Bupleurum chinense polysaccharide inhibit adhesion of human melanoma cells via blocking β1 integrin function

Accepted Manuscript Title: Bupleurum chinense polysaccharide inhibit adhesion of human melanoma cells via blocking ␤1 integrin function Author: Haibin Tong Guiquan Jiang Dake Qi Jingfeng Bi Dan Tian Xingang Guan Sheng Zheng Xin Sun PII: DOI: Reference:

S0144-8617(16)31088-8 http://dx.doi.org/doi:10.1016/j.carbpol.2016.09.034 CARP 11561

To appear in: Received date: Revised date: Accepted date:

4-2-2016 12-9-2016 12-9-2016

Please cite this article as: Tong, Haibin., Jiang, Guiquan., Qi, Dake., Bi, Jingfeng., Tian, Dan., Guan, Xingang., Zheng, Sheng., & Sun, Xin., Bupleurum chinense polysaccharide inhibit adhesion of human melanoma cells via blocking ␤1 integrin function.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.09.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

★BCP, water-soluble polysaccharide extracted from Bupleurum chinense, inhibited integrin-mediated adhesion of human melanoma A375 cells to fibronectin but had no effects on nonspecific adhesion to poly-L-lysine. ★ BCP-treatment

reduced

β1

integrin

ligand

affinity

and

inhibited

the

adhesion-dependent formation of F-actin stress fibers and focal adhesions. ★The inhibition of BCP on integrin-mediated signaling is probably through its dephosphorylatory effects on focal adhesion kinase (FAK) and paxillin. ★Our current findings indicated that BCP may be a potential therapy for melanoma metastasis due to its inhibitory effects on integrin signaling.

1

Bupleurum chinense polysaccharide inhibit adhesion of human melanoma cells via blocking β1 integrin function

Haibin Tong a,b,1,*, Guiquan Jiang b,1, Dake Qi c,1, Jingfeng Bi d, Dan Tian a, Xingang Guan a, Sheng Zheng e, Xin Sun a,*

a

Jilin Provincial Key Laboratory of Molecular Geriatric Medicine, Life Science

Research Center, Beihua University, Jilin 132013, China; b

Wood Material Science and Engineering Key Laboratory of Jilin Province, Beihua

University, Jilin 132013, China; c

Division of Biomedical Sciences, Faculty of Medicine, Memorial University of

Newfoundland, St. John’s, NL, Canada; d

Research Center for Clinical & Translational Medicine, 302 Hospital, Beijing

100039, China; e

School of Chemical Engineering, Northeast Dianli University, Jilin 132012, China.

1

Contributed equally to this work.

* Correspondence author: Tel: + 86-432-64608351; Fax: + 86-432-64608350; E-mail: [email protected] (H. Tong), [email protected] (X. Sun)

2

Abstract Adhesive interaction contributes toward tumor metastasis and the transmembrane glycoprotein receptor, integrin has been recognized to mediate the adhesion to extracellular matrix thus upregulating tumor metastasis. In the current study, we evaluated the anti-adhesive mechanisms of a water-soluble polysaccharide (BCP) extracted from Bupleurum chinense. BCP inhibited integrin-mediated adhesion of human melanoma A375 cells to fibronectin but had no effects on nonspecific adhesion to poly-L-lysine. BCP also reduced β1 integrin ligand affinity for GST-FNIII9-10 proteins. The adhesion-dependent formation of F-actin stress fiber and focal adhesion (FA) was also inhibited by BCP treatment. The inhibition of BCP on integrin-mediated signaling is probably through inhibiting phosphorylation of focal adhesion kinase (FAK) and paxillin. Collectively, our current findings indicated that BCP may be a potential therapy for melanoma metastasis due to its inhibitory effects on integrin function. Keywords: Bupleurum chinense; Polysaccharide; Integrin; Fibronectin; Focal adhesion

3

1. Introduction Melanoma is a common cancer with an increasing incidence, which can be found throughout the body including in the skin, iris and rectum, and its global incidence is 15-25 per 100,000 individuals (Schadendorf, & Hauschild, 2014). Melanoma possesses an aggressive and highly metastatic characteristic, with rapid systemic dissemination, and the prognosis of patients with metastatic malignant melanoma is grim, with a 5-year survival rate less than 15% (Damsky, Theodosakis, & Bosenberg, 2014; Gray-Schopfer, Wellbrock, & Marais, 2007). Sun (UV) exposure is still considered to be the most important risk factor for melanoma. As well as sun exposure, distinct genetic alterations have been identified as associated with melanoma (Lawrence et al., 2013). For example, BRAF and NRAS mutations are typically found in cutaneous melanomas, whereas KIT mutations are predominantly observed in mucosal and acral melanomas. Melanoma immunotherapy refers to a number of clinical approaches that can activate the immune system against cancer and specifically initiate or amplify a host response against evolving melanoma cells (Drake, Lipson, & Brahmer, 2014). Although vaccine approaches have had some clinical success, most cancer vaccines, in which tumour antigens are co-administered along with an adjuvant, fail to induce objective tumour shrinkage in patients (Hoos et al., 2010; Weber, Kahler, & Hauschild, 2012). In recent years, the blockade of immune checkpoints by antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD-1) or its ligand, PD-L1, becomes a promising immunotherapy for melanoma (Buchbinder, & Hodi, 2015; Hauschild, & 4

