Bisbibenzyl derivatives sensitize vincristine-resistant KB/VCR cells to chemotherapeutic agents by retarding P-gp activity

Article abstract of DOI:10.1016/j.bmc.2010.07.055

P-glycoprotein (P-gp) is known to mediate multidrug resistance (MDR) by acting as an efflux pump to actively transport chemotherapeutic agents out of carcinoma cells. Inhibition of P-gp function may represent one of the strategies to reverse MDR. We have

Full text of DOI:10.1016/j.bmc.2010.07.055

Bioorganic & Medicinal Chemistry 18 (2010) 6725–6733
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bisbibenzyl derivatives sensitize vincristine-resistant KB/VCR cells
to chemotherapeutic agents by retarding P-gp activity
b,
Guang-min Xi a, , Bin Sun b, , Hui-hui Jiang a, Feng Kong a, Hui-qing Yuan a, , Hong-xiang Lou
*
*
a Department of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan, Shandong 250012, PR China
b Department of Natural Product Chemistry, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
P-glycoprotein (P-gp) is known to mediate multidrug resistance (MDR) by acting as an efflux pump to
actively transport chemotherapeutic agents out of carcinoma cells. Inhibition of P-gp function may rep-
resent one of the strategies to reverse MDR. We have previously reported that marchantin C (MC), a mac-
rocyclic bisbibenzyl compound from liverworts, exerts anti-tumor activity as an antimitotic agent. This
study was designed to evaluate the possible modulatory effect of MC and its three synthetic derivatives
(MC1, MC2 and MC3) on P-gp in VCR-resistant KB/VCR cells. Results of the cytotoxicity assay revealed
that MC was the most potent inhibitor of cell proliferation in both KB and KB/VCR cells among these four
compounds, while the three MC-derived chemicals had little anti-proliferative activity under the same
condition. However, in P-gp-expressing MDR cells, analysis of potency of these compounds in enhancing
cytotoxicity of VCR led to the identification of MC2 as a more effective chemical on reversal of resistance.
Further study showed that MC2 was able to reduce efflux of rhodamine-123, and in turn, increase the
accumulation of rhodamine-123 and adriamycin in KB/VCR cells, indicating that MC2 re-sensitized cells
to VCR by inhibition of the P-gp transport activity. In addition, the combination of MC2 and VCR at a con-
centration that does not inhibit cell growth resulted in an induction of apoptosis in KB/VCR cells. These
results suggest that MC2, as a novel and effective inhibitor of P-gp, may find potential application as an
adjunctive agent with conventional chemotherapeutic drugs to reverse MDR in P-gp overexpressing can-
cer cells.
Received 8 June 2010
Revised 22 July 2010
Accepted 23 July 2010
Available online 29 July 2010
Keywords:
Marchantin C
Derivatives
Multidrug resistance
P-glycoprotein
Apoptosis
KB/VCR cells
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
terized mediator of MDR.4 P-gp is capable of extruding various
drugs from cells in an energy-dependent manner, leading to de-
Chemotherapy plays a very important role in the treatment of
malignant tumors. However, the occurrence of the multidrug resis-
tance (MDR) still represents a major obstacle for successful cancer
chemotherapy. Multidrug resistance is a phenomenon in which
cancer cells exposed to one anticancer drug become resistant to
other chemotherapeutic drugs whose structures and functions
are unrelated with each other.1–3 Although several cellular mecha-
nisms are associated with the development of MDR, including the
reduction of apoptosis, activation of DNA damage repair systems
and alteration of drug metabolism, P-glycoprotein (P-gp), an ATP-
binding cassette (ABC) transporter, is the most extensively charac-
creased drug concentrations within the cell and reduced efficacy
of drugs. Therefore, numerous efforts have been made to develop
compounds capable of modulating the activity of P-gp in order to
reverse the MDR phenotype.5,6 A number of noncytotoxic drugs
which sensitize MDR cells to chemotherapeutic drugs have been
identified, including calcium channel blockers (verapamil, nifedi-
pine), calmodulin antagonists (trifluoperazine chlorpromazine),
and immunosuppressive drugs (cyclosporine A, rapamycin). Natu-
rally occurring polyphenolic compounds, such as curcuminoids,
curcumin and epigallocatechin 3-gallate (EGCG) have also been re-
ported to block drug efflux and reverse MDR phenotype.7–9 How-
ever, these compounds failed in clinical trials because of their
severe side effects and poor pharmacokinetics in vivo.1 Therefore,
the development of safe and effective MDR reversal agents is an
important approach to reversing clinical MDR.
Abbreviations: ADR, adriamycin; MDR, multidrug resistance; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; P-gp, P-glycoprotein;
Rh123, rhodamine-123; VCR, vincristine; VRP, verapamil.
* Corresponding authors. Tel.: +86 531 88382092; fax: +86 531 88382019
(H.Q.Y.).
Macrocyclic bisbibenzyls, a class of liverwort-derived natural
chemicals, have diverse biological activities, including cytotoxic,
antioxidant, antibacterial and antifungal activity.10–12 Marchantin
C (MC) (Fig. 1), a natural macrocyclic bisbibenzyl, was first isolated
E-mail addresses: lyuanhq@sdu.edu.cn (H. Yuan), louhongxiang@sdu.edu.cn (H.
Lou).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.055

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G. Xi et al. / Bioorg. Med. Chem. 18 (2010) 6725–6733
Figure 1. Chemical Structures of MC and MC2.
from liverworts Marchantia polymorpha by Asakawa et al. in
1983.13 It was previous found to be an excellent cytotoxic agent
against KB and P388 leukemia cell lines, and also displayed anti-
HIV, antibacterial, DNA polymerase b inhibitory, iNOS inhibitory
and
a
-glucosidase inhibitory activities.14–18 Recently, our group
found marchantin C could induce apoptosis of tumor cells as an
antimitotic agent.19,20
The potent anti-tumor activity of MC and limited natural re-
sources motivated us to prepare it by total synthesis and analyzed
its possible structure-mediated anti-tumor activity. In the present
study, we synthesized MC (16) as well as its three cyclic synthetic
derivatives, dimethyl ether of dehydromarchantin C (MC1, com-
pound 13), dimethyl ether of marchantin C (MC2, compound 14),
and dehydromarchantin C (MC3, compound 15) in a satisfactory
yield, and examined their effects on both drug-sensitive and
drug-resistant cancer cells. The results indicated that these com-
pounds potentiated the cytotoxicity of VCR and inhibited P-gp
activity in KB/VCR cells.
