orally administered mucolytic drug l-carbocisteine...
TRANSCRIPT
JPET #224816
1
Orally administered mucolytic drug L-carbocisteine inhibits angiogenesis and tumor
growth in mice
Tomohiro Shinya, Tsubasa Yokota, Shiori Nakayama, Sayuri Oki, Junpei Mutoh, Satoru
Takahashi, and Keizo Sato
Department of Clinical Biochemistry, School of Pharmaceutical Science, Kyushu University
of Health and Welfare, Nobeoka, Miyazaki, Japan (TS, TY, SN, SO, KS)
Second Department of Pharmacology, School of Pharmaceutical Science, Kyushu University
of Health and Welfare, Nobeoka, Miyazaki, Japan (JM)
Department of Immunobiology, School of Pharmacy and Pharmaceutical Science, Mukogawa
Women’s University, Nishinomiya, Hyogo, Japan (ST)
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
2
Running title: L-Carbocisteine is a novel inhibitor of tumor angiogenesis
Corresponding author: Keizo Sato
Kyushu University of Health and Welfare, 1714-1 Yoshino-machi, Nobeoka-shi, Miyazaki
882-8508, Japan
Phone: +81-982-23-5557; Fax: +81-982-23-5559; E-mail: [email protected]
Number of text pages: 19
Number of tables: 0
Number of figures: 9
Number of references: 55
Number of words in the abstract: 191
Number of words in the introduction: 384
Number of words in the discussion: 1457
Nonstandard abbreviations: EGF, epidermal growth factor; ERK, extracellular
signal-related kinase; HUVECs, human umbilical vein endothelial cells; MAP kinase,
mitogen activated protein kinase; MEK, mitogen extracellular kinase; PKC, protein kinase C;
PLC, phospholipase C; ROS, reactive oxygen species; VEGF, vascular endothelial growth
factor
Recommended section assignment: Chemotherapy, Antibiotics, and Gene Therapy
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
3
Abstract
Angiogenesis, the formation of new blood vessels from pre-existing vessels, is essential for
the growth and metastasis of tumors. In this study, we found that L-carbocisteine, a widely
used expectorant, potently inhibits angiogenesis in vitro and in vivo. An in vivo Matrigel plug
assay revealed that L-carbocisteine (2.5 mg/kg administered intraperitoneally twice daily)
significantly inhibited VEGF-induced angiogenesis. L-Carbocisteine also suppressed
VEGF-stimulated proliferation, migration, and formation of capillary-like structures of
human umbilical vein endothelial cells (HUVECs). We examined the signaling pathways
affected in VEGF-stimulated HUVECs, and found that L-carbocisteine significantly inhibited
VEGF-induced phosphorylation of phospholipase C gamma (PLCγ), protein kinase C mu
(PKCµ), and extracellular signal-related kinase (ERK) 1/2, which has been shown to be
essential for angiogenesis. However, these inhibitory effects of L-carbocisteine were not
observed in the HeLa human cervical cancer cell line. An in vivo study of Colon-26
tumor-bearing mice found that tumor volumes were significantly smaller in mice treated with
L-carbocisteine (150 mg/kg administered orally twice daily) in comparison with
vehicle-treated mice. However, L-carbocisteine had no direct effect on Colon-26 cell
proliferation or ERK activation. Collectively, our results suggest that L-carbocisteine inhibits
tumor angiogenesis by suppressing PLCγ/PKC/ERK signaling.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
4
Introduction
Angiogenesis plays an important role in tumor growth (Thairu et al., 2011) because blood
vessels generated via this pathophysiological process supply oxygen and nutrients to cancer
cells and subsequently remove carbon dioxide and metabolites, both of which are
indispensable to the proliferation and survival of cells (McMahon, 2000; Bhat and Singh,
2008; Claesson-Welsh, 2012). Considerable evidence shows that appropriate suppression of
tumor angiogenesis can attenuate tumor growth (Bhat and Singh, 2008; Claesson-Welsh,
2012). Vascular endothelial growth factor (VEGF)-A is a key regulator of angiogenesis.
Angiogenesis-related VEGF signaling is mediated primarily by VEGF receptor 2
(VEGFR2/KDR) activation (Takahashi, 2011; Nagy et al., 2007; Shibuya, 2014), which
activates various cell-signaling molecules, such as phosphoinositide 3-kinase/Akt, Cdc42/p38
mitogen-activated protein (MAP) kinase, focal adhesion kinase (FAK), Src family kinase,
phospholipase C (PLC)/protein kinase C (PKC), and mitogen extracellular kinase
(MEK)/extracellular signal-related kinase (ERK) (Zachary and Gliki, 2001).
L-Carbocisteine (S-carboxymethylcysteine) is used widely as an expectorant (Rhinathiol®,
Mucodyne®) because it normalizes sialic acid and fucose contents in mucins through the
regulation of glycosyltransferase activity, and its use is not associated with serious side
effects. L-Carbocisteine removes phlegm and indications for its use include inflammation of
the upper respiratory tract, acute bronchitis, bronchial asthma, chronic bronchitis,
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
5
bronchiectasis, pulmonary tuberculosis, and chronic sinusitis (Hooper and Calvert, 2008). In
recent years, novel biological activities of L-carbocisteine have been reported in the context
of inhibition of inflammation associated with influenza virus infection and chronic
obstructive pulmonary disease (COPD) (Yamaya et al., 2010; Asada et al., 2012; Yasuda et al.,
2006; Zheng at al., 2008). Another report showed that L-carbocisteine possessed free
radical-scavenging properties in vitro (Nogawa, 2009). Various inflammatory cells, including
neutrophils, mast cells, natural killer cells, macrophages, and dendritic cells, are involved in
induction and promotion of angiogenesis (Noonan et al., 2008; Kim et al., 2013). Moreover,
generation of reactive oxygen species (ROS) is a primary function of activated inflammatory
cells, which serve as important stimuli for angiogenic signaling (Kim et al., 2013; Reuter et
al., 2010; Grote et al., 2011). However, the effects of L-carbocisteine on angiogenesis have
not been reported.
We hypothesized that L-carbocisteine produces anti-angiogenic activity, and tested this
hypothesis in vitro and in vivo, because an understanding of the molecular mechanisms and
targets of established drugs is essential for safe drug use and the development of novel
indications.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
6
Materials and Methods
Antibodies and reagents
L-Carbocisteine was a gift from Kyorin Pharmaceutical Co. (Tokyo, Japan).
L-2-Aminoadipic acid was obtained from TCI (Tokyo, Japan). Human recombinant VEGF165
and epidermal growth factor (EGF) were purchased from PeproTech (Rocky Hill, NJ, USA).
