Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2013, Article ID 285903, 10 pages
http://dx.doi.org/10.1155/2013/285903
Research Article
Cytotoxicity and Modes of Action of the Methanol Extracts of
Six Cameroonian Medicinal Plants against Multidrug-Resistant
Tumor Cells
Victor Kuete,1,2 Aimé G. Fankam,2 Benjamin Wiench,1 and Thomas Efferth1
1
Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University of Mainz,
Staudinger Weg 5, 55128 Mainz, Germany
2
Department of Biochemistry, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon
Correspondence should be addressed to Thomas Efferth; efferth@uni-mainz.de
Received 18 June 2013; Accepted 31 July 2013
Academic Editor: Shrikant Anant
Copyright © 2013 Victor Kuete et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Introduction. The present study aims at evaluating the cytotoxicity of twelve parts from six Cameroonian medicinal plants on
sensitive and drug-resistant cancer cell lines. We also studied the mode of action of the most active plants, Gladiolus quartinianus,
Vepris soyauxii, and Anonidium mannii. Methods. The cytotoxicity of the extracts was determined using a resazurin assay. Flow
cytometry was used for cell-cycle analysis and detection of apoptosis, analysis of mitochondrial membrane potential (MMP), and
measurement of reactive oxygen species (ROS). Results. At 40 g/mL, three extracts showed a growth of CCRF-CEM leukemia
cells by less than 50%. This includes the extracts from G. quartinianus (GQW; 25.69%), Vepris soyauxii leaves (VSL; 29.82%), and
Anonidium mannii leaves (AML; 31.58%). The lowest IC50 values below 30 g/mL were obtained with GQW, AML and VSL against
7/9, 8/9, and 9/9 tested cancer cell lines, respectively. The lowest IC50 values for each plant were 4.09 g/mL, and 9.14 g/mL (against
U87MG."EGFR cells), respectively, for VSL and AML and 10.57 g/mL (against CCRF-CEM cells) for GQW. GQW induced cell
cycle arrest between G0/G1 and S phases, whilst VSL and AML induced arrest in G0/G1. All three extracts induced apoptosis in
CCRF-CEM cells by loss of MMP, whilst AML also enhanced production of ROS. Conclusion. The three active plants may be a
source for the development of new anticancer drugs.
1. Introduction are oftenless thanhalf of those inmore developedcountries
[4]. It has been observed that throughout the continent,
Cancer is one of the major causes of death in humans though infectious diseases continue to burden African popu-
representing the third leading cause of death worldwide lation, noninfectious diseases require much more attention
(12.4%), the first being cardiovascular disease (30%) and [4]. Currently, limited funding is available to tackle cancer
the second being infectious diseases, including HIV/AIDS in African countries. Awareness of this impeding epidemic
(18.8%) [1]. Chemotherapy remains the treatment of choice in Africa deserves priority, and further resources should be
in many malignant diseases [2]. Nevertheless, the appearance mobilized to both prevent and treat cancer. Research on
of drug resistance, in particular multidrug resistance (MDR), anticancer agents has become a worldwide effort in both
can make many of the clinically established anticancer drugs developed and developing countries, since chemotherapy is
ineffective [3]. Thus, MDR is one of the major concerns a mainstay in the treatment of many malignancies [5]. The
preventing cure of many cancer patients. Also, malignancies majority of standard anticancer drugs has been isolated or
are increasingly recognized as a critical public health problem derived from natural sources, based on their use in traditional
in Africa [4]. Worldwide, the number of new cancer cases will medicine [6]. Screenings of medicinal plants used as anti-
annually reach 15 million by 2020, 70% of which will occur in cancer drugs have provided modern medicine with effective
developing countries, where governments are less prepared to cytotoxic pharmaceuticals. More than 60% of the approved
address the growing cancer burden and where survival rates anticancer drugs in USA were from natural origin [7 9].
2 Evidence-Based Complementary and Alternative Medicine
In Cameroon, the use of plants in traditional medicine sys- 2.5. Cell Cultures. Drug-sensitive CCRF-CEM and multi-
tems has been extensively documented in the Cameroonian drug-resistant CEM/ADR5000 leukemia cells were main-
pharmacopoeia [10]. Evidence highlighting the importance tained in RPMI 1640 medium (Invitrogen) supplemented
of these plants for cancer therapy has been provided [11 with 10% fetal calf serum in a humidified 5% CO2 atmosphere
16]. However, whether these plants are also effective in cells at 37"C. Sensitive and resistant cells were kindly provided
resistant to standard chemotherapy is largely unknown. It by Dr. Axel Sauerbrey (Department of Pediatrics, University
has been recommended that ethnopharmacological usages of Jena, Jena, Germany). The generation of the resistant
such as immune and skin disorders, inflammatory, infectious, subline was described [35]. The specific overexpression of
parasitic, and viral diseases should be taken into account P-glycoprotein, but not other ABC transporters, has been
when selecting plants used to treat cancer, since these reported [36, 37]. Breast cancer cells transduced with con-
reflect disease states bearing relevance to cancer or cancer- trol vector (MDA-MB-231-pcDNA3) or with cDNA for the
like symptoms [17, 18]. Though the plants selected in the breast cancer resistance protein, BCRP (MDA-MB-231-BCRP
present studied are used in the Cameroonian traditional clone 23) were maintained under standard conditions as
medicine to fight cancers, there is still a lack of published described previously for CCRF-CEM cells. Human wild-type
data regarding the use. Therefore, in our continuous search
HCT116 (p53+/+) colon cancer cells as well as knockout clones
of the cytotoxic candidates from Cameroonian plants with
HCT116 (p53-/-) derived by homologous recombination were
unpublished ethnopharmacological information related to
a generous gift from Dr. B. Vogelstein and H. Hermeking
cancer use, we investigated the antiproliferative potential of
(Howard Hughes Medical Institute, Baltimore, MD, USA).
six Cameroonian plants against cancer cell lines with different
Human glioblastoma multiforme U87MG cells (nontrans-
mechanisms of drug resistance, that is, ATP-binding cassette
duced) and U87MG cell line transduced with an expres-
(ABC) transporters (P-glycoprotein, breast cancer resistance
sion vector harboring an epidermal growth factor receptor
protein), tumor suppressors (p53), or oncogenes (epidermal
(EGFR) gene with a genomic deletion of exons 2 through 7
growth factor receptor). The most cytotoxic extracts from
(U87MG."EGFR) were kindly provided by Dr. W. K. Cavenee
Gladiolus quartinianus A. Rich. (Iridaceae), Vepris soyauxii
(Ludwig Institute for Cancer Research, San Diego, CA, USA)
Engl. (Rutaceae) and Anonidium mannii (oliv) Engl. et Diels.
