins D1, and E and on

the cyclin dependent kinase inhibitors (CKIs) p21 CIP1 , p27 KIP1 , p16 INK4a

and on cell death was

investigated.

------------------------

RESULTS

Comparative inhibition of cancer cell proliferation by a - and ? -tocopherol

Comparing the effects of a -, ß -, and ? -tocopherol on prostate cancer cell

proliferation, we

observed that all three forms of vitamin E significantly inhibited cell

proliferation at

concentrations =25 µM (Fig. 1). ? -Tocopherol, however, induced significantly

stronger growth

inhibition (P < 0.05) compared with either a - or ß -tocopherol [25 and 50 µM].

When treated

with 25 µM of ? -tocopherol, DU-145 cells grew only 14% relative to control

cells, whereas a -

tocopherol treated cells grew 50% and ß -tocopherol treated cells 59%.

In LNCaP cells (androgen-dependent prostate carcinoma), the effect of a -, ß -,

and ? -tocopherol

on growth inhibition was compared at 25 µM where cell proliferation was more

significantly

inhibited by ? -tocopherol (74%) than by ß - (42%) or by a -tocopherol (48%)

(Table 1, P<0.05).

In CaCo-2 cells (colon carcinoma), the analyzed a - and ? -tocopherols (25 µM)

significantly

inhibited cell proliferation as compared with controls (P<0.05). The growth

inhibition effect of

a -tocopherol in these cells was similar to that observed in the prostate

carcinoma cell lines

tested, whereas ? -tocopherol inhibited proliferation by 64% (Table 1). In

CaCo-2 cells the trend

for ? -tocopherol to inhibit cell proliferation more potently than a -tocopherol

was observed,

however, statistically significant differences between the two tocopherols could

not be drawn.

SaOs-2 cells (osteosarcoma cells) were insensitive to a - and ß -tocopherol. ?

-Tocopherol (25

µM) inhibited weakly but significant cell growth of SaOs-2 cells (P<0.05). They

grew 13% less

than control cells (Table 1).

We examined further whether the decrease in cell number induced by a - or ?

-tocopherol was the

result of apoptosis or a chemical disruption of the cell membrane (necrosis).

Neither significant

LDH-release nor differences in trypan blue uptake were observed when control and

tocopherol

treated DU-145 or CaCo-2 cells were compared (data not shown).

Additionally, DU-145 and LNCaP cells were tested for apoptosis or necrosis by

analysis

following staining with Hoechst 33342 and SYTOX Green. Only negligible amounts

of

apoptosis or necrosis were observed (less than 3%), thus, diminishment of cell

number is neither

a result of apoptosis nor necrosis.

? -Tocopherol inhibits cell-cycle progression and DNA synthesis

To assess the effect of ? -tocopherol on cell cycle progression and DNA

synthesis we used flow

cytometry analysis and a BrdU incorporation assay. ? -Tocopherol (25 µM) treated

cells

presented a higher G1-phase population and a decreased S-phase population than

control cells.

As shown in the cell cycle histogram (Fig. 2), the S-phase population was

significantly lower in

the ? -tocopherol treated cells (21.8% ± 6.3) than in the control cells (30% ±

1.8) 6 h after serum

re-stimulation (P<0.05). 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 a

decreased activity of DNA synthesis in the tested DU-145 and CaCo-2 cells (Table

2). In DU-

145 cells a -tocopherol (25 µM) inhibited DNA synthesis by 32% ± 9 and ?

-tocopherol (25 µM)

inhibited it by 55% ± 5 (P<0.05). In CaCo-2 cells, the activity of DNA synthesis

was inhibited

by 25% ± 11 following a -tocopherol treatment and 48% ± 22 after ? -tocopherol

treatment

(P<0.05). In the BrdU incorporation assay, as in the proliferation assay, the

inhibition effect of ? -

tocopherol was significantly stronger than that of a -tocopherol in the prostate

cancer cell line

tested.

? -Tocopherol down regulates cyclin D1 and cyclin E levels

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. 3a+. Similarly,

serum re-stimulation led

to an increase of cyclin E levels in control cells (Fig. 3c) and ? -tocopherol

both delayed and

inhibited this increase. The effect of a -tocopherol (50 µM) on cyclin D1 and

cyclin E levels was

assessed further in LNCaP cells where a similar, significant pattern of

down-regulation was

observed for both cyclins.

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 CKI

families was examined. Serum stimulation led to increased protein levels of all

the three tested

CKIs differently in time. From a baseline level observed at the end of serum

deprivation, p27 Kip1

protein increased in control cells by more 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

re-stimulation and decreased thereafter. ? -Tocopherol significantly

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

examined.

