Free Radical Biology and Medicine 86 (2015) 228–238

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Free Radical Biology and Medicine

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

Original Contribution

Role of cystathionine β-synthase in human breast Cancer

Suvajit Sen a,b,n, Brian Kawahara a, Divya Gupta a, Rebecca Tsai a, Marine Khachatryan a,
Sinchita Roy-Chowdhuri c, Shikha Bose d, Alexander Yoon e, Kym Faull b,e,
Robin Farias-Eisner a, Gautam Chaudhuri a,b,f
a
Department of Obstetrics and Gynecology, David Geffen School of Medicine at University of California at Los Angeles, Los Angeles, CA 90095, USA
b
The Jonsson Cancer Center, David Geffen School of Medicine at University of California at Los Angeles, Los Angeles, CA 90095, USA
c
Department of Pathology at MD Anderson School of Medicine, Houston, TX 77054, USA
d
Pathology and Laboratory Medicine at Cedars–Sinai Medical Center, Los Angeles, CA 90048, USA
e
Semel Institute for Neuroscience and Human Behavior at University of California at Los Angeles, Los Angeles, CA 90095, USA
f
Molecular and Medical Pharmacology, David Geffen School of Medicine at University of California at Los Angeles, Los Angeles, CA 90095, USA

art ic l e i nf o a b s t r a c t

Article history: Cystathionine β-synthase (CBS) is an enzyme in the transulfuration pathway that can catalyze the
Received 25 March 2015 condensation of homocysteine (Hcy) and cysteine (Cys) to hydrogen sulfide (H2S) and cystathionine
Received in revised form (CTH). CBS-derived H2S is important in angiogenesis and drug resistance in colon and ovarian cancers,
15 May 2015
respectively. However, the mechanisms by which cancer cell-derived H2S is utilized by cancer cells as a
Accepted 16 May 2015
Available online 4 June 2015
protective agent against host-derived activated macrophages are not yet investigated. This study in-
vestigated the mechanistic role of CBS-derived H2S in the protection of human breast cancer (HBC) cells
Keywords: against activated macrophages. HBC patient-derived tissue arrays and immunoblot analysis of HBC cells
Cystathionine β-synthase exhibited significantly increased levels of CBS when compared with their normal counterparts. This was
Hydrogen sulfide
associated with increased levels of H2S and CTH. Silencing of CBS in HBC cells caused a significant de-
Breast cancer
crease in the levels of H2S and CTH but did not affect the growth of these cells per se, in in vitro cultures.
4-Hydroxynonenal
However CBS-silenced cells exhibited significantly reduced growth in the presence of activated macro-
phages and in xenograft models. This was associated with an increase in the steady state levels of re-
active aldehyde-derived protein adducts. Exogenous addition of H2S countered the effects of CBS silen-
cing in the presence of macrophages. Conversely overexpression of CBS in human breast epithelial (HBE)
cells (which do not naturally express CBS) protected them from activated macrophages, which were
otherwise susceptible to the latter.
& 2015 Elsevier Inc. All rights reserved.

Introduction the transcription of CBS [7,8]. Furthermore, the enzyme activity of

It is well known that CBS regulates the steady state levels of
homocysteine (Hcy), a sulfur-containing amino acid, the accumu-
lation of which leads to cellular toxicity associated with a variety
of diseases including those of the cardiovascular and neurological
systems [1–3]. CBS, which can catalyze the condensation of Hcy
and cysteine (Cys) to hydrogen sulfide (H2S) and cystathionine
(CTH) [4,5], may play an important role in breast cancer as well.
Interestingly, gain of function mutations in the CBS gene have been
shown to be associated with increased risks for breast cancer [6].
Moreover estrogen, an independent risk factor for breast cancer,
activates the transcription factor SP1, which in turn upregulates
n
Corresponding author at: 650 Charles E Young Drive, University of Los Angeles,
CHS 22115, CA 90095, USA. Fax: þ1 3102063670;
E-mail address: ssen@mednet.ucla.edu (S. Sen).
CBS is increased by lactate, the metabolite that is ubiquitously
present in all cancers including HBC [9]. However the cellular
mechanism by which CBS modulates breast cancer homeostasis is
not yet known.
Endogenous H2S derived from CBS is a reductant and has been
shown to play important roles in the resolution of inflammation
and immune modulation [10–12]. However its role in the down-
regulation of host immune surveillance especially with respect to
the cross talk between cancer cells and macrophages has not been
addressed. In this study we assessed the role of the HBC cell-de-
rived CBS/H2S pathway in the mechanistic interaction between
cancer cells and macrophages. This is of significant physiological
relevance as macrophages comprise an important part of the host
immune surveillance against cancers including breast cancer
[13,14].
Infiltrated macrophages that compose a significant portion
of the tumor mass exhibit both tumor-eliminating and