[38]. MDA-MB-231-BCRP, U87MG."EGFR, and HCT116
(Annonaceae) were further analyzed to study their mode of
(p53-/-) were maintained in DMEM medium containing 10%
action regarding cell-cycle distribution, MMP, and ROS.
FBS (Invitrogen) and 1% penicillin (100 U/mL) streptomycin
(100 g/mL) (Invitrogen) and were continuously treated with
800 ng/mL and 400 g/mL geneticin, respectively. Human
2. Materials and Methods
HepG2 hepatocellular carcinoma cells and normal AML12
hepatocytes were obtained from American Type Culture
2.1. Plant Material. All medicinal plants used in the present
Collection (ATCC, USA). The previous medium without
work were collected in different areas of Cameroon between
geneticin was used to maintain MDA-MB-231, U87MG,
January and April 2012 (Table 1). The plants were identified
HCT116 (p53+/+), HepG2, and AML12 cell lines. The cells
at the National Herbarium (Yaounde, Cameroon), where
were passaged twice weekly. All experiments were performed
voucher, specimens were deposited under the references
with cells in the logarithmic growth phase.
numbers given in Table 1.
2.6. Resazurin Reduction Assay. Resazurin reduction assay
2.2. Extraction. The air-dried and powdered plant samples [16, 39] was performed to assess cytotoxicity of the studied
samples toward cancer cells. The assay is based on reduction
(1 kg) were soaked in methanol (3 L) for 48 h, at room
temperature. The methanol extracts were concentrated in of the indicator dye, resazurin, to the highly fluorescent
vacuum to obtain the crude extracts [12]. These extracts were resorufin by viable cells. Nonviable cells rapidly lose the
metabolic capacity to reduce resazurin and thus produce no
then stored at 4"Cuntil further use.
fluorescent signal. Briefly, adherent cells were detached by
treatment with 0.25% trypsin/EDTA (Invitrogen, Darmstadt,
2.3. Chemicals. Doxorubicin, vinblastine, and daunorubicin Germany) and an aliquot of 1 × 104 cells was placed in
were provided by the University Pharmacy of the Johannes each well of a 96-well cell culture plate (Thermo Scientific,
Gutenberg University (Mainz, Germany) and dissolved in Langenselbold, Germany) in a total volume of 200 L. Cells
PBS (Invitrogen, Eggenstein, Germany) at a concentration of were allowed to attach overnight and then were treated
10 mM. Geneticin (72.18 mM) was purchased from Sigma- with different concentrations of the studied sample. For
Aldrich (Munich, Germany). suspension cells, aliquots of 2 × 104 cells per well were seeded
in 96-wellplates in a total volume of 100 L. The studied
sample was immediately added in varying concentrations in
2.4. Preliminary Phytochemical Investigations. The major sec- additional 100 L of culture medium to obtain a total volume
ondary metabolites classes such as alkaloids, anthocyanins, of 200 L/well. After 24 h or 48 h, 20 L resazurin (Sigma-
anthraquinones, flavonoids, phenols, saponins, sterols, and Aldrich, Schnelldorf, Germany) 0.01% w/v in ddH2O was
triterpenes (Table 2) were determined according to a com- added to each well and the plates were incubated at 37"C
mon phytochemical methods previously described [34]. for 4 h. Fluorescence was measured on an Infinite M2000
Table 1: Pharmacognosy of Cameroonian medicinal plants.
Samples, family, and Part used in this study Area of plant Known bioactive (or Screened activity for crude plant
Traditional treatment
herbarium numbera and extraction yield (%)b collection potentially active) compounds extract
Antimicrobial against Gram-positive
Allan Xanthones A and D; and Gram-negative bacteria, yeasts,
Allanblackia gabonensis Dysentery, cold, toothache [19, 20]; Fruits (4.14%), leaves Lebialem, South
1,3,6,7-tetrahydroxy-2-(3- and mycelial fungus [22]; analgesic
Pellegr. (Clusiaceae); 17275 pain, rheumatism, inflammations [21], (10.16%), stem (8.42%) West region of
methylbut-2-enyl)xanthone and anti-inflammatory effect of
SRF/Cam cancer (personal information) and roots bark (8.69%) Cameroon
[22] aqueous extract of the stem bark [21].
Sore feet, spider bite, bronchitis,
dysentery, sterility caused by poison,
Anonidium mannii (oliv) Bafoussam, West
gastroenteritis [23]; syphilis, infectious
Engl. et Diels. Leaves (3.39%) region of Not reported Not reported
diseases [24]; diarrhea, snake bite,
(Annonaceae); 1918/SRFK Cameroon
malaria [25], cancer (personal
information)
Gladiolus quartinianus A. Lebialem, South
Gastrointestinal infection, cancer
Rich (Iridaceae); Whole plant (10.22%) West region of Not reported Not reported
(personal information)
17260/SRF/Cam Cameroon
Peperomia fernandopoiana Lebialem, South
Gastrointestinal infection, cancer
C.DC. Whole plant (7.28%) West region of Not reported Not reported
(personal information)
(Piperaceae); 7171 SRF/Cam Cameroon
Antimicrobial activity of the stem
bark against Streptococcus faecalis
E-ferulic acid octacosylate,
Cough, antidote, intestinal diseases, [30], Staphylococcus aureus, Bacillus
3-methylmethylorsellinate,
Recinodindron heudelotii dysentery [26 28]; malaria, anaemia, cereus, Escherichia coli, Shigella
Melon, littoral lupeol, heudoletinone,
(Baill.) ex Pax. stomach pain, easy delivery, yellow Leaves (5.18%) and stems dysenteriae, Shigella flexneri,
region of 1,2-dihydroheudoleunol [26],
(Euphorbiaceae); 19695 fever, aphrodisiac [29], cancer bark (5.72%) Salmonella typhi, Pseudomonas
Cameroon aleuritolic acid 1 and
SRF/Cam (personal information) aeruginosa, Klebsiella pneumoniae,
labda-8(17),13-dien 3 ,15-diol
Candida albicans [31], antioxidant
2[30]
[32].
