Involvement of mitochondrial permeability transition pore opening in alpha-bisabolol induced...

Involvement of mitochondrial permeability transition poreopening in a-bisabolol induced apoptosisElisabetta Cavalieri1,*, Christian Bergamini2,*, Sofia Mariotto1, Serena Leoni2, Luigi Perbellini3,Elena Darra1, Hisanori Suzuki1, Romana Fato2 and Giorgio Lenaz2

1 Dipartimento di Scienze Morfologico-Biomediche, Universita di Verona, Italy

2 Dipartimento di Biochimica ‘G. Moruzzi’, Universita di Bologna, Italy

3 Dipartimento di Medicina e Sanita Pubblica, Universita di Verona, Italy

Introduction

a-Bisabolol is a small oily sesquiterpene (m, 222.37 Da),

widely present in plants, shrubs and trees. It has been

reported to reduce inflammation and arthritis, prevent

the development of gastric ulcers and act as an

antibacterial and antifungal agent. It also exhibits

wound-healing activity and is used in cosmetic

products to treat dermatitis [1–5]. More recently, insec-

ticidal effects of this oil have also been reported

[6]. An antitumor action of a-bisabolol has only

recently been discovered [7]. Indeed, we reported that

Keywords

a-bisabolol; apoptosis; cyclosporine A;

mitochondria; mitochondrial permeability

transition pore

Correspondence

H. Suzuki, Dipartimento di Scienze

Morfologico-Biomediche, Universita di

Verona, Strada Le Grazie, 8, 37134 Verona,

Italy

Fax: +39 045 802 7170

Tel: +39 045 802 7167

E-mail: [email protected]

*These authors contributed equally to this

work

(Received 16 April 2009, revised 15 May

2009, accepted 21 May 2009)

doi:10.1111/j.1742-4658.2009.07108.x

a-Bisabolol is a natural monocyclic sesquiterpene alcohol. It has been used

in cosmetics for hundreds of years because of its perceived skin-healing

properties. a-Bisabolol is known to have anti-irritant, anti-inflammatory

and antimicrobial properties. In precedent studies, we described how

a-bisabolol exerts a selective pro-apoptotic action towards transformed cells

[Cavalieri E et al. (2004) Biochem Biophys Res Commun 315, 589–594] and

its uptake is mediated by lipid rafts on the plasma membrane [Darra E et al.

(2008) Arch Biochem Biophys 476, 113–123]. In this study, we hypothesize

that the intracellular target of a-bisabolol may be the mitochondrial perme-

ability transition pore (mPTP). To evaluate this hypothesis, we used one

transformed cell line (human glioma T67) in comparison with a nontrans-

formed one (human fibroblasts). We assessed the effect of a specific mPTP

inhibitor (cyclosporine A) on the toxic action of a-bisabolol. Results show

that the a-bisabolol-induced decrease in oxygen consumption is abolished

by the addition of cyclosporine A in T67 cells, indicating that a-bisabololmay target mPTP. The central role of mitochondria was also demonstrated

by using galactose to force cells to a more aerobic metabolism. In this con-

dition, we observed higher a-bisabolol toxicity. Furthermore, we studied the

effect of a-bisabolol on isolated rat liver mitochondria. This study expands

the notion of the specific action of a-bisabolol on transformed cells and sug-

gests that it may act by disturbing the structure and function of the mPTP.

a-Bisabolol toxicity is clearly related to its cellular uptake, which is higher

in transformed cell lines.

Abbreviations

CsA, cyclosporine A; DCF-DA, dichlorodihydrofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; FCCP, carbonyl cyanide

p-(trifluoromethoxy)-phenylhydrazone; mPTP, mitochondrial permeability transition pore; NAC, N-acetyl-cysteine; PARP, poly(ADP-ribose)

polymerase; ROS, reactive oxygen species; TMRM, tetramethylrodamine methyl ester.

3990 FEBS Journal 276 (2009) 3990–4000 ª 2009 The Authors Journal compilation ª 2009 FEBS

a-bisabolol is able to kill by apoptosis human and

mouse glioma cell lines such as U87, T67 and C6,

which are highly resistant to common antitumor treat-

ments. a-Bisabolol, at a concentration of 2 lm, which

is estimated to be the EC50 value, induces rapid apop-

tosis in these cells. At higher concentrations (10 lm),

a-bisabolol requires only a few hours to completely kill

these cell lines. a-Bisabolol quickly induces caspase-3

activation, poly(ADP-ribose) polymerase (PARP)

cleavage and DNA ladder formation, which are associ-

ated with an increase in the hypo-G1 fraction of the

cell population [7].

