# In Vitro Ultramorphological Assessment of Apoptosis Induced by Zerumbone on (HeLa )

Authors: Siddig Ibrahim Abdel Wahab; Ahmad Bustamam Abdul; Adel Sharaf Alzubairi; Manal Mohamed Elhassan; Syam Mohan
Journal: Journal of Biomedicine and Biotechnology
 (2009)

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## Abstract

Zerumbone (ZER), a potential anticancer compound, isolated from the fresh rhizomes ofZingiber zerumbet. In this investigation, the cytotoxic properties of ZER were evaluated, on cancer cells of human cervix (HeLa), breast and ovary, and normal cells of Chinese Hamster ovary, using MTT assay. Apoptogenic effects of ZER on HeLa were studied using fluorescence microscopy (AO/PI double staining), scanning and transmission electron microscopy (SEM and TEM), and colorimetric assay of the apoptosis promoter enzyme, caspase-3. The results of MTT assay showed that ZER has less effect on normal cells compared to cancer cells. The lowest  IC50 of ZER was observed on HeLa cells. Cytological observations showed nuclear and chromatin condensation, cell shrinkage, multinucleation, abnormalities of mitochondrial cristae, membrane blebbing, holes, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by double staining of AO/PI, SEM and TEM. Statistical analysis (two-tailed t-test) of differential counting of 200 cells under fluorescence microscope revealed significant difference in apoptotic cells populations between treated and untreated HeLa cells. In addition, ZER has increased the cellular level of caspase-3 on the treated HeLa cells. It could be concluded that ZER was able to produce distinctive morphological features of cell death that corresponds to apoptosis.


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## Body

## 1. Introduction

Natural products provide a great chemical structural
diversity. In this respect, sesquiterpines have been demonstrated to stimulate cytotoxicity
and apoptosis in several cancer cell lines at low micromolar concentrations
with an acceptable clinical range of new anticancer drugs [1, 2]. Sesquiterpene have been found abundantly in Zingiber
zerumbet, a plant which is commonly found in Malaysia [3, 4]. Z.
zerumbet has significant economic properties, as the rhizome can be used as
both a spice and a traditional medicine. Zerumbone (ZER: Figure 1) is a monosesquiterpine,
which is recognized as a main compound of this plant. ZER has shown antiproliferative
effects on different cancer cell lines such as HT-29, CaCo-2, and HepG2 cancer
cells, with extra investigations on apoptosis pathway and G2/M cell cycle
arrest [5, 6]. Moreover, cytotoxic
effects of ZER have been reported to be selective toward cancer cells compared
to normal cells [4, 7]. Furthermore, the compound has shown potential in vivo
chemopreventive properties on experimental skin cancer in mouse [8], colon
cancer in rats [9], and cervical intraepithelial neoplasia in Balb/c mice [10].
In addition, ZER has shown anti-inflammatory (anticycloxygenase-2), suppression
of free radical generation, anticholinesterase, iNOS expression, and TNF-a release
[9, 11, 12].Figure 1
Chemical structure of zerumbone (molecular weight: 218.34).Cervical cancer remains a critical public health problem
that is second only to breast cancer in overall disease burden for women
throughout the world [13]. This cancer has been targeted by researchers to
discover new anticancer drugs that can replace the current unsafe regimens for
such disease [14]. Natural products are suitable alternatives that can be used
instead of platinum-based drugs in control of cervical cancer, which show some
harmful side effects [15]; however, a recent study has demonstrated that ZER is
significantly less harmful when tested on human peripheral blood lymphocytes compared
to cisplatin [16]. On the other hand, ZER inhibited the proliferation of
several cancer cell lines, while the growth of Chang normal liver cells, normalhuman dermal (2F0-C25), and colon (CCD-18 Co) fibroblasts wasless
affected [4, 17]. With regards to the cytoselective toxicity and the versatile
biological activities of this compound, the present study was suggested to
explore its antiproliferative effects and the apoptogenic ontogeny on different
cancer cells and human cervical cancer cell (HeLa), respectively.


## 2. Materials and Methods

### 2.1. Isolation and Characterization of ZER

ZER was isolated using the hydrodistillation
(steam distillation) method. Briefly, fresh rhizomes ofZ. zerumbet were
initially cleaned and sliced and later placed in a glass flask containing
distilled water and heated immediately using the heating mantel. The flask was
immediately connected to special glassware (Dienstag) in order to collect
vaporized steam containing the volatile oil. Then volatile oil was crystallized
using circulating cool water. The crystals were collected and used. To obtain highly
pure ZER, recrystalization was performed using hexane and the solution was left
standing to evaporate. Thin layer chromatography was used to examine
purification of ZER at each step. The crystals of ZER were kept for further
chemical and pharmacological analyses.


### 2.2. ZER Structure Elucidation by NMR

Five milligrams of ZER
crystals were dissolved
in HPLC-grade methanol and the sample was subsequently sent for (nuclear
magnetic resonance) NMR analysis at the laboratory of Natural Products,
Institute of Bioscience, UPM.1Hydrogen and 13Carbon NMR
spectra were recorded in Varian Unity Inova spectrometer operated at 500 MHz,
and the chemical shifts of the respective compound were reported in ppm.


### 2.3. Cell Culture and MTT Cytotoxicity Assay

Human cancer cell lines of cervix (HeLa),
ovary (Coav-3) and breast (MCF-7), and Chinese Hamster ovary normal cells were obtained
from American Type Culture Collection (ATCC, Va, USA).  ATCC protocol recommended the use of RPMI
1640 (PAA, Cölbe, Germany)
as a media for culturing cells. Disposable items (75 mL tissue culture flask,
filter system, well plates) were purchased from (NUNC, Roskilde, Denmark). Trypsin EDTA, feotal calf
serum, amphotericin B, and penicillin streptomycin were obtained from FlowLab
(Australia). The microtetrazolium (MTT) powder was purchased from Amresco,
Ohio, USA, and the dimethylsulphoxide (DMSO) was purchased from Sigma Aldrich,
Germany.Cells
were trypsinized and counted using hemocytometer and plated in a microtiter
plate of 96 wells. After an overnight incubation to allow cells attachment,
medium was changed
and 0.2 mL of new supplemented medium was added in each well. Cells were then treated in
a dose- and time-dependent manners, with zerumbone, 0.1% ethanol (negative
control), and cisplatin (positive control), and were incubated at 37°C, 5% CO2
for 72 hours. Each concentration of the compounds was assayed in triplicates. MTT
assay reading was performed using ELISA plate reader (TECAN,
SunriseTM, Männedorf, Switzerland).