Anti-phospho-Akt (Ser473), anti-Akt, anti-phospho ERK1/2 (Thr202/Tyr204), anti-ERK1/2,
anti-phospho-stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK)
(Thr183/Tyr185), anti-SAPK/JNK, anti-MEK1/2, anti-phospho-PLCγ (Tyr783), anti-PLCγ,
anti-phospho-PKCμ/PKD (Ser744/748), anti-PKCμ/PKD, anti-phospho-VEGFR2 (Tyr1175),
anti-VEGFR2, and horseradish peroxidase (HRP)-conjugated anti-rabbit/mouse IgG
antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-CD31
antibodies were purchased from eBioscience (San Diego, CA, USA). Anti-phospho-p38 MAP
kinase (Thr180/Tyr182) antibodies, anti-p38 MAP kinase antibodies, anti-ERK1 antibodies, and
growth factor-reduced Matrigel basement membrane matrix were obtained from BD
Biosciences (Lexington, KY, USA). Protein G Sepharose was obtained from GE Healthcare
(Pittsburgh, PA, USA). Cellmatrix types I-A and I-C and reconstitution buffer were obtained
from Nitta Gelatin, Inc. (Osaka, Japan). Dulbecco’s modified Eagle’s medium (DMEM) and
RPMI-1640 medium were obtained from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan).
Cell culture
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
7
Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza (Basel,
Switzerland) and maintained in endothelial basement medium-2 (EBM-2) supplemented with
EGM-2 BulletKit™ (Lonza). HeLa human cervical cancer cells were cultured in DMEM
supplemented with 10% fetal bovine serum (FBS; Cell Culture Bioscience/Nichirei
Biosciences, Inc., Tokyo, Japan). Colon-26 murine colon carcinoma cells were obtained from
Riken BioResource Center (Ibaraki, Japan) and maintained in RPMI-1640 medium
supplemented with 10% FBS. Cells were cultured in a humidified atmosphere of 5% CO2 at
37 °C.
Animals
Specific pathogen-free inbred C57BL6/JJms mice (weighing 19–21 g) and BALB/cCr
mice (weighing 20–22 g) for use in this study were obtained from Japan SLC, Inc. (Shizuoka,
Japan) and housed in a laminar airflow room with a 12-h light–dark cycle under specific
pathogen-free conditions. All animals were allowed to acclimatize to their new environment
for 1 week before experimentation. The animal experiments were performed according to the
guidelines of the Kyushu University of Health and Welfare (Nobeoka, Japan), which
complied with the “Law Concerning the Protection and Control of Animals” and “Standards
relating to the care and management, etc. of experimental animals’’ (Office of the Prime
Minister of Japan; http://law.e-gov.go.jp).
In vivo angiogenesis assay
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
8
The in vivo anti-angiogenic activity of L-carbocisteine was assessed with a Matrigel plug
assay as described elsewhere (Suehiro et al., 2010). Matrigel was mixed with vehicle or 30
ng/mL of VEGF and injected subcutaneously in a 500-μL bolus into the flank of a 6-week-old
male C57BL/6JJms mouse. Injected mice were treated twice daily with or without
L-carbocisteine (2.5 mg/kg administered intraperitoneally). On day 14, mice were injected
with 50 μL of 1% Evans blue solution via the orbital vein. After 1 h, mice were perfused with
phosphate-buffered saline (PBS) containing 2 mM EDTA by intravenous injection into the
left ventricle of the heart. Matrigel pellets were harvested and incubated with formamide for
2 days to elute Evans blue dye. Neovascular densities were determined by measuring the
absorbance of pellets at 620 nm.
Cell viability assay
Cell viability was assessed with the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan),
according to the manufacturer’s instructions. Cells (200 μL) were seeded onto 96-well plates
at a density of 2500 cells/well. After 24 h, cells were starved overnight and treated with
vehicle or the indicated agent. After 48 h of incubation, 10 μL of WST-8 solution was added
to each well, and cells were incubated for 40 min at 37 °C. After incubation, absorbance was
measured at 450 nm.
Migration assay
HUVECs were seeded on 35-mm plates and allowed to form confluent monolayers. Cells
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
9
were starved overnight in VEGF and basic fibroblast growth factor (bFGF)-free EGM-2
medium and pretreated with L-carbocisteine for 30 min. Monolayers were subjected to
scratch wounding with a sterile 200-μL pipette tip in the presence or absence of VEGF. Cells
were incubated for 18 h before observation using a phase-contrast microscope. Four
randomly selected fields were photographed and the number of migrated cells was
determined manually.
Assay to measure formation of HUVEC tubular networks
HUVEC tubular networks were formed according to a published method (Uchiyama, 2010).
Two volumes of Cellmatrix Type I-A were mixed with 5 volumes of 0.1% acetic acid, 2
volumes of 5× NaHCO3-free DMEM, and 1 volume of reconstitution buffer, and the resulting
solution was placed on ice. This mixture (800 μL) was added to each well of a 12-well plate,
which was incubated at 37 °C for 30 min to allow formation of a bottom gel layer. HUVECs
were seeded into each gel-containing well at a density of 1.0 × 104 cells/well and incubated
for 6 h to allow adherence to the collagen gel. The cultured medium was removed gently, and
500 μL of the collagen mixture was added atop the bottom layer, followed by solidification at
37 °C for 30 min (top layer). After addition of 1 mL VEGF and bFGF-free EGM-2 medium
containing the vehicle or the indicated agents above the top layer, cells were incubated at
37 °C for 18 h. The vehicle and test agents were allowed diffused into the gel matrix for 1 h,
thereby diluting their concentrations 2-fold. Formation of tubular networks was observed via
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
10
phase-contrast microscopy: 4 randomly selected fields were photographed and total tubule
lengths were measured.
Immunoblotting
Preparation of cell lysates and immunoblotting were conducted as described previously
(Takeuchi et al., 2009). Briefly, cells were lysed with lysis buffer (20 mM Tris-HCl (pH 7.4)
containing 137 mM NaCl, 2 mM EGTA, 5 mM EDTA, 1% NonidetTM P-40, 1% Triton
X-100, 100 μg/mL phenylmethanesulfonyl fluoride, 1 μg/mL pepstatin A, 1 μg/mL
p-toluenesulfonyl-L-arginine methyl ester, 2 μg/mL leupeptin, 1 mM sodium orthovanadate,
50 mM sodium fluoride, and 30 mM sodium diphosphate). Lysates were incubated on ice for
30 min, insoluble materials were removed by centrifugation, and supernatants were subjected
to SDS-PAGE, followed by transfer to Immobilon-P membranes (Millipore, Bedford, MA,
USA) for immunoblotting with antibodies.