http://dx.doi.org/10.1016/j.freeradbiomed.2015.05.024
0891-5849/& 2015 Elsevier Inc. All rights reserved.
 S. Sen et al. / Free Radical Biology and Medicine 86 (2015) 228–238
tumor-promoting properties [15–17]. HBC cells have evolved to
counter the tumor-eliminating properties of macrophages as well
as increasing the population of tumor-promoting macrophages
[18,19]. One of the mechanisms by which tumoricidal macro-
phages induce cytotoxicity in tumor cells is by causing the for-
mation of adducts between proteins and reactive aldehydes
(formed due to oxidative damages on the plasma membrane li-
pids) [20–23].
Hence, we investigated a protective role of CBS in HBC cells on
exposure to activated macrophages and the underlying mechan-
ism(s).
Experimental procedures
Materials
L-Cysteine (W326305), DL-homocysteine (H4628), and pro-
tease inhibitor cocktail (P8340) and all other chemicals were
purchased from Sigma-Aldrich (St. Louis, MO) unless stated
otherwise. GYY 4137 (No. 13345), 4-hydroxynonenal or 4-HNE (No.
32100), and butathione sulfoxamine (BSO) (No. 83730-53-4) were
purchased from Cayman Chemicals (Ann Arbor, MI). The following
primary antibodies were used: anti CBS (sc-133208), anti CGL (sc-
100583), and anti GAPDH (sc-47724) from Santa Cruz Bio-
technology (Santa Cruz, CA); Na þ /K þ ATPase (No. 3010 S) from Cell
Signaling (Danvers, MA); and citrate synthase (No. 5771-1) from
Epitomics (Burlingame, CA).
Cell culture
MDA-MB-468, MCF-7, MCF-10 A, and Hs 578 T were obtained
from American Type Culture Collection (Manassas, VA). MDA-MB-
468, MCF-7, and Hs 578 T were grown in 1X DMEM supplemented
with 10 mM nonessential amino acids, 2 mM L-glutamine, 1 μg/ml
insulin, and 10% fetal bovine serum (FBS). MCF-10 A was grown in
DMEM/F-12 1:1 supplemented with 10 μg/ml insulin, 20 ng/ml
epidermal growth factor, 100 ng/ml cholera toxin, 0.5 μg/ml hy-
drocortisone, 5% horse serum, and 100 U/ml penicillin/strepto-
mycin. Human mammary epithelial cells (HMEC) were obtained
from Lonza (Cologne, Germany) [CC-2551]. HMEC were grown in
the media recommended by Lonza: MEGM Bullet kit (CC-3151 and
CC-4136). Cells were passaged no more than 10 times after being
procured from the company and their genetic characteristics were
tested regularly. We regularly checked for the presence of myco-
plasma with the MycoAlert mycoplasma detection kit (LT07-318)
from Lonza (Basel, Switzerland). For experimental purposes, cells
were allowed to seed overnight prior to all treatments.
RNA purification and quantitative reverse transcriptase-PCR
Total RNA was extracted using TRIzol reagent according to the
protocol provided by the manufacturer (Invitrogen: Carlsbad, CA).
RNA concentrations were quantified with a NanoDrop 2000 c
(Nanodrop: Wilmington, DE). Reverse transcription reaction was
performed using 1 μg of total RNA with Maxima First Strand cDNA
synthesis kit from Thermo Scientific (Pittsburgh, PA). We used
validated CBS primer (PPH13484B-200) from Qiagen (Valencia,
CA) and the sense and antisense primers: 5′-ATTGGCAAT-
GAGCGGTTC-3′ and 5′-GGATGCCACAGGACTCCAT-3′ for β-actin.
RT-qPCR was performed on the ABI (Applied Biosystems) 7900 HT
Thermal cycler in standard mode using SYBR Select Master Mix for
CFX from Life Technologies (Carlsbad, CA) for 40 cycles. Each re-
action was run in triplicates and experiments were performed at
least three times. Relative mRNA expression values (CBS mRNA
versus β-actin) were calculated by the ΔΔCt method.
229
Immunohistochemistry
Human breast tissue microarrays (NBP2-30212) were obtained
from Novus Biologicals (Littleton, CO). Briefly, paraffin-embedded
breast tissue sections were deparaffinized and washed with gra-
ded ethanol. Samples were incubated with CBS primary antibody
(1:250). An Envision/HRP system was used for detection, and Gill’s
hematoxylin was used for counterstaining. Slides were evaluated
with a light microscope and a semiquantitative scoring system
(scale 0–3þ : 0 being least intense, 3þ being most intense stain-
ing) was used to assess staining intensities as per published pro-
tocols [24] Three researchers that included a trained pathologist
performed this blindly.
Enrichment of plasma membrane fractions
Plasma membrane fractions were isolated as published earlier
[25]. Briefly, cultured cells (200  106) were harvested by scraping
into phosphate-buffered saline (0.14 M NaCl, 0.05 M NaH2PO4, pH
7.5) with a cell scraper (Costar). The cells were concentrated by
centrifugation for 10 min at 3000 rpm and were resuspended in
15 ml of 10 mM Hepes–KOH buffer, pH 7.5, that contained 15 mM
KCl, 1.5 mM Mg acetate, 1 mM PMSF, and 1 mM dithiothreitol, and
were allowed to swell for 10 min on ice. The mixture was cen-
trifuged for 10 min at 3000 rpm and resuspended in 2 ml of the
10 mM Hepes–KOH buffer. The cells were ruptured, by passing
thorough a 10 ml 25-gauge needle (20 strokes). The lysed sus-
pension was centrifuged at 1000 g for 5 min to remove intact cells
and the nucleus and then at 5000 g for 15 min to remove mi-
tochondria. The relatively clear supernatant from this step was
then centrifuged at 75,000 g for 40 min in an ultracentrifuge. The
supernatant was drained out completely and the pellets were
pooled together and washed two times with the Hepes–KOH
buffer and suspended in subsequent assay buffers as per
requirements.
Protein concentration in the membrane preparation
To determine the protein concentration, the membrane pre-
parations were dissolved in 0.5% SDS and amount of protein was
estimated using a BCA protein assay kit (from Pierce). Typically the
protein concentration was in the range of 0.5 mg/ml. Membranes
equivalent to approximately 15–20 mg protein per reaction were
used as starting membrane concentration during vesicle
preparation.
Coculturing of human breast cells with macrophages
Human breast cells (100,000) were seeded into 6-well plates
and cocultured with 300,000 THP-1 on 0.4 μm pore membrane
polycarbonate Transwell Permeable Support Inserts (No. 3412)
from Corning (Corning, NY) in the presence of 25 ng/ml phorbol
myristate acetate (PMA) to differentiate THP-1 monocytes into
macrophages. Media were changed after 48 h, and cells continued
to grow as per experimental conditions. Buthionine sulfoximine
(BSO) was added at 0.2 mM concentrations to the HBC cells prior
to culturing with activated macrophages, wherever relevant.
Hydrogen sulfide (H2S) measurements
Hydrogen sulfide production from human breast cells and
plasma membrane protein was measured using HSN2, a com-
pound developed and gifted by Prof. Pluth at the University of
Oregon, as reported earlier [26]. Briefly cells (5  106) were in-
cubated with 5 μM HSN2 for 30 min to allow development of
florescence, which was read in a plate reader at (Ex/Em: 432/
230 S. Sen et al. / Free Radical Biology and Medicine 86 (2015) 228–238
542 nm). All measurements were normalized against equal num-
