Stromal Cell–Derived Factor-1/Chemokine (C-X-C Motif)Ligand 12 Stimulates Human Hepatoma Cell Growth,Migration, and Invasion

Angela Sutton,1 Veronique Friand,1 Severine Brule-Donneger,1 Thomas Chaigneau,1

Marianne Ziol,2 Odile Sainte-Catherine,1 Aurelie Poire,1 Line Saffar,1 Michel Kraemer,1

Jany Vassy,3 Pierre Nahon,1,4 Jean-Loup Salzmann,1 Liliane Gattegno,1,5

and Nathalie Charnaux1,5

1UPRES 3410 and 2UPRES 3406, Universite Paris XIII, Bobigny, France; 3Institut National de la Santeet de la Recherche Medicale U553, Institut Federatif de Recherche Saint-Louis, Paris, France;and 4Service d’Hepatologie and 5Laboratoire de Biochimie, Hopital Jean Verdier,AP-HP, Bondy, France

AbstractIn addition to their physiologic effects in inflammation

and angiogenesis, chemokines are involved in cancer

pathology. The aim of this study was to determine

whether the chemokine stromal cell–derived factor 1

(SDF-1) induces the growth, migration, and invasion

of human hepatoma cells. We show that SDF-1 G

protein–coupled receptor, chemokine (C-X-C motif)

receptor 4 (CXCR4), and SDF-1 mRNA are expressed

in human hepatoma Huh7 cells, which secrete and

bind SDF-1. This binding depends on CXCR4 and

glycosaminoglycans. SDF-1 associates with CXCR4,

and syndecan-4 (SDC-4), a heparan sulfate proteoglycan

at the plasma membrane of Huh7 cells, induces

the growth of Huh7 cells by promoting their entry

into the cell cycle, and inhibits the tumor necrosis

factor-A–mediated apoptosis of the cells. SDF-1 also

reorganizes Huh7 cytoskeleton and induces tyrosine

phosphorylation of focal adhesion kinase. Finally, SDF-1

activates matrix metalloproteinase-9, resulting in

increased migration and invasion of Huh7 cells. These

biological effects of SDF-1 were strongly inhibited by the

CXCR4 antagonist AMD3100, by a glycosaminoglycan,

heparin, as well as by B-D-xyloside treatment of the

cells, or by c-jun NH2-terminal kinase/stress-activated

protein kinase inhibitor. Therefore, the CXCR4,

glycosaminoglycans, and the mitogen-activated protein

kinase signaling pathways are involved in these events.

The fact that reducing SDC-4 expression by RNA

interference decreased SDF-1– induced Huh7 hepatoma

cell migration and invasion strongly indicates that

SDC-4 may be an auxiliary receptor for SDF-1. Finally,

the fact that CXCR4 is expressed in hepatocellular

carcinoma cells from liver biopsies indicates that the

in vitro results reported here could be extended to

in vivo conditions. (Mol Cancer Res 2007;5(1):21–33)

IntroductionChemokines are chemotactic cytokines that govern multiple

aspects of host defense and inflammation such as hematopoiesis

and leukocyte trafficking (1). Chemokines also play an

important role in tumor biology because they may influence

tumor growth, invasion, and metastasis (2-6). Stromal cell–

derived factor 1 (SDF-1)/chemokine (C-X-C motif) ligand 12

(CXCL12), a CXC chemokine that exists mainly in two

alternative splicing variants, a and h, is a homeostatic chemo-kine that signals through chemokine (C-X-C motif) receptor 4

(CXCR4), a G protein–coupled receptor, which in turn plays an

important role in hematopoiesis, development, and organization

of the immune system (2, 7). However, like other chemokines,

this chemokine also binds to glycosaminoglycans (8, 9). Recent

studies have indicated that SDF-1 is expressed in some cancer

cells (i.e., malignant ovarian and breast cancer cell lines) and is

involved in tumor cell migration and metastasis (10, 11).

The syndecans are a family of proteoglycans, which,

together with the lipid-linked glypicans, are the major source

of heparan sulfate chains at cell surfaces (12, 13). By way of

their heparan sulfate, syndecans bind a wide variety of soluble

and insoluble ligands, such as extracellular matrix components,

cell adhesion molecules, growth factors, cytokines, proteinases,

or pathogens such as HIV-1 (13-15).

We recently showed that SDF-1 forms complexes on HeLa

cells and human primary lymphocytes or macrophages, which

comprise CXCR4 and syndecan-4 (SDC-4; ref. 16). We also

showed the occurrence of a heteromeric complex between

SDC-4 and CXCR4 at the plasma membrane of these cells.

Nevertheless, our data showed that SDF-1 binds directly

to SDC-4, which may be a signaling molecule for the chemo-

kine (17).

A number of in vitro and in vivo studies highlight the

importance of some chemokines in acute or chronic liver

Received 4/17/06; revised 10/3/06; accepted 10/23/06.Grant support: Direction de la Recherche et des Enseignements Doctoraux(Ministere de l’Enseignement Superieur et de la Recherche), Universite Paris XIII.The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.Note: L. Gattegno and N. Charnaux contributed equally to this work.Requests for reprints: Liliane Gattegno, Laboratoire de Biologie Cellulaire,Biotherapies Benefices et Risques, UPRES 3410, Universite Paris XIII, 74 rueMarcel Cachin, 93017 Bobigny, France. Phone: 33-1-48-38-77-52; Fax: 33-1-48-02-65-03. E-mail: liliane.gattegno@jvr.ap-hop-paris.frCopyright D 2007 American Association for Cancer Research.doi:10.1158/1541-7786.MCR-06-0103

Mol Cancer Res 2007;5(1). January 2007 21on March 1, 2021. © 2007 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

diseases (18-20) and indicate that chemokines may modulate

certain biological actions in hepatocytes, including proliferation

(20). CXCR4 expression has already been shown in hepatoma

cells (21-23).

The aim of the present study was to determine whether

SDF-1 induces the growth, migration, and invasion of human

hepatoma cells and elucidate the molecular mechanisms of

these effects, including the involvement of SDF-1 G protein–

coupled receptor, CXCR4, and glycosaminoglycans. We

extended our in vitro data by using immunohistochemistry to

provide the status of CXCR4 in the liver samples of patients

with hepatocellular carcinoma (HCC).

ResultsHuman Huh7 Cells Express SDF-1, CXCR4, Syndecan-1,Syndecan-2, and Syndecan-4

In basal culture conditions, SDF-1a (82.5 F 21.9 pg/mL)

was detected in the culture supernatant of human hepatoma

Huh7 cells whereas mRNA encoding for SDF-1 was observed

in these cells (Fig. 1A). mRNAs encoding for CXCR4,

syndecan-1 (SDC-1), syndecan-2 (SDC-2), and SDC-4 were

also observed (Fig. 1A) whereas CXCR4 and the heparan

sulfate proteoglycans SDC-1 and SDC-4 were detected at their

plasma membrane (Fig. 1B).

SDF-1 Binds to Huh7 CellsBiotinylated SDF-1a bound in a dose-dependent manner to

Huh7 cells (Fig. 2A). AMD3100, a CXCR4 antagonist (24),

strongly decreased this binding by 66 F 14% (P < 0.01; n = 3;

Fig. 2B) and heparin by 73 F 19% (P < 0.001; n = 3; Fig. 2C).

This suggests that both CXCR4 and glycosaminoglycans are

involved in the binding.

