Apoptosis
DOI 10.1007/s10495-014-0998-8
ORIGINAL PAPER
Reversal of multidrug resistance in vitro and in vivo
by 5-N-formylardeemin, a new ardeemin derivative
Xuelian Zheng • Daoxia Li • Chen Zhao • Qiong Wang
Hao Song • Yong Qin • Linchuan Liao • Lin Zhang •
Yong Lin • Xia Wang
Ó Springer Science+Business Media New York 2014
Abstract Because multidrug resistance (MDR) is a seri-
ous impediment to the use of chemotherapy in treating
cancer patients, great efforts have been made to search for
effective MDR-reversing agents. We have developed a
brand new synthetic ardeemin derivative, 5-N-formylar-
deemin, and investigated the activity of which in reversing
MDR in MDR cancer cell lines derived from human breast
cancer (MCF-7-R) or lung cancer (A549-R). 5-N-formyl-
ardeemin strongly enhanced the anti-cancer efficacy of
doxorubicin, vincristine through potentiation of apoptosis
in both MCF-7-R and A549-R at relatively noncytotoxic
concentrations in vitro. Mechanistic studies showed that
5-N-formylardeemin inhibited the expression of MDR-1
(P-gp) and increased the intracellular accumulation of
cytotoxic drugs in the MDR cells, suggesting that 5-N-
formylardeemin reverses MDR activities through inhibiting
Electronic supplementary material The online version of this
article (doi:10.1007/s10495-014-0998-8) contains supplementary
material, which is available to authorized users.
X. Zheng
Q. Wang
L. Zhang
Y. Lin
X. Wang (&)
Laboratory of Molecular and Translational Medicine, Key
Laboratory of Birth Defects and Related Diseases of Women and
Children (Sichuan University) of Ministry of Education,
Department of Obstetrics and Gynecology, West China Second
University Hospital, Sichuan University, Chengdu 610041,
China
e-mail: xiawang@scu.edu.cn
D. Li
C. Zhao
L. Liao
Department of Forensic Analytical Toxicology, West China
School of Preclinical and Forensic Medicine, Sichuan
University, Chengdu 610041, China
H. Song
Y. Qin
Department of Chemistry of Medicinal Natural Products,
West China School of Pharmacy, Sichuan University,
Chengdu 610041, China
•
MDR-1 expression. Interestingly, 5-N-formylardeemin also
sensitized the parent wild-type cancer cells toward these
chemotherapeutic agents to various extents. Importantly,
in vivo studies demonstrated that 5-N-formylardeemin
significantly improved the therapeutic effects of doxoru-
bicin in nude mice bearing A549-R xenografts, which was
associated with reduced expression of MDR-1 protein level
and increased apoptosis in tumor tissues. These results
underscore 5-N-formylardeemin as a potential sensitizer
for chemotherapy against multidrug resistant cancers.
Keywords Multidrug resistance
Chemotherapy

5-N-Formylardeemin
Apoptosis
Cancer xenograft
Introduction
Multidrug resistance (MDR) remains a main hurdle of
chemotherapy in the treatment of cancer patients. Cancer
cells acquired MDR phenotype are cross-resistant to a
variety of chemotherapeutic drugs that have unrelated
structures and different mechanisms of action. Although
the underlying mechanisms are multifactorial and compli-
cated, MDR is usually associated with the overexpression
of ATP-binding cassette (ABC) transporters, such as
MDR1 (also known as P-glycoprotein, P-gp), breast cancer
resistance proteins (BCRP), and multidrug resistance
associated proteins (MRPs) on cell membrane [1–3]. These
ABC transporters actively pump cytotoxic drugs out of
cell, resulting in decreased intracellular concentrations of
the drugs. It is intriguing how these transporters have a
remarkable ability to interact with, and to transport, such a
wide variety of anticancer compounds. The hydrophobic
vacuum cleaner model, which proposes that various
hydrophobic compounds are bound indiscriminately by
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MDR-1 due to their hydrophobicity for efflux, may partly
explain this phenomenon [4]. Thus, a number of natural
and synthetic compounds with various structures have been
developed to block the function of P-gp for reversing the
MDR phenotype [5]. These compounds include calcium
channel blockers (e.g., verapamil, nifedipine), quinolines
(e.g., quinidine, chloroquine), calmodulin antagonists (e.g.,
chlorpromazine, trifluoperazine), steroids (e.g., progester-
one, tamoxifen), and immunosuppressive drugs (e.g., rap-
amycin, cyclosporine). However, most of these modulators
are unfortunately poor P-gp inhibitors that have either low
MDR reversal efficacy or unacceptable toxicity, preventing
their clinical application [6, 7]. Thus, it is important to
search for new and more effective MDR-reversing agents.