### 2.4. Colorimetric Assays of Caspase-3

The
colorimetric protease assay of caspase-3 provides a simple and convenient means
for quantifying the enzyme activity that recognize the amino acid sequence,
DEVD (a synthetic tetrapeptide, (Asp-Glue-Val-Asp), which is the upstream amino
acid sequence of the caspase-3 cleavage site), coupled with p-nitroanilide,
which is released upon substrate cleavage. This assay was performed using
commercial kit (ApoTarget kit, code: KHZ0022: BioSource International, Inc.,
Calif, USA). 2×  106 cells were treated with IC50 of ZER and
incubated for 72 hours while untreated cells acted as control. The cells were
lysed by the addition of 50 μL of chilled cell lysis buffer and incubated on
ice for 10 minutes. The resulting cell lysate was centrifuged for 1 minute at
10 000 ×g, and the supernatant was collected.
Fifty microliters of 2X reaction buffer (containing 10 mM DTT) were added to each
sample. Then 5 μL of DEVD-pNA (caspase-3 substrate) was added and
incubated in the dark at 37°C for 1 hour. At the end of the incubation period,
the samples were read at 405 nm in a microplate reader (TECAN, SunriseTM,
Männedorf, Switzerland). Data was presented as optical density (405 nm; mean  ±  SD).


### 2.5. Quantification of Apoptosis Using Propidium Iodide and Acridine Orange Double Staining

ZER-induced cell death in HeLa cancer cells
was quantified using propidium iodide (PI) and acridine-orange (AO) double
staining according to standard procedures and examine under fluorescence microscope
(Lieca attached with Q-Floro Software) [18, 19]. Briefly, treatment was carried
out in a 25 mL culture flask (Nunc). HeLa cells were plated at concentration of
1 × 106 cell/mL, and treated with ZER at IC50  concentration. Flasks were incubated in atmosphere of 5% CO2 at  37°C   for 24, 48, and 72 hours. The cells were then spun down at 1000 rpm for 10 minutes.
Supernatant was discarded and the cells were washed twice using phosphate
buffer saline (PBS) after centrifuging at 1000 rpm for 10 minutes to remove the
remaining media. Ten microliters of fluorescent dyes containing acridine orange
(AO, 10 μg/mL) and propidium iodide (PI, 10 μg/mL) were added into the cellular pellet at equal
volumes of each. Freshly stained cell suspension was dropped into a glass slide
and covered by coverslip. Slides were observed under UV-fluorescence microscope
within 30 minutes before the fluorescence color starts to fade. The percentages
of viable, apoptotic, and necrotic cells were determined in more than 200
cells. Acridine orange (AO) and propidium iodide (PI) are intercalating nucleic
acid specific fluorochromes which emit green and orange fluorescences,
respectively, when they are bound to DNA. Of the two, only AO can cross the
plasma membrane of viable and early apoptotic cells. Viewed by fluorescence
microscopy, viable cells appear to have green nucleus with intact structure
while apoptotic cells exhibit a bright-green nucleus showing condensation of
chromatin as dense green areas. Late apoptotic cells and necrotic cells will
stain with both AO and PI. Comparatively, PI produces the highest intensity
emission. Hence, late apoptotic cells exhibited an orange nucleus showing
condensation of chromatin whilst necrotic cells display an orange nucleus with
intact structure. This assay provides a useful quantitative evaluation and was
done three times (n = 3).


### 2.6. Transmission Unltrastructural Effects of ZER on Hela Cells (TEM)

HeLa
cancer cells were cultured with the IC50 of ZER and cisplatin and
incubated for 24, 48, and 72 hours at 37°C. The cultured cells were harvested
using trypsin and centrifuged for 10 minutes at 3500 rpm and room temperature.
The pellets were fixed in 4% (v/v) glutaraldehyde in 0.1 M coccadylate buffer
(pH 7.4) for 4 hours at 4°C. The fixed cells were centrifuged, and the pellets
were blocked in serum which was later fixed in glutaraldeyde overnight at 4°C.
The specimens were washed in three changes of sodium coccadylate buffer (pH
7.4) for 10 minutes each, postfixed in 1% osmium tetraoxide at 4°C. The
specimens were then washed in three changes of sodium coccadylate buffer (pH
7.4) for 10 minutes each and dehydrated with a graded series of acetone (35%,
50%, 75%, 95%, and 100%). The cells were then infiltrated with acetone and
resin and embedded with 100% resin in beam capsule, and left to polymerize at
60°C for 48 hours. The area of interest in the embedded cells resin block was
chosen using the toulidine blue staining and later examined using light
microscope. The selected area was cut in ulltrathin sections using
ultramicrotome. The sections were placed into a grid and stained with uranyl
acetate for 10 minutes followed by 50% filtered acetone, and finally stained
using lead which was then washed twice with distilled water. The stained
samples were then viewed
under transmission electron microscopy (Phillips, Eindhoven, The Netherlands).


### 2.7. Exterior Unltrastructural Effects of ZER on Hela Cells (SEM)

The IC50 of ZER was used to induce death toward human cervical cancer cell lines (HeLa).
HeLa cells were cultured for 24, 48, and 72 hours. The cancer cells were
trypsinized and centrifuged for 10 minutes at 3000 rpm. The pellets were fixed
in 4% (v/v) glutaraldehyde in 0.1 M coccadylate buffer (pH 7.4) for 4 hours, 4°C.
The fixed cells were washed in three changes of sodium coccadylate buffer for
10 minutes each, postfixed in 1% osmium tetraoxide at 4°C. The specimens were then
washed in three changes of sodium coccadylate buffer (pH 7.4) for 10 minutes
each, dehydrated in ascending grades of acetone 35%, 50%, 75%, 95%, and 100%),
and brought to critical point of drying by the critical point drier (CPD 030,
Bal-TEC, Switzerland) for thirty minutes. The cells were affixed to a metal SEM
stub and sputter coated in gold by using SEM coating unit (E5100 Polaron, UK).
The coated specimens were viewed using scanning electron microscopy (JOEL 64000,
Japan) at accelerating voltage of 15–25 KV.


