Free Radical Biology and Medicine 110 (2017) 280–290

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Free Radical Biology and Medicine

journal homepage: www.elsevier.com/locate/freeradbiomed

Original article

Cold atmospheric plasma restores tamoxifen sensitivity in resistant MCF-7 MARK

breast cancer cell
Seungyeon Leea,1, Hyunkyung Leea,1, Dawoon Jeonga, Juyeon Hama, Sungbin Parka,
⁎
Eun Ha Choib, Sun Jung Kima,
a
Department of Life Science, Dongguk University-Seoul, Goyang 10326, Republic of Korea
b
Plasma Bioscience Research Center, Kwangwoon University, Seoul, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: Cancer recurrence, which is frequently accompanied by chemotherapy, has been a challenge in cancer treat-
Apoptosis ment. This study was carried out to examine the potential applications of the reactive oxygen species (ROS)-
Cold atmospheric plasma producing cold atmospheric plasma (CAP) to overcome the cancer cells’ drug resistance, which has been
Breast cancer emerging as an alternative therapeutic tool for cancer. For this, we developed a tamoxifen (Tam)-resistant MCF-7
Genome-wide expression
(MCF-7/TamR) breast cancer cell model and examined the effect of CAP on the recovery of Tam sensitivity at the
Reactive oxygen species
cellular and molecular level. The ROS level was increased 1.9-fold in CAP-treated MCF-7/TamR cells compared
to the non-treated cell. CAP was proven to restore sensitivity by up to 50% for MCF-7/TamR cells against Tam
after CAP treatment. The comparison of genome-wide expression between the acquisition of Tam resistance and
CAP treatment identified 20 genes that commonly showed significant expression changes. Notably, all the genes
except two have been oppositely dysregulated in the two cellular statuses, and the majority of them are known to
contribute to the acquisition of Tam resistance. The protein expression of selected genes, MX1 and HOXC6, was
recovered to that of their parental cell by CAP. Furthermore, the dysregulation of MX1 and HOXC6 in MCF-7/
TamR alleviated the drug sensitivity recovery effect of CAP. Taken together, CAP inhibited the growth of Tam-
resistant MCF-7 cancer cells and reset it to the Tam-sensitive status by restoring the expression of drug re-
sistance–related genes. These findings may lend credence to CAP as an alternative or complementary tool in the
treatment or prevention of Tam-resistant cancer.

1. Introduction signaling pathway, Rac1-PAK1 pathway [7], EGFR/HER2 regulatory

Drug resistance recurrence is a core clinical challenge in cancer
treatment, because in the case of breast cancer, approximately a quarter
of cancer patients treated with tamoxifen for five years acquired re-
currence, limiting its capability as a cancer treatment [1]. A few me-
chanisms have been explicated for the recurrence of tamoxifen re-
sistance (TamR), including the alteration of specific gene expressions
and signaling pathways. For example, the overexpression of transcrip-
tion factor YBX1 in sensitive cells conferred resistance against Tam,
which was associated with the accelerated degradation of estrogen re-
ceptor (ER) [2]. In another study, the Tam-specific activation of Oct-4
recruited ER onto its preferential genomic binding sites, eventually
enhancing tumor growth [3]. In addition, factors that interact with ER
such as FOXA1 [4] and constituents of ER transcriptome such as IL-8,
NRF-1, and HOTAIR contribute to resistance [5,6]. As instances of the
loops [8], and PI3K-AKT-mTOR pathway [9] have been identified as
playing key roles in Tam resistance, the alteration of which led to in-
creased migration and proliferation rates, hormone independent phe-
notypes, and cell autophagy.
A few studies have suggested experimental approaches to over-
coming TamR, including targeting ER with chemical compounds [10],
modulating the components of the ER signaling pathway, or controlling
the components of other signaling pathways that crosstalk with the ER
pathway [11]. Nonetheless, in the efforts to overcome TamR, the
identified molecular mechanisms are limited and the prognosis for
cancer patients remains poor.
Cold atmospheric plasma (CAP) is a specific type of partially ionized
gas defined as a non-thermal non-equilibrium plasma with complex
chemical composition such as NO-, NO2-, OH-, and O- [12]. By virtue of
its efficacy at inducing cell death in multiple cancer cell lines including

