C-(2-chloroquinoline-3-yl)-N-phenyl nitrone: New synthetic antioxidant inhibits proliferation and induces apoptosis of breast carcinoma MCF-7 cells

Article abstract of DOI:10.1002/ardp.200500250

In this work, a new quinoline nitrone derivative, C-(2-chloroquinoline-3- yl)-N-phenyl nitrone (CQPN) was successfully prepared and proved by spectral analysis. The antioxidant activity of CQPN against various radicals was investigated and its anti-cancer

Full text of DOI:10.1002/ardp.200500250

242
Arch. Pharm. Chem. Life Sci. 2006, 339, 242–249
Full Paper
C-(2-Chloroquinoline-3-yl)-N-phenyl Nitrone: New Synthetic
Antioxidant Inhibits Proliferation and Induces Apoptosis of
Breast Carcinoma MCF-7 Cells
Mohamed Ramadan1, Amira M. Gamal-Eldeen2, Mohamed Abdel-Aziz1, Gamal El-Din Abuo-
Rahma1, Hisham Abdel-Nabi3, Abdel-Hamid Nagib4
1 Department of Medicinal Chemistry, Faculty of Pharmacy, El-Minia University, El-Minia, Egypt
2 Department of Biochemistry, Division of Genetic Engineering and Biotechnology, National Research Centre,
Cairo, Egypt
3 Department of Organic Chemistry, Faculty of Science, El-Minia University, El-Minia, Egypt
4 Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut University, Assiut, Egypt
In this work, a new quinoline nitrone derivative, C-(2-chloroquinoline-3-yl)-N-phenyl nitrone
(CQPN) was successfully prepared and proved by spectral analysis. The antioxidant activity of
CQPN against various radicals was investigated and its anti-cancer properties against different
human tumor cell lines including the solid tumor cell lines hepatocarcinoma (Hep-G2) and breast
carcinoma (MCF-7); the hematopoietic tumor cell line lymphoblastic leukemia (1301) was also
explored. CQPN activities were compared to that of the known nitrone C-phenyl-N-tert-butyl
nitrone (PBN). Our results showed that although PBN was the stronger antioxidant than CQPN,
the latter was an effective scavenger of different non-physiological (1,1-diphenyl-2-picrylhyrazyl)
and physiological (peroxyl and hydroxyl) radicals. Both of CQPN and PBN possess a significant
inhibitory property against LPS-stimulated NO production in macrophage. CQPN and PBN treat-
ment resulted in a growth inhibition in Hep-G2 cells (IC50 31.42 lM and 18.6 lM, respectively).
Unlike PBN, CQPN strongly inhibited the growth of MCF-7 cells (IC50 14.01 lM) in a dose-dependent
manner. On contrary, CQPN and PBN exhibited a proliferative stimulatory activity of the immune
cells including macrophages and lymphocytes. Exploring the cytotoxic effect of CQPN against
MCF-7 cells indicated that CQPN led to a major time-dependent disturbance in the cell-cycle
phases including progressive arrest in both S- and G2/M-phases. This disturbance was found to be
associated with a kinetic induction of apoptosis. The novel nitrone derivative CQPN is a strong
antioxidant, though less than PBN, and it may be an effective anti-proliferative compound against
breast carcinoma.
Keywords: Anticancer / Antioxidant / Apoptosis / C-(2-Chloroquinoline-3-yl)-N-phenyl nitrone / Cell cycle /
Received: December 5, 2005; Accepted: December 9, 2005
DOI 10.1002/ardp.200500250
They are of particular importance in cycloaddition reac-
Introduction
tions. Nitrones act as 1,3-dipoles, which is leading to the
construction of a wide variety of nitrogen-containing bio-
logically active compounds, such as alkaloids, amino
sugars, and antibiotics (b-lactams) [1, 2]. Nitrones show
numerous biological activities including; anti-ulcer [3],
antioxidant [4], anti-ischemic [5], anti-bacterial [6], anti-
inflammatory, anti-aging [4], neuroprotective [7], and
anti-cancer activities [8, 9]. Several methods were pre-
viously developed for the synthesis of various types of
Nitrones are compounds with a general common moiety
of R1R2C=N(R3)fiO, as shown in the formula in Fig. 1A.
