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Apoptosis-Inducing Effects of Melissa officinalis L. Essential Oil in Glioblastoma Multiforme Cells

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Current therapies for glioblastoma multiforme (GBM) are not effective. This study investigated the activity of the M. officinalis essential oil (EO) and its major component (citral) in GBM cell lines. Both EO and citral decreased the viability and induced apoptosis of GBM cells as demonstrated by DNA fragmentation and caspase-3 activation. Antioxidant prevented citral-induced death, indicating its dependence on the production of reactive oxygen species. Citral downmodulated the activity and inhibited the expression of multidrug resistance associated protein 1 (MRP1). These results show that EO, through its major component, citral, may be of potential interest for the treatment of GBM.
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Cancer Investigation, Early Online:1–10, 2014
ISSN: 0735-7907 print / 1532-4192 online
Copyright C
2014 Informa Healthcare USA, Inc.
DOI: 10.3109/07357907.2014.905587
ORIGINAL ARTICLE
Apoptosis-Inducing Effects of Melissa officinalis L. Essential
Oil in Glioblastoma Multiforme Cells
Rafaela Muniz de Queiroz,1Christina Maeda Takiya,1L´
ıvia Paes Tavares Pacheco Guimar˜
aes,1Gleice da
Grac¸a Rocha,1Daniela Sales Alviano,2Arie Fitzgerald Blank,3Celuta Sales Alviano,2
and Cerli Rocha Gattass1
Laborat´
orio de Imunologia Celular, Instituto de Biof´
ısica Carlos Chagas Filho, Centro de Ciˆ
encias da Sa´
ude, Universidade Federal
do Rio de Janeiro, Cidade Universit´
aria, Rio de Janeiro, RJ, Brazil,1Laborat´
orio de Estruturas de Superf´
ıcie de Microorganismos,
Instituto de Microbiologia Professor Paulo de Gois, Centro de Ciˆ
encias da Sa´
ude, Universidade Federal do Rio de Janeiro, Cidade
Universit´
aria, Rio de Janeiro, RJ, Brazil,2Departamento de Engenharia Agronˆ
omica, Universidade Federal de Sergipe (UFS), S˜
ao
Crist´
ov˜
ao, SE, Brazil3
Current therapies for glioblastoma multiforme (GBM) are not
effective. This study investigated the activity of the M.
officinalis essential oil (EO) and its major component (citral) in
GBM cell lines. Both EO and citral decreased the viability and
induced apoptosis of GBM cells as demonstrated by DNA
fragmentation and caspase-3 activation. Antioxidant
prevented citral-induced death, indicating its dependence on
the production of reactive oxygen species. Citral
downmodulated the activity and inhibited the expression of
multidrug resistance associated protein 1 (MRP1). These results
show that EO, through its major component, citral, may be of
potential interest for the treatment of GBM.
Keywords: Chemotherapy, Gliobastoma multiforme, Melissa
officinali essential oil, Citral
INTRODUCTION
Lemon balm (M. ocinalis L.) is a medicinal plant largely
used in Europe and the Mediterranean region as a tea, in
aqueous and alcoholic extracts or steeped in wine. Its essen-
tial oil (EO) is considered to be responsible for the phar-
macological properties of M. ocinalis such as antitumoral
and antibacterial (1, 2). Interestingly, geranial and neral,
two isomers of the monoterpene citral (3,7-dimethyl-2,6-
octadienal), represent more than 85% of the M. ocinalis
EO (1). Citral, a key component of essential oils extracted
from several herbal plants, is used as a food additive and a
fragrance material in cosmetics. It also displays biological ac-
tivities, including antitumor activity (3, 4). Although the anti-
neoplastic activity of M. o cinalis essential oil and citral has
already been described, their activity on glioblastoma multi-
forme (GBM) cell lines has not been investigated.
Correspondenceto:CerliRochaGattass,PhD,Laborat
´
orio de Imunologia Celular, IBCCF, CCS Bloco G, UFRJ, Rio de Janeiro, RJ, Brazil, email:
cerli@biof.ufrj.br
Received 26 January 2013; revised 13 January 2014; accepted 13 March 2014.
In general, the cytotoxic effect of many chemotherapeutics
is mediated by apoptosis. The apoptotic process is character-
ized by a series of morphological alterations and biochemical
reactions leading to DNA fragmentation and breakdown of
thecellintoapoptoticbodies.Activationofinitiatorcaspases
(caspases 8 and 9), triggered either by activation of death re-
ceptors on the plasma membrane (extrinsic pathway) or by
stress signals/alterations of the mitochondrial membrane po-
tential (intrinsic pathway), induces the activation of effector
caspases(caspases3,6,and7),themainexecutorsofapopto-
sis (5). It was recently proposed that the generation of reac-
tive oxygen species (ROS) also plays an essential role in the
apoptotic cell death induced by cytotoxic drugs (6).
Glioblastoma multiforme (GBM) is the most common
and aggressive glioma, representing 50% of all gliomas and
more than 40% of all central nervous system (CNS) tumors.
The standard GBM treatment, which consists of surgical re-
section, radiation and/or chemotherapy, is rarely curative (7).
The location of GBM in the central nervous system and the
lack of clear margins prevent complete resection of the tumor.
Furthermore, tumor cell resistance to chemoradiation con-
tributes to the poor prognosis of the disease. Thus, despite
the significant improvement in early diagnosis, the tumor’s
aggressive growth and resistance to available therapies have
hindered changes in the outcome of GBM (8).
