Deoxypodophyllotoxin Isolated from Juniperus Communis Induces Apoptosis in Breast Cancer Cells.

Abstract

The study of anticancer properties from natural products has regained popularity as natural molecules provide a high diversity of chemical structures with specific biological and medicinal activity. Based on a documented library of the most common medicinal plants used by the indigenous people of North America, we screened and isolated compounds with anti-breast cancer properties from the Juniperus communis (common juniper) species. Using bioassay-guided fractionation of a crude plant extract, we identified the diterpene isocupressic acid and the aryltetralin lignan deoxypodophyllotoxin (DPT) as potent inducers of caspase-dependent programmed cell death (apoptosis) in malignant MB231 breast cancer cells. Further elucidation revealed that DPT, in contrast to isocupressic acid, also concomitantly inhibited cell survival pathways mediated by the MAPK/ERK and NF B signaling pathways within hours of treatment. Our findings emphasize the potential and importance of natural product screening for new chemical entities with novel anticancer activities. Natural products research complemented with the wealth of information available through the ethnobotanical and ethnopharmacological knowledge of the indigenous peoples of North America can provide new candidate entities with desirable bioactivities to develop new cancer therapies.

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Anti-Cancer Agents in Medicinal Chemistry, 2015, 15, 79-88 79
Deoxypodophyllotoxin Isolated from Juniperus communis Induces Apoptosis in
Breast Cancer Cells
Sami Benzina
1,$
, Jason Harquail
1,$
, Stéphanie Jean
1
, Annie-Pier Beauregard
1
, Caitlyn D. Colquhoun
2
,
Madison Carroll
3
, Allyson Bos
2
, Christopher A. Gray
1,2,3
and Gilles A. Robichaud
1,2,#,
*
1
Department of Chemistry and Biochemistry, Université de Moncton, Moncton, NB, Canada E1A 3E9 and the Atlantic
Cancer Research Institute, Moncton, NB, Canada E1C 8X3;
2
Department of Biology, University of New Brunswick, Saint
John, NB, Canada E2L 4L5;
3
Department of Chemistry, University of New Brunswick, Saint John, NB, Canada E2L 4L5
Abstract: The study of anticancer properties from natural products has regained popularity as natural molecules provide
a high diversity of chemical structures with specific biological and medicinal activity. Based on a documented library of
the most common medicinal plants used by the indigenous people of North America, we screened and isolated
compounds with anti-breast cancer properties from Juniperus communis (common Juniper). Using bioassay-guided
fractionation of a crude plant extract, we identified the diterpene isocupressic acid and the aryltetralin lignan deoxypodophyllotoxin
(DPT) as potent inducers of caspase-dependent programmed cell death (apoptosis) in malignant MB231 breast cancer cells. Further
elucidation revealed that DPT, in contrast to isocupressic acid, also concomitantly inhibited cell survival pathways mediated by the
MAPK/ERK and NFκB signaling pathways within hours of treatment. Our findings emphasize the potential and importance of natural
product screening for new chemical entities with novel anticancer activities. Natural products research complemented with the wealth of
information available through the ethnobotanical and ethnopharmacological knowledge of the indigenous peoples of North America can
provide new candidate entities with desirable bioactivities to develop new cancer therapies.
Keywords: Apoptosis, breast cancer, caspase, deoxypodophyllotoxin, ERK, isocupressic acid, Juniperus communis, NFκB.
#
Author’s Profile: Dr. Gilles Robichaud is an Associate Professor at the Université de Moncton (NB, Canada) and a Researcher at the
Atlantic Cancer Research Institute. Dr. Robichaud’s primary research entails the molecular elucidation of the breast cancer metastatic pathway.
Dr Robichaud is also the recipient of the prestigious New Investigator Award from the Canadian Institute of Health Research (CIHR).
INTRODUCTION
Cancer persistently ranks among the top causes of death
worldwide where mammary tumors are the most common form of
cancer in women. In general, cancer arises from a disruption in
cellular homeostasis between cell survival and programmed cell
death (or apoptosis) processes [1]. Given that apoptosis malfunction
is a key hallmark of cancer development and tumor-cell survival,
the potential to effectively target cancer cell apoptotic processes
offers new hope for patient survival.
Apoptosis can be subdivided into the intrinsic and extrinsic
pathways. The intrinsic pathway, or mitochondrial-induced
apoptosis, is initiated from within the cell and leads to changes in
mitochondrial permeability and release of pro-apoptotic mediators
such as cytochrome c. Cytochrome c forms an apoptosome complex
with other components that trigger effector caspases (caspases-3, -6
and -7) leading to DNA fragmentation, the classic late hallmark of
apoptosis (reviewed in [2]). Extrinsic apoptosis is induced by the
binding of ligands to their cognate death receptors such as members
from the tumor necrosis factor receptor (TNFR) gene superfamily
[3, 4]. This ligation leads to the activation of caspase-8 and/or
caspase-10 and eventually effector caspases. Adding complexity to
the distinction of intrinsic/extrinsic pathways, they do not
necessarily act in exclusivity from one another [5]. For example,
Fas from the death-receptor pathway can mediate caspase-8
cleavage of the pro-apoptotic Bcl-2 family member, Bid, resulting
in mitochondrial release of cytochrome c [6, 7].
