Antileukemic Effect of Zerumbone-Loaded Nanostructured Lipid Carrier on
Murine Leukemic (WEHI-3B) Model
Heshu, R.S. a b,c*, Abdullah, R. a, b*, Chartrand, M.S.d, Swee, K.Y.b, Ahmad, A.B. b,e, Tan,
S.W. b, Hemn, H.O. a,b , Zahra, A.f , Farideh, N.g, Arulselvan, P.b, Fakurazi, S. b,e
a
Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
b
c
Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Faculty of Veterinary Medicine, University of Sulaimany, Sulaimany City, Kurdistan Region, Northern Iraq
d
e
Director, DigiCare Behavioral Research, Casa Grande, Arizona, USA
Faculty of Medicine and Health Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
f
Faculty of Science & Technology, University Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia
g
Institute of Tropical Forestry and Forest Products (INTROP), Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia
Abstract
Cancer
nanotherapeutics
are
progressing
rapidly
systems to replace conventional delivery systems.
with
innovative
drug
delivery
Although, antitumor activity of
zerumbone (ZER) has been reported, there has been no available information of
ZER-loaded nanostructured lipid carrier (NLC) affects murine leukemia cells in
vitro and in vivo. In this study, in vitro and in vivo effects of ZER-NLC on murine
leukemia WEHI-3B cells were investigated. The results demonstrated that the
growth of leukemia cells in vitro was inhibited by ZER-NLC using MTT, Hoechst
33342, Annexin V, cell cycle and caspase activity assays. In addition, outcomes of
histopathology, TEM and TUNEL assays of BALB/c leukemia mice revealed that
the number of leukemia cells were significantly decreased in spleen tissue after four
weeks of oral administration of various doses of ZER-NLC. These in vivo results
were further confirmed by western blot and qRT-PCR assays. In conclusion, the
results
demonstrated that
ZER-NLC
induced mitochondrial
dysfunction
in
vivo
triggered events, which were responsible for mitochondrial dependent apoptosis
pathways. Furthermore, NLC is suggested as a promising carrier for ZER oral
delivery.
Keywords:
Zerumbone
nanoparticles,
leukemia,
WEHI-3B
cells,
BALB/c
mice,
apoptosis intrinsic pathway
*Corresponding
authors:
Haematology
Laboratory,
Department
of
Veterinary
Clinical Diagnosis, University Putra Malaysia, 43400Serdang, Selangor, Malaysia.
E-mail Address: rasedee@vet.upm.edu.my (A. Rasedee)
heshusr77@gmail.com (R.S. Heshu)
1. Introduction
Although leukemia, a blood or bone marrow cancer, is recognized as the seventh
most common cancer for all ages, its highest incidence among children aged
newborn to 14 years. In the United States, for instance, an estimated 48,610 new
cases of leukemia and 23,620 deaths from leukemia were reported in 2013 for all
ages, approximately 30% of whom were children up to 14 years [1].
Natural compounds have been an important source of drugs since ancient times.
In modern pharmacology, about half of the useful drugs have been derived from
natural sources where such drug discovery has been of substantial interest in
research.
Whereas, some of natural compounds were initially used as drugs, others
have provided the chemical templates for developing synthetic drugs. Others yet
were derivatives demonstrating powerful
chemopreventive activity. Since active
principles from natural sources have exhibited activity via apoptotic and signalling
2
pathways, and/or for various cancer targets, suggests that they are helpful starting
points in the design, development of novel and biologically active cancer preventive
agents [2].
Although many natural products have a strong therapeutic value, their poor
solubility
and
bioavailability
have
severely
limited
their
usage
[3].
Using
nanoparticle delivery systems is a new recently introduced method to overcome
these limitations turning potential by poor soluble drugs into effect therapeutic
agents. Due to less toxicity on normal cells and biodegradable property of natural
product
nanoparticles
(NPN),
the
nanotechnology-mediated
natural products have been recognized as safe and
NPN
delivery
system
of
effective method [4]. Therefore,
could be utilised with considerable advantage over currently employed
chemopreventive and chemotherapeutic approaches for cancer [5].
Zerumbone (ZER), a natural predominant compound is found in the rhizome of
Zingiber zerumbet. As a poorly soluble compound it can be utilized as an effective
drug-carrier with delivery capabilities offered by the NPN. The ZER-NLC was
developed and tested to be efficacious in the treatment of a human lymphoblastic
leukaemia cell line [6]. In a previous study, ZER was also found to possess
anticancer
properties
homogenization
(HPH)
and
was
incorporated
technique.
into
Physicochemical
NLC
by
high
characterization
pressure
included
particle size, polydipersity index, zeta potential, pH, entrapment efficiency, loading
capacity, stability study, and in vitro drug release, as well as physicochemical
stability after being autoclaved and stored at 4, 25 and 40˚C for 1 month, were
examined [7]. However, the effect ZER-NLC has on leukaemia has been uncertain,
because to-date a study verifying such effects has not yet been conducted.
3
Thus, the present study was conducted to evaluate the effect of ZER-NLC on
WEHI-3B cell-induced leukemia in a mice model within the guidelines for the care
of laboratory animals. The murine system was chosen due to low cost and the easy
establishment of cancer, in this case, leukemia. WEHI-3B leukemia cell line is a
myelomonocytic leukemia. This cell line was originally derived from BALB/c mice,
and was ideal for the study of leukaemias.
2. Materials and methods
2.1. Leukemia cell line and culture condition
The murine myelocytic leukemia cell line (WEHI-3B) was
purchased from
American Type Culture Collection (ATCC) (Maryland, USA). The cells were
maintained in a complete growing RPMI-1640 (Sigma Aldrich, USA) medium,
supplemented
with
10%
antibiotic-antimycotic
fetal
(Gibco
bovine
Invitrogen,
serum
UK)
(FBS)
in
75
(PAA,
cm2
Austria)
culture
and
flasks
1%
(TPP,
Switzerland) at 37˚C and 5% CO2 in a humidified incubator (Binder, Germany).
2.2. Zerumbone-loaded nanostructured lipid carrier
Pure (99.96%) colourless ZER crystals were extracted from essential oil of fresh
Zingiber zerumbet rhizomes by steam distillation according to the method described
earlier [8]. The ZER-NLC was prepared by a high-pressure homogenization method
and
characterized
by
zetasizer,
reverse
phase
high
performance
liquid
chromatography (RP-HPLC), transmission electron microscopy (TEM), wide angle
x-ray
diffraction
(WAXR),
differential
scanning
colorimeter
(DSC)
and
Franz
Diffusion Cell (FDC) system analyses. It has shown to be physically stable with
4
particle size (PS) of 52.68 ± 0.1 nm, zeta potential of ˗ 25.03 ± 1.24 mV and
polydipersity index (PI) of 0.29 ± 0.0041 µm [6, 8].