Drug resistance is one of the major obstacles to success-
ful GBM treatment. Among several mechanisms underlying
GBM drug resistance, the expression of transporter proteins
from the ABC superfamily is the most relevant. These pro-
teins actively remove drugs from cells, decreasing their in-
tracellular concentration and preventing death. Although all
transporter proteins are present in GBM cells, members of
the MRP family seem to be important for drug resistance,
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and their expression has been correlated with poor progno-
sis (9–11). Therefore, new therapies, able to overcome mech-
anisms of drug resistance, should be of interest for GBM
treatment. In recent years, several substances isolated from
plantswereshowntomodulatedrugresistanceincancercells
(12–14). We have previously shown that the M. ocinalis
EO was active against several cancer cell lines (1), including
a drug-resistant lung cancer cell line. Here, we investigated
the in vitro effects of M. ocinalis EO and of its major com-
ponent, citral, on human GBM cell lines. The data obtained
demonstrated that EO induces apoptosis of GBM cells and
that this effect may be due to its major component, citral.
These results support the potential usefulness of M. oci-
nalis EOandcitralforthetreatmentofpatientswithrecur-
rent GBM.
MATERIALS AND METHODS
Reagents
The essential oil (EO) from M. ocinalis L. (exsiccate
#46,008 deposited at the Herbarium of the Federal Univer-
sity of Uberlandia, MG, Brazil) was obtained as described
previously (1). Briefly, leaves from a culture of M. ocinalis
L. established at the Research Station “Campus Rural da
UFS,” Federal University of Sergipe, Brazil, were harvested
at 09:00 hr and dried at 40C until complete dehydration,
and the essential oil was obtained by hydrodistillation in
a Clevenger-type apparatus. The composition of the oil
has been described elsewhere (1). Citral (MW 152.23)
provided by Sigma-Aldrich Chemical Co. (Saint Louis, MO,
USA), was dissolved in dimethyl sulfoxide (DMSO, Sigma
Chemical Co., Saint Louis, MO, USA), stored at 20Cand
diluted in culture medium for use. 3-(4,5 dimethylthiozol-
2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, Sigma
Chemical Co., Saint Louis, MO, USA), verapamil, penicillin,
streptomycin, N-acetyl-L-cysteine (NAC), propidium iodide
(PI), and rhodamine 123 (Rho123) were purchased from
Sigma-Aldrich (Saint Louis, MO, USA). 5-carboxifluorescein
diacetate (5-CFDA) and 2,7-dichlorofluorescein diacetate
(H2DCFDA)wereobtainedfromCalbiochem(SanDiego,
CA, USA). MK-571 was provided by Enzo Life Science,
Inc. (Farmingdale, NY, USA). Dulbecco’s modified Eagle’s
medium (DMEM), fetal calf serum (FCS), and trypsin-EDTA
were from Gibco BRL (Carlsbad, CA, USA). Caspase-3 and -9
assaykits(CaspGlow)werefromBiovision(MountainView,
CA, USA). 4,6-Diamidino-2-phenylindole dihydrochloride
(DAPI) was from Santa Cruz Biotechnology (Santa Cruz,
CA, USA) and Apoptag Fluorescein Direct In Situ Apoptosis
Detection from Millipore (Billerica, MA, USA).
Cell cytotoxicity assay
Human glioblastoma multiforme cell lines A172 and U87
weregrowninDulbeccosmodifiedEaglesmedium(DMEM)
supplemented with 10% heat-inactivated fetal calf serum
(FCS), 100 U penicillin and 100 mg/mL streptomycin in dis-
posable plastic bottles at 37Cwith5%CO
2.Cellsweresub-
cultured using trypsin-EDTA every 3–4 days.
Cell cytotoxicity was evaluated using the MTT assay. Cells
were plated at a density of 1 ×104cells/well (for 48 hr as-
say) or 2 ×104cells/well (for 24 hr assay) in 96-well plate
overnight and then treated with medium or various dilu-
tions of the EO or citral (1:200,000; 1:100,000; 1:50,000 or
1:25,000). Four hours before the end of the treatment, cells
were incubated with MTT (2.5 mg/mL) and kept in the dark
at 37C until the end of the treatment. The formazan pro-
duced by the reduction of MTT by viable cells was dissolved
in DMSO, and the optical density was measured with an
ELISA reader (BenchMark, Bio-Rad, CA) at 570 nm (refer-
ence filter 630 nm). Experiments were repeated at least three
times. The results were expressed as percentage of the con-
trol, considered to be 100%. To determine whether the use
of DMSO in first dilution of EO and citral would interfere
with cell viability, the same assay was performed with DMSO
in the same dilutions used for the OE and citral. No inter-
ferenceoncellviabilitywasobservedwiththehighercon-
centration (0.2%) of DMSO used. For cotreatment experi-
ments, plated cells were incubated for 48 hr with 2.5 μg/mL
cisplatin (CIS), 100 nM vincristine (VCR) or 0.5 μMdoxoru-
bicin (DOX) in the presence of absence of different dilutions
of citral (1:100,000; 1:50,000, and 1:30,000) and cell viability
was assessed by MTT. The dilutions of OE used: 1:200,000;
1:100,000; 1:50,000; and 1:25,000 correspond to 4.6; 9.2; 18.4;
and 36.8 μg/mL, respectively, while the dilutions of citral:
1:200,000; 1:100,000; 1:50,000; 30,000; and 1:25,000 corre-
spond to 4.2; 8.4; 16.9; 28.1, and 33.7 μg/mL or 27.7; 55.4;
110.8; 182.8, and 221.6 μM, respectively.