2. Results and discussion
2.1. Synthesis of marchantin C and its derivatives
We have focused on the efficient total synthesis of marchantin C
for a long time. Although a total synthesis of marchantin C was re-
ported in Tetrahedron Letters recently,21 herein we report another
efficient method to marchantin C in 12 steps with a total yield of
23%.
The total synthesis of marchantin C was achieved as shown in
Scheme 1. The synthetic route began with the Ullmann coupling
of the protected 2-hydroxy-3-methoxybenzaldehyde (2) with
commercially available 3-bromo-benzaldehyde, resulting in the
formation of the diphenyl ether 3. The phosphonium salt 8, as
the second diphenyl ether building block, was synthesized in a
conventional sequence starting with SNAr displacement of methyl
4-bromobenzoate (5) by 3-hydroxy-4-methoxybenzaldehyde (4)
to give the diphenyl ether 6. Compound 6 was then reduced with
sodium borohydride to give the benzyl alcohol 7, followed by reac-
tion with triphenylphosphonium bromide, affording 8 in three
steps. The building blocks 3 and 8 were combined by Wittig reac-
tion in the presence of potassium carbonate and 18-crown-6, and
the stilbene 9 (obtained as E/Z mixture) was hydrogenated over
Pd/C to give the bibenzyl 10. Next, the carboxylic ester group of
10 was reduced with lithium aluminium hydride, followed by
deprotection with HCl/H2O to yield compound 11. Again the reac-
tion of 11 with triphenylphosphonium bromide afforded com-
pound 12. Cyclization of 12 by means of an intramolecular Wittig
reaction was achieved with sodium methoxide, leading to key
macrocyclic intermediate 13. Finally, 13 could be demethylated
Scheme 1. Synthesis of marchantin C. Reagents and conditions: (a) 1,3-propane-
diol, DMS–DMF, DCM; (b) 3-bromo-benzaldehyde, K2CO3, CuO, Pyridine, reflux; (c)
K2CO3, pyridine, CuO, reflux; (d) NaBH4, THF, rt; (e)PPh3ꢁHBr, CH3CN, reflux; (f)
K2CO3, 18-crown-6, CH2Cl2, reflux; (g) H2, 10% Pd/C, Et3N, AcOEt, rt; (h) (1) LiAlH4,
THF, 30 °C; (2) H+/H2O, rt; (i) NaOMe, CH2Cl2, rt; (j) H2, 10% Pd/C, AcOEt, rt; (k) BBr3,
CH2Cl2, ꢀ78 °C.
by boron tribromide at ꢀ78 °C to give phenol 15 or hydrogenated
to give dimethyl marchantin C (14), followed by demethylation
to afford marchantin C (16).
2.2. Cytotoxicity of marchantin C and its derivatives in KB and
KB/VCR cells
In our previous study, we reported that MC could trigger apop-
tosis in various cultured tumor cells.19,20 Therefore, we firstly
investigated the cytotoxic effects of MC and its derivatives on KB
and KB/VCR cells with MTT assay. As shown in Figure 2, treatment
of cells with MC led to a profound cell viability inhibition in a dose-

G. Xi et al. / Bioorg. Med. Chem. 18 (2010) 6725–6733
6727
Figure 2. (A) Effects of MC and it’s derivatives on the growth of KB cells. (B) Effects of MC and it’s derivatives on the growth of KB/VCR cells. The cells were treated with
various concentrations of these compounds for 48 h. Viable cell numbers were evaluated by MTT assay and were denoted as a percentage of DMSO treated controls at the
concurrent time point. (C) Reversal effect of MC and it’s derivatives on the KB/VCR cells. The cells were exposed to VCR at presence of these four compounds. IC50 values for
*
**
VCR were calculated. Data from three independent experiments were shown as means SD. P <0.05, P <0.01 versus VCR treatment alone.
Table 1
dependent manner. The IC50 value of MC in KB cells was
16.48 1.17 M, and in KB/VCR the value was 17.47 1.14 M.
However, unlike MC, MC1, MC2 and MC3 ranged from 5 to
50 M had no significant inhibitory effects on the growth of both
KB and KB/VCR cells. The IC50 values of MC1, MC2 and MC3 were
Effects of MC and its synthetic derivatives on the cytotoxicity of VCR in KB/VCR cells
l
l
Treatment
IC50 (nmol/L)
RFb
l
VCR
1483.48 50.18
743.54 65.18
625.03 51.69
354.01 28.97
600.79 41.13
575.62 34.66
478.78 40.54
310.06 47.51
219.40 42.49
196.95 25.06
688.71 37.87
423.89 28.41
250.32 25.70
167.52 14.83
—
VCR + MC (2
VCR + MC (4
VCR + MC (8
VCR + MC1 (4
VCR + MC2 (8
l
l
l
l
l
M)
M)
M)
M)
M)
1.99
2.37
4.19
2.47
2.57
3.10
4.78
6.76
7.53
2.16
3.51
5.95
8.85
93.48 3.71, 94.85 2.09, and 83.98 3.82
88.51 2.02, 95.38 1.92, and 86.88 2.06
l
l
M for KB cells, and
M for KB/VCR cells,
respectively. These results suggested that both drug-resistant and
parental cells were more sensitive to MC than MC derivatives. In
addition, KB/VCR cells showed slightly resistant to MC.
VCR + MC1 (16
l
M)
VCR + MC2 (4
VCR + MC2 (8
VCR + MC2 (16
VCR + MC3 (4
VCR + MC3 (8
VCR + MC3 (16
l
l
M)
M)
l
M)
2.3. Marchantin C and its derivatives potentiate the cytotoxicity
of chemotherapeutic agents in KB/VCR cells
l
l
M)
M)
M)
M)
l
To investigate the effect of MC and its derivatives on the sensi-
tivity of cells to chemotherapeutic agent, we performed MTT assay
to determine the VCR cytotoxic effect with or without pretreat-
ment of these compounds. The results showed that these four com-
pounds were potent to reverse the VCR-associated MDR phenotype
in KB/VCR cells (Table 1, Fig. 2C). VCR-mediated cytotoxicity was
drastically increased, and the IC50 values of VCR toward KB/VCR
were decreased in the presence of these compounds. The fold
VCR + VRP (16
l
a
Cell survival was determined by MTT assay. Values are means SD of three
experiments.
b
The reversal activity of target compounds was expressed reversal folds (RF).
RF = IC50 of VCR alone/IC50 of VCR in the presence of test compounds.
MC2 at 16 lM, producing a 7.5-fold reduction in the IC50 of VCR
in KB/VCR cells. The effect was dose-dependent—an increasing
concentration of MC2 correlated with a decreased IC50 of VCR.
However, this effect of MC2 was less evident in drug-sensitive KB
cells lacking P-gp (Table 2).
