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Introduction

Soft tissue sarcomas are rare tumors of mesenchymal origin with a frequent ability for distant metastasis (1). Fibrosarcomas, tumors belonging to this group, specifically originate from muscular fibrous tissues, fascia and tendons (2). Annually, fibrosarcomas represent 3% of all soft tissue sarcomas and 1% of new cancer cases diagnosed in the United States and Europe (2,3). The treatment of fibrosarcomas is mainly surgical and must be individualized due to the rarity and pleiotrophy of these tumors (2,3).

Unfractionated heparin (UFH) is a mixture of heavily sulfated, linear glycosaminoglycan (GAG) chains, which participate in the regulation of various cell biological functions (4), including the modulation of growth factor activities (5), the inhibition of heparanase (5,6) and changes in extracellular matrix (ECM) composition (7). Its size ranges from 3 to 30 kDa, with chains in the 12–15 kDa size range most commonly used. On the other hand, low-molecular-weight heparin (LMWH) consists of LMW fragments produced by the depolymerization (enzymatic or chemical) of UFH, which yields chains that are <18 saccharide units long (8). UFH and LMWH have been extensively used over the years as anticoagulant agents for the prevention of thromboembolism in cancer patients (9–12). The long-term utilization of heparin preparations as anti-coagulants has led to the understanding that UFH and LMWH positively affect the survival of cancer patients (9, 13–15). Moreover, the effects of high-molecular-weight haparin (HMWH) and LMWH were found to be discrete. Thus, according to clinical data, patients who had been treated with LMWH in order to minize the risk of thrombosis have an improved survival (3 months) as compared to those treated with UFH (15), and an increased long-term survival period (16). Specifically, the anticancer effect of LMWH has been ascribed to its inhibition of angiogenesis via the cellular release of tissue factor pathway inhibitor in endothelial cells (17) or its expression by cancer cells (18). The anticancer effect of heparin may be due to its non-anticoagulant derivatives (19), and novel heparin therapeutical approaches are proposed. Of note, heparin has been found to inhibit the proliferation of various normal cell types, including vascular smooth muscle cells, mesanglial cells, fibroblasts and epithelial cells (20–23). Likewise, the majority of studies have indicated that heparin inhibits cancer cell growth (24,25), even though exceptions to the rule have been reported (26,27). Moreover, using the B6FS fibrosarcoma cell model, we have previously demonstrated that heparin attenuates the growth ability of these cells (28).

By contrast, some early studies have suggested that heparin enhances the spread of cancer cells to other organs and tissues (29–31). Different mechanisms of action of heparin have been proposed. Thus, LMWH was found to inhibit the proliferation, migration, invasion and lung metastatic ability of HT1080 fibrosarcoma cells through a blockade of the RAGE axis (32). Chalkiadaki et al demonstrated that UFH, by activating p53/focal adhesion kinase (FAK)-dependent signaling, modulated melanoma cell adhesion and migration (33). The same authors also demonstrated that LMWH inhibited the ability of melanoma cells to adhere and to migrate, utilizing a protein kinase C (PKC)α/c-Jun N-terminal kinase (JNK) signaling axis and resulting in actin cytoskeletal changes (34).

Fibronectin (FN) is a key ECM component that affects cell attachment and migration (35). Importantly, FN expression has been shown to correlate with aggressive cancer progression (35–37). Fibrosarcoma cells have been demonstrated to specifically adhere to the FN substrate (38,39). In this study, we investigated the putative biological roles of UFH and LMWH in the migratory and adhesive properties of B6FS fibrosarcoma cells.