Immunoprecipitation
Cells were cultured exactly as described in the methods for immunoblotting and extracted
in lysis buffer. Protein concentrations were measured and approximately 200 μg of cell
extract from each sample was immunoprecipitated with antibodies against VEGFR2 or PLCγ
that had been conjugated to 20 μL of Protein G-Sepharose. Immunoprecipitates were
recovered by adding 2 volumes of Laemmli sample buffer to the immunoprecipitated samples.
Samples were analyzed by western blotting.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
11
Colon-26 tumor-bearing mice
A Colon-26 tumor-bearing mouse model assay was set up as described previously, with
some modifications (Acharyya et al., 2004). Six-week-old male BALB/cCr mice were given
vehicle or L-carbocisteine (15, 75, or 150 mg/kg) via the oral route twice daily from 2 days
before tumor injection. On the day of inoculation, cultured Colon-26 cells were harvested and
washed with PBS. Next, 1 × 106 cells in 100 μL of serum-free RPMI 1640 culture medium
was injected subcutaneously and dorsally into mice. From 6 days after injection, tumors were
measured with calipers once every other day, and tumor growth (in mm3) was calculated
using the following formula:
V = (narrow side)2 × (long side)/2.
Tumor tissues were fixed in 4% neutral buffered paraformaldehyde for 48 h, embedded in
Tissue-Tek® OCT™ compound (Sakura Finetek, Torrance, CA, USA), and cut into 9-μm
sections with a cryotome (CM1900; Leica, Nußloch, Germany). Sections were dried at room
temperature for 1 h, washed with PBS, and treated with an anti-mouse CD31 antibody. After
washing in PBS, sections were stained with fluorescein isothiocyanate (FITC)-conjugated
IgG (Invitrogen, Carlsbad, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI). After
washing the sections in PBS, they were mounted and observed using a fluorescence
microscope. Areas of positive staining were measured using ImageJ (National Institutes of
Health; available at http://imagej.nih.gov/ij/).
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
12
Statistical analysis
SPSS version 20 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. Data are
presented as mean ± S.E.M. Statistical differences in the dose-response study were evaluated
by applying Dunnett’s multiple comparison test. Student’s t-test was used for comparisons of
2 groups. A p-value <0.05 was regarded as significant.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
13
Results
L-Carbocisteine inhibits VEGF-induced proliferation, migration, and formation of tubular
structures of endothelial cells
To assess the anti-angiogenic properties of L-carbocisteine in vitro, we examined the
inhibitory effects of L-carbocisteine on HUVEC proliferation. L-Carbocisteine attenuated
VEGF-induced proliferation in a concentration-dependent manner and exerted a significant
inhibitory effect at concentrations greater than 100 μM (Fig. 1A). The effects of
L-carbocisteine on chemotactic motility were examined in a wound-healing migration assay.
Treatment with L-carbocisteine (100 μM) significantly inhibited VEGF-induced HUVEC
migration (Fig. 1B). We examined the potential effects of L-carbocisteine on the formation of
tubular structures using a collagen gel matrix assay, and found that HUVECs formed an
extended network of tubular structures in response to VEGF. Treatment with L-carbocisteine
significantly abrogated VEGF-stimulated formation of tubular networks in endothelial cells
(Fig. 1C).
L-Carbocisteine inhibits VEGF-induced angiogenesis in vivo
To ascertain the effects of L-carbocisteine on angiogenesis in vivo, we conducted a Matrigel
plug assay. VEGF-loaded Matrigel (30 ng/mL) was stained positively with Evans blue,
suggesting that new blood vessels formed within the Matrigel via VEGF-induced
angiogenesis (Fig. 2A). In contrast, treatment with 2.5 mg/kg L-carbocisteine almost
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
14
completely abolished angiogenesis, as evidenced by the remarkably reduced level of Evans
blue staining in the L-carbocisteine-treated group (Fig. 2B), suggesting that L-carbocisteine
effectively inhibited angiogenesis in vivo.
L-Carbocisteine inhibits VEGF-induced phosphorylation of ERK1/2 in HUVECs
To evaluate the molecular mechanisms associated with L-carbocisteine-induced inhibition
of VEGF-dependent angiogenesis, we measured phosphorylation of key proteins downstream
of VEGFR2 activation: Akt, ERK1/2, JNK, and p38 MAP kinase by western blotting
(Dellinger and Brekken, 2011; Song et al., 2012; Wu et al., 2006). L-Carbocisteine (100 μM)
potently suppressed VEGF-induced ERK1/2 activation in HUVECs, but had no effect on
activation of Akt, JNK, or p38 MAP kinase (Figs. 3A-D).
To determine whether L-carbocisteine inhibits ERK1/2 phosphorylation in non-endothelial
cells, we examined the effect of L-carbocisteine on ERK1/2 activation induced by 100 ng/mL
EGF in HeLa cells. However, L-carbocisteine did not affect ERK1/2 activation in epidermal
cells (Fig. 4).
L-Carbocisteine inhibits activation of VEGFR2/PLCγ/PKC/MEK signaling in endothelial
cells
To clarify the mechanisms underlying L-carbocisteine-mediated inhibition of the activation
of ERK, we examined the effects of L-carbocisteine on phosphorylation of VEGFR2, PLCγ,
PKC, and MEK. VEGFR2 phosphorylation in VEGF-stimulated HUVECs was not
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
15
suppressed by L-carbocisteine. In contrast, pretreatment with L-carbocisteine significantly
suppressed the phosphorylation of PLCγ and PKCμ (Figs. 5A-C). In addition, L-carbocisteine
inhibited MEK1/2 phosphorylation after VEGF treatment (Fig. 5D).
L-Carbocisteine attenuates the association of PLCγ with VEGFR2
To determine whether L-carbocisteine suppresses the formation of the PLCγ/VEGFR2
complex, cell lysates were immunoprecipitated with antibodies against VEGFR2 or PLCγ
and immunoblotted with reciprocal antibodies. VEGF stimulated complex formation in
HUVECs, whereas pretreatment with L-carbocisteine prevented complex formation (Fig. 6).
L-Carbocisteine suppressed signals for angiogenesis by inhibiting VEGF-induced formation
of the PLCγ/VEGFR2 complex (Fig. 7).
