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Anticancer Activity of a Broccoli
Derivative, Sulforaphane, in
Barrett Adenocarcinoma:
Potential Use in
Chemoprevention and as
Adjuvant in Chemotherapy
1
Aamer Qazi*
,2
, Jagannath Pal
†,‡,2
,Ma’in Maitah*
,2
,
Mariateresa Fulciniti
†,‡
,DheerajPelluru
†,‡
,
Puru Nanjappa
†,‡
,SaemLee
‡
, Ramesh B. Batchu*,
Madhu Prasad*, Christopher S. Bryant*,
Samiyah Rajput
†
, Sergei Gryaznov
§
, David G. Beer
¶
,
Donald W. Weaver*, Nikhil C. Munshi
†,‡
, Raj K. Goyal
‡
and Masood A. Shammas
†,‡
*Departments of Surgery/Obstetrics and Gynecology,
Wayne State University and Karmanos Cancer Institute,
Detroit, MI, USA;
†
Adult Oncology, Harvard (Dana Farber)
Cancer Institute, Boston, MA, USA;
‡
Harvard Medical School
and VA Boston Healthcare System, Boston, MA, USA;
§
Geron
Corporation, Menlo Park, CA, USA;
¶
University of Michigan,
Ann Arbor, MI, USA
Abstract
INTRODUCTION: The incidence of Barrett esophageal adenocarcinoma (BEAC) has been increasing at an alarming
rate in western countries. In this study, we have evaluated the therapeutic potential of sulforaphane (SFN), an
antioxidant derived from broccoli, in BEAC. METHODS: BEAC cells were treated with SFN, alone or in combination
with chemotherapeutic, paclitaxel, or telomerase-inhibiting agents (MST-312, GRN163L), and live cell number
determined at various time points. The effect on drug resistance /chemosensitivity was evaluated by rhodamine
efflux assay. Apoptosis was detected by annexin V labeling and Western blot analysis of poly(ADP-ribose) poly-
merase cleavage. Effects on genes implicated in cell cycle and apoptosis were determined by Western blot
analyses. To evaluate the efficacy in vivo , BEAC cells were injected subcutaneously in severe combined immuno-
deficient mice, and after the appearance of palpable tumors, mice were treated with SFN. RESULTS: SFN induced
both time- and dose-dependent decline in cell survival, cell cycle arrest, and apoptosis. The treatment with SFN
also suppress ed the expression of mult idrug resistance protein, reduced drug efflux, and increased anticancer
activity of other antipro liferative agents including paclitaxel. A significant reduction in tumor volume was also
observed by SFN in a subcutaneous tumor model of BEAC. Anticancer activity could be attributed to the induc-
tion of caspase 8 and p21 and down-regulation of hsp90, a molecular chaperon required for activity of several
proliferation-associated proteins. CONCLUSIONS: These data indicate that a natural product with antioxidant
properties from broccoli has great potential to be used in chemoprevention and treatment of BEAC.
Translational Oncology (2010) 3, 389–399
Address all correspondence to: Masood A. Shammas, PhD, Harvard (Dana Farber) Cancer Institute, Boston, MA, Harvard Medical School at VAMC, 1400 VFW Pkwy, Bldg 3,
Room 2A111, West Roxbury, MA 02132. E-mail: masood_shammas@dfci.harvard.edu
1
This work was supported by grants from R01CA125711 to MAS, from the Department of Veterans Affairs Merit Review Awards and from the National Institutes of Health grant
RO1-124929 to NCM and grants P50-100007 and PO1-78378 to NCM and Dr. K. C. Anderson and by funds from Karmanos Cancer Institute and Department of Surgery, Wayne
State University, Detroit, MI.
2
AQ, JP, and MM contributed equally to this work.
Received 22 September 2010; Revised 6 October 2010; Accepted 9 October 2010
Copyright © 2010 Neoplasia Press, Inc. All rights reserved 1944-7124/10/$25.00
DOI 10.1593/tlo.10235
www.transonc.com
Translational Oncology
Volume 3 Number 6 December 2010 pp. 389–399 389
Introduction
Va rious epidemiologic studies have indicated that consumption of
broccoli is associated with a lower risk of cancer [1], including breast [2],
prostate [3], lung, stomach [4], and colon [5] cancers. The anticancer
effect of broccoli has been attributed to sulforaphane (SFN), an isothio-
cyanate formed by hydrolysis of a precursor glucosinolate called “glu-
coraphanin” [1]. Although glucoraphanin is found in varying amounts
in all cruci ferous vegetables, the highest concentration of this co m-
pound is found in broccoli and its sprouts [6]. Among various parts
of mature broccoli, the florets have the maximum amount of SFN.