⁎
Corresponding author.
E-mail address: sunjungk@dongguk.edu (S.J. Kim).
1
These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.freeradbiomed.2017.06.017
Received 17 March 2017; Received in revised form 9 June 2017; Accepted 26 June 2017
Available online 27 June 2017
0891-5849/ © 2017 Elsevier Inc. All rights reserved.
S. Lee et al.
breast, cervical, glioblastoma, melanoma, and lung cancers, and in
xenograft model animals, CAP has received attention for its clinical
application as an alternative cancer treatment option [13]. One re-
markable advantage of CAP resides in its preference for acting on
cancer cells rather than normal cells. Cancer cells are known to have a
higher ROS level than normal cells, and CAP accumulates more ROS,
eventually inducing the specific death of cancer cells [14]. Recently, the
hampering effect of cholesterol on the permeation of ROS through the
phospholipids bilayer was suggested to explain the increased ingress of
ROS in cancer cells, due to the decreased cholesterol fraction in their
cell membranes [15]. One of the major phenomena caused by the sig-
nificant rise of intracellular ROS is the intense DNA double-strand
break, which results in the mitochondrial apoptosis pathway [14].
Several of CAP's alterations to the gene expression and signaling
pathways other than the apoptotic pathway have been identified: EGFR
dysfunction in oral squamous cell carcinoma [16], the induction of
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Free Radical Biology and Medicine 110 (2017) 280–290
Fig. 1. CAP increases ROS in the MCF-7/TamR cell.
Fluorescence microscopic images were taken for the
MCF-7 and MCF-7/TamR cells using a fluorochrome
DCF-DA after treating the cells with CAP for 0 s or 10
times for 30 s. NAC was treated to scavenge the in-
tracellular ROS. Black and white-colored images are
from bright-field microscopy. (B) The bar graph
shows the quantification of fluorescence intensity
(mean ± SE of three replicates).
inflammation-related genes in keratinocytes [17], epigenetic changes in
breast cancer cells [18], and the enhanced expression of cell cycle–re-
lated γH2A.X [19].
In this study, we hypothesized that CAP can reverse the drug-re-
sistance status of cancer cells provoked by continued exposure to Tam.
This was explored by developing MCF-7/TamR breast cancer cells and
then illuminating the cells with CAP. The appropriate recovery of
sensitivity against Tam was observed in the CAP-treated cells with cell
death. The molecular mechanism of sensitivity recovery was in-
vestigated, revealing that many of the genes that had been dysregulated
by Tam had been set back to their original sensitive status. The current
study is expected to provide insight into how to overcome the drug
resistance of cancer cells using CAP.
S. Lee et al. Free Radical Biology and Medicine 110 (2017) 280–290

Fig. 2. CAP inhibits the proliferation of MCF-7/TamR cells. (A) The reduced tumorigenicity of CAP-treated MCF-7/TamR. The effect of CAP on the tumorigenicity of MCF-7/TamR was
examined by colony formation assay. CAP was treated 10 times for 30 s each, and the colonies were counted using ImageJ after culturing for 14 days. (B) Suppressed proliferation of CAP-
treated MCF-7/TamR. The effect of CAP on proliferation was analyzed by crystal violet dye–based assay. CAP was treated for 600 s or 10 times for 30 s each, and the proliferation was
chased for six days. All assays were performed in triplicate, and the results are depicted as mean ± SE. (C) Increased apoptosis of CAP-treated MCF-7/TamR. The effect of CAP on the
apoptosis of MCF-7/TamR cells was analyzed on a flow cytometer after treating cells 10 times with CAP for 30 s each. All assays were performed in triplicate, and the results are depicted
as mean ± SE.

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Fig. 3. CAP recovers the drug sensitivity of the Tamoxifen-resistant MCF-7. (A) Expression of TamR marker genes in the MCF-7/TamR. The acquisition of resistance and recovery of
sensitivity against Tam was examined by the RT-PCR of previously known marker genes. The effect of CAP on the sensitivity of MCF-7 and MCF-7/TamR against Tam was examined by
colony formation (B) and dye-based proliferation assay (C). Cells were treated with CAP 10 times for 30 s each, followed by treatment with 0.1 and 0.5 μM of Tam. The number of colonies
was counted using ImageJ and indicated as a bar graph. All assays were performed in triplicate, and the results are depicted as mean ± SE.