Materials and methods

Reagents

UFH and LMWH were supplied by Sigma (St. Louis, MO, USA). Stock solutions of 10 mg/ml were prepared by dissolving heparin in sterile, RNase- and DNase-free DEPC water (Cayman Chemical Co., Ann Arbor, MI, USA). Human plasma FN (1 mg/ml) was obtained by Millipore Corp. (Billerica, MA, USA). RPMI medium and penicillin-streptomycin were obtained from Biosera (Sussex, UK) and gentamycin was supplied by Invitrogen Life Technologies (Carlsbad, CA, USA). Fetal bovine serum (FBS) was purchased by Gibco Life Technologies (Carlsbad, CA, USA). Fluorescein isothiocyanate (FITC)-conjugated unfractionated heparin (referred to as FITC-Heparin) was obtained from Invitrogen Life Technologies. D-[6-3H(N)]glucosamine hydrochloride was supplied by DuPont de Nemours (Dreiech, Germany). Heparin lyase II (heparinase II, no EC number) from Flavobacterium heparinum, chondroitinase AC II from Arthrobacter aurescens (EC 4.2.2.5), proteinase K and 2X crystallized papain (EC 3.4.22.2) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Heparin lyases I and III from Flavobacterium heparinum (EC 4.2.2.7 and EC 4.2.2.8, respectively), chondroitinase ABC from Proteus vulgaris (EC 4.2.2.4), keratanase II and the anti-heparitinase stubs antibody clone (3G10) were from Seikagaku Kogyo Co. (Tokyo, Japan).

Cell culture conditions and transfection with short intefering RNA (siRNA)

In this study we used the B6FS human fibrosarcoma cell line (40). The cells were obtained from the Cell Bank of the Karolinska Institute, Stockholm, Sweden and were a kind gift from Dr. Anders Hjerpe (Karolinska Institute). The cells were cultured at 37°C and humidity 5% CO2 in RPMI supplemented with 10% FBS and antimicrobial agents (100 IU/ml penicillin, 100 µg/ml streptomycin and 0.5% gentamycin). All experiments were conducted under serum-free conditions; specifically after 24 h of serum starvation, the cells were treated with UFH and LMWH (10 µg/ml and 30 µg/ml) for 48 h.

The B6FS cells (450,000 cells per T25 flask) were incubated with siRNA (100 nM) specific for the FAK gene (siFAK), as previously described by Hong et al (41) and with siRNA negative control sequences (siScramble) for 6 h. Specific RNA (Invitrogen Life Technologies) and Lipofectamine® 2000 (Invitrogen Life Technologies) (1/50 µl medium) were added to Opti-MEM© (Invitrogen Life Technologies) for 5 min at room temperature. In continuation, diluted Lipofectamine 2000 was mixed with the siRNA for 20 min to induce the formation of liposome-siRNA complexes. The medium containing siRNA or siScramble was removed after 6 h of incubation and fresh RPMI 0% FBS medium supplemented with antibiotics was added. The cells were harvested for the respective experiments, after 48 h of culture.

Western blot analysis

After 48 h of respective treatments, the harvested B6FS cells were lysed with RIPA buffer and electrophoresed on an 8% polyacrylamide gel. Protein bands were transferred onto nitrocellulose membrane in 10 nM CAPS, pH 11 and 10% methanol. All membranes were blocked and incubated overnight at 4°C with PBS containing 0,1% Tween-20 and 5% v/v low fat milk powder. The respective membranes were incubated with the primary antibodies diluted in PBS containing 0,1% Tween and 1% v/v low fat milk powder, for 1 h at room temperature. The following primary antibodies were used: p-FAK (1:200; MAB1141; Millipore Corp.), FAK (1:200; sc-557; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), nuclear factor-κB (NF-κB) p65 subunit (1:200; (sc-109; Santa Cruz Biotechnology, Inc.) and β-actin (1:2,500; MAB1501; Millipore Corp.). The immune complexes were detected by peroxidase-conjugated anti-mouse, anti-goat and anti-rabbit secondary antibodies (1:10,000; Millipore Corp.), using SuperSignal West Pico Chemiluminescent substrate (Pierce Biotechnology, Inc., Rockford, IL, USA).

