PEG-conjugated Hemoglobin Combination with

Cisplatin Enforced the Antiangiogeic Effect in a

Cervical Tumor Xenograft Model
Min Dai, Minghua Yu, Jianqun Han and Hongwei Li
Institute of Microcirculation, Peking Union Medical College & Chinese Academy of
Medical Sciences, Beijing, China
Peilin Cui
Beijing Tiantan Hospital, Capital University of Medical Sciences, Beijing, China
Qian Liu
Peking Union Medical College Hospital, Beijing, China
Ruijuan Xiu
Institute of Microcirculation, Peking Union Medical College & Chinese Academy of
Sciences, Beijing, China
Abstract: Angiogenesis, an essential event involved in a tumors progression and
metastasis, is regulated by hypoxia. Hypoxia widely exists in solid tumors due to the
abnormal vasculature of tumor tissue and insufficiency of tissue oxygenation. We
speculate that hemoglobin-based oxygen carriers (HBOCs) can attenuate tissue
hypoxia, thereby suppressingthe angiogenesis in solid tumor in the context that
HBOCs have the ability to increase tissue oxygenation. In the present study, PEG-
conjugated hemoglobin solution (0.3 g/kg i.v. or 0.6 g/kg i.v.) was intravenously
administrated to BALB/c nude mice bearing the cervical tumor twice a week with or
without the treatment of cisplatin (5mg/kg i.p.) to investigate whether PEG-
conjugated hemoglobin has a chemo-sensitization effect though anti-angiogenesis
We thank Prof. TMS Chang, McGill University, Canada, for serious review of this
manuscript. This work was supported by Prof. Ruijuan Xius UNESCO Award for
Women in Science 2000 and the grant of Knowledge Innovation Project Academy
of Science, China (No.KJCX1-SW-07).
Address correspondence to Ruijuan Xiu, Institute of Microcirculation, Chinese
Academy of Medical Sciences (CAMS) & Peking Union Medical College (PUMC), 5
Dong Dan San Tiao, Beijing, 100005, China. E-mail: xiurj@yahoo.com.cn
Artificial Cells, Blood Substitutes, and Biotechnology, 36: 487497, 2008
Copyright # Informa UK Ltd.
ISSN: 1073-1199 print / 1532-4184 online
DOI: 10.1080/10731190802554109
487
pathway. Tumor volume was measured every three days and tumor hypoxia was
detected by immunohistochemistry for Hypoxyprobe
TM
-1. Anti-angiogenic effect was
accessed by detection of mRNA and protein levels of vascular endothelial growth
factor (VEGF), the most important angiogenic factor. Our results showed that high
concentration of PEG-conjugated hemoglobin solution significantly impeded the
growth of tumor when compared with the control group. Moreover, VEGF expression
was declined when treated with PEG-conjugated hemoglobin, possibly through the
HIF regulation system. Collectively, treatment of PEG-conjugated hemoglobin
combination with cisplatin has an antiangiogeic effect, but the underlying mechanism
should be further studied.
Keywords: Articial oxygen carrier, tumor, hypoxia, cisplatin, angiogenesis
INTRODUCTION
Solid tumors frequently contain a hypoxic microenvironment, which is
uniquely different from that of normal tissues. The hypoxic microenvironment
induces adaptive changes to tumor cell metabolism, and this alteration can
further distort the local microenvironment [1]. As a result, the abnormal
microenvironment inhibits many standard cytotoxic anticancer therapies and
predicts for a poor clinical outcome. Angiogenesis, an essential event involved
in tumors progression and metastasis, is also induced by tumor hypoxia [2,3].
But unlike normal blood vessels, newly formed tumor vasculature has
abnormal organization, structure, and function [4,5]. Vascular hyper perme-
ability caused by the leaky vessels and the lack of functional lymphatic vessels
inside tumors causes elevation of interstitial fluid pressure in solid tumors. The
abnormal tumor environment makes it difficult to deliver therapeutic agents to
tumors [6] and makes tumor cells more resistant to chemotherapy [7].
Furthermore, elevated tumor interstitial fluid pressure increases fluid flow
from the tumor margin into the peri-tumor area and may facilitate tumor
metastasis [1,8]. Anti-angiogenic therapy is considered to be a very promising
strategy because it can normalize the tumor vascular net to limit the growth
of tumor and enforce the efficacy of chemotherapy [9,10].
