Tailor-made fluorescent trilobolide to study its biological relevance

Article abstract of DOI:10.1021/jm500690j

Trilobolide (Tb) is a potent natural counterpart of thapsigargin, which has shown promising results in cancer clinical trials. Here, we report a rational approach to study intracellular localization and biological activity of this sesquiterpene lactone. We conjugated Tb with a green-emitting Bodipy dye attached by alternative linkers of different lengths. The live-cell imaging of the prepared bioconjugates brought clear evidence that Tb-Bodipy localized in the endoplasmic reticulum (ER) of various cancer cell lines. The localization signal was compared with ER-specific dyes. Cytotoxicity of Tb conjugates and impact on the mitochondrial physiology and nitric oxide release were also studied. The nitric oxide production and cytokine secretion in rat peritoneal cells indicate immunobiological potential of these lactone bioconjugates. In summary, our Tb-Bodipy conjugates could help us to reveal the molecular mechanism of trilobolide for its further potential use in biomedical applications.

Full text of DOI:10.1021/jm500690j

Article
pubs.acs.org/jmc
Tailor-Made Fluorescent Trilobolide To Study Its Biological Relevance
†
†
,§,‡
,†
,†
Michal Juras
́
e
̌
k, Silvie Rimpelova,
́
Eva Kmoníck
̌
ova,
́
*
̌ ́ ̌
Pavel Drasar,* and Tomas Ruml*
†
‡
§
Institute of Chemical Technology in Prague, 16628 Prague, Czech Republic
Faculty of Medicine in Pilsen, Charles University in Prague, 30166 Pilsen, Czech Republic
Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, v.v.i., 14220 Prague 4, Czech Republic
S Supporting Information
*
ABSTRACT: Trilobolide (Tb) is a potent natural counterpart
of thapsigargin, which has shown promising results in cancer
clinical trials. Here, we report a rational approach to study
intracellular localization and biological activity of this
sesquiterpene lactone. We conjugated Tb with a green-emitting
Bodipy dye attached by alternative linkers of different lengths.
The live-cell imaging of the prepared bioconjugates brought
clear evidence that Tb−Bodipy localized in the endoplasmic
reticulum (ER) of various cancer cell lines. The localization
signal was compared with ER-specific dyes. Cytotoxicity of Tb
conjugates and impact on the mitochondrial physiology and
nitric oxide release were also studied. The nitric oxide
production and cytokine secretion in rat peritoneal cells indicate
immunobiological potential of these lactone bioconjugates. In
summary, our Tb−Bodipy conjugates could help us to reveal the molecular mechanism of trilobolide for its further potential use
in biomedical applications.
INTRODUCTION
restores the original activity. Because SERCA protein is present
in all body tissues, targeted delivery based on a Tb-based
prodrug form must be developed. This way, the tumor cells
should be specifically recognized and killed by the activated
inhibitor. In order to characterize the cellular uptake and
intracellular localization of Tb for further rational design of
effective drug forms, we focused on a preparation and testing of
variously modified fluorescently labeled Tb. We altered the
length of a linker between Tb and a fluorescent tag that could
be replaced with the prodrug counterpart of choice. We
synthesized a series of differently linked Tb with a green-
emitting Bodipy (boron dipyrromethane) dye, since it is a
relatively small, membrane permeable dye often used for
■
Trilobolide (Tb), a structurally related compound to
intensively studied thapsigargin (Tg), is a sesquiterpene lactone
isolated from Laser trilobum (L.) Borkh. Previous studies have
revealed that Tb is an effective inducer of nitric oxide (NO)
1
production as well as of cytokines. Both lactones cause striking
expression of interferon γ (INF-γ) cytokine in human and rat
macrophages. Further, it has been reported that stimulatory
potential of these lactones depends on activation of MAP
kinases p38, ERK 1/2, and transcription factor NF-κB. It is
generally accepted that these biological effects are most likely
caused by the presence of 7,11-dihydroxyguaianolide skeleton
and fixed acyl substituents at positions C-3, C-8, and C-10 (ref
2
,3
6,7
labeling of lipoid compounds. With the prepared compounds
4
) in both Tb and Tg. Another attractive pharmacological
feature of these substances is their inhibition of sarco/
we imaged their distribution in cells of various tumor origins
and confirmed the expected intracellular localization in the
endoplasmic reticulum (ER). The biological activities of Tb and
its novel bioconjugates such as induction of NO release and
INF-γ production in rat macrophages and cytotoxic potency
across the cancer cell lines of different histogenic origin have
been also evaluated.
