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Citation: Jadhao, M.; Chen, C.-L.;
Liu, W.; Deshmukh, D.; Liao, W.-T.;
Chen, J.Y.-F.; Urade, R.; Tsai, E.-M.;
Hsu, S.-K.; Wang, L.-F.; et al.
Endoglin Modulates TGFβR2
Induced VEGF and Proinflammatory
Cytokine Axis Mediated
Angiogenesis in Prolonged
DEHP-Exposed Breast Cancer Cells.
Biomedicines 2022,10, 417.
https://doi.org/10.3390/
biomedicines10020417
Academic Editor: Randolph
C. Elble
Received: 22 December 2021
Accepted: 3 February 2022
Published: 10 February 2022
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biomedicines
Article
Endoglin Modulates TGFβR2 Induced VEGF and
Proinflammatory Cytokine Axis Mediated Angiogenesis in
Prolonged DEHP-Exposed Breast Cancer Cells
Mahendra Jadhao 1, Chun-Lin Chen 2,3 , Wangta Liu 4, Dhanashri Deshmukh 1, Wei-Ting Liao 4,
Jeff Yi-Fu Chen 4, Ritesh Urade 2, Eing-Mei Tsai 5,6, Sheng-Kai Hsu 4, Li-Fang Wang 1,*
and Chien-Chih Chiu 2,4,6,7,8,*
1Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan;
mah.jadhao@yahoo.com (M.J.); dhanashrivdeshmukh1990@gmail.com (D.D.)
2Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan;
chunlinchen@mail.nsysu.edu.tw (C.-L.C.); uraderit@gmail.com (R.U.)
3Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University,
Kaohsiung 807, Taiwan
4Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan;
liuwangta@kmu.edu.tw (W.L.); wtliao@kmu.edu.tw (W.-T.L.); yifuc@kmu.edu.tw (J.Y.-F.C.);
b043100050@gmail.com (S.-K.H.)
5Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan;
tsaieing@kmu.edu.tw
6The Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University,
Kaohsiung 807, Taiwan
7Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan
8Center for Cancer Research, Kaohsiung Medical University Hospital, Kaohsiung Medical University,
Kaohsiung 807, Taiwan
*Correspondence: lfwang@kmu.edu.tw (L.-F.W.); cchiu@kmu.edu.tw (C.-C.C.);
Tel.: +886-67-312-1101 (ext. 2217) (L.-F.W.); +886-67-312-1101 (ext. 2368) (C.-C.C.);
Fax: +886-67-312-5339 (L.-F.W.)
Abstract:
Angiogenesis is the process of vascular network development and plays a crucial role in
cancer growth, progression, and metastasis. Phthalates are a class of environmental pollutants that
have detrimental effects on human health and are reported to increase cancer risk. However, the
interplay between phthalate exposure and angiogenesis has not been investigated thoroughly. In
this study, we investigated the effect of prolonged di (2-ethylhexyl) phthalate (DEHP) treatment
on the angiogenic potential of triple-negative breast cancer. MDA-MB-231 cells were exposed to
physiological concentrations of DEHP for more than three months. Prolonged DEHP exposure
induced angiogenesis in breast cancer cells. Endoglin (ENG)/CD105 is a membrane glycoprotein and
an auxiliary receptor of the TGF
β
receptor complex. In endothelial cells, ENG is highly expressed and
it is a prerequisite for developmental angiogenesis. A literature review highlights endoglin as a well-
known mesenchymal stem cell marker responsible for vascular development and angiogenesis. NGS
analysis showed that endoglin overexpression in DEHP-exposed MDA-MB-231 cells correlated with
tumor development and growth. An
in vivo
zebrafish xenograft assay showed that VEGFA induced
sprouting of the subintestinal vein (SIV) in embryos injected with DEHP-exposed cells. Endoglin
knockdown reduced SIV sprouting and VEGFA expression in zebrafish embryos. An
in vitro
HUVEC
tube formation assay showed that endoglin depletion reversed DEHP-induced VEGF-mediated
HUVEC tube formation in coculture. DEHP-induced endoglin activated TGF
β
/SMAD3/VEGF and
MAPK/p38 signaling in MDA-MB-231 cells. A cytokine angiogenesis antibody array showed induced
expression of the inflammatory cytokines IL1
α
, IL1
β
, IL6, and IL8, along with GMCSF and VEGF.
Endoglin knockdown reversed DEHP-induced activation of the TGF
β
/SMAD3/VEGF signaling
axis, MAPK/p38 signaling, and cytokine regulation, limiting angiogenesis potential both
in vivo
and
in vitro
. Targeting endoglin might serve as a potential alternative treatment to control angiogenesis,
leading to metastasis and limiting cancer progression.
Biomedicines 2022,10, 417. https://doi.org/10.3390/biomedicines10020417 https://www.mdpi.com/journal/biomedicines
Biomedicines 2022,10, 417 2 of 21
Keywords: DEHP; angiogenesis; endoglin; VEGF; TGFβsignaling; inflammatory cytokines
1. Introduction
In the growth and progression of breast cancer, new blood vessel generation from pre-
existing vessels is pivotal and is known as neovascularization. An increase in angiogenic
cues and mutation at a genetic level are some of the factors that accompany the growing
tumor. In quiescent vessels, endothelial cells (ECs) line up by forming a lumen surrounded
by a thick layer of pericytes or vascular smooth muscle cells (VSMCs), supporting the
structural integrity of the vessel [
1
]. Angiogenic cues or ischemia increase endothelial
permeability, giving rise to matrix metalloproteins for extracellular matrix degradation
and relieving pericyte-EC contact [
2
]. This provides space and a chance for the adjacent
cells to influx fluids and/or macromolecules due to the absence of cell-to-cell contact.
Through coordinated activation, endothelial cells tend to proliferate and migrate toward
promigratory phenomena (such as VEGF) to reach their final destination, where they
undergo morphogenesis to form a lumen and branches [
3
]. Several factors, such as VEGF,
thrombin, and sphingosine 1 phosphate, control EC permeability and lead to reversible
loss of junctional integrity [
4
]. Angiogenesis provides oxygen, nutrients, and metabolites to
growing tumors simultaneously, removes waste products from them, and nourishes them
to form a solid mass. Angiogenesis is mostly controlled by proangiogenic factors such as
VEGF and its tyrosine kinase receptors VEGFR1 and VEGFR2. Activation of the VEGF
receptor by its ligand leads to activation of a number of downstream signaling pathways,
such as PI3K, MAPK, and PLCγ, which mostly occur during angiogenesis [5].
Cancer highjacks and diverts the flow of blood vessels toward it to fulfil its require-
ments, leading to enhanced proliferation and metastatic spread. The angiogenesis niche
is triggered by hypoxia, leading to the production of angiogenic factors and immunosup-
pressive cytokines. For instance, in neuroblastoma (NB), extracellular microenvironment
hypoxia is reported to promote extracellular adenosine generation, which induces VEGF
production by binding with A3 adenosine receptor and activating HIF-1
α
/2
α
/VEGF
axis [
6
]. In addition, a recent study conducted by Shen demonstrated that HIF-1
α
expres-
sion is parallel with VEGF expression, and both expressions are positively correlated with
poor prognosis of ovarian cancer [
7
]. In contrast, pituitary adenylate cyclase-activating
polypeptide (PACAP) and its receptor PAC1R suppress angiogenic pathway and mesenchy-
mal markers, such as vimentin and MMP-2 by inhibition of MAPK/PI3K/Akt signaling
in the hypoxia niche of glioblastoma [
8
,
9
]. Maugeri et al. indicated that caffeine exerts
anti-tumor effects on glioblastoma multiforme (GBM), characterized by extensive hypoxia
through significant downregulation of HIF-1
α
as well as VEGF [
10
]. Metformin, widely
prescribed for type II diabetes, is also suggested to circumvent hypoxia-mediated overex-
pression of pro-angiogenic factors (VEGF) and aberrant angiogenesis through promoting
perfusion [
11
]. In our previous study, a reduced ROS level was observed in MDA-MB-231
under prolonged exposure to DEHP [
12
]. Chen et al. revealed that hypoxia serves as a
double-edged sword in ROS production. On one hand, HIF decreases ROS production
via suppressing the tricarboxylic acid (TCA) cycle; on the other hand, it promotes ROS
formation by NADPH oxidase (NOX) because NOX is a downstream gene of HIF [
13
].