2. Materials and methods
2.1. Cell culture and treatment with CAP
Breast cancer cell line MCF-7 was purchased from American Type
Culture Collection (ATCC; Manassas, VA, USA), and cultured in RPMI-
1640 medium supplemented with 10% FBS and 1% penicillin/strepto-
mycin under humidified 5% CO2 condition. The MCF-7/TamR cell line
was generated by sequential exposure to increasing doses of 4-hydro-
xytamoxifen (Sigma-Aldrich, St. Louis, MO, USA) up to 0.1 µM. A mesh-
type Dielectric Barrier Discharge (DBD) CAP device was manufactured
at the Plasma Bioscience Research Center (Kwangwoon University,

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Table 1
Genes of which expression was restored to the level of MCF-7 after CAP treatment in MCF7/TamR.

Gene symbol Accession No. Description Fold change (MCF-7 vs. MCF- Fold change (MCF-7/TamR vs. MCF-7/
7/TamR) TamR + CAP 600 S)

FKBP14 NM_017946.2 FK506 Binding Protein 14 3.231 −1.657

ZNF483 NM_001007169.1 Zinc Finger Protein 483 2.766 −1.602
DEM1 NM_022774.1 Exonuclease 5, Defects In Morphology Protein 1 Homolog 2.506 −1.518
PLIN5 NM_001013706.2 Perilipin 5 2.442 −1.828
XRCC2 NM_005431.1 X-Ray Repair Complementing Defective Repair In Chinese 2.405 −1.534
Hamster Cells 2
DST NM_001723.4 Dystonin 2.401 −1.640
SRPK2 NM_182691.1 SRSF Protein Kinase 2 2.290 −1.554
SULT1A1 NM_177530.1 Sulfotransferase Family, Cytosolic, 1A, Phenol-Preferring, 2.056 −1.579
Member 1
LOC440353 NR_002603.1 Nuclear Pore Complex Interacting Protein Family, Member 2.017 −1.523
A1 Pseudogene
HOXC6 NM_004503.3 Homeobox C6 1.944 −1.533
TNFSF15 NM_005118.2 Tumor Necrosis Factor (Ligand) Superfamily, Member 15 1.863 −1.512
CDK5RAP3 NM_176095.1 CDK5 Regulatory Subunit Associated Protein 3 1.640 −1.712
FLAD1 NM_025207.3 Flavin Adenine Dinucleotide Synthetase 1 1.593 −1.643
SOX9 NM_000346.2 SRY (Sex Determining Region Y)-Box 9 −1.556 1.594
CPM NM_001005502.1 Carboxypeptidase M −2.475 1.516
IFI6 NM_022872.2 Interferon, Alpha-Inducible Protein 6 −4.692 1.615
IFI6 NM_022873.2 Interferon, Alpha-Inducible Protein 6 −4.692 1.526
MX1 NM_002462.2 MX Dynamin-Like GTPase 1 −5.731 1.598

