RGD-FasL induces apoptosis in hepatocellular carcinoma.

Abstract

Despite impressive results obtained in animal models, the clinical use of Fas ligand (FasL) as an anticancer drug is limited by severe toxicity. Systemic toxicity of death ligands may be prevented by using genes encoding membrane-bound death ligands and by targeted transgene expression through either targeted transduction or targeted transcription. Selective induction of tumor cell death is a promising anticancer strategy. A fusion protein is created by fusing the extracellular domain of Fas ligand (FasL) to the peptide arginine-glycine-aspartic acid (RGD) that selectively targets avbeta3-integrins on tumor endothelial cells. The purpose of this study is to evaluate the effects of RGD-FasL on tumor growth and survival in a murine hepatocellular carcinoma (HCC) tumor model. Treatment with RGD-FasL displaying an obvious suppressive effect on the HCC tumor model as compared to that with FasL (p < 0.05) and resulted in a more additive effect on tumor growth delay in this model. RGD-FasL treatment significantly enhanced mouse survival and caused no toxic effect, such as weight loss, organ failure, or other treatment-related toxicities. Apoptosis was detected by flow cytometric analysis and TUNEL assays; those results also showed that RGD-FasL is a more potent inducer of cell apoptosis for H22 and H9101 cell lines than FasL (p <0.05). In conclusion, RGD-FasL appears to be a low-toxicity selective inducer of tumor cell death, which merits further investigation in preclinical and clinical studies. Furthermore, this approach offers a versatile technology for complexing target ligands with therapeutic recombinant proteins. To distinguish the anti-tumor effects of FasL in vivo, tumor and liver tissues were harvested to examine for evidence of necrotic cells, tumor cells, or apoptotic cells by Hematoxylin and eosin (HE) staining.

Full text

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Cellular & Molecular Immunology 285


Article
RGD-FasL Induces Apoptosis in Hepatocellular Carcinoma


Zhongchen Liu1, 3, 4, Juan Wang1, 2, 4, Ping Yin3, Jinhua Qiu1, Ruizhen Liu1, Wenzhu Li1, 2, Xin Fan1,
Xiaofeng Cheng1, Caixia Chen1, 2, Jiakai Zhang2 and Guohong Zhuang1, 5

Despite impressive results obtained in animal models, the clinical use of Fas ligand (FasL) as an anticancer drug is
limited by severe toxicity. Systemic toxicity of death ligands may be prevented by using genes encoding
membrane-bound death ligands and by targeted transgene expression through either targeted transduction or
targeted transcription. Selective induction of tumor cell death is a promising anticancer strategy. A fusion protein is
created by fusing the extracellular domain of Fas ligand (FasL) to the peptide arginine-glycine-aspartic acid (RGD)
that selectively targets avβ3-integrins on tumor endothelial cells. The purpose of this study is to evaluate the effects
of RGD-FasL on tumor growth and survival in a murine hepatocellular carcinoma (HCC) tumor model. Treatment
with RGD-FasL displaying an obvious suppressive effect on the HCC tumor model as compared to that with FasL
(p < 0.05) and resulted in a more additive effect on tumor growth delay in this model. RGD-FasL treatment
significantly enhanced mouse survival and caused no toxic effect, such as weight loss, organ failure, or other
treatment-related toxicities. Apoptosis was detected by flow cytometric analysis and TUNEL assays; those results
also showed that RGD-FasL is a more potent inducer of cell apoptosis for H22 and H9101 cell lines than FasL (p <
0.05). In conclusion, RGD-FasL appears to be a low-toxicity selective inducer of tumor cell death, which merits
further investigation in preclinical and clinical studies. Furthermore, this approach offers a versatile technology for
complexing target ligands with therapeutic recombinant proteins. To distinguish the anti-tumor effects of FasL in
vivo, tumor and liver tissues were harvested to examine for evidence of necrotic cells, tumor cells, or apoptotic cells
by Hematoxylin and eosin (H&E) staining. Cellular & Molecular Immunology. 2009;6(4):285-293.

