Induction of apoptosis and cytotoxicity by raphasatin in human breast adenocarcinoma MCF-7 cells

Article abstract of DOI:10.3390/molecules23123092

Glucoraphasatin (GRH), a glucosinolate present abundantly in the plants of the Brassicaceae family, is hydrolyzed by myrosinase to raphasatin, which is considered responsible for its cancer chemopreventive activity; however, the underlying mechanisms of action have not been investigated, particularly in human cell lines. The aims of this study are to determine the cytotoxicity of raphasatin, and to evaluate its potential to cause apoptosis and modulate cell cycle arrest in human breast adenocarcinoma MCF-7 cells. The cytotoxicity was determined following incubation of the cells with glucoraphasatin or raphasatin (0-100 μM), for 24,48, and 72 h. GRH displayed no cytotoxicity as exemplified by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. When myrosinase was added to the incubation system to convert GRH to raphasatin, cytotoxicity was evident. Exposure of the cells to raphasatin stimulated apoptosis, as was exemplified by cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation. Moreover, using Annexin V-FITC assay, raphasatin induced apoptosis, as witnessed by changes in cellular distribution of cells, at different stages of apoptosis; in addition, raphasatin caused the arrest of the MCF-7 cells at the G2 + M phase. In conclusion, raphasatin demonstrated cancer chemopreventive potential against human breast adenocarcinoma (MCF-7) cells, through induction of apoptosis and cell cycle arrest.

Full text of DOI:10.3390/molecules23123092

molecules
Article
Induction of Apoptosis and Cytotoxicity by
Raphasatin in Human Breast Adenocarcinoma
MCF-7 Cells
1
1
1
2
Muhammad Din Ibrahim , Saie Brindha Kntayya
, Nooraini Mohd Ain , Renato Iori ,
Costas Ioannides 3 and Ahmad Faizal Abdull Razis
4,5,6,
*
1
UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia; marc_dean89@yahoo.com (M.D.I.); saiebrindhak@yahoo.co.uk (S.B.K.);
noorainim@upm.edu.my (N.M.A.)
2
3
4
5
6
CREA Consiglio per la ricerca in agricoltura e l’analisi dell’economia agrarian, Centro di ricerca Agricoltura
e Ambiente (CREA-AA), Via di Corticella 133, 40128 Bologna, Italy; renato.iori48@gmail.com
Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK;
c.ioannides@surrey.ac.uk
Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia,
4
3400 UPM Serdang, Selangor, Malaysia
Laboratory of Food Safety and Food Integrity, Institute of Tropical Agriculture and Food Security, Universiti
Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia
*
Correspondence: madfaizal@upm.edu.my; Tel.: +603-89472115
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 18 October 2018; Accepted: 14 November 2018; Published: 27 November 2018
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Glucoraphasatin (GRH), a glucosinolate present abundantly in the plants of the Brassicaceae
family, is hydrolyzed by myrosinase to raphasatin, which is considered responsible for its cancer
chemopreventive activity; however, the underlying mechanisms of action have not been investigated,
particularly in human cell lines. The aims of this study are to determine the cytotoxicity of raphasatin,
and to evaluate its potential to cause apoptosis and modulate cell cycle arrest in human breast
adenocarcinoma MCF-7 cells. The cytotoxicity was determined following incubation of the cells
with glucoraphasatin or raphasatin (0–100 µM), for 24, 48, and 72 h. GRH displayed no cytotoxicity
as exemplified by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
When myrosinase was added to the incubation system to convert GRH to raphasatin, cytotoxicity
was evident. Exposure of the cells to raphasatin stimulated apoptosis, as was exemplified by cell
shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation. Moreover,
using Annexin V-FITC assay, raphasatin induced apoptosis, as witnessed by changes in cellular
distribution of cells, at different stages of apoptosis; in addition, raphasatin caused the arrest of the
MCF-7 cells at the G + M phase. In conclusion, raphasatin demonstrated cancer chemopreventive
2
potential against human breast adenocarcinoma (MCF-7) cells, through induction of apoptosis and
cell cycle arrest.
Keywords: glucoraphasatin; raphasatin; glucosinolates; isothiocyanates; human breast adenocarcinoma
cell line; cytotoxicity; apoptosis; cell cycle
1
. Introduction
Cancer is a failure of the mechanisms that regulate cell growth and division, and remains a leading
cause of death worldwide. Although chemo- and radio-therapies are currently used routinely in cancer
Molecules 2018, 23, 3092; doi:10.3390/molecules23123092
www.mdpi.com/journal/molecules