The amount of SFN in 1 g of dry broccoli florets ranges from 507
to 684 μg [7]. The sprouts of broccoli seem to have 20 to 30 times
higher concentration of glucoraphanin, an SFN precursor [8,9], indi-
cating that 1 oz of sprouts may have the amount of antioxidant present
in 20 oz of mature broccoli. Glucoraphanin is converted to SFN by
myrosinase, an enzyme released from broccoli during its consumption
and also found in our stomach [1]. The reduced risk of cancer after
consumption of broccoli is associated with the ability of SFN to inhibit
phase 1 enzymes (implicated in the conversion of procarcinogens to
carcinogens) and induce phase 2 enzymes (implicated in detoxification
and excretion of carcinogens from body) [1,6].
However, the anticancer activity of SFN is not limited to its ability
to promote detoxification and removal of carcinogens. SFN has been
shown to inhibit cell cycle progression, induce apoptotic cell death,
and inhibit angiogenesis in a variety of cancer cell types [1,6]. Expo-
sure to SFN (20 μM) has been shown to induce a chk2 kinase–
dependent arrest at the G
2
/M phase of cell cycle in prostate cancer
(PC3) cells [10]. A G
2
/M arrest after treatment with SFN has also
been demonstrated in human colon cancer (HCT116) cells [10].
Although cell cycle arrest at G
2
M has been frequently observed after
treatment of cancer cells with SFN, arrest at other phases of cell cycle
has also been reported for many cell lines. For example, the treatment
of colon cancer (HT-29) cells with this agent led to the induction of
p21, down-regulation of cyclin D1 and cyclin A, and a G
1
cell cycle
arrest [11], whereas prostate cancer (LnCap) cells arrested at G
1
/S
after treatment with 10 μM drug [12,13].
SFN has also been shown to induce apoptosis, or programmed cell
death, in cancer cells. Treatment with 15 μM SFN induced apoptosis
in both the p53-positive and p53-negative human colon cancer cell
lines [14]. Similarly, exposure to 10 μM SFN caused apoptosis in
prostate cancer DU145 cells [15]. The mechanisms of SFN-induced
apoptosis in these cell lines include activation of caspase 7 and 9 [14]
and/or release of cytochrome C from mitochondria [16].
Anticancer activity of SFN has also been demonstrated in vivo .SFN
has been shown to inhibit growth of human pancreatic cancer [17],
human prostate cancer [18], murine osteosarcoma xenografts [19], and
prevent intestinal polyposis [11], UV light–induced skin tumors [20],
carcinogen-induced skin tumors [21], and carcinogen-induced stomach
tumors [22] in vivo. SFN has also been reported to increase natural killer
cell activity and antibody-dependent cellular cytotoxicity in both the
control and tumor-bearing mice [23]. These and other modulations
of immune system by SFN have been shown to play important role
in the inhibition of metastatic spread of melanoma in mice [24].
Barrett esophageal adenocarcinoma (BEAC) develops in Barrett
esophagus, a precancerous condition associated with chronic esophageal
reflux. Because the cancer starts to spread before onset of clinical symp-
toms, BEAC patients usually have a dreary outcome with a poor sur-
vival rate [25]. Moreover, the incidence of this cancer has been
increasing at a disturbing rate in Europe and United States [26]. Be-
cause the effect of SFN on BEAC cells has not been demonstrated,
we studied the effect of this natural food ingredient, either alone or
in combination with other anticancer agents including paclitaxel, on
cell cycle and cell viability in BEAC cell lines. We have shown that
SFN induces cell cycle arrest and apoptosis in BEAC cells at concentra-
tions (3-7 μM) lower than (10-20 μM) those of SFN required to kill
other cancer cell lines. Moreover, the SFN also suppressed multidrug
resistance protein (MRP), reduced drug efflux, and significantly
enhanced the anticancer activity of telomerase inhibitors (MST-312,
GRN163L) and paclitaxel, a chemotherapeutic currently used to treat
BEAC, in BEAC cell lines tested. The treatment with SFN was also
associated with induction of caspase 8 and p21 and suppression of
hsp90, a molecular chaperone required for activity of several prolifera-
tion related proteins. A significant anticancer activity of SFN was also
observed in a subcutaneous tumor model in vivo. These data indicate a
marked anticancer activity of SFN in BEAC and provide a rationale for
clinical evaluation.
Materials and Methods
Chemicals
L-Sulforaphane (SFN) and paclitaxel were purchased from Sigma-
Aldrich (St Louis, MO) and dissolved in phosphate-buffered saline.
MST-312 [N ,N ′-bis(2,3-dihydroxybenzoyl)-1,3-phenylenediamine]
was purchased from Calbiochem/EMD Biosciences (Madison, WI) and
dissolved in DMSO. GRN163L is a palmitoyl (C16) lipid—attached
N3′-P5′ phosphoramidate oligonucleotide, targeting the template
region of RNA subunit of telomerase (hTR) and was obtained from
Geron Corporation (Menlo Park, CA). GRN140833 mismatch oligo-
nucleotide was also obtained from Geron Corporation and used as a
negative control.