Garbe, 2015). The metastatic melanoma cells express high levels of PD-L1, which bind to PD-1 expressed on T cells and drive their inactivation. The use of PD-1 or PD-L1 antibodies in patients with metastatic melanoma results in high remission rates of 30-40% and impressive overall survival improvements. However, adverse effects of immunotherapy can range from asymptomatic vitiligo or autoimmune thyroiditis, to symptomatic skin, gastrointestinal, hepatic and endocrine immune-related toxic effects,

many

of

them

relate

to

the

induction

of

autoimmunity

and

pro-inflammatory-like states. These effects might limit eligibility or become challenges in clinical application (Kaufman et al., 2013; Michot et al., 2016). Additionally, the expensive treatment costs of immunotherapy are unaffordable for patients in developing countries. For example, Keytruda, a fully humanized monoclonal antibody as the first PD-1 inhibitor approved in the US market will cost approximately $150,000 per patient for each year of treatment. Therefore, those shortcomings have led to the development of non-immunotherapy strategies or combination treatment regimens for patients with melanoma. Melanoma metastasis is a multi-step procedure and its metastasis is strongly related to adhesive interaction with involvement of integrins, a family of transmembrane glycoprotein receptors (Aladowicz, Ferro, Vitali, Venditti, Fornasari, & Lanfrancone, 2013). Integrins play a key role in mediating cell-matrix and cell-cell interactions. Integrin consists of α- and β-subunits, which together form a non-covalent heterodimer (Liddington 2014). So far, integrins have been identified to be involved in many physiological and pathological conditions including cancer 5

progression and metastasis (Seguin, Desgrosellier, Weis, & Cheresh, 2015). Generally, integrins function to bind with extracellular matrix (ECM) molecules, including fibronectin, vitronectin, laminin, and collagen, thereby facilitating cell motility and invasion (Iwamoto, & Calderwood, 2015; Kanchanawong et al., 2010). Specifically, melanoma cells are rapidly eliminated from circulation through their initial adhesion to vascular endothelial cells mediated by integrins (McGary, Lev, & Bar-Eli, 2002). Metastatic tumor cells then adhere to elements of the sub-endothelial basement membrane, also mediated by β1 and β4 integrins. β1 integrin eventually contributes to the adhesion of metastatic tumor cell to connective tissue elements such as fibronectin and collagen, which facilitates the movement of cancer cells into sub-endothelial stroma and subsequent growth at these new sites. Thus, blocking the adhesive function of integrins is important to inhibit melanoma metastasis (Pinon & Wehrle-Haller, 2011; Schwartz et al., 2008). Previous studies have showed that natural polysaccharides are effective inhibitors to block tumor cell adhesion and metastasis. Ascophyllan, a sulfated polysaccharide isolated from brown seaweed Ascophyllum nodosum, inhibited the migration, adhesion and metastasis of B16 melanoma cells by reducing the expression of N-cadherin and matrix metalloprotease-9 (MMP-9), and enhancing the expression of E-cadherin (Abu et al., 2015). An acidic polysaccharide from Phellinus linteus markedly inhibited melanoma cell metastasis in mice, and directly blocked cancer cell adhesion and invasion through the extracellular matrix (Han et al., 2006). SIP-SII, a sulfated Sepiella maindroni ink polysaccharide, suppressed the invasion and 6

migration of cancer cells via the inhibition of intercellular adhesion molecule 1 (ICAM-1) and proteolytic activity of MMP-2 (Zong et al., 2013). The above reports indicate a potential direction in the development of new anti-cancer drugs by studying natural polysaccharides. Bupleurum Chinese is a well-known traditional Chinese herb, widely planted in North China. The roots of this perennial plant are the main medicinal parts, referred as ―Chai Hu‖ in Chinese Traditional Medicine (Sun et al., 2010). In the present study, we evaluated whether the BCP could inhibit human melanoma cell adhesion and metastasis, and investigated the possible mechanism.