2
+
endoplasmic reticulum Ca -ATPase (SERCA). The top-
ography of SERCA binding site of Tg was described by Skytte
5
et al. It is assumed that Tb interacts in a similar way due to the
high similarity to Tg (there are only minor structural
differences). Empirical structure−activity relationship (SAR)
studies of Tg indicate that chemical modifications at O-8 and
O-2 positions do not hamper its biological activity. However,
Tb lacks the octanoyl moiety present in Tg at C-2 position;
therefore, derivatization of Tb molecule remains amenable
merely at O-8. Solvolysis of (S)-methylbutanoate yields a “triol”
RESULTS AND DISCCUSSION
■
Design and Synthesis of Tb−Bodipy Conjugates. A
sustainable and straightforward method to obtain fluorescent
5
which lacks the SERCA inhibitory activity. The subsequent
esterification of this product results in a regioselective insertion
Received: May 5, 2014
of an acyl group to preferred O-8 position. This partially
Published: September 8, 2014
©
2014 American Chemical Society
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Tb derivatives is the Cu(I)-catalyzed Huisgen 1,3-dipolar
linker. All azido derivatives were fully characterized by NMR,
HRMS, IR, and specific rotation (see Supporting Information
section 1.3).
Synthesized Tb-azido derivatives were introduced into
CuAAC cycloaddition with the prepared Bodipy dyes. All of
8,9
cycloaddition reaction taking place between terminal alkynes
and azides (“click chemistry”, CuAAC). Thus, both Tb and
Bodipy dye were tailored to intermediates suitable for click
chemistry. Generally, Tb was derivatized with linkers of various
lengths containing an azido moiety and a Bodipy dye with a
terminal acetylene group.
click reactions were catalyzed by CuSO ·5H O. Sodium
4 2
ascorbate served as a reducing agent, and the product formation
The synthesis of the Bodipy dye started with p-
was accelerated by catalytic amount of tris[(1-benzyl-1H-1,2,3-
10,11
16
hydroxybenzaldehyde (1) which was propargylated
(1a)
triazol-4-yl)methyl]amine (TBTA). The reactions were
and transformed to dipyrrole (1b) and subsequently to the
Bodipy dye (1c) according to a previously described
carried out in DMF under microwave irradiation at 60 °C for
1 h. All the prepared trilobolide−Bodipy conjugates (4c, 3b, 6a,
6b, and 7) were obtained in ≥95% purity. The spectral
characteristics and synthetic details are described in Supporting
Information, section 1.3, and fluorescence emission spectra are
presented in Figure S1. Prior to biological testing the samples
were repurified by short column chromatography. The purity
was verified by HPLC (see Supporting Information, section
1
2,13
method
(for further details see Supporting Information,
section 1.2). Polyethylene glycol (PEG) handles were
appended via click chemistry to further extend the variability
of linker‘s length (see Scheme 1).
a
Scheme 1. Synthesis of Bodipy Dyes
1
.4).
Cellular in Vitro Studies. The following in vitro
experiments were performed to evaluate the potential biological
activity of the compounds prepared in this work: live-cell
imaging of cellular uptake and intracellular localization of Tb−
Bodipy derivatives, cytotoxicity assay (WST-1), NO release,
and cytokine production.
Live-Cell Imaging of Tb−Bodipy Localization in Cancer
Cells. To elucidate the intracellular localization of Tb, its
fluorescent conjugates (3b−7) with Bodipy dye were prepared.