Antifibromyoma, stomachache, Leaves (10.16%), stems Melon, littoral
Vepris soyauxii Engl.
malaria [32], cancer (personal (5.18%), and roots bark region of Not reported Not reported
(Rutaceae); 18394 SFR/Cam
information) (9.26%) Cameroon
a b
Plants were identified at the Cameroon National Herbarium (HNC); The percentage of the methanol extract.
Evidence-Based Complementary and Alternative Medicine
3
4 Evidence-Based Complementary and Alternative Medicine
Table 2: Chemical constituents and extraction yield of the studied plant extracts.
Phytochemical constituents
Studied samples
Alkaloids Anthocyanins Anthraquinones Flavonoids Phenols Saponins Tannins Sterols Triterpenes
Allanblackia gabonensis
Leaves + + - + + - + - -
Stem bark + + + + + + + - +
Root bark + + + + + + + - +
Fruits + + - + + + + - +
Anonidium mannii
Leaves + -- - + + + + +
Gladiolus quartinianus
Whole plant + + - + + - + + +
Peperomia fernandopoiana
Whole plant + + - + + - + + -
Ricinodendron heudelotii
Leaves + -- - + + + - +
Stem bark + + - + + - + - -
Vepris soyauxii
Leaves + + - + + + + + +
Stem bark + + + + + + + - +
Roots bark + + - + + + + - +
(+): present; (-): absent.
Pro plate reader (Tecan, Crailsheim, Germany) using an that can selectively enter into mitochondria and exhibits
excitation wavelength of 544 nm and an emission wavelength an intense red fluorescence in healthy mitochondria with
of 590 nm. Each assay was done at least two times, with normal membrane potentials. In cells with reduced MMP, the
six replicate each. The viability was evaluated based on red fluorescence disappears. Briefly, 1 × 106 CCRF-CEM cells
a comparison with untreated cells. IC50 values represent treated with different concentrations of the test compounds
the sample s concentrations required to inhibit 50% of cell or DMSO (solvent control) for 24 h were incubated with
proliferation and were calculated from a calibration curve by JC-1 staining solution according to the manufacturer s
linear regression using Microsoft Excel. protocol for 30 min. Subsequently, cells were measured in
an LSR-Fortessa FACS analyzer (Becton-Dickinson). For
each sample, 1 × 104 cells were counted. The JC-1 signal was
2.7. Flow Cytometry for Cell Cycle Analysis and Detection
measured with 561 nm excitation (150 mW) and detected
of Apoptotic Cells. Cell-cycle analysis was performed by
using a 586/15 nm bandpass filter. The compounds signal
flow cytometry using The Vybrant DyeCycle (Initrogen).
was analyzed with 640 nm excitation (40 mW) and detected
The Vybrant DyeCycle Violet stain is a DNA-selective, cell
using a 730/45 nm bandpass filter. All parameters were
membrane-permeant, and nonfluorescent dye for DNA con-
plotted on a logarithmic scale. Cytographs were analyzed
tent analysis in living cells. The Vybrant DyeCycle Violet
using FlowJo software (Celeza, Switzerland). All experiments
stain is fluorescent upon binding to double-stranded DNA.
were performed at least in triplicate.
Leukemia CCRF-CEM cells (1 × 106) were treated with the
concentrations equivalent to the IC50 values of the crude
extract for 24 h, 48, and 72 h. Following incubation, 1 L
2.9. Measurement of Reactive Oxygen Species (ROS) by
of Vybrant DyeCycle Violet stain was added to 1 mL of
Flow Cytometry. 2 ,7 -Dichlorodihydrofluorescein diacetate
cell suspension and incubated for 30 min at 37"C. Cells
(H2DCFH-DA) (Sigma-Aldrich, Germany) is a probe used
were measured on an LSR-Fortessa FACS analyzer (Becton-
for the highly sensitive and quantifiable detection of
Dickinson, Germany) using the violet laser. Ten thousand
ROS. The nonfluorescent H2DCFH-DA diffuses into the
cells were counted for each sample. Vybrant DyeCycle Violet
cells and is cleaved by cytoplasmic esterases into 2 ,7 -
stain was measured with 440 nm excitation. Cytographs were
dichlorodihydrofluorescein (H2DCF) which is unable to
analyzed using FlowJo software (Celeza, Switzerland). All
diffuse back out of the cells. In the presence of hydrogen
experiments were performed at least in triplicate.
peroxide, H2DCF is oxidized to the fluorescent molecule
dichlorofluorescein (DCF) by peroxidases. The fluorescent
signal emanating from DCF can be measured and quantified
2.8. Analysis of Mitochondrial Membrane Potential (MMP).
The effects of extract on the MMP were analyzed by 5,5 ,6,6 - by flow cytometry, thus providing an indication of intra-
tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine cellular ROS concentration [40, 41]. Briefly, 2 × 106 CCRF-
iodide) (JC-1; Biomol, Germany) staining. JC-1 is a dye CEM cells were resuspended in PBS and incubated with
Evidence-Based Complementary and Alternative Medicine 5
70
2 MH2DCFH-DA for 20 min in the dark. Subsequently,
60
cells were washed with PBS and resuspended in RPMI
50
1640 culture medium containing different concentrations of
40
extract or DMSO (solvent control). After 1 h of incubation,
30
cells were washed and suspended in PBS. Subsequently,
20
cells were measured in an FACS Calibur flow cytometer
10
(Becton-Dickinson, Germany). For each sample 1 × 104
0
cells were counted. DCF was measured at 488 nm excitation
(25 mW) and detected using a 530/30 nm bandpass filter. All
parameters were plotted on a logarithmic scale. Cytographs
were analyzed using FlowJo software (Celeza, Switzerland).
All experiments were performed at least in triplicate.
3. Results
3.1. Chemical Composition of the Studied Extracts. The results
of the qualitative analysis showed that each of the studied
plant extract contained at least one class of secondary
metabolites such as alkaloids, anthocyanins, anthraquinones,
Figure 1: Growth (% of untreated control) of CCRF-CEM leukemia
flavonoids, phenols, saponins, and triterpenes. All studied
cells in the presence of plant extracts (40 g/mL) or doxorubicin
extracts contained alkaloids, phenols, and tannins (Table 2).
