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Scientific Report
regarding the implementation of the project PCE Ideas no. 342/2011 during the period
October 2011 – October 2013
Synthesis
Studies concerning the correlation between the quercetin fluorescence and its ability to trigger Ca2+ release from the Jurkat T cells. Characterization of quercetin as a fluorescent
indicator of ctivity of the ryanodinic receptor RyR3 in Jurkat lymphoblasts (I and III phases of the project)
INTRODUCTION
A wide number of studies perrormed in the last decades have evidenced the beneficial effects of natural
flavonoids on human health. The natural flavonoids are frequently found in fruits, vegetables and tea and it was
observed that they possess cardioprotective, anticancer, antiinflammatory and antialergic properties [1, review and
the references therein]. Among these flavonoids we have chosen to study the epigallocatechin 3-gallate (EGCG) and
quercetin (QC; 3,5,7,3’,4’-pentahydroxyflavone), namely the way they are interacting with the human leukaemia
cells, the Jurkat T lymphoblasts. It has been shown that these two flavonoids can inhibate the cell proliferation and
induce the apoptosis in various types of cancer [1-7]. Both EGCG and QC can exert a dual effect, pro- and anti-
oxidant, depending on the dose and the duration of the treatment and numerous studies indicate that the
malignant cells are more susceptible than the normal ones to the cytotoxicity of these two flavonoids [3,5-7]. This
property recommends the use of the two flavonoids either in the prevention of various cancer types, in particular
leukaemia, or in order to increase the chemotherapy efficience in the treatment of these illnesses. As, so far, the
available data concerning the effects of these compounds on the cell cycle and the apoptosis/necrosis in Jurkat T
cells are extremely limited and inhomogenous, we have proposed ourselves to undertake a series of studies with the
aim to elucidate the mechanisms underlying the interaction of the two flavonoids with leukaemia cells, alone or in
combination with chemotherapeutical agents. This kind of research has been initiated in a previous project (Ideas
no. 1138/2009) and the results we have obtained prompted us to continue this field of research and, moreover, they
have suggested the approach of a new line of research, namely the attempt to elucidate the role of the calcium
release channels from ER/SR in the induction of cellular apoptosis. Among the results obtained in the previous
project (published in 7 ISI papers and 9 BDI papers) we would like to mention those that evidence the role of QC and
EGCG in the induction of apoptosis in Jurkat T cells, the combined effects with those of the chemotherapeutic agent
menadione (MD) or the effect of the mitochondrial respiration inhibitor rotenone [4,8,9]. An extremely interesting
result, that, as previously mentioned, will constitute a new line of research refers to the triggering of a calcium signal
following the addition of 50 M QC in Jurkat cells[10].
In the first phase of the project we have proposed ourselves to examine the way in which quercetin, which
presents a weak fluorescence in aqueous solutions, modifies its fluorescence when enetring the Jurkat cells and to
examine more closely the correlation between the quercetin application and the resulted calcium signal. It has been
shown that QC binds itself to cellular proteins unidentified yet and is thus accumulated in large quantities in the
cytosol and the mitochondria of Jurkat T cells [10,11]. In the third phase of the project we have evidenced that using
quercetin asa a fluorescent probe it is possible to monitor the activity of the ryanodinic receptor RyR3 and to
calculate the probability of the channel opening in situ. The obtained results have been a premiere and have been
published in the ISI journal Pflügers Archiv, Europen Journal of Physiology [12]. This article has received the
distinction „Global Medical Discovery” as a „Key Scientific Articles” („Global Medical Discovery (ISSN 1929-8536)
MATERIALS AND METHODS Cell cultures. Human leukemia Jurkat T-cell lymphoblasts were cultured in MegaCell RPMI 1640 medium supplemented with 5%
fetal bovine serum, 2 mM L-
with a 5% CO2 atmosphere. Dihydrated quercetin (Sigma) and fura-2/AM (Invitrogen) were dissolved in dimethyl sulfoxide
(DMSO) and kept at -20ºC. Unless stated otherwise, reagents were purchased from Sigma. Cell density, viability and morphology
were examined under a phase contrast microscope. Viability was assessed by the trypan-blue exclusion test. Cell count was
performed with the use of a haemocytometer. Saline solutions. The extracellular-like solution (ECS) contained 140 mM NaCl, 5
mM KCl, 1 mM CaCl2, 1 mM MgCl2, 20 mM HEPES, 10 mM glucose, pH 7.2/NaOH. The intracellular-like solution (ICS) contained
140 mM KCl, 4 mM NaCl, 0.14 mM CaCl2, 0.5 mM MgCl2, 20 mM HEPES, pH 7.2/NaOH. A variant of ICS (ICSM) did not contain
MgCl2. When required, CaCl2 and MgCl2 solutions were freshly prepared at working concentrations of 50 mM, 1 M or 6 M
(CaCl2), and 4 M (MgCl2) in either ICS or ECS as needed. The level of free Ca2+
in solutions was calculated using the software
WEBMAXCLITE v1.15. Assessment of intracellular [Ca2+
]i was performed according to the method of Grynkiewicz et al. [12] as
described in [10] with minor modifications. Exponentially growing Jurkat cells were washed twice in ECS. The cells were then
incubated with 4 M fura-2/AM for 10-15 min. in the dark at room temperature (24-25ºC) with occasional agitation, then
washed twice in ECS, resuspended in 2 ml ECS, counted and adjusted to the desired cell concentration by addition of ECS as
necessary. Cells were then incubated at 37ºC for an additional 45 min. for complete de-esterification of the calcium indicator.
The cells were then transferred to the spectrofluorimeter into a 2 ml quartz cuvette maintained at 37ºC under continuous
stirring. Cell viability assessed immediately before the fluorescence recordings was >90% in all cell samples. Fluorescence was
recorded with a Horiba Jobin Yvon spectrofluorimeter, by sequential excitation at 340, 380 and 440 nm. The excitation pulses
were repeated every 20.28 s. Integration time was 10 ms. Emission/excitation parameters were 495 nm/340 nm and 495
nm/380 nm for fura-2, 560 nm/380 nm and 560 nm/440 nm for QC, 420 nm/380 nm for NADH and 470 nm/440 nm for flavins
(FLV). Quercetin was added after an initial pre-equilibration period (~45 min. of recording) when the fluorescence signal became
stable. The cytosolic Ca2+
concentration, [Ca2+
]cyt, was calculated from the equation [Grynkiewicz]:[Ca2+
]cyt = Kd (R - Rmin)/(Rmax -
R), where R = F340/F380 represents the ratio of the fluorescence emission at 340 nm and 380 nm excitation, respectively, Rmin is
the ratio F340/F380 obtained in a nominally Ca2+
-free solution, Rmax is F340/F380 obtained when the Ca2+
indicator is saturated with
Ca2+
, is the ratio between F380 in the Ca2+
-free solution and F380 at saturation, and Kd = 0.225 M is the Ca2+
-dissociation
constant of the indicator. F340 and F380 were corrected for autofluorescence. Calibration for Rmax was performed at the end of the
measurement by addition of 35 M digitonin for 15-20 min. The degree of cell permeabilization was 100% as evaluated by
trypan blue exclusion tests in separate determinations. Then 10 mM EGTA was added to calibrate the QC fluorescence signal in
15 nM Ca2+
. After 15-20 min., other 10 mM EGTA were added to reach a final concentration of 7 nM free Ca2+
and Rmin was
evaluated after an additional 15-20 min. The control cells have been treated in the same manner only instead of calcium
indicator an equal amount of DMSO has been added. Fluorescence spectroscopy. Cell suspensions were prepared at a density of
≈106 cells/ml in ECS after three washes in ECS. 2 ml of suspension were transferred to the quartz cuvette under continuous
stirring. Fluorescence recordings were done at 37ºC in a Horiba Jobin Yvon spectrofluorimeter, as described above for fura-2
loaded cells. Recordings were briefly interrupted for about 4 min. to collect fluorescence spectra at indicated parameters. The
first spectrum was recorded after a pre-equilibration period of 45 min. Excitation/emission spectra were collected before and
after addition of 50 M quercetin at indicated times. After 1 h of exposure to QC, 35 M digitonin was added to the cuvette.
Fluorescence spectra were collected after 15 min., then 10 mM EGTA was added and a new series of spectra were recorded
after an additional 15 min. In this final step, the calculated free Ca2+
concentration in solution was 15 nM. For autofluorescence
measurements, separate control cell samples were treated in the same manner with the exception that an equal amount of
DMSO was added instead of fura-2 or instead of quercetin, respectively.
RESULTS AND DISCUSSION (1st phase)
On the basis of previously obtained preliminary results [10], we have repeated into more detail the
experiments concerning the induction of a calcium signal following the entrance of quercetin in the intact Jurkat T
cells. Indeed, the fluorescence measurements of the fura 2/AM loaded cells indicate a sustained calcium release
from the intracellular stores, generating thus a bi-phasic Ca2+ signal evoked by 50 M QC (Fig. 1a). In order to
establish that this was really a true calcium signal and not an artifact, we have used an inhibitor of calcium channels,
namely ruthenium red (RR). As it can be noticed in the Fig. 1b,in the same experimental conditions, the calcium
signal is inhibited. Moreover, RR is an specific inhibitor for the ryanodinic receptor (RyR) and not of the inositol
trisphosphate receptor (IP3). The fluorescence spectra measured in a previous work [10], evidenced a decrease in the
NADH level in intact Jurkat T cells exposed at 50 M QC. We took a step further in this research and we measured the
NADH level when the cellular calcium concentration is modified, respectively, we compared the evolution of this level
in intact cells, in cells permeabilized with digitonin and then after adding EGTA. We found out that, in all the cases,
the NADH level invariably decrease. (Fig. 2). Farther, we have examinated, developing the previous research line [10]
the way in which the quercetin fluorescence is modified depending on the calcium concentration in the cells. n our
previous studies [10] we have observed that the wavelengths at which the excitation spectra of the intracellular
quercetin present maxima are found around 389 nm, and 449 num respectively.
Fig. 1. Ca2+
and QC fluorescence signals evoked by 50 M QC in Jurkat cells. a-b, Individual traces of Ca2+
signals induced by 50
M QC in fura-2 loaded Jurkat cells in the absence of ruthenium red (RR) in 14 separate experiments (a) or in the presence of 10
M RR in 5 independent experiments (b). The signals have been calibrated at the end of each experiment. The emission was collected at 420 nm with excitation at 360 nm. The signal was background-corrected.
The emission spectrum presents a maximum around 540 nm. We have proposed ourselves to examine into
more detail the evolution of these spectra as a function of calcium concentration in the cell. In order to better
characterize the fluorescent properties of the intracellular quercetin, we have monitored the modifications of the
fluorescence spectra when the intact Jurkat cells are exposed to 50 M QC and after the cell permeabilization by
digitonin. Fig. 3 illustrates an example of emission fluorescence spectra of QC in intact cells and in permeabilized
Jurkat T cells, obtained on the excitation with 380 nm and 440 nm respectively. All the spectra present a prominent
maximum at ~535-540 nm specific to the tautomeric forms of quercetin [13].
Fig. 3. Fluorescence emission spectra of QC in intact and permeabilized Jurkat cells. The excitation was done at 380 nm (a) and
440 nm (b). The spectra were recorded at 60 min. after exposure of the intact cells to 50 M QC (black line), after the permeabilization in 1 mM Ca
2+ (red line) or in 15 nM Ca
2+ (blue line). These results have been obtained by averaging in 4
independent representative experiments.
Fig. 4 presents a representative set of excitation spectra of quercetin in intact and permeabilized cells, and the
analysis by a Gaussian deconvolution. Our domain of interest lies in the two bands centered at ~380 nm and ~440
nm, respectively (Fig. 4, Table 1). The fluorescence data were fitted to the equation:
y = y0 + A1/w1/(/2)1/2
exp{-2[(ex - 1)/w1]2} + A2/w2/(/2)
1/2 exp{-2[(ex - 2)/w2]
2} +
+A3/w3/(/2)1/2
exp{-2[(ex - 3)/w3]2}
where y represents the fluorescence emitted at 540 nm, y0 represents a residual fluorescence component, A1, A2, A3
and 1, 2, 3 are the amplitudes and wavelengths of the three excitation maxima, respectively. The parameters
derived from the best fit are collected in Table 1.
a b
Flu
ore
sce
nc
e (
a.u
.)
em (nm)
60 min. QC
1 mM Ca2+
15 nM Ca2+
ex = 380 nm
Flu
ore
sce
nc
e (
a.u
.)
em (nm)
60 min. QC
1 mM Ca2+
15 nM Ca2+
ex = 440 nm
Table 1. Fluorescence excitation parameters of intracellular quercetin
Conditions y0 A1 w1
(nm)
1
(nm) A2
w2
(nm)
2
(nm) A3
w3
(nm)3 (nm)
60 min.
QC 765 8359997 45.7 312.1 22845346 53.4 381.8 12725602 55.6 434.8
1 mM
Ca2+
3570 4902427 34.5 314.9 20175523 49.9 381.6 12646700 51.1 438.4
15 nM
Ca2+
2751 n.d. n.d. n.d. 16938566 49.1 381.6 1753912 36.1 444.1
n.d., not determined
b
c d
60 min. QC
1 mM Ca2+
15 nM Ca2+
Flu
ore
sce
nc
e (
a.u
.)
ex (nm)
Flu
ore
sce
nc
e (
a.u
.)
ex (nm)
60 min. QC
a
ex (nm)
Flu
ore
sce
nc
e (
a.u
.)
1 mM Ca2+
Flu
ore
sce
nc
e (
a.u
.)
ex (nm)
15 nM Ca2+
Fig. 4. Fluorescence excitation spectra of quercetin in intact and permeabilized Jurkat cells. Emission was collected at 540 nm. a,
Spectra were taken 60 min. after exposure of intact cells to 50 M QC (black trace), after permeabilization in 1 mM Ca2+
(red trace) or in 15 nM Ca
2+ (blue trace). b-d, Gaussian deconvolution of the excitation spectra shown in a. The fitting curve (red) was
obtained according to Eq. 4 with parameters provided in Table 1. Dashed blue curves represent the corresponding Gaussian components described by Eq. 4. In d, the first spectral component could not be determined. These results are representative of 4 independent experiments. Fluorescence was corrected for NADH/FLV interference.
Our previous studies [10] have shown, in agreement with other studies in the literature [11], that quercetin
accumulates inside the Jurkat cells and it binds to intracellular proteins, presenting a specific fluorescence with
emission at ~540 nm. In the experiments performed at this stage of the project we have evidenced the specific
mode in which the excitation and emission fluorescence spectra of quercetin are modified as a function of
intracellular calcium concentration. Moreover, we could demonstrate that the addition of quercetin to intact Jurkat
cells a calcium signal is evoked, and the inhibition with ruthenium red indicates the activation of a ryanodinic
receptor. From the data presented here, we infer the possibility that the intracellular protein to which the quercetin
binds might be the ryanodinic receptor from the endoplasmic reticulum. In the following research we poropse
ourselves to study into more detail at which degree our hypothesis might be confirmed.
Thus, in the third phase of the project we have continued the previous investigations and we have shown,
for the first time to our knowledge, that the conventional spectrofluorimetry allows the direct measurement of the
calciul release channel activity, the ryanodinic of the type 3 (RyR3), inintact and permeabilized cells, and we have
performed the characteriztion of its regulation by various ligands in situ. . Central to this methodology is the finding
that the flavonoid quercetin (3,3’,4’,5,7-pentahydroxyflavone) can be used as an efficient fluorescent probe to
distinguish with high sensitivity between the open and the closed conformation of the RyR3 channel. To this end,
we used the human leukaemia Jurkat T-cell line, which has been reported by two independent laboratories to
express solely the type 3 isoform of the ryanodine receptor [14, 15].
RESULTS AND DISCUSSION (3d phase).
Following previous work and on its basis [10, 1st phase of the project], we investigated the correlation between the
QC-induced Ca2+ release signal in Jurkat cells and the fluorescence emitted by QC-bound cellular targets at 540 nm
upon excitation at two specific wavelengths, 380 nm and 440 nm, respectively (F380 and F440).
Fig. 5. The F380/F440 ratio of intracellular QC fluorescence is highly correlated with the Ca2+
signal evoked by QC in intact Jurkat
cells. a, The average Ca2+
signal induced by 50 M QC in fura-2 loaded Jurkat cells in the absence (-RR) or in the presence (+RR)
of 10 M ruthenium red. Agents were added at indicated time points (arrows). Data are presented as average s.d. of 14 and 5
independent determinations, respectively. The average resting level obtained in the absence of RR (yi = 122.0 42.3 nM) is
indicated (dashed line). The decay phase of the signal in the absence of RR was fitted to the function y = yf + A exp(-t/), with yf =
410.7 10.4 nM, A = 702.6 19.2 nM, and = 15.25 0.99 min. (continuous line). b, Representative traces of the QC fluorescence, and c, of the F380/F440 ratio (Q) elicited upon excitation at 380 nm (F380) and 440 nm (F440) by Jurkat cells after
addition of 50 M QC, 35 M digitonin (DIG) and 10 mM EGTA at indicated times. Cells were not loaded with fura-2. d, Time
course of the calibrated Q ratio (Popen) before and after addition of 50 M QC. The value before stimulation is derived from data
shown in g. The data recorded after stimulation are average s.d. from 19 independent determinations, of which 9 were done
with fura-2 loaded cells and 10 without fura-2. The decay phase of the signal was fitted to the function y = yf + A exp(-t/), with yf
= 0.3619 0.0017, A = 0.1686 0.0020, and = 14.76 0.51 min. (continuous line). In a-d, signals were corrected for autofluorescence. e, Cross-corelation, and f, Pearson-correlation between Popen and [Ca
2+]cyt, as a function of the delay between
the two signals. Data from panels a and d recorded after stimulation (t > 0) were used. The delay for which the maximum of each function is reached is indicated by arrows. g, Linear correlation between [Ca
2+]cyt and Popen after >4 min. from addition of
QC. The linear fit to the data (continuous line; y = 4.1294x - 1.0778) was used to compute Popenrest
= 0.2905 in resting cells (the “initial” state marked by a square), assuming a basal level of 122.0 nM Ca
2+. The coordinates of the asymptotic (“final”) state
indicated by the black circle were extracted from the yf values indicated in panels a and d. h, Normalized response of [Ca2+
]cyt and Popen. The relative increase normalized to its maximum was calculated for each quantity as (y - yi)/(A + yf - yi), with A, yf and yi indicated in panels a, d and g. i, Calibration curve of maximal Q (Qmax) as a function of the autofluorescence of fura-2-free resting
cells excited at 440 nm (A440rest). Data were fitted to the function y = (a + bx)-1
, with a = 0.01784 0.00521 and b = (2.627
0.163) 10-6
. The Pearson correlation coefficient associated with these data was -0.78. A number n = 10 of experiments were
conducted with 50 M QC as described in c, n = 45 experiments were conducted with ICS cell suspensions challenged with 5 M
QC as described in Fig. 2a-b, and n = 8 and 7 determinations were done with ICS cell suspensions challenged with up to 10 M QC as described in Fig. 7.