This type of fluorophore has already been successfully applied
to determine in vitro subcellular localization of a thapsigargin
1
7,18
derivative.
for staining of lipid droplets
Fluorescent Bodipy dyes commonly employed
19,20
are nontoxic. The intracellular
localization of Tb−Bodipy conjugates was examined in a
number of human cancer cell lines, such as U-2 OS
(
osteosarcoma), PC-3 and LNCaP (prostatic carcinomas),
MCF-7 (breast ductal carcinoma), HeLa (cervix carcinoma),
MiaPaCa-2 (pancreatic carcinoma), A549 (lung carcinoma)
cells, one mouse cancer cell line, C2C12 (myoblastoma), and
HEK 293T (human embryonic fibroblasts). Cellular uptake of
the tested derivatives was followed by means of fluorescence
microscopy in living cells at a number of time points: 10, 20,
and 40 min and 1, 3, 5, and 24 h. Interestingly, we found that
only compounds 3b and 6a efficiently localized inside the cells
(see Figure S2 (U-2OS cells) in Supporting Information). We
observed stable localization of fluorescence signal of these two
Tb derivatives (1 μM) already after 20 min of incubation with
all the tested cell lines. We have found almost no fluorescence
signal in any of the tested cell lines for conjugates 4c, 6b, and 7
used at 5 μM concentration even after 24 h of treatment (see
Supporting Information Figure S2). This might be due to the
extensive size of 6b and 7, which probably did not penetrate
through the cell plasma membrane. The lack of the
fluorescence signal of Tb conjugate 4c could be caused by
too short distance between both counterparts (Tb and Bodipy
dye) and/or by prompt clearance from cells. Cells stained only
with Bodipy dye were used as a control to exclude the
possibility that the localization of the Tb−Bodipy conjugates
inside the cells was driven by the Bodipy dye itself. We
observed localization of Bodipy dye in living cells in spherical
particles, which correspond to cellular lipid droplets. This
a
Reagents and conditions: (a) propargyl chloride, K CO , DMF−
2
3
toluene, 72 h, 60 °C; (b) (i) pyrrole, TFA, 3.5 h, rt; (ii) K CO , 30
min; (c) (i) DDQ, CHCl , 45 min, rt; (ii) BF Et O, Et N, 3 h, rt; (d)
azidoPEG -acid, CuSO ·5H O, sodium ascorbate, DMF, microwave,
6
2
2
3
3
3
2
3
4
4
2
0 °C, 1 h; (e) acetylenePEG -amine, EDCI, DMAP, HOBt, CH Cl ,
4
2
2
4 h, 0 °C → rt.
Trilobolide isolated from roots of Laser trilobum (L.) Borkh
as a white crystalline powder was used as a starting material for
the described syntheses. The available data published on a
closely related sesquiterpene lactone of Tb, i.e., Tg, show that
substitution at O-8 is the most effective option preserving the
biological potency of Tb. Solvolysis of trilobolide methyl-
butanoate at the O-8 position was carried out using N,N,N-
14
5
triethylamine in methanol according to a previously described
method.
5
,10,15
Selective acylation of triol 2a (see Scheme 2) at
the same position was accomplished by reaction with organic
acids containing azido moiety in the presence of N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride
(
EDCI) and p-N,N-dimethylaminopyridine (DMAP) in
dimethylformamide (DMF). This way, we synthesized
trilobolide-8-O azidovalerate (3a), azidoPEG -carboxylate
4
19
(
5a), and azidoPEG -carboxylate (5b). Azidoacetate (4b) was
observation is in coherence with Sook et al. and with the fact
that Bodipy is broadly employed for lipid droplets labeling. See
example of a Bodipy dye localization in U-2 OS cells in image L
of Figure S2 in Supporting Information.