Because neither the antagonist anti-Fas IgG nor the

caspase-8 inhibitor block a-bisabolol-induced cell

death, a-bisabolol has been presumed to trigger apop-

tosis in a Fas-receptor-independent manner, through

the intrinsic (i.e. mitochondrially related) pathway [7].

Furthermore, treatment of cells with a-bisabolol rap-

idly induces loss of mitochondrial inner transmem-

brane potential (DWm) and the release of cytochrome c

from mitochondria.

Another striking feature of a-bisabolol is that it is

less toxic to normal cells, such as astroglial cells [7],

than to transformed cells, in line with its nontoxicity

in adult mice and rats (reported LD50 values are

14–15 gÆkg)1) [8].

Apoptosis is a physiological, energy-requiring pro-

cess that is genetically programmed [9]. Tumor cells

are believed to be more resistant to apoptosis than

normal cells [10,11]. Unfortunately, a majority of the

antitumor compounds reported to date induce apopto-

sis indiscriminately in normal and tumor cells [12]. A

preferential apoptotic action of a-bisabolol towards

tumor cells, therefore, should represent a remarkable

advantage for therapeutics.

The higher toxicity of a-bisabolol towards tumor cell

lines may be related to a different uptake efficiency

between normal and tumor cells that might be medi-

ated by plasma membrane lipid composition. Because

a-bisabolol is a quite hydrophobic compound, it is rea-

sonable to hypothesize that the lipid composition of

the plasma membrane may play a crucial role in its

uptake. In a recent study, Darra et al. [13] described

how a-bisabolol can be rapidly incorporated into lipid

rafts, highly dynamic membrane domains enriched

with sterol and sphingolipid that play an important

role in intracellular protein transport and membrane

fusion [14]. In this regard, it has been reported that

the plasma membranes of tumor cells contain many

more lipid rafts than the plasma membranes of normal

cells. Moreover, Darra et al. [13] have demonstrated

that a-bisabolol induces the transfer of Bid from cyto-

plasm to lipid rafts and interacts with Bid in a highly

specific way, suggesting that Bid may be directly

involved in a-bisabolol transport from lipid rafts to

the mitochondrial membrane.

In this study, we confirmed the high a-bisabololuptake in a T67 glioma cell line in comparison with

normal fibroblasts and we investigated the molecular

mechanism by which a-bisabolol efficiently and rapidly

induces apoptosis in T67 cells. Based on the previous

observation that a-bisabolol induces apoptosis throughthe mitochondrial intrinsic pathway, rather than the

receptor-mediated pathway, we analyzed the relation

between mitochondrial damage induced by a-bisabololand the viability of the human glioblastoma cell line

and human fibroblasts.

Because cytochrome c release is an early event

induced by treatment with a-bisabolol and could be

mediated by mitochondrial permeability transition

pore (mPTP) opening or by members of the Bcl-2 fam-

ily, we analyzed both the effect of cyclosporine A

(CsA), a well-characterized inhibitor of mPTP opening,

and the amount of Bax and Bcl-2 on a-bisabololaction in glioblastoma cells and fibroblasts.

Bax and Bcl-2 have opposite effects on apoptotic

cell death: the first induces cell death and the second

inhibits it. Bax is a cytosolic, monomeric protein

that, following a death stimulus, translocates from

the cytoplasm to the mitochondria, where it becomes

an integral membrane protein. Bcl-2 is an integral

membrane protein, heavily localized in the mito-

chondria [15].

Results

a-Bisabolol uptake is higher in human glioma cell

line T67 than in fibroblasts

Our previous results [7] showed that 5 lm a-bisabololis able to induce apoptotic cell death in all examined

malignant cell lines and is far less effective in killing

human normal cells. Table 1 shows the effect of 5 lm

a-bisabolol on cell viability, DNA ladder and PARP

cleavage in different cell lines. The data clearly show

that a-bisabolol toxicity is higher in malignant than

nontransformed cell lines; this behavior may be related

to differences in cellular uptake. In fact, intracellular

concentration analysis shows that in the T67 cell line,

a-bisabolol is rapidly distributed in the membrane,

cytosolic and nuclear fractions, whereas in fibroblasts

its intracellular distribution is significative lower

(Fig. 1). These results are in line with previous

data obtained in other cell lines [13] and suggest that

a-bisabolol, once absorbed into plasma membrane

lipid rafts, is transported inside cells.