7

2. Materials and Methods 2.1. Materials and chemicals B. chinense roots were purchased from a local pharmaceutical market, and identified according to the identification standard of Pharmacopeia of the People’s Republic of China. The antibodies of FAK (C-20) and paxillin (H-114) were purchased from Santa Cruz Biotechnology, and phospho-FAK (Tyr397) and phospho-Paxillin (Tyr118) antibodies were obtained from Cell Signaling Technology. In addition, Anti-β1 integrin (BV7) and Anti-Vinculin (SPM227) antibodies were purchased from Abcam, antibody against GST (G7781) was obtained from Sigma-Aldrich. The negative isotype controls, depending on the species and subclasses of the primary antibodies used, were obtained from Santa Cruz Biotechnology. Protein A/G-Sepharose beads, glutathione-Sepharose 4B beads, and ECL western blotting chemiluminescent detection reagents were purchased from GE Healthcare Life Sciences. TIRTC-conjugated phalloidin were purchased from Sigma. All other chemical reagents used were analytical grade. 2.2. Isolation and purification of BCP The roots of B. chinense were ground and extracted with hot water. The whole extract was filtered, concentrated and centrifuged, and the crude polysaccharide was precipitated by treating the supernatant with 3 volumes of ethanol. The crude polysaccharides were dried under reduced pressure after washed with dehydrated alcohol and diethyl ether. The sample was deproteinated by a combination of proteinase and Sevag method. Then, the supernatant was collected, dialyzed and 8

lyophilized to obtain water-soluble B. chinense polysaccharide, BCP. 2.3. General analytical methods Total carbohydrate content was determined by phenol-sulfuric acid colorimetric method using glucose as standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Protein content was quantified according to the Bradford’s method using bovine serum albumin as standard (Bradford, 1976). Total uronic acid contents were measured by m-hydroxydiphenyl method using glucuronic acid as standard (Filisetti-Cozzi & Carpita, 1991). Ultraviolet–visible spectra were recorded with a Varian Cary-100 Ultraviolet Visible Spectrophotometer (Varian Inc., Victoria, Australia). IR spectra were recorded with a Nicolet 5700 Fourier transform infrared spectrometer (Nicolet Instrument, Thermo Company, Madison, USA) in the range of 4000-400 cm-1, using KBr-disk method. Monosaccharide compositions were identified and quantified using gas chromatography (GC). BCAP-1 was hydrolyzed with 2 M trifluoroacetic acid (2 ml) at 120°C for 2 h. The hydrolyzed product was converted into the alditol acetates as described (Lehrfeld, 1985) and analyzed by GC. GC was performed on a Varian 3400 instrument (Hewlett-Packard Component, USA) equipped with DM-2330 capillary column (30 m × 0.32 mm × 0.2 μm) and flame-ionization detector (FID). The homogeneity and molecular weight of BCP were evaluated and determined by HPGPC. BCP was dissolved in distilled water, and then applied to Shimadzu HPLC system equipped with a TSK-GEL G3000 PWXL column (7.8×300 mm), eluted with 0.1 M Na2SO4 solution at a flow rate of 0.6 ml/min and detected by a 9

refractive index detector. Dextran standard with different molecular weights was used to calibrate the column and establish a standard curve. Molecular weight of BCP was estimated by reference to the calibration curve. 2.4. Methylation analysis BCP was methylated twice according to the method described by Ciucanu and Kerek (1984). The methylated product was extracted by CHCl3, and showed no absorption peak in the region of 3600-3300 cm-1 in the IR spectrum analysis, which indicated the methylated product was methylated completely. The product was hydrolyzed with formic acid and 2 M TFA. The hydrolyzed product was reduced with NaBH4, and then acetylated with acetic anhydride-pyridine. The alditol acetates of the methylated sugars were analyzed by GC-MS. GC-MS was run on the instrument HP5890 (II) (Hewlett-Packard Component, USA) with a HP-5 quartz capillary column (25 m × 0.22 mm × 0.2 μm), and at temperatures programmed from 120°C (maintained for 2 min) to 260°C (kept for 40 min) at a rate of 15°C /min. 2.5. Cell Culture Human melanoma cell line A375 was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China). Cells were cultured in DMED (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL streptomycin at 37°C. 2.6. Cell viability assay A375 cells were initially treated with BCP and incubated for 48 h. Then, the medium was replaced with 100 μl fresh medium containing 0.5 mg/ml MTT. After 4 10

