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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
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
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
aria, Rio de Janeiro, RJ, Brazil,2Departamento de Engenharia Agronˆ
omica, Universidade Federal de Sergipe (UFS), S˜
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
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.
orio de Imunologia Celular, IBCCF, CCS Bloco G, UFRJ, Rio de Janeiro, RJ, Brazil, email:
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
(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
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
(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.
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
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
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
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
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-
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.
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,
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
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
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
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.
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-
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
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-
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
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
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
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
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.
Malignant central nervous system neoplasms, particularly
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.
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-
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-
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-
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-
(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-
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
The authors report no declarations of interest. The authors
alone are responsible for the content and writing of this
This study was supported by grants from Fundac¸˜
(FAPERJ) and Instituto Nacional para Pesquisa Transla-
cional em Sa´
ude e Ambiente na Regi˜
ao Amazˆ
Conselho Nacional de Desenvolvimento Cient´
ıfico e Tec-
ogico/MCT, Brazil (INCT-INPeTAm/CNPq/MCT). The
authors thank the Conselho Nacional de Desenvolvimento
ıfico e Tecnol´
ogico (CNPq) for the graduate fellowships
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]. ...
Glioblastoma (GBM) is the most common primary tumor of the central nervous system. Current treatments available for GBM entails surgical resection followed by temozolomide chemotherapy and/or radiotherapy, which are associated with multidrug resistance and severe side effects. While this treatment could yield good results, in almost all cases, patients suffer from relapse, which leads to reduced survival rates. Thus, therapeutic approaches with improved efficiency and reduced off-target risks are needed to overcome these problems. Regarding this, natural products appear as a safe and attractive strategy as chemotherapeutic agents or adjuvants in the treatment of GBM. Besides the increasing role of natural compounds for chemoprevention of GBM, it has been proposed to prevent carcinogenesis and metastasis of GBM. Numerous investigations showed that natural products are able to inhibit proliferation and angiogenesis, to induce apoptosis, and to target GBM stem cells, which are associated with tumor development and recurrence. This review gives a timely and comprehensive overview of the current literature regarding chemoprevention and therapy of GBM by natural products with a focus on essential oils and phenolic compounds and their molecular mechanisms.
... 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. ...
... 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. ...
Full-text available
Colorectal cancer (CRC) is one of the most frequently diagnosed cancers worldwide. Lifestyle-related factors, such as diet, are associated with the development of CRC. Cumulating evidence indicates noticeable chemopreventive effects of phytochemicals on CRC, suggesting that drinking herbal tea potentially reduces the risk of distal colon cancer via its antiproliferative and anti-angiogenic activities. We examine the antitumor effects of nine components frequently found in herbal tea and uncover the underlying molecular mechanism. Among them, the hot water extract of Melissa officinalis (MO) exhibited the highest anticancer activity on CRC cells. We revealed that MO reduced cell proliferation, induced cell cycle arrest at the G2/M phase, triggered caspase-dependent apoptotic cell death, and inhibited cell migration ability by modulating the epithelial–mesenchymal transition in HCT116 CRC cells. To examine the metabolite composition in the MO hot water extract, we applied mass spectrometry-based analysis and identified 67 compounds. Among them, the phenolic compounds, including lignans, phenylpropanoids, and polyketides, are widely found in natural products and possess various bioactivities such as anti-inflammatory, antioxidation, and anticancer effects. The results indicate that herbal tea consumption benefits CRC prevention and management.
... The essential oil was found to be inactive up to 1000 mg/ml on root tip meristem cells isolated from Hordeum vulgare [32]. Generally, essential oils and their components are safe in low concentrations while several researchers have reported that the higher dose (500 mg/ml) of geranial (citral A) and geraniol are responsible for cytotoxic and genotoxic effects in human cells [33,34]. Geraniol inhibits prostate cancer growth by targeting cell cycle and apoptosis pathways [35]. ...
Full-text available
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]. ...
