Carcinogenesis vol.24 no.10 pp.1665±1670, 2003
DOI: 10.1093/carcin/bgg123
Allyl isothiocyanate, a constituent of cruciferous vegetables, inhibits growth of
PC-3 human prostate cancer xenografts in vivo
Sanjay K.Srivastava , Dong Xiao , Karen L.Lew,
Pamela Hershberger, Demetrius M.Kokkinakis, Candace
S.Johnson1, Donald L.Trump1 and Shivendra V.Singh2
Introduction
Abbreviations: AITC, allyl isothiocyanate; Cdc25, cell division cycle 25;
Cdk1, cyclin-dependent kinase 1; ITCs, isothiocyanates.
These authors contributed equally.
Cruciferous vegetable-derived isothiocyanates (ITCs) are
highly effective in prevention of chemically induced cancers
in animals (reviewed in refs 1,2). The chemoprotective effect
for ITCs has been observed against diethylnitrosamine and
benzo[a]pyrene-induced pulmonary and forestomach cancer
in mice (3), N-nitrosobenzylmethylamine-induced esophageal
tumorigenesis in rats (4), 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone-induced pulmonary neoplasia in rats (5),
and azoxymethane-induced colonic aberrant crypt foci in rats
(6). Moreover, the thiol conjugates of ITCs retain their chemopreventive activity in some models (7,8). Inhibition of carcinogen activation due to modulation of cytochrome-450-dependent
monooxygenases and/or an increase in detoxification of the
activated carcinogenic metabolites through induction of
Phase II drug metabolizing enzymes, including glutathione
transferases and quinone reductase, are implicated in ITCmediated prevention of chemically induced cancers (1,2).
More recent studies have shown that certain ITCs can inhibit
proliferation of cultured cancer cells by inducing apoptosis
and/or causing cell cycle arrest (9±16). Yu et al. (9) were the
first to report time- and concentration-dependent induction of
apoptosis in ITC-treated cells that was associated with a rapid
and transient activation of caspase-3-like activity. Involvement
of caspases, including caspase-3 and caspase-8, in ITCinduced apoptosis has also been established in human
leukemia HL-60 cells (12,14). Furthermore, phenethyl-ITC
(PEITC), a natural analog of allyl isothiocyanate (AITC),
effectively blocked tumor promoter (12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor)-induced cell
transformation in mouse epidermal JB6 cells, which correlated
with apoptosis induction (11).
Recent studies from our laboratory have shown that AITC, a
constituent of cruciferous vegetables and one of the most
widely studied members of the ITC family, significantly inhibits survival of PC-3 and LNCaP human prostate cancer cells
in culture, whereas proliferation of a normal prostate epithelial
cell line is minimally affected by AITC even at concentrations
that are highly cytotoxic to the prostate cancer cells (16). We
also found that exposure of PC-3 and LNCaP cells to growth
suppressive concentrations of AITC results in apoptotic cell
death and cell cycle arrest in G2 /M phase (16). Based on these
observations, we predicted that AITC administration could
inhibit growth of human prostate cancer xenografts in vivo.
The present studies were designed to experimentally verify
this hypothesis. We demonstrate that AITC administration
significantly retards growth of PC-3 cells implanted in nude
mice. Furthermore, the results of the present study indicate that
the AITC-mediated growth inhibition of PC-3 xenografts is
associated with increased apoptosis and reduced mitotic activity. To the best of our knowledge, the present study is the first
published report to indicate in vivo anticancer activity of a
natural ITC compound in a human prostate cancer xenograft
model.
Carcinogenesis vol.24 no.10 # Oxford University Press; all rights reserved.