### 2.8. Statistical Analysis

Data was expressed as mean  ± SEM (mean ± SD).
Normality and homogeneity of variance assumptions were checked. One way ANOVA
followed by pairwise comparison analysis was used when needed. Independentt-test was used to analyze
the data of the colorimetric assay of caspase. SPSS 16.0 was utilized to
conduct statistical analysis. The value of 0.05 was considered as the cutoff
point of type one error (alpha).


## 2.1. Isolation and Characterization of ZER

ZER was isolated using the hydrodistillation
(steam distillation) method. Briefly, fresh rhizomes ofZ. zerumbet were
initially cleaned and sliced and later placed in a glass flask containing
distilled water and heated immediately using the heating mantel. The flask was
immediately connected to special glassware (Dienstag) in order to collect
vaporized steam containing the volatile oil. Then volatile oil was crystallized
using circulating cool water. The crystals were collected and used. To obtain highly
pure ZER, recrystalization was performed using hexane and the solution was left
standing to evaporate. Thin layer chromatography was used to examine
purification of ZER at each step. The crystals of ZER were kept for further
chemical and pharmacological analyses.


## 2.2. ZER Structure Elucidation by NMR

Five milligrams of ZER
crystals were dissolved
in HPLC-grade methanol and the sample was subsequently sent for (nuclear
magnetic resonance) NMR analysis at the laboratory of Natural Products,
Institute of Bioscience, UPM.1Hydrogen and 13Carbon NMR
spectra were recorded in Varian Unity Inova spectrometer operated at 500 MHz,
and the chemical shifts of the respective compound were reported in ppm.


## 2.3. Cell Culture and MTT Cytotoxicity Assay

Human cancer cell lines of cervix (HeLa),
ovary (Coav-3) and breast (MCF-7), and Chinese Hamster ovary normal cells were obtained
from American Type Culture Collection (ATCC, Va, USA).  ATCC protocol recommended the use of RPMI
1640 (PAA, Cölbe, Germany)
as a media for culturing cells. Disposable items (75 mL tissue culture flask,
filter system, well plates) were purchased from (NUNC, Roskilde, Denmark). Trypsin EDTA, feotal calf
serum, amphotericin B, and penicillin streptomycin were obtained from FlowLab
(Australia). The microtetrazolium (MTT) powder was purchased from Amresco,
Ohio, USA, and the dimethylsulphoxide (DMSO) was purchased from Sigma Aldrich,
Germany.Cells
were trypsinized and counted using hemocytometer and plated in a microtiter
plate of 96 wells. After an overnight incubation to allow cells attachment,
medium was changed
and 0.2 mL of new supplemented medium was added in each well. Cells were then treated in
a dose- and time-dependent manners, with zerumbone, 0.1% ethanol (negative
control), and cisplatin (positive control), and were incubated at 37°C, 5% CO2
for 72 hours. Each concentration of the compounds was assayed in triplicates. MTT
assay reading was performed using ELISA plate reader (TECAN,
SunriseTM, Männedorf, Switzerland).


## 2.4. Colorimetric Assays of Caspase-3

The
colorimetric protease assay of caspase-3 provides a simple and convenient means
for quantifying the enzyme activity that recognize the amino acid sequence,
DEVD (a synthetic tetrapeptide, (Asp-Glue-Val-Asp), which is the upstream amino
acid sequence of the caspase-3 cleavage site), coupled with p-nitroanilide,
which is released upon substrate cleavage. This assay was performed using
commercial kit (ApoTarget kit, code: KHZ0022: BioSource International, Inc.,
Calif, USA). 2×  106 cells were treated with IC50 of ZER and
incubated for 72 hours while untreated cells acted as control. The cells were
lysed by the addition of 50 μL of chilled cell lysis buffer and incubated on
ice for 10 minutes. The resulting cell lysate was centrifuged for 1 minute at
10 000 ×g, and the supernatant was collected.
Fifty microliters of 2X reaction buffer (containing 10 mM DTT) were added to each
sample. Then 5 μL of DEVD-pNA (caspase-3 substrate) was added and
incubated in the dark at 37°C for 1 hour. At the end of the incubation period,
the samples were read at 405 nm in a microplate reader (TECAN, SunriseTM,
Männedorf, Switzerland). Data was presented as optical density (405 nm; mean  ±  SD).


## 2.5. Quantification of Apoptosis Using Propidium Iodide and Acridine Orange Double Staining

ZER-induced cell death in HeLa cancer cells
was quantified using propidium iodide (PI) and acridine-orange (AO) double
staining according to standard procedures and examine under fluorescence microscope
(Lieca attached with Q-Floro Software) [18, 19]. Briefly, treatment was carried
out in a 25 mL culture flask (Nunc). HeLa cells were plated at concentration of
1 × 106 cell/mL, and treated with ZER at IC50  concentration. Flasks were incubated in atmosphere of 5% CO2 at  37°C   for 24, 48, and 72 hours. The cells were then spun down at 1000 rpm for 10 minutes.
Supernatant was discarded and the cells were washed twice using phosphate
buffer saline (PBS) after centrifuging at 1000 rpm for 10 minutes to remove the
remaining media. Ten microliters of fluorescent dyes containing acridine orange
(AO, 10 μg/mL) and propidium iodide (PI, 10 μg/mL) were added into the cellular pellet at equal
volumes of each. Freshly stained cell suspension was dropped into a glass slide
and covered by coverslip. Slides were observed under UV-fluorescence microscope
within 30 minutes before the fluorescence color starts to fade. The percentages
of viable, apoptotic, and necrotic cells were determined in more than 200
cells. Acridine orange (AO) and propidium iodide (PI) are intercalating nucleic
acid specific fluorochromes which emit green and orange fluorescences,
respectively, when they are bound to DNA. Of the two, only AO can cross the
plasma membrane of viable and early apoptotic cells. Viewed by fluorescence
microscopy, viable cells appear to have green nucleus with intact structure
while apoptotic cells exhibit a bright-green nucleus showing condensation of
chromatin as dense green areas. Late apoptotic cells and necrotic cells will
stain with both AO and PI. Comparatively, PI produces the highest intensity
emission. Hence, late apoptotic cells exhibited an orange nucleus showing
condensation of chromatin whilst necrotic cells display an orange nucleus with
intact structure. This assay provides a useful quantitative evaluation and was
done three times (n = 3).


