Effect of magnetic nanoparticles of Fe3O4 and 5-bromotetrandrine on reversal of multidrug resistance in K562/A02 leukemic cells.

Abstract

This study aims to evaluate the multidrug resistance (MDR) reversal activity by magnetic nanoparticles of Fe3O4 (MNPs-Fe3O4) and 5-bromotetrandrine (BrTet) MDR cell line K562/A02 solitarily or symphysially. The proliferation of K562 and K562/A02 cells and the cytotoxicity on peripheral blood mononuclear cells (PMBCs) were evaluated by MTT assay. Cellular accumulation of daunorubicin (DNR) was analyzed by flow cytometry. Real-time polymerase chain reaction and Western blotting analyses were performed to examine the mRNA and protein levels of mdr1, respectively. The results showed that the combination of MNPs-Fe3O4 and BrTet with effective concentrations significantly increased cytotoxicity against MDR cell line K562/A02. Both BrTet and MNPs-Fe3O4 increased the intracellular DNR accumulation in the K562/A02 cell line, and downregulated the level of mdr1 gene and expression of P-glycoprotein. Furthermore, the combination did not have significant cytotoxicity in PMBCs. We propose that MNPs-Fe3O4 conjugated with DNR and BrTet probably have synergetic effects on MDR reversal.

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effect of magnetic nanoparticles of Fe3O4
and 5-bromotetrandrine on reversal of multidrug
resistance in K562/A02 leukemic cells
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O R I g I N A L R e s e A R C h
Jian Cheng1*
Weiwei Wu1*
Bao-an Chen1
Feng gao1
Wenlin Xu2
Chong gao1
Jiahua Ding1
Yunyu sun1
huihui song1
Wen Bao1
Xinchen sun3
Cuirong Xu1
Wenji Chen1
Ningna Chen1
Lijie Liu4
guohua Xia1
Xiaomao Li5
Xuemei Wang6
1Department of hematology,
3Department of Oncology, The
Afiliated Zhongda hospital, southeast
University, Nanjing, People’s Republic
of China; 2Department of hematology,
The First People’s hospital of Zhengjiang,
Zhenjiang, People’s Republic of China;
4Institution of Physiology, 6state Key Lab
of Bioelectronics (Chien-shiung Wu
Laboratory), southeast University,
Nanjing, People’s Republic of China;
5Department of Physics, University of
saarland, saarbruechen, germany; *These
authors have contributed equally to this
work
Correspondence: Bao-an Chen
Department of Hematology, The Affiliated
Zhongda hospital, southeast University,
Nanjing 210009, People’s Republic
of China
Tel +86 25 8327 2006
Fax +86 25 8327 2011
email cba8888@hotmail.com
Abstract: This study aims to evaluate the multidrug resistance (MDR) reversal activity by
magnetic nanoparticles of Fe3O4 (MNPs-Fe3O4) and 5-bromotetrandrine (BrTet) MDR cell line
K562/A02 solitarily or symphysially. The proliferation of K562 and K562/A02 cells and the
cytotoxicity on peripheral blood mononuclear cells (PMBCs) were evaluated by MTT assay.
Cellular accumulation of daunorubicin (DNR) was analyzed by flow cytometry. Real-time
polymerase chain reaction and Western blotting analyses were performed to examine the mRNA
and protein levels of mdr1, respectively. The results showed that the combination of MNPs-Fe3O4
and BrTet with effective concentrations significantly increased cytotoxicity against MDR cell
line K562/A02. Both BrTet and MNPs-Fe3O4 increased the intracellular DNR accumulation
in the K562/A02 cell line, and downregulated the level of mdr1 gene and expression of
P-glycoprotein. Furthermore, the combination did not have significant cytotoxicity in PMBCs.
We propose that MNPs-Fe3O4 conjugated with DNR and BrTet probably have synergetic effects
on MDR reversal.