1665
Departments of Pharmacology, Pathology and Medicine, and University of
Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania, USA
1
Present address: Roswell Park Cancer Institute, Buffalo, NY, USA
2
To whom correspondence should be addressed at: 2.32A Hillman Cancer
Center Research Pavilion, University of Pittsburgh Cancer Institute,
University of Pittsburgh School of Medicine, 5117 Center Avenue, Pittsburgh,
PA 15213, USA
Email: [email protected]
We have shown previously that allyl isothiocyanate
(AITC), a constituent of cruciferous vegetables, significantly inhibits survival of PC-3 and LNCaP human prostate cancer cells in culture, whereas proliferation of a
normal prostate epithelial cell line is minimally affected
by AITC even at concentrations that are highly cytotoxic to
the prostate cancer cells. The present studies were designed
to test the hypothesis that AITC administration may retard
growth of human prostate cancer xenografts in vivo. Bolus
i.p. injection of 10 mmol AITC, three times per week
(Monday, Wednesday and Friday) beginning the day of
tumor cell implantation, significantly inhibited the growth
of PC-3 xenograft (P 5 0.05 by two-way ANOVA). For
example, 26 days after tumor cell implantation, the average
tumor volume in control mice (1025 205 mm3 ) was ~1.7fold higher compared with AITC-treated mice. Histological analysis of tumors excised at the termination of the
experiment revealed a statistically significant increase in
number of apoptotic bodies with a concomitant decrease in
cells undergoing mitosis in the tumors of AITC-treated
mice compared with that of control mice. Western blot
analysis indicated an ~70% reduction in the levels of antiapoptotic protein Bcl-2 in the tumor lysate of AITC-treated
mice compared with that of control mice. Moreover, the
tumors from AITC-treated mice, but not control mice,
exhibited cleavage of BID, which is known to promote
apoptosis. Statistically significant reduction in the expression of several proteins that regulate G2 /M progression,
including cyclin B1, cell division cycle (Cdc)25B and
Cdc25C (44, 45 and 90% reduction, respectively, compared with control), was also observed in the tumors of
AITC-treated mice relative to control tumors. In conclusion, the results of the present study indicate that AITC
administration inhibits growth of PC-3 xenografts in vivo
by inducing apoptosis and reducing mitotic activity.
S.K.Srivastava et al.
Materials and methods
Materials
AITC was purchased from Aldrich (Milwaukee, WI). Antibodies against Bax,
BID, Bcl-XL , cell division cycle (Cdc)25C and cyclin-dependent kinase 1
(Cdk1) were from Santa Cruz Biotechnology (Santa Cruz, CA), antibodies
against Bcl-2 were from DAKO (Carpenteria, CA), antibodies against Cdc25B
were from BD Transduction (San Diego, CA), and the antibodies against cyclin
B1 and actin were from Oncogene Research Products (Boston, MA). PC-3
cells were obtained from American Type Culture Collection (Rockville, MD).
Male athymic mice (6±10 weeks old) were purchased from Harlan Sprague
Dawley (Madison, WI).
Cell culture and colony formation assay
Monolayer cultures of PC-3 cells were maintained in F-12K Nutrient Mixture
(Kaighn's modification) supplemented with 7% (v/v) non-heat inactivated
fetal bovine serum and 10 ml/l PSN Antibiotic Mixture (Gibco BRL, Grand
Island, NY). PC-3 cells were cultured at 37 C in a humidified atmosphere of
5% CO2 and 95% air. The experiments described in this paper were conducted
using PC-3 cells that were passaged 525 times. The cytotoxic effect of AITC
was assessed by colony formation assay as described by us previously (17)
with slight modifications. Briefly, 2 103 cells were plated in 6-well plates,
and allowed to attach overnight. Subsequently, the medium was replaced with
fresh complete medium containing desired concentrations of AITC or equal
volume of DMSO, and the plates were incubated for 10 days at 37 C in a
humidified atmosphere of 95% air and 5% CO2 . AITC or DMSO containing
medium was replaced every 48 h. The colonies were fixed in ethanol and
stained with a Giemsa solution (Sigma, St Louis, MO). Colonies containing
450 cells were counted under an inverted microscope. The IC50 value was
determined from a plot of percentage of survival versus AITC concentrations.