A hemoglobin-based oxygen carrier (HBOC), designed with a purpose to
substitute blood transfusions [11], shows the potential to be used to cure some
hypoxic ischemic diseases as a kind of oxygen therapy in recent years. One of
the most important applications is that it can be used in cancer treatment as a
radio- or chemotherapy sensitizer [1215]. It is speculated that HBOC may
have a chemo-sensitivity effect when combined with chemo-therapy in cancer
treatment because it is possible to ameliorate tumor hypoxia microenviron-
ment, which is a characteristic of solid tumor. Some studies have reported that
intravenous administration of hemoglobin solution was effective in ameliorat-
ing tumor hypoxia conditions [12,15,16] and improving the efficacy of both
488 M. Dai et al.
irradiation [12,14,17,18] and chemotherapeutic agents [14,1820]. Until now
there has been no study to investigate the effect of hemoglobin solution on
tumor angiogenesis. The present study was performed to investigate the
influence of PEG-HB, a new kind of PEG-Hb solution, on the hypoxia
microenvironment and tumor angiogenesis.
MATERIALS AND METHODS
Polyethyleneglycol-conjugated Hemoglobin (PEG-Hb)
The PEG-Hb used in this study was a Chinese domestic sample. Its
preparation processes and physiochemical properties belong to the manufac-
turers proprietary information and will not be discussed in this paper. The
sample was kept at 4
o
C in the dark until use.
Cell Line
HeLa cells were obtained from the cell biology center of the Chinese
Academy of Medical Sciences (CAMS) and Peking Union Medical College
(PUMC). Cells were cultured in RPMI-1640 (Gibco, Life Technologies,
Vienna, Austria), supplemented with 10% heat-inactivated FBS (Gibco), 4
mM glutamine, 100 nM Na-pyruvate, 25 mM Hepes, 100 u/ml penicillin and
100 u/ml streptomycin, and incubated at 37 8C in a humidified atmosphere
containing 5% CO
2
atmosphere.
Xenograft Tumor Model and Drug Administration
We subcutaneously injected 0.2ml 510
6
HeLa cells into the armpits of 5-
week-old female BALB/c nude mice. We measured tumors with calipers twice
a week and calculated tumor volume as (lengthwidth
2
). After two weeks
of innoculation, we select the mouse whose tumor volume was around
6080 mm
3
. The nude mice were separated into five groups (n10) randomly
and treated differently as follows: physiological saline (group 1), cisplatin
(5mg/kg, group 2), cisplatin with PEG-HB at dose levels of 0.3 g/kg (group 3),
0.6 g/kg (group 4), or PEG-HB alone at dose level of 0.45 g/kg (group 5) was
administered to each group intravenously, twice a week, for four weeks. The
general health of mice was monitored daily. Tumor dimensions and body
weights were recorded two to three times a week starting with the first day of
treatment.
Influence of PEG-Hb on Tumor Angiogenesis 489
Hypoxia Detection
Hypoxyprobe
TM
-1 kit (Chemicon, CA, USA) was used to detect the tumor
hypoxia in vivo. Hypoxyprobe-1(pimonidazol hydrochloride) is a chemical
component that specifically binds to proteins in hypoxic cells at an oxygen
pressure equal to or lower than 10 mmHg [21]. The high water solubility of
Hypoxyprobe
TM
-1 permits small volume injections to be made, which is
convenient for studies with small animals. The formed protein adducts are
detected by staining with specific monoclonal antibodies and the amount of
adducts formed is proportional to the level of hypoxia.
To assess hypoxia regions, we intraperitoneally injected mice with 60
mg/kg Hypoxyprobe-1, 1h before sacrificed. After the mice were sacrificed,
tumors were rapidly removed and fixed in 10% formalin, dehydrated and
embedded in paraffin. Immunohistochemical studies were performed on
deparaffinized and rehydrated sections, according to the manufacturing
instructions. The slides were exposed to hypoxyprobe-1 Mab1 (diluted
1:50) for 40 min at RT, rinsed in phosphate-buffered saline and 0.2% Brij
35 for seven times at 08C, and incubated with biotinylated secondary
antibody. Finally, the DAB were performed according to the manufacturers
protocol (DAB Kit, Promega, Madison, WI, USA). Controls were performed
by processing slides in absence of the primary antibody. Each tumor was
scored semi-quantitatively section by section in all cases using a scoring
system where hypoxia in the ranges of 0; 05%; 515%; 1530%;
30% were assigned scores of 0, 1, 2, 3 and 4. The cumulative
scores of each group were calculated and averaged for use as descriptors.