2.3. In vitro cytotoxic effect of zerumbone-loaded nanostructure lipid carrier on
WEHI-3B
2.3.1.The colorimetric cytotoxicity effect of ZER-NLC on WEHI-3B cells
The antiproliferative effect of ZER-NLC at concentrations of 1 to 100 μg/mL on
treated
WEHI-3B
cells
were
assessed
using
3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) assay according to the method described earlier
[9]. Briefly, about 1 × 105 cells were seeded into each well of 96-well plates and
incubated for 24 hours to become attached. After treating with ZER-NLC for 24, 48
and 72 h, MTT was freshly prepared at a concentration of 5.5 mg/mL and incubated
with cells for 4 hours. Later on, the formazan crystals were dissolved in 100 μL of
DMSO. Consequently, the optical density (OD) was measured at 570 nm using
ELISA plate reader (Universal Microplate reader) (Biotech, Inc, USA). The IC50
value (the
concentrat ion, which inhibited 50%
of cellular growth) was determined
from the absorbance versus concentration curve. Finally, the values were compared
to those of the positive antineoplastic agent control; doxorubicin (Sigma Aldrich,
USA). DMSO (0.1%) was used as negative control. All experiments were carried
out in triplicate.
2.3.2. Morphological assessment of apoptotic cells by Fluorescent microscope
WEHI-3B cells (1 × 105 cells/mL) were seeded on a 25 cm2 culture flask and
treated with IC50 value of ZER-NLC for 24, 48, and 72 hours. Later, the cells were
collected and washed twice with cold PBS and stained in dark with 10 μL Hoechst
33342 (1 mM) and 5 μL PI (100 μg/mL) on glass slides. Morphological changes of
5
stained cells were observed under a fluorescence microscope (ZEISS, Germany) in
less than 30 min [10].
2.3.3. Early cell apoptosis detection by Annexin V-FITC/PI assay
Apoptosis was detected with an Annexin V/FITC kit (Sigma Aldrich, USA)
following instructions of manufacturer without modifications. Briefly, about 1 × 105
ZER-NLC pre-treated (12, 24 and 48 hours) WEHI-3B cells were harvested and
washed with pre-chilled PBS. Later, the cells were suspended in 500 μL of
1 ×
binding buffer and stained with annexin V (5 μL) and propidium iodide (10 μL),
then incubated on ice in the dark for 15 minutes. Flow cytometric analysis was
immediately conducted using laser emitting excitation light at 488 nm using BD
FACs Calibur flow cytometer equipped with an Argon laser (BD, USA). Lastly, the
analysis was carried out using Summit V4.3 software (Beckman Coulter, Inc., Brea,
CA, USA).
2.3.4. Determination of DNA content of the cells by cell cycle analysis
Cell
cycle
analysis
was
carried
out
on
ZER-NLC
treated
leukemic
cells
according to the method described previously with slight modification [11]. Briefly,
WEHI-3B cells were seeded at a density 1 × 105 cells/mL and incubated for 24
hours. Cells were then treated with ZER-NLC for 24,48 and 72 hours. After
incubation, the cell pellets were washed with washing buffer (cold PBS-BSAEDTA containing 0.1% sodium azide) and fixed in 500 μL of 80% cold ethanol and
kept at ˗ 20˚C for one week. After that, the cells were washed twice with washing
buffer and to which 1 mL of staining buffer that contained 0.1% Triton-100, 50 μL
RNase A (1.0 mg/mL), and 25 μL propidium iodide (PI) (1.0 mg/mL) was added to
6
the fixed cells for 30 minutes at dark on ice. The DNA content of cells was then
analyzed
and
Flow
cytometric
analysis
was
conducted
using
laser
emitting
excitation light at 488 nm using BD FACSCalibur flow cytometer equipped with an
Argon laser (BD, USA). Data analysis was performed using Summit V4.3 software
(Beckman Coulter, Inc., Brea, CA, USA).
2.3.5. Caspase activities assay
The protease activity of caspase -3 and -9 in WEHI-3B cells were performed
using fluorometric assay kit according to the instructions of manufacturer (Abcam,
USA). Briefly, 1 × 105 WEHI-3B cells were seeded overnight, treated with ZERNLC and incubated for 24, 48 and 72 hours. Then, cells were washed with cold
PBS, and were prepared to a final volume of 50 μL with dH2O in 96-well plate to
which 5 μL active caspase and 50 μL master mix that containing 2X reaction buffer
and 50 μM caspase substrate
were added. After incubation at 37˚C for exactly 1 h,
the samples were read in a fluorescence plate reader (Infinite M200, Tecan, USA)
equipped with a 400 nm excitation filter and 505 nm emission filter. Data was
presented as optical density (OD) and an histogram was plotted.
2.4. In vivo anti-leukemic effect of zerumbone-loaded nanostructured lipid carrier
2.4.1. Preparation of cancer cells and leukemia induction
Upon growing of the WEHI-3B cells and reaching a 90% confluence, the
medium was removed and cells were washed twice with PBS and count with trypan
blue
(Sigma
Aldrich,
USA)
staining
under
light
microscope
(Nikon,
Japan).
Eventually, the cells were suspended in 300 μL PBS and used within 1 hour of
preparation.
7
2.4.2. Animals
Adult female and male BALB/c mice aged 6 to 8 weeks were purchased from the
animal house of the Faculty of Medicine, Universiti Putra Malaysia. The mice were
housed in polypropylene plastic cages with wood chips as bedding. The rats were
acclimatized to the laboratory environment at 24 ± 1˚C under a 12 hours dark-light
cycle for at least five days before commencement of the experiment. The mice were
provided pellet and water ad libitum during the period of study. This study was
approved by the Animal Care and Use Committee (ACUC), Faculty of Veterinary
Medicine, Universiti P u t r a Malaysia (UPM/FPV/PS/3.2.1.551/AUP-R152).