Correspondence: Dr. Amira Gamal-Eldeen, Biochemistry Department,
Genetic Engineering and Biotechnology Division, National Research Cen-
tre, Dokki 12622, Cairo, Egypt.
E-mail: aeldeen7@yahoo.com, aeldeen@hotmail.com
Fax: +20 23 370-931
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Antioxidant and Anti-Proliferation by a New Nitrone Derivative
243
Figure 1. A. Structure moiety of nitrones. B. Molecular structure of PBN. C. Synthesis of
CQPN. (a) 2.5 mol DMF and 7 mol phosphorus oxychloride at 758C; (b) N-phenyl hydro-
xylamine in ethanol; compound 3 is CQPN.
nitrones derived from different compounds [10, 11]. Reac-
tive oxygen (ROS) and nitrogen species (RNS) play a signif-
Results
icant role in cancer occurrence based on electron spin
resonance [12]. Among ROS varieties, hydroxyl (OH) and
peroxyl radicals are the most potent radicals in dama-
ging the critical biological targets including protein oxi-
dation, DNA modification, i.e. single-base deletion, base-
ring opening, single and double strand breakage [13],
and lipid peroxidation [12].
C-phenyl-N-tert-butyl nitrone (PBN) (Fig. 1B) is one of
the synthetic antioxidants, which is capable of scaven-
ging many types of free radicals including both oxygen
and carbon based radicals [4, 5]. PBN has both hydrophilic
and lipophilic characteristics, having the ability to pene-
trate all tissues, including the brain. Addition of free radi-
cals to the PBN carbon-nitrogen double bond, yields a
stable nitroxide product, which is safely metabolized
and excreted in the urine. It was reported that PBN inhi-
bits both synthesis of nitric oxide (NO) and formation of
NO-mediated 3-nitrotyrosine adducts in activated astro-
cytes. Quinoline compounds have numerous biological
applications such as anti-malarial [14], anti-microbial
[15], anti-inflammatory [16], anti-cancer [17], anti-ulcer
[18], antioxidant [19], and antiviral [20] activities.
The objective of this work is to prepare a new quinoline
nitrone derivative, C-(2-chloroquinoline-3-yl)-N-phenyl
nitrone (CQPN) that may gather the biological activities
of both nitrones and quinolines and may provide a novel
active antioxidant and/or anti-cancer agent. To investi-
gate these activities, CQPN was submitted for several
experiments including testing its antioxidant activity
against various radicals and its anti-cancer properties
against different tumor cell lines, solid as well as hemato-
poietic lines. Additionally, CQPN results were compared
to the corresponding results of the known active nitrone
PBN.
CQPN proved to have a moderate antioxidant capacity
against different radicals compared to the strong capa-
city of PBN. CQPN exhibited a moderate scavenging activ-
ity against the artificial radical DPPH (SC50 39.01 lM)
compared to the potent scavenging activity of PBN (SC50
4.3 lM) and to ascorbic acid (SC50 8.2 lM), a known potent
DPPH scavenger. In the Oxygen Radical Absorbance Capa-
city (ORAC) assay, CQPN possessed 0.8-fold and 1.1-fold,
and PBN possessed 4.6-fold and 3.4-fold antioxidant capa-
city compared to trolox, a known reference scavenger,
against OH9 and peroxyl radicals, respectively.
To enhance inducible NO synthase (iNOS) expression
and NO production, we stimulated Raw 264.7 murine
macrophages with bacterial LPS. Further, we measure
the nitrite levels (as an NO index) in the cell culture
supernatants after treatment with 14 lM of CQPN or
PBN. Our findings proved that LPS effectively induced the
NO generation in macrophages, as indicated by the
nitrite concentration, which showed a 6.7-fold increase
in the nitrite concentration of the supernatants of LPS-
treated macrophage. Interestingly, the treatment with
both of CQPN and PBN resulted in a potential inhibition
of the generated NO close to the nitrite baseline of the
control supernatants (p a0.001, Fig. 2).