Collectively, there is an intimate interplay between hypoxic microenvironment, ROS, and
angiogenesis. Some of the angiogenic factors involved in tumor-induced angiogenesis
include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF),
fibroblast growth factor (FGF), and cytokines such as interleukins (ILs), tumor necrosis
factors (TNFs), interferons (INFs), and tumor growth factors (TGFs) [
14
]. The process of
angiogenesis is governed by activators and inhibitors [
15
]. Determination and localization
of angiogenic activators and inhibitors can lead us to design a drug to reduce the metastatic
spread rate of the respective cancer.
Biomedicines 2022,10, 417 3 of 21
Endocrine disrupters (EDs)/endocrine disrupting chemicals (EDCs) are naturally
occurring or synthetic compounds that mimic endocrine hormonal activity to affect the
normal physiological functioning of the endocrine system [
16
]. EDs are often found in
many products, such as plastic, cosmetics and hygiene products, personal care products,
packaging, medical devices, heavy metals, and furniture [
17
]. One ED/EDC is phthalates
or phthalic acid, which are used to enhance the durability and flexibility of plastic products
and are reported to exert detrimental effects on human health [
18
]. Some of the studies
showed its ubiquitous presence in urine in industrialized countries. Its effects range
from an imbalance in the hormonal system to human sexual and reproductive system
development [
19
]. Frequently found phthalates include diethyl phthalate (DEP), dimethyl
phthalate (DMP), di (2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DINP), and
dibutyl phthalate (DBP) [
20
]. Among all phthalates, DEHP is the most widely used; it is
noncovalently bound in plastics and results in leaching under different conditions, resulting
in ubiquitous environmental distribution [
18
,
21
]. Phthalates may result in human exposure
in one of several ways, such as dermal contact, inhalation, and ingestion, which could
pose a risk to potential health conditions, such as cancer progression and disturbance of
reproductive and respiratory systems [
22
,
23
]. DEHP exposure in uterine leiomyoma cells
increased cell viability and anti-apoptotic proteins [
24
]. In lung cancer cells, it increased
cell viability, cell proliferation, and inflammatory proteins [
25
]. Inhibition of angiogenesis
reduced placental growth and development, followed by DEHP exposure, in pregnant
mice [
26
]. Angiogenic factors such as sFlt-1 and PIGF are affected by treatment with
DEHP, suggesting a strong correlation between angiogenesis and phthalates [
27
]. An
increase in cancer cell viability and suppression of angiogenesis adversely both affect
cancer development. An increase in cancer cell viability spares a chance to proliferate
and metastasize and increases the niche to develop a solid tumor. At the molecular level,
several signaling pathways correlated with cancer growth and differentiation following
phthalate exposure have been recorded. Zu et al. reported that phthalate exposure activated
ERK5/p38 signaling, which promoted the spread of prostate cancer [
28
]. Activation of
MAPK (ERK1/2) and JNK signaling following DBP treatment induced testicular and liver
damage in a murine model [
29
,
30
]. Similarly, in our previous study, we demonstrated the
involvement and activation of PI3K/Akt signaling in continuous and long-term exposure
to DEHP-induced multidrug resistance in breast cancer cells [12].
Endoglin (CD105/ENG) is a transmembrane glycoprotein and a coreceptor of the
tumor growth factor-
β
(TGF
β
) family. Endoglin is highly expressed in proliferating vascu-
lar endothelial cells [
31
,
32
], and it interacts with their serine/threonine kinase receptors
(TGF
β
RI or TGF
β
RII), forming a heterotrimer along with ligand [
33
–
35
]. Other ligands in-
clude TGF
β
, activins, GDFs, and BMPs [
36
]. Ligand binding leads to receptor dimerization
and autophosphorylation at serine residues, recruiting R-SMAD and Co-SMAD proteins to
the nucleus and transcribing target genes, leading to several biological processes, namely,
proliferation, migration, differentiation, cell death, and angiogenesis [
37
]. Significant evi-
dence has shown increased endoglin in the TGF-
β
signaling pathway in tumor-associated
endothelial cells, making it ‘a marker’ for tumor-induced angiogenesis [
38
]. Solid tumors
such as prostate, cervical, and breast cancer showed an increased level of endoglin in
the endothelium [
39
–
41
]. The expression of endoglin increases during wound healing,
inflammation, vasculogenesis, and angiogenesis [
42
,
43
]. Liu et al. showed that endoglin
is indispensable for VEGF-induced angiogenesis. Endoglin-deficient mouse embryos die
due to malformation of blood vessels [
44
]. Proliferation and angiogenesis are inhibited in
ovarian cancer endothelial cells following endoglin depletion/knockdown [
45
]. In murine
mammary carcinoma, endoglin silencing reduces the growth and number of vessels formed
during tumor progression [
46
]. Endoglin interacts with VEGFR2 in a VEGF-dependent
manner to sustain and stabilize VEGFRII on the cell surface to promote tip cell formation
for tumor progression and growth [
47
]. In the current study, we demonstrated the detailed
mechanism of endoglin-mediated regulation of TGF
β
, MAPK/p38 signaling, and cytokines
Biomedicines 2022,10, 417 4 of 21
controlling angiogenesis in prolonged DEHP-exposed MDA-MB-231 cells
in vitro
and
in vivo.
2. Materials and Method
2.1. Cell Culture
MDA-MB-231 cells, human breast cancer cells, and human umbilical vein endothelial
cells (HUVECs) were used as experimental models for the study. MDA-MB-231 cells were
acquired from the American Type Culture Collection (ATCC) and maintained in DMEM
(Gibco, Grand Island, NE, USA) supplemented with penicillin, streptomycin antibiotics,
10% FBS, 0.03% glutamine, and 1 mM sodium pyruvate in a 5% CO
2
incubator at 37
◦
C in
humidified conditions. A sample of 293T cells were acquired from ATCC and maintained
in low antibiotic (0.1% penicillin/streptomycin) DMEM containing FBS (10%), glutamine
(0.03%), and sodium pyruvate (1 mM). Human umbilical vein endothelial cells (HUVECs)
were purchased from Lonza (CC2519, Basel, Switzerland) and maintained in EGM
TM
-
2 endothelial cell growth medium (CC-3121, Lonza, Basel, Switzerland) supplemented
with an EGM
TM
-2 supplement pack (CC-4122, Lonza, Basel, Switzerland) containing
bovine brain extract (BBE) and free of exogenous VEGF in a 5% CO2incubator at 37 ◦C in
humidified conditions.
2.2. Reagents and Antibodies
DEHP (Sigma-Aldrich, 36735, Saint Louis, MO, USA) was reconstituted in DMSO
(Sigma-Aldrich, Saint Louis, MO, USA). Doxorubicin (Dox) (Sigma-Aldrich, D1515, Saint Louis,
MO, USA) was dissolved in DMSO (Sigma-Aldrich, Saint Louis, MO, USA). Primary an-
tibodies for proteins: Endoglin/CD105 (71 kDa, Proteintech, 10862-1-AP, Rosemont, IL,
USA), MAPK (ERK1/2) (44 kDa, Cell Signaling, 137F5, Danvers, MA, USA), phospho-
MAPK (ERK1/2) (Thr202/Tyr204) (44 kDa, Cell Signaling, 9101 s, Danvers, MA, USA), p38
(38 kDa, Proteintech, 14064-1-AP, Rosemont, IL, USA), phospho-p38 (Tyr182) (38 kDa, Santa
Cruz, SC-166182, Santa Cruz, CA, USA), GAPDH (35.5 kDa, EMD Millipore, MAB374,
Burlington, MA, USA), Smad2/3 (52, 60 kDa, Cell Signaling, 8685, Danvers, MA, USA),
phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (52, 60 kDa, Cell Signaling, 8828, Dan-
vers, MA, USA),
β
actin (43 kDa, Santa Cruz, SC-47778, Santa Cruz, CA, USA), and VEGFA
(43 kDa, Proteintech, 66828-1-IG, Rosemont, IL, USA). Secondary antibodies: Goat anti-
rabbit IgG (Alexa Fluor 594, Invitrogen, A11012, Waltham, MA, USA) was used as the
secondary antibody.