Keywords: magnetic nanoparticles of Fe3O4, 5-bromotetrandrine, multidrug resistance K562/A02
Introduction
Multidrug resistance (MDR), defined as being induced by one kind of drug which
generates cross-tolerance to various anticancer drugs with different structures and
mechanisms. This cross-tolerance was noted to be the most important mechanism
of self-dependence in cytotoxic injury of chemotherapeutics, as well as a major
reason for relapse or failure of chemotherapy during treatment for hematological
malignancies. The mechanism which generates the MDR phenotype is complex.1
Mechanisms include drug excretion, antiapoptosis, activity changes in drug-
metabolizing enzymes such as serum glutathione S epoxide transferase (GST),
topoisomerase II (TOPO II), protein kinase C (PKC), and reinforcement of recovery
from DNA injury. However the major mechanism in the mediation of MDR was
P-glycoprotein (P-gp, P170), a kind of transmembrane glycoprotein.2 P-gp, a
member of ATP-binding cassette (ABC) transporter superfamily, encoded by mdr1
gene, mediated drug resistance to anthracycline, vinca alkaloids, etc, in the function
of the lipophilic drug excretion pump and leading to a decrease in cytotoxic drug
accumulation. Several patients with hematological malignancies, such as acute
leukemia,3 non-Hodgkin’s lymphoma,4 which were sensitive to chemotherapy at the
initial stage of morbidity, were detected showing a non- or hypo-expression of P-gp
pretherapy, but an over expression of P-gp after relapse. Some studies indicated that
mdr1/P-gp had a close correlation in the prognosis of the outcome of chemotherapy.
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Patients with mdr1 overexpression had shorter life span,
and a higher recurrence rate. P-gp inhibitors can improve
the curative effect of myelodysplastic syndrome with
overexpressed P-gp.5 Therefore, the analysis of mdr1/P-gp
could be considered an anticipation index for the outcome
of hematological malignancies.
At present, only the first and second generation of
P-gp inhibitors have been tested in clinic trials, but the
therapeutic effects and adverse reactions experienced
were not ideal. The unity of the reversal mechanism was
an important factor that interfered in clinic utilization.
In recent years, drugs packaged by liposome or multimer
have been developed. This could change the character of
such drugs by affecting tumor cells selectively, decreasing
toxicity, and augmenting the MDR reversal effect.6–8
Nanotechnology displayed feasible application perspec-
tives initially through improving administration routes of
chemotherapeutics, which rivaled neutralization excretion
drugs by tumor cells in order to increase the intracellular
accumulation of drugs. Magnetite, possessing a magnetic
positive charge group, was the essential component of the
magnetic particle. The present understanding is that the
magnetic compound of Fe3O4 could self-synthesize into
tela, suggesting that Fe3O4 has an existence physically. This
characteristic of Fe3O4 was the safeguard for using it as a
safe hypotoxic carrier.9
Tetrandrine (Tet), a type of hypotoxic calcium channel
antagonist,10 could inhibit P-gp overexpresssion through
competing activation of PKC and mdr1, and had a better
reversal effect for leukemia in vivo and in vitro. The Tet
may reverse MDR through downregulating the expression
of mdr1mRNA or P-gp, increasing drug accumulation
intracellularly, as well as reinforcing apoptosis induced by
anticancer drugs. 5-bromotetrandrine (BrTet), a bromized
derivative of Tet, has been shown to be more potent than
Tet in the modulation of MDR.11 In the prophase trial, we
applied magnetic nanoparticles of Fe3O4 (MNPs-Fe3O4) and
BrTet connected with adriamycin (ADM) respectively for
MDR reversal, and indicated that both of the drugs could
increase the reversal effect. However both of the drugs had
different targets, and the complexity of MDR mechanism
determined the effect of the synergy in MDR reversal.
Materials and methods
Main reagents
Adriamycin (Hisun Phamaceutical Co., Zhejiang, China)
stock solution 3.68 mM and daunorubicin (DNR) (Main
Luck Phamaceuticals Inc., Shenzheng, China) stock solution
3.55 mM were prepared with 0.01 M phosphate-buffered
saline (PBS, pH = 7.4). BrTet (Kanghong Phamaceuticals
Inc., Chengdu, China) was also diluted with 0.01 M
PBS (pH = 7.4). All reagents used in this trial were of
analytical grade.
