Asid
Betulinik ubat barah
Abstract
Betulinic Acid (BetA) and
its derivatives have been extensively studied in the past for their anti-tumor
effects, but relatively little is known about its precursor Betulin (BE). We
found that BE induces apoptosis utilizing a similar mechanism as BetA and is
prevented by cyclosporin A (CsA). BE induces cell death more rapidly as
compared to BetA, but to achieve similar amounts of cell death a considerably
higher concentration of BE is needed. Interestingly, we observed that
cholesterol sensitized cells to BE-induced apoptosis, while there was no effect
of cholesterol when combined with BetA. Despite the significantly enhanced
cytotoxicity, the mode of cell death was not changed as CsA completely
abrogated cell death. These results indicate that BE has potent anti-tumor
activity especially in combination with cholesterol.
Citation: Mullauer FB,
Kessler JH, Medema JP (2009) Betulin Is a Potent Anti-Tumor Agent that Is
Enhanced by Cholesterol. PLoS ONE 4(4): e1. doi:10.1371/journal.pone.0005361
Editor: Mikhail V.
Blagosklonny, Ordway Research Institute, United States of America
Received: February 12, 2009;
Accepted: March 23, 2009; Published: April 28, 2009
Copyright: © 2009 Mullauer
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.
Funding: The work is
supported by the “stichting nationaal fonds tegen kanker” http://www.tegenkanker.nl/.
The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing interests: The
authors have declared that no competing interests exist.
Introduction
Triterpenoids
are extensively studied for the potential use as anticancer agents. One of the
most promising compounds in this class is Betulinic Acid (BetA), but its effect
is limited by the poor solubility of the compound. A lot of effort is therefore
put into the development of derivatives of BetA with the goal to develop even
more powerful compounds and to achieve better solubility for enhanced in vivo
administration [1]–[3]. BetA has been modified at many
different positions including C1-4, C-20, C-28 and A-, D- and E ring with
different outcomes [2], [4]. For example, Kvasanica et al found
3beta-O-phthalic esters from BetA more cytotoxic and polar in comparison to
BetA itself [5]. In contrast, generation of different
C-28 ester derivatives did not result in enhanced cytotoxicity [4]. On the other hand, C-28 amino acid
conjugates made by Jeong et al showed improved selective toxicity and
solubility [6] and a C-3 modified BetA derivative has
shown promising results in a human colon cancer xenograft model [2].
BetA
can be found in numerous different plants, but it can also be obtained by a
simple 2 step reaction from its more abundantly available precursor molecule
Betulin (BE) [3]. BE is easily isolated and therefore
plays an important role as raw material for the production of BetA and other
biologically active compounds [7]. BE itself has been shown in the past
to only possess limited or no cytotoxic effects on cancer cells [5], [8]. For example it was shown to be
inactive against MEL-2 (melanoma) cells when compared to other BetA derivatives
[9]. Several other melanoma lines (G361,
SK-MEL-28) leukemia lines (HL60, U937, K562), and neuroblastoma (GOTO, NB-1)
cell lines were also found to be more resistant to BE than to other tested
lupane triterpenes [10]. In contrast, a recent report found BE
to be active against colorectal (DLD-1), breast (MCF7), prostate ( PC-3) and
lung (A549 ) cancer cell lines [11], and for A549 it was shown that
apoptosis was induced [12]. Apoptosis is one of the major cell
death pathways induced by anti tumor agents. In principle, two main pathways
can be distinguished, the extrinsic or death receptor pathway and the intrinsic
or mitochondrial pathway with the latter being regulated by the Bcl-2 family of
proteins [13]. Numerous studies have shown that BetA
induces apoptosis via the mitochondrial pathway [14]–[17], however, to our knowledge, it is
currently not clear how BE induces cell death. Here we show that apoptosis
induction by BE does not involve the death receptor pathway, but is dependent
on the mitochondria. Nevertheless, similar as we have previously shown for BetA
[17], cytochrome c release and caspase
activation occur independently of the Bcl-2 family proteins but are blocked in
the presence of cyclosporin A (CsA), an inhibitor of the mitochondrial
permeability transition (PT) pore. Furthermore we found that cholesterol
strongly enhances the cytotoxic effects induced by BE but not BetA. Our results
suggest that BE should not be regarded as an inactive precursor, but as a
potent anti-tumor agent.