ber of cells. For estimation of H2S, produced by plasma membrane
fractions, 20 μg of purified plasma membrane fractions was in-
cubated in 250 mM Tris–HCl, pH 8.6, with 20 mM L-Cys and DL-
Hcys, 100 μM PLP, 0.5 mg/ml BSA, and 5 μM HSN2. The reaction
was incubated at 37 °C for 30 min, and this was followed by the
measurement of HSN2-generated fluorescence. The measurements
were normalized against equal amounts of proteins in micro-
grams. Convincing evidence of the specific reactivity of HSN2 to-
ward H2S has shown that the major intracellular thiol glutathione
exhibits negligible reactivity toward HSN2 [26]. We too observed
that while there was significant decrease in HSN2-derived fluor-
escent in HBC cells on silencing the gene for CBS, no significant
decrease was observed when HBC cells were treated with BSO, a
specific inhibitor of glutathione synthesis (Supplementary Fig. 1).
This further confirmed the specificity of HSN2 toward H2S.
Estimation of CTH
Cells in the amount of 5  106 were suspended in 200 μl,
10 mM ammonium acetate, pH 7.4, and lysed via three cycles of
freeze–thaw. Next, 800 μl methanol was added to lysates. Samples
were immediately mixed vigorously, centrifuged (16,000 g, 5 min),
and then supernatants were transferred to microcentrifuge tubes
and dried in a vacuum centrifuge. Methanol (100 μl) was added to
the dried samples. They were dried again under a stream of ni-
trogen and benzene (100 μl) was added. Then the samples were
dried yet again under a stream of nitrogen. The samples were then
treated with methanolic HCl (3 N, 50 μl, 60 min, 60 °C) and dried
again under a nitrogen stream. Samples were redissolved in H2O
(100 μl) and centrifuged (16,000 g, 5 min), and the supernatant
was transferred to liquid chromatography (LC) injector vials. Ali-
quots of the solution (10 μl) were injected onto a reverse phase
column (Phenomenex Kinetex XB-C18, 100  2.1 mm, 1.7 um
particle size, 100 Å pore diameter) equilibrated with 85% eluant A
(1 mM perfluorooctanoic acid in H2O) and 15% eluant B (1 mM
perfluorooctanoic acid in acetonitrile) and eluted (100 ul/min)
with increasing concentration of eluant B (min/% B; 0/15, 5/15, 35/
50, 33/15, 45/15). The effluent from the column was directed to an
Agilent Jet Stream electrospray ionization (ESI) source connected
to a triple quadrupole mass spectrometer (Agilent 6460) operating
in the positive ion tandem mass spectrometric multiple reaction-
monitoring (MRM) mode in which the intensity of the CTH parent
to fragment transition (251@ 481, rt 22.57 min) was recorded
using previously optimized settings. With each set of samples, a
set of standards was prepared containing increasing concentra-
tions of CTH (0, 0.5, 2.5, 12.5, 25, 50 pmol/μl). The peak areas for
CTH in the standards were used to construct calibration curves,
and the amount of CTH in each biological sample was calculated
by interpolation from the curves.
Lentiviral transfection and generation of stable cell lines
Four stable cell lines, silenced for either CBS or scrambled
control, were generated in MCF-7 and MDA-MB-468 with shRNA,
using lentiviral particles obtained from Santa Cruz Biotechnology
(sc-60335- V and sc-108080, respectively) as per the manu-
facturer’s protocol. A set of four shRNAs was packed into each viral
particle to counter off target effects. Cells were selected with 1 μg/
ml puromycin until resistant colonies could be identified and
propagated. MCF-7 and MDA-MB-468 cells that survived pur-
omycin selection after transfection with shRNA against CBS were
given the names MCF7shCBS and 468shCBS, respectively. MCF-7
and MDA-MB-468 cells that survived puromycin selection after
transfection with scrambled control shRNA were given the names
MCF7scram and 468scram, respectively.
Overexpression of CBS
Human CBS overexpression plasmids (Ad-h-CBS) and corre-
sponding control plasmids (Ad-CMV-null), packaged in adeno-
virus, were obtained from Vector Biolabs (PA). Briefly, MCF-10 A
cells were seeded into 100 mm dishes and grown to  95% con-
fluency and then infected with the CBS overexpression or control
viral stocks of 1  105  106 PFU/ml. After 24 h, the cells were
washed of virus, fresh medium was added, and the cells were used
for subsequent experiments.
4-HNE/MDA adduct assay
Whole cell lysates were analyzed using the 4-HNE adduct ELISA
kit from Cell Biolabs, Inc. (San Diego, CA). Cell pellet samples were
sonicated for 15 s in alkaline lysis buffer (50 mM Tris, pH 8.6,
50 mM NaCl, protease inhibitor cocktail) and centrifuged at
10,000 g, 30 min, 4 °C. Assays were conducted as per the manu-
facturer’s protocols. Bovine serum albumin (BSA) standards and
protein samples were adsorbed onto a 96-well plate overnight at
4 °C. The HNE/MDA–protein adducts present in the sample or
standard were probed with an anti-HNE/MDA antibody, followed
by horseradish peroxidase (HRP)-conjugated secondary antibody.
The HNE/MDA–protein adduct content in an unknown sample was
determined by comparing with a standard curve that is prepared
from predetermined HNE/MDA–BSA standards.
Xenograft studies
Female Balb/c nude mice (were obtained from Charles River
Laboratories), 4–6 weeks, 18–22 g weight were housed in a specific
pathogen-free room. In our study, the models of xenografts of
MCF-7 cells, scramble controls (MCF7scram), and MCF-7 silenced
for CBS (MCF7shCBS) were grown to confluency and injected sc
(5  106cells in 100 ml of PBS þ100 ml of Matrigel [(BD,USA)]) in
nude mice (n ¼3) in the right and left flanks of each mice, re-
spectively. The animal’s weight was measured every 2 days. We
monitored tumor growth starting 2 days after injection up to
3 weeks. Tumors were measured twice a week with a caliper, and
tumor volumes were calculated using the following formula: tu-
mor volume ¼[length  width  (length þwidth/2)  0.56]. The
Office of Animal Research Oversight at the University of California
at Los Angeles approved all animal experiments.
Glutathione assay
Cells were plated at 1000 per well in white, opaque 96-well
plates and allowed to attach overnight. After addition of BSO/
sulfasalazine, where applicable, plates were incubated for 3 h.
Cells were then washed with PBS, and total and oxidized glu-
tathionine levels were measured using GSH/GSSG-Glo, according
to manufacturer’s instructions (Promega, Madison, WI). Lumines-
cence was measured with a BioTek plate reader.
Cell viability
Cell viability was determined by the trypan blue exclusion
method using a hemocytometer as well as the Vi-CELL XR cell
viability analyzer from Beckman Coulter (Brea, CA). The number of
viable cells for each treatment and time point was determined in
triplicate.
Treatment of HBCs with exogenous 4-HNE and GYY
Briefly, 6-well plates were seeded with 100,000 MCF7scram
and MCF7shCBS cells with 10 μM 4-HNE, 2 μM GYY, or vehicle
 S. Sen et al. / Free Radical Biology and Medicine 86 (2015) 228–238 231

