molecules
Article
Functional Mitochondria Are Important for the Effect
of Resveratrol
Anne L. Widlund, Kaushal Baral, Louise T. Dalgaard and Ole Vang *
Department of Science and Environment, Roskilde University, Roskilde DK-4000, Denmark;
[email protected] (A.L.W.); [email protected] (K.B.); [email protected] (L.T.D.)
* Correspondence: [email protected]; Tel.: +45-4674-2552
Academic Editor: Derek J. McPhee
Received: 13 March 2017; Accepted: 16 May 2017; Published: 20 May 2017
Abstract: Resveratrol (Resv) is a polyphenol reported to modulate mitochondrial activity. The aim
was to use HeLa and 143B cells to characterize the action of Resv on mitochondrial activity, cell size
and proliferation using wild type (WT) and Rho 0 cells deficient in mitochondrial DNA. In both HeLa
WT and Rho 0 cells, the oxygen consumption rate (OCR) was increased at 20 µM Resv after 24 h,
whereas only a non-significant increase of OCR was observed in 143B WT cells. Resv decreased cell
number concentration-dependently in both WT and Rho 0 cell types. An increased cell diameter
was observed in HeLa WT, but not in Rho 0 when treated with Resv. Overall, the findings presented
indicate that functional mitochondria are a prerequisite for cell enlargement by Resv.
Keywords: mitochondria; resveratrol; HeLa; 143B; wild type; Rho 0; oxygen consumption rate;
oxidative stress
1. Introduction
Mitochondria are highly dynamic organelles that are involved in the regulation of cellular
metabolism, redox regulation, and programmed cell death/apoptosis. Mitochondria constitute the
principal energy source of the cell and convert nutrients into energy through cellular respiration [1].
Resveratrol (3,40 ,5-trihydroxy-trans-stilbene) (Resv) is a naturally occurring polyphenol found
in a high number of unrelated plant species including grapes, cranberries and peanuts, and also in
a number of herbal remedies [2]. Resv shows cytostatic action, prolongs the cell cycle S phase [3],
enlarges cell size [4], and there is a clear anticarcinogenic activity of Resv in animal experiments [5,6].
Previously, an increase in mitochondrial size by Resv has been observed in vivo [7,8] and, in
isolated rabbit renal tubules, experiments have demonstrated increased basal respiration following
Resv exposure [9]. On the other hand, experiments with isolated mitochondria show that Resv
decreased the activity of several complexes in the electron transport chain (ETC) [10,11] and directly
interacted with complex I [12]. Therefore, inhibition of the cellular respiration is suggested to be one of
the primary targets of the cytotoxic effects of resveratrol [13].
A proper cellular metabolism is a prerequisite for cellular proliferation, differentiation and
apoptosis. Impaired mitochondria may alter cellular redox homeostasis, ATP production, the
biosynthetic and secretory function of cells as well as nuclear gene expression by changing
retrograde signaling pathways. Resv-induced mitochondrial biogenesis would act to overcome these
impairments [7,8,14]. When mitochondrial biogenesis is stimulated, as observed for Resv, a lower flow
rate of electrons per unit of mitochondria is obtained, which decreases the propensity for free radical
formation [15,16]. Resv protects mitochondria against oxidative stress through AMP-Activated Protein
Kinase-mediated Glycogen Synthase Kinase-3β inhibition downstream of the Poly (ADP-ribose)
polymerase-LKB1 pathway [17]. Since Rho 0 cells’ mitochondria are devoid of mtDNA and do not
have oxidative phosphorylation, they can be used as a model for testing the molecular effects of lack
Molecules 2017, 22, 847; doi:10.3390/molecules22050847
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lack of proper
mitochondrial
compared
to aThe
wild
type experiments
(WT). The present
of effects
properof
mitochondrial
activity
comparedactivity
to a wild
type (WT).
present
describe
describe
the effects
of Resv on several
mitochondrial
activity
in HeLa
WTIt is
theexperiments
effects of Resv
on several
mitochondrial
activity
parameters in
HeLaparameters
WT and Rho0
cells.
and
Rho0
cells.
It
is
demonstrated
that
Resv
treatment
decreases
cell
numbers
and
proliferation,
while
demonstrated that Resv treatment decreases cell numbers and proliferation, while an increase in cell
anin
increase
in cell
size in HeLa
WT is observed.
Resv
increased
in both
WT
size
HeLa WT
is observed.
Nevertheless,
ResvNevertheless,
increased OCR
in both
HeLaOCR
WT and
RhoHeLa
0, whereas,
whereas,
for and
the cell
line WT
143B
andOCR
derived
OCR
only
altered
by Resv
forand
theRho
cell 0,
line
WT 143B
derived
Rho0
cells,
wasRho0
onlycells,
altered
bywas
Resv
in WT
cells.
in WT cells.
2. Results
2. Results
2.1. Effect of Resveratrol on Cell Numbers, Proliferation and Diameter
2.1. Effect of Resveratrol on Cell Numbers, Proliferation and Diameter
Cell proliferation was studied in both HeLa WT and Rho 0 cells by following the cell
Cell
proliferation
washstudied
in without
both HeLa
WT treatment.
and Rho 0 cells
by decreased
following the
number
number
counts
up to 48
with or
Resv
Resv
thecell
cell
number
counts
up
to
48
h
with
or
without
Resv
treatment.
Resv
decreased
the
cell
number
concentrationconcentration-dependently in both HeLa WT and HeLa Rho 0 (Figure 1A). The cell number counts
dependently
in lower
both HeLa
WTWT
and
HeLa Rho
0 HeLa
(FigureRho
1A).
cell
counts
were
significantly
in HeLa
compared
with
0 atThe
both
10 number
and 50 µM
Resvwere
at 24 h
significantly lower in HeLa WT compared with HeLa Rho 0 at both 10 and 50 µM Resv at 24 h (p <
(p < 0.001 (***)) and 48 h (p < 0.01 (**), p < 0.05 (*)). As the number of cells exposed to 50 µM Resv at 24
0.001 (***)) and 48 h (p < 0.01 (**), p < 0.05 (*)). As the number of cells exposed to 50 µM Resv at 24 and
and 48 h still is higher than the numbers at 0 h, a cytostatic effect of Resv is more likely than a cytotoxic
48 h still is higher than the numbers at 0 h, a cytostatic effect of Resv is more likely than a cytotoxic
effect. This further indicates that the absence of functional mitochondria decreases the effect of Resv on
effect. This further indicates that the absence of functional mitochondria decreases the effect of Resv
cell proliferation. However, the stronger effect of Resv in HeLa WT cells could in part be explained by
on cell proliferation. However, the stronger effect of Resv in HeLa WT cells could in part be explained
a higher
proliferation
rate in
HeLa
WT WT
relative
to HeLa
RhoRho
0 cells,
alsoalso
shown
by by
thethe
impedance
data
by a higher
proliferation
rate
in HeLa
relative
to HeLa
0 cells,
shown
impedance
(Figure
1B).