Barrett Adenocarcinoma Cell Lines and Normal Cells
BEAC cell line FLO-1 and a lung adenocarcinoma cell line H460
have been described previously [27]. BEAC cell line OE33, from
European Collection Of Cell Cultures, was purchased through Sigma-
Aldrich and has been described previously [28,29]. Normal primary
human esophageal epithelial cells (HEEC) were purchased from Scien-
Cell Research Laboratories (Carlsbad, CA) and have been described
previously [30]. FLO-1 cells were cultured in Dulbecco modified Eagle
medium (Sigma) supplemented with 10% fetal bovine serum (H yClone,
South Logan, UT). OE33 cells were cultured in RPMI-1640 sup-
plemented with 2 mM
L-glutamine and 10% fetal bovine serum.
Normal HEE C (human esophageal epithe lia l cells) were cultured
in epithelial cell medium-2 (ScienCell Research Laboratories). Cells
were maintained in a state of logarithmic growth at 37°C in humidified
air with 5% CO
2
, as described previously [30]. For RNA and protein
analyses, cultures were harvested at the same final cell density (5 ×
10
5
/ml) and immediately processed.
Treatment and Growth of Cells
Cells (5 × 10
5
) were plated in 100-mm dishes and treated with
SFN alone or in combination with paclitaxel (PAC) and MST-312,
at concentrations described in the figure legends. Substrate-attached
viable cell number was counted, and cell viability was confirmed by
trypan blue exclusion or cell proliferation assays at various time points.
Cell proliferation assays were performed using Cell Counting Kit-8
(Dojindo Molecular Technologies, Inc, Gaithersburg, MD) according
to the manufacturer’s protocol. The method provides a highly sensitive
390 SFN Inhibition of Barrett Adenocarcinoma Qazi et al. Translational Oncology Vol. 3, No. 6, 2010
colorimetric assay for the determination of viable cell number and is
based on the production of a yellow product (formazan) after reduction
of a highly water-soluble tetrazolium salt by dehydrogenases in viable
cells. The amount of the formazan dye generated is measured by a plate
reader and is directly proportional to the number of viable cells.
Rhodamine Efflux Assay
FLO-1 cells, plated in a six-well plate, were pretreated with SFN
(3 or 5 μM) for 5 hours or with cyclosporin A (10 μM; a known
inhibitor of MDR1 gene product) for 1 hour . Rhodamine 123 (3 μM)
was then added into the same medium, and the cells were incubated for
an additional 1.5 hours. Rhodamine-containing medium was then re-
moved and cells were washed with PBS and incubated overnight at
37°C, 5% CO
2
with serum-free medium containing the same concen-
trations of drugs without rhodamine. Cells were harvested, washed with
PBS, and evaluated for rhodamine 123 florescence by flow cytometry.
Cell Cycle Analysis
The effect of SFN and PAC on progression of the cell cycle was
determined using the Cell Cycle Phase Determination Kit (Cayman
Chemical Company, Ann Arbor, MI). Briefly, the control and treated
cells were washed and resuspended in assay buffer at 10
6
/ml. Fixative
(1 ml) was then added, and cells were allowed to permeabilize for at
least 2 hours. The cells were centrifuged, the fixative was removed,
and cells were resuspended in 0.5 ml of staining solution containing
propidium iodide. After 30 minutes of incubation at room temper-
ature, the cells were analyzed in the FL2 channel of a flow cytometer
with a 488-nm excitation filter.
Figure 1. Effect of SFN on BEAC cell survival. BEAC cells were cultured in the medium containing no SFN or various concentrations of
SFN. Cells were harvested at different time points as indicated and proliferative potential was assessed by trypan blue exclusion and/or
proliferation assay, based on the production of a yellow product (formazan) after reduction of a highly water-soluble tetrazolium salt by
dehydrogenases in viable cells. The growth curves show the mean of three independent experiments, with SEM. (A) Barrett adenocar-
cinoma (FLO-1) cells treated with various concentrations of SFN. (B) BEAC (OE33) cells treated with various concentrations of SFN. (C)
Photomicrograph of BEAC (FLO-1 and OE33) cells treated with 3 μM SFN for 72 hours. (D) Photomicrograph of normal diploid fibroblasts
and primary normal esophageal epithelial cells (ScienCell Research Laboratories) treated with 3 μM SFN for 72 hours. (E) FLO-1 cells
were treated with SFN for 48 hours, detached floating cells from the medium and the attached cells (by trypsinization) were collected
separately and evaluated for number and viability using trypan blue exclusion. The number of cells detached after treatment with various
concentrations of SFN is expressed as percent of untreated FLO-1 cells. “Total” represents the total number of detached cells whereas
“Dead” reflects the fraction of dead cells in detached cell population. (F) Panel (I): FLO-1 cells were incubated with various concentra-
tions of SFN for 48 hours, and the expression of caspase 8 was detected by Western blot analysis, using anti–caspase 8 mouse mono-
clonal antibody (Cell Signaling, Danvers, MA). Panel (II): Bar graph showing caspase 8 expression relative to β-actin.
