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Differential Interactions of Falcarinol Combined with Anti-Tumour Agents on Cellular Proliferation and Apoptosis in Human Lymphoid Leukaemia Cell Lines

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Leukaemia is the most common childhood cancer, and whilst recent advances in therapy have improved survival, current treatments are still limited by their side effects. Thus, new therapies are urgently needed, this study investigated the effects of Falcarinol, a polyacetylene isolated from carrots (Daucus carota) in combination with chemotherapy agents, anti-cancer agents and other apoptosis inducers. Inhibition of cellular proliferation and induction of apoptosis were investigated in three human lymphoid Leukaemia cell lines. Cellular proliferation was determined via ATP quantification using the Cell Titer Glo assay. Induction of apoptosis was investigated using caspase 3 activity assay and confirmed by nuclear morphology using Hoechst 33342. The study demonstrated that CCRF-CEM cells failed to induce synergistic response with any of the investigated chemotherapies, but importantly no inhibition was observed either. Jurkat cells showed a significant synergistic induction of apoptosis following joint treatment with Falcarinol and a Death Receptor 5 agonist (DR5), whereas CCRF-CEM cells showed only an additive response. Conversely within MOLT-3 cells Falcarinol partially inhibited the induction of apoptosis by DR5 agonist although this failed to reach significance. However MOLT-3 cells demonstrated synergistic induction of apoptosis when Falcarinol was combined with either Bortezomib (proteosome inhibitor), or Sulforaphane (histone deacetylase inhibitor). Identification of interactions between natural bioactive compounds with anti-cancer drugs may provide new pathways to target cancerous cells. Furthermore, since some combinations enhance apoptosis but some inhibit apoptosis it may be important to consider these interactions for dietary advice during therapy.
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Differential Interactions of Falcarinol Combined with Anti-Tumour Agents on
Cellular Proliferation and Apoptosis in Human Lymphoid Leukaemia Cell Lines
Zaini R, Haywood Small SL, Cross NA and Le Maitre CL*
Biomolecular Sciences Research Centre, Sheffield Hallam University, Sheffield, S1 1WB, UK
*Corresponding author: Christine Le Maitre, Biomolecular Sciences Research Centre, Sheffield Hallam University, Sheffield, S1 1WB, UK, Tel: 0114 225 6163; Fax:
0114 225 3066; E-mail: c.lemaitre@shu.ac.uk
Received date: Feb 04, 2015, Accepted date: Mar 16, 2015, Publication date: Mar 19, 2015
Copyright: © 2015 Zaini R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Leukaemia is the most common childhood cancer, and whilst recent advances in therapy have improved survival,
current treatments are still limited by their side effects. Thus, new therapies are urgently needed, this study
investigated the effects of Falcarinol, a polyacetylene isolated from carrots (Daucus carota) in combination with
chemotherapy agents, anti-cancer agents and other apoptosis inducers. Inhibition of cellular proliferation and
induction of apoptosis were investigated in three human lymphoid Leukaemia cell lines. Cellular proliferation was
determined via ATP quantification using the Cell Titer Glo assay. Induction of apoptosis was investigated using
caspase 3 activity assay and confirmed by nuclear morphology using Hoechst 33342. The study demonstrated that
CCRF-CEM cells failed to induce synergistic response with any of the investigated chemotherapies, but importantly
no inhibition was observed either. Jurkat cells showed a significant synergistic induction of apoptosis following joint
treatment with Falcarinol and a Death Receptor 5 agonist (DR5), whereas CCRF-CEM cells showed only an additive
response. Conversely within MOLT-3 cells Falcarinol partially inhibited the induction of apoptosis by DR5 agonist
although this failed to reach significance. However MOLT-3 cells demonstrated synergistic induction of apoptosis
when Falcarinol was combined with either Bortezomib (proteosome inhibitor), or Sulforaphane (histone deacetylase
inhibitor). Identification of interactions between natural bioactive compounds with anti-cancer drugs may provide new
pathways to target cancerous cells. Furthermore, since some combinations enhance apoptosis but some inhibit
apoptosis it may be important to consider these interactions for dietary advice during therapy.
Keywords: Falcarinol; Leukaemia; Apoptosis; Synergistic;
Chemotherapy; Apoptotic inducers
Introduction
Leukaemia is the most common childhood cancer, whereby
abnormal white blood cells (leukocytes) are produced. These abnormal
cells accumulate in the bone marrow and prevent the production of
other vital blood cells resulting in anaemia and immunodeficiency.
Commonly, Leukaemia affects children who are between two and four
years old, but children and adults of any age can develop this blood
malignancy [1]. Although there are effective treatments for certain
types of Leukaemia, it remains a major cause of morbidity and
mortality with more than 4300 deaths from Leukaemia in the UK
annually [2]. Moreover, mortality rates for both men and women from
Leukaemia have shown only a very gradual decline between the late
1970's and 2008 in the UK [3], demonstrating the need to find
treatments. One potential source of novel therapeutic agents is
bioactive compounds isolated from natural sources. Our previous
work has implicated Falcarinol a natural polyacetylene from carrots
(
Daucus carota
) as a potential inducer of apoptosis and inhibitor of
cellular proliferation within Leukaemia cell lines [4,5]. A potential role
for natural agents in anti-cancer therapies is in the combination with
existing chemotherapy and anti-tumour agents. If these agents interact
synergistically, they could lead to a dramatic reduction in the dose of
chemotherapy agents required and thus decrease side effects and the
morbidity associated with their use.
Induction of apoptosis as a target for anti-cancer therapies holds
great promise as apoptosis leads to the permanent removal of tumour
cells without triggering an inflammatory response and causing nearby
tissue damage [6]. There are two main pathways which lead to
apoptosis firstly the extrinsic (death receptor) pathway where cell
membrane receptors known as the death receptors (FasR, TNFR1,
DR4 and DR5) are activated via cytotoxic ligands leading to caspase 8
activation, which activates the execution caspases 3, 6 and 7 [6-8].
