Altered regulation of extrinsic apoptosis pathway in HCV-infected HCC cells enhances susceptibility to mapatumumab-induced apoptosis

ArticleinHepatology Research 39(12):1178-89 · September 2009with25 Reads
Impact Factor: 2.74 · DOI: 10.1111/j.1872-034X.2009.00568.x · Source: PubMed
Abstract

Background: Hepatitis C virus (HCV)-infected patients, including those co-infected with human immunodeficiency virus (HIV), are at increased risk of developing hepatocellular carcinoma (HCC). We evaluated the ability of agonistic human monoclonal antibodies to tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors, mapatumumab and lexatumumab, respectively, to induce TRAIL-receptor mediated apoptosis (TRMA) in HCC (HCV-infected and -uninfected) cells and in peripheral blood cells (HIV-infected and -uninfected). Methods: Susceptibility to antibody-mediated TRMA was measured by caspase 3/7 activity and by confocal microscopy. Surface expression of receptors on HCV-uninfected and -infected Huh7.5 cells was measured by flow cytometry and confocal microscopy. Inhibitor of Apoptosis Protein (IAP) RNA levels were quantified by RT-PCR. DNA Microarray was performed using RNA isolated from Huh7.5 cells (HCV-infected and uninfected) using Affymetrix U133A chips. Results: Mapatumumab preferentially induces TRMA of HCV-infected Huh7.5 cells by binding to TRAIL-R1. Higher basal expression of TRAIL-R2 compared to that of TRAIL-R1 on HCV-uninfected Huh7.5 cells were observed. Lexatumumab induces TRMA of both HCV-infected and -uninfected cells by binding to TRAIL-R2. IFN-α has minimal effect on mapatumumab- and lexatumumab-induced TRMA. HCV infection of Huh7.5 cells up-regulates TRAIL-R1 expression and X-linked Inhibitor of apoptosis protein and survivin gene expression. Neither antibody had a pro-apoptotic effect on PBMCs from patients with HIV infection ex vivo. Conclusion: Both mapatumumab and lexatumumab are excellent candidates for therapy of HCC. HCV infection of Huh7.5 cells selectively up-regulates TRAIL-R1 receptor, associated with increased susceptibility to mapatumumab-mediated TRMA. HCV infection up-regulated IAP genes, offering promise for future combination therapy using TRAIL agonists and IAP inhibitors.

Full-text

Available from: Richard A Lempicki
Original Articlehepr_568 1178..1189
Altered regulation of extrinsic apoptosis pathway in
HCV-infected HCC cells enhances susceptibility to
mapatumumab-induced apoptosis
Xiaozhen Zhang,
1
Astrid C. Frank,
1
Christine M. Gille,
1
Marybeth Daucher,
1
Juraj Kabat,
2
Steven Becker,
2
Richard A. Lempicki,
3
Karoll J. Cortez,
4
Michael A. Polis,
1
G. Mani Subramanian
5
and Shyam Kottilil
1
1
LIR, NIAID, NIH, DHHS, Bethesda,
2
Biological Imaging facility, RTB, NIAID, NIH, DHHS, Bethesda,
3
SAIC-Frederick
Inc. Frederick, MD,
4
National Cancer Institute, NIH, DHHS, Bethesda,
5
Human Genome Sciences Inc, Rockville MD,
USA
Background: Hepatitis C virus (HCV)-infected patients,
including those co-infected with human immunodeficiency
virus (HIV), are at increased risk of developing hepatocellular
carcinoma (HCC). We evaluated the ability of agonistic human
monoclonal antibodies to tumor necrosis factor-related apo-
ptosis inducing ligand (TRAIL) receptors, mapatumumab and
lexatumumab, respectively, to induce TRAIL-receptor medi-
ated apoptosis (TRMA) in HCC (HCV-infected and -uninfected)
cells and in peripheral blood cells (HIV-infected and
-uninfected).
Methods: Susceptibility to antibody-mediated TRMA was
measured by caspase 3/7 activity and by confocal microscopy.
Surface expression of receptors on HCV-uninfected and
-infected Huh7.5 cells was measured by flow cytometry and
confocal microscopy. Inhibitor of Apoptosis Protein (IAP) RNA
levels were quantified by RT-PCR. DNA Microarray was per-
formed using RNA isolated from Huh7.5 cells (HCV-infected
and uninfected) using Affymetrix U133A chips.
Results: Mapatumumab preferentially induces TRMA of
HCV-infected Huh7.5 cells by binding to TRAIL-R1. Higher
basal expression of TRAIL-R2 compared to that of TRAIL-R1 on
HCV-uninfected Huh7.5 cells were observed. Lexatumumab
induces TRMA of both HCV-infected and -uninfected cells
by binding to TRAIL-R2. IFN-a has minimal effect on
mapatumumab- and lexatumumab-induced TRMA. HCV infec-
tion of Huh7.5 cells up-regulates TRAIL-R1 expression and
X-linked Inhibitor of apoptosis protein and survivin gene
expression. Neither antibody had a pro-apoptotic effect on
PBMCs from patients with HIV infection ex vivo.
Conclusion: Both mapatumumab and lexatumumab are
excellent candidates for therapy of HCC. HCV infection of
Huh7.5 cells selectively up-regulates TRAIL-R1 receptor,
associated with increased susceptibility to mapatumumab-
mediated TRMA. HCV infection up-regulated IAP genes, offer-
ing promise for future combination therapy using TRAIL
agonists and IAP inhibitors.
Key words: hepatitis C virus cell culture system
,
hepatocellular carcinoma, tumor necrosis factor-related
apoptosis inducing ligand agonistic antibodies
INTRODUCTION
C
HRONIC HEPATITIS C virus (CHCV) infection
affects approximately 180 million people world-
wide,
1
with cirrhosis and hepatocellular cancer develop-
ing in a significant proportion of CHCV patients.
Approximately 30% of individuals with human immu-
nodeficiency virus type 1 (HIV-1) infection in the US
and Europe are also co-infected with HCV, which is a
major reason for the morbidity seen in this popula-
tion.
2,3
Moreover, HCV/HIV co-infected subjects have a
more rapid progression of liver fibrosis, and lower
response rates to IFN-a based regimens when compared
to that of HCV mono-infected individuals.