2.3. DEHP Exposure and Stable Clone Establishment
MDA-MB-231 cells were exposed to DEHP at a low concentration (100 nM) for three
months in cell culture medium. DEHP stock was directly diluted in culture medium
to achieve a final concentration of 100 nM. Cells were cultured in 100 mm cell culture
dish supplemented with 10–12 mL of DEHP containing DEHP. Upon reaching 80–90%
confluence,
1
4
cells were passaged and continuous culture with DEHP treatment was
maintained for 3 months. After 3 months, DEHP treatment was terminated permanently,
and cells were treated with 10 nM Dox for 72 h. Colonies formed following dox treatment
were selected and maintained for further study. Untreated MDA-MB-231 cells are denoted
as the control (Ctrl), and DEHP-exposed cells are denoted as DEHP from here onward. Both
untreated and DEHP-treated cells were maintained and equally passaged (10–12 passages)
to reduce senescence effects.
2.4. Zebrafish Xenograft
Transgenic zebrafish strain Tg (fli1: EGFP) was raised and maintained at 28.5
◦
C in the
Zebrafish Core Facility at KMU. Embryos were obtained by pairwise mating and incubating
them in 0.03% phenylthiourea (PTU) at 28.5
◦
C in an incubator for 48 h. Forty-eight hpf
embryos were injected with Vybrant
®
DiI-stained control and DEHP-exposed MDA-MB-
231 cells using a microinjector setup. Following injection, embryos were maintained in
Biomedicines 2022,10, 417 5 of 21
an incubator at 28.5
◦
C for 24 h. Embryos were further observed for SIV sprouting, and
images were captured under a fluorescence microscope (MZ10F, Leica, Singapore) using
Metaview software (version 7.8.0.0).
2.5. RNA Sequencing
To evaluate the underlying mechanism of DEHP-induced angiogenesis in MDA-MB-
231 cells, next-generation sequencing (NGS) was performed on the control and DEHP-
exposed MDA-MB-231 cells as described in our previous publication [
12
]. Briefly, 3
µ
g of
isolated RNA from Biotools Co. Ltd. (Taipei, Taiwan) was used for sequencing. RNA was
sequenced, and data were analyzed by Illumina software. DEGs and GO were analyzed by
TopHat (v2.0.12) and GoSeq & topGO (2.12); KEGG analysis was performed by KOBAS
(v2.0). For data confirmation and validation, the log ratio of expression obtained by NGS
was further evaluated by QIAGEN Ingenuity Pathway Analysis (IPA
®
, QIAGEN Redwood,
Redwood City, CA, USA, Available online: www.qiagen.com/ingenuity (accessed on
21 December 2021).
2.6. Lentiviral Transfection
Envelop plasmid (pMD2. G), packaging plasmid (pCMV-dR8.91), and short hairpin
RNA (shRNA) containing hairpin-pLKO.1 vector was used for lentiviral particle prepa-
ration; pMD2. G, pCMV-dR8.91, scrambled/mock shRNA (clone ID: ASN0000000004)
and shENG (clone ID: TRCN0000083140) were purchased from the RNAi core facility
(Academia Sinica, Taipei, Taiwan). Scramble shRNA or sheng, along with pMD2. G and
pCMV-dR8.91, were transfected into 293T cells using Lipofectamine 2000 (Thermo Fisher,
11668019, Waltham, MA, USA) in OptiMEM for 18 h. Packaged lentiviruses were harvested
in FBS/BSA-enriched DMEM at 36 h and 48 h post-transfection. Collected lentiviruses
were concentrated using a 100 K molecular weight cutoff filter unit (MAP100C38, Pall
Corporation, New York, NY, USA). Lentiviral transfection with shScr (mock) or shENG con-
taining lentivirus was performed using Lipofectamine 2000 in the control and DEHP-treated
MDA-MB-231 cells for 24 h. Transfected cells were exposed to puromycin (
1–2 µg/mL
) for
selection and establishment of stable knockdown cells.
2.7. Quantitative Polymerase Chain Reaction (qPCR)
Total RNA was extracted from 25–30 noninjected and tumor cell-injected embryos
using TRIzol reagent. Similarly, cellular RNA from the control and DEHP-exposed (mock-
treated and ENG knockdown) cells was isolated using TRIzol reagent. Extracted RNA was
reverse transcribed to cDNA, and qPCR was performed using SYBR Green master mix
(Applied BiosystemsTM, Waltham, MA, USA). cDNA from zebrafish embryos or cells and
zebrafish-specific primers against VEGFa (XM_009292018.3) (5
0
-CAGTTATTTCTCGCGGCTCT-
3
0
; 5
0
- TCCCCCTTCTTTGGGTATGT-3
0
) and GAPDH (NM_001115114.1) (5
0
-GTGGAGTCTA
CTGGTGTCTTC-3
0
and 5
0
-GTGCAGGAGGCATTGCTTACA-3
0
) and human-specific primers
against endoglin (NM_001278138.2) (5
0
-CGCCAACCACAACATGCAG-3
0
and 5
0
- GCTCCA
CGAAGGATGCCAC-3
0
); TGF
β
RII (NM_003242.6) (5
0
-GCTTTGCTGAGGTCTATAAGGC-
3
0
and 5
0
-GGTACTCCTGTAGGTTGCCCT-3
0
); SMAD3 (NM_001145103.2) (5
0
-GTCTGCAAG
ATCCCACCAG-3
0
and 5
0
-AGCCCTGGTTGACCGACT-3
0
);
β
-actin (NM_001101.5) (5
0
-
TGAGACCTTCAACACCCCAGCCAT-30and 50-CGTAGATGGGCACAGTGTGGGTG-30)
was purchased from Genomics (New Taipei, Taiwan) and analyzed on a QuantStudio
TM
5 Real-Time PCR System (A28574, Applied Biosystems
TM
, Waltham, MA, USA). The data
analysis was performed using QuantStudio
TM
5 Design & Analysis Software (version 1.5.1,
Applied Biosystems
TM
, Waltham, MA, USA), and relative mRNA expression was calculated
by the equation 2
−∆∆Ct
. Data quantification was performed by SigmaPlot (version 12.3,
Systat Software, Inc., Erkrath, Germany).
Biomedicines 2022,10, 417 6 of 21
2.8. Western Blotting
Protein samples (30–40
µ
g) were separated by SDS–PAGE, followed by transfer to
PVDF membranes. PVDF membrane blocking was performed in 5% non-fat milk and
subsequent incubation with specific primary antibodies against ENG, MAPK (ERK1/2),
phospho-MAPK (ERK1/2), phospho-p38, and GAPDH at 4
◦
C. The following day, mem-
branes were washed with blocking buffer to remove unbound antibodies, and host-specific
secondary antibody treatment was performed. Finally, membranes were washed, and
chemiluminescence signal detection was performed using an ECL
TM
detection kit and
luminescent image analyzer (Amersham Imager 680, GE Healthcare and Bioscience ab,
Princeton, NJ, USA).