All
animal
intraperitonal
groups,
excluding
the
first
group,
were
anesthetized
by
an
injection of a mixture of ketamine-HCl and xylazine. The remaining
groups were injected intraperitonally with 300 μL of WEHI-3B cells (1 × 106
cells/animal) in PBS using a tuberculin (TB) syringe and 26 G needle. Beginning the
next day, a drop of blood from the tail veins of four mice per group were collected
for 5 consecutive days. Blood smears were observed under Wright stain to check
leukemia development in the mice.
2.4.3. Experimental design and drug treatment
Group 1 comprised untreated normal, healthy mice and served as the negative
controls (animals without leukemia burden). Group 2 comprised of mice that were
induced to develop leukemia and served as the leukemia control, while groups 3, 4
and 5 were leukemic mice treated daily with 60 mg/kg body weight with blank NLC
(vehicle), 30 mg/kg body weight ZER-NLC and 60 mg/kg body weight ZER-NLC
respectively. Group 6 were treated with 4 mg/kg body weight all trans-retinoic acid
(ATRA) (Sigma Aldrich, USA), an anticancer chemotherapy drug, dissolved in
8
olive oil (Sigma Aldrich, USA) and served as positive control. The animals were
deprived of feed 12 hours prior to treatment. The treatments were given orally
exactly after four days of injection and after confirmation of leukemia induction for
four consecutive weeks to the animals through gastric intubations using a balltipped stainless steel gavage needle attached to a syringe. All animals were
euthanised by an overdose of intraperitonal injection of a mixture of ketamine-HCl
and xylazine, and spleen samples were collected for further analysis.
2.4.4. Histopathology
Collected spleen samples were cut into small pieces and fixed in 10% formalin
for at least 48 hours. The fixed samples were dehydrated using an automated tissue
processor (Leica ASP300, Germany), embedded in paraffin wax (Leica EG1160,
Germany), and then blocks trimmed and sectioned using a microtome (Leica
RM2155). The tissue sections were mounted on glass slides using a hot plate
(Leica HI1220, Germany) and subsequently treated in order with 100, 90 and 70%
ethanol for 2 min each. Finally, tissue sections were stained with the Harris’s
haematoxylin and eosin (H & E) [12] and examined under light microscope
(Nikon, Japan).
Leukemia scoring was conducted on H & E slides based on the number of
leukemic cells in the spleen tissue. Score 0 = normal (no leukaemic cells), score 1 =
mild (leukaemic cells were between 0 to 25 cells), score 2 = moderate (leukemic
cells were between 25-50 cells), score 3 = severe (leukemic cells were between 5075 cells) and score 4 = more severe (leukaemic cells were between 75 to100 cells)
[13].
9
2.4.5. Transmission Electron Microscopy (TEM)
Spleen samples were collected, cut into sections of about 0.5 cm2 sizes and fixed
in 4% glutaraldehyde in a cocodylate buffer overnight. The specimens were washed
in sodium cocodylate buffer and post-fixed in 1% osmium tetra-oxide. Then, the
specimens were washed again in sodium cocodylate buffer, dehydrated in ascending
grades (35, 50, 75, 95 and 100%) of acetone, infiltrated with a mixture of acetone
and resin (50: 50), embedded with 100% resin in beam capsule, and then
polymerized. Then, the area of interest was chosen from the thick sections, stained
with toulidine blue and examined under lighted microscope. Then, the selected area
was cut into ultrathin sections using ultra-microtome, placed on copper grids and
stained with uranyl acetate and citrate. The tissue was finally washed twice with
distilled water and viewed under TEM (Phillips, Eindhoven, Netherlands).
2.4.6. TUNEL assay
Apoptosis in spleen tissues was evaluated with the TUNEL (Tdt-mediated dUTP
Nick-end
labeling)
Fluorometric
deparaffinized,
TUNEL
kit
according
System).
rehydrated,
fixed,
to
Briefly,
and
manufacturer’s
the
spleen
equilibrated.
After
protocol
tissue
that,
(DeadEndTM
sections
rTdT
were
incubation
buffer was added to the equilibrated area, covered with plastic cover slips and
incubated at 37ºC for 60 minutes in the humidified chamber away from direct light.
Then, the reactions were terminated by immersing the slides in 2X SSC and stained
by freshly prepared PI solution in PBS in the dark. The slides were washed with
PBS between each step. Finally, the samples were mounted using glass cover slip
and viewed under fluorescent microscope using a standard fluorescent filter set to
10
view the green fluorescence at 520 ± 20 nm: view red fluorescence of PI at > 620
nm.
Leukemia scoring was conducted on TUNEL slides based on the apoptosis
progression of leukemic cells in the spleen tissue. Score 0 = no apoptosis, score 1 =
mild apoptosis (apoptotic cells were between 0 to 25 cells), score 2 = moderate
apoptosis (apoptotic cells were between 25 to 50 cells), score 3 = highly moderated
apoptosis (apoptotic cells were between 50 to 75 cells) and score 4 = marked
apoptosis (apoptotic cells were between 75 to 100 cells) [14].
2.4.7. Western blotting analysis
Protein was extracted from mice spleen tissues by snap-freezing in liquid
nitrogen
and
adding
RIPA
lysis
buffer
(Thermo
Scientific,
USA)
containing
protease inhibitor cocktail (Sigma Aldrich, USA). The concentration of extracted
proteins were quantified using Bradford protein assay kit, and later on protein
suspensions were aliquoted into PCR tubes (Bio-Rad, USA) and stored at ˗ 80˚C
until use. Equal amounts of protein (25 µg) were resolved and separated based on
molecular weight via electrophoresis in an electric field at 10% SDS-PAGE system
(Bio-Rad,
USA).
The
broad
pre-stained
protein
molecular
weight
ladder
(GeneDirex, USA) was used to monitor protein migration. Then, proteins were
transferred and blotted on to the PVDF membrane (Bio-Rad, USA) and were
blocked sequentially for 1 hour in blocking solution at room temperature on the
Belly
Dancer®
(Stovall,
Life
Science
Incorporated,
North
Carolina,
USA).
Membranes were washed with PBST and probed with specific primary antibodies to
subunits of Bcl-2, Bax, Cyt-c, PARP, FasL and β-actin as internal control (Abcam,
USA) overnight at 4˚C on the roller mixer. The following day, membranes were
11
washed
several
times
with
PBST
and
incubated
with
goat–anti-rabbit
IgG
conjugated to horseradish peroxidase secondary antibody (Abcam, England) room
temperature for 1 hour. Then, the membranes were washed again with PBST. The
immunoreacted
protein
chemiluminescence
England)
and
blotting
bands
were
substrate
chemiluminescence
developed
kit (ECL
image
western
analyzer
and
detected
using
blot
substrate,
Abcam,
system
(Chemi-Smart,
Vilber
Lourmat, Germany) were used to view the membranes. The results were expressed
in standard units and intensity of bands quantitated using the image J software
(BioTechniques, USA).