h incubation, the supernatants were removed, and 150 μl of DMSO was added to each well to dissolve the crystals. Cell viability was determined with a microplate reader at a wavelength of 490 nm. 2.7. Cell adhesion assay Fibronectin or poly-L-lysine was diluted in sterile water and applied to 96-well plates overnight at 4°C. Nonspecific binding sites were blocked with 1% heat-inactivated bovine serum albumin (BSA) for 1 h at room temperature. Following 45 min starvation, cells were detached and suspended in serum-free medium, with or without BCP treatment for 30 min. Then, the cells were plated into wells and allowed to adhere for indicated time period at 37°C. Adherent cells were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet, and the dye was extracted with 33% acetic acid and quantified by an ELx800 absorbance reader (Bio-Tek, USA). 2.8. F-actin content assay Following BCP treatment for 30 min, A375 cells were seeded in fibronectin-coated 24-well plates for the indicated time. After washing with PBS, and 400 μl of fixative buffer containing rhodamine phalloidin (20 mM KPO4, 10 mM Pipes, 5 mM EGTA, 2 mM MgCl2, 0.1% Triton X-100, 3.7% formaldehyde, and 2 μM rhodamine phalloidin) was added and incubated for 1 h. Then, cells were washed with washing buffer (0.1% saponin, 20 mM KPO4, 10 mM Hepes, 5 mM EGTA, and 2 mM MgCl2). 200 μl of methanol was added to extract the rhodamine phalloidin, which was further measured by Molecule Devices CytoFluor II plate reader. F-actin content was expressed as a relative fold change in the mean fluorescence intensity. 11

2.9. Immunofluorescence for focal adhesion Following BCP incubation for 30 min, A375 cells were allowed to adhere on fibronectin-coated glass slides for 2 h. Cells were then fixed and treated with primary antibody against vinculin for 1 h. Following incubation of secondary antibody, cells were observed under an Nikon Eclipse 80i fluorescence microscope and the number of focal adhesion were quantified by using Image J software. 2.10. Expression and purification of recombinant GST-FNIII9-10 proteins The expression vector of GST-FNIII9-11 was transformed into E. coli strain BL-21 (DE3) and cultured overnight. GST-fusion protein expression was induced with IPTG for 3 h. Cells were then harvested and homogenized by sonication. After centrifugation, GST-fusion proteins in supernatant were purified by affinity chromatography

with

glutathione-Sepharose

4B

beads

according

to

the

manufacturer’s instructions (GE Healthcare Life Sciences, USA). 2.11. Binding assay between β1 integrin and GST-FNIII9-10 proteins After stimulation with 1 mmol/L MnCl2 for 1 hour, A375 cells were lysed and centrifuged. The supernatants were then incubated with the anti-β1 integrin antibody at 4°C for 2 h followed with 20 μl of protein A/G-Sepharose beads (50% slurry) for 1 h. The immunoprecipitated beads were treated with or without BCP at 4°C for 1h. After washing, the beads were incubated with GST-FNIII9-11 at 4°C for 2 h and the binding of GST-FNIII9-11 was immunoblotted with GST antibody. 2.12. Immunoblotting Cells were washed twice with ice-cold PBS and lysed in the lysis buffer (50 mM 12

Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 2.5 mM sodium pyrophosphate, 1 mM NaF, 1 mM Na3VO4, 1 mM β-glycerophosphate, 1 mM PMSF, and 20 μg/ml aprotinin/leupeptin). After incubation on ice for 15 min, the lysates were centrifuged at 15,000× g for 25 min, then mixed with Laemli buffer and boiled at 95°C for 5 min. Cell lysates were loaded to 10% polyacrylamide gels, and then proteins were transferred to a nitrocellulose membrane. The membranes were incubated with 5% nonfat milk in TBST, and then with the indicated primary antibodies and the HRP-conjugated secondary antibodies. Chemiluminescent detection was performed by using ECL Plus Western blotting reagents according to the manufacturer's protocol (Amersham Pharmacia Biotech). The quantification of densitometry was performed using ImageJ software. 2.13. Statistical analysis Data shown represent mean ± SD. Statistical significance was determined by Student t test. Statistical analysis was performed using SPSS version 13.0. P value less than 0.05 was considered statistically significant.

13

3. Results 3.1. Physicochemical and structural characterization of BCP The total sugar, protein, and uronic acid contents, as well as monosaccharide composition of BCP are summarized in Table 1. BCP had a negative response to Bradford protein assay, and no absorption was detected by the UV spectrum at 280 and 260 nm, indicating the absence of protein and nucleic acid. The total carbohydrate content of BCP was 98.5% determined by phenol-sulfuric acid method, and BCP did not contain uronic acid evaluated by m-hydroxydiphenyl colorimetric method. The HPGPC profile of BCP (Figure 1) showed a single and symmetrical peak revealing that BCP was a homogeneous polysaccharide with an average molecular weight of 131.7 kDa calculated according to the calibration curve with standard dextrans. Moreover, monosaccharide composition analysis of polysaccharide is an important procedure to control quality standard and give basic information of polysaccharide. GC analysis showed BCP was composed of four kinds of monosaccharides, including arabinose, xylose, mannose, and glucose with a molar ratio of 2.1:1:1.7:3.2, and this result indicated glucose was the predominant monosaccharide in BCP. IR spectrum of BCP (Figure 2) revealed a typical major broad stretching peak at 3444 cm-1 for the hydroxyl group, and a weak band at 2931 cm-1 showed C-H stretching vibration. The broad band at 1643 cm-1 was due to the bound water. The bands at 844 cm-1 and 889 cm-1 indicated α- and β-configurations of the sugar units simultaneously existing in the polysaccharide.

