Inuence of PEG-conjugated Hemoglobin on Tumor

Oxygenation and Response to Chemotherapy

Minghua Yu, Jianqun Han and Min Dai
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
Hongwei Li
Institute of Microcirculation, Peking Union Medical College & Chinese Academy of
Medical Sciences, Beijing, China
Qian Liu
Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing,
China
Ruijuan Xiu
Institute of Microcirculation, Peking Union Medical College & Chinese Academy of
Medical Sciences, Beijing, China
Abstract: Hypoxic tumors are significantly more malignant, metastatic, radio- and
chemoresistant. The use of artificial oxygen carriers represents a new approach to the
problem of hypoxia. In the present study, female athymic BALB/c nude mice bearing
the cervical carcinoma were untreated or treated with cisplatin to determine whether
administration of artificial oxygen carrier (PEG-conjugated Hemoglobin, PEG-Hb)
could improve the tumor oxygenation and enhance the anti-tumor efficacy of cisplatin.
Pimonidazole staining was employed to detect tumor tissue oxygenation status. We
We thank Prof. T.M.S. Chang, McGill University, Canada, for serious review of this
manuscript. This work was supported by Prof. Ruijuan Xius UNESCO Award for
Woman 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: 551561, 2008
Copyright # Informa UK Ltd.
ISSN: 1073-1199 print / 1532-4184 online
DOI: 10.1080/10731190802556674
551
found that the application of a higher dose (0.6 g/kg) PEG-Hb could significantly
ameliorate the hypoxic condition in cervical carcinoma xenograft models. Co-
administration of PEG-Hb (0.6 g/kg) with cisplatin produced significant tumor growth
inhibition and pro-apoptotic and anti-proliferative effects as compared to cisplatin
alone. These suggest the evaluated PEG-Hb in this experiment has positive effects on
cisplatin or cisplatin-based chemotherapy, and further work to optimize its application
is warranted.
Keywords: articial oxygen carrier, tumor, hypoxia, cisplatin
INTRODUCTION
The presence of hypoxia in solid tumors has been recognized for more than
50 years. Hypoxic cells are more resistant to standard chemotherapy and
radiotherapy, are more invasive and metastatic, resistant to apoptosis, and
genetically unstable [19]. Therefore, hypoxia has a key negative role in tumor
prognosis, both because it causes resistance to standard therapies and because
it promotes a more malignant phenotype. So it is then not surprising that
hypoxia has been considered an attractive target for the development of novel
anti-cancer therapies, including prodrugs activated by hypoxia, hypoxia-
specific gene therapy, targeting the hypoxia-inducible factor-1(HIF-1a)
transcription factor, and recombinant anaerobic bacteria [3]. However, to
date, most of these strategies are still in preclinical or early clinical
development. The potential to improve local control and survival by hypoxia
modification was demonstrated by a meta-analysis of 83 clinical trials [21] and
a number of therapeutic strategies have also been established to overcome
tumor hypoxia by improving oxygen supply either by oxygen or carbogen
breathing or by increasing the hemoglobin level and oxygen delivery [13, 15].
The use of artificial oxygen carriers represents a new approach to solving the
problem of hypoxia.
Previous studies have demonstrated that intravenous administration of
ultrapurified polymerized bovine hemoglobin solution was effective in
increasing the oxygenation throughout experimental tumors under normal
air breathing conditions [24, 33, 35]. Therefore, an enhancement of tissue
oxygenation (tpO2) in rodent tumors associated with an increased tumor
growth delay was reported for purified bovine hemoglobin solutions combined
with irradiation [24, 28, 30, 32], as well as with chemotherapeutic agents [30
32, 34] such as carmustine (BCNU), cyclophosphamide, ifosfamide, adria-
mycin, TNP-470, minocycline, melphalan. When carbogen breathing was
added to administration of the hemoglobin preparations, further increased
therapeutic response and decreased tumor hypoxia were achieved. Various
modified hemoglobin prepared from bovine, human, or mouse Hb, for
example the polyethyleneglycol-conjugated bovine hemoglobin, could also
552 M. Yu et al.
increase tumor oxygen content [18, 20] and improve the effectiveness of
radiotherapy in rodent models [20]. Perhaps spurred by these encouraging
results, a clinical phase I/II study on the effect of polyethyleneglycol-
conjugated hemoglobin (Enzon Corp., USA) for radiosensitization of tumors
has been performed [26].