This work was preceded by an evaluation of the effect of
different concentrations of citral on GBM cell viability. We
did not include aldehydes control experiments in this study
because an antitumoral effect of citral with concentration
higher than that used here was shown previously (3,4).
Apoptosis assays
Apoptosis was assessed by DNA fragmentation and TUNEL
assays. DNA fragmentation was evaluated by cell cycle anal-
ysis using flow cytometry. Twenty-four hours after plating,
the cells (1 ×104cells/well - 96-well plate) were treated with
medium (control) or various dilutions of citral (1:200,000;
1:100,000; 1:50,000 or 1:25,000 equivalent to 4.2; 8.4; 16.9 and
33.7 μg/mL or 27.7; 55.4; 110.8 and 221.6 μM, respectively)
and incubated for different times. Next, cells were harvested,
resuspended in a hypotonic fluorescent solution (50 mg/mL
PI and 0.1% Triton X-100 in 0.1% Na citrate buffer) for 1 hr
in the dark at 4C. The cell cycle was analyzed by flow cytom-
etry (FL-2) (FACSCalibur, Becton Dickinson, San Jose, CA)
to determine the sub-G0/G1 DNA content. Subdiploid popu-
lations were considered to be apoptotic. Data acquisition and
analysis were controlled by CellQuest software, version 3.1f.
Theresultsarepresentedasrepresentativehistogramsandas
the mean ±SDofthepercentageofthefragmentedDNA.
The TUNEL assay was performed using a commercial kit
(ApopTag RFluorescein Direct In Situ Apoptosis Detection
Kit, cat. #S7160) according to the instructions of the man-
ufacturer (Millipore, Billerica, MA, USA). An image analy-
sis system composed of a digital camera (Evolution Media
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M. OFFICINALIS EO I A  G
Cybernetics Inc., Bethesda, MD) and a computer with the
graphical interface software Q-Capture 2.95.0, version 2.0.5,
(Silicon Graphic Inc., USA) coupled to an epifluorescence
microscope was used to obtain high-quality images (2048 ×
1536 pixel buffer) using the 40×objective lens. At least a hun-
dred cells per cover slip were captured, and the percentage of
TUNEL-positive nuclei was calculated from the total DAPI-
positive nuclei. The results are expressed as the percentage of
reactive tissue in the total area (mean ±SD).
Caspase activation assay
Activation of caspase-3 and -9 were assayed using CaspGlow
commercial kits according to the instructions of the man-
ufacturer (Biovision, Mountain View, CA, USA). In brief,
cells (5 ×104/well) were incubated for 24 hr with medium
(control) or different dilutions of EO (1:25,000 equivalent to
36.8 μg/mL) or citral (1:50,000 or 1:25,000 equivalent to 16.9
or 33.7 μg/mL or to 110.8 or 221.6 μM, respectively) be-
fore being harvested, centrifuged, and suspended in the cas-
pase assay solution. This solution contained a molecule con-
jugated to FITC that is cell permeable, nontoxic, and binds
irreversibly to the activated caspase. After 1 hr of incubation
(37C, 5% CO2), cells were washed twice with washing buffer,
andthepercentageofcaspase-activatedcellswasanalyzedby
flow cytometry (FL-1). The results are presented as represen-
tative histograms and as the mean ±SD of the Ratio of Flu-
orescence Intensity (RIF) compared with the control.
Quantification on Reactive Oxygen Species (ROS)
ROS was determined by flow cytometry in cells treated with
H2-DCFDA. Cells (2 ×104cells/well) were plated for 24 hr
and then exposed to medium (control) or citral (1:25.000)
for different times. After the desired time, cells were har-
vested, washed with PBS, pH 7.4, and resuspended in 0.16 mL
PBS containing 20 μMH
2-DCFDA. After 30-min incuba-
tion at 37C, the production of ROS was evaluated by flow
cytometry (FL-1). The results are presented as the mean ±
SD of Ratio of Fluorescence Intensity (RIF) compared with
the control. To assess the role of ROS in citral citotoxicity
A172cellsweretreatedfor24hrwithmedia,pretreatedornot
for 2 hr with the ROS inhibitor N-acetyl-L-cysteine (NAC,
10 mM) and then incubated with citral (1:30.000 equivalent
to 28.1 μg/mL or 182.8 μM). Cell viability was estimated by
the MTT assay and results were presented as percentage of
control.
Activity and expression of MDR proteins
The activity of the MDR proteins was determined by the
accumulation of specific substrates as described previously
(15). Rho123 and 5-carboxyfluorescein diacetate (CFDA),
a nonfluorescent molecule that is converted into the fluo-
rescent carboxy-fluorescein (CF) by intracellular esterases,
were used to measure the activity of Pgp/ABCB1 and
MRP1/ABCC1, respectively. For each experiment, cells (2
×104/well)wereplatedfor24hrat37
C/5% CO2to allow
the culture to stabilize. Cells were incubated for 30 min with
medium (to measure autofluorescence), 400 nM Rho123 or
3μM CFDA in the presence of medium, inhibitors of Pgp
(50 μM verapamil) or MRP1 (50 μM MK-571), or the indi-
cated concentrations of citral (1:50,000; 1:30,000 or 1:25,000
equivalent to 16.9; 28.1 or 33.7 μg/mL, or to 110.8, 182.8 or
221.6 μM, respectively). The cells were washed in PBS, har-
vested, and kept on ice until flow cytometry analysis. The
results are presented as representative histograms or as the
mean ±SDofthemeanfluorescenceintensity(MIF).