An early study showed that extracts of the fungus
Aspergillus fischeri (var. brasiliensis) exerted the activity
to reverse MDR in human cancer cell lines [8]. Isolation of
the active components from the extracts led to discovery of
three structurally related compounds named ardeemin,
15bb-hydroxy-5-N-acetylardeemin, and 5-N-acetylardee-
min. These compounds belong to the natural products
family of ‘‘reverse prenyl’’ hexahydropyrroloindole alka-
loids, with the presence of a ‘‘reverse prenyl’’ (a,a-dime-
thallyl) group at the junction of rings B and C of the
cyclized tryptophan. Among the three compounds, 5-N-
acetylardeemin is the major and most active constituent in
the A. fischeri extracts for MDR reversal. 5-N-acetyl-
ardeemin is able to potentiate the anticancer activity of
vinblastine, doxorubicin or paclitaxel in multidrug resistant
human tumor cells in vitro and in mice bearing tumors
in vivo [8, 9]. Mechanistic studies showed that reversal of
MDR by 5-N-acetylardeemin is mainly through the direct
inhibition of the P-glycoprotein-mediated drug efflux [10].
Because of considerable difficulty in isolation and
purification of ardeemins from fungus fermentation mix-
ture, chemical synthesis serves as a better recourse to
access significant quantities of these compounds. A series
of ardeemins and its derivatives and analogues have been
synthesized and their MDR reversing activities were sys-
tematically compared [10–15], however, a clinical appli-
cable ardeemin derivative has not been found. In this study,
we synthesized a new ardeemin derivative, 5-N-formylar-
deemin, and investigated its MDR reversing activity. The
results show that 5-N-formylardeemin strongly enhanced
the anti-cancer efficacy of several anticancer drugs
including vincristine and doxorubicin in MDR cancer cell
lines derived from human breast cancer (MCF-7-R) or lung
cancer (A549-R). The MDR reversing activity of 5-N-
formylardeemin is associated with inhibiting MDR-1
expression, which increase the intracellular accumulation
of cytotoxic drugs in the MDR cells. Importantly, 5-N-
formylardeemin strongly enhanced the anti-cancer efficacy
of doxorubicin in mice bearing A549-R xenograft. These
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Apoptosis
Fig. 1 Structures of ardeemin, 5-N-acetylardeemin, and 5-N-
formylardeemin
results strongly suggest 5-N-formylardeemin as a potential
chemotherapy sensitizer for reversing MDR in cancer cells.
Materials and methods
Reagents
5-N-Formylardeemin was synthesized in a related way as
described [13], which has a similar structure as the natural
occurring compound 5-N-acetylardeemin except substitut-
ing the 5-N-acetyl group with 5-N-formyl group (Fig. 1).
Antibodies against active caspase-3 and poly (ADP-ribose)
polymerase (PARP) were from BD bioscience (San Diego,
CA). Antibodies against Mdr-1 and b-actin were from
Santa Cruz Biotechnology (Santa Cruz, CA) and Protein
Tech Group (Chicago, IL), respectively. Vincristine and
doxorubicin were purchased from Sigma (St. Louis, MO).