controls. The cells were incubated overnight and then harvested
and cell viability was assessed. The data were plotted as fold
change from control.
Western blot analysis
Cytoplasmic extracts were prepared from cells after various
treatments by lysis in buffer (50 mM Hepes, pH 7.5, 1 mM DTT,
150 mM NaCl, 1 mM EDTA, 0.1% Tween 20, 10% glycerol, 10 mM β-
glycerophosphate, 1 mM NaF, 0.1 mM orthovanadate, 10 mg/ml leu-
peptin, 10 mg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl
fluoride (PMSF) and kept at 4 °C for 30 min. Cytosolic protein con-
centrations were quantified by the Bradford assay. Equal amounts
(20 μg protein) were separated on a 10% SDS-PAGE gel and trans-
ferred onto polyvinylidene difluoride (PVDF) membranes. Incuba-
tions with primary and HRP-conjugated secondary antibodies were
done at 4 °C overnight and 1 h at RT, in 1:1000 and 1:10,000 dilu-
tions, respectively. Immunoreactivity was detected by using en-
hanced chemiluminescence [Amersham Biosciences].
Cell cycle analysis
We determined phases of cell cycle using flow cytometry. Cells
were trypsinized, washed with 1  PBS, fixed, permeabilized with
cold 70% ethanol, and finally incubated in the dark for 30 min with
1 ml of propidium iodide (containing NP-40) (Biosure, Grass Val-
ley, CA, USA). The DNA content of these cells was measured based
on the presence of propidium iodide (PI) staining. Flow cytometric
analysis was done on at least 10,000 cells from each sample, and
cell cycle data were analyzed using a FACSCalibur flow cytometer
(BD BioSciences, San Jose, CA, USA) with an excitation wavelength
of 488 nm and emission wavelength of 530 nm.
Statistical analysis
Data are presented as mean 7SEM. Student’s t test was per-
formed for comparison between two groups. For statistical ana-
lysis of more than two groups, data were analyzed by ANOVA, and
pairwise comparisons were performed by Tukey’s post hoc ana-
lysis. P values o0.05 (*) were considered statistically significant.
Results
HBC is associated with an increased expression of CBS
We first compared CBS expression in tissues obtained from breast
cancer patients, and in HBC cells, with matched controls and human
breast epithelial (HBE) cells, respectively. A tissue array analysis from
60 breast cancer patients demonstrated significantly increased ex-
pression of CBS in HBC tissues compared with adjacent normal
controls. The scoring of the tissue array was performed by a total of
three blinded researchers including one pathologist, according to the
0/1þ/2þ /3þ scoring scheme, 0 being least intense and 3þ being
the highest. We observed that the average score for normal tissue
sections was 1.2þ/– 0.43 compared to 3.0 þ/– 0.5 for the trans-
formed sections (Fig. 1A,B). Moreover, the magnitude of CBS ex-
pression correlated with the progression of the disease. Im-
munohistochemical intensity was in the order: Normal oStage IIo
Stage IIIolymph node metastasis (Fig. 1A,B). Expression of CBS was
independent of the estrogen receptor (ER), progesterone receptor
(PR), HER2, and p53 status of the HBC tissues studied (Fig. 1A). A
representative panel of the tissue array staining is seen in Fig. 1A,
while the complete panel along with annotations is included in
supplementary figures (Figs. 2,3).