E. Cavalieri et al. a-Bisabolol, an efficient apoptosis inducer

FEBS Journal 276 (2009) 3990–4000 ª 2009 The Authors Journal compilation ª 2009 FEBS 3991

a-Bisabolol-induced apoptosis is independent of

Bax and Bcl-2 and of reactive oxygen species

Cytochrome c release and loss of mitochondrial mem-

brane potential are early events in the apoptotic cell

death induced by a-bisabolol treatment [7]; both events

may be related to a change in the amounts of pro- ⁄antiapoptotic proteins of the Bcl-2 family or to a direct

involvement of the mPTP.

To investigate the molecular mechanism involved in

a-bisabolol toxicity, we examined the change in the

amounts of Bcl-2 family proteins and the role of

mPTP in a-bisabolol-induced apoptosis. Expression of

these two proteins in T67 cells was examined by wes-

tern blotting after a-bisabolol treatment over periods

of 15–60 min, corresponding to the period in which we

observed cytochrome c release [7]. As shown in Fig. 2,

there was no change in the amounts of cytosolic and

mitochondrial Bax, or in total Bcl-2 proteins at any

time during treatment.

However, cell treatment with CsA, a specific inhibi-

tor of mPTP, induced an increase in cell survival in

the presence of a-bisabolol, as confirmed by the retain-

ing of membrane potential (Fig. 7A,B). Because mPTP

opening can be induced by reactive oxygen species

(ROS), we studied their contribution to a-bisabolol-induced apoptosis. For this purpose, we measured the

ROS level in both control and a-bisabolol-treated T67

and fibroblast cell lines. Treatment with 5 lm a-bisab-olol increases the ROS level in T67 cells. This effect

was counteracted by adding 10 mm N-acetyl-cysteine

(NAC). Moreover, we did not observe any increase in

ROS levels in treated fibroblasts (Fig. 3A). Neverthe-

less, in T67 cells, NAC did not prevent PARP cleavage

or DNA ladder formation after 3 or 5 h of a-bisabololtreatment (Fig. 3B,C). These data indicate that oxida-

tive damage, although present, is not involved in the

apoptotic process induced by a-bisabolol.

a-Bisabolol affects oxygen consumption in intact

cells and rat liver mitochonria

Mitochondrial involvement in a-bisabolol-induced cell

death was confirmed by measuring oxygen consump-

tion by intact cells. Because this (Table 1) and previous

studies [7] have shown that the toxic effect is higher in

cancer cells than in normal cells, we measured oxygen

consumption in two different cell types: T67 and fibro-

blasts. We measured the endogenous respiration rate

in the presence of glucose. The maximum respiration

rate was achieved by adding carbonyl cyanide p-(triflu-

oromethoxy)-phenylhydrazone (FCCP) as an electron

transport uncoupler.

Figure 4A shows that the oxygen consumption in T67

cells exposed to 5 lm a-bisabolol for 20 min was � 50%

Table 1. a-Bisabolol (5 lM) induced apoptotic cell death in a

number of transformed and normal human cells. PARP,

poly(ADP-ribose) polymerase.

Cells

% Cell

deatha

DNA

ladderb

PARP

cleavagec

T67 glioblastoma 90 + +

U87 glioblastoma 60 + +

MDA-MB231 breast cancer 86 + +

LNCap prostate cancer 73 + +

PC3 prostate cancer 90 + +

HL-60 leukemia 67 + +

Lymphocytes T normal cell 2 ) )Neutrophiles normal cell 1 ) )Fibroblasts normal cell 20 ) )

a Cell viability was examined after 5 h. b DNA ladder formation was

examined after 5 h. c PARP cleavage was analyzed after 3 h.

Fig. 1. Measurement of a-bisabolol concentration in T67 cells and

fibroblasts. Concentration of a-bisabolol in human fibroblasts and

T67 cell lines after 30 min of treatment. a-Bisabolol measured in

cytosolic, membrane and nuclear fractions using the gas chroma-

tography method. Data are presented as ± SD of six independent

measurements.

Fig. 2. Mitochondrial and cytosolic levels of Bax and Bcl-2 are unaf-

fected by a-bisabolol treatment. T67 cells were treated with 5 lM

a-bisabolol or with vehicle solvent over a period of 15–60 min. The

amounts of cytosolic and mitochondrial Bax and total Bcl-2 were

visualized by western blot using anti-Bax and anti-Bcl-2 IgG, respec-

tively. The results are indicative of three independents blots.

a-Bisabolol, an efficient apoptosis inducer E. Cavalieri et al.


with respect to controls and was almost completely

insensitive to FCCP. The decrease in oxygen consump-

tion became more pronounced after 90 min of incuba-

tion (5% of retained respiration, data not shown).