## 2.6. Transmission Unltrastructural Effects of ZER on Hela Cells (TEM)

HeLa
cancer cells were cultured with the IC50 of ZER and cisplatin and
incubated for 24, 48, and 72 hours at 37°C. The cultured cells were harvested
using trypsin and centrifuged for 10 minutes at 3500 rpm and room temperature.
The pellets were fixed in 4% (v/v) glutaraldehyde in 0.1 M coccadylate buffer
(pH 7.4) for 4 hours at 4°C. The fixed cells were centrifuged, and the pellets
were blocked in serum which was later fixed in glutaraldeyde overnight at 4°C.
The specimens were washed in three changes of sodium coccadylate buffer (pH
7.4) for 10 minutes each, postfixed in 1% osmium tetraoxide at 4°C. The
specimens were then washed in three changes of sodium coccadylate buffer (pH
7.4) for 10 minutes each and dehydrated with a graded series of acetone (35%,
50%, 75%, 95%, and 100%). The cells were then infiltrated with acetone and
resin and embedded with 100% resin in beam capsule, and left to polymerize at
60°C for 48 hours. The area of interest in the embedded cells resin block was
chosen using the toulidine blue staining and later examined using light
microscope. The selected area was cut in ulltrathin sections using
ultramicrotome. The sections were placed into a grid and stained with uranyl
acetate for 10 minutes followed by 50% filtered acetone, and finally stained
using lead which was then washed twice with distilled water. The stained
samples were then viewed
under transmission electron microscopy (Phillips, Eindhoven, The Netherlands).


## 2.7. Exterior Unltrastructural Effects of ZER on Hela Cells (SEM)

The IC50 of ZER was used to induce death toward human cervical cancer cell lines (HeLa).
HeLa cells were cultured for 24, 48, and 72 hours. The cancer cells were
trypsinized and centrifuged for 10 minutes at 3000 rpm. The pellets were fixed
in 4% (v/v) glutaraldehyde in 0.1 M coccadylate buffer (pH 7.4) for 4 hours, 4°C.
The fixed cells were washed in three changes of sodium coccadylate buffer for
10 minutes each, postfixed in 1% osmium tetraoxide at 4°C. The specimens were then
washed in three changes of sodium coccadylate buffer (pH 7.4) for 10 minutes
each, dehydrated in ascending grades of acetone 35%, 50%, 75%, 95%, and 100%),
and brought to critical point of drying by the critical point drier (CPD 030,
Bal-TEC, Switzerland) for thirty minutes. The cells were affixed to a metal SEM
stub and sputter coated in gold by using SEM coating unit (E5100 Polaron, UK).
The coated specimens were viewed using scanning electron microscopy (JOEL 64000,
Japan) at accelerating voltage of 15–25 KV.


## 2.8. Statistical Analysis

Data was expressed as mean  ± SEM (mean ± SD).
Normality and homogeneity of variance assumptions were checked. One way ANOVA
followed by pairwise comparison analysis was used when needed. Independentt-test was used to analyze
the data of the colorimetric assay of caspase. SPSS 16.0 was utilized to
conduct statistical analysis. The value of 0.05 was considered as the cutoff
point of type one error (alpha).


## 3. Results

### 3.1.13C NMR and 1H NMR Analyses of ZER

NMR analyses were performed to verify the chemical
structure of ZER before conducting further biological investigations.1H-NMR
spectrum showed four singlets
which resonate at δ  1.573, δ 1.770, δ 1.086, and δ 1.238, and they were
due to the methyl group at carbon C-12, C-13, C-14, and C-15 (Figure 1). A
broad duplet was found between δ 6.083 and δ 6.106 at C-6 position. Three
multiplets between δ 2.243 and δ 2.544 were attributed to the methylene groups
at positions C-1,
C-4, and C-5, respectively. The 13C-NMR spectrum gave a total of
fifteen carbons. The most downfield signal at δ 205.528 was due to the carbonyl
group at position C-8. The signals
of methyl group at positions
C-12, C-13, C-14, and C-15 were observed at δ 14.228, δ 10.724, δ 23.360, and δ
28.659, respectively. These 1H-NMR and 13C-NMR analyses coincide
to the chemical structure that resembles ZER (Figure 1).


### 3.2. Cytotoxic Effects of ZER on Normal and Cancer Cells

As
depicted in Table1, ZER was clearly found to exert antiproliferative effects
toward HeLa, Coav-3, and MCF-7. The IC50 value, which is the
concentration required for 50% growth inhibition, is determined to be 20.30±1.1,
24.30±0.9, or
27.7±1.2 μM against HeLa, Coav-3, or MCF-7, respectively. Comparatively, the anti-neoplastic
drug, cisplatin, was used as a positive control in this study. Cisplatin
revealed an inhibitory effect on HeLa, MCF-7, and Coav-3 cancer cells with IC50 values of 5.45±0.44, 4.9±1.8, and 3.7±0.44 μM, respectively. ZER reveals a
lower cytotoxicity (High IC50) toward normal cells Chinese hamster ovary
(Table 1).Table 1
Effects of ZER and cisplatin on the viability of cancer and normal
cells. Cells were cultured in RPMI 1640 media maintained at 37°C and 5% CO2. The lowest IC50 of ZER was observed on HeLa cells.
TreatmentIC50HeLaMCF-7Coav-3CHOZER20.30±1.1 μM27.7±1.2 μM24.30±0.9 μM35.80±5.1 μMCisplatin5.45±0.44 μM4.9±1.8 μM3.7±0.44 μM4.15±1.23 μM


### 3.3. Colorimetric Assay of Caspase-3

Since
HeLa were the most sensitive cells to ZER, an in vitro colorimetric assay of
caspase-3 was conducted to assess apoptosis between control and treated cells. As
shown in Figure2, ZER significantly (independent t-test, P < .05)
stimulated caspase-3, the hallmark enzyme of apoptosis. The level of this
enzyme is higher in treated HeLa cells as compared to nontreated cells, this
concludes that ZER induces cell death toward human cervical cancer cells, HeLa.Figure 2
The colorimetric assay of caspase-3 in human cervical cancer cells treated and untreated with ZER (IC50) for 72 hours. Cells were cultured in RPMI 1640 (75 mL flask) media maintained at 37°C and 5% CO2. Independent t-test showed a significant difference (P*<.05) between treated and untreated cells in the activity of caspase-3.


