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LIVER INJURY/REGENERATION
Effect of Sorafenib on Murine Liver Regeneration
Caroline Hora,
1
Pamela Romanque,
1,2
and Jean-Franc¸ois F. Dufour
1,3
Hepatocellular carcinoma (HCC) is a common cause of cancer-related death. Sorafenib pro-
longs survival of patients with advanced disease and is approved for the systemic treatment of
unresectable HCC. It possesses antiangiogenic and antiproliferative properties by way of inhi-
bition of the receptor tyrosine kinases vascular endothelial growth factor receptor 2 (VEGFR-
2) and platelet-derived growth factor receptor-beta 1/2 (PDGFR-b)andthekinaseRAF.Sora-
fenib represents a candidate compound for adjuvant therapy in HCC patients. The aim of our
study was to investigate whether sorafenib affects liver regeneration. C57BL6 mice received sor-
afenib orally at 30 mg/kg/day or its vehicle either for 14 days until the day before hepatectomy
or starting the day after surgery or both. Animals were sacrificed 24, 72, and 120 hours after
hepatectomy. Liver regeneration was calculated as a percent of initial liver weight. Bromodeox-
yuridine (BrdU) incorporation and phospho-extracellular signal-regulated kinase (pERK1/2)
were determined by immunohistochemistry on liver sections. VEGF-A, PDGF-BB, and hepato-
cyte growth factor (HGF) levels were measured in liver tissue homogenates. Histological analy-
sis of scar tissue was performed. Treatment stopped 1 day before surgery had no impact on
liver regeneration. Continuous sorafenib treatment and treatment started 1 day after surgery
had statistically significant effects on liver regeneration at 120 hours compared to vehicle-
treated control animals (72% 612 versus control 88% 615 and 70% 613 versus control
86% 65at120hours,bothP0.02). BrdU incorporation showed decreased numbers of
positive nuclei in both groups receiving sorafenib after surgery. Phospho-ERK levels were
reduced in sorafenib-treated animals. An increase of VEGF-A levels was observed in mice
receiving sorafenib. Wound-healing complications were observed in animals receiving sorafenib
aftersurgeryandconfirmedonhistologicalsections.Conclusion: This preclinical study shows
that sorafenib did not impact on liver regeneration when ceased before surgery; however,
administration after hepatectomy affected late liver regeneration. (HEPATOLOGY 2011;53:577-586)
Hepatocellular carcinoma (HCC) belongs to the
six most commonly diagnosed cancers world-
wide and represents the third most common
cause of cancer-related death
1
; moreover, its incidence is
rising in the Western world.
2-4
Conventional chemo-
therapy yields only marginal benefits and patients with
unresectable or metastatic HCC have a poor progno-
sis.
5,6
Curative strategies such as liver resection or local
ablation are only amenable to patients with small
tumors and preserved liver function. These approaches
are associated with a reduction of the hepatic functional
mass and are followed by compensatory liver regenera-
tion. Sorafenib is a multikinase inhibitor with antiangio-
genic properties; it has been shown to significantly
improve the survival of patients with advanced HCC
and preserved liver functions.
7,8
Given these beneficial
results, the indication for sorafenib could become
extended to other HCC patients, i.e., as a neoadjuvant
or adjuvant therapy given before or after local ablation/
resection, respectively. This may increase the number of
patients eligible for curative treatment and/or prolong
survival of patients with more advanced disease.
Sorafenib acts by blocking the receptor tyrosine ki-
nases VEGFR (vascular endothelial growth factor recep-
tor) 1, 2, and 3, PDGFR-b(platelet derived growth
Abbreviations: BrdU, bromodeoxyuridine; EGF, epidermal growth factor;
ERK1/2, extracellular signal-regulated kinase; HCC, hepatocellular carcinoma;
HGF, hepatocyte growth factor; JNK, c-Jun N-terminal kinase; MAPK,
mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/
extracellular signal-regulated kinase kinase; PDGFR-b, platelet-derived growth
factor receptor-beta; 1/2; PI3-kinase, phosphatidylinositol 3-kinase; TGFa,
transforming growth factor alpha; IL-6, interleukin-6; TNF, tumor necrosis
factor; VEGFR-2, vascular endothelial growth factor receptor 2.
From the
1
Department of Clinical Research, University of Bern, Switzerland;
2
Biomedical Sciences Institute, Faculty of Medicine, University of Chile, Santiago, Chile;
3
University Clinic for Visceral Surgery and Medicine, Inselspital, Bern, Switzerland.
Received May 21, 2010; accepted September 28, 2010.