(10 g/mL).
3.2. Cytotoxicity of the Studied Samples. The growth inhibi-
extracts showed higher IC50 values in normal AML12 hepa-
tion of CCRF-CEM cells induced by 12 extracts belonging
tocytes compared to HepG2 liver cancer cells. Furthermore,
to six medicinal plants is depicted in Figure 1. The extracts
AML12 normal hepatocytes were more doxorubicin resistant
from Gladiolus quartinianus (whole plant; GQW; 25.69%),
than HepG2 cancer cells towards doxorubicin. None of the
Vepris soyauxii (leaves; VSL; 29.82%), and Anonidium mannii
extracts inhibited normal AML12 hepatocytes by more than
(leaves; AML; 31.58%) inhibited cell growth by more than 50%
50%.
at 40 g/mL.
To investigate these extracts in more detail, their IC50
3.3. Cell Cycle Distribution and Apoptosis. The cell-cycle
values were determined in a panel of cancer cell lines. The
VSL extract was active (IC50 < 40 g/mL) against all 9 sensi- distribution and induction of apoptosis of CCRF-CEM cells
tive or drug-resistant cell lines. IC50 values below 30 g/mL upon treatment with GQW, VSL AML, are depicted in
Figure 2. Upon 72 h treatment, the GQWextract induced cell
were obtained with GQW, AML, and VSLagainst 7/9, 8/9,
and9/9 testedcancer cell lines, respectively. The IC50 values cycle arrest between G0/G1 and S phases whilst VSL and
were in a range from 4.09 g/mL (U87MG."EGFR cells) to AMLextracts induced G0/G1 arrest. The three extracts led
13.60 g/mL (HepG2 cells) for VSL from 10.57 g/mL (CCRF- to a time-dependent increase of sub-G0/G1 cells, indicat-
CEM) to 34.01 g/mL (U87MG."EGFR) for GQW, andfrom ing induction of apoptosis. CCRF-CEM cells treated with
9.14 g/mL (U87MG."EGFR) to 32.02 g/mL (MDA-MB- concentrations equivalent to the IC50 value of each studied
extracts progressively underwent apoptosis, with percentages
231-BCRP) for AML. For the control drug doxorubicin,
the IC50 values were in a range from 0.11 g/mL (CCRF- in sub-G0/G1 phase ranging from 11.2% (24 h) to 44.3% (72 h)
for GQW, from 19.7% (24 h) to 53.2% (72 h) for VSL, and
CEM cells) to 195.12 g/mL (CEM/ADR5000 cells) (Table 3).
from 22.7% (24 h) to 76.2% (72 h) for AML. The values of
High degrees of resistance to doxorubicin were observed
for CEM/ADR 5000 cells (1772-fold), MDA-MB-231-BCRP the sub-G0/G1 phase recorded with AMLwere higher than
thoseobtainedwithnontreatedcells(rangefrom3.82%(24h)
cells (7.11-fold), and U87MG."EGFR (5.76-fold) compared to
to 9.37% (72 h)), but were comparable to those obtained for
their corresponding parental cell lines. HCT116 (p53-/-) cells
the control drug, doxorubicin (range from 59.4% (24 h) to
were weakly resistant to doxorubicin (2.84-fold) compared
71.9% (72 h)) (see Supplementary Material available online at
to HCT116 (p53+/+) cells. Interestingly, the drug-resistant
http://dx.doi.org/10.1155/2013/285903, Figure S1).
cell lines were not or only weakly resistant to the tested
extracts (d"2.53-fold). Remarkably, none of the tested extract
inhibited the growth of more than 50% normal AML12 hepa- 3.4. Effect onthe Mitochondrial Membrane Potential (MMP).
tocytes at a concentration of 40 g/mL. Collateral sensitivity, We assessed the effect of the GQW, VSL, and AML extracts on
which means that resistant cells are more sensitive than MMPinCCRF-CEMcells. As showninFigure 3, percentage
sensitive cells, was observed with the three extracts against alterations of 13.5%, 28.9%, and 32.3% were induced by GQW,
U87MG."EGFR with degree of resistances below 1. This was VSL, and AML extracts, respectively, after 24 h of treatment
alsonotedfor theVSLandAMLextracts against HepG2cells with twofold IC50. The MMP value for untreated cells was
and AML extract against CEM/ADR5000 cells. All the plant 4.81%. Under similar experimental conditions, these values
Growth (% of control)
Doxorubicin
Vepris soyauxii
(leaves)
Vepris soyauxii
(root bark)
Vepris soyauxii
(stem bark)
Anonidium mannii
(leaves)
Allanblackia gabonensis
(fruits)
Allanblackia gabonensis
(leaves)
Ricenodindron heudelotii
(leaves)
Allanblackia gabonensis
(root bark)
Allanblackia gabonensis
(stem bark)
Gladiolus quartinianus
(whole plant)
Ricenodindron heudelotii
(stem bark)
Peperomia fernandopoiana
(whole plant)
6 Evidence-Based Complementary and Alternative Medicine
Table 3: Cytotoxicity of the studied extracts towards sensitive and drug-resistant cancer cell lines and normal cells as determined by the
resazurin assay.
Studied samples, IC50 values ( g/mL)a and degree of resistance (in bracket)
Cell lines
Gladiolus quartinianus (Whole plant) Vepris soyauxii (Leaves) Anonidium mannii (Leaves) Doxorubicin
CCRF-CEM 10.57 Ä… 2.08 9.28 Ä… 1.01 17.32 Ä… 2.27 0.11 Ä… 0.01
CEM/ADR5000 26.14 Ä… 1.97 (2.47) 11.72 Ä… 1.43 (1.26) 16.44 Ä… 1.76 (0.95) 195.12 Ä… 14.30 (1772)
MDA-MB-231 16.11 Ä… 1.62 7.52 Ä… 0.84 12.65 Ä… 1.49 1.10 Ä… 0.01
MDA-MB-231-BCRP 29.6 Ä… 3.19 (1.49) 12.93 Ä… 1.69 (1.71) 32.02 Ä… 3.16 (2.53) 7.83 Ä… 0.01 (7.11)
HCT116 53+/+ 19.83 Ä… 1.66 8.59 Ä… 0.88 13.61 Ä… 1.79 1.43 Ä… 0.02
HCT116 53-/- 22.15 Ä… 1.97 (1.12) 9.70 Ä… 0.72 (1.12) " (>2.94) 4.06 Ä… 0.04 (2.84)
U87MG " 8.75 Ä… 1.21 22.25 Ä… 2.76 1.06 Ä… 0.03
U87MG"EGFR 34.01 Ä… 2.78 (<0.85) 4.09 Ä… 0.56 (0.47) 9.14 Ä… 1.77 (0.41) 6.11 Ä… 0.04 (5.76)
HepG2 " (n.a) 13.60 Ä… 1.22 (<0.34) 22.09 Ä… 2.42 (0.55) 1.41 Ä… 0.12 (<0.04)
AML12 "" " "
a
The degree of resistance was determined as the ratio of IC50 value of the resistant/IC50 sensitive cell line.
