Synthesis and Cell Death Mechanism of a Militarin Derivative

Article abstract of DOI:10.1002/bkcs.10667

Militarin, a novel anticancer substance from a caterpillar-grown traditional insect-flower (Cordyceps militaris), was previously isolated and characterized as an ether compound of 4-tert-octylphenol and a nonaethylene glycol. Among militarin analogs with different numbers of ethylene glycol substructure, the analog with four ethylene glycol substructure (M4EG) exhibited potent anticancer activities. A facile and scalable synthetic pathway of the M4EG was developed and the mechanism of cell death by the M4EG against lung adenocarcinoma epithelial cell line (A549) was explored. The results of this study showed that apoptosis of M4EG toward A549 cells is mediated by a caspase-dependent death-signaling pathway.

Full text of DOI:10.1002/bkcs.10667

Article
DOI: 10.1002/bkcs.10667
BULLETIN OF THE
F. Chen et al.
KOREAN CHEMICAL SOCIETY
Synthesis and Cell Death Mechanism of a Militarin Derivative
Fei Chen,†, Deok Hyo Yoon,‡, Dinneswara Reddy,† Changwoo Han,† Sunga Choi,§ Jae Youl Cho,¶ Gi-
Ho Sung,k Ki-Chul Hwang,k Tae Woong Kim,‡ and Haeil Park†,
*
†College of Pharmacy, Kangwon National University, Chunchon 200-701, Korea.
*E-mail: haeilp@kangwon.ac.kr
‡College of Natural Science (Biochemistry), Kangwon National University, Chunchon 200-701, Korea
§Department of Physiology, School of Medicine, Chungnam National University, Daejeon 301-747, Korea
¶Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Korea
kInstitute for Bio-Medical Convergence, International St. Mary’s Hospital and College of Medicine, Catholic
Kwandong University, Incheon 404-834, Korea
Received July 17, 2015, Accepted November 2, 2015, Published online March 4, 2016
Militarin, anovelanticancersubstance fromacaterpillar-growntraditionalinsect-flower(Cordyceps militaris),
was previously isolated and characterized as an ether compound of 4-tert-octylphenol and a nonaethylene gly-
col. Among militarin analogs with different numbers of ethylene glycol substructure, the analog with four eth-
ylene glycol substructure (M4EG) exhibited potent anticancer activities. A facile and scalable synthetic
pathway of the M4EG was developed and the mechanism of cell death by the M4EG against lung adenocar-
cinoma epithelial cell line (A549) was explored. The results of this study showed that apoptosis of M4EG
toward A549 cells is mediated by a caspase-dependent death-signaling pathway.
Keywords: Cordyceps militaris, Militarin derivative, Polyethylene glycol substructure, Cell death
mechanism
Introduction
carbohydrates15,16 were subsequently synthesized and tested
their anticancer activity against several tumor cell lines. It
was proved that the militarin derivative with four tetraethylene
glycol substructure (M4EG) exhibited strong anticancer activ-
ities against the following cancer cell lines (B16F10, Hela, and
HT-29) as well as anti-inflammatory activity. These results
encourage us to investigate its cell death mechanism against
human lung adenocarcinoma cell line (A549) and scalable
synthetic conditions for further development as anticancer
agents.
Cordyceps militaris (C. militaris), a caterpillar-grown tradi-
tional insect-flower (medicinal mushroom), is a fungus that
parasitizes Lepidoptera Larvae and is known to possess
diverse biological activities in the human body including
immune, respiratory, circulatory, and glandular systems.