Western Blot Analysis
For HIF-1a expression analysis, tumors were grinded in liquid nitrogen and
suspended in 100200ml lysis buffer (50mmol/L Tris, 150mmol/L NaCl,
5mmol/LEDTA, 5mmol/L EGTA, 1%SDS, pH7.5), then ultrasonicated on ice
until the solution became clear. The total protein concentrations were
measured with the Bradfold method. Samples were heated at 1008C for
5min with 2SDS loading buffer and briefly cooled on ice. 50mg total
proteins from each sample were subjected to 8% or 12%SDS-PAGE to detect
HIF-1a (120KD) and b-actin (43KD), respectively. Proteins were electro-
phoretically transferred to PVDF membranes (Millipore Corp., Bedford, MA,
USA) in transfer buffer (25 mmol/L Tris, 200mmol/L glycerin, 20% methanol,
pH8.5) at 100V for 3 hours, and then membranes were blocked with 5% skim
milk in PBS for 1h at room temperature. Specific immunodetections were
carried out by incubation with primary antibodies (rabbit polyclonal antibody
to HIF-1a, sc-10790, Santa Cruz; goat polyclonal antibody to b-Actin,
490 M. Dai et al.
sc-1616, Santa Cruz) diluted 1:500 in skim milk overnight at 48C. After three
washes with PBS, the membranes were incubated for another 1h with
horseradish peroxidase-conjugated goat anti-rabbit or rabbit anti-goat IgG
(BeiJing ZhongShan Goldbridge Biotechnology Co. Ltd.) and diluted 1:10000
in PBS at room temperature. Antigens were revealed using a chemilumines-
cence assay (Western Blotting Luminol Reagent, sc-2048, Santa Cruz).
RT-PCR Analysis
Tumors were grinded in liquid nitrogen, and then total RNA was extracted
separately from tumor tissues of each group with TRIZOL

reagent following
the manufacturers instructions. A 2mg (treated in 8 mL DEPC water in an Ep
tube) sample of total RNA (added 1ml 10mM dNTPmix and 1ml 0.5mg/mL
Oligo (dt) 1218) was denatured by incubating at 658C for 5 min, and the tube
was placed on ice for 2 min, and then reverse-transcribed into complementary
DNA (cDNA) using the following procedures: briefly, the denatured RNA
was incubated for 428C for 2 min with 2ml 10RT buffer, 4ml 25mM MgCl
2
,
2ml 0.1M DTT and 1ml RNaseOUTTM Recombinant Rnase inhibitor (50u/
mL), then we added 1ml (50units) SuperScriptTM II RT, and incubated it at
428C for 50min and terminated at 708C for 15min in a total volume of 20ml;
finally, samples were chilled on ice, 1ml Rnase H was added and incubated for
20 min at 378C before PCR. For PCR, 2ml of the resulting cDNA, 36.75ml of
tripled-distilled H
2
O, 5ml of 10PCR buffer, 3ml of MgCl
2
(25 mmol/L), 1ml
of dNTPs (10mmol/L), 1ml of each of sense and antisense primers (10 mmol/
L), and 0.25ml Taq DNA polymerase (5 u/ul) in a total volume of 50ml were
added. Amplifications for VEGF were performed for 30 cycles and b-actin
was amplified for 21 cycles. Each amplification consisting of denaturation
at 948C for 45s, primer annealing at 548C for 45s and extension at 728C for
1 min. Cycles were preceded by incubation at 948C for 5 min to ensure full
denaturation of the target gene, followed by an extra incubation at 728C for
7min to ensure full extension of the products. PCR products were analyzed on
1% agarose gel containing ethidium bromide. The sequences of the primers for
VEGF165 were sense 5?-GGGCAGAATCATCACGAAGT-3? and antisense
5?-AAATGCTTTCTCCGCTCTGA-3? (359 bp) and for b-actin, sense 5?-
GTGCGTGACATTAAGGAG-3? and antisense 5?-CTAAGTCATAGTCCG
CCT-3? (520 bp).