Full-text available
Lavandin essential oil (LEO), a natural sterile hybrid obtained by crossbreeding L. angustifolia × L. latifolia, is mainly composed by active components belonging to the family of terpenes endowed with relevant anti-proliferative activity, which can be enhanced by proper application of nanotechnology. In particular, this study reports the chemical characterization and the screening of the anti-proliferative activity on different human cell lines of pure and nano-formulated lavandin essential oil (EO). LEO and its formulation (NanoLEO) were analyzed by HS/GC-MS (Headspace/Gas Chromatography-Mass Spectrometry) to describe and compare their chemical volatile composition. The most abundant compounds were linalool and 1,8-cineole (LEO: 28.6%; 27.4%) (NanoLEO: 60.4%; 12.6%) followed by α-pinene (LEO: 9.6%; NanoLEO: 4.5%), camphor (LEO: 6.5%; NanoLEO: 7.0%) and linalyl acetate (LEO: 6.5%; NanoLEO: 3.6%). The cytotoxic effects of LEO and NanoLEO were investigated on human neuroblastoma cells (SHSY5Y), human breast adenocarcinoma cells (MCF-7), human lymphoblastic leukemia cells (CCRF CEM), human colorectal adenocarcinoma cells (Caco-2) and one normal breast epithelial cell (MCF10A) by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)-assay. Caco-2, MCF7 and MCF10A normal cells resulted more resistant to the treatment with LEO, while CCRF-CEM and SHSY5Y cells were more sensitive. The antiproliferative effect of LEO resulted amplified when the essential oil was supplied as nanoformulation, mainly in Caco-2 cells. Scanning and transmission electron microscopy investigations were carried out on Caco-2 cells to outline at ultrastructural level possible affections induced by LEO and NanoLEO treatments.
... 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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With strong antimicrobial properties, citral has been repeatedly reported to be the dominant component of lemongrass essential oil. Here, we report on a comparison of the antimicrobial and anticancer activity of citral and lemongrass essential oil. The lemongrass essential oil was prepared by the vacuum distillation of fresh Cymbopogon leaves, with a yield of 0.5% (w/w). Citral content was measured by gas chromatography/high-resolution mass spectrometry (GC-HRMS) and determined to be 63%. Antimicrobial activity was tested by the broth dilution method, showing strong activity against all tested bacteria and fungi. Citral was up to 100 times more active than the lemongrass essential oil. Similarly, both citral and essential oils inhibited bacterial communication and adhesion during P. aeruginosa and S. aureus biofilm formation; however, the biofilm prevention activity of citral was significantly higher. Both the essential oil and citral disrupted the maturated P. aeruginosa biofilm with the IC50 7.3 ± 0.4 and 0.1 ± 0.01 mL/L, respectively. Although it may seem that the citral is the main biologically active compound of lemongrass essential oil and the accompanying components have instead antagonistic effects, we determined that the lemongrass essential oil-sensitized methicillin-resistant S. aureus (MRSA) and doxorubicin-resistant ovarian carcinoma cells and that this activity was not caused by citral. A 1 mL/L dose of oil-sensitized MRSA to methicillin up to 9.6 times and a dose of 10 µL/L-sensitized ovarian carcinoma to doxorubicin up to 1.8 times. The mode of multidrug resistance modulation could be due to P-glycoprotein efflux pump inhibition. Therefore, the natural mixture of compounds present in the lemongrass essential oil provides beneficial effects and its direct use may be preferred to its use as a template for citral isolation.
Medicinal plants serve as a rich source of therapeutic modalities against various diseases especially cancer, which gained attention by researchers, due to the problems associated with available treatments. Indian traditional medicinal systems have a rich repository of medicinal plants and could serve as a source of safe and cost-effective alternative therapy. A major limitation with natural products is meager information about their mode of action, hindering their wider clinical use. Herein, we are focusing on anticancer potential of few medicinal plants based on common chemical constituents, viz. Peganum harmala, Quercus infectoria, Melissa officinalis, and Plumbago zeylanica, with well-cited use in folk medicines. Anticancer mechanism of these plants/active components has been well deciphered with targeted pathways involved in their anticancer potential. Peganum harmala and its active constituents showed involvement of various signaling pathways for anticancer effect. Cancer cell death by Quercus infectoria and its constituents also involved pathways such as AKT, NF-κB, and JAK/STAT. Melissa officinalis showed anticancer potential by affecting various transcription factors such as NF-κB, TNF-α, and COX-2. Anticancer activity mediated by plumbagin isolated from Plumbago also showed effect on various transcription factors/signaling pathways as pTEN, mTOR, and Akt pathways, thereby inhibiting survival signaling. However, overall data suggested the need for more extensive studies focused on clinical investigation for pharmacokinetics, bioavailability, and toxicity of these plants/plant products. We propose in this chapter that studies based on computational biology using identified pure compounds and their target to develop pharmacophore model for drug discovery would be a fast and feasible way to design new potential derivatives.