Translational Oncology Vol. 3, No. 6, 2010 SFN Inhibition of Barrett Adenocarcinoma Qazi et al. 391
Apoptosis Assay
Apoptotic cells were detected using the Annexin V–Biotin Apoptosis
Detection Kit (Oncogene Research Products, San Diego, CA). Un-
treated or treated BEAC cells (1 × 10
6
cells/ml) were mixed with annexin
V–biotin and medium-binding reagent and incubated in the dark for
15 minutes at room temperature. Cells were then centrifuged, and the
medi um was replaced with 1× binding buffer (Oncogene Research
Products) containing fluorescein isothiocyanate (FITC)–streptavidin
(Amersham Life Sciences, Inc, Arlington Heights, IL). A portion of
the cell suspension (50 μl) was placed onto a glass slide, covered with
a coverslip, and viewed immediately using a fluorescence microscope
equipped with FITC (green) filter. Two hundred cells, representing
at least five distinct microscopic fields, were analyzed to assess the frac-
tion of FITC-labeled cells for each sample.
Western Blot Analysis
Approximately 50 mg o f protein was suspended in Laemmli
sample buffer (0.1 M Tris-HCl buffer pH 6.8, 1% SDS, 0.05% β-
mercaptoethanol, 10% glycerol, and 0.001% bromphenol blue), boiled
for 2 minutes, and electrophoresed on 4% to 20% glycerol gradient
SDS–polyacrylamide gel for 4 hours at 120 V. Gels were electroblotted
onto Trans-Blot nitrocellulose membrane (Bio-Rad Laboratories,
Hercules, CA) at 40 V for 3 hours in a Tris-glycine buffer system. In-
cubation with indicated antibodies was performed for 2 hours in PBS–
Tween 20 (PBST) containing 1% BSA with constant rocking. Blots
were washed with PBST and incubated in either antirabbit or anti-
mouse horseradish peroxidase (Santa Cruz Biotechnology, Inc, Santa
Cruz, C A) conjugates for 2 hours in PBST containing 3% nonfat
dry milk. After washing, specific proteins were detected using an en-
hanced chemiluminescence, according to the instructions provided in
the manual (Amersham Life Sciences, Inc).
In Vivo Study
The in vivo efficacy of SFN was tested in a murine xenograft model
of BEAC in which FLO-1 cells were injected subcutaneously in severe
combined immunodeficient (SCID) mice. After detection of tumors,
mice were treated with either 0.75 mg of SFN or 10% DMSO sub-
cutaneously daily for 2 weeks. Tumor growth was measured in two
perpendicular dimensions once every 3 days using a caliper and the
following formula: V =(a
2
× b) / 2, where a is the width of the tumor
(smaller diameter) and b is the length (larger diameter).
Results
SFN Induces Time- and Dose-Dependent Decline in Survival
of BEAC Cells
FLO-1 and OE33 cells were cultured in the presence or absence of
SFN at various concentrations and for variable length of time, substrate-
Figure 1. (continued).
392 SFN Inhibition of Barrett Adenocarcinoma Qazi et al. Translational Oncology Vol. 3, No. 6, 2010
attached viable cell number was counted, and cell viability was con-
firmed by trypan blue exclusion or CCK 8 assays (Figure 1, A and B).
SFN induced both ti me- and dose-dependent decline in survival of
BEAC cells (Figure 1). Exposure to 7 μM SFN led to a complete cell
death in both BEAC cell lines tested, in a period of 3 to 5 days (Figure 1,
A and B). A substantial antiproliferative activity was also seen by SFN
at 5 μM, which killed 100% of OE33 cells in 3 days and 83% ± 4% of
FLO-1 cells in 5 days. The treatment of FLO-1 cells with 3 μMSFN
ledto74%±4%celldeathin5days.At1μM, SFN was less affective
and killed 54% of OE33 cells in 3 days and 39% of FLO-1 cells in 5 days
(Figure 1). These data indicate that SFN inhibits the proliferation of
BEAC cells. To evaluate the effect of SFN on the morphology of treated
cells, both the cancer (FLO-1 , OE33) and normal (fibroblasts, primary
esophageal epithelial) cells were treated with 3 μMSFNfor72hoursand
photographed under a phase-contrast microscope. As shown in Fig ure 1
(C and D), whereas the cancer (FLO-1 and OE33) cells started to detach
and/or reduce in size and number , both types of normal cells (fibroblasts
and esophageal epithelial cells) remained unaffected by the treatment.
To evaluate the nature of detached cells, FLO-1 cells were treated
with SFN for 48 hours. Detached floating cells from the medium
and the attached cells (by trypsinization) were collected separately
and evaluated for the number and viability using trypan blue exclu-
sion. Figure 1E shows the relative proportion of cells detached after
treatment with various concentrations of SFN, expressed as percent
of untreated FLO-1 cells. The total number of detached cells in-
creased with increasing concentrations of SFN. Although majority
(∼60%) of detached cells were dead, approxi mately 40% appeared
alive, indicating detachment before complete death (Figure 1E ).