VEGF ELISA Assay
VEGF protein levels in blood were quantified by ELISA methods. The blood
wasw collected before the mice were sacrificed and centrifuged at 12,000 rpm
Influence of PEG-Hb on Tumor Angiogenesis 491
at 48C for 15 min, and then VEGF concentration in serum was accessed
by ELISA according to the manufacturers instructions (VEGF ELISA kit,
Promega, Madison, WI, USA). The values of OD (A
450
values) were
measured at 450 nm. The standard curve was worked out by the SPSS
statistical software. Serum was harvested with 6 replicated and the experiment
was performed three times.
STATISTICAL ANALYSIS
Statistical analysis was performed using the statistical program SPSS 10.0 for
windows (SPSS Inc., Chicago, IL, USA). All data are presented as mean9
S.E.M and are analyzed by One-Way ANOVA. P valuesB0.05 were
considered as statistically significant.
RESULT
Influence of PEG-HB on Tumor Growth
The result showed that administration PEG-HB combined with cisplatin
caused a inhibition of tumor growth and resulted in a reduction of tumor
volume in comparison with administration cisplatin only (group 2) ( Figure 1).
While co-administration of cisplatin and lower dose (0.3 g/kg) of PEG-HB
0
100
200
300
400
500
600
700
14 17 21 24 28 31 35 38 42
Days post-implant
T
u
m
o
r

v
o
l
u
m
e
(
m
m
3
)
group1 group2 group3 group4 group5
Figure 1. Representative photographs of serial changes in pre-established tumor
volume during each treatment in BALB/c nude mice mice.
492 M. Dai et al.
(group 3) showed no significant gained anti-tumor efficacy as compared with
cisplatin, co-administration of cisplatin and lower dose (0.6 g/kg) of PEG-HB
(group 4) has a significant difference with group 1 (PB0.01). It is surprising
that administration with PEG-HB alone to the tumor-burden mice in group 5
also exhibited slight anti-tumor efficacy compared with the control group
(group 1).
Influence of PEG-HB on Tumor Hypoxia
To access hypoxia in tumor tissues of the five groups, animals were
intravenously injected with hypoxyprobe-1 (pimonidazole hydrochloride)
1 hour before the animals were sacrificed, then the tissues were processed
according to Hypoxyprobe
TM
-1 Kit protocol. The hypoxyprobe-1 immunohis-
tochemistry staining of hypoxic tumor cells is scored for quantitative
assessment (Figure 2A). The results showed that there exist more binding of
pimonidazole hypoxyprobe to tumors harvested from animals that were
administered with PEG-HB (group 3, 4, 5), which means PEG-HB enhanced
the oxygenation in tumor tissues.
We also chose HIF-1a as another indicator to reflect the influence of
PEG-HB on tumor oxygenation. HIF-1a, the heart role of hypoxia regulation
system, exists only in hypoxia tissues. The western blot of HIF-1a showed the
similar results of the immunostaining of hypoxyprobe-1. Administration of
PEG-HB significant reduced the expression of HIF-1a in tumor tissues (Figure
2B).
Influence of PEG-HB on Expression of Angiogenic Factor VEGF
VEGF, the most active endogenous pro-angiogenic factor and the specific
endothelial cell mitogen, was selected to be a reliable index to investigate the
antiangiogenesis effect of PEG-HB. VEGF mRNA levels in tumor tissues
were detected by RT-PCR method. The results showed that 0.6 g/kg PEG-HB
downregulated the expression of VEGF mRNA compared with other groups
(Figure 3A).
We next assessed whether the influence of VEGF mRNA by melatonin
resulted in a decreased production of VEGF protein by ELISA method. The
results (Figure 3B) showed that levels of VEGF protein were notably
decreased in the serum of mice administrated with PEG-HB (group 3, 4, 5)
(PB0.05). But it seems there is no significant difference between these three
groups treated with different concentrations of PEG-HB.
Influence of PEG-Hb on Tumor Angiogenesis 493
C
Group 1 2 3 4 5
PEG Hb
HIF-1
-actin
0
0.5
1
1.5
2
2.5
3
3.5
1 2 3 4 5
Groups
S
c
o
r
e
s
*
**
**
A
B
G1
G5 G4
G2 G3
Figure 2. Tumor hypoxia reected by (A) images of immunocytochemical staining of
Hypoxyprobe-1 in tumor tissue, (B) Pimonidazole binding scores, and (C) expression
of HIF-1a. *PB0.05, **PB0.05.