4
While early
Correspondence: Dr Shyam Kottilil, Immunopathogenesis Section,
NIH/NIAID/LIR, Bldg.10. Rm. 11N204, 9000 Rockville Pike,
Bethesda, MD 20892, USA. Email: skottilil@niaid.nih.gov
Disclaimer: The content of this publication does not necessarily
reflect the views of policies of the Department of Health and
Human Services, nor does mention of trade names, commercial
products or organization imply endorsement by the US
Government.
Conflict of Interest Statement: G. Mani Subramanian is an
employee of Human Genome Sciences. No other authors have a
conflict of interest.
Received 3 February 2009; revision 22 May 2009; accepted 29 May
2009.
Hepatology Research 2009; 39: 1178–1189 doi: 10.1111/j.1872-034X.2009.00568.x
© 2009 The Japan Society of Hepatology
No claim to original US government works
1178
Page 1
studies with maintenance interferon (IFN)-a therapy
showed a beneficial effect in reducing fibrosis progres-
sion or the development of HCC, the recently concluded
HALT-C study showed no beneficial effects.
5
These data
suggest that a substantial number of HCV-infected sub-
jects, especially those who are co-infected with HIV, are
at a risk of progressive liver fibrosis and its complica-
tions such as cirrhosis and HCC in the future.
6,7
Chronic hepatitis B and C virus infections have been
the most prominent predisposing factors for hepatocar-
cinogenesis.
8
HCC, the fifth most common malignancy
worldwide, is associated with a poor prognosis due to
late diagnoses and a lack of effective therapy options.
9
Carcinogenesis is associated with suppression of apop-
tosis pathways resulting in uncontrolled proliferation
of abnormal cells.
10,11
Induction of apoptosis of cancer
cells has been suggested as a viable therapeutic option
for the treatment of malignancies.
10,12
Mapatumumab
and lexatumumab are monoclonal TNF-related apopto-
sis inducing ligand (TRAIL)-agonistic human mono-
clonal antibodies (mAbs), which have been shown to
induce apoptosis in several cancer cell lines.
13,14
Binding
of mapatumumab and lexatumumab to TRAIL receptors
R1 and R2 respectively results in the initiation of the
extrinsic apoptosis pathway signaling cascade, leading
to caspase 8 and caspase 3 activation and subsequent
cell death.
15–19
However, mapatumumab and lexatu-
mumab have not yet been extensively evaluated for their
abilities to induce apoptosis of human liver cancer cells
infected with HCV or HBV in vitro or in vivo. A recent
study demonstrated that a specific HCV isolate, JFH-1, is
capable of inducing apoptosis of LH86 hepatoma cells
probably by triggering an intracellular anti-viral defense
mechanism mediated by enhanced susceptibility to
TRAIL-mediated apoptosis.
20
The effect of TRAIL and/or TRAIL receptor agonists
on the susceptibility of HCV-infected Huh7.5 cells to
TRAIL-mediated apoptosis (TRMA) has not been previ-
ously addressed. Since TRAIL agonist-mediated apopto-
sis induced by antibodies could provide a way of
specifically targeting HCV-infected, poorly differenti-
ated human HCC cells, we examined the apoptosis
inducing abilities of mapatumumab and lexatumumab
on Huh7.5 cells. Furthermore, while the role of IFN-a
in preventing liver carcinogenesis is still debated, the
molecular mechanisms underlying IFN-a activity
include the up regulation of cell death receptors
21
and
modulation of the central apoptosis regulator NF-kB.
22,23
Recent therapeutic advances that target apoptosis
directly or indirectly have provided a rationale for com-
bination therapy with IFN-a.
22
We therefore explored
the interactions of IFN-a2b and albinterferon alfa-2b
(alb-IFN), a long acting fusion polypeptide (24), with
the TRAIL pathway in this system. Finally, the impact
of these antibodies in HIV-infected peripheral blood
mononuclear cells (PBMC) was investigated given pre-
vious reports of enhanced sensitivity to TRAIL-mediated
apoptosis in HIV-infected T cells.
24,25
MATERIALS AND METHODS
Study subjects
P
ERIPHERAL BLOOD MONONUCLEAR Cells
(PBMCs) were obtained from HIV seronegative
healthy volunteers and HIV-infected patients after
signing informed consents approved by the Institutional
Review Boards (IRB) of the Department of Transfusion
Medicine (DTM) and the National Institute of Allergy
and Infectious Diseases (NIAID) at the National Insti-
tutes of Health (NIH) respectively.
Isolation of PBMCs and culture conditions
PBMCs were isolated by Ficoll Hypaque density gradient
centrifugation (GE Healthcare, South Burlington, VT)
and resuspended in RPMI 1640 medium (Invitrogen
Corporation, Carlsbad, CA) supplemented with 10%
fetal bovine serum (FBS), 1% penicillin/streptomycin,
2% glutamine and incubated at 37°C in a 5% CO
2
containing incubator for 24 h in the presence or absence
of control mouse anti-human IgG1 antibody (R&D
Systems, Minneapolis, MN), or mapatumumab, lexatu-
mumab and/or TRAIL –R1-Fc and TRAIL-R2-Fc proteins
(R&D Systems).
Cell line and culture conditions
The Huh7.5 HCC cell line was provided by Apath LLC
(Saint Louis, MO). Cells were cultured in Dulbecco’s
modified eagle medium (DMEM; Invitrogen Corpora-
tion) supplemented with 10% FBS and 100 U/mL
penicillin/streptomycin at 37°C in a 5% CO
2
containing
incubator.
HCV infectious clone
The infectious plasmid clone for the HCV J6/JFH1 infec-
tious cell culture system was provided by Apath LLC.
Transfection and subsequent infection of Huh 7.5
cells using this plasmid was performed as described
elsewhere.
26,27
Antibodies
Mapatumumab and Lexatumumab are agonistic, fully
human IgG1 antibodies specific to TRAIL receptors
Hepatology Research 2009; 39: 1178–1189 TRAIL-Receptor-mediated apoptosis in HCV-infected HCC 1179
© 2009 The Japan Society of Hepatology
No claim to original US government works
Page 2
TRAIL-R1 and TRAIL-R2 respectively and were provided
by Human Genome Sciences (Rockville, MD). Mouse
anti-human IgG1 antibody (R&D Systems) and TRAIL-
R1-Fc and TRAIL-R2-Fc fusion proteins (R&D Systems)
were used as a control and to block receptor binding
of mapatumumab and lexatumumab to TRAIL-R1 and
TRAIL-R2 respectively.