Consistent with decline in cell viability and attachment, Western
blot analysis indicated that treatment of BEAC cells with SFN leads
to a marked induction of caspase 8, an initiator caspase implicated in
death receptor–induced apoptosis (Figure 1F ).
SFN Increases Intracellular Accumulation of Drug in
BEAC Cells
To evaluate if SFN can increase intracellular drug accumulation and
chemosensitivity by inhibiting drug efflux, we used rhodamine efflux
assay. Rhodamine 123 is a substrate of MRP and P-glycoprotein, a prod-
uct of multidrug resistance gene (MDR), implicated in the extrusion of
drugs outside the cell. We therefore exposed BEAC cells, pretreated with
SFN or cyclosporin A (broad-spectrum inhibitor of multidrug resistance
gene products), to rhodamine 123. Rhodamine-containing medium
Figure 2. SFN increases intracellular accumulation of drug and enhances antiproliferative activity of other anticancer agents in BEAC cells.
(A and B) Effect of SFN on intracellular drug accumulation. BEAC (FLO-1) cells, pretreated with SFN or cyclosporin A, were exposed to
rhodamine 123. Rhodamine was then removed, and cells were incubated overnight in the presence of corresponding drugs as described.
Cells were harvested, washed, and evaluated for rhodamine 123 florescence by flow cytometry. (A) The amount of rhodamine 123 fluo-
rescence retained was measured using a fluorescence-activated cell flow analyzer. (B) Bar graph showing dose-dependent increases in
the accumulation of intracellular rhodamine 123 in FLO-1 cells treated with different concentrations of SFN. (C) FLO-1 cells were treated
with various concentrations of SFN for 24 hours, and the expression of MRP was monitored by Western blot analysis using MRP (E-19)
antibody (Santa Cruz Biotechnology, Inc).
Translational Oncology Vol. 3, No. 6, 2010 SFN Inhibition of Barrett Adenocarcinoma Qazi et al. 393
was then removed, and cells were washed with PBS and incubated
overnight in the presence of corresponding drugs without rhodamine.
Cells were harvested, washed with PBS, and evaluated for rhodamine
123 florescence by flow cytometry. Figure 2 (A and B)showsadose-
dependent i ncrease in the accumulation of intracellular rhodamine
123 in FLO-1 cells treated with different concentrations of SFN. These
data indicate th at SFN inhibits the drug efflux, thus increasing the
intracellular concentration and chemosensitivity. Consistent with these
data, Western blot analysis showed down-regulation of MRP in SFN-
treated, relative to untreated FLO-1 cells (Figure 2C ).
SFN Significantly Enhances the Antiproliferative Effect of
Chemotherapeutic and Telomerase-Inhibiting Agents in
BEAC Cells
We next evaluated if SFN can enhance the antiproliferative affect
of other anticancer agents. For this purpose, we chose paclitaxel (a
chemotherapeutic agent used for treatment of BEAC), MST-312 (a
chemical inhibitor of telomerase), and GRN163L (a lipidated oligo-
nucleotide targeting RNA component of telomerase). For SFN-PAC
combination study, we first cultured the FLO-1 and OE33 cells in the
presence of paclitaxel at various concentrations and for variable length
of time and evaluated for cell viability as described (not shown). Con-
centrations of SFN and paclitaxel inducing similar cell death at a given
time point were used for combination study. As shown in Figure 3A,
the treatment of FLO-1 cells with 1 μM paclitaxel and 3 μM SFN for
3 days led to 57% ± 6% and 61% ± 6% cell death, respectively. How-
ever, a combination of both drugs led to 92% ± 3% cell death, indi-
cating a significant (P > .0003) 35% increase in cell death relative to
paclitaxel alone (Figure 3 A). Similarly, in OE33 cells, paclitaxel alone
induced 42% ± 5% cell death, whereas a combination of paclitaxel and
SFN led to 79% ± 4% cell death, showing a significant (P <.01)37%
increase in cell death relative to paclitaxel alone (Figure 3B). These data
show that SFN can significantly enhance the antiproliferative effect of
paclitaxel in BEAC cells.
To evaluate the effect of SFN on antiproliferative effect of telomerase
inhibiting agents, FLO-1 cells were pretreated with a telomerase inhib-
itor MST-312 (1 μM) for 10 days to initiate telomere shortening and
were then treated with SFN (3 or 5 μM) for 48 hours. As shown in
Figure 3C, the treatment of FLO-1 cells pretreated with MST-312
to SFN led to a significant (P ≤ .002) decrease in cell growth, relative
Figure 3. Effect of SFN on the antiproliferative activity of other agents. BEAC cells, FLO-1 (A) or OE33 (B), were cultured in the medium
containing no drug, 1 μM paclitaxel, 3 μM SFN, or a combination of both 1 μM paclitaxel and 3 μM SFN. Cells were harvested at indi-
cated time points, substrate-attached live cell number was determined, and cell viability was confirmed by trypan blue exclusion and/or
proliferation assays described. Bar graphs show the mean of three independent experiments, with SEM. (C and D) Effect of SFN on
antiproliferative activity of FLO-1 and H460 adenocarcinoma cells pretreated with telomerase inhibitors. FLO-1 cells (C), pretreated with
MST-312 (1 μM) for 10 days, were treated with SFN (3 or 5 μM) for 48 hours, and live cell number was determined. H460 lung adeno-
carcinoma cells (D), treated with GRN163L or mismatch oligonucleotide at 2 μM for 10 days, were exposed to SFN (2 μM) and evaluated
for live cell number everyday for the next 3 days. Error bars represent SEMs of triplicate experiments.