In vivo xenograft assay
Mice were fed AIN-76A semi-purified diet (ICN Biomedicals, Aurora, OH) for
8 days prior to the start of the experiment. The animals were maintained on this
diet throughout the duration of the experiment. PC-3 cells were mixed in a 1:1
ratio with Matrigel (Becton Dickinson, Bedford, MA), and a 0.1 ml suspension
containing 106 cells was injected subcutaneously on both left and right flank of
each mouse. Mice were randomized into two groups of five mice per group
(two tumors per mouse). The experimental group of mice received bolus i.p.
injection of 10 mmol AITC in 0.1 ml cottonseed oil three times per week
(Monday, Wednesday and Friday) beginning the day of tumor cell implantation. Control mice received an equal volume of vehicle (cottonseed oil) three
times per week. We used cottonseed oil as a vehicle for AITC administration
not only because this ITC compound is insoluble in aqueous solution but also
because cottonseed oil has been used previously for administration of other
ITCs to mice (18). AITC dose of 10 mmol was selected because (i) it was
within the range (3±12 mmol) of concentrations used previously for other ITCs
(1,2,18,19) and corresponded to the IC50 for AITC in PC-3 cells (~15 mM), and
(ii) 20 mmol AITC (three times per week) exhibited toxicity to nude mice
(Srivastava et al., unpublished observations). Body weights of the mice of both
groups were determined periodically to assess non-specific toxicity of AITC.
Tumor measurement began once each mouse had palpable tumor. Tumor size
was determined three times per week using a caliper. Tumor volume was
calculated as described by us previously (20). The experiment was terminated
26 days after tumor cell implantation because tumors of control mice began to
show sign of possible necrosis. The tumor tissues of control and AITC-treated
mice were harvested, washed with ice-cold phosphate-buffered saline and
divided into two pieces. One piece was processed for histology and immunohistochemistry, whereas the second portion was used for western blotting.
Histological analysis of mitosis and apoptosis
Tumor tissues were fixed in 10% neutral-buffered formalin overnight. Subsequently, the tissues were dehydrated, embedded in paraffin and sectioned
(6 mm) every 100 mm intervals. Sections were stained with hematoxylin and
eosin and mitotic activity was defined as the number of cells undergoing
mitosis per high-power view (400). For apoptosis, paraffin-embedded sections (6 mm) were de-paraffinized and immunostained using ApoTag plus
peroxidase in situ apoptosis detection kit (Intergen, NY) according to manufacturer's instructions. The sections were counterstained with methylene green
and brown color apoptotic bodies were counted at 400 magnification.
Western blot analysis
Tumor tissues from control and AITC-treated mice were minced and suspended in a lysis solution described by us previously (16). Tumor tissues
were homogenized using a Polytron, and the homogenate was centrifuged at
1666
14 000 g for 15 min. The supernatant fraction was collected and used for
sodium dodecyl sulfate polyacrylamide gel electrophoresis (21). The proteins
were transferred onto PVDF membrane. After blocking for 1 h with 10% nonfat dry milk in Tris-buffered saline containing 0.05% Tween-20, the membrane
was probed for 1 h at room temperature with the desired primary antibody. The
membrane was then treated with appropriate peroxidase-conjugated secondary
antibody, and the immunoreactive proteins were visualized using enhanced
chemiluminescence kit (NEN Life Science Products, Boston, MA). Each
membrane was stripped and re-probed with antibodies against actin to correct
for differences in protein loading.
Statistical analysis
Statistical significance of difference in tumor volume and body weight
between control and AITC-treated mice was assessed by two-way ANOVA.
Statistical significance of differences in apoptotic and mitotic cells and protein
expression between control and AITC-treated tumors were determined by
Student's t-test.
Results
Cytotoxic effect of AITC on PC-3 cells was determined
by colony formation assay, and the results are shown in
Figure 1A. The colony formation by PC-3 cells was significantly inhibited in the presence of AITC with an IC50 of ~2.2
mM (Figure 1A).