Cell survival cascades are also well characterized in cancer
processes. The extracellular signal regulated kinase (ERK) pathway, a
major component of mitogen-activated protein kinases (MAPKs),
*Address correspondence to this author at the Université de Moncton,
Moncton, NB, Canada, E1A3E9; Tel: (506)858-4320; Fax: (506)858-4541;
E-mail: gilles.robichaud@umoncton.ca
$
Authors have equally contributed to the writing of the manuscript
plays an essential role in the development and progression of cancer
by promoting cell survival and proliferation [8, 9]. Following its
phosphorylation and activation by numerous extracellular signals, it
phosphorylates a number of downstream target proteins, including
metabolic enzymes, structural proteins and transcriptional factors
[9]. Another prominent pro-survival regulator is the NFκB transcription
factor which has been largely studied in mammalian cells for its
role in inflammation, proliferation, differentiation, apoptosis and
cell survival [10]. Many stimuli induce survival responses mediated by
NFκB. In fact, the overall reduction in NFκB activity is associated
with an increase of apoptotic index in many cell types. Thus, NFκB
directed survival response is associated with increased expression
of anti-apoptotic proteins [11]. Mechanistically, NFκB is usually
sequestered in the cytoplasm by its natural NFκB inhibitor (IκB)
which is regulated by IκB kinases (IKK). Upon activation, IKK
phosphorylates IκB which mediates its degradation. Thereafter,
NFκB is liberated and imports to the nucleus to exert its role as a
transcription factor (reviewed in [11]). Given the role of MAPK and
NFκB on apoptosis and cell survival, it is not surprising that their
activation levels are generally targeted by anti-cancer treatments
[12-14].
The alarming incidence of morbidity and mortality associated
with cancer has generated a constant interest for the identification
and development of new effective therapeutic strategies. A
promising source for anticancer drug discovery is the use of
bioactive compounds from natural products (reviewed in [15, 16]).
Naturally occurring lead molecules present unparalleled structural
diversity and provide the building blocks for innovative
therapeutics bearing an array of structure-activity relationships. As
a result, bioactive molecules with anticancer activity have strongly
influenced the pharmaceutical industry and occupy a primary role
in anticancer drug research. Accordingly, in 2008, an estimated
60% of commercially available pharmaceutical drugs stemmed
from natural products [17]. More specifically, approximately half of
all anticancer drugs approved originate, or directly derive from
naturally occurring molecules [18]. Natural dietary supplements
187-5/15 $58.00+.00 © 2015 Bentham Science Publishers

80 Anti-Cancer Agents in Medicinal Chemistry, 2015, Vol. 15, No. 1 Benzina et al.
have also garnered popularity for their anticancer activity either
alone or in combination with chemotherapeutic regimes [19, 20].
Altogether, natural product research has uncovered an array of
biologically potent chemicals capable of modulating specific cancer
processes such as: growth, angiogenesis, migration, invasion,
autophagy and apoptosis.
Natural products research complemented with the wealth of
information available through the ethnobotanical and ethnopharma-
cological knowledge of the indigenous natives of North America
provide new opportunities to screen for candidate entities with
desirable bioactivity. In this study, we turned our attention to a
group of plants traditionally used in ancient medicines to study
anticancer effects on mammary tumour cells. We performed
bioassay-guided fractionations of crude plant material to isolate
pro-apoptotic compounds and identified isocupressic acid and
deoxypodophyllotoxin (DPT) as specific and potent inducers of
breast cancer cell apoptosis. Interestingly, DPT, a naturally
occurring cyclolignan, has previously been shown to convey
pharmacological properties including antiproliferative, antitumor,
antiviral, anti-inflammatory, anti-platelet aggregation and anti-
allergic (reviewed in [21]). Our mechanistic evaluation of DPT has
revealed that DPT induces apoptosis predominantly through the
extrinsic pathway while concomitantly suppressing tumor cell
survival signaling mediated by the MAPK and NFκB activities. To
the best of our knowledge, the mechanistic actions of DPT on
NFκB survival and extrinsic apoptotic pathways in breast cancer
cells have never been explored. In addition, our studies provide a
straightforward systematic approach for the identification of
biologically active compounds from natural products. We also
present further validation for DPT as a potent anticancer drug.
EXPERIMENTAL PROCEDURES
Cell Culture and Treatments
The MDA-MB-231 (MB231) and the MCF-10A mammary
epithelial cell lines were obtain from the American Type Culture
Collection (ATCC, Manassas, VA, USA). MB231 cells were
cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), 2mM L-
glutamine, 100 units/mL of penicillin and 100μg/mL streptomycin.
MCF10A cells were cultivated in DME/F12 media with 5% FBS, 2
mM L-glutamine, 1 mM sodium pyruvate, 10 μg/ml human insulin,
20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin and 500
ng/ml hydrocortisone. Cell culture media DMEM and DME/F12
were obtained from HyClone (Thermo Scientific, Rockford, IL,
USA); FBS from PAA Laboratories (ON, Canada); and other
reagents from Sigma-Aldrich (St. Louis, MO, USA).
Treatments consisted of plant extracts reconstituted in DMSO
at the indicated concentrations and incubated with cells seeded in
96 well microplates. Cells were then incubated at 37ºC for the
specified time points. In the specified experiments, cells were
submitted to chemical inhibitors of NFκB such as IKK-2 Inhibitor
IV (CAS 507475-17-4, EMD Millipore, Billerica, MA, USA) or
pre-treatments with neutralizing antibodies against Fas-L (AF126,
R&D Systems, Minneapolis, MN, USA) or TNF-α (#7321, Cell
signaling, Boston, MA, USA) in addition to their respective host
irrelevant immunoglobulins used as controls.