Melissa officinalis L., Quercus infectoria G. Oliviera, and Ceratonia siliqua L. are medicinal plants that have been used in different ethno-medical systems especially in the Iranian Traditional Medicine for the treatment of several diseases. Their several biological activities were well-documented, however, their anti-Varroa activities are unknown. Varroa destructor, the most important ectoparasite of Apis mellifera, threatens the honey bee populations all over the world so the search for novel control methods is an essential task for researchers. In this study, various concentrations (5, 10, 15, 20, and 25 μL/l air) of Quercus infectoria, Melissa officinalis, and Ceratonia siliqua essential oils at various exposure times (5, 10, 15, 20, and 25 h) were evaluated for their anti-Varroa activity. After exposure, mortality rate and oxidative/nitrosative stress biomarkers including superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GSH-Px], protein carbonylation [PCO], malondialdehyde [MDA], total antioxidant status [TAS], nitric oxide contents [NO] in V. destructor were measured. The obtained results indicated that mite mortality was increased in parallel with increase in essential oils concentration and exposure time. The concentrations of 15, 20 and 25 μL/l air of all essential oils resulted in the induction of oxidative/nitrosative stress (decreased SOD, GST, CAT and GSH-Px, and increased MDA, PCO and NO), and mortality of V. destructor compared to the control. These essential oils did not cause a large amount of mortality in A. mellifera compared to the control group. Also, GC/MS analysis of the oils showed that carvacrol (24.97%), γ –Terpinene (20.68%) in M. officinalis oil, β-pinene oxide (21.09), β-pinene (14.31%) in Q. infectoria, Nonadecane (23.34%), 1,2-Benzenedicarboxylic acid, dibutyl ester (15.95%) in C. siliqua oil were the major chemical constituents. In conclusion, our experiment indicated that these essential oils could be a great agent of choice to manage V. destructor.
Cancer retains a central place in fatality rates among the wide variety of diseases known world over, and the conventional synthetic medicaments, albeit used until now, produce numerous side effects. As a result, newer, better, and safer alternatives such as natural plant products, are gravely required. Essential oils (EOs) offer a plethora of bioactivities including antibacterial, antiviral, antioxidant, and anticancer properties, therefore, the use of EOs in combination with synthetic drugs or aromatherapy continues to be popular in many settings. In view of the paramount importance of EOs and their potential bioactivities, this review summarizes the current knowledge on the interconnection between EOs and cancer treatment. In particular, the current review presents an updated summary of the chemical composition of EOs, their current applications in cancer treatments based on clinical studies, and the mechanism of action against the cancer cell lines. Similarly, an overview of using EOs in aromatherapy and enhancing immunity during cancer treatment is provided. Further, this review focuses on the recent technological advancements such as the loading of EOs using protein microspheres, ligands, or nanoemulsions/nanoencapsulation, which offer multiple benefits in cancer treatment via site-specific and target-oriented delivery of drugs. The continuing clinical studies of EOs implicate that their pharmacological applications are a rewarding research area.
Pre-sowing seed priming and seedling inoculation with bioelicitor improve growth and phytochemical constituents of medicinal plants. This study was aimed to investigate the role of seed priming with silicon nanoparticles (nSi, 0, 100 and 500 mg/L) and inoculation of seedling originated from primed seeds with rhizobacteria strains (Pseudomonas fluorescens and P. putida) on physiological and metabolic attributes of Melissa officinalis L. Foliar application of P. putida on plants raised from nSi-primed (at 500 mg/L) seeds showed the wider deposition of nSi on root surface compared with that of P. fluorescens, however, P. fluorescens application on plants grown from nSi-primed (at 100 mg/L) seeds showed more open stomata on leaf surface than non-inoculated controls. Individual treatments of seed priming and seedling inoculation significantly (P < 0.05) increased plant biomass indices, leaf relative water content, photosynthetic pigments values, total soluble protein and phenolic contents, essential oil yield and their all (except thymol) major constituents, neral, geranial, and geranyl acetate compared with the control. However, the maximum increase in measured traits was recorded in mixed treatment of seed priming and inoculation of plants raised from primed seeds with rhizobacteria. Furthermore, the employed treatment combinations enhanced the free radical scavenging activities of plant extracts compared with the individual treatment and untreated control samples. According to the multivariate analyses, the treatments applied in combinations (especially seed priming at 100 mg nSi/L and seedling inoculation with P. putida) scores were substantially farther from the other treatments mean. Overall, seed priming with nSi along with seedling inoculation with pseudomonas strains play vital role in the increase in both primary and secondary metabolites of lemon balm plants.