50 M QC evoked a strong biphasic and sustained Ca2+ signal in fura-2 loaded cells (Fig. 5a), consistently
depressed by the RyR, but not IP3R antagonist, ruthenium red (RR) (Fig. 5a), indicating that the key role in the QC-
induced Ca2+ release is played by the ryanodine receptor. During continuous exposure of Jurkat cells to QC, F380 and
F440 increased progressively up to a plateau level, then declined upon permeabilization of the cells in 1 mM CaCl2
and decreased further after addition of 10 mM EGTA (Fig. 5b). The ratio Q = F380/F440 recorded in intact cells after
a b c
d e f
0.21
0.22
0.23
0.24
0.25
0.26
0.27
-11 -7 -3 1 5 9 13
delay (min.)
Cro
ss
-co
rre
lati
on
3.38 min.
0
0.2
0.4
0.6
0.8
1
-20 -10 0 10 20
delay (min.)
Pears
on
co
eff
icie
nt
2.37-2.70 min.
-0.1
0.1
0.3
0.5
0.7
0.9
1.1
-10 0 10 20 30 40 50
No
rmalized
re
sp
on
se
[Ca2+]cyt
Popen
t (min.)
0
0.2
0.4
0.6
0.8
-20 -10 0 10 20 30 40 50
t (min.)
50 M QC
Po
pe
n
0
0.2
0.4
0.6
0.8
1
0.25 0.3 0.35 0.4 0.45 0.5
[Ca
2+] c
yt,
M
Popen
Initial
Final
Qm
ax
A440rest (a.u.)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
-20 -10 0 10 20 30 40 50
[Ca
2+] c
yt,
M
t (min.)
50 M QC
10 M RR
RR
+ RR
-50000
50000
150000
250000
350000
450000
550000
-20 0 20 40 60 80 100
F440F380
QC
flu
ore
sce
nce (
a.u
.)
QC
DIG
EGTA
t (min.)
0
1
2
3
4
5
-20 0 20 40 60 80 100
QC
DIG
EGTA
t (min.)
Qg ih
addition of QC (Fig. 5c,d, F) presented in a highly reproducible way a time course which was strikingly similar to that
of the cytosolic Ca2+ level ([Ca2+]cyt). The Q ratio was then normalized to its maximal value (Qmax) which was obtained
after cell permeabilization in 1 mM CaCl2 and subsequent addition of 10 mM EGTA (Fig.5c). Q/Qmax, represents a
good estimate of the open probability of the RyR3/Ca2+ release channel. Therefore, in following we denote Q/Qmax as
Popen. We can notice a strong correlation between Popen and [Ca2+]cyt (Fig. 5e,f).
The Ca2+-dependence of F380/F440 reveals the RyR3-characteristic bell shape. Some typical recordings at various
decreasing Ca2+ levels in permeabilized cells are shown in Fig. 6a.
Fig. 6. The Ca2+
-dependence of Popen reveals the RyR3-characteristic bell shape. a, Typical recording of the Q ratio in
permeabilized cells at various levels of bulk cytosolic Ca2+
(indicated near the horizontal bars) adjusted by sequential addition of
CaCl2 or EGTA. Cells were first permeabilized for 15 min. in ICS containing 140 M Ca2+
and then challenged with 5 M QC at t = 0 (arrow). The quasi-steady state values of Q extracted from the data were normalized to the maximal value (Qmax = 5.19) observed after addition of QC, which was consistent with the standard calibration curve displayed in Fig. 1i. b, Ensemble data of Popen as a function of [Ca
2+]bulk, obtained from 45 experiments conducted similarly to that described in a. The data were fitted to
the equation y = Pmax xHA1
/(xHA1
+ KdA1HA1
) KdIHI
/(xHI
+ KdIHI
), with Pmax = 0.758 0.012, KdA1 = 2.72 0.22 nM, HA1 = 0.94 0.04,
KdI = 1.13 0.07 mM, HI = 1.10 0.12. c, Ensemble data of Popen as a function of [Ca2+
]bulk, obtained from 42 separate
experiments in which [Ca2+
]bulk was either increased or decreased from an initial level of 1-140 M. The data were fitted to the
same equation as in b, with Pmax = 0.457 0.008, KdA1 = 57.0 7.0 pM, HA1 = 0.90 0.09, KdI = 29.4 4.7 mM, HI = 0.34 0.03. d, The fraction of QC-bound channels depends biphasically on Ca
2+. Total fluorescence F440 obtained in experiments conducted as
described in b (rectified channel, n = 24) or in c (hindered channel, n = 31) was divided to the maximal value F440max indicated in Fig. 3c. Fitting curves were obtained by summing up the corresponding open- and closed-conformation curves shown in e and f, respectively, followed by normalization to F440max. e, Ca
2+-dependence of the fluorescence emitted by QC-bound channels in
open conformation (F440open). The data were fitted to the equations y = FOm xHA1
/(xHA1
+ KdA1HA1
) KdIHI
/(xHI
+ KdIHI
), with FOm =
28555.6 237.6, KdA1 = 12.3 4.1 pM, HA1 = 0.46 0.01, KdI = 487.8 25.5 mM, HI = 0.31 0.02 (hindered channel), and y = FO1
xHA1
/(xHA1
+ KdA1HA1
) + (FOm - FO1) xHA2
/(xHA2
+ KdA2HA2
), with FOm set to 28555.6, and FO1 = 16091.9 119.3, KdA1 = 298.6 77.0
pM, HA1 = 0.81 0.01, KdA2 = 1.40 0.13 mM, HA2 = 2.23 0.08 (rectified channel). f, Ca2+
-dependence of the fluorescence emitted by QC-liganded channels in closed conformation (F440closed). The hindered-channel data were fitted to the equation y =
FCm {1 - [PC1 xHA1
/(xHA1
+ KdA1HA1
) + (1 - PC1) xHA2
/(xHA2
+ KdA2HA2
)] KdIHI
/(xHI
+ KdIHI
)}, with FCm = 334603.0 7617.3, PC1 =
0.9068 0.0053, KdA1 = 8.27 1.15 pM, HA1 = 0.95 0.02, KdA2 = 30.3 6.0 mM, HA2 = 0.59 0.08, KdI = 3.45 0.17 M, HI = 0.34
0.01. The rectified-channel data were fitted to the equation y = FCm [1 - PC1 xHA1
/(xHA1
+ KdA1HA1
) KdIHI
/(xHI
+ KdIHI
)], with FCm
= 325302.3 10923.1, PC1 = 0.9854 0.0019, KdA1 = 105.8 10.4 pM, HA1 = 0.89 0.01, KdI = 8.25 0.47 mM, HI = 1.23 0.04. In
d-f, samples with similar cell densities were selected; the median s.e.m. of Qmax was 7.85 1.36 in all n = 24 rectified-channel
determinations, and 7.34 1.03 in n = 22 hindered-channel experiments performed with increasing [Ca2+
]bulk before calibration. When rectification was not achievable, Qmax was calculated using the calibration curve in Fig. 5i. All fluorescence signals were corrected for NADH/FLV interference.
After sequential addition of EGTA, Q displayed transient responses which were stable within <10 min., on a timescale
which was significantly faster than that observed in intact cells. Remarkably, the dependence of the steady-state
t (min.)
Q/Q
1m
M
EGTA
a d g
b e h
c if
0.0
0.2
0.4
0.6
0.8
1.0
1.E-11 1.E-09 1.E-07 1.E-05 1.E-03 1.E-01
corrected for autofluorescencecorrected for NADH interference
Po
pe
n
[Ca2+]bulk, M
-20000
20000
60000
100000
140000
180000
220000
-15 15 45 75 105 135 165
F380F440
QC
1.1 mM
490 M
241 M
142 M
45 M
271 nM
27 nM
10 nM
3 nM
t (min.)
Flu
ore
sc
en
ce
(a
.u.)
-2.5
-1.5
-0.5
0.5
1.5
2.5
3.5
4.5
-15 15 45 75 105 135 165
t (min.)
Q
QC
1.1 mM
490 M
241 M
142 M
45 M
271 nM
27 nM
10 nM
3 nM
Po
pen
[Ca2+]bulk, M
Po
pen
[Ca2+]bulk, M
[Ca2+]bulk, M
F440
op
en
(a.u
.)
Hindered
Rectified
[Ca2+]bulk, M
F440
clo
se
d(a
.u.)
HinderedRectified
Hindered channel
Rectified channel
[Ca2+]bulk, M
Fra
cti
on
of
QC
-bo
un
d c
ha
nn
els Hindered
Rectified
Popen on the bulk Ca2+ level, [Ca2+]bulk, (Fig. 6b) appeared to be closely similar to the bell-shape dependence which is
specific to the RyR3 receptor in lipid bilayers, with a high Popen (~0.8-1) reached at optimal Ca2+ levels and a steep
Ca2+-dependence on both sides of the curve [16-19]. In order to reach lower levels of cytosolic Ca2+ and also to
reduce the recording time, a slightly different type of experiments were conducted in which the cells were
permeabilized in 1-140 M CaCl2. Unexpectedly, any subsequent addition of EGTA or CaCl2 did not replicate the
results described above. The corresponding Popen curve remained bell-shaped; however, the maximal Popen was
reduced to half, the activating domain of [Ca2+]bulk enlarged considerably, and Ca2+ binding to its inhibitory site(s)
exhibited negative cooperativity (Fig. 6c). Together, these observations identify a novel regulatory mechanism by
which the channel activity under physiological conditions is partially suppressed (hindered channel) whereas the
channel becomes maximally activated after exposure to millimolar concentrations of cytosolic bulk Ca2+ followed by
chelation of Ca2+ (rectified channel).
RyR3 affinity for Ca2+ and QC in open/closed conformation. We have scrutinized the molecular properties of these
unique facets of RyR3 regulation by assessing the dependence of the fluorescence emitted by the QC-liganded
channel on [Ca2+]bulk (Fig. 6d-f) and on the cytosolic concentration of quercetin, [QC]cyt (Fig. 7), in either closed or
open configuration.
Fig. 7. Biphasic regulation of Popen by quercetin. a, Typical traces of F380 (red) and F440 (blue) fluorescence emitted by QC-liganded channels, and b, of the Q ratio recorded in permeabilized cells at increasing levels of cytosolic QC (indicated near the horizontal bars) adjusted by sequential addition of quercetin. Cells were first permeabilized for 15 min. in ICS containing 80 nM Ca
2+ and then challenged with QC at t = 0. c, QC-dependence of the fluorescence emitted by QC-bound channels in open or in
closed conformation (open and closed circles, respectively). Data were obtained as median s.e.m. from 8-16 determinations
similar to those described in a. All samples had similar cell densities. The median s.e.m. of Qmax was 7.41 1.27 in n = 8
experiments in which Qmax could be assessed after rectification in a final level of 100 nM Ca2+
and 10 M QC. In the remaining experiments Qmax was calculated using the calibration curve in Fig. 1i. The closed conformation data were fitted to the equation:
y = F440max [PC1’ xH1
/(xH1
+ Kd1H1
) + (1 - PC1’) xH2
/(xH2
+ Kd2H2
)], with F440max = 647652.3 17184.9, PC1’ = 0.0469 0.0039, Kd1 = 1.54
0.19 M, H1 = 1.04 0.06, Kd2 = 194.4 9.2 M, H2 = 1.63 0.13. The data obtained in open conformation were fitted to the
equation: y = F440max xH/(x
H + Kd
H) [PC1’ x
H1/(x
H1 + Kd1
H1) + (1 - PC1’) x
H2/(x
H2 + Kd2
H2)], with F440max set to 647652.3, and Kd = 255.1
30.7 M, H = 0.79 0.02, PC1’ = 0.623 0.052, Kd1 = 3.52 0.23 M, H1 = 0.52 0.02, Kd2 = 88.9 4.2 M, H2 = 3.66 0.51. d,
Dependence of Popen on [QC]cyt in the presence (+DAN) or in the absence (-DAN) of 10 M dantrolene. Data are median s.e.m. from 4-16 and 8-16 separate determinations, respectively. Both sets of data were fitted to the equation y = [P0 + (1 - P0) x
HA/(x
HA
+ KdAHA
)] KdIHI
/(xHI
+ KdIHI
), with P0 = 0.268 0.017, KdA = 60.8 3.3 M, HA = 1.60 0.07, KdI = 2.24 0.31 M, HI = 1.54 0.06 (-
DAN), and P0 = 0.137 0.021, KdA = 73.2 5.2 M, HA = 1.49 0.15, KdI = 1.70 0.47 M, HI = 1.63 0.16 (+DAN). e,
Representative trace of the Q ratio recorded in permeabilized cells in the presence of 10 M dantrolene after sequential addition of QC as indicated. The signal was calibrated by addition of CaCl2 and EGTA at indicated time points. The free Ca
2+ and
QC levels at various steps are indicated. Cells were first permeabilized for 15 min. in ICS containing 80 nM Ca2+
, then 10 M DAN was added, and after an additional 15 min. the cells were challenged with QC (at t = 0). f, The fraction of QC-bound channels increases with [QC]cyt. Measurements were done with two different levels of cytosolic Ca
2+, 80 nM (red) and 1.2 mM (black).
Total fluorescence F440 obtained from the data shown in c or in Fig. S8a was divided to F440max indicated in c or in the Supplementary Material (Fig. S8a), respectively. Continuous curves for 80 nM or 1.2 mM Ca
2+ were calculated by summing up
the two fitting curves shown in c or in Fig. S8a, respectively, and then by dividing the result to the corresponding F440max. All fluorescence signals were corrected for NADH/FLV interference.
-4
-2
0
2
4
6
-10 0 10 20 30 40 50
0.5
2.5
10
20
50
75
t (min.)
1
100
200
300Q
-50000
50000
150000
250000
350000
450000
550000
-10 0 10 20 30 40 50
F380F440
0.5
2.5
10
20
50
75
t (min.)
Flu
ore
sc
en
ce
(a
.u.)
1
100
200
300
0
2
4
6
8
10
-10 0 10 20 30 40 50 60 70
t (min.)
QC
QC
QC
QC
CaCl2
EGTA
QC
Q
80 nM Ca2+
0.35 1 2.5 5 10 QC (M)
1.2 mM Ca2+
730 nM Ca2+
0
a b c
d ef
[QC]cyt, M
Fra
cti
on
of
QC
-bo
un
d c
ha
nn
els
1.2 mM Ca2+
80 nM Ca2+
Flu
ore
sc
en
ce (
a.u
.)
[QC]cyt, M
Po
pe
n
+ DAN
- DAN
[QC]cyt, M
To discriminate between the fluorescence emitted by the channel in the open or in the closed conformation,
we should first point out that when all RyR3 channels in a given cell sample are fully activated, the ratio F380/F440 is
equal to Qmax. Based on the invariability of the 380 nm excitation band width with the treatment conditions (2011
report, [12]) an important implication with general applicability is that the ratio F380/Qmax, denoted F440open,
represents the contribution to F440 of the channels found in open configuration, and the corresponding contribution
of the channels in closed configuration is then F440closed = F440 - F440open. Data analysis, corroborated by several
lines of evidence, suggests that F380 and F440 are proportional to the number of quercetin-liganded channels rather
than to the number of QC molecules bound to the channels. Consequently, the fraction of channels found in open or
closed conformation can be readily determined by dividing F440open or F440closed to the maximal F440 value, F440max.
This upper bound could be obtained at saturating levels of quercetin (>300 M) and appeared to be invariable at
activating or inhibitory [Ca2+]bulk (Fig. 7c). The data indicate the existence of two distinct classes of Ca2+ binding sites
that affect the binding of quercetin to the receptor in its closed conformation in opposite ways: a high-affinity Ca2+
site (A1) which inhibits, and a low-affinity Ca2+ site (I) which stimulates QC binding to the receptor. This working
mechanism appears to operate in the hindered channel as well as in the rectified channel (Fig. 6f). In open
configuration, the hindered channel presented an inverted image of the fluorescence dependence on [QC]cyt (Fig. 6e)
which is also consistent with the allosteric coupling scenario. A surprisingly different picture could be observed with
the open rectified channel, which revealed a second class of operational, low-affinity activating Ca2+ sites (A2), while
no apparent inhibitory site could be distinguished (Fig. 6e). The dependence of F380 and F440 on the flavonoid level
(Fig. 3a-c) uncovered interesting features of channel regulation by quercetin. The data obtained with 80 nM cytosolic
Ca2+ indicate that in the closed conformation of the receptor two distinct classes of QC inhibitory binding sites (IQ1
and IQ2) are functional, with corresponding dissociation constants (Kd) of 1.54 M and 194.4 M, and Hill
coefficients (H) of 1.04 and 1.63, respectively (Fig. 7c). The consistent difference in the relative abundance of the two
corresponding closed states of the receptor indicates that at saturation with quercetin the average time spent in the
low-affinity closed state is 20.3 times longer than the average dwell-time spent in the counterpart high-affinity
closed state The fluorescence data show that in closed conformation no activating QC binding site is available for
binding, whereas in open conformation an activating site (AQ) with Kd = 255.1 M and H = 0.79 seems to operate
(Fig. 7c). The characteristic parameters of the two inhibitory binding sites in open conformation (Kd: 3.52 M and
88.9 M; H: 0.52 and 3.66) indicate a significant change in the affinity and the number of QC binding molecules,
suggesting that a conformation change may take place upon channel opening. It should be noted that the binding
parameters of IQ2 in both open and closed conformation are similar to those predicted for the inhibitory QC-binding
site of RyR1 (Kd = 86-210 M, H = 4) [20]. Moreover, the affinity of AQ established here is also similar to that
estimated for RyR1 (Kd = 300 M) in one of its four activity modes [20]. As a consequence of the manifested dual
regulation by quercetin, Popen exhibited a biphasic dependence on the cytosolic level of quercetin (Fig. 7d).
Dantrolene (DAN), which is an inhibitor of the RyR1 and RyR3 but not of the cardiac RyR (RyR2) (RyR2) [21, 22],
reduced Popen at all tested levels of quercetin (Fig. 7d,e). Interestingly, the inhibitory effect of DAN could be
overcome upon channel rectification, when a robust increase of the Q ratio was recorded (Fig. 7e). With an
inhibitory level of 1.2 mM bulk Ca2+, the QC-dependence of F380, F440, Popen and the fraction of QC-bound channels
was similar to that described above (Fig. 7f). However, significant differences could be noticed, in particular the
reduced cooperativity of QC associated with IQ2 in the open conformation, and the increased affinity of AQ.
Additional informations can be found in the papers resulted from this studies [12, 23].