Figure 1B shows the effect of AITC treatment on growth of
PC-3 tumor xenografts in nude mice. Each mouse of both the
groups had measurable tumor on day 7 after tumor cell implantation. AITC treatment resulted in a statistically significant
inhibition (P 5 0.05 by two-way ANOVA) of PC-3 xenograft
growth, and the growth inhibitory effect of AITC was evident
in terms of tumor volume (Figure 1B) as well as wet tumor
weight (data not shown). For example, on day 21, the average
tumor volume in control mice (640 95 mm3 , n ˆ 10) was
~1.8-fold higher compared with AITC-treated mice. Similarly,
the average tumor volume in control group was ~1.9-fold
higher than that in AITC-treated group (464 58 mm3 ) on
day 24. These results indicated that the growth of PC-3 xenograft in nude mice was retarded upon AITC administration.
The body weights of the control and treated mice were
determined periodically to assess non-specific toxicity of
AITC. The average body weights of the control and AITC
treated mice did not differ significantly by two-way ANOVA
suggesting that AITC administration did not cause weight loss
(Figure 1C). The mice in AITC-treated group appeared healthy
and did not show any other sign of non-specific toxicity, such
as food and water withdrawal and impaired movement.
We have shown previously that reduced survival of PC-3
cells following exposure to AITC is associated with G2 /M
arrest and apoptosis induction (16). To determine whether
AITC-mediated in vivo growth inhibition of PC-3 xenografts
was caused by increased apoptosis and/or reduced mitotic
activity, the tumors from control and AITC-treated mice
were harvested at the termination of the experiment for analysis of cells undergoing apoptosis and mitosis. As shown in
Figure 2, histological analysis revealed a statistically significant decrease in the number of cells undergoing mitosis in
tumors of AITC-treated mice compared with control tumors.
Adjacent sections from the same tumor were analyzed for
apoptotic bodies using TUNEL assay, which showed a significantly higher count of apoptotic bodies in tumors of AITCtreated mice compared with controls (Figure 2).
To gain insights into the mechanism for increased apoptosis
in tumors of AITC-treated mice, the tumors of both groups
were analyzed for the expression of Bcl-2 family of proteins
AITC inhibits PC-3 xenograft growth
Fig. 2. Histological analysis of mitotic activity and apoptotic bodies in tumors
of control and AITC-treated mice. Tumors from control and AITC-treated
mice were harvested 26 days after tumor cell implantation, and processed for
scoring of mitotic activity and apoptotic bodies as described in `Materials and
methods'. Three separate tumors from control and AITC-treated mice were
analyzed. At least five separate randomly selected fields on each slide were
analyzed. Data are mean SE. *Significantly different compared with control,
P 5 0.05 by Student's t-test.
Fig. 1. Effect of AITC on growth of PC-3 cells in culture and PC-xenografts
in vivo. (A) Colony formation by PC-3 cells following continuous exposure to
different concentrations of AITC. Results are expressed as percentage of
vehicle-treated control cells, and are mean SE of three determinations.
(B) Effect of AITC administration on growth of PC-3 tumor xenografts in nude
mice. Data are mean SE, n ˆ 10 (5 mice/group; 2 tumors/mouse). Tumor
volume between control and AITC-treated group was statistically significantly
different at P 5 0.05 by two-way ANOVA. (C) Body weights of the control
and AITC-treated mice. Data are mean SE (n ˆ 5 mice/group). Body
weights of the control and AITC-treated mice did not differ significantly at
P ˆ 0.05 by two-way ANOVA.
that are known to regulate programmed cell death (22,23).