14

Table 1. Physicochemical characterization of BCP Sample

BCP

Mw

Total sugar

Protein

Uronic acid

Molar ratios of monosaccharide

(kDa)

(%)

(%)

(%)

(mol%)

131.7 a

98.5

nd a

nd

Ara

Xyl

Man

Glc

2.1

1.0

1.7

3.2

nd: not detected

Figure 1. HPGPC profile of BCP. Homogeneity and molecular weight of BCP was evaluated and determined by HPGPC. Sample was loaded onto Shimadzu HPLC system equipped with a TSK-GEL G3000 PWXL column, eluted with 0.1 M Na2SO4 solution and detected by a refractive index detector.

15

120

100

2931

889 844

80

1643

60

3444 4000

3000

2000

W a v e n u m b e r (c m

1000 -1

)

Figure 2. FT-IR spectra of BCP. FT-IR spectra were recorded with a Nicolet 5700 Fourier transform infrared spectrometer in the range 4000-400 cm-1, using KBr-disk method. To obtain more structural information, BCP was subjected to linkage analysis by permethylation. The fully methylated BCP was hydrolyzed with acid, converted into alditol acetates, and analyzed by GC-MS using HP-5 capillary column. Peaks of methylated sugars were identified by their retention time and mass spectra. Their relative molar ratios were estimated from the peak areas of GC and corresponding response factors. Data from the methylation analysis (Table 2) indicated that the backbone of BCP mainly consisted of (1→3)-linked arabinose, (1→4)-linked mannose and (1→4)-linked glucose with branch point located at C-6 position of (1→4)-linked mannose and glucose residue. The non-reducing termini were composed of arabinosea and a trace of mannose.

16

Table 2. GC-MS analysis of methylated BCP

Methylated sugars Type of linkage

Molar ratio

2,3-Me2-Ara

→3)-Araf-(1→

2.4

2,3-Me2-Xyl

→4)-Xylp-(1→

trace

2,3,4-Me3-Xyl

Xylp-(1→

1

2,3,6-Me3-Man

→4)-Manp-(1→

1.4

2,3-Me2-Man

→4,6)-Manp-(1→

0.4

2,3,4,6-Me4-Man

Manp-(1→

trace

2,3,6-Me3-Glc

→4)-Glcp-(1→

2.7

2,3-Me2-Glc

→4,6)-Glcp-(1→

0.9

(as alditol acetates)

a

a

2,3,4,6-Me4-Glc = 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl- galactose, etc.

3.2. BCP inhibit integrin-mediated A375 cell adhesion Cell-ECM adhesion is an essential step in cancer metastasis. In our current study, we initially investigated the effects of BCP on mediating A375 cell adhesion to fibronectin, which is the most common component of ECM and also an integrin ligand. As shown in Figure. 3A, the percentage of adherent A375 cells on fibronectin increased gradually from time 0 to 6 h, and reached 76.6% at 4 h. While, our data suggested that BCP treatment could block the A375 cell adhesion on fibronectin. The adherent percentage of BCP-treated cells increased slowly, and reached only 49.6% and 27.6%, respectively, at 6 h with the BCP concentration of 100 and 400 μg/ml. 17

Interestingly, cell adhesion to poly-L-lysine, a non-integrin binding protein, was not affected (Figure. 3B) except at the time point of 4 h (p<0.05, both of the BCP treatment groups at the concentration of 100 and 400 μg/ml compared to control group), suggesting that BCP may specifically block integrin-dependent A375 cell adhesion. Importantly, our data also indicated that the effective concentrations of BCP (100 to 400 μg/ml for 48h) to inhibit cell adhesion did not impact cell viability (Figure. 4), suggesting that BCP may have an anti-adhesive efficacy without any toxicity. A A d h e r e n t c e lls ( % )

100

F ib ro n e c tin

C o n tro l B C P 1 0 0 μ g /m l

80

B C P 4 0 0 μ g /m l 60

40

20

0 0

1

2

3

4

5

6

t im e ( h o u r s )

B A d h e r e n t c e lls ( % )

100

P o ly - L - ly s in e

C o n tro l B C P 1 0 0 μ g /m l

80

B C P 4 0 0 μ g /m l 60

40

20

0 0

1

2

3

4

t im e ( h o u r s )

18

5

6

Figure 3. BCP inhibits the adhesion of A375 cells to fibronectin. A375 cells were plated on 96-well plates with pre-coated by fibronectin (A) or poly-L-lysine (B). The adhesion was allowed as indicated time following vehicle or 100 to 400 μg/ml of BCP treatment. Adherent cells were then stained with crystal violet.