Expression of MRP1 protein was assessed by western
blotting. Cells (5 ×105) were platted in 6-wells plates and
24 hr later incubated with medium or citral (1:50,000 or
1:30,000 equivalent to 16.9 or 28.1 μg/mL or to 110.8 or
182.8 μM,respectively)foranother24hr.Totalcelllysates
were made by lysing harvested cells in lysis buffer (50 mM
Tris pH 7.4, 0.5% Nonidet P-40, 1mM EDTA, 150 mM NaCl,
and proteases inhibitors added at the time of preparation).
Samples were fractionated by 8% polyacrylamide gel. The
proteins were transferred on to nitrocellulose membrane
(Bio-Rad Laboratory Inc., Hercules, CA, USA) which was
blocked for 1 hr with Tris-buffered saline containing 0.1%
Tween 20–milk 5%. Next, the membrane was incubated
overnight at 4C, with anti-MRP1 (A23, Alexis Biochemicals,
Figure 1. Eects of M. ocinalis essential oil (EO) and citral on the
viability of GBM cells. A172 and U87 cells (104/well) were incubated
with medium or dierent dilutions of EO or citral [1:200,000 (200),
1:100,000 (100), 1:50,000 (50), 1:25,000 (25)]. Aer 48 hr, cell viability
was assessed by MTT as described in the M&M. Results are expressed
as a percentage of the control and represent the mean ±SD of three
independent experiments. ,p<.05; ∗∗,p<.01. OE dilutions corre-
spond to 4.6; 9.2; 18.4; and 36.8 μg/mL respectively, while citral dilu-
tions correspond to 4.2; 8.4; 16.9 and 33.7 μg/mL or 27.7; 55.4; 110.8
and 221.6 μM, respectively.
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SanDiego, CA) or anti-β-actina (Sigma Chemical Co., Saint
Louis, MO, USA) antibodies. Following a series of washes
with Tris-buffered saline - 0.1% Tween 20, a secondary
horseradish peroxidase antibody was added and incubated
for 1 hr at room temperature. After washing the blot was
visualized by chemiluminescence detection using the en-
hanced chemiluminescence (ECL) system (GE Healthcare,
Tokyo) according to manufacture’s instructions.
Figure 2. EO and citral induce apoptosis in GBM cells. Cells (104/well) were treated with medium or citral [1:200,000 (200), 1:100,000 (100), 1:50,000
(50), 1:25,000 (25)] for the indicated times. DNA fragmentation was assessedbyowcytometryincellsstainedwithPI,andthesub-G1peakwas
considered apoptotic. (a) Representative histograms of A172 cells treated with increasing dilutions of citral aer 48 hr. (b and c) Quantitative
analysis of DNA fragmentation of A172 and U87 cells treated with the indicated doses of citral for 12, 18, 24, and 48 hr. (d) Quantitative analysis
of apoptotic A172 cells treated with EO or citral at 1:25,000 for 24 hr then submitted to the TUNEL assay as described in M&M. e data represent
the average of at least three independent experiments ±SD. ,p<.05; ∗∗,p<.01. Citral dilutions correspond respectively to: 27.7, 55.4, 110.8, and
221.6 μM.
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M. OFFICINALIS EO I A  G
Statistical analysis
All the data reported here are expressed as the mean ±
SD from three independent experiments. A significant dif-
ference from the respective control for each experimental
test condition was assessed by one-way analysis of variance
(ANOVA) using GraphPad Prism 4.0 software. Values of p<
.05 were considered statistically significant.
RESULTS
M. officinalis essential oil and citral inhibited the viability
of GBM cells
The cytotoxic effect of M. ocinalis EOandcitralonGBM
cell lines A172 [Figure 1(A)] and U87 [Figure 1(B)] were an-
alyzedbytheMTTassay.Cellsweretreatedwithmediaand
different dilutions of the oil (1:200,000; 1:100,000; 1:50,000
or 1:25,000 equivalent to 4.6; 9.2; 18.4 or 36.8 μg/mL, re-
spectively) for 48 h, and cell viability was measured by the
MTT assay. Treatment with EO reduced the number of viable
cells in both cell lines in a dose-dependent manner (Figure
1 - white bars). Because neral and geranial, the two isomers
of citral, represent more than 85% of the EO, we investigated
whether the cytotoxic effect of EO is due to the action of these
components.Todothis,theviabilityofGBMcellstreated
with commercial citral (1:200,000; 1:100,000; 1:50,000 or
1:25,000 equivalent to 4.2; 8.4; 16.9; 28.1 or 33.7 μg/mL or to
27.7, 55.4, 110.8, or 221.6 μM, respectively) was determined
under the same conditions used for the EO (Figure 1 - black
bars). The data showed that the effect of citral on the GBM
cells lines U87 and A172 is similar to or even higher than that
of the EO, suggesting that the activity of the EO against the
tumor cells may be due to the action of citral.
Essential oil and citral induce apoptosis in GBM cells
Morphological analysis of GBM cells treated with EO or cit-
ral (1:50,000 equivalent to 18.4 μg/mL or 110.8 μM) for 48
hr showed a decrease in cell number accompanied by the
presence of “blebs” in the plasmatic membrane, a cellular
cyto-architecture indicative of apoptosis (data not shown).
