ONCOLOGY LETTERS 28: 471, 2024

Characteristics of sphingomyelin metabolism
in the MCF7 and BT474 radiotherapy‑resistant
HER2‑positive breast cancer cell lines
MEGUMI KIKUCHI1, SATORU MONZEN1,2, MAI HORIKOSHI1, SHURI TSUDA1,
YOTA TATARA3, ANDRZEJ WOJCIK4 and YASUSHI MARIYA5
1

Department of Radiation Science, Hirosaki University Graduate School of Health Sciences, Hirosaki, Aomori 036‑8564, Japan;
2

3

Research Center for Biomedical Sciences, Hirosaki University, Hirosaki, Aomori 036‑8564, Japan;

Department of Stress Response Science, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine,
Hirosaki, Aomori 036‑8562, Japan; 4Department of Molecular Biosciences, The Wenner‑Gren Institute, Stockholm University,

Stockholm, Stockholm SE‑10691, Sweden; 5Department of Radiology, Aomori Rosai Hospital, Hachinohe, Aomori 031‑0822, Japan

Received January 18, 2024; Accepted June 18, 2024
DOI: 10.3892/ol.2024.14604
Abstract. Breast cancer is the most common cancer globallyin terms of incidence. This cancer is classified into subtypes
based on histological or immunological characteristics.
HER2‑positive cases account for 15‑25% of breast cancer
cases, and one of the first events in breast carcinogenesis is
HER2 upregulation. Furthermore, HER2 expression increases
the detection rate of metastatic or recurrent breast cancers by
50‑80%. The epidermal growth factor receptor family includes
HER2, which is a transmembrane receptor protein. In ourprevious case report, patients who were resistant to anti‑HER2
monoclonal antibody therapy, chemotherapy and radiotherapy
had higher concentrations of phospholipid metabolites suchas phosphatidylcholine and sphingomyelin (SM), which was
associated with cancer recurrence progression. To better understand the relationship between radiotherapy resistance and SM
expression, breast cancer cell lines with and without HER2
expression (MCF7 and BT474) after exposure to ionizing
radiation (IR) were examined. In the cell culture supernatant,similar levels of SM in MCF7 cells were identified after 1‑4 Gy
exposure. However, SM levels in BT474 cells were upregulatedcompared with those of in the control group. Intracellular SM
levels were upregulated in BT474 cells exposed to 1 and 4 Gycompared with the non‑irradiated control group. Furthermore,significantly increased mRNA expression levels of sphingomyelin synthase 2 (SGMS2) in BT474 cells exposed to IR wereobserved compared with those in nonirradiated cells; however,Correspondence to: Professor Satoru Monzen, Department ofRadiation Science, Hirosaki University Graduate School of Health
Sciences, 66‑1 Hon‑cho, Hirosaki, Aomori 036‑8564, JapanE‑mail: monzens@hirosaki‑u.ac.jp

Keywords: HER2‑positive breast cancer, sphingomyelin,sphingomyelin synthase 1, sphingomyelin synthase 2, radioresistant

the SGMS2 levels in MCF7 cells did not differ significantly
among the 0, 2 and 4 Gy groups. These findings suggested thata higher dose of IR induced the secretion of SM and its associated gene expression in HER2‑positive breast cancer cells.
Introduction
Breast cancer is the most common cancer in the world,
according to the International Agency for Research on
Cancer (1). According to the World Health Organization's
report in 2020, approximately 2.3 million new cases of breast
cancer surpassed the incidence of lung cancer, which waspreviously the most common cancer (2). According to cancer
statistics in Japan and the United States, the 5‑year survival
rates of patients with breast cancer (all stages combined)
were approximately 92.3% (2009‑2011) and 91% (2012‑2018),
respectively (3,4). These findings appear to indicate
higher survival rates than for other serious types of cancer.
Furthermore, if this cancer is not treated or becomes resistant
to treatment, it can spread to distant sites. The histological or
immunological characteristics of this cancer are classified as
subtypes. Breast cancer is classified into four types based on
the immunohistochemical expression of hormone receptors:
estrogen receptor positive (ER+), progesterone receptor positive (PR+), human epidermal growth factor receptor 2‑positive(HER2+), and triple‑negative, which is defined by the absenceof expression of any of the above receptors (5). HER2 expression accounts for 15‑25% of breast cancers, and during breast
carcinogenesis, its overexpression is one of the first events tooccur (6,7). HER2 also increases the detection rate of metastatic or recurrent breast cancers by 50‑80%. The epidermal
growth factor receptor (EGFR) family includes HER2, which
is a transmembrane receptor protein. It is a protein found on
the cell surface that promotes cell proliferation. It is encodedby the gene erb‑b2 on the long arm of chromosome 17 (8).
In a previous study using serum metabolome analysis ofpatients who were resistant to anti‑HER2 monoclonal antibody therapy, chemotherapy, and radiotherapy, we discovered