Cell Viability and Apoptosis Assays
5x10
3
cells were seeded in 96 well plates and analyzed at the
indicated time points for cellular viability and apoptosis using a
multiplex assay formed with the CellTiter Blue
®
and Apo-ONE
®
assay kits (Promega, Madison, WI, USA) according to the
manufacturer’s instructions. In brief, 20μL of CellTiter Blue
®
substrate was added to 100μL of media containing the cells and
incubated at 37°C for 1h. Then, the microplates were subjected to
analysis on a fluorescence microplate reader (FLUOstar Optima,
BMG Lab technologies, 544
Ex
/590
Em
). Apoptosis was then
measured on the same microplate by removing 80μL of the total
media and adding 40μL of the Apo-ONE
®
substrate. Next, the
microplate was incubated at room temperature for 1h on a plate
shaker and analyzed by fluorescence reading (485
Ex
/520
Em
).
Plant Material and Extraction
Plant material was collected by hand from various sites in
southern New Brunswick (Table 1) and immediately cleaned by
hand with deionised water, freeze dried and stored at −20 °C. Plants
were identified by Dr Stephen Clayden and voucher specimens
have been deposited in the New Brunswick Museum Herbarium.
Freeze dried samples were ground into powders, exhaustively
extracted in methanol for eight hours using a Soxhlet extractor and
the resulting extracts were concentrated in vacuo to give crude
methanolic extracts.
Isolation of Isocupressic Acid and DPT from Juniperus
communis
The fractionation of J. communis was guided by the induction
of apoptosis activity in the MB231 breast cancer cell line as
Table 1. Medicinal plant collection data.
Plant Organ/Tissue Extracted Collection Coordinates Voucher Number Collection Date
Acorus calamus Rhizome and root N 45° 32.476’ W 65° 50.516’ NBM VP-37476 2010/11/07
Aralia nudicaulis Rhizome N 45° 18.334’ W 66° 05.513’ NBM VP-37477 2011/07/18
Empetrum nigrum Aerial parts N 45° 11.932’ W 66° 13.803’ NBM VP-37479 2010/05/25
Fragaria virginiana Leaves and fruit N 45° 18.375’ W 66° 05.616’ NBM VP-37478 2010/06/08
Geum macrophyllum Aerial parts N 47° 62.481’ W 67° 91.267’ NBM VP-37480 2010/06/22
Heracleum maximum Root N 45° 30.794’ W 65° 53.956’ NBM VP-37481 2010/11/12
Hypericum perforatum Aerial N 45° 18.319’ W 66° 05.200’ NBM VP-37566 2010/07/12
Juniperus communis Branches and needles N 45° 17.815’ W 66° 03.666’ NBM VP-37482 2010/05/25
Moneses uniflora Whole plant N 47° 90.586’ W 68° 15.071’ NBM VP-37097 2010/06/22
Nuphar lutea Root N 45° 17.782’ W 66° 03.554 NBM VP-37483 2010/05/25
Populus tremuloides Bark N 45° 40.737’ W 66° 30.234’ NBM VP-37484 2010/05/20
Symplocarpus foetidus Root N 45° 26.324’ W 65° 55.027’ NBM VP-37564 2010/05/13

Deoxypodophyllotoxin Induces Breast Cancer Apoptosis Anti-Cancer Agents in Medicinal Chemistry, 2015, Vol. 15, No. 1 81
previously described by Carpenter et al. 2012 [22]. Briefly, freeze
dried branches and needles (20 g) were exhaustively extracted in
methanol (400 mL) for 6.5 hours using a Soxhlet extractor and the
resulting solution concentrated in vaccuo to give a dark green solid
(5.69 g). A further portion (9.95 g) of the freeze dried branches and
needles were extracted with methanol (overnight, 3 x 250 mL) at
−20ºC in the dark. The extract was concentrated in vaccuo to give a
dark green oil (2.26 g). The crude extract was initially fractionated
by a modified Kupchan solvent−solvent partition protocol to give
five fractions as follows: The organic extract (5.50 g) was taken up
in 9:1 MeOH/H
2
O (400 mL) and extracted with hexanes (3 x 150
mL) before being diluted with H
2
O (200 mL) and extracted with
CH
2
Cl
2
(3 x 150 mL). The aqueous fraction was then concentrated,
taken up in H
2
O (400 mL) and extracted with EtOAc (3 x 150 mL)
and nBuOH (3 x 150 mL). The five partition fractions were
concentrated in vaccuo, and the hexanes (1.62 g), CH
2
Cl
2
(850 mg),
and EtOAc (693 mg) fractions exhibited apoptosis-inducing ability.
The hexanes fraction (1.62 g) was subjected to silica gel flash
chromatography using a stepwise gradient of hexanes to 4:1
hexanes/EtOAc (5% increments of EtOAc, 250 mL per elution) to
afford 4 fractions. The CH
2
Cl
2
(850 mg) and EtOAc (693 mg) fractions
were combined and subjected to silica gel flash chromatography
using a stepwise gradient of hexane to 1:1 hexanes/EtOAc (5%
increments of EtOAc, 250 mL per elution). Selected bioactive
fractions from the flash chromatography column were further
purified by isocratic normal phase HPLC using a Phenomenex Luna
silica column (10μm, 100Å, 250 × 10 mm) eluted with 65:35
hexanes/EtOAc at a flow rate of 4 mL/min. Pure compounds of
interest (isocupressic acid and DPT) were identified and characterized
by spectrometric methods as previously described [22].