Cell culture
Human chronic myeloid leukemia in blast crisis cell line,
K562, and its resistant cell line to ADM, K562/A02, were
both cultured in a RPMI 1640 medium (Gibco/BRL,
Gaithersburg, MD, USA) supplemented with 10% fetal
calf serum (FCS) (Sijiqing, Hangzhou, China), 100 U/mL
penicillin and 100 µg/mL streptomycin at 37 °C in a
humidified atmosphere of 5% CO2, and passaged every two
days. The resistant cell line was incubated in the presence
of ADM (1 µg/mL) until at least three days before starting
the experiments. Peripheral blood mononuclear cells
(PBMCs) were assembled and separated from healthy cells,
and then cultured in a RPMI 1640 medium supplemented
with 15% FCS, 100 ng/mL granulocyte colony-stimulating
factor (G-CSF)12 at 37 °C in a humidified atmosphere of
5% CO2 for one week before the commencement of the
experiments.
Cytotoxicity assay
The in vitro chemosensitivity was measured by 3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
(MTT) (Sigma Aldrich, St. Louis, MO, USA) assay. Tumor
cells (1.5 × 106/mL) were suspended in 100 µL of culture
medium on 96-well culture plate (Costar; Fisher Scientific,
Hampton, NH, USA) per well. For determining the reversal
effect of MNPs-Fe3O4, BrTet was used alone or symphysially
in graded concentrations of DNR with or without the reversal
agents added. The concentration of BrTet was 0.5 µM, which
is half of the recommended reversal concentration according
to Chen and colleagues.13 MNPs-Fe3O4, 0.1 (V/V), was
conjugated with graded concentrations of DNR and kept at
4 °C for 48 hours before being applied to the experiment.14
PBMCs (2.0 × 105/mL) were also suspended in 100 µL of
culture medium in 96-well culture plate per well using the
same concentration. To determine the antiproliferative effect
of BrTet or MNPs-Fe3O4, various concentrations of these
two reagents in 100 µL dilute of the culture medium were
added into every well. Meanwhile, RPMI 1640 medium
was regarded as the bank control and cells without reagents
were the negative control. The cells were then incubated
for 48 hours at 37 °C, following which, MTT (0.5 mg/mL)
20 µL were added to each well and cultured for an additional

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four hours. The formazan was dissolved with 150 µL
dimethyl sulfoxide (Sigma Aldrich) after blotting the culture
medium. The plates were shaken lightly for 10 minutes, and
the reduction of MTT was quantified by absorbance at a
wavelength of 490 nm using a microplate reader (Model-550;
Bio-Rad Laboratories, Hercules, CA, USA). The relative
growth rates (RGR) of PMBCs, evaluating the antiprolifera-
tive effect of BrTet or MNPs-Fe3O4, were transformed into
six bands according to Table 1.
Cellular accumulation of DNR
Cellular accumulation of DNR was analyzed by flow
cytometry (FCM) assay. Briefly, both K562/A02 cells
(1.5 × 106/mL) and PMBCs (2.0 × 105 mL) were exposed to
2 µM DNR in the absence or presence of BrTet at 0.5 µM or
MNPs-Fe3O4 at 0.1 (V/V) for 24 hours at 37 °C to determine
the cytotoxicity of BrTet and MNPs-Fe3O4. The PMBCs were
sieved and incubated with CD34-FITC for 15 minutes. After
being washed by PBS three times, the cells were suspended
by 400 µL PBS and determined by a FACS Calibur flow
cytometry (Becton Dickinson, Franklin Lakes, NJ, USA)
assay at excitation and emission wavelengths of 488 nm
and 575 nm, respectively.
Western blotting of P-gp
As described before, K562/A02 cells (1.5 × 106/mL)
were treated, harvested, and then lysed in a lysis buffer
containing 25 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1%
Triton X-100, 5 mM EDTA, 5 mM EGTA, 10 mM NaF,
1 mM PMSF, 0.5% NP-40, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 1 mM pepstatin for 15 minutes. The 150 µg
of total protein was electrophoresesed on 10% SDS-
polyacrylamide gels, and transferred onto a nitrocellulose
membrane. Nonspecific binding sites were blocked by 5%
milk powder dissolved in Tris-buffered saline (TBS) at
room temperature for one hour. The primary P-gp antibody
was mouse monoclonal anti-human antibody (Neomarkers,
Fremont, CA, USA). The secondary antibody was alkaline
phosphatase-labeled rabbit-mouse IgG. Membranes were
exposed in nitroblue tetrazolium chloride (NBT)/5-bromo-
4-chloro-3-indoxylphosphate (BCIP) were used as color
detection reagents. After normalization by the correspond-
ing expression of β-actin, the levels of P-gp protein expres-
sion were determined by densitometry scans (ECL system,
Amersham, UK). Meanwhile, K562 cells without any
reagents were negative controls.