Fig. 1. HBC is associated with an increased expression of CBS. (A) Immunohistochemistry utilizing monoclonal antibody against CBS on a representative panel from a tissue
array of 60 human samples showing receptor and p53 status and disease progression. (B) Mean 7SD of the scoring of CBS staining of the tissue array comprising 60 human
samples. (C) mRNA expression of CBS in HMEC, MCF-10 A, Hs 578 T, MCF-7, and MDA-MB-468 by RT-qPCR. (D) Immunoblot of CBS in breast cell line panel; GAPDH was a
loading control. (E) Comparison of steady levels of H2S among MCF-10 A, MCF-7, and MDA-MB-468 utilizing the florescent probe HSN2 normalized against equal number of
cells. (F) Steady state levels of cystathionine in normal breast cells, MCF-10 A, and in breast cancer cells, MCF-7 and MDA-MB-468, measured by HPLC-MS (*Po 0.05).
232 S. Sen et al. / Free Radical Biology and Medicine 86 (2015) 228–238

Fig. 2. Silencing of CBS-sensitized HBC cells to activated macrophages. (A) Immunoblot of CBS in the human breast cancer cells, MCF7 and MDA-MB-468, on silencing of CBS.
MCF7shCBS and 468shCBS denote MCF7 and MDA-MB-468 cells, respectively, stably silenced for CBS utilizing shRNA-mediated gene knockout via lentiviral particles.
MCF7scram and 468scram denote the corresponding scrambled controls for MCF7 and MDA-MB-468 cells, respectively. Rat liver extract was a positive control for CBS.
GADPH was utilized as the loading control. (B) Comparison of intracellular H2S levels between CBS-silenced MCF7 and MDA-MB-468 cells and their corresponding scrambled
controls shown by the fold differences in fluorescence measured by HSN2 normalized against equal number of cells (C) Cystathionine levels in the scrambled controls and
CBS-silenced MCF7 and MDA-MB-468 cells measured by HPLC-MS. (D) Viable cell count of MCF7scrambled controls (MCF7scram) and CBS-silenced MCF7 cells (MCF7shCBS)
over 5 days utilizing trypan blue exclusion methods. (E) Viable cell count of MDA-MB-468 scrambled controls (468scram) and CBS-silenced MDA-MB-468 cells (468shCBS)
over 5 days utilizing trypan blue exclusion methods. (F) Viable cell count of MCF7scrambled controls (MCF7scram) and CBS-silenced MCF7 cells (MCF7shCBS) over 5 days in
cocultures with activated macrophages, utilizing trypan blue exclusion methods. (G) Viable cell count of MDA-MB-468 scrambled controls (468scram) and CBS-silenced
MDA-MB-468 cells (468shCBS) over 5 days in cocultures with activated macrophages, utilizing trypan blue exclusion methods. (H) Representative picture of tumors excised
from mouse xenografts following injection of MCF7 scrambled controls (MCF7scram) (left panel) and CBS-silenced MCF7 (MCF7shCBS) cells (right panel), at 6 weeks fol-
lowing injection. The average volumes of the excised tumors (derived from MCF7scram and MCF7shCBS) from 3 experiments have been shown alongside (I) cell cycle
distribution of MCF7scram/shCBS cells at 5 days in cocultures with activated macrophages. (J) Cell cycle distribution of 468scram/shCBS cells at 5 days in cocultures with
activated macrophages (* Po 0.05).
 S. Sen et al. / Free Radical Biology and Medicine 86 (2015) 228–238
Fig. 3. Overexpression of CBS-protected HBE cells from activated macrophages. (A) Immunoblot utilizing antibody against CBS to show its overexpression in MCF-10 A cells
following adenoviral transduction of plasmids carrying the CBS gene. (B) Fold change in the steady state levels of H2S production (detected by change in HSN2-derived
fluorescence) in CBS overexpressed (10 AoxCBS) MCF10A cells in comparison with MCF10A empty vector controls (10Anull). (C) Cell viability of MCF-10A with or without the
overexpression of CBS at 5 days on coculturing with activated macrophages (* Po 0.05).
We next assessed the basal expression of CBS in a panel of HBC
cells along with two normal HBE cells. All three HBC cells, MCF-7
(ERþ ve), MDA-MB-468 (ER-ve), and Hs 578 T (ER-ve) expressed
significantly increased levels of CBS, both at the level of mRNA and
at the level of protein when compared with the HBE cells, HMEC
and MCF-10 A (Fig. 1C,D). Fold increase in the mRNA expression of
CBS was 6.2,  21.3, and 20.3 in MCF-7, MDA-MB-468, and
Hs578T, respectively, when compared with HMEC.
We observed that the increased CBS expression in cancer cells
was associated with significantly elevated levels of endogenous
H2S and CTH in MCF-7 and MDA-MB-468 cells, respectively, when
compared to MCF-10 A (Fig.1E,F). HBC cells, MDA-MB-468, ex-
hibited decreased levels of H2S when compared with MCF7cells
but samples had similar levels of CTH. This could be due the
production of CTH from other reactions catalyzed by CBS such as
serine þ Hcy - CTH þH2O. Following this we proceeded to assess
the effects of genetically silencing CBS in HBC cells.
Silencing of CBS-sensitized HBC cells to activated macrophages
We established a CBS-silenced stable cell lines to ascertain the
role of CBS in HBC cell growth. For this purpose we utilized both
ER þve, MCF-7 and ER-ve, MDA-MB-468 cells. Henceforth, the
scrambled control and CBS-silenced MCF-7 and MDA-MB-468 cells
would be denoted as MCF7scram and MCF7shCBS and 468scram
and 468shCBS, respectively (Fig. 2A). Silencing of CBS in HBC cells
per se did not elicit any changes either in the growth or in the
viability in in vitro cell cultures despite a significant decrease in
the steady state levels of H2S and CTH (Fig. 2B–E). On the other
hand, CBS silencing significantly compromised the growth of these
cells only in cocultures with activated macrophages and in in vivo
xenograft models which indicated an average size of CBS silent
tumors (derived from MCF7shCBS) to be  8 mm3 versus  125
mm3 for scrambled controls (derived from MCF7scram), following
3 weeks of growth in nude mice (Fig. 2F–H).
Cell cycle analysis of HBC cells, cocultured with activated mac-
rophages, indicated that MCF7shCBS exhibited a  9% decrease in the
S-phase population as well as a  10% increase in the G0/G1 phase
when compared to MCF7scram and 468shCBS exhibited a  11%
increase in the sub-G1 population when compared to 468scram
(Fig. 2I,J). This indicated that silencing of CBS inhibited cell cycle
progression in both HBC cells, irrespective of their ER status.
We further wanted to confirm the protective role of CBS and
therefore overexpressed it in the HBE cells, MCF-10 A (that
233
otherwise exhibited undetectable CBS expression). The empty
vector and CBS overexpressed MCF10A cells are indicated as
10Anull and 10AoxCBS, respectively. We utilized increasing con-
centrations (105 and 106 plaque forming units [PFU]/ml) of viral
particles, overexpressing CBS to confirm specificity (Fig. 3A). This
was associated with a  4.5-fold increase in steady state levels of
H2S in these cells (Fig. 3B). Interestingly, MCF-10 A cells, which
normally exhibited decreased growth in the presence of activated
macrophages, became resistant to them once overexpressed with
CBS. This was indicated by viable cell counting at 5 days following
coculturing with activated macrophages. Equal numbers of 10An-
ull and 10AoxCBS cells were seeded in the presence of activated
macrophages to start the experiment (Fig. 3C).
CBS silencing increased the steady state levels of reactive aldehyde-