DISCUSSION

The present study demonstrates that ? -tocopherol is more potent than a - or ß

-tocopherol in

inhibiting proliferation of DU-145, LNCaP, and CaCo-2 cells and that ?

-tocopherol inhibits cell

cycle progression via reduction of cyclin D1 and cyclin E levels. Parallel to

the inhibition of

proliferation, DNA synthesis was more strongly inhibited by ? - than by a

-tocopherol in DU-145

and CaCo-2 cells. Because ? -tocopherol delayed DU-145 in G1-phase, as seen from

flow

cytometry analysis, we examined the effect of ? -tocopherol on the regulation of

the G1-phase

cyclins D1 and E, as well as the cdk-inhibitors (CKIs) p21 CIP1 , p27 Kip1 , and

p16 INK4a . Western-

blot analysis of DU-145 and LNCaP cells showed that serum stimulation led to an

increase of

cyclin D1 levels and ? -tocopherol inhibited this increase. DU-145 cells,

however, have been

described to have a mutated retinoblastoma protein (pRB) with a deletion in the

B-motif of the

pocket for the binding to E2Fs (22). As the cyclin E promoter contains multiple

E2F sites, and

disruption of pRB function was shown to up-regulate cyclin E levels (23), we

investigated

whether the down-regulation of cyclin D1 in DU-145 and LNCaP cells was connected

with a

down-regulation of cyclin E levels. Like cyclin D1 protein levels, cyclin E

increased in control

cells after serum stimulation and ? -tocopherol both delayed and decreased this

effect. In a similar

way, protein levels of the three analyzed CKIs increased after serum stimulation

in control cells,

but remained significantly lower in ? -tocopherol treated cells. The three CKIs

were up-regulated

differently in time after serum stimulation. p27 KIP1 protein level was

up-regulated three to

fourfold already after 2 h and decreased after 8 h. p21 CIP1 protein level

increased 5 h after serum

stimulation and decreased again after 8 h. p16 Ink4a increased only after 8 and

11 h in control cells,

and ? -tocopherol nearly abolished this increase. The analyzed CKIs belong to

two different

families of cdk-inhibitors. p16 Ink4a is a representative of the INK

cdk-inhibitor family that binds

and blocks specifically the early G1 phase cdk4 and cdk6, whereas p21 CIP1 and

p27 KIP1 belong to

a second family of cdk inhibitors. They are broad-spectrum CKIs and bind to

different cyclin/cdk

complexes. p21 CIP1 and p27 KIP1 act mostly in the regulation of the G1-S

transition where they

function as inhibitors as well as activators (24). Taken together, not one of

the tested CKIs

p16 Ink4a , p21 CIP1 , or p27 KIP1 was up-regulated in the presence of ?

-tocopherol; rather, in direct

contrast to the corresponding CKI levels of control cells, they remained near

initial levels for the

time interval evaluated. To our knowledge, this study demonstrates for the first

time, the down-

regulation of cancer cell growth by ? -tocopherol on the level of DNA synthesis,

G1-S transition

delay, and on protein levels for proteins that are important in G1-S transition,

resulting in a

diminishment of cell proliferation without apparent apoptosis or necrosis.

It is well known that ? -tocopherol possesses unique features that distinguish

it from a -tocopherol

(8). For example, ? -tocopherol was shown to be superior to a -tocopherol in

inhibiting neoplastic

transformation of C3H/10T1/2 embryonic fibroblasts (25). Furthermore,

cyclooxygenase activity

in macrophages and epithelial cells was shown to be inhibited by ? -tocopherol

and the ? -

tocopherol metabolite 2,7,8-trimethyl-2-( -carboxyethyl)-6-hydroxychroman (

-CEHC), but not

by a -tocopherol (18). a -Tocopherol similarly has specific non-antioxidant

properties that are not

shared by the other tocopherols. In rat A7r5 smooth muscle cells the control of

cell proliferation

is mediated by inhibition of PKC- activity, a function not shared by ? - or d -

tocopherol (26).

Although a specific target for a - and ? - tocopherol has not yet been

identified, our results

indicate a more potent growth inhibition effect of ? - than of a -tocopherol in

prostate and colon

cancer cell lines. Recalling that non-antioxidant functions have been described

for both a - and ? -

tocopherol (8, 27), ? -tocopherol has a weaker antioxidant capacity than a

-tocopherol, and

combining this with our findings that DU-145, LNCaP, and CaCo-2 cell growth was

inhibited

more significantly by ? - than by a -tocopherol, we suggest a novel

non-antioxidant function to be

at the basis of this ? -tocopherol control of cell proliferation.