Resv
increased
the
cell
diameter
in
HeLa
WT
already
at
5
µM
Resv
lasting
up
to
40
data (Figure 1B). Resv increased the cell diameter in HeLa WT already at 5 µM Resv lasting up to 40µM
Resv,
Resv treatment
did notdid
increase
cell diameter
in HeLa
0 (Figure
1C). The
obtained
µMwhereas
Resv, whereas
Resv treatment
not increase
cell diameter
in Rho
HeLa
Rho 0 (Figure
1C).
The
data
from
HeLa
WT
and
HeLa
Rho
0
cells
were
supported
by
experiments
using
human
osteosarcoma
obtained data from HeLa WT and HeLa Rho 0 cells were supported by experiments using human
cells
(143B). Thecells
143B
WT cells
wereWT
more
to sensitive
Resv treatments
(IC50 = 7.3(IC
µM
h) in
osteosarcoma
(143B).
The 143B
cellssensitive
were more
to Resv treatments
50 =at
7.348µM
contrast
143B
0. This
indicates
strongerstronger
reduction
in cell number
for 143B
at 48 h)toinIC
contrast
to µM,
IC50 =for
13.0
µM,Rho
for 143B
Rho
0. This indicates
reduction
in cell number
50 = 13.0
WT
forWT
Rhothan
0 (Supplemental
Figure S1). Therefore,
functional
mitochondria
are a prerequisite
forthan
143B
for Rho 0 (Supplemental
Figure S1).
Therefore,
functional mitochondria
are afor
for the effect
cell enlargement
theprerequisite
cell enlargement
of Resv. effect of Resv.
A
B
4
3
2
WT 0 M
WT 10 M
WT 20 M
Rho 0 0 M
Rho 0 10 M
Rho 0 20 M
1
0
20
40
60
80
Time (hours)
Figure 1.
Figure
1. Cont.
Cont.
100
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Figure
EffectofofResveratrol
Resveratrolon
on cell
cell number,
number, proliferation
proliferation and
normalized
cellcell
Figure
1. 1.Effect
andcell
celldiameter.
diameter.(A)
(A)
normalized
counts
of
HeLa
wild
type
(WT)
and
HeLa
Rho
0
cells
treated
with
Resv
(10
and
50
µM
for
24
and
48 h).
counts of HeLa wild type (WT) and HeLa Rho 0 cells treated with Resv (10 and 50 µM for 24 and 48
h). HeLa WT 10, 50 µM Resv vs. HeLa Rho 0 10, 50 µM Resv, p < 0.001 (***) at 24 h Cell counts in HeLa
HeLa WT 10, 50 µM Resv vs. HeLa Rho 0 10, 50 µM Resv, p < 0.001 (***) at 24 h Cell counts in HeLa
WT 10 µM Resv vs. HeLa Rho 0 10 µM Resv, p < 0.01 (**) and HeLa WT 50 µM Resv vs HeLa Rho 0
WT 10 µM Resv vs. HeLa Rho 0 10 µM Resv, p < 0.01 (**) and HeLa WT 50 µM Resv vs. HeLa Rho 0
50 µM Resv, p < 0.05 (*) at 48 h; (B) impedance curves of HeLa WT and Rho 0 cells treated with Resv
50 µM Resv, p < 0.05 (*) at 48 h; (B) impedance curves of HeLa WT and Rho 0 cells treated with Resv in
in long-term exposure (110 h); and (C) cell diameter in HeLa WT and HeLa Rho 0 cells treated for 24
long-term exposure (110 h); and (C) cell diameter in HeLa WT and HeLa Rho 0 cells treated for 24 h
h with 5–40 µM Resv. HeLa WT compared with Rho 0 when treated with 5 to 40 µM Resv, p < 0.01(**).
with 5–40 µM Resv. HeLa WT compared with Rho 0 when treated with 5 to 40 µM Resv, p < 0.01(**).
All values are a pool of three independent experiments with a determination of four replicates in (A),
All values are a pool of three independent experiments with a determination of four replicates in (A),
(C) and two replicates in (B). ANOVA/Bonferroni used for statistical analysis.
(C) and two replicates in (B). ANOVA/Bonferroni used for statistical analysis.
2.2. Effect of Resveratrol Exposure for 24 h on Oxygen Consumption Rate in HeLa Cells
2.2. Effect of Resveratrol Exposure for 24 h on Oxygen Consumption Rate in HeLa Cells
To determine if Resv has an effect on mitochondrial OCR, we used the XF24 Extracellular Flux
To determine
if Resv
has an
effect ontomitochondrial
OCR,
Extracellular
Flux
Analyzer
(Agilent,
Glostrup,
Denmark)
characterize the
effectwe
ofused
Resvthe
on XF24
the electron
transport
Analyzer
(Agilent,
characterize
the effect of Resv on the electron transport
chain (ETC)
by Glostrup,
applying Denmark)
oligomycin,tocarbonyl
cyanide-p-(trifluoromethoxy)phenylhydrazone
chain
(ETC)
byrotenone/antimycin
applying oligomycin,
carbonyl
cyanide-p-(trifluoromethoxy)phenylhydrazone
(FCCP)
(FCCP)
and
A. Extra
glucose
and sodium pyruvate were provided as additional
energy
sources.
An
overall
higher
OCR
signal
is
observed
in
HeLa
Rho
0
compared
with
HeLa
WT
and rotenone/antimycin A. Extra glucose and sodium pyruvate were provided as additional energy
when treated
with Resv,
which
evidentisfrom
the XF24
respiration
(Figurewith
2A,B)HeLa
and the
basal
sources.
An overall
higher
OCRissignal
observed
in HeLa
Rho 0traces
compared
WT
when
OCR (Figure
2C).which is evident from the XF24 respiration traces (Figure 2A,B) and the basal OCR
treated
with Resv,
with Resv for 24 h showed a concentration-dependent increase in basal OCR, p < 0.05
(FigureTreatment
2C).