Western Blot Analysis
Cells (6 x 10
5
) were lysed using 0.1mL of 2X whole cell lysate
(WCL) buffer (0.125M Tris pH 6.8, Glycerol 0.2g/mL, 4.0% SDS)
along with freshly added phenylmethylsulfonyl fluoride (PMSF,
5ng), 0,5μL of protease inhibitor cocktail set III (Calbiochem
Gibbstown, NJ, USA), 1μL sodium orthovanadate (100mM). Cells
treated with the complete WCL solution were scraped and passed 5
times through a 27G syringe. Protein concentration was then
determined with a bicinchoninic acid (BCA) quantification assay
(Thermo Scientific, Rockford, IL, USA). Next, 20μg of protein was
mixed in 2X laemmli buffer, heated at 95°C for 5 minutes and
separated on a 10% polyacrylamide gel. Proteins were then
transferred to a PVDF (polyvinylidene fluoride) membrane
(Millipore, Billerica, MA, USA). The membranes were blocked
with 5% non-fat milk for 1 hour at room temperature and incubated
thereafter with either anti-ERK (#9107, Cell Signaling, Boston,
MA, USA), anti-phospho-ERK (#9106, Cell Signaling), anti-IκBα
(ab32518, Abcam, Toronto, ON, Canada), anti-cleaved caspase-3
(#9664, Cell Signaling) or anti-G3PDH (#2275-PC-100, Trevigen,
Gaithersburg, MD, USA) antibodies overnight at 4°C. Next,
horseradish peroxidase (HRP)-conjugated secondary antibodies
(Pierce, Thermo Scientific) were incubated with the membrane
for 1 hour at room temperature and the signal was revealed by
chemiluminescence according to the manufacturer’s protocol
(SuperSignal West Dura, Thermo Scientific).
Transfections and Reporter Gene Assays
Transfections for intracellular protein expression were carried
out using the XtremGENE reagent (Roche, Branford, CT)
according to the manufacturer’s guidelines. Briefly, cells were
seeded in six-well plates 24h pre-transfection at a density of 3 × 10
5
cells/well. Cells were then incubated with a DNA-reagent complex
(ratio of 2 μg of DNA/5 µ L reagent) for 24 h in OPTI-MEM in
reduced serum without antibiotics. Luciferase-based reporter gene
assays were conducted using the Luciferase Assay System
(Promega) as described previously [23]. Briefly, cells were
transfected with 2 μg of the NFκB-luciferase construct [24], lysed
and analyzed for luciferase activity using a luminometer (BMG
Fluostar, Fisher Scientific, Ottawa, ON, Canada). Relative reporter
activity was calculated using experimental triplicates.
Mitochondrial Membrane Permeability Assays
Mitochondrial membrane potential (Δψm) was assessed using
the MitoProbe™ JC-1 Assay Kit as indicated by the manufacturer
(#M34152; Invitrogen, Carlsbad, CA, USA). Briefly, MB231 cells
were incubated with JC-1 for 15 min at 37°C. After two washes,
cells were trypsinised and resuspended in PBS and then examined
for red fluorescence (extra-mitochondrial) at 544
Ex
/590
Em
or green
fluorescence (intra-mitochondrial) at 485
Ex
/520
Em
using flow
cytometry (FACSCalibur, BD Biosciences, Mississauga, ON,
Canada) or a fluorescence microplate reader (FLUOstar Optima,
BMG Lab technologies). Where applicable, red/green fluorescence
ratios were measured and calculated based on replicate
sampling and compared to cells treated with carbonyl cyanide 3-
chlorophenylhydrazone (CCCP) used as positive control for
mitochondrial membrane depolarization.
RESULTS
Screening of Traditionally used Medicinal Plants for Anti-
Breast Cancer Activity
In order to evaluate the potential of traditionally used medicinal
plants as a source of anti-breast cancer agents, we assessed cell
viability from an aggressive breast cancer cell line (MB231)
exposed to crude extracts from a series of North American plants
reported to bear various medicinal properties (reviewed in [25]).
Plant extracts included: Acorus calamus; Aralia nudicalis;
Empetrum nigrum; Fragaria virginiana; Geum macrophyllum;
Heracleum maximum; Hypericum perforatum; Juniperus communis;
Moneses uniflora; Nuphar lutea; Populus tremuloides; and
Symplocarpus foetidus (Table 1). Using a microscale fluorometric
test, MB231 viability was monitored in time (days 1, 3 and 5)
following the exposure of cells to 10 μg/ml of crude extracts
(Fig. 1A). In general, all samples proliferated up to day 3 (including
untreated and DMSO control samples) which then slightly
decreased at day 5 most likely due to elevated cell density and
suboptimal long term culturing conditions. Interestingly, cells
treated with the J. communis extract displayed a drastic growth
arrest in comparison to all other samples tested.
To determine whether the observed growth arrest was due to
apoptosis, a fluorometric assay based on the activities of caspase
effectors 3 and 7 was performed on plant samples that displayed the
highest relative growth suppression in MB231 breast cancer cells.