Key Words: FasL, RGD-FasL, apoptosis, tumor targeting


Introduction

Hepatocellular carcinoma (HCC) is one of the most common
cancers and has an extremely poor prognosis because of its
aggressive invasion, resistance to existing chemotherapeutic
agents, and lack of specific symptoms. Hepatitis B virus
(HBV) infects approximately two billion individuals
Volume 6 Number 4 August 2009
worldwide and causes an estimated 320,000 deaths annually.
Approximately 30%-50% of HBV-related deaths are
attributable to HCC (1). Traditionally, chemotherapy is the
primary option for the treatment of unresectable solid tumors;
however, these drugs are not effective in promoting tumor
regression and prolonging survival in HCC (2). Moreover,
conventional therapeutic modalities, such as transcatheter
arterial embolization, radiofrequency
microwave coagulation therapy are no longer recommended
because of their low efficacy and potential complications (3).
Therefore, a new effective modality is needed to treat HCC.
Apoptosis, or programmed cell death, is a key mechanism
that multicellular organisms utilize to regulate cell growth
and to prevent pathological processes, such as autoreactivity,
cancer, and immunodeficiency (4). Fas ligand (FasL), a type
II membrane protein, belongs to the tumor necrosis factor
(TNF) superfamily. FasL is expressed by activated T
lymphocytes, natural killer (NK) cells, and a small number of
ablation, and
1Anti-Cancer Research Center, Medical College, Xiamen University, 422
Siming South Road, Xiamen 361005, China;
2School of Life Science, Xiamen University, 422 Siming South Road,
Xiamen 361005, China;
3Zhongshan Hospital, Xiamen University, Lakeside South 201-209, Xiamen
361004, China;
4These authors contributed equally to this work;
5Corresponding author: Dr. Guohong Zhuang, Anti-Cancer Research Center,
Medical College, Xiamen University, 422 Siming South Road, Xiamen
361005, China. Tel: +86-592-2180-587; Fax: +86-592-2186-731; E-mail:
zhuangguohong@yahoo.com.cn
Received Feb 20, 2009. Accepted Aug 10, 2009.

© 2009 Chinese Society of Immunology and University of Science & Technology
of China
Abbreviations: FasL, Fas ligand; RGD, arginine-glycine-aspartic acid; HCC,
hepatocellular carcinoma; H22, hepatocellular carcinoma 22; H9101, human
hepatocellular carcinoma 9101; ADM, adriamycin; mAb, monoclonal
antibody; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-
biotin nick end-labeling; TNF, tumor necrosis factor.

