Báo cáo khoa học: Ixocarpalactone A isolated from the Mexican tomatillo shows potent antiproliferative and apoptotic activity in colon cancer cells pot

Ixocarpalactone A isolated from the Mexican tomatillo
shows potent antiproliferative and apoptotic activity
in colon cancer cells
Juliana K. Choi
1
, Genoveva Murillo
2
, Bao-Ning Su
3
, John M. Pezzuto
4
, A. D. Kinghorn
3
and
Rajendra G. Mehta
2
1 Department of Surgical Oncology, College of Medicine, University of Illinois at Chicago, IL, USA
2 Carcinogenesis and Chemoprevention Division, Life Sciences Group, IIT Research Institute, Chicago, IL, USA
3 Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Ohio State University, Columbus, OH, USA
4 Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Purdue University,
West Lafayatte, IN, USA
As the third leading cause of cancer deaths, colon
cancer continues to be a major cause of mortality in
the United States [1]. Several epidemiological studies
have indicated a correlation between diet and colon
cancer risk [2–4]. Diet is considered one of the most
important environmental factors in colon cancer
development, particularly those characterized by
decreased consumption of fruits and vegetables and
increased intake of meats and fats [5–7]. Westerniza-
tion of diets, or greater intake of meats and fats, has

been linked with an increased incidence of colon can-
cer, providing support for the influence of diet on
colon cancer development [6,8,9]. Therefore, the dis-
covery of novel chemopreventive agents of natural
origin has been targeted, with fruits and vegetables
being of key interest.
Keywords
apoptosis; colon cancer; ixocarpalactone A;
Physalis philadelphica; tomatillo
Correspondence
R. G. Mehta, Carcinogenesis and
Chemoprevention Division, IIT Research
Institute, 10 West 35th Street, Chicago,
IL 60616, USA
Fax: +1 312 567 4931
Tel: +1 312 567 4970
E-mail:
(Received 20 September 2006, accepted
27 October 2006)
doi:10.1111/j.1742-4658.2006.05560.x
Physalis philadelphica Lam, commonly known as a tomatillo, is a staple
of the Mesoamerican cuisine. In our laboratory, an ethyl acetate-soluble
extract and four withanolides [ixocarpalactone A (IxoA), ixocarpalac-
tone B, philadelphicalactone B, and withaphysacarpin] were isolated. Stud-
ies conducted on Hepa-1c1c7 hepatoma cells revealed that withanolides
were potent inducers of quinone reductase, suggesting possible cancer chemo-
protective activity. Here we evaluated the antiproliferative properties of the
withanolides in SW480 human colon cancer cells. IxoA, which is present in
the edible part of the tomatillo, was selected for further evaluation. SW480
cells treated with IxoA showed cell cycle arrest in the G2⁄ M phase, up-regu-

lation of hyper-phosphorylated retinoblastoma, and down-regulation of
E2F-1 and DP-1. On the basis of flow cytometry analysis, ethidium bro-
mide ⁄ acridine orange, and 4¢,6-diamidino-2-phenylindole staining, it was
found that IxoA induces apoptosis in SW480 cells. Moreover, increased
concentrations of the pro-apoptotic protein, BIM ⁄ BOD, were found by
western blot analysis and immunocytochemistry. Morphological examina-
tion revealed vacuole formation in cells treated with IxoA, and Oil Red O
staining showed that the vacuole content was nonlipid. Furthermore,
immunocytochemistry demonstrated increased concentrations of mucin 3 in
IxoA-treated SW480 cells. These findings suggest that chemicals present in
tomatillos (e.g. IxoA) may have cancer chemopreventive properties.
Abbreviations
DAPI, 4¢,6-diamidino-2-phenylindole; IxoA, ixocarpalactone A; IxoB, ixocarpalactone B; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide; PhilB, philadelphicalactone B; pRb, hyperphosphorylated retinoblastoma; Rb, retinoblastoma; Withpc, withaphysacarpin.
5714 FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS
The beneficial effects of fruits and vegetables have
been attributed among other things to the high content
of bioactive compounds [10]. Studies conducted in the
last two decades have shown that these bioactive
compounds have important roles in the prevention
of chronic diseases, including cancer, diabetes and
hypercholesterolemia [11]. Noteworthy examples of
plant-derived substances that have been shown to
reduce experimental colon carcinogenesis are indole-3-
carbinol from cruciferous vegetables such as brussel
sprouts and broccoli [12], curcumin from the root of
Curcuma [13], and epigallocatechin gallate from tea
[14]. Some of these agents are currently being investi-
gated in clinical trials for the prevention or treatment
of cancer [15].