Apoptosis activity was thus monitored over time in cells treated
with crude extracts from F. virginiana; J. communis; G. macrophyllum;
and A. nudicalis (Fig. 1B). Surprisingly, only MB231 cells treated
with the J. communis extract demonstrated a strong apoptotic signal
within 24h of treatment in comparison to other plant material tested
and control samples. We extended our study to verify if J. communis-
induced apoptosis was a general effect in mammary epithelials or
specific to cancer cells. We thus evaluated the apoptosis inducing
ability of the latter selected plants to a non-cancerous mammary
epithelial cell model: MCF10A cells (Fig. 1C). As expected, plant
extractions that did not mediate apoptosis in MB231 cells had no
effect on MCF10A cells. However, apoptotic levels in MCF10A
cells treated with the J. communis extract were only slightly raised
over basal levels. Altogether, we demonstrate that the methanol
extract of J. communis is a potent apoptosis inducer that appears to
be specific to malignant breast cancer cells.
Bioassay-Guided Fractionation of Juniperus communis Plant
Tissue
To reveal and identify the bioactive ingredients from J.
communis in breast cancer apoptosis, we developed a working

82 Anti-Cancer Agents in Medicinal Chemistry, 2015, Vol. 15, No. 1 Benzina et al.
platform utilizing a series of fractionation methods and apoptosis-
based bioassays. Using specific organic solvents and chromato-
graphy, a series of fractions was generated (fractions A, B and C)
and tested for MB231 apoptosis (Fig. 2A). Apoptosis-inducing
compounds were then tracked within the partitioned series and
further isolated for characterization. We identified two major
derivatives from J. communis capable of initiating MB231 apoptosis;
isocupressic acid and DPT (Fig. 2B). To validate caspase-
dependent apoptosis from isocupressic acid and DPT, we examined
the activation of effector caspase-3 upon treatment for 2 hours of
MB231 cells by Western blot (Fig. 2C). As expected, we detected
elevated levels of active (cleaved form) caspase-3 in MB231 cells
treated with either isocupressic acid or DPT in comparison to
control samples.
To evaluate the potency of isocupressic acid and DPT in breast
cancer apoptosis, ten-fold serial dilutions were next performed with
the two compounds and assessed for caspase 3/7 activity in MB231
cells treated for 2 hours. Isocupressic acid demonstrated a dose-
dependent pattern in MB231 apoptosis as the concentration
decreased (Fig. 3). On the other hand, DPT showed more potency
as an apoptosis inducer as the dilutions increased. DPT induced the
highest apoptotic levels in MB231 at a final concentration of
100 ng/ml (approximately 0.25 nM) and was thus further investigated
to elucidate its mechanistic action on breast cancer cell apoptosis.
Fig. (1). Bio-effects of medicinal plant crude extracts on breast cancer cell viability. (A) Plant crude extracts (10μg/ml) were incubated with MB231breast
cancer cells and evaluated for cell viability over time (days 1, 3 and 5) using a microscale viability assay (Cell-Titer Blue, Promega). Plant extracts that exerted
the highest growth suppression were thereafter evaluated for caspase 3/7 activities over time (days 1, 2 and 3) in (B) MB231 and (C) MCF10A non-cancerous
mammary epithelial cells to determine apoptotic events (Apo-ONE, Promega). Control samples include non-treated (NT) and solvent (DMSO) treated cells.
Results and standard deviations are representative of biological and experimental triplicates.
A
4000
D1
3000
3500
n
ce)
D
ay
1
Day 3
Day 5
2000
2500
i
ty (fluoresce
n
500
1000
1500
Viabil
i
0
500
BC
MB231
MCF10A
0.6
0.7
0.8
s
tivity)
Day 1
Day 3
0.6
0.7
0.8
s
ctivity)
Day 1
Day 2
0.2
0.3
0.4
0.5
Apoptosis
Caspase 3/7 ac
0.2
0.3
0.4
0.5
Apoptosis
Caspase 3/7 ac
0.0
0.1
(C
0.0
0.1
(C
Day

Day 3

Deoxypodophyllotoxin Induces Breast Cancer Apoptosis Anti-Cancer Agents in Medicinal Chemistry, 2015, Vol. 15, No. 1 83
Deoxypodophyllotoxin Attenuates Survival Cascades in MB231
Cells
Given that apoptosis and survival cascades are tightly regulated
events leading to cell fate outcomes, we set out to examine how
DPT could influence commonly known survival pathways in breast
cancer cells. We turned our attention to expose the activation
status of extracellular-regulated kinases 1/2 (ERK1/2) and NFκB
signaling cascades following DPT treatment. Using Western blots,
we first observed that the expression levels of total ERK1/2 were
slightly induced in breast cancer cells treated with DPT in comparison
to control cells (untouched and DMSO-treated) (Fig. 4A). However,
DPT-treated cells demonstrated lower phosphorylation of ERK1.
On the other hand, when NFκB components were studied, DPT-
treated MB231 cells demonstrated elevated expression levels of
the NFκB inhibitor, IκBα, suggesting NFκB sequestering and
inactivation. Further analysis of NFκB upstream signaling through
IκB kinases α and β (IKK-α and IKK-β) revealed no changes in
expression levels between DPT-treated and control lysates. To
validate whether DPT treatment could result in the suppression of
NFκB transactivation, luciferase-based reporter gene assays were
performed using transfections of the NFκB-luciferase reporter
vector into the MB231 breast cancer cell line. We observed that
Fig. (2). Bioassay-guided fractionation of Juniper communis. (A) Schematic representation of the experimental workflow of plant fractionation guided by
caspase 3/7-dependant apoptosis. Fractions from series A, B and C were subsequently tested for apoptosis in MB231 (corresponding right panels) treated with
10μg/ml of extracts for 2h. Control samples include non-treated (NT), solvent (DMSO) and cells treated with apoptosis-inducing agent Melphalan (5μM) as a
positive control (Ctrl+). Results and standard deviations are representative of biological and experimental replicates. (B) Pure active compounds were
subsequently characterized and identified as isocupressic acid and deoxypodophyllotoxin (DPT). (C) Levels of active (cleaved) caspase-3 (C3) was examined
by Western blot in treated MB231 cells (10μg/ml) and compared to loading control GAPDH.