( f o ld o f c o n t r o l)

C e ll v ia b ility

1 .2 1 .0 0 .8 0 .6 0 .4 0 .2 0 .0 C o n tro l

100

400

μ g /m l

BCP

Figure 4. Cytotoxic effects of BCP on A375 cells. A375 cells were treated with various concentrations of BCP for 48 h.

3.3. BCP blocks the interaction between β1 integrin and fibronectin Integrins are the major receptors of ECM proteins. Thus, recognition and interaction between integrins and ECM is an important step to initiate cell attachment and adhesion that further contribute to cancer metastasis. β1 integrin is a major prototypic fibronectin receptor in human melanoma A375 cells (Kato et al., 2013; Pocheć, Lityńska, Amoresano, & Casbarra, 2003) and GST-FNIII9-11 protein contains a β1 integrin-binding RGD motif (Brown, Dysart, Clarke, Stabenfeldt, & Barker, 2015). Therefore, our current study introduced an in vitro ligand affinity binding 19

assay to investigate the effect of BCP on blocking the interaction between β1 integrin and GST-FNIII9-11 protein. As shown in Figure 5, the immunoprecipitated β1 integrin exhibited high affinity with GST-FNIII9-11. However, the affinity between GST-FNIII9-11 and immunoprecipitated β1 was significantly inhibited with the β1 integrin incubated by BCP. The blocking effects reached 75.2% and 93.8%, respectively, at the concentrations of 100 and 400 μg/ml, suggesting that BCP can directly block the interaction between β1 integrin and its physiological ligand fibronectin.

Figure 5. BCP inhibits the binding between β1 integrin and GST-FNIII9-10 proteins. Data were representative of three independent experiments.

3.4. BCP inhibits the formation of F-actin stress fibers The remodeling of cytoskeleton and the formation of F-actin stress fibers are two critical events involved in cancer cell adhesion and metastasis (Yamaguchi & Condeelis, 2007). Our study subsequently investigated the effects of BCP on 20

mediating fibronectin-dependent actin polymerization. As shown in Figure 6, following seeding in fibronectin-coated 24-well plates, A375 cells had increased actin polymerization in a time-dependent manner, the fluorescence intensity increased gradually and the relative F-actin content was elevated above approximately 1.8-fold at 6 h compared to time 0. However, following 100 or 400 ug/ml of BCP treatment, the fluorescence intensity of rhodamine phalloidin in treated cells were significant lower than the intensity in control cells at 6 h, and the F-actin contents increased only 54% and 28%, respectively, compared to the time 0.

( F o ld o f c o n t r o l)

R e la t iv e F - a c t in c o n t e n t

2 .0

1 .5

1 .0

C o n tro l B C P 1 0 0 μ g /m l B C P 4 0 0 μ g /m l

0 .5

0

60

120

180

240

300

360

t im e ( m in u t e s )

Figure 6. Effect of BCP on actin polymerization in A375 cells. A375 cells were seeded on fibronectin-precoated plates, and then treated with 100 or 400 μg/ml of BCP as time indicated at 37°C. F-actin was calculated by measuring the fluorescence of extracted rhodamine phalloidin with a Molecule Devicer CytoFluor II plate reader. Bar graphs show the changes in F-actin content expressed as a relative fold change in the mean fluorescence intensity of the cells at time 0. Error bars represent the standard deviation of the mean (SD) from three independent experiments per time point. 21

3.5. BCP inhibits the formation of focal adhesion (FA) Focal adhesion (FA) is a large and dynamic macromolecular assembly, through which integrins and scaffold proteins link the actin cytoskeleton to the extracellular matrix. FA is an important sub-cellular structure regulating the interaction and adhesion between ECM and cancer cells (Geiger, Bershadsky, Pankov, & Yamada, 2001). Thus, we further examined the effects of BCP on mediating the formation of focal adhesion. A375 cells were plated onto fibronectin-coated glass slides for 2 h, and then fixed and stained for vinculin, a marker of focal adhesion. As shown in Figure 7A and B, the mean number of focal adhesion in A375 cells was 103 per cell, while BCP treatment caused a significant reduction in the number of the focal adhesion in a dose-dependent manner, the mean numbers were 78 and 66 per cell, respectively, at the concentration of 100 and 400 ug/ml, suggesting that BCP significantly reduced integrin-dependent focal adhesion formation.