Molecules 2018, 23, 3092
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therapy [
1], these are accompanied by serious side-effects, as a result of damage to normal tissues
surrounding the tumors [
2
]. Moreover, tumors may demonstrate resistance to these treatments [ ].
3
Breast cancer is a very common disease among Malaysian women and worldwide, with about 150–200
new cases being diagnosed, annually, in the Breast Clinic in Kuala Lumpur Hospital, alone. It is
estimated that 1 in 20 women will be diagnosed with breast cancer [
4,5]. Breast cancer accounted for
16.5% of all cancer cases registered with the Malaysian National Cancer Registry in 2006, and 34.4% of
the registered breast cancer cases were in the working age of 15–49 years [6].
During the last few decades the importance of diet in the etiology of human cancer has
been well-documented [7]. Many phytochemicals, including ascorbic acid and phenolics, such as
anthocyanins and flavonoids, possess antioxidant activity and are considered important contributors
to the beneficial effects linked to the consumption of vegetable-rich diets. Broccoli (Brassica oleracea),
radish (Raphanus sativus L.), kale, and cabbage (B. oleracea L.) are examples of Brassica vegetables that
are rich in these antioxidants, as well as glucosinolates, which have attracted much attention, in recent
years, due to the remarkable anti-carcinogenic activity of isothiocyanates, their major hydrolysis
products [8,9].
In a number of epidemiological studies, the consumption of cruciferous vegetables is associated
with lower cancer incidence, at a number of sites [10,11]. The chemopreventive property of
these vegetables is recognized to be due to glucosinolates, a class of sulfur-containing glycosides
that are present in significant amounts [10]. Glucosinolates (GLs) are secondary metabolites of
Brassica vegetables that are related to their health-promoting benefits, as their hydrolysis products,
isothiocyanates, possess chemopreventive and antioxidant properties [12].
4
-methylsulfanyl-3-butenyl glucosinolate, also referred to as GRH, is a glucosinolate, the major
source of which is Raphanus sativus (Kaiware Daikon), a white radish that is extensively consumed
in Japan and increasingly in Europe and North America [13 14]. Raphanus sativus L. radishes have
,
lately attracted interest in the scientific community because of their health-promoting phytochemical
content. The interest focused, particularly, on the isothiocyanate raphasatin (Figure 1), which is
released by myrosinase hydrolysis, upon chewing, cutting, or other mechanical disruption of the
fresh (uncooked) vegetable tissues [15]. Along with contributing to the distinctive pungent taste of
radish, isothiocyanates are powerful indirect antioxidants possessing chemopreventive activity and
potentially therapeutic activity [16].
The ability to induce apoptosis is an important property of anti-cancer candidate drugs.
Many methods have been developed to evaluate the potential of chemical agents to stimulate
apoptosis, including morphological studies, biochemical assays, ELISA, flow cytometry analysis and
others, of which morphological studies appear to be the most reliable [17]. As previous studies
have demonstrated that isothiocyanates manifest their chemopreventive activity, at least partly,
by modulating favorably the processes of apoptosis and cell proliferation [18–21], the present study
was undertaken to establish the potential of GRH and its degradation product raphasatin, to initiate
apoptosis and cell cycle arrest in human breast adenocarcinoma (MCF-7) cells.
Figure 1.
The hydrolysis process of glucoraphasatin by myrosinase produces
4
-methylsulfanyl-3-butenyl isothiocyanate, raphasatin. Adapted from Razis et al. [20].

Molecules 2018, 23, 3092
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2
. Results
2
.1. Effects of GRH and Raphasatin on Cell Viability of MCF-7 Cells
In this study, the viability of MCF-7 cells was investigated, following exposure to GRH and
raphasatin. At the concentrations studied (1–100 M), GRH had no effect on the MCF-7 cells (Table 1).
µ
Conversely, its degradation product, the isothiocyanate raphasatin was cytotoxic, following incubation
for 24, 48, and 72 h. Paclitaxel (0–25 nM) served as the positive control and was highly cytotoxic to
the cells.
Table 1. Cytotoxicity of GRH, raphasatin, and paclitaxel in human breast adenocarcinoma MCF-7 cells,
following incubation for 24, 48, and 72 h, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay.
independent experiments.
Data are expressed as mean
±
standard deviation (SD) of three
IC50
Compound
2
4 h
48 h
72 h
GRH
>100 µM
>100 µM
>100 µM
Raphasatin
Paclitaxel
9.84 ± 0.55 µM
9.64 ± 0.24 µM
5.61 ± 0.23 µM
7.53 ± 0.68 nM
5.21 ± 0.28 nM
5.09 ± 0.07 nM
2
.2. Morphological Analyses of Apoptosis Using the Terminal Deoxynucleotidyl Transferase dUTP Nick End
Labelling (TUNEL) Assay, Acridine Orange/Propidium Iodide (AO/PI) Staining, and
0
4
,6-Diamidino-2-phenylindole (DAPI) Staining
In morphological studies, the TUNEL assay, AO/PI staining and DAPI staining assays were
employed to assess the morphological changes in MCF-7 cells, following exposure to raphasatin,
for different periods of time, namely 24, 48, and 72 h. In the TUNEL assay the untreated cells (Figure 2A)
were healthy, characterized by a round-shape, and unstained by TUNEL. After treatment for 24 h
(
Figure 2B), the cells began to be stained with dark brown color indicating that some cells underwent
apoptosis at an early stage. After a 48 h treatment (Figure 2C), the number of dark brown-stained cells
increased; about 50% of the cell population had undergone apoptosis, which caused nuclear DNA
fragmentation. Finally, the 72 h-treated cells exhibited the highest number of apoptotic cells, covering
almost 70% of the cell population (Figure 2D).