("): >40 g/mL; n.a: not applicable.
Gladiolus quartinianus Vepris soyauxii
Anonidium mannii
0.23
5.41 1.51 0.74 0.39 0.39
2.63 0.92 2.11
100 100 100
7.59 6.65
12
21.2 22.4
26.2
17.5
25.9 27.7
18.3
32.7
34.5
31.1
34.7
36.7
50 50 50
53.1 45.5
76.2
47.9 73.4
53.2
41.1
44.3
32.6
22.7
19.7
11.2
0 0 0
24 48 72 24 48 72 24 48 72
Experimentation time (h) Experimentation time (h) Experimentation time (h)
G2/M G0/G1 G2/M G0/G1 G2/M G0/G1
S Sub-G1 S Sub-G1 S Sub-G1
(a) (b) (c)
Figure 2: Cell-cycle distribution of CCRF-CEM cells treated with plant extractsordoxorubicin at their corresponding IC50 values for 72 h.
Data of control and doxorubicin obtained under similar experimental conditions were previously reported [33]. Flow cytometry histograms
are available as supportive information (Figure S1).
were lower than that of the reference compound, vinblastine with fatal outcome for cancer patients. Secondary metabolites
which yielded 48.6% as previously reported [33]. play an important role in plant defense against herbivores,
microbial infections, and other interspecies defenses and
can be exploited to fight human diseases, including cancer
3.5. Effects on Reactive Oxygen Species (ROS). The effects of
[44]. Their antiproliferative properties have been broadly
the GQW, VSL, andAMLextracts onROS levels were inves-
discussed [45]. In the present study, the classes of secondary
tigated in CCRF-CEM cells after 24 h treatment (Figure 4).
metabolites detected in the tested plant extracts (Table 2)
The control agent, H2O2, increased ROS level to 10.4%, while
provide a preliminary explanation on their activities. The
ROS production in nontreated cells was 0.94%. Only AML
obtained results represent the first phytochemical data on
induced significant ROS production in CCRF-CEM cells
the cytotoxic activity of G. quartinianus, V. soyauxii, and A.
treated with a concentration equivalent to 2 × IC50 (8.42%).
manni.
According to the criteria of the ATCC, 30 g/mL repre-
4. Discussion
sent the upper IC50 limit considered promising for purifi-
cation of a crude extract [46]. In the present work, the
Drug resistance is a complex multifactorial phenomenon
highest concentration tested (40 g/mL) in our screening was
that can result from a number of biochemical mechanisms,
slightly above this limit. Herein, we recorded IC50 values
including decreased drug uptake or increased drug efflux,
below 30 g/mL for GQW, AML, and VSL extracts towards
perturbed expression of target enzymes or altered target
the majority of the tested cancer cell lines (Table 3). This
enzymes, altered metabolism of drugs, increased repair of
drug-induced DNA damage, or failure to undergo apoptosis demonstrates that the crude extracts of GQW, AML, and VSL
[42, 43]. Resistance phenomena may lead to failure of therapy could serve as potential sources of cytotoxic compounds.
Cells (%)
Cells (%)
Cells (%)
Evidence-Based Complementary and Alternative Medicine 7
Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2
105 3.92% 96.1% 8.27% 91.7% 105 12.7% 87.3% 105 13.5% 86.4%
105
104
104
104 104
GQW-4
GQW-2
GQW-1
103 103 103 GQW-3 103
102 102 102
102
Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3
0
0 0 0
0.00% 0.00%
0.009% 0.00% 0.038% 0.00% 0.047% 0.00%
0 102 103 104 105
0 102 103 104 105 0 102 103 104 105 0 102 103 104 105
YG561 nm 586 15-A YG561 nm 586 15-A YG561 nm 586 15-A YG561 nm 586 15-A
Q1 Q2 Q1 Q2
Q1 Q2 Q1 Q2
105 105 105 14.5% 85.4% 105 32.3% 65.2%
7.85% 92.0% 12.7% 87.3%
104
104 104 104
VSL-1 VSL-2 VSL-3
VSL-4
103 103 103
103
102 102 102 102
Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3
0 0 0 0
0.131% 0.00% 0.009% 0.009% 0.056% 0.00% 2.50% 0.00%
0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105
YG561 nm 586 15-A
YG561 nm 586 15-A
YG561 nm 586 15-A
YG561 nm 586 15-A
Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2
105 11.0% 89.0% 105 20.0% 79.9% 105 105 28.9% 71.0%
16.7% 83.3%
104 104 104
104
AML-2
AML-4
AML-3
103 AML-1 103 103 103
102 102 102 102
Q4 Q3 Q4 Q3
Q4 Q3 Q4 Q3
0
0 0 0
0.035% 0.00%
0.00% 0.00% 0.043% 0.00% 0.140% 0.00%
0 102 103 104 105
0 102 103 104 105 0 102 103 104 105 0 102 103 104 105
YG561 nm 586 15-A YG561 nm 586 15-A YG561 nm 586 15-A YG561 nm 586 15-A
Figure 3: Effect of plant extracts and vinblastine (VIN) on the MMP of CCRF-CEM cells after 24 h of treatment. Data of control and
vinblastine under similar experimental conditions were previously reported [33]. Samples were tested at their 1/4 × IC50 (1), 1/2 × IC50 (2),
IC50 (3), and 2 × IC50 (4) values. The IC50 values are 0.20 M for VIN, 10.57 g/mL (Gladiolus quartinianus whole plant, GQW), 9.28 g/mL
(Vepris soyauxii leaves, VSL), and 17.32 g/mL (Anonidium mannii leaves, AML).
