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Bromelain-Induced Apoptosis in GI-101A Breast Cancer Cells
Sivanesan Dhandayuthapani,
1
Honey Diaz Perez,
2
Alexandra Paroulek,
2
Panneerselvam Chinnakkannu,
1
Umadevi Kandalam,
1
Mark Jaffe,
2
and Appu Rathinavelu
1,3
1
Rumbaugh Goodwin Institute for Cancer Research, Health Professions Division;
2
Division of Math, Science,
and Technology, Farquhar College of Arts and Sciences; and
3
College of Pharmacy; Nova Southeastern University,
Fort Lauderdale, Florida, USA.
ABSTRACT Bromelain is a proteolytic enzyme extracted from the stems and the immature fruits of pineapple that was
found to be antitumorigenic in different in vitro models. Bromelain has been reported to promote apoptosis, particularly in
breast cancer cells, with the up-regulation of c-Jun N-terminal kinase and p38 kinase. Our study was designed to determine if
bromelain could induce apoptosis in GI-101A breast cancer cells. GI-101A cells were treated with increasing concentrations
of bromelain for 24 hours. The effect of bromelain for inducing cell death via activation of the apoptosis mechanism in GI-
101A cells was further determined by using caspase-9 and caspase-3 assays along with the M30-Apoptosense assay to
measure cytokeratin 18 (CK18) levels in the cytoplasm of the cultured cancer cells. A dose-dependent increase in the activities
of caspase-9 and caspase-3 coinciding with elevation of CK18 levels was found in bromelain-treated cells compared with
control cells. Furthermore, the apoptosis induction by bromelain was confirmed by DNA fragmentation analysis and 4,60-
diamino-2-phenylindole dihydrochloride fluorescence staining of the nucleus. Our results indicate an increase in apoptosis-
related cell death in breast cancer cells with increasing concentrations of bromelain.
KEY WORDS: apoptosis breast cancer bromelain caspase-3caspase-9 4,60-diamino-2-phenylindole staining
DNA fragmentation
INTRODUCTION
For several centuries plants containing a high con-
tent of proteolytic enzymes have been used in the tra-
ditional practice of medicine among the native people living
in Central and South America. Even now plants containing
these proteolytic enzymes are being studied for their thera-
peutic use in a variety of ailments.
1–3
One such proteolytic
enzyme that has been used for many years to reduce fever
and relieve indigestion is bromelain.
4
This enzyme is ex-
tracted from the stems and immature fruits of pineapple
(Ananas comosus) and has been shown to possess anti-
inflammatory, anti-edematous, and antithrombotic proper-
ties.
4–6
Several recent research reports have also pointed out
bromelain’s possible antimetastatic and antitumorigenic
activities.
6
So far, there is a sufficient amount of literature
evidence to show that under in vitro conditions, bromelain
can inhibit the growth of a variety of cancer cells, including
MCF-7 and MDA-MB-231 breast cancer cells, KB squa-
mous carcinoma cells, and SK-MEL-28 melanoma cells. In
addition, bromelain was shown to induce differentiation of
leukemic cells that eventually resulted in the cells under-
going apoptosis.
6–8
The apoptotic pathway is controlled by a
series of tightly regulated biochemical processes in which a
cell, once triggered, goes through consecutive phases of cell
shrinkage, chromatin condensation, DNA fragmentation,
nuclear disintegration, cell blebbing, and finally the forma-
tion of ‘‘apoptotic bodies.’’ As the physiological counterpart
of cell growth, apoptosis plays an important role in the
balance of tissue dynamics. Disturbances in this balance
result in diseases such as cancer.
The GI-101A breast cancer cell line used in this study was
derived from a poorly differentiated mammary carcinoma
that was also metastatic to the lungs and lymph nodes.
9,10
These cells are positive for estrogen receptors but are resis-
tant to anti-estrogen drugs such as tamoxifen.
10
The aim of
this study was to explore the effect of bromelain at different
concentrations on the estrogen receptor–positive GI-101A
breast cancer cell line. The extent of apoptosis was assessed
by measuring the activities of caspase-9 and caspase-3, the
level of caspase-cleaved cytokeratin 18 (CK18)-Asp396
neo-epitope using the M30-Apoptosense enzyme-linked
immunosorbent assay (ELISA) kit, and DNA fragmentation.