In conclusion, we show that the calibrated F380/F440 ratio of the fluorescence emitted at 540 nm by
quercetin-bound cellular targets constitutes a faithful measure of the open probability of the RyR3/calcium
release channel in Jurkat cells. This study presents the first direct, non-invasive measurement of the ensemble
activity of the RyR3 channel in intact and in permeabilized cells and advances a simple, but robust and sensitive
method to assess the functional and molecular properties of this ionic channel in situ. The future development of
this novel assay can bring significant contributions to our understanding of the RyR function in physiological and
pathological conditions. The data raise the attractive possibility that new molecular probes with high specificity
for the RyR3 receptor could be designed in order to optimize measurement protocols amendable to fluorescence
microscopy, flow-cytometry or other fluorescent detection techniques, and encourages further studies oriented
towards different ionic channels.
The results of the studies presented here have been published [12], communicated at conferences [24, 25]
or accepted for publication [23].
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4. Baran I, Ganea C, Scordino A, Musumeci F, Barresi V et al. 2010. Cell Biochem Biophys 58: 169-179
5. Chen D et al. 2005. Biochem Pharmacol 69: 1421-1432
6. Jeong JH et al. 2009. J Cell Biochem 106: 73-82
7. Yen GC et al. 2003. Biosci Biotechnol Biochem 67: 1215
8. Baran, I., C. Ganea, et al., Activity Report Istituto Nazionale Di Fisica Nucleare Laboratori Nazionali Del Sud, acceptat; Edit. Arti Grafiche Le
Ciminiere Catania, Italia; ISSN: 1827-1561
9. Baran, I., C. Ganea, et al., Activity Report Istituto Nazionale Di Fisica Nucleare Laboratori Nazionali Del Sud, acceptat; Edit. Arti Grafiche Le
Ciminiere Catania, Italia; ISSN: 1827-1561
10. Baran, I, C. Ganea I. Ursu, V. Baran, O Calinescu, A. Iftime, R. Ungureanu, I.T. Tofolean, Rom. J. of Physics Volume 56, no. 3-4, 388-398,
2011
11. M. Fiorani, A. Guidarelli, M. Blasa, C. Azzolini, M. Candiracci, E. Piatti, O. Cantoni, J. Nutr. Biochem. 21, 397 – 404 (2010)
12. Irina Baran, Eva Katona, Constanta Ganea, Quercetin as a fluorescent probe for the ryanodine receptor activity in Jurkat cells, Pfluegers
Archiv, European Journal of Physiology, 2013, DOI 10.1007/s00424-013-1235-y, 465:1101–1119.
13. Sengupta, B., Sengupta, P. K. Binding of quercetin cu human serum albumin: a critical spectroscopic study. Biopolymers 72, 427-434 (2003)
14. Hakamata, Y., Nishimura, S., Nakai, Y. J., Nakshima, Y., Kita, T., Imoto, K. Involvement of the brain type ryanodine receptor in T-cell proliferation. FEBS Lett. 352, 206-210 (1994).
15. Guse, A. H., da Silva, C. P., Berg, I., Skapenko, A. L., Weber, K., Heyer, P., Hohenegger, M., Ashamu, G. A., Schulze-Koops, H., Potter, B. V., Mayr, G. W. Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose. Nature 398, 70-73 (1999).
16. Chen, S. R. W., Li, X., Ebisawa, K., Zhang, L. Functional characterization of the recombinant type 3 Ca2+
release channel (ryanodine receptor) expressed in HEK293 cells. J. Biol. Chem. 272, 24234-24246 (1997).
17. Jeyakumar, L. H., Copello, J. A., O’Malley A. M., Wu, G.-M., Grassucci, R., Wagenknecht, T., Fleischer, S. Purification și characterization of ryanodine receptor 3 from mammalian tissue. J. Biol. Chem. 273, 16011-16020 (1998).
18. Murayama, T., Oba, T., Katayama, E., Oyamada, H., Oguchi, K., Kobayashi, M., Otsuka, K., Ogawa, Y. Further characterization of the type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm. J. Biol. Chem. 274, 17297-17308 (1999).
19. Murayama, T., Ogawa, Y. Characterization of type 3 ryanodine receptor (RyR3) of sarcoplasmic reticulum from rabbit skeletal muscles. J. Biol. Chem. 272, 24030-24037 (1997).
20. Baran, I., Ganea, C., Baran, V. A two-gate model for the ryanodine receptor cu allosteric modulation by caffeine și quercetin, Eur. Biophys. J. 37, 793-806 (2008).
21. Zhao, F., Li, P., Chen, S. R., Louis, C. F., Fruen, B. R. Dantrolene inhibition of ryanodine receptor Ca2+
release channels. Molecular mechanism și isoform selectivity. J. Biol. Chem. 276, 13810-13816 (2001).
22. Vaithianathan, T., Narayanan, D., Asuncion-Chin, M. T., Jeyakumar, L. H., Liu, J., Fleischer, S., Jaggar, J. H., Dopico, A. M. Subtype identification și functional characterization of ryanodine receptors in rat cerebral artery myocytes. Am. J. Physiol. Cell Physiol. 299, C264-278 (2010).
23. Irina Baran and Constanta Ganea, RyR3 in situ regulation by Ca2+
and quercetin and the RyR3-mediated Ca2+
release flux in intact Jurkat cells, Archives of Biochemistry and Biophysics, 2013, (acceptat)
24. Ganea C. ; Katona, E.; Baran, I., Quercetin activates the RyR3 receptor in Jurkat human leukemia cells, conferință invitată, Max Planck Institut for Biophysics, Franfurt/Main, Germania, 24 oct. 2011
25. Baran, I.; Katona, E.; Ganea, C., Quercetin fluorescence reveals the open probability of the RyR3 Ca2+ channel in intact cells, 22nd
IUBMB Congress/37th FEBS, Seville, SPAIN, SEP 04-09, 2012, FEBS JOURNAL Volume: 279 Special Issue: SI Suppl: 1, p: 269-270, SEP 2012
Detailed analysis of apoptosis and delayed luminescence of human leukemia Jurkat T-cells after proton-
irradiation and treatments with oxidant agents and flavonoids
(2nd phase)
Introduction. As we have already shown in the first phase of the project, epigallocatechine-3-gallate (EGCG) and
quercetin (QC; 3,5,7,3’,4’-pentahydroxyflavone) are two well-investigated flavonoids which inhibit cell proliferation
and induce apoptosis in various cancer cell types [1-6]. Continuing our previous studies concerning the effects of
these two compounds on the human leukemia Jurkat-T [7], we have investigated into more detail the relationship
between apoptosis and delayed luminescence (DL) in this cell type, following various treatments. In order to induce
the oxidative stress we have used menadione (MD) and hydrogen peroxide (H2O2) as well as the two flavomoids, QC
and EGCG, applied alone or in combination with MD or H2O2. Because in the therapy of various cancer types there
are often used combined treatments, drugs-radiations, 62 MeV proton beams were used to irradiate cells under a
uniform dose of 2 or 10 Gy, respectively. We have assessed the apoptosis, the cell cycle distributions and the
delayed luminescence. We have shown that menadione, H2O2 and quercetin were potent inducers of apoptosis and
DL inhibitors. Quercetin decreased clonogenic survival and the NAD(P)H level in a dose-dependent manner. Proton-
irradiation with 2 Gy but not 10 Gy increased the apoptotic rate. However, both doses induced a substantial G2/M
arrest. Quercetin reduced apoptosis and prolonged the G2/M arrest induced by radiation. DL spectroscopy indicated
that proton-irradiation disrupted the electron flow within Complex I of the mitochondrial respiratory chain, thus
explaining the massive necrosis induced by 10 Gy of protons, and also suggested an equivalent action of menadione
and quercetin at the level of the Fe/S center N2, which may be mediated by their binding to a common site within
Complex I, probably the rotenone binding site.
Materials and methods. The method has been described into detail in the scientific report in extenso of the project, as well as in
the publication resulted from the present study [8]. In short, we have performed measurements of cellular viability with trypan
blue solution, and the apoptosis and the cell cycle have been assessed with a flow cytometer, for cells incubated with a
propidium iodide. The measurements have been performed on cell cultures of human leukemia Jurkat T-cell lymphoblasts in
suspension, in MegaCell RPMI 1640 medium supplemented with 5% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100
units/ml penicillin and 100 g/ml streptomycin, at 37ºC in a humidified incubator with a 5% CO2 atmosphere. Proton-
irradiation. Cell suspensions (7 ml) were irradiated in 50 ml- centrifuge tubes fixed in a vertical position. 62 MeV proton beams
accelerated by the superconducting cyclotron at LNS-INFN, Catania (Italy) were used for proton-irradiation at a dose rate of
11.76 Gy/min. A plane-parallel advanced PTW 34045 Markus ionization chamber was adopted as a reference dosimeter.
Delayed luminescence spectroscopy. We used an improved version of the ARETUSA set-up [39], a highly sensitive equipment
able to detect single photons. The cell samples were excited by a Nitrogen Laser source (Laser Photonics LN 230C; wavelength
337 nm, pulse-width 5 ns, energy 100 ± 5 µJ/pulse). A multi-alkali photomultiplier tube (Hamamatsu R-7602-1/Q) was used as a
detector for photoemission signals with wavelengths in the visible range (VIS, 400-800 nm), in single photon counting mode.
Spectrofluorimetry. For determination of the relative level of intracellular nicotinamide adenine dinucleotide and nicotinamide
adenine dinucleotide phosphate in their reduced form (NADH and NADPH, respectively), denoted generically as NAD(P)H,
exponentially growing cells were washed twice in a standard saline solution (SS) containing 140 mM NaCl, 5 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, 20 mM HEPES, 10 mM glucose, pH 7.2/NaOH, resuspended in SS at ~106 cells/ml and transferred into a 2 ml
quartz cuvette maintained at 37ºC under continuous stirring in a Horiba Jobin Yvon spectrofluorimeter. Every 22 s the cell
sample was excited at 340 nm and emission was collected at 450 nm. After stabilization of the fluorescence signal, QC at the
indicated dose was added directly to the cuvette and the kinetic recording was carried on for an additional 45-60 min. Statistics.
Unless indicated otherwise, the data are presented as median s.e.m. of at least three different measurements. Statistically
significant differences were determined using Student’s t-test. A level of P < 0.05 was considered significant in all statistical
tests. Other details in [8].
Results and discussion
Effects of proton radiation, MD, H2O2, QC and EGCG on apoptosis and cell cycle
Continuing a previous preliminary study, we performed more extensive measurements and we have done a more
detailed analysis of the experimental results. We have firstly assessed the apoptosis and the cell cycle distributions in
the Jurkat cells under various treatments. In agreement with our previous investigations [7, 10], quercetin induced
apoptosis in Jurkat cells in a dose- and time-dependent manner, while 0.5 M EGCG applied for 24 h did not affect
the apoptotic rate or cell cycle distribution, but could enhance the apoptosis induced by MD or H2O2. Consistently
with the already mentioned studies, in the present study we have found that proton irradiation with high energy
protons of 2 Gy but not with 10 Gy produced a significant increase of the apoptotic rate at 48 hr. after irradiation.
Trypan blue exclusion tests confirmed high rates of cell death, namely 18.4 3.2% and 46.6 6.8% at 24 h and 48 h
after irradiation with 10 Gy of protons, respectively.
0
20
40
60
80
100
0 24 48
Ap
op
toti
c c
ells (
%)
t (h)
A
0
20
40
60
80
0 24 48
G0/G
1cells (
%)
t (h)
B
0
20
40
60
80
0 24 48
t (h)
S c
ells (
%)
C
0
20
40
60
80
0 24 48
Q50
H100
Q50+H100
t (h)
G2/M
cells (
%)
D
0
20
40
60
80
100
120
0 50 100 150 200
[QC], M
Clo
no
ge
nic
su
rviv
al (%
)
A
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 10 25 50
[QC], M
Rela
tiv
e [
NA
D(P
)H] B
0
0.2
0.4
0.6
0.8
1
1.2
-10 0 10 20 30
10 M
25 M
50 M
Re
lati
ve
[N
AD
(P)H
]
t (min.)
C
Fig. 1. Time course of the apoptotic rate and cell-cycle distribution
after treatment of Jurkat cells with 50 M QC for 24 h (Q50, solid
circles), 100 M H2O2 for 20 min. (H100, open circles) or combination
of the two (50 M QC pre-incubation followed by addition of 100 M
H2O2 for 20 min.; treatment denoted as Q50 + H100, gray circles).
Apoptotic rates (A), G0/G1 (B), S-phase (C) and G2/M (D) cell fractions
are indicated. The dashed line represents the average obtained from
control cell samples.
From kinetic measurements one can notice that quercetin can
arrest the Jurkat cells in the G2/M phase (Fig. 1). Moreover,
the G2/M cell fraction in the case of the cells treated with 50
M QC for 24 h decreased from 39.8 6.4% at 9 h, to 10.3 3.0% at 48 h after the treatment (Fig. 1D). The G2/M
block was associated with a significant reduction in the G0/G1 cell fraction (Fig. 1B),
whereas the S-phase distribution was unaltered (Fig. 1C). The cells also displayed a
consistent apoptotic rate (52.2 7.3%) 9 h after drug removal, which then
increased gradually up to 81.5% during the probing interval (Fig. 1A).
In treatments with duration of 1 h, QC decreased clonogenic survival in an
exponential manner, with an estimated dose for reduction of clonogenicity to 50%,
D50% =109.8 M (Fig. 2A). In separate spectrofluorimetry experiments, QC also
decreased the cellular content of NAD(P)H in a dose-dependent manner, with an
effective dose for half-maximal effect IC50 = 39.5 M (Fig. 2B). In Fig. 2C we present
some examples of NAD(P)H fluorescence recordings in Jurkat cell suspensions
exposed to different concentrations of QC. After addition of QC, the NAD(P)H
fluorescence signal decreased slowly (in up to ~15 min.) to a steady value which
appeared to be dose-dependent. Fig. 2B summarizes the steady state data obtained
from recordings like those in Fig. 2C.
Fig. 2. Quercetin decreases clonogenic survival and the cellular content of NAD(P)H in Jurkat
cells. (A) Dose-response of clonogenicity (S) was fitted to an exponential function (curve) of
the form S (%) = 100 exp(-D/D0), where D represents the dose of QC applied for 1 h and the
characteristic dose derived from the fit was D0 = 158.5 M. Data are expressed as mean
standard deviation of 4 - 6 separate determinations. (B) The ratio between NAD(P)H
fluorescence of treated vs. control cells (relative NAD(P)H) obtained in steady state after
addition of QC to cell suspensions decreases with the level of QC. (C) Representative
recordings of NAD(P)H fluorescence relative to the resting value in cell suspensions before and after addition of QC at various
levels indicated near each trace.
Fig. 3. Apoptosis and cell-cycle distributions assessed at 6, 24 and 48
h after treatment of Jurkat cells with the vehicle (Ctrl), with 50 M
QC for 1 h (QC), with 2 Gy of proton radiation (IR) or with 2 Gy of
proton radiation after preincubation with 50 M QC for 1 h (QC + IR).
Apoptotic rates (A), G0/G1 (B), S-phase (C) and G2/M (D) cell fractions
are illustrated. The star denotes significant difference between the
treatments IR and QC + IR.
0
10
20
30
40
CTRL QC IR QC+IR
Ap
op
tos
is r
ate
(%
)
6 h
24 h
48 h
*
A
0
20
40
60
80
100
CTRL QC IR QC+IR
G0/G
1 c
ell
fra
cti
on
(%
)
6 h
24 h
48 h
B
0
20
40
60
80
100
CTRL QC IR QC+IR
S c
ell
fra
cti
on
(%
)
6 h
24 h
48 h
C
*
0
20
40
60
80
100
CTRL QC IR QC+IR
G2/M
cell
fra
cti
on
(%
)
6 h
24 h
48 h
D
*
I/I C
trl
0
0.4
0.8
1.2
1.6
2
0.00001 0.0001 0.001 0.01
E0.5Q0.5Q5Q50
0
0.4
0.8
1.2
1.6
0.00001 0.0001 0.001 0.01
E0.5 + H500
Q10* + H500
H500
I/I C
trl
t (s)
t (s)
A
B
1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
0.00001 0.0001 0.001 0.01 0.1
VIS
460 nm
645 nm
DL
in
ten
sit
y (
a.u
.)
t (s) t (s)
I IR
/ I C
trl
0
0.5
1
1.5
2
2.5
0.00001 0.0001 0.001 0.01 0.1
1 h
24 h
t (s) t (s)
I IR
/ I C
trl
I IR
/ I C
trl
460 nm
VIS
0
0.5
1
1.5
2
0.00001 0.0001 0.001 0.01 0.1
1 h
24 h
645 nm
A
C
B
D
0
0.5
1
1.5
2
0.00001 0.0001 0.001 0.01 0.1
1 h
24 h
In a different set of experiments, we investigated the effects of pre-incubating Jurkat cells with 50 M QC for 1 h on
apoptosis and cell cycle distribution after irradiation with 2 Gy of protons (Fig. 3). Quercetin exercised an inhibitory
effect on apoptosis (Fig. 3A) and appeared to prolong significantly the G2/M arrest induced by proton-irradiation (Fig.
3D), which may indicate an enhanced capacity for DNA repair and maintenance of the G2/M checkpoint active. The
parallel reduction in the S cell pool (Fig. 3C) and conservation of the G0/G1 cell fraction (Fig. 3B) suggest that cells
surviving irradiation may experience an additional G0/G1 but not S-phase arrest after 48 h from irradiation.
Effects of proton radiation, MD, H2O2, QC and EGCG on delayed luminescence
DL of Jurkat cells irradiated with 10 Gy of high energy protons exhibited different characteristics when
probed at 1 h or 24 h after irradiation. Hence, a reduction of 34.1 9.6% in the DL-III relative quantum yield in VIS
was observed after 1 h from irradiation, whereas the cell samples probed at 24 h after irradiation exhibited an
increase of 27.3 8.5% in the DL-II relative quantum yield and an increase of 41.8 14.3% in the time domain 10 -
100 ms, while all the other components of the DL emission in VIS were not significantly different from the resting DL
emission (Fig. 4B).
Fig. 4. Kinetics of DL emission of Jurkat cells under control conditions (A) or after irradiation with 10 Gy of protons (B-D). In (A) some representative photoemission curves are shown for the entire visible domain (VIS), as well as for detection of 460 nm and 645 nm light emitted by the same cell sample. In (B-D) the intensity of light emission of irradiated cells (IIR) is normalized to the DL intensity of sham-irradiated cultures (ICtrl). Measurements were done after 1 h and 24 h from irradiation, as indicated. Results are presented for VIS (B), 460 nm (C) and 645 nm (D) emitted light.