Secondly, the intrinsic (mitochondrial) pathway is activated by
induction of the pro-apoptotic members of the Bcl-2 family [8]. This
induces cytochrome c release and subsequent activation of caspase 9
which can then activate the effector caspases [9,10]. Many anti-cancer
drugs induce apoptosis by the activation of both the extrinsic and
intrinsic pathways [6-9,11]. For example, treatment of cancer cell lines
with chemotherapy agents, such as Cisplatin, Etoposide and
Methotrexate, induce activation of Fas receptors via over expression of
FasL [6,12]. These agents can also increase production of Bax in
response to DNA damage and p53 activation which leads to
cytochrome c release from the mitochondria and activation of caspase
3 [6]. However, many agents in current use or under investigation as
potential chemotherapeutic agents selectively target specific agents
within apoptotic pathways. This study assesses whether selected anti-
tumour agents act synergistically with the known pro-apoptotic and
anti-proliferative effects of the natural bioactive compound: Falcarinol,
in Leukaemia cell lines.
Chemotherapy drugs
Cisplatin is one of the most widely used anti-cancer drugs and is a
DNA-damaging agent used in the treatment of head, neck, lung,
ovarian cancers and lymphoma [13,14]. Nevertheless, the clinical use
Blood Disorders & Transfusion Zaini et al., J Blood Disorders Transf 2015, 6:2
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Research Article Open Access
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ISSN:2155-9864 JBDT, an open access journal Volume 6 • Issue 2 • 1000258
of Cisplatin is associated with a number of side effects including;
nausea and vomiting, whilst the most serious side effects are
neurotoxicity [15], and nephrotoxciity [16]. Cisplatin works by
crosslinking DNA interstrand and intrastrands preventing DNA
transcription, replication and cellular division (mitosis) [17,18]. The
damaged DNA is then unable to undergo DNA repair and thus
initiates apoptosis [18-20]. Etoposide is one of the most widely used
chemotherapy agents [21-23]. Etoposide is a member of DNA
topoisomerase II (topo2) inhibitors, which inhibit the ligation and
joining of the DNA strands together. Thus generating DNA double-
strand breaks and induce a progressive inhibition of DNA replication
[23,24]. Induction of DNA double-strand breaks by Etoposide has
been considered as the key mechanism responsible for its pro-
apoptotic and anti-tumour properties [24-26]. Chlorambucil is a DNA
alkylating agent, which has been used as a standard chemotherapy
treatment for lymphomas and CLL for more than 40 years [27]. The
mechanism of action of this drug is thought to be as a result of binding
to RNA, proteins and DNA. Resistance to Chlorambucil could be seen
as a result of increased drug metabolism or increased DNA repair [28].
5-Fluorouracil (5-FU) has been widely used in the treatment of cancer
over the past 30 years [29,30]. However, the response rates to the
single-agent treatments have not been satisfactory and drug resistance
remains a significant limitation to its clinical use. 5-FU induces
apoptosis via induction of DNA strand breaks [31], leading to
induction of p53, hence mutations in p53 can lead to 5-FU resistance
[32].
6-Mercaptopurine (6-MP) is an immunosuppressive drug used to
treat Leukaemia and lymphoma particularly childhood lymphoblastic
Leukaemia (CLL) [33,34]. 6-MP inhibits purine nucleotide metabolism
and synthesis which leads to dysfunctional synthesis and function of
RNA and DNA [33]. The DNA and RNA damage that results triggers
activation of intrinsic apoptosis via p53. However, because of its severe
side effects including inhibition of normal haematopoiesis which can
lead to increased risk of infection, anaemia and reduced blood clotting
and bleeding, the use of this therapeutic strategy is problematic [33].
As such the use of dual therapies which could reduce the
chemotherapy dose used, and thus lead to reduced side effects would
be beneficial [35].
Apoptotic inducer agents
Bortezomib (Velcade, PS-341) is a proteasome inhibitor which
induces apoptosis in several blood cancers and solid malignancies,
including mantle cell lymphoma, myeloma, and T cell Leukaemia cell
line (Jurkat) [36-40]. Combination of histone deacetylase inhibitors
(HDACis) with Bortezomib within Leukaemia cell lines has
demonstrated potential to overcome resistance [41,42]. However,
certain natural agents have also been shown to inhibit the actions of
Bortezomib, of particular note were the inhibitory actions of the
dietary Flavonoid: Quercetin, in B cell lines and primary CLL cells
[43]. These potential inhibitory actions highlight the importance of
identifying whether natural agents act synergistically with therapeutic
agents but also to ensure that they do not inhibit their actions.
Leptomycin B (LMB) is a nuclear export inhibitor, which prevents
protein transfer from the nucleus to the cytoplasm through nuclear
pores leading to accumulation of proteins within the nucleus such as
p53 and NFκB leading to modulation of apoptotic signalling [44,45].
LMB acts synergistically with a number of anti-cancer agents such as
ABL kinase inhibitor: Imatinib mesylate by excluding BCR/ABL from
the cytosol leading to accumulation of death signals in the nucleus of
CML cells [46]. Sulforaphane a HDACi increases acetylated histones
H3 and H4, leading to re-expression of silenced genes such as p21 and
Bax [47]. Sulforaphane also induces apoptosis and cell cycle arrest by
downregulation of NF-κB, and inhibits cancer stem cell properties
such as self-renewal and ALDH1 expression [48,49].
Specific agonists to death receptors are in clinical trials and have
shown promise in tumour therapies either as single agents or in
combination with cytotoxic chemotherapy [50]. Mouse anti-human
DR5 monoclonal antibody (AD5–10) has been shown to induce
apoptosis in T cell Leukaemia cell line (Jurkat) [50]. However, a
number of Leukaemia cell lines display partial resistance to death
receptor agonists, within which, a number of Methoxyflavone
derivatives have been shown to enhance death receptor induced
apoptosis [51]. Here, we investigated whether Falcarinol could act
synergistically with death receptor agonists to induce apoptosis.