Real-time PCR

For real-time PCR, mRNA was isolated using TRIzol reagent (Invitrogen Life Technologies) according to manufacturer's instructions. RNA (1 µg) was used for cDNA synthesis, using the Takara reverse transcription reagent kit (Takara Bio, Dalian, China). Real-time PCR was performed using the Mx300P cycler (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA). The KAPA SYBR® FAST Universal qPCR kit (Kapa Biosystems, Inc., Wilmington, MA, USA) was used for real-time PCR reactions in a total volume of 20 µl and with suitable specific gene primers (Table I). The PCR conditions used for amplification were: 94°C for 15 min followed by 40 cycles at 94°C for 20 sec, 55°C for 30 sec and 72°C for 30 sec, followed by 72°C for 10 min. Standard curves were run and produced a linear plot of threshold cycle (Ct) against dilution (log). Gene levels were quantified according to the concentrations of a standard curve and are presented as arbitrary units. GAPDH was used as a housekeeping gene, for comparison between samples.

Table I Primer sequences used for real-time PCR.

Table I

Primer sequences used for real-time PCR.

Gene	Primer sequences
FAK	F: 5′-GTGCTCTTGGTTCAAGCTGGAT-3′
	R: 5′-ACTTGAGTGAAGTCAGCAAGATGTGT-3′
GAPDH	F: 5′-GGAAGGTGAAGGTCGGAGTCA-3′
	R: 5′-GTCATTGATGGCAACAATATCCACT-3′

[i] FAK, focal adhesion kinase. F, forward, R, reverse.

Determination of heparan sulfate (HS) content

The determination of the HS content was carried out as previously described by Karamanos et al (42). Briefly, in order to determine the amount of HS production by the B6FS cells, we performed metabolic labeling of GAGs by supplementing the cell cultures with D-[6-3H(N)]glucosamine hydrochloride (10 µCi/ml) during the period of 16 h prior to the respective harvesting time. Upon the termination of the incubation period, the cells were harvested and cell-associated proteoglycans (PGs) were extracted with 50 mM Tris-HCl, pH 8.0, containing 1% (v/v) Triton X-100 and 0.1% (w/v) NaCl and the following proteinase inhibitors: phenylmethanesulfonyl flouride, benzamidine hydrochloride and hexanoic acid at final concentrations of 2, 5 and 50 mM, respectively. The collected conditioned medium was concentrated to 1:100 of its original volume on an YM-10 membrane (Amicon/Millipore). The PGs were then precipitated by the addition of 4 vol. of 95% (v/v) ethanol containing 2.5% (w/v) sodium acetate with 40 µl chondroitin sulfate (CSA; 0.2 mg/l) added as a carrier. Following centrifugation (11,000 × g for 10 min at 25°C), the precipitates of Pgs were digested with 2 U/ml proteolytic enzyme papain in 100 mM phosphate buffer (pH 7.0) at 65°C for 60 min. The GAGs liberated in this manner were precipitated by the addition of 10 vol. 1% (w/v) cetylpyridium chloride (CPC) and centrifuged at 10,000 × g for 10 min. The pellets obtained were dissolved in 500 µl of 60 (v/v) propanol-1 containing 0.4% (w/v) CPC. The liberated GAGs were reprecipitated by the addition of 6 vol. of 95% (v/v) ethanol containing 2.5% (w/v) sodium acetate. The precipitates were then washed with ethanol and allowed to dry. For the identification of galactosaminoglycans (GalAGs)., i.e., chondroitin sulfate (CS) and/or dermatan sulfate (DS), the GAG preparation was dissolved in water and digested with an equi-unit mixture (0.2 U/ml) of chondroitinases ABC and AC II. Aliquots from the supernatant were analyzed by reversed polarity high-performance capillary electrophoresis (HPCE), as previously described (42). The determination of HS was carried out in the GAG preparations which were digested with heparin lyases I, II and III in combination (0.05 U/ml) in 20 mM acetate buffer, pH 7.0, containing 1 µmol calcium acetate at 37°C for 90 min (43). In all cases, the amount of GAGs was determined from the integrated peak area of the GAG-derived Δ-disaccharides.