ADR, another substrate of P-gp with different modes of action
that is structurally unrelated to VCR, was employed to confirm
the reversal activity of MC2 in KB/VCR cells. As shown in Table 2,
the IC50 value of ADR was reduced 6.1-fold in KB/VCR cells exposed
reversal of resistance to VCR upon exposure to MC (8
lM), MC1,
MC2, MC3 (16 M) was 4.2, 3.1, 7.5, 6.0, respectively. The data
l
indicated that MC and its derivatives had the ability to sensitize
drug-resistant cells to VCR. Moreover, this led to the selection of
MC2 as a potent modulator for further mechanistic studies. Table
2 showed the IC50 of VCR and the fold reversal of resistance in
the presence of MC2. The data revealed that the IC50 of VCR de-
creased drastically from 1483.48 to 196.95 nM in the presence of

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G. Xi et al. / Bioorg. Med. Chem. 18 (2010) 6725–6733
Table 2
The results in Figure 3D demonstrated that without MC2, a rapid
decrease of intracellular Rh123 was observed in KB/VCR cells after
incubation in Rh123-free medium for 120 min. However, in the
presence of MC2, Rh123 efflux was drastically suppressed in KB/
VCR cells. In addition, like VRP, duration of action of MC2 was short
and reversible because the intracellular Rh123 was not retained for
120 min (Fig. 3D). These results suggest that MC2 increased che-
motherapeutics accumulation inside KB/VCR cells through inhibit-
ing outward transport function of P-gp.
Potency of MC2 in promoting cytotoxicity of VCR and ADR in KB and KB/VCR cellsa
Treatment
KB
KB/VCR
IC50
RF
IC50
RF
VCR (nM)
VCR + MC2 (4
VCR + MC2 (8
VCR + MC2 (16
VCR + VRP (16
ADR (lg/ml)
ADR + MC2 (4
ADR + MC2 (8
ADR + MC2 (16
ADR + VRP (16
6.07 0.68
5.20 0.45
4.56 0.32
3.93 0.19
4.70 0.18
1.66 0.53
1.62 0.44
1.44 0.25
1.83 0.16
1.56 0.40
—
1489.58 50.18
310.05 47.51
219. 40 42.49
196.95 25.06
176.84 14.83
49.66 2.61
12.53 1.46
11.82 1.31
8.19 0.87
—
l
l
l
l
M)
M)
M)
1.17
1.33
1.54
1.29
—
1.02
1.15
0.91
1.06
4.78
6.76
7.53
8.85
—
3.96
4.20
6.06
5.32
M)
l
l
M)
M)
2.7. Treatment with MC2 and VCR induces apoptosis in KB/VCR
cells
l
M)
l
M)
9.34 0.77
a
To determine whether the observed cell death caused by the co-
treatment of the cells with VCR and MC2 was due to apoptosis, typ-
ical markers of apoptosis were analyzed in response to VCR and/or
MC2. Then we analyzed nuclei fragmentation by DAPI staining and
investigated the expression level of Bcl-2, Bax and PARP by wes-
KB/VCR cells were exposed to anticancer drugs in the presence of MC2 or VRP
for 48 h and IC50 (concentration inhibiting 50% growth) was determined by MTT
assay. Values are means SD of three experiments.
to MC2 at a concentration of 16 lM. However, neither of the VRP
tern-blot. As shown in Figure 4A, at a concentration of 16
lM of
nor MC2 significantly affected the IC50 of ADR in the parental KB
cells. These observations suggested that these compounds were
able to reverse MDR via P-gp inhibition.
MC2 and 0.2 M VCR, neither drug caused significant morpholog-
l
ical changes of apoptosis in KB/VCR cells. In contrast, treatment
of KB/VCR cells with the combination of MC2 and VCR resulted
in increase in the fraction of apoptotic cells with condensed and
fragmented DNA (as indicated by a stronger blue fluorescence). Gi-
ven the profound roles of the Bcl-2 family in triggering apoptosis,22
we next examined if antiapoptotic protein Bcl-2, and proapoptotic
protein Bax were involved in combination treatment-induced
apoptosis. The results in Figure 4B indicated that neither MC2
nor VCR alone had any detectable effect on the expression of Bcl-
2 and Bax, however, the combination treatment of MC2 with VCR
led to noticeable down-regulation of Bcl-2 and up-regulation of
Bax in KB/VCR cells. In addition, alterations in expression of Bcl-2
family members may trigger the activation of caspases, which, in
turn, lead to cleavage of poly (ADP-ribose) polymerase (PARP), an-
other hallmark of the apoptotic response.23 Accordingly, the results
in Figure 4B indicated that the native 116 kDa PARP protein was
cleaved into its characteristic 85 kDa fragment upon treatment
with MC2 and VCR. These results suggested that the enhanced
inhibitory effect on KB/VCR cells by the combination of MC2 and
VCR was achieved through a concomitant increase in apoptosis.
2.4. The effect of MC2 on P-gp expression
The reversal of P-gp-mediated MDR can be achieved either by
decreasing P-gp expression or by inhibiting its drug-transport
activity. We incubated KB/VCR cells with MC2 at 4, 8, and 16 lM
for 48 h, and examined the change in P-gp protein abundance.
The results of the Western blot shown in Figure 3A, revealed that
the protein level of P-gp was not altered by MC2 treatment in
KB/VCR cells. These results indicated that reversal of resistance
by MC2 may be attributed to the inhibition of P-gp function.
2.5. MC2 causes accumulation of ADR and Rh123 in KB/VCR cells
The fluorescence characteristics of ADR were utilized to deter-
mine intracellular drug concentration by flow cytometry. The abil-
ity of MC2 to inhibit P-gp-mediated transport was examined by
determining the intracellular ADR-associated fluorescence inten-
sity in KB/VCR cells. As shown in Figure 3B, the accumulation of
ADR in KB cells was evident in either untreated or treated cells.
Whereas, the accumulation of ADR in KB/VCR cells was signifi-
cantly lower than that in KB cells. After the treatment with 4, 8,
3. Discussions
In the past two decades, a number of natural and synthetic com-
pounds have been tested for their ability to reverse MDR. Although
several chemosensitizers have been shown to be very potent in
reversal of MDR in both in vitro and in vivo experiments, the re-
sults of clinical trials with these compounds were disappointing
because of the side effects and/or weak potency.4,24,25 The aim of
this study was to continue our work to identify novel naturally-
occurring chemicals that exert anti-tumor activity and/or reverse
multidrug resistance.