L-Carbocisteine suppresses the tumor growth and angiogenesis
To determine the effects of L-carbocisteine on tumor growth and angiogenesis in vivo, we
evaluated the effect of L-carbocisteine in Colon-26 tumor-bearing mice. For this purpose, we
injected Colon-26 tumor cells into male BALB/c mice, following which they were orally
administered various concentration of L-carbocisteine (experimental group) or vehicle
(control group) daily for 26 days (Fig. 8A). On day 22, mice treated orally twice daily with
150 mg/kg L-carbocisteine presented with considerably smaller tumors than those observed in
control mice (Fig. 8B). At 15 mg/kg and 75 mg/kg doses, L-carbocisteine was associated with
slight retardation of tumor growth in comparison with the control treatment. From 10 days
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
16
after inoculation with tumor cells, tumor volumes were significantly smaller in mice treated
with 150 mg/kg L-carbocisteine in comparison with the control group (Fig. 8C). No apparent
toxic effects were observed in any of the treatment groups. Capillary density in the
peritumoral region was determined by staining sections with anti-CD31 antibodies. Treatment
with 150 mg/kg L-carbocisteine significantly reduced the number of capillary microvessels
(Fig. 8D). From 12 days after the injection of tumor cells, tumor volume was significantly
smaller in mice treated intraperitoneally twice daily with 10 mg/kg L-carbocisteine in
comparison with the control group (Figs. S1A and S1B).
To determine whether L-carbocisteine directly induces apoptosis in tumor cells, we tested the
effect of L-carbocisteine on Colon-26 cell viability. We found that treatment with a high
concentration (approximately 500 µM) of L-carbocisteine had no effect on Colon-26 cell
proliferation (Fig. S2A). Subsequently, we used western blot analysis to examine the effect of
L-carbocisteine on growth factor-induced phosphorylation of ERK1/2 in tumor cells, and
found that L-carbocisteine did not suppress EGF-induced activation of ERK1/2 in Colon-26
cells (Fig. S2B).
L-2-Aminoadipic acid inhibits VEGF-induced proliferation and activation of ERK1/2 in
endothelial cells
L-2-Aminoadipic acid is a substitution product of L-carbocisteine (sulfur to carbon). To
confirm whether sulfur is important for the anti-angiogenic effect of L-carbocisteine, we
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
17
evaluated the effects of L-2-aminoadipic acid in VEGF-stimulated endothelial cells.
L-2-aminoadipic acid suppressed VEGF-induced proliferation in a concentration-dependent
manner and suppressed VEGF-induced ERK1/2 activation in HUVECs (Figs. 9A and 7B).
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
18
Discussion
Angiogenesis plays a crucial role in the tumor growth and metastasis (Fischer et al., 2008;
Zetter, 2008). Therefore, inhibition of tumor angiogenesis has become an important strategy
for cancer treatment. Several inhibitors of tumor angiogenesis have been shown to prevent
the growth and metastasis of solid tumors (Argyriou, 2009), and such findings have spurred
efforts to discover novel angiogenic inhibitors. Human umbilical endothelial cells (HUVECs)
are derived from the endothelium of large veins in the umbilical cord and are used as a model
system for angiogenesis studies (Wang et al., 2015; Mu et al., 2011). L-Carbocisteine was
synthesized in the 1930s and was first used as a mucoregulatory agent (Rhinathiol®,
Mucodyne®) in the treatment of respiratory diseases in the 1960s (Hooper and Calvert, 2008).
In recent years, novel biological activities of L-carbocisteine have been reported.
L-Carbocisteine inhibits inflammation associated with influenza virus infection and COPD
(Yamaya et al., 2010; Asada et al., 2012; Yasuda et al., 2006; Zheng et al., 2008), suppresses
oxaliplatin-induced hepatocyte toxicity by inhibiting oxaliplatin-induced decreases in the
Bcl2/Bim ratio, and inhibits oxaliplatin-induced apoptosis in vitro (Zhai et al., 2012).
Moreover, L-carbocisteine possesses free radical-scavenging and anti-inflammatory
properties in vitro (Zheng et al., 2008; Nogawa et al., 2009).
Based on recent studies showing that L-carbocisteine inhibits multiple steps of
VEGF-induced angiogenesis, we hypothesized that it is a promising novel anti-cancer agent.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
19
This is the first report to demonstrate comprehensively that L-carbocisteine inhibits
angiogenesis and tumor growth. Unlike conventional anti-cancer agents, the uses of which
are complicated by various side effects and/or severe cytotoxicity, L-carbocisteine produces
exceptional anti-angiogenic activity without cytotoxicity or side effects.
Angiogenesis is a complex, multistep process that involves the proliferation, migration,
and tubular-network formation of endothelial cells (Patan, 2004), and inhibition of any step
of this process has been shown to prevent formation of new blood vessels (Tournaire et al.,
2004). In this study, we showed that L-carbocisteine significantly inhibits endothelial cell
proliferation in a concentration-dependent manner (Fig. 1A). Moreover, L-carbocisteine
inhibits VEGF-induced angiogenic responses such as cell migration and formation of
capillary-like structures (Figs. 1B and C). Furthermore, L-carbocisteine inhibited
angiogenesis in a Matrigel plug assay in mice (Fig. 2), showing that L-carbocisteine inhibits
angiogenesis in vitro and in vivo.
VEGFR2-mediated activation of Akt, ERK, JNK, and p38 MAP kinase contributes to
VEGF-induced survival, proliferation, migration, and tubular-network formation of
endothelial cells (Zachary and Gliki, 2001; Dellinger and Brekken, 2011; Song et al., 2012;
Wu et al., 2006). Our data showed that L-carbocisteine significantly abrogated ERK
activation specifically in endothelial cells; no effect was observed in epidermal cells (Figs.
3B and 4). Reports have noted that, unlike other representative growth factor receptor
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
20
tyrosine kinases, VEGFR2 forms a complex with and subsequently phosphorylates PLCγ,
which is critical for ERK activation (Takahashi et al., 2001; Takahashi and Shibuya 1997; Wu,
2000). In contrast, Ras is weakly activated by VEGF (Takahashi et al., 1999). VEGF
stimulates activation of PKCµ (PKD) via the VEGFR2/PLCγ/PKC pathway. PKCµ in
endothelial cells is rapidly phosphorylated at Ser744/Ser748 in response to VEGF, and PKCµ
is involved in VEGF-induced ERK signaling and endothelial cell proliferation (Wong et al.,
2005). In the present study, L-carbocisteine had no effect on VEGFR2 phosphorylation.
However, L-carbocisteine significantly attenuated VEGF-induced phosphorylation of ERK
and PLCγ, as well as upstream formation of VEGFR2/PLCγ complexes (Figs. 5 and 6).