Fibroblasts showed a very different behavior: incu-

bation with 5 lm a-bisabolol induced limited uncou-

pling without loss of respiratory capacity over the first

40 min of incubation (Fig. 4B). At longer incubation

times (120 min), the respiratory capacity remained at

50% with respect to the control (data not shown).

Under the described conditions, fibroblasts retained

good viability for as long as 60 min, with some decline

over the next hour. Even for incubation times longer

than 2 h, fibroblast viability did not drop below 70%.

By contrast, under the same culture conditions, T67

viability dropped to � 30–40% within the first hour,

and declined to almost zero in the second hour

(Fig. S1).

B

A

C

++ __10 mM NAC

5 h 3 h _ +_ +

116 kDa

85 kDa

+ + + +__αα-bisabolol _ _

Fig. 3. ROS involvement in a-bisabolol-induced apoptosis. (A) ROS

production in T67 cells and fibroblasts in the presence or absence

of 5 lM a-bisabolol and 10 mM NAC, using DCF-DA as the fluores-

cent probe. Data (expressed in fluorescence arbitrary units) were

collected after 40 min of incubation and are the mean of four differ-

ent experiments (± SD). (B,C) T67 cells were pre-treated with

10 mM NAC for 30 min and then treated with 5 lM a-bisabolol or

with vehicle solvent. Apoptosis were evaluated analyzing the DNA

ladder formation (B) and PARP cleavage (C). Figures show repre-

sentative results from three independent experiments.

A

B

Fig. 4. Oxygen consumption rate of the T67 cell line and human

fibroblasts in the presence of glucose. Effect of a-bisabolol treat-

ment on coupled and uncoupled respiration in the T67 cell line (A)

and human fibroblasts (B). Uncoupling was achieved by adding

FCCP to a final concentration of 600 nM. Data are the mean of

three different determinations ± SD.



In experiments performed on isolated rat liver mito-

chondria, we observed that the decrease in oxygen con-

sumption induced by a-bisabolol treatment was not

related to a direct inhibitory effect on the mitochon-

drial respiratory enzymes. In fact, the respiration rate

observed in the uncoupled condition was insensitive to

a-bisabolol treatment (Table 2). The decrease in state 3

respiration in the presence of a-bisabolol is partially

counteracted by adding more ADP. This behavior sug-

gests adenine nucleotide translocator or ATP syntase

impairment. Preliminary results show that a-bisabololcan affect adenine nucleotide translocator activity

(data not shown).

To further test whether the toxic action of a-bisabo-lol affects mPTP, we examined the effect of CsA on

cellular respiration and apoptosis in T67 cells.

Figure 5A shows that the toxic effect of a-bisabolol oncellular respiration was abolished by the presence of

10 lm CsA in the culture medium. Moreover, T67 cells

preincubated with CsA and then exposed to 5 lm a-bi-sabolol, showed no PARP cleavage after 3 h and no

DNA ladder formation after 5 h (Fig. 5B). At this

concentration, CsA has numerous nonspecific effects,

including inhibition of calcineurin, a protein phospha-

tase differently involved in apoptotic process. There-

fore, to exclude that the effect of a-bisabolol could be

mediated by calcineurin, the same experiments were

performed in the presence of the calcineurin inhibitor

FK506. Pretreatment of T67 cells with 1 lm FK506

for 30 min was not able to prevent the apoptotic effect

of a-bisabolol (Fig. 5C).

a-Bisabolol toxicity is higher under aerobic

metabolic conditions

To confirm the involvement of mitochondria in

a-bisabolol toxicity, we pushed cells towards more

aerobic metabolism by growing them in Dulbecco’s

modified Eagle’s medium (DMEM) containing galac-

tose instead of glucose. Galactose is a carbon source

known to favor the oxidative phosphorylation path-

way [16]. T67 cells cultured in DMEM–galactose

showed measurably greater oxygen consumption in

state 3 and were also more sensitive to a-bisabololtoxicity (Fig. 6A).

Even fibroblasts became more sensitive to a-bisabololtoxicity when pushed to a more aerobic state. In

Fig. 6B, we show that the normal respiration rate is

maintained for only 20 min. Under these culture condi-

tions, 40 min of incubation with 5 lm a-bisabololresulted in a 50% loss of viability in normal fibroblasts,

whereas T67 cells show 100% mortality (Table S1). The

increase in a-bisabolol toxicity in cells forced to an

aerobic metabolism strengthens the hypothesis of a

mitochondrial involvement in a-bisabolol toxicity.

a-Bisabolol affects mitochondrial membrane

potential in intact cells

The effect of a-bisabolol on mitochondrial membrane

potential in digitonin-permeabilized cells was evaluated

using the fluorescent cationic probe tetramethylrod-

amine methyl ester (TMRM). T67 cells and fibroblasts,

preincubated for 30 min with or without CsA, were

treated for 40 min with a-bisabolol.In T67 cells, a-bisabolol treatment results in a

decreased membrane potential and this effect was

counteracted by CsA (Fig. 7A). As shown in Fig. 7B,

a-bisabolol did not affect fibroblasts membrane poten-

tial within 40 min of treatment.