### 3.4. Quantification of Apoptosis Using Propidium Iodide and Acridine Orange Double Stainin

Apoptotic, necrotic, and viable
HeLa cells were scored under fluorescence microscope. This is also included the
control cells (untreated) whereby, 200 cells were randomly and differentially
counted. The study revealed that ZER triggered morphological features that
relates to apoptosis in a time-dependent manner (Figures3(a), 3(b), 3(c), and
3(d)). Whereby, early apoptosis is obvious by intercalated AO within the
fragmented DNA. In several of such cases, the fluorescent bright-green color could
be seen in treated HeLa cells only. In contrast, untreated cells were observed
with a green intact nuclear structure. At 48-hour treatment with ZER, blebbing
and nuclear margination were noticed (moderate apoptosis). In addition, late
stages of apoptosis, such as apoptotic body separation and presence of reddish-orange
color due to the binding of AO to denatured DNA, were observed after 72-hour
treatment with ZER. Differential scoring of treated HeLa cells (200 cells population) showed
that there is a statistical significant (P  <  .05) difference in
apoptosis positive cells, which indicates clearly that ZER has a time-dependent
apoptogenic effect. On the other hand, there was no statistical significant (P > .05)
difference in necrotic counts at different treatment times (24, 48, and 72
hours) (Figure 4).Fluorescent micrograph of acridine orange and propidium iodide double-stained human cervical cancer cells lines (HeLa). HeLa was treated at IC50  of ZER at time-dependent manner. Cells were cultured in RPMI 1640 media (25 mL flask) maintained at 37°C and 5% CO2. (a) Untreated cells showed normal structure without prominent apoptosis and necrosis. (b) Early apoptosis features were seen after 24 hours representing intercalated acridine orange (bright green) amongst the fragmented DNA. (c) Blebbing and nuclear margination were noticed in 48-hour treatment of ZER. (d) Late apoptosis was seen in 72 hours incubated cells, whereby positive staining with orange color represents the hallmark of late apoptosis (magnification 400X).
(a)(b)(c)(d)Figure 4
Percentages of viable, apoptotic, and necrotic cells after ZER treatment.  HeLa cell death via apoptosis increased significantly (P*<.05) in time-dependent manner. However, no significant (P > .05) difference was observed in the cell count of necrosis. Cells were cultured in RPMI 1640 (25 mL flask) media maintained at 37°C and 5% CO2.


### 3.5. Effects of ZER on Human Cervical Cancer Cells (Hela) Using Scanning Electron Microscope (SEM)

The
surface ultrastructure of HeLa cells treated with ZER was examined.
Interpretation of SEM electromicrographs showed distinct morphological changes
corresponding to a typical cellular surface morphology of apoptosis, including
cell membrane blebbing, microvilli disappearance or reduction (Blunt
microvillus), and separated apoptotic bodies. The effects of ZER on HeLa cells
were found to be time-correlated phenomena (Figures5(b), 5(c), and 5(d)) and this
was noticed when considering the number of blebs (cytoplasmic extension) as an
indicator of cell death via apoptosis. Untreated HeLa cells have shown a
restoration of the typical morphological features of cervical cancer cells
which include numerous microvilli on its surface with membrane connections (Figure 5(a)). However, occasional existence of rounded
cells and blebbing on the cell surface are characteristics of HeLa cells in
culture but this phenomenon is not rampant. Although a few cells from the
control groups resembled those from treated groups, ZER caused morphological
alterations in a larger proportion of cells.SEM micrographs of surface ultrastructural characteristics of HeLa cells treated with ZER in time-dependent manner (0, 24, 48, and 72 hours) and cultured in RPMI 1640 media maintained at 37°C and 5% CO2.  (a) The characteristic of untreated HeLa cells surface showing the restoration of a typical morphological feature of a cancer cell such as numerous microvilli (as shown in grey arrow) with several membrane connections. (b), (c), and (d) ZER-treated HeLa cells (24, 48, and 72 hours, IC50: 20.41 μM) showed distinct morphological changes corresponding to typical apoptosis, including cell membrane blebbing (b), microvilli disappearance or reduction (blunt microvillus (s)), and separated apoptotic bodies (X2500).
(a)(b)(c)(d)


### 3.6. Effects of ZER on Cervical Human Cancer Cells (Hela) Using Transmission Electron Microscopy

ZER-treated HeLa cells showed
death corresponded very well to the cross-sectional classical signs of
apoptosis: cell shrinkage, increased cellular granularity, the formation of
apoptotic bodies, and dilated nuclear membranes (Figures6(b), 6(d), and 6(e)).
Mitochondrial cristae in ZER-treated cancer cells were ruptured and condensed (Figure 6(c)). The
evidences which suggested that the apoptogenic effect of ZER on HeLa cells is
time-dependent manner were obtained through observation of dynamic micrographs
images using transmission electron microscopy. The initial induction of cell
death indicates primary (early) stage of apoptosis that involved cell shrinkage
and chromatin condensation. Nuclear membrane dilatation and cell membrane
blebbing (Figure 6(d)) were the next stage in this sequence of apoptosis
followed by nuclear collapse and continual blebbing stage (48 hours). HeLa
cells treated with ZER after 72 hours were observed to show late stages of apoptosis
phenomena, which included apoptotic bodies formation (Figure 6(e)). Cisplatin-treated
HeLa cells have shown similar morphological aspects of apoptosis, however, with
fewer necrotic characteristics also observed.(a) Electromicrograph of untreated human cervical cancer cells (HeLa) demonstrates the normal structure of HeLa cancer cell. Nucleus (N), nucleolus (NL), and the cytoplasm appeared without abnormal changes (X4600).  (b) ZER-treated (24 hours) human cervical cancer cells (HeLa) demonstrate morphological features of early apoptosis: cell shrinkage, chromatin condensation (arrow), and integrity of plasma membrane (X6000). (c) ZER-treated human cervical cancer cells (HeLa) demonstrate the condensed cristae of mitochondria (MC) as a typical morphological feature in apoptosis (arrow) (X27500).  (d) ZER-treated (48 hours) human cervical cancer cell line (HeLa) demonstrates morphological features of intermediate apoptosis: cell shrinkage, chromatin condensation (white arrow), and membrane blebbing (small white arrow) (X6000). (e) ZER-treated (72 hours) cervical cancer cells (HeLa) demonstrate morphological feature of late apoptosis: nuclear collapse, continuing blebbing, and apoptotic body formation (arrow) (X10 000). Cells were cultured in RPMI 1640 (25 mL flask) media maintained at 37°C  and 5% CO2.
(a)(b)(c)(d)(e)