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286 Apoptosis and cancer therapy
Volume 6 Number 4 August 2009
non-lymphoid cells (5), suggesting that Fas-FasL system may
play an important role in the privileged status (6). These
findings may indicate that gene transfer of FasL induces
apoptotic responses.
Despite the impressive results obtained with various
animal models, the clinical use of FasL as an anticancer drug
is limited by potential systemic toxicity. For this reason, FasL
can be administered to patients only regionally. Regional
administration of relatively high doses of TNF in
combination with chemotherapeutic drugs has produced
high-response rates in patients with advanced limb tumors
(7-9), as well as regression of primary and metastatic tumors
confined to the liver (10). These results are of particular
interest, because they show that, in principle, the antitumor
effects of FasL may be exploited therapeutically in humans
with greater success if sufficient dose levels can be attained
locally, and if the organism can be exempted from the
systemic toxic effects.
The tripeptide sequence Arg-Gly-Asp (RGD) is a well
known motif that recognizes and interacts with integrins, a
family of transmembrane heterodimeric glycoproteins
composed of one α and one β subunit (11). Proteins that
contain the RGD attachment site, together with the integrins
that serve as receptors for them, constitute a major
recognition system for cell adhesion (12). The RGD
sequence is the cell attachment site of a large number of
adhesive proteins in the extracellular matrix, blood, and cell
surface, and nearly half of the more than 20 known integrins
recognize and bind to related sequences in their adhesion
protein ligands (13). The integrin-binding activity of
adhesion proteins can be reproduced by short synthetic
peptides containing the RGD sequence. Such peptides
promote cell adhesion when insolubilized onto a surface and
inhibit it when presented to cells in solution. As the
integrin-mediated cell attachment influences and regulates
cell migration, growth, differentiation, and apoptosis, RGD
peptides and mimics can be used to probe integrin functions
in various biological systems (14).
Kuroda K., Miyata K. and others have reported that the
rhTNF mutant V29 has potent antitumor activity with
reduced toxicity in mice (15), whereas another mutant,
RGD-T, showed decreased gastrointestinal toxicity (16).
RGD-T also overcames the detrimental metastasis-enhancing
activity of TNF (17). To eliminate systemic toxicity,
including the hypotension associated with human TNF-, we
constructed mutant proteins (muteins) by means of genetic
engineering. A novel mutein, F4614, which resulted in the
introduction of cell-adhesive Arg-Gly-Asp and 29Arg-->Val,
had remarkably reduced hypotensive effects and lower
lethality (18).
We have prepared a fusion protein RGD-FasL by
recombinant DNA technology and investigated its in vitro
and in vivo antitumor activity. Since RGD-based anti-
angiogenic therapies are currently under development, and
cyclic Arg-Gly-Asp peptide can also serve as a ligand in
human nuclear medicine, optimization of its specificity and
drug delivery options is of major importance for clinical
applications.
Materials and Methods

Cell culture and mouse tumor model
Hepatoma H22 cells were obtained from the China Center for
Type Culture Collection, (CCTCC, Wu Han, China); H9101
cells (Human hepatocellular carcinoma cells) were
maintained in our laboratory. H22 and H9101 cells were
cultured in RPMI 1640, 1% L-glutamine, 1% penicillin-
streptomycin, and 10% fetal calf serum (Wako Pure
Chemical Ind., ltd., Japan). The cells were maintained at
37C in an atmosphere of 5% CO2 in humidified air. Inbred
BALB/c wild-type (WT) mice were purchased from Animal
Center for SIAS (Shanghai, China). They were bred and
maintained at the Animal Research Central of Xiamen
University. All experiments were performed in accordance
with guidelines set out by the animal experimental ethics
committee.

Expression and purification of recombinant FasL and
RGD-FasL
First, the FasL fragment was released from the FasL-pET22b(+)
plasmid by polymerase chain reaction (PCR) amplification
using the upstream primer F1 (5’-CAG CAG CCC TTC AAT
TAC CC-3’) and the downstream primer F18 (5’-GCT GCT
CGA GCT TAT ATA AGC-3’). Overlapping PCR was
performed to link RGD with FasL. Finally, the full-length
RGD-FasL gene was obtained by PCR using primers F2
(5’-GTA GGA TCC TCA TGG GCT GCG ATT GTC G-3’)
and F18 (5’-GCT GCT CGA GCT TAT ATA AGC-3’). The
PCR products and the plasmid were digested with BamHI
and XhoI restriction enzymes, respectively. This RGD-FasL
cDNA was purified by agarose gel electrophoresis and
subcloned into the responding sites of the eukaryotic
expression plasmid pGEX-5X-1 with T4 DNA ligase. After
amplification, the recombinant plasmid DNA was transformed
into competent Escherichia coli (E. coli) BL21 (DE3) and
screened for ampicillin sensitivity. Subsequently, the plasmid
DNA was extracted. After identification of the plasmid
restriction fragments, the positive recombinant clones were
sequenced at Shanghai Handsome Biotechnology Ltd. A
single positive E. coli Bl21 (DE3) pGEX-5X-1/ RGD-FasL
clone was incubated in shaker flasks at 37°C in 3 mL Luria
Bertani (LB) medium for 18-24 h. For large-scale pre-
parations, a 3 ml aliquot of the overnight culture was added
to 250 mL of fresh LB medium and incubated in shaker
flasks shaken at 250 rpm at 37°C. When the cell density
reached an A600 = 0.6~0.8, recombinant protein expression
was induced with isopropylthiogalactoside (IPTG). This
induction was optimized under a variety of conditions, such
as IPTG concentration, induction time and temperature.
The fusion protein was purified by glutathione-S-
transferase-affinity column chromatography (according to the
manufacturer’s protocols provided by GenScript Corporation)
and was identified by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) electrophoresis.