Using MTT assay, we studied the possible anti-prolifera-
tive effect of CQPN (Fig. 3A) and PBN (Fig. 3B) on the
growth of three different tumor cell lines after incuba-
tion for 24 h. The treatment of Hep-G2 cells with CQPN
and PBN dramatically inhibited the cell growth in a dose-
dependent manner, with IC50 values of 31.42 lM and
18.60 lM, respectively. The cytotoxic affinity of CQPN
was stronger against MCF-7 cells as indicated by the low
IC50 14.01 lM, while PBN showed no influence on the via-
bility of MCF-7 cells. On contrary, the treatment of lym-
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Figure 2. Inhibition of NO generation: The effect of nitrones
treatment on NO generation from LPS-stimulated Raw 264.7
was investigated by measuring nitrite concentration as an index
of NO production, (using Griess assay) comparing with the level
of nitrite in control cells (untreated with LPS).
phoblastic leukemia cells 1301 with CQPN resulted in a
strong dose-dependent enhancement in the lymphocyte
proliferation, starting from the lowest dose, while PBN
induced the 1301 cells proliferation to a low extent even
at the highest dose. Similarly, we noticed during the
nitrite assay that the treatment of Raw 264.7 macro-
phages with CQPN and PBN (14 lM) enhanced the macro-
phage proliferation to 143% and 131% of the DMSO-trea-
ted cells, so that we normalized the nitrite concentration
to the protein content of the cells.
Cell cycle checkpoints can be targeted for cancer ther-
apy either by activating checkpoint-mediated apoptosis
or by exploiting chemical sensitivity because of loss of
checkpoint function [21]. To investigate further the nat-
ure of growth inhibition by CQPN in MCF-7 cells, flow
cytometric analysis was performed. The growing cells
were treated with IC50 value of CQPN (14 lM) for different
intervals (0, 12, and 24 h), and then subjected to flow
cytometric analysis after staining their DNA. The distri-
bution of cells in different phases of the cycle is illu-
strated in Fig. 4. The untreated cells showed the expected
pattern for continuously growing cells, whereas the cells
treated with CQPN for 12 h showed a progressive accu-
mulation in the S phase and G2/M phase of the cell cycle
correlating with decreased number of cells in the G0/G1
phase.
Figure 3. The cytotoxicity of CQPN (A) and PBN (B) against
Hep-G2 cells (open circle), MCF-7 cells (closed circle), and 1301
cells (square) was assessed using MTT assay. The data are
represented as the mean € S.D. of the percentage of control
(DMSO-treated cells).
population at S-phase, as compared with 12 h treated
cells. These results suggest that CQPN appears to affect
processes that are essential to allow cells to progress
through the S phase and G2/M phase of the cell cycle, pre-
sumably by perturbation of the cell progression into the
G1 phase. An insignificant low sub-G0/G1 peak was
Moreover, cells treated with CQPN for longer interval
(24 h) showed an accumulation of cells in the G2/M phase
of the cell cycle, concomitantly to unchangeable cell
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Arch. Pharm. Chem. Life Sci. 2006, 339, 242–249
Antioxidant and Anti-Proliferation by a New Nitrone Derivative
245
tion and promotion, which might ultimately lead to car-
cinogenesis [22]. Similarly, overproduction of NO and
other RNS during inflammation and infection induced
nitrosative deamination of DNA bases and induction of
lipid peroxidation, which are associated with initiated
cellular injury and carcinogenesis [23]. Therefore, the
anti-carcinogenic potential of the antioxidants repre-
sents an effective strategy in preventing tumor initiation
and promotion. In this respect, we examined the scaven-
ging capacity of CQPN against physiologically relevant
ROS such as OH9 and peroxyl radicals, and RNS such as
NO. NO is a highly reactive free radical and it also can
form a number of oxidation products that play an impor-
tant role in the initiation and promotion of cancer, such
as NO2, NO29 and ONOO– [24]. The NO inhibitory property
of CQPN and PBN suggested an essential effective role of
the nitrone moiety and a possible anti-inflammatory
activity and it may have different explanations including
a direct scavenging capacity against NO, an inhibition of
the iNOS expression, and/or a modulation of other fac-
tors in the NO cascade such as transcriptional factors.