derived adducts in HBC cells in the presence of activated macrophages
Several lines of evidence have established that tumoricidal mac-
rophages induce oxidative stress in target cells including cancer cells,
to eliminate them [15,16]. One of the major mechanisms by which
macrophages do so is by producing oxidants that eventually oxidizes
lipids of the plasma membranes generating reactive aldehydes such as
4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) [15,16,21].
These are oxidizing species that subsequently exert cellular toxicity by
forming stable adducts with various biomolecules including proteins
[20,21]. Hence, we evaluated whether the activated macrophage-in-
duced cell growth inhibition of HBC cells was associated with the
formation of adducts between cellular proteins and reactive lipid al-
dehydes such as 4-HNE and MDA in HBC cells and whether their
steady state levels were dependent on the presence of CBS.
We observed that HBC cells, silenced for CBS, exhibited sig-
nificantly increased steady state levels of 4-HNE and MDA protein
adducts compared to scrambled controls. MCF-7 and MDA-MB-468
cells exhibited 6- and  3-fold enhancement, respectively, in the
levels of 4-HNE and 2.5- and2-fold enhancement, respectively, in
the steady state levels of MDA adducts as evidenced from ELISA-
based assays (Fig. 4).
CBS-derived H2S protected HBC cells against reactive aldehyde-de-
rived adduct formations in cocultures with activated macrophages
We assessed whether exogenously administered H2S could com-
pensate for the lack of its endogenous production by HBC cells si-
lenced for CBS, in terms of regulating 4-HNE–protein adduct
234 S. Sen et al. / Free Radical Biology and Medicine 86 (2015) 228–238
Fig. 4. CBS silencing increased the steady state levels of reactive aldehyde-derived
adducts in HBC cells in the presence of activated macrophages. (A) Comparison of
protein–4-HNE adduct formations between MCF7 scrambled controls (MCF7scram)
and MCF7 cells silenced for CBS (MCF7shCBS) and between MDA-MB-468 scram-
bled controls (468scram) and MDA-MB-468 silenced for CBS (468shCBS) at 5 days
in cocultures with activated macrophages. (B) Protein–MDA-adduct formations in
MCF7scram/shCBS and 468scram/shCBS at 5 days in cocultures with activated
macrophages. (* Po 0.05).
formations following incubation with activated macrophages. We
observed a significant decrease in the steady state levels of 4-HNE–
protein adducts in CBS-silenced HBC cells when exposed to GYY4137
(a slow releaser of H2S) kept in cocultures with activated macro-
phages over a period of 5 days. There was a 40% and  50% de-
crease in the levels of 4-HNE–protein adducts in MCF7shCBS and
468shCBS, respectively (Fig. 5A,B). This was associated with a re-
covery of cell growth of 60% and  80% in MCF7shCBS and
468shCBS, respectively as assessed from viable cell counts at 5 days
following coculturing with activated macrophages (Fig. 5C,D).
The H2S/HS– system has been shown to reduce oxidants in-
cluding reactive aldehydes directly [27]. Oxidants from macro-
phages initially accumulate at the vicinity of plasma membranes to
cause oxidation of phospholipids [28]. Since H2S has a short half-
life [29], we hypothesized it was being produced in the vicinity of
the production of oxidants, i.e., the plasma membrane. We
therefore examined whether the CBS–H2S system was associated
with the plasma membrane fractions in HBC cells.
The CBS/H2S machinery was detected on the plasma membrane
fractions of HBC cells
Plasma membrane fractions from HBC cells, MCF7 [ER þve] and
MDA-MB-468 [ER-ve] along with HBE cells, MCF10A, were
purified. Increased band intensity of Na þ /K þ ATPase (the typical
plasma membrane residential protein) in plasma membrane frac-
tions compared with whole cells validated the enrichment of the
membrane fraction. Conversely the decreased intensity of the
band intensity of citrate synthase (the typical mitochondrial re-
sidential protein), in plasma membrane fractions compared with
whole cells, validated the absence of mitochondrial fractions in the
plasma membrane preparation (Fig. 6A). We observed that pur-
ified plasma membrane fractions from HBC cells (MCF-7 [ER þve]
and MDA-MB-468 [ER-ve]) not only expressed CBS but also pro-
duced H2S in the presence of Hcy and Cys. Interestingly, HBE cells,
MCF-10 A, were not found to express CBS in their plasma mem-
brane fractions and expectedly the latter did not produce H2S in
the presence of Cys and Hcy (Fig. 6B,C).
Exogenous H2S decreased growth inhibition in CBS-silenced HBC cells
on exposure to 4-HNE
We observed that the direct addition of 4-HNE (  10 μM for
48 h as reported to be toxic) [30] decreased the cell growth of CBS-
silenced HBC cells by  1.7-fold when compared with  0.2-fold in
scrambled controls (Fig. 6D). However, preincubation with GYY
4137, 2 μM (H2S donor), decreased the sensitivity of CBS-silenced
HBC cells ( 2-fold recovery) toward the growth retarding effects
of 4-HNE (Fig. 6E).
Glutathione status was independent of CBS expression in HBC cells
In addition to catalyzing the production of H2S, CBS also pro-
duces CTH, which in turn is metabolized by cystathionine γ-lyase
(CGL) to produce cysteine required for the endogenous production
of glutathione GSH, an important component of the cellular anti-
oxidant system. We therefore wanted to assess whether in our
model system GSH in addition to H2S played a role toward reg-
ulating the levels of the 4-HNE-protein adducts. To our surprise,
we observed that silencing of CBS did not affect the levels of either
the total GSH or the ratio of oxidized /reduced GSH (Fig. 7A,B).
Moreover, we observed that CGL was undetectable in tissues de-
rived from breast cancer patients as well in HBC cells (Fig. 7C).
Furthermore exposure of HBC cells both scrambled controls and
those silenced for CBS, to 0.2 mM BSO, a specific inhibitor of glu-
tathione synthesis as reported earlier [31], did not have any effect
on cellular viability over time in the presence of activated mac-
rophages (Fig. 7D,E).
Discussion
Our primary objective of this study was to investigate whether
CBS played an important role in the maintenance of HBC home-
ostasis. We also wanted to assess the mechanistic link between the
downregulation of macrophage-induced oxidative stress and CBS-
derived H2S in breast cancer.
The increased expression of CBS in tissues derived from HBC
patients and in HBC cells, irrespective of their molecular subtype
and receptor status (at least in the tissue samples that we studied),
indicated an important role of CBS in this disease. Moreover, the
undetectable levels of CBS mRNA and protein in normal HBE cells
suggested that it catered to a specific cellular function that was
associated only with the event of transformation.
Similar to that reported for colon and ovarian cancers, we ob-
served that CBS-catalyzed condensation of Hcy þCys to CTH and
H2S, was important in breast cancer as well [32, 33]. This was
evidenced by a significant decrease in the levels of H2S and CTH on
silencing of CBS in HBC cells. The decrease in the steady state le-
vels of H2S was more pronounced in MCF7 cells compared to
 S. Sen et al. / Free Radical Biology and Medicine 86 (2015) 228–238 235