Colon cancer growth was shown to be related to enhanced levels of prostaglandin

E2 (produced

by COX-2) that led to transactivation of EGF receptor (28). Should ? -tocopherol

inhibit COX-2

activity in colon cancer cells, as it does in macrophages and epithelial cells,

this could partially

explain the anti-proliferative effect of ? -tocopherol in CaCo-2 cells. In

prostate cancer, however,

COX-2 is not up-regulated and is not inducible by phorbol ester in DU-145 or

LNCaP cells (29).

Thus, in the tested DU-145 and LNCaP cells another mechanism must be responsible

for the

anti-proliferative effect of the tocopherols.

Not all tested cancer cell lines were sensitive to vitamin E. In SaOs-2

osteosarcoma cells a - and

ß -tocopherol had no significant effect on proliferation and DNA synthesis,

whereas ? -tocopherol

only weakly inhibited cell proliferation. Such a finding suggests that ?

-tocopherol has tumor-

specificity and that such an event may depend on the pathways used by different

tumors to

sustain proliferation.

A possible explanation for the down-regulation of cyclin D1 and p21 CIP1

observed in our system

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-3 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 un-inhibiting GSK-3

activity (30,

31). In LNCaP cells, the PI3K pathway was shown to be a dominant growth-factor

activated cell

survival pathway (32), whereas LNCaP and DU-145 cells are known to have a

mutation in the

PTEN gene (33). PTEN is a phosphatase that deposphorylates 3-phosphorylated

inositol

phospholipids, the triggering substrates of the PI3K pathway (34). Serum

stimulation activates

the PI3K pathway and in turn leads to increased cyclin D1 and p21 CIP1

concentrations. In LNCaP

cells, treatment with the PI3K inhibitors Ly294002 and wortmannin induced

apoptosis (32).

Therefore, we speculate that ? -tocopherol, rather than blocking the PI3K

pathway, interacts by a

homeostatic mechanism in inhibiting this pathway. The CKI p16 Ink4a was

up-regulated only 8 h

after serum stimulation and only in control cells. We propose that this CKI is

activated as a part

of a negative feedback loop, when in serum-stimulated control cells cyclins D1

and E

concentrations were significant enough for entry into S-phase.

A possible cancer growth-regulation mechanism for ? -tocopherol could also be

related to the

maintenance of intracellular tocopherol concentration and/or to tocopherol

transport, both

believed to be governed by the tocopherol-associated protein (TAP) (35). TAP is

highly

expressed in prostate, liver, and brain tissues and binds both a - and ?

-tocopherol, although

binding affinity is higher for ? - than for a -tocopherol (36). The primary

reason for ? -tocopherol

action in tumor cells is not known as yet; however, several hypotheses can be

made at this time.

Some components of the proliferation pathways of some tumors may be more

sensitive to ? -

tocopherol than to other tocopherols. Alternatively, ? -tocopherol may be taken

up and/or

accumulated more efficiently in some, relative to other cells. The latter event

may be related to

the presence of the newly discovered TAPs, which have been suggested to play a

role in the

uptake, transfer, and protection of tocopherols in cells (27). It may be

interesting to recall that

TAP is highly expressed in normal prostate, although its regulation and

expression in tumors has

not yet been described.

In accordance with the fact that a - and ? -tocopherol can have different

functions in cells, also

their uptake and metabolism by the human body is regulated differently and

depends on both the

diet and the activity of cytochrome P450 3A (37). In muscle, adipose and brain

tissues, ? -

tocopherol levels depend more directly on the diet and can reach the same levels

as a -tocopherol

(38). In plasma, ? -tocopherol concentrations are lower than a -tocopherol, and

a -tocopherol

supplementation diminishes ? -tocopherol levels (20). ? -Tocopherol

supplementation, on the other

hand, increases ? -tocopherol plasma concentration without significantly

lowering a -tocopherol

levels (39). Diet can vary in total amount of vitamin E and also in the

properties of a -, ? -, and

other tocopherols. For example oil from corn, soybean, sesame, nuts, walnuts,

pecans, or peanuts

are rich sources of ? -tocopherol, and a typical U.S. diet contains ~70% ?

-tocopherol in the total

vitamin E consumed (40), whereas other oils, like sunflower oil, contain almost

exclusively a -

tocopherol (41).

Our findings, that ? -tocopherol inhibits proliferation of prostate and colon

cancer cells more

potently than a -tocopherol, provide a cellular mechanism supporting the concept

brought forth

from epidemiological studies, that a greater magnitude of risk reduction for

prostate and colon

cancer may occur if both total vitamin E consumed and concentrations 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 effects

on the turnover of cyclin D1 are ongoing studies. By addressing these issues the

molecular

characterization of the ? -tocopherol induced cell cycle control demonstrated in

this study will be

further developed.