22

N u m b e r o f fo c a l a d h e s io n s p e r c e ll

B

p < 0 .0 1

250

p < 0 .0 5 200

150

100

50

0 C o n tro l

100

400

μ g /m l

BCP

Figure 7. BCP affects focal adhesion assembly in A375 cells. (A) A375 cells incubated with or without BCP were seeded on fibronectin-coated glass slides, then allowed to adhere for 2 h. Cells were fixed and stained for endogenous vinculin. (B) Quantification of number of focal adhesions per cell. Three independent experiments assessing focal adhesions from at least twenty cells were performed for each condition.

3.6. BCP inhibits downstream integrin signaling Previous studies have reported that β1 integrin binds with ECM proteins, leading to the activation of downstream factors, such as FAK and paxillin. We therefore further investigate whether BCP treatment downregulates β1 integrin-associated signaling pathways in A375 cells. Western blot analysis (Figure 8A) using phosphospecific antibodies targeting FAK and paxillin, respectively, suggested after seeding A375 cells on fibronectin, the phosphorylation levels of FAK on the Tyr-397 site and paxillin on the Tyr-118, which were early consequences of the β1 23

integrin-ligand interaction, were increased with integrin engagement. As shown in Figure 8B and C, the BCP treatment significantly suppressed the ratio of p-FAK/total FAK and p-paxillin/total paxillin quantified by ImageJ software in a dose-dependent manner. These results further demonstrated that BCP may down-regulate β1 integrin downstream signaling, including FAK and paxillin, eventually resulting in the subsequent loss of cell adhesion to fibronectin.

R a t io o f p - F A K /t o t a l F A K

B

1 .5

1 .0

0 .5

0 .0 C o n tro l

100

400

BCP

24

μ g /m l

R a t io o f p -p a x illin /t o ta l p a x illin

C

1 .5

1 .0

0 .5

0 .0 C o n tro l

100

400

μ g /m l

BCP

Figure 8. BCP inhibits the phosphorylation of FAK and paxillin. (A) A375 cells treated with or without BCP were seeded on fibronectin-coated 6-well plates, and then allowed to adhere for 2 h. Phosphorylated FAK (p-FAK) and paxillin (p-paxillin ) were detected by Western blot analysis. The ratio of p-FAK/total FAK (B) and p-paxillin/total paxillin (C) were quantified by ImageJ software respectively. Results represented the mean (±SD) of three independent experiments.

25

4. Discussion Morbidity and mortality of melanoma are continuously rising around the world, and the mortality is majorly associated with high risk of melanoma metastasis. Each year, there are almost 132,000 new patients diagnosed melanoma worldwide and 48,000 of them die from advanced melanoma metastasis. Thus, interfering with metastasis has been considered as an important strategy to improve the therapy of melanoma. Melanoma metastasis is related to adhesive interaction with involvement of integrins. Our present study evaluated the anti-adhesive effects of water-soluble polysaccharide (BCP) extracted from Bupleurum chinense and found that BCP inhibited integrin-mediated adhesion of human melanoma A375 cells to fibronectin. BCP also reduced β1 integrin ligand affinity with GST-FNIII9-10 proteins. The inhibition of BCP on integrin-mediated signaling is probably through its dephosphorylatory effects on focal adhesion kinase (FAK) and paxillin. The adhesion-dependent formation of F-actin stress fibers and focal adhesion (FA) were also impacted by BCP. Overall, our current findings indicated that BCP may be a potential pharmacologic therapy for melanoma metastasis due to its inhibitory effects on integrin signaling. β1 integrin has aberrant expression and improper functions in human melanoma cells, which significantly contribute to melanoma metastasis (Kuphal, Bauer, & Bosserhoff, 2005; Seftor, Seftor, & Hendrix, 1999; Seguin, Desgrosellier, Weis, & Cheresh, 2015). Cancer metastasis involves many stage-specific adhesive interactions, whereas β1 integrin has been also reported to modulate malignant melanoma cell 26

adhesion and invasion (Barkan, & Chambers, 2011; Ganguly, Pal, Moulik, & Chatterjee, 2013; Tsuji et al., 2002). During the progression of human melanoma A375 cells from a low to high metastatic phenotype, the α4β1 integrin expression level appeared to increase several fold, resulting in increased adhesion to vitronectin or fibronectin substrates, which may facilitate interactions with platelets, endothelial cells and specific extracellular matrix proteins to promote metastasis (Gehlsen, Davis, &

Sriramarao,

1992).