Taken together, our data suggest that L-carbocisteine affects formation of VEGFR2 and PLCγ
complexes without inhibiting VEGFR2 phosphorylation, which subsequently affects
signaling cascades in a manner that may be responsible for the anti-angiogenic effects of
L-carbocisteine. VEGF-induced VEGFR2/PLCγ complex formation and activation of PLCγ
evoke Ca2+ mobilization, phosphatidylinositol 4,5-biphosphate (PIP2) breakdown, and
inositol 1,4,5-triphosphate (IP3) production, which are signaling events upstream of PKC
(Ayada et al., 2009). Therefore, the results of our study suggest that L-carbocisteine
suppressed Ca2+ mobilization, PIP2 breakdown, and IP3 production.
Inhibition of tumor angiogenesis represents a novel therapeutic modality for controlling
tumor metastasis (Kruger et al., 2001; Yi et al., 2008). In this report, we elucidated some
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
21
mechanisms underlying the inhibitory effect of L-carbocisteine on VEGF-induced
angiogenesis by using matrigel containing VEGF. However, we also studied the effects of
L-carbocisteine in a tumor-bearing mouse model, because malignant cells release a wide
range of growth factors in addition to VEGF. In our in vivo Colon-26 tumor-bearing mouse
model, we demonstrated the effectiveness of oral administration of 150 mg/kg
L-carbocisteine as a tumor suppressor (Figs. 8B and C). Related immunohistochemical
analyses further revealed that expression of the endothelial marker CD31 was reduced
markedly in tumor sections from L-carbocisteine-treated mice (Fig. 8D). Furthermore, we
determined that 500 μM L-carbocisteine did not directly induce apoptosis or inhibit
proliferation of Colon-26 cells (Fig. S2A). These results suggest that L-carbocisteine inhibits
tumor growth indirectly by inhibiting tumor angiogenesis.
It has been reported that L-carbocisteine suppresses tumor necrosis factor
(TNF)-alpha-induced activation of phosphatidyl inositol-specific phospholipase C (PI-PLC)
in NCH-H292 epithelial cells (Ishibashi et al., 2006; Ishibashi et al., 2012). L-Carbocisteine
has also been shown to attenuate N-formyl-Met-Leu-Phe (FMLP)-stimulated neutrophil
activation by inhibiting PI-PLC-mediated signal transduction (Ishii et al., 2002). In this study,
we demonstrated for the first time that L-carbocisteine directly inhibits formation of VEGFR2
and PLCγ complexes in endothelial cells.
Similar to L-carbocisteine, N-acetylcysteine (NAC) is a cysteine-derivative mucolytic drug
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
22
that acts by breaking disulfide bridges between macromolecules (Mallet et al., 2011). At the
cellular level, NAC inhibits endothelial cell invasion and angiogenesis, probably by
inhibiting metalloproteinase activities (Albini et al., 1995). NAC has also been shown to exert
direct cytoprotective and anti-genotoxic effects on endothelial cells (Aluigi et al., 2000).
Given the possible association between NAC treatment and reduced tumor-dependent
angiogenesis, a reported and potentially important aspect of the effectiveness of NAC is its
ability to limit VEGF expression (Albini et al., 2001; Agarwal et al., 2004), and this effect
may be related to its suppression of ROS and hypoxia-induced transcription via hypoxia
inducible factor-1 (Agarwal et al., 2004; Albini et al., 1995; Sceneay et al., 2013). Therefore,
the anti-angiogenic effects of NAC are due to its anti-oxidant activity and are distinct from
the anti-angiogenic effects of L-carbocisteine reported in the present study.
VEGF stimulates ROS production (Ushio-Fukai, 2007) and ROS play a critical role in
stimulation of angiogenic signaling, including ERK and JNK signaling (Lee et al., 2014).
Because sulfur compounds have strong anti-ROS activity, we considered whether the
inhibitory effect of L-carbocisteine on VEGF-induced ERK activation was based on anti-ROS
activity. We showed that L-2-aminoadipic acid inhibited proliferation and activation of
ERK1/2 in VEGF-stimulated endothelial cells (Fig. 9), indicating that the anti-angiogenic
effect of L-carbocisteine is not conferred by its constituent sulfur. We believe that steric
effects associated with L-carbocisteine and L-2-aminoadipic acid are important to their
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
23
inhibitory effects. Additionally, we expect that addition of another carboxymethyl or amino
group to L-carbocisteine could enhance its interference with VEGFR2 and suppression of
ERK activation in endothelial cells. The effect of L-carbocisteine does not seem to be
stronger than other available anti-angiogenic agents, such as bevacizumab, sunitinib, and
sorafenib, which attenuate VEGFR2- and VEGFR2-mediated phosphorylation and activation
of ERK, Akt, JNK, and p38 MAP kinase (Reddy et al., 2012; Okines et al., 2011). In the
present study, we found that L-carbocisteine suppressed VEGF-induced ERK1/2 activation
but had no effect on activation of Akt, JNK, or p38 MAP kinase (Figs. 3A-D). Furthermore,
the usual oral dose of L-carbocisteine prescribed to adults is 500 mg of L-carbocisteine (3
times daily). In our study, L-carbocisteine inhibited angiogenesis, but did so at a dose about
10 times greater than the normally prescribed dose. One of the reasons why a higher
concentration of L-carbocisteine was required is its short biological half-life (t1/2) (about 2 h;
from a medical package insert of Mucodyne®). However, anti-angiogenic effects might be
produced with lower doses of L-carbocisteine by reducing the dosing interval. Currently used
anti-angiogenic drugs such as the anti-VEGF antibody bevacizumab can induce transient
functional normalization of tumor vasculature that can potentiate the activity of
co-administered chemoradiotherapeutics (Jie et al., 2008). We believe that the combination of
L-carbocisteine with conventional chemotherapeutic agents might increase their efficacy.
To our knowledge, this is the first report to demonstrate that the mucolytic drug
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
24
L-carbocisteine inhibits angiogenesis in vitro and in vivo. Moreover, L-carbocisteine was
found to attenuate endothelial cell proliferation, as well as to inhibit formation of
VEGFR2/PLCγ complexes and ERK activation in endothelial cells. These findings suggest
that L-carbocisteine inhibits tumor angiogenesis and growth by inhibiting cellular
PLCγ/PKC/ERK activity in vivo; however, this specific effect of L-carbocisteine does not
occur in epidermal cells, which suggests that L-carbocisteine could serve as a useful selective
anti-angiogenic therapy with few side effects. Our discovery of this novel action of
L-carbocisteine supports the notion that it is a promising anti-angiogenic agent and a valuable
lead compound in the development of anti-cancer therapies.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
25
Acknowledgments
We thank Dr. Akinori Sugiyama (School of Pharmaceutical Science, Iwate Medical
University), Shou Hasegawa, Tomohiro Arima, Eri Toyota, Ryota Morisawa, Ryudai Mizobe,
Yuya Ito, Makoto Hamada, and Kayo Nakao (Kyushu University of Health and Welfare) for
their assistance.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
26
Authorship Contributions
Participated in research design: Shinya, Takahashi, and Sato
Conducted experiments: Shinya, Yokota, Nakayama, Oki, and Mutoh
Performed data analysis: Shinya, Yokota, Nakayama, and Mutoh
Wrote or contributed to the writing of the manuscript: Shinya, Takahashi, and Sato
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
27
References
Acharyya S, Ladner KJ, Nelsen LL, Damrauer J, Reiser PJ, Swoap S, and Guttridge DC
(2004) Cancer cachexia is regulated by selective targeting of skeletal muscle gene products.