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that the concentrations of phospholipid metabolites, such as
phosphatidylcholine and sphingomyelin (SM), increase withcancer recurrence (9). Furthermore, immunological analysisof resected recurrent cancer tissue revealed that HER2+ areasand neutral sphingomyelinase‑2 were compressed. In breast
cancer, SM‑containing lipids may be involved in cancer
growth and remodeling of the signaling along with the HER2
protein (10). To confirm the dynamics of SM observed in thepatient, the present study examined SM expression in breast
cancer cell lines with or without HER2 expression (MCF7
and BT474) after radiation, as well as the relationship between
radioresistant cancer and SM.
Materials and methods
Cell preparation and culture. The human breast cancer celllines MCF7 (ER+, PR+, and HER2-) and BT474 (ER+, PR+, andHER2+) were obtained from the RIKEN BioResource Centerand the National Institute of Biomedical Innovation, Health, andNutrition, respectively. MCF7 cells were cultured in Minimum
Essential Media (Nacalai Tesque Co., Ltd.) containing 10%
heat‑inactivated fetal bovine serum (FBS; Japan Bioserum)and 1% penicillin/streptomycin in a humidified atmosphere at
37˚C under 5% CO2. BT474 cells were grown in RPMI‑1640(Thermo Fisher Scientific, Tokyo, Japan) supplemented with
10% heat‑inactivated FBS and 1% penicillin/streptomycin
in a humidified atmosphere of 37˚C under 5% CO2. Viable
cells were determined using the trypan blue (Nacalai Tesque)exclusion assay, which were then counted using a Burker‑Turk
hemocytometer (SLGC, Saitama, Japan).
Irradiation. X‑ray irradiation (150 kVp, 20 mA with 0.5 mm
aluminum and 0.3 mm copper filters) was carried out using
an X‑ray generator (MBR‑1520R‑3; Hitachi Medical Co. Ltd.)
with a 45 cm distance between the focus and target. During
irradiation, the dose was monitored using a thimble ionizationchamber next to the samples. The dose rate was 1 Gy/min. The
cell viability test using the trypan blue exclusion assay was
performed post 24 h after exposure to IR.
Clonogenic potency assay after irradiation. The clonogenic
potency assay was performed using colony forming cells.
It was tested in a basic medium enriched with 2.6% methyl
cellulose (Nacalai Tesque Inc.). The cells (5.0x102) following
irradiation were suspended in 1 ml of methylcellulose medium.
This mixture was transferred to 24‑well cell culture plates(Corning Inc.) at 0.3 ml/well and incubated at 37˚C for 7 daysin a humidified atmosphere of 95% air/5% CO2. Colonies withmore than 50 cells were counted using an inversion microscope.
Flow cytometry for cell cycle distribution analysis. MCF7
and BT474 cells were seeded in a 60 mm dish containing
4 ml of culture medium. These cells were irradiatedat 1 to 4 Gy and incubated for 12 h. The harvested cells(5x105 cells) were treated with cold 70% ethanol for over
5 min on ice before adding RNaseI. These cells were
stained with propidium iodide (50 µg/ml, FUJIFILM Wako
Pure Chemical Co. Ltd.) for 30 min in the dark. Cell cycle
distribution analysis was done with a Cell Lab Quanta™ Sc