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Figure 2. Raphasatin-mediated apoptosis in MCF-7 cells, as determined using the DeadEnd
Colorimetric terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. MCF-7
cells were treated with 9.84 M (IC50) concentration of raphasatin for various periods of time. Images
represent one of three independent experiments. Morphological changes after the treatment for 24 h ( ),
8 h ( ), and 72 h ( ) were compared with the untreated cells ( ), which acted as control. The arrows
in denote the viable unaffected cells, and in , and are the typical apoptotic cells (dark brown
µ
B
4
C
D
A
A
B
,
C
D
color stained in B, C, and D) resulting from nuclear DNA fragmentation.
In AO/PI staining, the untreated cells (Figure 3A) did not show any morphological changes,
and only bright intact green-colored, round-shaped, viable cells were observed, indicating no signs of
apoptosis. However, apoptosis was clearly evident upon 24 h (Figure 3B) treatment with raphasatin,
where early apoptosis with fragmented nucleus and chromatin condensation features were evident,
and the round-shaped cells also began to shrink. After the 48 h treatment (Figure 3C), apoptosis
was clearly present, with membrane blebbing and chromatin condensation being visible. The blebs
surrounding the single cell signified that apoptosis had developed. In addition, some necrotic cells
were clearly seen, with the red-stained color cells. Cells exposed to raphasatin for 72 h (Figure 3D)
exhibited more severe apoptosis, including membrane blebbing, late apoptosis, secondary necrosis,
and formation of apoptotic bodies. Furthermore, in the late stages of apoptosis, changes such as the
presence of reddish-orange color, due to the binding of AO to the denatured DNA, were observed after
the 72 h treatment of the cells.

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Figure 3. Raphasatin-induced apoptosis in the MCF-7 cells assessed by acridine orange/propidium
iodide (AO/PI) staining. MCF-7 cells were treated with 9.84 M (IC ) concentration of raphasatin,
for 24 h ( ), 48 h ( ), and 72 h ( ), and were compared with the untreated cells (
of the three independent experiments. Viable cells (VI), early apoptosis (EA), chromatin condensation
CC), blebbing (BL), late apoptosis (LA), necrosis (N), and secondary necrosis (SN).
µ
5
0
B
C
D
A). Images represent one
(
As shown in Figure 4, DAPI staining indicated that the treatment of MCF-7 cells with raphasatin,
for the different periods of time (24, 48, and 72 h), induced morphological features typical of
apoptotic cells, including nuclear fragmentation, micronuclei formation, and chromatin condensation,
as compared with the control cells that maintained the round shape. In contrast, the treated cells
exhibited the morphological characteristics of early apoptosis. Apoptotic cells and pyknotic nuclei with
bright blue color intensity were noted. After the 24 h treatment with raphasatin (Figure 4B), the cells’
morphology began to alter. Some of the cells had undergone nuclear fragmentation, an indicator of
apoptosis. Some viable cells were still visible as the apoptosis was at an early stage. After treatment
for 48 h (Figure 4C), more fragmented and apoptotic cells were visible. These features appeared more
frequently, as the duration of incubation increased to 72 h (Figure 4D), when the treated cells showed
chromatin condensation indicative of late apoptosis. The normal round-shaped cells had condensed
and the majority of the cells had completely undergone apoptosis. Figure 5 shows the significant
differences between the control (untreated) with the treated cells at 24, 48, and 72 h of incubation for
all the morphological analyses.