Cell culture and in vitro cytotoxicity assay
The non-small cell lung cancer cell line A549 and breast
cancer cell line MCF-7 were purchased from American
Type Culture Collection (ATCC, Manassas, VA) and
grown in RPMI 1640 and DMEM, respectively, supple-
mented with 10 % fetal bovine serum (Hyclone), 100 units/
mL penicillin, and 100 lg/mL streptomycin under standard
incubation condition (37 °C, 5 % CO2). The cell lines with
MDR phenotype (MCF-7-R and A549-R), which are cross-
resistant to vincristine and overexpress MDR-1 (Table 1
and data not shown), were established by a multistep
selection with exposure to increasing concentrations of
doxorubicin up to 20 lM, The MDR cell lines were
maintained in culture medium with doxorubicin (2 lM)
and were cultured in medium without doxorubicin 1 week
prior to experiments. Cell death was detected quantitatively
Apoptosis
Table 1 Sensitivity of A549 and MCF-7 cells and their MDR cells to doxorubicin and vincristine
Cell line DOX
Cytotoxicity-IC50 (lM)
A549-WT 1.750 ± 0.305
A549-R 121.936 ± 2.875
MCF-7-WT 1.412 ± 0.202
MCF-7-R 98.639 ± 1.781
The IC50 values, which mean concentrations causing 50 % of cell death by in vitro cytotoxicity assay, were determined from 5 to 6 concen-
trations of each compound and the dose–effect curves were analyzed with SPSS statistics software package. Resistance fold was calculated using
the following formula: Resistance fold = IC50 resistant/IC50 parent
by lactate dehydrogenase (LDH) releasing assay using a
cytotoxicity detection kit (Promega, Madison, WI) as
described previously [16]. All the experiments were repe-
ated three to five times and the average was shown in each
figure. The IC50 value was calculated as the concentration
of anticancer drug yielding a 50 % rate of cell death, which
was calculated using probit regression using SPSS statistics
software package.
Apoptosis analysis by flow cytometry
Apoptosis in cultured cells was measured by flow cytom-
etry using an Annexin V-FITC Apoptosis Detection Kit
(Nanjing KeyGen Biotech, Nanjing, China). In brief, after
designated treatment, cells were double staining with
annexin V-FITC and propidium iodide (PI), and apoptosis
was analyzed by flow cytometry (BD bioscience, Franklin
Lakes, NJ). Early apoptosis is defined by Annexin V?/PI-
staining (Q4) and late apoptosis is defined by Annexin V?/
PI? staining (Q2).
Western blot
Cell extracts were prepared by lysing cells in M2 buffer
[20 mmol/L Tris–HCl (pH 7.6), 0.5 % NP40, 250 mmol/L
NaCl, 3 mmol/L EDTA, 3 mmol/L EGTA, 2 mmol/L DTT,
0.5 mmol/L phenylmethylsulfonyl fluoride, 20 mmol/L
h-glycerophosphate, 1 mmol/L sodium vanadate, and 1 lg/
mL leupeptin]. Cell extracts (*50 lg) were resolved in
SDS-PAGE and analyzed by Western blot. The specific
proteins were visualized by enhanced chemiluminescence
(Millipore, Billerica, MA) using BIO-RAD Image station.
Each experiment was repeated at least three times and rep-
resentative results were shown.