SDF-1 Associates with CXCR4 and SDC-4 at the Huh7Cell Plasma Membrane

To characterize SDF-1 ligands or receptors expressed by

Huh7 cells, SDF-1a–containing complexes were collected on

anti-SDF-1a–coated beads. Immunoblotting the complexes

with anti-CXCR4 monoclonal antibody (mAb) 12G5 revealed

48-kDa proteins (Fig. 2D, lane 1), characterized by an apparent

molecular mass close to that reported for CXCR4 (45-48 kDa;

refs. 25, 26). Neither immunoreactivity with anti-CCR5 2D7

nor with the isotype was detected (Fig. 2D, lane 2 , and data not

shown). If this complex was treated with glycosaminidases,

32- and 45-kDa proteins immunoreactive with anti–SDC-4

mAb 5G9 were observed but not with anti–SDC-1 mAb

DL-101 nor with the isotype (Fig. 2D, lanes 3-5 , and data

not shown). The 32 kDa apparent molecular mass is close to

that predicted for SDC-4 protein core (13, 27), whereas the

45 kDa molecular mass may represent proteoglycan oligomer-

ization. Such eluted proteins were not detected if the cells were

incubated in SDF-1–free buffer (data not shown). Therefore,

CXCR4 and SDC-4 coimmunoprecipitate with SDF-1. Whether

CXCR4 and SDC-4 associate on Huh7 cells was then

investigated; the cells were stimulated or not by SDF-1 and

lysed. Lysates were incubated with protein G coated with anti–

SDC-4 mAb 5G9. In both cases, proteins immunoreactive with

anti-CXCR4 12G5 mAb coimmunoprecipitated with SDC-4

(data not shown). Therefore, a heteromeric complex between

SDC-4 and CXCR4 occurs even in the absence of SDF-1.

Moreover, we observed that biotinylated SDF-1 directly binds

to electroblotted SDC-4 (data not shown), which is consistent

with our previous studies (16, 17).

SDF-1 Induces Free Radical Production and ActivatesMitogen-Activated Protein Kinases in Huh7 Cells

Stimulation of the cells with SDF-1a resulted in a

significant, marked, and rapid increase in free radical formation

after a 1-min stimulation. Heparin, AMD3100, or heparitinase

treatment of the cells abolished this SDF-1a–induced reactive

oxygen species production (P < 0.05; Fig. 3A). None of these

cell treatments significantly affected basal reactive oxygen

species levels.

Stimulation of Huh7 cells with SDF-1a (3 and 125 nmol/L)

also significantly increased phosphorylated forms of both extra-

cellular signal–regulated kinase 2 (Erk2; p42) and c-jun NH2-

terminal kinase/stress-activated protein kinase (JNK/SAPK; p54/

p46) in a time-dependent manner, reaching a maximum after

15 min of stimulation (Fig. 3B and data not shown). As a posi-

tive control, phorbol 12-myristate 13-acetate also significantly

activated Erk1/2 and JNK/SAPK kinases (data not shown).

SDF-1 Induces Huh7 Cell ProliferationSDF-1a (3 and 125 nmol/L) significantly stimulated

Huh7 cell proliferation, as assessed by both crystal violet and

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

FIGURE 1. Huh7 cells express CXCR4, SDF-1, and heparan sulfateproteoglycans. A. Semiquantitative RT-PCR analysis for the mRNAexpression of CXCR4, SDF-1, SDC-1, SDC-2, SDC-4, and glyceraldehyde3-phosphodehydrogenase (GAPDH ). B. Immunocytochemistry analysisof CXCR4, SDC-4, and SDC-1. Cells were incubated with anti-CXCR412G5 mAb, anti –SDC-4 5G9 mAb, anti –SDC-1 B-B4 mAb, or theirisotypes (IgG2a or IgG1), and then with Alexa Fluor 488– labeledsecondary antibodies. Microscopy images are representative of threeindependent experiments. Bar, 5 Am.

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(MTT) assays (P < 0.05; n = 3; Fig. 4A and B). This was partly,

but significantly, inhibited by AMD3100 (47 F 4% inhibition;

P < 0.05; n = 3; Fig. 4C). Preincubating SDF-1a with heparin

(100 Ag/mL) also strongly decreased this effect (83 F 6%

inhibition; P < 0.05; n = 3). Furthermore, in Huh7 cells

depleted of proteoglycans by a 3-day incubation in

1 mmol/L 4-methylumbelliferyl-h-D-xyloside (hDX; Fig. 4D),the proliferation induced by SDF-1a was strongly attenuated

(67 F 7% inhibition; P < 0.05; n = 3; Fig. 4C). Therefore,

both CXCR4 and glycosaminoglycans are involved in this

SDF-1–dependent Huh7 cell growth.

SP600125, a JNK/SAPK pharmacologic inhibitor, partly

prevented the proliferative effect induced by 3 nmol/L SDF-1a(54 F 9% inhibition; P < 0.05; n = 3) whereas the mitogen-

activated protein kinase (MAPK)/Erk kinase inhibitor PD98059

had no effect (Fig. 4C).

None of these compounds significantly affected basal cell

proliferation.

SDF-1 Affects Huh7 Cell CycleTo discriminate G0 from G1 phase, Huh7 cells stimul-

ated with SDF-1a were analyzed for expression of Ki67

proliferation–associated nuclear antigen. This antigen is

undetectable in G0 resting cells; it is expressed in cells entering

G1 and its expression increases with changes in staining pattern

during the cell cycle (28). SDF-1a (125 nmol/L) significantly

increased the proliferative Ki67 labeling index (Ki67/4¶,6-diamidino-2-phenylindole (DAPI)–positive stainings) in these

cells, which was 75 F 4% for untreated cells versus 95 F 1%

for SDF-1a–treated cells (P < 0.05; Table 1); moreover, the

Ki67 flow cytometry index [Ki67/isotype immunoglobulin G1

(IgG1) mean fluorescence intensities] increased 3-fold in SDF-

1a–treated cells (Table 1).

SDF-1a also affects Huh7 cell cycle status: the addition of

SDF-1a (125 nmol/L) to the cells significantly prevented

spontaneous DNA degradation in Huh7 cells because their

proportion in sub-G1 decreased from 51.5 F 9.4% to 25 F6.8% (P < 0.001; n = 3). SDF-1a also increased the percent-

age of Huh7 cells in G0-G1 from 28.7 F 8.4% to 39.4 F 7.2%,

and in S + G2-M phases from 19.8 F 4.8% to 35.6 F 6.9%

(P < 0.001; n = 3; Table 2). Therefore, SDF-1 triggers

quiescent Huh7 cells from G0 into cycle, whereas it stimulates

the transition of cells already engaged in G1 to S + G2-M.

We consequently investigated the chemokine-mediated

effect on apoptosis using previously established tumor necrosis

factor a (TNFa)–mediated apoptosis in Huh7 cells (29). The

FIGURE 2. SDF-1a binds to Huh7 cells and associates with CXCR4 and SDC-4 at their plasma membrane. A. Binding of biotinylated SDF-1a to Huh7cells. Cells were incubated with biotinylated SDF-1a (at 0, 12.5, 40, or 125 nmol/L). Binding was analyzed by flow cytometry using streptavidin-Alexa Fluor488. Reactivity was compared with streptavidin-Alexa Fluor 488. B. Some cells were preincubated with AMD3100 (12 Amol/L) before the addition ofbiotinylated SDF-1a (40 nmol/L), or biotinylated SDF-1a (40 nmol/L) was preincubated with heparin (100 Ag/mL; C), and the suspension was added to thecells. Representative of five independent experiments. D. Huh7 cells were incubated with SDF-1a (0.5 Amol/L) and lysed. The SDF-1a– interacting proteinswere collected on anti-SDF-1a–coated beads. The immunocomplexes, immobilized on protein-G coated beads, were treated (lanes 3-5 ) or not (lanes 1 and2) with heparitinase I, heparitinase III, and chondroitinase ABC, electroblotted and revealed with anti-CXCR4 12G5 (lane 1), anti-CCR5 2D7 (lane 2), anti –SDC-4 5G9 (lane 3), anti –SDC-1 DL-101 (lane 4 ) mAbs, or IgG2a (lane 5). Representative of three individual experiments.