HS digestion

Heparitinase treatments were performed for the digestion of B6FS cell HS chains, as previously described (24,26). In brief, cells seeded in 24-well plates were serum-starved for 24 h and then treated with heparitinase (0.001 U/ml) for 24 and 48 h in 0% FBS medium. The cell extracts treated with heparitinase were electrophoresed on 8% polyacrylamide Tris/glycine gels and transferred onto nitrocellulose membranes. After blocking, the membranes were incubated for 1 h at room temperature with primary antibodies [mouse anti-heparitinase stubs antibody (clone 3G10; 1:500; CF500913; Seigakagu, Tokyo, Japan); goat anti-actin (1:200; sc-1616; Santa Cruz Biotechnology, Inc.) and mouse anti-CSA (1:200; C8035; Sigma)]. The immune complexes were detected following incubation with peroxidase-conjugated anti-goat or anti-mouse antibody, 1:4,000 or 1:2,000, respectively, with the SuperSignal West Pico Chemiluminescent substrate (Pierce Biotechnology, Inc.)

Cell adhesion assay

For the cell attachment assay, we used cells transfected with siFAK or cells treated with UHF and LMWH (for 48 h). The cells were detached with 5 mM PBS/EDTA. In continuation 5,000 cells/well were seeded onto a flat-bottom 96-well black plate. The bottom of the wells was coated with FN (5 µg/ml) for 1 h at 37°C. BSA 1% (30 min at room temperature) was added for the blocking of non-specific binding sites. The cells were allowed to adhere for 30 min at 37°C. The number of attached cells was determined using the CyQUANT fluometric assay (Molecular Probes; Invitrogen Life Technologies) according to the manufacturer's instructions.

Cell migration assay

To investigate the motility of cells, 24-well plates were used. Cells (60,000 cells/well) were seeded for 24 h in RPMI 10% FBS and subsequently treated (as described above) for 48 h. The cell stroma was wounded by scratching with a sterile 10 µl pipette tip. The detached cells were washed twice by using fresh RPMI (serum-free). The wound closure was monitored at 0 and 6 h using a digital camera (Canon Inc., Tokyo, Japan) connected to a microscope (Leica, Mannheim, Germany). The quantification of the wound area was measured using ImageJ software.

Heparin internalization assay

The B6FS cells were seeded onto a 96-well plate in RPMI supplemented with 10% FBS for 24 h incubation and subsequently, FITC-Heparin was added at 1 and 10 µg/ml for 24 h, in serum-free RPMI. Following this incubation, FITC-Heparin was removed and the cells were gently washed with RPMI (0% FBS). The concentration and the incubation times were chosen selected following optimization experiments (data not shown). FITC-Heparin internalization was visualized under a fluorescence microscope utilizing Leica DM2500 to acquire images.

Immunofluorescence

The B6FS cells were seeded onto glass coverslips into 24-well plates (65,000 cells/well) and incubated with 10% FBS RPMI for 24 h. Following 24 h of serum starvation, the cells were treated with UFH and LMWH for 48 h. Subsequenlty, the cells were fixed in 5% formldehyde and 2% sucrose in PBS (incubation for 10 min at room temperature). Following 3 washes with PBS, Triton X permeabilizing agent was appled for 10 min at room temperature and washed prior to the addition of fluorescent phalloidin (1:100; Molecular Probes; Invitrogen Life Technologies) for 20 min in the dark. Fluorescent phalloidin was used for the detection of actin filaments. TO-PRO-3 (T3605; Molecular Probes/Thermo Fisher Scientific, Waltham, MA, USA) was then used for the nuclear staining (20 min in the dark). The coverslips were placed onto slides using glycerol and images were collected using a laser-scanning spectral confocal microscope (TCS SP2; Leica), LCS Lite software (Leica) and a 63 Apochromat 1.40 numerical aperture oil objective.