MPL (Beckman Coulter). Kaluza analysis software (version
2.1; Beckman Coulter) was used to identify the sub‑G1,
G 0/G1, S, and G2/M phases.
Quantitation of SM. The total SMs in the cell culture
supernatant and within the cell were quantified using a
Sphingomyelinase Fluorometric Assay kit (Cayman Chemical
Co. Ltd.). The experimental samples that reacted withsphingomyelinase were broken down into ceramide and phosphorylcholine. Resorufin, a fluorescent molecule, was thenproduced from ceramide using alkaline phosphatase, choline
oxidase, and an H2O2 reaction. The fluorescence (Ex530/Em590)
of these samples was measured with a microplate reader(TriStar LB 941; Berthold Tech.).
Total RNA extraction and reverse transcription‑quantitative
polymerase chain reaction. Total RNA was obtained from
culture cells using the RNeasy® Plus Mini kit (Qiagen Inc.)and measured using a NanoDrop system (Thermo Fisher
Scientific, Inc.). Total RNA quality was determined using a
2100 Bioanalyzer (Agilent Technologies Inc.), and first‑strand
cDNA was synthesized using ReverTra Ace ® qPCR RT
Master Mix (Toyobo Co. Ltd.) following the manufacturer's
protocol. mRNA expression was then assessed using quantitative polymerase chain reaction (qPCR) with the PowerSYBR™ Green PCR Master Mix (Life Technologies Inc.)and a SmartCycler® II (Takara Bio Inc.). Thermocycler conditions were 95˚C for 10 min, followed by 40 cycles of 95˚C
for 15 sec and 60˚C for 1 min. The relative levels of sphingomyelin synthase 1 (SGMS1) and sphingomyelin synthase
2 (SGMS2) were determined using the 2‑ΔΔCq method (11,12)in cells subjected to X‑irradiation after 24 h of no irradiation, after normalization with the housekeeping gene ACTB.
The genetic sequences for SGMS1, SGMS2 and ACTB werereferred by NCBI Gene database (https://www.ncbi.nlm.nih.
gov/gene/). The accession numbers were as follows: SGMS1(NM_147156.4), SGMS2 (NM_001136257.2) and ACTB
(NM_001101.5). The oligonucleotide primer sets used forreverse transcription‑quantitative polymerase chain reaction
were designed by Primer3 software (13), and supplied by
Eurofins Genomics Inc. (Table I). ACTB was selected as thehousekeeping gene for normalization based on MIQE guidelines.
Statistical analysis. The statistical analysis was carriedout using OriginLab software version 9.1 (OriginLab) and
Office 365 (Microsoft) with an add‑in software (OMS
Publishing, Inc.). Cell damage analysis, sphingomyelin
quantitation and mRNA expression data were obtained
from 4 independent experiments and two replications, andwere compared using the Tukey‑Kramer test after one‑way
ANOVA. The clonogenic surviving curves were fitted by
the Levenberg‑Marquardt algorithm, which combines theGauss‑Newton and steepest‑descent methods, non‑linear
models based on the equation y = 1‑(1‑exp(‑x/D 0)) n, and thevalues for D 0 (37% survival dose) and n (number of targets)were determined using a single‑hit multitarget equation (14).
Sphingomyelin quantitation data and mRNA expression data
were compared with the non‑irradiated condition. Statisticalsignificance was determined at a P<0.05.

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Table I. Sequences of human SGMS1, SGMS2 and ACTB real time polymerase chain reaction primers.
Accession number

Primer namePrimer sequence (5'‑3')

NM_147156.4

SGMS1 forward
SGMS1 reverse
SGMS2 forward
SGMS2 reverse
ACTB forward
ACTB reverse

TCGGAACAGTGACTGCTGACGAAATGCTCCAGAGGCTCACTGGAAAACATCCCCAAATGT
AGCACCAAAGGATGTTGACCGGACTTCGAGCAAGAGATGG
AGCACTGTGTTGGCGTACAG

NM_152621.6
NM_001101.5
SGMS, sphingomyelin synthase; ACTB, actin β.