MATERIALS AND METHODS
Cell line and reagents. The GI-101A human breast
carcinoma cells were derived from a xenograft of a patient
with recurrent ductal adenocarcinoma at the Rumbaugh
Manuscript received 25 May 2011. Revision accepted 6 September 2011.
Address correspondence to: Dr. Appu Rathinavelu, Rumbaugh Goodwin Institut e for
Cancer Research, 1850 NW, 69th Avenue, Site #5, Plantation, FL 33313, USA, E-mail:
appu@nova.edu
JOURNAL OF MEDICINAL FOOD
J Med Food 15 (4) 2012, 344–349
#Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2011.0145
344
Goodwin Institute for Cancer Research, Plantation, FL,
USA. The RPMI-1640 growth medium, amphotericin B, l-
glutamine, and antibiotic–antimycotic solution containing
penicillin and streptomycin were obtained from Atlanta
Biologicals (Lawrenceville, GA, USA). Fetal bovine serum
was purchased from Hyclone (Logan, UT, USA), and bro-
melain was purchased from Sigma Chemical Co. (St. Louis,
MO, USA). The M30-Apoptosense ELISA kit was pur-
chased from Peviva AB (Bromma, Sweden). The substrates
and inhibitors of caspase-9 and caspase-3 were purchased
from Enzo Life Sciences AG (Lausen, Switzerland). All
other chemicals used in our research were of research grade
purchased through standard suppliers.
Cell culture and bromelain treatment
The human breast carcinoma cell line GI-101A was
grown as a complete monolayer in RPMI-1640 growth
medium supplemented with 10% fetal bovine serum,
10,000 U/mL penicillin, 10,000 lg/mL streptomycin, 1%
(+)-l-glutamine, and 1% amphotericin B. The cells were
grown at 37C under a humidified air/CO
2
(19:1 vol/vol)
atmosphere. All experiments were conducted using cells in
logarithmic phase. In each well of a six-well plate, ap-
proximately 1 ·10
6
cells per well were grown for 24 hours.
The cells in the experimental groups were treated with 5, 10,
20, 40, or 50 lg/mL concentrations of bromelain, and the
control group was untreated with bromelain. After 24 hours
of drug treatment, the cells were trypsinized for harvesting
and washed with phosphate-buffered saline (PBS). After
centrifugation the number of live cells per well was counted
using the trypan blue dye exclusion method to calculate the
percentage of viable cells.
Assay of caspase-9 and caspase-3 activities
Breast cancer cells were treated with 5, 10, and 20 lg/mL
bromelain for 24 hours, and control cells were incubated
without any bromelain. After the incubation interval, cells
were harvested, washed, resuspended in cell lysis buffer,
and kept on ice for 10 minutes. The cell lysate was centri-
fuged for 1,400 gfor 5 minutes, and after centrifugation the
supernatant was assayed for protein concentration and then
diluted with the lysis buffer to adjust the protein concen-
tration. Equal amounts of protein from each sample were
added to 96-well plates and mixed with the 2 ·reaction
buffer (100 mMHEPES [pH 7.4], 200 mMNaCl, 20 mM
dithiothreitol, 2 mMEDTA, and 0.2% CHAPS) and the re-
spective substrates acetyl-Leu-Glu-His-Asp-p-nitroaniline
and acetyl-Asp-Glu-Val-Asp-p-nitroaniline at 2 mM. For
measuring the specific activity of caspase-9 and caspase-3,
the respective inhibitors acetyl-Leu-Glu-His-Asp-CHO and
acetyl-Asp-Glu-Val-Asp-CHO were used. Release of the
cleaved p-nitroanilide from the tetrapeptide substrates was
measured using the 96-well microplate reader at 405 nm.
M30-Apoptosense ELISA immunoassay
During the process of apoptosis, cleavage of CK18 by
caspase 3 occurs, resulting in fragments containing the
CK18-Asp396 neo-epitope. This neo-epitope is recognized
by the M30 antibody provided with the Apoptosense ELISA
kit, allowing for the quantification of apoptotic cells.