Statistical analysis

Statistical significance was evaluated by one-way ANOVA analysis with Turkey's post-test, using GraphPad Prism (version 4.0) software. A value of p<0.05 was considered to indicate a statistically significant difference.

Results

Effect of heparin in B6FS fibrosarcoma cell functions

Initially, we examined the effects of UFH and LMWH on FN-dependent B6FS cell adhesion. As shown in Fig. 1A, both UFH and LMWH enhanced B6FS cell adhesion (p<0.01, p<0.05); the maximal effect being evident at the concentration of 10 µg/ml. Of note, the molecular weight of the heparin preparations did not modify their effects on cellular function. Thus, UFH and LMWH had the same promoting effect on B6FS cell adhesion. This suggests that the effects of heparin are attributed to its oligosaccharide structure.

Figure 1 Effect of unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) on B6FS cell adhesion and migration. (A) B6FS cells were treated with UFH and LMWH (10 µg/ml) for 48 h prior to harvesting and reseeding for 1 h on 96-well plates coated with fibronectin. The number of attached cells was determined using the fluometric CyQUANT assay kit; (B) B6FS cells were treated with UFH and LMWH (10 µg/ml) for 48 h prior to wounding. The cells were allowed to migrate for 6 h; (C) the wound closure was monitored at 0 and 6 h using a digital camera connected to microscope. The quantification of the wound area was measured by using ImageJ software. The results represent the average of 3 separate experiments in triplicate. The means ± standard error of the mean are plotted. *p<0.05 and **p<0.01, indicate statistically significant differences.

In continuation, we analyzed the effects of heparin on B6FS fibrosarcoma cell migration. Subsequent to pre-treatment with UFH and LMWH (10 µg/ml) an enhancement of fibrosarcoma cell migration was observed (p<0.05; Fig. 1B and C). To exclude false-positive results due to traces of heparin-binding growth factors, heat treatment was applied for the utilized heparin preparation. The effects of heparin on cell adhesion/migration were not affected by heat treatment (data not shown).

The participation of FAK is necessary for heparin-induced cell adhesion

FAK is a 125-kDa cytoplasmic tyrosine kinase protein which is primarily positioned at adhesion sites and plays a key role in the processes of cell adhesion and migration (44). Importantly, FAK expression has been shown to be closely associated with (even in early reports) aggressive cancer behavior (45). To examine the putative participation of FAK in heparin-dependent adhesion, the B6FS cells were transfected with siRNA specific for the FAK gene as previously described (41). This approach resulted in an efficient downregulation of FAK both at the mRNA (p<0.01) and protein level (p<0.05) (Fig. 2A–C). Our results demonstrated that the FAK-deficient cells exhibited a marked decrease in their basal level ability to adhere to the FN substrate (p<0.05; Fig. 2D). Importantly, both the UFH and LMWH stimulatory effects on B6FS cell adhesion were abolished in the FAK-deficient cells (p<0.05 and p<0.01, respectively) (Fig. 2D and E). Therefore, the effect of heparin on fibrosarcoma cell adhesion was FAK-dependent.

Figure 2 Effect of focal adhesion kinase (FAK) on unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH)-dependent B6FS cell adhesion. (A) B6FS cells were transfected with FAK short interfering RNA (siFAK), whereas scramble RNA (siScr) was used as a negative control. The cells were cultured for 48 h prior to harvesting. Representative blot of FAK protein (125 kDa band) is presented; (B) FAK protein bands were densitometrically analyzed and adjusted against actin; (C) B6FS cells were transfected with siFAK with the use of siScr as a negative control. The cells were cultured for 48 h prior to harvesting and the FAK expression level was verified by real-time PCR. Subsequenlty, 24 h after transfection, the B6FS cells were treated with (D) UFH (10 µg/ml) and (E) LMW heparin (10 µg/ml) for 48 h prior ot harvesting and reseeding for 1 h on 96-well plates coated with fibronectin. The number of attached cells was determined using the fluometric CyQUANT assay kit. The results represent the average of 3 separate experiments in triplicate. The means ± standard error of the mean are plotted. *p<0.05 and **p<0.01, indicate statistically significant differences.