(*) Treatment
at 20 and 30
µM
Resvfor
treatments
(Figure
2C). Neither the OCR related
to ATP
production
with Resv
24 h showed
a concentration-dependent
increase
in basal
OCR, p(Figure
< 0.05 (*)
2D),
or
to
non-mitochondrial
respiration
(Figure
2E),
nor
proton
leak
(Figure
2F)
was
affected
by Resv
at 20 and 30 µM Resv treatments (Figure 2C). Neither the OCR related to ATP production (Figure
2D),
treatment. Despite optimization of FCCP levels used for the OCR assay, it was not possible to
or to non-mitochondrial respiration (Figure 2E), nor proton leak (Figure 2F) was affected by Resv
uncouple the respiration of HeLa cells, which is evident from the traces shown in Figure 2A,B.
treatment. Despite optimization of FCCP levels used for the OCR assay, it was not possible to uncouple
To evaluate whether this relatively high OCR in HeLa Rho 0 cells could be specific for this cell
the respiration of HeLa cells, which is evident from the traces shown in Figure 2A,B.
line or a general phenomenon, a similar set of experiments was performed using 143B WT and the
To evaluate whether this relatively high OCR in HeLa Rho 0 cells could be specific for this cell
corresponding 143B Rho 0 cells. The OCR is increased in 143B WT compared with HeLa WT also
line or a general phenomenon, a similar set of experiments was performed using 143B WT and the
when treated with Resv (Figure 2A and Supplemental Figure S2A). The OCR in both HeLa WT and
corresponding
0 cells.(Figure
The OCR
is Supplemental
increased in 143B
WT
compared
with the
HeLa
WT also
143B Rho 0 cell143B
linesRho
are similar
2B and
Figure
S2A).
Furthermore,
increased
when
treated
with
Resv
(Figure
2A
and
Supplemental
Figure
S2A).
The
OCR
in
both
HeLa
and
OCR observed for HeLa Rho 0 following exposure to Resv was not observed in 143B Rho 0 cells WT
when
143B
Rho
0
cell
lines
are
similar
(Figure
2B
and
Supplemental
Figure
S2A).
Furthermore,
the
increased
tested up to 48 h Resv exposure. Non-significant changes of the various parts of the OCR are observed
OCR
observed
HeLa
Rho
0 following
exposure
to10
Resv
observed
in 143B Rho
0 cells
for 143B
WT orfor
Rho
0 cells
following
exposure
to 5 and
µM was
Resv not
up to
48 h (Supplemental
Figure
when
tested
up
to
48
h
Resv
exposure.
Non-significant
changes
of
the
various
parts
of
the
OCR
S2C-E), even though the cell number is significantly lower when exposed to 10 µM Resvare
observed
for 143BFigure
WT or1).Rho 0 cells following exposure to 5 and 10 µM Resv up to 48 h (Supplemental
(Supplemental
Figure S2C–E), even though the cell number is significantly lower when exposed to 10 µM Resv
(Supplemental Figure S1).
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Figure 2. Mitochondrial activity of HeLa WT and HeLa Rho 0 following 24 h exposure to resveratrol.
Figure 2. Mitochondrial activity of HeLa WT and HeLa Rho 0 following 24 h exposure to resveratrol.
(A) Oxygen consumption rates (OCR), HeLa WT trace; (B) OCR, HeLa Rho 0 trace; (C) average of
(A) Oxygen consumption rates (OCR), HeLa WT trace; (B) OCR, HeLa Rho 0 trace; (C) average of
basal respiration measurements, HeLa WT and Rho 0, were HeLa WT 20 and 30 µM Resv compared to
basal respiration measurements, HeLa WT and Rho 0, were HeLa WT 20 and 30 µM Resv compared
HeLa WT Ctrl (*: p < 0.05); (D) relative OCR related to ATP production of HeLa WT and Rho 0
to HeLa WT Ctrl (*: p < 0.05); (D) relative OCR related to ATP production of HeLa WT and Rho 0
calculated data after addition of oligomycin, were HeLa WT ctrl compared to HeLa Rho 0 Ctrl
calculated data after addition of oligomycin, were HeLa WT ctrl compared to HeLa Rho 0 Ctrl (***: p
(***: p < 0.001); (E) relative rate of non-mitochondrial respiration of HeLa WT and Rho 0, calculated data
< 0.001); (E) relative rate of non-mitochondrial respiration of HeLa WT and Rho 0, calculated data
after addition of rotenone/antimycin A, were HeLa WT ctrl compared to HeLa Rho 0 Ctrl (***: p < 0.001);
after addition of rotenone/antimycin A, were HeLa WT ctrl compared to HeLa Rho 0 Ctrl (***: p <
(F) relative OCR related to proton leak of HeLa WT and Rho 0 calculated after addition of oligomycin
0.001); (F) relative OCR related to proton leak of HeLa WT and Rho 0 calculated after addition of
minus non mitochondrial respiration. Data are presented as mean of three experiments ± SEM.
oligomycin minus non mitochondrial respiration. Data are presented as mean of three experiments ±
ANOVA/Bonferroni used for statistical analysis.
SEM. ANOVA/Bonferroni used for statistical analysis.
2.3. Resveratrol Effect on Mitochondrial Mass, Membrane Potential and Reactive Oxygen Species in HeLa WT
2.3. Resveratrol Effect on Mitochondrial Mass, Membrane Potential and Reactive Oxygen Species in HeLa
and Rho 0 Cells
WT and Rho 0 Cells
The mitochondrial membrane potential estimated as Tetramethylrhodamine methyl ester,
The mitochondrial membrane potential estimated as Tetramethylrhodamine methyl ester,
perchlorate (TMRM) relative to Mitotracker Green (MTG), is not changed following exposure to Resv
perchlorate (TMRM) relative to Mitotracker Green (MTG), is not changed following exposure to Resv
nor when comparing HeLa Rho 0 with HeLa WT (Figure 3A). Furthermore, no changes in oxidative
nor when comparing HeLa Rho 0 with HeLa WT (Figure 3A). Furthermore, no changes in oxidative
stress were observed after Resv treatment or when comparing the oxidative stress in HeLa Rho 0
stress were observed after Resv treatment or when comparing the oxidative stress in HeLa Rho 0 and
and HeLa WT cells (Figure 3B). The MTG fluorescence was not increased by Resv treatment, but a
HeLa WT cells (Figure 3B). The MTG fluorescence was not increased by Resv treatment, but a
significantly increased MTG signal was observed in HeLa Rho 0 relative to HeLa WT.
significantly increased MTG signal was observed in HeLa Rho 0 relative to HeLa WT.
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Figure 3. Mitochondrial membrane potential and cellular reactive oxygen species in HeLa WT and
Rho
0 cells.
Cells were treated
with or
withoutand
20 µM
Resvreactive
for 24 h,oxygen
and mitochondria
wereWT
labeled
Figure
3. Mitochondrial
membrane
potential
cellular
species in HeLa
and
Figure
3.
Mitochondrial
membrane
potential
and
cellular
reactive
oxygen
species
in
HeLa
WT
and
using
Tetramethylrhodamine,
methyl
ester, perchlorate
as anmitochondria
estimate of mitochondrial
Rho 0(A)
cells.
Cells were treated with
or without
20 µM Resv(TMRM),
for 24 h, and
were labeled
Rho
0
cells.