In addition to the identification of crude extracts with The GQW, VSL, and AML extracts were more cytotoxic
reasonable low IC50 values, we identified extracts capable of towards HepG2 liver carcinoma cells and the other cancer cell
killing otherwise drug-resistant cancer cells. Having in mind lines tested than towards normal AML12 hepatocytes. This
that drug resistance is a major obstacle of chemotherapy highlights at least some specificity of the three plant extracts
in the clinic, the search for novel noncross-resistant cyto- towards target malignant cells with little effects on normal
toxic compound from natural sources is urgently warranted. cells.
Drug-resistant cell models overexpressing P-glycoprotein, We further found that the GQW, VSL, and AML extracts
BCRP, or "EGFR as well as p53 knockout cells were used induced apoptosis by disruption of MMP, whilst in addition
to assess the suitability of the studied extracts to tackle AML produced ROS. To the best of our knowledge, the
multifactorial drug resistance. The degrees of resistance of cytotoxicity of GQW, VSL, and AML is being reported
the three extracts were generally lower than that of dox- here for the first time. Therefore, the isolation of the active
orubicin in corresponding drug-resistant cell lines (Table 3), constituents from these plants is worthwhile for the better
clearly highlighting their possible role fighting multidrug understanding of their activities towards cancer cells.
resistance. It was pleasing that even collateral sensitivity was In conclusion, the present study provides evidence of the
observed in several cases. This phenomenon is character- cytotoxic potential of GQW, VSL, and AML extracts on sen-
ized by the fact that drug-resistant cells are more sensitive sitive and drug-resistant cancer cell lines. The three extracts
to a test compound than the parental sensitive cells [47, induced apoptosis in CCRF-CEM cells by loss of MMP and,
48]. in the case of AML, also enhanced ROS production. These
The objective of cancer chemotherapy is to kill cancer plant extracts merit more detailed investigations to improve
cells with as little damage as possible to normal cells [49]. therapy of drug-resistant and refractory tumors in the future.
BL
488
nm 530 30-A
BL
488
nm 530 30-A
BL
488
nm 530 30-A
BL
488
nm 530 30-A
BL
488
nm 530 30-A
BL
488
nm 530 30-A
BL
488
nm 530 30-A
BL
488
nm 530 30-A
BL
488
nm 530 30-A
BL
488
nm 530 30-A
BL
488
nm 530 30-A
BL
488
nm 530 30-A
8 Evidence-Based Complementary and Alternative Medicine
400
BL488 nm BL488 nm BL488 nm BL488 nm
530 30-A- 530 30-A+ 530 30-A- 530 30-A+
99.1% 0.942% 89.6% 10.4%
300
300
Control H2O2
200
200
100
100
0 0
101 102 103 104 105 101 102 103 104 105
BL488 nm 530 30-A BL488 nm 530 30-A
BL488 nm BL488 nm BL488 nm BL488 nm BL488 nm BL488 nm
BL488 nm BL488 nm
120
530 30-A- 530 30-A+ 530 30-A- 530 30-A+ 530 30-A- 530 30-A+ 120
120 530 30-A- 530 30-A+
60
99.8% 0.190% 99.5% 0.512% 97.0% 3.02%
96.2% 3.79%
GQW-2
GQW-1 GQW-3
90
90
90
GQW-4
40
60
60 60
20
30 30 30
0
0 0 0
101 102 103 104 105 101 102 103 104 105 101 102 103 104 105
101 102 103 104 105
BL488 nm 530 30-A BL488 nm 530 30-A BL488 nm 530 30-A
BL488 nm 530 30-A
BL488 nm BL488 nm BL488 nm BL488 nm BL488 nm BL488 nm BL488 nm BL488 nm
400
400
530 30-A- 530 30-A+ 530 30-A- 530 30-A+ 400 530 30-A- 530 30-A+ 530 30-A- 530 30-A+
300
99.3% 0.652% 99.1% 0.893% 98.8% 1.20% 98.5% 1.47%
VSL-1
300 VSL-2 VSL-3 VSL-4
300
300
200
200
200
200
100
100 100
100
0 0 0 0
101 102 103 104 105 101 102 103 104 105 101 102 103 104 105 101 102 103 104 105
BL488 nm 530 30-A BL488 nm 530 30-A BL488 nm 530 30-A BL488 nm 530 30-A
BL488 nm BL488 nm BL488 nm BL488 nm
400 BL488 nm BL488 nm 400 BL488 nm BL488 nm
530 30-A- 530 30-A+ 400 530 30-A- 530 30-A+
530 30-A- 530 30-A+
530 30-A- 530 30-A+
300
98.8% 1.25%
99.6% 0.360% 98.7% 1.25% 91.6% 8.42%
300 300
AML-4
300
AML-2
AML-1
AML-3
200
200 200
200
100
100 100
100
0
0 0 0
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105 101 102 103 104 105
BL488 nm 530 30-A
BL488 nm 530 30-A BL488 nm 530 30-A BL488 nm 530 30-A
Figure 4: Effect of plant extracts and H2O2 (at 50 M) on the ROS production of CCRF-CEM cells after 24 h treatment. Samples were tested
at their 1/4 × IC50 (1), 1/2 × IC50 (2), IC50 (3), and 2 × IC50 (4) values. The IC50 values are 10.57 g/mL (Gladiolus quartinianus whole plant,
GQW), 9.28 g/mL (Vepris soyauxii leaves, VSL), and 17.32 g/mL (Anonidium mannii leaves, AML).
Conflict of Interests Efferth supervised the work and provided the facilities for the
study. All authors read and approved the final paper.
The authors declare that there is no conflict of interests
regarding the publication of this paper.
Acknowledgment
Authors Contribution
Victor Kuete is very grateful to the Alexander von Humboldt
Victor Kuete, Aimé G. Fankam, and Benjamin Wiench foundation for an 18 months fellowship in Germany through
carried out the experiments. Victor Kuete and Thomas Efferth the Georg Foster Research Fellowship for Experienced
designed the study. Victor Kuete wrote the paper. Thomas Researcher Program.