Real time PCR analysis of mdr1
mRNA expression
Total cellular RNA was extracted from K562/A02 cells with
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and quanti-
fied by 1.5% agarose gel. Each group contained RNA from
the cells incubated with DNR (2 µM), MNPs-Fe3O4 (0.1 v/v)
loaded with DNR (2 µM), DNR (2 µM) connected with BrTet
(0.5 µM); MNPs-Fe3O4 (0.1 v/v) loaded with DNR (2 µM)
and conjugated with BrTet (0.5 µM), respectively. K562/A02
cells and K562 cells without reagents were regarded as posi-
tive and negative controls. The reverse transcription reactions
were performed using TaKaRa RNA PCR Kit (avian myelo-
blastosis virus, version. 3.0; Dalian, Liaoning, China). The
newly synthetic cDNA was amplified by SYBR Premix Ex
TaqTm real-time polymerase chain reaction (PCR) (TaKaRa)
at ABI 7300 Real-Time PCR system (Applied Biosystems,
Foster City, CA, USA). Primers involved were the mdr1
primers (forwards 5′-TTGTTTGCCACCACGATA-3′,
reverse 5′-GAACCACTGCTTCGCTTT-3′) and the GAPDH
primers (forwards 5′-TGAACGGGAAGCTCACTGG-3′,
reverse 5′-TCCACCACCCTGTTGCTGTA-3′). The ampli-
fied PCR products were 274bp and 205bp for mdr1 and
GAPDH, respectively. The conditions for real-time PCR
were 50 °C for 20 minutes and 45 cycles of 95 °C for
10 minutes, 94 °C for 15 seconds, 59 °C for 20 seconds, and
72 °C for 30 seconds. The Ct of both mdr1 and GAPDH were
detected after each cycle. The expression of mdr1 was given
as follows: Ct = Ctmdr1 - CtGAPDH.
statistical analysis
Data was analyzed using the Statistical Package for Social
Science (v. 15.0; SPSS Inc., Chicago, IL, USA). The
significance of differences in the mean value between groups
was analyzed using one-way ANOVA; P values 0.05
were considered statistically significant.
Table 1 The RgR and cytotoxicity gradation of PMBCs incubated
with BrTet or MNPs-Fe3O4 for 48 hours
Cytotoxicity gradation RGR (%)
Band 0
100
Band 1
75∼99
50∼74
25∼49
1∼24
0
Band 2
Band 3
Band 4
Band 5
Notes: The RgR was calculated as follow: ODtest cells/ODnegative control × 100%. The
band 0 or 1 was qualified, band 2 should be evaluated with morphology, and band
3∼5 was unqualified.23
Abbreviations: OD, optical density; PMBCs, peripheral blood mononuclear cells;
RgR, relative growth rate.

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Results
effect of cytotoxicity of BrTet or Fe3O4
The cytotoxicity of BrTet or MNPs-Fe3O4 in PBMCs was
assayed by the MTT assay. The transformation of RGR
and the cytotoxicity gradation were evaluated according to
Table 1. The data in Table 2 clearly indicates that BrTet at
0.25∼2 µM and MNPs-Fe3O4 at 0.025∼0.1 (V/V) did not
generate significant cytotoxicity.
Cell survival
According to the MTT assay, the ability of BrTet or
MNPs-Fe3O4 used alone or in conjunction to reverse DNR
resistance was compared in K562/A02 cell line. BrTet and
MNPs-Fe3O4 symphysially showed significant reversal
effect on DNR resistance in the K562/A02 cell line, and its
potency was greater than using BrTet and Fe3O4 alone. The
inhibitory concentration at 50% (IC50) of DNR decreased
from 32.33 ± 8.40 µM to 1.80 ± 0.30 µM (P  0.001)
at the combination of BrTet 0.5 µM and MNPs-Fe3O4
0.1 (V/V), while the values were down to 7.49 ± 0.85 µM
and 4.25 ± 2.16 µM for Fe3O4 and BrTet, respectively
(P  0.001). The fold reversals were 17.96 of the synergia
compared with the 4.32 of MNPs-Fe3O4 and 7.61 of
BrTet alone. In contrast, there were no significant differences
between those in K562 cell line (Table 3).