Caspase Glo 3/7 assay (Promega)
Uninfected and J6/JFH1-infected Huh 7.5 cells were
plated in 96-well solid white plates (1.8 ¥ 10
4
cells/well)
in 100 ml of DMEM. After overnight incubation at 37°C,
100 ml of fresh DMEM containing serial dilutions of
TRAIL-R antibody were added to the cells. After 24 h of
incubation, Caspase-Glo 3/7 reagent (Promega Corpo-
ration, Madison. WI) was added to each well. The con-
tents of the plate were gently mixed using a plate shaker
at 300–500 rpm for 30 s. The cells were then lysed at
room temperature for 1 h. The luminescence activities
(relative Caspase 3 and 7 activities) were measured
using an OPTIMA plate reader (BMG Labtech, Durham,
NC).
DNA microarray analysis
J6/JFH-infected and uninfected Huh7.5 cells were used
for DNA microarray analysis. Total RNA and labeled
cRNA synthesis and hybridization to the Affymetrix
U133A human microarray were performed according to
the manufacturer’s recommended protocol. Gene
expression values (log2) were determined by GC-Robust
Multi-Array algorithm (Wu ZJ and Irizarray R: http://
www.bioconductor.org) followed by a Loess normaliza-
tion using an R package (http://www.elwood9.net/
spike). Expression of TRAIL-R1, R2, R3 and R4 were
compared between HCV-infected and uninfected
Huh7.5 cells from four separate experiments. TRAIL-R3
mRNA signal was not detected.
Flow cytometry
The number of lymphocytes undergoing apoptosis was
defined by surface staining with CD3-APC, CD4-PE-Cy7
as well as PE-conjugated Annexin-V antibody (BD
Pharmingen, San Jose, CA) utilizing BD FacsArray bio-
analyzer (BD Biosciences, San Jose, CA). TRAIL-R2 PE
antibody (BD Pharmingen) staining of Huh 7.5 cells
resuspended in EDTA-containing PBS was performed
and analyzed using the FACS Canto bioanalyzer (BD
Biosciences). FlowJo software (Tree Star, Ashland, OR)
was used to analyze the data.
Confocal microscopy
Approximately 20 000 J6/JFH-1- infected and unin-
fected Huh7.5 cells were plated in Lab-Tek eight well
Nunc Glass Chamber Slide (Krackeler Scientific, Albany,
NY) and incubated at 37°C overnight. Cells were
washed three times with PBS, and permeabilized with
0.05% Triton for 20 min. Cells were incubated with two
primary antibodies (10 mg/mL goat IgG1 anti-human
TRAIL-R1 (R&D Systems) and mouse IgG1 anti-HCV
core antigen (Abcam)) with 1% BSA for one hour at
room temperature. Cells were washed again with 1%
BSA containing PBS, and incubated for 30 min at room
temperature with first secondary antibody (Alexa Fluor
594 Fab fragment of rabbit anti-goat IgG (Invitrogen,
Carlsbad, CA)) and subsequently with second fluores-
cent conjugates secondary antibody (Alexa Fluor 488
Goat anti-mouse IgG1 (Invitrogen)). Finally, cells were
washed in PBS and mounted with DAPI containing
mounting medium. Samples were observed under a
confocal microscope (Leica SP2, Leica Microsystems,
Exton, PA) using a 63X oil immersion objective NA
1.32. Images were captured with Huygens Essential
software, version 3.3 (Scientific Volume Imaging BV,
Hilversum).
Time-lapse DIC Microscopy
Approximately 20 000 Huh 7.5 cells were plated in Lab-
Tek two well Nunc Glass Chamber Slide (Krackeler Sci-
entific) and incubated at 37°C and 5% CO
2
overnight.
Differential interference contrast image sequences were
collected every 5 min on an AF 6000LX, Leica Microsys-
tems (Wetzlar) with a 20¥ oil immersion objective NA
0.7.
Real time PCR for HCV copy number
and IAPs
The culture supernatants from HCV-infected cells were
collected 48 h after treatment with the antibody and/or
interferon-a and HCV copy numbers were determined
using a quantitative real-time PCR as described
elsewhere.
28
Total RNA was extracted from HCV-
uninfected, HCV-infected as well as Huh7.5 cells in the
presence or absence of IFN-a2b or alb-IFN for 24 h
using the mirVAna miRNA isolation kit (Applied Bio-
systems, Foster City, CA). 5 mg of total RNA were used
for cDNA synthesis with the High-capacity cDNA
Archive kit (Applied Biosystems). Primers and probes
for the detection of cIAP1, cIAP2, XIAP and survivin
mRNA (Applied Biosystems) by RT-PCR (ABI 7500
RT-PCR System) were used according to the manufac-
1180 X. Zhang et al. Hepatology Research 2009; 39: 1178–1189
© 2009 The Japan Society of Hepatology
No claim to original US government works
Page 3
turer’s guidelines. The glyceraldehyde-3-phosphate
dehydrogenase primer and probe set served to provide
an endogenous control. Results were analyzed using
the comparative Ct method for the relative quantita-
tion of gene expression.
Statistical analysis
Analysis of variance with Tukey’s multiple comparison
test was used to compare means of the independent
data. The paired Student’s t-test with the Bonferroni
adjustment for multiple testing was used to compare
paired responses.
RESULTS
Mapatumumab preferentially induces
apoptosis of HCV-infected Huh7.5 cells
W
E PERFORMED A caspase 3/7 assay on HCV-
infected and uninfected Huh7.5 cells treated
with serial concentrations of mapatumumab or lexatu-
mumab. As shown in Figure 1A and B, mapatumumab
and lexatumumab both induced dose-dependent apop-
tosis of Huh7.5 cells when compared to that seen with
control antibody. Mapatumumab preferentially induced
higher levels of apoptosis of HCV-infected Huh7.5 cells
when compared to that seen with HCV-uninfected
Huh7.5 cells, which was statistically significant at higher
concentrations (P < 0.05; Fig. 1A). With lexatumumab,
the degree of apoptosis induced was not affected
whether the cells were infected with HCV or not
(P > 0.05; Fig. 1B). Relative potency of both mAb
against HCV-uninfected and infected Huh 7.5 cells as
expressed as EC50 values of Caspase 3/7 activity were
252 ng/mL and 137.3 ng/mL respectively for mapatu-
mumab and 112 ng/mL and 121 ng/mL respectively for
lexatumumab.