2.9. In Vitro-Angiogenesis/Tube Formation Assay
In vitro
angiogenesis/tube formation was performed by coculturing HUVECs with
the control and DEHP-treated MDA-MB-231 cells. Briefly, 1
×
10
4
HUVECs were seeded
on Matrigel (Culturex
®
5X BME)-coated 24-well plates. A total of 1
×
10
5
of the control and
DEHP-exposed cells were seeded in 0.4
µ
m Transwell inserts (3413, Corning Incorporated,
Corning, NY, USA) and placed in HUVEC seeded plates. Cultures were incubated at 37
◦
C
in an incubator, and images were captured 8 h after seeding. Tube formation data analysis
was performed by web-based WIMASIS Image Analysis software (Onimagin Technologies
SCA, Cordoba, Spain). Data quantification was performed by SigmaPlot.
2.10. Quantitative Enzyme-Linked Immunosorbent Assay (ELISA)
Approximately 2
×
10
5
of the control and DEHP-exposed (mock-treated and ENG
knockdown) MDA-MB-231 cells were seeded in 6-well plates and incubated overnight.
After incubation, serum-free culture medium was replaced, and cells were incubated for
48–72 h. Culture medium was collected and used to perform VEGF quantification using a
Human VEFG
165
Standard TBM ELISA Development kit (900-T10, PEPROTech, Hamburg,
Germany), as described previously [48].
2.11. Immunofluorescence
Approximately 2
×
10
4
of the control and DEHP-exposed (mock-treated and ENG
knockdown) MDA-MB-231 cells were seeded in cell culture chamber slides (30114, SPL
Lifesciences, Pocheon-si, Korea) and incubated overnight at 37
◦
C in an incubator. Cells
were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.1% Triton X-100, and
blocked with blocking buffer (3% BSA in PBS). Cells were incubated with primary anti-
body (SMAD3), washed with blocking buffer, and incubated with host-specific secondary
antibody (goat anti-rabbit IgG, Alexa Fluor 594), along with nuclear staining with DAPI.
Fluorescence imaging was performed on a fluorescence microspore (Olympus, 1X71, Shin-
juku, Japan). Data analysis and quantification were performed by SigmaPlot (version 12.3,
Systat Software, Inc., Erkrath, Germany).
2.12. Antibody Angiogenesis Array
An antibody angiogenesis microarray was performed using a Human Angiogene-
sis Antibody Array-membrane kit (ab193655, Abcam, MA, USA), according to the man-
ufacturer’s instructions. Briefly, control and DEHP-exposed (mock-treated and ENG
knockdown) MDA-MB-231 cells were lysed, and the protein concentration was estimated;
200–250 µg
of total protein was used. The protein-conjugated membranes provided in the
kit were blocked with blocking buffer for 30 min. After incubation, half of the blocking
buffer was removed and replaced with an equal volume of protein sample and then incu-
bated for 1.5–5 h at room temperature. Membranes were washed and conjugated with a
biotinylated antibody cocktail for 1.5–2 h at room temperature. Membranes were washed
and incubated with HRP-conjugated streptavidin for 2 h at room temperature. Finally,
membranes were incubated with detection buffer, and chemiluminescence signals were
detected by a luminescence imager analyzer (Amersham Imager 680). Quantification of
Biomedicines 2022,10, 417 7 of 21
signal intensity was performed by ImageJ (version 1.53, NIH, USA). Data quantification
was performed using SigmaPlot.
2.13. Statistical Analysis
Statistical analysis of data was performed by SigmaPlot. Standard one-way ANOVA
and Student’s t tests were used to compare different groups. Data presented is as a
±
standard deviation (SD). A pvalue was considered statistically significant if it was <0.05.
3. Results
3.1. Prolonged DEHP Exposure Enhances the Angiogenesis Potential of MDA-MB-231 Cells
In Vivo
To evaluate the effect of prolonged DEHP exposure at physiological concentrations,
a zebrafish xenograft assay was performed. Control and DEHP-treated MDA-MB-231
cells injected in 48 hpf Tg (fli1: EGFP) embryo sacs showed enhanced SIV sprouting in
DEHP-treated MDA-MB-231-injected embryos compared to control MDA-MB-231-injected
embryos (Figure 1A). On average, 55% and 12% of embryos (n= 50) showed SIV sprouting
in DEHP-treated and control MDA-MB-231-injected embryos, respectively (Figure 1B). A
43% difference in SIV sprouting is indicative of pro-angiogenic effects of DEHP exposure in
MDA-MB-231 cells.
Biomedicines 2022, 10, x FOR PEER REVIEW 7 of 23
detected by a luminescence imager analyzer (Amersham Imager 680). Quantification of
signal intensity was performed by ImageJ (version 1.53, NIH, USA). Data quantification
was performed using SigmaPlot.
2.13. Statistical Analysis
Statistical analysis of data was performed by SigmaPlot. Standard one-way ANOVA
and Student’s t tests were used to compare different groups. Data presented is as a ±stand-
ard deviation (SD). A p value was considered statistically significant if it was <0.05.
3. Results
3.1. Prolonged DEHP Exposure Enhances the Angiogenesis Potential of MDA-MB-231 Cells In
Vivo
To evaluate the effect of prolonged DEHP exposure at physiological concentrations,
a zebrafish xenograft assay was performed. Control and DEHP-treated MDA-MB-231
cells injected in 48 hpf Tg (fli1: EGFP) embryo sacs showed enhanced SIV sprouting in
DEHP-treated MDA-MB-231-injected embryos compared to control MDA-MB-231-in-
jected embryos (Figure 1A). On average, 55% and 12% of embryos (n = 50) showed SIV sprout-
ing in DEHP-treated and control MDA-MB-231-injected embryos, respectively (Figure 1B). A
43% difference in SIV sprouting is indicative of pro-angiogenic effects of DEHP exposure
in MDA-MB-231 cells.
Figure 1. Representative and quantitative results of the zebrafish xenograft angiogenesis assay. (A)
DEHP-exposed MDA-MB-231 cells enhance SIV sprouting in Tg (fli1: EGFP) zebrafish embryos at
24 hpi. Red fluorescence: DiI stained breast cancer cells; Green fluorescence: vascular network of
zebrafish embryo. Scale bar = 1000 µm. White arrow: Indicate site of SIV sprouting. (B) Quantitative
results of angiogenesis in zebrafish showing SIV sprouting (n = 50); ** p < 0.001.
Figure 1.
Representative and quantitative results of the zebrafish xenograft angiogenesis assay.
(
A
) DEHP-exposed MDA-MB-231 cells enhance SIV sprouting in Tg (fli1: EGFP) zebrafish embryos at
24 hpi. Red fluorescence: DiI stained breast cancer cells; Green fluorescence: vascular network of
zebrafish embryo. Scale bar = 1000
µ
m. White arrow: Indicate site of SIV sprouting. (
B
) Quantitative
results of angiogenesis in zebrafish showing SIV sprouting (n= 50); ** p< 0.001.
3.2. Endoglin Predicted as a Regulator of DEHP-Induced Angiogenesis in Breast Cancer Cells
To identify the underlying mechanism and regulators of DEHP-induced angiogenesis
potential in MDA-MB-231 cells, RNA sequencing was performed. Gene ontology (GO)
enrichment analysis of differentially expressed genes (DEGs) was performed for control and
DEHP treated MDA-MB-231 cells. GO enrichment analysis showed the 30 most significantly
enriched terms related to biological process, cellular component, and molecular function.
Biomedicines 2022,10, 417 8 of 21
Regulation of cell growth, organ development, regulation of cell development, system
development, locomotion, response to wounding, and cell growth were identified among
the top 30 enriched GO terms (Figure 2A). GO terms such as cellular growth, organ
development, system development, and locomotion are closely related to angiogenesis
or vascular development, which is the initial phase of system or organ development.
During cancer progression, angiogenesis is a prerequisite for tumor growth and metastasis.