The use of plant-derived agents to prevent the onset
or delay progression of the carcinogenic process has
attracted considerable interest, with much attention
aimed at understanding the mode of action by which
they function. Several cellular signaling pathways
involved in apoptosis, proliferation, cell cycle, and
angiogenesis, all processes implicated in many cancers,
have been shown to be modulated by chemopreventive
agents. Natural agents derived from dietary sources,
unlike conventional single-site agents, offer the ability
to exhibit multisite mechanisms of action. Moreover, a
role for these compounds in combinatorial therapy
with more traditional chemotherapeutics has been
suggested, with the aim of lowering the toxicity and
enhancing the efficacy of treatments of more advanced
cancers.
As part of our continuing search for novel, plant-
derived cancer chemopreventive agents [16,17], we have
evaluated a number of plants originating from differ-
ent parts of the world. Physalis philadelphica is an
example of such a plant. The fruit of P. philadelphica
(Fig. 1A), commonly known as tomatillos, husk toma-
toes, ground cherries, jamberries or fresadillas [18], are
everyday components of the Mexican and Guatemalan
diet [19]. Several medicinal properties have been attrib-
uted to P. philadelphica, e.g. antibacterial properties
against respiratory infections caused by Staphylococ-
cus aureus, Streptococcus pneumoniae, and Streptococ-
cus pyogenes [20]. Moreover, in Guatemala, the
tomatillo was believed to have health benefits against

gastrointestinal disorders [21].
Previously in our laboratory, an ethyl acetate-soluble
extract and four withanolides [ixocarpalactone A
(IxoA)], ixocarpalactone B (IxoB), philadelphicalac-
tone B (PhilB), and withaphysacarpin (Withpc)] were
isolated in pure form. All four have been shown to
be present in the leaves and stems of P. philadelphica
[22]. Furthermore, IxoA and Withpc have been found
in the fresh fruits of P. philadelphica [23]. Earlier
studies demonstrated that IxoA possessed quinone
reductase activity with an IC
50
(concentration that
produces 50% inhibition) of 7.54 lm in Hepa-1c1c7
mouse hepatoma cells. IxoA was also shown to
inhibit the transformation of the murine epidermal
JB6 cell with an IC
50
of 0.26 lm [23]. On the basis of
these results, we selected IxoA (Fig. 1B) for further
investigations.
Results
Treatment with P. philadelphica extract and
withanolide isolates inhibits growth of human
colon cancer cells
The antiproliferative effects of ethyl acetate-soluble
extract from P. philadelphica were evaluated in a
human colon cancer cell line (SW480). For these stud-
ies, cells were treated at a concentration range of 1–
20 lgÆmL

)1
for 2–7 days. As shown in Fig. 2A, extract
treatment demonstrated significant growth inhibition
in treated SW480 cells, with 85% inhibition at
5 lgÆmL
)1
and 100% at doses ‡ 10 lgÆmL
)1
. Next the
effects of the four isolates, IxoA, IxoB, PhilB and
A
B
OH
OH
O
O
OH
O
OH
O
Fig. 1. (A) Tomatillo fruit. (B) Structure of IxoA.
J. K. Choi et al. Tomatillo and colon carcinogenesis
FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS 5715
Withpc, were evaluated using the same cell line. As
illustrated in Fig. 2B, all four compounds significantly
suppressed cell proliferation in a dose-dependent man-
ner ranging from 80% to 99%, at 1 lm and 10 lm,
respectively. Similar findings were observed in the HT-
29 and SW620 human colon cancer cell lines (data not
shown).