This in vitro antioxidant activity may suggest that CQPN
can provide the normal cells prevention from initiation
and transformation processes.
Figure 4. Cell cycle analysis: MCF-7 cells were treated with
14 lM CQPN for different intervals 0, 12, and 24 h. The percen-
tages of the cell populations were presented as sub-G0/G1
phase (black bar), G0/G1 phase (white bar), S-phase (gray bar),
and G2/M phase (hatched bar). Values are means € S. D., n = 3.
The findings from the cytotoxicity assay proved a
tumoricidal activity of CQPN against human hepatocarci-
noma and breast carcinoma as indicated by the relatively
low IC50 values. These findings suggested that CQPN pos-
sessed more specific anti-cancer activity against solid
than hematopoietic tumor cells, with more potency
against breast carcinoma.
detected after treatment at both of the incubation inter-
val 12 and 24 h. The analysis of the cells treated with vehi-
cle at different studied intervals showed unchangeable
percentages of cell population at different cell cycle
stages.
Most solid tumor cells are less sensitive to the inhibi-
tion of cell growth induced by anticancer drugs than
hematopoietic cancer cells [23]. That was not the case
with CQPN, which exhibited an effective cytotoxic prop-
erty against examined solid tumor cells. This specific
property was also explored previously in a comparative
report [25] using two apoptosis inducers, bactobolin and
cytostatin, against solid (B16 melanoma) and hemato-
poietic (EL-4 lymphoma) cancer-derived cell lines. In this
report, bactobolin was found to enhance apoptosis only
in solid tumor cells by a specific caspases/cyclins pattern,
while cytostatin enhanced apoptosis only in hematopoie-
tic tumor cells through a different caspases/cyclins pat-
tern. However, in vitro efficacy of CQPN as growth inhibi-
tor of the solid tumor cells needs to be proved by in vivo
models, since the solid tumor environment is a more
complicated system than the monolayer culture in deli-
vering drugs to all of tumor cells. The observed non-cyto-
toxic effect of PBN with MCF-7 cells suggested that the
cytotoxic effect of PBN and CQPN against Hep-G2 cells
may be attributed to the nitrone moiety, and that the qui-
This disturbance of the cell cycle phases aroused our
interest to investigate the CQPN modulation of the necro-
sis and apoptosis ratio. Using a dual flow cytometry that
investigate both of the apoptosis via tracing the presence
of annexin-V protein and the necrosis via binding to PI
(Fig. 5). The results of the analysis demonstrated that
treatment with CQPN induced apoptosis ratio from
almost 15 to 40% of the cell count, in comparison with
the vehicle treated cells, which showed unchanged pat-
tern through time increment. The growth inhibition of
MCF-7 cells, S-phase and G2/M arrest and the induction of
apoptosis ratio are suggesting that the apoptosis is asso-
ciated to the block of DNA replication and/or mitosis.
Discussion
In oxidative stress, excessive production of ROS and RNS,
results in DNA damage and contributes to tumor initia-
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Figure 5. Analysis of apoptosis and necrosis (annexin-V-FITC/PI): MCF-7 cells treated with 14 lM CQPN. Representative flow cyto-
metric histograms at different intervals 0, 12, and 24 h are demonstrated in panels A, B, and C, respectively. The percentages of the
cell populations (D) were presented for the regions of lower left (R1, gray segment), lower right (R2, black segment), higher left (R3,
white segment), and higher right (R2, pointed segment). Values are means € S.D., n = 3.
noline molecule of CQPN may be responsible for its cyto- the chromatin is condensed and partitioned into multi-
toxicity against MCF-7 cells. On contrary, the observed ple fragments, and the cells are finally broken into multi-
concomitant enhanced proliferation in both of Raw ple membrane-surrounded bodies (apoptotic bodies) [26].