Mapatumumab and lexatumumab induce
apoptosis by direct interaction with
respective cell surface receptors
As shown in Figure 1C, mapatumumab-induced apop-
tosis of Huh7.5 cells was blocked by addition of
Fc-TRAIL-R1 (P < 0.05 for uninfected and P < 0.02 for
HCV-infected Huh7.5 cells) and lexatumumab-induced
apoptosis of Huh7.5 cells was blocked by Fc-TRAIL-R2
(P < 0.02 for HCV uninfected and P < 0.03 for HCV-
infected Huh7.5 cells, Fig. 1D). Thus induction of
apoptosis of Huh7.5 cells by mapatumumab and lexa-
tumumab resulted from direct interactions with
TRAIL-R1 and TRAIL-R2 receptors respectively.
IFN-a formulations have no effect on
mapatumumab and lexatumumab-mediated
apoptosis of Huh7.5 cells
We performed caspase 3/7 assays on Huh7.5 cells
treated with mapatumumab or lexatumumab in the
absence or presence of IFN-a2b or alb-IFN. As shown in
Figure 2A and B, 24 h treatment of both IFN-a formu-
lations had no significant effect on mapatumumab- and
lexatumumab -mediated apoptosis.
Susceptibility of HCV-infected Huh7.5 cells
to mapatumumab-mediated apoptosis
correlated with up regulation of TRAIL-R1
receptor
To address the effects of mapatumumab treatment of
HCV-infected Huh7.5 cells on HCV replication, we per-
formed real time qPCR to quantitate the copy numbers
of HCV. As shown in Figure 3A, exposure of HCV-
infected Huh 7.5 cells to mapatumumab did not have
any effect on levels of HCV copy numbers in the super-
natants. This could be due to the release of HCV virions
from apoptotic cells into the supernatant, which is then
detected by PCR.
To explore underlying mechanisms of enhanced
susceptibility of HCV-infected Huh7.5 cells to
mapatumumab- mediated apoptosis, we analyzed the
levels of mRNA of TRAIL receptors on HCV-uninfected
and HCV-infected Huh7.5 cells. As shown in Figure 3B,
HCV-infected Huh7.5 cells expressed higher levels of
TRAIL-R1 mRNA (P < 0.02; Fig. 3B) compared to unin-
fected cells, while the levels of expression of TRAIL-R2
(Fig. 3B) mRNA remained unchanged. Likewise, surface
expression of TRAIL-R1 receptor on HCV-infected
Huh7.5 cells were higher compared to uninfected cells
(Fig. 3C). Modulation of expression of TRAIL decoy
receptors (DcR), TRAIL-R3 and R4 has also been sug-
gested as a mechanism that may result in altered sus-
ceptibility of tumor cells to TRMA.
23
As shown in
Figure 3B and C, HCV infection of Huh7.5 cells did not
modulate the expression of TRAIL DcR (Fig. 3B,C).
Given the suppression of HCV replication by IFN-a,
one might anticipate a decrease in TRAIL-R1 expression
and TRMA in IFN-a-treated HCV-infected Huh7.5 cells.
However, the levels of TRAIL-R1 expression before and
after IFN-a treatment remain unchanged (data not
shown).
Hepatology Research 2009; 39: 1178–1189 TRAIL-Receptor-mediated apoptosis in HCV-infected HCC 1181
© 2009 The Japan Society of Hepatology
No claim to original US government works
Page 4
Confocal microscopy confirms HCV-infection
of Huh7.5 cells up-regulates TRAIL-R1 and
are susceptible to Mapatumumab-mediated
apoptosis
To visualize and confirm the effects of HCV infection on
TRAIL-R1 expression of Huh 7.5 cells, we performed
confocal microscopy of HCV-uninfected and infected
Huh7.5 cells after labelling cells for HCV core protein
and TRAIL-R1 simultaneously. As shown in Figure 4A,
TRAIL-R1 (red) expression was increased on HCV-
infected (green) as compared to that seen on HCV unin-
fected Huh7.5 cells (left panel). Figure 4B shows the
(a) (b)
(d)
Uninfected Huh7.5 cells
J6JFH infected Huh7.5 cells
Uninfected Huh7.5 cells
J6JFH infected Huh7.5 cells
8
6
4
2
0
(c)
150000
100000
50000
150000
100000
200000
50000
0
0
*
*
*
*
*
*
*
#
#
#
#
Median fold increase in caspase
3/7 activity
Median fold increase in caspase
3/7 activity
Median caspase 3/7 activity
Median caspase 3/7 activity
5
4
3
2
1
0
No treatment control
No treatment control
No treatment control
No treatment control
Mapa 0.041 ug/ml
Lexa 0.041 ug/ml
Lexa 0.123 ug/ml
L
e
x
a 0.37 ug/ml
Lexa 1.1 ug/ml
Lexa 3.3 ug/ml
Lexa 10 ug/ml
Lexa 30 ug/ml
Mapa 0.123 ug/ml
Mapa 0.37 ug/ml
Mapa 1.1 ug/ml
3 ug/ml Mapa
3 ug/ml Lexa
5 ug/ml TRAIL-R1-F
c
5 ug/ml TRAIL-R2-Fc
5
ug/ml Mapa + 5 ug/ml
TRAIL-R1-Fc
3 ug/ml Lexa + 5 ug/ml
T
R
AIL-R2-Fc
Mouse IgG1
(negative control)
Mouse IgG1
(negative control)
Mapa 3.3 ug/ml
Mapa 10 ug/ml
Mapa 30 ug/ml
Figure 1 The levels of apoptosis of hepatitis C virus (HCV)-uninfected (blue line) or HCV–infected Huh7.5. cells (red line) in the
absence or presence of serial concentrations of mapatumumab or lexatumumab or isotype control antibody (mouse anti-human
IgG1) were determined as described in Material and Methods. The level of apoptosis is expressed as median fold increase in Caspase
3/7 activity of each experimental condition over that seen with control antibody. (a) Mapatumumab -induced dose-dependent
apoptosis of both HCV-uninfected and infected Huh 7.5 cells with higher levels seen with HCV-infected Huh7.5 cells. There was
statistical significance for values marked with * (P < 0.05). (b) Lexatumumab-induced dose-dependent apoptosis of both HCV-
uninfected and infected Huh 7.5 cells. There was no statistical significance for any values (P > 0.05). The (median) levels of (c)
mapatumumab or (d) lexatumumab -induced apoptosis of HCV-uninfected (blue bars) or infected Huh7.5 cells (red bars) in the
absence or presence of tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-R1-Fc were determined as described in
Material and Methods. (c) The results revealed a statistically significant decline in the Caspase3/7 activity when the experiments were
performed in the presence of TRAIL-R1-Fc for both HCV-uninfected (*P < 0.05) and HCV-infected (#P < 0.02) implying a direct
interaction between mapatumumab and TRAIL-R1 to account for mapatumumab -induced apoptosis. (d) The results revealed a
statistically significant decline in the Caspase3/7 activity when the experiments were performed in the presence of TRAIL-R2-Fc for
both HCV-uninfected (*P < 0.02) and HCV-infected (#P < 0.03) implying a direct interaction between lexatumumab and TRAIL-R2
to account for lexatumumab-induced apoptosis.