This study tested the hypothesis that Falcarinol isolated from
Daucus carota
demonstrates synergistic actions on chemotherapeutic
or anti-cancer agents within human lymphoid Leukaemia cell lines.
Materials and Methods
Cell culture
Three human lymphoid Leukaemia cell lines were used in this
study, which we have previously shown have high sensitivity to
Falcarinol [5], CCRF-CEM (acute lymphoblastic Leukaemia) (ATCC:
CCL-119, Middlesex, UK); Jurkat (peripheral blood T cell Leukaemia)
(ATCC: TIB-152, Middlesex, UK); and MOLT-3 (acute lymphoblastic
Leukaemia patient relapsed following chemotherapy) (ATCC:
CRL-1552, Middlesex, UK) [5]. All cell lines used in this study were
routinely tested against mycoplasma bacterial infection using the
MycoAlert® Mycoplasma Detection Kit (Lonza, USA). Cells were
cultured in RPMI 1640 medium (Invitrogen, Paisley, UK)
supplemented with 10% (v/v) fetal bovine serum, 100 µg/ml penicillin/
streptomycin and 1.5 mM L-Glutamine (complete RPMI) and
incubated at 37ºC with 5% CO2. Prior to stimulation, cells number
were transferred into 12 well plates at a cell density of 1x106 cells per
ml.
Individual treatment with chemotherapy agents
Cisplatin, Chlorambucil and 5-FU were investigated at 2.5 to 10
µM, whilst Etoposide was investigated at 5, 50 and 5000 nM on CCRF-
CEM cells to assess their ability to inhibit ATP levels with less than
20% to be used in the combination treatment to investigate synergistic,
additive or inhibitory effects.
Individual treatment with anti-tumour agents
A dose response curve for different concentrations of Falcarinol, 6-
MP, Bortezomib, human Death Receptor 5 (DR5) agonistic
monoclonal antibody, LMB and Sulforaphane was determined using
Jurkat cells. Doses which induced less than 20% apoptotic cell death
were then used in combination treatments to investigate synergistic,
additive or inhibitory effects.
Citation: Zaini R, Small SLH, Cross NA, Le Maitre CL (2015) Differential Interactions of Falcarinol Combined with Anti-Tumour Agents on
Cellular Proliferation and Apoptosis in Human Lymphoid Leukaemia Cell Lines. J Blood Disorders Transf 6: 258. doi:
10.4172/2155-9864.1000258
Page 2 of 9
J Blood Disorders Transf
ISSN:2155-9864 JBDT, an open access journal Volume 6 • Issue 2 • 1000258
Effects of individual and combination treatment on
apoptotic induction
Half a million CCRF-CEM cells per well were treated with
Cisplatin, Etoposide, Chlorambucil and 5-FU with or without
Falcarinol for 24 and 48 h. Moreover, three lymphocytic leukaemia
cells; CCRF-CEM, Jurkat and MOLT-3 were treated with 6-MP,
Bortezomib, DR5, LMB and Sulforaphane in the presence or absence
of 6 μM Falcarinol in 12 well plates, following 24 h cells were stained
with caspase 3 activity assay (Cambridge Biosciences, Cambridge, UK)
and analyzed on the flow cytometer using a BD FACS Calibur
instrument (BD, Oxford, UK) as previously described. Data, from at
least 10,000 events per sample, were recorded and processed using the
Cell Quest software (Becton–Dickinson, UK). Dot-plots were analyzed
using Flow Jo software (Tree Star, Inc). To confirm synergistic actions
200,000 cells per well were treated as above for 24 h. Following
treatments cellular content from each culture well was transferred to
eppendorf tubes and centrifuged for 5 minutes at 400g at 4ºC. The
supernatant was removed, and cells washed in 100 µl DPBS. Cells were
then resuspended in 100 µl 4% (w/v) paraformaldhyde/DPBS and
stored at 4ºC overnight. One hundred microlitres of cell suspension
was transferred to slides via 10 minute cytospin (Shandon Cytospin 3
Centrifuge) at 1000 rpm. Samples were air dried and washed twice
with DPBS then stained in 100 µl of 10 μg/ml Hoechst 33342 (Sigma,
Poole, UK) for 10 minutes in the dark. Slides were mounted in 90%
(v/v) glycerol/PBS and coverslips sealed with nail varnish. Nuclear
morphology was examined using an Olympus BX61 fluorescence
microscope using a 350 nm U-MNV filter (Olympus, Essex, UK).
Images were captured using Q Capture- Pro 8.0 (UVP BioImaging
Systems, Loughborough, UK).
Effects of individual and combination treatment on
proliferation
Inhibition of cellular proliferation was assessed using Cell Titer Glo
Luminescent Cell Viability Assay kit (Promega, Southampton, UK) to
measure the ATP level of metabolically active cells following 24 h of
combination treatment on CCRF-CEM, Jurkat and MOLT-3 cell lines
as per manufactures instructions.
Statistical analysis
Average and Standard error of the mean (SEM) was calculated and
Stats Direct (Cheshire, UK) was used to test whether data followed a
normal distribution using a Shapiro Wilke test. Data were non-
parametric and thus a Kruskal-Wallis test and a Conover-Inman Post
hoc test was used to investigate significant differences. Results were
considered statistically significant when P 0.05. In order to
investigate synergistic responses the expected additive responses from
the combination treatment were calculated by adding the percentage
of apoptotic cells induced from the Falcarinol treatment alone to the
percentage of apoptotic cells induced by the apoptotic inducer agent
under investigation. The effect was considered synergistic when the
result of combination treatment was significantly higher than the
expected additive effect and also to each single agent treatment, the
additive response seen when the result of combination treatment was
not significant to the expected additive effect, while for an inhibitory
effect, the combination treatment showed a lower significant effect
than the expected response.