Western blot analysis
FasL protein was resolved on a 15% SDS-PAGE gel under

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Cellular & Molecular Immunology 287
Volume 6 Number 4 August 2009
reducing conditions and was transferred to PVDF membranes.
After blocking with 5% non-fat milk for 1 h, the PVDF
membrane was incubated with anti-human FasL monoclonal
antibody (reserved by our lab). Then, the membrane was
washed three times with phosphate buffered saline Tween-20
(PBST), followed by incubation for 1 h with HRP conjugated
anti-goat antibody. The membrane was washed and then
developed with an enhanced chemiluminescence (ECL)
detection system (Cell Signaling, USA), followed by
exposure to X-ray film.

Cell adhesion assay
Polyvinyl chloride microtiter plates were coated with
RGD-FasL or FasL solutions at various concentrations
(60,000, 30,000, 15,000, 7,500, 3,750 and 1,875 ng/ml) (100
µl/well in 150 mM sodium chloride, 50 mM sodium
phosphate, pH 7.3) at 4°C overnight, then each well was
washed with 0.9% sodium chloride, filled with DMEM
containing 2% BSA at 37°C for 45 min, and washed again.
Cells were harvested and washed three times with 0.9%
sodium chloride, resuspended in incomplete DMEM, and
added to FasL or RGD-FasL-coated plates (3  104 cells/100
µl well). Then the cells were incubated for 1-1.5 h at 37°C,
5% CO2. Unbound cells were removed by washing with
incomplete DMEM. Adherent cells were fixed with 3%
paraformaldehyde, 2% sucrose in PBS (pH 7.3), stained with
0.5% crystal violet, and then quantified by reading the
absorbance of each well at 540 nm using a microplate reader.

Analysis of Fas and CD61 expression in tumor cell lines by
flow cytometry
H22 cells and H9101 cells (l × 106 cells/ml) were washed
twice with PBS containing 1% (w/v) bovine serum albumin
(BSA). They were incubated on ice for 40 min with
anti-human Fas (Sigma, USA) and anti-CD61 mAb (Sigma,
USA). The cells were washed in cold PBS three times,
followed by incubation with (FITC)-conjugated affinity-
purified goat anti-rabbit IgG (Sigma, USA) for 30 min on ice.
The cells were washed and were then analyzed by a
FACSCalibur flow cytometer (Becton Dickinson, USA).

In vitro cytolytic assay
The effects of FasL and RGD-FasL on cell proliferation were
measured using an MTT-based assay (Microtiter-tetrazolium,
Sigma, USA). Briefly, the cells (5,000/ml) were incubated in
triplicate in a 96-well plate (Costar, Cambridge, MA, USA)
in the presence of various concentrations of FasL and
RGD-FasL (1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 g/ml)
in a final volume of 200 l for the indicated times. Thereafter,
20 l of MTT solution (5 g/L) was added to each well and
then incubated for 12 h. After centrifugation, the supernatant
was removed from each well. The colored formazan crystal
produced from MTT was dissolved in 150 ml DMSO. Then,
the optical density (OD) value was measured at 490 nm by a
multiscanner autoreader (Dynatech MR 5000, Chantilly, VA).
The following formula was used: cell proliferation inhibited
(%) = [1 - (OD of the experimental samples / OD of the
control)] × 100%.