Chemicals
Betulin (≥98% pure; Sigma-Aldrich, St
Louis, MO, USA) and Betulinic Acid (≥99% pure; BioSolutions Halle, Germany)
were dissolved in DMSO at 4 mg/ml, cholesterol (Sigma-Aldrich) was dissolved at
5 mM in DMSO. Aliquots were kept frozen. Propidium iodide (PI), zVAD.fmk
(benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone),etoposide
and cyclosporin A were purchased from Sigma-Aldrich, Mitosox was obtained from
Invitrogen (Carlsbad, CA, USA).
Antibodies
Anti-PARP (#9542; Cell Signaling
Technology, Danvers, MA, USA) and anti-cytochrome c (clone 6H2.B4; BD
Biosciences, San Diego, CA, USA) were used.
Cell lines: A549 and Hela were obtained
from the ATCC, FADD-deficient, Caspase 8- deficient and control Jurkat cells
(JA3) were kindly provided by Dr John Blenis (Harvard Medical School, Boston),
Jurkat cells over-expressing Bcl-2 by Dr Jannie Borst (NKI, Amsterdam) and
Bax/Bak double knockout (DKO) mouse embryonic fibroblasts (MEFs) and wild-type
control MEFs were from Dr Stanley Korsmeyer.
Cell death analysis
Overall cell death was assessed as
previously described [18] by PI exclusion assay. Briefly, cells
were incubated with 1 µg/ml PI and measured by flow cytometry.
DNA fragmentation
Cells were incubated in Nicoletti buffer
containing 50 µg/ml PI for at least 24 hours before analysis via flow
cytometry.
Western blot analysis
(immunoblotting)
Cells were lysed using Triton X-100
buffer and for protein quantification a BCA kit from PIERCE was used. SDS-PAGE
was performed and proteins were transferred onto a PVDF transfer membrane
(Amersham Biosciences). Blocking of unspecific binding sites was achieved by
incubation of the membrane in 5% low fat milk powder in PBS/0.2% Tween-20
(blocking buffer) for 1 hour at room temperature. Primary antibody incubation
was performed overnight at 4°C and secondary antibody (HRP labeled) incubation
for 2 hours at room temperature. For chemiluminescent detection ECL from
Amersham Biosciences was used in combination with a LAS-3000 imaging system.
ROS detection
For ROS measurements the highly
selective dye for mitochondrial superoxide Mitosox was used. Cells were
incubated with 5 µM Mitosox in pre-warmed tissue culture medium at 37°C for 10
min before flow cytometry analysis.
Cytochrome c release by FACS
staining
Cytochrome c release was measured as
previously described by Waterhouse et al [19]. First, outer cell membrane
permeabilization was achieved by incubation for 5–10 minutes with 50 µg/ml
digitonin in PBS containing 100 mM KCl. Cells were then fixed in 4%
paraformaldehyde for 30 minutes at room temperature, washed and incubated in
blocking buffer (3% BSA, 0.05% saponin, 0.02% azide in PBS supplemented with
normal goat serum, dilution 1:200). Anti cytochrome c incubation was done
overnight at 4°C and for flow cytometric detection a FITC conjugated secondary
antibody was applied.
MTT assay
cells were incubated in the presence of
40 µg/ml MTT reagent for 2 hours at 37°C. During the incubation period
appearance of purple formazan structures was followed by phase-contrast light
microscopy.
Cholesterol strongly
enhances cytotoxic effects of BE but not BetA
Previously we have shown that BetA
induces cell death in Jurkat T leukemia cells in a concentration and
time-dependent fashion [18]. Here we show that low concentrations
(5 µg/ml) of BetA are non toxic up to 48 hours incubation and show limited cell
death after 72 hours (Figure 1A). In contrast, when 7.5 µg/ml BetA
or more is used almost all cells are PI positive after 48 to 72 hours (Figure 1A). To analyze whether Betulin
(BE), the precursor of BetA, is capable of inducing cell death we titrated BE
on Jurkat T Leukemia cells. In contrast to previous reports we show here that
BE is capable of killing cells, but required higher concentrations than BetA.