2.4.8. Relative quantitative gene transcription assay (qRT-PCR)
Total RNA was extracted from spleen specimens using RNeasy® lipid tissue
mini kit (Qiagen, Valencia, CA) for exactly two weeks before conducting the qPCR analysis. The extracted RNA was quantified using Nanophotometer (IMPLEN,
GmbH, Germany) and the extracted RNA was aliquoted and stored at ˗ 80˚C. qRTPCR assay for targeted genes (Bcl-2, Bax, Cyt-c, PARP and FasL) and reference
genes
(GAPDH and β-actin) was run for extracted RNA of spleen samples in all
animal groups using QIAGEN® One Step RT-PCR SYBR Green kit (Qiagen,
Valencia, CA). The cycles were 50˚C for 10 min (reverse transcription), 1 cycle of
95˚C for 5 min (initial activation), and 39 cycles of 95˚C for 10 sec (denaturation),
50 – 60˚C for 30 sec (combined annealing and extension). The fluorescence was
measured after each extension step. The threshold was set manually at the
exponential phase of the amplification process and the melting curve analysis was
performed from 70 ˗ 95˚C, with 0.5˚C/5 sec increments. All reactions were
performed in triplicate and relative expression of genes were performed using CFX
12
Managertm
software,
version
1.6
(BioRad
Laboratories,
Inc.,
Hercules,
CA)
incorporated with the real-time PCR thermal cycler (BioRad®, CFX96, BioRad
Laboratories, Inc., Hercules, CA) in order to calculate Ct values for each sample by
comparing Ct values of the target gene with Ct values of the GAPDH and β-actin
constitutive gene product.
2.5. Statistical analysis
The experiments were carried out in triplicate, and the results were expressed as
mean ± SD. Statistical analysis was accomplished using SPSS version 20.0 (SPSS
Inc., Chicago, USA). Data were analyzed using comparison test-one way ANOVA,
following with post hoc by Tukey’s-b test. Probability values of less than 0.05 (P <
0.05) were considered statistically significant.
3. Results
3.1. In vitro cytotoxicity of ZER-NLC
3.1.1. Cell viability of ZER-NLC treated WEHI-3B leukemia cells
Under the experimental conditions, cytotoxicity results revealed that various
concentrations of ZER-NLC exhibited marked and
significant (P < 0.05) inhibition
effects on the survival of WEHI-3B cells with calculated
IC50 as 14.25 ± 0.36
μg/mL, 10.42 ± 0.77 μg/mL and 7.5 ± 0.55 μg/mL time and dose dependently for
24, 48 and 72 hours treatment, respectively (Figure 1A). Similarly, doxorubicin
imposed a significant (P < 0.05) cytotoxic effect against WEHI-3B cells time
dependently with an IC50 value of 1.3 ± 0.15 μg/mL, 1.09 ± 1.24 μg/mL and 0.82 ±
1.5 μg/mL after 24, 48 and 72 hours incubation (Figure 1B). On the other hand,
13
DMSO as a negative control did not exhibit any inhibitory effects toward treated
WEHI-3B cells.
3.1.2. Induction of apoptosis achieved by Hoechst 33342 staining
The occurrence of apoptosis was identified with Hoechst 33342 staining time
dependently in the treated cells. Staining the 24 hours ZER-NLC treated WEHI-3B
cell showed the typical features of apoptosis such as chromatin condensation and
morphology changes, as well as cell shrinkage and membrane blebbing.
hours
ZER-NLC
treated
cells
showed
smaller
nuclei;
some
had
The 48
peripherally
condensed or clumped chromatin whereas others had fragmented nuclear chromatin.
Apoptotic body formation was more prominent at 72 hours post-treatment of ZERNLC. In contrast, the cells in control group, without treatment demonstrated normal
nuclear and cellular morphology (Figure 2).
3.1.3. Phosphatidylserine externalization
In order to quantify the apoptosis, we performed an Annexin V-FITC/PI staining
experiment to examine the occurrence of phosphatidylserine externalization onto the
cell surface. The percentage of Annexin V-FITC stained cells both the early and late
apoptotic cells increased gradually and significantly (P < 0.05) in all groups with
the time applied, while the percentage of viable cells consequently decreased
gradually (Figure 3). At 12 hours treatment, an abundant amount of cells was
primarily in the early phase of apoptosis (18.50 ± 0.91), while
increasing
incubation time to 48 and 72 hours, respectively, induced more cell apoptosis in late
stage (19.79 ± 0.62 and 27.36 ± 0.10 consequently).
14
3.1.4. Cell cycle assay
The cell cycle analysis demonstrated that the untreated cells showed normal
DNA content and cell cycle distribution. On the other hand, ZER-NLC induced a
concomitant and significant (P < 0.05) accumulation of WEHI-3B cell populations
with apoptotic peak in the subG0/G1 phase especially at 72 hours of treatment
(21.22 ± 0.66). Moreover, ZER-NLC induced cell cycle arrest in the G2/M phase
with values of 10.54 ± 0.45, 19.62 ± 0.37 and 30.56 ± 0.53 after 24, 48 and 72 hours
of treatment, respectively (Figure 4).
3.1.5. Introduction of apoptosis by caspase protease family
To
investigate
the
involvement
of
caspases
in
ZER-NLC-induced
apoptosis,
WEHI-3B cells were treated for various times and protease enzymatic activity were
determined.
ZER-NLC significantly (P < 0.5) stimulated both caspase -3 and -9
enzyme activities with more than one fold activity in a time-dependent manner in
the treated WEHI-3B cells, compared to untreated control groups (Figure 5).
3.2. In vivo anti-leukaemic effect of ZER-NLC
3.2.1. Blood smear
Mice induced to developed leukaemia showed increased immature myeloid and
monocytic cells in circulation. The cells appeared large with high cytoplasm to
nucleus (Figure 6).