In the present study, we showed for the first time that MC and
its synthetic derivatives significantly increased the sensitivity of
KB/VCR cells toward VCR. Especially MC2, showed the largest po-
tency to enhance VCR cytotoxicity in drug resistance cells with
the reversal fold of 4.7–7.5. In order to understand the underlined
mechanism of reversal of MDR of this class of compounds, the most
active compound MC2 was employed as the tested drug. The re-
sults showed that MC2 enhanced the cytotoxicity of VCR and
ADR toward KB/VCR cells, whereas MC2 in concentrations ranging
16 lM of MC2 for 1 h, respectively, intracellular accumulation of
ADR was increased 2.1-, 2.8-, and 5.7-fold in comparison with
DMSO-treated control cells (p <0.05). These results suggested that
MC2 elevated the sensitivity of KB/VCR cells toward chemothera-
peutics through increasing chemotherapeutics accumulation in-
side KB/VCR cells.
To further confirm the ability of MC2 to inhibit P-gp-mediated
transport, we investigated the effect of MC2 on transport of
Rh123, another well-established fluorescent substrate of P-gp.
Consistent with the results shown in Figure 3B, Rh123 in KB cells
was not affected by MC2 or VRP treatment (Fig. 3C), however,
MC2 treatment induced a marked accumulation of Rh123 in KB/
VCR cells in a dose-dependent manner. These observations sug-
gested that the reversal of resistance to VCR and ADR is P-gp
specific.
2.6. MC2 inhibits P-gp-mediated efflux
from 5 to 50 lM did not display any significant anti-proliferative
Subsequently, we performed experiments to determine
whether the increased accumulation of Rh123 in the KB/VCR cells
was due to inhibition of Rh123 efflux. The time course of Rh123 ef-
effect on both drug-sensitive KB and drug-resistant KB/VCR cells.
Thus, the low cytotoxicity provides the foundation property for
MC2 because MDR modulators would better not have cytotoxic
or off-target effects.
flux in the presence MC2 (16 lM) was analyzed by flow cytometry.

G. Xi et al. / Bioorg. Med. Chem. 18 (2010) 6725–6733
6729
Figure 3. (A) Effect of MC2 on the expression of P-gp. KB/VCR cells were treated with MC2 at 4, 8, 16
lM for 48 h. Total cell proteins (50
lg) were separated by SDS–PAGE, and
then transferred to PVDF membrane. The membranes were immunoblotted with P-gp antibody. Immunoreactive proteins were visualized using an ECL. (B) Effect of MC2 on
ADR accumulation in KB and KB/VCR cells. Cells were treated with 3 g/ml ADR in the absence and presence of MC2 for 1 h. Intracellular accumulation of ADR was evaluated
by measuring the mean fluorescence intensity. (C) MC2 increased the accumulation of Rh123 in KB/VCR cells. Cells were pretreated with 4, 8, 16 M of MC2 for 1 h and then
were exposed to 2 mg/mL of Rh123 for another 1 h. Rh123-associated fluorescence intensity was determined by flow cytometry. Data were expressed as means S.D. of three
l
l
*
**
independent experiments. P <0.05 and P <0.01 versus DMSO treated KB/VCR cells. (D) MC2 blocked intracellular Rh123 efflux in KB/VCR cells. Cells were exposed to 5
lM of
Rh123 in the presence of MC2 or VRP at 37 °C for 60 min. The cells were then washed three times with medium without Rh123, Cells were collected at 15, 30, 45, 60, 90,
120 min, respectively, and resuspended in 1 mL of ice-cold PBS. Rh123-associated fluorescence intensity was analyzed by flow cytometry.
Figure 4. (A) MC2 treatment increased VCR-induced apoptosis in drug-resistant KB/VCR cells. Cells were treated with 16
MC2 and 0.2 M VCR for 48 h. After fixation, cells were stained with DAPI and cell morphological characterization was analyzed using fluorescence microscope. (B) The
expression levels of Bax, Bcl-2 and PARP in KB/VCR cells after treatment with 16 M MC2, 0.2 M VCR, or the combination of 16 M MC2 and 0.2 M VCR for 48 h. Total cell
proteins (50 g) were separated by SDS–PAGE, and then transferred to PVDF membrane. The membranes were immunoblotted with desired antibody. Immunoreactive
lM MC2, 0.2 lM VCR, or the combination of 16 lM
l
l
l
l
l
l
proteins were visualized using an ECL.
The results above indicated that MC2 could enhance the sensi-
tivity of P-gp-overexpressing cells to certain chemotherapeutic
agents. The mechanism by which this occurs is unknown. The
reversal of P-gp-mediated MDR can be achieved either by decreas-
ing P-gp expression or by inhibiting its activity.26 We firstly stud-
ied the effect of MC2 on P-gp expression. The result of Western
blot showed that the protein level of P-gp in KB/VCR cells was
not altered by MC2 treatment. This experiment suggested that
MC2 didn’t affect the expression of P-gp in KB/VCR cells. So we
then examined its effect on the drug-transport function of P-gp.

6730
G. Xi et al. / Bioorg. Med. Chem. 18 (2010) 6725–6733
As shown in Figure 3, the transport of ADR and Rh123 by P-gp is
significantly inhibited by MC2 in a concentration-dependent man-
ner. The treatment of MC2 resulted in a remarkable increment of
the fluorescent intensity from ADR and Rh123 in KB/VCR cells,
indicating that MC2 elevated accumulation of ADR and Rh123 in
KB/VCR cells. However, the accumulation level of ADR and Rh123
was hardly varied in KB cells no matter with or without MC2 treat-
ment. These supported the notion that the enhanced cytotoxicity
of chemotherapeutic agents by MC2 was attributed to its inhibition
activity to P-gp.
Bruker Spectospin spectrometer at 300 or 600 MHz, using TMS as
an internal standard. The chemical shifts are reported in parts
per million (ppm d) referenced to the residual 1H resonance of
the solvent (CDCl3, 7.26 ppm). Abbreviations used in the splitting
pattern were as follows: s = singlet, d = doublet, t = triplet,
quin = quintet, m = multiplet, and br = broad. Reagents were used
as purchased without further purification. Solvents (THF, CH2Cl2
and CH3CN) were dried and freshly distilled before use according
to procedures reported in the literature.
The above results showed that MC2 could inhibit the function of
P-gp. The question if MC2 may act as direct inhibitor of P-gp func-
tion or as P-gp substrate was addressed by Rh123 efflux assay. The
time kinetics of MC2-mediated inhibition of Rh123 efflux was
investigated in KB/VCR cells. Whereas P-gp inhibitors commonly
show prolonged activity even after a washing out period, the activ-
ity of P-gp substrates rapidly declines after substance removal.27,28
To examine this, cells were incubated with Rh123 in the presence
of MC2 or VRP for 60 min. Then, cells were washed and Rh123 fluo-
rescence was detected after 0, 15, 30, 45, 60, and 120 min. Results
indicated that Rh123 fluorescence decreases after wash-out of MC2
or VRP. Noteworthy, MC2-induced effects on Rh123 accumulation
were similar to those induced by VRP that is known to inhibit P-gp
being a P-gp substrate. Taken together, these results suggest that
MC2 acts as a P-gp transport substrate.