Spectrofluorimetric data are confirmed by fluores-

cence microscopy imaging, using the same fluorogenic

probe (TMRM). T67 images collected after 20 and

40 min of a-bisabolol incubation showed a progressive

loss of mitochondrial membrane potential (Fig. 7C).

Preincubation with CsA protected T67 cells from

depolarization. Treated fibroblasts images revealed

only a slight effect of a-bisabolol on mitochondrial

membrane potential.

Discussion

The different effect of a-bisabolol on tumor versus nor-

mal cells is unusual and knowledge of the molecular

basis for this difference should be very important for the

research into and development of targeted antitumor

Table 2. Effect of 5 lM a-bisabolol on oxygen consumption in coupled rat liver mitochondria energized with glutamate–malate. State 3 of

respiration was measured in the presence of two different ADP concentrations (180 and 500 lM). 2,4-Dinitrophenol (60 lM) was added to

achieve the maximal respiration rate.

Substrate

State 4 State 3(ADP 180 lM) State 3(ADP 500 lM) 2,4-Dinitrophenol

ng AoxygenÆmin)1Æmg)1 ng AoxygenÆmin)1Æmg)1 ng AoxygenÆmin)1Æmg)1 ng AoxygenÆmin)1Æmg)1

Glutamate ⁄ malate 19.4 ± 1.7 93.9 ± 10.9 118.8 ± 5.3

Glutamate ⁄ malate + a-bisabolol 5 lM 23.5 ± 2.7 59.8 ± 7.2* 72.05 ± 6.5* 105.2 ± 12

*P < 0.05.



drugs. One possible reason for this selectivity was

exploited in a recent study by Darra et al. [13] in which

they studied the uptake of a-bisabolol in lipid rafts from

tumor and normal cells. Usually tumor plasma mem-

branes contain more lipid rafts than the normal plasma

membranes and this may explain the higher toxicity

exerted by a-bisabolol [17,18]. Moreover, a-bisabololtrafficking between the plasma membrane and the

mitochondria may be mediated by Bid, as suggested by

the binding experiments described by Darra et al. [13].

Furthermore, once absorbed in the plasma membrane,

a-bisabolol is rapidly distributed in the cytosol

and nuclei. We confirm the different accumulation of

a-bisabolol inside cells using T67 and fibroblasts.

The increase in cytochrome c release [7] and the

resistance to cell death conferred by CsA strongly sug-

gest mitochondrial involvement in a-bisabolol-inducedapoptosis; moreover our results show that neither

Bax ⁄Bcl-2 nor ROS production are involved. In this

scenario, ROS production observed in the T67 cell line

is not the direct cause of apoptosis, but could be a

secondary effect because of the loss of mitochondrial

integrity. In fact, using NAC as a radical scavenger,

we observed a decrease in ROS production, but we did

not prevent cell death. In T67 cell lines, death seems to

be caused by a massive mPTP opening with a conse-

quent release of mitochondrial constituents and a loss

A

B

C

Fig. 5. CsA protects T67 cells from apoptotic death induced by a-

bisabolol. (A) Pretreatment with 10 lM CsA prevented a decrease

in the of oxygen consumption rate induced by 30 min of incubation

with 5 lM a-bisabolol in both coupled and uncoupled respiration.

Uncoupling was achieved by adding FCCP to a final concentration

of 600 nM. (B) CsA prevented PARP cleavage and DNA ladder for-

mation induced by a-bisabolol. (C) FK506 did not prevent a-bisabo-

lol-induced PARP cleavage and DNA ladder formation. Cells were

treated with 10 lM CsA (B) and 1 lM FK506 (C) for 30 min and then

with 5 lM a-bisabolol or vehicle solvent. Figures show representa-

tive results from four independent experiments.

A

B

Fig. 6. Oxygen consumption rate of the T67 cell line and human

fibroblasts in the presence of galactose. Effect of a-bisabolol treat-

ment on coupled and uncoupled respiration in the T67 cell line (A)

and human fibroblasts (B). Uncoupling was achieved by adding

FCCP to a final concentration of 600 nM. Data are the mean of

three different determinations ± SD.



of respiratory activity. The inference that mPTP is a

target in the mechanism of action of this small terpene

is supported by the prevention of cell death by CsA, a

specific inhibitor of mPTP opening [19]. CsA preincu-

bation prevents a-bisabolol-induced mitochondrial

membrane depolarization both in permeabilized and

adherent T67 cells. In normal fibroblasts, 40 min of

a-bisabolol incubation did not exert any significant

effect on mitochondrial potential.