IxoA was selected for further evaluation for the fol-
lowing reasons: it is found in the edible fruit of the
tomatillo plant, it has previously been reported to have
potent quinone reductase activity in hepatoma cells,
and because it has been shown to inhibit the transfor-
mation of murine epidermal JB6 cells with an IC
50
of
0.26 lm [23]. Therefore, cell growth studies with IxoA
were conducted in three additional human colon can-
cer cell lines (HT-29, Caco-2 and HCT116 in addition
to SW480). As shown in Fig. 2C, dose-dependent inhi-
bition was evident in all four cell lines studied after
5 days of treatment. IxoA showed equal or greatest
inhibition in SW480 cells, with percentage inhibitions
ranging from 19.0 to 100% at concentrations of 0.1–
10.0 lm, respectively.
Subsequently, the time-dependent effects of IxoA
were evaluated in the SW480 cells. For these experi-
ments, cells were treated for 1–7 days with doses ran-
ging from 250 nm to 10.0 lm. As shown in Fig. 2D,
by day 2 after treatment, > 60%, > 83%, and 100%
growth inhibition was noted for cells treated with
IxoA at 1.0 lm, 2.5 lm, and 7.5 lm, respectively. The
growth inhibition remained evident until day 7 after
treatment.
SW480 cells treated with IxoA for 1 day also
showed a large percentage of growth inhibition; how-
ever, consistent with a time-dependent pattern, the per-
centage inhibition was not as great as observed at

longer time points. After 1 day of IxoA treatment,
58.1%, 67.6%, 87.1% and 90.3% inhibition was
observed at 2.5, 5.0, 7.5 and 10.0 lm IxoA, respect-
ively. The IC
50
for 1 day of IxoA treatment was
1.66 lm. To ensure a minimum of 50% inhibition of
SW480 proliferation at shorter time points (1 day), a
concentration of 5.0 lm IxoA was used for subsequent
studies.
IxoA treatment induces G2/M cell cycle arrest
in SW480 cells
To determine whether the antiproliferative actions of
IxoA were mediated by an arrest in the cell cycle,
SW480 cells were treated with 5 lm IxoA for 12–24 h
and analyzed by flow cytometric analysis. Cell cycle
analysis demonstrated that 5 lm IxoA treatment resul-
ted in an accumulation of cells in the G2 ⁄ M phase of
the cell cycle, as shown in Fig. 3A,B. At 12 h, a 20.0%
increase in SW480 cells arrested in G2 ⁄ M was
observed, and at 24 h a 20.2% increase (Fig. 3C).
Flow cytometric analysis was repeated in HT-29 cells,
and similar results were obtained (data not shown).
AB
CD
Fig. 2. Percentage growth inhibition of
human colon cancer cells treated with
P. philadelphica extract and ⁄ or withanolide
isolates. (A) Effect of ethyl acetate-soluble
extract from P. philadelphica on SW480

human colon cancer cells. Cells were
seeded in 96-well plates as described in
Experimental procedures and treated with
the indicated concentrations of treatment or
vehicle (Me
2
SO). Cell proliferation was
determined by MTT assay at 2, 3, 4, 5 and
7 day time points by measuring the absorb-
ance of formazan at 570 nm. The data are
mean ± SD from triplicate wells. (B) The
effects of IxoA, IxoB, PhilB and Withpc on
SW480 cells were measured at 5 days.
(C) The effect of IxoA on HT29, Caco-2,
HCT116 and SW480 human colon cancer
cell lines was measured at 5 days. (D)
Effect of IxoA on SW480 cells at 1, 2, 4, 5,
6 and 7 days. The experimental procedures
for (B), (C) and (D) were the same as in (A).
Tomatillo and colon carcinogenesis J. K. Choi et al.
5716 FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS
Hyperphosphorylated retinoblastoma (pRb) is
up-regulated, whereas E2F-1 and DP-1 are down-
regulated in SW480 cells treated with IxoA
Given that IxoA induced G2 ⁄ M cell cycle arrest, west-
ern blot analysis was used to examine the effects of
this compound in G2-related proteins. These studies
revealed an increased expression of pRb with a simul-
taneous decrease in the expression of retinoblastoma
(Rb) in SW480 cells exposed to 5 lm IxoA for 24–