However, it appeared that cell death induced by BE is more efficient after 12
hours when compared to BetA and maximum cell death is achieved after 24 hours (Figure 1C).
Figure 1. Cholesterol
strongly enhances cytotoxic effects of BE but not BetA.
Jurkat cells were treated
with the indicated concentrations of BetA (A), BetA in combination with 5 µM
cholesterol (B), BE (C), BE in combination with 5 µM cholesterol (D) or various
concentrations of cholesterol only (E). Cell death was monitored after 12, 24,
48 and 72 hours using PI exclusion. A549 lung cancer (F) and HeLa cervix
carcinoma (G) cell lines were treated with 5 µM cholesterol (chol), 5 µg/ml BE
(5 BE) or the combination of 5 µg/ml BE with 5 µM cholesterol (5 BE+chol) and
after 24 hours cell death was analyzed via PI exclusion.
doi:10.1371/journal.pone.0005361.g001
We have found previously that when using
the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)
assay to measure BetA [18] or BE (unpublished data) induced
cytotoxicity, results were much more pronounced when compared to other assays
such as PI exclusion and clonogenic survival [18]. This decrease in MTT conversion is
likely the result of a direct effect of BetA on the mitochondria and was
accompanied by a different morphological appearance of the formazan
precipitates. While normal formazan formation shows a punctuate appearance,
BetA and BE-induced formazan formation shows the rapid appearance of needle-like
structures on the cell surface (Figure S1). Interestingly, cholesterol,
which shares some structural similarities with BE and BetA, has been reported
to have a comparable effect in the MTT assay [20]–[22] (Figure S1). This suggests that cholesterol,
BetA and BE may share common targets in the cell. To clarify if this feature is
related to the cytotoxicity of these compounds we decided to analyze the effect
of cholesterol on cell death and combine cholesterol with either BetA or BE and
measure PI exclusion after various time points. Cholesterol itself did not
induce cell death in Jurkat cells (Figure 1E) and it did not enhance
cytotoxicity of BetA at all time points measured (Figure 1B). However, the combination of BE
with cholesterol resulted in massive cell death in Jurkat cells even when very
small concentrations of BE were used (2.5 and 5 µg/ml BE, Figure 1D). To rule out that this is a cell
type specific effect we analyzed cell death in A549 (lung carcinoma) and HeLa
(cervical carcinoma) cells exposed to either BE or BE in combination with
cholesterol. Similar to what was observed with the Jurkat cells, both solid
cancer cell lines displayed massive cell death when treated with the
combination of BE and cholesterol, whereas BE by itself showed only minor
toxicity at the concentration used (Figure 1F and 1G).
BE/Cholesterol induces
apoptosis in Jurkat cells
To identify the nature of cell death
induced by BE/Cholesterol we investigated the apoptotic pathway. Apoptosis has
been previously reported to be the cell death pathway induced by BE in A549
lung cancer cells [12]. We assessed DNA fragmentation as an
apoptosis read-out in Jurkat cells treated for 24 hours with either
cholesterol, BE or the combination of both. In cells treated with cholesterol
only, DNA fragmentation was completely absent (Figure 2A), consistent with the lack of
cell death. BE, at 5 µg/ml, induced only moderate DNA fragmentation. However,
when combined with cholesterol DNA was clearly fragmented (Figure 2A). To verify these results we
performed immunoblotting for the classical caspase target PARP and observed
similar effects: Upon BE treatment PARP was processed to some extent and this
was strongly enhanced by addition of cholesterol (Figure 2B). Importantly, both, DNA fragmentation
and PARP cleavage were blocked when cells were pre-treated with zVAD.fmk (a
pan-caspase inhibitor) confirming that both are caspase-mediated events (Figure 2A and 2B).