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Study of resistance to chemotherapy mediated by abc transporters in biopsies of glioblastoma multiforme Background: Mortality rate is dramatically high in high-grade brain tumors. The presence of multiple drug resistance transporters in glioblastoma multiforme, has contributed largely to the poor efficacy of targeted therapy against cancer in the central nervous system. Aim: To analyze the percentage of survival and mortality of patients with glioblastoma multiforme in a cohort of patients in Chile and to correlate the chemo-resistance of these cells with the expression level of multiple drug resistance transporters. Materials and Methods: Eighteen biopsies of glioblastoma multiforme were obtained from patients at the Institute of Neurosurgery Dr. Asenjo (INCA). The tumor cells were obtained from primary cultures and the expression and activity of multiple drug resistance transporters was assessed by RT-PCR and immunohistochemistry. Population-based study was performed using the databases of the Department of Neurosurgery of INCA. Results: The number of patients with glioblastoma multiforme increased between 2007 and 2009, from 3.5% to 7.9% of total brain tumors. Mortality of these tumors is 90 % at three years. A high expression and activity of the multiple drugs resistance associated protein 1 (Mrp1) transporter was observed in primary cultures of biopsies. Conclusions: We propose that Mrp1 activity is responsible for the chemo-resistance of the glioblastoma multiforme and inhibition of this transporter could represent a plausible strategy for the treatment. (Rev Med Chile 2011; 139: 415-424).
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Multidrug resistance (MDR) is considered the main cause of cancer chemotherapy failure and patient relapse. The active drug efflux mediated by transporter proteins of the ABC (ATP-binding cassette) family is the most investigated mechanism leading to MDR. With the aim of inhibiting this transport and circumventing MDR, a great amount of work has been dedicated to identifying pharmacological inhibitors of specific ABC transporters. We recently showed that 3β-acetyl tormentic acid (3ATA) had no effect on P-gp/ABCB1 activity. Herein, we show that 3ATA strongly inhibited the activity of MRP1/ABCC1. In the B16/F10 and Ma104 cell lines, this effect was either 20X higher or similar to that observed with MK571, respectively. Nevertheless, the low inhibitory effect of 3ATA on A549, a cell line that expresses MRP1-5, suggests that it may not inhibit other MRPs. The use of cells transfected with ABCC2, ABCC3 or ABCC4 showed that 3ATA was also able to modulate these transporters, though with an inhibition ratio lower than that observed for MRP1/ABCC1. These data point to 3ATA as a new ABCC inhibitor and call attention to its potential use as a tool to investigate the function of MRP/ABCC proteins or as a co-adjuvant in the treatment of MDR tumors.
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Glioblastoma multiforme (GBM) is the most malignant and aggressive type of brain tumor with an average life expectancy of less than 15 months. This is mostly due to the highly mutated genome of GBM, which is characterized by the deregulation of many key signaling pathways involving growth, proliferation, survival, and apoptosis. It is critical to explore novel diagnostic and therapeutic strategies that target these pathways to improve the treatment of malignant glioma in the future. This review summarizes the most common and important pathways that are highly mutated or deregulated in GBM and discusses potential therapeutic strategies targeting these pathways.