C. militaris has been used in Chinese medicine to treat numer-
ous illnesses, promote longevity, relieve exhaustion, and
increase athletic prowess. Precedent literatures showed vari-
ous biological activities such as antitumor and antidiabetic,1
antiangiogenesis,2 antimutagenic,3 and hypoglycemic effect.4
It was also reported by us5 that butanol fraction of C. militaris
produces a number of bioactive constituents and one of the
major components was identified as cordycepin which mark-
edly inhibited the phosporylation of Akt and MAP kinase in
LPS-activated macrophage, suppressed tumor necrosis factor
(TNF-α) expression. Cordycepin was reported to possess var-
ious biological properties in precedent literatures.6–12
Bioactivity-guided further fractionation of butanol fraction
of C. militaris yielded a novel anticancer substance, which
was characterized as 4-tert-octylphenol ether compound with
a nonaethylene glycol substructure, and named as militarin
(Figure 1). Militarin was proved to possess anticancer activity
against several tumor cell lines and a concise synthetic method
was developed.13 A series of militarin derivatives bearing
different numbers of ethylene glycol (Figure 1)14 or
Experimental
Synthesis (General). All chemicals were obtained from com-
mercial suppliers, and used without further purification. All
solvents used for reaction were freshly distilled from proper
dehydrating agent under nitrogen gas. All solvents used for
chromatography were purchased and directly applied without
further purification. 1H-NMR spectra were recorded on a Bru-
ker DPX 400 (400 MHz; Germany) spectrometers. Chemical
shifts arereportedinpartspermillion(ppm)downfieldrelative
to tetramethylsilane as an internal standard. Peak splitting pat-
ternsareabbreviatedasm(multiplet), s(singlet), bs(broadsin-
glet), d (doublet), bd (broad doublet), t (triplet), dd (doublet of
doublets), and ddd (doublet of double doublet). 13C-NMR
spectra were recorded on a Bruker DPX 400 (100 MHz) spec-
trometer, fully decoupled and chemical shifts are reported in
parts per million (ppm) downfield relative to tetramethylsilane
as an internal standard. Mass spectra were recorded on an API
†† These authors contributed equally to this work.
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solution with isopropanol for extraction of dye. After incuba-
tion for 30 min, absorbance was measured on micro plate
reader at a test wavelength of 570 nm.
For the trypan blue dye exclusion assay, cells were plated in
6-well culture plates (5 × 103 cells/well) and allowed to attach
overnight. The medium was replaced with fresh complete
medium containing different concentration of the M4EG,
and incubation continued for 24 h. At the end of the incuba-
tion, both floating and adherent cells were collected and sus-
pended in PBS. The cells were then mixed with equal volume
of trypan blue solution, and live (unstained) and dead (stained)
cells were counted under the microscope.
Figure 1. Structures of militarin, synthetic militarin derivatives
including M4EG.
3200 MS system of AB SCIEX (Canada). Analytical thin-
layer chromatography (TLC) was performed using commer-
cial glass plate with silica gel 60F254 purchased from Merck
KGaA (Germany). Chromatographic purification was carried
out by flash chromatography using Kieselgel 60 (230–400
mesh; Merck).
Apoptosis Assay. Apoptotic cell death mediated by the
M4EG was analyzed by (1) DNA fragmentation assay with
agarose gel electrophoresis, (2) DNA fragmentation using a
sandwich-type ELISA, which determines cytoplasmic
histone-associated DNA fragments, (3) nuclear morphology
observed by DAPI staining and TUNEL assay, (4) DNA-
strand breaks during apoptosis by TUNEL assay, and (5) flow
cytometric analysis of cells with sub-G0/G1 DNA content fol-
lowing staining with propidium iodide. For agarose gel elec-
trophoresis, cells were lysed in a buffer containing 10 mM
Tris–Hcl pH 7.4, 150 mM NaCl, 5 mM EDTA and 0.5% Tri-
ton X-100 for 30 min on ice. The lysates were vortexed, then
cleared by centrifugation at 10 000 × g for 20 min. The DNA
in the supernatant was extracted using a 25:24:1 (vol/vol/vol)
equal volume of neutral phenol:chloroform:isoamyl alcohol
and analyzed electrophoretically on 1.2% agarose gel contain-
ing 0.1 μg/mL ethydium bromide. The effect of the M4EG on
DNS fragmentation and TUNEL assays was determined using
a kit according to the manufacturer’s recommendations.