Figure
2. BE/cholesterol induces apoptosis in Jurkat cells.
(A) Jurkat cells were
pretreated with 20 µM zVAD.fmk for at least one hour prior to addition of either
DMSO, 5 µM cholesterol (5 Chol), 5 µg/ml BE (5 BE) or 5 µg/ml BE in combination
with 5 µM cholesterol (5 BE+chol). After 24 hours DNA fragmentation was
assessed by FACS analysis of propidium iodide (PI) stained nuclei. (B) Jurkat
cells were treated as described in (A) but after 24 hours PARP cleavage was
assessed by immunoblotting. The protein kinase ERK was used as a loading
control.
The death receptor pathway
is not involved in BE/cholesterol induced apoptosis
Cholesterol is an important constituent
of cell membranes where it plays a crucial role in maintaining integrity and
fluidity [23]. In addition, cholesterol-enriched
micro-domains, so called lipid rafts, are important signal transduction
platforms [24], which have been related to apoptosis [25] and changes in plasma cholesterol
levels have been associated with Fas-FADD complex formation and caspase-8
activation [26], [27]. BetA has been shown to induce
apoptosis independently of the extrinsic pathway [28]. However, because of the strong
apoptosis-enhancing effects of cholesterol when combined with BE, we decided to
investigate the involvement of this pathway by applying BE/cholesterol on
Jurkat cells either deficient for FADD or caspase-8. Recently we showed that
the FADD and caspase-8 deficient cells were completely resistant to Fas-induced
apoptosis [17]. Here this resistance was further
confirmed using TRAIL (Figure 3A). Despite the resistance towards
the extrinsic pathway, neither cell line showed decreased DNA fragmentation
when treated with BE/cholesterol (Figure 3B), indicating that the death
receptor pathway is not involved in BE/cholesterol-induced apoptosis.
Figure
3. The death receptor pathway is not involved in BE/cholesterol induced apoptosis.
Jurkat control (JA3), FADD
deficient (FADD ko) or caspase-8 deficient (Casp-8 ko) cells were treated with
TRAIL (0.5 µg/ml plus 1 µg/ml anti-FLAG) (A) or with either 5 µM cholesterol
(chol) or 5 µg/ml BE in combination with 5 µM cholesterol (5 BE+chol) and after
24 hours DNA fragmentation was analyzed.
BE/cholesterol induced
apoptosis is mechanistically related to BetA induced apoptosis
BetA induced apoptosis has been clearly
linked to the mitochondria [14]–[17] with the consistently described
features of cytochrome c release and induction of reactive oxygen species (ROS)
[28]–[31]. These events were initially described
to be Bcl-2 family dependent [15], [16], however, our recent evidence suggests
only a minor role for the Bcl-2 family proteins. Instead we proposed a direct
effect on the PT-pore [17]. To test if BE/cholesterol induces
apoptosis via similar mechanisms as BetA we investigated the mitochondrial
pathway of apoptosis.
BE/cholesterol showed clear cytochrome c
release in Jurkat cells. Importantly, there was only a slight difference in
cytochrome c release in the Bcl-2 over-expressing cells (Figure 4A), but this difference was statistically
not significant (paired t-test). Jurkat cells over-expressing Bcl-2 were
completely resistant to etoposide (Figure. 4A). In contrast to the lack of
effect of Bcl-2 over-expression, CsA provided almost complete protection (Figure 4A). To determine if ROS are
produced upon BE/cholesterol treatment we used a dye specifically detecting
mitochondrial superoxide. Both wildtype as well as Bcl-2 over-expressing cells
showed clear increase in ROS, strikingly this was again abolished in the
presence of CsA (Figure 4B). To verify that these events
resemble the amount of apoptosis and overall cell death we measured DNA
fragmentation and PI exclusion respectively. Bcl-2 over-expression did not provide
any protection whereas CsA effectively prevented both, apoptosis and cell death
(Figure 4C and 4D). In order to find out if
Bcl-2 over-expression causes a delay in apoptosis as is the case with BetA [17], we performed a kinetic analysis. Cell
death and DNA fragmentation were measured after various time points from 0–24
hours. At all time points we did not observe any difference in sensitivity to
BE/cholesterol, further underscoring the lack of inhibition by Bcl-2 (Figure 4E and 4F). These results suggest
that BE/cholesterol kills Jurkat cells by inducing mitochondrial damage that
leads to cytochrome c release and apoptosis which is completely independent of
Bcl-2.