72 h. Densitometric analysis revealed 28.5–51.9%
increase in pRb and 4.5–41.4% reduction in Rb com-
pared with the control band (b-actin). Western blot
analysis demonstrated that E2F-1 expres-
sion was down-regulated by 7.3–54.3% when com-
pared with b-actin. DP-1 expression varied from
29.3% up-regulated at 24 h to 51.8% down-regulated
at 48 h (compared with b-actin). No significant chan-
ges in cyclin A and cdk1 concentrations were observed
(Fig. 4).
IxoA induces apoptosis in SW480 cells
The effects of IxoA on apoptosis were measured by
four independent assays. Initially, acridine orange ⁄ ethi-
dium bromide staining was used to evaluate apoptosis
in SW480 cells treated with IxoA. SW480 cells were
treated with 5 lm IxoA for 24 h, stained with acridine
orange ⁄ ethidium bromide and examined by fluorescent
microscopy. Morphological changes characteristic of
apoptosis, including fragmented nuclei, blebbing, and
irregular cytoplasmic membranes, were evident in the
nuclei of IxoA-treated cells. Treatment with IxoA for
24 h revealed 54% of the SW480 cells were orange
in color (late apoptosis), 36% were observed to be
AC
B
Fig. 3. Effect of IxoA on cell cycle distribu-
tion in SW480 cells. Cells were prepared for
flow cytometry analysis as described in
Experimental procedures. (A) SW480 cells
were treated with vehicle (Me

2
SO) as con-
trol for 12 h. (B) or with IxoA 5 l
M (C)
Percentages of cells in each cell cycle phase
at 12, 18 and 24 h. An increase in the num-
ber of cells arrested in the G2 ⁄ M phase of
the cell cycle is observed at each time
point.
Fig. 4. Western blot analyses of G2-related proteins. SW480 cells
were treated with 5 l
M IxoA for 24-72 h. As described in Experi-
mental procedures, cell lysate was collected, and western blot ana-
lysis was conducted to determine the protein expression of pRb
and Rb, E2F-1, DP-1, cdk1 and cyclin A. Cell lysate was also collec-
ted from untreated (Untxd) SW480 cells at each time point, and
protein expression was compared between untreated and treated
SW480 cells. All bands were compared with b-actin bands using
densitometric analysis. The percentage change for each protein
compared with b -actin bands is indicated as up-regulation (+) or
down-regulation (–).
J. K. Choi et al. Tomatillo and colon carcinogenesis
FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS 5717
blebbing (early apoptosis), and 9% were a green color
(live cells) (Fig. 5B–D) The control cells, treated with
vehicle (Me
2
SO) were 19% orange, 7% blebbing, and
73% green in color (Fig. 5A).
To better evaluate nuclear fragmentation, a feature

of apoptotic cells, the fluorescent DNA-binding dye,
4¢,6-diamidino-2-phenylindole (DAPI) was used. As
shown in Fig. 6, cells treated with 5 lm IxoA for 24 h
displayed the typical morphological features, con-
densed and fragment nuclei, of apoptotic cells.
Members of the BH3 domain-only pro-apoptotic
proteins, including BIM ⁄ BOD, have been shown to
have a critical role in initiating the apoptotic program
by antagonizing the function of the antiapoptotic
BCL-2 and activating BAX and BAK [24]. Therefore,
the expression of BIM ⁄ BOD was evaluated by western
blot analysis. As shown in Fig. 7A, exposure of
SW480 cells to IxoA increased expression of BIM ⁄
BOD. SW480 cells treated with 5 lm IxoA for 24, 48
and 72 h revealed a 20%, 35%, and 64% up-regula-
tion, respectively, in BIM ⁄ BOD compared with the
control band (b-actin) upon evaluation by densito-
metry. After these studies, the effects of IxoA on
BIM ⁄ BOD were examined by immunocytochemistry
(Fig. 7B,C). Treatment with 5 lm IxoA for 24 h
increased BIM ⁄ BOD protein staining. This result
complements those obtained by western blot analysis.
Vacuole content detection
Treatment of SW480 cells with 5 lm IxoA for 24 h
induced the formation of multiple vacuoles within
each cell. To determine the content of these vacuoles,
Oil Red O, a red stain specific for lipids, was used to
stain the SW480 cells. Figure 8A,B show that the
vacuole content was not positive for the presence of
A