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percentage of Annexin V–positive cells decreased from 6.8 F0.5% to 1.1 F 0.3% (n = 3; P < 0.05) after a 48-h incubation

with the chemokine, suggesting that SDF-1 promotes the

survival of these cells (Table 3).

SDF-1 Stimulates Huh7 Cell MigrationDue to the important role of the CXCR4/SDF-1 axis in

cancer metastasis, we investigated whether SDF-1 induces

Huh7 cell migration. In these experiments, hepatocyte growth

factor (20 ng/mL) was used as a positive control (30). As shown

in Fig. 5A, SDF-1a induced Huh7 cell migration in a dose-

dependent manner (P < 0.05). This induction was significantly

reduced by incubating Huh7 cells with AMD3100 (65 F 9%

inhibition; P < 0.05; n = 3; Fig. 5B). In some experiments,

SDF-1a preincubated with heparin was added to the lower

chamber. Blocking the heparin-binding site of the chemokine

or treating the cells with hDX strongly decreased SDF-1a–induced migration (78 F 8% and 92 F 12% inhibition,

respectively; P < 0.05; n = 3; Fig. 5B).

SP600125 slightly but significantly reduced this SDF-1a–induced migratory effect by 27 F 3% (P < 0.05; n = 3;

Fig. 5B). PD98059 had no effect.

None of these compounds significantly affected basal

migration (data not shown).

Therefore, SDF-1a–induced Huh7 cell migration depends

on CXCR4, glycosaminoglycans, and, at least partly, JNK/

SAPK pathway activation.

To further characterize the molecular events involved

in SDF-1a–induced Huh7 cell migration, SDF-1a–treatedcells were examined by indirect immunostaining of phosphotyr-

osine residues with an anti-Tyr(P) mAb (4G10) and of tyrosine

phosphorylation of focal adhesion kinase (FAK) at Tyr397 with a

polyclonal anti–FAK-(P)-Tyr397 antibody (Fig. 6B).

The respective stainings occur especially on focal adhesion

plaques and were much more intense in SDF-1a–stimulatedHuh7 cells compared with controls (Fig. 6A and B). Whereas

untreated cells displayed slight actin stress fiber networks and

smooth, regular cell borders, SDF-1a causes a change in the

reorganization of filamentous actin and induces mostly

membrane ruffling at the cell periphery (Fig. 6C).

Finally, FAK was immunoblotted from lysates of SDF-

1a–stimulated or unstimulated control cells with anti-FAK

antibodies and with anti-FAK-(P)-Tyr577 phosphospecific anti-

bodies. The level of tyrosine phosphorylation at FAK-Tyr577

FIGURE 3. SDF-1 induces free radical production and MAPK activation in Huh7 cells. A. Flow cytometry analysis of reactive oxygen species (ROS )production. Huh7 cells were stimulated with SDF-1a (3 nmol/L) and loaded with dichlorofluorescein diacetate. Alternatively, cells were preincubated withAMD3100 (12 Amol/L) or with heparitinase I (100 mIU/mL) and heparitinase III (200 mIU/mL) before their stimulation with SDF-1a, or SDF-1a waspreincubated with heparin (100 Ag/mL) and the suspension was added to the cells. Unstimulated control cells were incubated in parallel for each time point.Points, mean of three different experiments; bars, SE. * and &, P < 0.05, versus unstimulated cells or versus SDF-1a-treated cells in the absence of theinhibitor, respectively. B. SDF-1a induces Erk2 and JNK/SAPK signaling activation. Western blot analysis of phosphorylated (P ) and total forms of Erk1/2(p44/p42) or JNK/SAPK (p54/p46) in Huh7 cells that were either untreated (UT) or stimulated with SDF-1a (3 or 125 nmol/L). Whole-cell extracts wereseparated on 12% SDS-PAGE and immunoblotted with either phosphospecific anti-p44/p42 Erk1/2 or phosphospecific anti-p46 and anti-p54-JNK/SAPKantibodies (top ). Parallel immunoblottings with anti – total p44/p42 Erk1/2 or anti – total p46-JNK/SAPK and p54-JNK/SAPK antibodies, respectively, werecarried out (bottom ). Quantification of p44/p42 MAPK and JNK/SAPK phosphorylations was done by using the Scion Imager after autoradiography scanning.For each sample, data were expressed as ratio of p44/p42 MAPK–phosphorylated and JNK/SAPK–phosphorylated proteins over total proteins.Representative of three individual experiments. *, P < 0.05, versus unstimulated cells.

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from SDF-1a–treated cells was higher as compared with that ofSDF-1a–untreated control cells (Fig. 6D; P < 0.05; n = 3).

Exposure to SDF-1a Increases the Invasive Potential ofHuh7 Cells

SDF-1a (3 nmol/L) inducedHuh7 invasion intoMatrigel (P <

0.05; Fig. 7A). This was inhibited by incubating the

cells with AMD3100 (51 F 17% inhibition; P < 0.05; n = 3;

Fig. 7B). AMD3100 itself did not affect basal invasion (data not

shown). Strikingly, heparin itself increased it by 50% (data not

shown). However, preincubation of SDF-1a with heparin

strongly reduced SDF-1a–induced cell invasion (70 F 15%

inhibition;P < 0.05; n = 3).Whereas treatment of cells with hDXdid not affect basal invasion, it reduced SDF-1a–dependent cellinvasion (82 F 12% inhibition; P < 0.05; n = 3; Fig. 7B).

Pretreatment of Huh7 cells with PD98059 or SP600125

abolished the increased invasion induced by SDF-1a (Fig. 7B).

Therefore, CXCR4, glycosaminoglycans, and MAPK pathways

are involved in this SDF-1 biological effect.

Interestingly, SDF-1a (3 nmol/L) increases matrix metal-

loproteinase-9 (MMP-9) mRNA levels in Huh7 cells (Fig. 7C),

as well as MMP-9 precursor, as assessed by zymography

(Fig. 7D). Huh7 cell preincubation with anti-MMP-9 mAb

resulted in a 42 F 14% inhibition (P < 0.05; n = 3) of SDF-

1a–dependent cell invasion (Fig. 7B).

SDC-4 Is Required for SDF-1–Induced Migration andInvasion of Huh7 Cells

Because SDF-1 binds to SDC-4 on Huh7 cells, we

investigated whether the down-regulation of SDC-4 by RNA

interference affects SDF-1–induced biological effects. Huh7

cells were transfected with SDC-4 double-strand RNA (SDC-4

dsRNA) or with a small interfering negative control RNA

(snc-RNA) for up to 3 days. Specific SDC-4 RNA

interference significantly reduced SDC-4 mRNA and protein

levels in Huh7 whereas SDC-1 expression remained un-

changed (Fig. 8A and data not shown). SDF-1a–inducedHuh7 cell growth was not affected by SDC-4 RNA

interference (data not shown). In contrast, in cells transfected

with SDC-4 dsRNA, SDF-1a–induced cell migration and

invasion were strongly reduced (Fig. 8B and C) compared

with snc-RNA–treated control cells (P < 0.05; n = 3). SDC-4

RNA interference did not affect basal migration or invasion

(data not shown).