Assessment of doxorubicin content in cells
Doxorubicin is intrinsically fluorescent and can be excited
at wavelength around 480 nm. Thus doxorubicin content in
VCR
Resistance fold Cytotoxicity-IC50 Resistance fold
69.67 19.437 ± 0.594 (nM) 626.54
12.178 ± 0.333 (lM)
69.86 17.752 ± 0.218 (nM) 312.02
5.539 ± 0.144 (lM)
cells can be detected through monitoring the cellular
doxorubicin fluorescence intensity. For flow cytometric
determination of doxorubicin content, MCF-7-R cells
treated as indicated in figure legend were washed and
suspended in PBS and cellular fluorescence was detected
by flow cytometry (BD bioscience, Franklin Lakes, NJ). A
minimum of 10,000 cells were analyzed for each histogram
generated. For fluorometric determination of cellular
doxorubicin content, cells were washed twice with an
excess volume of ice-cold PBS and lysed with 1 % sodium
dodecyl sulfate (SDS). Fluorescence intensity in superna-
tant recovered after centrifugation of the cell suspension
was measured using a microplate reader (Tecan) at exci-
tation and emission wavelengths of kex = 480 nm and
kem = 570 nm, respectively.
In vivo xenograft and chemotherapy study
Athymic male nude mice (6 weeks old) were obtained
from the Animal Center of Sichuan University (Chengdu,
China). All procedures involving animals and their care
were conducted in accordance with the guidelines of the
Institutional Animal Care and Use Committee of Sichuan
University. A549-R cells (2 9 106) resuspended in
0.05 mL of PBS were mixed with 0.05 mL of matrigel
(BD bioscience, Franklin Lakes, NJ) and then injected
subcutaneously into mice. When the xenograft tumors
were palpable, the mice were randomly divided into four
groups and administrated with the following agents by i.p.
injection every 3 days: (a) vehicle control; (b) 1 mg/kg of
doxorubicin; (c) 50 mg/kg of 5-N-formylardeemin
(d) combination of 1 mg/kg of doxorubicin and 50 mg/kg
of 5-N-formylardeemin. Tumor sizes were measured
using a micrometer caliper and tumor volume (V) were
calculated using the following formula:
V = length 9 width2/2. The body weight of the mice
were also measured during the course of treatment. At
the end of experiment, animals were euthanized and
sacrificed. Excised tumors were measured and weighed.
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Fig. 2 5-N-formylardeemin enhanced the cytotoxicity of doxorubicin
and vincristine in MDR cancer cells and their parental wild-type cells
in vitro. a, b A549-R cells were treated with doxorubicin (DOX,
10 lM) or vincristine (VCR, 0.3 lM) in the absence or presence of
increasing concentrations of 5-N-formylardeemin as indicated in the
figures. c, d MCF-7-R cells were treated with doxorubicin (10 lM) or
vincristine (0.3 lM) in the absence or presence of increasing
concentrations of 5-N-formylardeemin as indicated in the figures. e,
TUNEL assay
Apoptosis in tumor tissues was detected by terminal
deoxynucleotidyl transferase-mediated nick end labeling
(TUNEL) assay with the in situ cell death detection kit
(Roche Diagnostics, Mannheim, Germany) according to
the manufacturer’s instructions. Briefly, paraffin-embedded
tumor tissue sections were dewaxed, rehydrated, treated
with protease K, and permeabilized. Then the sections were
incubated with the terminal deoxynucleotidyl transferase
labeling reaction mixture for 60 min at 37 °C. After
incubated with the converter-AP (anti-fluorescein antibody
conjugated with alkaline phosphatase) for 30 min at 37 °C,
the slides were stained with AP-red and haematoxylin for
color development. The apoptotic cells (TUNEL-positive
cells) in five fields (409) were counted under a microscope
and the average number of TUNEL-positive cell per field
was calculated.
Statistical analysis
Data were expressed as mean ± SD. Statistical signifi-
cance was determined by paired Student’s t test using SPSS
statistics software package. A P \ 0.05 was considered as
statistically significant.