A
Crude extract from
dried material
Relative a
p
o
p
tosis
CH
2
Cl
2
and
EtOAc fraction
pp
(caspase 3/7 activity)
1.0
1.5
2.0
1
st
silica column
0.0
0.5
Fractions A1
A2 A3 A4
A5
2
nd
silica column
05
1.0
1.5
Fractions B1
B2 B3 B4
B5
0.0
0
.
5
Fractions C1 C2 C6C3 C4 C5
HPLC
1.0
2.0
3.0
4.0
0.0
Characterization
B
B
DeoxypodophyllotoxinIsocupressic Acid
C
C
Cleaved C3
GAPDH

84 Anti-Cancer Agents in Medicinal Chemistry, 2015, Vol. 15, No. 1 Benzina et al.
DPT-treated cells exhibited suppressed NFκB transcriptional
activity (up to 47%) in comparison to DMSO solvent control
samples (Fig. 4B). Overall, our findings validate DPT as a potent
inducer of breast cancer cell apoptosis. In addition, we also observe
that DPT concomitantly represses survival signaling mediated by
ERK and NFκB-dependent cascades.
Elucidation of the Apoptotic Pathway Induced by J. Communis
Derivatives
To examine whether DPT-induced apoptosis is dependent on
the mitochondrial pathway (or intrinsic pathway), we assessed
the mitochondrial membrane potential (Δψm) following breast
cancer cell treatment. MB231 cells were thus stained with JC-1,
Fig. (3). Isocupressic acid and DPT are potent inducers of caspase-dependent apoptosis in MB231 cells. Caspase 3/7 activity was determined in MB231
cells 2 hours following treatment with shown serial dilutions of either isocupressic acid or DPT and compared to corresponding concentrations of solvent
(DMSO) and non-treated (NT) control samples. Results and standard deviations are representative of experimental replicates.
Fig. (4). DPT inhibits cell survival cascades during apoptosis. MB231 cells were treated (2h) with DPT and examined for (A) protein expression levels of
extracellular-regulated kinases (ERK), phosphorylated ERK (P-ERK), IκBα, ΙκΒ kinases α and β (ΙΚΚ−α and −β) and GAPDH using Western blotting. (B)
NFκB transactivation was also evaluated in DPT-treated MB231 cells using a NFκB-luciferase reporter construct. Control samples include non-treated cells
(NT), solvent (DMSO) and NFκB chemical inhibitor (IKK-2 Inhibitor IV/10uM) used as a positive control (Ctrl+). Results and standard deviations are
representative of experimental replicates.
90
v
ity)
60
70
80
Apoptosis
C
aspas 3/7 acti
v
30
40
50
60
(
C
10
20
30
NT DMSO Isocupressic Ac. Deoxypodophyllotoxin
(ng/ml)
0
10
4
10
3
10
2
10
1
10
4
10
3
10
2
10
1
A
B
A
B
NF
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p-ERK
ERK
25000
30000
35000
a
ctivity
NF
N
B
-
luc
IkB-
10000
15000
20000
25000
v
e luciferase
a
(RLU)
IKK-
IK
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0
5000
Relati
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GAPDH

Deoxypodophyllotoxin Induces Breast Cancer Apoptosis Anti-Cancer Agents in Medicinal Chemistry, 2015, Vol. 15, No. 1 85
a selective mitochondrial dye, and studied for alteration in
Δψm following DPT treatment for 2 hours by cell cytometry.
Interestingly, DPT did not alter mitochondrial membrane potential
of breast cancer cells in contrast to cells treated with carbonyl
cyanide 3-chlorophenylhydrazone (CCCP) used as a positive
control (Fig. 5A). To further validate our findings, we also assessed
Δψm by calculating fluorescence ratios to determine intra- (green)
versus extra-mitochondrial (red) JC-1 staining. As expected,
MB231 cells treated with DPT did not show any alteration in
mitochondrial membrane potential in contrast to control samples
treated with CCCP (Fig. 5B). Our results thus suggest that DPT
induces breast cancer cell apoptosis through an extrinsic pathway
(mitochondrial independent).
Given that the most characterized initiators of extrinsic
apoptosis involve Fas-ligand (Fas-L) and TNF-α, we set out to
determine whether DPT could mediate programmed cell death
through these soluble mediators. To do so, we examined DPT-
induced apoptosis in MB231 cells pretreated with anti-Fas-L
and anti-TNF-α neutralizing antibodies in comparison to their
respective irrelevant immunoglobins used as controls. We found
that blockade of Fas-L and TNF-α signaling had no suppressive
effect upon DPT-induced apoptosis (Fig. 5C). Our observations
thus suggest an alternative route for DPT-induced extrinsic
apoptosis which appears to be independent from Fas-L and TNF-α
signaling.