The present study was designed to determine whether co-administration
of polyethyleneglycol-conjugated hemoglobin (PEG-Hb) and cisplatin could
alter the extent of oxygenation in cervical carcinoma xenograft model and
influence the response to chemotherapy.
MATERIALS AND METHODS
Polyethyleneglycol-conjugated Hemoglobin (PEG-Hb)
PEG-Hb used in this study was a Chinese domestic sample. Its preparation
processes and physiochemical properties belong to the manufacturers
proprietary information and will not be discussed in this paper. The sample
was kept at 48C in the dark until use.
Tumor Cell Line
HeLa cells were obtained from the Cell Bank of the Chinese Academy of
Medical Sciences, Beijing, China. The cell line was maintained and
propagated in RPMI-1640 (Gibco, Life Technologies, Vienna, Austria)
supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and
100 U/ml penicillin0.1 mg/ml streptomycin in 75 cm
2
tissue plastic flasks
(Corning, USA). Cells were maintained at 378C in a humidified 5% CO
2
atmosphere.
In Vivo Experiments with Subcutaneous Tumor
Female athymic BALB/c nude mice, 5 weeks old, weighing 1923 g at the
start of the study, were implanted subcutaneously with 0.2 ml 510
6
HeLa
cells into right flank. Mice were housed at SPF laboratory in accordance with
state guidelines for humane treatment and care of the laboratory animals. Mice
received food and water ad libitum. Treatment began when tumors reached a
volume of 6080 mm
3
. Animals were randomly assigned to five groups (n
10 per group). 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. Tumor size was
PEG-Hb Can Sensitize Cisplatins Efficacy in Cervical Cancer Model 553
calculated using the equation (l w
2
)/2, where l and w refer to the larger and
smaller dimensions collected at each measurement. Efficacy was measured as
percent tumor growth inhibition (TGI) relative to saline-treated group. TGI is
calculated by the equation [1(T/C)] 100, where T and C represent the
mean tumor mass on the last day of therapy in the treatment (T) and saline
control (C) groups, respectively. 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.
Immunohistochemistry
On the last day of therapy, tumors were removed, weighed and fixed in 10%
formalin and embedded in paraffin for immunohistochemical (IHC) analysis.
5mm sections were deparaffinized and rehydrated in a graded series of ethanol
following standard protocol. Sections were treated with 3% H
2
O
2
for 10 min
to eliminate endogenous peroxidase activity, and then immunostaining of
tumor sections (n10 tumors in each group) was performed with an anti-
PCNA antibody (Zymed Laboratories) diluted 1:100 for determination of
the proliferation index in HeLa tumors. After incubation for 30 min at
room temperature they were exposed to biotinylated secondary antibodies for
10 min. Antigen-antibody complexes were visualized using the substrate-
chromogen mixture (Zymed Laboratories Inc., San Francisco, CA) and
counterstained with hematoxylin. Omission or substitution of the primary
antibody with preimmune serum was used as a negative control. Immunor-
eactivity was assessed by the intensity and percentage of positive staining.
Staining intensity was scored as 0 (negative), 1 (faint), 2 (moderate), and
3 (strong) positive staining in the ranges of 525%, 26%50%, 51%75%,
75% were assigned scores of 1, 2, 3, and 4. The cumulative scores
of each group was calculated and averaged for using as descriptors.
The extent of apoptosis in the tumors was measured by terminal
deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)
staining using the In Situ Cell Death Detection Kit (Roche Applied Science,
Penzberg, Germany), following the manufacturers protocol. This system end-
labels the fragmented DNA of apoptotic cells. Omission of the enzyme in the
TUNEL reaction was used as a negative control and cells treated with DNase I
were used as a positive control. The number of TUNEL-positive cells was
counted in five different fields under a light microscope at 200 magnification.
Tumor hypoxia was determined using Hypoxyprobe
TM
-1 Kit for the
Detection of Tissue Hypoxia (Chemicon International, Temecula, CA) in
which, 1 h before tumor collection, mice were injected intravenously
with 60 mg/kg pimonidazole hydrochloride. Then the formalin-fixed,
554 M. Yu et al.
paraffin-embedded tumor sections were incubated with 150 ml of a 1:50
dilution of Hypoxyprobe
TM
-1 Mab1, and biotin-conjugated F(ab)
2
(1:500)
was used to reveal Hypoxyprobe
TM
-1 adducts in the hypoxic tumor tssues.