3.2.2. Histopathology
Histopathological examination indicated that there was a significant (P < 0.05)
and massive proliferation of pleomorphic neoplastic cells in the spleens parenchyma
15
(red and white pulp) of untreated leukemic animals, which led to disappearance of
the sinusoids. These neoplastic cells were characterised by large irregular nuclei
with clumped chromatin. Same lesions with minor changes were also found in NLC
treated animals. On the other hand, spleen tissues of treated animals (low and high
dose ZER-NLC or ATRA) demonstrated a significant reduction (P < 0.05) in
leukemic cells when comparing to the untreated normal control group (Figure 7).
3.2.3.Transmission electron microscopy
The untreated normal control spleen showed normal cell features. The malignant
cells in leukemic spleen, without treatment, showed pleomorphism, large-sized with
markedly irregular surfaces and abnormal nuclear features. On the other hand, the
apoptotic changes in cells of the treated animal groups (low and high dose ZERNLC
and
ATRA)
included
the
appearance
of
peripheral
nuclear
chromatin
condensation (margination of chromatin), lobulation of nuclei, and cell membrane
blebs. Some apoptotic cells showed fragmented nucleus that formed apoptotic
bodies (Figure 8).
3.2.4.TUNEL assay
Sections of spleen tissues of leukemic mice in low and high doses of ZER-NLC
and ATRA treated groups showed significant (P < 0.05) increase in the number of
apoptotic cells indicated by higher signal of green fluorescence under fluorescent
microscope. The spleen of untreated control and untreated leukemia groups showed
non significant (P > 0.05) apparent apoptosis. Spleen tissues of leukaemic mice
treated with NLC alone showed presence of only a few apoptotic cells (Figure 9).
16
3.2.5.Western blot
β-actin was used as the loading control, with relatively equal intensity bands,
confirming that the samples have equal protein concentrations. This study showed
that there is a significant (P < 0.05) increase in expression (up-regulation) of Bcl-2
protein in leukemia and NLC-treated groups. While, non-significant (P > 0.05) Bax,
Cyt-c, and PARP protein expression was observed in leukemia and NLC treated
leukaemia. On the other hand, significant (P < 0.05) increased (up-regulation) in
expression of Bax, Cyt-c, and PARP proteins in low and high doses of ZER-NLC,
and ATRA treated groups were found. While, a significant (P < 0.05) decreased
(down-regulation) in suppression of Bcl-2 protein in low and high doses of ZERNLC, and ATRA treated groups were found. Simultaneously, in these low and high
doses of treated ZER-NLC and ATRA groups, the PARP protein cleaved from 116
kDa to 85 kDa. In addition, FasL, a type II transmembrane protein did not indicate
any up-regulation or down-regulation in all treatment groups (Figure 10).
3.2.6. Relative expression levels of genes transcripts using qRT-PCR
The expression levels of each gene were calculated and normalized to the
expression level of the reference genes. Similarly, the relative expression levels of
each gene in the untreated control group were calculated and compared to that of the
other groups. As a result, it was found that the relative expression of Bcl-2 was
significantly (P < 0.05) up-regulated in leukemia control and NLC group comparing
to the untreated control group. Whereas the relative expression of Bcl-2 was
significantly (P < 0.05) down-regulated in low and high ZER-NLC- and ATRAtreated groups.
In case of Bax, Cyt-c and PARP, their relative expression were
none-significantly (P > 0.05) down-regulated in leukemia control and NLC-treated
groups, while they were significantly up-regulated (P < 0.05) in the ZER-NLC- and
17
ATRA-treated groups. On the other hand, FasL did not show any up-regulation or
down-regulation in all treatment groups (Figure 11).
4. Discussion
The use of the above described drug-delivery systems were implemented to
improve drug efficacy through increased solubility, sustained release, and prolonged
effect and tissue targeting [15]. To improve the bio-distribution and bio-availability
of
cancer
drugs,
nanoparticles
were
designed
for
optimal
size
and
surface
characteristics to increase circulation time. Thus, the water-insoluble ZER can be
solubilised for parenteral applications to increase half-life and improve tumour
targeted delivery [16, 17]. To ascertain the potential of ZER-NLC as an anticancer
compound, its anticancer effects were investigated both in vitro and in vivo. For the
in vitro study, WEHI-3B leukemia cell line, which is a myelomonocytic leukemia
cell line originally derived from BALB/c mice, was used, [18] since the murine in
vivo model possess the advantages of low cost and ease of establishment [19, 20].
For in vivo study, mice were injected with WEHI-3B to develop leukaemia and
treated with various doses of ZER-nanoparticles.
In this study, results indicated that various concentrations of ZER-NLC exhibited
marked and significant (P < 0.05) inhibition effect towards WEHI-3B cells for 24,
48 and 72 hours timelines. Thus, we confirmed that WEHI-3B cells were sensitive
to ZER-NLC and cell growth inhibition occurred in a time- and dose-dependent
manner. We have also shown in a previous study that the cytotoxicity of ZER-NLC
is primarily due to the ZER itself and not to NLC as we confirmed previously that
the anticancer activity of ZER is not affected or impaired by its incorporation into
NLC.[8] On the other hand, doxorubicin, which is classified as an antitumor
18
antibiotic made from natural products produced by species of the soil fungus
Streptomyces, [21] was used as control positive. Doxorubicin has IC50 value of 1.3 ±
0.15 μg/mL, 1.09 ± 1.24 μg/mL and 0.82 ± 1.5 μg/mL on WEHI-3B after 24, 48 and
72
hours
incubation.
Thus,
our
results
confirmed
that
ZER-NLC
had
lower
cytotoxicity than doxorubicin.
In an attempt to elucidate whether the loss in the WEHI-3B cell viability induced
by ZER-NLC was associated with apoptosis and cell morphology, using Hoechst
33342 staining were analyzed. WEHI-3B cells treated with ZER-NLC presented
with typical characteristic morphological changes of apoptosis, such as shrinking
cytoplasm,
condensed
chromatin
and
nuclear
fragmentation
with
intact
cell
membrane. Thus, the observations of Hoechst 33342 staining suggest that the
progression of cell death is time-dependent.
Results from the morphological assessment were further supported by AnnexinV/PI
and
cell
cycle
evaluations
using
flow
cytometer.
Phospholipid
phosphatidylserine (PS), which can be bound by Annexin V, is located in the
cytosolic leaflet of the plasma membrane lipid bilayer where its redistribution from
the inner to the outer leaflet is an early and widespread event during apoptosis, [22]
thus allowing for detection, quantification, and discrimination of apoptotic, necrotic,
and dead cells.