394 SFN Inhibition of Barrett Adenocarcinoma Qazi et al. Translational Oncology Vol. 3, No. 6, 2010
to cells treated with MST-312 or SFN alone. SFN at 3 and 5 μMin-
creased the cell death by 36% and 48%, respectively, in FLO-1 cells pre-
treated with MST-312. Similar observations were also made in H460
lung adenocarcinoma cells treated with telomerase inhibitor GRN163L,
a lipid-attached oligonucleotide-targeting RNA component of telome-
rase. H460, pretreat ed with GRN163L or mismatch oligonucleotid e at
2 μM for 10 days, was exposed to SFN (2 μM) and evaluated for live
cell number everyday for next 3 days. Addition of SFN to cells pretreated
with 163L reduced the live cell number to 16.3% ± 3.2% in 3 days,
whereas ∼40% cells were still alive when drugs were used separately
(Figure 3D). These data indicate that SFN significantly enhances the
anticancer activity of chemotherapeutic and other antiproliferative agents.
SFN Inhibits Cell Cycle Progression and Enhances the Ability
of Paclitaxel to Induce Cell Cycle Arrest
Paclitaxel is a microtubule inhibitor and is known to induce cell
cycle arrest at G
2
-M. In this study, we evaluated the effect of SFN on
cell cycle progression and on paclitaxel-induced cell cycle arrest in
BEAC cells. FLO-1 cells were treated with SFN (3 μM), paclitaxel
(1 μM), or both for 3 days, and the effect on cell cycle progression was
determined using the Cell Cycle Phase Determination Kit (Cayman
Chemical Company). Briefly, the control and treated cells were fixed,
stained with propidium iodide, and analyzed using a flow cytometer.
Although 41% of control cells were in the S-phase, the fraction of cells
in the S-phase after treatment with paclitaxel and SFN was only 18%
and 24%, respectively (Figure 4A). Consistent with cell viability data,
a more powerful inhibition of the S-phase was observed when pacli-
taxel was combined with SFN (Figure 4A, panel IV ). Cells treated
with paclitaxel were arrested at G
2
-M (55%) or G
1
(27%), whereas
those treated with SFN were mostly at G
1
. However, when SFN was
added along with paclitaxel, majority of cells were arrested at the G
1
phase of the cell cycle (Figure 4A).
Consistent with cell cycle arrest, the treatment with paclitaxel,
SFN, and the combination of both drugs led to 5-fold, 9-fold, and
Figure 4. Effect of SFN, paclitaxel, and combination treatments on cell cycle in BEAC cells. (A) Barrett adenocarcinoma (FLO-1) cells,
untreated or treated with SFN (3 μM), paclitaxel (1 μM), or both for 72 hours, were processed for evaluation of cell cycle using the Cell
Cycle Phase Determination Kit (Cayman Chemical Company). Phases of cell cycle were analyzed in the FL2 channel of a flow cytometer
with a 488-nm excitation filter. Panels: (I) untreated BEAC (FLO-1) cells, (II) BEAC (FLO-1) cells treated with SFN, (III) BEAC (FLO-1) cells
treated with paclitaxel, and (IV) BEAC (FLO-1) cells treated with both the paclitaxel and SFN. (B) Induction of p21 by SFN. Panels: (I)
Western blot showing protein levels of p21 after the above treatments and (II) bar graph showing fold induction in p21 protein levels in
those treated relative to control cells, after normalization with β-actin. (C) Suppression of hsp90 by SFN: Transcript levels of hsp90 in
FLO-1 cells after treatments described above. Panels: (I) Protein levels of hsp90 after the above treatments and (II) bar graph showing
fold reduction in hsp90 protein levels in treated relative to control cells after normalization with β-actin.
Translational Oncology Vol. 3, No. 6, 2010 SFN Inhibition of Barrett Adenocarcinoma Qazi et al. 395
57-fold induction of cell cycle inhibiting protein, p21 (Figure 4B,
panels I and II ). These data not only confirm that p21 was induced
after SFN treatment but also demonstrate a marked effect of the com-
bination treatment in BEAC cells. SFN treatment also led to t he
down-regulation of hsp90, a molecular chaperone required for activity
of several proliferation-associated proteins (Figure 4C ). The suppres-
sion of hsp90 was also the strongest when SFN was used in combina-
tion with paclitaxel.