Figure
4. BE/cholesterol induced apoptosis is not affected by Bcl-2 over-expression
but is inhibited in the presence of cyclosporin A.
(A) Jurkat control (wt) or
Bcl-2 over-expressing cells (Bcl-2) were treated as indicated (5BE = 5 µg/ml
BE; chol = 5 µM cholesterol; CsA = 5 µg/ml cyclosporin A), after 24 hours
intracellular staining for cytochrome c release was performed. (B, C, D) Jurkat
control (wt) or Bcl-2 over-expressing cells were treated with 5 µg/ml BE/ 5 µM
cholesterol either in the absence or presence of 5 µg/ml cyclosporin A. After
24 hours ROS (B), DNA fragmentation (C) and overall cell death (D) were
assessed by FACS analysis. (E, F) Jurkat control (wt) or Bcl-2 over-expressing
cells were treated with 5 µg/ml BE/ 5 µM cholesterol and PI exclusion (E) or
DNA fragmentation (F) were measured after 0, 4, 8, 16 and 24 hours.
To further determine the efficacy of
BE/cholesterol and to find out if Bax and Bak are involved in BE/cholesterol
induced cytotoxicity we used Bax/Bak double-knockout (DKO) mouse embryonic
fibroblasts (MEFs). DKO MEFs are resistant to drugs such as etoposide,
staurosporine, UVC or actinomycin D, all targeting the Bcl-2 family regulated
mitochondrial pathway [32]. We measured PI exclusion and found
DKO MEFs to be sensitive to BE/cholesterol, as a control for the functionality
of the cells etoposide was included (Figure 5A). We assessed if apoptosis was
induced like in BetA treated cells by analyzing PARP cleavage. PARP was clearly
processed in wildtype as well as in DKO MEFs, suggesting that Bax and Bak are
not essential in BE/cholesterol induced apoptosis (Figure 5B). Also cytochrome c release was
not prevented in DKO MEFs (Figure 5C), further substantiating that Bax
and Bak are not required for BE/cholesterol mediated cytotoxicity. Similar to
Jurkat cells, CsA provided complete protection against cell death (Figure 5A), apoptosis (Figure 5B) and cytochrome c release (Figure 5C), confirming the crucial role for
the mitochondrial permeability transition in BE/cholesterol induced
cytotoxicity.
(A) Wildtype (wt) or Bax/Bak
double knockout (DKO) mouse embryonic fibroblasts (MEFs) were treated as
indicated and after 24 hours cell death was assessed by PI exclusion. Etoposide
was included as a control for functionality of the cells. (B) Wt or DKO MEFs
were treated as indicated and after 24 hours cells were subjected to immunoblotting
to determine PARP processing. ERK was used as control for equal protein
amounts. (C) Wt and DKO MEFs were treated as indicated for 24 hours before
measuring cytochrome c release by intracellular FACS staining.
Discussion
BE
is a natural compound, which contains derivatives that have been shown to possess strong anti-tumor properties [7], [33]. Here we provide evidence that BE
itself, especially in combination with
cholesterol (BE/cholesterol), is very
potent in killing cancer cells in vitro (Figure 1). BE/cholesterol induces apoptosis
in a similar manner as BetA and does not involve the extrinsic pathway of
apoptosis (Figure 3), but instead apoptosis depends on
the mitochondrial pathway (Figure 4). However, as we reported for
BetA, this pathway is activated in an unconventional manner as cytochrome c
release and apoptosis are induced in cells over-expressing Bcl-2 (Figure 4) or in cells deficient for Bax/Bak
(Figure 5), while both events are blocked by
CsA (Figure 4 and Figure 5). This indicates that permeability
transition is pivotal in the process of BE/cholesterol induced cytotoxicity.
Despite
the strong similarities, and the almost identical structure of BE and BetA,
there are also important differences in comparison to BetA induced apoptosis.