In our study, the percentage of viable cells gradually fell in all groups, while the
percentage of early and late apoptotic cells consequently increased gradually and
significantly (P < 0.05). Taken together with previous experiment, the results
suggested that ZER-NLC induced growth suppression of WEHI-3B cells was via
the induction of apoptosis. In terms of cell cycle evaluation, shifts in the
redistribution of phases of the cell cycle in response to various stimuli, including a
19
response to growth factors, drugs, mutations or nutrients, was readily assessed by
flow cytometry via the stain of DNA using dye such as PI [23].
The
cell
cycle
analysis
demonstrated
that
ZER-NLC
was
induced
with
a
significant (P < 0.05) accumulation of apoptotic WEHI-3B cells in the sub G0/G1
phase and G2/M cell cycle arrest after 24, 48 and 72 hours of treatment. This
indicated that these cells had undergone apoptosis. This result is consistent to the
hypothesis that the appearance of sub-G1 cells is the marker of cell death by
apoptosis [24]. Previously, ZER-NLC have been shown to induce apoptosis by their
ability to arrest cells in the G2/M phase of the cell cycle in Jurkat cells. In this
respect there was accumulating evidence that ZER-NLC can naturally inhibit the
cell cycle process [25].
The activation of caspase proteases is a critical event in the induction of
apoptosis. Caspase is produced as inactive zymogens and undergo proteolytic
activation during apoptosis. Caspase -9 is upstream initiator caspase; caspase-3 is
one of the downstream effector that plays the central role in the initiation of
apoptosis [26]. Recent studies have also shown that a number of anti-cancer drugs
induce apoptosis through the activation of caspases pathway and the mitochondrial
membrane dysfunction. In line with our previous findings, where the ZER-NLC
showed a time dependent increase in caspase -3 and -9 activities in Jurkat cells,
ZER-NLC also demonstrated the same caspase- activities in WEHI-3B cells over
time in this study. Thus, these results suggest that ZER-NLC decreased WEHI-3B
cell viability mainly due to the induction of apoptosis via intrinsic mitochondrial
pathway.
WEHI-3B leukemia cell line was first established in 1969 and demonstrated
identifiable characteristics of myelomonocytic leukemia. It has since been used
20
successfully to induce leukemia in syngenic BALB/c mice and has become a useful
animal model for evaluation of the anti-leukemia effects of drugs and natural
compounds [27]. At the end of in vivo experiments the mice belonging to the
normal control group were healthy, whereas all the mice injected with leukaemia
eventually developed symptoms of anemia, weakness, and labored breathing with
no symptoms of vomission or diarrhea. In this study, the stained blood smears of
leukemic mice revealed increased numbers of immature myeloid and monocytic
cells in peripheral blood and the cells appeared large with a high cytoplasm to
nucleus ratio. These manifestations were observed in the mice as early as four days
following intraperitonal inoculation of WEHI-3B cell line.
The spleen, as a secondary immune organ, has shown to be the best organ with
which
to
examine
leukemia,
because
of
its
high
lymphocyte
population.
Histological examinations showed an uncontrolled proliferation of neoplastic cells
in the spleens in the untreated leukemia group and in those treated with blank NLC.
On the other hand, the spleen tissues of mice treated with high dose of ZER-NLC
and ATRA displayed significant (P < 0.05) and markedly lower numbers of
neoplastic cells than those treated with low dose ZER-NLC. Similar findings were
likewise reported in other BALB/c mice leukemia model studies [28, 29].
TEM is an analytical tool that detects ultrastructural characteristic of the cells.
Leukemic cells in the spleen of untreated leukaemic mice appeared large in size and
with a markedly irregular surface. The nucleus to cytoplasm ratio was high and the
cytoplasm
showed
occasional
organelles.
Distinctive
ultrastructural
features
of
malignant cells were also observed in spleens of leukemic mice treated with NLC.
On the other hand, the spleen of leukaemic mice treated with low and high doses of
ZER-NLC and ATRA showed neoplastic cells in apoptosis, with characteristic
21
peripheral nuclear chromatin condensation (margination of chromatin), lobulation of
nuclei, and cell membrane blebbing. Some apoptotic cells showed fragmented
nucleus that formed apoptotic bodies.
Fragmentation of DNA resulting from the induction apoptosis signaling pathway
is usually associated with effects of anticancer agents on treated cells or tissues. In
this study, analysis using TUNEL assay showed that ZER-NLC, like ATRA the
anticancer drug, had antileukemic activity by inducing significant (P < 0.05)
apoptosis leukemic cells in the spleen of the BALB/c mice. The normal and
leukemia control treated group showed little evidence of apoptosis in the spleen,
while
and
NLC-treated
group
showed
non
significant
evidence
of
apoptosis.
Previously we established the apoptotic effect of ZER-NLC on Jurkat cells using
TUNEL assay [25]. These findings were confirmed in the current study in the in
vivo mice model.
In this study, the expression of pro- and anti-apoptotic proteins was analysed.
The Bcl-2 family is major apoptotic proteins that govern mitochondrial membrane
permeability [30]. In our study, the expression of apoptotic proteins in the spleen of
untreated and treated leukemic mice was determined by western blotting. This assay
may give some insight into the mechanism of apoptosis induction by ZER-NLC. It
was found that low and high doses of ZER-NLC and ATRA were significantly (P <
0.05) down-regulated Bcl-2 proteins, and significantly (P < 0.05) up-regulated Bax,
Cyt-c and PARP proteins. FasL protein did not show any up-regulation or downregulation on all treatment groups.
Reverse transcription quantitative PCR (RT-qPCR) assay is considered the gold
standard for gene expression analysis in various cells because of its reliability,
accuracy, sensitivity and fast quantification of results [31]. Using PCR assay it was
22
found that relative expression of Bcl-2 was significantly (P < 0.05) up-regulated in
leukemia control and NLC group in comparison with the untreated control group.
Whereas, the relative expression of Bcl-2 was significantly down-regulated in group
ZER-NLC- and ATRA-treated groups.
In the case of Bax, Cyt-c and PARP, their
relative expression were insignificantly (P > 0.05) down-regulated in leukemia
control and NLC-treated groups, while significantly up-regulated (P < 0.05) in the
ZER-NLC- and ATRA-treated groups. FasL protein did not show any up-regulation
or
down-regulation
in
all
treatment
groups.
Collectively,
the
result
of
this
experiment revealed the same and consistent result as that of western blotting.