SFN Induces Apoptotic Cell Death and Enhances the
Proapoptotic Activity of Paclitaxel
BEAC cells (FLO-1 and OE33) were treated with SFN (3 μM)
and paclitaxel (1 μ M) and analyzed for apoptotic cell death. Both
untreated or BEAC cells were sequentially treated with annexin V–
biotin and FITC-streptavidin and apoptotic cells were evaluated by a
fluorescence microscope. Approximately 200 cells, representin g at
least five distinct microscopic fields, were analyzed to assess the frac-
tion of annexin-positive cells for each sample. After a 3-day exposure,
43% ± 6% of cells treated with paclitaxel, 50% ± 7% of cells treated
with SFN, and 80% ± 8% of cells treated with both paclitaxel and
SFN were annexin V–positive (Figure 5A). These data indicate that
both paclitaxel and SFN induce apoptosis in BEAC cells, and the
addition of SFN significantly increases the fraction of cells undergoing
apoptosis. To further evaluate the effect of these treatments and their
combination on apoptosis, we analyzed poly(ADP-ribose) polymerase
(PARP), a protein which undergoes caspase 3–mediated cleavage
during apoptosis. At an earlier time point of 48 hours, the cells treated
with paclitaxel or SFN alone had only 5% to 6% of cleaved PARP
product, whereas a substantial fraction (35%) of PARP was found
to be cleaved in the cells treated with combination of both drugs
(Figure 5B).
In Vivo Efficacy of SFN
In vivo efficacy of SFN was demonstrated in a murine model in
which SCID mice were subcutaneously inoculated in the interscap-
ular area with human Barrett adenocarcinoma (BEAC; FLO-1) cells.
After detection of tumors, mice were treated with either 0.75 mg of
SFN or 10% DMSO subcutaneously daily, for 2 weeks. Tumor
growth was measured in two perpendicular dimensions, once every
3 days, using a caliper. As shown in Figure 6A, the tumor size was
Figure 5. Effect of SFN, paclitaxel, and combination treatments on apoptosis in BEAC cells. (A) BEAC (FLO-1) cells, untreated or treated
with SFN (3 μM), paclitaxel (1 μM), or both for 72 hours, were analyzed for apoptosis using the Annexin V–Biotin Apoptosis Detection Kit.
Cells were sequentially treated with annexin V–biotin and FITC-streptavidin. FITC-streptavidin–labeled apoptotic cells within the same
microscopic field were viewed and photographed by phase-contrast (PC) or by fluorescence emitted at 518 nm (FITC filter). Using the
FITC filter, apoptotic cells appear bright green. Panels: (I) Annexin labeling of FLO-1 cells, untreated or treated as described and (II)
bar graph showing percent apoptotic cells after each treatment. (B) FLO-1 cells were treated as described for panel A but for a duration of
48 hours and analyzed for cleavage of PARP, a marker for apoptosis. Panels: (I) PARP was identified by Western blot analysis using a rabbit
polyclonal antibody against PARP (Santa Cruz Biotechnology, Inc) and (II) bar graph showing percentage of cleaved 89-kDa PARP fragment.
396 SFN Inhibition of Barrett Adenocarcinoma Qazi et al. Translational Oncology Vol. 3, No. 6, 2010
significantly reduced in the mice treated with SFN, relative to control
mice. In Figure 6B, the tumor sizes in individual mice show that tumors
in three of four treated mice were smaller than any control mice. These
data show that SFN has significant antitumor activity in vivo.
Discussion
In this study, we have evaluated the therapeutic potential of SFN, an
antioxidant derived from broccoli, in BEAC. We have found that: 1)
SFN induc es both the cell cycle arrest and apoptosis in BEAC cell
lines tested and inhibits tumor growth in a subcutaneous model of
BEAC in vivo. 2) The concentration of SFN (3-7 μM) required to
induce cell cycle arrest and apoptosis in BEAC cells is lower than that
(10-20 μM) reported for similar affects in several other cancer cell types. 3)
Treatment of FLO-1 cells with SFN is associated with down-regulation
of MRP and reduction in drug efflux. 4) SFN significantly enhances the
antiproliferative activity of other antiproliferative agents (paclitaxel, a
chemotherapeutic used in the treatment of BEAC) and telomerase in-
hibitors MST-312 and GRN163L. 5) Addition of SFN with paclitaxel
leads to a marked increase in the expression of p21, cell cycle arrest, and
apoptosis in BEAC cells. 6) Anticancer activity of SFN could be attrib-
uted, at least in part, to the induction of caspase 8 and p21 and the
suppression of MRP and hsp90, a molecular chaperone required for
activity of several proliferation associated proteins.
Anticancer effects of SFN have been demonstrated in several malig-
nancies including human colon, bladder, prostate, ovarian, lympho-
blastoid, pancreatic, cervical cancer, and lung cancers [15,17,31–36].