C.H. is supported by the Stiftung fu
¨r die Leberkrankheiten. J.F.D. is a recipient of
grants from the Swiss National Foundation, Oncosuisse, and the Sassella Foundation.
Address reprint requests to: Jean-Franc¸ois F. Dufour, University Clinic for
Visceral Surgery and Medicine, Inselspital, Bern, Switzerland. E-mail: jf.
dufour@insel.ch; fax: þ41 31 632 97 65.
Copyright V
C2010 by the American Association for the Study of Liver Diseases.
View this article online at wileyonlinelibrary.com.
DOI 10.1002/hep.24037
Potential conflict of interest: Dr. Dufour advises and receives grants from
Bayer. He also receives grants from Novartis.
Additional Supporting Information may be found in the online version of
this article.
577
factor receptor-beta), Flt-3, c-Kit, fibroblast growth fac-
tor receptor-1, and the serine/threonine kinase RAF,
9,10
thereby repressing tumor cell proliferation and angio-
genesis. These same enzymes, however, also belong to
pathways involved in liver regeneration, orchestrating
the complex interplay of growth factor and cytokine sig-
naling leading to restoration of liver mass.
11,12
The aim of our study was therefore to investigate
the effects of sorafenib on liver regeneration. We per-
formed our experiments using a murine model of par-
tial hepatectomy. Our results show a mild effect on
liver regeneration in animals that received sorafenib af-
ter liver resection.
Materials and Methods
Animals and Surgery. Experiments were performed
in male C57/BL6 mice (Charles Rivers Laboratories,
Germany) weighing 20-25 g. Animals were housed on
a 12-hour light/dark cycle and were provided with
mice chow and water ad libitum. Experimental animal
protocols and animal procedures complied with the
Guide for the Care and Use of Laboratory Animals
(National Academy of Sciences, NIH Publication 86-
23, revised 1996) and were approved by local regula-
tory authorities.
BAY 54-9085 (sorafenib tosylate) (Bayer HealthCare
Pharmaceuticals, Montville, NJ) 30 mg/kg/day or its
vehicle (Cremophor/ ethanol/ distilled water) was
administered by gavage. The dosing volume used was
0.1 mL/10 g body weight. The proportions of Cremo-
phor/ ethanol/ distilled water were 12.5% Cremophor,
12.5% ethanol, and 75% distilled water. For the ani-
mals receiving sorafenib, the drug was first dissolved in
a 50% Cremophor / 50% ethanol mixture and water
was then added to reach the final volume. Animals
treated with the vehicle only received the analog fluid
mixture without the drug. Cremophor EL was pur-
chased from Sigma (Sigma Cat. No. C-5135). Animals
were divided into three groups and their controls. For
the first group, treatment was started 14 days before 2/
3 hepatectomy and stopped 1 day before surgery; the
second group received continuous sorafenib treatment
beginning 14 days prior to surgery until the time of
harvest; and the third group started treatment the day
after 2/3 hepatectomy. Two-thirds hepatectomy was
performed according to the method described by Hig-
gins and Anderson.
13
Under isoflurane anesthesia the
left lateral and median lobes were ligated and resected.
The abdominal muscular and skin walls were sutured
separately with nonabsorbable material until harvest-
ing. Animals were euthanized with Nembutal (50 mg/
kg intraperitoneal) 24, 72, and 120 hours after partial
hepatectomy for the first two groups and at 72 and
120 hours for the third group starting sorafenib after
surgery; liver, scar tissue, and blood samples were
taken at endpoints (n ¼7-14 animals/group). Liver
regeneration was determined as the ratio of liver
weight (g) at harvesting time/liver weight (g) at the
time of partial hepatectomy. Liver weight at the time
of hepatectomy was calculated using five animals sacri-
ficed for this purpose.
Cell Proliferation. At harvesting time, liver sections
were fixed in 10% buffered formalin and processed for
staining with hematoxylin and eosin or for immuno-
histochemistry. The remaining liver was snap-frozen in
liquid nitrogen and kept at 80C until further use.
For determination of hepatocyte proliferation, 1 mg
bromodeoxyuridine (BrdU) was injected intraperitone-
ally 2 hours before sacrifice and BrdU incorporation
was measured using the BrdU In-Situ Detection kit
obtained from BD Pharmingen (BD Biosciences, San
Jose, CA). BrdU incorporation was expressed as num-
ber of BrdU-positive nuclei/mm
2
.