9
obtained from bromoacetate (4a) by a nucleophilic substitution
using sodium azide (see Scheme 2). The yields of the products
were inversely related to increasing length of the appended
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a
Scheme 2. Synthesis of Trilobolide−Bodipy Conjugates
a
Reagents and conditions: (a) bromoacetyl bromide, DMAP, CH Cl , 0 °C, 30 min; (b) NaN , DMF, 55 °C; (c) Bodipy 1c (compounds 4c, 3b, and
a)/Bodipy dye 1e (compound 7), CuSO ·5H O, sodium ascorbate, TBTA, DMF, microwave, 60 °C, 1 h; (d) 5-azidovaleric acid (3a)/azidoPEG -
4 2 4
2
2
3
6
acid (5a)/azidoPEG -acid (5b), EDCI, DMAP, DMF, rt.
9
Figure 1. Trilobolide−Bodipy 6a localization in the endoplasmic reticulum of human osteosarcoma cells (U-2 OS). Shown are fluorescent live-cell
images of the endoplasmic reticulum labeled by (A) compound 6a (1 μM, 1 h), (B) ER-Tracker Blue-White DPX (100 nM, 20 min), (C) merge.
Interestingly, the fluorescence signal of 3b and 6a in U-2 OS
started to slowly vanish after incubation longer than 40 min;
see Figure 1 (strong fluorescence signal of 6a was apparent only
in few cells). We observed this phenomenon only in U-2 OS
cells but not in other tested cell lines, as it can be seen from the
example in Figure S3 in Supporting Information, where
localization of 3b and 6a is depicted in two prostatic cancer
cell lines, PC-3 and LNCaP, and one pancreatic cancer cell line,
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Figure 2. Time-dependent localization of trilobolide−Bodipy (1 μM, 1 h) derivatives in human osteosarcoma cells (U-2 OS). Fluorescent live-cell
images of U-2 OS were taken after 1, 3, and 5 h of incubation (in columns). Derivatives 3b and 6a are in the upper and bottom rows, respectively.
Red arrows depict stressed and damaged the endoplasmic reticulum (notice small pouches), and pink arrows depict numerous of nuclear speckles.
MiaPaCa-2. In Figure S4 in Supporting Information, local-
with 6a (1 μM) for 60 min resulted in almost complete
vanishing of the fluorescence signal from the cell interior in
most of the cells (at some regions of interest, there were still
cells present with Tb−Bodipy intracellular localization). We
have also used fluorescence microscopy analysis to determine
the saturation plateau of these compounds’ efflux in U-2 OS
cells. After assessment of wide range of time points (0.5−24 h)
and 3b and 6a concentrations, we have found that fluorescence
signal inside U-2 OS cells reached the plateau at 2 μM
concentration (data not shown). This phenomenon was
independent of the time period tested. We assume that above
ization of Tb−Bodipy derivative 3b is depicted in C2C12
(
mouse myoblastoma cells), HEK 293T (human embryonic
kidney cells), HeLa (cells from cervical carcinoma), and A549
lung carcinoma cells). This difference between the cell lines
(
might be caused by the presence of a multidrug resistance
protein (e.g., P-glycoprotein), which could result in efflux of Tb
and its Tb derivatives. We have previously observed similar
behavior for U-2 OS cells labeled by mitochondria-specific dye,
21
MitoTracker Green.
Thus, we performed time- and concentration-based local-
ization study of 3b and 6a in U-2OS cells. From Figure S5,
showing the localization kinetics of 6a, it is apparent that at
constant concentration (1 μM) this Tb-derivative was localized
inside the cells after 10 min, and stable fluorescence signal was
observed after 20 min. Nevertheless, already after 30 min, some
of the U-2 OS cells lacked any detectable fluorescence signal
2
μM concentration the capacity of the efflux pumps might be
saturated.
To determine precise organelle localization of Tb−Bodipy
conjugates, we costained 3b- and 6a-treated U-2 OS, PC-3,
LNCaP, and HeLa cells with organelle-specific dyes. We found
that conjugates 3b and 6a colocalized with both ER-Tracker
Blue-White DPX and ER-Tracker Red. Thus, we conclude that
both 3b, 6a, and most probably also trilobolide, localized in ER
of the examined cell lines (see Figure 1 and Figure S6 in
Supporting Information; data for HeLa cells are not shown).