Shortly after irradiation, DL emitted at 460 nm was similar to
that of control cells; however, the surviving cells exhibited 24 h later
a significant overall DL enhancement with about 35% of the control
intensity (Fig. 4C). A remarkable augmentation, up to ~1.7 fold, of blue light emission was detected for a DL
component with an established time constant of 178 s (value derived from fitting analysis, not shown; however,
the distinctive peak centered on ~180 s is clearly visible in Fig. 4C). 1 h after irradiation, delayed emission of red
light presented a significant reduction of the DL-III component, with 38.3 11.5%, whereas 24 h later DL-I decreased
to 76.1 13.8% of control emission and there was a significant increase of a DL component with an estimated time
constant of 379 s (Fig. 4D, and data analysis not shown). At increasing doses, quercetin inhibited DL progressively
(Fig. 5A). The most sensitive DL region was DL-III, which decreased by one order of magnitude after the treatment
with 50 M QC for 24 h, whereas DL-I was only slightly affected by QC. EGCG exerted a qualitatively different effect
on DL by producing a fairly uniform reduction of the photoemission intensity along the entire timescale .
Fig. 5. Kinetics of DL emission of Jurkat cells after various treatments with flavonoids (A) or with H2O2 alone or in combination with EGCG or QC (B). Treatments are labeled as in Fig. 1. The intensity of light emission of treated cells (I) is normalized to the DL intensity of control cells (ICtrl) [7].
500 M H2O2 applied for 20 min. reduced DL significantly over the regions DL-I and DL-
II (Fig. 5B, Fig. 6A, B). Pre-treatment with 0.5 M EGCG for 24 h was able to induce a
significant recovery of DL-II emission, whereas preincubation with 10 M QC for 1 h
further reduced the DL-III intensity. The lower dose of 100 M H2O2 had a modest
effect on DL and inhibited photoemission by ≈22% over the entire timescale (Fig. 6A-
C). Preincubation with 50 M QC for 24 h restored DL-I emission, but inhibited
substantially DL-II and DL-III.
Fig. 6. DL-quantum yield relative to control (A-C) and its correlation to the
apoptotic cell fraction (D-F) under various treatments indicated in Fig. 1.
Q, E, M/MQ/ME, H/HQ/HE and IR denote single QC- or EGCG-treatments,
MD-treatments with or without QC-or EGCG-preincubation, H2O2-
treatments with or without QC- or EGCG-preincubation, and irradiation
with 10 Gy of protons, respectively. Pearson correlation coefficients are
shown for all treatments (rall), for the M/MQ/ME/IR treatments
(rM/MQ/ME/IR) and for the M/Q/MQ treatments (rM/Q/MQ). Results obtained
for separate DL time-domains indicated inside boxes are displayed
individually for DL-I (A, D), DL-II (B, E) and DL-III (C, F).
Menadione also inhibited DL in a dose-dependent manner,
and, at variance with the modest effect of QC on DL-I, MD reduced
substantially photoemission in the DL-I region (Fig. 6A).This
inhibition was strong even at the lowest dose of 25 M
menadione. DL-II was inhibited to a similar extent by high doses of
MD (Fig. 6B), whereas DL-III exhibited a drastic reduction and thus,
in the M250 treatment, the DL-III quantum yield reached 15.5 6.1% of its resting value (Fig. 6C). Preincubation with
the two flavonoids generally induced partial recovery of DL-III up to ~25% of the resting value, except in the case of
pre-treatment with 5 M QC for 24 h, when a further reduction to 9.2 3.8% was recorded.
At the moment, the effects of QC or EGCG on apoptosis induced in Jurkat T-cells by the flavonoids
themselves or in conjunction with menadione and hydrogen peroxide are poorly known. Here we found that a 24-
hour treatment with physiological levels (0.5 - 5 M) of QC and EGCG can potentiate the antiproliferative activity of
menadione by enhancing drug-induced apoptosis in human leukemia Jurkat T cells. In agreement with previous
reports that QC is a more potent inhibitor of hydroxyl radical formation than a scavenger of superoxide anions [11],
none of the quercetin-based treatments used in the present work exercised protective effects against MD, whereas
a short incubation with 10 M QC for 1 h offered consistent protection against H2O2 and induced G2/M cell cycle
arrest, hence allowing time for repair of H2O2-induced damage. In addition, preincubation for 24 h with a very low
level (0.5 M) of EGCG increased significantly the G2/M cell fraction after exposure to 250 M MD. Nevertheless,
albeit long-term administration of QC or EGCG may improve significantly the menadione-based treatment of
leukemia, it is important to establish the critical level of flavonoid that is no longer beneficial to normal cells. Our
investigations suggest a connection between the ability of quercetin to decrease the level of NAD(P)H and the
induction of apoptosis, which is probably mediated by the failure to maintain the ATP-dependent electrochemical
gradient across the inner mitochondrial membrane, and the consequent dissipation of the mitochondrial membrane
potential. Similarly to the protective effect of QC against H2O2 discussed above, our results suggest that short
treatments with quercetin could be able to improve cell survival after proton-irradiation, most likely by inhibiting
hydroxyl radical formation after irradiation and protecting against cellular oxidative DNA damage. The data
presented here indicate that DL of proton-irradiated cells probed shortly after irradiation was dominated by light
emission in the red region of the spectrum and was characterized by a significant reduction in the millisecond DL-III
region. On the contrary, DL emission of irradiated cells that survived the subsequent 24 h was dominated by light
emission in the blue region of the spectrum and exhibited a significant increase in the sub-millisecond DL-II region.
Moreover, cells that survived 1 day post-irradiation revealed two distinctive DL states, namely a blue-light emitting
state with a characteristic lifetime of 178 s, and a red-light emitting state with a characteristic lifetime of 379 s. In
agreement with our previous results [7] and a series of data we have obtained with rotenone-treated cells (not
shown), as well as with established electron transfer rates within Complex I [12], we propose that the red-light
emitting state is characteristic to the Fe/S center N2 in reduced form. Having in view the growing interest of using DL
spectroscopy in clinical applications [13-16], our results lend further support for the development of this
D
F
0
20
40
60
80
100
120
140
0 20 40 60 80
Apoptosis (%)
DL
-I Q
ua
ntu
m y
ield
(%
)
EQH/HQ/HEM/MQ/MEIR
0
20
40
60
80
100
120
140
0 20 40 60 80
Apoptosis (%)
DL
-II
Qu
an
tum
yie
ld (
%)
0
20
40
60
80
100
120
140
0 20 40 60 80
Apoptosis (%)
DL
-III
Qu
an
tum
yie
ld (
%)
rall = -0.36
rM/MQ/ME/IR = -0.76
rM/Q/MQ = -0.54
rall = -0.61
rM/MQ/ME/IR = -0.98
rM/Q/MQ = -0.92
rall = -0.54
rM/MQ/ME/IR = -0.84
rM/Q/MQ = -0.82
0
20
40
60
80
100
120
140
IR 1
0 Gy
E0.
5Q0.
5Q5
Q10
*Q50
M25
x20
'
M25
x4h
M25
0
E0.
5+ M
250
Q0.
5+ M
250
Q5+
M25
0
Q10
*+ M
250
H10
0
Q50
+ H10
0
H50
0
Q0.
5+ H
500
Q10
*+ H
500
E0.
5+ H
500
DL-I relative quantum yield (% of control) 10 – 100 s
A
0
20
40
60
80
100
120
140
IR 1
0 Gy
E0.
5Q0.
5Q5
Q10
*Q50
M25
x20
'
M25
x4h
M25
0
E0.
5+ M
250
Q0.
5+ M
250
Q5+
M25
0
Q10
*+ M
250
H10
0
Q50
+ H10
0
H50
0
Q0.
5+ H
500
Q10
*+ H
500
E0.
5+ H
500
DL-II relative quantum yield (% of control) 100 s – 1 ms
B E
0
20
40
60
80
100
120
140
IR 1
0 Gy
E0.
5Q0.
5Q5
Q10
*Q50
M25
x20
'
M25
x4h
M25
0
E0.
5+ M
250
Q0.
5+ M
250
Q5+
M25
0
Q10
*+ M
250
H10
0
Q50
+ H10
0
H50
0
Q0.
5+ H
500
Q10
*+ H
500
E0.
5+ H
500
DL-III relative quantum yield (% of control) 1 – 10 ms
C
methodology as a valuable tool of investigation and diagnosis The results of this study have been published in the ISI
journal Oxidative Medicine and Cellular Longevity, having an impact factor of 2.8 [8].
Selected references
1. D. W. Han, M. H. Lee, H. H. Kim et al., “Epigallocatechin-3-gallate regulates cell growth, cell cycle and phosphorylated nuclear factor-kappa B in human dermal fibroblasts”, Acta Pharmacologica Sinica, vol. 32, pp. 637-646, 2011.
2. H. Nakagawa, K. Hasumi, J. T. Woo, K. Nagai, și M. Wachi, “Generation of hydrogen peroxide primarily contributes to the induction of Fe(II)-dependent apoptosis in Jurkat cells by (-)-epigallocatechin gallate”. Carcinogenesis, vol. 25, pp. 1567-1574, 2004.
3. S. Matzno, Y. Yamaguchi, T. Akiyoshi, T. Nakabayashi, și K. Matsuyama, „An attempt to evaluate the effect of vitamin K3 using as an enhancer of anticancer agents”, Biological și Pharmaceutical Bulletin, vol. 31, pp. 1270-1273, 2008.
4. D. Chen, K. G. Daniel, M. S. Chen, D. J. Kuhn, K. R. Landis-Piwowar, și Q. P. Dou, „Dietary flavonoids as proteasome inhibitors and apoptosis inducers in human leukemia cells”, Biochemical Pharmacology, vol. 69, pp. 1421-1432, 2005.
5. J. H. Jeong, J. Y. An, Y. T. Kwon, J. G. Rhee, și Y. J. Lee, „Effects of low dose quercetin: cancer cell-specific inhibition of cell cycle progression”, Journal of Cellular Biochemistry, vol. 106, pp. 73-82, 2009.
6. G. C. Yen, P. D. Duh, H. L. Tsai, și S. L. Huang, “Pro-oxidative properties of flavonoids in human lymphocytes”, Bioscience, Biotechnology and Biochemistry, vol. 67, pp. 1215-1222, 2003.
7. Baran, C. Ganea, A. Scordino et al., “Effects of menadione, hydrogen peroxide and quercetin on apoptosis and delayed luminescence of human leukemia Jurkat T-cells”, Cell Biochemistry și Biophysics, vol. 58, pp. 169-179, 2010.
8. Baran, Irina; Ganea, Constanta; et al., Detailed Analysis of Apoptosis and Delayed Luminescence of Human Leukemia Jurkat T Cells after Proton Irradiation and Treatments cu Oxidant Agents and Flavonoids, OXIDATIVE MEDICINE and CELLULAR LONGEVITY Article Number: 498914 DOI: 10.1155/2012/498914 Published: 2012
9. S. Tudisco, A. Scordino, G. Privitera, I. Baran, și F. Musumeci, “ARETUSA – advanced research equipment for fast ultraweak luminescence analysis: new developments”, Nuclear Instruments și Methods in Physics Research Section A, vol. 518, pp. 463-464, 2004.
10. I. Baran, C. Ganea, A. Scordino et al., “Apoptosis, cell cycle și delayed luminescence of human leukemia Jurkat T-cells under proton-irradiation and oxidative stress conditions”, in Activity Report Istituto Nazionale Di Fisica Nucleare Laboratori Nazionali Del Sud, Arti Grafiche Le Ciminiere Catania, Italia, pp. 246-249, 2010.
11. L. C. Wilms, J. C. Kleinjans, E. J. Moonen, și J. J. Briedé, "Discriminative protection against hydroxyl and superoxide anion radicals by quercetin in human leucocytes in vitro", Toxicology In Vitro, vol. 22, pp. 301-307, 2008.
12. M. L. Verkhovskaya, N. Belevich, L. Euro, M. Wikström, și M. I. Verkhovsky, “Real-time electron transfer in respiratory complex I”, Proceedings of the National Academy of Sciences of the United States of America, vol. 105, pp. 3763-3767, 2008.
13. M. A. Ortner, B. Ebert, E. Hein et al., “Time gated fluorescence spectroscopy in Barrett's oesophagus”, Gut, vol. 52, pp. 28-33, 2003. 14. F. Musumeci, L. A. Applegate, G. Privitera, A. Scordino, S. Tudisco, and H. J. Niggli, “Spectral analysis of laser-induced ultraweak delayed
luminescence in cultured normal and tumor human cells: temperature dependence”, Journal of Photochemistry și Photobiology. B, Biology, vol. 79, pp. 93-99, 2005.
15. H. W. Kim, S. B. Sim, C. K. Kim, J. Kim, C. Choi, H. You, și K. S. Soh, "Spontaneous photon emission and delayed luminescence of two types of human lung cancer tissues: adenocarcinoma and squamous cell carcinoma”, Cancer Letters, vol. 229, pp. 283-289, 2005.
16. W. Kemmner, K. Wan, S. Rüttinger et al., “Silencing of human ferrochelatase causes abundant protoporphyrin-IX accumulation in colon cancer”, FASEB Journal, vol. 22, pp. 500-509, 2008.
Evaluation of the quercetine effect on cellular viability and mitochondrial metabolism of
the Jurkat lymphoblasts (faza a III-a)
INTRODUCTION In this study we have evaluated the in vitro effects of short treatments of Jurkat cells with
quercetin, menadione (MD) or combination of the two. In view of the known ability of quercetin to manifest a dual,
anti- or pro-oxidant character, depending on dosage and treatment conditions [1], as well as to activate or inhibit
mPTP (mitochondrial permeability transition pore)[2], we intended to verify whether the antiproliferative effect of
quercetin, as well as its capacity to enhance MD-induced cell death in Jurkat cells are mediated by an increased
production of reactive oxygen species (ROS) and/or by mitochondrial dysfunction caused by disruption of Δm. We
have investigated the clonogenic survival, mitochondrial superoxide levels and cellular H2O2 content, as well as the
mitochondrial polarization state. In some experiments we also referred to the effects of rotenone (ROT), a widely
used inhibitor of respiratory Complex I, the first component in the mitochondrial electron transfer chain. Both
menadione and rotenone can produce large quantities of superoxide anion (O2) [3-5], which can be then
dismutated to hydrogen peroxide (H2O2) by cytosolic or mitochondrial superoxide dismutases. The results obtained
here indicate that short (1 h) treatments with QC exert a significant dose-dependent antiproliferative effect in Jurkat
cells that is nevertheless accompanied by a considerable reduction in ROS levels, and support the idea that calcium
release is an early and critical step in QC-induced cell-death, which is followed by a transient loss of Δm. However,
the subsequent large variations in Δm (switching from complete depolarization to considerable hyperpolarization)
that were observed after 30 min. from drug removal were insensitive to inhibition of QC-induced Ca2+ release.
These observations suggest that, under our conditions, the crucial events that triggered the commitment to
(apoptotic) cell death occurred during, not after the one-hour exposure to quercetin. In addition, the data indicate
that treatments based on a combination of quercetin and menadione are more effective in decreasing clonogenic
survival of Jurkat cells than a single treatment regimen, hence encouraging further studies toward the
characterization of quercetin as a potential chemotherapeutic agent that could be used in the treatment of
leukemia.
MATERIALS AND METHODS
Cell cultures. Human leukemia Jurkat T cell lymphoblasts (clone E6.1 from ECACC) were cultured in RPMI 1640 medium (Invitrogen 72400-021) containing Glutamax-I and 25 mM HEPES, and supplemented with 10% fetal bovine serum, 100 units/ml
penicillin and 100 g/ml streptomycin, at 37ºC in a humidified incubator with a 5% CO2 atmosphere. Cell density, viability and morphology were examined under a phase contrast microscope. Viability was assessed by the trypan-blue exclusion test. Assessment of the cellular concentration and of the average cell volume was performed using a Countess Automated Cell Counter (Invitrogen). For preparing the stock solutions, menadione sodium bisulphite was dissolved in phosphate buffer saline (PBS), whereas dihydrated quercetin and rotenone were dissolved in dimethyl sulfoxide (DMSO). The fluorescent indicators, JC-1, MitoSOX Red and CM-H2DCFDA (all from Invitrogen), were dissolved in DMSO as recommended by the manufacturer. All stock solutions were kept at -20ºC. Unless specified otherwise, chemicals were from Sigma-Aldrich. Control cells were always treated with the corresponding vehicle. In experiments involving prolonged recordings a standard saline solution (SS, containing 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 20 mM HEPES, 10 mM glucose, pH 7.2/NaOH) was used. Clonogenic survival assay. After the treatment, cells were washed thoroughly with warm PBS twice and plated in 96 well plates at a plating density
of 3 or 6 cells/well in 100 l of complete medium per well. After 3-4 weeks of incubation, the plates were inspected by microscopy and wells containing colonies with >50 cells were counted. The plating efficiency was calculated as ln[96/(no. of
negative wells)]/(plating density) 100. Clonogenic survival was calculated as the ratio between the plating efficiency of treated and control cells, respectively. Fluorescence spectroscopy. Cell suspensions (1.5 ml) prepared at a density of ≈10
6 cells/ml were
measured in a quartz cuvette under continuous stirring at 37ºC, in a Horiba Jobin Yvon spectrofluorimeter. For determination of
the mitochondrial polarization state, cells were incubated with JC-1 (1 g/ml) for 20 min. in complete medium at 37C, then washed twice with warm PBS, resuspended in PBS and transferred immediately to the spectrofluorimeter. After 5 min. allowed for thermal equilibration, emission spectra were collected in triplicate, with excitation at 490 nm. Fluorescence intensity was averaged and corrected for background and autofluorescence. The degree of mitochondrial polarization was evaluated as the ratio F594/F534 between the fluorescence emitted by JC-1 at 594 nm and that emitted at 534 nm, which was then normalized to
the ratio F594/F534 obtained with control cells. The latter was obtained as 12.3 1.5% (n = 21). For evaluation of changes in the
mitochondrial level of superoxide, not-treated cells were washed twice with warm SS, incubated for 15 min. at 37C with the
specific indicator MitoSOX Red (5 M) in SS, washed once with warm SS, resuspended in SS and immediately transferred to the spectrofluorimeter. Fluorescence was recorded in the kinetic mode, with excitation at 380 nm and emission at 580 nm to select the specific superoxide product of MitoSOX Red oxidation [24], then corrected for background and autofluorescence, and normalized to cellular mass (i.e., the cell concentration multiplied by the average cell volume). The excitation pulses were repeated every 22 s. At indicated time points the drug (QC or ROT) was added directly to the cuvette, without interrupting the recording. For assessing the variations in the cellular content of H2O2, cells were washed with warm PBS, collected by centrifugation and the supernatant was carefully discarded with the use of a Pasteur pipette. Cells were then resuspended in
warm PBS containing 0.5 M CM-H2DCFDA, incubated for 10 min. at 37C, washed as before, then resuspended in warm SS, incubated for an additional 10 min. and then transferred to the spectrofluorimeter. After 5 min. allowed for thermal equilibration, emission spectra were collected in triplicate, with excitation at 490 nm. Fluorescence intensity was averaged, corrected for background and autofluorescence, and then normalized to the cellular mass. The cellular content of H2O2 was quantified by the fluorescence emitted by CM-H2DCFDA at 522 nm. In kinetic recordings, the excitation pulses were repeated every 20 s. At indicated time points the drugs (QC or MD) were added directly to the cuvette, without interrupting the recording.