Immunohistology. Immunohistochemistry for phos-
pho-extracellular signal-regulated kinase (pERK) was
performed after deparaffinization, rehydration, and anti-
gen retrieval in citrate buffer by incubation in blocking
solution (Tris-Hcl buffer saline Tween-20 [TBST]/5%
goat serum) for 1 hour, incubation with primary anti-
body (phospho-p44/p42 MAPK [Thr202-Tyr204] rab-
bit monoclonal antibody [mAb], 1:150, Cell Signaling)
overnight at 4C, incubation with secondary antibody
(polyclonal goat antirabbit immunoglobulin/biotinyl-
ated; Dako) and signal detection by the avidin-biotin
system. Imaging was performed with an Olympus
microscope and analysis with the MetaMorph imaging
software (Molecular Devices) in at least 3 fields per slide.
Nuclear phospho-ERK expression was expressed as
pERK-positive nuclei/total nuclei/mm
2
.
Liver Homogenate for Enzyme-Linked Immuno-
sorbent Assay (ELISA). Thirty mg of frozen liver tis-
sues was weighed and lysed in 300 lL HEPES buffer
(20 mM HEPES, pH 7.4; 1.5 mM EDTA; 0.5 mM
PMSF; 1protease inhibitor mix [complete mini tab-
lets, Roche]; 1phosphatase inhibitor [PhosStop,
Roche]). Homogenate was collected after homogeniza-
tion and centrifugation at 14,000 rpm for 10 minutes at
4C. Protein concentration was measured according to
Lowry et al.
14
The amount of VEGF-A, PDGF-BB, and
hepatocyte growth factor (HGF) present in whole liver
protein extracts were measured using ELISA assays
(VEGF-A, PDGF-BB measured with Quantikine im-
munoassay, R&D; HGF measured with RayBio ELISA
578 HORA, ROMANQUE, AND DUFOUR HEPATOLOGY, February 2011
Kit, RayBiotech) following the manufacturer’s instruc-
tions. Protein concentration of each liver homogenate
was used to normalize the hepatic VEGF-A, PDGF-BB,
and HGF levels.
Scar Tissue Analysis. Scar tissue of the peritoneal
and muscular abdominal wall were collected at harvest
and embedded in paraffin. Tissue was stained with the
chromotrope-aniline blue method (CAB trichromic
assay).
15
Microscopic evaluation was performed with an
Olympus microscope by a blinded investigator. In order
to evaluate wound healing in the different treatment
groups, the scar margins of the abdominal wall were
assessed for bridging reactions. Both the 72-hour and the
120-hour timepoints were studied. Bridging reactions
were defined as loci where inflammatory cells transvade
the thin layer of collagen formed on the cut edge, partici-
pating in the granulation tissue that fills the wound cleft,
and eventually linking up opposite scar margins.
Statistical Analysis. Data were analyzed with
GraphPad Prism 4.0 software. Kruskal-Wallis and the
Mann-Whitney test assessed the statistical significance
of differences between mean values; Pless than 0.05
was considered significant.
Results
Mice which were treated with sorafenib for 14 days
and stopped treatment 1 day before partial hepatec-
tomy showed no impairment in liver regeneration
when compared to the control group that received the
vehicle only (Figs. 1, 2A). In contrast, the animals
receiving continuous sorafenib treatment presented sig-
nificantly lower liver mass restoration at 120 hours in
comparison to the animals treated with the vehicle
(72% 612% versus vehicle 88% 615%, P<0.02).
No significant differences were observed at earlier
timepoints (24 hours, 62% 68% versus vehicle 61%
616%; 72 hours, 74% 622% versus vehicle 72% 6
22%) (Fig. 2B). A similar effect on late regeneration
was observed in the group starting sorafenib treatment
1 day after 2/3 hepatectomy, with significantly reduced
liver regeneration at 120 hours (70% 613% versus
vehicle 86% 65%, P<0.003; 72 hours, 62% 69%
versus vehicle 70% 612%, not significant [n.s.]) (Fig.
2C).
Cell proliferation was assessed by BrdU incorpora-
tion. At 24 and 72 hours after surgery the number of
positive nuclei was significantly decreased in the liver
of animals continuously treated with sorafenib in com-
parison to their controls (24 hours, 6 63 versus vehi-
cle 17 69 nuclei/mm
2
P<0.001; 72 hours, 74 6
25 versus vehicle 144 667 nuclei/mm
2
,P<0.02)
(Fig. 3B). BrdU incorporation also revealed reduced
cell proliferation at 72 hours in the group of mice
starting treatment after surgery compared to their con-
trols (23 68 versus vehicle 99 640 nuclei/mm
2
,
P<0.001) (Fig. 3C). Both groups showed no signifi-
cant difference at 120 hours after hepatectomy. Fur-
ther, no differences were observed when comparing
animals stopping sorafenib 1 day before surgery and
their controls at any timepoint (Fig. 3A).