We previously showed that Bcl-2 over-expression delayed BetA-induced apoptosis[17], but curiously in the case of
BE/cholesterol it has very limited effect on the amount of cytotoxicity induced
(Figure 4). Furthermore, CsA by itself
provides much stronger protection in the case of BE/cholesterol in Jurkat
cells, while BetA treated Jurkat cells are only completely protected when a
combination of CsA with Bcl-2 over-expression is used.
This
difference between BetA and BE/cholesterol is even more remarkable when
considering the time dependency of cytotoxicity of both molecules: For BetA the
maximum effect requires around 48–72 hours and a dose of 7.5–10 µg/ml (Figure 1A and 1B), while BE/cholesterol
induced death is already maximum at 24 hours. Nevertheless, CsA is capable of
providing efficient protection.
Striking
is the fact, that cholesterol strongly enhances the cytotoxic effects of BE but
not BetA (Figure 1B and 1D) whilst being completely
non-toxic on its own, even at very high concentrations (Figure 1E). Currently we do not know the
mechanism by which cholesterol acts as a “cytotoxicity-amplifier” for BE but it
likely involves membrane integrity. Cholesterol is abundantly present in the
plasmamembrane and it is possible that changes in cholesterol content can affect
the amount of BE that is taken up by a cell.
The
effect on MTT conversion to formazan (MTT measures mitochondrial enzymatic
activity [20], [34]) by all three compounds, BetA, BE and
cholesterol, suggests a common target in the mitochondria. Even though this is
clearly not directly related to cytotoxicity, as cholesterol on its own is
completely non-toxic, it may point to a mechanism that sensitizes cells to BE.
It is not clear how this is orchestrated but it could involve the mitochondrial
membrane, for instance mitochondrial PT pore opening. The exact composition of
the pore has yet to be established but adenine-nucleotide-translocator (ANT),
voltage-dependent-anion-channel (VDAC) and cyclophilin D are discussed as core
components in the currently accepted model [35]. PT pore opening is influenced by the
amount of cholesterol present in the mitochondrial membrane, cholesterol
affects VDAC function [35] and impairs ANT mediated PT through
altered membrane fluidity [36]. So cholesterol-induced effects on the
PT pore may facilitate BE-induced opening. Why this then does not influence
BetA-induced opening is unclear at this point and will require further
investigation. In this light it is also important to realize that Bcl-2
over-expression delays BetA-induced apoptosis [17], while CsA can only partially prevent
the induction of apoptosis. This suggests that BetA may has a direct effect on
the PT pore, which is blocked by CsA and maybe also induces a more classical
Bcl-2-dependent pathway to cytochrome c release. This latter seems absent when
using BE and may be the reason these compounds react slightly different to CsA
and potentially also cholesterol.
To
further evaluate the anti-tumor properties of BE/cholesterol in vivo studies
will be required. Preliminary results from a pharmacokinetic study using
triterpene extract (TE) mainly consisting of Betulin suggest that it is safe;
no signs of toxicity were observed in rats or dogs in a subchronic toxicity
study [37]. Another study investigated the
effects of BE on the central nervous system (CNS) with the conclusion that
there was no effect of BE on muscle tone and coordination in mice; doses up to
100 mg/kg bodyweight were used [38]. Interestingly another study explored
the antinociceptive properties of Betulin in mice and results suggest that it
is even more active than aspirin and paracetamol [39].
It
will be interesting to explore the combined effects of BE and cholesterol in
vivo. Because cholesterol is ubiquitously present in the body it is unlikely
that additional applied cholesterol is useful for in vivo effects of BE as an
anti-tumor agent. Our results indicate that the amount of cholesterol necessary
(5 µM) for enhanced in vitro effects of BE are about 1000 times lower than
normal plasma cholesterol levels in humans (5 mM). However the fast majority of
this cholesterol is contained in LDL or HDL and it is therefore difficult to
assess whether there is sufficient free cholesterol available to potentiate
BE-induced apoptosis in vivo. Adding more cholesterol may not bear any
significance though, but application of cholesterol containing Betulin-liposomes
may be an interesting mode of applying this cytotoxic agent. In summary we
conclude that Betulin by itself and in combination with cholesterol is a potent
anti-cancer agent in vitro and warrants further investigation in vivo.
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