4.2. Synthesis
4.2.1. 2-(1,3-Dioxan-2-yl)-6-methoxyphenol (2)
2-Hydroxy-3-methoxybenzaldehyde (1, 9.12 g, 0.06 mol) was
added to a well-stirred solution of DMS–DMF adduct (15.99 g,
0.08 mol) and 1,3-propanediol (14.05 g, 0.18 mol) in CH2Cl2
(40 mL), the resulting mixture was then stirred for 24 h at room
temperature, and slowly quenched with Et3N (11 mL, 0.08 mol)
at 0 °C. The reaction mixture was extracted with Et2O
(50 mL ꢂ 5) and the combined organic layer was washed with
NaOAc–saturated 5% aqueous NaHSO3 (25 mL ꢂ 3) and NaOAc-sat-
urated brine (25 mL ꢂ 2), and then dried over anhydrous sodium
sulfate. The solvent was evaporated to yield
a white solid
(10.83 g, 86%) that was recrystallized from CH2Cl2 and hexane:
mp white solid; mp 105–106 °C. 1H NMR (300 MHz, CDCl3) d
7.00 (dd, J = 4.2 Hz, 5.4 Hz, 1H), 6.86 (s, 1H), 6.84 (d, J = 1.8 Hz,
1H), 5.78 (s, 1H), 4.29 (dd, J = 4.8 Hz, 12.0 Hz, 2H), 4.03 (dt,
J = 2.4 Hz, 12.6 Hz, 2H), 3.87 (s, 3H), 2.33–2.20 (m, 1H), 1.49–1.46
(m, 1H); MS (ESI) 211 (M+H)+.
An important aspect of cancer chemotherapy is based on the
abilities of chemotherapeutic agents to induce apoptosis in cancer
cells.29,30 In addition to evaluate the effect of MC2 on P-gp trans-
port function, we examined the effect of MC2 on VCR cytotoxicity.
The combination treatment of MC2 with VCR markedly induced
apoptosis. However, treatment of the cells with MC2 (16
l
M) or
4.2.2. 3-(2-(1,3-Dioxan-2-yl)-6-methoxyphenoxy)benzaldehyde
(3)
VCR (0.2 M) alone did not trigger apoptosis under the same con-
l
dition. The result suggested that the increased inhibitory effect on
KB/VCR cells from the combination of MC2 with VCR was achieved
through the action of MC2, which enhanced the VCR-induced
apoptosis. To further investigate the possible mechanism of action
by which MC2 potentiated cytotoxicity of VCR, we evaluated the
expression level of Bcl-2 and Bax through Western blot analysis.
The results showed that the expression of proapoptotic protein
Bax was significantly up-regulated and antiapoptotic protein Bcl-
2 was decreased by the combination treatment of VCR with MC2.
This result suggested that combination treatment-induced apopto-
sis in KB/VCR cells is partly mediated by the Bcl-2 family. Addition-
ally, typical processing of PARP into the 85-kDa fragment was also
observed when KB/VCR cells were treated with VCR in the presence
of MC2. These phenomena confirmed the results of the MTT assay.
Future research is required to analyze the action of MC2 in other
MDR cell lines as well as in animal models.
In conclusion, the present study shows that marchantin C and
its synthetic derivatives significantly reverse P-gp-mediated MDR
in cancer cells. Of four derivatives, MC2 exhibited more potent ef-
fects on MDR reversal by inhibiting the drug efflux function of P-
gp, resulting in the increase of the intracellular accumulation of
the anticancer drugs. The low cytotoxicity, non-interference on
the expression of P-gp, and reversible blocking action of P-gp sug-
gest that MC2 is a promising candidate for further investigation.
A mixture of 2-(1,3-dioxan-2-yl)-6-methoxy-phenol (2, 10 g,
0.05 mol), 3-bromo-benzaldehyde (8.8 g, 0.05 mmol), potassium
carbonate (13.5 g, 0.09 mol) and cupric oxide (0.95 g, 5.9 mmol)
in pyridine (50 mL) was stirred under reflux for 12 h. The pyridine
was distilled off in vacuo and the residue was extracted with ethyl
acetate (200 mL). The solution was concentrated and the residue
was purified by flash column chromatography (Al2O3), eluting with
a 2:1 solution of petroleum ether–CH2Cl2 to afford 3 (12.6 g, 81%)
as
a
yellow solid; white solid; mp 105–106 °C. 1H NMR
(300 MHz, CDCl3) d 9.95 (s, 1H), 7.55 (d, J = 7.2 Hz, 1H), 7.43 (t,
J = 7.8 Hz, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.30 (t, J = 7.8 Hz, 1H), 7.15
(d, J = 5.4 Hz, 1H), 7.02 (d, J = 7.8 Hz, 1H), 5.69 (s, 1H), 4.14 (d,
J = 7.8 Hz, 2H), 3.85 (t, J = 12.0 Hz, 2H), 3.74 (s, 3H), 2.21–2.16 (m,
1H), 1.37 (d, J = 13.2 Hz, 1H); MS (ESI) 315 (M+H)+.
4.2.3. Methyl 4-(5-formyl-2-methoxyphenoxy) benzoate (6)
This compound was prepared from methyl 4-bromobenzoate
(15.05 g, 69.98 mmol) and 3-hydroxy-4-methoxybenzaldehyde
(10.63 g, 69.98 mmol) by means of a procedure similar to that used
for 3. After concentration of the solution, the residue was purified
by flash column chromatography (SiO2), eluting with a 3:2 solution
of petroleum ether–CH2Cl2 to afford 6 (15.86 g, 79%) as an orange
solid; mp 118–120 °C. 1H NMR (300 MHz, CDCl3) d 9.87 (s, 1H),
8.02–7.99 (m, 2H), 7.76 (dd, J = 8.2, 2.1 Hz, 1H), 7.59 (d, J = 2.1 Hz,
1H), 7.14 (d, J = 8.3 Hz, 1H), 6.94 (d, J = 8.7 Hz, 2H), 3.91 (s, 3H),
3.90 (s, 3H); MS (ESI) 287 (M+H)+.