Sorafenib inhibits the serine/threonine kinase RAF;
therefore, the inhibitory effect on the mitogen-acti-
vated protein kinase (MAPK) pathway was assessed by
immunohistochemistry for pERK. At the time of hep-
atectomy (0 hours) (Fig. 4), vehicle-treated animals
and mice receiving sorafenib after surgery showed
comparable numbers of pERK-positive nuclei (7.3%
65 and 7.5% 64.7 positive nuclei / total nuclei,
respectively). Both groups starting sorafenib treatment
2 weeks prior to surgery showed significantly lower
pERK levels when compared to the control group (0
hours, sorafenib presurgery 3.4% 62.6 versus vehicle
7.3% 65, P0.01; 0 hours, sorafenib pre- and
postsurgery 3.0% 62.1 versus vehicle 7.3% 65,
P0.01) and to the group starting sorafenib 1 day
postsurgery (0 hours, sorafenib presurgery 3.4% 62.6
versus postsurgery 7.5% 64.7, P0.05; 0 hours,
sorafenib pre- and postsurgery 3.0% 62.1 versus
postsurgery 7.5% 64.7, P0.05). Twenty-four
Fig. 1. Treatment scheme. In the ‘‘sorafenib pre-surgery’’ group sora-
fenib treatment was started 14 days prior to hepatectomy and stopped
1 day before the intervention. In the ‘‘sorafenib pre- and post-surgery’’
group sorafenib was started 14 days before surgery and continued until
harvest. For those two groups animals were harvested at 24 hours, 72
hours, or 120 hours after hepatectomy. In the ‘‘sorafenib post-surgery’’
group sorafenib treatment was started 24 hours after hepatectomy and
continued until the time of harvest at 72 or 120 hours.
HEPATOLOGY, Vol. 53, No. 2, 2011 HORA, ROMANQUE, AND DUFOUR 579
hours after partial hepatectomy, pERK levels in the ve-
hicle-treated control animals increased more than 4-
fold; in contrast, pERK levels did not increase in the
animals administered sorafenib before surgery only (24
hours, 4.3% 65.6 versus vehicle 33.6% 610.6, P
0.001). Moreover, mice administered continuous sora-
fenib had even lower pERK levels (24 hours, 0.6% 6
0.8 versus vehicle 33.6% 610.6, P0.001). Note
that the group starting sorafenib after surgery could
not be assessed at 24 hours because this timepoint
coincided with beginning of treatment. At 72 hours
(Fig. 4; Supporting Information Fig. 1) the group that
had stopped sorafenib 1 day before surgery showed
comparable pERK levels as the vehicle-treated animals
Fig. 2. Liver regeneration. Graphs representing liver regeneration at
different timepoints; liver mass was calculated as percentage of total
initial liver weight. (A) Animals treated with sorafenib or vehicle until 1
day before surgery. (B) Animals treated with sorafenib or vehicle con-
tinuously. (C) Animals treated with sorafenib or vehicle after surgery.
Black bars: vehicle-treated mice; gray bars: sorafenib-treated mice.
(A) No differences in liver regeneration were observed between vehicle-
and sorafenib-treated animals. (B) Mice treated with sorafenib show
impaired liver regeneration 120 hours after hepatectomy. (C) Sorafenib
treatment compromises liver mass restoration 120 hours after surgery.
Values are mean 6SD. *Statistical significance (P<0.05) versus
vehicle-treated control mice.
Fig. 3. Cell proliferation assessed by BrdU incorporation. (A) Mice
treated before surgery. (B) Animals receiving continuous treatment.
(C) Mice treated after surgery. No differences in cell proliferation were
noted when treatment was stopped before hepatectomy (A). In the
mice receiving continuous sorafenib, cell proliferation was reduced at
24 and 72 hours compared to controls (B). Similarly, BrdU incorpora-
tion was reduced at 72 hours in mice starting treatment 1 day after
surgery (C). Black bars: vehicle-treated mice; gray bars: sorafenib-
treated mice. Values are mean 6SD. *Statistical significance (P<
0.05) versus vehicle-treated control mice.