Effect of heparin on FAK activation

Heparin has been shown to antagonize the function of integrin adhesion receptors (46), which trigger FAK clustering (47), and to have an effect on FAK expression and activation (33,34). Thus, we investigated the possible role of heparin in FAK expression and activation. To this end, the heparin-treated B6FS cell extracts were probed with antibodies against FAK and p-FAK. As shown in Fig. 3A and B, both UFH and LMWH significantly activated FAK (Y397) at 10 µg/ml, whereas no effect of heparin on total FAK protein expression was evident (Fig. 3A and B).

Figure 3 Role of unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) in focal adhesion kinase (FAK) activation. (A) B6FS cells were treated with UFH (10 µg/ml) and LMWH (10 µg/ml) for 48 h prior to harvesting. Representative blots of FAK and p-FAK protein (125 kDa) are presented; (C) B6FS cells were treated with UFH (10 µg/ml) and LMWH (10 µg/ml) for 48 h prior to harvesting. Representative blot of nuclear factor-κB (NF-κB) (65 kDa) is presented; (D) NF-κB (65 kDa) protein band was densitometrically analyzed and adjusted against actin. The results represent the average of 3 separate experiments in triplicate. The means ± standard error of the mean are plotted. *p<0.05, indicates a statistically significant difference

NF-κB has been previoulsy demonstrated to stimulate FAK promoter activity and upregulation (41). Heparin has been suggested to modulate NF-κB translocation to the nucleus and its transcriptional activity (48). In this study, we examined the possible role of NF-κB in heparin-induced FAK activation by using a specific antibody for the p65 subunit of NF-κB. As shown in Fig. 3C and D, UFH and LMWH did not affect the protein expression of the p65 subunit of NF-κB. Thus, p65 does not participate in the heparin-induced upregulation of FAK (Fig. 3C and D).

Heparin affects cytoskeleton organization in B6FS cells

The phenotype of cancer cells is directly associated with their adhesive and migratory properties, which are of utmost importance for the process of cancer metastasis (49). FAK, has been postulated as the key conduit point which regulates the flow of signals from the ECM to the actin cytoskeleton (50). In this study, we investigated the possible effect of heparin on actin cytoskeleton organization in B6FS fibrosarcoma cells. To this end, rhodamine-conjugated phalloidin staining was used. The results of confocal miscroscopy indicated that B6FS cell actin cytoskeleton organization was enhanced. Thus, both the HMW- and LMWH-treated cells were found to have the fully spread, adhesive phenotype with the cells being stretched by the tensile forces of actin stress fibers (Fig. 4).

Figure 4 Role of unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) on actin polymerization. B6FS cells were treated with UFH and LMWH (10 µg/ml) for 48 h, were seeded onto round coverslips, fixed, permeabilized and then stained then stained using phalloidin to visualize the actin filaments (red color). The nuclei were stained using TO-PRO-3. The signals against phalloidin and TO-PRO-3 were superimposed. Slides were analyzed by confocal microscopy, and images were acquired at a magnification of ×40.

Heparin internalization in B6FS fibrosarcoma cells

In continuation, we hypothesized that heparin affects fibrosarcoma cell motility and cell attachment through its internalization. Indeed, the cellular internalization of heparin has been demonstrated in previous studies (51,52). In this study, FITC-Heparin was utilized to investigate the internalization of heparin in B6FS fibrosarcoma cells. As shown in Fig. 5, FITC-Heparin was taken up by the B6FS cells in a dose-dependent manner, with the majority of cells exhibiting strong staining. The results of immunoflourescence demonstrated that FITC-Heparin was located, not only in the cytoplasmic region, but also in the nucleus of fibrosarcoma cells.