Molecules 2018, 23, 3092
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1
0 µm
10 µm
1
0 µm
1
0 µm
Figure 4. The fluorescent micrographs showing the effect of raphasatin on apoptosis, assessed by DAPI
in MCF-7 cells. MCF-7 cells were treated with 9.84 M (IC ) concentration of raphasatin for 24 h ( ),
8 h ( ), and 72 h ( ), and compared with the untreated cells ( ). Images represent one of the three
µ
B
5
0
4
C
D
A
independent experiments. The arrows in B, C, and D show the nuclear fragmentation and chromatin
condensation commensurate, with apoptosis.
Figure 5. Bar graph showing the effect of raphasatin on apoptosis assessed using TUNEL, AO/PI, and
DAPI in the MCF-7 cells. The cells were treated with 9.84
4 h, 48 h, and 72 h, and compared with the untreated cells. Data are expressed in mean
deviation (SD). *** p < 0.001.
µM (IC ) concentration of raphasatin for
50
2
±
standard

Molecules 2018, 23, 3092
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2
.3. Raphasatin-Induced Apoptosis in the MCF-7 Cells Determined by Annexin V-FITC/PI Double Staining
and Using Flow Cytometry
The qualitative assessment of apoptosis was carried out utilizing the Annexin V-FITC/PI staining
assay and using flow cytometry in order to determine the cell distribution at four different quadrants,
namely viable cells, early apoptosis, late apoptosis, and necrotic cells. The histogram illustrates the
time course of Annexin V-FITC/PI binding to the phosphatidylyserine (PS), in the apoptotic cells
(Figure 6).
Figure 6. Annexin V-FITC/PI staining to evaluate apoptosis. The MCF-7 cells were treated with
IC50 concentration of raphasatin (9.84 M), incubated for 24 (B), 48 (C), and 72 h (D) and compared
with untreated cells ( ). The rate of apoptosis was determined by flow cytometry, using the Annexin
µ
A
V-FITC/PI staining assay. The data represent one of three independent experiments. (i) Lower left
quadrant represents the viable cells, (ii) lower right quadrant shows the early apoptosis cells, (iii) upper
right quadrant signifies the late apoptosis cells, and (iv) the upper left quadrant indicates dead cells.
As shown in Figure 7, the percentage of apoptotic cells increases with the length of the incubation
time. The untreated cells which served as the control in the experiment exhibited the least percentage
of necrotic cells, with a value of 0.08 0.02%. During the first 24 h treatment, the percentage of necrotic
cells rose to 2.31 0.25%, whereas after 48 h, the percentage of necrotic cells climbed to 4.58 0.14%.
±
±
±
The treated cells incubated for 72 h, displayed a drop in the percentage of necrotic cells, with a value
of 0.16 ± 0.01%.

Molecules 2018, 23, 3092
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Figure 7. The effect of exposure time on the raphasatin-induced rate of apoptosis in the MCF-7 cells.
The cells were treated with 9.84 M (IC50) concentration of raphasatin for 24–72 h. The data comparing
µ
the percentage of cells between the untreated with treated, in terms of viable, early, and late apoptosis
and necrotic for 24, 48, and 72 h of incubation. The data expressed as mean ± SD. *** p < 0.001.
As expected, the untreated cells exhibited the highest value of viable cells, 99.6
that the majority of the cells were still healthy and visible. Following treatment with raphasatin for
±
0.13%, indicating
2
3
4, 48, and 72 h, the percentage of viable cells decreased significantly to a value of 54.26
1.97 ± 1.03%, and 2.09 0.24%, respectively. In the early apoptosis stage, the control incubations
0.02%, which was elevated to 7.68 0.41%, following
exposure to raphasatin for 24 h. Following the 48 h treatment, cell numbers declined to 5.12 ± 0.11%,
and lastly, in contrast, the cell population increased to 22.90 2.50%, at 72 h. Finally, in the late
apoptosis stage, the fraction of apoptotic cells rose with the time of exposure to the isothiocyanate.
The percentage of apoptotic cells was 35.45 1.25%, 58.33 0.87%, and 74.84 2.37% at 24, 48, and 72
h, respectively; in the untreated MCF-7 cells, the number of apoptotic cells was 0.28 ± 0.09%.
±
1.73%,
±
contained the lowest number of cells, 0.04
±
±
±
±
±
±
2
.4. Cell Cycle Analysis by Flow Cytometry
Cell cycle arrest analysis through flow cytometry was performed in order to evaluate whether the
anti-proliferative effect of raphasatin in MCF-7 cells is related to changes in the cell cycle. The cellular
distribution of the cells in different phases is illustrated in relation to the intracellular DNA content.
The phases were divided into four, namely G /G , S, G + M, and Sub G /G phase (Figure 8).
0
1
2
0
1
MCF-7 cells were treated with raphasatin (9.84
in the Sub G /G phase increased from 22.16 0.16% to 35.48
8 and 72 h respectively compared with the untreated cells (6.24
The percentage of cells in the G /G phase showed a decreasing trend which was associated with an
µ
M) for 24, 48 and 72 h. The percentage of cells
0.71% and 49.89 0.71% at 24,
0.18%) as shown in Figure 9.
±
±
±
0
1
4
±
0
1
increase in percentage of cells at the G + M phase. The proportion of cells in the G /G phase dropped
2
0
1
to 61.01
±
0.43%, 30.37
±
0.58% and 19.56
±
0.30% at 24, 48 and 72 h respectively, compared to the
control (91.57 ± 0.40%). At the G + M phase, the percentage of cells recorded were 9.41 ± 0.34%
,
2
2
5.21 ± 0.19% and 23.66
±
0.54% at 24, 48 and 72 h respectively, compared to the control cells
(1.64 ± 0.09%). Exposure to raphasatin led to the arrest of the cells at the G + M phase. Clearly,
2
treatment of the MCF-7 cells with raphasatin inhibited cell cycle progression at the G + M phase so
2
that it may be inferred that raphasatin impaired cell growth by arresting the cells at the G + M phase.
2