Cells
were
treated
with
or
without
20
µM
Resv
for
24
h,
and
mitochondria
were
labeled
membrane
potential; (B) H2DCFDA
to visualize
reactive oxygen
species.
The
estimates
are shown as
using (A) Tetramethylrhodamine,
methyl
ester, perchlorate
(TMRM),
as an
estimate
of mitochondrial
using (A)
Tetramethylrhodamine,
methyl
ester, perchlorate
(TMRM),
as an estimate
of mitochondrial
relative
to
a
simultanous
estimate
of
mitochondrial
mass
by
Mitotracker
green
(MTG).
Data
are
membrane potential; (B) H2 DCFDA to visualize reactive oxygen species. The estimates are
shown
membraneaspotential;
(B) H(n2DCFDA
to visualize
reactive oxygen species. The estimates are shown as
presented
mean
±
SEM
=
3)
analyzed
using
t-test.
as relative to a simultanous estimate of mitochondrial mass by Mitotracker green (MTG). Data are
relative to a simultanous estimate of mitochondrial mass by Mitotracker green (MTG). Data are
presented as mean ± SEM (n = 3) analyzed using t-test.
presented
as mean ±Exposure
SEM (n =for
3) analyzed
using t-test. Acidification Rate (ECAR)
2.4. Effect
of Resveratrol
24 h on Extracellular
2.4.
Effect
of Resveratrol
Resveratrol
Exposure
for
hh on
Acidification
Rate
Extracellular
acidification
(ECAR)
is predominantly
the result
of glycolytic activity. It is
2.4. Effect
of
Exposurerate
for 24
24
on Extracellular
Extracellular
Acidification
Rate (ECAR)
(ECAR)
investigated
if Resvacidification
affected ECAR
HeLa WT
and HeLa Rho 0, especially
based on the
high OCR
Extracellular
ratein
(ECAR)
is predominantly
Extracellular acidification
rate
(ECAR) is
predominantly the
the result
result of
of glycolytic
glycolytic activity.
activity. It
It is
is
rates
seen
in
the
HeLa
Rho
0
cells
(Figure
2B).
Resv
concentration-dependently
increased
basal
ECAR
investigated
if
Resv
affected
ECAR
in
HeLa
WT
and
HeLa
Rho
0,
especially
based
on
the
high
OCR
investigated if Resv affected ECAR in HeLa WT and HeLa Rho 0, especially based on the high OCR
but
had noin
effect
on oligomycin-induced
(Figure
4). Overall, HeLa WT
cells showed
higher
rates
the HeLa
HeLa
Rho 00 cells
cells (Figure
(Figureglycolysis
2B). Resv
Resv concentration-dependently
concentration-dependently
increased
basal
ECAR
rates seen
seen
in the
Rho
2B).
increased
basal ECAR
ECAR
ECAR
compared
to
HeLa
Rho
0,
also
when
treated
with
Resv,
which
is
evident
form
the
XF24
but
had
no
effect
on
oligomycin-induced
glycolysis
(Figure
4).
Overall,
HeLa
WT
cells
showed
higher
but had
no effect
on and
oligomycin-induced
glycolysis
(Figure
4). Overall,
HeLa
WTThis
cellsindicates
showed ahigher
traces
(Figure
4A,B)
the average
basal
ECAR with
measurements
(Figure
4C).
high
ECAR
alsoof
when
treated
Resv, which
ECAR compared
compared to
to HeLa
HeLa Rho
Rho 0,
0, also
when
treated
with Resv,
which is
is evident
evident form
form the
the XF24
XF24 ECAR
ECAR
reliance
on glycolysis
for energy
production
in both HeLa WT and(Figure
Rho 0, evenThis
when treateda with
traces
traces (Figure
(Figure 4A,B)
4A,B) and
and the
the average
average of
of basal
basal ECAR
ECAR measurements
measurements (Figure 4C).
4C). This indicates
indicates a high
high
Resv.
reliance
on
glycolysis
for
energy
production
in
both
HeLa
WT
and
Rho
0,
even
when
treated
with Resv.
reliance on glycolysis for energy production in both HeLa WT and Rho 0, even when treated
with
Resv.
Figure 4.
4. Glycolytic
Glycolyticactivity
activityofof
HeLa
WT
and
HeLa
Rho
0 following
h exposure
to resveratrol.
Figure
HeLa
WT
and
HeLa
Rho
0 following
24 h24exposure
to resveratrol.
(A)
(A) extracellular
acidification
(ECAR),
HeLa
trace;
ECAR,
HeLa
0 trace;
average
extracellular
acidification
raterate
(ECAR),
HeLa
WTWT
trace;
(B) (B)
ECAR,
HeLa
RhoRho
0 trace;
(C)(C)
average
of
Figure
4.
Glycolytic
activity
of
HeLa
WT
and
HeLa
Rho
0
following
24
h
exposure
to
resveratrol.
(A)
of
basal
ECAR,
HeLa
WT
and
Rho
0;
(D)
relative
ECAR
of
HeLa
WT
and
Rho
0
determined
after
basal ECAR, HeLa WT and Rho 0; (D) relative ECAR of HeLa WT and Rho 0 determined after
extracellularaddition.
acidification
(ECAR), HeLa
WTof
trace;
ECAR, HeLa
Rho 0 trace; (C) average of
oligomycin
addition.
Datarate
are presented
presented
as mean
mean
of
three(B)
experiments
SEM.
oligomycin
Data
are
as
three
experiments
±±SEM.
basal ECAR, HeLa WT and Rho 0; (D) relative ECAR of HeLa WT and Rho 0 determined after
oligomycin addition. Data are presented as mean of three experiments ± SEM.
Molecules 2017, 22, 847
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2.5. Molecules
Effect of2017,
Resveratrol
on Mitochondria Related mRNA Transcript Levels
22, 847
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To evaluate further if HeLa WT and HeLa Rho 0´s mitochondria were affected by Resv treatment,
2.5. Effect of Resveratrol on Mitochondria Related mRNA Transcript Levels
the expression of several key mRNA transcripts related to mitochondrial function was measured
To Since
evaluate
further
if HeLa
WT and HeLa
Rho 0´s
weremetabolic
affected by
Resv by
(Figure 5).
Resv
improves
mitochondrial
function
andmitochondria
protects against
diseases
treatment, the expression of several key mRNA transcripts related to mitochondrial function was
activating NAD-dependent deacetylase sirtuin-1 (SIRT1) [7], the mRNA expression level of Sirt1
measured (Figure 5). Since Resv improves mitochondrial function and protects against metabolic
was also measured. In general, expression levels of the measured transcripts are lower in HeLa WT
diseases by activating NAD-dependent deacetylase sirtuin-1 (SIRT1) [7], the mRNA expression level
than HeLa Rho 0, except for SIRT1 (Figure 5). In HeLa WT, Resv treatment does not change SIRT1
of Sirt1 was also measured. In general, expression levels of the measured transcripts are lower in
(Figure
a trend
for upregulation
with
Resv
is WT,
observed
for SIRT1,
HeLa5A).