580 HORA, ROMANQUE, AND DUFOUR HEPATOLOGY, February 2011
(72 hours, 28% 612.9 versus vehicle 22.1% 615.5,
n.s.), whereas pERK levels remained barely detectable
in the group receiving continuous sorafenib (72 hours,
0.8% 60.3 versus vehicle 22.1% 615.5, and versus
sorafenib presurgery 28% 612.9, P0.05). In the
group of mice starting sorafenib treatment after sur-
gery, pERK levels were not increased at 72 hours (72
hours, 8.9% 67.1 versus vehicle 22.1% 615.5, P
0.05). Finally, the 120-hour timepoint revealed the
highest number of pERK positive nuclei in animals
treated before surgery only (120 hours, sorafenib pre-
surgery 33% 60.9 versus vehicle 18.1% 613.6,
n.s.). The group administered sorafenib pre- and post-
surgery still showed very low pERK levels at 120 hours
(120 hours, 1.4% 61.7 versus vehicle 18.1% 613.6,
P0.05); moreover, in the group starting sorafenib 1
day after surgery, pERK-positive nuclei were barely de-
tectable (120 hours, 0.3% 60.2 versus vehicle 18.1%
613.6, P0.001).
Next, hepatic VEGF-A levels were quantified from
whole liver lysates by ELISA in the three treatment
groups. After 2 weeks of sorafenib treatment, a signifi-
cant increase in VEGF-A was observed at baseline (0
hours, measured at the time of hepatectomy). A 1.5-
fold and 2-fold increase was measured in the mice
receiving sorafenib prior to hepatectomy and in mice
administered continuous sorafenib treatment compared
to vehicle-treated animals (0 hours, sorafenib presur-
gery 38.2 66.7 pg/lg versus vehicle 25.4 63.0 pg/
lg, P<0.0001; and 0 hours, continuous sorafenib
42.6 66.6 pg/lg versus vehicle 20.5 65.0 pg/lg,
P<0.0001, respectively) (Fig. 5A,B). In the group
that stopped sorafenib before partial hepatectomy, the
initial increase in VEGF levels was not maintained and
no differences were seen at any of the timepoints post-
surgery (Fig. 5A). The group receiving continuous sor-
afenib and the group starting treatment after surgery
had significantly higher hepatic VEGF levels compared
to vehicle control animals at 72 and 120 hours (con-
tinuous sorafenib group: 72 hours, 44.8 63.6 pg/lg
versus vehicle 21.4 63.9 pg/lg, P<0.01, and 120
hours, 60.0 612.0 pg/lg versus vehicle 20.7 63.8
pg/lg, P<0.05; in the sorafenib postsurgery group:
72 hours, 43.8 611.1 pg/lg versus vehicle 23.3 6
6.4 pg/lg, P<0.001, and 120 hours, 32.9 64.1 pg/
lg versus vehicle 17.0 63.5 pg/lg, P<0.0001) (Fig.
5B,C). Surprisingly, continuous sorafenib administra-
tion did not alter hepatic VEGF levels measured at 24
hours compared to controls (24 hours, 25.8 65.1 pg/
lg versus vehicle 24.0 611.7 pg/lg) (Fig. 5B).
No differences were observed for PDGF-BB protein
levels measured by ELISA in whole-liver lysates. The
sorafenib-treated animals showed similar levels of
PDGF as the vehicle-treated mice at all timepoints in
all three treatment groups (data not shown).
HGF protein levels revealed a modest increase of liver
HGF protein levels at 24 hours after hepatectomy in the
control animals receiving vehicle treatment (Supporting
Information Fig. 2). This increase was not observed in
animals treated with sorafenib prior to 2/3 hepatectomy;
HGF levels at 24 hours were even decreased in mice
receiving continuous sorafenib treatment (24 hours,
Fig. 4. Liver pERK levels. Quantification of immunohistochemical staining for pERK, percentage of pERK-positive nuclei per total nuclei. At the
time of hepatectomy, both groups treated with sorafenib before surgery had a significant reduction of pERK when compared to controls and/or
to animals starting treatment after surgery. At 24 hours, pERK levels failed to increase in the pre-surgery group and decreased in the continuous
treatment group. Seventy-two hours after hepatectomy, ERK activity was comparable to controls in the pre-surgery group. It remained very low in
the continuous treatment group, whereas activity was comparable to baseline (0 hours) in the post-surgery animals. For the late timepoint, most
phosphor-ERK was measured in the pre-surgery group. In contrast, both groups treated with sorafenib after surgery showed minimal expression.
Values are mean 6SD. *Statistical significance (P<0.05) versus vehicle-treated control mice. §Statistical significance (P<0.05) versus
mice starting sorafenib after surgery.