We further used IPA, a bioinformatics tool to identify and predict the profile of gene
expression and pathways underlying the impact of prolonged DEHP exposure to breast
cancer cells, and IPA analysis showed the significant enrichment of endothelial tissue
development and cell development under disease and function (Figure 2B). Downstream
effect analysis highlighted the involvement of endoglin with a log expression ratio of
4.457 (Figure 2C). Further disease and function analysis found enrichment of growth of
malignant tumors and growth of solid tumors (Figure 2D). Interestingly, endoglin was
found to be positively correlated with both the growth of malignant tumors and the growth
of solid tumors. To our surprise, the matrix metalloproteins MMP1 and MMP3, along
with the inflammatory cytokines IL1
β
and IL1
α
, were predicted to be activated with a
high log expression ratio (Figure 2E,F). The expression of endoglin was further confirmed
through Western blotting and qPCR. Prolonged DEHP exposure increased the expression
of endoglin almost 2-fold at protein and 3-fold at mRNA levels (Figure 2G,H). Collectively,
endoglin was predicted to be involved in endothelial tissue and cell development, along
with malignant and solid tumor growth, indicating a central role of endoglin. Considering
the pathophysiological conditions, angiogenesis/vascular development plays a crucial role
in normal tissue development or tumor growth [
49
,
50
]. An increase in endoglin expression
consistent with prediction of NGS data points towards its involvement in regulating DEHP-
induced angiogenesis, tumor growth, and ultimately metastasis.
3.3. Endoglin Depletion Reverses the DEHP-Induced Angiogenic Potential of MDA-MB-231 Cells
To confirm and validate the involvement of endoglin in DEHP-induced angiogenesis in
MDA-MB-231 cells, endoglin was knocked down using lentivirus-mediated shRNA trans-
fection. Western blotting showed upregulation of endoglin in scrambled/mock shRNA-
treated DEHP-exposed MDA-MB-231 cells. However, endoglin expression was completely
depleted in shENG-treated control and DEHP-exposed MDA-MB-231 cells (Figure 3A).
Next, control and DEHP-exposed MDA-MB-231 cells (mock- and shENG-treated) were
injected into the embryo sacs of 48 hpf Tg (fli1: EGFP) embryos. SIV sprouting evaluation at
24 h after injection showed that endoglin knockdown significantly reduced SIV sprouting
(Figure 3B). The number of embryos showing SIV sprouting following xenografting was
reduced from 50% to 12.5% in endoglin knockdown DEHP-exposed MDA-MB-231 cell-
injected mock-treated embryos compared with DEHP-exposed MDA-MB-231 cell-injected
embryos (Figure 3C). However, no change was observed in mock- and shENG-treated
control MDA-MB-231 cells. To understand the mechanism of cancer cell-induced SIV
sprouting of Tg (fli1: EGFP) embryos, mRNA levels of zebrafish VEGFA were investigated.
qPCR results showed that VEGFA mRNA levels were significantly increased in breast
cancer cell-injected embryos compared to embryos without injection. The highest VEGFA
mRNA levels were recorded in mock-treated DEHP-exposed MDA-MB-231 cell-injected
embryos, followed by embryos injected with mock-treated control MDA-MB-231 cells.
Interestingly, DEHP knockdown significantly reduced VEGFA mRNA levels in embryos
injected with shENG-treated DEHP-exposed MDA-MB-231 cells (Figure 3D). Surprisingly,
ENG knockdown in control cell-injected embryos showed no significant change in VEGFA
mRNA levels. Overall, endoglin controls and regulates DEHP-induced SIV sprouting and
VEGFA expression in Tg (fli1: EGFP) embryos injected with breast cancer cells.
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Figure 2. Representative results of RNA sequencing and IPA of control and DEHP-treated MDA-
MB-231 cells. (A) Bar chart of significantly enriched GO terms and the number of DEGs enriched in
Figure 2.
Representative results of RNA sequencing and IPA of control and DEHP-treated MDA-
MB-231 cells. (A) Bar chart of significantly enriched GO terms and the number of DEGs enriched in
biological processes (green), cellular components (orange), and molecular function (violet). (
B
) IPA-
derived heatmap analysis of development under cellular diseases and functions of the DEGs involved.
(
C
) Downstream analysis of genes involved in the development of endothelial tissue and cell devel-
opment. (
D
) IPA-derived heatmap analysis of cancer growth under cellular diseases and functions of
the DEGs involved. (
E
,
F
) Downstream analysis of genes involved in the growth of malignant tumors
and the growth of solid tumors in control and DEHP-exposed MDA-MB-231 cells, highlighting the
involvement of endoglin/CD105 and its prediction as a regulator of DEHP-induced angiogenesis.
(
G
,
H
) Prolonged DEHP exposure increased endoglin expression at the protein and mRNA level in
MDA-MB-231 cells; ** p< 0.001.
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Figure 3. Representative and quantitative results showing that DEHP-induced endoglin-controlled
VEGFA-mediated angiogenesis in vivo. (A) Prolonged DEHP treatment upregulated endoglin ex-
pression in MDA-MB-231 cells, and shENG treatment depleted endoglin expression in control and
DEHP-exposed MDA-MB-231 cells. (B) Endoglin knockdown reduced DEHP-induced SIV sprout-
ing in Tg (fli1: EGFP) zebrafish embryos at 24 hpi. Scale bar =1000 µ m. (C) Quantitative results of
angiogenesis showing a reduced number of zebrafish embryos showing SIV sprouting following
endoglin knockdown (n = 50). (D) Depletion of VEGFA mRNA levels in zebrafish embryos follow-
ing endoglin knockdown cell xenografts of DEHP-exposed MDA-MB-231 cells; ** p < 0.001.
3.4. Endoglin Regulates HUVEC Tube Formation through VEGF Production in Prolonged
DEHP-Treated MDA-MB-231 Cells
To validate the results of the in vivo zebrafish angiogenesis assay, we performed an
in vitro angiogenesis assay by coculturing HUVECs with control and DEHP-exposed
MDA-MB-231 cells (mock- and shENG-treated). Induced tube formation was observed
under all coculture conditions compared to HUVECs alone, and mock-treated DEHP-ex-
posed MDA-MB-231 cell coculture showed the highest tube formation potential (Figure 4A).
Endoglin knockdown in DEHP-exposed cells significantly reduced HUVEC tube
Figure 3.
Representative and quantitative results showing that DEHP-induced endoglin-controlled
VEGFA-mediated angiogenesis
in vivo
. (
A
) Prolonged DEHP treatment upregulated endoglin ex-
pression in MDA-MB-231 cells, and shENG treatment depleted endoglin expression in control and
DEHP-exposed MDA-MB-231 cells. (
B
) Endoglin knockdown reduced DEHP-induced SIV sprouting
in Tg (fli1: EGFP) zebrafish embryos at 24 hpi. Scale bar =1000
µ
m. (
C
) Quantitative results of
angiogenesis showing a reduced number of zebrafish embryos showing SIV sprouting following
endoglin knockdown (n= 50). (
D
) Depletion of VEGFA mRNA levels in zebrafish embryos following
endoglin knockdown cell xenografts of DEHP-exposed MDA-MB-231 cells; ** p< 0.001.
3.4. Endoglin Regulates HUVEC Tube Formation through VEGF Production in Prolonged
DEHP-Treated MDA-MB-231 Cells
To validate the results of the
in vivo
zebrafish angiogenesis assay, we performed an
in vitro
angiogenesis assay by coculturing HUVECs with control and DEHP-exposed MDA-
MB-231 cells (mock- and shENG-treated). Induced tube formation was observed under all
coculture conditions compared to HUVECs alone, and mock-treated DEHP-exposed MDA-
MB-231 cell coculture showed the highest tube formation potential (Figure 4A). Endoglin
knockdown in DEHP-exposed cells significantly reduced HUVEC tube formation, consis-
Biomedicines 2022,10, 417 11 of 21
tent with the
in vivo
results. The number of tubes, average tube length, and number of
nodes showed similar observations. The average number of tubes, average tube length, and
number of nodes formed were highest in mock-treated DEHP-exposed MDA-MB-231 cells,
and HUVECs without coculture were lowest among all groups. Endoglin knockdown
in DEHP-exposed MDA-MB-231 cells significantly reduced the average number of tubes,
average tube length, and number of nodes formed in coculture. Interestingly, endoglin
knockdown in control MDA-MB-231 cells showed a slight increase in the average number
of tubes and average tube length; however, it did not affect the number of nodes formed
in coculture (Figure 4B–D). To understand the mechanism of HUVEC tube formation
in coculture, quantitative ELISA was performed. Cell culture medium of control and
DEHP-exposed MDA-MB-231 cells (mock- and shENG-treated) was used to evaluate VEGF
concentrations. The results showed that mock-treated DEHP-exposed MDA-MB-231 cell
culture medium contained the highest VEGF concentration of 432 pg/
µ
L, followed by
320 pg/
µ
L in mock-treated control MDA-MB-231 cell culture medium. Endoglin knock-
down in DEHP-exposed MDA-MB-231 cells resulted in significant depletion of VEGF levels,
as low as 97 pg/
µ
L. However, endoglin knockdown did not affect VEGF levels in control
MDA-MB-231 cells (Figure 4E). Collectively, prolonged DEHP exposure induced HUVEC
tube formation in coculture through endoglin-mediated VEGF production.