To further investigate this hypothesis, GBM cells were treated
with different concentrations of citral, and DNA fragmenta-
tion was evaluated by flow cytometry. The sub-G1 peak of
the cell cycle was considered apoptotic. As depicted in Fig-
ure 2(a), cell-cycle histograms of A172 showed that treatment
with citral for 48 hr led to a significantly dose-dependent
increase in the sub-G1 phase [Figure 2(b)]. The DNA frag-
mentation induced by treatment with citral was also time-
dependent [Figure 2(c)]. Because similar results were ob-
tained with U87 [Figure 2(c)], only the A172 cell line was
used in the next experiments. To confirm the indication of
induction of apoptosis by citral and EO, A172 cells were sub-
mittedtotheTUNELassay.Thedataobtaineddemonstrated
that treatment with both EO and citral (1:25,000 equivalent
to 36.8 and 33.7 μg/mL for EO and citral, respectively) for
Figure 3. Activation of caspases by EO and citral. Plated A172 cells (5 ×104/well) were treated for 24 hr with medium, EO [1:25,000 (25)] or citral
[1:50,000 (50) and 1:25,000 (25)]. Aer treatment, cells were harvested, and caspase activity was measured using a commercial kit as described
in M&M. e results show representative histograms and quantitative analysis of caspase-9 (a) and -3 (b). Results represent the average of three
independent experiments ±SD. ,p<.05; ∗∗,p<.01. For OE and citral concentrations see legend for Figure 1.
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24 hr causes an increase in the number of cells positively la-
beled for TUNEL, confirming that both compounds induce
apoptosis in GBM cells [Figure 2(d)].
To determine whether the induction of apoptosis in GBM
cells was dependent on caspase activation, A172 cells were
treated with EO (1:25,000 equivalent to 36.8 μg/mL) or cit-
ral (1:50,000 or 1:25,000 equivalent to 18.4 or 36.8 μg/mL or
to 110.8 or 221.6 μM) for 24 hr, and the levels of activated
caspase-9 and -3 were measured by a commercial kit (Casp-
Glow, Biovision) containing a fluorochrome conjugated to a
molecule that irreversibly binds to the activated caspase. As
showninFigure3(aandb),activationofcaspase-9and-3
were detected in cells treated with either citral or EO. Citral
induces a dose-dependent activation of caspases.
Apoptosis induced by citral is mediated by ROS
To evaluate whether the generation of ROS was involved in
citral-induced apoptosis, A172 cells were treated with citral
(1:25,000 equivalent to 33.7 μg/mL or 221.6 μM) for differ-
ent times, and H2DCF-DA was used to examine ROS gener-
ation. As shown in Figure 4(a), treatment with citral led to a
rapid increase in ROS generation that peaked at 6 hr and re-
turned to normal levels at 24 hr. Evaluation of cell death, per-
formed in a parallel experiment, revealed that DNA fragmen-
tation begins to increase after 8 hr of citral treatment. Thus,
the increase in the ROS levels precedes cell death [Figure
4(a)], suggesting a role for oxidative stress in citral-induced
cell death. To confirm this hypothesis, cells were treated
for 24 hr with citral (1:30,000 equivalent to 28.1 μg/mL or
182.8 μM)inthepresenceorabsenceoftheantioxidantN-
acetyl-cysteine (NAC), and cell viability was measured by
MTT. As shown in Figure 4(b), cellular death induced by cit-
ral is completely blocked by the ROS scavenger, indicating
that the generation of ROS is an essential process in citral-
induced apoptosis.
Citral inhibits MRP1 activity and expression
Next, we investigated whether citral was able to modulate the
activity of MDR transporter proteins expressed by A172 and
U87 cells. The activity of Pgp/ABCB1 and MRP1/ABCC1 in
these cells was evaluated by their ability to retain substrates
ofthepumps.Todothis,cellswereloadedwithsubstratesfor
Pgp (Rhodamine-123) or MRP1 (5-CFDA), in the presence
or absence of specific inhibitors (verapamil or MK571, re-
spectively), and the cell fluorescence, indicative of the pump
activity, was measured by flow cytometry [Figure 5(a and b)].
While no increase in fluorescence was observed when cells
were incubated with Rho-123 in the presence of verapamil
[Figure 5(b)], an increase in fluorescence was observed when
cells were incubated with 5-CFDA in the presence of MK571
[Figure 5(a)]. These data demonstrated that MRP1/ABCC1 is
activeinA172cellsbutPgp/ABCB1isnot.U87cellsshowed
thesamepatternofactivityforthesepumps[Figure6(aand
b)]. To investigate the effect of citral on MRP1, cells were
treated with 5-CFDA and different concentrations of citral
(1:50,000, 1:30,000 or 1:25,000 equivalent to 16.9, 28.1 or
33.7 μg/mL or to 110.8, 182.8, 221.6 μM) for 30 min, and the
intracellular fluorescence was measured [Figure 5(C)]. The
Figure 4. Involvement of ROS in citral-induced cell death. (a) ROS
generation and DNA fragmentation. A172 cells (2 ×104/well) were
treated with medium or citral (1: 25,000) for dierent times, and ROS
generationandDNAfragmentationweremeasuredbyowcytome-
try. Results are presented as the ratio of the uorescence of citral over
control cells [ROS production ()] and the percentage of DNA frag-
mentation (). (b) Eect of NAC. A172 cells were treated with citral
(1:30.000) for 24 hr in the presence or absence of NAC (10 mM), and
cell viability was estimated by the MTT assay as described in Section
2 of the M&M. Results are expressed as percentage of the control and
represent the mean ±SD of three independent experiments. ,p<.05;
∗∗,p<.01; and ∗∗∗ ,p<.001. Citral dilutions correspond respectively
to: 221.6 and 182.82 μM.
increase in fluorescence observed in presence of citral [Figure
5(d)], indicates that this substance is able to downmodulate
the activity of MRP1. Indeed, the modulatory effect of citral is
approximately 50% of that observed with the commercial in-
hibitor MK571. To probe if the modulation of MRP1 by citral
was due to a decrease in protein expression, A172 cells were
treated with medium or citral (1:50,000 or 1:30,000 equiva-
lent to 16.9 or 28.1 μg/mL or to 110.8 or 182.8 μM) for 24 hr
and protein expression was evaluated by western blotting. As
shown in Figure 5(e) citral decrease the expression of MRP1.