4. Experimental
4.1. General procedures
4.2.4. Methyl 4-(5-(hydroxymethyl)-2-methoxyphenoxy)
benzoate (7)
Column chromatography was carried out on silica gel or alu-
mina (200–300 mesh). Reactions were monitored by thin-layer
chromatography, using Merck plates with a fluorescent indicator.
Melting points were determined on an X-6 melting-point appara-
tus and are uncorrected. The 1H NMR spectra were recorded on a
Sodium borohydride (0.68 g, 18.32 mmol) was added to a solu-
tion of 6 (12.87 g, 45.73 mmol) in THF (40 mL) over 15 min at 0 °C.
The reaction mixture was then stirred at room temperature for 3 h.
Water (10 mL) and 1 M HCl (18 mL) were added and the THF was
evaporated in vacuo. The resulting mixture was extracted with

G. Xi et al. / Bioorg. Med. Chem. 18 (2010) 6725–6733
6731
CH2Cl2 (20 mL), washed with satd aq NaCl (10 mL ꢂ 3), and dried
over sodium sulfate. The solution was concentrated to obtain a
crude oil that was purified by flash column chromatography
(SiO2), eluting with CH2Cl2 to afford 7 (11.87 g, 91%) as a white so-
lid; mp 77–79 °C. 1H NMR (300 MHz, CDCl3) d 7.97 (d, J = 8.7 Hz,
2H), 7.21 (dd, J = 8.1, 1.8 Hz, 1H), 7.09 (d, J = 1.8 Hz, 1H), 7.00 (d,
J = 8.1 Hz, 1H), 6.92 (d, J = 8.7 Hz, 2H), 4.63 (s, 2H), 3.89 (s, 3H),
3.80 (s, 3H), 1.67 (br s, 1H); MS (ESI) 289 (M+H)+.
(9.74 g, 87%) as colorless oil. Compound 11 (10 g, 20.7 mmol) then
reacted with triphenylphosphonium bromide (7.08 g, 20.7 mmol)
by means of a procedure similar to that used for 8 to yield 12
(13.93 g, 83%) as
a
white solid; mp 229–230 °C. 1H NMR
(600 MHz, CDCl3) d 10.22 (s, 1H), 7.77–7.65 (m, 15H), 7.53 (d,
J = 6.6 Hz, 1H), 7.33–7.29 (m, 3H), 7.14 (t, J = 7.8 Hz, 1H), 7.04 (d,
J = 7.8 Hz, 2H), 6.86 (s, 2H), 6.81 (d, J = 7.2 Hz, 1H), 6.71 (s, 1H),
6.68 (d, J = 7.8 Hz, 2H), 6.63 (s, 1H), 5.40 (d, J = 13.8 Hz, 2H), 3.80
(s, 3H), 3.77 (s, 3H), 2.80 (s, 4H); MS (ESI) 533 (MꢀBr)+.
4.2.5. (4-Methoxy-3-(4-
(methoxycarbonyl)phenoxy)benzyl)triphenylphosphonium
bromide (8)
4.2.8. Dimethyl ether of dehydromarchantin C (13)
A solution of 12 (1.13 g, 1.41 mmol) in anhydrous CH2Cl2
(150 mL) was added dropwise to a stirred suspension of sodium
methoxide (152 mg, 2.82 mmol) in anhydrous CH2Cl2 (200 mL)
over 7 h and the reaction mixture was stirred for 5 h at room tem-
perature. The reaction mixture was filtered, concentrated and the
residue was purified by silica gel column chromatography, eluting
with CH2Cl2 to yield 13 (0.54 g, 86%) as a white solid; mp 225–
226 °C. 1H NMR (600 MHz, CDCl3) d 7.23–7.15 (m, 3H), 7.09 (d,
J = 7.5 Hz, 1H), 6.98–6.6.89 (m, 4H), 6.84 (d, J = 7.8 Hz, 2H), 6.77
(d, J = 8.4 Hz, 1H), 6.65 (d, J = 15.9 Hz, 1H), 6.53 (d, J = 9.0 Hz, 1H),
6.47 (d, J = 15.9 Hz, 1H), 6.10 (d, J = 8.7 Hz, 1H), 5.66 (s, 1H), 3.95
(s, 1H), 3.89 (s, 1H), 2.97–2.87 (m, 4H); MS (ESI) 451 (M+H)+.
Methyl 4-(5-(hydroxymethyl)-2-methoxyphenoxy) benzoate
(7, 7.06 g, 24.5 mmol) and triphenylphosphonium bromide
(8.40 g, 24.5 mmol) was dissolved in anhydrous CH3CN (50 mL),
the resulting mixture was then refluxed for 3 h, the solvent was re-
moved in vacuo and the residue was purified by silica gel column
chromatography, eluting with 5% ethanol in dichloromethane, to
yield 8 (13.21 g, 88%) as a white solid; mp 217–219 °C. 1H NMR
(300 MHz, CDCl3) d 7.90 (d, J = 9.0 Hz, 2H), 7.80–7.73 (m, 9H),
7.65–7.58 (m, 6H), 7.27 (s, 1H), 6.84 (d, J = 8.4 Hz, 1H), 6.69 (d,
J = 8.7 Hz, 2H), 6.51 (s, 1H), 5.46 (d, J = 14 Hz, 2H), 3.90 (s, 3H),
3.73 (s, 3H); MS (ESI) 533 (MꢀBr)+.
4.2.6. Methyl4-(5-(3-(2-(1,3-dioxan-2-yl)-6-
4.2.9. Dimethyl ether of marchantin C (14)
methoxyphenoxy)phenethyl)-2-methoxyphenoxy)benzoate
(10)
Pd/C 10% (250 mg) were added to a solution of 13 (2.25 g,
5.34 mmol) in ethyl acetate (150 mL). The suspension was stirred
under H2 for 24 h at room temperature. The reaction mixture
was filtered, and the solution was concentrated to provide the
crude product that was recrystallized from ethanol to yield 14
(2.2 g, 95%) as a white solid; mp 150–151 °C. 1H NMR (600 MHz,
CDCl3) d 7.23 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 7.8 Hz, 1H), 6.95 (d,
J = 7.8 Hz, 2H), 6.92 (t, J = 7.8 Hz, 1H), 6.86 (t, J = 8.4 Hz, 2H), 6.80
(d, J = 8.4 Hz, 1H), 6.64 (d, J = 7.2 Hz, 2H), 6.62 (s, 1H), 6.45 (d,
J = 8.4 Hz, 1H), 6.26 (d, J = 7.2 Hz, 1H), 5.49 (s, 1H), 3.93 (s, 3H),
3.70 (s, 3H), 3.06 (s, 3H), 2.85–2.79 (m, 4H); MS (ESI) 453 (M+H)+.