Korea). Cells with 50% confluence in a 60 mm plate were irradiated 10
times for 30 s each hour, with at a distance of 5 mm from the DBD disc
to the media surface. The discharged voltage was measured at 0.46 kV
when the argon gas flow rate was set to 1.5 L/min with 0.12 kV supply
voltage.
2.2. Cell transfection
siRNAs were synthesized by Bioneer (Korea), and transiently
transfected into the MCF-7 and MCF-7/TamR cells at approximately
50% confluence and a final concentration of 30 nM using Lipofectamine
RNAiMAX (Invitrogen, Carlsbad, CA, USA). cDNA was overexpressed by
transiently transfecting 1 µg of recombinant pEZ-M02 plasmid vector
(GeneCopoeia, Rockville, MD, USA) using Lipofectamine 3000 and
PLUS reagents (Invitrogen), and the cells were then cultured for 24 h
for further analysis. Information about the siRNAs and plasmid clones is
indicated in Supplementary Table S1.
2.3. Cell proliferation and apoptosis analysis
The cell growth rate was measured once 1×103 cells were seeded in
each well of 96-well plates, incubated for 12 h, and then exposed to
CAP 10 times for 30 s each. After 24 h, Tam was added to the culture
media at a final concentration of 0, 0.1, and 0.5 µM. At day two, four,
and six after Tam treatment, 10 µl of CCK reagent from a cell counting
kit-8 assay kit (Dojindo Laboratories, Japan) was added to each well,
and absorbance at 450 nm was measured after 1 h using a microplate
reader. In the case of colony formation assay, 4×103 cells were seeded
in a 60 mm culture dish and cultured for two weeks. The colonies were
then fixed with acetic acid/methanol (1:7), stained with 0.5% crystal
violet (Sigma-Aldrich), and counted with Image J software (National
Institutes of Health, Bethesda, MD, USA). Apoptosis analysis was car-
ried out once cells had been harvested with trypsin, washed twice with
PBS, and then resuspended in 1x binding buffer from FITC Annexin V
Apoptosis Detection Kit I (BD Biosciences, Franklin Lakes, NJ, USA) at a
concentration of 1×106 cells/ml. The cell suspension was incubated as
200 µl samples with 5 µl of Annexin V FITC for 15 min, and then with
2 µl of propidium iodide (PI) at room temperature in the dark for 3 min.
All samples were analyzed with a Becton Dickinson Accuri C6 flow
cytometer (BD Biosciences) within 1 h.
2.4. Tam sensitivity assay
The sensitivity against Tam was evaluated by seeding 5×103 cells
per 60 mm dish, or 2×103 cells per well of a 96-well plate and cul-
turing for 12 h. Then, transient transfection of siRNA or recombinant
plasmid vectors was conducted, followed by CAP treatment. The cells
were exposed to either 100 or 500 nM of 4-hydroxytamoxifen or
ethanol (Sigma-Aldrich) as a vehicle control. Cell viability was mea-
sured using CCK-8 reagent or colony counting through the aforemen-
tioned procedures.
2.5. Genome-wide expression assay
The total RNA was isolated from MCF-7 and MCF-7/TamR, and
CAP-treated MCF-7/TamR using the ZR-Duet DNA/RNA MiniPrep kit
(Zymo Research, Irvine, CA, USA). For the experiment, 750 ng of
Biotin-labeled cRNA was synthesized from the RNA and was hybridized
on the Illumina HumanHT-12 v4 Expression BeadChip (Illumina, San
Diego, CA, USA) by following the supplier's protocol. All arrays were
quantile normalized, and data processing was performed using Illumina
GenomeStudio v2011.1. All raw data were deposited in the Gene
Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) with series
entry number GSE 95208.
2.6. Pathway and clustering analysis
Relevant networks were constructed through the use of Ingenuity
Pathway Analysis (IPA, Qiagen, Redwood City, CA, USA) (http://www.
qiagen.com/ingenuity) using differentially expressed genes (|fold
change| ≥ 1.5, P-value < 0.05) from the microarray analysis.
Clustering analysis was conducted using Cluster 3.0 software (http://
bonsai.hgc.jp/~mdehoon/software/cluster/) and visualized through a
TreeView program (http://jtreeview.sourceforge.net/) with genes dif-
ferentially expressed in both comparisons, MCF-7 vs. MCF-7/TamR and
MCF-7/TamR vs. CAP-treated MCF-7/TamR.
2.7. Real-time RT-PCR
Total RNA was reverse transcribed using ReverTra Ace qPCR RT
Master Mix with gDNA remover (Toyobo, Japan) following the sup-
plier's instruction. An ABI 7300 system (Applied Biosystems, Foster
City, CA, USA) was utilized to perform quantitative real-time PCR using

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Fig. 4. Clustering of genes affected by Tam and CAP in MCF-7. (A) Genome-wide expression analysis identified 20 genes that commonly showed significant expression changes (> 1.5
fold) during the acquisition of Tam resistance and CAP treatment. The heatmap was constructed with genes from MCF-7/Tam vs. MCF-7 and CAP-treated MCF-7/Tam vs. MCF-7/Tam. (B)
The RT-PCR analysis of genes affected by Tam and CAP. RT-PCR was carried out for 10 genes that appeared in (A). Black bar, MCF-7 parent cell; red bar, Tam-resistant MCF-7; cyan bar,
CAP-treated MCF-7/Tam. All assays were performed in triplicate, and the results are depicted as mean ± SE.