These data accord with similar observations of Bodipy
conjugated to Tb counterpart, thapsigargin, reported by Ong
(
see left bottom part of image D in Figure S5; space with cells
without fluorescent signal is marked with red arrow). We may
hypothesize that this is due to loss of the integrity of the plasma
membrane, as the bright field image shows aberrant cell
morphology in the lower left corner of the corresponding panel
(
image C of Figure S5 in Supporting Information). The
compounds 3b and 6a might be also effluxed from the U-2 OS
cells (e.g., hypothetically via multidrug resistance proteins),
since after 45 min of incubation at 1 μM concentration, we
observed translocation of most of the fluorescence signal of 6a
from intracytoplasmic structures to the plasma membrane
17
et al.
After prolonged incubation (3 and 5 h) of 3b and 6a with the
tested cells, we observed that both compounds caused ER stress
manifested by formation of small vesicles originating from the
ER network. This effect was more pronounced after treatment
with Tb−Bodipy 6a; see depiction by red arrows in lower panel
of Figure 2. This was probably a consequence of Tb active
conjugates accumulation in ER. These data are in agreement
(
image F of Figure S5). A minor proportion of the cells still
exhibited fluorescence in the intracellular space in the
endoplasmic reticulum (upper corner of image F in Figure S5
and some other images not shown). The U-2 OS treatment
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Figure 3. Trilobolide−Bodipy 3b localization in the endoplasmic reticulum of human osteosarcoma cells (U-2 OS). Shown are fluorescent live-cell
images of (A) the endoplasmic reticulum labeled by derivative 3b (1 μM, 1 h), (B) the mitochondria stained by red-emitting MitoTracker Red FM
(
100 nM, 10 min), (C) merge, where fragmentation of the mitochondrial network is apparent in the enlarged areas.
a
Table 1. Evaluation of in Vitro Dark Cytotoxicity of Trilobolide and Its Fluorescent Conjugates
b
IC50 [μM]
compd
time [h]
24
LNCaP
PC-3
C2C12
U-2 OS
MCF-7
MiaPaCa-2
HT-29
2
3.4
9.6
4.0
12.5
0.2
0.2
17.4
6.0
41.4
9.8
4
7
8
2
0.5
1.3
1.0
2.5
0.4
0.5
4.2
0.3
0.7
2.1
1.5
9.5
3
4
6
6
7
b
24
12.7
9.7
>50
7.5
25.6
13.2
9.1
>50
12.8
10.8
>50
>50
>50
7.4
>50
43.5
38.3
>50
>50
>50
>50
9.9
>50
>50
>50
>50
>50
>50
>50
14.2
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
4
7
8
2
4.6
>50
>50
3.5
3.8
2.2
15.1
>50
>50
>50
8.0
c
24
>50
>50
>50
>50
5.4
4
7
8
2
2.9
>50
7.4
3.2
2.5
a
b
24
4
7
8
2
0.4
3.1
6.5
6.3
20.5
>50
>50
>50
>50
>50
>50
>50
>50
>50
4.1
>50
>50
>50
>50
>50
>50
1.5
14.3
>50
>50
>50
>50
>50
>50
>50
>50
>50
9.1
6.0
6.5
24
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
48.0
47.3
>50
>50
>50
>50
>50
>50
>50
50.0
41.1
>50
>50
>50
>50
>50
>50
4
7
8
2
24
4
7
8
2
Bdp
24
4
7
8
2
3.1
5.6
a
IC50 values are expressed as concentrations of the tested compounds required for 50% inhibition of the cell viability without light exposure after 24,
8, and 72 h of incubation with the tested compound. See text for explanation of the boldface values. SD is up to 10%.
b
4
with action of thapsigargin, known as an ER stress inducer in
HepG2 cells by Matsumoto et al.
mitochondria, which have only limited buffering capacity for
elevated level of calcium ions (reviewed by Demaurex et al. ).