Biological Effects of SDF-1 on HepG2 or Hep3BHepatoma Cell Lines

CXCR4 mRNAwas clearly expressed in HepG2 and faintly

so in Hep3B cells, whereas SDF-1 mRNAwas only detected in

HepG2 cells (Fig. 9A). CXCR4 was present at the plasma

membrane of both cell lines (Fig. 9B). SDF-1a bound to these

FIGURE 4. SDF-1 increases Huh7 cell proliferation. A. Cells were cultured for 48 h in the absence or presence of SDF-1a (3 or 125 nmol/L) andincubated with crystal violet. Data are expressed as absorbance at 595 nm. B and C. Cell viability was evaluated by MTT reduction assay. B. Cells werecultured for 48 h in the absence or presence of SDF-1a (at 3 or 125 nmol/L) and then assayed. Data are expressed as absorbance at 595 nm. *, P < 0.05,versus untreated cells. C. SDF-1a– induced Huh7 cell proliferation was decreased by AMD3100 (12 Amol/L) or by SP600125 (1 Amol/L) whereas PD98059(1 Amol/L) had no effect. Preincubation of SDF-1a with heparin (100 Ag/mL) or treatment of the cells with hDX (1 mmol/L) significantly reduced SDF-1a–induced cell proliferation. SDF-1a– induced proliferation of the cells in the absence of inhibitor (control) was set to 100%. SDF-1a– induced proliferation in thepresence of inhibitor is shown as a percentage of control. Columns, mean of triplicate determinations in three individual experiments; bars, SE. *, P < 0.05,versus SDF-1a– treated cells in the absence of inhibitor. D. Expression of heparan sulfate chains in hDX-treated Huh7 cells. Cells, treated or not with hDX (1mmol/L) for 72 h, were stained with anti –heparan sulfate 10E4 mAb or murine IgM. Fluorescence-activated cell sorting analysis revealed a significantlydecreased amount of positive cells for membrane-bound heparan sulfate on hDX treatment.

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cells (Fig. 9C). However, whereas SDF-1a induced a slight

but significant activation of JNK/SAPK in Hep3B cells, this

effect was not detected in HepG2 cells (data not shown).

Finally, SDF-1a did not induce the growth or the migration

of HepG2 or Hep3B cells (data not shown). In contrast,

whereas SDF-1a increased the invasion of Hep3B cells

(P < 0.05; n = 3), it did not exert such an effect on HepG2

cells (Fig. 9D).

Immunohistochemical Staining of CXCR4 in LiverSamples of Patients with HCC

Liver biopsies from two patients (with hepatitis C or B

virus–related cirrhosis and HCC) exhibited a strong membra-

nous CXCR4 staining of the carcinoma cell, irregularly

distributed throughout the samples (Fig. 10). In two other

patients (one with hepatitis C virus–related cirrhosis and one

with alcoholic-related cirrhosis, and HCC), a moderate nuclear

staining of scattered carcinoma cells with anti-CXCR4 mAb

was detected. No membranous staining was concurrently

observed. One patient (hepatitis B virus–related cirrhosis) did

not show any staining. Taking all samples into account, no

cytoplasmic staining was observed. Isotypic controls were

negative.

DiscussionIn the liver, chemokines are leukocyte chemoattractants and

can also stimulate key biological processes in hepatic stellate

cells, such as activation, proliferation, and migration (31, 32).

Recently, the CCL3/CCR1 axis has been shown to be involved

in HCC progression (33). CXCR4 expression has been shown

in hepatoma cells (21-23). Mitra et al. (21) showed that the

human HCC cell line HepG2 expresses CXCR4 but is

unresponsive to SDF-1 stimulation because of a defect of the

receptor. More recently, it has been shown that a strong

expression of CXCR4 in HCC specimens is significantly

associated with progressed HCC (23).

Table 1. SDF-1 Triggers Quiescent Huh7 Cells from G0 intoCycle

Untreatedcells

SDF-1 stimulatedcells

Proliferative labeling index: Ki67/DAPIpositive cells (immunocytochemistry), %

75 F 4 95 F 1*

Ki67 fluorescence intensity(flow cytometry)

7 F 0.5 21 F 1.2*

NOTE: Expression of Ki67 proliferation – associated nuclear antigen wasdetermined by immunocytochemistry and flow cytometry, as indicated. Theproliferative labeling index was as follows: (number of cells with moderate tostrong nuclear reactivity for anti-Ki67 mAb / the number of 4¶,6-diamidino-2-phenylindole (DAPI) – positive cells) � 100. The Ki67 fluorescence intensity wasthe difference between the mean fluorescence intensity of the cells labeled withanti-Ki67 mAb and that of the cells labeled with the isotype IgG1. Data are meanF SE of at least three independent experiments.*P < 0.05, versus untreated cells.

Table 2. SDF-1 Prevents Huh7 Cell Spontaneous DNADegradation and Stimulates the Transition of CellsAlready Engaged in G1 to S + G2-M

SDF-1 treatment % Cells in cell cycle phases (mean F SD)

Sub-G1 G0-G1 S + G2-M

� 51.5 F 9.4 28.7 F 8.4 19.8 F 4.8+ 25.0 F 6.8* 39.4 F 7.2* 35.6 F 6.9*

NOTE: Cells were serum deprived for 24 h and incubated for 48 h in mediumsupplemented or not (control) with SDF-1a (125 nmol/L). Cells were pretreatedwith RNase, stained with propidium iodide, and analyzed by flow cytometry.The cell histogram FL-2 was divided into three regions according to the cellcycle phases, sub-G1, G0-G1, and S + G2-M. Data are mean F SE of threeindependent experiments.*P < 0.05, versus untreated cells.

Table 3. SDF-1 Prevents TNFA–Mediated Apoptosis

TNFa-treatedcells

TNFa-treated cellsincubated with SDF-1

Annexin V–negative cells(flow cytometry), %

93.1 F 6 98.9 F 3*

Annexin V–positive cells(flow cytometry), %

6.8 F 0.5 1.1 F 0.3*

NOTE: Cells were serum deprived for 24 h, incubated for 48 h in mediumsupplemented with TNFa (50 nmol/L), and coincubated or not with SDF-1a (125nmol/L) for 48 h. Cell staining with Annexin V was analyzed by flow cytometry.Data are mean F SE of three independent experiments.*P < 0.05, versus SDF-1a–untreated cells.

FIGURE 5. SDF-1 induces the migration of Huh7 cells. A. SDF-1a at 0to 125 nmol/L concentrations, as indicated, or 20 ng/mL hepatocyte growthfactor (HGF ) was used for this experiment, and SDF-1a induced a dose-dependent Huh7 cell migration. Columns, mean of cells counted by fieldfor three independent experiments; bars, SE. *, P < 0.05, versus cellschemoattracted with 10% FCS (control). B. SDF-1a– induced Huh7 cellmigration was decreased by AMD3100 (12 Amol/L) or SP600125(1 Amol/L) whereas PD98059 (1 Amol/L) had no effect. Preincubationof SDF-1a with heparin (100 Ag/mL) or treatment of cells with hDX(1 mmol/L) significantly reduced SDF-1a– induced cell migration. Migra-tion induced in the controls (SDF-1a alone minus background ofunstimulated cells) was set to 100%. SDF-1– induced migration in thepresence of inhibitor is shown as a percentage of control. *, P < 0.05,versus SDF-1a– treated cells in the absence of inhibitor.