SYBR FAST qPCR Kit (Kapa Biosystems, Wilmington, MA, USA) in tri-
plicate. The expression of genes was normalized using GAPDH and
calculated according to 2−ΔΔCt method. The primers used for RT-PCR
are detailed in Supplementary Table S2.
2.8. Western blot analysis
Total protein was collected using an ice-cold RIPA lysis buffer that
contained a protease inhibitor cocktail (Thermo Fisher Scientific,
Waltham, MA, USA). After concentration quantification by BCA assay
(Thermo Fisher Scientific), 50 µg of protein from each sample was
subjected to SDS-PAGE. The separated proteins were transferred to a
PVDF membrane (Whatman, UK), and blocked with 5% non-fat milk
solution diluted in 0.1% Tween-20 TBS for 1 h at room temperature.
The blots were then incubated with anti-MX1 (1:500, Santa Cruz,
Dallas, Texas, USA) and anti-HOXC6 (1:500, Novus, St. Charles, MO,
USA) antibodies, followed by incubation with HRP-conjugated goat
anti-rabbit secondary antibodies (1:1000, Genetex, Pennsylvania, USA)
for 1 h at room temperature. Rabbit polyclonal beta actin antibody
(1:800, Novus) was used to normalize the quantification of target
proteins. The bands were detected with West Save ECL reagents
(Abfrontier, Korea) and quantified with Image Lab software (Bio-Rad,
Hercules, CA, USA).
2.9. ROS and NO detection assay
To detect ROS, CAP exposure was performed on 5×105 cells per
well of 6-well plates 10 times for 30 s every hour, and these were