HEPATOLOGY, Vol. 53, No. 2, 2011 HORA, ROMANQUE, AND DUFOUR 581
continuous sorafenib 16.5 64.0 ng/mg versus vehicle
30.7 64.8 ng/mg, P<0.01). Further, we noted a con-
sistent increase of HGF levels at 120 hours after hepatec-
tomy in the mice receiving continuous sorafenib treat-
ment and the mice starting sorafenib after surgery,
although this was not significant.
At the time of sacrifice, the abdominal scar was
excised and the suture removed carefully. Although the
scar margins of the control animals remained sealed,
mice receiving sorafenib until harvest had more fragile
scars, i.e., the margins were not sealed or separated in
a zip-like fashion upon minimal traction. Histological
analysis of scar tissue revealed differences in the scar of
vehicle- and sorafenib-treated animals. The scars of ve-
hicle control animals presented tissue remodeling of
the muscular wall with dense granulation tissue filling
the wound cleft (Fig. 6, top panel). Quantification of
bridging reactions revealed no significant differences
72 hours after surgery. However, at 120 hours we
observed significantly less bridges in animals that had
received sorafenib treatment after surgery compared to
vehicle controls (120 hours, continuous sorafenib 1.8
61.1 versus vehicle 4.2 61.8, P<0.05; sorafenib
postsurgery 1.8 61.4 versus vehicle 4.2 61.8, P<
0.01) (Fig. 7). Moreover, the scars of animals that
were treated with sorafenib after surgery showed less
intense tissue remodeling and granulation tissue was
less dense or barely present (Fig. 6, lower panels).
Discussion
Our preclinical results show that sorafenib adminis-
tration that is stopped 1 day before hepatic resection
had no effect on liver regeneration in this study,
whereas liver regeneration was impaired at the late
timepoint examined (120 hours) when sorafenib was
administered postoperatively.
Liver regeneration is a complex process that depends
on the activation of several growth signal pathways.
Sorafenib inhibits the serine/threonine kinase activity
of RAF in the RAF/MEK/ERK signaling pathway and
the receptor tyrosine kinase activity of the VEGF re-
ceptor-2.
9
Liver regeneration studies have shown that a
variety of growth factors and cytokines, acting by way
of their respective receptors, activate complementary
signaling pathways that elicit cellular proliferation and
liver mass restoration. Among these intracellular medi-
ator is the RAS/RAF/MEK pathway, resulting in the
activation of ERK1/2.
12
Growth factors such as EGF,
HGF, and TGFaand different cytokines (interleukin-6
[IL-6], TNF [tumor necrosis factor]) trigger ERK1/2
activation.
16-18
This mitogenic cascade is inhibited by
sorafenib at the level of RAF. Our analysis of phospho-
rylated ERK by immunohistochemistry showed
decreased levels in the sorafenib-treated animals, with
an important inhibition of ERK activation after
Fig. 5. VEGF hepatic levels. VEGF protein content measured by
ELISA from whole liver lysate, showing baseline (hepatectomy) and
protein levels at sacrifice. (A) Animals treated with sorafenib or vehicle
until 1 day before surgery. (B) Animals treated with sorafenib or vehi-
cle continuously. (C) Animals treated with sorafenib or vehicle after
surgery. Black bars: vehicle-treated mice; gray bars: sorafenib-treated
mice. Baseline levels increased after 2 weeks of sorafenib treatment
but returned to control levels 24 hours after hepatectomy in the group
stopping the drug before surgery (A). VEGF levels are higher in the sor-
afenib-treated animals at baseline (0 hours). No difference was
observed after 24 hours, but at 72 and 120 hours, higher VEGF levels
were measured in the mice receiving sorafenib, with a 3-fold increase
at 120 hours compared to controls (B). Equal amounts of hepatic
VEGF were seen at baseline; VEGF levels were increased at 72 and
120 hours in mice starting sorafenib after surgery compared to control
animals (C). Values are mean 6SD. *Statistical significance (P<
0.05) versus vehicle-treated control mice.
582 HORA, ROMANQUE, AND DUFOUR HEPATOLOGY, February 2011
hepatectomy but also diminished baseline phospho-
ERK contents at the time of hepatectomy in the ani-
mals that had received sorafenib treatment prior to
surgery. It is interesting that liver regeneration still
occurred reasonably effectively in spite of almost com-
plete pERK ablation in the mice treated with sorafenib
after resection. The importance of phospho-ERK1/2
activity in hepatocyte proliferation is not univocal.
Although some reports support a key role of the
MAPK pathway in regulating hepatocyte prolifera-
tion,
19-21
others observed a discrepancy between
ERK1/2 activity and cellular proliferation.
22
Further
and similar to our findings, Borowiak et al.