WT However,
than HeLa Rho
0, except
for SIRT1 (Figure
5). In
HeLa
Resv treatment
doesespecially
not changewith
10 µM
Resv
treatments
(Figure
5A).
SIRT1
and
peroxisome
proliferator-activated
receptor
gamma
SIRT1 (Figure 5A). However, a trend for upregulation with Resv is observed for SIRT1, especially
coactivator-1α
both induce
genes
oxidative
phosphorylation
and show
trends for
with 10 µM(PGC-1α)
Resv treatments
(Figure
5A).related
SIRT1 to
and
peroxisome
proliferator-activated
receptor
increase
in transcript
levels
with Resv
(Figure
5A,G).
Mitochondrial
transcription
factor A
gamma
coactivator-1α
(PGC-1α)
bothtreatment
induce genes
related
to oxidative
phosphorylation
and show
trends
for increase
in mitochondrial
transcript levels
with Resvfactor,
treatment
5A,G). Mitochondrial
(TFAM),
a nuclear
encoded
transcription
which(Figure
is indispensable
for expression of
transcription factor
A (TFAM),
a nuclear
encoded
mitochondrial
transcription
key mitochondrial
encoded
genes (17),
is increased
in HeLa
Rho 0 compared
to HeLafactor,
WT, pwhich
< 0.001is (***)
indispensable
for respiratory
expression of
key mitochondrial
(17), is is
increased
in HeLa
0 in
(Figure
5B). Nuclear
factor
1 (NRF-1, alsoencoded
known genes
as NFE2L1)
expressed
in lowRho
levels
compared
to
HeLa
WT,
p
<
0.001
(***)(Figure
5B).
Nuclear
respiratory
factor
1
(NRF-1,
also
known
as
HeLa WT, and >10 fold upregulated in HeLa Rho 0 (Figure 5C) (p < 0.001, (***)). Treatment of HeLa
is expressed in low levels in HeLa WT, and >10 fold upregulated in HeLa Rho 0 (Figure 5C)
RhoNFE2L1)
0 with 10
µM Resv decreased NRF1 mRNA levels (p < 0.05 (*)). Interestingly, Resv treatment
(p < 0.001, (***)). Treatment of HeLa Rho 0 with 10 µM Resv decreased NRF1 mRNA levels (p < 0.05
reduced NRF1 mRNA levels of Rho 0 to HeLa WT levels. Cox5b show no changes in mRNA levels
(*)). Interestingly, Resv treatment reduced NRF1 mRNA levels of Rho 0 to HeLa WT levels. Cox5b
(Figure 5D). Estrogen-related receptor alpha (ERR-α) is markedly decreased in HeLa Rho 0 compared
show no changes in mRNA levels (Figure 5D). Estrogen-related receptor alpha (ERR-α) is markedly
withdecreased
WT (p < 0.001
(***)).
HeLa
cells,(***)).
ResvMoreover,
concentration-dependently
in HeLa
RhoMoreover,
0 comparedinwith
WTRho
(p <00.001
in HeLa Rho 0 cells,increased
Resv
mRNA
levels of ERR-α (10 µM
Resv; pmRNA
< 0.05 levels
(*), 20of
µM
Resv;
< 0.001
Cytochrome
complex
concentration-dependently
increased
ERR-α
(10p µM
Resv;(***)).
p < 0.05
(*), 20 µM Resv;
(CytpC)
was (***)).
increased
by 2 fold
in HeLa
Rho
0 compared
(pin
< HeLa
0.001 Rho
(***)0(Figure
5F),
< 0.001
Cytochrome
complex
(Cyt
C) was
increasedto
byWT,
2 fold
compared
towhereas
WT,
Resv(phad
no
effect
on
Cyt
C
mRNA
levels.
PGC-1
α
transcript
levels
were
similar
in
HeLa
WT and
< 0.001 (***) (Figure 5F), whereas Resv had no effect on Cyt C mRNA levels. PGC-1 α transcript
Rholevels
0 cellswere
andsimilar
were unaffected
Resv
5G).
in HeLa WTby
and
Rhotreatment
0 cells and(Figure
were unaffected
by Resv treatment (Figure 5G).
Figure 5.
5. Cont.
Figure
Cont.
Molecules
2017,
22, 22,
847847
Molecules
2017,
7 of 127 of 12
Figure
5. 5.
Effect
ofofResveratrol
levelsofofmRNA
mRNAtranscripts
transcripts
related
to mitochondrial
Figure
Effect
Resveratrolon
on expression
expression levels
related
to mitochondrial
function.
Relative
gene
expression
of
key
genes
related
to
mitochondrial
function
function. Relative gene expression of key genes related to mitochondrial function measured measured
by Q-RT- by
Q-RT-PCR
(quantitative
reverse
transcriptase
polymerase
chain
reaction)
in
HeLa
WT
PCR (quantitative reverse transcriptase polymerase chain reaction) in HeLa WT and HeLa Rho and
0 cellsHeLa
Rhotreated
0 cellswith
treated
with
10 and
Resv
for 24 h.
Expression
of (A) NAD
(nicotineamide
10 and
20 µM
Resv20
forµM
24 h.
Expression
of (A)
NAD (nicotineamide
dinucleotide)dependent deacetylase deacetylase
sirtuin-1 (SIRT1);
(B) mitochondrial
transcription factor
A (TFAM),
HeLa
dinucleotide)-dependent
sirtuin-1
(SIRT1); (B) mitochondrial
transcription
factor
A WT
(TFAM),
compared
to
HeLa
Rho
0
(***:
p
<
0.001)
untreated;
(C)
nuclear
respiratory
factor
1
(NRF-1
also
known
HeLa WT compared to HeLa Rho 0 (***: p < 0.001) untreated; (C) nuclear respiratory factor 1 (NRF-1
NFE2L1),
WT compared
to HeLa
Rho 0 to
(***:
p < 0.001)
HeLauntreated
Rho 0 untreated
alsoasknown
as HeLa
NFE2L1),
HeLa WT
compared
HeLa
Rho untreated
0 (***: p <and
0.001)
and HeLa
compared
to
HeLa
Rho
0
10
µM
Resv
(*:
p
<
0.05);
(D)
cytochrome
C
oxidase
5b
(COX5b);
(E) estrogenRho 0 untreated compared to HeLa Rho 0 10 µM Resv (*: p < 0.05); (D) cytochrome
C oxidase 5b
related(E)
receptor
alpha (ERR-α),
HeLa alpha
WT compared
HeLaWT
Rhocompared
0 (***: p < 0.001)
untreated
and HeLa
(COX5b);
estrogen-related
receptor
(ERR-α),toHeLa
to HeLa
Rho 0 (***:
p < 0.001)
Rho 0 untreated compared to HeLa Rho 0 10 µM Resv (*: p < 0.05), compared to 20 µM (***: p < 0.001);
untreated and HeLa Rho 0 untreated compared to HeLa Rho 0 10 µM Resv (*: p < 0.05), compared
(F) cytochrome complex (Cyt C), HeLa WT compared to HeLa Rho 0 (***: p < 0.001) untreated, (G)
to 20 µM (***: p < 0.001); (F) cytochrome complex (Cyt C), HeLa WT compared to HeLa Rho 0
proliferator-activated receptor coactivator-1α (PGC-1α). All data are presented as relative to levels of
(***: p < 0.001) untreated, (G) proliferator-activated receptor coactivator-1α (PGC-1α). All data are
RPL23a and are shown as mean ± SEM (n = 6). Statistical analysis performed using a t-test.