Fluorescence intensity
of endocellular DNR
After repeating the trial three times, at a wavelength of
488 nm, DNR was excited to emit at 575 nm wavelength
spontaneously where fluorescence intensity (FI) of intra-
cellular DNR could be recorded by FCM. The mean
fluorescence intensity of K562/A02 cells preincubated
with 2 µM DNR for 48 hours was 44.49 ± 2.57; with DNR-
Fe3O4, 117.54 ± 2.53; with DNR-BrTet, 140.61 ± 4.32;
and with DNR-Fe3O4-BrTet, 117.34 ± 3.54. The differ-
ences were significant when compared with control group
(P  0.001). Furthermore, the fluorescence intensity of
intracellular DNR of PMBCs had no dramatic variations
(Figures 1, 2).
expression of P-gp
The expression of P-gp determined by western blotting
analysis was downregulated to some extent after treatment
with DNR in the presence of MNPs-Fe3O4 or BrTet alone
or in combination for 48 hours, compared with K562/A02
blank group (P  0.05). But there were no significant
differences between the DNR in company with BrTet
group and the group of DNR loaded with MNPs-Fe3O4 and
BrTet (P  0.05). The relative expression was calculated
as follows: gradationP-gp/gradationβ-actin (Figure 3).
Mdr1 mRNA level
DNR in the absence or presence of MNPs-Fe3O4 or
BrTet alone or in combination downregulated the mdr1
mRNA levels. The mdr1 relative expression of K562/A02
was 19.68, which was 3.23 times lower compared with
K562 cells. The value of mdr1 expression was significantly
Table 2 The cytotoxicity of BrTet or MNPs-Fe3O4 on PBMCs
for 48 hours determined by MTT assay
Groups OD
(mean ± SD)
RGR (%) Cytotoxicity
gradation
BrTet (µM)
Control
1.218 ± 0.154
––
0.25
1.247 ± 0.111*
102.38Band 0
0.5
1.261 ± 0.079*
103.50Band 0
1
1.222 ± 0.104*
100.27 Band 0
2
1.221 ± 0.195*
100.22 Band 0
MNPs-Fe3O4 (V/V)
Control
0.763 ± 0.023
––
0.025
0.741 ± 0.033**
97.16 Band 1
0.05
0.737 ± 0.045**
96.63Band 1
0.1
0.697 ± 0.075**
91.39Band 1
0.2
0.552 ± 0.028**
72.30Band 2
Notes: *P  0.05, compared with control group; **P  0.05, compared with control
group.
Abbreviations: BrTet, 5-bromotetrandrine; MNPs-Fe3O4, magnetic nanoparticles
of Fe3O4; OD, optical density; PBMCs, peripheral blood mononuclear cells; RgR,
relative growth rate; sD, standard deviation.
Table 3 The cytotoxicity of BrTet or MNPs-Fe3O4 on K562/
A02 and K562 cells for 48 hours determined by MTT assay
(mean ± sD)
Groups
IC50 of DNR (µM)
K562/A02K562
DNR
32.33 ± 8.40
7.49 ± 0.85 (4.32)*
4.25 ± 2.16 (7.61)*
1.80 ± 0.30 (17.96)*
2.74 ± 0.19
2.31 ± 0.27 (1.19)
2.80 ± 0.27 (0.98)
2.39 ± 0.20 (1.15)
DNR – Fe3O4 ( 0.1 V/V)
DNR + BrTet (0.5 µM)
DNR – Fe3O4 + BrTet
Notes: The values in parentheses were FR calculated as follows: FR = IC50 DNR alone/
IC50 DNR + agent. *P  0.05, compared with DNR group.