Results
Individual effects of chemotherapy agents on CCRF-CEM
cells
Cisplatin, Etoposide, Chlorambucil and 5-FU were used at different
concentrations to assess their ability to induce anti-proliferative effects
on CCRF-CEM cells assessed by the Cell Titer Glo assay. All
concentrations of Cisplatin, Etoposide, Chlorambucil and 5-
Fluorouracil investigated showed a significant (P ≤ 0.05) decrease in
the percentage of ATP level following 24 h (data not shown). From
these investigations doses of Cisplatin (2.5 µM), Etoposide (5 nM),
Chlorambucil (2.5 µM) and 5-Fluorouracil (5 µM) were selected for
further treatments as these induced less than 20% decrease in the ATP
levels.
Individual effects of anti-tumour agents on Jurkat cell line
Jurkat cells were treated with different concentrations of anti-
tumour agents individually to assess their ability to induce low levels
of apoptotic cells as measured by caspase 3 activation. Treatment of
Jurkat cells for 24 h with 6 µM Falcarinol, 10 µM 6-MP, 2.5 nM
Bortezomib, 25 ng/ml DR5, 0.5 nM LMB and 25 µM Sulforaphane
were identified as inducing less than 20% apoptosis (Figure 1) and
thus were selected for combination treatments.
Figure 1: Induction of apoptosis detemined via caspase 3 activity
assay within Jurkat cells following 24 h of treatment with Falcarinol
(A), 6-MP (B), Bortezomib (C), DR5 agonist (D), Leptomycin B
(LMB) (E) and Sulforaphane (F). Percentage cells positive for
caspase 3 activitiy assay displayed as means ± SEM, *P<0.05
compared to untreated cells.
Citation: Zaini R, Small SLH, Cross NA, Le Maitre CL (2015) Differential Interactions of Falcarinol Combined with Anti-Tumour Agents on
Cellular Proliferation and Apoptosis in Human Lymphoid Leukaemia Cell Lines. J Blood Disorders Transf 6: 258. doi:
10.4172/2155-9864.1000258
Page 3 of 9
J Blood Disorders Transf
ISSN:2155-9864 JBDT, an open access journal Volume 6 • Issue 2 • 1000258
Combination effects on induction of apoptosis determined
via caspase 3 activity and morphological analysis
Combination therapies with chemotherapy agents: CCRF-CEM
cells showed significant (P ≤ 0.05) yet small decrease (3%) in the
number of live cells following the treatment with 5 µM Falcarinol in
combination with 2.5 µM Cisplatin when assessed after 24 h (Figure
2A). However, the treatment with Cisplatin alone showed a greater
decrease (5.5%) in the number of live cells after 24 h (Figure 2A).
Similarly, following 48 h the reduction in live cell population and
increase in caspase 3 activity was significantly higher following the
treatment with Cisplatin alone than in combination with Falcarinol
(Figure 2A). Similarly individual Etoposide treatment showed a
greater induction of apoptosis than seen following combination
treatment with Falcarinol, although this was not significant to the
individual treatment with Etoposide (Figure 2B). On the other hand,
an additive response was seen in CCRF-CEM cells following the
treatment with 5 µM Falcarinol combined with 2.5 µM Chlorambucil
or 5 µM 5-Fluorouracil following 24 h (Figure 2C and 2D).
Figure 2: Induction of apoptosis detemined via caspase 3 activity assay within CCRF-CEM cell line following the treatment with Falcarinol
together with Cisplatin (A), Etoposide (B), Chlorambucil (C) and 5-Fluorouracil (D) after 24 and 48 h. Percentage cells positive for caspase 3
activitiy assay displayed as means ± SEM, *P<0.05 compared to untreated cells.
All cell lines displayed an additive response when Falcarinol was
combined with 6-MP (Figure 3A). MOLT-3 cells showed a significant
(P ≤ 0.05) synergistic induction of caspase 3 activity following 24 h
incubation with Falcarinol and Bortezomib (Figure 3B). However,
only an additive response was seen within CCRF-CEM and Jurkat cells
(Figure 3B). These results were confirmed using morphological
assessment of apoptosis (Figure 4).
CCRF-CEM cells showed no significant increase in apoptosis as
determined by percentage of caspase 3 positivity with either Falcarinol
or DR5 agonist alone, combination therapy was not significantly
different than the expected additive effect (Figure 3C). However,
Jurkat cells showed significant (P 0.05) synergistic response
following treatment with Falcarinol and DR5 agonist following 24 h
(Figure 3C).
Morphological assessment confirmed these results (Figure 5). In
contrast, an inhibitory effect was observed following the treatment
with Falcarinol combined with DR5 agonist in MOLT-3 cells
compared to single agent treatments (Figure 3C).
However, no synergistic response were seen following combination
of Falcarinol with Leptomycin B (Figure 3D). MOLT-3 cells showed a
significant (P 0.05) synergistic induction of caspase 3 activity
following 24 h incubation with Falcarinol and Sulforaphane (Figure
3E). Conversely CCRF-CEM and Jurkat cells only demonstrated
additive responses when Falcarinol was combined with Sulforaphane
(Figure 3E).
Citation: Zaini R, Small SLH, Cross NA, Le Maitre CL (2015) Differential Interactions of Falcarinol Combined with Anti-Tumour Agents on
Cellular Proliferation and Apoptosis in Human Lymphoid Leukaemia Cell Lines. J Blood Disorders Transf 6: 258. doi:
10.4172/2155-9864.1000258
Page 4 of 9
J Blood Disorders Transf
ISSN:2155-9864 JBDT, an open access journal Volume 6 • Issue 2 • 1000258
Figure 3: Induction of apoptosis detemined via caspase 3 activity
assay within CCRF-CEM, Jurkat and MOLT-3 cells following
combination treatments with Falcarinol together with 6-
mercaptopurine (A), Bortezomib (B), DR5 agonist (C), Leptomycin
B (D) and Sulforaphane (E) after 24 h. Percentage cells positive for
caspase 3 activitiy assay displayed as means ± SEM, *P<0.05
compared to untreated cells.