1182 X. Zhang et al. Hepatology Research 2009; 39: 1178–1189
© 2009 The Japan Society of Hepatology
No claim to original US government works
Page 5
levels of apoptosis induced by mapatumumab as
detected by direct visualization and quantitation
was higher in HCV-infected than uninfected
Huh7.5 cells. We also obtained time-lapse video of
mapatumumab exposed Huh7.5 cells (HCV un-
infected and infected) over time, which shows a
more extensive and rapid induction of apoptosis of
HCV-infected Huh7.5 cells (Supplemental video
S1,S2).
Increased basal expression of TRAIL-R2
receptors on Huh7.5 cells
Our results have demonstrated that HCV infection
of Huh7.5 cells neither increases the expression of
TRAIL-R2 receptor nor enhances the susceptibility to
lexatumumab mediated apoptosis. As shown in
Figure 5, both mRNA (P < 0.001) and cell surface
expression of TRAIL-R2 was significantly higher on the
surface of HCV uninfected Huh7.5 cells. These results
Figure 2 Levels of apoptosis of HCV-
uninfected or infected Huh7.5 cells in
the presence of (a) mapatumumab or
(b) lexatumumab and in the absence or
presence of different interferon (IFN)-a
formulations were determined as
described in Material and Methods.
Different IFN-a formulations in vitro
had minimal effect on mapatu-
mumab-induced apoptosis (a) and
lexatumumab-induced apoptosis (b)
levels of Huh7.5 cells. There was no sta-
tistical significance for any values
(P > 0.05).
(a)
(b)
400000
300000
200000
100000
3 ug/ml Mapa +
3 ug/ml Lexa
3 ug/ml Lexa +
50 IU alb-IFNa-2b
3 ug/ml Lexa +
50 IU IFNa-2b
3 ug/ml Lexa +
5
00 IU IFNa-2b
3 ug/ml Lexa +
500 IU alb-IFNa-2b
50 IU alb-IFN
500 IU alb-IFN
50 IU IFNa-2b
500 IU IFNa-2b
50 IU alb-IFN
3 ug/ml Mapa +
50 IU IFNa-2b
3 ug/ml Mapa +
500 IU IFNa-2b
500 IU alb-IFN
50 IU IFNa-2b
500 IU IFNa-2b
3 ug/ml Mapa +
50 IU alb-IFNa-2b
3 ug/ml Mapa +
500 IU alb-IFNa-2b
Median caspase 3/7 activity
Median caspase 3/7 activity
0
400000
300000
200000
100000
0
Uninfected Huh7.5 cells
J6JFH infected Huh7.5 cells
Uninfected Huh7.5 cells
J6JFH infected Huh7.5 cells
Hepatology Research 2009; 39: 1178–1189 TRAIL-Receptor-mediated apoptosis in HCV-infected HCC 1183
© 2009 The Japan Society of Hepatology
No claim to original US government works
Page 6
suggest that level of expression and density of TRAIL
receptors on Huh7.5 cells may explain the differential
susceptibility of Huh7.5 cells to mapatumumab and
lexatumumab mediated apoptosis.
Gene expression of IAPs XIAP and survivin
is significantly up-regulated in HCV-infected
compared to HCV-uninfected Huh7.5 cells
To investigate whether HCV infection of HCC cells
modulated the expression of IAP genes, we performed
real time PCR to quantitate the relative expression
of different IAP genes in HCC cells infected or
uninfected with HCV, in the presence or absence
of IFN-a. As illustrated in Figure 6, HCV
infection of Huh7.5 cells was associated with an
up-regulation of gene expression for XIAP (P < 0.01)
and survivin (P < 0.03). Both IFN-a and albin-
terferon had no significant effect on IAP gene
expression of Huh 7.5 cells after 24 h exposure
(P > 0.05).
(a)
(b)
(c)
010
2
10
3
10
4
10
5
010
2
10
3
10
4
10
5
TRAIL-R1-PE TRAIL-R2-PE
010
2
10
3
10
4
10
5
TRAIL-R3-PE
010
2
10
3
10
4
10
5
TRAIL-R4-PE
1.
1.
Huh 7.5
cells
P < 0.02 P > 0.05
P > 0.05
HCV-infected
huh 7.5 cells
Huh 7.5
cells
HCV-infected
huh 7.5 cells
Huh 7.5
cells
HCV-infected
huh 7.5 cells
2. 3.
2. 3. 4.
100
80
60
40
20
0
100
80
60
40
20
0
300
200
100
0
300
200
100
0
3000
2000
1000
0
100
80
60
40
20
0
100
80
60
40
20
0
20000
10000
0
Huh 7.5
IgG1 control
IFNa 10 IU
IFNa 50 IU
IFNa 10 IU + mapa 3 ug
IFNa 50 IU + mapa 3 ug
Mapa 3 ug
HCV VL (log) Copies/mL
Mean signal intensity of TRAIL-R1 mRNA
Mean signal intensity of TRAIL-R2 mRNA
Mean signal intensity of TRAIL-R4 mRNA
Uninfected Huh 7.5
Huh 7.5 infected with HCV
Figure 3 (a) Levels of hepatitis C virus (HCV) replication in J6/JFH HCV-infected Huh 7.5 cells in the presence or absence of
control antibody, interferon (INF)-a and/or mapatumumab (3 mg/mL) was measured as described in the Materials and Methods.As
expected, IFN-a suppressed HCV replication in JFH-infected Huh7.5 cells, while Mapatumumab did not have any effect on HCV
replication. Representative data from 4 separate experiments shown. (b) The levels of mRNA transcripts for tumor necrosis
factor-related apoptosis inducing ligand (TRAIL)-receptors (R1 to R4) in Huh 7.5 cells (HCV-uninfected and infected). While there
was a statistically significant increase in the levels of TRAIL-R1 mRNA transcripts in infected Huh7.5 cells (P < 0.02), there were no
differences in the levels of transcripts of TRAIL-R2, and R4 detected in HCV uninfected and infected Huh7.5 cells (P > 0.05 for
both). TRAIL-R3 mRNA signal was not detected in this microarray experiment. (c) The level of expression of TRAIL receptors (TRAIL
R1 and 2 and TRAIL DcR 3 and 4) on the surface of HCV-uninfected or infected Huh7.5 cells was measured as described in Material
and Methods. TRAIL-R1 surface expression levels were substantially up regulated in HCV-infected compared to uninfected Huh7.5
cells (C (1)), while HCV-infection had no effect on the levels of expression of TRAIL-R2 (C (2)), TRAIL-R3 (C (3)) and TRAIL-R4
(C (4)).