Briefly, cells were plated and exposed to DMSO or M4EG
2-(2-(2-(2-(4-(2-Methylpentan-2-yl)phenoxy)ethoxy)-
ethoxy)ethoxy)ethanol (M4EG). The reaction mixture of tet-
raethylene glycol ditosylate (2.51 g, 5 mmol),13 4-tert-
octylphenol (1.03 g, 5 mmol) and NaH (0.36 g, 15 mmol) in
10 mL of anhydrous tetrahydrofuran (THF) was refluxed for
6 h. The reaction was quenched with aqueous 2-N NaOH
(4 mL). The quenched reaction mixture was refluxed for
4h, cooledtoroomtemperature, andcarefully neutralizedwith
aqueous 10% HCl. The reaction mixture was extracted with
ethyl acetate (3 × 100 mL), washed with saturated aqueous
NaHCO3 solution (50 mL), brine (100 mL), and dried with
anhydrous MgSO4. Evaporation of the filtrate under reduced
pressure gave pale yellow crude product. Purification of the
crude product by column chromatography (Hexane:EA =
3:1) afforded the M4EG (1.81 g, 82%) as colorless liquid.
1H NMR (400 MHz, CDCl3): δ 7.36 (d, 2H, J = 1.9 Hz),
6.95 (d, 2H, J = 1.8 Hz), 4.15–4.08 (t, 2H, J = 4.7Hz),
3.85–3.05 (t, 2H, J = 5 Hz), 3.75–3.62 (m, 10H), 3.60–3.57
( t, 2H, J = 1.7 Hz), 3.39 (brs, 1H), 1.70 (s, 2H), 1.31 (s,
6H), 0.65 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 156.6,
142.7, 127.3, 114.1, 73.0, 71.0, 70.9, 70.8, 70.6, 70.1, 67.6,
61.9, 57.3, 38.2, 32.6, 32.1, 32.0; ESIMS: m/z 405 [M+Na]+.
Cell Culture. Human NSCLC cell line A549 (American Type
Culture Collection, Manassas, VA, USA) was cultured in
RPMI-1640 medium supplemented with 10% fetal bovine
serum (FBS), 100 IU/mL penicillin and 100 μg/mL strepto-
ꢀ
at the indicated concentration for 24 h at 37 C. The attached
cells were harvested, and reacted with antihistone antibody in
96-well plates. The histone–DNA complexes are recognized
by anti-DNA-peroxidase antibodies. This binding, which is
quantifiable by a colorimetric reaction, represents a measure
of apoptotic cell death. For the TUNEL assay, the fixed and
permeabilized cells were resuspended in a TUNEL reaction
mixture. Positive images were observed under a confocal
microscope at ×400 magnification. The amount of Sub-G0/
G1 fraction in control and M4EG-treated cells was analyzed
by flow cytometry.
ꢀ
mycin. The cells were incubated at 37 C in a humidified
Immunoblotting. Cells (A549) in 60 mm culture dish
(4 × 105 cells) were incubated for time dependent in RPMI-
1640 with the M4EG. Incubation was terminated by rapid
aspiration of the cell supernatant followed by washing with
ice-cold PBS. Cells were harvested, lysed in cell lysed buffer
(50 mM Tris–HCl, 1% Triton X-100, 1% SDS, 150 mM
NaCl) with Protease inhibitor cocktail solution BD Bios-
ciences (San Jose, CA, USA). The supernatant was obtained
atmosphere containing 5% CO2. Before M4EG treatment,
cells were adapted in conditioned medium with 1% FBS.
Cell Proliferation and Viability Assay. MTT (3-(4-5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and
trypan blue dye exclusion assay were used to test the effect
of the M4EG on cell proliferation and viability. Briefly, cells
were plated in 96-well culture plates (1 × 104 cells/well).
After 24-h incubation, the cells were treated various concen-
tration with the M4EG for 24 h. At the end of the incubation,
50 μL of the MTT solution (3 mg/mL in phosphate-buffered
saline, PBS), was then added to each well. After 2-h incuba-
tion, medium was replaced to the one containing MTT
ꢀ
from the suspension after 12 000 × g centrifugation at 4 C
for 20 min. The protein concentration in cell lysated superna-
tant was determined using Thermo Scientific Pierce BCA Pro-
tein AssayTM kit (Thermo Scientific, Pittsburgh, PA, USA).