22
26
Interestingly, we have found that 3b and 6a caused
accumulation of small speckles in nuclei (nuclear speckles) of
U-2 OS cells. Unfortunately, because of the various ranges and
dynamic character of nuclear compartments (Cajal body, PML
bodies, gems, paraspeckles, nucleoli, etc.) we were not able to
determine the exact type of these nuclear speckles. Never-
theless, it is known that other natural compounds from
actinomycetes cause enlargement and induce formation of
To visualize the mitochondria, MitoTracker Red FM was
employed for costaining studies with Tb−Bodipy 3b and 6a. It
is unlikely that Tb derivatives reside in the mitochondria, as the
mitochondria-specific red-emitting dye did not colocalize with
green-emitting Tb−Bodipy conjugates (see Figure 3 and Figure
S7 in Supporting Information). Importantly, we detected
mitochondrial fragmentation in U-2 OS and PC-3 cells treated
with 3b and 6a (1 μM) for at least 40 min (see Figure 3 and S7
in Supporting Information). Compound 6a caused more
pronounced disruption of the mitochondrial network in these
cells (Figure S7).
2
3
nuclear speckles; thus, Tb and Tb−Bodipy derivatives could
act similarly.
Impact of Tb and Tb−Bodipy on the Mitochondrial
Network. It is known that Tg has a large impact on balance of
Cytotoxicity Assay. In the next step, we assessed cytotoxic
activity of Tb and its derivatives in vitro. The dark cytotoxicity
of the compounds (concentrations up to 50 μM) was measured
using WST-1 assay based on conversion of tetrazolium salt into
2
4,25
calcium ions between ER and cytoplasm.
Because Tb is
expected to perform similarly, the calcium ion imbalance caused
by Tb treatment should affect the physiology of the
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Figure 4. NO production in rat peritoneal cells induced by the tested compounds. The cells were treated with fluorescent conjugates for 24 h. For
−1
evaluation of potential effects of compounds 3b, 4c, 6a, 6b, and 7, lipopolysaccharide (LPS, 100 pg·mL ) and nonfluorescent trilobolide
(
3
4
compound 2, 0.1 μM) were used. The results represent the mean ± SEM of three independent experiments, n = 6. Values for 10 μM compounds
b and 6a are significantly different from untreated cells ((∗) P < 0.05 and (∗∗) P < 0.01, respectively). All values of 40 and 100 μM compounds 3b,
c, 6a, 6b, 7 are significantly different from untreated cells ((∗∗) P < 0.01).
a colored product, formazan, by the mitochondrial activity.
Only Tb and its derivatives 3b and 6a, which localized inside
various cell lines, exhibited a cytotoxic effect. This experiment
was performed using LNCaP, PC-3, C2C12, U-2 OS, MCF-7,
MiaPaCa-2, and HT-29 after 24, 48, and 72 h of treatment;
results are summarized in Table 1. As expected, compounds 4c,
at 40 μM concentration to primary immune cells. Mild decrease
(by 20%) of viability was found only for compound 3b. Other
compounds including Bodipy itself were not cytotoxic (4c, 6a,
6b, 7); see Figure S8 in Supporting Information.
Microscopic Evaluation of Nitric Oxide Evaluation Release
in Cancer Cell Lines. Nitric oxide, a very important effector of
the immune system but also a source of free radicals, plays a
pivotal role in a number of essential cellular processes.
Monitoring of NO is very difficult because, in biological
systems, it is unstable and present only in very low
concentrations. For NO imaging in living cells, we have chosen
a nitric oxide specific red-emitting fluorescent probe, DAR-2.
An apparent increase of NO was observed in cells (PC-3,
LNCaP, MCF-7, and U-2 OS) stimulated by Tb or its
fluorescent derivatives 3b and 6a (see Figure S9 and S10 in
Supporting Information). These results confirm our data on
NO release determined by spectrophotometric measurement
(next paragraph). The induction of NO production is probably
6b, and 7 that did not penetrate into the cells were not
cytotoxic. The cytotoxicity of fluorescent conjugates 3b and 6a
was slightly lower compared to Tb itself. This might be caused
by the enlargement of the original Tb molecule by the linkers
and the fluorescent dye.