To confirm the effect of citral on MRP1, U87 cells were
treated with 5-CFDA and citral (1:25,000 equivalent to
33.7 μg/mL or 221.6 μM)for30min,andtheintracellu-
lar fluorescence presence of citral [Figure 6(d)] indicates that
this substance is able to downmodulate the activity of MRP1.
The effect of citral was similar to that observed for A172.
Effects of combination of citral with chemotherapy drugs
Next, we evaluate if the combination of citral with an-
tineoplastic drugs would decrease the survival of GBM
cells. For this, A172 cells were cocultured for 48 hr with
different dilutions of citral (1:100,000, 1:50.00, and 1:30,000
corresponding to 8.4, 16.9, and 22.1 μg/mL or to 55.4,
110.8, and 182.8 μM) and 2.5 μg/mL cisplatin (CIS), 100
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M. OFFICINALIS EO I A  G
Figure 5. Activity of the eux pumps. A172 cells (2 ×104/well) were incubated for 30 min with medium (autouorescence), 3 μM5-CFDAor
400 ng/mL Rho-123 in the presence or absence of 50 μM MK751 (MK) or 50 μM verapamil (VRP), respectively. e retention of specic substrates
by the cells was evaluated by ow cytometry. (a and b) are representative histograms of the activity of MRP1 (a) and Pgp (b). (c and d) show the
eect of citral on the MRP1 pump. Cells were incubated for 30 min with medium (autouorescence) or 3 μMCFDAinthepresenceorabsence
of 50 μM MK751 (MK) or increasing concentrations of citral [1:50,000 (Cit 50); 1:30,000 (Cit 30) or 1:25,000 (Cit 25)], and cell uorescence was
measured by ow cytometry. e results are expressed as representative histograms of cell uorescence for each condition (c) and by the mean ±
SD of the mean uorescence intensity (MIF) obtained in three dierent experiments (d). (e) shows the eect of citral on MRP1 expression. Cells
were incubated medium or citral 1:50,000 (50) or 1:30,000 (30) for 24 hr and lysates were submitted to western blot as described in M&M. Citral
dilutions correspond respectively to: 110.8, 182.82, and 221.6 μM.
nM vincristine (VCR) or 0.5 μg/mL doxorubicin (DOX)
and cell viability was assessed by MTT. Results show that
cotreatments with citral do not seem to sensitize GBM cells
to antineoplastic drugs.
DISCUSSION
Malignant central nervous system neoplasms, particularly
GBM,areamongthemostlethalandintractableofhuman
tumors. These tumors have defied all current therapeutic
modalities, and patient prognosis is poor (8), emphasizing
the need to explore new therapeutic strategies for treatment.
Thus,thesearchfornewdrugsthatareabletoovercome
GBM’s drug resistance mechanisms and prevent tumor re-
currence is of great interest for GBM therapy (16).
In the last few years, natural products have been recog-
nized as an important source of new antineoplastic drugs
(17). Accumulating evidence of the antitumor activity of es-
sential oils (18,19) supports this material as one of these drug
sources. In a previous paper (1), we showed that the EO of
M. ocinalis was cytotoxic to several cancer cell lines but its
effect on GBM cells was not investigated. Although the com-
position of the M. ocinalis EO was described in this paper,
the component responsible for the observed antitumor activ-
ity was not identified. The data presented in Figure 1 demon-
strated that MocinalisEO is also able to cause cell death
of established GBM cell lines (U87 and A172) and indicated
that this effect is due to its major component citral. The ca-
pacity to induce cell death in cancer cell lines is not a property
particular to M. ocinalis EO. This effect has already been
shown for EO from other sources (18, 19) as well as for citral
(3, 4).
Microscopic observation suggested that the cytotoxic ef-
fects of EO and citral on the GBM cell lines (U87 and A172)
were due to the induction of apoptosis. Measurement of
DNA fragmentation by cell cycle analysis [Figure 2(a–c)]
and TUNNEL assays [Figure 2(d)] corroborated this hy-
pothesis. The apoptotic effect of both EO and citral is dose-
dependent. M. ocinalis EO and citral also induced the acti-
vation of caspase-9 and -3 [Figure 3 (a and b)], indicating that
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R. M  Q  .
Figure 6. Activity of the eux pumps. U87 cells (2 ×104/well) were incubated for 30 min with medium (autouorescence), 3 μM5-CFDAor
400 ng/mL Rho-123 in the presence or absence of 50 μM MK751 (MK) or 50 μM verapamil (VRP), respectively. e retention of specic substrates
by the cells was evaluated by ow cytometry. (a and b) are representative histograms of the activity of MRP1 (a) and Pgp (b). (c and d) show the
eect of citral on the MRP1 pump. Cells were incubated for 30 min with medium (autouorescence) or 3 μMCFDAinthepresenceorabsence
of 50 μM MK751 (MK) or citral [1:25,000 (Cit 25)], and cell uorescence was measured by ow cytometry. e results are expressed in (c) as
representative histograms of cell uorescence for each condition and in (d), as mean uorescence intensity of the experiment shown in c. Results
represent two experiments. Citral dilution corresponds, respectively, to 221.6 μM.
their apoptotic effect is caspase-dependent. These data are in
agreement with studies showing the activation of caspase-3
by essential oils from other plants (19) and citral (3).