Lysate were separated by SDS–PAGE on polyacrylamide
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gel and transferred onto PVDF membrane. Membrane was
blocked with 5% nonfat milk in TBS–Tween 20 (0.1%) for
1 h and incubated with primary antibody in TBS-T containing
5% bovine serum albumin for 2 h at room temperature. After
washing thrice with TBS-T, the membrane was incubated with
horseradish peroxidase-conjugated secondary antibody for1 h
atroomtemperature andwashedthricewith TBS-T. Themem-
brane was incubated with ECL reagent for 2 min and exposed
to X-ray film. Thereafter, the same membrane was stripped
and reprobed with mouse anti-β-actin antibody.
Immunocytochemical Analysis of NF-κB. Cells (A549)
were cultured on coverslips and treated with DMSO (control)
or 50 μM M4EG for 0–12 h. The cells were then washed with
PBS. Cellswerefixed with4% paraformaldehydeand permea-
bilized with 0.1% Triton X-100. The cells were incubated with
normal goat serum in PBS for 45 min. Cells were then treated
with nuclear factor-κB (NF-κB) antibody for 2 h and Alexa
Fluor 568-conjugated secondary antibody for 1 h. After wash-
ing, cells were counterstained with DAPI (10 ng/mL) for
10 min. The cells were visualized under a confocal
microscope.
blue dye exclusion assay, respectively. The results indicate
that the M4EG has potential anticancer effect.
M4EG Treatment-induced Apoptotic Cell Death in A549
Cells. To find whether the inhibition of cell proliferation
and viability is directly related to the apoptosis in the
M4EG-treated A549 cells, four different assays were deter-
mined to test the effects of the M4EG on apoptosis cell death.
First of all, treatment of the M4EG resulted in the DNA ladder-
ing in A549 cells, which is a hallmark of apoptotic cell
death.17,18 As shown in Figure 3(a), the increased concentra-
tion of the M4EG induced a progressive accumulation of frag-
mented DNA, which appeared as a typical ladder pattern of
DNA fragmentation due to internucleosomal cleavage associ-
ated with apoptosis. Also, results from the cytoplasmic
histone-associated DNA fragmentation were similar to
Figure 3(a) (Figure 3(b)). In addition, M4EG treatment
resulted in a dose-dependent increase in fragmented nuclei
and fluorescence-positive lung cancer cells, as shown by
DAPI staining and TUNEL assay, respectively (Figure 3(c)).
To examine the mechanism responsible for M4EG-
mediated cell death, the cell cycle distribution of A549 was
analyzed by the flow cytometry. The results showed that
50 μM M4EG-treated A549 cells showed a significantly
slower cell cycle progression than did the DMSO-treated con-
trols, and showed a statistically significant increase in the frac-
tion of cells with sub G0/G1 DNA content, which is
characteristic of cells undergoing apoptosis (Figures 3
(d) and 4(e)). These data suggest that the M4EG possesses
the ability to reduce the viability of A549 cells via apoptotic
induction.
Statistical Analysis. All data are presented as mean ꢁ
S.E. and representative of three or more independent experi-
ments. Statistical significance of differences in measured var-
iable between control and M4EG-treated groups was
determined by t-test or one-way ANOVA followed by Dun-
nett’s test for multiple comparisons. A value of p < 0.05
was considered significant.
Results and Discussion
M4EG Treatment Leads to Caspase-3 Activation. It has
been shown in recent studies that caspases are very important
regulators of apoptosis induced by apoptosis stimuli.19,20 The
effect of treatment of the M4EG on proteolytic cleavage of
caspase-3 was tested. As shown in Figure 4, Western blot data
showed that treatment with the M4EG significantly increased
cleaved-caspase-3 (active form) proteins in a concentration-
dependent manner. Also, the level of cleaved PARP, a sub-
strate for caspase-3, was correspondingly increased in
M4EG-treated A549 cells. We also found that treatment with
the M4EG significantly increased APAF-1 protein level.
APAF-1 is a central component of the intrinsic pathway of
apoptosis. These data demonstrate that M4EG-induced apop-
tosis in A549 cells mainly involves the mitochondria-
dependent pathway.