J Clin Invest 114: 370–378.
Agarwal A, Muñoz-Nájar U, Klueh U, Shih SC, and Claffey KP (2004) N-acetyl-cysteine
promotes angiostatin production and vascular collapse in an orthotopic model of breast
cancer. Am J Pathol 164: 1683–1696.
Albini A, D'Agostini F, Giunciuglio D, Paglieri I, Balansky R, and De Flora S (1995)
Inhibition of invasion, gelatinase activity, tumor take and metastasis of malignant cells by
N-acetylcysteine. Int J Cancer 61: 121–129.
Albini A, Morini M, D'Agostini F, Ferrari N, Campelli F, Arena G, Noonan DM, Pesce C, and
De Flora S (2001) Inhibition of angiogenesis-driven Kaposi's sarcoma tumor growth in
nude mice by oral N-acetylcysteine. Cancer Res 61: 8171–8178.
Aluigi MG, De Flora S, D'Agostini F, Albini A, and Fassina G (2000) Antiapoptotic and
antigenotoxic effects of N-acetylcysteine in human cells of endothelial origin. Anticancer
Res 20: 3183–3187.
Argyriou AA, Giannopoulou E, and Kalofonos HP (2009) Angiogenesis and anti-angiogenic
molecularly targeted therapies in malignant gliomas. Oncology 77: 1–11.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
28
Asada M, Yoshida M, Hatachi Y, Sasaki T, Yasuda H, Deng X, Nishimura H, Kubo H,
Nagatomi R, and Yamaya M (2012) L-carbocisteine inhibits respiratory syncytial virus
infection in human tracheal epithelial cells. Respir Physiol Neurobiol 180: 112–118.
Ayada T, Taniguchi K, Okamoto F, Kato R, Komune S, Takaesu G, and Yoshimura A (2009)
Sprouty4 negatively regulates protein kinase C activation by inhibiting
phosphatidylinositol 4,5-biphosphate hydrolysis. Oncogene 28: 1076–88.
Bhat TA and Singh RP (2008) Tumor angiogenesis—a potential target in cancer
chemoprevention. Food Chem Toxicol 46: 1334–1345.
Claesson-Welsh L (2012) Blood vessels as targets in tumor therapy. Ups J Med Sci 117:
178–186.
Dellinger MT and Brekken RA (2011) Phosphorylation of Akt and ERK1/2 is required for
VEGF-A/VEGFR2-induced proliferation and migration of lymphatic endothelium. PLoS
One 6: e28947.
Fischer C, Mazzone M, Jonckx B, and Carmeliet P (2008) FLT1 and its ligands VEGFB and
PlGF: drug targets for anti-angiogenic therapy? Nat Rev Cancer 8: 942–956.
Grote K, Schütt H, and Schieffer B (2011) Toll-like receptors in angiogenesis. Scientific
World Journal 11:981–991.
Hooper C and Calvert J (2008) The role for S-carboxymethylcysteine (carbocisteine) in the
management of chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
29
3: 659–669.
Ishibashi Y, Imai S, Inouye Y, Okano T, and Taniguchi A (2006) Effects of carbocisteine on
sialyl-Lewis x expression in an airway carcinoma cell line stimulated with tumor necrosis
factor-alpha. Eur J Pharmacol 530: 223–238.
Ishibashi Y, Inouye Y, and Taniguchi A (2012) Expression and role of sugar chains on airway
mucus during the exacerbation of airway inflammation. Yakugaku Zasshi 132: 699–704.
Ishii Y, Kimura T, Morishima Y, Mochizuki M, Nomura A, Sakamoto T, Uchida Y, and
Sekizawa K (2002) S-carboxymethylcysteine inhibits neutrophil activation mediated by
N-formyl-methionyl-leucyl-phenylalanine. Eur J Pharmacol 449: 183–189.
Kim YW, West XZ, and Byzova TV (2013) Inflammation and oxidative stress in angiogenesis
and vascular disease. J Mol Med (Berl) 91: 323–328.
Kruger EA, Duray PH, Price DK, Pluda JM, and Figg WD (2001) Approaches to preclinical
screening of antiangiogenic agents. Semin Oncol 28: 570–576.
Ma J and Waxman DJ (2008) Combination of anti-angiogenesis with chemotherapy for more
effective cancer treatment. Mol Cancer Ther 7: 3670–3684.
Mallet P, Mourdi N, Dubus JC, Bavoux F, Boyer-Gervoise MJ, Jean-Pastor MJ, and
Chalumeau M (2011) Respiratory paradoxical adverse drug reactions associated with
acetylcysteine and carbocysteine systemic use in pediatric patients: a national survey. PLoS
One 6: e22792.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
30
Maraldi T, Prata C, Caliceti C, Vieceli Dalla Sega F, Zambonin L, Fiorentini D, and Hakim G
(2010) VEGF-induced ROS generation from NAD(P)H oxidases protects human leukemic
cells from apoptosis. Int J Oncol 36:1581–1589.
McMahon G (2000) VEGF receptor signaling in tumor angiogenesis. Oncologist 5 Suppl 1:
3–10.
Nagy JA, Dvorak AM, and Dvorak HF (2007) VEGF-A and the induction of pathological
angiogenesis. Annu Rev Pathol 2: 251–275.
Nogawa H, Ishibashi Y, Ogawa A, Masuda K, Tsubuki T, Kameda T, and Matsuzawa S
(2009) Carbocisteine can scavenge reactive oxygen species in vitro. Respirology 14:
53–59.
Noonan DM, De Lerma Barbaro A, Vannini N, Mortara L, and Albini A (2008) Inflammation,
inflammatory cells and angiogenesis: decisions and indecisions. Cancer Metastasis Rev 27:
31–40.
Okines AF, Reynolds AR, Cunningham D (2011) Targeting angiogenesis in esophagogastric
adenocarcinoma. Oncologist 16: 844–858.
Patan S (2004) Vasculogenesis and angiogenesis. Cancer Treat Res 117: 3–32.