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Here, we show that Huh7 hepatoma cells express CXCR4

and secrete SDF-1a under basal conditions. Exogenous

SDF-1a binds to these cells through CXCR4 and glycosami-

noglycans, just as the CXCR4 antagonist AMD3100, or

heparin, strongly decreased this binding. Moreover, the SDF-

1/CXCR4 axis activates signaling pathways such as Erk2

and JNK/SAPK kinases as previously observed in other

tumor cells (34, 35). SDF-1 also induces reactive oxygen

species production through CXCR4 in Huh7 cells. Such

production may be an intracellular message involved in the

chemokine signaling pathways (36). However, glycosamino-

glycans interfere with SDF-1a– induced reactive oxygen

species production because preincubation of the chemokine

with heparin or treatment of the cells with heparitinases reduced

ROS level.

We next investigated whether SDF-1 modulates key

biological functions in Huh7 cells and explored the under-

lying molecular mechanisms. As recently described (23),

we observed that SDF-1a stimulates Huh7 cell prolifera-

tion as assessed by both crystal violet and MTT assays. We

showed that this stimulation depends on CXCR4, glycosami-

noglycans, and the JNK/SAPK kinase transduction path-

way. That SDF-1 triggers G0 quiescent Huh7 cells in G1 and

S + G2-M phases of cell cycle, as assessed by the expression

of the proliferation-associated nuclear antigen Ki67 and

propidium iodide-DNA binding assays, may also explain, at

least partly, the proliferative effect of SDF-1a in Huh7

cells. The fact that SDF-1 prevents spontaneous DNA degra-

dation in these cells and decreases the percentage of Annexin

V–positive cells in a TNFa-mediated apoptosis experimental

model also suggests that SDF-1 behaves as a survival factor for

these cells.

The effect of SDF-1 in Huh7 was considerably stronger on

migration and invasion than on proliferation and was also

mediated through CXCR4. Moreover, whereas SDF-1a–induced Huh7 cell migration, to some extent, depends on

JNK/SAPK signaling pathway, SDF-1a invasive effect is

very dependent on both Erk2 and JNK/SAPK kinase activa-

tions. This involvement of MAPK signaling pathways has

been reported in SDF-1 stimulatory effect on human pan-

creatic (37) or ovarian (38) cancer cell migration and inva-

sion. Cancer cell mobility depends on their interactions with

their microenvironment (39). HCC occurs mainly on fibrotic

liver and is associated with altered extracellular matrix

composition (40). The cleavage of basement membrane

boundaries by tumoral cells and invasion is mediated by

proteases, such as MMPs (39). MMP-9 overexpression,

associated with capsular infiltration and growth of HCC, has

been reported (41, 42).

Here, we show that SDF-1a increases MMP-9 expression in

Huh7 cells and that MMP-9 inhibition decreases SDF-1a–induced Huh7 cell invasion. Cell attachment to fibrillar

FIGURE 6. SDF-1 induces phos-phorylation of FAK at Tyr397 andreorganizes Huh7 cytoskeleton.Huh7 cells, incubated with SDF-1a(125 nmol/L), were examined byindirect immunostaining for phospho-tyrosine residues using anti-Tyr(P)mAbs (4G10; A) and for tyrosinephosphorylation of FAK at Tyr397

using anti-FAK-(P)-Tyr397 antibodies(B). The original fluorescent imageswere converted into negative ones.For visualization of filamentous actin(C), cells were labeled with Alexa-Fluor 568-phalloidin. Bar, 5 Am. D.Left, Western blot analysis of phos-phorylated ((P)Y577) and total formsof FAK in Huh7 cells that were eitheruntreated or stimulated with SDF-1a(3 and 125 nmol/L). Representative ofthree individual experiments. Right,quantification of FAK phosphorylationwas done by using the Scion Imagerafter autoradiography scanning. Foreach sample, data were expressed asa ratio of FAK-phosphorylated pro-teins over total proteins. Representa-tive of three individual experiments. *,P < 0.05, versus untreated cells.

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extracellular matrix and migration are mediated by structures

called ‘‘focal adhesions,’’ which connect the extracellular

matrix with the plasma membrane and the underlying actin

cytoskeleton (39). Moreover, SDF-1a enhances the tyrosine

phosphorylation of FAK, and probably that of the focal

adhesion complex components, and reorganizes Huh7 cyto-

skeleton.

These data strongly suggest the involvement of SDF-1 in the

highly regulated processes of HCC invasion and metastasis,

which are major determinants in HCC progression and

prognosis (43).

Our study also shows that CXCR4 and SDC-4 coim-

munoprecipitate with SDF-1 at the plasma membrane of

Huh7 cells. Whether two separate complexes or a trimole-

cular complex is formed on these cells can be hypothesized.

However, the fact that CXCR4 coimmunoprecipitates with

SDC-4 in the absence or presence of SDF-1 strongly argues

for the formation of one complex that comprises SDF-1,

CXCR4, and SDC-4. Because biotinylated SDF-1 binds to

electroblotted SDC-4, this also suggests a direct binding of

SDF-1 to SDC-4.

All biological effects, cell growth, migration, and invasion,

induced by SDF-1 on Huh7 cells were significantly affected by

preincubating the chemokine with heparin or by treating the

cells with h-DX, suggesting the involvement of glycosamino-

glycans. Moreover, SDF-1–induced migration and invasion

were reduced when SDC-4 cell expression was specifically

down-regulated by RNA interference. These results, which

agree with those previously observed in HeLa cells (17, 18),

suggest that SDC-4 expressed on Huh7 cells may be an

auxiliary receptor for SDF-1. Interestingly, previous studies

have shown that protein kinase Cy–dependent phosphorylationof SDC-4 regulates cell migration and that SDC-4 is required

for focal adhesion formation (44).

Strikingly, SDC-4 RNA interference did not affect SDF-

1–induced Huh7 cell growth, whereas hDX treatment of

the cells reduced it. It can therefore be hypothesized that

heparan sulfate chains expressed by other proteoglycans may

be involved in this effect. In fact, our immunoprecipitation

data do not exclude other heparan sulfate proteoglycans,

such as SDC-2, from binding to SDF-1. Indeed, SDC-2 is

expressed in Huh7 cells and has been shown to play an

important role in the tumorigenic activity of numerous tumor

cells (45, 46).

Otherwise, we previously showed that SDF-1 induces the

shedding of SDC-1 and SDC-4 ectodomains from HeLa cells

and that MMP-9 is involved in these events (47). Whether SDF-

1 also induces syndecan ectodomain shedding from Huh7 cells

is currently under investigation. In this context, the MMP-9

activation induced by SDF-1 in Huh7 cells could be part of an

autoregulatory/down-regulation cycle mediated by SDF-1/

MMP-9 syndecan ectodomain shedding (48).

In the present study, we observed that the HepG2 and

Hep3B hepatoma cell lines, characterized by different p53

status compared with Huh7 cells (49), expressed CXCR4.

Indeed, it was recently shown that CXCR4 expression does

not depend on p53 status (23). However, in agreement with

earlier published data (21, 23), HepG2 cells are unresponsive

FIGURE 7. SDF-1 induces the invasion of Huh7 cells.A. SDF-1a (3 nmol/L) induced Huh7 cell invasion into Matrigel. Hepatocyte growth factor (20 ng/mL)was used as positive control. Columns, mean of cells counted by field for three independent experiments; bars, SE. *, P < 0.05, versus cells chemoattractedwith 10% FCS (control). B. SDF-1a– induced Huh7 cell invasion was decreased by AMD3100 (12 Amol/L), SP600125 (1 Amol/L), and PD98059 (1 Amol/L) orby anti –MMP-9 mAb. Preincubation of SDF-1a with heparin or treatment of cells with hDX reduced SDF-1a– induced cell invasion. Invasion induced in thecontrols (SDF-1a alone minus background of unstimulated cells) was set to 100%. SDF-1a– induced invasion in the presence of inhibitor is shown as apercentage of control. *, P < 0.05, versus SDF-1a– treated cells in the absence of inhibitor or, in the case of anti-MMP-9 mAb experiments, versus SDF-1a–treated cells preincubated with murine IgG1.C andD. SDF-1a activates MMP-9.C.HuH7 cells were incubated or not for 16 h with SDF-1a (3 and 125 nmol/L).The effect on MMP-9 mRNAs synthesis was studied by RT-PCR. PCR products were analyzed on agarose gel stained with ethidium bromide. D. Analysis ofgelatinolytic activity of MMP-9 in the conditioned media of Huh7 cells. Cells were either untreated or treated with phorbol 12-myristate 13-acetate (PMA ; as apositive control) or SDF-1 (3 and 125 nmol/L) for 24 h. Conditioned media were collected and analyzed by gelatin zymography, done with equal amounts ofprotein loaded. Representative of three experiments.