M4EG Treatment-induced Regulation of Bcl-2 Family
Proteins in A549 Cells. It is known that there are two major
pathways for apoptosis that have been elucidated so far: the
intrinsic and the extrinsic pathways,21 and cellular stress often
triggers mitochondria-dependent apoptotic way.22 In order to
test whether the M4EG induces A549 cells apoptosis through
mitochondrial pathway, we determined important apoptosis-
related proteins byWestern blot analysis. The protein amounts
of antiapoptotic Bcl-2 and Bcl-xL decreased accordingly after
different doses of M4EG treatment for 24 h, while pro-
apoptotic Bak protein level increased (Figure 5(a)). Histogram
Synthesis. The militarin derivative with M4EG was prepared
by the following synthetic pathway as shown in Scheme 1.13
Reaction of 4-(tert-octyl)phenol, tetraethylene glycol di-
tosylate and NaH in THF at refluxing condition followed by
hydrolysis with aqueous NaOH in situ provided the M4EG
in one step with excellent yield (82%).
M4EG Inhibits Cell Proliferation and Viability in A549
Cells. For the investigation of the potential cell growth inhi-
bition of the M4EG in A549 cells, MTT assay was tested for
the effect of the M4EG on the proliferation of A549 cells.
A549 cells were treated with 10, 30, 50, 70, and 100 μM
M4EG for 24 h, respectively. Results showed that the
M4EGexhibitedsignificantlyinhibitioneffectoncellviability
in A549 cells with a dose-dependent manner (Figure 2(a)).
Trypan blue dye exclusion assay showed a similar result com-
pared to the MTT assay (Figure 2(b)). The IC50 values of the
M4EG were 31.1 μM in MTT assay and 27.6 μM in trypan
Scheme 1. Synthesis of a militarin derivative with atetraethylene gly-
col substructure (M4EG).
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Figure 2. The M4EG inhibited cell proliferation and viability in A549 cells. Effect of the M4EG treatment on proliferation and viability of A549
cells wasdetermined by MTTassay(a), trypanblue dye exclusion assay(b). Cells were exposedtodifferent concentrations ofthe M4EG for24 h.
Data (a, b) represent the mean ꢁ S.E. (n = 3). **p < 0.01 and ***p < 0.001 compared to control group.
Figure 3. Induction of apoptosis by the M4EG in A549 cells. After the treatment of different concentrations of the M4EG for 24 h, A549 cells
apoptosis was measured. (a) DNA fragmentation in the form of a laddering pattern, genomic DNA was extracted and analyzed on 1.2% agarose
gel. M, Marker; St, 1 μM staurosporine. (b) Cytoplasmic histone-associated DNA fragmentation. (c) Nuclear morphology was observed
by DAPI staining and TUNEL assay. (d, e) Representative flow cytometric analysis of sub-G0/G1 cell phase. Data (b, e) represent the mean
ꢁ S.E. (n = 3). **p < 0.01 and ***p < 0.001 compared to control group.
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analyses of Bcl-2/Bak (Figure 5(b)) and Bcl-xL/Bak (Figure 5
(c)) show that the balance between antiapoptosis protein and
apoptosis protein was altered, and inclined to apoptosis. At
the dose of 10 μM M4EG, downregulation of both Bcl-2/
Bak (fourfold) and Bcl-xL/Bak ratios (6.3-fold) was observed
compared to DMSO-treated control cells. These results indi-
cated that M4EG treatment altered the ratio pro-apoptotic to
antiapoptotic Bcl-2 family proteins in A549 cells.
nucleus. The free NF-κB translocates to the nucleus, where
it activated gene transcription.23 Thioredoxin has been
demonstrated to activate NF-κB through c-Jun N-terminal
kinase (JNK; stress-activated protein kinase) signaling cas-
cade.24 Furthermore, p21Cip1/Waf1 is activated through
NF-κB and is an important mediator of this growth arrest
response.25 Cyclooxygenase-2 (COX-2) is one of the novel
targets under evaluation for NSCLC therapy and chemopre-
vention. The expression of COX-2 is tightly regulated by
the binding activity of NF-κB on COX-2 promoter structure.26
To examine NF-κB response in M4EG-induced apoptosis,
the cells were cultured for 0, 4, 8, and 12 h in 50 μM,
apoptosis-inducing concentration of M4EG. As shown in
Figure 6(a), in untreated conditions (Control group) NF-κB
p65 subunit was detected in the nucleus, while treatments with
50 μM M4EG in A549 cells after 8- and 12-h block nuclear
translocation of NF-κB in a time-dependent manner.