C
E
B
D
Fig. 5. Cell apoptosis and morphological
changes in the nuclei of SW480 cells trea-
ted with or without IxoA were identified by
fluorescent staining with acridine
orange ⁄ ethidium bromide. Non-viable cells
had orange-stained nuclei, and viable cells
had green-stained nuclei under fluorescent
microscopy. (A) Control SW480 cells were
treated with Me
2
SO. The green color indi-
cates viability. (B–D) SW480 cells were trea-
ted with 5 l
M IxoA for 24 h. Blebbing of the
membrane, chromatin aggregation, and nuc-
lear condensation (B and C) were criteria
used to identify apoptotic cells. (D) The
orange color indicates non-viable cells. Ori-
ginal magnification, 40·. (E) Percentage
distribution is presented for control and
treatment.
AB
Fig. 6. Morphological evidence of apoptosis
in SW480 cells stained with DAPI. (A) Con-
trol cells treated with Me
2

SO had intact
nuclei. (B) After 24 h, the nuclei of SW480
cells treated with 5 l
M IxoA showed nuclear
fragmentation and chromatin condensation
characteristic of apoptosis. Magnification,
40·.
Tomatillo and colon carcinogenesis J. K. Choi et al.
5718 FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS
lipid, as no red color was detected in the IxoA-treated
cells. Increased concentrations of mucin 3, however,
were observed by immunocytochemical analysis
(Fig. 8C,D). Increased mucin 3 protein staining was
observed in SW480 cells treated for 24 h with 5 lm
IxoA, suggesting that the vacuole content includes
mucin.
Discussion
This study was part of a large-scale investigation of
the efficacy of natural products as chemopreventive
agents, particularly those found in the diet [25]. Thus
far, over 200 active compounds have been identified as
chemopreventive agents, including resveratrol [26,27],
brassinin [28,29], and deguelin [30,31]. Resveratrol is
present in grapes, red wine and peanuts, brassinin is
from Chinese cabbage, and deguelin is from an Afri-
can plant, Mundule sericea. The success of these nat-
ural products as anticancer agents led us to evaluate
an additional plant, P. philadelphica or more com-
monly known as the tomatillo, to determine its efficacy
as a chemopreventive agent. Because of the efficacy of

fruits and vegetables against colon cancer [2–4,8,9], we
elected to study the effects of tomatillos against colon
cancer in vitro.
A
BC
Fig. 7. BIM ⁄ BOD, a BH3-region only pro-apoptotic protein, was investigated to further characterize the apoptosis observed in IxoA-treated
SW480 cells. (A) SW480 cells were treated with 5 l
M IxoA for 24–72 h. Cell lysate was collected (also from untreated SW480 cells at each
time point), and western blot analysis was conducted to determine the protein expression of BIM ⁄ BOD. Protein expression was compared
between untreated and treated SW480 cells, and BIM ⁄ BOD expression was shown to increase at each time point. All bands were com-
pared with b-actin bands using densitometric analysis. (B) Immunocytochemistry was also performed on SW480 cells to confirm BIM ⁄ BOD
expression. Control cells were treated with Me
2
SO for 24 h. (C) SW480 cells treated with 5 lM IxoA for 24 h confirmed up-regulation of
BIM ⁄ BOD. Magnification, 40·.
AB
C
D
Fig. 8. Identification of vacuole content in
treated SW480 cells. The formation of mul-
tiple vacuoles within the cells was observed
after treatment of SW480 cells with 5 l
M
IxoA for 24 h. SW480 cells were plated,
treated and fixed as described in Experimen-
tal procedures. Oil Red O staining counter-
stained with hematoxylin was then
performed to distinguish the vacuole con-
tent as lipid or non-lipid. (A) SW480 cells
treated with Me