Potassium carbonate (8.89 g, 64.44 mmol) and a trace of 18-
crown-6 were added to a solution of 3 (10.12 g, 32.22 mmol) and
8 (19.72 g, 32.22 mmol) in anhydrous CH2Cl2 (80 mL), the resulting
mixture was stirred under reflux for 24 h. The insoluble material
was then filtered off and the filtrate was concentrated to provide
the orange oil that was purified by flash column chromatography
(Al2O3), eluting with a 3:1 solution of hexane–CH2Cl2 to afford 9
(16.87 g, 92%) as a yellow oil. Pd/C 10% (1.5 g) and triethylamine
(35 mL) were then added to a solution of 9 (15 g, 26.41 mmol) in
ethyl acetate (200 mL). The suspension was stirred under H2 for
24 h at room temperature. The mixture was filtered, and concen-
tration provided a crude yellow solid that was purified by precip-
itating from ethyl ether-petroleum ether to afford 10 (14.7 g,
97%) as a white solid; mp 89–90 °C. 1H NMR (600 MHz, CDCl3) d
7.99 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 7.8 Hz, 1H), 7.27 (d, J = 8.4 Hz,
1H), 7.15 (t, J = 7.8 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 7.01 (d,
J = 8.4 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.89 (d, J = 8.4 Hz, 2H),
6.86 (d, J = 1.8 Hz, 1H), 6.77 (d, J = 7.8 Hz, 1H), 6.70 (dd, J = 2.4 Hz,
8.4 Hz, 1H), 5.68 (s, 1H), 4.17–4.14 (m, 2H), 3.91 (s, 3H), 3.83 (s,
2H), 3.78 (s, 3H), 3.71 (s, 3H), 2.85 (s, 4H), 2.24–2.16 (m, 1H),
1.35 (d, J = 13.2 Hz, 1H); MS (ESI) 571 (M+H)+.
4.2.10. Dehydromarchantin C (15)
A solution of boron tribromide (1.95 g, 7.83 mmol) in anhy-
drous CH2Cl2 (10 mL) was added dropwise to a stirred solution of
13 (0.44 g, 0.98 mmol) in anhydrous CH2Cl2 (10 mL) at ꢀ78 °C.
The reaction mixture was stirred at ꢀ78 °C for 3 h, and then was
allowed to warm up to room temperature within 12 h. The ice-cold
water was added, and the reaction mixture was stirred vigorously
for 1 h. The solution was then diluted with CH2Cl2 (50 mL), washed
with satd aq NaCl (20 mL ꢂ 3) and dried over sodium sulfate. The
solution was concentrated, and the residue was purified by flash
column chromatography (SiO2), eluting with CH2Cl2 to yield 15
(0.31 g, 73%) as
a
white solid; mp 101–102 °C. 1H NMR
4.2.7. (4-(5-(3-(2-Formyl-6-methoxyphenoxy)phenethyl)-2-
methoxyphenoxy)benzyl)triphenylphosphonium bromide (12)
A solution of 10 (13.35 g, 23.86 mmol) in anhydrous THF
(25 mL) was added dropwise to a stirred suspension of lithium alu-
minum hydride (1.79 g, 46.98 mmol) in anhydrous THF (30 mL).
The resulting mixture was stirred at room temperature for 2.5 h
and carefully hydrolysed with satd aq ammonium chloride
(10 mL). THF was removed in vacuo and the resulting mixture
was diluted with CH2Cl2 (100 mL), washed with satd aq NaCl
(20 mL ꢂ 3) and dried over sodium sulfate. The solution was con-
centrated to obtain a crude oil that was subsequently dissolved
in a solution of ethanol (100 mL) and 10% aq HCl (20 mL). The
resulting mixture was then stirred at room temperature for 12 h.
Satd aq sodium bicarbonate (150 mL) was added and the ethanol
was removed in vacuo. The resulting mixture was extracted with
CH2Cl2 (200 mL), washed with satd aq NaCl (25 mL ꢂ 3) and dried
over sodium sulfate. The solution was concentrated to yield 11
(600 MHz, CDCl3) d 7.18 (d, J = 9.0 Hz, 2H), 7.12 (t, J = 9.0 Hz, 1H),
7.04 (t, J = 9.6 Hz, 2H), 6.93 (d, J = 3.0 Hz, 2H), 6.91 (d, J = 3.0 Hz,
2H), 6.89 (d, J = 7.8 Hz, 1H), 6.77 (d, J = 7.8 Hz, 1H), 6.70 (d,
J = 16.2 Hz, 1H), 6.57 (d, J = 7.8 Hz, 1H), 6.36 (d, J = 16.2 Hz, 1H),
6.19 (dd, J = 1.8 Hz, 8.4 Hz, 1H), 5.65 (s, 1H), 6.53 (d, J = 9.0 Hz,
1H), 6.47 (d, J = 15.9 Hz, 1H), 6.10 (d, J = 8.7 Hz, 1H), 5.66 (s, 1H),
3.95 (s, 1H), 3.89 (s, 1H), 3.02–2.93 (m, 4H); 13C NMR (600 MHz,
CDCl3) d 157.9, 154.6, 148.9, 148.0, 143.2, 143.1, 139.3, 137.9,
134.1, 131.8, 131.6, 131.1, 128.8, 127.0, 125.8, 122.8, 122.6,
120.3, 117.4, 117.0, 115.1, 114.8, 113.8, 112.9, 33.8, 30.5; MS
(ESI) 423 (M+H)+.
4.2.11. Marchantin C (16)
This compound was prepared from compound 13 (0.60 g,
1.33 mmol) by means of a procedure similar to that used for 15.
Compound 16 (0.37 g, 89%) was obtained as a white solid; mp
148–149 °C. 1H NMR (600 MHz, CDCl3) d 7.11 (t, J = 7.8 Hz, 1H),

6732
G. Xi et al. / Bioorg. Med. Chem. 18 (2010) 6725–6733
7.05 (dd, J = 7.8 Hz, 1.8 Hz, 1H), 6.98 (d, J = 8.5 Hz, 2H), 6.97 (t,
J = 7.8 Hz, 1H), 6.85 (dd, J = 7.8 Hz, 1.8 Hz, 1H), 6.83 (d, J = 8.1 Hz,
1H), 6.58 (t, J = 2.1 Hz, 1H), 6.56 (d, J = 8.5 Hz, 2H), 6.55 (dt,
J = 7.8 Hz, 2.1 Hz, 1H), 6.35 (d, J = 7.8 Hz, 1H), 5.64 (d, J = 2.0 Hz,
1H), 3.01–3.05 (m, 4H), 2.85 (m, 2H), 2.75 (m, 2H); 13C NMR
(600 MHz, CDCl3) d 156.7, 152.8, 148.6, 146.1, 143.4, 143.0,
139.6, 139.1, 136.1, 132.6, 129.7, 128.9, 126.1, 123.4, 122.4,
122.0, 121.3, 115.6, 115.4, 114.9, 114.3, 112.0, 35.8, 35.3, 34.0,
30.3; MS (ESI) 425 (M+H)+.
treated with MC2 (4, 8, 16
lM) or DMSO for 1 h before the addition
of Rh123 (5 M) at 37 °C, and the cells were maintained for an
l
additional 1 h in the dark. After washing with cold PBS, the cells
were then resuspended in 1 ml of ice-cold PBS for analysis using
flow cytometry. Fluorescence intensity associated with Rh123
was measured using FACScan caliber equipped with a 488 nm ar-
gon laser. The emitted fluorescence was measured by a 530 nm
band-pass filter.