M4EG-induced Inactivation of NF-κB Signaling Pathway
in A549 Cells. NF-κB is generally inhibited through its
sequestration in the cytoplasm by IκBs and is active in the
In order to find the mechanisms of the M4EG against pro-
liferation and cell cycle arrest in A549 cells, the effects of the
M4EG on NF-κB-related protein expression by Western blot
analysis were studied. The results showed that the enhanced
protein expressions of endogenous cyclin-dependent kinase
inhibitor p21, p-JNK, and peroxisome proliferator-activated
receptor-γ (PPAR-γ) protein in A549 cells were found
(Figure 6(b)). Activation of PPAR-γ plays an inhibitory role
in cell proliferation and growth, and it has the ability to induce
differentiation.27 In addition, 50 μM M4EG-treated A549
cells showed suppression of the COX-2 protein expression
in a time-dependent manner (Figure 6(c)). These observations
indicate that the increase of p21 and decrease of COX-2 may
be correlated to the downregulation of NF-κB activity in A549
cells after M4EG treatment.
Figure 4. The M4EG-induced cell death contains caspase-dependent
pathway. Western blotting of PARP, caspase-3, and Apaf-1, using
lysate from A549 cells treated with various concentrations of the
M4EG for 24 h. β-Actin was used to confirm the equal amount of pro-
teins loaded in each lane. Western blotting for each protein was per-
formed twice using independently prepared lysates and the results
were similar.
Figure 5. The M4EG-induced apoptosis through the initiation of the mitochondrial pathway. (a) Western blotting for Bcl-2 family proteins using
lysatedfromA549cellstreatedwithvariousconcentrationsfor24h. Theblotswerestrippedandreprobedwithanti-β-actinantibodytocorrectfor
differences in protein loading. (b) The densitometric analysis of the bands normalized to β-actin is reported in the histograms.
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the ability to reduce the viability of A549 cells via apoptotic
induction. Also, treatment with the M4EG significantly
increased cleaved-caspase-3 proteins in a concentration-
dependent manner. And, the level of cleaved PARP, a sub-
strate for caspase-3 was correspondingly increased in
M4EG-induced A549 cells. These results show that M4EG-
induced apoptosis in A549 cells mainly involves the
mitochondria-dependent pathway. The M4EG also induced
mitochondria-dependent apoptosis resulted in the increase
of Bak expression, the decreases of Bcl-2 and Bcl-xL expres-
sions, and the M4EG induced activation of NF-κB. Further-
more, the M4EG induced activations of p21 and JNK,
increase of PPAR-γ, decrease of COX-2, and apoptosis of
A549 cells by a caspase-dependent pathway. Thus, the results
indicate that the M4EG may be a potential candidate of bioac-
tive ingredients as anticancer agents.
Acknowledgments. This research was supported by the Con-
vergence of Conventional Medicine and Traditional Korean
Medicine R&D project funded by the Ministry of Health &
Welfare through the Korea Health Industry Development
Institute (HI15C0094). The authors also thank to Pharmacal
Research Institute and Central Laboratory of Kangwon
National University for the use of analytical instruments and
bioassay facilities.
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Conclusion
The militarin derivative with four ethylene glycol substructure
(M4EG) was successfully synthesized in one step and via a
concise and scalable synthetic pathway from commercial
available starting materials. The M4EG exhibited significant
inhibitory effects on cell viability in A549 cells with a dose-
dependent manner in Trypan blue dye exclusion assay and
the MTT assay. Also, the M4EG possesses the ability to
reduce the viability of A549 cells via apoptotic induction,
proved by DNA fragmentation, FACs, DAPI staining, and
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