11
For
this assay approximately 1 ·10
4
cells were grown in a 96-
well plate, using complete RPMI growth medium. Once the
cells attached to the plate, the cells were treated for 24 hours
with increasing concentrations of bromelain at 5, 10, or
20 lg/mL, and the control was incubated similarly without
bromelain. At the end of the incubation period the cells were
lysed, and aliquots of the lysates were transferred to the
CK18 monoclonal antibody (M30)–coated microplate wells
and assayed per the manufacturer’s instructions. The M30
conjugate dilution buffer was added to each well, and the
plate was placed on a shaker for 4 hours at room tempera-
ture. The wells were washed and then incubated with tetra-
methylbenzidine substrate for 20 minutes in the dark at
room temperature. The reaction was stopped by adding 1.0
Msulfuric acid, and the absorbance was read at 450 nm
using a microplate reader. A standard curve was run using
the standards provided with the Apoptosense assay kit.
Quantification of the cleaved CK18 was achieved according
to the manufacturer’s directions.
DNA fragmentation assay
Monolayers of GI-101A cells were grown in T-25 culture
flasks and incubated with different concentrations of bro-
melain and without bromelain for 24 hours. After incubation
the cells were harvested and washed with PBS. Cells were
resuspended in 200 lL of PBS, and 20 lL of proteinase K
was added. The DNA was extracted using the Qiagen
(Chatsworth, CA, USA) DNeasy kit by following the
manufacturer’s protocol. The samples were subjected to
electrophoresis at 80 V for 2 hours in 1.5% agarose gel
containing 5 lL of ethidium bromide. Separated DNA
fragments were viewed with a UVP (Upland, CA, USA)
image analyzer.
4,60-Diamino-2-phenylindole fluorescence staining
4,60-Diamino-2-phenylindole (DAPI) is a nuclear stain
that binds to DNA, allowing for DNA visualization when
the stain fluoresces. This staining also enables the viewing
of chromatin structures that are condensing in cells that are
undergoing apoptosis. For conducting this experiment the
GI-101A cells were plated at a density of 1 ·10
5
cells per
well in a six-well plate, grown in complete RPMI growth
medium, and treated with increasing bromelain concentra-
tions: 0, 5, 10, and 20 lg/mL. Following the 24-hour treat-
ment the cells were fixed with 3.7% paraformaldehyde and
incubated with 0.1% Triton X-100 in PBS at room tem-
perature for 20 minutes. After this incubation, the cells were
washed gently with PBS and then incubated for another 10
minutes at room temperature with 200 ng/mL DAPI solu-
tion. At the end of the incubations, the cells were re-
suspended in PBS and examined quickly under an Olympus
(Center Valley, PA, USA) fluorescent microscope (model
BX51) with appropriate fluorescence filters and differential
interference contrast optics. Images were captured at ·100
BROMELAIN-INDUCED APOPTOSIS 345
magnification using an Olympus DP70 digital camera and
associated imaging software.
Statistical analysis
The data presented here represent mean –SD values from
at least four individual experiments. Statistical analyses
were performed using a one-way analysis of variance fol-
lowed by Student–Newman–Keuls multiple comparisons
tests. Values of P<.05 were considered as significant and
are presented in Results.
RESULTS
Bromelain-induced cell death and caspase activities
A decrease in viable cell number was observed in GI-
101A breast cancer cells after treatment with different
concentrations of bromelain (5, 10, 20, 40, and 50 lg/mL)
following 24 hours of incubation (Fig. 1a). A bromelain
concentration of 20 lg/mL was found to effectively reduce
the percentage of viable cells to 36% after a 24-hour treat-
ment. Doses higher than 20 lg/mL caused cell death in the
range of 95% or greater within 24 hours (Fig. 1b). Because
caspase-9 is an initiator caspase in the apoptotic pathway,
activation of this isoform leads to cleavage of procaspase-3
into its active form. Therefore, we analyzed the activities of
caspase-9 and caspase-3. Results in Figure 2 clearly show
that the cells treated with bromelain exhibited a significant
increase in the activities of both caspase-9 and caspase-3
after 24 hours with 5, 10, and 20 lg/mL concentrations.
M30-Apoptosense ELISA immunoassay
The M30-Apoptosense results showed that the cells
treated with bromelain contained significantly higher levels
of cleaved CK18 containing the CK18-Asp396 neo-epitope
than the control cells. Additionally, as the bromelain con-
centration increased, the level of CK18 containing CK18-
Asp396 neo-epitope also increased as shown in Figure 3.