Figure 1. Viable cell numbers of (A) MCF7 and (B) BT474 cells as assessed using the trypan blue exclusion assay, as well as (C) radiation dose‑response curves
for MCF7 and BT474 cells. The data are presented as the mean ± standard error of the mean of three separate experiments. *P<0.05 vs. 0 Gy, #P<0.05 vs. 1 Gy,$
P<0.05 vs. 2 Gy, &P<0.01 vs. 4 Gy.

Results
Cell viability under exposure to ionizing radiation (IR). To
clarify the proliferation potency under IR exposure, both
MCF7 and BT474 cells were exposed to IR until 8 Gy, andcell numbers were calculated after 24 h, as well as a clonogenic potency assay after incubation. MCF7 cells showeda significant decrease of viable cells in a dose‑dependent

manner (nonirradiation control, 2.6±0.1x106 cells/ml; 1 Gy,
2.1±0.2x106 cells/ml, P<0.05; 2 Gy, 1.9±0.1x106 cells/ml, P<0.05;
4 Gy, 1.5±0.2x106 cells/ml, P<0.01; 8 Gy, 0.8±0.2x106 cells/ml,
P<0.01) (Fig. 1A). BT474 cells exposed to 1‑8 Gy irradiation
had a similar number of viable cells as the nonirradiatedcontrol (1.2±0.1x106 cells/ml) (Fig. 1B). Furthermore, BT474
demonstrated a higher clonogenic potency when exposed to IR
than MCF7 (Fig. 1C).

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Figure 2. Cell cycle distribution analysis of (A) MCF7 and (B) BT474 cells exposed to IR. Each phase (sub‑G1, G1, S and G2/M phase) was examined 12 h after
exposure to IR (1‑8 Gy). (C) Typical histogram of DNA contents using PI staining. IR, ionizing radiation.

Alteration of cell cycle distribution by IR. Cell cycle distribution in MCF7 and BT474 cells was assessed using flow
cytometry. In the G2/M phase, both MCF7 and BT474 cells
exposed to 4‑8 Gy IR had a significant upregulation (MCF7
exposed to 4Gy, 10.2±2.7%, P<0.05; MCF7 exposed to 8 Gy,
11.7±3.6%, P<0.05; BT474 exposed to 4 Gy, 11.3±2.6%, P<0.05;
BT474 exposed to 8Gy, 19.9±4.9%, P<0.05) in comparison to
the nonirradiated control (MCF7, 3.4±0.1%; BT474, 6.6±0.3%)(Fig. 2). In the sub‑G1 phase, which indicates apoptotic cells,
MCF7 cells exposed to 1‑8 Gy were upregulated by approximately 35% (P<0.05), compared to nonirradiated controls(13.3%). In contrast, BT474 cells exposed to 2‑8 Gy showed an
approximate 40% upregulation (P<0.05) compared to nonirradiated controls (16.6%).
Quantitation of intra and extracellular SM. We analyzed theconcentrations of cellular SM and cell culture supernatant released
from each cell 48 h after exposure to IR. The concentration ofphosphocholine, a marker for SM, was 1‑5 µM in the cell culture
supernatant and 10‑15 µM in the nonirradiated control. In thecell culture supernatant and in cells, a similar level of SM wasfound after 1‑4 Gy exposure in MCF7 cells. However, SM levels
in BT474 cell culture supernatants were significantly upregulatedcompared with those in the 0 Gy control group (1 Gy, 4.7±1.2‑fold,P<0.05; 2 Gy, 4.2±0.9‑fold, P<0.05; 4 Gy, 6.1±0.5‑fold, P<0.05)(Fig. 3A). In addition, the SM concentration in the cell culture
supernatant of BT474 cells was increased in the 4 Gy groupcompared with the 2 Gy group (P<0.05). Upregulation of intracellular SM levels was detected in BT474 cells exposed to 1 and4 Gy compared with the 0 Gy control group (1 Gy, 4.3±2.5‑fold,P<0.05; 4 Gy, 2.7±2.0‑fold, P<0.05) (Fig. 3B).
mRNA expression related to SM synthesis. To see if radiation
affects the expression of mRNAs involved in SM synthesis,
SGMS1 and SGMS2 mRNAs were quantified. In both types ofcells, the expression of SGMS1 exposed to IR was comparable