presented as relative to levels of RPL23a and are shown as mean ± SEM (n = 6). Statistical analysis
using a t-test.
3.performed
Discussion
The mitochondrion is a central organelle for cellular proliferation and development and one of
the proposed cellular targets for the effect of Resv. To analyze this in real time, a comparison of the
effect
Resv on cells with
without
functional
mitochondria
was made
using
the cervical and
cancer
The of
mitochondrion
is aand
central
organelle
for cellular
proliferation
and
development
one of
line HeLa
with supporting
osteosarcoma
cells.
the cell
proposed
cellular
targets fordata
the from
effect143B
of Resv.
To analyze
this in real time, a comparison of the
decreased
proliferation
ratefunctional
in HeLa Rho
0 compared to
HeLa
WT using
is consistent
with less
effect ofThe
Resv
on cellscell
with
and without
mitochondria
was
made
the cervical
cancer
ATP
being
produced
via
oxidative
phosphorylation
in
the
mitochondria,
but
data
also
showed
(1) a
cell line HeLa with supporting data from 143B osteosarcoma cells.
higher mitochondrial mass; (2) increased expression of most of the tested mitochondria related
The decreased cell proliferation rate in HeLa Rho 0 compared to HeLa WT is consistent with
mRNAs in HeLa Rho 0; and (3) an increased basal respiration in HeLa Rho 0. These features of HeLa
less ATP being produced via oxidative phosphorylation in the mitochondria, but data also showed
Rho 0 cells indicate that the increased mitochondrial mass and TFAM expression could be a result
(1) a higher mitochondrial mass; (2) increased expression of most of the tested mitochondria related
from a compensatory survival strategy. In turn, a larger amount of mitochondria, which are
mRNAs
in HeLa may
Rho result
0; andin(3)
increased
basal respiration
HeLa
Rho observed
0. These features
of HeLa
dysfunctional,
anan
increased
oxidative
stress level. in
This
is partly
in HeLa Rho
Rho0 0cells
cells when
indicate
that
the
increased
mitochondrial
mass
and
TFAM
expression
could
be
a
result
calculating the absolute values but not the oxidative stress level relative to thefrom
a compensatory
survival
strategy.
a larger amount
of subunits
mitochondria,
which
are dysfunctional,
mitochondrial mass. Absence
of In
theturn,
mitochondria
encoded
prevents
the assembly
and
mayfunction
result in
increased oxidative
level.[18].
ThisHowever,
is partly HeLa
observed
HeLa Rho 0retain
cells when
of an
mitochondrial
respiratorystress
complexes
Rho 0inmitochondria
a
proton gradient,
whichvalues
is essential
forthe
protein
import
intolevel
mitochondria
counteractingmass.
calculating
the absolute
but not
oxidative
stress
relative toand
thefor
mitochondrial
apoptosis
[19].
This proton gradient
generatedprevents
by hydrolyzing
ATP imported
from the
Absence
of the
mitochondria
encodedissubunits
the assembly
and function
of cytoplasm
mitochondrial
by
the
ATP-ADP
shuttle.
ATP
is
hydrolyzed
by
the
ATP
synthase
working
in
reverse
mode to
respiratory complexes [18]. However, HeLa Rho 0 mitochondria retain a proton gradient,
which
generate
a
transmembrane
proton
gradient
[20].
The
enhanced
expression
of
TFAM,
Cyt
c
and
most
is essential for protein import into mitochondria and for counteracting apoptosis [19]. This
proton
of the is
analyzed
mitochondria
relatedATP
transcripts
therefore
indicates
increased
mitochondrial
mass or ATP
gradient
generated
by hydrolyzing
imported
from the
cytoplasm
by the
ATP-ADP shuttle.
volume in HeLa Rho 0.
is hydrolyzed by the ATP synthase working in reverse mode to generate a transmembrane proton
The enhanced basal respiration in HeLa Rho 0 compared with HeLa WT can partially be
gradient [20]. The enhanced expression of TFAM, Cyt c and most of the analyzed mitochondria+ related
explained by the increased non-mitochondrial respiration. In Rho 0 cells, NADH2 (reduced NAD ) is
transcripts
therefore
indicates
increased
mitochondrial mass
or volume
in to
HeLa
RhoNAD
0. + for use
re-oxidized
and electrons
delivered
to plasma-membrane
oxidases,
in order
recycle
The
enhanced
basal
respiration
in
HeLa
Rho
0
compared
with
HeLa
WT
can
in glycolysis [21]. This flow of electrons will provide an increased non-mitochondrial OCR aspartially
seen in be
explained
by However,
the increased
respiration.
In Rho
0 cells,
NADH
NAD+ ) is
2 (reduced
Figure 2E.
basalnon-mitochondrial
respiration in Rho 0 is
increased above
what
can be
explained
by increased
+
re-oxidized
and electrons
delivered
to plasma-membrane
oxidases, inunaccounted
order to recycle
NAD
forbe
use in
non-mitochondrial
respiration,
leaving
some oxygen consumption
for. It
should
glycolysis
[21]. This
flow
of electrons
will provide
an consumption
increased non-mitochondrial
OCR
as seen in
noted, however,
that
substantial
cell membrane
oxygen
has been observed for
different
3. Discussion
Figure 2E. However, basal respiration in Rho 0 is increased above what can be explained by increased
non-mitochondrial respiration, leaving some oxygen consumption unaccounted for. It should be noted,
however, that substantial cell membrane oxygen consumption has been observed for different cell
Molecules 2017, 22, 847
8 of 12
lines, including HelA subtypes [21]. For comparison, 143B Rho 0 cells had a lower OCR (much lower
than the 143B WT counterpart), which was unaffected by Resv treatment. To gain mechanistic insight
into unaccounted non-mitochondrial respiration in HeLa Rho0 cells, these should be further studied
using inhibitors, since it is evident from other studies that plasma membrane redox system is a Resv
target [22]. Moreover, when comparing the OCR/ECAR ratios of the Hela WT and HelA Rho 0 versus
the 143B WT and 143B Rho 0, there were similar ratios for the two WT cell lines (4.8 ± 1.6) in contrast to
a much higher ratio in HeLa Rho 0 cells (8.4 ± 1.0) and a much lower ratio for 143B Rho 0 cells (2.7 ± 0.9).