Figure 4: Induction of apoptosis determined via nuclear
morphology following Hoechst 33342 stain following treatment of
MOLT 3 cells for 24 h with Falcarinol and Bortezomib alone and in
combination. Scale bar=25 µm.
Figure 5: Induction of apoptosis determined via nuclear
morphology following Hoechst 33342 stain following treatment of
Jurkat cells for 24 h with 25 ng/ml DR5 and 6 µM Falcarinol, as
either single agents or in combination. Scale bar=25 µm.
Combination effects on cellular viability determined via ATP
levels
6-MP demonstrated a direct inhibitory effect on ATP production
when assessed with Cell Titer Glo (Figure 6A). This was also
confirmed with Trypan blue staining assay to determine the level of
live cells, which showed a significant (P ≤ 0.05) decrease in the number
of viable cells after treatment with Falcarinol alone and 6-MP alone
and significant increase (P ≤ 0.05) following combination treatment.
CCRF-CEM, Jurkat and MOLT-3 cells showed a significant
decrease in ATP level compared to control cells following combined
treatment with Falcarinol and Bortezomib for 24 h (P ≤ 0.05) (Figure
6B).
Single and combination treatment with Falcarinol alone and
Falcarinol with DR5 agonist showed no significant decrease in ATP
levels in CCRF-CEM. However, Jurkat cell line showed a significant
decrease in the level of metabolically active cells after combination
treatments compared to the treatment with either DR5 agonist or
Falcarinol alone (Figure 6C). Moreover, MOLT-3 cells showed a 35%
decrease with Falcarinol, no effect with DR5 and only 17% decrease in
ATP levels with combination treatment (Figure 6C).
All three Leukaemia cell lines showed a significant decrease in ATP
level when assessed after 24 h treatment with Falcarinol in
combination with Leptomycin B (Figure 6D). Similarly, the effect of
Falcarinol and Sulforaphane treatment on all examined cells
significantly decreased ATP levels (P ≤ 0.05).
For example, in CCRF-CEM the ATP level decreased ~25% in cells
treated individually with Falcarinol or Sulforaphane whereas the
combined treatment showed approximately 40% reduction in ATP
levels (Figure 6E).
Citation: Zaini R, Small SLH, Cross NA, Le Maitre CL (2015) Differential Interactions of Falcarinol Combined with Anti-Tumour Agents on
Cellular Proliferation and Apoptosis in Human Lymphoid Leukaemia Cell Lines. J Blood Disorders Transf 6: 258. doi:
10.4172/2155-9864.1000258
Page 5 of 9
J Blood Disorders Transf
ISSN:2155-9864 JBDT, an open access journal Volume 6 • Issue 2 • 1000258
Figure 6: Cellular viability investigated using Cell Titre Glo assay to
measure the ATP levels as a measure of cell viability within three
human Leukaemia cell lines (CCRF-CEM, Jurkat and MOLT-3)
following 24 h of individual and combied treatment with
Falcarinol, DR5, Bortezomib, Leptomycin B, Sulforaphane and 6-
MP. Percentage ATP levels normalized to untreated controls
displayed as means ± SEM, *P<0.05 compared to untreated cells.
Discussion
Falcarinol, a natural polyacetylene from carrots (
Daucus carota
) has
been previously indicated as an inducer of apoptosis and inhibitor of
cellular proliferation within Leukaemia cell lines [4,5]. The key
potential for natural agents in anti-cancer therapies is in the
combination with existing chemotherapy and anti-tumour agents. If
chemotherapy and anti-tumour agents interact synergistically with
Falcarinol, this could lead to a dramatic reduction in the dose of
chemotherapy agents required and thus decrease side effects and the
associated morbidity with their use. Here, we demonstrated that the
combination of Falcarinol with several apoptotic inducer agents or
chemotherapy drugs resulted in differential responses within three
human lymphoid Leukaemia cell lines.
CCRF-CEM cells failed to show any synergistic response with either
of the investigated chemotherapies (Cisplatin, Etoposide,
Chlorambucil and 5-Fluorouracil). Cisplatin treatment in CCRF-CEM
cells in combination with Falcarinol showed a lower cytotoxic effect
toward live cells as well as apoptotic induction when compared to the
individual treatment with 2.5 µM Cisplatin following 24 and 48 h.
Similarly, apoptotic induction within CCRF-CEM cells was greater
following 24 h treatment with Etoposide alone, than in combination
with Falcarinol. However, an additive response in CCRF-CEM was
seen when treated with Falcarinol combined with Chlorambucil or 5-
Fluorouracil following 24 h. Similar to our results, Kwa and colleagues
(2010) found that the treatment with Chlorambucil with the HDACi,
sodium butyrate resulted in an additive induction of apoptosis within
lymphoid leukaemia cell lines LP-1 after 3 days post treatment [52]. In
addition, treatment with 5-Fluorouracil in combination with fish oil-
based lipid emulsion (FO) which was rich in omega-3 fatty acids
showed a greater effect on growth inhibition of the colon cancer cell
line Caco-2 than 5-FU alone [53].
6-MP demonstrated additive actions with Falcarinol, inducing
caspase-3 activity within all three Leukaemia cell lines. In contrast, the
ATP level of combined treatment was significantly higher than single
agent treatment, this suggests that the combination of 6-MP with
Falcarinol results in a reduced effectiveness of 6-MP.
Within CCRF-CEM and Jurkat cells, no significant increase in
caspase-3 activity was observed either with individual or combined
treatment with Falcarinol and Bortezomib after 24 h although
increases were observed which did not reach significance. MOLT-3
cells showed a significant synergistic response to Falcarinol and
Bortezomib treatment with 15% higher apoptotic cells compared to
the expected additive effect, in contrast the ATP level of metabolically
active cells showed similar decreases following Falcarinol alone and in
combination with Bortezomib (67%), demonstrating a specific
synergistic response to induction of apoptosis as opposed to cellular
proliferation which warrants further investigation. The targeted
synergy to apoptosis suggests increased sensitivity to unfolded
proteins or accelerated accumulation of proteasome degraded pro-
apoptotic factors such as the FOXO transcription factors.