Effect of bromelain treatment on DNA fragmentation
To determine whether bromelain treatment induced DNA
fragmentation, DNA was isolated from treated and untreated
control GI-101A cells and separated by agrose gel electro-
phoresis. A typical ladder pattern of internucleosomal
fragmentation of DNA was observed in cells treated
with bromelain (Fig. 4, lanes 3–5), whereas in untreated
control cells no fragmentation of DNA was observed (Fig. 4,
lane 2).
FIG. 1. (a) Representative photographs showing control cells and
cells treated with different concentrations of bromelain after 24 hours
of incubation. (b) Dose–response graph showing GI-101A cell death
following a 24-hour treatment with various concentrations of bro-
melain as determined by the trypan blue dye exclusion method. Data
are mean –SD values of four or more experiments. **P<.01 for
comparison with respective controls.
FIG. 2. Activities of caspase-9 and caspase-3 in GI-101A cells
following a 24-hour bromelain treatment. Caspase-9 and caspase-3
activities were measured using the synthetic tetrapeptide substrates
acetyl-Asp-Glu-Val-Asp-p-nitroaniline and acetyl-Leu-Glu-His-Asp-
p-nitroaniline, respectively. Data are mean –SD values of four or
more experiments. **P<.01 for comparison with respective controls.
346 DHANDAYUTHAPANI ET AL.
Determination of apoptosis using DAPI staining
The DAPI staining method was used to assess the extent
of apoptosis in control and bromelain-treated GI-101A
cells. The DAPI staining results, shown in Figure 5, re-
vealed good contrast between the control group (lightly
stained) and the cells treated with bromelain (brightly
stained). The bromelain-treated cells, especially those
treated with 10 and 20 lg/mL, showed notable chromatin
condensation and fragmentation compared with the control
sample.
DISCUSSION
Over the past two decades, determination of the phar-
macological effects of bioactive compounds used for cancer
treatment and prevention has increased dramatically.
12
Therefore, evaluation of the potential benefits of bioactive
compounds that are derived from consumable fruits and
vegetables may lead to the identification of additional
chemopreventive tools and strategies. Bromelain, a proteo-
lytic enzyme isolated from pineapple, has been reported to
possess antimetastatic and antitumorigenic activity.
6
In the
present study, GI-101A breast cancer cells treated with
bromelain resulted in a significant decrease in proliferation
by inducing apoptosis. Because disrupted apoptotic pro-
gression could lead to cancer growth, induction of apoptosis
in cancer cells by bioactive compounds is a key target for
chemotherapeutic and chemopreventive applications. Our
study demonstrates that bromelain may induce apoptosis
in breast cancer cells through activation of a caspase-
dependent pathway. Bromelain induced a maximum of 67%
cell death at a concentration of 20 lg/mL with a 24-hour
treatment interval, and it was almost 100% beyond that
concentration.
Induction of apoptosis observed in bromelain-treated GI-
101A cells is consistent with previous reports that bromelain
induces apoptosis by activating the preexisting apoptosis
machinery through caspase-3. Once activated, the effector
caspase-3 appears to stimulate the DNase and cause DNA
fragmentation. From the results obtained in our study, it is
evident that the increase in the activity of caspase-3 and
caspase-9 plays a pivotal role in apoptotic cell death induced
by bromelain. Apoptotic protease activating factor 1, cyto-
chrome c, and caspase-9 are the actual important partici-
pants in a complex pathway necessary for caspase-3
activation. Some of the in vitro studies have shown that
depletion of caspase-9 from cytosolic fractions could result
in the failure of caspase-3 activation.
13
The results from this
study have demonstrated that bromelain significantly in-
duced the activation of pro-caspase-9 to its active form and
furthermore amplified the activity of caspase-3 to cause
apoptosis.
A consequence of induction of apoptosis is the damage to
the cytoskeleton, where the cytokeratins are found in
abundance, resulting in the release of CK18 to the extra-
cellular environment (caspases).
14,15
Recently, it has been
shown that the determination of cell death (e.g., apoptosis
mode) is possible by measuring the soluble CK18 fragments
FIG. 3. M30-Apoptosense levels by enzyme-linked immunosorbent
assay in the GI-101A cell line following a 24-hour bromelain treat-
ment. Data are mean –SD values of four or more experiments.