However, to our knowledge, this is the first report demonstrating the
ability of SFN to induce apoptotic cell death, increase chemosensitivity,
and significantly enhance the antiproliferative effects of chemothera-
peutic and telomerase-inhibiting agents in BEAC cells. Moreover, the
SFN could induce apoptosis in BEAC cell lines at 3 to 7 μM, a con-
centration lower than 10 to 40 μM, reported to induce apoptotic cell
death in most other cancers including colon, ovarian, non–small lung,
cervical, breast, l ung ade nocarcinoma, hepat oma, and prostate
[10,15,18,31–39], indicating a greater sensitivity of this agent for BEAC.
SFN also significantly enhanced the antiproliferative activity of other
anticancer agents including a chemotherapeutic agent, paclitaxel, used
for the treatment of BEAC. A combination of SFN and paclitaxel led to
a significant increase in the fraction of apoptotic cells, as indicated by
annexin labeling (Figure 5). To further confirm the apoptosis and the
increased efficacy of combination treatment, we analyzed the treated
cells for PARP cleavage at an earlier time point of 48 hours. In the cells
treated with paclitaxel, SFN, or both drugs, the fraction of PARP
cleaved into the 89-kDa fragment was 6%, 5%, and 35%, respectively.
These data indicate that the combination of both the drugs leads to a
remarkable increase in apoptotic activity and is consistent with the sig-
nificant increase in cell death and annexin labeling after exposure of
BEAC cells to both the paclitaxel and SFN.
SFN also significantly enhanced the antiproliferative activity of a
chemical inhibitor of telomerase (MST-312) in BEAC cells. A similar
effect was also observed in lung adenocarcinoma H460 cells in which
SFN significantly enhanced the antiproliferative activity of GRN163L,
an oligon ucleotide targeting the RNA component of telomerase.
Consistent with these observations, we have found that SFN down-
regulates MRP and reduces drug efflux in BEAC cells. Because MRPs
have been implicated in the export of chemotherapeutic drugs includ-
ing paclitaxel [40], it is possible that SFN enhances the chemosensitivity
Figure 6. In vivo efficacy of SFN in BEAC. SCID mice were inoculated subcutaneously in the interscapular area with FLO-1 (BEAC) cells,
and after the appearance of tumors, mice were treated intraperitoneally with PBS alone or SFN 25 mg/kg (5 times per week). Average
tumor sizes in all treated and control mice (A) and tumor sizes in individual mice (B) are shown.
Translational Oncology Vol. 3, No. 6, 2010 SFN Inhibition of Barrett Adenocarcinoma Qazi et al. 397
of other antiproliferative agents, at least in part, by increasing intracel-
lular drug concentration.
Exposure of BEAC cells to SFN was associated with both the cell
cycle arrest and apoptosis, and it is consistent with the observations
made in other cancer cell types including, osteosarcoma, ovarian carci-
noma, colon, prostate, and lung cancers [1,14,19,32,41,42]. As shown
in Figure 4, the treatment with SFN led to the accumulation of FLO-1
cells at the G
1
phase of the cell cycle. Although a G
2
/M cell cycle arrest is
more commonly seen after exposure of cancer cells to SFN [15,18,43],
arrest at G
1
has been observed for human colon cancer (HT-29) cells
[44]. Consistent with our observations in BEAC cells, the G
1
arrest in
colon HT-29 cells was also associated with induction of p21.
Several mechanisms have been proposed for induction of apoptosis
after treatment of cancer cells with SFN. SFN-induced apoptosis in
colon cancer (HT-29) cells is attributed to up-regulation of Bax and
release of cytochrome C from mitochondria [16]. In both the prostate
and colon cancer cells, SFN treatment was associated with inhibition
of histone deacetylase, leading to increased histone acetylation and
apoptosis [42,45]. SFN has also been reported to induce apoptosis
through activation of AP-1 [46], activation of MAPK pathways (ERK,
JNK, p38) [44,47], and down-regulation of nuclear factor κB [48]. In
our study, the apoptosis in BEAC cells could be attributed, at least in
part, to the induction of caspase 8 and p21 and suppression of hsp90.
Exposure of BEAC cells to SFN led to a marked induction of caspase 8,
an initiator caspase implicated in death receptor–mediated apoptosis
[45]. Consistent with this, the transcript levels of caspase 8 and several
death receptors were also elevated in SFN-treated BEAC cells (not
shown). SFN, paclitaxel, and the combination of both led to 9-fold,
5-fold, and 57-fold induction of p21 in FLO-1 cells, respectively. These
data indicate that there could be a remarkable effect of adding SFN
with paclitaxel in treating BEAC.
In summary, these studies demonstrate that SFN inhibits the prolif-
eration o f BEAC cells at a nontoxic concentration, reduces tumor
growth in vivo, and significantly enhances the anticancer activity of
other chemotherapeutic and antiproliferative agents in BEAC. These
data therefore indicate that a natural product with antioxidant properties
from broccoli has a specific activity against BEAC, making it an ideal
compound for therapy and possible chemoprevention of this disease.
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