23
showed
only mild effects on liver regeneration for conditional
Met mutant mice 5-7 days after hepatectomy, despite
low phospho-ERK1/2 levels and reduced cell prolifera-
tion. These data and our findings, together with
reports assessing the roles of other signal transducers
such as JNK1/2, p38, and the PI3-kinase,
22,24,25
indi-
cate a redundancy of the system rather than exclusive
roles of selected pathways. HGF is an important player
in the liver regeneration process; its receptor Met is
Fig. 6. Wound healing: histology.
Wound healing was assessed on
histological sections of the abdomi-
nal scar. Examples of scarring tis-
sue for each group at 120 hours
after surgery are shown. The top
panel shows wound healing in a
control animal; dense granulation
tissue links up the wound edges,
appearing as a bridge (inset). The
granulation tissue is looser or
scarce in the animals receiving sor-
afenib after surgical intervention
(bottom panels); there are fewer
bridges and the muscular tissue
appears less reactive.
HEPATOLOGY, Vol. 53, No. 2, 2011 HORA, ROMANQUE, AND DUFOUR 583
rapidly activated after partial hepatectomy.
11
Down-
stream signaling is mediated in part by PI3K/Akt,
RAS/RAF/MEK/ERK, and the transcription factor
STAT3, resulting in cell survival and cell prolifera-
tion.
26,27
Overall, our analyses of liver HGF protein
levels showed minimal effects of sorafenib on HGF
levels. However, our experimental setup did not focus
on the very early events of liver regeneration (before
24 hours). Apart from a reduction of HGF protein
content at 24 hours in the mice continuously treated
with sorafenib, HGF levels did not appear to be
reduced by sorafenib treatment. These data suggest
that the regenerative process could still occur by way
of other signaling cascades than RAS/RAF/MEK, pro-
viding in part an explanation as to why regeneration
occurred despite minimal phospho-ERK induction.
Liver regeneration depends not only on hepatocyte
proliferation but also on endothelial cell proliferation
and angiogenesis.
28
VEGF is a key mediator of angio-
genesis and also participates in the induction of
growth factors in the regenerating liver.
29,30
It indi-
rectly promotes hepatocyte proliferation by stimulating
HGF production in sinusoidal endothelial cells (via
VEGF receptor 1),
31-33
the transient inhibition of
HGF observed at 24 hours in the continuously treated
animals may reflect the inhibition of endothelial
VEGFR-1 by sorafenib. Endothelial cell proliferation,
migration, and survival is mediated by VEGFR-2.
34,35
Mice heterozygous for VEGFR-2 were reported to
maintain normal proliferative capacity of the paren-
chyma and the sinusoidal endothelial cells following
partial hepatectomy.
36
We observed a pharmacody-
namic effect of elevated VEGF levels in the liver of
animals treated with sorafenib. Interestingly, sorafenib
treatment alone, prior to surgical intervention, had al-
ready induced an increase in VEGF levels at baseline
(0 hours). Hepatocytes are the source of VEGF in the
regenerating liver, but VEGF can be produced by
most cells in mammals.
29
The increase in VEGF levels
observed at 3 and 5 days after hepatectomy in the
mice receiving sorafenib treatment are likely produced
by the replicating hepatocyte population; the source of
elevated VEGF prior to liver resection may be diverse,
accounting for a systemic adaptation of impeded
VEGF signaling elicited by sorafenib. Similarly, suniti-
nib, a multitarget receptor tyrosine kinase inhibitor,
was reported to induce increases in VEGF levels and
other proangiogenic factors in mice.
37
Sorafenib treatment did have an effect on liver mass
restoration in the animals receiving the drug postoper-
atively, independent of drug administration prior to
surgery or starting the day after the operation. Liver
regeneration was impaired in these mice, albeit mildly
and only at a late timepoint. Similar observations were
noted in a study looking at the effects of anti-VEGF
therapy after partial hepatectomy.
38
For the earlier
timepoints studied, the difference in liver mass recu-
peration was not significant, suggesting that inhibition
of the RAS/MAPK/ERK pathway and the VEGFR ki-
nase is not critical to initiate liver regeneration, but
plays a role in sustaining the process. A possible expla-
nation for the late appearance of the antiangiogenic
effect is that, chronologically, replication of endothelial
cells follows replication of hepatocytes.
39
On the other
hand, an earlier effect concerning hepatocyte prolifera-
tion was observed, as assessed by BrdU incorporation.