Figure 5 Internalization of fluorescein isothiocyanate (FITC)-conjugated unfractionated heparin (FITC-Heparin). B6FS cells were incubated with 1 and 10 µg/ml FITC-Heparin) and heparin internalization was visualized at 24 h at a magnification of ×20. Cells incubated in 0% RPMI (control) were utilized as negative controls.

Effect of cell-associated HS chains on B6FS cell adhesion

HS exhibits structural similarities with heparin, but does not have as many 'highly sulfated' sequences consisting of tri-sulfated disaccharide units, which are sequences with the highest binding affinity to heparin/HS-binding proteins (53). Biochemical analyses of the metabolically [3H]-labeled CS/DS/HS/PGs produced by the B6FS cells demonstrated that the HS amount was 63.1% of the total B6FS cell-associated GAGs (data not shown). Therefore, we examined the possible involvement of endogenous HS chains on B6FS cell adhesion. Initially, treatment of the cells with heparitinase and concomitant blotting with the monoclonal antibody 3G10, which recognizes a neoepitope generated by heparitinase digestion, demonstrated an efficient removal of HS chains at the 24 h time point (Fig. 6A and B). The specific digestion of cell-associated HS with heparitinase did not affect their adhesion to FN (Fig. 6C). These results suggest that B6FS cell adhesion is independent of their cell-associated HS content.

Figure 6 Role of heparan sulfate (HS) chains on B6FS cell adhesion. (A) B6FS cell extracts, treated with heparitinase (0.001 U/ml) for 0, 24 and 48 h, were immunoblotted with the monoclonal antibody 3G10 which recognizes a neoepitope generated by heparitinase digestion (lane 1, untreated cells; lane 2, 24-h heparitinase-treated cells; lane 3, 48-h heparitinase-treated cells. Representative blot of HS stubs is presented; (B) HS epitope band band was densitometrically analyzed and adjusted against actin; (C) B6FS cells were treated with heparitinase (0,001 U) for 48 h prior to harvesting and reseeding for 1 h on 96-well plates coated with fibronectin. The number of attached cells was determined using the fluometric CyQUANT assay kit. The results represent the average of 3 separate experiments in triplicate. The means ± standard error of the mean are plotted. **p<0.01, indicates a statistically significant difference.

Discussion

In the present study, the effects of UFH and LMWH on fibrosarcoma cell motility functions were examined. We demonstrated that both heparin preparations affected fibrosarcoma cell motility and adhesion. The stimulatory effects of both UFH and LMWH on B6FS cell adhesion were FAK-dependent and resulted in actin cytoskeleton reorganization.

Fibrosarcoma is a tumor of mesenchymal origin that is mostly composed of transformed fibroblasts and abundant is in ECM microenvironment (2,54). The B6FS cell line originates from a poorly differentiated fibrosarcoma (40). In poorly differentiated tumors, cancer cells are characterized by scant phenotypic similarity with cells of origin, mild pleomorphism, as well as intense mitotic activity (55,56). It is noteworthy that fibrosarcoma is a particularly heterogeneous tumor type, taking into account morphology, differentiation and behavior (57).