In the efflux study, KB and KB/VCR cells were cultured with
medium containing 5 lM of Rh123 in the presence of MC2 or
4.3. Chemical preparation
VRP at 37 °C for 60 min, respectively. The cells were then washed
three times with normal growth medium without Rh123. Cells
were collected at 15, 30, 45, 60, 90, 120 min, respectively, and
resuspended in 1 mL of ice-cold PBS. Rh123-associated fluores-
cence intensity was analyzed by flow cytometry.
MC (16) and its derivatives (13, 14, and 15, namely MC1, MC2
and MC3) were dissolved in dimethylsulfoxide (DMSO, Sigma-Al-
drich) and stored as small aliquots at ꢀ20 °C. Rhodamine123
(Rh123), 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bro-
mide (MTT), and verapamil (VRP) were purchased from Sigma
Co, USA. Adriamycin (ADR) and vincristine (VCR) were purchased
from Zhejiang Haizheng Pharmaceutical Factory (Zhejiang, China),
and prepared in sterile deionized H2O. The stock solutions were
stored as aliquots at ꢀ20 °C.
4.8. Western blot analysis
After treatment with MC2 or VCR as indicated for 24 h, cells were
washed with ice-cold PBS and cell lysates were prepared using RIPA
buffer containing fresh protease inhibitor mixture (50 lg/mL apro-
tinin, 0.5 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM so-
dium orthovanadate, 10 mM sodium fluoride and 10 mM b-
glycerolphosphate). Proteins were quantified using the BCA protein
4.4. Cell culture
Human epidermoid carcinoma KB cells and its VCR resistant KB/
VCR cells31 with characteristics of MDR attributable to expression
of P-gp (a gift from Dr. J. Ding, Shanghai Institute of Materia Med-
ica, Chinese Academy of Sciences, Shanghai, China) were routinely
cultured in RPMI-1640 medium supplemented with 10% fetal bo-
assay. Samples containing equal amounts of protein (100 lg) from
lysates were separated by SDS–PAGE and electrophoretically trans-
ferred onto polyvinylidene fluoride (PVDF) membranes. After being
blocked with 5% fat-free dry milk in TBST (20 mM Tris–HCl (pH 7.6),
150 mM NaCl and 0.1% Tween 20) for 1 h at room temperature, the
membranes were immunoblotted overnight with specific antibod-
ies against P-gp, Bcl-2, Bax, or poly (ADP-ribose) polymerase (PARP)
(Santa Cruz Biotech, USA), respectively, followed by incubation with
HRP-conjugated secondary antibodies for 1 h at room temperature.
Membranes were stripped and reprobed with Glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology) as
a protein loading control. Membranes were visualized by enhanced
chemiluminescence detection system (Millipore) and followed by
exposure to X-ray films.
vine serum, 100 U/mL penicillin-G, and 100 lg/mL streptomycin
at 37 °C in a humidified atmosphere containing 5% CO2. KB/VCR
cells were, maintained in medium containing 200 ng/mL VCR and
incubated in drug-free medium for at least one week prior to
experiments.
4.5. Cytotoxicity and multidrug resistance reversal assay
Cells were seeded into 96-well plates and exposed to varying
concentrations of MC, MC1, MC2 and MC3, respectively. The cyto-
toxicity was analyzed after treatment of cells with these com-
pounds for 48 h by MTT assay. Control cells were subjected to
DMSO treatment. In MDR reversing assay, cells seeded in 96-well
plates were treated with VCR alone, or VCR combined with various
concentrations of target compounds for 48 h, cell proliferation was
assessed using MTT assay. Verapamil (VRP) was included as a ref-
erence agent. The IC50 values were defined as the drug concentra-
tion that resulted in a 50% inhibition of cell growth using untreated
cells as 100%. Reversal fold index (RF) was calculated as the ratio,
RF = IC50 of VCR alone/IC50 of VCR combined with desired
chemicals.
4.9. DAPI staining
Nucleic morphological changes of apoptosis were determined
by staining cell with DAPI. Cells were seeded into 24-well plates
and treated with MC2 (16
l
M), VCR (0.2
lM) or a combination
of MC2 (16 M) with VCR (0.2
l
l
M) for 48 h. After washing with
PBS, cells were fixed with 4% paraformaldehyde (Sigma) in PBS
for 10 min at room temperature. The fixed cells were then washed
with PBS and permeabilized with 0.1% Triton X-100 for 10 min
prior to staining with DAPI (1:2000 dilution, in 1 ꢂ PBS) for
10 min. The cells were washed with PBS and mounted. Images of
DAPI fluorescence were collected using a Nikon phase-fluores-
cence microscope.
4.6. Intracellular adriamycin accumulation
4.10. Statistical analysis
Accumulation of adriamycin (ADR) was determined by incubat-
ing tumor cells with ADR (3
lg/mL) alone or in combination with
Data were expressed as means SD. Statistical analysis of the
data was performed using the Student’s t test. p <0.05 was consid-
ered statistically significant.
MC2 (4, 8, 16 M, respectively) for 1 h at 37 °C. Cells were then
l
placed in ice-water bath to stop the reaction followed by harvest-
ing and washing twice with ice-cold PBS. The intracellular ADR
concentration was determined by monitoring a change in fluores-
cence intensity associated with ADR using FACScan flow cytome-
try. Data analysis was completed with Cell Quest software.
Acknowledgments
This work was supported by the National Natural Science Foun-
dation of China (30973551), Shandong Provincial Key Research
Foundation (2006GG1102023 and 2008GG10002042), and Founda-
tion for the Young Researcher Program of the Health Department
of Shandong Province (2006J39).
4.7. Rh123 accumulation and efflux assay
Effect of MC2 on the intracellular accumulation of Rh123 was
determined as previously described. KB and KB/VCR cells were

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