The results obtained on isolated rat liver mitochon-

dria suggest that the damage is related to a loss of mem-

brane integrity. Nevertheless, in rat liver mitochondria,

the effect of a-bisabolol on mPTP opening was slight; at

first glance, this behavior seems to be in disagreement

with the strong cell death induced (Fig. S2).

The reasons for this apparent discrepancy may be

related to the different experimental models (cell-free

vs. cell models). In a cell model, even a partial loss of

mitochondrial membrane integrity may trigger an

amplified response (e.g. cytochrome c release and

apoptosis). Loss of mitochondrial integrity may be

more dangerous for cells that depend mainly on aero-

bic rather than glycolytic metabolism. In the latter

case, the energy requirement is less dependent on mito-

chondrial activity, and so cells can survive even with

mitochondrial impairment.

Because a-bisabolol seems to be preferentially

adsorbed in lipid rafts, we speculate that its effect

may be mediated by perturbation of the lipid bilayer.

In pilot experiments, we prepared dipalmitoylphos-

phatidylcholine vesicles in the presence of 5 lm

a-bisabolol and examined them using differential scan-

ning calorimetry. Analysis showed that a-bisabololinduced a decrease in the transition temperature of the

lipid phase without broadening the differential scan-

ning calorimetry spectra (Fig. S3). This is indicative of

a change in the more external part of the lipid vesicles

and suggests that a-bisabolol is able to induce disorga-

nization on the membrane surface. In mitochondria,

such a change may be involved in the PTP regulation,

as shown by Ricchelli et al. [20].

In conclusion, our results confirm that a-bisabolol isable to induce apoptotic cell death preferentially in

tumor cells and suggest that its toxicity is strictly

related to its intracellular concentration. Because of its

high lipophilicity, a-bisabolol uptake should be medi-

ated by membrane lipid rafts and its intracellular traf-

ficking may involve Bid, a protein of the Bcl-2 family.

This would explain the increased uptake in cancer

cells, whereas the results obtained using CsA confirm

the involvement of mPTP opening in the cell death

mechanism.

Materials and methods

Reagents

All chemicals used were of the highest analytical grade and

purchased from Sigma Chemical Company (Milan, Italy),

unless otherwise specified.

Fibroblasts αα-bisabolol α-bisabolol

T67

0 min

20 min

40 min

α-bisabolol+ CsA

α-bisabolol+ CsA

A

B

C

Fig. 7. a-Bisabolol reduces the mitochondrial DW in T67 cells, but

not in fibroblasts. Effects of 5 lM a-bisabolol (40 min of incubation)

on mitochondrial membrane potential in T67 cells (A) and fibro-

blasts (B) in the presence or absence of 10 lM CsA. Data are

expressed as fluorescence arbitrary unitsÆ106 cells)1 and are the

mean of at least three different determinations (± SD). (C) Fluores-

cence microscopy images of fibroblasts and T67 cells in the pres-

ence of 5 lM a-bisabolol and 5 lM a-bisabolol + 10 lM CsA. Images

were collected at 0, 20 and 40 min of incubation, using 100 nM

TMRM as the mitochondrial membrane potential probe.



Cell culture

The T67 human glioma cell line was derived by Lauro et al.

[21] from a World Health Organization (WHO) Grade III

gemistocytic astrocytoma. The primary culture of human

fibroblasts was a generous gift from U. Armato (Verona

University, Italy). T67 cells and normal human fibroblasts

were cultured in DMEM (BioWhittaker, Cambrex BioSci-

ence, Verviers, Belgium), supplemented with 10% fetal

bovine serum (BioWhittaker, Cambrex BioScience),

100 UIÆmL)1 penicillin, 100 lgÆmL)1 streptomycin and

40 lgÆmL)1 gentamycin, in a 5% CO2 atmosphere at 37 �C,with saturating humidity. Cell stocks were cryopreserved

using standard methods, and stored in liquid nitrogen. Cell

viability was measured by Trypan blue exclusion [22].

a-Bisabolol treatment

Cells were treated with a-bisabolol (purum, ‡ 95%, Fluka

and Riedel-de Haen, Sigma Chemical Co.) from a freshly

prepared stock solution in absolute ethanol (1 : 8 v ⁄ v). Thea-bisabolol concentration indicated in cell experiments rep-

resents its soluble fraction in culture medium, measured as

described previously [7].

