Bromelain in Pineapples inhibits the growth and proliferation of cancer cells
it impairs survival of cancer cells by inhibiting Akt signaling and attenuating Bcl2 and MUC1 Also appeared to activate caspase-dependent apoptotic pathways and transcription-independent p53 apoptosis
Pineapple,
botanically named Ananas comosus, has been used for centuries as a
folk medicine by the indigenous inhabitants of Central and South America to
treat a range of ailments. The medicinal qualities of the plant are attributed
to bromelain, a pineapple stem extract, which has been available as a
pharmaceutical product since 1956.1 The beneficial
effects of bromelain are attributable to its multiple constituents. Although
primarily comprised of sulfhydryl-containing proteolytic enzymes, bromelain
also contains escharase (a nonproteolytic component with debriding effects),2 peroxidase, acid
phosphatase, glucosidases, cellulases, several protease inhibitors,
glycoproteins, carbohydrates, and organically bound calcium.3,4 Bromelain has shown
an ability to interact with a variety of effectors and pathways involved in
physiological processes, including inflammation, the immune response, and
coagulation. Thus, the therapeutic potential of bromelain, alone or in
combination with other agents, has been tested in preclinical and clinical
settings, suggesting a number of clinical indications.5 However, as an
anticancer agent, bromelain has been the subject of limited preclinical and
clinical observations.6
Gastrointestinal
cancers account for more than one third of all deaths from malignant neoplasms
worldwide,7 among which
colorectal and stomach cancers are the most common.8 In the present study,
as an initial attempt towards implementing a novel approach to the management
of advanced gastrointestinal carcinoma, the anticancer efficacy of bromelain
was investigated in a range of gastrointestinal carcinoma cells. To explore the
efficacy of bromelain in a more comprehensive way, gastrointestinal cancer
cells of different origins and various sensitivities to cytotoxic agents were
employed. These included poorly differentiated human gastric cancer cell lines
(MKN45 and KATO-III) and chemoresistant subpopulations of the HT29 human colon
cancer cell line (HT29-5F12 and HT29-5M21).
Materials and
methods
Cell culture
The human gastric
carcinoma cell lines (MKN45 and KATO-III) were obtained from the Cancer
Research Campaign Laboratories (University of Nottingham, NG7 2RD, UK) and the
American Type Culture Collection (Manassas, VA, USA), respectively. The
HT29-5F12 and HT29-5M21 cells were a kind gift from Dr Thécla Lesuffleur
(Université Pierre et Marie Curie, Paris, France). All cell lines were
maintained in a humidified atmosphere of 95% air and 5% CO2 at
37°C in their respective media as follows: MKN45 in RPMI-1640, KATO-III cells
in Iscove’s Dulbecco’s modified Eagle’s medium, and HT29-5F12 and HT29-5M21
cells in Dulbecco’s modified Eagle’s medium (all from Invitrogen, Carlsbad, CA,
USA). All of the culture media used were supplemented with 10% (v/v) fetal
bovine serum and 1% (v/v) penicillin-streptomycin (Invitrogen), with the
exception of Iscove’s Dulbecco’s modified Eagle’s medium, for which 20% fetal
bovine serum supplementation was used.
Drug
preparation
Bromelain and
cisplatin were purchased from Sigma-Aldrich (St Louis, MO, USA). Bromelain and
cisplatin stock solutions were prepared at concentrations of 1000 μg/mL and
15,000 μg/mL, respectively. For each experiment, bromelain stock solution was
freshly made, filtered, and diluted with the appropriate culture medium to
achieve the final treating concentrations.
Proliferation
assay
The effect of
bromelain on growth and proliferation of MKN45, KATO-III, HT29-5F12, and
HT29-5M21 cells was determined using the sulforhodamine B assay. In brief, the
cells were seeded into 96-well plates and cultured in complete culture medium
at densities of 1500–5000 cells per well. At the desired confluence, the cells
were treated for 72 hours with a range of concentrations of bromelain (5–1000
μg/mL) in serum-free medium, as well as with cisplatin as the positive control.
At the end of the treatment period, the cells were fixed by 30 minutes of
incubation with 10% (w/v) trichloroacetic acid at 4°C. This was followed by
five washes with slow-running tap water. The plates were then stained with 0.4%
(w/v) sulforhodamine B (Sigma-Aldrich) dissolved in 1% acetic acid. Unbound dye
was removed by rinsing the plates with 1% acetic acid, and the plates were then
allowed to dry out at room temperature. Bound sulforhodamine B was solubilized
with 10 mM Tris base (Sigma-Aldrich) and absorbance was read using a microplate
reader (PowerWaveX, Bio-Tek Instruments Inc, Winooski, VT, USA) with a working
wavelength of 570 nm.