3.5. Endoglin Maintains the TGFβ/SMAD3/VEGF Signaling Axis in Prolonged DEHP-Treated
MDA-MB-231 Cells
To understand the underlying mechanism of DEHP-induced angiogenesis potential
through VEGF expression, gene set enrichment analysis (GSEA) was performed. TGF
β
signaling was enriched and found to be positively correlated with DEHP-exposed MDA-
MB-231 cells, with an enrichment score of 0.28 compared to
−
0.67 of the control MDA-MB-
231 cells (Figure 5A). Individual DEGs of the TGF
β
gene set showed core enrichment in
DEHP-treated MDA-MB-231 cells (Figure 5B). Endoglin is a TGF
β
coreceptor, and endoglin
upregulation following DEHP treatment is postulated to stabilize and induce TGF
β
signal-
ing, supporting the results. mRNA levels of TGF
β
signaling markers TGF
β
RII and SMAD3,
along with endoglin and VEGF, were evaluated and showed that DEHP treatment increased
mRNA levels of endoglin, TGF
β
RII, SMAD3, and VEGF; however, endoglin knockdown
resulted in a significant decline in mRNA levels of endoglin, TGF
β
RII, SMAD3, and
VEGF (Figure 5C–F). qPCR results show that activation of canonical TGF
β
/SMAD3/VEGF
signaling regulated through endoglin in DEHP-exposed MDA-MB-231 cells, consistent
with GSEA results. To verify the transcriptional regulatory activity of SMAD3, IF was
performed, and consistent with prior observations, SMAD3 expression was highest in
DEHP-exposed MDA-MB-231 cells. SMAD3 was observed to be colocalized in the nucleus
of DEHP-exposed cells, indicating SMAD3 activation and nuclear translocation. Interest-
ingly, endoglin knockdown not only reduced the expression of SMAD3 but also depleted
nuclear translocation, limiting its activation and SMAD3-mediated transcriptional reg-
ulation of VEGF (Figure 5G–I). The effect of DEHP exposure and endoglin knockdown
was evaluated on the expression of total and phospho-SMAD3 and showed that DEHP
exposure upregulates total and phospho-SMAD3. Consistent with qPCR and IF findings,
endoglin knockdown downregulates expression of both total and phospho-SMAD3 in
DEHP-exposed MDA-MB-231 cells (Figure 5J). Overall, prolonged DEHP exposure induces
endoglin expression, which stabilizes TGFβRII and activates TGFβsignaling, resulting in
increased VEGF production and secretion and enhancement of the angiogenesis potential
of MDA-MB-231 cells.
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Figure 4. Representative and quantitative results of endoglin-mediated HUVEC tube formation. (A)
Induced HUVEC tube formation in coculture with control and DEHP-exposed MDA-MB-231 cells
at 8 h after seeding; endoglin knockdown reversed DEHP-induced HUVEC tube formation. Scale
bar = 250 µ m. (B) Quantitative evaluation of the number of tubes formed in coculture at 8 h after
cell seeding. (C) Quantitative evaluation of average tube length in coculture at 8 h after cell seeding.
(D) Quantitative evaluation of the number of nodes formed in coculture at 8 h after cell seeding. (E)
Results of quantitative ELISA for VEGF levels in the cell culture medium of control and DEHP-
exposed MDA-MB-231 cells (mock- and shENG-treated); ** p < 0.001.
Figure 4.
Representative and quantitative results of endoglin-mediated HUVEC tube formation.
(
A
) Induced HUVEC tube formation in coculture with control and DEHP-exposed MDA-MB-231
cells at 8 h after seeding; endoglin knockdown reversed DEHP-induced HUVEC tube formation.
Scale bar = 250
µ
m. (
B
) Quantitative evaluation of the number of tubes formed in coculture at 8 h
after cell seeding. (
C
) Quantitative evaluation of average tube length in coculture at 8 h after cell
seeding. (
D
) Quantitative evaluation of the number of nodes formed in coculture at 8 h after cell
seeding. (
E
) Results of quantitative ELISA for VEGF levels in the cell culture medium of control and
DEHP-exposed MDA-MB-231 cells (mock- and shENG-treated); ** p< 0.001.
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Figure 5. Representative and quantitative results of endoglin-mediated regulation of the
TGFβ/SMAD3/VEGF signaling axis. (A) GSEA of TGFβ signaling showing a positive correlation
with DEHP-treated MDA-MB-231 cells (enrichment score). (B) Blue–Pink O’ gram showing core
Figure 5.
Representative and quantitative results of endoglin-mediated regulation of the
TGF
β
/SMAD3/VEGF signaling axis. (
A
) GSEA of TGF
β
signaling showing a positive correla-
tion with DEHP-treated MDA-MB-231 cells (enrichment score). (
B
) Blue–Pink O’ gram showing core
enrichment of individual genes in the TGF
β
signaling gene set (red: upregulation; blue: downregula-
tion). (
C
–
F
) qPCR analysis showing the mRNA expression of endoglin, TGF
β
RII, SMAD3, and VEGF
in control and DEHP-exposed MDA-MB-231 cells (mock- and shENG-treated). (
G
) IF results showing
expression changes and nuclear localization of p-SMAD3. Scale bar = 100
µ
m. (
H
,
I
) Quantitative
analysis of p-SMAD3 IF showing total and nuclear p-SMAD3 expression levels. (
J
) SMAD3 and
phospho-SMAD3 expression evaluated by Western blotting; ** p< 0.001.
Biomedicines 2022,10, 417 14 of 21
3.6. Endoglin-Mediated MAPK/p38 Signaling and Secretory Cytokine Production May Contribute
to DEHP-Induced Angiogenesis in MDA-MB-231 Cells
Western blotting was performed to evaluate the effect of prolonged DEHP treatment on
MDA-MB-231 cells and showed activation of MAPK/p38 signaling. Upregulation of MAPK,
phospho-MAPK, and phospho-p38 was observed in DEHP-treated cells compared to control
MDA-MB cells. Endoglin knockdown reduced the expression of MAPK, phospho-MAPK,
and phospho-p38 in DEHP-exposed MDA-MB-231 cells; however, endoglin knockdown in
control MDA-MB-231 cells upregulated all three proteins (Figure 6A). Overall, these results
indicate that endoglin can positively regulate MAPK/p38 signaling in DEHP-exposed cells
and that MAPK/p38 may not be involved in angiogenesis progression in control MDA-
MB-231 cells. Next, to understand the molecular mechanism of MAPK/p38 signaling,
we performed an angiogenesis antibody array to screen multiple candidate proteins that
might be involved in ENG-mediated angiogenesis in MDA-MB-231 cells. The results
showed six proteins involved in endoglin-mediated angiogenesis among the panel of
43 angiogenesis-associated markers evaluated. The inflammatory cytokines IL1
α
, IL1
β
,
IL6, IL8, GMCSF, and VEGF were highly expressed in DEHP-treated cells compared to
control MDA-MB-231 cells. Endoglin knockdown reversed the expression of all six markers
in both control and DEHP-exposed MDA-MB-231 cells (Figure 6B–H). Overall, it can be
predicted that endoglin-mediated MAPK/p38 signaling may regulate the expression of the
abovementioned inflammatory cytokines and that GMCSF contributes to DEHP-induced
angiogenesis potential in MDA-MB-231 cells. Moreover, in control MDA-MB-231 cells,
endoglin knockdown resulted in MAPK/p38 activation, but it reduced the expression of
cytokines that might not be interrelated with each other. Endoglin depletion still affected
the expression of secretory cytokines in control MDA-MB-231 cells, which may be regulated
by different signaling mechanisms, warranting further investigation.