Measurement of a-bisabolol in membrane,

cytosol and nuclear fraction

T67 cells and fibroblasts treated for 15 min with a-bisabololwere resuspended and homogenized using a hand-held glass

homogenizer in 300 lL of ice-cold buffer A (20 mm

Tris ⁄HCl, pH 7.5; 2 mm EDTA; 0.5 mm EGTA; complete

EDTA-free protease inhibitor cocktail; Roche Applied

Science, Mannheim, Germany). Lysates were centrifuged at

800 g for 5 min at 4 �C in order to remove nuclei and super-

natants were centrifuged at 1 000 000 g for 20 min at 4 �C.The obtained supernatants were collected and used as the

cytosolic fraction. The membrane pellets were solubilized in

200 lL buffer A plus 1% Triton X-100 by bath sonication

and centrifuged at 12 000 g for 20 min at 4 �C in a micro-

centrifuge. The obtained supernatants were used as the

membrane fraction. a-Bisabolol concentration was analyzed

in each fraction by the gas chromatography method [23].

Western blot analysis

Bax analysis

Cell pellets were suspended in 1 mL of solution containing

10 mm NaCl, 1.5 mm MgCl2, 10 mm Tris ⁄HCl, pH 7.5,

1 mm sodium orthovanadate and complete EDTA-free pro-

tease inhibitor cocktail (Boehringer-Mannheim). Cells were

then chilled on ice for 10 min and gently lysed by adding

0.3% (v ⁄ v) Nonidet-P40. In order to restore an isotonic

environment, a solution containing 525 mm mannitol,

175 mm sucrose, 12.5 mm Tris ⁄HCl, 2.5 mm EDTA,

pH 7.5 and protease inhibitor cocktail was added. Lysates

were centrifuged at 17 000 g for 30 min at 4 �C. The cyto-

solic and mitochondrial fractions so obtained were sepa-

rated on a 15% SDS polyacrylamide gels.

Bcl-2 and PARP analysis

Cells were homogenized at 4 �C in 50 mm Tris ⁄HCl, pH 8,

containing 0.1% Nonidet-P40, 200 mm KCl, 2 mm MgCl2,

50 lm ZnCl2, 2 mm dithiothreitol and EDTA-free protease

inhibitor cocktail (Boehringer-Mannheim). Aliquots of the

homogenates were loaded on 15% (for Bcl-2) or 7.5% (for

PARP) SDS polyacrylamide gels.

Electrophoresis was performed at 100 V with a running

buffer containing 0.25 m Tris ⁄HCl, pH 8.3, 1.92 m glycine

and 1% SDS. The resolved proteins were electroblotted onto

a poly(vinylidene difluoride) membrane (Immobilon P; Milli-

pore, Bedford MA, USA). Membranes were incubated with

a rabbit polyclonal IgG antibody to Bax (Upstate, Lake

Placid, NY, USA), a mouse monoclonal IgG antibody to Bcl-

2 (Upstate) and a mouse monoclonal IgG antibody to PARP

(Zymed, South San Francisco, CA, USA) and incubated with

the appropriate secondary antibody IgG–peroxidase conju-

gate (Amersham Biosciences, Little Chalfont, UK). The blots

were successively incubated with enhanced chemiluminescent

detection reagents (ECL; Amersham Biosciences) according

to the manufacturer’s instructions and proteins were detected

by exposing the blots to Kodak X-AR film.

DNA ladder

For internucleosomal DNA laddering, 3 · 106 cells, resus-

pended in 0.3 mL of culture medium containing 10% fetal

bovine serum, were incubated for 45 min at 65 �C, and then

overnight at 37 �C in the presence of 0.4 m NaCl, 5 mm

Tris ⁄HCl, pH 8, 2 mm EDTA, 4% SDS and 2 mgÆmL)1 pro-

teinase K. The lysates, brought to a final concentration of

1.58 m NaCl, were centrifuged twice for 10 min at 6000 g to

separate the DNA fragments from intact DNA. The superna-

tants were recovered and DNA was precipitated by addition

of three volumes of absolute ethanol at )80 �C for 1 h. DNA

pellets were recovered by microcentrifugation (10 min at

12 000 g), and resuspended in a minimal volume of 40 lL of

10 mm Tris ⁄HCl, pH 7.4, 1 mm EDTA, 1 mgÆmL)1 DNase-

free ribonuclease A. Aliquots of 5 lg of DNA were then

loaded onto a 1% agarose gel containing 0.25 lgÆmL)1

ethidium bromide. After electrophoresis, the DNA was

visualized by UV light and recorded photographically.