Mitochondria play a central role in the apoptotic pro-
cess induced by several drugs. The activation of caspase-9
[Figure 3(a)] indicated that the effects of M. ocinalis EO
and citral on GBM cells are mediated by the intrinsic (mi-
tochondrial) pathway of apoptosis. In addition to the release
of proapoptotic proteins, the generation of reactive oxygen
species (ROS) is used by the intrinsic pathway to induce
apoptosis in tumor cells. Due to the high content of ROS, it
has been proposed that insults leading to further ROS gener-
ation would turn cancer cells very susceptible to ROS dam-
age. Indeed, many cytotoxic agents employed in chemother-
apyseemtoexerttheireffectsthroughROSgeneration(6,
20, 21). Treatment of GBM cells with citral resulted in a rapid
generation of ROS that preceded the induction of cell death
[Figure 4(a)]. The addition of the antioxidant NAC prevented
citral-induced death, corroborating the dependence of citral-
induced death on ROS [Figure 4(b)].
Expression of transporter proteins from the ABC super-
family is one of the most relevant mechanisms of drug re-
sistance. Drug resistance is a severe limitation of the ef-
fect of chemotherapy on various malignancies. A 1998 study
(22) showing that GBM patients subjected to chemother-
apyhaveanincreaseincellspositiveforPgp/ABCB1and
MRP1/ABCC1, suggested a role for these ABC pumps in re-
sistance to treatment. The expression of MRP1 is considered
a negative prognostic factor for patients with GBM (10, 11).
Indeed, high levels of transporter proteins were also identi-
fied in cancer stem cells, a cell population described in recur-
rentGBMpatients(23).Inapreviouspaper(1),weshowed
that the EO of M. ocinalis caused cell death in several can-
cer cell lines including A549, a lung cancer cell line that ex-
pressed several members of the MRP family.
Data presented in this paper show that M. ocinalis OE
and its major component, citral, induced apoptosis of GBM
cell lines (A172 and U87) that expressed active MRP1. They
also indicated that citral induces production of ROS and in-
hibits MRP1 expression and activity [Figures 4 and 5 (c, d, e)].
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M. OFFICINALIS EO I A  G
Figure 7. Eects of cotreatment of GBM cells with citral and
chemetherapics. A172 cells (104/well) were incubated with medium
or dierent dilutions of citral [1:100,000 (100), 1:50,000 (50), 1:30,000
(30), equivalent to 55.4, 110.8, and 182.82 μM, respectively] in the
presence or absence or 2.5 μg/mL cisplatin (CIS), 100 nM vincristine
(Vin) or 0.5 μM doxorubicin (DOX). Aer 48 hr, cell viability was as-
sessed by MTT as described in the M&M. Results are expressed as a
percentage of the control and represent the mean ±SD of three inde-
pendent experiments.
Recent studies showed that temozolomide, the first choice of
chemotherapy drug for GBM treatment induces ROS pro-
duction(21)andthatresistancetothisdruginvolvedmi-
tochondriadysfunctionandadecreaseinROSproduction
(24). Other studies showing that inhibition of MRP1 activity
or expression (25–27) lead to an increase in the response of
GBM cell lines to chemotherapeutic drugs indicating the im-
portance of this protein in GBM drug resistance. Combina-
tion of citral and antineoplastic drugs does not seem to sen-
sitize GBM cells as the resulting decrease in cell viability is
equivalent to the added effect of both drugs (Figure 7). How-
ever, if a sensitizing effect does exist, it is hard to demonstrate
since citral modulation of MRP1 activity was only observed
at cytotoxic concentrations. In addition, it should be consid-
ered that the modulatory effect of citral on MRP1 activity is
quite low [Figure 5 (c, d)] when compared to inhibitors used
by other researchers (26,27). However, M. ocinalis EO and
citral are able to kill resistant cells that express MRP1 (this
work) or overexpress Pgp (1) but have no effect on noncancer
cells (3,28). Although the relationship between ROS produc-
tion and inhibition of MRP1 activity by citral was not investi-
gated in this work, data from the literature showed that com-
poundsabletoaffectROSorGSHproduction,alteringthe
cell oxidative stress, may also affect MRP1 expression and/or
its activity (29,30).
Efficient therapeutic treatments for highly malignant
gliomas, such as GBM, are lacking and patient prognosis
is poor. Thus, despite the inclusion of targeted drugs in
clinical protocols, no significant improvement over the
standard protocol has been observed (7, 31). As cancer cells
use several mechanisms to evade death, the simultaneous
attack to multiple targets is important for a successful cancer
therapy. Therefore, the results presented in this paper, show-
ing that M. ocinalis OE and citral affect two pathways of
drug resistance in GBM cell points to the potential use of
these compounds for the development of new drugs for this
neoplasia and as adjuvants for the treatment of glioblastoma
multiforme.
DECLARATION OF INTEREST
The authors report no declarations of interest. The authors
alone are responsible for the content and writing of this
article.