Abbreviations: BrTet, 5-bromotetrandrine; DNR, daunorubicin; FR, fold reversal;
IC50, inhibitory concentration at 50%; MNPs-Fe3O4, magnetic nanoparticles of Fe3O4;
OD, optical density; PBMCs, peripheral blood mononuclear cells; RgR, relative growth
rate; sD, standard deviation.

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decreased to 12.2 and 10.12 in the cells pretreated with
DNR and loaded with MNPs-Fe3O4 and the combination
of MNPs-Fe3O4 and BrTet, respectively, while BrTet con-
nected with DNR showed a less significant amount of
mdr1 compared with BrTet only. The values were 17.89
(Figure 4).
Discussion
MDR is considered to be a major problem for clinical
therapy related to hematological malignancies. Several
calcium channel inhibitors, such as verapamil, nifedipine,
azidopine, or tetrandrine, can inhibit the excretion of anti-
cancer drugs, thus overcome MDR,15–16 but the toxicity
of these drugs constrains both their application and their
therapeutic effect. BrTet, a modified production of Tet, sig-
nificantly reversed resistance to ADM in MCF-7/Dox cell
line in vivo, and restrained tumor growth in athymic mice
bearing cancer.17 In our former study, reversal effect of BrTet
was more effective than that of Tet in K562/A02 cell line
in vivo and in vitro.11 In this experiment, we also studied the
Figure 1 Cellular accumulation of DNR in K562/A02 cells after treatment with different reversal agents for 24 hours. A) Incubated with 2 µM DNR; B) Incubated with 2 µM
DNR loaded with MNPs-Fe3O4 (0.1 v/v); C) Incubated with 2 µM DNR in conjunction with BrTet (0.5 µM); D) Incubated with 2 µM DNR loaded with MNPs-Fe3O4 (0.1 v/v)
synergia with BrTet (0.5 µM).
Abbreviations: DNR, daunorubicin; MNPs-Fe3O4, magnetic nanoparticles of Fe3O4.
Counts
M1
Marker Left, Right % Gated Mean
All
1, 9910
10, 9910
M1
100.00
97.83
40.78
41.55
120
0
Counts
FL2-H
100
102
104
103
101
M1
Marker Left, Right % Gated
All 1, 9910
10, 9910 M1
Mean
113.17
115.42
100.00
97.98
120
0
Counts
FL2-H
100
102
104
103
101
M1
Marker Left, Right % Gated Mean
1, 9910
10, 9910
M1
139.51
145.00
All
FL2-H
100
102
104
103
101
0
Counts
120
M1
100.00
96.10
Marker Left, Right % Gated
All1, 9910
10, 9910
M1
Mean
165.08
168.49
100.00
97.91
FL2-H
100
102
104
103
101
120
0
A
CD
B
Marker Left, Right % Gated Mean
All
1, 9910
10, 9910
M1
104
100.00
100.00
320.77
320.77
Marker Left, Right % Gated Mean
All
1, 9910
10, 9910 M1
100.00
100.00
326.24
326.24
Marker Left, Right % Gated
All1, 9910
10, 9910
M1
Mean
305.24
305.24
100.00
100.00
Marker Left, Right % Gated Mean
All
1, 9910
10, 9910
M1
100.00
100.00
319.69
319.69
A
CD
B
60
0
Counts
60
0
Counts
60
0
Counts
60
0 10 20 304050
Counts
FL2-H
100
102
104
103
101
FL2-H
100
102
103
101
FL2-H
100
102
104
103
101
FL2-H
100
102
104
103
101
M1
M1
M1
M1
Figure 2 Intracellular accumulation of DNR in PMBCs after treated with different reversal agents for 24 hours. A) Incubated with 2 µM DNR; B) Incubated with 2 µM
DNR loaded with MNPs-Fe3O4 (0.1 v/v); C) Incubated with 2 µM DNR in conjunction with BrTet (0.5 µM); D) Incubated with 2 µM DNR loaded with MNPs-Fe3O4 (0.1 v/v),
synergia with BrTet (0.5 µM).
Abbreviations: BrTet, 5-bromotetrandrine; DNR, daunorubicin; MNPs-Fe3O4, magnetic nanoparticles of Fe3O4; PBMCs, peripheral blood mononuclear cells.