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to SDF-1, and SDF-1 increases Hep3B cell invasion into

Matrigel, but not proliferation or migration. Therefore, SDF-1

biological effects on hepatoma cells strongly depend on the

cell type.

Finally, to evaluate to what extent our in vitro results

may be extrapolated to the in vivo situation, we explored

CXCR4 expression in liver samples of patients with HCC.

Immunohistochemical staining of HCC liver biopsies displayed

either membrane expression or nuclear localization of CXCR4

in hepatocarcinoma cells. In our preliminary study, we did

not find any reduced CXCR4 expression compared with the

surrounding nontumor tissue, in contrast to others (50).

Schimanski et al. (23) recently showed that a strong cyto-

plasmic expression of CXCR4 in HCC specimens is signifi-

cantly associated with progressed HCC. In contrast, an earlier

published study showed that the degree of CXCR4 expression

did not correlate with the clinicopathologic features of HCC

(51). Considering the fact that SDF-1 signals through CXCR4

and that SDC-4 may be an auxiliary receptor for the chemokine

on hepatoma cells, the relevance of the SDF-1/CXCR4 axis

and also of SDC-4 in the liver biopsies of HCC patients could

therefore be of interest.

In summary, our data indicate that the SDF-1/CXCR4

ligand receptor axis may play an important role in the

pathogenesis of HCC and that a CXCR4 receptor antagonist,

such as AMD3100, could inhibit cell growth, migration,

and invasion of hepatoma cells. Moreover, glycosaminogly-

cans modulate the effects of SDF-1 in hepatoma cells. A

better understanding of this chemokine effect on HCC

development and progression may enable novel chemokine

glycosaminoglycan mimetic–based immunomodulating drugs.

Materials and MethodsCell Culture

Huh7, HepG2, and Hep3B human hepatoma cell lines were

grown as described (52). For proteoglycan biosynthesis

inhibition, cells were incubated with hDX (Sigma-Aldrich,

Saint-Quentin Fallavier, France) for 72 h as described (53).

Flow Cytometry AnalysisCells (105) were incubated with biotinylated SDF-1a

(0, 12.5, 40, or 125 nmol/L; gift of F. Baleux, Laboratoire de

Chimie, Institut Pasteur, Paris, France) as described (17). In

parallel, cells were preincubated for 1 h at 37jC with

AMD3100 (1.2-12 Amol/L; Sigma-Aldrich) or biotinylated

SDF-1a was preincubated for 2 h at 20jC with heparin

(100 Ag/mL; low molecular weight heparin, H3149, Sigma-

Aldrich). After washing, cells were labeled for 30 min at +4jCwith streptavidin-Alexa Fluor 488 complex (1:100; Molecular

Probes, Invitrogen, Cergy-Pontoise, France). Flow cytometry

data of SDF-1 binding to the cells (B) were expressed as the

mean fluorescence intensity of the cells, incubated in the

presence of biotinylated SDF-1 (test) minus that of the cells,

incubated in the absence of the chemokine (negative control).

The percentage of inhibition of SDF-1 binding to the cells,

induced by AMD3100 or heparin, was calculated by dividing

the difference between B of the cells in the absence of the

inhibitor and B of the cells in the presence of the inhibitor (BI)

by B of the cells in the absence of the inhibitor, and then

multiplying by 100. Results were expressed as the mean

percentage of inhibition of at least three independent experi-

ments F SD. Statistical analysis of the coupled differences

between B and BI was done with Student’s t test.

FIGURE 8. SDC-4 is in-volved in the migration andinvasion of Huh7 cells, inducedby SDF-1a. Cells were trans-fectedwith eitherSDC-4 dsRNAor small interfering negativecontrol dsRNA (snc-dsRNA) orwere mock transfected. A.Left, Huh7 cells were analyzedfor SDC-1 and SDC-4 mRNAexpression by semiquantitativeRT-PCR, 3 d posttransfection.To normalize for input of totalRNA, glyceraldehyde 3-phos-phodehydrogenasemRNA levelwas also determined. Right,Huh7 cells were analyzed forSDC-4 protein expression byflow cytometry 3 d posttransfec-tion. Reactivity was comparedwith an isotype-matched controlantibody (murine IgG2a). Band C. SDF-1a– induced Huh7cell migration (B) or invasion(C) was abolished in SDC-4dsRNA– transfected cells ascompared with cells transfectedwith small interfering negativecontrol dsRNA (snc-dsRNA) ormock-transfected cells. Repre-sentative of three independentexperiments. Migration or inva-sion of the mock-transfectedcells induced by SDF-1a wasset to 100%. *, P < 0.05, versusmock-transfected cells.

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CXCR4 or SDC-4 immunostaining for flow cytometric

analysis was done using anti-CXCR4 mAb (clone 12G5, BD

Bioscience PharMingen, Pont de Claix, France; 10 Ag/mL) oranti–SDC-4 mAb (mouse IgG2a; clone 5G9, Santa Cruz

Biotechnology, Inc., Santa Cruz, CA; 10 Ag/mL) or murineIgG2a (BD Bioscience PharMingen) as described (17).

To assess hDX treatment efficiency, 105 Huh7 cells,

pretreated or not with hDX (1 mmol/L) for 72 h, were

incubated for 30 min on ice with 10 Ag/mL anti–heparan

sulfate mAb (clone 10E4, Seikagaku Corporation, Tokyo,

Japan) or murine IgM (BD Bioscience PharMingen). Cells

were then labeled with FITC-labeled goat anti-mouse

immunoglobulin (BD Bioscience PharMingen) and fixed in

1% paraformaldehyde and analyzed on a FACScan (Becton

Dickinson, Le Pont-de-Claix, France).

Immunofluorescence Staining and Microscopy AnalysisAdherent Huh7 cells were incubated for 1 h at 4jC,

with anti-CXCR4 mAb 12G5 (15 Ag/mL), anti–SDC-4 mAb

5G9 (10 Ag/mL) or murine IgG2a, then labeled as described

(17). Alternatively, cells were fixed with paraformaldehyde

(1%) and incubated for 1 h at 20jC with anti–SDC-1 mAb

B-B4 (10 Ag/mL; Serotec, Oxford, United Kingdom) or

murine IgG1 (BD Bioscience PharMingen). Cells were

observed under a fluorescence microscope (Olympus, Rungis,

France).

Reverse Transcription-PCRCXCR4, SDF-1, SDC-1, SDC-2, SDC-4, MMP-9 mRNAs

and glyceraldehyde 3-phosphodehydrogenase mRNA were

amplified by reverse transcription-PCR (RT-PCR; ref. 18).