This study was supported by grants from Fundac¸˜
ao
deAmparoaPesquisadoEstadodoRiodeJaneiro
(FAPERJ) and Instituto Nacional para Pesquisa Transla-
cional em Sa´
ude e Ambiente na Regi˜
ao Amazˆ
onica/
Conselho Nacional de Desenvolvimento Cient´
ıfico e Tec-
nol´
ogico/MCT, Brazil (INCT-INPeTAm/CNPq/MCT). The
authors thank the Conselho Nacional de Desenvolvimento
Cient´
ıfico e Tecnol´
ogico (CNPq) for the graduate fellowships
awardedtoRafaelaMunizdeQueiroz,L
´
ıvia Paes Tavares
Pacheco Guimar˜
aes and Gleice da Grac¸a Rocha.
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... Subsequently, EOs cause a release of cytochrome C and induce a dysfunction of the B-cell lymphoma 2 (Bcl2)/Bcl2 associated X-protein (Bax) ratio with activation of caspases pathway and apoptotic cell death. However, these secondary metabolites can also induce cell cycle arrest by blocking the cyclin-dependent kinase 1 (CDK1)/cyclin complex or by suppressing mammalian target of rapamycin (mTOR) and protein pyruvate dehydrogenase kinase 1 (pPDK1) expression with subsequent phosphorylation of protein kinase B (PKB), blocking of oncogene MDM2 and increase of p21 expression [16,[67][68][69]. ...
... The primary focus is now to find bioactive natural products, which are able to target deregulated cellular signaling pathways in order to induce a maximized therapeutic effect. As a matter of fact, there is an excellent potential of plant-based products to inhibit proliferation, invasion, metastasis and induce apoptosis in experimental GBM models, either alone or in combination with other natural products or as adjuvants to chemotherapeutic drugs [67,69,82,91,100,101]. Several investigations explored the potential use of natural products (EOs and phenolic compounds) as therapeutic approaches for GBM together with their mode of action and molecular target (e.g., key signaling pathways involved in the regulation of cell proliferation, cell cycle regulation, and apoptosis inducement) ( Table 4). ...
... Most of these phytochemicals from EOs and/or phenolic compounds induced cell (Table 4). Others have the capacity to induce apoptosis by activating caspase-3, enhancing the expression of p53 and Bax, and decreasing the expression of Bcl-2, as well as reducing proliferation and invasion of GBM cells by promoting downregulation of matrix metalloproteinase-2 and − 9 (MMP-2 and − 9), hypoxia-inducible factor-1α (HIF-1α) and inhibition of nuclear factor-kappa B (NF-κB) or activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT [67,[104][105][106][107][108] (Table 4). Additionally, another huge advantage of natural products as therapeutic agents is their capability to modulate and cross the BBB to increase the treatment effect [109,110]. ...
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... Following the cell cycle arrest, MO induced apoptosis in several types of cancer cell lines in the forms of hydroethanolic, ethanolic, and essential oil extracts. 19,34,46 The same property was observed in the hot water extract that the MO treatment triggered apoptosis in CRC cells and caused PARP cleavage in a caspase-dependent fashion ( Figure 4). Although MO ethanolic extracts have been reported to inhibit cell migration in MDA-MB-231 breast cancer cells, 47 the anti-metastasis effect of the MO water extract remains unclear. ...
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Background Rosa alba L. belongs to the family Rosaceae. This species is widely cultivated in Europe, Asia, North America, and Northwest Africa due to its fragrance, ornamental, and medicinal values. It is commonly known as white oil-bearing rose, white rose, white rose of York, backyard rose, and sufaid gulab. Main text Rosa alba L. has many biological properties like antioxidant, antimicrobial, antifungal, antifertility, teratogenic, memory enhancing, cytotoxic, and genotoxic activities. The essential oil of Rosa alba L. possesses good antimicrobial activity and consists of many chemical constituents like- citronellol, geraniol, nerol, linalool, citral, carvacrol, eugenol, etc. Conclusion This article briefly reviews the cultivation, traditional uses, phytochemistry, and biological activities of Rosa alba L. Many research papers have been published on the proposed plant and still, there is a very vast scope of research on it. Therefore, this review will be very fruitful for those scientists who are doing or plan to do research work on this plant. All the scientific findings written in this review are explored from Google web, Google Scholar, PubMed, ScienceDirect, Medicinal and Aromatic Plants Abstracts (MAPA), and SciFinder. To date, it is the first systematic review article of such kind, on this plant.
... Russo et al. [39], reported that different essential oils showed anticancer activities in vitro and in vivo models underlining among them those whose activities are due to the phytocomplex. Clear evidences were reported on apoptosis induction mechanism exerted by essential oils: Melissa officinalis essential oil induced apoptosis of glioblastoma multiforme cells [40], Salvia milthiorriza treatment reduced the proliferation of HepG2 hepatoma cells, changing their morphology and inducing cell death by apoptosis [41], Artemisia annua essential oil induced apoptosis in SMMC-7721 hepatocarcinoma cells [42] and essential oil of the conifer tree Tetraclinis articulata showed pro apoptotic activity in human melanoma and ovarian cancer cell lines [43]. In our previous paper, LEO treatment has been reported to induce apoptosis on HL60 human leukemia cells and, among the main LEO compounds, both terpinen-4-ol and linalyl acetate possess antiproliferative activity [28]. ...
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... Moreover, citral should not affect ATPase activity in any of these pumps [38]. On the other hand, Queiroz et al. [40] observed that citral downmodulated the activity and inhibited the expression of multidrug resistance associated protein 1 (MRP1). In contrast to lemongrass essential oil, citral up to a concentration of 50 mL/L was unable to inhibit pure P-gp in the in vitro system and did not affect the DOX sensitivity of doxorubicin-resistant ovarian carcinoma over 72 h of incubation. ...
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Chapter
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