Apoptosis is an important and well-controlled form of cell death that occurs
under a variety of physiological and pathological conditions. Experimental studies
have revealed that the apoptotic pathway of cell death induction is one of the most
sorted mechanisms of action for reliable and potential anticancers. It is well
accepted today that apoptosis has become an important issue in biomedical research
and chemopreventive agents, which are able to modulate induction of apoptosis, and
are often useful anticancer therapies [32]. The intrinsic pathway (mitochondria
related pathway) is regulated through Bcl-2 family proteins.
In mammals, the Bcl-2 family includes anti-apoptotic protein such as Bcl-2, proapoptotic protein such as Bax. Bcl-2 associated with the mitochondrial outer
membrane and the endoplasmic reticulum/nuclear membrane and maintained their
integrity. Bax is a cytosolic monomer in the normal state, but it changes formation
during
apoptosis,
integrity, provokes
oligomerizes
in
the
the permeabilization
mitochondrial
membrane,
breaches
of outer mitochondrial membrane,
its
and
dissipates the mitochondrial membrane potential (△Ψm), allowing the efflux of
pro-apoptotic proteins such as cytochrome c, which allows activation of caspase-9
23
and caspase-3 and consequently cleavage of PARP protein. The extrinsic pathway
(death receptor-related pathway) was triggered by the binding of extracellular death
ligands, such as Fas L with death receptor, in turn, activated the caspase -8 [30].
The mechanisms of the ZER-NLC induced apoptosis in the WEHI-3B cell are
unknown. To obtain a better mechanism of ZER-NLC action, we investigated the
expression of some Bcl-2 family members. Our data demonstrated that ZER-NLC
treatment resulted in up-regulation of the level of Bax with a concomitant downregulation of the level of Bcl-2, suggesting that the changes in the ratio of antiapoptosis
and
pro-apoptosis
promotion
activity.
Bcl-2
Caspase-3
family
activation
proteins
was
might
dominant,
involve
and
apoptosis-
reflected
in
the
cleavage of PARP, a well-known caspase-3 substrate. We found that ZER-NLC also
resulted in a cleavage of 116 kDa PARP into 85 kDa in WEHI-3B cells. As shown
previously, ZER-NLC induced apoptosis in Jurkat cells via intrinsic mitochondrial
pathway [25]. Taken together, our data suggest that ZER induces apoptosis through
the mitochondrial-dependent pathway in WEHI-3B cells in vitro and in vivo as well.
In
conclusion,
leukemia
cells.
this
The
study
confirmed
apoptotic
effect
of
that
ZER-NLC
ZER-NLC
on
induces
mice
apoptosis
of
myelomonocytic
leukaemia is similar to that of human T-lymphoblastic leukemia through the
activation of mitochondrial pathway of apoptosis.
Collectively, we supported that
the use of NLC opens up new perspectives for the formulation of poorly-soluble
drugs and has potential in parenteral administration of drugs.
5. Conflict of interest statement
All authors declare no conflict of interest.
24
Acknowledgements
We would like to extend appreciation to the Institute of Bioscience (IBS) and
Faculty of Biotechnology /UPM for their kind help and support. Grateful thanks to
the Faculty of Veterinary Medicine/ UPM and to DigiCare Behavioral Research for
providing technical expertise for this study. Special thanks to MOSTI (Grant No.
5495308) for funding this project.
References
[1] American Cancer Society (2013). Cancer Facts & Figures 2013. Retrieved on
January
10,
2014
from
http://www.cancer.org/acs/groups/content/@epidemiologysurveilance/document
s/document/acspc-036845.pdf
[2]M.H. Pan, C.T. Ho, Chemopreventive effects of natural dietary compounds on
cancer development. Chem. Soci. Rev. 37 (2008) 2558-2574.
[3]S.I. Abdelwahab, B.Y. Sheikh, M.M.E. Taha, C.W. How, R. Abdullah, U.
Yagoub, R. El-Sunousi, E.E.M. Eid, Thymoquinone-loaded nanostructured
lipid
carriers:
preparation,
gastroprotection,
in
vitro
toxicity,
and
pharmacokinetic properties after extravascular administration. Int. J.
Nanomed. 8 (2013) 2163-2172.
[4]I.A.
Siddiqui, V.M. Adhami,
nanotechnology in cancer:
Nanomed. 7 (2012) 591-605.
J.C. Chamcheu, H. Mukhtar, Impact
emphasis on nanochemoprevention. Int.
of
J.
[5]D.J. Bharali, I.A. Siddiqui, V.M. Adhami, J.C. Chamcheu, A.M. Aldahmash, H.
Mukhtar, S.A. Mousa, Nanoparticle delivery of natural products in the
prevention and treatment of cancers: current status and future prospects.
Cancers 3 (2011) 4024-4045.
[6]A. Rasedee, S.R. Heshu, A. Ahmad Bustamam, C.W. How, K.Y. Swee, A
Composition for Treating Leukaemia, in, Malaysian Patent Application,
Malaysia, 2013.
[7]H.S. Rahman, R. Abdullah, B.A. Ahmad, A.Z. Nazariah, H.O. Hemn, K.Y. Swee,
W.H. Chee, W.G. Wan Nor Hafiza, Zerumbone-loaded Nanostructured Lipid
Carrier Induces G2/M Cell Cycle Arrest and Apoptosis via Mitochondrial
Pathway in human lymphoblastic leukemia cell Line. Int. J. Nanomed. 9
(2014) 527-538.
25
[8]H.S. Rahman, A. Rasedee, W.H. Chee, A.B. Abdul, N.A. Zeenathul, H.H.
Othman, I.S. Mohamed, K.Y. Swee, Zerumbone Loaded Nanostructured
Lipid Carrier: Preparation, Characterization and Antileukemic Effect. Int J
Nanomed 8 (2013) 2769-2781.
[9]A.A. Van de Loosdrecht, R.H.J. Beelen, G.J. Ossenkoppele, M.G. Broekhoven,
M.A.C. Langenhuijsen, A tetrazolium-based colorimetric MTT assay to
quantitate human monocyte mediated cytotoxicity against leukemic cells
from cell lines and patients with acute myeloid leukemia. J. Immunol. Meth.
174 (1994) 311-320.
[10]S.-Y. Park, S.-J. Cho, H.-c. Kwon, K.-R. Lee, D.-K. Rhee, S. Pyo, Caspaseindependent cell death by allicin in human epithelial carcinoma cells:
involvement of PKA. Cancer Lett. 224 (2005) 123-132.