Qian HP, Zhi RC, and Zhong NS (2008) Effect of carbocisteine on acute exacerbation of
chronic obstructive pulmonary disease (PEACE Study): a randomised placebo-controlled
study. Lancet 371: 2013–2018.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
31
Reddy S, Raffin M, and Kaklamani V (2012) Targeting angiogenesis in metastatic breast
cancer. Oncologist 17: 1014–1026.
Reuter S, Gupta SC, Chaturvedi MM, and Aggarwal BB (2010) Oxidative stress,
inflammation, and cancer: how are they linked? Free Radic Biol Med 49: 1603–1616.
Sceneay J, Liu MC, Chen A, Wong CS, Bowtell DD, and Möller A (2013) The antioxidant
N-acetylcysteine prevents HIF-1 stabilization under hypoxia in vitro but does not affect
tumorigenesis in multiple breast cancer models in vivo. PLoS One 8: e66388.
Shibuya M (2014) VEGF-VEGFR signals in health and disease. Biomol Ther (Seoul) 22:1–9.
Song Y, Dai F, Zhai D, Dong Y, Zhang J, Lu B, Luo J, Liu M, and Yi Z (2012) Usnic acid
inhibits breast tumor angiogenesis and growth by suppressing VEGFR2-mediated AKT
and ERK1/2 signaling pathways. Angiogenesis 15: 421–432.
Takahashi S (2011) Vascular endothelial growth factor (VEGF), VEGF receptors and their
inhibitors for antiangiogenic tumor therapy. Biol Pharm Bull 34: 1785–1788.
Takahashi T and Shibuya M (1997) The 230 kDa mature form of KDR/Flk-1 (VEGF
receptor-2) activates the PLC-gamma pathway and partially induces mitotic signals in
NIH3T3 fibroblasts. Oncogene 14: 2079–2089.
Takahashi T, Ueno H, and Shibuya M (1999) VEGF activates protein kinase C-dependent, but
Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary
endothelial cells. Oncogene 18: 2221–2230.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
32
Takahashi T, Yamaguchi S, Chida K, and Shibuya M (2001) A single autophosphorylation site
on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC-gamma and DNA
synthesis in vascular endothelial cells. EMBO J 20: 2768–2778.
Takeuchi K, Shin-ya T, Nishio K, and Ito F (2009) Mitogen-activated protein kinase
phosphatase-1 modulated JNK activation is critical for apoptosis induced by inhibitor of
epidermal growth factor receptor-tyrosine kinase. FEBS J 276: 1255–1265.
Thairu N, Kiriakidis S, Dawson P, and Paleolog E (2011) Angiogenesis as a therapeutic target
in arthritis in 2011: learning the lessons of the colorectal cancer experience. Angiogenesis
14: 223–234.
Tournaire R, Simon MP, le Noble F, Eichmann A, England P, and Pouysségur J (2004) A short
synthetic peptide inhibits signal transduction, migration and angiogenesis mediated by Tie2
receptor. EMBO Rep 5: 262–267.
Uchiyama T, Toda K, and Takahashi S (2010) Resveratrol inhibits angiogenic response of
cultured endothelial F-2 cells to vascular endothelial growth factor, but not to basic
fibroblast growth factor. Biol Pharm Bull 33: 1095–1100.
Ushio-Fukai M (2007) VEGF signaling through NADPH oxidase-derived ROS. Antioxidants
& Redox Signaling 9: 731–739.
Wang B, Yu W, Guo J, Jiang X, Lu W, Liu M, and Pang X (2015) The antiparasitic drug,
potassium antimony tartrate, inhibits tumor angiogenesis and tumor growth in
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
33
nonsmall-cell lung cancer. J Pharmacol Exp Ther 352: 129–138.
Wong C and Jin ZG (2005) Protein kinase C-dependent protein kinase D activation modulates
ERK signal pathway and endothelial cell proliferation by vascular endothelial growth
factor. J Biol Chem 280: 33262–33269.
Wu G, Luo J, Rana JS, Laham R, Sellke FW, and Li J (2006) Involvement of COX-2 in
VEGF-induced angiogenesis via P38 and JNK pathways in vascular endothelial cells.
Cardiovasc Res 69: 512–519.
Wu LW, Mayo LD, Dunbar JD, Kessler KM, Baerwald MR, Jaffe EA, Wang D, Warren RS,
and Donner DB. (2000) Utilization of distinct signaling pathways by receptors for vascular
endothelial cell growth factor and other mitogens in the induction of endothelial cell
proliferation. J Biol Chem 275: 5096–5103.
Wu Y, He L, Zhang L, Chen J, Yi Z, Zhang J, Liu M, and Pang X (2011) Anacardic acid
(6-pentadecylsalicylic acid) inhibits tumor angiogenesis by targeting Src/FAK/Rho
GTPases signaling pathway. J Pharmacol Exp Ther. 339: 403–411.
Yamaya M, Nishimura H, Shinya K, Hatachi Y, Sasaki T, Yasuda H, Yoshida M, Asada M,
Fujino N, Suzuki T, Deng X, Kubo H, and Nagatomi R (2010) Inhibitory effects of
carbocisteine on type A seasonal influenza virus infection in human airway epithelial cells.
Am J Physiol Lung Cell Mol Physiol 299: L160–168.
Yasuda H, Yamaya M, Sasaki T, Inoue D, Nakayama K, Yamada M, Asada M, Yoshida M,
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
34
Suzuki T, Nishimura H, and Sasaki H (2006) Carbocisteine inhibits rhinovirus infection in
human tracheal epithelial cells. Eur Respir J 28: 51–58.
Yi T, Cho SG, Yi Z, Pang X, Rodriguez M, Wang Y, Sethi G, Aggarwal BB, and Liu M
(2008) Thymoquinone inhibits tumor angiogenesis and tumor growth through suppressing
AKT and extracellular signal-regulated kinase signaling pathways. Mol Cancer Ther 7:
1789–1796.
Zachary I and Gliki G (2001) Signaling transduction mechanisms mediating biological
actions of the vascular endothelial growth factor family. Cardiovasc Res 49: 568–581.
Zetter BR (2008) The scientific contributions of M. Judah Folkman to cancer research. Nat
Rev Cancer 8:647–654.
Zhai Q, Bian XL, Lu SR, Zhu B, and Yu B (2012) Carbocisteine reduces the cytotoxicity of
oxaliplatin. Z Naturforsch C 67: 215–221.