Fig. 5. CBS-derived H2S protected HBC cells against reactive aldehyde-derived adduct formations in cocultures with activated macrophages. (A,B) Protein–4-HNE adduct
formations in MCF7scram/shCBS and 468scram/shCBS, þ /– 2 mM GYY4137, at 5 days in the presence of activated macrophages. (C) Cell viability of MCF7shCBS þ 2 mM
GYY4137 in the presence of macrophages versus scrambled controls. (D) Cell viability of 468shCBS þ 2 mM GYY4137 in the presence of macrophages versus scrambled
controls (* P o 0.05).

MDA-MB-468 cells, suggesting that there could be other sources of
endogenous H2S in these cells that were not investigated in this
study. The decrease in CTH was also more pronounced in MCF7
cells compared with MDA-MB-468 following CBS silencing. This
could be due to other reactions of CBS such as serine
þhomocysteine -CTH þH2O [4] being more prevalent in MCF7
cells. Kinetic data utilizing purified CBS have also suggested the
preferential condensation of Hcy and Cys in the production of H2S
and CTH [4, 5].
Silencing of CBS in colon cancer cells significantly decreased
the levels of CTH and H2S, which was associated with inhibition of
cell growth in in vitro cultures. However, unlike that observed in
colon cancer [32], CBS silencing per se did not affect the growth of
HBC cells in in vitro cultures, although there was a decrease in the
levels of H2S and CTH. This indicated that the mechanism of CBS
action in breast cancer is likely to be different from that reported
in colon cancer.
As CBS is mechanistically linked to the antioxidant capacity of a
cell [4,5], we hypothesized that HBC cells utilized CBS to protect
themselves against oxidative insults induced exogenously by im-
mune cells including infiltrated tumoricidal macrophages.
In order to test our hypothesis, we cocultured HBC cells (both
ER þve and ER-ve), with activated macrophages (that produce
reactive oxygen and nitrogen species). Under these conditions si-
lencing of CBS compromised the growth of HBC cells, confirming
its protective role in HBC. The growth curves generated by viable
cell counting over a period of time correlated with that of cell cycle
analysis, indicating delayed cell cycle progression for CBS-silenced
cells. ER þve, MCF-7 cells exhibited a decrease in S-phase popu-
lation as well an increase in the GO/G1 phase, whereas the ER-ve,
MDA-MB-468 cells exhibited an increase in the sub-G1 population.
This difference in the cell cycle population most likely suggested
that MDA-MB-468 cells tended more toward apoptosis at the end
of 5 days in cocultures with activated macrophages compared to
MCF-7 cells. However both cell types indicated a general decrease
in cell cycle progression. This difference may be attributed to their
different ER status. HBE cells, MCF-10 A, did not express CBS and
hence similar to CBS-silenced HBC cells were susceptible to
growth inhibition induced by activated macrophages. As expected,
overexpression of CBS in these cells made them resistant to
macrophage-induced inhibition of cell growth. Our in vivo data
utilizing the xenograft model that showed compromised growth of
tumors on CBS silencing further confirmed our hypothesis, as nude
mice that were injected with HBC cells were capable of producing
macrophages.
We next evaluated the mechanism by which CBS protected HBC
cells, against activated macrophages. Our data demonstrated that
silencing of CBS increased the steady state levels of adduct formed
between reactive aldehydes and proteins in HBC cells when cul-
tured in the presence of activated macrophages. It is well estab-
lished that macrophages that inhibit tumor growth release oxi-
dants that oxidize lipids on the plasma membrane to produce
reactive aldehydes which in turn cause cytotoxicity by adduct
formations with various biomolecules including proteins [27].
4-HNE and MDA–protein adducts are stable compounds that re-
presents footprints of oxidative damage and are known to be
important in macrophage-induced oxidation [27]. We therefore
monitored the changes in the levels of 4-HNE and MDA–protein
adduct formations in HBC cells following cocultures with activated
macrophages on silencing of CBS. We observed that the steady
236 S. Sen et al. / Free Radical Biology and Medicine 86 (2015) 228–238

Fig. 6. The CBS/H2S machinery was detected on the plasma membrane fractions of HBC cells. (A) Immunoblot of Naþ/K þ ATPase and citrate synthase in plasma membrane
fractions [20 mg] versus whole lysates [20 g] of MCF-7, MDA-MB-468, and MCF-10 A. (B) Cysteine/homocysteine-dependent H2S production by plasma membrane fractions of
MCF-7, MDA-MB-468, and MCF-10 A. (C) Immunoblot of CBS in 20 mg plasma membrane fractions of MCF-7, MDA-MB-468, and MCF-10 A. Na K ATPase was used as loading
controls for membrane fractions. (D) Viable cell count of MCF7scram at 48 h on exposure to 10 mM 4-HNE versus vehicle controls. (E) Viable cell count of MCF7shCBS at 48 h
on exposure to 10 mM 4-HNE and/or 2 mM GYY4137 versus control (* Po 0.05).