2
SO for 24 h served as
controls. (B) SW480 cells treated with 5 l
M
IxoA for 24 h did not stain red, indicating a
non-lipid vacuole content. Also, immunocyto-
chemistry was performed on SW480 cells
to investigate mucin 3 expression. (C)
Me
2
SO-treated SW480 cells served as con-
trols. (D) SW480 cells treated with 5 l
M
IxoA for 24 h showed increased mucin 3
protein staining. Magnification, 40·.
J. K. Choi et al. Tomatillo and colon carcinogenesis
FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS 5719
Our rationale for selecting tomatillos for evaluation
has been described previously [16,17,23]. Briefly, plants
are selected on the basis of information about its
expected antiproliferative activity, nontoxic nature,
and from information received from the population
that uses the plant for medicinal purposes. Then, selec-
ted plant parts are extracted with ethyl acetate and
evaluated for activity using select in vitro bioassays,
including induction of quinone reductase with Hepa
1c1c7 cells and inhibition of transformation with JB6
cells [23,32]. After in vitro bioassays, the mouse mam-
mary gland organ culture (MMOC) model is used to
select active agents for further evaluation [33]. Chosen

extracts are then fractionated using an HPLC solvent
system, and all fractions are evaluated in in vitro bioas-
says specific to the extract. Pure compounds are then
isolated from active fractions and the structure of the
compound is determined. The activity of the chemo-
preventive agent is then confirmed in experimental
carcinogenesis models as shown in this study.
Using the above screening process, we have identi-
fied and evaluated a novel chemopreventive agent.
Upon fractionation of the ethyl acetate-soluble toma-
tillo extract, we discovered four compounds with
chemopreventive potential. Although all four com-
pounds showed significant antiproliferative activity in
colon cancer cells, we elected to focus on IxoA because
of its abundance in the leaves and stems of the plant
and its presence in the fruits.
To examine the mechanism that might account for
the effects of IxoA in colon cancer cells, we investi-
gated its effects on cell cycle distribution. A noticeable
accumulation of colon cancer cells in the G2 ⁄ M phase
of the cell cycle occurred, with a concomitant decrease
of cells in the G0 ⁄ G1 phase. This suggests that IxoA
has a pronounced effect on colon cancer proliferation
which is due to cell cycle arrest. In support of this
observation, SW480 cells cultured with IxoA (5 lm)
for 24–72 h showed an increased level of expression of
pRb and a decreased level of expression of E2F-1 and
DP-1 (at 48 h). E2F-1 and DP-1 are known to exist in
a complex and act synergistically in E2F site-depend-
ent transcriptional activation [34], and pRb can inhibit

transcriptional activation of E2F [35]. Therefore,
IxoA-induced pRb inhibition of E2F-1 and DP-1 may
account for the accumulation of cells at the G2 ⁄ M
phase of the cell cycle. We show here that sustained
G2 ⁄ M arrest induced by IxoA may be E2F-1-depend-
ent and involves an increase in expression of the mito-
tic regulator, pRb.
In addition, IxoA was shown for the first time to
induce apoptosis in SW480 human colon cancer cells.
To investigate the effects of IxoA on apoptosis, we
used acridine orange ⁄ ethidium bromide staining and
DAPI staining and found a marked increase in the per-
centage of apoptotic cells in SW480 cells exposed to
IxoA for 24 h. Additional apoptotic studies focused on
BIM ⁄ BOD, a BH3 region-only pro-apoptotic Bcl-2
family member [36–38]. Pro-apoptotic proteins are divi-
ded into two subgroups, those that possess BH1, BH2
and BH3 regions and those that only possess the BH3
region [36,39]. Pro-apoptotic proteins induce the release
of cytochrome c from the mitochondria, and their abil-
ity to achieve this depends on a hydrophobic pocket
and an amphipathic a-helix. The BH1, BH2 and BH3
regions form a hydrophobic pocket, which binds to a
BH3 region of another protein, and the BH3 region
consists of an amphipathic a-helix. Furthermore, some
Bcl-2 family members have exposed BH3 regions,
whereas other members have buried BH3 regions that
require cleavage to expose the BH3 region and activate
cytochrome c release. BH3 region-only proteins with
exposed BH3 regions, such as BIM ⁄ BOD, appear to