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Figure 6. Representative and quantitative results of endoglin-mediated MAPK/p38 signaling and
cytokine regulation. (A) Western blotting results showing upregulation of MAPK, phospho-MAPK,
p38, and phospho-p38 in DEHP-treated MDA-MB-231 cells, and endoglin reversed protein expres-
sion. (B) Representative results of the antibody angiogenesis array showing endoglin-mediated ex-
pression of the inflammatory cytokines GMCSF and VEGF. (C–H) Quantification of the expression
of IL1α, IL1β, IL6, IL8, GMCSF, and VEGF in control and DEHP-exposed MDA-MB-231 cells (mock-
and shENG-treated). +ve ctrl: positive control; ** p < 0.001.
Figure 6.
Representative and quantitative results of endoglin-mediated MAPK/p38 signaling and
cytokine regulation. (
A
) Western blotting results showing upregulation of MAPK, phospho-MAPK,
p38, and phospho-p38 in DEHP-treated MDA-MB-231 cells, and endoglin reversed protein expression.
(
B
) Representative results of the antibody angiogenesis array showing endoglin-mediated expression
of the inflammatory cytokines GMCSF and VEGF. (
C
–
H
) Quantification of the expression of IL1
α
,
IL1
β
, IL6, IL8, GMCSF, and VEGF in control and DEHP-exposed MDA-MB-231 cells (mock- and
shENG-treated). +ve ctrl: positive control; ** p< 0.001.
Biomedicines 2022,10, 417 16 of 21
4. Discussion
Di-2-ethylhexyl phthalate (DEHP) is widely used as a plasticizer in plastic products
and is ubiquitous in daily life [
51
]. In particular, Chinese populations are exposed to higher
phthalate concentrations than Western populations, leading to exposure from contami-
nated food and air [
52
]. A large amount of evidence suggests that DEHP exposure is
intimately correlated with several disorders, such as Alzheimer’s disease, male infertility,
and increased risk of breast cancer [
53
–
55
]. The effects of DEHP exposure
in vivo
has
been evaluated in several studies. Barkat et al. demonstrated that high oral DEHP doses
in pregnant mice resulted in infertile male offspring [
56
]. However, other groups found
prenatal DEHP exposure affected female reproduction in F1–F3 generations, leading to
reproductive aging [
57
]. These studies indicate that DEHP exposure exerts significant
effects on reproductive system
in vivo
; however, long-term effects with low concentrations
of DEHP have not been demonstrated. A strategic study of cancer progression in long-term
DEHP-exposed mice might provide an insight into physiological condition and the effects
of DEHP exposure on cancer progression. Moreover, this type of study might lack the
actual prolonged DEHP exposure to tumor cells, considering the proliferation rate of tumor
implants
in vivo
. To overcome this issue, we exposed breast cancer cells with physiological
DEHP concentration (100 nM) long term in an attempt to mimic real life conditions. In
addition, phthalate has been confirmed to promote cancer progression through induced
proliferation, drug resistance, and angiogenesis. Crobeddu et al. demonstrated that DEHP
and its primary metabolite mono (2-ethylhexyl) phthalate (MEHP) enhance the prolifer-
ation of human breast ductal carcinoma cells via upregulation of progesterone receptor
(PR) [55].
In our previous study, we showed that long-term DEHP exposure at a concentra-
tion can induce multidrug resistance through increased expression of ABC transporters
and reduced intracellular ROS generation [
12
]. Over the past few decades, most studies
have reported the role of DEHP in placental development [
26
,
58
]. However, the associ-
ation between DEHP and angiogenesis in cancer remains largely unknown. Tsai et al.
reported that benzyl butyl phthalate (BBP) can induce angiogenesis in hepatocellular car-
cinoma (HCC) through the aryl hydrocarbon receptor AhR/ERK/VEGF axis [
59
]. This
finding indicates that phthalate has the potential to promote angiogenesis. Consistent
with previous reports, our data showed that SIV sprouting was remarkably increased
in zebrafish embryos implanted with prolonged DEHP-exposed MDA-MB-231 cells. We
demonstrated the proangiogenic activity of DEHP treatment to mimic physiological DEHP
exposure for an extended period of time because most DEHP-related studies emphasize
high-concentration DEHP treatment for a short period and research on low-dose and
prolonged DEHP exposure–mediated tumor progression is relatively insufficient.
Endoglin, also known as CD105, is a transmembrane glycoprotein that serves as a
coreceptor for TGF-
β
receptors I and II and is important for angiogenesis [
60
]. Kasprzak
et al. revealed that endoglin plays a significant role in angiogenesis in HCC. It cannot only
promote the proliferation of liver sinusoidal endothelial cells (ECs) but also enhance the
resistance of ECs to apoptosis [
61
]. Endoglin is also reported to promote colorectal cancer
liver metastasis by cancer-associated fibroblast (CAF)-expressing endoglin and TGF-
β
signaling [
62
]. Consistent with these findings, our study showed that endoglin knockout
reduced SIV sprouting and VEGFa expression in zebrafish embryos. Moreover, endoglin
depletion seriously disrupted DEHP-induced VEGF-mediated HUVEC tube formation.
Collectively, DEHP-induced angiogenesis is mainly mediated by endoglin.
A literature review revealed that endoglin is the auxiliary receptor of the TGF-
β
recep-
tor that facilitates the initiation of the TGF-
β
/SMAD signaling pathway. Phosphorylated
SMAD serves as a transcription factor (TF) to promote the expression of the downstream
effector genes responsible for cell proliferation, migration, and angiogenesis [
63
]. SMAD3
functions as a TF once phosphorylated following TGF
β
signaling activation, which in-
duces the expression of its downstream transcripts, especially VEGFA [
64
,
65
]. Our results
indicated that prolonged DEHP exposure activated the TGF
β
/SMAD3/VEGF signaling
Biomedicines 2022,10, 417 17 of 21
axis. Moreover, DEHP treatment induced nuclear localization of SMAD3, indicating the
transcriptional regulator activity of SMAD3. Endoglin depletion reversed the activation of
TGFβsignaling and reduced the nuclear translocation of SMAD3.
Mitogen-activated protein kinase (MAPK) is an important signaling pathway involved
in several cellular events, such as cell differentiation, proliferation, inflammation and
apoptosis. There are three major components of the MAPK cascade, including c-Jun N-
terminal kinases (JNKs), p38, and extracellular signal-regulated kinases
(ERKs) [66,67].
Preliminary evidence indicates that VEGFA overexpression is triggered by both the PI3K
and MAPK/p38 signaling pathways, which further drives angiogenesis [
68
]. Our results
corroborated the activation of MAPK/p38 in DEHP-exposed MDA-MB-231 cells. Con-
versely, endoglin knockdown inhibited phosphorylation and activation. Taken together,
DEHP exposure can induce angiogenesis through ENG-controlled TGF-
β
/SMAD3/VEGF
and MAPK/p38 signaling.