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Figure 3. Quantitative analysis of total sphingomyelin levels. (A) Cell culture supernatant and (B) intracellular sphingomyelin levels were examined using the
ELISA method. The data are presented as the mean ± standard error of the mean of three separate experiments. *P<0.05.

Figure 4. Expression levels of sphingomyelin synthesis‑related mRNAs. (A) SGMS1 and (B) SGMS2 were examined using reverse transcription‑quantitative
PCR. The data are presented as the mean ± standard error of the mean of three separate experiments. *P<0.05.

to that in the nonirradiated control (Fig. 4A). Conversely, asignificantly higher expression of SGMS2 in BT474 cells
exposed to IR was detected compared to nonirradiated cells(2 Gy, 4.8±1.1‑fold, P<0.05; 4 Gy, 3.8±0.9‑fold, P<0.05). In
MCF7 cells, the SGMS2 mRNA levels did not differ significantly among the 0, 2 and 4 Gy groups (Fig. 4B).
Discussion
In the current study, we focused on a breast cancer cell model(MCF7 and BT474) with or without HER2 expression andconfirmed the release of SM when exposed to a higher doseof IR, which is used in radiotherapy. It was demonstrated
that HER2+ cells (BT474) exposed to IR have a higher
proliferation potential than HER2− cells (MCF7). Generally,

exposure to high‑dose rate IR causes apoptosis via DNA
cluster damage (15). The cell cycle distribution can be assessedusing flow cytometry, including the sub‑G1, G1, S, and G2/M
phases. Apoptotic cells are present in the sub‑G1 phase (16).
An increase in the G2/M phase in both cells exposed to IR
could indicate that the cell cycle checkpoint for DNA repair
from IR damage, such as strand breaks, is triggered by natural
reactions. Our presented cell cycle results revealed that thetwo cell lines were all arrested in G2/M, and sub‑G1 levels
increased significantly when exposed to IR. However, theBT474 was radioresistant at the same radiation dose (4 and8 Gy). These findings suggest that BT474 (HER2+) retains astrong proliferative potency even at high doses of IR, which
may explain its radioresistance. Concurrently, it is interestingthat SM was secreted outside of HER2+ BT474 cells after

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Figure 5. Schematic of the interaction between phospholipids and cellular survival pathways in the present study. EMT, epithelial‑mesenchymal transition;IR, ionizing radiation; p‑, phosphorylated; S1P, sphingosine‑1‑phosphate; SM, sphingomyelin.