represent a death ligand, which can neutralize certain
pro-survival members of the Bcl-2 family [38,39]. On
the basis of immunoblotting and immunocytochemistry
results obtained to date, the mechanism of IxoA-
induced apoptosis appears to involve the interaction of
IxoA with BIM ⁄ BOD death receptors.
Treatment of SW480 cells with IxoA also caused the
formation of numerous vacuoles. To identify the con-
tent of these vacuoles, we began by staining for lipids
using Oil Red O. We were interested in lipid build-up
because several studies have indicated that fatty acids
such as linoleic acid may hold anticancer properties
[40,41], and perhaps a build-up of fatty acids was
triggering apoptosis or cell cycle arrest. However,
Oil Red O staining revealed that the vacuole content
was not lipid. Next, the vacuole content was tested
for mucin formation. A common feature of colonic
neoplasia is altered concentrations of mucin. Compared
with normal colon, colon cancers have been reported
to express decreased concentrations of mucin 3 [42,43].
Secreted isoforms of mucin 3 have been reported to
protect the colonic epithelial surface [44], and immuno-
cytochemical analysis performed on SW480 cells
treated with IxoA demonstrated increased mucin 3
concentrations compared with untreated cells. This
suggests that the vacuole content may include mucin 3,
which may play a protective role.
In summary, the data reveal that IxoA shows potent
antiproliferative and apoptosis activity in human colon
cancer cells. The evidence presented here suggests for

the first time that IxoA present in tomatillos may have
chemopreventive or therapeutic value in the manage-
ment of colon cancer.
Tomatillo and colon carcinogenesis J. K. Choi et al.
5720 FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS
Experimental procedures
Physalis philadelphica extract and withanolide
isolates
An ethyl acetate-soluble extract of P. philadelphica as well
as four withanolides, IxoA, IxoB, PhilB, and Withpc, were
isolated as previously described [23,32].
Antibodies
The mouse monoclonal antibodies used for these studies
included Rb (clone 1F8), E2F-1 Ab-7 (clone KH129),
cdk1 ⁄ p34cdc2 Ab-3 (clone A17.1.1 + POH-1), cyclin A
(clone CYA06), and mucin 3 Ab-1 (clone M3.1) as well as
rabbit polyclonal antibody against Bcl-2-related ovarian
death gene (BIM ⁄ BOD; clone 1F8), which were purchased
from NeoMarkers (Fremont, CA, USA). Rabbit polyclonal
DP-1 (clone K-20) sc-610 antibody and goat polyclonal
b-actin (clone I-19) antibody were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA, USA).
Cell lines and culture conditions
SW480, SW620, HT-29, Caco-2 and HCT116 human colon
cancer cells were obtained from the American Tissue Cul-
ture Collection (Manassas, VA, USA). The cells were cul-
tured in RPMI 1640 medium supplemented with 10% fetal
bovine serum, 2 mml-glutamine and 1% antibiotic ⁄
antimycotic solution (10 UÆlL
)1

penicillin, 10 lgÆlL
)1
streptomycin and 25 lgÆmL
)1
amphotericin B) at 37 °Cina
5% CO
2
humidified atmosphere.
Growth inhibition assay
The antiproliferative effects of IxoA, IxoB, PhilB, Withpc,
and ethyl acetate-soluble extract were evaluated in human
colon cancer cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide (MTT) assay (TACS MTT cell
proliferation assay kit; Trevigen, Gaithersburg, MD, USA).
Cells were seeded in 96-well plates at a density of 5 · 10
2
per
well. Cell viability was analyzed at various time points
between 1 and 7 days after treatment with extract or vehicle
(Me
2
SO). After treatment with extract, cells were incubated
with MTT tetrazolium reagent for 2 h at 37 °C, and the
absorbance of formazan was then measured at 570 nm. Each
treatment was performed in triplicate, and the percentage cell
growth inhibition was calculated by comparison of the
absorbance readings of the control and treated cells.
Fluorescence-activated cell sorter analyses
SW480 cells were treated with or without IxoA for 12, 18
and 24 h, harvested with trypsin, and washed with