Measurement of ROS

Confluent T67 cells and fibroblasts washed with fresh

DMEM were incubated for 2 h in DMEM premixed with



10 lm dichlorodihydrofluorescein diacetate (DCF-DA).

Cells loaded with DCF-DA were subsequently washed with

NaCl ⁄Pi and incubated for 40 min in DMEM mixed with

5 lm a-bisabolol. Fluorescence was recorded in a fluores-

cence plate reader using a six-well microtiter plate after

excitation at 485 nm and emission at 535 nm at 30 �C.Cells were treated with 10 lm CsA and 10 mm NAC

during DCF-DA incubation to test their effect on ROS

production. To normalize the fluorescence detection each

well was carefully seeded with the same number of cells

[24–26].

Mitochondrial membrane potential in intact cells

Confluent T67 cells and fibroblasts were incubated for

40 min with 5 lm a-bisabolol, with or without 10 lm CsA

preincubation. Briefly, after trypsinization, cells were

collected and resuspended in 0.25 m sucrose, 10 mm

Hepes, 5 mm KH2PO4, 1 mm EDTA, 50 lm EGTA,

pH 7.4 with KOH. Assays were carried out in 2 mL resus-

pension medium, containing 1 · 106 cells, in the presence

of 100 nm TMRM, 1 lgÆmL)1 oligomycin and 1 lgÆmL)1

rotenone. After 10 min for stabilization, succinate ⁄Triswas added to 12 mm to energize mitochondria. Then

5 lgÆmL)1 digitonin pulses were added stepwise until

complete cell permeabilization [27]. Depolarization was

achieved by 200 nm FCCP addition. TMRM fluorescence

was measured in a Jasco FP-777 spectrofluorometer, using

558 nm as excitation wavelength and 578 nm as emission

wavelength.

Cell images were obtained using an IX50 inverted fluo-

rescence microscope (Olympus, Tokyo, Japan). Confluent

cells were washed with DMEM, incubated with 100 nm

TMRM for 20 min, then washed again with DMEM and

treated with 5 lm a-bisabolol in the presence or absence of

10 lm CsA. Images were collected at 20 and 40 min after

a-bisabolol treatment.

Oxygen consumption

Cells

Intact cells (1 · 106) were assayed for oxygen consumption

at 37 �C using a thermostatically controlled oxygraph and

Clark electrode. The respiration rate was measured in cells

treated with 5 lm a-bisabolol in DMEM. Maximal respira-

tion rate was empirically determined by stepwise addition

of the uncoupler FCCP.

For respiration assays in the presence of galactose, cells

were incubated for 24 h in DMEM without glucose, con-

taining 10% (v ⁄ v) dialyzed fetal bovine serum. This glu-

cose-free medium was supplemented with galactose (5 mm)

and pyruvate (110 mgÆL)1) as the oxidizable substrate. Res-

piration rates were measured in this medium, and expressed

as nmol O2Æmin)1Æ106 cells)1.

Rat liver mitochondria

Coupled rat liver mitochondria were prepared according to

Costantini et al. [28]. Oxygen consumption was assayed

according to Slater [29]. The maximal electron transport

capability (maximal respiration rate) was achieved by add-

ing 60 lm 2,4-dinitrophenol as the electron transfer uncou-

pler. The respiratory rates were expressed in ng atoms

oxygenÆmin)1Æmg)1 of protein referencing to 250 nmo-

l O2Æml buffer)1 as 100% at 30 �C [30].

Acknowledgements

We thank Dr Sergio Bonora for performing the differ-

ential scanning calorimetry experiments. This work

was supported financially by grant COFIN2002 of Ital-

ian Ministry of University and Scientific and research

and grant of CARIVERONA 2003 to HS.

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Supporting information

The following supplementary material is available:

Fig. S1. Effect of different incubation times on the via-

bility of T67 cells and human fibroblasts treated with

a-bisabolol.Fig. S2. Effect of a-bisabolol on mitochondrial swelling.

Fig. S3. Effect of a-bisabolol on transition temperature

of dipalmitoylphosphatidylcholine vesicles.

Table S1. Effect of different incubation times on the

viability of T67 cells and human fibroblasts treated

with a-bisabolol, using galactose ⁄pyruvate as a respira-

tory substrate.



This supplementary material can be found in the

online article.

Please note: As a service to our authors and read-

ers, this journal provides supporting information sup-

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reviewed and may be re-organized for online deliv-

ery, but are not copy-edited or typeset. Technical

support issues arising from supporting information

(other than missing files) should be addressed to the

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