123
Apoptosis
f A549-WT cells were treated with doxorubicin (0.5 lM) or
vincristine (5 nM) in the absence or presence of increasing concen-
trations of 5-N-formylardeemin as indicated in the figures. g, h MCF-
7-WT cells were treated with doxorubicin (0.5 lM) or vincristine
(5 nM) in the absence or presence of increasing concentrations of
5-N-formylardeemin as indicated in the figures. The cells were treated
for 72 h, and cell death was measured by lactate dehydrogenase
(LDH) release assay. Columns mean of three experiments; bars SD
Results
5-N-Formylardeemin enhanced the cytotoxicity
of doxorubicin, vincristine in both MDR cell lines
and their parental cell lines in vitro
The effects of 5-N-formylardeemin on drug sensitivity
were investigated in two MDR cell lines. A549-R cells
were treated with 10 lM of doxorubicin in the absence or
presence of increasing concentrations of 5-N-formylar-
deemin. The 5-N-formylardeemin concentrations were not
toxic, which caused \10 % cell death in A549-R cells
(Fig. 2a). As expected, A549-R cells were resistant to
doxorubicin as \5 % cell death induced in the cells treated
with 10 lM of doxorubicin alone. However, co-treatment
with 5-N-formylardeemin and doxorubicin resulted in a
synergistic cytotoxicity in a 5-N-formylardeemin dose-
dependent manner (Fig. 2a). 5-N-Formylardeemin also
similarly enhanced vincristine-induced cytotoxicity in
A549-R cells (Fig. 2b). Synergistic effects were seen in
MCF-7-R cells treated with doxorubicin or vincristine in
combination (Fig. 2c, d). Interestingly, 5-N-formylardee-
min also exhibited synergistic effects on doxorubicin and
vincristine against the parent wild-type cells that have
not acquired MDR (Fig. 2e–h). The IC50 values of
Apoptosis
Table 2 Reversal of resistance Compound
to doxorubicin and vincristine in
A549 and MCF-7 MDR cells by
5-N-formylardeemin
DOX (A)
DOX ? F-Ard (5 lM) (B)
VCR (D)
VCR ? F-Ard (5 lM) (F)
doxorubicin or vincristine in A549-R cells and MCF-7-R
cells were determined with a fixed dose of 5-N-formylar-
deemin (at 5 lM). As shown in Table 2, 5-N-formylar-
deemin sensitized the MDR cells about 5.67- to 5.86-fold
in cytotoxic response to doxorubicin and about 45.78- to
69.19-fold to that of vincristine. It is noteworthy that 5-N-
formylardeemin had little effect on doxorubicin- or vin-
cristine-induced cell death in untransformed human bron-
chial epithelial cell lines HBEC-2, which are immortalized
by insertion of cyclin-dependent kinase 4 and human tel-
omerase reverse transcriptase (Fig. S1, S2). These results
provided strong in vitro evidence supporting that 5-N-
formylardeemin effectively sensitizes MDR cancer cells to
different chemotherapeutics.
5-N-Formylardeemin enhanced doxorubicin-induced
apoptosis in MDR cancer cells
To investigate whether the reversal of drug resistance by
5-N-formylardeemin is through potentiation of apoptosis,
A549-R cells were treated with doxorubicin in the absence
or presence of 5-N-formylardeemin and apoptosis was
analyzed by flow cytometric assay. The results showed that
both early apoptotic and late apoptotic cells were signifi-
cantly increased after doxorubicin and 5-N-formylardeemin
co-treatment (Fig. 3a). Additionally, the activation of
apoptotic pathway in co-treated cells was potentiated as
detected by Western blot showing that both the generation
of active form of caspase-3 and cleavage of the caspase-3
substrate PARP were increased (Fig. 3b). These results
suggest that the enhanced cytotoxicity induced by doxo-
rubicin in the presence of 5-N-formylardeemin likely
occurs through potentiation of doxorubicin-induced
apoptosis.