Detection of apoptosis by flow cytometric analysis
To determine cell of apoptosis, FasL/RGD-FasL treated or
untreated cells (50 μg/ml, 6 h) were washed in PBS and
resuspended in binding buffer at a concentration of 1 × 106
cells/ml. After incubation, 195 μl of the solution was
transferred into a 5 ml culture tube with 5 μl annexin V-FITC
(BD, USA). The tube was then incubated for 30 min at room
temperature in the dark. The cells were washed with binding
buffer and resuspended in 190 μl binding buffer, with 10 μl
PI added. Finally, the tube was gently vortexed and incubated
for another 30 min in the dark. Cells were analyzed by
FACSCalibur with CellQuest software.

In vivo animal tumor model experiment
All experiments were performed in accordance with
guidelines set out by the Animal Experimental Ethics
Committee. Single-cell suspensions (1.0 × 105 cells in 100 μl)
of H22 were injected s.c. into the right anterior flank of male
BALB/c mice (9-12 weeks old). Tumor volume (V) was
measured using the formula (L × W2 × 0.52), where L and W
are the length and width, respectively. Measurements were
made with a vernier caliper. Tumor growth was allowed to a
volume of 40-50 mm3. At this point, mice were randomly
assigned to different experimental groups. Group 1 (n = 6)
received 0.9% NaCl (200 l), group 2 (n = 6) received FasL
(30 g/mouse in 200 l of 0.9% NaCl), group 3 (n = 6)
received RGD-FasL (30 g/mouse in 200 l of 0.9% NaCl),
and group 4 (n = 6) received ADM (30 g/mouse in 200 l
of 0.9% NaCl) via i.v. tail vein injection. Injections were
administered once daily. According to our project license,
animals had to be sacrificed when tumors became too large,
if mice lost >20% of body weight, or at signs of pain.
Blood samples were immediately taken retro-orbitally
from all groups to observe the levels of serum alanine
transaminase (ALT) and aspartate aminotransferase (AST).
Standard procedures were used to determine blood
parameters using an automated analyzer according to
manufacturer’s instructions (Cobas Integra 400, Roche
Analytics, Germany).
Tumors and livers were excised and fixed in 4% buffered
paraformaldehyde solution before embedding in paraffin.
Tumor sizes are shown as mean ± SE (6 animals/group).
Antitumor activity was assessed by the inhibition rate (IR) of
tumor growth 18 days after implantation. The tumor growth
inhibition rate (IR) was calculated as follows: IR = (1-T/C) ×
100%. Where T is the mean tumor volume in the group
treated with FasL or RGD-FasL, and C is the mean tumor
volume in the control group treated with saline.

Hematoxylin-and-eosin staining (H&E) analysis
To distinguish antitumor effect of FasL in vivo, HE staining
was used. Tumor and liver tissues were harvested after the
mice died (n = 6/group). The HE stained tissues were
examined for evidence of necrotic cells, tumor cells, or
apoptotic cells. HE-stained sections were viewed using an

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288 Apoptosis and cancer therapy
Volume 6 Number 4 August 2009
Olympus BHT microscope (Melville, NY, USA).

In situ terminal deoxynucleotidyl transferase assay (TUNEL)
Cell apoptosis, were examined by TUNEL method using the
Apop Tag in situ apoptosis detection Kit (Chemicon
International, Inc., Temecula, CA). This method can detect
fragmented DNA ends of apoptotic cells. Briefly, the paraffin
embedded sections were deparaffinized in xylene and
rehydrated in a graded series of ethanol baths. The sections
were treated with 20 mg/ml of proteinase K in distilled water
for 10 min at room temperature. The tumor tissues were fixed
in 1% paraformaldehyde for 10 min. To block endogenous
peroxidase, the slides were incubated in methanol containing
0.3% hydrogen peroxide for 20 min. The remaining
procedures were performed according to the instructions
provided by the manufacturer. To quantify the degree of
apoptosis, apoptotic cells were counted by three independent
observers who were blinded to the experimental protocol.
The apoptotic index was expressed as the percent of the total
number of myocardial cells.