NaCl ⁄ P
i
. After the final wash, the cells were resuspended in
1.0 mL NaCl ⁄ P
i
and 9.0 mL ice-cold 70% ethanol. The
samples were stored at )20 °C until staining. In preparation
for staining, cells were washed three times with NaCl ⁄ P
i
and resuspended in 0.3 mL citrate buffer [250 mm sucrose,
40 mm trisodium citrate, 0.05% (v ⁄ v) Me
2
SO, pH 7.6]. The
samples were then stained with propidium iodide using a
previously described method [45].
Apoptosis studies
Acridine orange ⁄ ethidium bromide staining
SW480 cells with or without IxoA treatment were centri-
fuged and suspended in NaCl ⁄ P
i
, followed by the addition
of the acridine ⁄ ethidium mixture. Fluorescent microscopy
was used to distinguish nonviable cells with orange-stained
nuclei from viable cells with green-stained nuclei, which do
not absorb ethidium bromide. The percentage of apoptotic
cells and those with highly condensed or fragmented nuclei
was determined quantitatively.
DNA-binding dye, DAPI staining
SW480 cells with or without IxoA treatment were also eval-
uated by DAPI staining. For this, cells were grown on glass

microscope slides, fixed in formalin and methanol, and
stained with DAPI. Stained nuclei were visualized using a
fluorescent microscope. Blebbing of the membrane, chroma-
tin aggregation, and nuclear condensation were used as cri-
teria to identify cells undergoing apoptosis.
Western blot analysis
Treated and control SW480 cells were lysed in freshly pre-
pared extraction buffer (20 mm Hepes, pH 7.9, 400 mm
NaCl, 0.1% Nonidet P-40, 10% glycerol, 1 mm sodium
vanadate, 1 mm NaF, 1 mm dithiothreitol, 1 mm phenyl-
methanesulfonyl fluoride, 10 lgÆmL
)1
aprotinin, 10 lgÆmL
)1
leupeptin) for 45 min on ice. Lysate was centrifuged at
15 000 g for 10 min using the Eppendorf 5417R centrifuge,
supernatant collected, and protein concentration was deter-
mined using a modified Lowry method (Bio-Rad, Hercules,
CA, USA). Samples were separated using 7.5–12.0% poly-
acrylamide gels and ⁄ or ready-made gradient gels from
Bio-Rad, and transferred to nitrocellulose membranes. The
membranes were blocked in 5% milk followed by incuba-
tion with appropriate primary antibodies for 2 h at room
temperature. The membranes were then washed and incuba-
ted for 45 min at room temperature with the corresponding
secondary antibodies. The chemiluminescence reaction was
performed using the ECL system and protocol from Amer-
sham Pharmacia Biotech (Piscataway, NJ, USA). Using
Un-Scan-It Image Digitizing Software (Silk Scientific; Orem,
UT, USA), the bands of interest were compared with those

of b-actin, and the relative intensity ratios were calculated.
J. K. Choi et al. Tomatillo and colon carcinogenesis
FEBS Journal 273 (2006) 5714–5723 ª 2006 The Authors Journal compilation ª 2006 FEBS 5721
Immunocytochemistry
SW480 cells were plated on coverslips and allowed to
adhere for 24 h before treatment with IxoA or vehicle for
appropriate time points. The cells were washed with
NaCl ⁄ P
i
, and then fixed with 10% buffered formalin and
cold methanol. Staining was then conducted using a BIM ⁄
BOD rabbit polyclonal antibody or a mucin 3 mouse
monoclonal antibody. The immunoperoxidase reaction was
performed using the Dako LSAB2 System kit (Dako Cor-
poration, Carpinteria, CA, USA). Briefly, the biotinylated
link IgG was applied for 10 min, followed by incubation of
horseradish peroxidase-linked streptavidin. After the sec-
tions had been washed with NaCl ⁄ P
i
, 3-amino-9-ethyl-
carbazole (AEC) substrate ⁄ chromogen solution was
applied. The cells were then counterstained with hematoxy-
lin and examined for antibody localization.
Oil Red O staining
Levels of lipid accumulation, a classic differentiation mar-
ker, were measured in IxoA-treated SW480 cells by histo-
chemical analysis using Oil Red O staining. Briefly, SW480
cells were plated, treated, and fixed as previously described
for immunocytochemistry studies. The samples were then
placed in propylene glycol for 2 min, followed by 1 h incu-

bation in Oil Red O at room temperature. Counterstaining
was performed with hematoxylin.
Acknowledgements
The studies were supported in part by Public Health
Grants P01 CA48112 and CA103861 from the National
Cancer Institute, National Institutes of Health, and
Department of Health and Human Services.
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