5-N-Formylardeemin increased cellular doxorubicin
accumulation by inhibiting MDR-1 expression in MDR
cells
Cell killing has been reported to be closely correlated with
the intracellular concentration of doxorubicin in cancer
cells, especially in doxorubicin-resistant cell lines that
express high levels of MDR-1 [17]. Thus, we investigated
A549-R MCF-7-R
Cytotoxicity-IC50 Ratio Cytotoxicity-IC50 Ratio
(lM) (lM)
121.936 ± 2.875 98.639 ± 1.781
20.808 ± 0.962 A/B = 5.86 17.386 ± 0.456 A/B = 5.67
12.178 ± 0.333 5.539 ± 0.144
0.176 ± 0.059 D/F = 69.19 0.121 ± 0.007 D/F = 45.78
whether 5-N-formylardeemin enhances doxorubicin-
induced cell killing through increasing drug accumulation
in the MDR cells. By flow cytometric analysis, 5-N-
formylardeemin was found to effectively increase the
intracellular concentration of doxorubicin in MCF-7-R
cells, which was demonstrated by the rightward shift of the
peak of cells co-treated with doxorubicin and 5-N-formy-
lardeemin compared with that of the cells treated with
doxorubicin alone (Fig. 4a). Consistently, fluorometric
determination of cellular doxorubicin content showed that
5-N-formylardeemin significantly promoted the retention
of doxorubicin in A549-R cells, and the intracellular
fluorescence intensity increased with the increase of con-
centrations of doxorubicin (Fig. 4b). When different doses
of 5-N-formylardeemin were used, 5-N-formylardeemin
exerted the doxorubicin retention effect in a dose-depen-
dent manner (Fig. 4c). Importantly, 5-N-formylardeemin
was shown to inhibit the expression of MDR-1 in A549-R
cells in a time- and dose-dependent manner (Fig. 4d).
Therefore, it is likely that 5-N-formylardeemin enhances
chemotherapeutic-induced cytotoxicity through increasing
cellular drug accumulation by inhibiting MDR-1 expres-
sion in MDR cells.
5-N-Formylardeemin enhanced the anti-cancer activity
of doxorubicin in A549-R xenograft mouse model
The aforementioned in vitro data prompted us to further
examine whether 5-N-formylardeemin is able to enhance
the in vivo anticancer efficacy of doxorubicin in a mouse
model with A549-R xenografted tumors. As expected,
doxorubicin caused only moderate retardation of tumor
growth compared with that of the tumors in the vehicle
treated animals. A pronounced tumor growth inhibition
was seen in mice treated with doxorubicin plus 5-N-
formylardeemin (Fig. 5a, b), which was also supported by
the results of tumor weight assessed at the end of experi-
ment (Fig. 5c). Thus, the synergistic anticancer effects of
combination of doxorubicin and 5-N-formylardeemin were
validated in vivo. It was noted that body weight was
moderately decreased in the animals co-treated with 5-N-
formylardeemin and doxorubicin. This result implies
that the combined treatment of 5-N-formylardeemin and
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Fig. 3 5-N-Formylardeemin enhanced doxorubicin-induced apopto-
sis in cultured MDR cancer cells. a A549-R cells were treated with
5-N-formylardeemin (10 lM) or doxorubicin (10 lM) alone or both
for 24 h. The cells were then stained with annexin V and PI followed
by flow cytometry analysis. The percentage of cell population in Q2
and Q4 were shown. b A549-R cells were treated with 5-N-
formylardeemin (10 lM) or doxorubicin (10 lM) alone or both for
indicated times. Caspase-3 and PARP were detected by Western blot.
b-Actin was detected as an input control
doxorubicin may have some toxicity in mice with the
regime tested, which may be related to drug doses and
frequency. This issue should be clarified in future studies
including pharmacological and histopathological evalua-
tion and biochemical analysis in animals.