1184 X. Zhang et al. Hepatology Research 2009; 39: 1178–1189
© 2009 The Japan Society of Hepatology
No claim to original US government works
Page 7
Mapatumumab does not induce apoptosis
of PBMCs from HIV-negative and HIV
viremic individuals
Since recent studies have suggested that HIV-infection
may trigger TRAIL-mediated apoptosis of peripheral T
cells in vitro, we evaluated the degree of apoptosis in
PBMCs from HIV-infected viremic and HIV negative
subjects resulting from exposure to mapatumumab in
vitro. As shown in Figure 7A through 7D there was no
significant degree of mapatumumab-mediated apopto-
sis of B, T, NK cells or monocytes from both HIV-
negative and HIV viremic individuals. Similar results
were observed with lexatumumab (data not shown).
DISCUSSION
I
N THIS STUDY we show that HCV infection of
Huh7.5 cells results in preferential up regulation of
TRAIL-R1 receptor, which correlates with enhanced sus-
ceptibility to mapatumumab-induced apoptosis com-
pared to HCV-uninfected cells. Although lexatumumab
is capable of inducing apoptosis of Huh7.5 cells
more effectively than mapatumumab, apoptosis was not
increased by HCV infection. However, HCV-infected
Huh 7.5 cells have increased basal expression of
TRAIL-R2 than TRAIL-R1, which could explain the
greater potency of lexatumumab-mediated apoptosis
than mapatumumab in uninfected Huh7.5 cells. HCV
infection of Huh7.5 cells up regulates the surface expres-
sion of TRAIL-R1 receptor as well as gene expression of
IAPs, XIAP and survivin. IFN-a treatment does not
appear to affect the susceptibility of Huh7.5 cells to
undergo TRMA. Further, mapatumumab does not
induce apoptosis of PBMCs from HIV-negative and HIV-
infected viremic individuals.
Previous studies have shown that HCV infection of
primary HCC cells results in apoptosis of HCC cells in a
TRAIL receptor dependent and independent manner.
20,28
One study showed that HCV infection of primary
hepatoma cells up-regulate gene expression of TRAIL
receptors and enhance their susceptibility for TRMA.
20
Our results demonstrate that the susceptibility of HCV-
infected hepatoma cells to undergo TRMA is associated
with up-regulation of TRAIL-R1 receptor. We were able
to demonstrate an increase in TRAIL-R1 mRNA and cell
surface expression in HCV-infected Huh7.5 cells by flow
cytometry as well as by confocal microscopy. However,
it is not known whether HCV-infection can also affect
down-stream events that could enhance the susceptibil-
ity of hepatoma cells to TRMA. Elevated apoptosis levels
in HCV-infected Huh7.5 cells might also result from
down regulation of TRAIL decoy receptor (DcR) surface
expression.
29
However our results show that HCV infec-
tion of Huh7.5 cells does not have any effect on TRAIL-
DcR expression.
Interestingly, mapatumumab or lexatumumab had no
effect on the levels of HCV copy numbers in the super-
natant of HCV-infected Huh 7.5 cells in vitro. This likely
reflects the HCV virions that are released from Huh 7.5
(a)
(b)
J6/JFH-1 uninfecte
d
Overlay
TRAIL-R1 TRAIL-R1
Overlay
4.3.
1. 2.
HCV-uninfected HCV-infected
Cells undergoing apoptosis (%)
J6/JFH-1 infected
Mapa
untreated
Mapa
treated
15
10
5
0
Figure 4 A. The levels of tumor necrosis factor-related apop-
tosis inducing ligand (TRAIL)-R1 and intracellular hepatitis C
virus (HCV) core proteins were determined using confocal
microscopy as described in the Materials and Methods. HCV-
uninfected Huh7.5 cells (A (1)) had higher expression of
tumor necrosis factor-related apoptosis inducing ligand
(TRAIL)-R1 expression (in red) when compared to that seen
with HCV-uninfected cells (a(2)). Further, when the images for
intracellular HCV core protein and TRAIL-R1 surface expres-
sion were overlaid, the image clearly showed that HCV-
infected (a(4)) and not HCV uninfected Huh7.5 cells (a(3)),
that express high levels of TRAIL-R1 receptor. (b) When the
number of HCV uninfected and infected Huh7.5 cells under-
going apoptosis was counted visually using confocal micros-
copy and using a software as described in the Materials and
Methods there was an increased percentage of HCV-infected
Huh7.5cells per field undergoing apoptosis when compared to
that seen with HCV uninfected cells.
Hepatology Research 2009; 39: 1178–1189 TRAIL-Receptor-mediated apoptosis in HCV-infected HCC 1185
© 2009 The Japan Society of Hepatology
No claim to original US government works
Page 8
cells undergoing TRMA into the culture supernatant.
However, the impact of reducing HCV viremia in vivo
by eliminating HCV-infected hepatocytes remains
unknown. Thus, efforts to investigate whether HCV-
infected primary hepatocytes are also susceptible to
TRMA will help define the potential role of TRAIL-
agonists in vivo for enhancing rates of HCV eradication
in combination with IFN-based regimens for the treat-
ment of chronic HCV infection.
IAPs XIAP and survivin, have been shown to be asso-
ciated with protection from apoptosis through both
extrinsic and intrinsic pathways and have generated
interest as potential targets for chemotherapeutic agents
for cancer.
30
Recent studies have suggested roles for both
XIAP and survivin expression on regulating apoptosis of
hepatoma cells in vitro.