264.7 macrophages and lymphoblastic leukemia cells (T-
In the present study, the kinetic induction of apoptosis
lymphocytes) after treatment with CQPN and PBN may in CQPN-treated MCF-7 cells was triggered by a coincident
reveal a nitrone moiety-associated immunoproliferative disturbance in the cell cycle phases. Apoptosis was asso-
or immunostimulatory activity, an activity that needs to ciated with a detected sub-G0/G1 population and an accu-
be investigated in vivo.
mulation of cells in S-phase and a subsequent G2/M phase
Apoptosis and necrosis are two different forms of cell block, which suggested a mechanistic modulation in the
death, which are distinguished by well defined morpho- apoptosis factors. There are several possible speculated
logical and biochemical features. Necrosis is character- mechanisms that may explain the shifted mode of CQPN-
ized by cell swelling, disruption, and rapid disintegra- treated MCF-7 to apoptosis. Bcl-2 proteins and caspases
tion of the cell membrane, leading to the release of the play a crucial role in apoptosis, accordingly CQPN may
cellular content, which may result in an inflammatory increase the expression of bax, which in turn can induce
response [26], and lysis of intracellular organelles. Apop- apoptosis by suppressing the activity of Bcl-2, and/or
tosis on the other hand is a tightly regulated process con- modulate caspase expression [27]. CQPN may modulate
trolled by a hierarchical set of molecules. During apopto- the cyclin-dependent kinases responsible for the control
sis the cells undergo nuclear and cytoplasmic shrinkage, of both G1 and G2/M checkpoints and/or lead to deregula-
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Arch. Pharm. Chem. Life Sci. 2006, 339, 242–249
Antioxidant and Anti-Proliferation by a New Nitrone Derivative
247
nal reference. Accurate masses were obtained on HP mass spec-
trometer, which were recorded in the positive ion mode with
leucine enkephalin as an internal lock mass standard. Elemental
microanalysis was performed on a Perkin Elmer 2400 CHN Ele-
mental analyzer (Perkin Elmer Beaconsfield, UK).
tion of the transcriptional protein p53 that can induce
p21/WAF1 expression, which in turn inhibits cyclin-
dependent kinases for the control of both G1 and G2/M
checkpoints and triggers apoptosis [28]. However, addi-
tional mechanistic investigations of the molecular and
biochemical signals, which activate the cell cycle-asso-
ciated apoptosis are in need to be explored. Clinical uti-
lity of anticancer drugs may require a demonstrated in
vitro efficacy in the nanomolar range. Considering that
CQPN showed an anti-proliferative activity against MCF-7
cells in a micromolar range, one could suggest that addi-
tional modification of CQPN may increase its cytotoxicity
in a range that could be more promising in clinical trials.
This work is a novel trial to successfully synthesize a
new derivative of quinoline nitrone. Our results proved
an antioxidant activity of CQPN against different physio-
logical and non-physiological radicals. Similarly, CQPN
possesses a significant strong inhibitory property against
LPS-stimulated NO production in macrophage, which
suggested a possible anti-inflammatory activity of CQPN
that needs to be explored. CQPN treatment, in a micro-
molar range, resulted in a dose-dependent inhibition in
the growth of both of the solid tumor cell lines Hep-G2
and MCF-7 cell with higher potency against MCF-7 cells.
On contrary, CQPN exhibited a proliferative stimulatory
activity of immune cells including macrophages and
lymphocytes. Moreover, CQPN treatment led to a major
time-dependent disturbance in the cell cycle phases of
MCF-7 cells including arrest in both S- and G2/M-phases.
This disturbance was found to be associated with a kineti-
cally induced apoptosis.