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Gum resins obtained from trees of the Burseraceae family (Boswellia sp.) are important ingredients in incense and perfumes. Extracts prepared from Boswellia sp. gum resins have been shown to possess anti-inflammatory and anti-neoplastic effects. Essential oil prepared by distillation of the gum resin traditionally used for aromatic therapy has also been shown to have tumor cell-specific anti-proliferative and pro-apoptotic activities. The objective of this study was to optimize conditions for preparing Boswellea sacra essential oil with the highest biological activity in inducing tumor cell-specific cytotoxicity and suppressing aggressive tumor phenotypes in human breast cancer cells. Boswellia sacra essential oil was prepared from Omani Hougari grade resins through hydrodistillation at 78 or 100 °C for 12 hours. Chemical compositions were identified by gas chromatography-mass spectrometry; and total boswellic acids contents were quantified by high-performance liquid chromatography. Boswellia sacra essential oil-mediated cell viability and death were studied in established human breast cancer cell lines (T47D, MCF7, MDA-MB-231) and an immortalized normal human breast cell line (MCF10-2A). Apoptosis was assayed by genomic DNA fragmentation. Anti-invasive and anti-multicellular tumor properties were evaluated by cellular network and spheroid formation models, respectively. Western blot analysis was performed to study Boswellia sacra essential oil-regulated proteins involved in apoptosis, signaling pathways, and cell cycle regulation. More abundant high molecular weight compounds, including boswellic acids, were present in Boswellia sacra essential oil prepared at 100 °C hydrodistillation. All three human breast cancer cell lines were sensitive to essential oil treatment with reduced cell viability and elevated cell death, whereas the immortalized normal human breast cell line was more resistant to essential oil treatment. Boswellia sacra essential oil hydrodistilled at 100 °C was more potent than the essential oil prepared at 78 °C in inducing cancer cell death, preventing the cellular network formation (MDA-MB-231) cells on Matrigel, causing the breakdown of multicellular tumor spheroids (T47D cells), and regulating molecules involved in apoptosis, signal transduction, and cell cycle progression. Similar to our previous observations in human bladder cancer cells, Boswellia sacra essential oil induces breast cancer cell-specific cytotoxicity. Suppression of cellular network formation and disruption of spheroid development of breast cancer cells by Boswellia sacra essential oil suggest that the essential oil may be effective for advanced breast cancer. Consistently, the essential oil represses signaling pathways and cell cycle regulators that have been proposed as therapeutic targets for breast cancer. Future pre-clinical and clinical studies are urgently needed to evaluate the safety and efficacy of Boswellia sacra essential oil as a therapeutic agent for treating breast cancer.
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Temozolomide (TMZ) is an alkylating agent used for treating gliomas. Chemoresistance is a severe limitation to TMZ therapy; there is a critical need to understand the underlying mechanisms that determine tumor response to TMZ. We recently reported that chemoresistance to TMZ is related to a remodeling of the entire electron transport chain, with significant increases in the activity of complexes II/III and cytochrome c oxidase (CcO). Moreover, pharmacologic and genetic manipulation of CcO reverses chemoresistance. Therefore, to test the hypothesis that TMZ-resistance arises from tighter mitochondrial coupling and decreased production of reactive oxygen species (ROS), we have assessed mitochondrial function in TMZ-sensitive and -resistant glioma cells, and in TMZ-resistant glioblastoma multiform (GBM) xenograft lines (xenolines). Maximum ADP-stimulated (state 3) rates of mitochondrial oxygen consumption were greater in TMZ-resistant cells and xenolines, and basal respiration (state 2), proton leak (state 4), and mitochondrial ROS production were significantly lower in TMZ-resistant cells. Furthermore, TMZ-resistant cells consumed less glucose and produced less lactic acid. Chemoresistant cells were insensitive to the oxidative stress induced by TMZ and hydrogen peroxide challenges, but treatment with the oxidant L-buthionine-S,R-sulfoximine increased TMZ-dependent ROS generation and reversed chemoresistance. Importantly, treatment with the antioxidant N-acetyl-cysteine inhibited TMZ-dependent ROS generation in chemosensitive cells, preventing TMZ toxicity. Finally, we found that mitochondrial DNA-depleted cells (ρ°) were resistant to TMZ and had lower intracellular ROS levels after TMZ exposure compared with parental cells. Repopulation of ρ° cells with mitochondria restored ROS production and sensitivity to TMZ. Taken together, our results indicate that chemoresistance to TMZ is linked to tighter mitochondrial coupling and low ROS production, and suggest a novel mitochondrial ROS-dependent mechanism underlying TMZ-chemoresistance in glioma. Thus, perturbation of mitochondrial functions and changes in redox status might constitute a novel strategy for sensitizing glioma cells to therapeutic approaches.
Evasion of apoptosis, the cell's intrinsic death program, is a hallmark of human cancers including neuroblastoma. Also, failure to undergo apoptosis may cause treatment resistance, since the cytotoxic activity of anticancer therapies commonly used in the clinic, e.g. chemotherapy, gamma-irradiation or immunotherapy, is predominantly mediated by triggering apoptosis in tumor cells. Therefore, a better understanding of the signaling pathways and molecules that govern apoptosis in neuroblastoma cells is expected to open new avenues for the design of molecular targeted therapies for neuroblastoma.