[11]D. Schloffer, M. Horky, V. Kotala, J. Wȩsierska-Ga̧dek, Induction of cell cycle
arrest and apoptosis in human cervix carcinoma cells during therapy by
cisplatin. Cancer Detec. Preven. 27 (2003) 481-493.
[12]L.G. Luna, Manual of histologic staining methods of the Armed Forces Institute
of Pathology 3rd ed., Mc Graw-Hill, New York, 1968.
[13]A.-S. Hutheyfa, Effects of Morinda citrifolia on N-methyl N-nitroosourea
induced peripheral T cell non-Hodgkin's lymphoma in male Sprague Dawely
rats, in, Microbiology and Pathology, University Putra Malaysia, Serdang,
2010, pp. 142.
[14]S.I. Abdelwahab, A.B. Abdul, N. Devi, T.M. Ehassan, A.S. Al-Zubairi, S.
Mohan, A.A. Mariod, Regression of cervical intraepithelial neoplasia by
zerumbone in female Balb/c mice prenatally exposed to diethylstilboestrol:
Involvement of mitochondria-regulated apoptosis. Exp. Toxicol. Pathol. 62
(2010) 461-469.
[15]S.N. Mistry, P.K. Patel, P.D. Bharadia, V.M. Pandya, D.A. Modi, Novel Drug
delivery system for lipophilic agents: Solid Lipid Nanoparticles. IJPI’s
Journal of Pharmaceutics and Cosmetology 1 (2011) 76-89.
[16]D.J. Bharali, I.A. Siddiqui, V.M. Adhami, J.C. Chamcheu, A.M. Aldahmash, H.
Mukhtar, S.A. Mousa, Nanoparticle delivery of natural products in the
prevention and treatment of cancers: current status and future prospects.
Cancers 3 (2011) 4024-4045.
[17]N.V. Cuong, M.F. Hsieh, C.M. Huang, Recent development in nano-sized
dosage forms of plant alkaloid camptothecin-derived drugs. Recent Patents
on Anti-Cancer Drug Discovery 4 (2009) 254-261.
[18]C.C. Su, J.S. Yang, S.Y. Lin, H.F. Lu, S.S. Lin, Y.H. Chang, W.W. Huang, Y.C.
Li, S.J. Chang, J.G. Chung, Curcumin inhibits WEHI-3 leukemia cells in
BALB/c mice in vivo. In Vivo 22 (2008) 63-68.
[19]D. Zips, H.D. Thames, M. Baumann, New anticancer agents: in vitro and in vivo
evaluation. In Vivo 19 (2005) 1-7.
26
[20]Y. Kimura, New anticancer agents: in vitro and in vivo evaluation of the
antitumor and antimetastatic actions of various compounds isolated from
medicinal plants. In Vivo 19 (2005) 37-60.
[21]D.K. Mehta, British National Formulary 52 ed., Pharmaceutical Press: UK,
London, 2006.
[22]S. Martin, C. Reutelingsperger, A.J. McGahon, J.A. Rader, R. Van Schie, D.M.
LaFace,
D.R.
Green,
Early
redistribution
of
plasma
membrane
phosphatidylserine is a general feature of apoptosis regardless of the
initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp.
Med. 182 (1995) 1545-1556.
[23]J.K. Ko, W.C. Leung, W.K. Ho, P. Chiu, Herbal diterpenoids induce growth
arrest and apoptosis in colon cancer cells with increased expression of the
nonsteroidal anti-inflammatory drug-activated gene. Eur. J. Pharmacol. 559
(2007) 1-13.
[24]C. Park, D.-O. Moon, C.-H. Rhu, B.T. Choi, W.H. Lee, G.-Y. Kim, Y.H. Choi,
β-Sitosterol induces anti-proliferation and apoptosis in human leukemic
U937 cells through activation of caspase-3 and induction of Bax/Bcl-2 ratio.
Biol. Pharm. Bull. 30 (2007) 1317-1323.
[25]S.R. Rahman, R. Abdullah, B.A. Ahmad, A.Z. Nazariah, H.O. Hemn, K.Y.
Swee,
W.H.
Chee,
W.G.
Wan
Nor
Hafiza,
Zerumbone-loaded
Nanostructured Lipid Carrier Induces G2/M Cell Cycle Arrest and Apoptosis
via Mitochondrial Pathway in human lymphoblastic leukemia cell Line. Int J
Nanomed In Press (2013).
[26]D.R. Green, Apoptotic pathways: paper wraps stone blunts scissors. Cell 102
(2000) 1-4.
[27]M.-F. Tsou, C.-T. Peng, M.-C. Shih, J.-S. Yang, C.-C. Lu, J.-H. Chiang, C.-L.
Wu, J.-P. Lin, C. Lo, M.-J. Fan, Benzyl isothiocyanate inhibits murine
WEHI-3 leukemia cells in vitro and promotes phagocytosis in BALB/c mice
in vivo. Leuk. Res. 33 (2009) 1505-1511.
[28]S. Mohan, A.B. Abdul, S.I. Abdelwahab, A.S. Al-Zubairi, M. Aspollah Sukari,
R. Abdullah, M.M.E. Taha, N.K. Beng, N.M. Isa, Typhonium flagelliforme
inhibits the proliferation of murine leukemia WEHI-3 cells in vitro and
induces apoptosis in vivo. Leuk. Res. 34 (2010) 1483-1492.
[29]J.-P. Lin, J.-S. Yang, C.-C. Lu, J.-H. Chiang, C.-L. Wu, J.-J. Lin, H.-L. Lin, M.D. Yang, K.-C. Liu, T.-H. Chiu, Rutin inhibits the proliferation of murine
leukemia WEHI-3 cells in vivo and promotes immune response in vivo. Leuk.
Res. 33 (2009) 823-828.
[30]J. Adams, S. Cory, The Bcl-2 apoptotic switch in cancer development and
therapy. Oncogene 26 (2007) 1324-1337.
[31]S. Derveaux, J. Vandesompele, J. Hellemans, How to do successful gene
expression analysis using real-time PCR. Methods 50 (2010) 227-230.
27
[32]Y. Aragane, D. Kulms, D. Metze, G. Wilkes, B. Pöppelmann, T.A. Luger, T.
Schwarz, Ultraviolet light induces apoptosis via direct activation of CD95
(Fas/APO-1) independently of its ligand CD95L. J. Cell Biol. 140 (1998)
171-182.
28