Count
Count
Count
Count
Count
Count
Count
Count
Count
Count
Count
Count
Count
Count
Evidence-Based Complementary and Alternative Medicine 9
References [17] G. A. Cordell, C. W. W. Beecher, and J. M. Pezzuto, Can
ethnopharmacology contribute to the development of new
[1] C. Mathers, C. Boschi-Pinto, A. Lopez, and C. Murray, Cancer
anticancer drugs? Journal of Ethnopharmacology, vol. 32, no.
Incidence, Mortality and Survival by Site for 14 Regions of the
1 3, pp. 117 133, 1991.
World, World Health Organization, Lyon, France, 2001.
[18] J. Popoca, A. Aguilar, D. Alonso, andM. L. Villarreal, Cytotoxic
[2] H. Lage, N. Duarte, C. Coburger, A. Hilgeroth, and M. J. U.
activity of selected plants used as antitumorals in Mexican
Ferreira, Antitumor activity of terpenoids against classical and
traditional medicine, Journal of Ethnopharmacology, vol. 59, no.
atypical multidrug resistant cancer cells, Phytomedicine, vol. 17,
3, pp. 173 177, 1998.
no. 6, pp. 441 448, 2010.
[19] A. Raponda-Walker and R. Sillans, Les plantes utiles du Gabon,
[3] E. Borowski, M. M. Bontemps-Gracz, and A. Piwkowska,
Paul Lechevalier, Paris, France, 1976.
Strategies for overcoming ABC-transporters-mediated mul-
[20] J. Vivien and J. Faure, Fruitiers sauvages d Afrique: especes du
tidrug resistance (MDR) of tumor cells, Acta Biochimica
Cameroun, Coopération Française, Paris, France, 1995.
Polonica, vol. 52, no. 3, pp. 609 627, 2005.
[21] E. V. Ymele, A. B. Dongmo, and T. Dimo, Analgesic and anti-
[4] V. Kuete, Recent advances in medicinal plant research in
inflammatory effect of aqueous extract of the stem bark of
sub-saharan Africa, in Recent Progress in Medicinal Plants:
Allanblackia gabonensis (Guttiferae), Inflammopharmacology,
Ethnomedicine and Therapeutic Validation, J. Govil and G.
vol. 21, no. 1, pp. 21 30, 2013.
Kaushik, Eds., vol. 32, pp. 73 131, Studium Press, Houston, Tex,
[22] A. G. B. Azebaze, B. M. W. Ouahouo, J. C. Vardamides et al.,
USA, 2012.
Antimicrobial and antileishmanial xanthones from the stem
[5] S. Carter and R. Livingston, Principle of cancer chemotherapy,
bark of Allanblackia gabonensis (Guttiferae), Natural Product
in Principles of Cancer Treatment, S. Carter, E. Glatstein, and R.
Research, vol. 22, no. 4, pp. 333 341, 2008.
Livingston, Eds., pp. 95 110, McGraw-Hill, New York, NY, USA,
[23] J. Thomas, S. Bahuchets, A. Epelboin, and S. Furniss, Ency-
1982.
clopédie des Pygmées Aka: techniques, langage et société des
[6] M. Cragg and D. Newman, Natural Product Drugs Discovery
chasseurs-cueilleurs de la foręt centrafricaine (Sud-Centrafrique
in Next Millennium, National Cancer Institute, Frederick, Md,
et Nord-Congo), Peeters-SELAF, Paris, France, 2003.
USA, 2001.
[24] E. Noumi and M. Eloumou, Syphilis ailment: prevalence
[7] C. Stévigny, C. Bailly, and J. Quetin-Leclercq, Cytotoxic and
and herbal remedies in Ebolowa subdivision (South region,
antitumor potentialities of aporphinoid alkaloids, Current
Cameroon), International Journal of Biomedical and Pharma-
Medicinal Chemistry Anti-Cancer Agents, vol. 5, no. 2, pp. 173
ceutical Sciences, vol. 2, no. 1, pp. 20 28, 2011.
182, 2005.
[25] J. Betti, An ethnobotanical study of medicinal plants among
[8] D. J. Newman and G. M. Cragg, Natural products as sources of
the Baka Pygmies in the Dja Biosphere Reserve, Cameroon,
new drugs over the last 25 years, Journal of Natural Products,
African Study Monographs, vol. 25, pp. 1 27, 2004.
vol. 70, no. 3, pp. 461 477, 2007.
[26] S. F. Kimbu, F. Keumedjio, L. B. Sondengam, and J. D. Connolly,
[9] D. J. Newman and G. M. Cragg, Natural products as sources
Two dinorditerpenoids from Ricinodendron heudelotii, Phyto-
of newdrugs over the 30 years from1981 to 2010, Journal of
chemistry, vol. 30, no. 2, pp. 619 622, 1991.
Natural Products, vol. 75, no. 3, pp. 311 335, 2012.
[27] E. Noumi and A. Yomi, Medicinal plants used for intestinal
[10] J. Adjanohoun, N. Aboubakar, K. Dramane et al., Traditional
diseases in Mbalmayo Region, Central Province, Cameroon,
Medicine and Pharmacopoeia: Contribution to Ethnobotanical
Fitoterapia, vol. 72, no. 3, pp. 246 254, 2001.
and Floristic Studies in Cameroon, OUA/STRC, Lagos, Nigeria,
[28] J. Momeni, M. Akam, S. Kimbu, andW. Kreiser, Diterpenoids
1996.
from Ricinodendron heudelotii (Euphorbiaceae), Journal of the
[11] V. Kuete, A. T. Mbaveng, M. Tsaffack et al., Antitumor,
Cameroon Academy of Science, vol. 6, no. 1, pp. 119 124, 2006.
antioxidant and antimicrobial activities of Bersama engleriana
[29] J. Fondoun, T. Tiki, and J. Kengue, Recinodindron heudelotii
(Melianthaceae), Journal of Ethnopharmacology, vol. 115, no. 3,
(Djansang): ethnobotany and importance for forest dwellers in
pp. 494 501, 2007.
Southern Cameroon, Plant Genetic Resources Newsletter, vol.
[12] V. Kuete, B. Krusche, M. Youns et al., Cytotoxicity of some
118, pp. 1 6, 1999.
Cameroonian spices and selected medicinal plant extracts,
[30] J. Momeni, R. D. Djoulde, M. T. Akam, and S. F. Kimbu,
Journal of Ethnopharmacology, vol. 134, no. 3, pp. 803 812, 2011.