## 3.1.13C NMR and 1H NMR Analyses of ZER

NMR analyses were performed to verify the chemical
structure of ZER before conducting further biological investigations.1H-NMR
spectrum showed four singlets
which resonate at δ  1.573, δ 1.770, δ 1.086, and δ 1.238, and they were
due to the methyl group at carbon C-12, C-13, C-14, and C-15 (Figure 1). A
broad duplet was found between δ 6.083 and δ 6.106 at C-6 position. Three
multiplets between δ 2.243 and δ 2.544 were attributed to the methylene groups
at positions C-1,
C-4, and C-5, respectively. The 13C-NMR spectrum gave a total of
fifteen carbons. The most downfield signal at δ 205.528 was due to the carbonyl
group at position C-8. The signals
of methyl group at positions
C-12, C-13, C-14, and C-15 were observed at δ 14.228, δ 10.724, δ 23.360, and δ
28.659, respectively. These 1H-NMR and 13C-NMR analyses coincide
to the chemical structure that resembles ZER (Figure 1).


## 3.2. Cytotoxic Effects of ZER on Normal and Cancer Cells

As
depicted in Table1, ZER was clearly found to exert antiproliferative effects
toward HeLa, Coav-3, and MCF-7. The IC50 value, which is the
concentration required for 50% growth inhibition, is determined to be 20.30±1.1,
24.30±0.9, or
27.7±1.2 μM against HeLa, Coav-3, or MCF-7, respectively. Comparatively, the anti-neoplastic
drug, cisplatin, was used as a positive control in this study. Cisplatin
revealed an inhibitory effect on HeLa, MCF-7, and Coav-3 cancer cells with IC50 values of 5.45±0.44, 4.9±1.8, and 3.7±0.44 μM, respectively. ZER reveals a
lower cytotoxicity (High IC50) toward normal cells Chinese hamster ovary
(Table 1).Table 1
Effects of ZER and cisplatin on the viability of cancer and normal
cells. Cells were cultured in RPMI 1640 media maintained at 37°C and 5% CO2. The lowest IC50 of ZER was observed on HeLa cells.
TreatmentIC50HeLaMCF-7Coav-3CHOZER20.30±1.1 μM27.7±1.2 μM24.30±0.9 μM35.80±5.1 μMCisplatin5.45±0.44 μM4.9±1.8 μM3.7±0.44 μM4.15±1.23 μM


## 3.3. Colorimetric Assay of Caspase-3

Since
HeLa were the most sensitive cells to ZER, an in vitro colorimetric assay of
caspase-3 was conducted to assess apoptosis between control and treated cells. As
shown in Figure2, ZER significantly (independent t-test, P < .05)
stimulated caspase-3, the hallmark enzyme of apoptosis. The level of this
enzyme is higher in treated HeLa cells as compared to nontreated cells, this
concludes that ZER induces cell death toward human cervical cancer cells, HeLa.Figure 2
The colorimetric assay of caspase-3 in human cervical cancer cells treated and untreated with ZER (IC50) for 72 hours. Cells were cultured in RPMI 1640 (75 mL flask) media maintained at 37°C and 5% CO2. Independent t-test showed a significant difference (P*<.05) between treated and untreated cells in the activity of caspase-3.


## 3.4. Quantification of Apoptosis Using Propidium Iodide and Acridine Orange Double Stainin

Apoptotic, necrotic, and viable
HeLa cells were scored under fluorescence microscope. This is also included the
control cells (untreated) whereby, 200 cells were randomly and differentially
counted. The study revealed that ZER triggered morphological features that
relates to apoptosis in a time-dependent manner (Figures3(a), 3(b), 3(c), and
3(d)). Whereby, early apoptosis is obvious by intercalated AO within the
fragmented DNA. In several of such cases, the fluorescent bright-green color could
be seen in treated HeLa cells only. In contrast, untreated cells were observed
with a green intact nuclear structure. At 48-hour treatment with ZER, blebbing
and nuclear margination were noticed (moderate apoptosis). In addition, late
stages of apoptosis, such as apoptotic body separation and presence of reddish-orange
color due to the binding of AO to denatured DNA, were observed after 72-hour
treatment with ZER. Differential scoring of treated HeLa cells (200 cells population) showed
that there is a statistical significant (P  <  .05) difference in
apoptosis positive cells, which indicates clearly that ZER has a time-dependent
apoptogenic effect. On the other hand, there was no statistical significant (P > .05)
difference in necrotic counts at different treatment times (24, 48, and 72
hours) (Figure 4).Fluorescent micrograph of acridine orange and propidium iodide double-stained human cervical cancer cells lines (HeLa). HeLa was treated at IC50  of ZER at time-dependent manner. Cells were cultured in RPMI 1640 media (25 mL flask) maintained at 37°C and 5% CO2. (a) Untreated cells showed normal structure without prominent apoptosis and necrosis. (b) Early apoptosis features were seen after 24 hours representing intercalated acridine orange (bright green) amongst the fragmented DNA. (c) Blebbing and nuclear margination were noticed in 48-hour treatment of ZER. (d) Late apoptosis was seen in 72 hours incubated cells, whereby positive staining with orange color represents the hallmark of late apoptosis (magnification 400X).
(a)(b)(c)(d)Figure 4
Percentages of viable, apoptotic, and necrotic cells after ZER treatment.  HeLa cell death via apoptosis increased significantly (P*<.05) in time-dependent manner. However, no significant (P > .05) difference was observed in the cell count of necrosis. Cells were cultured in RPMI 1640 (25 mL flask) media maintained at 37°C and 5% CO2.