Molecules 2018, 23, 3092
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Figure 8. The time-dependent distribution of the MCF-7 cells exposed to raphasatin at the various
stages of the cell cycle. MCF-7 cells were treated with IC50 concentration of raphasatin (9.84 M) for 24 h
), 48 h ( ) and 72 h ( ) compared with untreated cells ( ). Raphasatin arrested the MCF-7 cells at the
µ
(B
C
D
A
G + M phase as analyzed by flow cytometry. The data represent one of three independent experiments.
2
Figure 9. Time-dependent distribution of the raphasatin-treated MCF-7 cells for the different cell cycle
stages. Cells were treated with 9.84
comparing the proportion of cells between the untreated with the treated, in terms of cell cycle for 24,
8, and 72 h of incubation. The data expressed as mean ± SD. *** p < 0.001.
µM (IC50) concentration of raphasatin for 24, 48, and 72 h. The data
4

Molecules 2018, 23, 3092
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Finally, the lowest number of cells was observed at the end of the cell cycle. At the S phase,
the proportion of cells recorded was 7.32 0.51%, 8.18 0.55% and 6.12 0.39% at 24, 48 and 72 h
respectively compared to the control cells (0.59 ± 0.08%).
±
±
±
3
. Discussion
Extensive experimental studies have established that isothiocyanates, both aliphatic and aromatic,
such as sulforaphane, erucin, and phenethyl isothiocyanate, owe their potent cancer chemopreventive
activity to the fact that they act through a number of mechanisms, modulating favorably the
initiation and promotion stages of carcinogenesis [18,22–30]. Raphasatin, present in substantial
amounts in Daikon (Japanese white radish) as the glucosinolate glucoraphasatin, is consumed
extensively in Japan [31]. Indeed, studies on rodents, conducted in Japan, demonstrated that
raphasatin afforded protection against N-nitrosobenzylamine-induced esophageal cancer in rats [32
]
and N-nitrosobis(2-oxopropyl)amine-induced pancreatic cancer in hamsters [31], the effect being
more pronounced when administered to the animals during the initiation stage, in comparison with
the post-initiation stage. The reported antimutagenic in vitro activity of raphasatin [31,33], indicate
that the isothiocyanate may protect DNA from the injury mediated by the reactive intermediates.
This is generated following the bioactivation of chemical carcinogens and supports a beneficial role
for isothiocyanate, at the initiation stage. This premise is reinforced by studies emanating from
our laboratory where raphasatin, generated by the action of myrosinase on glucoraphasatin, was a
potent inducer of enzyme systems involved in the deactivation of carcinogenic reactive intermediates,
such as glutathione S-transferase, quinone reductase, and epoxide hydrolase, when incubated with
precision-cut rat liver slices [20]. Similar observations have been made in vivo where rats maintained
on a diet of a glucosinolate-rich Japanese Daikon extract had markedly higher levels of hepatic
glutathione S-transferase activity, and to a lower extent of other detoxification enzymes [19]. As such,
the antioxidant role of raphasatin is another key mechanism of its chemopreventive activity via
antioxidant responsive elements (AREs) pathway, thus, the data on the antioxidant status would be
beneficial in future.
When administered to rats, raphasatin could also antagonize the chemically-induced
carcinogenesis, when administered post-initiation, albeit less effectively, compared with when given
during initiation, and the same workers also showed immunologically that the isothiocyanate could
induce apoptosis, as well as impair cellular proliferation [32]. Moreover, hexane extracts of Daikon
inhibited the proliferation in human cancer cells and stimulated apoptosis by influencing genes
involved in apoptosis [14,15]. The commercial availability of raphasatin allowed us to extend these
studies and evaluate, in detail, its potential to enhance apoptosis and arrest cell proliferation in human
breast adenocarcinoma MCF-7 cells.
Apoptosis was monitored using the TUNEL assay, AO/PI, and DAPI staining, and by Annexin
V-FITC/PI double-staining, followed by flow cytometry. In all assays raphasatin clearly elevated
apoptosis, as was exemplified by the increased membrane blebbing, chromatin condensation, nuclear
fragmentation, and the formation of micronuclei; furthermore, the effect was related to the time of
exposure. These findings demonstrate that increased apoptosis is a viable mechanism contributing to
the cancer chemopreventive effect of raphasatin.
The potential of raphasatin to perturb the cell cycle was assessed in the present study, using
flow cytometry, in order to establish whether the anti-proliferative effect of this isothiocyanate is
related to changes in the cell cycle. In a previous study by Papi et al. [15], conducted in CaCo-2 cells,
an increase in the G /G population was observed in the presence of raphasatin. In the present study,
0
1
flow cytometry analysis revealed that raphasatin significantly arrested the cell cycle in the MCF-7 cells,
at the G + M phase. The percentage of cells in the G + M phase increased, significantly, probably
2
2
reflecting apoptosis induced by raphasatin, resulting in DNA degradation. Anticancer agents may
modify the regulation of the cell-cycle machinery, resulting in an arrest of cells in various phases of the
cell cycle, thus, decreasing the cancerous cells growth and proliferation [34].