In this study, we demonstrated a promoting effect of UFH and LMWH on the motility of B6FS cells. Specifically, both UFH and LMWH were found to enhance cell adhesion onto FN, as well as the migration of B6FS fibrosarcoma cells. Heparin and the closely resembling HS chains, via the activation of key cell signaling pathways (for fibrosarcoma cells), act as extracellular regulators for many cell functions (5,58,59). Previously, we demonstrated a negative regulatory effect of LMWH on melanoma cell adhesion and migration via the PKCα/JNK signaling pathway (34). Furthermore, LMWH was found to inhibit the proliferation, migration, invasion and the induction of lung metastases of HT1080 fibrosarcoma cells, through a blockade of the RAGE axis (32). Likewise, in the present study, we examined the mechanisms of action of heparin in B6FS cells by focusing on FAK (60). FAK, as its name implies, is a key constituent of the focal adhesion complex, which is an actin-based anchoring junction limited to the cell-ECM interface in mammalian tissues. It is used by motile cells, such as fibroblasts and metastatic cancer cells for their attachment and movement under physiological or pathological conditions (61). By generating FAK-deficient cells, as previously described (33,41) we demonstrated that the effects of UFH and LMWH on B6FS cell adhesion were FAK-dependent. As regards the effect of heparin on cell adhesion, in other cell models, it has been shown that the interaction of heparin activates integrin and results in the tyrosine-phosphorylation of focal adhesion-associated proteins, such as FAK, Src and paxillin in endothelial cells (62). On the other hand, heparin has been shown to block osteoblast adhesion onto osteoactivin in an FAK/ERK-dependent manner (63). In the present study, however, both UFH and LMWH were was found to enhance FAK (Y397) phosphorylation in B6FS fibrosarcoma cells. FAK activation is closely associated with actin cytoskeleton organization. Importantly, both heparin preparations were demonstrated to induce morphological changes in the B6FS cell actin cytoskeleton. Indeed, actin polymerization results in the formation of the actin stress fibers, which are necessary for efficient cell motility functions. Treatment of fibrosarcoma cells with both UFH and LMWH enhanced the formation of actin filaments and induced the well 'spread' migratory active phenotype. This effect of heparin on the organization of the cytoskeleton is well correlated with the heparin-dependent enhancement of B6FS cell adhesion and migration.

The internalization of heparin has been demonstrated in various cell models. Thus, heparin stabilizes lens epithelium-derived growth factor (LEDGF) and facilitates its translocation from the ECM to the nucleus (64). In a murine macrophage cell line, heparin was found to initiate the phogocytosis of gelatin-latex particles (65), whereas we previously demonstrated an internalization of UFH in melanoma cells (33). In continuation, in the present study, we investigated the putative internalization of heparin in the B6FS cell model utilizing FITC-Heparin. This approach showed a dose-dependent internalization of FITC-Heparin and its subsequent localization into the B6FS cell cytoplasm and nucleus. Importantly, other authors have provided data which showed the heparin nuclear localization and regulation of gene expression of several cell types (66,67), even though the mechanism of heparin uptake remains unspecified (48). Thus, heparin is suggested to regulate the transactivation of transcription factors, such as Jun/c-Fos/AP-1 (68) and to inhibit NF-κB transcriptional activity (48). Moreover, NF-κB was suggested to stimulate FAK activation (41). In the B6FS cells, no effect of heparin on NF-κB, subunit p63, transactivation was evident and the intracellular mechanism of heparin action resulting in FAK/actin cytoskeleton-dependent enhanced adhesion and migration requires further study.

In the present study, the enzymatic cleavage of endogenous HS chains did not affect the adhesion of B6FS cell line. These data suggests that the exogenous addition of heparin specifically activates signaling pathways responsible for the promotion of cell adhesion and migration and highlights the putative role of heparin/HS content of the cancer microenvironment. The effects of heparin seem to be tissue and cell line-specific. Thus, studies have suggested that heparin exerts an inhibitory effect on both cancer and normal cell proliferation (21–23,28). On the other hand, a dose-dependent stimulatory role of heparin on the proliferation of HT29, SW1116 and HCT116 human colon cancer cells, with no involvement of endogenous HS chains was proposed (27).

In this study, we report that both UFH and LMWH through a FAK/actin cytoskeleton axis, enhance the adhesion and migration of B6FS fibrosarcoma cells. The responsiveness of fibrosarcoma cell motility to the exogenous heparin/HS content of the cancer microenvironment may play a role in their ability to metastasize. The exact mechanisms of action of heparin require further investigation, which is currently under way in our laboratories.

Abbreviations:

References