## 3.5. Effects of ZER on Human Cervical Cancer Cells (Hela) Using Scanning Electron Microscope (SEM)

The
surface ultrastructure of HeLa cells treated with ZER was examined.
Interpretation of SEM electromicrographs showed distinct morphological changes
corresponding to a typical cellular surface morphology of apoptosis, including
cell membrane blebbing, microvilli disappearance or reduction (Blunt
microvillus), and separated apoptotic bodies. The effects of ZER on HeLa cells
were found to be time-correlated phenomena (Figures5(b), 5(c), and 5(d)) and this
was noticed when considering the number of blebs (cytoplasmic extension) as an
indicator of cell death via apoptosis. Untreated HeLa cells have shown a
restoration of the typical morphological features of cervical cancer cells
which include numerous microvilli on its surface with membrane connections (Figure 5(a)). However, occasional existence of rounded
cells and blebbing on the cell surface are characteristics of HeLa cells in
culture but this phenomenon is not rampant. Although a few cells from the
control groups resembled those from treated groups, ZER caused morphological
alterations in a larger proportion of cells.SEM micrographs of surface ultrastructural characteristics of HeLa cells treated with ZER in time-dependent manner (0, 24, 48, and 72 hours) and cultured in RPMI 1640 media maintained at 37°C and 5% CO2.  (a) The characteristic of untreated HeLa cells surface showing the restoration of a typical morphological feature of a cancer cell such as numerous microvilli (as shown in grey arrow) with several membrane connections. (b), (c), and (d) ZER-treated HeLa cells (24, 48, and 72 hours, IC50: 20.41 μM) showed distinct morphological changes corresponding to typical apoptosis, including cell membrane blebbing (b), microvilli disappearance or reduction (blunt microvillus (s)), and separated apoptotic bodies (X2500).
(a)(b)(c)(d)


## 3.6. Effects of ZER on Cervical Human Cancer Cells (Hela) Using Transmission Electron Microscopy

ZER-treated HeLa cells showed
death corresponded very well to the cross-sectional classical signs of
apoptosis: cell shrinkage, increased cellular granularity, the formation of
apoptotic bodies, and dilated nuclear membranes (Figures6(b), 6(d), and 6(e)).
Mitochondrial cristae in ZER-treated cancer cells were ruptured and condensed (Figure 6(c)). The
evidences which suggested that the apoptogenic effect of ZER on HeLa cells is
time-dependent manner were obtained through observation of dynamic micrographs
images using transmission electron microscopy. The initial induction of cell
death indicates primary (early) stage of apoptosis that involved cell shrinkage
and chromatin condensation. Nuclear membrane dilatation and cell membrane
blebbing (Figure 6(d)) were the next stage in this sequence of apoptosis
followed by nuclear collapse and continual blebbing stage (48 hours). HeLa
cells treated with ZER after 72 hours were observed to show late stages of apoptosis
phenomena, which included apoptotic bodies formation (Figure 6(e)). Cisplatin-treated
HeLa cells have shown similar morphological aspects of apoptosis, however, with
fewer necrotic characteristics also observed.(a) Electromicrograph of untreated human cervical cancer cells (HeLa) demonstrates the normal structure of HeLa cancer cell. Nucleus (N), nucleolus (NL), and the cytoplasm appeared without abnormal changes (X4600).  (b) ZER-treated (24 hours) human cervical cancer cells (HeLa) demonstrate morphological features of early apoptosis: cell shrinkage, chromatin condensation (arrow), and integrity of plasma membrane (X6000). (c) ZER-treated human cervical cancer cells (HeLa) demonstrate the condensed cristae of mitochondria (MC) as a typical morphological feature in apoptosis (arrow) (X27500).  (d) ZER-treated (48 hours) human cervical cancer cell line (HeLa) demonstrates morphological features of intermediate apoptosis: cell shrinkage, chromatin condensation (white arrow), and membrane blebbing (small white arrow) (X6000). (e) ZER-treated (72 hours) cervical cancer cells (HeLa) demonstrate morphological feature of late apoptosis: nuclear collapse, continuing blebbing, and apoptotic body formation (arrow) (X10 000). Cells were cultured in RPMI 1640 (25 mL flask) media maintained at 37°C  and 5% CO2.
(a)(b)(c)(d)(e)