Statistical analysis
Data were presented as the mean ± SD per group. Statistical
analysis was made for multiple comparisons using analysis of
variance and Student’s t-test. A p-value < 0.05 was
considered to be statistically significant.

Results

Production of recombinant FasL and RGD-FasL
The E. coli expression system was used to prepare the
inclusion form of the RGD-FasL fusion protein. After
isolation and purification, the purity of the target protein was
more than 95%. The mobility of the purified protein, as
determined by SDS-PAGE, was observed at a molecular
weight of 62 kDa (Figure 1A). As shown in Figure 1A, the
RGD-FasL migrated at the expected molecular weight of 62
kDa, and no degradation was observed. Western blot analysis
confirm the production of recombinant RGD-FasL protein
using a goat anti-human FasL antibody.

RGD-FasL promotes cell adhesion and spreading
To assess whether the RGD domain of RGD-FasL is
functional and accessible to integrins, we compared their cell
pro-adhesive properties with a cell adhesion assay. This
assay takes advantage of the fact that RGD is the minimal
recognition sequence of many integrins and serves as an
adhesion motif for them. To this aim, microtiter wells were
coated with various amounts of RGD-FasL or FasL and
seeded with H22 and H9101 cells. After 1.5 h, cell adhesion
was measured. We observed that adhesion and spread of the
H22 and H9101 cells on RGD-FasL-coated plates was much
greater than that on FasL-coated plates (Figure 1B). These
results strongly suggest that the RGD domain of RGD-FasL
was sufficiently folded and able to interact with adhesion
receptors, most likely integrins, on the cell membrane.

Surface expression of Fas and CD61 in different cell lines
The tripeptide sequence Arg-Gly-Asp (RGD) is a well known
motif recognizing and interacting with integrins, a family of
transmembrane heterodimeric glycoproteins composed of
one  and one  subunit; the  subunits are also called CD61.
We examined the cell-surface expression of Fas and CD61 in
different cell lines by flow cytometry. As represented in
A
M 1 2 3 4 5 6 7 8
66.2
66.2
42.7
42.7
20.4
20.4
B
SDS-PAGEWB
H22
H9101
RGD-FasLFasL
Control


Figuer 1. Recombinant RGD-FasL protein purification and
activity identification. (A) Purified RGD-FasL proteins were
analyzed by SDS-PAGE or Western blot. Lane M, molecular weight
markers; Lane 1, empty vector induced without IPTG; Lane 2,
empty vector induced with isopropylthiogalactoside (IPTG); Lane
3, recombinant plasmid induced without IPTG; Lane 4, recombinant
plasmid induced with 0.5 mM IPTG; Lane 5, supernatant protein of
the recombinant plasmid subjected to IPTG induction and ultrasonic
treatment; Lane 6, insoluble protein of the recombinant plasmid
subjected to IPTG induction and ultrasonic treatment; Lane 7,
recombinant protein purified by glutathione-S-transferase chromato-
graphy column; Lane 8, Western blot analysis of recombiant RGD-
FasL protein. (B) Cell adhesion assay was performed as described
in Materials and Methods. Microtiter wells were coated with 30
mg/ml RGD-FasL or FasL and seeded with H22 and H9101 cells.
After 1.5 h, cell adhesion was measured.

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Cellular & Molecular Immunology 289
Volume 6 Number 4 August 2009
Figure 2, approximately 24.2% of H22 cells and 13.5% of
H9101 cells expressed Fas, and 26.3% of H22 cells and
15.5% of H9101 cells expressed CD61 (Figure 2A).