Western
blotting
The efficacy of
bromelain in activating apoptotic processes and modulating expression of
apoptosis-associated proteins was explored in MKN45 cells using Western
blotting. Briefly, the cells were homogenized in protein lysis buffer (RIPA)
containing 10% protease inhibitor (Sigma-Aldrich), and the protein
concentrations were then quantified using a BioRad protein assay (Bio-Rad
Laboratories, Hercules, CA, USA). Equal amounts of proteins were separated by
sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to
polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The following
primary antibodies were then applied to the membranes according to the
manufacturers’ protocols: rabbit polyclonal anticaspase 3, anti-Bcl2, anti-p53
(Santa Cruz Biotechnology, Santa Cruz, CA, USA), anticaspase 8 (R&D
Systems, Minneapolis, MN, USA), anticaspase 9, anti-PARP, anticytochrome C,
anti-Akt, rabbit monoclonal antiphospho-Akt, and mouse monoclonal anti-MUC1
(Cell Signaling Technology Inc, Danvers, MA, USA). The membranes were washed
and treated with the appropriate horseradish peroxidase-conjugated secondary
antibodies (Cell Signaling Technology Inc). A similar process was carried out
for β-actin as a loading control using mouse monoclonal anti-β-actin antibody
(Santa Cruz Biotechnology). The antigen-antibody reaction was visualized using
ImageQuant LAS 4000 Biomolecular imager and ImageQuant software (GE Healthcare,
Chalfont, UK). Band densitometry was quantified and the data were normalized
against the values of β-actin protein expression.
Statistical
analysis
All data presented
are representative of three independent experiments. Statistical analyses were
performed using GraphPad InStat (GraphPad Prism 5, San Diego, CA, USA). The
Student’s t-test was applied for unpaired samples and P values
< 0.05 were considered to be statistically significant.
Results
Bromelain
inhibited proliferation of human gastrointestinal carcinoma cells
Using the
sulforhodamine B assay, we investigated the potential of bromelain to inhibit
growth of gastrointestinal cancer cells. As shown in Figure 1,
bromelain significantly inhibited cell proliferation in MKN45 (P =
0.0018, 0.0010, 0.0002, and <0.0001 using concentrations of 100, 200, 400,
and 600 μg/mL, respectively), KATO-III (P < 0.0001 using
concentrations of 100, 200, and 400 μg/mL, respectively), and 5F12 and 5M21 (P <
0.0001 using concentrations of 40 and 50 μg/mL, respectively). Fifty percent
inhibitory concentration (IC50)
values were calculated from concentration-response curves plotting growth
percentage versus drug concentration using GraphPad Prism 5 (Figure 1).
Our data revealed IC50 values
of 29, 34, 94, and 142 μg/mL for HT29-5F12, HT29-5M21, MKN45, and KATO-III
cells, respectively.
Sulforhodamine
B assay in MKN45 (A and B), KATO-III (C and D), HT29-5M21 (E and F),
and HT29-5F12 (G and H) cells after 72 hours of treatment with
bromelain concentrations ranging from 5 μg/mL to 600 μg/mL, and with different
concentrations of cisplatin used as a positive control (shown as smaller graphs
a, c, e and g).
Bromelain
induced caspase-dependent apoptosis
To investigate the
inhibitory effects of bromelain observed in the proliferation assay further,
the implication of caspase-driven apoptotic events was evaluated by examining
expression of cytochrome C, caspases 3, 8, and 9 in MKN45 cells after 72 hours
of treatment with concentrations ranging from 25 to 200 μg/mL (Figure 2).
Along with overexpression of cytochrome C, the appearance of immunoreactive
subunits of caspase 3 and caspase 8, as well as withering of procas-pase 9, was
observed in a concentration-dependent manner. Moreover, the functionality of
activated caspase 3 was confirmed by cleavage of PARP Expression of cleaved
PARP was more prominent at a concentration of 200 μg/mL, where the precursor
protein was no longer detectable.
Western
blot imaging (A) and densitometric quantification (B–E) for a range of proteins
involved in apoptotic death of MKN45 cells treated for 72 hours with bromelain
concentrations of 25, 50, 100, and 200 μg/mL.
Bromelain
caused cleavage of p53, removal of MUC1, and attenuation of phospho-Akt and
Bcl2
Exploring the role
of other mediators of apoptosis, our results showed cleavage of p53 protein and
emergence of cleaved fragments at concentrations of 100 μg/mL and 200 μg/mL.
Moreover, disappearance of MUC1, along with attenuation of phospho-Akt and
Bcl2, occurred when the MKN45 cells were treated with a bromelain concentration
of 200 μg/mL (Figure 2).