The proliferation assay showed significantly reduced
DNA synthesis at early timepoints (24 and 72 hours),
pointing to an inhibitory effect on parenchyma restitu-
tion. Considering clinical settings, these findings may
be of importance for patients receiving sorafenib while
being treated with a local ablative therapy such as
transarterial chemoembolization (TACE) or radiofre-
quency ablation (RFA). It also may be of relevance for
patients who are subjected to portal vein ligation to
induce a compensatory hypertrophy in view of a
hemihepatectomy.
An important finding of our work is that sorafenib
stopped the day before surgery had no impact on liver
regeneration in this preclinical study. It did not impair
hepatocyte proliferation nor ERK phosphorylation;
only the hepatic VEGF levels were increased at base-
line, returning to control values as early as 1 day
Fig. 7. Wound healing: quantification. The number of bridging reac-
tions per tissue slide was counted. Quantification revealed most bridg-
ing reactions in the control animals as compared to the three
treatment groups, with significantly less sealing observed at 120 hours
in the two groups receiving sorafenib after surgery. Values are mean
6SD. *Statistical significance (P<0.05) versus vehicle-treated con-
trol mice.
584 HORA, ROMANQUE, AND DUFOUR HEPATOLOGY, February 2011
postoperatively. The compound sorafenib is a competi-
tive inhibitor, implying reversibility of its actions, and
has a half-life of 25-48 hours
40,41
in man. These find-
ings suggest that patients receiving sorafenib while
waiting for liver transplantation may receive a small-
for-size liver, without having a negative effect of prior
sorafenib treatment on liver size adaptation. Further,
considering an indication for sorafenib as a neoadju-
vant treatment, our data suggest that drug administra-
tion may be continued until the day preceding surgery
without compromising liver mass recuperation.
Wound healing was affected in mice treated with
sorafenib after surgical intervention. Scar tissue of the
muscular abdominal wall revealed histological differen-
ces that we were able to quantify. Wound healing was
also markedly affected at the skin level. The inhibitory
effect of sorafenib on vasculogenesis is a plausible ex-
planation for these observations; another mechanism
may be the inhibition of ERK phosphorylation. Mice
carrying a conditional c-Met mutant show impaired
wound healing of the skin.
42
The c-Met mutated kera-
tinocytes are unable to form a hyperproliferative epi-
thelium, essential for the reepithelialization of the
wound. These keratinocytes show altered signal trans-
duction, including markedly reduced ERK1/2 phos-
phorylation, upon growth factor stimulation. In order
to determine the impact of sorafenib on wound heal-
ing in a clinical setting, prospective studies are needed;
however, it is recommended that sorafenib be discon-
tinued prior to surgery. The findings of this preclinical
study suggest there is a potential for an increased rate
of complications if treatment is not discontinued prior
to, or started rapidly after, surgery. The complications
we observed consisted mainly of scar dehiscence pre-
senting as incomplete sealing of the abdominal wall.
This study could not evaluate parameters relevant for
transplantation such as vessel suture remodeling of the
hepatic branches and the biliary system.
We showed that sorafenib does have an effect on cyto-
kine levels after partial hepatectomy. Interestingly, the
levels of VEGF increased under sorafenib treatment.
This is an important finding because plasma VEGF lev-
els have been reported to correlate with tumor VEGF
expression and to have a prognostic signification in
HCC patients treated by resection or TACE.
43-46
The
interpretation of VEGF as a parameter to predict recur-
rence and patient outcome should take into account a
concomitant therapy with sorafenib.
Our study has limitations. It was performed in an
animal model and it is unclear how it will translate in
the clinic. It was performed in noncirrhotic animals,
when most of the patients affected by HCC have
underlying cirrhosis. Cirrhosis impairs liver regenera-
tion; whether this is further affected by sorafenib is
unknown and is not addressed by our experimental
model. According to guidelines, HCC resection is pos-
sible only in highly selected cirrhosis patients with nor-
mal bilirubin and no portal hypertension. Our data do
not allow us to predict the effect of sorafenib in such
a setting. Also, our model does not take into account
other patient characteristics such as concomitant meta-
bolic disorders, i.e., fatty liver disease or diabetes, sus-
ceptible to interfere with normal liver regeneration or
wound healing.
In conclusion, this is the first preclinical study ana-
lyzing sorafenib and liver regeneration. To date there
have been no clinical reports on liver regeneration
under sorafenib treatment. We show that sorafenib
affects late liver mass restoration when it is adminis-
tered after surgery, although this effect is mild in our
murine model. Liver regeneration is not impaired by
sorafenib when treatment is discontinued before
intervention.
Acknowledgment: We thank Monika Ledermann
for surgical expertise, Scott Wilhelm for editorial sup-
port, and Bayer for providing sorafenib.
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