Molecules 2018, 23, 3092
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It is pertinent to point out that raphasatin has caused a marked increase in apoptosis and arrest
in cell cycle, at a concentration lower than 10 M. Although to our knowledge the pharmacokinetic
µ
behavior of raphasatin has not been investigated, studies performed with other isothiocyanates,
such as sulforaphane and phenethyl isothiocyanate, in rodents, noted that such concentrations in the
plasma may be achieved following exposure to doses simulating the human dietary intake [35–42].
However, the distribution of isothiocyanates to tissues, following exposure to dietary doses, has not
been evaluated, so that the concentration of the parent compound in the breast cell has not been
defined. It is pertinent to point out that intracellular concentrations of isothiocyanates, including
their dithiocarbamate metabolites, may be much higher than those encountered in the plasma [43,44].
Moreover, cruciferous vegetables strains with high glucosinolate content may be developed to increase
the level of exposure to isothiocyanates [45].
4
. Materials and Methods
4
.1. Isolation of Glucoraphasatin (GRH)
GRH was isolated from the sprouts of Raphanus sativus L. (Daikon), as previously described [15].
Briefly, GRH was isolated from the seven-day-old Raphanus sativus L. sprouts which were freeze-dried
through two sequential steps, including ion exchange and size exclusion chromatography. GRH
◦
(
10 mg/mL) was dissolved in water and kept in a freezer, at
myrosinase-catalyzed hydrolysis [46]. In each experiment, 5
from the seeds of Sinapsis alba L., was added into the cell culture medium to generate raphasatin.
−
20 C. Raphasatin was produced through
µ
L of myrosinase (0.3 units/mL), isolated
One myrosinase unit is defined as the amount of enzyme capable of hydrolyzing 1
min, at pH 6.5 and 37 C.
µmol sinigrin, per
◦
4
.2. Cell Viability
MCF-7 (Human breast adenocarcinoma) cell line was obtained from the American Type Culture
Collection (ATCC, Manassasa, VA, USA). The cells were cultured in a flask containing RPMI 1640
Sigma-Aldrich, Munich, Germany) supplemented with 10% FBS (fetal bovine serum, Sigma-Aldrich,
(
Munich, Germany), 1% of antibiotics (Streptomycin & Penicillin) (Sigma-Aldrich, Munich, Germany),
◦
and incubated in a humidified incubator supplemented with 5% CO , at a temperature of 37 C.
2
Cell viability was determined using the MTT assay. The MCF-7 cells were seeded in 96-well
5
flat-bottomed tissue culture plates, at a concentration of 1
×
10 cells/well, and allowed to grow
for 24 h. Upon 50–70% of confluency, the cells were treated with GRH at concentrations ranging
from 0–100 M. Five L of myrosinase (0.3 units/mL) (Sigma Aldrich, St. Louis, MO, USA) was
added into each well to hydrolyze the GRH into the raphasatin, before the cell treatment. The cells
µ
µ
were then incubated for 24, 48, and 72 h. At the end of the incubation, 20
reagent (Sigma-Aldrich, Munich, Germany) was added into each well, and incubated for 3 h, at 37 C.
µ
L (5 mg/mL) of the MTT
◦
The medium was removed and 100 µL of dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) and
was added into each well, resulting in the solubilization of the formazan salt. The absorbance was
measured at 570 nm, using an ELISA reader (TECAN, Männedorf, Zürich, Switzerland) and the IC50
(50% inhibitory concentration) was calculated [47].
4
.3. Acridine Orange/Propidium Iodide Staining
2
6
The MCF-7 cells were seeded in 25 cm culture flasks, at a concentration of 1
×
10 cells/mL, and
incubated for 24 h. Following incubation, the cells were treated with 9.84
µM (IC ) concentration
50
of raphasatin. After the treatment, the supernatant was collected and transferred into a falcon tube,
whereas, the remaining cells in the flask were trypsinized and collected, using a scraper. The cells were
mixed with 2 mL PBS, resuspended, and mixed with the supernatant in the falcon tube. The cells were
centrifuged at 300
×
g for 5 min. The supernatant was discarded and cells were washed again, using
5
mL PBS. The centrifugation step was repeated and the falcon tube was put on ice. One ml Acridine