Jurkat cells demonstrated sensitivity to DR5 agonists, inducing
apoptosis and inhibiting cellular proliferation which agrees with
previous studies [50]. A number of agents have been shown to act as
sensitizers to DR5 ligands, including TRAIL [54], here we
demonstrated Falcarinol induced apoptosis in a synergistic manner
with DR5 agonists suggesting its role as a TRAIL sensitizer. Here, we
show for the first time that MOLT-3 cells are sensitive to DR5
agonists, which has been shown previously for MOLT-4 cells [55]
which are derived from the same patient. Importantly however within
these cells an antagonistic effect was seen in combination with
Falcarinol. The differential effects seen between cell lines highlights
key differences in apoptotic signalling between leukaemia cell lines
[56]. Jurkat cells which have been shown to be highly sensitive to DR5
agonists previously [57], classically undergo type I rapid apoptosis and
a synergistic induction of apoptosis was observed in these cells when
combined with Falcarinol. In contrast, most tumour cell lines undergo
type II apoptosis, which could account for differential affects between
Jurkat and MOLT-3 cells. Interestingly other agents which
synergistically induce apoptosis in leukaemia cells have been shown to
act via increasing the expression of DR5 but not DR4 via activation of
JNK signalling [58], further studies are required to determine if this is
the mechanism of synergy induced by Falcarinol.
In this study, we demonstrated that Leptomycin B (LMB)
significantly induced apoptosis and decreased proliferation in all cell
lines, although synergistic interactions were not observed additive
responses were seen. Leptomycin B is known to induce apoptosis
through blocking CRM-1 mediated nuclear exports leading to
accumulation of pro-apoptotic proteins such as p53, FOXO
transcription factors and HSP 27 within the nucleus [44,59]. In 2006, a
study showed synergistic actions of Leptomycin B with the ABL kinase
inhibitor Imatinib Mesylate [46]. These actions are thought to be
brought about by Leptomycin B trapping BCR/ABL in the nucleus and
thus BCR/ABL cannot induce proliferation/survival as this signal is
normally passed on to cytoplasmic mediators [46]. Nuclear
accumulation of p53, FOXO transcription factors and HSP 27 within
Citation: Zaini R, Small SLH, Cross NA, Le Maitre CL (2015) Differential Interactions of Falcarinol Combined with Anti-Tumour Agents on
Cellular Proliferation and Apoptosis in Human Lymphoid Leukaemia Cell Lines. J Blood Disorders Transf 6: 258. doi:
10.4172/2155-9864.1000258
Page 6 of 9
J Blood Disorders Transf
ISSN:2155-9864 JBDT, an open access journal Volume 6 • Issue 2 • 1000258
the nucleus may induce apoptosis, however the lack of synergy
observed within the current study with Leptomycin suggests that
Falcarinol induced apoptosis is unlikely to be via increased signalling
through CRM-1 responsive pro apoptotic transcription factors such as
FOXO.
Combination treatment with Falcarinol and the HDACi,
Sulforaphane showed only an additive response in CCRF-CEM and
Jurkat cells after 24 h whereas a significant synergistic effect was
observed in MOLT-3 cells as determined by caspase-3 activity and
nuclear morphology. Sulforaphane has been previously demonstrated
to induce apoptosis within U937 cells and Jurkat cells [60,61].
Moreover, Sulforaphane significantly inhibited cellular growth within
CCRF-CEM and Jurkat cells following combined treatment with
Falcarinol. HDACi inhibit proliferation and induce apoptosis via re-
expression of tumour suppressor genes, such as p21 that have been
epigenetically silenced by chromatin modifications [60]. HDACi
prevents histone deacetylation and since acetylated chromatin is more
transcriptionally active it is associated with tumour suppressor gene
re-expression. Thus the synergistic responses seen within MOLT-3
highlight the potential role of Falcarinol in modulating tumour
suppressor gene expression, or their downstream pro-apoptotic
actions.
For the first time this study has shown that combination treatments
with the bioactive compound: Falcarinol, isolated from Daucus carota
together with a number of classical inducers of apoptosis and
chemotherapy agents, could act in a synergistic, additive or inhibitory
manner dependent on cell line and combination treatments.
Interestingly the related aliphatic C17Polyactelene Panaxynol [62],
isolated from Panax notoginseng has also been shown to induce
apoptosis in the leukaemia cell line HL-60 via activation of PKCδ,
caspase 3 activation and cleavage of poly(ADP[adenosine
diphosphate]-ribose) polymerase (PARP) [63]. Here we also show
Falcarinol results in activation of caspase 3 and induction of apoptosis.
However to date, the direct actions of polyacetylenes in the induction
of apoptosis have not been elucidated, many studies demonstrate
induction of classical pathways involved in apoptosis, however these
do not indicate whether these are the primary targets for
polyacetylenes or merely products of the apoptotic cascade. The
synergistic responses observed following combination of Falcarinol
with classical apoptosis inducers seen here, highlight differential
mechanism of apoptosis in leuakemia cell lines. CCRF-CEM, the most
sensitive cell line to Falcarinol, failed to demonstrate any synergistic
responses when combined with apoptotic inducers. Jurkat cells treated
with Falcarinol and DR5 agonist showed synergistic induction of
apoptosis suggesting Falcarinol may increase expression of DR5 within
Jurkat cells but not CCRF-CEM and MOLT-3 cells. However as Jurkat
cells are known to undergo type I apoptosis unlike most tumour cells,
Falcarinol may be increasing DR5 activation via the caspase 8
activation of caspase 3 whilst not affecting the intrinsic (mitochondrial
pathways), hence the lack of response seen in CCRF-CEM and MOLT
3 cells [64]. Whilst synergistic induction of apoptosis was observed in
MOLT-3 cells when Falcarinol was combined with proteosome
inhibitors and HDACi, suggesting direct actions on proteins involved
in apoptotic signaling. Thus highlighting the need for further
investigation into the mechanisms of apoptotic induction by
Falcarinol alone and in combination across a spectrum of leukaemia
cell lines and primary leukaemia cells.