Discussion
In this study, we
observed the efficacy of bromelain in inhibiting growth and proliferation of
four human gastrointestinal cancer cell lines in vitro. Clinical evaluation of
the efficacy of bromelain as an anticancer agent, alone or in combination with
other agents, has been confined to few anecdotal observations.6 This may have
stemmed from inadequate preclinical studies. Our literature search of PubMed
yielded a limited number of investigations which had explored the anticancer
effects of bromelain in preclinical settings, few of which had used cancer
cells of human origin. Taussig et al reported bromelain-induced growth
inhibition in three mouse tumor cell lines.9 Byrnes et al
indicated a role of bromelain in reversibly inhibiting the invasive properties
of glioma cells.10 In a study by Beuth
et al, treatment with bromelain led to significant reduction of tumor growth in
mice inoculated by murine sarcoma L-1 cells.11 In vivo antitumoral
and antimetastatic activity of bromelain against a panel of murine cancer cell
lines has been shown by Baez et al.12 Kalra et al
reported reduced formation of mouse skin tumor with bromelain,13 and Paroulek et al
demonstrated the antitumor effects of bromelain against the GI101A human breast
cancer cell line.14 Bhui et al also
demonstrated that pretreatment with bromelain reduced the number and volume of
murine skin tumors,15 and recently showed
that bromelain had an antiproliferative effect against human A431 epidermoid
carcinoma and A375 melanoma cells.16
Our study of MKN45
cells demonstrates that bromelain induces caspase-dependent apoptotic cell
death. Earlier studies have reported the proapoptotic effects of bromelain in a
number of in vitro and in vivo cancer models. In their mouse models, Kalra et
al and Bhui et al observed that treatment with bromelain resulted in
upregulation of p53 and subsequent activation of caspase 3 and caspase 9.13,15 Using the M30 Apoptosense®
assay (Peviva, Bromma, Sweden), Paroulek et al reported increased levels of the
cytokeratin 18 protein, along with a large number of apoptotic cell bodies
following bromelain treatment of GI-101A breast cancer cells.14 They recently
indicated a concentration-dependent increase in the activities of caspase 9 and
caspase 3 coinciding with elevation of cytokeratin 18 levels.17
An interesting
feature of bromelain-induced apoptosis in our study was the cleavage of p53.
Apart from the known anti-tumorigenic role of p53 as a sequence-specific
transcription factor activating several proapoptotic genes, there is some
evidence suggesting a direct, extranuclear apoptotic function of p53,
activating so-called transcription-independent p53 apoptosis.18,19 Leu et al showed
that p53 could interact with Bak, a proapoptotic mitochondrial membrane
protein, resulting in release of cytochrome C from mitochondria.20 Sayan et al
demonstrated that p53 could be targeted and cleaved by caspases, generating two
cytosolic fragments that translocate to the mitochondria and induce
depolarization of the mitochondrial membrane.21
In our study,
bromelain also exhibited an intriguing aspect of its anticancer function by
eliminating the MUC1 oncoprotein, which could be of particular therapeutic
importance. Being expressed as a transmembrane mucin in normal epithelial cells
of various organs, MUC1 is overexpressed and aberrantly glycosylated in most
carcinomas.22 Of the 1.4 million
tumors diagnosed each year in the US, about 900,000 show overexpression of
MUC1. MUC1 is exploited by malignant cells to induce transformation and
tumorigenicity. It also plays an important role in cancer cell survival, tumor
invasion, and metastasis, which is why MUC1 has emerged as a particularly
attractive target for the development of anticancer agents.23,24
Diminished
expression of phospho-Akt, but not total Akt, represents another molecular
event of bromelain-induced apoptosis in the present study. This might be due to
at least two mechanisms, ie, bromelain modulation of upstream kinase versus
phosphatase activity or bromelain enhancement of protein phosphatase activity,
and thus reduced phosphorylation status of phospho-Akt. Given that constitutive
activation of Akt via crucial phosphorylation events promotes survival of
cancer cells as well as resistance to treatment,25 inhibition of
phosphorylation may play an important role in the cytotoxicity of bromelain. In
our study, bromelain appeared to impair survival of cancer cells by inhibiting
Akt signaling and attenuating Bcl2 and MUC1.
Conclusion
Here, we report
the anticancer effects of bromelain, a pineapple stem extract, in a panel of
gastrointestinal cancer cell lines in vitro. Treatment with bromelain inhibited
the growth and proliferation of cancer cells. Bromelain not only appeared to
activate caspase-dependent apoptotic pathways and transcription-independent p53
apoptosis, but also induced concomitant inhibition of cell survival. The role
of bromelain in removing MUC1 oncoprotein and attenuating phospho-Akt could be
of particular therapeutic importance and warrants further investigation.
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