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treated with 20 µM of DCFH-DA (Sigma-Aldrich) for 30 min at 37 °C.
Intracellular ROS was quantified by measuring the level of the fluor-
escent DCF which was converted from the cell permeable DCFH-DA. To
detect NO, cells were loaded with 5 µM of DAF-FM diacetate
(Invitrogen) and incubated for 20 min at 37 °C in the dark. Two mM of
N-Acetyl-L-Cysteine (NAC) (Sigma-Aldrich) and 2 mM of N(G)-Nitro-L-
Arginine Methyl Ester (L-NAME) (Sigma-Aldrich) were used to inhibit
the generation of intracellular ROS and NO, respectively. After in-
cubation for 6 h, the cells were exposed to CAP and the intracellular
ROS or NO levels were measured with aforementioned procedure.
Fluorescence was detected by a fluorescence microscope (Leica
Microsystems, Germany) and quantified using an Infinite 200 Pro
fluorescence plate reader (Tecan, Switzerland).
2.10. Statistical analysis
Student's t-test was carried out to compare expressions between
CAP-treated TamR and control cells using SPSS for Windows, release
17.0 (SPSS, Chicago, IL, USA). Experimental graphs were plotted with
Microsoft Excel as mean ± standard error, and the expression differ-
ences of selected genes among three groups, MCF-7, MCF-7/TamR, and
CAP-treated MCF-7/TamR, were determined by ANOVA comparisons. A
value of P < 0.05 was considered statistically significant.
3. Results
3.1. CAP inhibited the proliferation and promoted the apoptosis of MCF-7/
TamR
Interrogating the impact of cold atmospheric plasma on cancer cells
for its potential application in the treatment of drug-resistant cancer
required that we first developed a modified MCF-7 cell line that had
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Free Radical Biology and Medicine 110 (2017) 280–290
Fig. 5. CAP regulates genes related with cell death and cancer.
The highest confidence IPA networks of genes displaying altered
expression as a consequence of CAP treatment of MCF-7/TamR.
The top networks were “Cell death and survival, Neurological
Disease, and Cancer.” Upregulated and downregulated genes are
respectively shaded in red and green, with the color intensity
indicating the magnitude of the expression change. Solid and
dashed lines respectively represent the direct and indirect inter-
actions.
obtained resistance against Tam. The detailed characteristics of the
developed MCF-7/TamR are beyond the scope of the current study and
shall be described in a separate manuscript. When CAP was used to
illuminate the MCF-7/TamR cell, it appropriately increased the level of
ROS (Fig. 1) and RNS (Supplementary Fig. S1) at a similar rate as the
parental MCF-7, although MCF-7/Tam showed a higher level of ROS
than MCF-7 before CAP treatment. The effect of CAP on the prolifera-
tion of MCF-7/TamR was chased by colony formation and dye-based
growth assay. The MCF-7 cell became more tumorigenic during the
acquisition of drug resistance, as judged by the colony formation assay
(Fig. 2A). CAP retarded the growth rate of MCF-7/TamR approximately
to the level of the parental MCF-7, as shown by the colony formation
assay and proliferation assay (Fig. 2A and B). FACS analysis revealed an
increase in both early (32% increase) and late (33% increase) apoptosis
of MCF-7/TamR when CAP was treated (Fig. 2C).
3.2. CAP resensitized MCF-7/TamR cells to Tam
During the acquisition of Tam resistance in cancer cells, specific
metabolic and/or signaling pathways were known to be affected,
especially those of drug transportation and metabolism [20–22]. We
hypothesized that CAP could re-sensitize TamR cells to the drug at least
in part by reversing the expression of affected genes. We first measured
the expression of four genes that had been previously identified as
being upregulated (BAG1, CD24) or downregulated (HDAC4, and
NGX6) during TamR acquisition to examine this, and the results con-
firmed the appropriate deregulation (Fig. 3A). All genes except NGX6
showed expression recovery after CAP treatment, suggesting that CAP
could reverse the expression of TamR-related genes even though the
recovery level was not the same as that in the parental MCF-7 (Fig. 3A).
Next, the MCF-7/TamR cell was examined for the restoration of Tam
sensitivity by CAP through colony formation assay. The results
S. Lee et al.
indicated that the Tam-treated parental MCF-7 showed a sharp decrease
in colony size, while the MCF-7/TamR cells showed a much lesser de-
crease (Fig. 3B). Notably, when CAP was illuminated to the MCF-7/
TamR, sensitivity against the drug appeared again approximately at the
level of MCF-7. The effect of CAP on drug sensitivity was also mon-
itored using a dye-based cell proliferation assay. The result supported
the data of the colony formation assay by showing a retarded growth
rate for CAP-treated MCF-7/TamR compared to non-treated MCF-7/
TamR (Fig. 3C).
3.3. MX1 and HOXC6 mediated the restoration of sensitivity against Tam
The molecular mechanism of how CAP recovers sensitivity against
Tam was interrogated using genome-wide expression analysis, which
was carried out for MCF-7/TamR and CAP-treated MCF-7/TamR cells.
In total, 322 genes with expression changes higher than 1.5-fold from
MCF-7 vs. MCF-7/TamR and 39 genes from MCF-7/TamR vs. CAP-
treated MCF-7/TamR were identified, with 20 genes hitting both
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Free Radical Biology and Medicine 110 (2017) 280–290
Fig. 6. CAP sets the expression of TamR-related MX1 and HOXC6
genes back to their MCF-7 status. The expression of MX1 (A) and
HOXC6 (B) in CAP-treated MCF-7/TamR cells was examined by
Western blot analysis. MX1 and HOXC6 were selected because
they respectively showed downregulation and upregulation in the
MCF-7/Tam compared to MCF-7. Expression was compared
among MCF-7, MCF-7/Tam, and CAP-treated MCF-7/Tam. All
assays were performed in triplicate and a representative image is
shown. Results are depicted as mean ± SE. (C) The effect of CAP
on the expression of MX1 and HOXC6 in the presence of NAC, an
ROS inhibitor, was measured by qPCR. The assay was performed
in triplicate and the result is depicted as mean ± SE.
compared groups. Notably, of the 20 genes, 18 showed the opposite
expression pattern, i.e., they were once upregulated in the MCF-7/
TamR, but the gene had been downregulated by CAP and vice versa
(Table 1 and Fig. 4A). Ten of the 18 genes were randomly selected, and
their expressions were examined by quantitative RT-PCR to further
confirm their involvement in drug resistance and sensitivity. Thus, all
genes recovered their expression with regard to MCF-7 when CAP was
treated to MCF-7/TamR (Fig. 4B). This fact strongly implies sensitivity
restoration in the MCF-7/TamR cells for the drug through CAP resetting
many of the genes that had undergone expression changes during their
resistance acquisition. Ingenuity pathway analysis was carried out to
identify the highest confidence network for the CAP-treated MCF-7/
TamR, and this resulted in the “Cell death and survival, Neurological
disease and Cancer” pathway (Fig. 5).
Interrogating the role of genes mediated by CAP activity to recover
drug sensitivity revealed two genes, MX1 and HOXC6, that respectively
showed up- and down-regulation at the RNA level by CAP; these were
selected and their protein levels were analyzed by Western blot. As
S. Lee et al. Free Radical Biology and Medicine 110 (2017) 280–290