irradiation, but not in MCF7 cells. Furthermore, it is interesting that IR induced SGMS2 mRNA, which could provide anenvironment for SM production. This is the first study to showthat higher doses of IR in the clinical radiation range induce
mRNA expression of SGMS2 and promote SM productionin HER2+ breast cancer cells. In our previous report (9), we
identified that chemo/radio‑therapeutic‑resistant cancer tissue
connected to reduced expression of nSMase in HER2‑positive
breast cancer are strongly co‑expressed with the sphingomyelin family which can be detected at the peripheral blood
serum level. The behavior of the cell line models in this study
appears to support this patient data.
HER2 overexpression in breast cancer cells activates
signaling pathways that promote cell proliferation, tumor
growth, and lymph node metastasis (17). Furthermore,
several reports, including our previous case report, show thatmost radioresistant cells express HER2 (18‑20). There are
distinctive molecular mechanisms like ‘a shift from ER to
EGFR signaling pathways with increased MAPK and PI3K
activity’ (21), ‘HER2 overexpression in cells induces NF‑kB
expression’ (22,23), as well as ‘HER2 knockdown in breast
cancer cells is induced to radiosensitivity’ (24). Therefore,
this information suggests that radioresistant breast cancer isundoubtedly linked to HER2 expression.
SM is the most abundant sphingolipid in mammalian cell
membranes, with concentrations particularly high in the plasma
membrane, endocytic recycling compartment, and trans Golgi
network. SM regulates endocytosis, receptor‑mediated ligand
uptake, ion channel and G‑protein‑coupled receptor function,and protein sorting, and serves as a receptor for various bacterial and nonbacterial pore‑forming toxins (25). According toBilal et al (26), SM is produced through the chemical reactionof SGMS1 and SGMS2 with ceramide and phosphocholine.
Although there have been no reports demonstrating that SM
promotes radioresistance in HER2+ breast cancer, it is criticalto study the behavior of SGMS2 mRNA, which increases SM
in a radioresistant environment.
According to Zheng et al (27), SGMS2 is a critical regulator of ceramide and SM homeostasis that promotes cancer
cell proliferation by suppressing apoptosis via a Cer‑associatedpathway. Also, SGMS2 increases cancer cell invasiveness bypromoting epithelial‑to‑mesenchymal transition initiation via
the TGF‑β/Smad signaling pathway. Furthermore, SGMS2 activates the TGF‑β/Smad signaling pathway mainly by increasing
TGF‑β1 secretion, which is likely associated with abnormal
SM expression. TGF‑β1 plays a key role in radiation‑induced
fibrosis, which primarily leads to radioresistance; therefore,a higher concentration of SM is associated with increased
radioresistance (28). In one study on colorectal cancer, LARP6
protein binding to RNA inhibits SGMS2 expression and slows
cancer progression by causing ceramide and SM imbalance (29). According to these findings, because IR increases
SM and promotes SGMS2 expression, an intracellular signaltransduction pathway exists between the HER2+ environment
and IR induced SGMS2 expression (Fig. 5). It is critical tomonitor the radioresistance of HER2+/SGMS2+ breast cancer
cells during radiotherapy, and SM may be used as a biomarker
for this. Kozar et al (30) attempted to use serum SM frompatients with epithelial ovarian cancer as a diagnostic marker,
and we hope to further understand its molecular mechanism.
One limitation of this study is the lack of a functional analysis
of SGMS2 expression in BT474, including the intracellularsignaling pathway, such as knock down of SGMS mRNA. In
addition, we could not validate whether SM regulates EMTof these cells under exposure of IR and affects the cell cycle.
Therefore, it is requiring that additional analysis for this issue.
Future studies using this analysis may reveal the relationshipbetween SGMS2, HER2 and radiosensitivity.
In conclusion, these findings indicate that a higher dose of
IR causes secretion of SM and associated gene expression inONCOLOGY LETTERS 28: 471, 2024

HER2+ breast cancer cells. This molecule may also function asa marker of radioresistance in cells.
Acknowledgements
Not applicable.
Funding
The present study was supported by KAKENHI, Grants‑in‑Aid
for Scientific Research (B; grant no. 21H02861), Fund for the
Promotion of Joint International Research (Fostering Joint
International Research; grant no. 17KK0181), Grant‑in‑Aid for
Challenging Research (Exploratory; grant no. 19K22731) andTakeda Science Foundation (grant no. 2022‑Satoru Monzen).
Availability of data and materials
The data generated in the present study may be requested fromthe corresponding author.
Authors' contributions
MK and SM designed the study, drafted the manuscript andactively participated in its revision. MK, SM, MH, ST, YT, AW
and YM examined and analyzed the experimental data. MK, SMand MH confirmed the authenticity of all the raw data. SM, AW
and YM oversaw the study, critically reviewed the manuscript,
and gave final approval of the version submitted and published.
All authors read and approved the final version of the manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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