The combined effect of Falcarinol on induction of cell death and
inhibition of cellular proliferation indicate that this agent could be
beneficial in improving Leukaemia therapy, however combination
therapies could also prevent the actions of some anti-tumour agents.
Identification of interactions between natural bioactive compounds
with anti-cancer drugs may provide new pathways to target cancerous
cells. Furthermore, since some combinations enhance apoptosis but
some inhibit apoptosis it may be important to consider these
interactions for dietary advice during therapy.
Funding
This study is funded by the Saudi Ministry of Health.
Acknowledgements
The Authors would like to thank Dr Kerstin Brandt, Newcastle
University, Newcastle, NE1 7RU, UK for supply of the Falcarinol.
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Citation: Zaini R, Small SLH, Cross NA, Le Maitre CL (2015) Differential Interactions of Falcarinol Combined with Anti-Tumour Agents on
Cellular Proliferation and Apoptosis in Human Lymphoid Leukaemia Cell Lines. J Blood Disorders Transf 6: 258. doi:
10.4172/2155-9864.1000258
Page 9 of 9
J Blood Disorders Transf
ISSN:2155-9864 JBDT, an open access journal Volume 6 • Issue 2 • 1000258
... Additional safety issues Potential herb-related augmenting effect of chemotherapy proliferation in human lymphoid leukaemia cell lines (Zaini et al., 2015); oil extract fractions possess potential anti-cancer activity on the human breast adenocarcinoma cell lines (Shebaby et al., 2014) ...
... In addition the extract of the plant has also a strong immunomodulatory and interferon-like activity; it inhibits the progression of tumor and endothelial cell, and reduces the production of the angiogenic protein fibroblastic growth factor by tumor cells (Medenica et al., 1997). Increased cell death has been observed with exposure of ginger leaf to human colorectal cancer cells (HCT116, SW480 and LoVo cells) ; galbanic acid isolated from Ferula asafoetida exerts anti-cancer activity in association with anti-angiogenic and anti-proliferative actions (Kim et al., 2011); Falcarinol (a polyacetylene isolated from Daucus carota) showed a significant induction of apoptosis of Human Lymphoid Leukaemia Cell Line (Jurkat cells) with a Death Receptor 5 agonist (DR5), while CCRF-CEM cells showed only an additive response (Zaini et al., 2015); nanovesicles isolated from Citrus lemon have shown to activate a TRAIL-mediated apoptotic cell death leading to inhibition of cancer cell proliferation in different tumor cell lines (Raimondo et al., 2015). Herbal compounds also have been found to prevent and reduce chemotherapy-induced mucositis through anti-inflammatory and antioxidant mechanisms (e.g. ...
... The anticancer effects of FA are its best characterized bioactive property [76][77][78][79][80][81][82][83]. FA also has positive metabolic effects. ...
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6-Mercaptopurine (6MP) and methotrexate are the backbone of continuation therapy for childhood acute lymphoblastic leukemia (ALL). In studies of oral 6MP and methotrexate, indices of chronic systemic exposure to active metabolites of these agents, namely, red blood cell (RBC) concentrations of methotrexate polyglutamates (MTXPGs) and thioguanine nucleotides (TGNs) have positively correlated with event-free survival (EFS). Our objective was to evaluate whether MTXPGs, TGNs, and the dose intensity of administered methotrexate and 6MP were prognostic in the setting of a treatment protocol in which all treatment was coordinated through a single center, and the weekly doses of methotrexate were given parenterally. On protocol Total XII, 182 children achieved remission and received weekly methotrexate 40 mg/m2 parenterally and daily oral 6MP, interrupted every 6 weeks during the first year by pulse chemotherapy. A total of 709 TGN, 418 MTX-PG, and 267 thiopurine methyltransferase (TPMT) measurements, along with complete dose intensity information (dose received divided by protocol dose per week) for 19,046 weeks of 6MP and methotrexate, were analyzed. In univariate analyses, only higher dose intensity of 6MP and of weekly methotrexate were significant predictors of overall EFS (P = .006 and .039, respectively). The occurrence of neutropenia was associated with worse outcome (P = .040). In a multivariate analysis, only higher dose intensity of 6MP (P = .020) was a significant predictor of EFS, with lower TPMT activity (P = .096) tending to associate with better outcome. 6MP dose intensity was also associated (P = .007) with EFS among patients with homozygous wild-type TPMT phenotype. Lower 6MP dose intensity was primarily due to missed weeks of therapy and not to reductions in daily dose. We conclude that increased dose-intensity of oral 6MP is an important determinant of EFS in ALL, particularly among those children with a homozygous wild-type TPMT phenotype. However, increasing intensity of therapy such that neutropenia precludes chemotherapy administration may be counterproductive.
Article
Apoptosis triggered by exogenous and endogenous stimuli such as ultraviolet radiation, oxidative stress, and genotoxic chemicals is a crucial phenomenon within biological systems. DNA damage activates and stabilizes p53 in nucleus and cytoplasm and regulates other proteins that stimulate intrinsic and extrinsic apoptotic pathways. Apoptosis is morphologically distinct from that of necrosis and both the phenomena depend on the types, developmental stages, physiological environment of tissues and the nature of death signal. Malfunctioning of apoptotic pathway may cause human diseases like cancer, neurodegenerative and autoimmune disorders. Recently, potent apoptosis-inducing compounds associated with human health have been recorded that prevent tumor promotion, progression, and the occurrence of cellular inflammatory responses. Certain photosensitizing drugs are being employed in photodynamic therapy to induce apoptosis for the treatment of cancer and non-cancerous cells. This review emphasizes the molecular mechanisms of apoptosis, associated diseases and certain therapeutic agents implicated in the elimination of malignant cells.