Fig. 7. Recovery of CAP-mediated Tam sensitivity is hampered by the dysregulation of MX1 and HOXC6 in the MCF-7/TamR cell. (A) Effect of the downregulation of MX1 on the recovery
of Tam sensitivity. MX1 was downregulated with siRNA in MCF-7/TamR cells, and the sensitivity against Tam was examined by colony formation assay after treating cells with CAP. The
colonies were counted using ImageJ and denoted with a bar graph. siNC, negative-control siRNA. All assays were performed in triplicate, and the results are depicted as mean ± SE. (B)
The effect of the upregulation of HOXC6 on the Tam-sensitivity recovery. HOXC6 was upregulated with a HOXC6 cDNA-containing recombinant plasmid by transiently transfecting the
MCF-7/TamR cell, and the sensitivity against Tam was examined.

shown in Fig. 6A, MX1 was decreased in the MCF-7/TamR compared to
MCF-7, but increased after CAP treatment. In the case of HOXC6, the
expression trend was opposite to that of MX1 (Fig. 6B). The regulation
of the two genes by CAP was abrogated when NAC, an ROS inhibitor,
was treated to the cell before CAP exposure judged by qRT-PCR, in-
dicating the role of ROS relaying the CAP activity (Fig. 6C). Next, the
effect of CAP on drug sensitivity was monitored in the genes’ dysre-
gulated conditions through colony formation assay. When MX1 was
induced to be downregulated by the siRNA (Supplementary Fig. S2A),
MCF-7/TamR grew at a higher rate than in control siRNA-treated cells,
implying a lower sensitivity against Tam (Fig. 7A). Meanwhile, higher
cell growth was observed when HOXC6 was induced to be upregulated
with an ORF-containing recombinant plasmid (Fig. 7B and
Supplementary Fig. S2B). The growth rate of MCF-7/TamR was re-
versed when ORF of MX1 and siRNA of HOXC6 were used
(Supplementary Figs. S2 and S3). These observations were also found
with a similar pattern in the parental MCF-7 cell (Supplementary Figs.
S4 and S5). To see whether MX1 or HOXC6 affects the ROS production,
the genes were deregulated in the MCF-7/TamR cell, and ROS level was
measured after CAP treatment. This resulted in no significant change of
ROS level, suggesting that the genes were regulated downstream of the
ROS (Supplementary Fig. S6). Cumulatively, these results suggest that
MX1 and HOXC6 are strongly involved in mediating the effect of CAP at
least in part through ROS on recovering Tam sensitivity.