Cytotoxic effects of FasL/RGD-FasL on tumor cell lines
To quantify apoptosis, we utilized the annexin V-fluorescein
isothiocyanate (FITC) staining assay, which reports the loss
of the phosphatidylserine asymmetry of the plasma
membrane at an early stage of apoptosis. Both H22 and
H9101 cell lines underwent synergistic apoptosis upon 24-h
treatment with RGD-FasL or FasL. As shown in Figure 2B a
significant increase in apoptosis was induced in cells treated
with RGD-FasL or FasL compared to those that were
untreated.
To test whether our products were functionally active and
capable of inducing cell death and apoptosis, we measured
their activity on H22 and H9101 cells. The cytotoxic effects
of FasL and RGD-FasL on both of the tumor cell lines were
dose-dependent. The 50% inhibition concentration (IC50)
values of H9101 cells at 12 h were 26.53 μg/ml (RGD-FasL)
and 33.67 μg/ml (FasL) (Figure 2C). The IC50 values of H22
cells at 12 h were 59.63 μg/ml (RGD-FasL) and 92.34 μg/mL
(FasL). H9101 cells showed the same sensitivity to
RGD-FasL as to FasL (p > 0.05), whereas H22 was more
sensitive to RGD-FasL than to FasL (p < 0.05) (Figure 2D).
Both RGD-FasL and FasL have no effect on normal mouse
liver cells. However, the sensitivities of the H22/H9101
tumor cell lines to FasL/RGD-FasL differed among
experimental groups.

Antitumor effects of FasL and RGD-FasL on mice with
H22 tumor
In order to determine whether RGD-FasL had a more potent
antitumor activity and reduced toxicity compared with FasL,
we evaluated the antitumor effects of the FasL molecules
determined in BALB/c mice bearing 40-50 mm3 H22 tumors
(Figure 3). The groups were injected daily (i.v.) with 30 μg of
recombinant human FasL or RGD-FasL. Both RGD-FasL
and FasL clearly demonstrated antitumor activity in this
experiment. Tumor growth was monitored daily by
measuring the tumor volumes with calipers, as described
previously. The tumor inhibition rates (IR) of RGD-FasL and
Annexin V Annexin V
PI PI
99.5 99.5
99.5 99.5
3.30 22.4 3.30 22.4
74.2 0.04 74.2 0.04
0.15 3.850.15 3.85
33.8 61.4 33.8 61.4
3.70 3.70
20.6 74.720.6 74.7
0.140.14
65.8 32.465.8 32.4
BB
ControlControlFasLFasL
RGD-FasLRGD-FasL
H22H22
H9101H9101
A
Isotype
24.2 ±± 3.0
26.3 ±± 3.1
Fas
CD61
15.5 ±± 1.2
Counts
13.5 ±± 1.6
Fluorescence
H22
H9101
FasL (H22)
RGD-FasL (H22)
FasL (normal)
RGD-FasL (normal)
FasL
RGD-FasL
1.56 3.13 6.25 12.5 25 50 100
Concentration ( g/ml)
1.56 3.13 6.25 12.5 25 50 100
Concentration ( g/ml)
Cell death (%)
C D
100
80
60
40
20
0
80
70
60
40
20
0
50
30
10
Cell death (%)

Figure 2. Analysis of in vitro activity of RGD-FasL and FasL. (A) Cell surface expression of Fas and H9101 on cell lines H22 and H9101
measured by flow cytometry. (B) Treatment with FasL or RGD-FasL induces apoptosis in H22 and H9101 cells. Treatment of FasL (50 g/ml)
or RGD-FasL (50 g/ml) for 24 h induced apoptosis in H22 and H9101 cells, as revealed by annexin V-FITC staining assay. (C, D) The
inhibition rate of H9101 cells (C) and H22 cells and mouse normal hepatic cells (D) treated with RGD-FasL and FasL. The cells line were
incubated for 12 h with 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 g/ml RGD-FasL or FasL. Cell growth was determined by the MTT assay. Data
are expressed as mean ± SD.