Statistics. All data are presented as median s.e.m. of at least three different measurements. Other details are found in the publication resulted from this study.
RESULTS AND DISCUSSION
Effects of QC, MD and their combination on clonogenic survival
Both quercetin and menadione decreased clonogenic survival of Jurkat cells in a dose-dependent manner (Fig. 1A).
The characteristic dose (D50%) required for decreasing clonogenicity to 50% by quercetin applied for 1 h or
menadione applied for 20 min. was 107.4 M or 84.2 M, respectively. Since in former works [6, 7], we have shown
that quercetin induces a strong and sustained Ca2+ release signal in Jurkat cells, triggered by the activation of the
ryanodinic receptor of type 3 (RyR3) [7], we have investigated here the involvement of calcium release in QC-
induced apoptosis in this cell system. To this end, we used dantrolene, a RyR3 inhibitor, which was applied for 20?
min. before addition of quercetin for 1 h. The results shown in Fig. 1A clearly indicate that dantrolene, which reduces
consistently the cytosolic Ca2+ increase triggered by quercetin [7], has a protective effect against apoptosis induction.
Thus, the characteristic dose required for decreasing clonogenicity to 50% by quercetin in dantrolene-pretreated
Jurkat cells was 282.9 M, which is substantially higher than the dose indicated above in the absence of dantrolene
(107.4 M).
Fig. 1. Quercetin and menadione decrease clonogenic survival in Jurkat cells in a dose-dependent manner. (A) Quercetin was
applied for 1 h in the absence (-DAN) or presence (+DAN) of 20 M dantrolene, which was added 15 min. before quercetin. The
data were fitted to an exponential function (curves) and yielded the characteristic doses D50% of 107.4 M and 282.9 M, respectively. (B) Menadione was applied for 20 min. in the absence or presence of quercetin at indicated doses, which was
added 1 h before menadione. The data were fitted to the equation y = y0 KdH/(x
H + Kd
H) with y0 = 100 for 0 and 5 M QC, and y0 =
67.4 for 50 M QC. Kd and H values are given in Table 1. (C)-(D) Isobologram diagrams constructed for QC-MD combinations at various effect intensities corresponding to indicated values of clonogenic survival (S). All data points were derived using data from the fitting curves presented in panel B. Combination indexes are provided in Table 2.
Menadione applied for 20 min. also decreased, in a dose-dependent manner, the clonogenic survival of
Jurkat cells with an apparent Kd = 66.8 M and a Hill coefficient, H = 1.37 (Fig. 1B), suggesting a cooperative
interaction probably of two MD binding sites to a specific macromolecule. It is also interesting that QC in
combination with menadione has manifested a dual effect. Thus, at a low doses (5 M) QC exerted a protective
effect against the cellular death induced by MD with moderate effects (clonogenic survival ~10-50%), while at large
doses (50 M) QC enhanced significantly the cell death induced by MD with high levels of effects (clonogenic survival
20%) (Fig. 1B).
Table 1. Binding parameters of menadione derived from clonogenic survival data.
Parameter [QC], M
0 5 50
Kd (M) 66.8 151.8 72.7
H 1.37 2.17 2.45
0
20
40
60
80
100
120
0 50 100 150 200
[QC], M
Clo
no
gen
ic s
urv
ival (%
)
-DAN
+DAN
A B
0
100
200
300
400
500
0 100 200 300 400
S = 50%
S = 20%
S = 10%
QC dose, M
MD
do
se, M
C
[MD], M
Clo
no
ge
nic
su
rviv
al (%
)
0
20
40
60
80
100
120
0 50 100 150 200 250 300
0 μM QC
50 μM QC
5 μM QC
0
500
1000
1500
2000
0 200 400 600 800
S = 10%
S = 1%
QC dose, M
MD
do
se, M
D
0
40
80
120
160
0 2 4 6
-DAN
+DAN
t (h)P
ola
riza
tio
n (
% o
f c
on
tro
l)
100
120
140
160
180
0 50 100 150 200 250 300
[QC], M
Po
lari
za
tio
n (
% o
f c
on
tro
l)
0
25
50
75
100
125
150
175
0 2 4 6
QC
QC+MD
MD
Po
lari
zati
on
(%
of
co
ntr
ol)
t (h)
0
20
40
60
80
100
0 2 4 6
30 min.
2 h
Po
lari
zati
on
(%
of
co
ntr
ol)
t (h)
Po
lari
zati
on
(%
of
co
ntr
ol)
Treatment time (h)
0
20
40
60
80
100
120
0 1 2 3
MD
ROT
Fig. 2. Effects of QC, MD and ROT on Δm in Jurkat cells. (A) Kinetic variations of Δm, expressed as the polarization degree
relative to control, were monitored after treatments with 150 M QC for 1 h as a function of the time elapsed from QC removal.
The results obtained in the absence (-DAN) or presence (+DAN) of 20 M dantrolene added 15 min. before quercetin were closely similar. (B) The mitochondrial polarization state (relative to control) assessed 3 h after a one-hour exposure to QC was determined in function of the dose of quercetin. The data were fitted to the function P (%) = 100 + (Pmax - 100) [QC]
h/([QC]
h +
Kdh), where P represents the relative polarization degree, Kd dissociation constant of QC, and h Hill coefficient. The best fit
(curve) was obtained with Pmax = 157.0, Kd = 7.05 M and h = 0.85. (C) Kinetic variations of Δm induced by treatments with 150
M QC for 1 h (QC), 250 M MD for 20 min. (MD) or combination of the two agents added consecutively (QC+MD), as a function of the time elapsed after drug removal. The dotted line represents the 100% level corresponding to control cells. In (A) and (C), the first data point was obtained by loading JC-1 during the last 20 min. of the drug treatment. (D) Recovery from mitochondrial
depolarization following treatments with 250 M ROT for 30 min. and 2 h, respectively. (E) Mitochondrial depolarization
assessed 1 h after drug removal as a function of the treatment time. Cells were treated with 250 M MD or 250 M ROT, respectively, washed and loaded immediately with JC-1 as described under Materials and Methods.
Table 2. Combination indexes in MD combinations with 5 M or 50 M QC. Parentheses indicate synergism (+) or
antagonism (-). The biological effect (clonogenic survival, S) for which the combination index was computed is
indicated.
Effect 5 M QC 50 M QC
S = 50% 2.32 (-) 1.17 (-)
S = 20% 1.58 (-) 0.77 (+)
S = 10% 1.27 (-) 0.59 (+)
S = 1% 0.66 (+) 0.32 (+)
Irrespective of the dose, quercetin appeared to increase the Hill coefficient of menadione (Table 1), suggesting an
allosteric interaction between QC and MD. Thus, in the presence of quercetin, it is possible that three molecules of
menadione to interact cooperatively with a specific macromolecule in order to trigger the signal of cell death in
Jurkat lymphoblasts, while, in the absence of the flavonoid, two molecules of menadione are required to bind
cooperatively the respective molecuar target. Moreover, isobologram and combination index analysis [8] Moreover,
isobologram and combination index analysis [8] indicates that at low and medium effect levels (i.e., clonogenic
survival 10% ≤ S ≤ 50%) quercetin at low or high doses can manifest a differential antagonistic or
synergistic effect, respectively, in combination with menadione (Fig. 1C, Table 2). combination with menadione
(Fig. 1C, Table 2). Nevertheless, at high effect levels (i.e., S ≤ 1%), which are relevant to therapy, there is
significant synergism at both low and high doses of quercetin. Hence, it appears that with a proper dosage,
a combined treatment quercetin-menadione can be more effective in reducing clonogenicity than a treatment
with menadione alone.
Effects of QC, MD and ROT on Δm
Quercetin (150 M) applied for 1 h produced a complex kinetic pattern of mitochondrial depolarization in
the first hour after drug removal, followed by a consistent hyperpolarization phase starting at 2 hours after QC
removal, which persisted for at least 4 hours (Fig. 2A). Pretreatment with dantrolene had no significant effect on the
degree of mitochondrial polarization (Fig. 2A), suggesting that mitochondrial Ca2+ overload is not a primary cause of
the observed variations in the mitochondrial membrane potential. We assessed the polarization degree at the
moment of maximal hyperpolarization (2.5 h after drug removal) as a function of the concentration of quercetin. The
results (Fig. 2B) indicate that QC binds to a molecular target, presumably found inside the mitochondria, to induce
hyperpolarization of the inner membrane, with a dissociation constant (Kd) of 7.05 M and Hill coefficient 0.85.
The results presented so far also suggest that most likely the hyperpolarized state of the mitochondria is not
involved in the (apoptotic) cell death induced by quercetin in Jurkat cells, since the characteristic dose D50% is much
higher than Kd, and, in addition, the effects of dantrolene on clonogenic survival or mitochondrial hyperpolarization
differ significantly. We next compared the effects of QC, MD and combination of the two on the variations of Δm.
Quercetin (50 M) applied for 1 h produced a similar pattern as described above, whereas menadione applied for 20
min. had a biphasic depolarizing effect (Fig. 2C). Thus, after an initial strongly depolarized state observed 30 min.
after MD removal, there was a partial recovery within the next 30 min., which was followed by a gradual
depolarization recorded over the next five hours (Fig. 2C). The combination of the two agents produced an
intermediate effect, with extensive depolarization in the first hour after the wash, subsequent recovery to the
resting state for the next 3 h, and finally considerable depolarization observed at 6 h after removal of the drugs (Fig.
2C). To evaluate the significance of our measurements using the Δm indicator JC-1, we also used a familiar
mitochondrial respiratory chain inhibitor, rotenone. Exposure of Jurkat cells to 250 M rotenone for 20? min.
produced clonogenic survival of 27.8 7.5% (n = 5) of the cells. We also assessed by flow-cytometry that rotenone
(250 M) induced an early apoptotic cell fraction of 23 8% (n = 4) and a late apoptotic/necrotic cell fraction of 56
7% (n = 4) at 48 h after the treatment (not shown), indicating a slow progression of the ROT-induced apoptotic
process in these cells. ROT also produced extensive mitochondrial depolarization within 1 h from the treatment
which then recovered progressively during the next 5 h (Fig. 2D). The degree and kinetics of the recovery were
similar between treatments of either 20 min. or 2 h (Fig. 2D). Moreover, the degree of mitochondrial depolarization
evaluated 1 h after drug removal was virtually identical for treatment durations of 20 min., 1 h, 2 h or 3 h (Fig. 2E).
Consistent with this, apoptosis was not significantly different between treatments of either 30 min. or 1 h. Together,
these findings suggest that ROT effects on apoptosis induction and mitochondrial depolarization are completed
within 20 min. of exposure, which is in very good agreement with previous reports that rotenone disrupts Δm and
induces mPTP opening within 20 min. [9]. Our results also indicate that under our conditions the ROT-induced loss
of Δm is reversible, and that Δm recovery observed after ROT removal may reflect the gradual weakening of
rotenone binding to Complex I, which was found to be quite stable, hence persisting a long time upon washing [10].
Both quercetin and menadione were found to decrease the level of NAD(P)H in Jurkat cells [6, 7, 11] which could
suggest that under our conditions both agents stimulated the activity of respiratory Complex I (submitted
manuscript [12]). In the absence of other additional mechanisms this would lead to the mitochondria
hyperpolarization. However, by monitoring the variations in the mitochondrial transmembrane potential it appears
most likely that the early loss of Δm is a consequence of MPT pore opening. In QC-treated cells, this event seems to
be reversed within 2 h from drug removal, suggesting that mitochondrial permeability transition pores return to
their resting closed state, whereas the accelerated respiratory rate leads to the installation of the observed
hyperpolarized state via an increased rate of proton pumping by the electron transfer chain. It is therefore
conceivable that the two specific doses obtained here, namely 107 M and 7 M, characterize the binding of
quercetin to mPTP and mitochondrial Complex I, respectively. In addition, QC has been found to accumulate in high
concentration in mitochondria, and a slow redistribution to the cytosol has been observed in Jurkat cells after
removal of the drug from the extracellular medium [13]. Consequently, it is possible that quercetin is retained in a
bound form in sufficient quantities inside the mitochondria to sustain the long-term hyperpolarized state observed
here. The different effect of menadione on Δm, corroborated by the reduction effect of MD on the level of
NAD(P)H, suggests a long-term, gradual opening of mPTP following MD removal. Treatments with menadione for
increased duration led to progressive loss of Δm, which was very well fitted by an exponential function (Fig. 2E). The
results show that exposure to 250 M MD for 3 h abolished Δm almost completely, and that a mitochondrion
depolarization event occurs after an average time of 45.7 min. in the presence of 250 M MD.
QC-induced hyperpolarization is inhibited by respiration inhibitors
To strengthen the idea that observed QC-induced mitochondrial hyperpolarization originates primarily from
Complex I stimulation, we employed the Complex I specific inhibitor, rotenone, which was added to QC-
treated Jurkat cells at the moment of maximal mitochondrial hyperpolarization reached after removal of
quercetin.
Table 3. Rotenone binding parameters derived from spectrofluorimetric measurements.
Parameter Measured quantity
m* [NADH]m**
w1 0.42 0.33
Kd1 (M) 3.68 3.58
H1 1.22 1.35
w2 0.58 0.67
Kd2 (M) 215.2 164.1
H2 2.81 3.57
* Data presented in Fig. 3A ** Data presented in Fig. 3B
Table 4. Antimycin A binding parameters derived from spectrofluorimetric measurements.
Parameter Measured quantity
m* [NADH]m**
Kd (M) 16.2 79.3
H 1.23 1.66
* Data presented in Fig. 3D,
** Data presented in Fig. 3E
Following this treatment, the polarization degree decreased dose-dependently in two distinct phases
(Fig. 3A), suggesting that ROT binds to two classes of binding sites, with parameters indicated in Table 3.
The mitochondrial level of NADH, which is the specific substrate of Complex I, increased with the ROT
concentration in two phases as well (Fig. 3B, Table 3), so that a very strong anticorrelation was obtained
between the mitochondrial polarization degree and [NADH]m under these conditions (Fig. 3C), suggesting a
direct link between the QC-induced hyperpolarized state of the mitochondria and the activity of Complex I.
Fig. 3. QC-induced mitochondrial hyperpolarization is inhibited by ROT (A-C) and AM (D-F). Polarization degree (relative to control) as a function of the dose of ROT (A) or AM (D). Data were fitted to the equation (A) y = y0 [w1 Kd1
H1/(x
H1 + Kd1
H1) + w2
Kd2H2
/(xH2
+ Kd2H2
)] with y0 = 147.0, or (D) y = y0 KdH/(x
H + Kd
H) with y0 = 155.2; other parameter values are given in Table 3 and
Table 4, respectively. The mitochondrial level of NADH (relative to control) as a function of the dose of ROT (B) or AM (E). Data were fitted to the equation (B) y = y0 + (ym - y0) [w1 x
H1/(x
H1 + Kd1
H1) + w2 x
H2/(x
H2 + Kd2
H2)] with y0 = 0.98 and ym = 2.33, or (D) y = y0
+ (ym - y0) xH/(x
H + Kd
H) with y0 = 0.85 and ym = 9.61; other parameter values are given in Table 3 and Table 4, respectively. Strong
anticorrelation between Δm and [NADH]m in (C) ROT-, or (F) AM-treated cells. The Pearson correlation coefficient (r) is indicated. Data points were fitted to a linear (C) or a Hill-type nonlinear (F) equation. In panel F - inset, the data points presented in panel C were superimposed on the graph obtained in panel F.
The Complex III inhibitor, antimycin A, also inhibited dose-dependently the QC-induced mitochondrial
hyperpolarization (Fig. 3D). However, this inhibition appeared to be monophasic (Fig. 3D, Table 4), suggesting
that a single class of AM binding sites is involved in the process. This idea was also supported by the
dependence of [NADH] m on the concentration of AM (Fig. 3E), which led to a strong anticorrelation between
the mitochondrial polarization degree and [NADH]m (Fig. 3F). Moreover, the notable superposition of the two
curves obtained with the two, Complex I and Complex III, inhibitors, which can be clearly noticed within a
certain domain (Fig. 3F, inset), most likely reflects the effect of Complex I inhibition, since inhibition of
Complex III results in inhibition of the entire respiratory chain. As a consequence of the contribution of
different respiratory complexes to the overall effect, the dependence of the mitochondrial polarization
degree on [NADH] m in AM-treated cells deviates from linearity at high levels of NADH (Fig. 3F). This should
also be regarded in relation to the fact that NADH is the specific substrate of Complex I, not Complex III.
Taken together, all these findings strongly suggest that the QC-induced mitochondrial hyperpolarization is due
to stimulation of Complex I activity.
0
40
80
120
160
200
0.0001 0.01 1 100
[ROT], M
Po
lari
zati
on
(%
of
co
ntr
ol)
A
0
40
80
120
160
0 40 80 120 160
[AM], M
Po
lari
za
tio
n (
% o
f c
on
tro
l)
0
2
4
6
8
10
0 40 80 120 160
[AM], MR
ela
tiv
e [
NA
DH
] m
0
1
2
3
0.0001 0.01 1 100
[ROT], M
Re
lati
ve
[N
AD
H] m
B
0
50
100
150
0 0.5 1 1.5 2 2.5 3
Pola
rization (
% o
f contr
ol)
Relative [ NADH] m
r = -0.95
C
D
E
F
0
50
100
150
0 2 4 6 8 10
Po
lari
za
tio
n (
% o
f c
on
tro
l)
Relative [NADH]m
r = -0.83
Po
lari
za
tio
n (
%)
Relative [NADH]m
0
50
100
150
0 2 4 6 8 10
AM
ROT
Effects of QC, MD and ROT on ROS production
To establish whether the antiproliferative effect of quercetin is linked to an increased ROS production, we
employed two fluorescent ROS indicators, MitoSOX Red and CM-H2DCFDA, to monitor the variations in the
mitochondrial level of superoxide and the cellular content of H2O2, respectively. These investigations revealed that
quercetin applied for 1 h manifests a strong antioxidant character in Jurkat cells. Thus, the fluorescence of MitoSOX
Red decreased immediately after addition of 50 M QC to a relatively steady level which was one third of the resting
fluorescence (Fig. 4A). This reduction could be observed for a long time (≈80 min.), and was followed by a well
defined recovery phase (Fig. 4A), probably reflecting the activation of some endogenous antioxidant mechanisms. To
test the reliability of our experimental procedure, we also investigated the effect of rotenone on MitoSOX Red
fluorescence. It is widely recognized that ROT produces large quantities of superoxide inside the mitochondria via
inhibition of respiratory Complex I [5]. Indeed, our fluorimetric measurements showed that a substantial, 6-fold
increase in the fluorescence of MitoSOX Red was reached within 45-60 min. from addition of 50 M ROT, which was
then followed by a steady and abrupt decay phase (Fig. 4B), suggesting, as before, that some endogenous
antioxidant mechanisms are activated. Taken together, all these results argue against the possibility that the QC-
induced decrease in MitoSOX Red fluorescence is due to the dispersion of the indicator from the mitochondria into
the cytosol due to loss of Δm, and support the notion that the observed decrease is attributable to the actual
reduction of the mitochondrial superoxide level.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
-1 0 1 2 3 4 5 6 7
t (h)
QC
no QC
Re
lati
ve
lev
el o
f H
2O
2
*
-100000
0
100000
200000
300000
400000
500000
600000
0 20 40 60 80 100
t (min.)