state levels of these adducts in HBC cells were dependent on the
expression of CBS. Several lines of evidences have reported a major
role for 4-HNE in biology [34]. Hence we chose to monitor changes
in 4-HNE–protein adducts in further investigations.
Our observation that exogenous H2S could deplete the levels of
4-HNE–protein adducts in CBS-silenced HBC cells confirmed that
the CBS-derived H2S was responsible for protection against re-
active aldehydes. Our data indicated that the percentage decrease
in the steady state levels of protein–4HNE adducts was 44% and
60% in MCF7shCBS and 468shCBS, respectively. This correlated
to a lesser degree of recovery of cell growth on exogenous treat-
ment of H2S in MCF7shCBS (i.e.,  40%) compared with  80% re-
covery in 468shCBS.
Our results are in agreement with other studies performed in
neuronal cells where exogenous H2S was shown to inactivate
4-HNE adducts as well to downregulate cytotoxicity induced by Aβ
peptides [27].
Interestingly, we observed that the CBS/H2S machinery was
associated with the plasma membrane in HBC cells. It is possible
that CBS localizes to the plasma membrane during macrophagic
attack to release H2S at the site of accumulation of oxidants re-
leased by macrophages. Our findings are supported by other stu-
dies that reported the localization of CBS in the plasma membrane
of tissues from the gall bladder [35]. We, however, did not in-
vestigate the relative proportions of CBS distribution between the
cytosol and the plasma membrane nor did we assess its localiza-
tion in the mitochondrial fractions as is reported in colon cancer
cells [32].
To assess the regulation of 4-HNE–protein adducts by H2S in a
more direct manner, we exposed HBC cells with or without intact
CBS to toxic concentrations of pure 4-HNE. As expected, CBS-si-
lenced HBC cells were more sensitive to cell growth inhibition by
4-HNE when compared with scrambled controls. Moreover, pre-
incubation with exogenous H2S partially rescued cell growth in
CBS-silenced HBC cells. The fact that the rescue was not 100%
could be due to the differential kinetics of the reaction of 4-HNE
with cellular proteins compared with the initial kinetics of the
reaction between 4-HNE and the slowly released H2S by its donor.
Finally our data demonstrated that CBS was not a major reg-
ulator of the endogenous levels of GSH in HBC. The lack of CGL
created a truncation in the transulfuration pathway, which in-
hibited the conversion of CTH to Cys and subsequently its in-
corporation into GSH. This phenomenon was also reported in
neuroblastomas as well as in the early developing stages of the
fetus [36,37]. Moreover our observation that viability of either CBS
intact or silenced HBC cells (in the presence of activated macro-
phages) was not affected by BSO, a specific inhibitor of glutathione
synthesis, further confirmed that glutathione did not play an im-
portant role in the regulation of macrophage-induced reactive
aldehyde-mediated cytotoxicity in HBC cells.
In this study we did not assess the role of other possible re-
ducing agents such as Cys SSH (cysteine persulfides) that can be
synthesized by CBS from cystines [38]. Thus the antioxidant ca-
pacity promoted by CBS may also include persulfides and H2S
derived from persulfides, as recently reported in lung cancer cells
[38].
This work may provide rationale for the utilization of tissue-
specific CBS blockers as adjuvants in the treatment of breast can-
cer. Compromising CBS may significantly downregulate the cancer
cell’s ability to counter oxidative stress induced by many che-
motherapeutic drugs. This may thus increase drug efficacy in
breast cancer treatment.
 S. Sen et al. / Free Radical Biology and Medicine 86 (2015) 228–238 237

8.0 30.0

(µM GSH/103 cells)

GSH/GSSG Rao
n.s. n.s. n.s.
6.0

Total GSH
20.0
4.0
10.0
2.0 n.s.
0.0 0.0

7s ram
7s ram

8s ram

8s ram
46 8sc S
46 8sc S

BS
BS

46 hCB
46 hCB

hC
hC

CF sc
CF sc

M C F7
M F7
C

M
M

CGL
GAPDH

MCF7scram 468scram
MCF7scram+BSO 468scram+BSO
MCF7shCBS 468shCBS
MCF7shCBS+BSO 468shCBS+BSO
0.8 0.3
Viable Cell Count
Viable Cell Count

(1X106 cells/mL)
(1X106 cells/mL)

0.6
0.2
0.4
0.1
0.2

0.0 0.0
0 2 4 6 0 2 4 6
Time (Days) Time (Days)
Fig. 7. Glutathione status was independent of CBS expression in HBC cells. (A) Measurement of total glutathione (GSH) in MCF7scram/shCBS and 468scram/shCBS. (B) GSH/
GSSG ratio in MCF7scram/shCBS and 468scram/shCBS. (C) Immunoblot utilizing antibodies against cystathionine γ-lyase [CGL] in whole cell lysates of MCF-10 A, MCF-7, and
MDA-MB-468. (D) Viable cell count of MCF7 cells either scrambled controls (MCF7scram) or silenced for CBS (MCF7shCBS) in cocultures with activated macrophages with or
without pretreatment with buthione sulfoximine (BSO). (E) Viable cell count of MDA-MB-468 cells either scrambled controls (468scram) or silenced for CBS (468shCBS) in
cocultures with activated macrophages with or without pretreatment with buthione sulfoximine (BSO) the inhibitor of glutathione synthesis (* P o 0.05).

Acknowledgments References

We acknowledge The Roberta Deutsch Foundation and Kelly
Day for their financial support and Svetlana Roberts for technical
help. We sincerely thank Prof. Pluth at the University of Oregon,
for gifting us HSN2 for intracellular H2S measurements. We also
acknowledge Ana Maria Cruz, M.S., for her contribution toward
editing the language in this manuscript.
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.
2015.05.024
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