Chemistry
Preparation of C-(2-chloroquinoline-3-yl)-N-Phenyl
nitrone (CQPN)
The preparation of 2-chloroquinoline-3-carboxaldehyde (2 in
Fig. 1C) was performed as reported previously [29]. In brief, phos-
phorus oxychloride (107.31 g) was added to a solution of acetani-
lide (13.5 g; 1 in Fig. 1C) in dimethylformamide (18.25 g). The
mixture was heated at 75 8C for 6 h, then allowed to cool to
room temperature and poured on 1 L of ice-water. The obtained
precipitate was collected and recrystallized from ethyl acetate to
give 10.92 g of 2 as pale yellow crystals in 57% yield, m.p. 145–
147 8C. A solution of N-phenylhydroxylamine (0.11 g) in absolute
ethanol was added to a stirred solution of 2 (0.19 g) in absolute
ethanol (5 mL) at 70 8C. After 2 h of continuous stirring at room
temperature, the obtained crystals were filtered, washed, dried
and re-crystallized from absolute ethanol to give CQPN (3 in
Fig. 1C). The structure of CQPN was confirmed by elemental
analysis and spectral data.
The preparation of CQPN afforded 0.19 g (68% yield) of pure
yellow crystals with m.p. 124–126 8C. The spectral analysis data
1
indicated that H-NMR (400 MHz, CDCl3-d6) d: 7.24–8.04 (m, 9H,
Ar-H), 8.59 (s, 1H, CH), 10.48 (s, 1H, CH). 13C-NMR (125 MHz,
CDCl3) d:121.66 (CH), 122.35 (CH), 124.45 (CH), 127.03 (CH),
127.13 (CH), 127.44 (CH), 129.36 (CH), 129.68 (CH), 130.46 (CH),
132.08 (C), 136.45 (C), 146.36 (C), 147.55 (C), 149.17 (C). IR mmax
/
cm–1: 765 (C–Cl), 1020 (N–O), 1540, 1562 (C=C), 1608, 1648 (C=N),
3050 (Ar C–H). FAB-MS m/z: 284 [M+1]. C16H11ClN2O Anal. calcd.:
C16H11ClN2O; C, 67.97; H, 3.92; N, 9.91; Found: C, 67.72; H, 3.72;
N, 9.81.
Biology
We thank Prof. Mohammad A. Ali, Virology laboratory, NRC,
for his support, cooperation, and supply of facilities. This work
was funded by The National Research Centre (NRC), Cairo,
Egypt, and The Faculty of Pharmacy, El-Minia University, El-
Minia, Egypt.
Cell culture
Several cell lines were used throughout this study including:
human lymphoblastic leukemia (1301), a generous gift from The
Training Center of DakoCytomation, Elly, UK; human hepatocar-
cinoma (Hep G2); human breast carcinoma (MCF-7), both was a
generous gift from Prof. Mohamad A Ali, Virology laboratory,
NRC, Cairo, Egypt; and Raw murine macrophage (RAW 264.7) a
generous gift from Prof. Volker Schirrmacher, DKFZ, Heidelberg,
Germany. Cells were routinely cultured in Dulbeco’s Modified
Eagle’s Medium (DMEM), except RAW 264.7 cells, which were
grown in RPMI-1640. Media were supplemented with 10% fetal
bovine serum (FBS), 2 mM L-glutamine, containing 100 U/mL
penicillin G sodium, 100 U/mL streptomycin sulphate, and
250 ng/mL amphotericin B. Cells were maintained in humidi-
fied air containing 5% CO2 at 37 8C. Tested compounds were dis-
solved in DMSO (99.9%) and diluted 1000-fold in the assays. In all
the cellular experiments, results were compared with DMSO-
treated cells. Monolayer cells were harvested using trypsin/
EDTA, except RAW 264.7 cells, which were collected by scraping.
All experiments were repeated four times, unless mentioned,
and the data was represented as mean €S. D. All cell culture
material was obtained from Cambrex, BioScience (Copenhagen,
Denmark).