FIG. 4. A representative photograph of the agarose gel showing
DNA fragmentation following bromelain treatment: lane 1, DNA
marker; lane 2, DNA from control cells; and lanes 3–5, DNA from
bromelain-treated cells, 5, 10, and 20 lg/mL respectively. The DNA
was separated by electrophoresis using 1.5% agarose gel. DNA
fragments stained with ethidium bromide were visualized using a
UVP image analyzer.
BROMELAIN-INDUCED APOPTOSIS 347
(CK18-Asp396 neo-epitope, also called M30 antigen) that
are formed by caspase activation.
16
CK18 is a major com-
ponent of the intermediate filament of simple epithelial cells
and epithelial-derived tumors and makes up approximately
5% of total protein.
17
It undergoes proteolytic cleavage
during apoptosis into fragments,
11,18
exposing the CK18-
Asp396 neo-epitope.
19,20
Treatment with the proteolytic
enzyme bromelain was found to enhance the level of CK18
in the GI-101A breast cancer cell line in our study. Recently
it has been reported that an increased level of CK18 in a
patient’s serum with breast cancer, after treatment with
docetaxel, was indicated as the primary measure of cell
death due to apoptosis.
21
Our results are similar to the ob-
servation reported earlier in terms of intracellular cleavage
and increase in the levels of CK18 during apoptosis. How-
ever, we did not measure the extracellular levels of CK18 in
our samples, and therefore it is not reported here. It appears
that the intracellular cleavage of CK18 in estrogen receptor–
positive GI-101A cells may occur by a mechanism similar
to the one that exists in estrogen receptor–negative MDA-
MB-231 cells, indicating that the apoptotic mechanism
leading to CK18 cleavage was probably estrogen receptor
independent.
The characteristics of apoptotic cell death are the induc-
tion of chromatin condensation, fragmentation of nuclei,
fragmentation of DNA, and cleavage of specific pro-
teins.
22,23
Several anticancer agents are being tested these
days that are expected to kill cancer cells by inducing pro-
apoptotic signals. In this regard, we have so far determined
that bromelain treatment induces chromatin condensation
and internucleosomal fragmentation of DNA in GI-101A
cells. The morphological changes of apoptosis in most of the
cell types are contraction in cell volume and condensation of
the nucleus, which allows the intracellular organelles to
retain their normal morphology. This change is followed by
the plasma membrane blebbing and nuclear fragmentation
to form apoptotic bodies.
24
A closer look at the pattern of
DAPI staining in bromelain-treated GI-101A cells in our
study suggests that DNA fragmentation is initiated at nu-
clear periphery and progresses toward the center. Although
DAPI staining enables the determination of cells undergoing
apoptosis, DNA from the cells forms a characteristic ladder
pattern on agarose gel that also confirms the biochemical
changes involved in the fragmentation of chromosomes into
nucleosome units.
25
As shown in our results, multiple units
of apoptotic DNA ladder were detected in bromelain-treated
GI-101A cells, whereas in the control, there was no such
fragmentation.
Finally, our results confirm the cytotoxic effects of
bromelain on the GI-101A breast cancer cells in a dose-
dependent manner. Furthermore, bromelain increased the
level of CK18, one of the markers indicating the cell death
via apoptosis. Our results also suggest that bromelain in-
duced the apoptotic signal through activation of a caspase
pathway, resulting in nuclear condensation and disintegra-
tion, which are hallmarks of apoptotic cell death.
ACKNOWLEDGMENTS
Financial support from Chancellor’s Faculty Research
and Development Grant of Nova Southeastern University is
gratefully acknowledged. We also would like to thank the
Royal Dames of Cancer Research, Ft. Lauderdale Inc., for
their generous support in conducting this research.
FIG. 5. Representative photographs of
control and bromelain (5, 10, and 20 lg/
mL)-treated GI-101A cells stained with
4,60-diamino-2-phenylindole after 24
hours of treatment: (a) control; (b) 5lg/
mL; (c) 10 lg/mL; and (d) 20 lg/mL.
Arrows indicate chromosome condensa-
tion and nuclear fragmentation. Magni-
fication used was ·100 in an Olympus
DP70 microscope system.
348 DHANDAYUTHAPANI ET AL.
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist with this work.
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