Chemical constituents and antibacterial activities of the stem
[13] J. P. Dzoyem, A. H. L. N. Kuete, V. Kuete et al., Cytotoxicity and
bark extracts of Ricinodendron heudelotii (Euphorbiaceae),
antimicrobial activity of the methanol extract and compounds
IndianJournal of Pharmaceutical Sciences, vol. 67, no. 3, pp. 386
from Polygonum limbatum, Planta Medica, vol. 78, no. 8, pp.
389, 2005.
787 792, 2012.
[31] E. Tekwu, A. Pieme, and V. Beng, Investigations of antimi-
[14] A. T. Choumessi, M. Danel, S. Chassaing et al., Characteriza- crobial activity of some Cameroonian medicinal plant extracts
tion of the antiproliferative activity of Xylopia aethiopica, Cell against bacteria and yeast with gastrointestinal relevance,
Division, vol. 7, article 8, 2012.
Journal of Ethnopharmacology, vol. 142, no. 1, pp. 265 273, 2012.
[15] J. Dzoyem, S. Guru, C. Pieme et al., Cytotoxic and antimi- [32] J. Momeni, W. P. D. D. Ntchatchoua, F. Fadimatou, M. T. Akam,
crobial activity of selected Cameroonian edible plants, BMC and M. B. Ngassoum, Antioxidant activities of some cameroo-
Complementary and Alternative Medicine, vol. 13, article 78, nian plants extracts used in the treatment of intestinal and
2013. infectious diseases, Indian Journal of Pharmaceutical Sciences,
vol. 72, no. 1, pp. 140 144, 2010.
[16] J. D. Tamokou, J. Chouna, E. Fischer-Fodor et al., Anticancer
and antimicrobial activities of some antioxidant-rich cameroo- [33] V. Kuete, P. D. Tchakam, B. Wiench et al., Cytotoxicity and
nian medicinal plants, PLoS ONE, vol. 8, no. 2, Article ID modes of action of four naturally occuring benzophenones:
e55880, 2013. 2,2 ,5,6 -tetrahydroxybenzophenone, guttiferone E, isogarcinol
10 Evidence-Based Complementary and Alternative Medicine
and isoxanthochymol, Phytomedicine, vol. 20, no. 6, pp. 528
536, 2013.
[34] J. Harbone, Phytochemical Methods: A Guide to Modern Tech-
niques of Plant Analysis, Chapman & Hall, London, UK, 1973.
[35] A. Kimmig, V. Gekeler, M. Neumann et al., Susceptibility
of multidrug-resistant human leukemia cell lines to human
interleukin 2-activated killer-cells, Cancer Research, vol. 50, no.
21, pp. 6793 6799, 1990.
[36] T. Efferth, A. Sauerbrey, A. Olbrichet al., Molecular modes of
action of artesunate in tumor cell lines, Molecular Pharmacol-
ogy, vol. 64, no. 2, pp. 382 394, 2003.
[37] J. Gillet, T. Efferth, D. Steinbach et al., Microarray-based
detection of multidrug resistance in human tumor cells by
expression profiling of ATP-binding cassette transporter genes,
Cancer Research, vol. 64, no. 24, pp. 8987 8993, 2004.
[38] H. S. Huang, M. Nagane, C. K. Klingbeil et al., The enhanced
tumorigenic activity of a mutant epidermal growth factor
receptor common in human cancers is mediated by threshold
levels of constitutive tyrosine phosphorylation and unattenu-
ated signaling, The Journal of Biological Chemistry, vol. 272, no.
5, pp. 2927 2935, 1997.
[39] J. O Brien, I. Wilson, T. Orton, and F. Pognan, Investigation of
the Alamar Blue (resazurin) fluorescent dye for the assessment
of mammalian cell cytotoxicity, European Journal of Biochem-
istry, vol. 267, no. 17, pp. 5421 5426, 2000.
[40] D. A. Bass, J. W. Parce, and L. R. Dechatelet, Flow cytometric
studies of oxidative product formation by neutrophils: a graded
response to membrane stimulation, Journal of Immunology, vol.
130, no. 4, pp. 1910 1917, 1983.
[41] A. Cossarizza, R. Ferraresi, L. Troiano et al., Simultaneous
analysis of reactive oxygen species and reduced glutathione
content in living cells by polychromatic flow cytometry, Nature
Protocols, vol. 4, no. 12, pp. 1790 1797, 2009.
[42] E. Teodori, S. Dei, C. Martelli, S. Scapecchi, and F. Gualtieri,
The functions and structure of ABC transporters: implications
for the design of new inhibitors of Pgp and MRP1 to control
multidrug resistance (MDR), Current Drug Targets, vol. 7, no.
7, pp. 893 909, 2006.
[43] G. Szakács, J. K. Paterson, J. A. Ludwig, C. Booth-Genthe, and
M. M. Gottesman, Targeting multidrug resistance in cancer,
Nature Reviews Drug Discovery, vol. 5, no. 3, pp. 219 234, 2006.
[44] N. Stamp, Out of the quagmire of plant defense hypotheses,
Quarterly Review of Biology, vol. 78, no. 1, pp. 23 55, 2003.
[45] V. Kuete and T. Efferth, Pharmacogenomics of Cameroonian
traditional herbal medicine for cancer therapy, Journal of
Ethnopharmacology, vol. 137, no. 1, pp. 752 766, 2011.
[46] M. Suffness and J. Pezzuto, Assays related to cancer drug dis-
covery, in Methods in Plant Biochemistry: Assays for Bioactivity,
K. Hostettmann, Ed., pp. 71 133, Academic Press, London, UK,
1990.
[47] K. M. Pluchino, M. D. Hall, A. S. Goldsborough, R. Callaghan,
and M. M. Gottesman, Collateral sensitivity as a strategy
against cancer multidrug resistance, Drug Resistance Updates,
vol. 15, no. 1-2, pp. 98 105, 2012.
[48] M. Saeed, H. Greten, and T. Efferth, Molecular mechanisms of
tumor cell resistance to chemotherapy, in Collateral Sensitivity
in Drug-Resistant Tumor Cells, B. Bonavida, Ed., pp. 187 211,
Springer, NewYork, NY, USA, 2013.
[49] B. Halliwell and J. Gutteridge, Free Radicals in Biology and
Medicine, Charendon Press, Oxford, UK, 2nd edition, 1988.
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