F
luo
res
ce
nc
e(a
.u.)
MD
QC
MD
-90000
-70000
-50000
-30000
-10000
10000
30000
0 20 40 60 80 100
t (min.)
F
luo
res
ce
nc
e(a
.u.)
MD
QC
MD
QC
Fig. 4. Effects of QC, MD and ROT on cellular ROS production. QC decreases (A), whereas ROT increases (B) the mitochondrial
level of superoxide. A representative fluorimetric recording of MitoSOX Red fluorescence before and after addition of 50 M QC
(A) or 50 M ROT (B) is illustrated. (C) The cellular content of H2O2, quantified by the fluorescence of CM-H2DCFDA relative to
control, decreases after treatment with 50 M QC for 1 h. The measurement at the end of the treatment (marked by a star) was obtained from kinetic recordings like that shown in panel D. (D) Absolute variation in CM-H2DCFDA fluorescence relative to
control cells, in exposures to 250 M MD alone (blue trace) or in sequential addition of 50 M QC and 250 M MD (red trace) at
indicated time points. (E) Variation in CM-H2DCFDA fluorescence relative to control cells after sequential addition of 50 M QC
and 250 M MD at indicated time points.
In favor of this, some different but relevant insights could also be gained by determining the variations in the cellular
level of H2O2 elicited by quercetin. After one hour of exposure to 50 M QC, the fluorescence of the H2O2 indicator,
CM-H2DCFDA, was decreased by 29%, and even after removal of the drug, it remained below the control level for at
least 6 h (Fig. 4C). Moreover, quercetin demonstrated an impressive capacity of preventing the massive cellular
production of H2O2 induced by a high dose of menadione. Thus, 250 M MD elicited a 7-fold increase in the
fluorescence of CM-H2DCFDA, which was reached within about 40 min. from MD addition and was then followed by
a modest and very slow decrease during the next 60 min. (Fig. 4D). When 50 M QC was added to the cuvette, the
fluorescence of CM-H2DCFDA decreased constantly with about 30% (relative to control) in 1 h. Moreover,
subsequent addition of 250 M MD did not seem to affect the decrease rate imposed by quercetin, so that CM-
H2DCFDA fluorescence continued to decline steadily for at least 25 min. after addition of menadione (Fig. 4D). When
menadione (250 M) was added after a shorter time (30 min.) of exposure to quercetin, the level of H2O2 continued
to diminish for about 20 min., however at a somewhat lower rate, then stabilized for the subsequent 20 min., and
finally seemed to increase again (Fig. 4E). To facilitate comparison, the data extracted from the lower (red) trace
shown in Fig. 4D are also illustrated in Fig. 4E. Our current investigations prove that exposure of Jurkat cells for 1 h to
50 M QC diminishes the mitochondrial level of superoxide and the cellular content of H2O2, and prevents the
extensive production of H2O2 induced by menadione. In spite of the strong antioxidant character manifested by the
flavonoid, this QC-treatment enhanced significantly the antiproliferative effect of menadione. The data also indicate
that calcium release is a critical step in QC-induced cell death, most likely via prolonged Ca2+ overload of the
mitochondria. In previous works [11, 14] we found that a short-term pretreatment with 10 M QC for 1 h enhanced
significantly apoptosis induced by 250 M MD. In line with those reports, our current results indicate that
pretreatment with 50 M QC for 1 h enhances substantially cell death induced by 250 M MD. However, we show
here that, contrary to our expectations, this enhancement is not due to increased ROS production, but is most likely
the result of a more severe and prolonged disruption of Δm as observed in the initial phase following drug removal
(Fig. 2C).
Investigations in our laboratory showed that QC and MD [11, 14] as well as ROT are potent apoptogens in
human leukemia Jurkat cells. The current finding that cells treated separately with each of the three agents exhibited
at similar time points a trend of recovery from the initially induced mitochondrial dysfunctionality is consistent with
the idea that, after proceeding through the very early phases of apoptosis, ATP is nevertheless required to incline
the balance in favor of subsequent progression through apoptosis, not necrosis. Thus, ROT-induction of apoptosis
was reported to occur irreversibly within 20 min. from exposure [9], which is in agreement with our current results.
Current data also suggest that two molecules of antimycin A bind cooperatively to Complex III (Table
4), consistent with the notion that Complex III is a homodimer that can bind one AM molecule per dimer
[15]. Moreover, cooperative AM binding to Complex III has been reported before [15–17]. Taken together, all
these data and observations further strengthen our conclusions and support the idea that QC-induced
mitochondrial hyperpolarization observed in Jurkat cells is mediated primarily by enhancement of the
respiratory activity.
The current findings that quercetin can act either in synergy or in antagonism with menadione,
depending on the dose, time of treatment and effect level, also encourage further investigations to find
optimal dosage windows for QC-MD combinations so that a protective or a cytotoxic effect could be
selectively targeted to normal or cancerous cells, respectively. In addition, in vitro research suggests that
malignant cells may be more susceptible than normal cells to the cytotoxicity of some flavonoids. Thus,
only a few agents, including quercetin, are currently known to possess such potential for selective
elimination of cancer cells while exerting cytoprotective effects on normal cells [18-20]. Therefore, the
aforementioned beneficial properties of quercetin could be used to improve standard chemotherapies for
leukemia, by maximizing the distructive effects of the main chemotherapeutical agents targeting the cancerous
cells, while minimizing the secondary effects of the treatment. Indeed, the participation of quercetin in
combination could allow a significant reduction of the chemotherapeutic agent dosage together with an enhanced
protection of the healthy tissues and with a potentially increased rate of cancer cells apoptosis, limiting thus the
risk of immediate inflammatory effects and also reducing the risk of long term effects such as secondary cancer.
The results of this study have been presented in a paper submitted to a ISI journal [21] and at a international
conference [22].
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2. Biasutto, L., Dong, L.F., Zoratti, M., Neuzil, J., 2010. Mitochondrially targeted anti-cancer agents. Mitochondrion 10, 670-681. 3. Floreani, M., Carpenedo, F., 1992. One- și two-electron reduction of menadione in guinea-pig și rat cardiac tissue 4. Li, N., Ragheb, K., Lawler, G., Sturgis, J., Rajwa, B., Melendez, J.A., Robinson, J.P., 2003. Mitochondrial complex I inhibitor rotenone
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5. Xu, X., Arriaga, E.A., 2009. Qualitative determination of superoxide release at both sides of the mitochondrial inner membrane by capillary electrophoretic analysis of the oxidation products of triphenylphosphonium hydroethidine. Free Rad. Biol. Med. 46, 905-913.
6. Baran, I., Ganea, C., Ursu, I., Baran, V., Calinescu, O., Iftime, A., Ungureanu, R., Tofolean, I.T., 2011. Fluorescence properties of quercetin in human leukemia Jurkat T-cells, Rom. J. Phys. 56, 388-398.
7. Baran, I., Katona, E., Ganea, C., 2013. Quercetin as a fluorescent probe for the ryanodine receptor activity in Celulele Jurkat. Pflügers Arch. 465, 1101-1119.
8. Chou, T.C., 2010. Drug combination studies și their synergy quantification using the Chou-Talalay method. Cancer Res. 70, 440-446. 9. Isenberg, J.S., Klaunig, J.E., 2000. Role of the mitochondrial membrane permeability transition (MPT) in rotenone-induced apoptosis in
liver cells. Toxicol. Sci. 53, 340-351. 10. Gutman, M., Singer, T.P., Casida, J.E., 1969. Role of multiple binding sites in the inhibition of NADH oxidase by piericidin și rotenone.
Biochem. Biophys. Res. Commun. 37, 615-622. 11. Baran, I., Ganea, C., Privitera, S., Scordino, A., Barresi, V., Musumeci, F., Mocanu, M.M., Condorelli, D.F., Ursu, I., Grasso, R., Gulino, M.,
Garaiman, A., Musso, N., Cirrone, G.A.P., Cuttone, G., 2012. Detailed analysis of apoptosis și delayed luminescence of human leukemia Jurkat T cells după proton-irradiation și treatments cu oxidant agents și flavonoids. Oxid. Med. Cell. Longev. 2012, Article ID 498914.
12. Irina Baran, Diana Ionescu, et al., Mitochondrial respiratory Complex I probed by delayed luminescence spectroscopy, Journal of Biomedical Optics, 2013, (trimis, sub revizie)
13. Fiorani, M., Guidarelli, A., Blasa, M., Azzolini, C., Candiracci, M., Piatti, E., Cantoni, O., 2010. Mitochondria accumulate large amounts of quercetin: prevention of mitochondrial damage și release upon oxidation of the extramitochondrial fraction of the flavonoid. J. Nutr. Biochem. 21, 397-404.
14. Baran, I., Ganea, C., Scordino, A., Musumeci, F., Barresi, V., Tudisco, S., Privitera, S., Grasso, R., Condorelli, D.F., Ursu, I., Baran, V., Katona, E., Mocanu, M.M., Gulino, M., Ungureanu, R., Surcel, M., Ursaciuc, C., 2010. Effects of menadione, hydrogen peroxide și quercetin on apoptosis și delayed luminescence of human leukemia Jurkat T-cells. Cell Biochem. Biophys. 58, 169-179.
15. Covian, R., Gutierrez-Cirlos, E.B., Trumpower, B.L., 2004. Anti-cooperative oxidation of ubiquinol by the yeast cytochrome bc1 complex. J. Biol. Chem. 279, 15040-15049.
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17. Lenaz, G., Genova, M.L., 2007. Kinetics of integrated electron transfer in the mitochondrial respiratory chain: random collisions vs. solid state electron channeling. Am. J. Physiol. Cell Physiol. 292, C1221-1239.
18. Chen, D., Daniel, K.G., Chen, M.S., Kuhn, D.J., Landis-Piwowar, K.R., Dou, Q.P., 2005. Dietary flavonoids as proteasome inhibitors și apoptosis inducers in human leukemia cells. Biochem. Pharmacol. 69, 1421-1432.
19. Jeong, J.H., An, J.Y., Kwon, Y.T., Rhee, J.G., Lee, Y.J., 2009. Effects of low dose quercetin: cancer cell-specific inhibition of cell cycle progression. J. Cell. Biochem. 106, 73-82.
20. Yen, G.C., Duh, P.D., Tsai, H.L., Huang, S.L., 2003. Pro-oxidative properties of flavonoids in human lymphocytes. Biosci. Biotechnol. Biochem. 67, 1215-1222
21. Irina Baran, Diana Ionescu, et al., Novel insights into the antiproliferative effect of quercetin, menadione and rotenone in human leukemia Jurkat T cells, Leukemia Research, 2013 (trimis, sub revizie)
22. Ionescu D., Ganea C, et al., Quercetin exerts its antitumoral effect while manifesting a strong antioxidant character, 9th EBSA European Biophysics Congress, Eur Biophys J (2013) 42 (Suppl 1):S1–S236
Investigation of the correlation between the delayed luminescence (DL) and mitochondrial respiratory Complex I (3d phase of the project)
In this study we have performed a detailed analysis of the properties of ultra-weak photon-induced delayed
photon emission (Delayed Luminescence, DL) of Jurkat cells by employing treatments with rotenone (ROT), as well as
with menadione (MD) and quercetin (QC). All the three drugs share net structural similarities with ubiquinone and
therefore can bind Complex I, inside or close to the Q-binding pocket. Rotenone, a familiar specific inhibitor of
mitochondrial respiration [1,2], is known to bind to two distinct, non-interacting sites on Complex I [3-5]. These ROT
sites, designated ROT site 1 and ROT site 2, are most likely situated in the 49-kDa and ND1 subunits of Complex I,
respectively [3]. The two ROT sites, which display different affinities for the inhibitor [6], appear to be involved in
the forward and reverse electron transfer, respectively [5]. It is widely accepted that in the forward mode (site 1
involved) rotenone disrupts the electron flow at the level of protein-bound ubiquinone adjacent to center N2 (QNf
[7]), specifically by destabilizing the ubisemiquinone produced after acceptance of the first electron from center N2
[7]. As a direct consequence of the rotenone blockage, respiration ceases and the intracellular level of NADH
augments due to lack of consumption by the mitochondria. Moreover, the accumulating electrons are deviated from
Complex I to the surrounding molecular oxygen, thereby producing superoxide (O2) at elevated rates, which is then
released into the matrix [1]. The superoxide produced by the mitochondrial electron transport chain is rapidly
converted to hydrogen peroxide (H2O2) by the action of mitochondrial superoxide dismutases. Rotenone and
rotenoids in general have demonstrated anticancer activity which was attributed to the induction of apoptosis [8,9].
The apoptotic effects of rotenone on human leukemia Jurkat T cells have been reported in several studies involving
particular doses and application times [9,10].
Fig. 1. Modular representation of Complex I architecture. The main seven hydrophilic and seven membranous subunits, labeled according to human Complex I subunit nomenclature [5,15], are schematically depicted. For simplicity, subunits ND3, ND6 and ND4L are comprised in a unique module. The relative positions of various subunits and Fe/S centers are only qualitatively pictured. Dashed arrows indicate possible electron transfer reactions (based on refs. [76,77]). The two prosthetic FMN groups and their interaction with NADH/NADPH are illustrated. The two ROT (1 and 2) and two Q (QNf and QNs) sites are specified. The dashed circle encompasses the Q-binding pocket at the interface between the hydrophobic and the hydrophilic Complex I domains. Q may slide along the putative hydrophobic tunnel formed between the 49-kDa and PSST subunits, and interact with center N6a. The Nnt - Complex I interaction may regulate the reduction of FMN-a via the control of the local NADPH/NADP
+ ratio
by Nnt [5] (here, only one Nnt monomer is illustrated; the three specific domains of the monomer, I, II (extramembrane domains) and III (membrane domain), are shown).
The second drug used in our investigations - menadione, a common stimulator of mitochondrial superoxide
production, is a quinone analog also used as a chemotherapeutic agent in the treatment of leukemia The second
drug used in our investigations - menadione, a common stimulator of mitochondrial superoxide production, is a
quinone analog also used as a chemotherapeutic agent in the treatment of leukemia [11,12]. Menadione reduction
at mitochondrial Complex I [26,27], which accounts for ~50% of its metabolism [13,14], may result in the formation
of a semiquinone with consequent superoxide production [1,14]. Menadione was also reported to increase the
activity of Complex I and hence the NADH consumption by mice liver submitochondrial fractions, although it did not
affect the rate of O2 uptake in those preparations [13]. In other studies menadione was found to stimulate cellular
oxygen consumption [15,16]. Consistent with these reports, our current results indicate that menadione decreases
the concentration of mitochondrial NADH in Jurkat cells by stimulating Complex I activity.
To further substantiate the link between DL and Complex I which was originally suggested by the partial outcomes of
our ROT- and MD-based experiments, we also employed the flavonoid quercetin, on account of its ability to behave
as a ubiquinone-like molecule allowing thus its binding to Complex I [17]. In previous works we determined that QC
decreases the cellular content of NADH [18-20], yet the underlying mechanism is unclear, having in view the
conflicting reports regarding the effect of quercetin on the activity of Complex I. It is likely that in our experiments
QC, as well as menadione, stimulated the activity of Complex I in Jurkat cells, hence leading to an increased rate of
NADPHNADPH
Matrix
Intermembrane space
Nnt Complex I
NADPNADP++
NADHNADH NADNAD++
N1a
II
IIII
IIIIII
FMNFMN--bb
FMNFMN--aa
ROTROT
QQNfNf ND
1
ND
3,6
,4L
ND
2
ND
4
ND
5
QQNsNs
22
N6b
N2
N6a
N4
N3
N5
N1b
N7
11
24k
51k
75k
49k
30k
TY
KY
PS
ST
NADHNADH NADNAD++
Hydrophobic
tunnel
NADH consumption. In view of the common effects of these drugs at the level of Complex I, we could investigate in
more detail the connection between delayed luminescence and the mitochondrial metabolism. Our current results
clearly demonstrate that the ultra-weak photon-induced delayed photon emission of Jurkat cells is quantitatively
related to the mitochondrial level of NADH and that of oxidized FMN, supporting the notion that DL is mainly
produced within the mitochondrial electron transfer system at the level of Complex I. The data provide novel insights
into the structural and functional organization of respiratory Complex I, which appears to function mainly as a dimer
in its native environment. Moreover, we present evidence for an active role of secondary NAD(P)H and FMN binding
sites in the functional dimer.