31,32
Since these proteins can spe-
cifically block TRMA mediated by TRAIL or TRAIL ago-
nists, their inhibitors could have a significant role as
adjuvant therapeutic agents with TRAIL agonists in vivo
in the treatment of HCC.
33
Our data suggest that HCV
infection up-regulates the expression of IAP genes, XIAP
and survivin. This up regulation of IAPs (XIAP and sur-
vivin) is an escape/survival mechanism adopted by the
tumor cells. Hence the net effect is a balance of both the
effect of TRMA and IAP-mediated inhibition of apopto-
sis. At the time frame of the experiments, we performed,
TRMA dominates the effect of IPA-mediated inhibition
of apoptosis, however, this could be different when the
IAP expression increases over time and Huh 7.5 cells
could become resistant to TRMA. Hence blocking of IAP
function could synergistically aid in further augmenting
TRMA mediated by mapatumumab or lexatumumab.
Future studies will provide insights into whether IAP
protein expression and function in HCC cells are modu-
lated by HCV infection.
Our in vitro results do not suggest synergy of mapatu-
mumab or lexatumumab with IFNa-2b or alb-IFN.
Within the time frame of our experiments, IFNa formu-
Figure 5 The levels of mRNA tran-
scripts and cell surface expression of
tumor necrosis factor-related apoptosis
inducing ligand (TRAIL) receptors
(TRAIL R1 and 2) in hepatitis C virus
(HCV)-uninfected Huh7.5 cells was
measured as described in Material and
Methods. Huh7.5 cells expressed signifi-
cantly higher levels of TRAIL mRNA
((a) P < 0.001) and (b) TRAIL-R1 recep-
tor expression on the surface (b).
(a)
(b)
10000
1000
100
80
60
40
20
0
100
10
0
TRAIL-R1
Mean signal intensity of mRNA
TRAIL-R1/R2-PE
010
2
10
3
10
4
10
5
TRAIL-R1
TRAIL-R2
TRAIL-R2
P < 0.001
XIAP
1. 0 0
0.50
0.00
–0.50
Relative fold change in the expression
of IAP Genes (over Huh 7.5)
Survivin
#
Huh 7.5 +
IFN-alpha
Huh 7.5 +
HCV
Huh 7.5 +
HCV + IFN-α
Huh 7.5 +
HCV + Alb-IFN
#
#
#
*
*
*
Figure 6 The gene expression levels of all four inhibitors of
apoptosis protein (IAP) genes were performed by RT-PCR as
described in the Materials and Methods. The relative fold change
in IAP gene expression in hepatitis C virus (HCV)-infected
compared to uninfected Huh7.5 cells revealed significant
up-regulation of X-linked IAP (*P < 0.01) and survivin
(#P < 0.03) gene expression. Interferon formulations had
minimal effect on the IAP gene expression on HCV-uninfected
(P > 0.05 for both) and HCV-infected Huh7.5 cells (P > 0.05
for both).
1186 X. Zhang et al. Hepatology Research 2009; 39: 1178–1189
© 2009 The Japan Society of Hepatology
No claim to original US government works
Page 9
lations did not affect expression of TRAIL-R1 or XIAP and
survivin genes. Further investigations will be needed to
determine whether inhibition of viral replication by
IFNa
26,27
impacts different components of the apoptotic
pathway. It remains to be determined, whether novel
agents such as IL-21 or mAbs agonistically mimicking the
effect of CD40 or CD137 ligands, which have recently
been found to exhibit synergistic effects with agonistic
mAbs against TRAIL-R2 in mice
13
will exhibit synergy
with mapatumumab or lexatumumab in vitro.
An increase in the incidence of HCC among HCV/HIV
co-infected patients is anticipated over the coming
decade. Since peripheral immune cells of HIV-infected
patients have been described to be more susceptible to
TRAIL-mediated apoptosis,
24,25
TRAIL agonists in vivo
could result in worsening of HIV-associated immunode-
ficiency. Our results suggest that mapatumumab has
minimal effect on the degree of apoptosis in PBMCs from
healthy and HIV viremic individuals. However, whether
HCC from HIV-infected individuals exhibit the same
degree of susceptibility to TRMA is not clearly under-
stood. Further clinical studies will be required to ascer-
tain in vivo safety of mapatumumab and lexatumumab
in the treatment of HCC in uninfected, HCV-mono-
infected and HCV/HIV co-infected patients. Future
studies will also address whether HCV-infection of a wide
spectrum of HCC cell lines and primary hepatocytes may
have similar effect of TRAIL-mediated apoptosis.
In summary, our study shows in vitro evidence for
the therapeutic potential of mapatumumab and lexatu-
mumab TRAIL-receptor agonistic antibodies in the treat-
ment of HCC. To date, there are several studies that have
demonstrated toxicological profiles of these antibodies
in Phase I clinical trials.
14,17,34
Both antibodies are under
investigation in various experimental cancer combina-
tion therapies
35–37
and this study suggests that mapatu-
mumab has the potential to be a therapeutic agent for
specifically targeting HCC in HCV-infected subjects.
ACKNOWLEDGMENT
T
HIS RESEARCH WAS supported in whole by the
Intramural Research Program of the National Insti-
tute of Allergy and Infectious Diseases. We would also
Figure 7 In vitro apoptosis levels of
PBMCs from human immunodefi-
ciency virus (HIV)-negative or HIV-
infected viremic individuals were
determined by staining for Annexin V
as described in Material and Methods in
the absence or presence of mapatu-
mumab or control isotype antibody
(mouse anti-human IgG1). Mapatu-
mumab did not induce apoptosis of (a)
CD8 T cells, (b) CD4 T cells, (c) Mono-
cytes, (d) B cells/NK cells.
15
10
5
0
15
10
5
0
15
10
5
0
CD8+ T cells
(a) (b)
(c) (d)
Monocytes
No treatment
Control antibody
Mapa 3ug/ml
Mapa 300ng/ml
Mapa 30 ng/ml
Mapa 3ug/ml + TRAIL-R1-Fc
TRAIL-R1-Fc
No treatment
Control antibody
Mapa 3ug/ml
Mapa 300ng/ml
Mapa 30 ng/ml
Mapa 3ug/ml + TRAIL-R1-Fc
TRAIL-R1-Fc
Annexin V -positive cells (%) Annexin V -positive cells (%)
B Cells/NK cells
CD4+ T cells
HIV negative
HCV viremic
15
10
5
0
Hepatology Research 2009; 39: 1178–1189 TRAIL-Receptor-mediated apoptosis in HCV-infected HCC 1187
© 2009 The Japan Society of Hepatology
No claim to original US government works
Page 10
like to thank Apath LLC (St. Louis, MO) and Toray
Industries, Japan for providing us with the Huh7.5cells
and J6/JFH-1 HCV strains.