5-N-Formylardeemin suppressed MDR-1 expression
and enhanced doxorubicin-induced apoptosis
in xenografted A549-R tumors
Apoptosis in xenografted A549-R tumors was determined
by TUNEL assay. While only a few TUNEL-positive cells
(apoptotic cells) were seen in doxorubicin- or 5-N-
formylardeemin-treated tumors, the number of TUNEL-
positive cells in the tumor tissues from animals receiving
doxorubicin and 5-N-formylardeemin co-treatment was
significantly increased (Fig. 6a, b). In addition, 5-N-
formylardeemin treatment significantly down-regulated
the expression of MDR-1 in tumor tissues (Fig. 6c), which
is consistent with the in vitro data (Fig. 4d). These results
suggest that the in vivo synergistic anticancer effects of
combination of doxorubicin and 5-N-formylardeemin are
likely through MDR-1 suppression and apoptosis
potentiation.
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Apoptosis
Fig. 4 5-N-Formylardeemin increased cellular doxorubicin concen-
tration and inhibited MDR-1 expression in MDR cancer cells. a MCF-
7-R cells were pretreated with 10 lM of 5-N-formylardeemin for 16 h
or left untreated, followed by doxorubicin treatment (10 lM) for
another 4 h, and cellular fluorescence was analyzed by flow
cytometry. The histogram overlays showed the results of treated
cells (2, doxorubicin, 3, doxorubicin and 5-N-formylardeemin)
compared with untreated cells (1). b A549-R cells were pretreated
with 10 lM of 5-N-formylardeemin for 16 h or left untreated,
followed by treatment with increasing concentration of doxorubicin
(10–160 lM) for another 4 h. c A549-R cells were pretreated with
increasing concentration of 5-N-formylardeemin (1.25–20 lM) for
16 h or left untreated, followed by treatment with 20 lM of
doxorubicin for another 4 h. Fluorescence intensity in cell lysate
was measured using a microplate reader. The mean fluorescence
intensity (columns) and SD (bars) of three experiments were shown in
b and c. d A549-R cells were treated with 10 lM of 5-N-
formylardeemin for indicated times (upper panel) or increasing
concentration of 5-N-formylardeemin (2.5–20 lM) for 16 h (lower
panel). The expression of MDR-1 was detected by Western blot. b-
Actin was detected as an input control
Discussion
In our attempts in screening for ardeemin derivatives that
are able to reverse drug resistance in MDR cancer cell
lines, 5-N-formylardeemin was found to be potent in
enhancement of the toxicity of vincristine and doxorubicin
in cultured MCF-7-R and A549-R cells and potentiation of
the anti-cancer activity of doxorubicin in mice bearing
A549-R xenograft tumors. Importantly, 5-N-formylardee-
min inhibited the expression of MDR-1 and increased the
intracellular concentration of doxorubicin in the MDR
cancer cells, which suggests that this compound may serve
as a potential MDR-reversing agent.
While it is a crucial to find MDR-reversing agents for
anticancer chemotherapy, the discovery of the MDR
reversal activity of natural ardeemins is intriguing. As the
major and most active constituent isolated from A.
fischeri fermentation mixture, 5-N-acetylardeemin could
Apoptosis
Fig. 5 5-N-Formylardeemin enhanced the antitumor activity of
doxorubicin in A549-R xenograft in vivo. a Athymic male nude
mice were injected s.c. with A549-R cells (2 9 106) for the
development of xenograft tumors. The mice were randomly divided
into four groups for treatment: vehicle control; 1 mg/kg doxorubicin;
50 mg/kg of 5-N-formylardeemin; combination of 1 mg/kg of
doxorubicin and 50 mg/kg of 5-N-formylardeemin. The volume of
tumors was measured and the mean tumor size of each group was
shown. b, c Xenograft tumors were excised and then weighed.