Materials and methods
Cell cultures. Human leukemia Jurkat T cell lymphoblasts were cultured in suspension in RPMI 1640 medium
supplemented with 5% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 g/ml
streptomycin, at 37ºC in a humidified incubator with a 5% CO2 atmosphere. Exponentially growing cells were adjusted to a
density of 0.2 106 cells/ml the day before the experiment. We used hydrogen peroxide 30% solution and stock solutions of
menadione sodium bisulphite dissolved in phosphate buffer saline (PBS), or dihydrated quercetin dissolved in dimethyl sulfoxide
(DMSO). Unless specified otherwise, all chemicals were from Sigma-Aldrich. After each treatment, cells were washed thoroughly
with PBS and resuspended in PBS at room temperature (20C) (for DL samples, ~40 106 cells/ml, or for spectrofluorimetry, ~1
106 cells/ml) or in complete medium for apoptosis assessment (~0.2 10
6 cells/ml). DL and fluorimetric samples were assessed
immediately by DL and fluorescence spectroscopy, respectively. Cell density, viability and morphology were examined with a
CCD camera Logitech QuickCam Pro 4000, connected to an Olympus CK30 phase contrast microscope. For cell density
assessment, 25 l- aliquots of the samples were diluted in PBS, stained with 0.4% trypan blue solution and ~1500-2000 cells
were imaged on a Bürker haemocytometer at the time of the DL assay. Cell count evaluation was performed both during
experiments, directly by visual inspection under the microscope, and later on, by analyzing the micrographs with the use of the
software ImageJ. Clonogenic survival assay. After the treatment, cells were washed thoroughly with warm PBS and plated in 96
well plates at a plating density of 3, 4 or 10 cells/well in 100 l of complete medium per well. After 4 weeks of incubation, the
plates were inspected by microscopy and the wells containing colonies with >50 cells were counted. The plating efficiency was
calculated as ln[96/(no. of negative wells)]/(plating density) 100. Clonogenic survival was calculated as the ratio between the
plating efficiency of treated and control cells, respectively. Flow cytometry. At specified times after the treatment, 106 cells
were collected, washed twice in PBS, and then incubated with 5 μl Annexin V-FITC (Apoptosis detection kit, BD Pharmingen) and
2.5 μl 7-AAD (BD Pharmingen) in 100 μl Annexin V Binding Buffer for 15 minute at room temperature in the dark. 400 μl of
Annexin V Binding Buffer were then added to the samples, as recommended by the manufacturer, which were then analyzed on
a Becton Dickinson FACS Calibur flow-cytometer. The excitation wavelength was 488 nm (air-cooled argon-ion laser, 15 mW) and
emission was collected with 530 nm (FITC) and >670 nm (7-AAD) bandpass filters. Data acquisition and analysis was performed
with the use of the software CellQuest and WinMDI 2.9, respectively. Cells negative for both Annexin V-FITC and 7-AAD were
considered as living cells, those positive for Annexin V-FITC and negative for 7-AAD were considered as early apoptotic cells and
those positive for both dyes were considered as late apoptotic/necrotic cells. Delayed luminescence spectroscopy. We used an
improved version of the ARETUSA set-up [49], a highly sensitive equipment able to detect single photons. Cell samples were
excited by a Nitrogen Laser source (Laser Photonics LN 230C; wavelength 337 nm, pulse-width 5 ns, energy 100 ± 5 µJ/pulse). A
multi-alkali photomultiplier tube (Hamamatsu R-7602-1/Q) was used as a detector for photoemission signals with wavelengths
in the visible range (VIS, 400-800 nm), in single photon counting mode. In some determinations, two broad band (about 80 nm
FWHM) Lot-Oriel interferential filters, disposed in a wheel between the sample and the photomultiplier, were used to select
photons with wavelength of ~460 nm (Blue) and ~645 nm (Red), respectively. The detected signals were acquired by a Multi-
channel Scaler (Ortec MCS PCI) with a minimum dwell-time of 200 ns. The laser power was reduced, in some cases, to prevent
the dimpling of the photomultiplier. DL measurements were done on at least 3 different drops from each cell sample (drop
volume 15-25 l) at room temperature (20 ± 1ºC). PBS luminescence was subtracted from all the recordings. Photoemission was
recorded between 11 s and 100 ms after laser-excitation. DL intensity (I) was expressed as the number of photons recorded
within a certain time interval divided to that time interval, to the number of living cells in the drop and to the energy of the
laser. The intensity of Yellow/Green DL was estimated by subtracting the additive contribution of Blue and Red DL intensities
from the VIS DL intensity. The time decay data of DL photoemission curves were fitted to an equation of the type:
)/exp( ii tAy with a variable number of exponential components. For each set of VIS, Blue, Green/Yellow or Red DL
emission data, the time decay constants (i) and the minimal number of exponential DL components were established from the
best simultaneous fit to all DL curves obtained with control- and rotenone-treated cells in the respective spectral data set. Then
0
20
40
60
80
100
0 50 100 150 200 250 300
[ROT], M
Clo
no
gen
ic s
urv
ival
(%)
A
0
20
40
60
80
0 25 50 75 125 250
Earl
y/l
ate
ap
op
tosis
(%
) Early, 24 h
Late, 24 h
Early, 48 h
Late, 48 h
B
[ROT], M
the DL yield corresponding to each kinetic component was calculated for each individual DL curve as the product Aii. To
facilitate comparison between different spectral DL components, which exhibited some significant differences in emission
kinetics, the DL yield was calculated in some cases in three time domains of the DL emission, corresponding to three main
classes of light emitting states: 11-100 s (S1 states), 100 s - 1 ms (S2) and 1-10 ms (S3), as the integral of the I-fitting function
over the respective time domains. This analysis could not be performed in a consistent manner in the time domain 10-100 ms,
as in some cases the signal-to-noise ratio was too high within this region. Spectrofluorimetry. For determination of NADH and
FMNox levels, 2 ml cell samples prepared in PBS as described above were placed into a covered quartz cuvette maintained at
20ºC under continuous stirring in a Horiba Jobin Yvon spectrofluorimeter equipped with two monochromators. All slits were set
to 5 nm. After 5 min. allowed for thermal equilibration, emission spectra were collected in triplicate, with excitation at 340 nm
for NADH [55,56] and at 450 nm for FMNox [57]. Fluorescence intensity was averaged, corrected for background and then
normalized to cellular density. The mitochondrial level of NADH, [NADH]m, was quantified by the ratio of fluorescence emitted at
450 nm to that emitted at 374 nm and is considered to be not affected by inherent artifacts related to cell movements, sample
inhomogeneities etc. [58,59]. In control (non-treated) cells, this ratio was 1.86 ± 0.31 (n = 18). The NADH fluorescence ratio
obtained with treated cells was normalized to the corresponding mean value of control cells (1.86). We should mention that in
the current experiments, the cellular level of NADH, assumed to be proportional to the fluorescence emission at 450 nm with
excitation at 340 nm, displayed features that were relatively similar to those of mitochondrial NADH, indicating that
mitochondrial NADH constitutes the major component of cellular NADH under our conditions, in agreement with other reports
[56,58-60; see also [55] for a review]. It is also assumed that the concentration of NADPH (reduced nicotinamide adenine
dinucleotide phosphate), whose photo-physical properties are virtually identical to those of NADH, is much lower than that of
NADH [55,61]. This idea is also strongly supported by the strong correlation found in the present study between the
fluorescence of NADH and that of FMN in cells treated with Complex I-targeting agents (discussed below). The level of FMNox is
expressed as the fluorescence emission at 515 nm with excitation at 450 nm [56,57]. In quercetin-based experiments, due to
consistent spectral overlap between the two fluorescent species, FMN fluorescence was recorded with excitation/emission at
485 nm/515 nm, where quercetin fluorescence is very weak [33,35]. This assessment may somewhat overestimate emission
from flavine mononucleotide, due to contribution from quercetin. Nevertheless, this further strengthens the current conclusion
that quercetin decreases the level of FMNox. As discussed below, the results obtained in this study suggest that the fluorescence
we detected at 515 nm originates mainly from FMN rather than FAD (flavin adenine dinucleotide) or other mitochondrial flavins,
which, due to their similar optical properties, are virtually indistinguishable by spectrofluorimetric means. Statistics. Unless
indicated otherwise, the data are presented as median s.e.m. of at least three different measurements. Data fitting was
performed using the program Origin, version 7.5. Further details can be found in the publication under revision at a ISI
journal[23].
RESULTS AND DISCUSSION
Effect of ROT on clonogenic survival and apoptosis. Rotenone decreased the
clonogenic survival of Jurkat cells in a dose-dependent manner (Fig. 2A).
Rotenone also induced apoptosis dose-dependently, which correlated well
with the clonogenic survival (Fig. 2B). The kinetics of apoptosis induction
appeared to be relatively slow, since at 48 h after the treatment a significant
number of cells were found in early apoptotic phases. The specific evolution of
apoptosis through early/late stages strongly indicated that rotenone
preferentially induced apoptosis, not necrosis in Jurkat cells. This conclusion
was further substantiated by forward and side scatter analysis
Fig. 2. Dependence of clonogenic survival (A) and apoptosis (B) on the dose of rotenone. Jurkat cells were exposed for 30 min. to the indicated doses of rotenone and then processed for assessment of clonogenic survival and early/late stages of apoptosis as described in Materials and Methods. Data in panel A were fitted to the
equation y = 100 × KdH/(x
H + Kd
H) with Kd = 114.7 M and H = 1.35. In B, early and late
apoptotic cell fractions were evaluated at 24 h and 48 h after drug removal.
Effect of ROT, QC, MD and H2O2 on NADH and FMNox levels. We investigated by spectrofluorimetric means
the effect of rotenone on the mitochondrial level of NADH and oxidized FMN (FMNox). As expected, steady state
measurements (Fig. 3) and kinetic recordings (not shown) showed that in ROT-treated Jurkat cells, the NADH level
increased substantially. Moreover, inhibition of Complex I by ROT was additionally confirmed by separate
fluorimetric determinations of the level of mitochondrial superoxide, which displayed a consistent increase after
addition of rotenone. The level of FMNox also increased after addition of rotenone (Fig. 3), probably reflecting a
direct effect of NADH on the rate of FMN oxidation at the level of Complex I in this cell type. From steady-state
measurements carried out after treatments with increasing doses of rotenone (Fig. 3A), the microscopic dissociation
constant associated with ROT binding to Complex I was estimated to be ≈33 M, and the Hill coefficient of 2.0-2.4
must indicate cooperative binding of at least two molecules of rotenone to a Complex I oligomer, since the Hill
coefficient for rotenone binding in Complex I in the forward mode (site 1 involved) was determined to be 1 [24].
Importantly, our data, corroborated by additional results presented below, suggest that in intact Jurkat cells,
Complex I functions mainly as a dimer in which two molecules of rotenone bind cooperatively to the corresponding
ROT sites 1. The data presented in Fig. 3B indicate that the effect of rotenone was complete in about 60 min. Indeed,
treatments with 50 M ROT for 30 min., 60 min. or 90 min. had similar effects on NADH and FMNox levels. However,
the slight reduction of ≈10% which was visible after the 90-min. treatment, corroborated by results from DL
spectroscopy (discussed below), indicate that a conformational change in Complex I may be induced after longer
treatments with rotenone.
Fig. 3. Effects of 30 min. rotenone-treatment on NADH and FMNox levels in Jurkat cells. (A) The relative ligand concentration (normalized to control) depended in a Hill-like fashion on the dose of rotenone. The data were fitted to the equation: c = 1 + cmax
[ROT]h/([ROT]
h + Kd
h). For the NADH data set, Kd = 32.5 M, h = 1.97 and cmax = 1.18. For the FMNox data set, Kd = 33.6 M, h =
2.37 and cmax = 0.96. (B) Dependence of NADH and FMNox levels on the duration of rotenone exposure, for a fixed dose of 50 M rotenone. (C) NADH and FMNox concentrations after rotenone-, menadione-, quercetin- and H2O2-treatments are strongly correlated. The line (y = 0.916 x - 0.0456) was obtained by linear fit to the data. Inset, comparison between effects of ROT-, MD-, QC-, and H2O2 and the effect of antimycin A (AA, indicated by arrow). The data used in C are indicated in Table 1.
We also investigated the effect of quercetin, menadione and H2O2 on the mitochondrial concentration of NADH or
oxidized FMN. In agreement with our previous reports [18-20], current measurements confirmed that QC decreases
the NADH level in Jurkat cells. In addition, quercetin also depressed the level of oxidized FMN (Table 1). Menadione
had similar effects, decreasing considerably the concentration of both NADH and FMNox in Jurkat cells (Table 1). High
C
1.0
1.4
1.8
2.2
2.6
3.0
0 50 100 150 200 250 300
NADH
FMN
Re
lati
ve
lig
an
d c
on
ce
ntr
ati
on
[ROT], M
A
ox
m
0
1
2
3
0 1 2 3
Relative [NADH]m
Re
lati
ve
[F
MN
ox]
0
1
2
3
0 30 60 90
NADH
FMN
B
Re
lati
ve
lig
an
d c
on
ce
ntr
ati
on
Treatment duration (min.)
ox
m
0
1
2
3
0 2 4 6 8
Relative [NADH]m
Rela
tive [
FM
No
x]
AA
concentrations of H2O2 (100 M and 500 M) applied for 20 min. (which were found to induce substantial apoptosis
in Jurkat cells did not cause significant variations in [NADH]m and produced only a slight decrease in [FMNox] (Table
1). Taken together, the steady-state fluorimetric data presented here demonstrate a strong linear correlation
between the level of NADH and that of FMNox in Jurkat cells treated with ROT, MD, QC, combination of QC and MD,
and H2O2 (Fig. 3C, Table 1).
Table 1. The relative levels of NADH și FMNox and DL quantum yield in Jurkat cells exposed to ROT, MD, H2O2, QC,
combinations of QC and MD or H2O2, and AA.
Spectrofluorimetry DL Spectroscopy
Treatment Relative [NADH]m
Relative [FMNox]
DL yield (VIS)
DL yield (blue)
DL yield (green/ yellow)
DL yield (red)
Control 1 1 0.970 0.276 0.335 0.359
25 M ROT 30 min. 1.46 ± 0.24 1.32 ± 0.22 1.547 0.534 0.537 0.476
50 M ROT 30 min. 1.74 ± 0.29 1.65 ± 0.25 1.939 0.658 0.738 0.543
75 M ROT 30 min. 2.13 ± 0.30 1.95 ± 0.21 4.868 1.318 2.764 0.785
150 M ROT 30 min. 2.08 ± 0.33 1.73 ± 0.28 n.d. n.d. n.d. n.d.
200 M ROT 30 min. 1.98 ± 0.20 2.06 ± 0.32 n.d. n.d. n.d. n.d.
250 M ROT 30 min. 2.33 ± 0.34 2.01 ± 0.40 n.d. n.d. n.d. n.d.
10 M ROT 60 min. 1.40 ± 0.20 1.28 ± 0.22 1.224 0.372 0.510 0.341
25 M ROT 60 min. 1.60 ± 0.12 1.29 ± 0.18 2.346 0.731 0.622 0.525
50 M ROT 60 min. 1.99 ± 0.31 1.81 ± 0.26 4.979 1.586 2.100 1.293
50 M ROT 90 min. 1.83 ± 0.32 1.62 ± 0.26 2.535 0.677 1.157 0.701
25 M MD 20 min. 0.82 ± 0.10 0.87 ± 0.10 0.577 n.d. n.d. n.d.
25 M MD 4 h 0.50 ± 0.08 0.82 ± 0.13 0.332 n.d. n.d. n.d.
250 M MD 20 min. 0.55 ± 0.18 0.68 ± 0.15 0.319 n.d. n.d. n.d.
100 M H2O2 20 min. 0.96 ± 0.17 0.86 ± 0.16 0.852 0.237 0.378 0.237
500 M H2O2 20 min. 0.91 ± 0.06 0.77 ± 0.06 0.677 n.d. n.d. n.d.
10 M QC 1 h 0.85 ± 0.09 0.52 ± 0.27 0.702 n.d. n.d. n.d.
5 M QC 24 h 0.76 ± 0.03 0.46 ± 0.12 0.794 n.d. n.d. n.d.
50 M QC 24 h 0.27 ± 0.09 0.13 ± 0.03 0.556 0.220 0.183 0.152
10 M QC 1 h, apoi
250 M MD 20 min.
0.86 ± 0.16 0.28 ± 0.16 0.362 n.d. n.d. n.d.
10 M QC 1 h, apoi
500 M H2O2 20 min.
0.86 ± 0.16 0.28 ± 0.16 0.647 n.d. n.d. n.d.
150 M AA 30 min. 6.53 ± 1.80 0.90 ± 0.27 n.d. n.d. n.d. n.d.
n.d. - not determined
The Pearson correlation coefficient associated with these data was r = 0.93. In addition, we found that under similar
conditions, antimycin A (AA), an inhibitor of respiratory Complex III, induced a substantial increase of [NADH]m but
had no measurable effect on [FMNox] (Fig. 3C, inset). Collectively, these data strongly suggest that quercetin,
menadione and rotenone act at the level of mitochondrial Complex I and most likely trigger the conversion to a new
conformation of the enzyme, in which NADH bound to Complex I accelerates the oxidation of reduced FMN. These
data also strongly suggest that the main flavoprotein detected under our conditions is most likely FMN bound to
Complex I.
Effect of ROT on delayed luminescence. DL spectroscopy in VIS revealed a notable enhancement of DL following
ROT-treatments of Jurkat cells. Light emission following UV-excitation presented a complex kinetic profile, which was
well fitted by a linear combination of four exponentials, with characteristic decay times of 22.9 s, 140 s, 1.40 ms
and 8.50 ms, respectively (Fig. 4), indicating the existence of four main classes of light-emitting states. With
treatments of 30 min. duration, the corresponding yields associated with the total number of excited states of each
component increased with the dose of ROT (Fig. 4, insets). Under the same treatment conditions, the kinetics of
delayed red light emission exhibited six dominant exponential components, with characteristic decay times of 13.3
s, 129 s, 388 s, 1.70 ms, 3.87 ms and 8.25 ms, respectively (Fig. 5). Some of these components provided an
extremely low DL yield in certain treatments. Therefore, to obtain a unitary description and gain relevant insights
into the effect of rotenone on red DL, we collected the contributions of some adjacent DL components as shown in
the insets of Fig. 5. Together, these data show that the overall effect of ROT was to stimulate red DL.
Next, we maintained the ROT dose constant and varied the treatment duration from 30 min. to 60 min. and 90 min.,
respectively. Delayed luminescence in VIS (Fig. 6) as well as in the Red region of the spectrum (Fig. 7) presented a
biphasic dependence on the treatment time, exhibiting a maximum in 60 min. treatments. In particular, a marked
(~12 fold) increase was observed in the delayed red-light emission by a distinctive component with decay time
constant of 129 s.
Fig. 4. Kinetics of delayed photoemission in VIS by Jurkat cells exposed for 30 min. to DMSO (Control) or to the indicated doses of rotenone. The time elapsed after UV-excitation of the cell sample is presented on the abscissa. Each curve was obtained by fitting the data to the
equation:
5
1)/exp(
i ii tAy . Insets present the ROT-dose
dependence of the DL yield (i.e., the product Aii) corresponding to
each kinetic component. The characteristic times (i) of individual
exponential components are indicated. The first component (1 < 10-5
s) is not specified and was disregarded in further analysis.
Fig. 5. Kinetics of delayed photoemission in Red by Jurkat cells exposed for 30 min. to DMSO (Control) or to the indicated doses of rotenone. Each curve was obtained by fitting the data to the equation:
7
1)/exp(
i ii tAy . Insets, the ROT-dose dependence of the DL
yield (i.e., Aii) produced by three dominant classes of kinetic
components. The characteristic times (i) of individual exponential
components are indicated. The first component (1 < 10-5
s) is not specified and was disregarded in further analysis.
DL
in
ten
sit
y (
a.u
.)
1.E-01
1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
1.E-05 1.E-04 1.E-03 1.E-02 1.E-01
t (s)
Control
25 M ROT
50 M ROT
75 M ROT
0
0.3
0.6
0.9
0 25 50 75
0
0.1
0.2
0.3
0.4
0.5
0 25 50 75
1.40 ms
Yie
ld (
a.u
.)