Molecules 2018, 23, 3092
12 of 16
Orange/Propidium Iodide (AO/PI) solution (Sigma-Aldrich, St. Louis, MO, USA) was diluted with
PBS (1 mL), the process being carried out under dark conditions, due to the sensitivity of both solutions
to light. AO and PI solutions (10 mg/mL of each) were transferred into an Eppendorf tube and the
mixture was resuspended, repeatedly, until a homogeneous solution was obtained. An aliquot of
the cell preparation (10
transferred on to a slide and viewed under a UV-fluorescent microscope, using 20
Axio Scope A1, Zeiss, Oberkochen, Germany).
µ
L) was mixed with the prepared AO/PI solution. The mixture (10
µL) was
×
objective lens
(
4
.4. DAPI Staining
Cells were grown on coverslips. After a 24 h treatment with raphasatin, the cells were fixed
with 4% paraformaldehyde and permeabilized with Triton X-100 (0.1% in PBS). Cells were stained
0
with 2.5 mg/mL DNA dye, 4 ,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO,
◦
USA) in PBS, for 30 min, at 20 C, and examined by fluorescence microscopy. Cells with condensed
or fragmented nuclei were counted on the adjacent four fields, of each coverslip. The percentage
of cells with chromatin condensation and nuclear fragmentation was determined in triplicates,
for each treatment, and compared with the controls [47]. Morphological changes were viewed
using a UV-fluorescent microscope (Axio Scope A1, Zeiss, Oberkochen, Germany) employing a
2
0× objective lens.
4
.5. DeadEnd Colorimetric TUNEL Assay
6
2
The MCF-7 cells were seeded at a concentration of 1
×
10 cells/mL, in 25 cm culture flasks, and
incubated for 24 h. The terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay
was carried out using the DeadEndTM colorimetric apoptosis detection system (Promega, Madison, WI,
USA), according to the manufacturer’s protocol. After the treatment with raphasatin, the cells were
harvested and grown onto poly-L-lysine slides, for 6 h. Slides were immersed in 4% paraformaldehyde
®
and then twice in PBS. Subsequently, slides were immersed in 0.2% Triton X-100 in PBS for 5 min and
washed twice in PBS. Equilibration Buffer (100 µL) was added on to the slides, followed by 100 µL of
terminal deoxynucleotidyl transferase (TdT) reaction mix. Slides were covered with plastic coverslips
◦
and incubated for 60 min, at 37 C, in a humidified chamber. After incubation, slides were immersed
in 2
Thereafter, 100
×
Saline Sodium Citrate (SSC), for 15 min, followed by 0.3% hydrogen peroxide for 3–5 min.
µ
L Streptavidin Peroxidase (diluted 1:500 in PBS) was added, and slides were incubated
0
for a further 30 min, at room temperature. 3,3 -Diaminobenzidine (DAB) (100
µL) was added until
a light brown background appeared; slides were then immersed several times in deionized water.
Lastly, slides were mounted in permanent mounting medium and staining was observed, under a
light microscope (Microscope Compound Olympus) (Olympus, Tokyo, Japan) using a 20
lens [47].
×
objective
4
.6. Annexin V-FITC/PI by Flow Cytometry
Annexin V-FITC assay was carried out using the Annexin V-FITC assay kit (Sigma-Aldrich,
St. Louis, MO, USA), according to the protocols provided. The MCF-7 cells at a concentration of 1
×
6
2
1
0 cells/mL were seeded in 25 cm culture flasks, and incubated for 24 h. At the end of the incubation,
the cells were treated with the IC50 concentrations of raphasatin and incubated for 24, 48, and 72 h.
After the treatment, the supernatant was collected and transferred into a falcon tube. The remaining
cells in the flask were trypsinized, using 1 mL trypsin, incubated for 5 min, and the cells were collected
and transferred into a falcon tube. The cells were mixed with 2 mL PBS, resuspended, and mixed with
the supernatant in the falcon tube. The cells were centrifuged at 300
×
g, for 5 min. The supernatant
was discarded and the precipitate was washed again, using 5 mL PBS. The centrifugation step was
repeated and the falcon tube was put into an ice box. Flow cytometric analysis was carried out using
a FACS Canto II Becton–Dickinson (Franklin Lakes, NJ, USA) flow cytometer, by analyzing at least
10,000 cells per sample. The binding buffer supplied by the manufacturer was used to bring the