## 4. Discussion

The
results of current study reveal that zerumbone possesses promising antiproliferative
properties against HeLa, MCF-7, and Coav-3 cancer cells. However, the lowest IC50 of ZER is obtained toward human cervical cancer cells (HeLa); therefore
all investigations regarding the apoptogenic property of this natural product
were carried on this cell line. On the other hand, ZER has shown a weaker
cytotoxic effect on normal cells of CHO. These cytoselective properties of ZER
have been reported earlier [4, 17].Apoptosis
is biological phenomena that involved in process ranging from embryogenesis to
ageing, from normal tissue homoeostasis to many human diseases. Apoptotic cells
share a number of common features such as cell shrinkage, nuclear condensation,
membrane blebbing, chromatin cleavage, and formation of pyknotic bodies of condensed
chromatin [20, 21]. These distinctive morphological features form the basis of
some of the most widely used techniques for the identification and
quantification of apoptosis, and thus morphologic description using light or
electron microscopy remains one of the best ways to define apoptosis [21]. The
programmed cell death (apoptosis) compared to necrosis is a desired somatic
defense mechanism against cancer cells [22]. Moreover, apoptosis is reported
earlier to be triggered by natural products [23, 24]. Zerumbone is one of the
promising natural products that possess potentiality to induce apoptosis on
different cancer cell lines [25]. The current study was intended to provide an
evidence of apoptosis on HeLa cells induced by ZER.Cancer
cell death phenomenon could be induced through different pathways [26]. A major
part of this phenomenon could be mediated by caspase-3, the promoter of
apoptosis [27]. This enzyme could be measured in vitro using colorimetric assay
that recognize the amino acid sequence, DEVD, coupled with p-nitroanilide,
which is released upon substrate cleavage and can be detected at 405 nm. This
approach of measuring the caspases has been used previously in HeLa cells [28].
The present study revealed that ZER induced significantly (two-tailed t-test, P  < .05) the elevation of caspase-3 on HeLa as compared to untreated
HeLa cells. These findings confirmed that ZER is capable of inducing apoptosis
in HeLa cells by stimulating the enzymes that play a central role in the
initiation of apoptosis. At this moment of writing, this study is the first to
demonstrate that caspase-3 activation is required for ZER-induced apoptosis in
human cervical cancer cells (HeLa).Nucleic
acid specific fluorochromes emit fluorescent color when bound to DNA and this phenomenon can be viewed
under fluorescence microscopy [18, 19]. The fluorescent color of that stain is
obtained by specific properties of different flourostain such as the ability to
cross plasma membrane of viable cells and dead cells [18]. Using properties of AO/PI,
HeLa cells were treated with ZER to study the antiproliferative and apoptogenic effects of ZER. The
antiproliferative properties of ZER could then be evaluated by counting the
numbers of viable cancer cells, while the apoptogenic effects were determined
through observing typical morphological changes of apoptosis. This technique
was previously applied to human colonic adenocarcinoma cells treated with ZER [11]
and also toward HeLa treated with different compounds [29]. The cell viability
results showed an obvious decrease of living cells in ZER-treated group. HeLa
cells with ZER stimulation had typical morphological changes that implicate
apoptosis such as DNA fragmentation, membrane blebbing, and apoptotic body
formation, which could be evidenced by the visual bright-green and orange color
of AO color of stained cells under fluorescence microscopy. Apoptotic cells
exhibit bright-green nucleus showing condensation of chromatin as dense-green
areas. Quantification of apoptosis with differential scoring of treated and
control HeLa cells revealed a significant (P < .05) difference in the
number of apoptotic cells in a time-dependent manner but with insignificant
difference between numbers of necrosis positive cells in control and treated
cells, concluding that ZER did not induce necrotic effects. Confocal microscopy examination
confirmed the onset of apoptosis features. These morphological criteria that
implicate apoptotic cell death were further confirmed by scanning electron microscopy
and transmission electron microscopy.Apoptosis
possesses distinctive morphological features which form the basis of some of
the most widely used techniques for the identification and quantification of
apoptosis. Thus, morphologic description of apoptosis using electron microscopy
still remains one of the best ways to define apoptosis and to contrast it with
necrosis [21]. Scanning electron microscopy (SEM) has been applied previously
to study surface ultrastructural morphology of HeLa cells [25, 30]. In the
current investigation, SEM was employed to obtain detailed information about
the cell surface of HeLa upon treatment with ZER at IC50 in a time-dependent
manner (0, 24, 48, and 72 hours). SEM electromicrographs showed distinct
morphological changes that correspond
to a typical cellular surface morphology implicating apoptosis, including cell
membrane blebbing, microvilli disappearance or reduction (blunt microvillus),
and separated apoptotic bodies after treatment. These findings were similar
that confirms to previous reported cytological features of HeLa cells
undergoing apoptosis [30]. Untreated HeLa cells have shown a restoration of the
typical morphological features of cervical cancer cells, which included
numerous microvilli (hairing rods) on the cell surface with membrane
connections. These results strongly provide evidence that ZER has promising
anticancer activity toward human cervical cancer based on the observation of
distinctive morphological features to surface HeLa cells upon treatment with
ZER.In this
study, the ultrastructural changes in HeLa cells after treatment with ZER were
studied using transmission electron microscopy (TEM). TEM has been utilized
commonly as the tool to study the morphological ultrastructure of cells [31].
The application of TEM technology is also used in the detection of associated
cellular microlesions of apoptosis and cell cycle arrest [32, 33]. In addition,
TEM allows the opportunity to view cytoplasmic changes [34] which cannot be
viewed using SEM. TEM is a powerful technique for differentiating between
apoptosis and necrosis [35], allows the analysis of sectioned specimens, and
provides a qualitative bidimensional image of the inner cell, often after
fixation, embedding, and staining [36, 37]. The TEM electromicrograph exhibited
defined chromatin margination, followed by the cell compaction toward the
nuclear periphery. In the residual diffuse chromatin, remnants of deeply
modified nucleoli can still be revealed in treated HeLa cells. The nucleus
appears strongly rearranged, if compared to normal, which the later shows
uncondensed heterochromatin, clearly distinguishable from the diffuse interchromatin
of treated cells. Plasma membrane and organelles are preserved for long, except
for cytosol condensation and blebbing phenomena, which characterize apoptotic models.
Subsequently, the nucleus generates numerous compact electron dense
micronuclei, frequently released in the extracellular space. Cell splitting in
a number of apoptotic bodies represents generally the final stage of apoptosis.Apoptosis
as a desired biodefense cell death can be induced in cancer tissues [38]. This
induction is made possible using natural products from a variety of plants-derived
compounds [39]. One of these natural compounds of plant derived is terpenoids [40],
which included mono-, sesqui-, di-, and triterpenoids. These biocompounds are
synthesized by tandem reactions of the phosphorylated isoprene unit [41]. Some
of these dietary compounds possess chemopreventive properties that have been
proven in vivo and in vitro. For example, d-limonene from the peel oil of
orange or lemon has a chemopreventive activity against mammary, skin, liver,
lung, and fore stomach carcinogenesis [42]. Clinical trials results also
demonstrated that some terpenoids have the potentiality for treating cancers
without major toxicity [43]. Thus, further understanding of their biological
and physiological mechanisms may lead to the identification of more effective
compounds in this category for the prevention and treatment of targeted cancer
types. ZER (mono-sesquiterpene) has been reported previously to induce
apoptosis in several cancer cell lines such as liver cancer [4] and leukemia
cell lines [25]. ZER has also been reported having the capability to induce
apoptotic and morphological changes in different types of leukemic cells [44]. On
the other hand, ZER has shown less cytotoxic effect on normal Chang liver
cells, normal human dermal (2F0-C25), and colon (CCD-18 Co) fibroblasts [4, 17]. The current results of this study are in agreement with previous studies in terms of the potential
anticancer of this compound and the typical features of apoptosis of HeLa [4, 14, 20]. This current study of using TEM technique has been concluded for the first time, the
compound ZER, having promising anticancer activity toward HeLa cancer cells. In
addition, TEM has never been used and reported before to include the study that
relates to ultrastructural changes occurring to the HeLa cancer cells upon
treatment by ZER.It has been reported that zerumbone inhibits
the proliferation of cancer cells, while having less effect on normal-cell
proliferation [17]. This report pointed to the α,β-unsaturated
carbonyl group in ZER as the likely source of the activity, and this group may
play an important role. The α,β-unsaturated carbonyl group effectively removes
the intracellular Glutathione (GSH) by forming the Michael adduct with it,
thereby raising intracellular redox potential (E), which in turn stops
the proliferation of the cancer cells [45, 46]. However, average intracellular
redox potential of normal fibroblasts is about −220 mV, and that
in proliferating fibrosarcoma cells is about −210 mV, this difference
could explained the weaker effect of ZER on the proliferation of normal cells.
There is, thus, a margin of ~10 mV in the adjustability of E between
these normal cells and the cancer cells [45, 46].In
conclusion, ZER has stronger cytotoxic effects toward cancer cells compared to
normal cells. This compound can stimulate apoptosis of HeLa cells. ZER would
have a bright future in the treatment of tumors and further work may lead to
relative antitumor agents to be used in clinical settings.



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*Source: 769568-2009-03-24.xml*