Specific primers were designed as follows: SDF-1/CXCL12,

5¶-CCATGAACGCCAAGGTCGTGGTC-3¶ (forward) and

5¶-GGGCATGGATGAATATAAGCTGC-3¶ (reverse); CXCR4,

FIGURE 9. Hep3B and HepG2 cells express SDF-1 and CXCR4 and bind exogenous SDF-1. A. Semiquantitative RT-PCR analysis for the mRNAexpression of CXCR4, SDF-1, and glyceraldehyde 3-phosphodehydrogenase in Hep3B and HepG2 cells. B. HepG2 and Hep3B cells were stained for flowcytometry analysis with anti-CXCR4 12G5 mAb or mouse IgG2a. C. Biotinylated SDF-1a binds to HepG2 and Hep3B cells. Cells were stained withbiotinylated SDF-1a (125 nmol/L) and analyzed by flow cytometry. Reactivity was compared to streptavidin-Alexa Fluor 488. D. Exposure to SDF-1a(3 nmol/L) induced invasion of Hep3B but not of HepG2 cells. Columns, mean of cells counted by field for three independent experiments; bars, SE.*, P < 0.05, versus cells chemoattracted with 10% FCS.

FIGURE 10. Microphotographs representative of CXCR4 immunos-taining in HCC samples. A. In patient no. 1, an exclusively membranousstaining was irregularly distributed throughout the tumor sample. B. Inpatient no. 2, no staining was observed. C. In patient no. 3,immunostaining was similar to that observed in patient no. 1. D. In patientno. 4, scattered positive nuclei were detected whereas there was nocytoplasmic or membranous staining. A and B, �400. C and D, �1,000.

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5¶-AGTATATACACTTCAGATAAC-3¶ (forward) and 5¶-CCA-CCTTTTCAGCCAACAG-3¶ (reverse); SDC-1, 5¶-TCTGACA-ACTTCTCCGGCTC-3¶ (forward) and 5¶-CCACTTCTGGCA-GGACTACA-3¶ (reverse); SDC-2, 5¶-GGGAGCTGATGAGG-ATGTAG-3¶ (forward) and 5¶-CACTGGATGGTTTGCGTTCT-3¶(reverse); SDC-4, 5¶-CGAGAGACTGAGGTCATCGAC-3¶ (for-ward) and 5¶-CGCGTAGAACTCATTGGTGG-3¶ (reverse); andMMP-9, 5¶-AAGATGCTGCTGTTCAGCGGG-3¶ (forward)

and 5¶-GTCCTCAGGGCACTGCAGGAT-3¶ (reverse). In some

experiments, optimum semiquantitative RT-PCR conditions were

established to remain in the linear phase of amplification curve.

SDF-1a Quantification by ELISAHuh7 cells were serum deprived for 48 h. Culture

supernatants were tested by ELISA for SDF-1a (R&D Systems,

Villejust, France).

Coimmunoprecipitation of CXCR4 and SDC-4 withSDF-1a

Huh7 cells (106) were incubated in the presence or absence

of SDF-1a (2 Ag) and lysed. Lysates were subjected to

immunoprecipitation on protein G-Sepharose beads (Phar-

macia, Paris, France), precoated with anti–SDF-1a mAb

(goat IgG) or its isotype (both from R&D Systems; each at

2.5 Ag), or with anti–SDC-4 mAb 5G9 or its isotype (16).

The complexes were electroblotted (17) and revealed with

anti-CXCR4 12G5 mAb or, as a negative control, with anti-

CCR5 2D7 (BD Bioscience PharMingen). Alternatively, the

complexes were treated with heparitinase I (1 units/mL),

heparitinase III (15 units/mL), and chondroitinase ABC

(5 units/mL) mixture (Sigma-Aldrich) and were revealed

with anti–SDC-1 DL-101 mAb (Santa Cruz Biotechnology),

anti–SDC-4 5G9 mAb, or their isotypes (all at 1:1,000-

1:5,000). After washing, strips were incubated with horseradish

peroxidase–conjugated antimouse IgG (1:5,000-1:20,000) and

revealed by enhanced chemiluminescence reagent (Amersham

Biosciences, Buckinghamshire, United Kingdom). In some

experiments, strips were revealed with biotinylated SDF-1a, asdescribed (17).

Reactive Oxygen Species ProductionCells (105) were stimulated with SDF-1a (3 nmol/L). In

parallel, cells were incubated with both AMD3100 (1.2-12

Amol/L) and SDF-1a (3 nmol/L). Alternatively, SDF-1a was

preincubated for 2 h at 20jC with heparin (100 Ag/mL) andthe suspension was added to the cells. In some experiments,

cells were pretreated for 2 h with heparitinase I (EC 4.2.2.8;

100 mIU/mL) and heparitinase III (EC 4.2.2.7.; 200 mIU/mL;

Sigma-Aldrich; refs. 16, 17).

Cells were then incubated for 30 min at 37jC in the dark

with a 10 Amol/L PBS-dichlorofluorescein diacetate solution

(Molecular Probes). Unstimulated control cells were incubated

in parallel.

Activation of Erk1/2, JNK/SAPK Kinases, and FAK bySDF-1a

Huh7 cells (2.5 � 105) were cultured for 48 h in 0.1% FCS-

DMEM and incubated at 37jC for 15 min with SDF-1a (3 and

125 nmol/L). MAPKs were revealed (17) using antibodies

specific for phospho-Erk1/2 (p44/p42) [Thr202/Tyr204] or

phospho-JNK/SAPK (p54/p46) [Thr183/Tyr185] or for their total

counterparts (all from Cell Signaling, Danvers, MD). Phos-

phorylated FAK was revealed using polyclonal anti-FAK-(P)-

Tyr577 antibodies (Cell Signaling). Parallel immunoblotting

with anti total FAK polyclonal antibodies (Cell Signaling) was

done to confirm equal loading of samples. Quantification of

Erk1/2, JNK/SAPK, and FAK phosphorylation was done by

using the Scion program after autoradiography scanning.

Crystal Violet AssayCells (5 � 103) were treated for 48 h at 37jC with SDF-1a

(0, 3, and 125 nmol/L), then fixed and incubated for 2 min

with 0.08% crystal violet (Sigma). Cell proliferation was

assessed by colorimetric assay. Absorbance was read at

595 nm with a microplate reader (model 680, Bio-Rad,

Ivry-sur-Seine, France).

MTT AssayCell viability was measured using the reduction of MTT

(Sigma-Aldrich). Cells (5 � 103) were treated for 48 h with

SDF-1a (0, 3, and 125 nmol/L). In parallel, cells were pre-

treated for 1 h at 37jC with AMD3100 (1.2-12 Amol/L) or for30 min at 37jC with PD98059 (1 Amol/L) and SP600125

(1 Amol/L; Calbiochem, Fontenay-sous-Bois, France) before

the addition of SDF-1a (3 nmol/L). Alternatively, cells were

treated with 1 mmol/L hDX for 24 h and 5 � 103 hDX-pretreated cells were further incubated with 1 mmol/L hDXand 125 nmol/L SDF-1a for 48 h. Cells were then incubated

with 0.5 mg/mL MTT for 1 h at 37jC. After MTT withdrawal,

the resulting blue formazan cristae were solubilized in DMSO

(Merck, Fontenay-sous-Bois, France). Absorbance was read at

595 nm.

Ki67 Proliferation–Associated Nuclear Antigen Immuno-staining and Cell Cycle Analysis

Cells were serum deprived for 24 h, incubated for 4 h at

37jC in 10% FCS-DMEM supplemented or not with SDF-1a(125 nmol/L), washed with PBS-0.1% bovine serum albumin,

fixed with paraformaldehyde (1%), and permeabilized in 0.05%

Triton X-100 (Sigma-Aldrich). Expression of Ki67 prolifera-

tion–associated nuclear antigen was assessed with an anti-Ki67

mAb (IgG1; 1:500; Santa Cruz Biotechnology). Cells were then

incubated with Alexa Fluor 488 goat anti-mouse IgG (1:400).