This indicates different adaptive strategies for Rho 0 cells to obtain the necessary energy input.
Cells in general, and especially tumor cells, require a sufficient amount of ATP in order to
synthesize bioactive compounds for rapid cell proliferation [23] and different tumor cells use different
strategies to gain sufficient amounts of ATP for the increased cell proliferation. Whereas some have
increased glycolysis, others have an enhanced oxidative phosphorylation pathway [24]. Our present
data indicate that HeLa Rho 0 and WT have a different source of ATP production, due to the lack of
well-functioning mitochondria, which also have an impact on regulation of glycolysis.
Numerous studies show a clear reduction in cell proliferation by Resv, which is confirmed
by present data. Here, we significantly extend these findings by the observation that functional
mitochondria are necessary for the cellular response to Resv and a Resv-induced cell enlargement.
Previous findings show that Resv does not have an effect on cell doubling time in PC3 Rho 0 cells
compared to the corresponding WT cells [25]. Cell size is coupled to cell cycle progression and is
affected by both internal and external cues. Cells must reach a certain size, before they progress in
the cell cycle. Here is presented that Resv increases cell size in HeLa WT but not in the mitochondria
deficient Rho 0 cells. In some cell types, Resv activates different pathways e.g., mechanistic target of
rapamycin (mTOR) [26], which could be the explanation to the increased cell size observed by Resv
treatment in HeLa WT cells.
Resv increases basal OCR in both HeLa cell lines, but to a greater extent in HeLa WT, suggesting
that mitochondrial function is indeed increased by Resv. On the other hand, the non-mitochondrial
respiration is highly increased but only in HeLa Rho 0 cells, which indicates that Resv regulates a
non-mitochondrial target as well. In contrast to the increased oxygen consumption rates, Resv did not
increase ROS levels (Figure 3B). Previous data reports that Resv increases the mitochondrial mass in
HeLa WT cells [7,8,14,24], which this experiment was unable to replicate.
Resv shows an increase in both mitochondrial oxidative as well as glycolytic activity for HeLa WT
and Rho 0 (only significant for WT). Our data support the notion that HeLa WT has a propensity for
using glycolysis for energy production, which underpins the importance of mitochondrial recycling of
NADH (Figure 4C). Catabolite repression is an important part of a cell’s global control system. It is
assumed that interplay of Resv with membranes of wild type cells probably can cause a negative effect
on the catalytic region of ETC proteins and F0 F1 -ATP, which is the opposite for Rho 0 cells. It is also
clear that this cell model has decreased oligomycin response and absent FCCP response (Figures 2A,B
and 4A,B).
Previous reports showed an inhibition of ETC by Resv using isolated mitochondria [10,11].
Such inhibition was not observed in the present experimental setup using intact cells. Even cells
exposed to 10 µM Resv for 48 h showed the same OCR even though the cell number was decreased
significantly, which indicates an increased ETC activity per cell.
Most of the mitochondria related mRNAs levels, except Sirt1 and PGC-1α, are increased in
HeLa Rho 0 cells. Thus, there appears to be nuclear compensatory transcription in response to the
dysfunctional mitochondria. The reference gene used for normalization showed similar Ct values
between cell lines. Resv only has a very minor impact on the level of these mRNAs, and if so, at a
lower concentration of Resv than the concentration which increases basal OCR (Figure 2C). The minor
effect of Resv on mRNA levels in treated HeLa WT could indicate that cellular modulation by Resv
primarily acts at protein level [26].
Molecules 2017, 22, 847
9 of 12
HeLa Rho 0 show more than expected oligomycin linked reduction of OCR. Both HeLa Rho 0 and
WT do need a functional oligomycin-sensitive ATP synthase as discussed above, but it is more likely
that the HeLa Rho 0 cells used may not have a completely malfunctioning electron transporting chain.
Therefore, in order to confirm the current findings in HeLa cells, additional Rho 0 cell types should
be investigated.
The described experiments showed cellular effects of Resv in the range of 5 to 50 µM after 24 h.
According to Yang et al., more than 90% of the administrated Resv is degraded after 24 h in sodium
bicarbonate containing media, and the actual concentrations of Resv are assumed to be lower than
the initial dose [27]. As Resv binds to proteins and lipid structures in the media, the high degree of
degradation described by Yang et al. is likely an overestimation.
The partly lacking effect of Resv in HeLa Rho 0 could be explained by the fact that Resv inhibits
ATP synthase. Recent experiments have shown that mitochondrial calcium overload and the apposite
is triggered in HeLa cells and other cancer cell types [28], which support the notion; functional
mitochondria are important for the effect of Resv.
4. Materials and Methods
4.1. Materials
HeLa WT and Rho 0 derived from cervical cancer cells were a gift from Dr. Claus Desler,
Department of Cellular and Molecular Medicine, University of Copenhagen, Denmark [29]. Dulbecco’s
modified Eagle’s medium (DMEM), Hank's Balanced Salt Solution (HBSS), Penicillin/streptomycin
and uridine were obtained from Sigma-Aldrich (Broendby, Denmark). Fetal bovine serum (FBS) was
from Merck Millipore (Hellerup, Denmark). Resveratrol was acquired from Fluxome Science (Stenloese,
Denmark)—now Evolva (Reinach, Switzerland)—and dissolved in DMSO.
4.2. Cell Culture
HeLa WT and HeLa Rho 0 were grown in DMEM supplemented with 10% fetal bovine serum
and 1% Penicillin/Streptomycin. HeLa Rho 0 medium was supplemented with 200 µM uridine. Cells
were cultured in Thermo Fisher Scientific NuncTM microplates (Roskilde, Denmark).
4.3. Determination of Cell Number and Size
To determine cell number and diameter in HeLa WT and HeLa Rho 0, a Beckman Coulter,
Z2 Coulter particle counter and size analyzer (Pasadena, CA, USA) was used [30]. Particles with
diameter size > 7 µm were included for data analysis. All cell counts were carried out in quadruplicate
for each treatment with three independent experiments. The cells were treated with DMSO (as solvent)
and 10–50 µM Resv for 24 and 48 h.