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4. Discussion
This study aimed to examine CAP for its potential applications for
overcoming drug resistance in breast cancer. When CAP was applied to
Tam-resistant MCF-7 cells, it appropriately set the expression of many
genes back to the status of the original MCF-7 cells, which had been
dysregulated by Tam. A previous study reported the restoration of re-
sponsiveness in temozolomide-resistant glioma cells against the drug by
CAP [23]. However, the molecular mechanism of how CAP relieves
drug-resistance is relatively unexplored.
It is speculated that two main streams of CAP activities exist that
induce cell death of TamR cells. First, CAP might induce cell death in
TamR cells via the typical apoptotic pathway. In detail, most apoptosis
pathways observed in CAP-treated cancer cells are based on the mi-
tochondrial pathway triggered by DNA damage and mitochondrial da-
mage [12,24]. Accordingly, a double-strand break marker, γ-H2AX, has
been commonly observed after CAP treatment [25]. The tumor necrosis
factor receptor (TNFR)-based apoptosis pathway can also be activated
by the rise of intracellular ROS in a few cancers such as melanoma [26]
and head and neck cancer cells [27]. In the current study, this was
supported by the results that the TamR cells showed a similar apoptotic
rate as the parental cells, and that many apoptosis-related genes were
dysregulated by CAP. In this sense, CAP seems to have a similar effect
on proliferation inhibition, regardless of the drug sensitivity status.
TamR cells showed more cancerous and stem-cell like characteristics
[28]; these alterations may increase the ROS level and decrease the
amount of membrane cholesterol, possibly making TamR cells more
vulnerable to CAP.
Second, CAP might have notably contributed to the proliferation
inhibition of TamR cells by restoring the expression of genes that were
involved in cancer cell proliferation, such as MX1 and HOXC6, which
had been deregulated during the acquisition of drug resistance. MX1 is
a dynamin family GTPase that strongly interacts with tubulin, sug-
gesting its involvement in mitosis. The protein is known to have tumor-
suppressive activity for several cancers, and the loss of its expression
results in increased metastasis and decreased sensitivity to Docetaxel
[29]. HOXC6 is a homeobox-containing gene associated with mammary
gland development and is overexpressed in a variety of cancers in-
cluding breast and prostate cancers [30]. HOXC6 expression was
strongly detected in multidrug-resistance (MDR) cancer [31]. An siRNA
of HOXC6 significantly reduced the intracellular level of MDR-1, and
inhibited MDR cancer cells in a xenograft tumor.
A remarkable observation during the acquisition of TamR in cancer
cell is the function of estrogen receptors and crosstalk with growth
factor–receptor pathways [32]. A few genes in our genome-wide ana-
lysis such as FKBP14, FGF21, and TNFSF15 in those pathways re-
presented a significant expression change. Furthermore, drug re-
sistance–related genes such as SULT1A1, XRCC, and SOX9 were found
in the network and in the list of deregulated genes, where their ex-
pression had changed to the opposite of TamR. SULT1A1 is a phase II
enzyme responsible for the sulfation of 4-hydroxytamoxifen in breast
tumors [33]. XRCC2- [34] and SOX9-deficient [35] cells were found to
be hypersensitive to the alkylating anticancer drug, temozolomide. The
molecular mechanism of how these proteins are associated with re-
sistance and sensitivity by Tam should be revealed in further studies.
It should be mentioned that only 20 genes appeared in common
between MCF-7/TamR and TamR/CAP, while 39 genes underwent
significant expression changes in TamR/CAP. Therefore, other path-
ways should be also responsible for the restoration of drug sensitivity,
which have yet to be elucidated. Another limitation of this study is the
lack of various strength conditions in the CAP treatment, because dif-
ferent cellular response, perhaps even opposite cellular phenomena,
would be expected for different strengths of CAP [36]. A precise stan-
dardization of the CAP condition would help establish appropriate
option for the treatment of drug-resistant cancer cells.
289
Free Radical Biology and Medicine 110 (2017) 280–290
5. Conclusion
CAP has successfully demonstrated its capacity to return the TamR
of MCF-7 breast cancer cells to the original drug-sensitive status by
setting back the expression of cell death and survival pathway–related
genes such as MX1 and HOXC6. The involvement of MX1 and HOXC6 in
the recovery of drug sensitivity was proven by showing the deteriora-
tion of the recovery effect upon dysregulation. The identified genes
could be developed as diagnostic markers, and could be molecular
targets for the clinical treatment of Tam-resistant breast cancer.
Furthermore, the successful restoration of sensitivity against Tam pro-
vides insights into the potential use of CAP for the treatment of TamR
cancer.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by the Science Research Center Program
(NRF-2010–0027963) and the Basic Science Research Program (NRF-
2016R1D1A1B01009235) of the National Research Foundation of
Korea funded by the Ministry of Education, Science, and Technology.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at http://dx.doi.org/10.1016/j.freeradbiomed.2017.06.
017.
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