Ki67 immunostaining was either analyzed directly by flow

cytometry or counterstained with 0.1 Ag/mL 4¶,6-diamidino-2-phenylindole (Sigma-Aldrich) to evaluate nuclei number in

each cell field counted.

Cell cycle studies were done by means of DNA-propidium

iodide binding. Cells were serum deprived for 24 h and

stimulated with SDF-1a (125 nmol/L) for 48 h. After fixation

with ethanol, cells were incubated with RNase A (200 Ag/mL;Sigma-Aldrich) for 30 min and resuspended in propidium

iodide-PBS (10 Ag/mL) for flow cytometric analysis.

The percentage of cells undergoing apoptosis was determined

using Annexin V detection assay (R&D Systems). Briefly,

Huh7 cells were incubated for 48 h under apoptosis-inducing

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conditions (50 ng/mL TNFa) with or without SDF-1a (125

nmol/L). Cells (105) were then incubated at room temperature

for 15 min with fluorescein-conjugated human Annexin V and

analyzed.

Cell Migration and Invasion AssaysCell migration or invasion was done using Bio-coat cell

migration chambers (Becton Dickinson). Inserts containing

8-Am pore size filters were coated with fibronectin (100 Ag/mL;Santa Cruz Biotechnology) for migration or Matrigel

(320 Ag/mL BD PharMingen) for invasion assay. After filter

blockage with 1 mg/mL bovine serum albumin for 1 h, 2.5 �105 cells in 0.1% bovine serum albumin-DMEM were added.

The chemokine SDF-1a was added to 500 AL of DMEM

supplemented with 10% FCS in the lower chamber. After 24 h,

cells that had migrated through the filter pores were fixed with

methanol, stained with hematoxylin, and counted. In parallel,

cells were preincubated for 2 h at 37jC with inhibitors

AMD3100 (12 Amol/L), anti-MMP-9 mAb (10 Ag/mL, IgG1;Santa Cruz Biotechnology), murine IgG1 (10 Ag/mL; BD

Bioscience PharMingen), PD98059 (1 Amol/L), or SP600125(1 Amol/L). Alternatively, cells were treated with 1 mmol/L

hDX for 48 h, and for each insert, 2.5 � 105 cells in 0.1%

bovine serum albumin-DMEM were further incubated with

1 mmol/L hDX for 24-h migration or invasion assay. The

percentage of inhibition was [(D1 � D2) / D1] � 100; D1 was

the difference between the number of untreated cells that

migrated toward SDF-1a and that of untreated cells that

migrated toward the culture medium without SDF-1; D2 was

the difference between the number of treated cells that migrated

toward SDF-1a and that of treated cells that migrated toward

culture medium (D2). Alternatively, SDF-1a was preincubated

for 2 h at 20jC with heparin (100 Ag/mL). Heparin alone or

SDF-1a preincubated with heparin was added to the lower

chamber of culture. The percentage of inhibition was [(D1 �D3) / D1] � 100, where D3 was the difference between the

number of cells that migrated toward SDF-1a preincubated

with heparin and the number of cells that migrated toward

heparin alone.

Gelatin ZymographyGelatin zymography was done as described (47). Briefly,

Huh7 cells were incubated for 24 h in serum-free medium

supplemented or not with phorbol 12-myristate 13-acetate

(0.5 Amol/L), used as a positive control, or SDF-1a (3 and

125 nmol/L). Conditioned media were resolved on 10%

SDS-PAGE, 0.1% gelatin (Sigma-Aldrich), with equal amounts

of proteins loaded. After SDS extraction, gelatinolytic acti-

vity was developed in buffer [50 mmol/L Tris-HCl (pH 7.4),

5 mmol/L CaCl2, 200 mmol/L NaCl, and 0.05% Brij 35;

Sigma-Aldrich] at 37jC for 24 h. The gel was stained with

Coomassie blue R-250, destained, and scanned.

Phosphotyrosine Residue ImmunostainingHuh7 cells were serum deprived for 24 h, incubated for

20 min at 37jC in 10% FCS-DMEM supplemented or not with

SDF-1a (125 nmol/L), fixed with paraformaldehyde (1%), and

permeabilized in 0.05% Triton X-100 (Sigma-Aldrich). Cells

were immunostained on phosphotyrosine residues using Tyr(P)

mAb (4G10; 10 Ag/mL, Cell Signaling) and Alexa Fluor 488

goat anti-mouse IgG (1:400). Cells were also examined by

indirect immunostaining for tyrosine phosphorylation of FAK at

Tyr397 using polyclonal anti–FAK-(P)-Tyr397 antibody (Cell

Signaling) and Cy3-conjugated goat anti-rabbit polyclonal

antibodies (1:400). For visualization of filamentous actin, cells

were then exposed to Alexa Fluor 568-phalloidin (1:1,000;

Molecular Probes) for 30 min at 37jC.

RNA InterferenceSDC-4 gene–specific sense and antisense 21-nucleotide

single-stranded RNAs with symmetrical two-nucleotide 3¶(2¶-deoxy)thymidine overhangs were designed as described (17).

For RNA interference experiments, dsRNAs were generated by

mixing equimolar amounts (50 Amol/L) of sense and antisensesingle-stranded RNAs in annealing buffer as described (17).

Huh7 cells were transfected with 150 nmol/L dsRNA in serum-

free medium using Jetsi transfectant reagent (Eurogentec,

Seraing, Belgium) following the manufacturer’s instructions.

Mock cells were cultured in parallel and transfected with the

transfection mixture lacking dsRNA. In each experiment, a

snc-RNA (Eurogentec) was used. Cells transfected with SDC-4

dsRNA or small interfering negative control dsRNA were used

3 days posttransfection for further analysis.

Immunohistochemical Staining of CXCR4 in HCCHCC samples were obtained from five patients with

cirrhosis who underwent ultrasound guided biopsy of hepatic

nodules. Two patients had hepatitis C virus–related cirrhosis,

two had hepatitis B virus–related cirrhosis, and one had

alcoholic-related cirrhosis. Four-micrometer-thick paraffin-

embedded, alcohol/formalin/acetic acid– fixed liver biopsy

sections were deparaffinized and hydrated. Heat antigen

retrieval was done by incubating the slides in 10 mmol/L

sodium citrate buffer (pH 6) for 20 min. After preincubation

with hydrogen peroxide, the slides were incubated overnight at

4jC with primary antibody to CXCR4 (clone 12G5, Zymed,

San Francisco, CA) at 1:100 dilution. Labeling was visualized

using a streptavidin-peroxidase complex and diaminobenzidine

as the chromogen. Slides were counterstained with Mayer’s

hemalum. Isotypic (IgG2a) negative controls were done on each

sample. For positive controls, paraffin-embedded tissue sam-

ples of metastatic breast carcinoma were used.

Statistical AnalysisFor the determination of statistical significance, ANOVA test

was done with the Statview software. P < 0.05 was used as the

criterion of statistical significance.

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Role of SDF-1 in Hepatocellular Carcinoma Progression

Mol Cancer Res 2007;5(1). January 2007

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2007;5:21-33. Mol Cancer Res   Angela Sutton, Veronique Friand, Severine Brulé-Donneger, et al.   Migration, and InvasionLigand 12 Stimulates Human Hepatoma Cell Growth,

Derived Factor-1/Chemokine (C-X-C Motif)−Stromal Cell

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