Western blots for the expression of Bcl-2, Bcl-XL , Bax and
BID in tumors of control and treated mice are shown in
Figure 3. Densitometric scanning of the immunoreactive
bands, followed by correction for differences in protein loading, indicated an ~70% reduction in Bcl-2 expression in
Fig. 3. Western blot analysis for expression of Bcl-2, Bcl-XL , BID and Bax
using tumor lysates from control and AITC-treated mice. The blots were
stripped and re-probed with antibodies against actin to correct for differences
in protein loading.
the tumors of AITC-treated mice compared with controls
(P 5 0.05 by Student's t-test). On the other hand, the expression of Bcl-XL did not differ significantly between tumors of
control and AITC-treated mice. Similar to Bcl-XL , the expression of Bax was not affected by AITC treatment. On the other
hand, AITC treatment resulted in cleavage of BID, which is
known to promote apoptosis (24). These results indicated that
increased apoptosis in tumors of AITC-treated mice may be
due to down-regulation of Bcl-2 and increased cleavage
of BID.
To gain insights into the mechanism for reduced mitotic
activity in tumors of AITC-treated mice, the expression of
cyclin B1, Cdk1, Cdc25B and Cdc25C was compared
by western blot analysis, and the results are shown in
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S.K.Srivastava et al.
Fig. 4. Western blot analysis for expression of cyclin B1, Cdk1, Cdc25B
and Cdc25C in tumors of control and AITC-treated mice. The blots
were stripped and re-probed with antibodies against actin to correct for
differences in protein loading.
Figure 4. The above proteins play critical roles in G2 /M progression (25). Expression of cyclin B1, Cdc25B and Cdc25C
proteins were significantly reduced in the tumors of AITCtreated mice compared with control tumors. Densitometric
scanning of the immunoreactive bands revealed a reduction
of ~44, 45 and 90% in expression of cyclinB1, Cdc25B and
Cdc25C, respectively, in the tumors of AITC-treated mice
when compared with control tumors (P 5 0.05 by Student's
t-test). Interestingly, the expression of Cdk1 was significantly
higher in the tumors of AITC-treated mice relative to control
tumors (Figure 4).
Discussion
Prostate cancer is the second leading cause of cancer-related
deaths among men in the US (26). The incidence of prostate
cancer has steadily increased over the years, and accounts for
~28% of all cancers in American men (27). While molecular
mechanisms underlying the onset and/or progression of human
prostate cancers are poorly defined, race, age, androgen secretion and metabolism, and diet are the identifiable risk factors
associated with this malignancy (27±30). Clinical management
of human prostate cancer has been challenging mainly due to
limited treatment options (31). Thus, identification of agents
that can delay onset and/or progression of human prostate
cancers could have a profound impact on clinical management
of this deadly malignancy. Recent epidemiological studies
have suggested that increased intake of cruciferous vegetables
may be protective against prostate cancer risk (32,33). Despite
compelling epidemiological correlation, however, no attempts
have yet been made to identify the anticancer agents in cruciferous vegetables.
We have demonstrated previously that AITC is highly effective in suppressing proliferation of PC-3 cells in culture (16).
Growth suppressive effect for sulforaphane, a natural analog of
AITC, has also been documented in LNCaP cells (34). Based
on the results of our cellular studies (16), we hypothesized that
AITC may be effective in retarding growth of PC-3 cells
1668
in vivo. The present study was undertaken to systematically
test this hypothesis. We found that the growth of PC-3 xenografts in nude mice is significantly retarded upon AITC administration. To the best of our knowledge, the present study is the
first published report on in vivo anticancer activity of a natural
ITC compound in a human prostate cancer xenograft model.