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Table 1

Comparison of growth inhibition effect between prostate carcinoma,

colon adenocarcinoma, and osteosarcoma cells

DU-145 LNCaP CaCo-2 SaOs-2

a -Tocopherol 50% ± 8 * 48% ± 2 * 50% ± 11 * 4% ± 9

ß -Tocopherol 41% ± 2 * 43% ± 3 * 13% ± 9

? -Tocopherol 86% ± 4 * 74% ± 6 * 64% ± 25 * 13% ± 4 *

Growth inhibition effect of a -tocopherol (25 µM) and ? -tocopherol (25 µM)

compared with control (0.05% ethanol) in the

cancer cell lines DU-145 (prostate cancer), LNCaP (prostate cancer), CaCo-2

(colon cancer), and SaOs-2 (osteosarcoma).

DU-145 and CaCo-2 cells were counted after 24 h; LNCaP and SaOs-2, after 48 h.

Each experiment was performed in

triplicate, and each well was counted twice. Values are means ± SD of three

independent experiments. *P < 0.05,

compared with control. P < 0.05, compared with a - and ß -tocopherol at the

respective concentration.

Table 2

DNA synthesis as measured by BrdU incorporation

DU-145 CaCo-2 SaOs-2

Control 100% 100% 100%

a -Tocopherol (25 µM) 68% ± 9 * 75% ± 10 * 94% ± 11

? -Tocopherol (25 µM) 45% ± 5 * 52% ± 19 * 88% ± 8

BrdU incorporation of tocopherol-treated cells compared with controls (ethanol

0.5% treated cells). Cells grew

exponentially in 96-well plates for 24 h before they were deprived of serum

(0.2% FCS, 24 h). Cells were then treated

either with tocopherol (25 µM) or ethanol (0.5%) and were stimulated with serum

(10% FCS) 1 h later, considered as time

0. Four hours after serum re-stimulation BrdU (10 µM) was added for 2 h, and

cells were fixed and analyzed. Values are

means ± SD of three independent experiments. *P < 0.05, compared with control.

P < 0.05, compared with a - and ß -tocopherol

at the respective concentration.

Table 2

DNA synthesis as measured by BrdU incorporation

DU-145 CaCo-2 SaOs-2

Control 100% 100% 100%

a -Tocopherol (25 µM) 68% ± 9 * 75% ± 10 * 94% ± 11

? -Tocopherol (25 µM) 45% ± 5 * 52% ± 19 * 88% ± 8

BrdU incorporation of tocopherol-treated cells compared with controls (ethanol

0.5% treated cells). Cells grew

exponentially in 96-well plates for 24 h before they were deprived of serum

(0.2% FCS, 24 h). Cells were then treated

either with tocopherol (25 µM) or ethanol (0.5%) and were stimulated with serum

(10% FCS) 1 h later, considered as time

0. Four hours after serum re-stimulation BrdU (10 µM) was added for 2 h, and

cells were fixed and analyzed. Values are

means ± SD of three independent experiments. *P < 0.05, compared with control.

P < 0.05, compared with a - and ß -tocopherol

at the respective concentration.

Fig. 2

Figure 2. Cell cycle distribution in DU-145 cells after 6h serum stimulated

growth. Exponentially growing DU-145

cells were synchronized in G1 phase by serum deprivation (0.2% FCS, 24h). Cells

were then treated either with

? -tocopherol (25 µM) or ethanol (0.05%). Serum stimulation (10% FCS) 1h later

was considered as time 0. After 6h, cells

were harvested and fixed in ethanol. For flow cytometry analysis cells were

stained with propidium iodide (5 µg/ml).

(A) Cell cycle histogram of a representative experiment of ethanol (0.05%) or ?

-tocopherol (25 µM) treated DU-145 cells.

( Cell cycle distribution of ethanol (0.05%) or ? -tocopherol (25 µM) treated

DU-145 cells. Values are means ± SD of

three independent experiments; * P < 0.05, as compared to control.

Fig. 3

Figure 3. Effects of g-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 indicated

times. (A) Cyclin D1; values are ß -actin

corrected means ± SD of four independent experiments. * p < 0.05, as compared to

control at the corresponding time (

Cyclin D1, Western-blot © Cyclin E Western-blot; representative blots from one

of three experiments.

Fig. 4

Figure 4. Effects of g-Tocopherol (25 µM) on CKI protein levels. Whole DU-145

cell lysates were immunoblotted

with antibodies against cyclin p16INK4a, p21CIP1, and p27Kip1at the indicated

times after re-stimulation with serum. A

representative blot from one of three experiments is shown.