Interestingly,

lipid

rafts

can

effectively

regulate

β1-integrin-mediated spreading and migration of A375 cells on fibronectin by modulating β1 integrin clustering and the interaction between β1 integrin and nucleolin, and MβCD treatment can disrupt the location and interaction of β1 integrin and nucleolin in lipid rafts, which may be responsible for disturbing the spreading and migration of A375 cells on fibronectin (Bi et al., 2013; Wang et al., 2013). Lumican, a small leucine-rich proteoglycan of the extracellular matrix, can inhibit the migration of A375 cells, and β1 integrin is identified as mediator of the anti-invasive effect depending on a direct binding between the core protein of lumican and β1 integrin (Brézillon et al., 2009; D'Onofrio et al., 2008; Zeltz et al., 2010). Therefore, β1 integrin is a promising anti-tumor target, and the inhibition of β1 integrin function may have potential as therapeutic strategy especially in preventing human melanoma metastasis. Currently available inhibitors of β1 integrin are functionally blocking monoclonal antibodies, peptide antagonists and cyclic peptides which mimic matrix, and some of them have been even tested in clinical patients (Desgrosellier & Cheresh, 2010; Ley, Rivera-Nieves, Sandborn, & Shattil, 2016; Millard, Odde, & Neamati, 27

2011; Tucker, 2002). Integrin inhibitors have not only revealed impressive therapeutical efficacy in tumor growth and metastasis but also represent an alternative strategy for targeting multidrug-resistant tumors that do not responded to conventional chemotherapy (Kuphal, Bauer, & Bosserhoff, 2005; Stupp, & Ruegg, 2007). However, so far there are very few β1 integrin antagonists isolated from herbs with a low cost and high anti-tumor efficacy. Our study evaluated the effects of water-soluble polysaccharide (BCP) isolated from Bupleurum chinense on integrin signaling in melanoma cells. We found that BCP significantly inhibited the adhesion of human melanoma A375 cells to fibronectin and reduced β1 integrin ligand affinity with GST-FNIII9-10 proteins, and impacted adhesion-dependent formation of F-actin stress fibers and focal adhesions, suggesting that BCP may be a potential drug to inhibit integrin signaling and downstream adhesion. Cell attachment to ECM components leads to the clustering of the integrins and the formation of FA. Accumulating evidence has indicated that FA is involved in metastatic adhesion and cancer cell invasion (Sakamoto, McCann, Dhir, & Kyprianou, 2010). More importantly, FA serves not only as a mechanical linkage to the ECM, but also as a biochemical signaling hub to regulate numerous signaling proteins to bind and cluster with integrins.

Many of these FA proteins through tyrosine

phosphorylation or activation of FA adaptor molecules eventually mediate cell adhesion and cancer metastasis (Eke & Cordes, 2015). Focal adhesion kinase (FAK) is an essential FA protein and key member of integrin-signaling pathway. FAK is a non-receptor protein tyrosine kinase that colocalizes with integrins in FA where 28

associated protein complexes regulate the assembly and integration of the actin cytoskeleton. FAK becomes phosphorylated on five different tyrosine residues (Y397, Y407, Y576, Y577, and Y925), in which Y397 becomes autophosphorylated upon integrin engagement to ECM proteins. The phosphorylated FAK Y397 becomes a binding site of tyrosine kinase Src and following binding, the complex of FAK and Src further activates downstream pCAS, Crk or paxillin, which regulates cancer metastasis (Kratimenos et al., 2014; Luo & Guan, 2010; Schwock, Dhani, & Hedley, 2010). Our current study showed that BCP significantly reduced the size and number of FAs in A375 cells which are paralleled with inhibited phosphorylation of FAK and paxillin. Paxillin is a well-known adhesion-dependent adaptor and key scaffolding protein located at the focal adhesion. The function of paxillin can be turned on via its Tyr-118 site phosphorylation, which further recruits SH2 domain-containing molecules such as CrkII leading to activation of Rac1 signaling and RhoA inactivation (Kwak et al. 2012). Thus, cell adhesion-dependent tyrosine phosphorylation of paxillin regulates actin reorganization and morphological changes that are involved in both cancer cell migration and invasion (Panetti 2002). Thus, BCP regulated paxillin signaling pathway might inhibit the formation of F-actin stress fibers and the de novo actin polymerization provides a driving force for cancer cell adhesion, spreading and migration. In summary, BCP inhibited integrin-mediated melanoma cell-ECM attachment, reduced β1 integrin affinity and adhesion-dependent formation of F-actin stress fibers 29

and FAs. BCP also down-regulated the functions of β1 integrin signaling via inhibiting the phosphorylation of FAK and paxillin. Our research provides insight into how BCP provides an anti-metastatic effect in melanoma and give direct evidence that BCP may be a potential anti-cancer drug.

30

Acknowledgements This study was supported by the National Natural Science Foundation of China (31401203 and 51503003), the Key Project of Science and Technology Department of Jilin Province (20140204039YY), the Youth Foundation of Health and Family Planning Commission of Jilin Province (2014Q040), the Scientific Foundation of Education Department of Jilin Province (2014-173), Jilin Provincial Key Laboratory of Wooden Materials Science and Engineering (Beihua University), Jilin Provincial Key Laboratory of Molecular Geriatric Medicine (20130624003 JC).

31

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