The results of the present study indicate that AITC-mediated
in vivo growth inhibition of PC-3 tumor xenografts is associated with an increase in apoptosis as well as a reduction in
cells undergoing mitosis in the tumor mass. These observations are consistent with cellular studies where treatment of
PC-3 as well as LNCaP cells with AITC resulted in apoptosis
induction and G2 /M phase cell cycle arrest (16). Mechanisms
for AITC-induced apoptosis and cell cycle arrest based on our
observations in cultured PC-3 cells (16) and PC-3 xenografts
(present study) are summarized in Figure 5. The present study
reveals that increased apoptosis in the tumors of AITC-treated
mice is associated with down regulation of Bcl-2. These results
are consistent with our observations in cultured PC-3 cells
(16). Similarly, in agreement with our observations in PC-3
cells (16), the expression of Bcl-XL or Bax in vivo is not
altered upon AITC administration (present study). It is interesting to point out that apoptosis induction by sulforaphane in
HT29 human colon cancer cell line has been shown to be
associated with over-expression of Bax but independent of a
change in Bcl-2 expression (13). While reasons for this discrepancy are not yet clear, it is possible that the mechanism for
ITC-induced apoptosis may be cell line or ITC compound
specific.
The tumors from AITC-treated mice exhibited cleavage of
p23 BID protein to a p15 fragment. BID cleavage in human
leukemic HL-60 cells exposed to 10 mM PEITC, another
analog of AITC, has been observed previously (14). The proapoptotic protein BID upon processing by caspase-8 to a p15
fragment is shown to initiate loss of mitochondrial membrane
permeability (24) (Figure 5). Thus, it is reasonable to postulate
that AITC treatment may activate caspase-8, but studies are
needed to validate this possibility.
We have shown previously that a 24 h exposure of PC-3 cells
to 10 or 20 mM AITC results in accumulation of cells in G2 /M
phase (16). The present study revealed a statistically significant decrease in cells undergoing mitosis in the tumor mass
from AITC-treated mice compared with control tumors.
Eukaryotic G2 /M progression is regulated by Cdk1±cyclin
B1 kinase complex, which is maintained in an inactive form
due to phosphorylations at Thr14 and Tyr15 of Cdk1 (25)
(Figure 5). Dephosphorylation, and hence activation of Cdk1
is mediated by dual specificity phosphatases Cdc25B and
Cdc25C (25). Present study indicates that the expression of
cyclin B1, Cdc25B and Cdc25C is reduced significantly in the
tumors of AITC-treated mice compared with that of control
mice. The expression of cyclin B1, Cdc25B and Cdc25C is
also reduced upon treatment of cultured PC-3 cells with AITC
(16). Thus, it seems reasonable to postulate that reduced mitotic activity in AITC-treated tumors may be due to accumulation of inactive Cdk1±cyclin B kinase complex.
In conclusion, the results of the present study indicate that
AITC, a constituent of cruciferous vegetables, significantly
inhibits growth of PC-3 xenografts in vivo by inducing apoptosis and decreasing proportion of cells undergoing mitosis.
The present study is the first published report on in vivo
activity of an ITC analog against human prostate cancer.
AITC inhibits PC-3 xenograft growth
Fig. 5. Proposed mechanisms for AITC-induced apoptosis and G2 /M arrest based on our observations in AITC-treated PC-3 cells (16), and in tumors from
AITC-treated mice (present study). (A) Apoptosis induction in AITC-treated PC-3 cells and in tumors of AITC-treated mice is associated with a statistically
significant decrease in expression of Bcl-2 protein but independent of a change in Bcl-XL or Bax expression (16, and present study). An increase in caspase-3-like
activity in AITC-treated PC-3 cells (16) and cleavage of BID in tumors of AITC-treated mice (present study) was also observed suggesting involvement of
caspases in AITC-induced apoptosis. (B) G2 /M arrest in AITC-treated PC-3 cells (16) and reduced mitotic activity in the tumors of AITC-treated mice (present
study) is associated with a statistically significant decrease in expression of cyclin B1, Cdc25B and Cdc25C suggesting that AITC treatment may cause
accumulation of inactive Cdk1±cyclin B1 kinase complex leading to cell cycle arrest.
Acknowledgements
This investigation was supported in part by USPHS grants CA55589 and
CA101753 (to S.V.S.), awarded by the National Cancer Institute.
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Received February 28, 2003; revised July 1, 2003; accepted July 13, 2003