Accumulating studies have suggested that MAPK/p38 is also involved in the produc-
tion of cytokines, including IL-1 and IL-6, and granulocyte-macrophage colony-stimulating
factor (GM-CSF) [
69
–
71
]. In proteome profiling, the relationship between inflammation
and angiogenesis was apparently observed; in addition, IL-1
β
and VEGF had considerable
overlaps on biological functions and signal transduction of pro-angiogenic effects, espe-
cially with both of them activating MAPK in HUVECs [
72
]. Another finding suggested that
IL-1
β
also plays a crucial role in invasiveness and metastasis. Angiogenesis is capable of
providing sufficient nutrients because metastasis requires abundant energy to complete [
73
].
Interleukin-6 (IL-6), a pleiotropic cytokine, has been suggested to induce angiogenesis in
mounting studies. IL-6 can contribute to not only the upregulation of VEGF but also in-
creased endothelial tip cell sprouting through the pSTAT3 and jagged-1/Notch-3 signaling
pathways, respectively [
74
]. Gopinathan et al. highlighted that IL-6 shares similarities with
VEGF on pro-angiogenic function; however, the difference lies in the fact that a vessel with
aberrant pericytes coverage was observed in IL-6-induced angiogenesis, but was absent
in the VEGF-stimulated one [
75
]. Our results indicated that prolonged DEHP exposure
induced the expression of the inflammatory cytokines IL1
α
, IL1
β
, IL6, IL8, and GM-CSF
through endoglin regulation in MDA-MB-231 cells.
5. Conclusions
In conclusion, our study confirmed that prolonged DEHP exposure can promote an-
giogenesis in TNBC via the ENG/TGF
β
/SMAD3/VEGF signaling axis
in vitro
and
in vivo
.
Alternatively, long-term DEHP exposure can also induce upregulation of the inflammatory
cytokines IL1
α
, IL1
β
, IL6, and GM-CSF through the MAPK/p38 signaling pathway to
facilitate breast cancer progression. Hence, targeting ENG and its downstream signaling
pathway might be a promising strategy to normalize vasculature or curb angiogenesis and
subsequently inhibit the progression of breast cancer (Figure 7).
Biomedicines 2022,10, 417 18 of 21
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Figure 7. Schematic representation of prolonged DEHP exposure-induced angiogenesis potential in
breast cancer cells. Prolonged DEHP exposure at physiological concentrations upregulate the ex-
pression of endoglin (ENG). Endoglin overexpression activates TGFβ and MAPK/p38 signaling-
mediated production of VEGF; inflammatory cytokines IL1α, IL1β, IL6, IL8; and GMCSF, contrib-
uting to enhanced DEHP-induced angiogenesis potential in breast cancer cells. shRNA mediated
ENG knockdown reversed ENG induced angiogenesis through downregulation of TGFβ and
MAPK/p38 signaling along with reduced expression of inflammatory cytokines IL1α, IL1β, IL6, IL8;
and GMCSF.
Author Contributions: Conceptualization, M.J., C.-C.C., E.-M.T., and L.-F.W.; data curation, M.J.,
D.D., and R.U.; formal analysis, M.J., D.D., W.-T.L., and J.Y.-F.C.; funding acquisition, C.-L.C., L.-
F.W., and C.-C.C.; investigation, M.J., J.Y.-F.C., and C.-C.C.; methodology, M.J., W.L., D.D., W.-T.L.,
R.U., and S.-K.H.; project administration, L.-F.W. and C.-C.C.; resources, C.-L.C., W.L., W.-T.L., J.Y.-F.C.,
E.-M.T., L.-F.W., and C.-C.C.; software, W.L.; supervision, L.-F.W. and C.-C.C.; validation, M.J., D.D.;
writing—original draft, M.J., R.U., and S.-K.H.; writing—review and editing, C.-C.C. All authors
have read and agreed to the published version of the manuscript.
Funding: We thank the following institutions for providing financial support: Ministry of Science
and Technology, Taiwan (grants MOST 107-2320-B-110-002-MY3, 109-2320-B-037-017-MY3, 109-
2314-B-037-069-MY3 and 110-2320-B- 110-004); NSYSU-KMU joint grants (grant NSYSUKMU 110-
I006, 111-P25 and 111-P12), the Kaohsiung Medical University Research Center, Taiwan (KMU-
TC109A04), and the Kaohsiung Medical University, Taiwan (KMU-DK(A)111001).
Institutional Review Board Statement: All animal experiments were conducted according to the
guidelines of the Institutional Animal Care and Use Committee (IACUC), Kaohsiung Medical Uni-
versity, Kaohsiung, Taiwan. KMU, IACUC Approval No: 108182 Date of approval: 27 February
2020.
Informed Consent Statement: Not applicable.
Data Availability Statement: The authors confirm that the data supporting the findings of this
study are available within the article and will be provided on request.
Acknowledgments: We are grateful to the Center for Research Resources and Development
(Kaohsiung Medical University, Kaohsiung, Taiwan) for instrument support (real-time PCR and
fluorescence microscopy) and for providing access to Ingenuity Pathway Analysis Software (IPA®, QI-
AGEN Redwood, Redwood City, CA, USA, www.qiagen.com/ingenuity accessed on 21 January
2022).
Figure 7.
Schematic representation of prolonged DEHP exposure-induced angiogenesis potential
in breast cancer cells. Prolonged DEHP exposure at physiological concentrations upregulate the
expression of endoglin (ENG). Endoglin overexpression activates TGF
β
and MAPK/p38 signaling-
mediated production of VEGF; inflammatory cytokines IL1
α
, IL1
β
, IL6, IL8; and GMCSF, contributing
to enhanced DEHP-induced angiogenesis potential in breast cancer cells. shRNA mediated ENG
knockdown reversed ENG induced angiogenesis through downregulation of TGF
β
and MAPK/p38
signaling along with reduced expression of inflammatory cytokines IL1
α
, IL1
β
, IL6, IL8; and GMCSF.
Author Contributions:
Conceptualization, M.J., C.-C.C., E.-M.T. and L.-F.W.; data curation, M.J.,
D.D. and R.U.; formal analysis, M.J., D.D., W.-T.L. and J.Y.-F.C.; funding acquisition, C.-L.C., L.-F.W.
and C.-C.C.; investigation, M.J., J.Y.-F.C. and C.-C.C.; methodology, M.J., W.L., D.D., W.-T.L., R.U.
and S.-K.H.; project administration, L.-F.W. and C.-C.C.; resources, C.-L.C., W.L., W.-T.L., J.Y.-F.C.,
E.-M.T., L.-F.W. and C.-C.C.; software, W.L.; supervision, L.-F.W. and C.-C.C.; validation, M.J. and
D.D.; writing—original draft, M.J., R.U. and S.-K.H.; writing—review and editing, C.-C.C. All authors
have read and agreed to the published version of the manuscript.
Funding:
We thank the following institutions for providing financial support: Ministry of Science
and Technology, Taiwan (grants MOST 107-2320-B-110-002-MY3, 109-2320-B-037-017-MY3, 109-2314-
B-037-069-MY3 and 110-2320-B- 110-004); NSYSU-KMU joint grants (grant NSYSUKMU 110-I006,
111-P25 and 111-P12), the Kaohsiung Medical University Research Center, Taiwan (KMU-TC109A04),
and the Kaohsiung Medical University, Taiwan (KMU-DK(A)111001).
Institutional Review Board Statement:
All animal experiments were conducted according to the
guidelines of the Institutional Animal Care and Use Committee (IACUC), Kaohsiung Medical Uni-
versity, Kaohsiung, Taiwan. KMU, IACUC Approval No: 108182 Date of approval:
27 February 2020.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The authors confirm that the data supporting the findings of this study
are available within the article and will be provided on request.
Acknowledgments:
We are grateful to the Center for Research Resources and Development (Kaohsi-
ung Medical University, Kaohsiung, Taiwan) for instrument support (real-time PCR and fluorescence
microscopy) and for providing access to Ingenuity Pathway Analysis Software (IPA
®
, QIAGEN
Redwood, Redwood City, CA, USA, www.qiagen.com/ingenuity accessed on 21 December 2021).
Biomedicines 2022,10, 417 19 of 21
Conflicts of Interest: The authors report no conflict of interest.
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