494 M. Dai et al.
DISCUSSION
HBOCs have been tried to elevate the efficacy of radio- and chemotherapy
though incensement of oxygenation in virtue of its potent oxygen carry and
delivery capability [22]. Besides, it is regarded that HBOCs are easier to get
into the abnormal microvessels of tumors compared with red blood cells
because of their smaller diameter [23]. Teicher et al. reported that ultrapurified
polymerized bovine hemoglobin solution could result in decreased tumor
hypoxia [12,15,16] and increased both irradiation [12,13,14,17] and che-
motherapeutic response [14,15,19,20], which were confirmed by the results of
our present study. Administration of high concentration (0.6 g/kg) of PEG-
HB, which is derived from PEG-conjugated hemoglobin, can effectively
increase the anti-tumor effect of cisplatin, one of the most important
chemotherapeutic drugs in clinical use. The chemo sensitize effect is attributed
to its amelioration of tumor hypoxia.
A
B
0
500
1000
1500
2000
2500
3000
3500
4000
1 2 3 4 5
Groups
V
E
G
F

p
r
o
t
e
i
n

(
p
g
/
m
l
)
*
* *
Group 1 2 3 4 5
PEG Hb
VEGF
-actin
Figure 3. VEGF mRNA levels in nude mice tumor tissues and VEGF protein
concentrations in the blood serum of the nude mice were assessed by RT-PCR (A) and
ELISA (B), respectively. Data shown are representative of three independent
experiments. *PB0.05.
Influence of PEG-Hb on Tumor Angiogenesis 495
As we know, widespread hypoxia within solid tumors is one of the most
potent stimuli of angiogenesis, which is essential for tumors growth,
development and metastasis [24,25]. Increased angiogenesis has been shown
to be associated with the tumor development of metastases, poor prognosis
and reduced survival [26]. Tumor hypoxia will induce the expression of
angiogenic factors such as VEGF, SDF and PDGF, hence broke the balance
between angiogenic factors and antiangiogenic factors [27]. VEGF, the most
important mediator of tumor angiogenesis, is crucially concerned with cancer
development and high vascularization among these angiogenic factors [28].
Tumor hypoxia has been a therapeutic target to overcome tumor angiogenesis.
Ultrapurified polymerized bovine hemoglobin solutions have improved their
capability to overcome tumor hypoxia. Until now, there has been no data
documenting the possible effect of hemoglobin solution on tumor angiogen-
esis. We are the first to show that HBOC can suppress the tumor angiogenesis
through downregulation of VEGF. Administration of PEG-HB was found to
suppress VEGF mRNA expression in tumor tissues. And the VEGF protein
levels also decreased after treatment with PEG-HB. Moreover, the decrease of
VEGF seems to have a positive correlation of the concentration of PEG-HB.
We speculated that downregulation of VEGF might be associated with
decreased HIF-1a protein levels. It is well established that HIF-1, the key
mediator of the hypoxia response, is the most important regulator of VEGF
[29,30]. HIF-1 is a ubiquitous transcription factor consisting of HIF-1a and
HIF-1b subunits [31]. Under normoxic conditions, HIF-1a is rapidly degraded
through the ubiquitin-proteasome system, whereas HIF-1b is constitutively
expressed. When the oxygen is insufficient, HIF-1a is released from the von
Hippel-Lindau tumor suppressor protein and translocates into the nucleus,
where it heterodimerizes with HIF-1b and binds on the hypoxia responsive
element (HRE) to regulate hypoxia-driven gene expression [30]. In our
experiment, HIF-1a protein levels were decreased in PEG-HB administration
groups, which indicated that PEG-HB may suppress the VEGF expression
through the inhibition of the accumulation of HIF-1a.
In summary, our present study shows that PEG-HB inhibits the
expression of endogenous HIF and VEGF in a rodent tumor model, which
may be a very innovative and challenging method of cancer anti-angiogenesis
therapeutics.
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This paper was first published online on iFirst on 9 December 2008.
Influence of PEG-Hb on Tumor Angiogenesis 497