4.4. Impedance Readings
Evaluating the effects of Resv on cell proliferation was based on real-time cell-based electronic sensor
technology iCELLigence System [31] (ACEA Biosciences, Inv., San Diego, CA, USA). Measurements
were performed according to manufacturer´s protocol. Briefly: in an E-plate L8 dish, each well was filled
with 200 µL medium and placed in the iCELLigence station in a 37 ◦ C incubator. Before seeding the cells
into the E-Plate L8 (15.000 cells per well), a background measurement was done with media. The HeLa
WT and HeLa Rho 0 were cultured in E-plates L8 in duplicates for 24 h before treatment with Resv was
initiated. The impedance signal is expressed as the cell index at each time point per concentration of
Resv. The cells were treated with DMSO (as solvent), 10 or 20 µM Resv for up to 110 h.
4.5. Respirometry in Intact Cells
The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements
were made using Seahorse Bioscience XF-24 instrument (Seahorse Bioscience, Copenhagen, Denmark).
Molecules 2017, 22, 847
10 of 12
HeLa WT and HeLa Rho 0 were grown to 80–90% confluence and then treated with 0, 10, 20 or
30 µM Resv 24 h prior to the day of the respirometry. On the day of the assay, the cell medium
was removed from the well and running medium (XF-DMEM assay buffer, Seahorse Bioscience)
was placed into the wells and warmed to 37 ◦ C for at least 30 min without 5% CO2 atmosphere.
The injection ports of the sensor cartridge were filled with Glucose/Sodium Pyruvate (25 mM/1 mM),
Oligomycin (10 µM), FCCP (30 µM), and rotenone/antimycin A (20 µM for both) (Seahorse Bioscience).
The measurement cycle consisted of a 3-min mix, a 3-min wait and a 3-min measurement. Eight basal
rate measurements were followed by injections, and each injection was followed by three to four
measurement cycles. Activity estimates for any given treatment were based on the rates from three
wells to calculate the investigated parameters. Basal OCR represents the net sum of all processes in the
cell capable of consuming O2 including mitochondria and other oxidases. Basal ECAR is established
with the first measurements, as well as OCR. Non-mitochondrial respiration is estimated by average
of OCR measurements after addition of antimycin A and rotenone. Proton leak is calculated as
the first measurement after addition of oligomycin subtracted from non-mitochondrial respiration.
The OCR-related ATP production is calculated as proton leak subtracted from basal OCR. Basal
ECAR represent the net sum of predominantly measure of lactic acid formed during glycolytic energy
metabolism. After addition of oligomycin, increased glycolytic rate in response to loss of mitochondrial
related ATP production is calculated as the average of oligomycin stimulated rate of ECAR.
4.6. Detection of Mitochondrial Mass, Mitochondrial Membrane Potential and Oxidative Stress by Flow Cytometry
After 24 h of treatment with 0 or 20 µM Resv., HeLa WT and HeLa Rho 0 cells were detached
and re-suspended in HBSS buffer (10 mM HEPES, 140 mM NaCl and 25 mM CaCl2, pH 7.4). Cells
were incubated with Mitotracker green (MTG) (Invitrogen M7514, Thermo Fisher Scientific, Roskilde,
Denmark) (0.01 µM), Tetramethylrhodamine methyl ester (TMRM) (Invitrogen T668) (0.01 µM) at room
temperature (RT) or 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) (Invitrogen C399) (0.1 µM)
at 37 ◦ C, for 30 min. Analysis was performed using flow cytometry (FACS Calibur, Becton-Dickinson,
NJ, USA) and analyzed by BD CellQuest™ Pro Analysis software (version 4.0.2, Becton Dickinson,
Khs. Lyngby, Denmark). Data were normalized to the HeLa WT solvent condition due to variation in
absolute signal strength between experiments.
4.7. Quantitative Real Time RT-PCR Assay
Total RNA was extracted using TRI-Reagent (Sigma-Aldrich, Broendby, Denmark) according
to manufacturer’s instructions. Concentration and purity of RNA were determined by NanoDrop
spectrophotometer ND 1000 (Fisher Scientific, Roskilde, Denmark) and stored at −80 ◦ C until cDNA
synthesis. In addition, 1 µg of total RNA was reverse transcribed using a Superscript III Reverse
transcription (Invitrogen) according to manufacturer’s instructions. The cDNA was subjected to real
time PCR using QuantiTect SYBR® Green PCR Kit (Qiagen, Venlo, Netherlands). A reaction contained
500 nM of each forward and reverses primer, and 2 µL of 10 fold diluted cDNA with final reaction
volume 10 µL. The reaction was initiated by 95 ◦ C for 15 min. and 40 amplification cycles were
carried out with 10 s denaturation at 95 ◦ C and 60 s annealing/elongation at 60 ◦ C. Subsequently,
a melting curve was carried out. Gene expression was normalized to mean of RPL23a rRNA signals.
Oligonucleotide sequences are available on request.
4.8. Statistical Analysis
All data are expressed as mean ± SEM and data are presented as a pool of replicate measurements
from 2–4 independent experiments. Multiple groups were analyzed using one-way ANOVA, with
Bonferroni post-test correction for multiple comparisons using GraphPad Prism (version 6.07, GrapPad,
La Yolla, CA, USA). A t-test was used to analyze differences in mRNA levels. A probability of less
than 0.05 was considered to indicate a significant difference.
Molecules 2017, 22, 847
11 of 12
5. Conclusions
In conclusion, functional mitochondria are a prerequisite for Resv to have an effect on cell size
and in part for the modulation of cell proliferation. Presented data also show that Resv increases
oxygen consumption rates as well as glycolysis in HeLa cells. Functional mitochondria are important
for the effect of resveratrol.
Supplementary Materials: The following are available online. Figure S1: Effect of Resveratrol on cell number of
143B cells (WT and Rho 0); Figure S2: Mitochondrial activity of 143B WT and 143 Rho 0 following exposure for
48 h to resveratrol.
Acknowledgments: We are very grateful for the skilled technical assistance of Marianne Lauridsen,
Roskilde University, for help with protocols, reagents and advice. This work was supported in part by the Danish
Council for Strategic Research (Grant 10-093499) to the Long-term Investigation of Resveratrol on Management
of Metabolic syndrome, Osteoporosis and Inflammation, and Identification of plant derived anti-inflammatory
compounds (LIRMOI) research program (www.LIRMOI.com) to O.V. and in part by the Danish Council for
Independent Research in Health and Disease (Grant DFF–1331-00033) to L.T.D.
Author Contributions: A.L.W., L.T.D. and O.V. conceived and designed the experiments; A.L.W. and K.B.
performed the experiments; A.L.W., K.B., L.T.D. and O.V. analyzed the data; A.L.W. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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