Zheng JP, Kang J, Huang SG, Chen P, Yao WZ, Yang L, Bai CX, Wang CZ, Wang C, Chen
BY, Shi Y, Liu CT, Chen P, Li Q, Wang ZS, Huang YJ, Luo ZY, Chen FP, Yuan JZ, Yuan
BT, Suehiro J, Hamakubo T, Kodama T, Aird WC, and Minami T (2010) Vascular
endothelial growth factor activation of endothelial cells is mediated by early growth
response-3. Blood 115: 2520–2532.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
35
Footnotes
Financial support
This work was supported by Kyorin Pharmaceutical Co.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
36
Legends for Figures
Figure 1. L-Carbocisteine inhibits VEGF-induced changes in viability, migration, and
capillary-structure formation in endothelial cells. A, HUVECs were pretreated with various
concentrations of L-carbocisteine and incubated with VEGF. After 2 d of incubation, cell
viability was quantified using a Cell Counting Kit-8. B, HUVECs were pretreated with 100
µM L-carbocisteine for 30 minutes and then incubated with 30 ng/mL VEGF. Migrated cells
were quantified by manual counting. C, HUVECs were pretreated with 100 µM
L-carbocisteine and incubated with 30 ng/mL VEGF. After 18 h of incubation, total tubule
length was assayed using a phase-contrast microscope (100x magnification). Values are mean
± S.E.M. *P < 0.05; **P < 0.01 vs. the VEGF-treated group. Similar results were obtained
from 3 independent experiments.
Figure 2. L-Carbocisteine inhibits VEGF-induced angiogenesis in an in vivo Matrigel model.
C57BL6/J mice were injected with 0.5 mL of Matrigel mixed with vehicle or VEGF.
Matrigel-bearing mice were treated with or without L-carbocisteine via the intraperitoneal
route twice per day. After 14 d, Evans blue dye was administered and Matrigel pellets were
harvested. A, Representative Matrigel plugs were photographed. B, Neovascular density was
determined. Data points represent mean ± S.E.M. (n = 6). *P < 0.05 vs. VEGF alone. Similar
results were obtained from 3 independent experiments.
Figure 3. L-Carbocisteine attenuated VEGF-induced ERK1/2 phosphorylation in endothelial
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
37
cells. A-D, HUVECs were pretreated with L-carbocisteine for 30 minutes and stimulated with
30 ng/mL VEGF for the indicated periods, and cellular lysates were analyzed by SDS-PAGE
and immunoblotting with phosphorylation site-specific antibodies, after which the
membranes were reprobed with antibodies against unmodified proteins. Protein levels of
p-Akt (A), p-ERK (B), p-JNK (C), and p-p38 MAPK (D) were determined.
Figure 4. L-Carbocisteine had no effect on EGF-induced ERK activation in epithelial cells.
HeLa cells were pretreated with L-carbocisteine for 30 minutes and incubated with EGF for
the indicated periods. The cells were harvested and equal aliquots of protein were analyzed
for anti-phospho-ERK1/2 by immunoblotting. Results are from an experiment representative
of 3 independent experiments. Data are presented as mean ± S.E.M. *P < 0.05.
Figure 5. L-Carbocisteine inhibits VEGF-induced PLCγ/PKC/ERK signaling in HUVECs.
A-D, HUVECs were pretreated with L-carbocisteine and stimulated with VEGF for the
indicated periods. Lysates were subjected to SDS-PAGE and the membranes were hybridized
with phospho-specific antibodies, after which the membranes were reprobed. Protein levels
of p-VEGFR2 (A), p-PLCγ (B), p-PKCµ (C), and p-MEK1/2 (D) were determined.
Quantitative results were obtained by densitometry. Data are presented as mean ± S.E.M.
from 3 independent experiments. *P < 0.05.
Figure 6. L-Carbocisteine attenuated VEGF-induced formation of PLCγ/VEGFR2 complexes.
HUVECs were pretreated with L-carbocisteine and stimulated with VEGF for the indicated
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
38
periods. The cells were harvested and equal aliquots of protein extracts were
immunoprecipitated with antibodies against VEGFR2 or PLCγ. Immunoprecipitates were
subjected to SDS-PAGE and blotted with antibodies against PLCγ or VEGFR2 as indicated.
Total cell extracts were prepared and subjected to SDS-PAGE for detection of VEGFR2 and
PLCγ. The blot was reprobed with beta-actin antibodies as a loading control. Data are
presented as mean ± S.E.M. from 3 independent experiments. *, P < 0.05.
Figure 7. Schematic representation of the mechanism by which L-carbocisteine inhibits
VEGF-stimulated angiogenesis. VEGF stimulates formation of complexes between VEGFR2
and PLCγ, and this phenomenon induces angiogenesis. Conversely, L-carbocisteine
suppresses VEGFR2/PLCγ complex formation and downstream signaling.
Figure 8. L-Carbocisteine inhibits tumor growth and angiogenesis in Colon 26-bearing mice.
A, Experimental schedule of in vivo tumor growth (schematic). B, Typical example of
tumor-bearing mice from the groups treated with vehicle or 150 mg/kg L-carbocisteine on day
6 and day 22. C, Tumor growth was measured with calipers once every other day and
calculated in mm3. All data are presented as mean tumor volume ± S.E.M. (n = 8 animals per
group). D, Representative photomicrographs of CD31 capillaries in tumor sections stained
with antibodies against CD31 (green fluorescence), an endothelial marker. Nuclei were
counterstained with DAPI (blue). The area of CD31-stained capillaries was measured using
Image J software. Data are the mean ± S.E.M. of 4 experiments. *P < 0.05 vs. the vehicle
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
39
group.
Figure 9. L-2-aminoadipic acid inhibited VEGF-induced proliferation and activation of
ERK1/2 in endothelial cells. A, HUVECs were pretreated with various concentrations of
L-2-aminoadipic acid and incubated with VEGF. After 2 d of incubation, cell viability was
quantified using a Cell Counting Kit-8 (n = 6). Similar results were obtained from 3
independent experiments. B, HUVECs were pretreated with 100 µM L-2-aminoadipic acid
for 15 minutes and treated with VEGF for the indicated periods. Lysates were prepared from
the treated cells, and phospho-ERK1/2 protein was measured by immunoblotting.
Immunoblots are from an experiment representative of 3 similar experiments. Quantitative
results were obtained by densitometry. Data are presented as mean ± S.E.M. *P < 0.05.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
Figures
Fig. 1
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
Fig. 2
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
Fig. 3
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
Fig. 4
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
Fig. 5
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
Fig. 6
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
Fig. 7
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
Fig. 8
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from
JPET #224816
Fig. 9
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 30, 2015 as DOI: 10.1124/jpet.115.224816
at ASPE
T Journals on M
ay 15, 2018jpet.aspetjournals.org
Dow
nloaded from