8.50 ms
Yie
ld (
a.u
.)
[ROT], M [ROT], M
0
0.5
1
1.5
2
2.5
0 25 50 75
0
0.4
0.8
1.2
1.6
0 25 50 75
22.9 s
Yie
ld (
a.u
.)
140 s
Yie
ld (
a.u
.)
[ROT], M [ROT], M
1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E-05 1.E-04 1.E-03 1.E-02
Control
25 M ROT
50 M ROT
75 M ROT
DL
in
ten
sit
y (
a.u
.)
t (s)
0
0.2
0.4
0.6
0 25 50 75
13.3 s
Yie
ld (
a.u
.)
[ROT], M
0
0.05
0.1
0.15
0.2
0 25 50 75
129 s
388 s
Yie
ld (
a.u
.)
[ROT], M
0
0.05
0.1
0.15
0 25 50 75
1.70 ms3.87 ms
8.25 ms
Yie
ld (
a.u
.)
[ROT], M
After analyzing, in the same manner, the delayed light emission in the Blue and the Green/Yellow regions of
the spectrum, which displayed similar features with regard to the dependency on the ROT dose and treatment
duration, we could notice that some kinetic components were not common to all spectral components of DL.
Therefore, to obtain a more general description of the DL characteristics and perform a consistent comparison
between different spectral domains, we considered three distinct classes of light-emitting states, namely one class of
fast-decay states (S1, lifetime ~10 s) and two classes of intermediate- (S2, lifetime ~100 s) and slow-decay states
(S3, lifetime ~1 ms); this approach, together with the individual decay times determined above, proved particularly
useful in identifying specific electron transfer rates in Complex I. The results collected in Fig. 8 show that the fast-
decay (S1) states emitted predominantly blue light, with somewhat lower contributions from red- and green/yellow-
light emitting states, whereas states S2 and S3 appeared to emit mostly in the Green/Yellow region, with some
significant blue- and red-light emission from S2 recorded only after ROT treatments of 60 min. The data presented in
Fig. 3B indicate that under our conditions, the effect of rotenone on both NADH and FMNox levels was complete in
about 60 min. The slight attenuation of the effect that was observable after longer treatments (90 min.) was
reflected more prominently by the DL yield (Figs. 6-8). It is conceivable that mitochondrial Complex I may become
insensitive to rotenone after long exposure to the inhibitor, via a slow de-activated/activated state transition [62]
which could be regulated metabolically in intact cells. Hence, this mechanism would explain the apparent recovery
from inhibition, which under our conditions seems to manifest in exposures of increased duration.
Fig. 6. Kinetics of delayed photoemission in VIS by Jurkat cells exposed for
indicated periods to DMSO (Control) or to 50 M rotenone. Each curve was obtained by fitting the data as in Fig. 4, using the characteristic decay times indicated therein. Insets, the DL yield dependence on treatment duration. Other details are as in Fig. 4.
Fig. 7. Kinetics of delayed photoemission in Red by Jurkat cells exposed
for indicated periods to DMSO (Control) or to 50 M rotenone. Each curve was obtained by fitting the data as in Fig. 5, using the characteristic decay times indicated therein. Insets, the DL yield dependence on treatment duration. Other details are as in Fig. 5.
DL
in
ten
sit
y (
a.u
.)
1.E-01
1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
1.E-05 1.E-04 1.E-03 1.E-02 1.E-01
t (s)
0
0.1
0.2
0.3
0 30 60 90
0
0.2
0.4
0.6
0 30 60 90
1.40 ms
Yie
ld (
a.u
.)
t (min.)
8.50 ms
Yie
ld (
a.u
.)
t (min.)
0
1
2
3
0 30 60 90
0
0.6
1.2
1.8
0 30 60 90
22.9 s
Yie
ld (
a.u
.)
t (min.)
140 s
Yie
ld (
a.u
.)
t (min.)
0 min.
30 min.
60 min.
90 min.
1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E-05 1.E-04 1.E-03 1.E-02
DL
in
ten
sit
y (
a.u
.)
0 min.
30 min.
60 min.
90 min.
0
0.2
0.4
0.6
0.8
0 30 60 90
13.3 s
Yie
ld (
a.u
.)
t (min.)
0
0.2
0.4
0.6
0 30 60 90
129 s
388 s
Yie
ld (
a.u
.)
t (min.)
0
0.05
0.1
0.15
0 30 60 90
1.70 ms
3.87 ms
8.25 ms
Yie
ld (
a.u
.)
t (min.)
t (s)
Fig. 8. Spectral analysis of the DL yield as a function of (A) the ROT dose in Jurkat cells exposed for 30 min. to the indicated
concentrations of rotenone, or (B) the ROT-treatment duration, at a fixed dose of 50 M ROT. Blue, Green/Yellow and Red DL yields were computed for three different classes of states, S1, S2 and S3, which emit on different timescales (see text for further details).
Correlation between DL and the level of NADH and FMNox DL spectroscopy and NADH/FMNox fluorimetric data
were collected after treatments with ROT, QC, MD and H2O2, or QC in combination with MD or H2O2. Consistent with
our previous reports [19,25], quercetin and menadione inhibited significantly the ultra-weak photon-induced
delayed photoemission of Jurkat cells, in a dose- and time dependent manner (Table 1).
Fig. 9. The dependence of the VIS (A), Blue (B), Green/Yellow (C) and Red (D) total DL yield on the relative concentration of NADH or FMNox, obtained after treatments of Jurkat cells with rotenone, menadione, quercetin or H2O2. The total DL yield was
calculated from emission over the time interval 11 s - 10 ms. The data (collected in Table 1) were fitted to the equation: y = Y0 +
Ymax ch/(c
h + Kd
h) (curves), where c is the relative ligand concentration. The fit to VIS-DL data was done with Ymax set to 21.0, the
maximal value obtained for the first exponential component of the photoemission curves in Figs. 4-5. The fit to spectral components was done with fixed Ymax and Y0, which were considered to be a certain fraction of the corresponding VIS-values. The Blue and Red fractions were set to 0.291 and 0.318, respectively, which were equal to the maximal values observed from all measurements. The remaining fraction, 0.391, was attributed to Green/Yellow DL. Parameters derived from the fit are presented in Table 2.
0
0.4
0.8
1.2
0 25 50 75
Blue
Green/Yellow
Red
S1
Yie
ld (
a.u
.)
[ROT], M
A
0
0.4
0.8
1.2
0 25 50 75
[ROT], M
S2
Yie
ld (
a.u
.)
0
0.5
1
0 25 50 75
S3
[ROT], M
Yie
ld (
a.u
.)
0
0.4
0.8
1.2
1.6
0 30 60 90
S1
Yie
ld (
a.u
.)
t (min.)
B
0
0.2
0.4
0.6
0.8
0 30 60 90
S2
Yie
ld (
a.u
.)
t (min.)
0
0.2
0.4
0.6
0.8
0 30 60 90
S3
Yie
ld (
a.u
.)
t (min.)
0
2
4
6
0 0.5 1 1.5 2 2.5
FMN
NADH
DL
yie
ld (
a.u
.)
Relative ligand concentration
A
ox
VIS
m
0
0.5
1
1.5
2
0 0.5 1 1.5 2 2.5
FMN
NADH
DL
yie
ld (
a.u
.)
Relative ligand concentration
B
ox
Blue
m
0
0.5
1
1.5
0 0.5 1 1.5 2 2.5
FMN
NADH
DL
yie
ld (
a.u
.)
Relative ligand concentration
D
ox
Red
m
0
1
2
3
0 0.5 1 1.5 2 2.5
FMN
NADH
DL
yie
ld (
a.u
.)
Relative ligand concentration
C
ox
Green/Yellow
m
H2O2 elicited as well an inhibitory effect on DL, but to a much lesser extent (Table 1). Combination of quercetin and
menadione at high dose also decreased consistently the VIS-DL yield, whereas combination of quercetin with a high
dose of H2O2 exerted a more reduced effect (Table 1). Taken together, the data revealed a strong correlation
between the total DL yield in VIS and the mitochondrial concentration of NADH or FMNox (Fig. 9A), which also
manifested robustly for all classes of states S1, S2 and S3 (not shown). The dependence of the total VIS-DL yield on
both [NADH]m and [FMNox] was very well fitted by a modified Hill equation (Fig. 9A). Interestingly, the apparent Hill
coefficient of both ligands was estimated to be 4.3-4.4, which is approximately two times higher than the value
estimated above for the binding of rotenone to oligomeric Complex I. The total yield of delayed emission of blue,
green/yellow or red light exhibited a qualitatively similar dependence on [NADH]m and [FMNox] (Fig. 9B-D). However,
the corresponding individual Hill coefficients varied from 3.7-4.0 (Blue) to 6.9-7.4 (Green/Yellow) and 2.7-2.8 (Red)
(Table 2). All these figures substantiate the idea that the dominant structural arrangement of Complex I in Jurkat
cells is the dimeric form, which binds cooperatively two ROT molecules, and that a significant contribution comes
also from tetrameric Complex I, in which at least three (most likely four) molecules of rotenone bind to ROT site 1 in
a cooperative manner.
From a quick inspection of the spectral DL domains we can gain some relevant insights concerning the relative
frequency of Complex I dimers and tetramers. Thus, the DL data indicate that green/yellow light emission
(presumably associated with tetrameric Complex I) contributes 39%, and blue- and red-light emission (associated
with dimeric Complex I) contributes 61% to total DL. So, our results suggest that about 60% and 40% of Complex I
oligomers in Jurkat cells are in dimeric and tetrameric form, respectively.
Table 2. Fit-derived parameters of the DL-yield dependence on relative [NADH]m and [FMNox ]. The DL yield in the
indicated spectral domains was calculated in the time interval of 11 μs to 10 ms.
Parameter DL yield (VIS)
DL yield (blue)
DL yield (green/ yellow)
DL yield (red)
NADH
Y0 0.446 0.130 0.174 0.142
Ymax 21.0 6.11 8.21 6.68
Kd 2.92 2.95 2.37 4.15
h 4.26 3.98 7.42 2.84
FMNox
Y0 0.473 0.138 0.185 0.150
Ymax 21.0 6.11 8.21 6.68
Kd 2.65 2.79 2.19 4.01
h 4.39 3.68 6.94 2.66
In conclusion, based on the main current findings that: 1) DL is closely related to the level of oxidized FMN,
which is found primarily in the mitochondria [22], 2) DL is also linked to the level of NADH, the substrate of
mitochondrial Complex I, 3) ROT, a specific inhibitor of Complex I, affected DL considerably, and 4) MD and QC,
which interact robustly with Complex I, also affected DL significantly, all our results reinforce the idea [26] that
mitochondrial Complex I plays a major role in the ultra-weak photon-induced delayed photoemission in Jurkat
cells.
Other details can be found in the publication sent to a ISI journal (under review) [23].
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18. I. Baran, C. Ganea, I. Ursu, V. Baran, O. Calinescu, A. Iftime, R. Ungureanu, I.T. Tofolean, Fluorescence properties of quercetin in human leukemia Jurkat T-cells, Rom. J. Phys. 56 (2011) 388-398.
19. I. Baran, C. Ganea, S. Privitera, A. Scordino, V. Barresi, F. Musumeci, M.M. Mocanu, D.F. Condorelli, I. Ursu, R. Grasso, M. Gulino, A. Garaiman, N. Musso, G.A.P. Cirrone, G. Cuttone, Detailed analysis of apoptosis and delayed luminescence of human leukemia Jurkat T cells after proton-irradiation and treatments with oxidant agents and flavonoids, Oxid. Med. Cell. Longev. 2012 (2012) 498914:14
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22. L.D. Gaspers, A.P. Thomas, Calcium-dependent activation of mitochondrial metabolism in mammalian cells, Methods 46 (2008) 224-232. 23. Irina Baran, Diana Ionescu, et al., Mitochondrial respiratory Complex I probed by delayed luminescence spectroscopy, Journal of
Biomedical Optics, 2013, (trimisă, sub revizie) 24. H. Suzuki, T.E. King, Evidence of an ubisemiquinone radical(s) from the NADH-ubiquinone reductase of the mitochondrial respiratory
chain, J. Biol. Chem. 258 (1983) 352-358. 25. A.D. Vinogradov, V.G. Grivennikova, The mitochondrial Complex I: progress in understanding of catalytic properties, IUBMB Life 52 (2001)
129-134. 26. I. Baran, C. Ganea, A. Scordino, F. Musumeci, V. Barresi, S. Tudisco, S. Privitera, R. Grasso, D.F. Condorelli, I. Ursu, V. Baran, E. Katona,
M.M. Mocanu, M. Gulino, R. Ungureanu, M. Surcel, C. Ursaciuc, Effects of menadione, hydrogen peroxide and quercetin on apoptosis and delayed luminescence of human leukemia Jurkat T-cells, Cell Biochem. Biophys. 58 (2010) 169-179.
DISSEMINATION OF THE RESULTS
ISI Papers
23. Baran, Irina; Ganea, Constanta; et al., "Detailed Analysis of Apoptosis and Delayed Luminescence of Human
Leukemia Jurkat T Cells after Proton Irradiation and Treatments with Oxidant Agents and Flavonoids",
OXIDATIVE MEDICINE AND CELLULAR LONGEVITY, Article Number: 498914, DOI: 10.1155/2012/498914
Published: 2012 (IF:3.393, is 0.691)
24. Irina Baran, Eva Katona, Constanta Ganea, Quercetin as a fluorescent probe for the Ryanodine receptor activity
in Jurkat cells, Pfluegers Archiv, European Journal of Physiology, 2013, DOI 10.1007/s00424-013-1235-y,
465:1101–1119, (IF 4.866, is. 1.8227)
25. Irina Baran and Constanta Ganea, RyR3 in situ regulation by Ca2+ and quercetin and the RyR3-mediated Ca2+
release flux in intact Jurkat cells, Archives of Biochemistry and Biophysics, 540 (2013) 145–159 (IF. 3.370, is
1.112)
26. M.M. Mocanu, M. Surcel, C. Ursaciuc, E. Katona, C. Ganea, Antiproliferative effect of quercetin in mammary and
epidermoid cancer, ROMANIAN BIOTECHNOLOGICAL LETTERS, 2013, acceptatd (IF. 0.363, is. 0.115)
27. Irina Baran, Diana Ionescu, Maria Magdalena Mocanu, Adrian Iftime, Ioana Teodora Tofolean, Ruxandra Irimia,
Alexandru Goicea, Alexandru Dimancea, Andrei Neagu, Constanta Ganea, Novel insights into the antiproliferative
effect of quercetin, menadione and rotenone in human leukemia Jurkat T cells, Leukemia Research, 2013 (under
review)(IF: 2.764, is 1.006)
28. Irina Baran, Diana Ionescu, Simona Privitera, Agata Scordino, Maria Magdalena Mocanu, Francesco Musumeci,
Rosaria Grasso, Marisa Gulino, Adrian Iftime, Ioana Teodora Tofolean, Alexandru Garaiman, Alexandru Goicea,
Ruxandra Irimia, Alexandru Dimancea, Constanta Ganea, Mitochondrial respiratory Complex I probed by
delayed luminescence spectroscopy, Journal of Biomedical Optics, 2013, accepted (IF. 2.881, is 2.107)
29. M.M. Mocanu, C. Ganea, et al., Epigallocatechin-gallate determines ErbB proteins downregulation, cell death
mediated by 67kDa laminin receptor and altered lipid order in mammary and epidermoid carcinoma cells, J.
NAT. PROD., (under review)(IF. 3.285, is. 1.867)
Conferences
2. Ganea C. ; Katona, E.; Baran, I., Quercetin activates the RyR3 receptor in Jurkat human leukemia cells, invited
lecture, Max Planck Institut for Biophysics, Franfurt/Main, Germania, 28 oct. 2011
3. Baran, I.; Katona, E.; Ganea, C., Quercetin fluorescence reveals the open probability of the RyR3 Ca2+ channel in
intact cells, 22nd IUBMB Congress/37th FEBS, Seville, SPAIN, SEP 04-09, 2012, FEBS JOURNAL Volume: 279
Special Issue: SI Suppl: 1, p: 269-270, SEP 2012
4. Agata Scordino, I. Baran, V. Baran, R. Bonfanti, A. Campisi, C. Ganea, R. Grasso, F. Musumeci, R. Parenti - New
insights into application of Delayed Luminescence in cancer research - Photodynamic Therapy and
Photodiagnosis in Clinical Practice: 9th International Symposium 2012 (October 16-20, 2012 - Brixen /
Bressanone, Italy)
5. Ionescu D., Ganea C, Iftime A., Tofolean I. ,Irimia R., Goicea A., Dimancea A., Neagu A., Baran I., Quercetin exerts
its antitumoral effect while manifesting a strong antioxidant character, 9th EBSA European Biophysics Congress,
Eur Biophys J (2013) 42 (Suppl 1):S1–S236
6. M.M. Mocanu, P. Nagy, L. Georgescu, T. Varadi, D. Shrestha, I. Baran, E. Katona, J. Szollosi, C. Ganea, The effect
of flavonoids in mammary and epidermoid tumor cells with ErbB proteins overexpression, 8th International
Conference "Structure and Stability of Biomacromolecules - SSB 2013", 2013, Kosice, Slovakia.
7. Ionescu D.,The influence of quercetin on lipid membranes with cholesterol, 8th International Conference
"Structure and Stability of Biomacromolecules - SSB 2013", 2013, Kosice, Slovakia.
8. Agata Scordino, Irina Baran, Roberta Bonfanti, Agata Campisi, Costanta Ganea, Rosaria Grasso, Marisa Gulino,
Adrian Iftime, Diana Ionescu, Maria Magdalena Mocanu, Francesco Musumeci, Rosalba Parenti - Delayed
Luminescence spectroscopy to monitor mitochondrially targeted effects of cell proliferation inhibitors - Ultra-
weak Photon Emission From Living Systems Conference (June 21-23, 2013, Olomouc, Czech Republic)
9. Agata Scordino, I. Baran, R. Bonfanti, A. Campisi, C. Ganea, R. Grasso, M. Gulino, A. Iftime, D. Ionescu, M. M.
Mocanua, F. Musumeci, R. Parenti - Delayed Luminescence spectroscopy in cancer research - XCIX Congresso
Nazionale Societa' Italiana di Fisica (23 - 27 settembre 2013, Trieste, Italy) – Book of Abstracts p. 120
Distinctions:
As an international appreciation of the value of the obtained results, the scientific paper published in the ISI journal
Pfluegers Archiv, European Journal of Physiology, 2013, received the distinction „Global Medical Discovery” at „Key
Scientific Articles” („Global Medical Discovery (ISSN 1929-8536) features breaking research judged to be of key
importance in science and medicine”).
Project Manager, Prof. Dr. Constanța Ganea
