Orally administered mucolytic drug L-carbocisteine ...jpet.aspetjournals.org/content/early/2015/06/30/jpet.115.224816... · Orally administered mucolytic drug L-carbocisteine inhibits

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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

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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


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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.


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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,


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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.


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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


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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


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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


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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


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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.


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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/).


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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.


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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


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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


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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


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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


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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).


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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.


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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


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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


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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


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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


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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


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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.


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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.


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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


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Footnotes

Financial support

This work was supported by Kyorin Pharmaceutical Co.


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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


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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


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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


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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.


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