Reactive oxygen species (ROS) are constantly generated and eliminated in the biological system, and play important roles in a variety of normal biochemical functions and abnormal pathological processes. Growing evidence suggests that cancer cells exhibit increased intrinsic ROS stress, due in part to oncogenic stimulation, increased metabolic activity, and mitochondrial malfunction. Since the mitochondrial respiratory chain (electron transport complexes) is a major source of ROS generation in the cells, the vulnerability of the mitochondrial DNA to ROS-mediated damage appears to be a mechanism to amplify ROS stress in cancer cells. The escalated ROS generation in cancer cells serves as an endogenous source of DNA-damaging agents that promote genetic instability and development of drug resistance. Malfunction of mitochondria also alters cellular apoptotic response to anticancer agents. Despite the negative impacts of increased ROS in cancer cells, it is possible to exploit this biochemical feature and develop novel therapeutic strategies to preferentially kill cancer cells through ROS-mediated mechanisms. This article reviews ROS stress in cancer cells, its underlying mechanisms and relationship with mitochondrial malfunction and alteration in drug sensitivity, and suggests new therapeutic strategies that take advantage of increased ROS in cancer cells to enhance therapeutic activity and selectivity.
The frequent development of multidrug resistance (MDR) hampers the efficacy of available anticancer drugs in treating cervical cancer. In this study, we aimed to use formononetin (7-hydroxy-4'-methoxyisoflavone), a potential herbal isoflavone, to intensify the chemosensitivity of human cervical cancer HeLa cells to epirubicin, an anticancer drug. The reactive oxygen species (ROS) levels were correlated with MDR modulation mechanisms, including the transporter inhibition and apoptosis induction. Our results revealed that formononetin significantly enhanced the cytotoxicity of epirubicin. Co-incubation of epirubicin with formononetin increased the ROS levels, including hydrogen peroxide and superoxide free radicals. Epirubicin alone markedly increased the mRNA expression of MDR1, MDR-associated protein (MRP) 1, and MRP2. In contrast, formononetin alone or combined treatment decreased the mRNA expression of MRP1 and MRP2. This result indicates that efflux transporter-mediated epirubicin resistance is inhibited at different degrees by the addition of formononetin. This isoflavone significantly intensified epirubicin uptake into HeLa cells. Apoptosis was induced by formononetin and/or epirubicin, as signified by nuclear DNA fragmentation, chromatin condensation, increased sub-G1 and G2/M phases. The cotreatment triggered the mitochondrial apoptotic pathway indicated by increased Bax-to-Bcl-2 expression ratio, loss of mitochondrial membrane potential, and significant activation of caspase-9 and -3. In addition, extrinsic/caspases-8 apoptotic pathway was also induced by the cotreatment. N-acetyl cysteine abrogated these events induced by formononetin, supporting the involvement of ROS in the MDR reversal mechanism. This study pioneered in demonstrating that formononetin may potentiate the cytotoxicity of epirubicin in HeLa cells through the ROS-mediated MRP inhibition and concurrent activation of the mitochondrial and death receptor pathways of apoptosis. Hence, the circumvention of pump and non-pump resistance using formononetin and epirubicin may pave the way for a powerful chemotherapeutic regimen for treating human cervical cancer.
This review is an updated and expanded version of the three prior reviews that were published in this journal in 1997, 2003, and 2007. In the case of all approved therapeutic agents, the time frame has been extended to cover the 30 years from January 1, 1981, to December 31, 2010, for all diseases worldwide, and from 1950 (earliest so far identified) to December 2010 for all approved antitumor drugs worldwide. We have continued to utilize our secondary subdivision of a "natural product mimic" or "NM" to join the original primary divisions and have added a new designation, "natural product botanical" or "NB", to cover those botanical "defined mixtures" that have now been recognized as drug entities by the FDA and similar organizations. From the data presented, the utility of natural products as sources of novel structures, but not necessarily the final drug entity, is still alive and well. Thus, in the area of cancer, over the time frame from around the 1940s to date, of the 175 small molecules, 131, or 74.8%, are other than "S" (synthetic), with 85, or 48.6%, actually being either natural products or directly derived therefrom. In other areas, the influence of natural product structures is quite marked, with, as expected from prior information, the anti-infective area being dependent on natural products and their structures. Although combinatorial chemistry techniques have succeeded as methods of optimizing structures and have been used very successfully in the optimization of many recently approved agents, we are able to identify only one de novo combinatorial compound approved as a drug in this 30-year time frame. We wish to draw the attention of readers to the rapidly evolving recognition that a significant number of natural product drugs/leads are actually produced by microbes and/or microbial interactions with the "host from whence it was isolated", and therefore we consider that this area of natural product research should be expanded significantly.