DISCUSSION
There is an obvious and urgent need to develop new drugs
with novel modes of action to combat cancer. The failure of
combinatorial, genomic and rational approaches to provide lead
chemical structures and the current renaissance of natural product
chemistry as a major contributor to the drug discovery process
emphasizes the importance of screening biodiversity for new
chemical entities with novel biological activities. In this study, we
examined the potential efficacy of traditionally used medicinal
plants in the treatment of cancer. After our initial screening, crude
extracts from J. communis demonstrated the most anti-tumorigenic
properties amongst a series of plants tested. More specifically,
Fig. (5). DPT-induced apoptosis in breast cancer cells is independent of the mitochondrial (intrinsic) pathway. MB231 mitochondrial membrane
integrity (Δψm) was examined in MB231 cells treated (2h) with either DPT or CCCP (positive control/50μM) before mitochondrial staining with JC-1
(Molecular Probes/2μM). Fluorescence from depolarized membranes was evaluated by cell cytometry (A) while fluorescence ratios from intra (485
Ex
/520
Em
)
versus extra-mitochondria (544
Ex
/590
Em
) dyes were monitored by (B) a fluorescence microplate reader. In (C), MB231 cells were pretreated 1h with 8ng/ml of
neutralizing antibodies against Fas-L and/or TNF-α before the examination of DPT-induced apoptosis (Apo-ONE/Promega). Control samples include isotype-
matched irrelevant antibodies from rabbit or goat hosts (IgG-G and IgG-R). Results and standard deviations are representative of experimental and biological
triplicates.
A
NT
NT
NT
DMSO
CCCP
DPT
Count
49 %
0.3 %
51 %
Fluorescence
B
1.5
e
ntial
t
io)
10
12
14
DMSO
DPT
C
v
ity)
0.5
1
o
chondrial pot
e
F
luorescence ra
t
4
6
8
10
Apoptosis
aspase 3/7 acti
v
0
Mit
o
(
F
0
2
(C

86 Anti-Cancer Agents in Medicinal Chemistry, 2015, Vol. 15, No. 1 Benzina et al.
extracts from J. communis induced apoptosis in the MB231
aggressive breast cancer cell line with little damaging effects in the
non-cancerous MCF10A mammary cell model.
J. communis, from the Cupressaceae (or cypress) family, has
long been ingrained in the history of traditional medicines. Frequently
called the common juniper, this tree has many documented
traditional uses originating from many aboriginal peoples including
the Malecite, Dene, Mi’kmaq, Algonquin, Cree, Montagnais,
Chippewa, Chipewyan and Métis [25]. Several parts of the tree
have been documented to be used in traditional medicine to cure a
myriad of ailments [22, 25, 26]. Using apoptosis-guided fractionation
of J. communis extracts, we isolated and identified two compounds
capable of inducing caspase-dependant apoptosis in malignant
breast cancer cells: isocupressic acid and deoxypodophyllotoxin
(DPT). Interestingly, isocupressic acid has been previously reported
to possess anti-mycobacterial activity [22] in addition to causing
abortifacient events in cattle [27, 28]. To our knowledge, this is the
first time that isocupressic acid has been shown to induce apoptosis
in breast cancer cells. On the other hand, DPT has been extensively
studied for its antitumor properties in lung, cervical, prostate and
breast cancers [29-33].
In our examination of apoptotic modes of action, we found that
both isocupressic acid and DPT were strong inducers of caspase-3
and -7 activities in MB231 breast cancer cells. Our findings
corroborate previous reports showing that DPT possesses antitumor
activity as a result of caspase-dependent apoptosis in cervical,
prostate and breast carcinoma [31-34]. In contrast to isocupressic
acid (data not shown), we also observed that DPT concomitantly
inhibited pro-survival cascades mediated by the MAPK/ERK and
NFκB pathways. MAPK/ERK and NFκB cascades play essential
roles in the development, maintenance and progression of cancer
cells [8, 9]. In fact, activation of the Ras-Raf-MEK-ERK pathway is
associated with protection of cells from apoptosis and inhibition of
caspase-3 activation [35, 36]. Equally, NFκB not only promotes
neoplastic transformation; but also, activates antiapoptotic proteins
and signaling (reviewed in [10, 11]). Thus, the synergy of apoptosis
induction and concomitant blockade of survival signaling reveal the
potency of DPT as an anticancer drug. Our findings are supported
by a recent study by Jung et al. in 2013 which has also demonstrated
the capacity of DPT to inhibit the AKT/mTOR cascade, another
major survival pathway, which leads to apoptosis of breast cancer
cells [33]. Interestingly, both the Ras/MAPK/ERK and the PI3K/
AKT/mTOR survival pathways are circuitously connected. On one
hand, upstream regulators of ERK and AKT, Ras and PI3K
respectively, cross-talk and can activate each other [37]. Another
example is the capacity of NFκB to downregulate the tumor
suppressor gene PTEN which functions as a PI3K negative
regulator (reviewed by [38]). Accordingly, our observations suggest
that DPT-induced suppression of NFκB activity (Fig. 4) could
result in subsequent down modulation of the PI3K/AKT/mTOR
through the upregulation of PTEN activity. Moreover, DPT has
previously been reported to upregulate PTEN expression in carcinoma
cells [31]. Globally, the mounting evidence strongly supports DPT
as a strong inhibitor of cell survival cascades (AKT, ERK and
NFκB) in addition to its pro-apoptotic control in cancer cells.