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SUPPORTING INFORMATION
A
DDITIONAL SUPPORTING INFORMATION may
be found in the online version of this article:
Video Clip S1 and S2 Time lapse DIC microscopy was
performed of HCV-uninfected and infected Huh 7.5
cells in the presence of mapatumumab was performed
over a period of 7 h. The video is condensed data for the
complete 7 h for HCV-uninfected (file1) and HCV-
infected (file2). The unstained cells undergoing apopto-
sis was monitored and counted. As clearly demonstrated
in the time-lapse video, HCV-infected Huh7.5 cells
undergo apoptosis (defined as membrane blebbing and
nuclear condensation) much rapidly and profoundly
when compared to that observed with HCV-uninfected
cells after exposure to mapatumumab.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials
supplied by the authors. Any queries (other than
missing material) should be directed to the correspond-
ing author for the article.
Hepatology Research 2009; 39: 1178–1189 TRAIL-Receptor-mediated apoptosis in HCV-infected HCC 1189
© 2009 The Japan Society of Hepatology
No claim to original US government works
Page 12
    • "However, some HCC cells remain resistant to TRAIL-induced apoptosis. It has been shown that the death ligand TRAIL is a critical IFN-induced apoptosis mediator in this scenario [29]. DR4 and DR5 are the main apoptosis-inducing receptors, and the receptors have an intracellular death domain to transmit the apoptotic signal. "
    [Show abstract] [Hide abstract] ABSTRACT: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. Interferon-alpha (IFN-α) has recently been recognized to harbor therapeutic potential in the prevention and treatment of HCC, but it remains controversial as to whether IFN-α exerts direct cytotoxicity against HCC. Cyclooxygenase-2 (COX-2) is overexpressed in HCC and is considered to play a role in hepatocarcinogenesis. Therefore, we aimed to elucidate the combined effect of a COX-2 inhibitor, celecoxib, and IFN-α on in vitro growth suppression of HCC using the hepatoma cell line HLCZ01 and the in vivo nude mouse xenotransplantation model using HLCZ01 cells. Treatment with celecoxib and IFN-α synergistically inhibited cell proliferation in a dose- and time-dependent manner. Apoptosis was identified by 4',6-diamidino-2-phenylindole dihydrochloride and fluorescent staining. IFN-α upregulated the expression of TRAIL, while celecoxib increased the expression of TRAIL receptors. The combined regimen with celecoxib and IFN-α reduced the growth of xenotransplanted HCCs in nude mice. The regulation of IFN-α- and COX-2 inhibitor-induced cell death is impaired in a subset of TRAIL-resistant cells. The molecular mechanisms of HCC cells resistant to TRAIL-induced apoptosis were explored using molecular biological and immunological methods. Interferon-α and the COX-2 inhibitor celecoxib synergistically increased TRAIL-induced apoptosis in hepatocellular carcinoma. These data suggest that IFN-α and celecoxib may offer a novel role with important implications in designing new therapeutics for TRAIL-resistant tumors. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Feb 2015 · Experimental Cell Research
    0Comments 1Citation
    • "Wealth of information verifies the fact that Mapatumumab preferentially induces apoptosis in HCV-infected Huh7.5 cells by binding to DR4. Lexatumumab induces apoptosis in both HCV-infected and uninfected cells by binding to DR5 [68]. Dual-regulated oncolytic adenoviral vector encoding TRAIL gene has lately been shown to be effective against liver carcinoma [1]. "
    [Show abstract] [Hide abstract] ABSTRACT: Apoptotic response in hepatocellular carcinoma (HCC) cells is impaired because of interconnectivity of proteins into complexes and signaling networks that are highly divergent in time and space. TNF-related apoptosis-inducing ligand (TRAIL) has emerged as an attractive anticancer agent reported to selectively induce apoptosis in cancer cells. Although diametrically opposed roles of TRAIL are reported both as an inducer of apoptosis and regulator of metastasis, overwhelmingly accumulating experimental evidence highlighting apoptosis inducing activity of TRAIL is directing TRAIL into clinical trials. Insights from TRAIL mediated signaling in HCC research are catalyzing new lines of study that should not only explain molecular mechanisms of disease but also highlight emerging paradigms in restoration of TRAIL mediated apoptosis in resistant cancer cells. It is becoming progressively more understandable that phytochemicals derived from edible plants have shown potential in modelling their interactions with their target proteins. Rapidly accumulating in vitro and in-vivo evidence indicates that phytonutrients have anticancer activity in rodent models of hepatocellular carcinoma. In this review we bring to limelight how phytonutrients restore apoptosis in hepatocellular carcinoma cells by rebalancing pro-apoptotic and anti-apoptotic proteins. Evidence has started to emerge, that reveals how phytonutrients target pharmacologically intractable proteins to suppress cancer. Target-based small-molecule discovery has entered into the mainstream research in the pharmaceutical industry and a better comprehension of the genetics of patients will be essential for identification of responders and non-responders.
    Full-text · Article · Aug 2014 · Molecular Biology Reports
    0Comments 3Citations
  • [Show abstract] [Hide abstract] ABSTRACT: HtrA1, a serine protease, is down-regulated in various human solid tumors. Overexpression of HtrA1 in human cancer cells inhibits cell growth and proliferation in vitro and in vivo, suggesting its possible role as a tumor suppressor. Immunohistochemistry was used to determine the expression of HtrA1 in 50 hepatocellular carcinoma specimens and adjacent liver tissues. The correlation between the expression of HtrA1 and the clinico-pathologic data were analyzed. The levels of HtrA1 were lower in tumor tissues than in their adjacent liver tissues. Moreover, an inverse relationship was found between HtrA1 expression and the differentiation of hepatocellular carcinoma. Loss of HtrA1 was more frequently found in tumors in Edmondson grade III-IV, especially in those with venous invasion, compared to tumors in Edmondson grade I-II. Most importantly, patients with higher HtrA1 expression had a better survival rate. All these data suggest an important role of HtrA1 in hepatocellular carcinoma development and progression, which may be a new target for its treatment.
    No preview · Article · Oct 2010 · Hepatobiliary & pancreatic diseases international: HBPD INT
    0Comments 17Citations
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