Molecules 2018, 23, 3092
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reaction volume to at least 500
µL, for the flow cytometry analysis. Unstained cells were classified as
viable cells; cells stained for Annexin-V were early apoptotic; cells stained with both Annexin-V and PI
were late apoptotic; cells stained with PI were indicated as necrotic cells [47]. Results are presented as
percentage (%) of the total number of cells.
4
.7. Cell Cycle Arrest Analysis by Flow Cytometry
Cell cycle arrest evaluation was carried out using a Cell Cycle Arrest Detection Kit (Sigma-Aldrich,
Becton Dickinson, Franklin Lakes, NJ, USA), according to the protocol provided. MCF-7 cells at a
6
concentration of 1
×
10 cells/mL were harvested and transferred into 17
×
100-mm tubes. The cells
were centrifuged for 5 min at 300
Buffer solution (1 mL) was added and the cells were resuspended by gentle vortexing, at low speed.
×
g, at room temperature, and the supernatant was aspirated.
6
The concentration was adjusted to 1.0
centrifuged at 300
Solution A (containing trypsin buffer), at a volume of 250
mixed by hand tapping the tube. Solution A was allowed to react for 10 min at room temperature.
Two hundred l of Solution B (containing trypsin inhibitor and RNase buffer) was added into each
tube and gently mixed by hand tapping, and a further 10 min incubation was carried out, at room
temperature. Cold Solution C (containing propidium iodide stain solution, 200 L) was added into
×
10 cells/mL, with buffer solution. The cell suspensions were
×
g for 5 min at room temperature, and the supernatant was decanted carefully.
L, was added to each tube and gently
µ
µ
µ
each tube. The solution was mixed, as above, and incubated once again for 10 min, on ice, in the dark.
◦
The tubes were finally capped and stored at 2–8 C, in the dark, until flow cytometric analysis was
performed. The samples were tested within 3 h, following the addition of Solution C. After storage,
the samples in the tubes were mixed by hand tapping, to resuspend the cells. The cells were then
analyzed using the FACS Canto II Becton-Dickinson (Franklin Lakes, NJ, USA) flow cytometer, by
analyzing at least 10,000 cells per sample. The percentage of cells in G1, S, and G2 phases were
analyzed by the ModFit LT software (Verity Software House, Topsham, ME, USA).
4
.8. Statistical Analysis
Data are expressed as mean
two-way ANOVA, using Dunnett’s test. Differences were considered significant at p < 0.05. All the
±
standard deviation (SD). Statistical evaluation was performed by
samples were measured in triplicates.
5
. Conclusions
In conclusion, the present studies performed in the MCF-7 cells have established that
raphasatin, at concentrations potentially achieved in the plasma, following dietary intake, stimulates
apoptosis and impairs cell proliferation, and that these mechanisms may be integral to its cancer
chemopreventive activity.
Author Contributions: Conceived and designed the experiment: M.D.I. and A.F.A.R. performed the experiment
and analysed data: M.D.I., S.B.K., N.M.A., and A.F.A.R. Drafted and proofread the paper: M.D.I., R.I., C.I., and
A.F.A.R. All authors finally reviewed and approved the manuscript.
Funding: This work was funded by the Ministry of Higher Education, Malaysia and Universiti Putra Malaysia
through GP-IPM (Vot No. 9400600).
Conflicts of Interest: The authors declare no conflict of interest.
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©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(