Experimental
General
All used reagents and solvents were obtained from Merck (Darm-
stadt, Germany) and Sigma (St. Louis, MO, USA). Melting points
were determined on Stuart electrothermal melting point appa-
ratus (England) and were uncorrected. TLC was performed on
analytical Merck 9385 silica glass plates with F254 indicator; TLCs
were viewed either under 254 nm UV or by staining with iodine
crystals. IR spectral data were obtained as KBr disks on a Bruker
Vector 22 IR spectrophotometer (Bruker, Rheinstetten, Ger-
1
many). H-NMR spectra were obtained on JNM-LA 400 FT-NMR
spectrophotometer (400 MHz; JEOL Ltd., Tokyo, Japan) using
TMS as internal reference. 13C-NMR spectra were performed on
Lambard NMR spectrophotometer (500 MHz), using TMS as inter-
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Arch. Pharm. Chem. Life Sci. 2006, 339, 242–249
30 min at room temperature. The stained cells were analyzed
using a MoFlo sorter (DakoCytomation, Glostrup, Denmark).
Antioxidant activity
Scavenging of DPPH radicals
The antioxidant activity of CQPN and PBN were studied preli-
minary through its scavenging activity against a non-physiologi-
cal radical, 1,1-diphenyl-2-picrylhyrazyl (DPPH), using the
method of [30] as modified from [31]. The percentage of DPPH
bleaching utilized for SC50 (half maximal scavenging concentra-
tion) was calculated, as being 0% is the absorbance of DPPH with
solvents (DMSO) and 100% is the absorbance of DPPH with an
efficient known scavenger (10 mM ascorbic acid, AA).
Apoptosis assay
Annexin V is a protein that binds to phosphatidylserine (PS) resi-
dues, which are exposed on the cell surface of apoptotic, but not
normal cells. In living cells, the distribution of the PS group in
the plasma membrane is asymmetrical, such that the groups are
directed toward the inside of the cell. During apoptosis, this
asymmetry is lost, and the PS groups are exposed to the exterior
of the cell membrane. The binding of PS with annexin V is there-
fore an established biochemical marker of apoptosis.
The oxygen radical absorbance capacity
Loss of phospholipid asymmetry and exposure of PS in the
outer leaflet of the cell membrane of MCF-7 cells was assayed
using FITC conjugated annexin V and propidium iodide (PI) dou-
ble staining using apoptest kit (DakoCytomation, Denmark). PI
can be used to assess plasma membrane integrity and cell viabi-
lity, where it is excluded from cells with intact plasma mem-
branes. MCF-7 cells (56105) growing on 6-cm culture dishes were
treated with IC50 concentration of CQPN (14 lM) for 0, 12 ,or
24 h, then collected by trypsinization, washed with PBS, spooled
with floating cells, and submitted to the kit procedure, as manu-
facturer instructions, and flow cytometric analysis using MoFlo
sorter.
The Oxygen Radical Absorbance Capacity (ORAC) of tested
nitrones against peroxyl, and hydroxyl (OH9) radicals, was inves-
tigated by the ORAC assay [32] adapted to a 96-well plate format
and modified at [33]. b-Phycoerythrin (b-PE) was used as a radical-
sensitive fluorescent indicator protein, 2,2-azobis-(2-amidino-
propane) dihydrochloride (AAPH) as a peroxyl-radical generator,
and a mixture of H2O2 – CuSO4 as a hydroxyl-radical generator. A
final concentration of CQPN and PBN (1 lM) was used, and the
reaction was initiated by addition of one of the radical genera-
tors. The decay of b-PE fluorescence was measured kinetically, at
37 8C or 2 h using a microplate fluorescence reader FluoStarOp-
tima, (BMG Labtechnologies, Durham, NC, USA). One ORAC unit
equals the net protection of a-PE produced by 1.0 lM trolox.
Nitrite assay
References
The accumulation of nitrite is an indicator of the synthesis of
the cellular nitric oxide (NO), which was measured in the culture
medium by Griess reaction as described in [30]. Raw 264.7 cells
were grown in phenol red-free RPMI, containing 10% FBS. Cells
were incubated for 2 h with bacterial lipopolysaccharide (LPS,
1 lg/mL) before being incubated for another 24 h with CQPN
(14 lM), PBN (14 lM) or with DMSO. Fresh culture medium was
used as the blank in all experiments. A standard curve was
blotted using serial concentrations of freshly prepared sodium
nitrite in culture medium. Results were normalized to the cell
protein content as measured by bicinchoninic acid [34].
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