Interestingly, the cytotoxic effect of compounds 3b and 6a
fluctuated over time in LNCaP cell line; e.g., the IC values for
50
24, 48, and 72 h were 7.41, 0.39, 20.53, respectively. A possible
explanation of this phenomenon might be related to the long
generation time of LNCaP cells which is approximately 60 h,
both according to our experience and consistent with data
27
published by Horoszewicz et al. We present the IC50 values
based on the data collected using the WST-1 assay that reflects
the cell metabolic activity. It seems likely that the intracellular
concentration of the compound may be affected by the cell
number that doubles between the 48 and 72 h time periods.
Also the overall metabolic activity could be affected by the cell
division step during the transition to mitosis occurring in the
same time frame. This is supported by the fact that a similar
discrepancy was observed also for the only other slowly
dividing cell line used in this study, i.e., HT-29. Nevertheless,
explanation of the real cause of this phenomenon would require
extensive experimental work exceeding the scope of this paper.
As numerous published papers present the IC values for 72 h
29
regulated by intracellular calcium ions, whose release was
caused by Tb and its derivatives treatment, which is a subject of
further investigation.
Nitric Oxide Release in Primary Macrophages. Within a
group of sesquiterpene lactones, Tg and Tb possess strong
activity in stimulating nitric oxide (NO) production by immune
2
8,30
cells.
Recently, we investigated modified trilobolide
molecules prepared as fluorescent conjugates in rat peritoneal
cells. We found a dose-dependent (0.01−100.0 μM) NO
production induced by fluorescent conjugates (see Figure 4).
While the potency of Tb to induce NO release was in the
submicromolar range, the ability of fluorescent conjugates to
induce NO production started in the micromolar range. All
fluorescent conjugates significantly stimulated NO release at 40
μM or higher concentration ((∗∗) P < 0.01). Of these
compounds, mainly 3b and 6a showed evident and significant
effects in the range of 1−10 μM concentration to induce
production of NO by cells when compared to untreated
controls ((∗) P < 0.05 for 4 μM 3b and (∗∗) P < 0.01 for 4 μM
5
0
of treatment, we refer to these values as the relevant ones
(
indicated in bold in Table 1).
In primary peritoneal rat cells, cytotoxic activity of Tb
significantly declined (by 65% for 10 μM Tb). It is in
accordance with our previous data obtained for murine and rat
28
peritoneal cells treated with Tg. On the basis of results with
cancer cell line cultures, the fluorescent conjugates were applied
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Figure 5. Secretion of cytokine TNF-α in rat peritoneal cells induced by conjugates 3b, 4c, 6a, 6b, and 7. The cells were treated with fluorescent
−1
compounds (10 μM) for 24 h. For evaluation of potential effects of compounds, lipopolysaccharide (LPS, 1000 pg·mL ) and nonfluorescent
trilobolide (compound 2, 1 μM) were used as positive controls. Cytokine secretion was analyzed by ELISA. The results represent the mean ± SEM
of two independent experiments, n = 4. (∗∗) Values are significantly different from untreated cells (P < 0.01).
of 6a). Values of NO production in the presence of compound
a reached the commonly achieved values induced by Tb or
trilobolide molecules 3b and 6a to induce cytokine secretion in
immune cells.
Overall, our study provides new fluorescently labeled natural
compound that could be used for live-cell imaging and to
further expand the scarce knowledge about the molecular
mechanism of Tb action.
6
30
lipopolysaccharide in rat resident peritoneal cells. The effects
of fluorescent compounds on nitric oxide release were not
observed for Bodipy itself during 24 h (data not shown).