In an attempt to elucidate the signaling pathway of DPT-
induced apoptosis, we evaluated the involvement of surface
membrane death receptor ligation (extrinsic) and the mitochondrial
(intrinsic) cascades. The complete absence of fluctuations in
mitochondrial membrane potential following DPT treatment
suggests that DPT predominantly mediates apoptosis through a
mitochondrial-independent manner (or extrinsic cascade) in breast
cancer cells. These observations are in contrast with previous
reports of DPT-induced apoptosis in human prostate cancer cells
[32] and human cervical cancer cells [31] which primarily involved
the mitochondrial pathway. However, in the latter study (Kim et al.
2013) they report that changes in mitochondrial membrane polarity
are time-dependent following DPT treatment [32]. Another
consideration is DPT kinetics. Whilst others have examined DPT-
induced apoptosis at 8h, 24h and 48h post-DPT treatment, our
studies were performed at 2h post-DPT treatment. In addition,
Shin’s group (2010) demonstrated that DPT treatment led to an
increase of p53 protein expression and phosphorylation resulting in
intrinsic apoptosis [31]. P53 activation can lead to mitochondrial-
dependent apoptosis through the activation of pro-apoptotic element
Bax or, the release of cytochrome c [39, 40].
We, on the other hand,
are working with p53 deficient (mutated p53) cells; the MB231 cell
model [41]. Altogether, our observations may elucidate the early
events of apoptosis triggered by DPT in carcinoma in a
mitochondrial and p53-independent manner. These findings are of
particular relevance when considering that inactivation or mutation
of the p53 tumor suppressor gene is the most common alteration
found in human cancers (up to 64%) (reviewed in [40]). DPT in this
case could represent a potent anticancer drug in cancers with p53
mutations.
Further investigation using TNF-α and Fas-L neutralizing
antibodies also suggests that DPT-initiated apoptosis is independent
from FasL/FasR and TNF-α/TNFR1 interaction. Alternatively,
DPT could signal apoptosis through other death ligand/receptor
interactions such as: Apo2L/DR4; Apo2L/DR5; or, Apo3L/DR3
[42-45]. Another possibility is that DPT could mediate apoptosis
through oxidative stress and the production of reactive oxygen
species (ROS). ROS is a collective term that broadly describes O
2
-
derived free radicals that can induce extrinsic apoptosis through the
activation of JNK or attack DNA directly (reviewed in [46, 47]). A
recent study by Kim and colleagues (2013) brings support to this
latter hypothesis through the demonstration that DPT induces the
accumulation of ROS in prostate carcinoma cells before the
engagement of apoptosis [32].
Our technical approach provides an efficient platform to
identify and characterize unprecedented natural product lead
structures with desirable bioactivities and potencies that are
desperately required to develop new cancer therapies. Indeed, our
apoptosis-guided fractionation assays quickly led to the discovery
of DPT; an intensively studied compound known for its potency to
induce cancer cell apoptosis. These latter pharmacological
properties are highly sought as they provide an effective, non-
inflammatory approach to eliminate cancer cells to regain tissue
homeostasis [48]. Accordingly, there has been an increasing
amount of focus to produce DPT and derivatives to generate
compounds with superior bioactivity and reduced toxicity in vitro
and in vivo [21, 49]. Also, the clinical use of DPT is limited by its
insolubility in water. However, researchers have recently succeeded
at complementing DPT with a β -cyclodextrin (β-CD) derivative,
sulfobutyl ether-β-CD (SBE-β-CD), to facilitate DPT solubility and
chemical stability [50]. The inclusion complex of SBE-β-CD and
DPT demonstrated up to 54% tumor inhibition when tested in vivo
using murine sarcoma tumor models and human carcinoma
xenografted mice. DPT in clinical use is also challenged by its
limited availability from natural sources. Given the strong interest
for the pharmacological properties of DPT, reports have also
shown alternative methods to produce DPT such as fermentation
technologies, genetically modified plants and endophytic
microorganisms (reviewed in [21]). In this regard, plants from the
Juniper species could offer additional natural sources of bioactive
DPT for future anticancer drug development.
Altogether, our findings validate the potential of DPT as an
anticancer agent. Investigations on DPT over the past decade have
clearly described this compound as a selective inducer of cancer
cell apoptosis. Despite the known current limitations of DPT
for clinical use, we strongly believe that the structure-activity
relationships of DPT warrant further investigation to adapt this
promising drug for therapeutic application.

Deoxypodophyllotoxin Induces Breast Cancer Apoptosis Anti-Cancer Agents in Medicinal Chemistry, 2015, Vol. 15, No. 1 87
CONFLICT OF INTEREST
The author(s) confirm that this article content has no conflict of
interest.
ACKNOWLEDGEMENTS
The authors would like to acknowledge the financial assistance
from various agencies. GAR is supported by grants from the New
Brunswick Innovation Foundation, the New Brunswick Health
Research Foundation, the Canadian Breast Cancer Foundation-
Atlantic Chapter, and the Canadian Breast Cancer Society/QEII
Foundation. GAR is also supported by a Canadian Institutes of
Health Research (CIHR) New Investigator Award. SB is supported
by TELUS in partnership with the Canadian Breast Cancer
Foundation. JH is supported by a trainee award from the Beatrice
Hunter Cancer Research Institute with funds provided by the
Cancer Research Training Program as part of The Terry Fox
Foundation Strategic Health Research Training Program in Cancer
Research at CIHR. Financial support for CAG’s research was
provided by the Natural Sciences and Engineering Research
Council of Canada (Discovery Grant to CAG, CGS-M to CDC and
USRA to MC), the New Brunswick Health Research Foundation,
and UNB and is gratefully acknowledged
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Received: February 11, 2014 Revised: June 04, 2014 Accepted: June 05, 2014