Columns, mean of tumor weight in each group, bars, SD. **P \ 0.01.
d Body weight of the mice was measured during the course of
treatment. The result was presented as mean ± SD
Fig. 6 5-N-Formylardeemin enhanced doxorubicin-induced apopto-
sis and suppressed MDR-1 expression in A549-R xenograft tumors.
a In Situ TUNEL staining in tumor tissues. Representative images for
each group were shown. b TUNEL-positive cells (apoptotic cells)
were counted in five fields (409) for tumor tissues from each group
and average TUNEL-positive cell numbers per field were shown as
mean ± SD. **P \ 0.01. c Expressions of MDR-1 in tumor tissues
from each group were detected by Western blot and b-actin was
measured as an input control
significantly increase the therapeutic activity of doxoru-
bicin in B6D2F1 mice inoculated with wild-type and
doxorubicin-resistant P388 tumor cells as well as in nude
mice bearing human MX-1 mammary carcinoma xeno-
grafts [10]. Another worth noting property of 5-N-
acetylardeemin is its low toxicity in mice, as quantities of
5-N-acetylardeemin as high as 300 mg/kg per day for
3 days or 150 mg/kg per day for 8 days were well tol-
erated by mice. However, verapamil is far more toxic;
150 mg/kg of which per day for 3 days resulted in 40 %
lethality to mice [10]. The potent MDR-reversing activity
of the ardeemin coupled with its low level of toxicity
aroused great interest in the study of ardeemin analogues
for reversing drug insensitivity and led to the finding of
several members of ardeemin family with different extent
of MDR reversal activity. The detailed assessment of the
structure–activity relationships of these compounds
showed that the pyrazinoquinazoline DEF portion of 5-N-
acetylardeemin is the pharmacophoric moiety [18]. This
conclusion was in agreement with the potent MDR-
modulating activity of the simplified ardeemin analogue
AV-200 that contains the DEF portion of the natural
compound 5-N-acetylardeemin [11]. The ‘‘reverse pre-
nyl’’ (a,a-dimethallyl) group at the junction of rings B
and C also seems be important for the activity of ardee-
mins. In this report, 5-N-formylardeemin bearing formyl
substituent at 5-N site exerted comparable MDR-revers-
ing activity as 5-N-acetylardeemin in MDR cancer cells,
which is consistent with previous report showing that the
5-N-acetyl group is not essential for reversal power of
ardeemins [15]. Importantly, 5-N-formylardeemin sig-
nificantly increased the therapeutic activity of doxorubi-
cin in mice bearing A549-R xenograft without markedly
increasing overall systemic toxicity.
The exact mechanisms of MDR-modulating action of ar-
deemins in the MDR cells have not been well elucidated.
Previously, it was reported that 5-N-acetylardeemin and its
analogues reversed MDR likely through inhibiting MDR-1-
mediated drug efflux. As expected, we found that 5-N-
formylardeemin effectively increased cellular doxorubicin
concentration in both A549-R and MCF-7-R cells. Further-
more, 5-N-formylardeemin inhibited MDR-1 expression in
MDR cancer cells both in vitro and in vivo. These data indi-
cate that the downregulation of the expression of MDR-1 by
5-N-formylardeemin may account for the inhibition of MDR-
1 function and increasing of doxorubicin concentration in the
MDR-1 expressing MDR cancer cells. However, the detailed
mechanisms are warranted for future study.
It is noted that 5-N-formylardeemin also potentiated the
anticancer effects of doxorubicin and vincristine against
the wild-type cancer cells that have not acquired MDR.
This may be due to the suppression of the basal MDR-1
expression in these cells. Alternatively, a MDR-1-inde-
pendent mechanism of 5-N-formylardeemin may be
involved, which remains to be elucidated.
In summary, our studies show that 5-N-formylardeemin,
a brand new analogue of ardeemins, suppresses MDR
expression both in vitro and in vivo, substantiating this
123

compound as a potential sensitizer for chemotherapy
against multidrug resistant cancers.
Acknowledgments This study was supported by grants from
National Natural Science Foundation of China (81172111 and
81372377) and by Program for New Century Excellent Talents in
University (NCET-11-0349) from Chinese Ministry of Education and
also partly supported by Program for Changjiang Scholars and
Innovative Research Team in University from Chinese Ministry of
Education (IRT0935).
Conflict of interest The authors declare that they have no conflict
of interest.
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