Cytokine Production. On the basis of findings that Tb and
its conjugates induced NO release in primary immune cells, we
continued with immunobiological characteristics. To compare
the immunostimulatory potential of fluorescently modified Tb,
rat peritoneal cells were used for the evaluation of secretion of
TNF-α, which is a critical cytokine involved in regulation of
cancer and immune system. Lipopolysaccharide and Tb served
as positive controls. Bodipy itself had no effect on cytokine
secretion at 0.01−40 μM concentrations (data not shown). In
contrast, secretion of TNF-α (see Figure 5) was significantly
induced by conjugate 6a (10 μM, 24 h culture, (∗∗) P < 0.01 vs
untreated cells). This correlated well with substantial effect of
the compound 6a on NO release and justified our original
ASSOCIATED CONTENT
■
*
S
Supporting Information
Synthesis and compound characterization (NMR, HRMS, IR);
detailed cell culture and microscopy procedures; supplementary
results from trilobolide−Bodipy intracellular localization,
colocalization studies, mitochondrial fragmentation, cytotox-
icity, microscopic and biochemical evaluation of nitric oxide
release, and cytokine secretion. This material is available free of
charge via the Internet at http://pubs.acs.org.
31
AUTHOR INFORMATION
choice of NO for immunomodulatory screening.
■
Corresponding Authors
CONCLUSIONS
*
*
*
E.K.: e-mail, eva.kmonickova@lfp.cuni.cz.
P.D.: e-mail, pavel.drasar@vscht.cz; phone, +420 220 443 698.
T.R.: e-mail, tomas.ruml@vscht.cz.
■
In summary, five fluorescent conjugates of a potent anticancer
compound, sesquiterpene lactone trilobolide, with a green-
emitting dye Bodipy were designed, synthesized, and properly
characterized. Their biological relevance was also examined in
vitro using several cancer cell lines.
Author Contributions
All authors have given approval to the final version of the
manuscript.
We found that two of these conjugates, 3b and 6a, are
functional derivatives of Tb. These two Tb conjugates localized
in the endoplasmic reticulum of LNCaP, PC-3, U-2 OS, HeLa,
MCF-7, MiaPaCa-2, and C2C12 cells. These findings
correspond with the fact that Tb’s primary target, SERCA,
resides in the endoplasmic reticulum of eukaryotic cells. We
also demonstrated that 3b and 6a conjugates caused
fragmentation of the mitochondrial network in a similar
manner as Tb does. These two conjugates induced also nitric
oxide release in both cancer cell lines and primary immune
cells. Moreover, we confirmed the ability of the modified
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Czech Science Foundation
̌
(GACR) Grant 303/14/04329-S and MSMT No. 20/2014.
We thank Dr. Juraj Harmatha from the Institute of Organic
Chemistry and Biochemistry (The Academy of Sciences of The
Czech Republic) for generously providing trilobolide for this
study.
7
953
dx.doi.org/10.1021/jm500690j | J. Med. Chem. 2014, 57, 7947−7954

Journal of Medicinal Chemistry
Article
(
17) Ong, H. L.; Liu, X. B.; Tsaneva-Atanasova, K.; Singh, B. B.;
ABBREVIATIONS USED
■
Bandyopadhyay, B. C.; Swaim, W. D.; Russell, J. T.; Hegde, R. S.;
Sherman, A.; Ambudkar, I. S. Relocalization of STIM1 for activation of
store-operated Ca entry is determined by the depletion of subplasma
membrane endoplasmic reticulum Ca store. J. Biol. Chem. 2007, 282,
12176−12185.
ERK, extracellular signal-regulated kinase; TFA, trifluoroacetic
acid; DDQ, 2,3-dichloro-5,6-dicyano-p-benzoquinone; HRMS,
high-resolution mass spectrometry; IR, infrared spectroscopy;
MAP, mitogen activated protein; NF-κB, nuclear factor κ light-
chain enhancer of activated B cells; TNF-α, tumor necrosis
factor α; NMR, nuclear magnetic resonance; NO, nitric oxide;
PEG, polyethylene glycol; SAR, structure−activity relationship;
2+
2+
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SERCA, sarco/endoplasmic reticulum Ca -ATPase
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Pharmacological ER stress promotes hepatic lipogenesis and lipid
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