Article
Resistance to chemotherapy is still a challenge for the treatment of acute myeloid leukemia. Combination use of histone deacetylase inhibitors (HDACIs) and proteasome inhibitors may provide a potential way to overcome drug resistance. One of the HDACIs, valproic acid (VPA), and a proteasome inhibitor, bortezomib (BOR), were assessed. Co-exposure of cells to VPA and BOR inhibited proliferation, arrested the cell cycle in G0-G1 phase and induced apoptosis in both HL60 and HL60A cells. These events were accompanied by the inhibition of cyclin D1 and human telomerase reverse transcriptase (hTERT) as well as telomerase activity. Moreover, synergism of proliferation inhibition was found in HL60A, superior to the additivity in HL60. The effects of combination treatment on cell cycle arrest and telomerase activity inhibition in HL60A were also more striking than those in HL60. In summary, our findings provide an insight into future clinical applications of the VPA-BOR combination regimen for AML, especially in those cases which are resistant to conventional chemotherapy.
Article
In vitro studies with drug combinations of carboplatin and other anticancer agents were carried out in MOLT-3 human lymphoblastic leukemia and HL-60 human promyelocytic leukemia cell lines. Cells were incubated for 3 days in the presence of various concentrations of carboplatin and other drugs and cell growth inhibition was determined by MTT assay. The antitumor effects of the drug combinations at id50 were analyzed using an improved isobologram.In MOLT-3 cells, supra-additive (synergistic) effects were observed for carboplatin in combination with cytosine arabinoside, mitoxantrone and CPT-11. Additive effects were observed for combinations of carboplatin with bleomycin, daunorubicin, doxorubicin, etoposide, 6-mercaptopurine, and vincristine. Sub-additive and protective (antagonistic) effects were observed with methotrexate. Synergistic or antagonistic effects for combinations of carboplatin and CPT-11, cytosine arabinoside, mitoxantrone and methotrexate were also observed in HL-60 cells.These findings suggest that carboplatin has additive or synergistic cytotoxic effects with most of the agents tested. Determination of the usefulness of these drug combinations awaits appropriate in vivo experiments that should assess both tumorcidal effects and possible increased toxicity. The simultaneous administration of carboplatin and methotrexate would be of little effect. To find optimal schedules for this combination, further pre-clinical studies of various combinations schedule would appear to be warranted.
Article
Leptomycin B (LMB), which is originally isolated from Streptomyces, possesses anti-tumor properties in vivo and in vitro. Though it was previously reported that LMB induces cell cycle arrest and p53-mediated apoptosis in certain cancer cells, however, the mechanism by which LMB induces apoptosis remains poorly understood. Here, we investigated the mechanisms of apoptosis induced by LMB in U937 cells. Treatment with LMB concentration-dependently induced cytotoxicity and apoptosis in U937 cells that correlated temporally with activation of caspases and down-regulation of Mcl-1 and XIAP. LMB did not change the expressions of Bcl-2 or Bax. A broad spectrum caspase inhibitor, z-VAD-fmk, blocked caspase-3 activation and elevated the survival in LMB-treated U937 cells, suggesting that caspase-3 activation is critical for LMB-induced apoptosis. Interestingly, Bcl-2 overexpression that blocked cytochrome c release by LMB effectively attenuated the apoptotic response to LMB, suggesting that LMB-induced apoptosis is mediated through the mitochondrial pathway. Antioxidants or antioxidant enzymes had no effects on LMB-induced apoptosis. Data of flow cytometry analysis using 2′,7′-dichlorofluorescein-diacetate further revealed no reactive oxygen species (ROS) generation by LMB, indicating that apoptosis induced by LMB is ROS-independent. However, the apoptotic response to LMB was not shown in U937 cells pretreated with the sulfhydryl group-containing antioxidant N-acetylcysteine (NAC). Further analysis suggested that NAC directly binds LMB and abolishes the apoptotic effects of LMB. Collectively, these findings suggest that LMB potently induces apoptosis in U937 cells, and LMB-induced apoptosis in U937 cells is related with cytochrome c release, activation of caspases, and selective down-regulation of Mcl-1 and XIAP.
Article
New therapies for leukaemia are urgently needed. Carrots have been suggested as a potential treatment for leukaemia in traditional medicine and have previously been studied in other contexts as potential sources of anticancer agents. Indicating that carrots may contain bioactive compounds, which may show potential in leukaemia therapies. This study investigated the effects of five fractions from carrot juice extract (CJE) on human lymphoid leukaemia cell lines, together with five purified bioactive compounds found in Daucus carota L, including: three polyacetylenes (falcarinol, falcarindiol and falcarindiol-3-acetate) and two carotenoids (beta-carotene and lutein). Their effects on induction of apoptosis using Annexin V/PI and Caspase 3 activity assays analysed via flow cytometry and inhibition of cellular proliferation using Cell Titer Glo assay and cell cycle analysis were investigated. Treatment of all three lymphoid leukaemia cell lines with the fraction from carrot extracts which contained polyacetylenes and carotenoids was significantly more cytotoxic than the 4 other fractions. Treatments with purified polyacetylenes also induced apoptosis in a dose and time responsive manner. Moreover, falcarinol and falcarindiol-3-acetate isolated from Daucus carota L were more cytotoxic than falcarindiol. In contrast, the carotenoids showed no significant effect on either apoptosis or cell proliferation in any of the cells investigated. This suggests that polyacetylenes rather than beta-carotene or lutein are the bioactive components found in Daucus carota L and could be useful in the development of new leukemic therapies. Here, for the first time, the cytotoxic effects of polyacetylenes have been shown to be exerted via induction of apoptosis and arrest of cell cycle.