Multiple biopsies were taken per tumor and one section was stained per
biopsy. Each tumor was scored semi-quantitatively section by section in all
cases, using a scoring system where hypoxia in the ranges of 0, 05%, 5
15%, 1530%, 30% were assigned scores of 0, 1, 2, 3, and 4.
The cumulative scores of each group was calculated and averaged for using as
descriptors.
DATA ANALYSIS
Data were analyzed statistically with one-way ANOVA or with two-tail
students t test. A value of PB0.05 was considered significant.
RESULTS
Effect of Cisplatin Alone or in Combination with PEG-Hb on Tumor
Growth In Vivo
Co-administration of cisplatin and 0.6 g/kg PEG-Hb (group 4) caused a
significant inhibition of tumor growth and resulted in a significant reduction of
tumor volume in compared with groups at the end of each treatment (PB0.01;
Figure 1).
The results in Table 1 showed that cisplatin alone or in conbination
with PEG-Hb exhibited an antitumor effect against the subcutaneous model
of human cervical carcinoma when administrated by intravenous administra-
tion. The inhibition rate reached 81.1% (group 4) when cisplatin co-
administrated with PEG-Hb at a higher dose of 0.6 g/kg, much higher than
the anti-tumor activity of cisplatin alone (group 2) as 73.47%. However, co-
administration of cisplatin and lower dose (0.3 g/kg) of PEG-Hb (group 3)
showed no significant gained anti-tumor efficacy as compared with cisplatin
alone. To our surprise, PEG-Hb alone administrated to the tumor-burden mice
in group 5 also exhibited slight anti-tumor efficacy at a inhibition rate of
20.54%.
On the other hand, cisplatin showed considerable side effects as indicated
by negative increase of animals body weight in groups 2, 3, and 4. When
administrated in combination with PEG-Hb at the indicated doses, cisplatin
led to more pronounced, though not significant, loss in body weight of mice,
which indicated that the tested sample PEG-Hb itself may have some side
effects.
PEG-Hb Can Sensitize Cisplatins Efficacy in Cervical Cancer Model 555
PEG-Hb Can Ameliorate Tumor Hypoxia
We chose the exogenous hypoxia specific Hypoxyprobe 1 kit that consists of
pimonidazole hydrochloride and a mouse monoclonal IgG antibody directed
against 2-nitroimidazole reduction products. Pimonidazole immunostaining
of hypoxic tumor cells is scored for quantitative assessment and shown in
Table 2.
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
group1 group2 group3 group4 group5
Figure 1. Representative photographs of the harvested tumors from each group and
serial changes in pre-established tumor volume during each treatment in athymic mice.
Table 1. Effect of cisplatin, or cisplatin with PEG-Hb, or PEG-Hb alone on HeLa
tumor cell growth in nude mice by intravenous administration (twice a week for four
weeks)
Increase of body
weight (g)
Tumor weight
(g)
Tumor Growth
Inhibition (%)
Group 1 1.1590.31 0.6690.32
Group 2 2.6590.98 0.1790.12 73.47
Group 3 2.8591.53 0.1790.12 74.52
Group 4 3.5392.09 0.1290.11 81.11
Group 5 0.9390.16 0.4890.36 20.54
556 M. Yu et al.
Hypoxia is a very common phenomenon in cervical cancer models. As
shown in Table 2, tumors harvested from animals that administrated with
saline (group 1) or cisplatin (group 2) without the oxygen-carrying agent
exhibited more hypoxic as revealed by higher scores of pimonidazole binding.
While tumor-burden animals that received oxygen carrier PEG-Hb (groups 3,
4, and 5) demonstrated decreased tumor hypoxia, especially in group 4, which
received more PEG-Hb at a higher dose of 0.6 g/kg.
PEG-Hb Can Increase the Therapeutic Response of Cisplatin
Since co-administration of cisplatin and 0.6 g/kg PEG-Hb can cause a
significant inhibition of tumor growth and gain an inhibition rate of 81.1%. To
further elucidate these performances, a proliferative immunocytochemical
marker, the Proliferating Cell Nuclear Antigen (PCNA) and TUNEL assay,
were used to determine the proliferative activity and apoptosis status in tumor
sections, respectively.
As presented in Table 3, the intensity and positive staining of PCNA, a
proliferative marker reflect cell proliferative activity in tumors harvested from
each group, has significant difference in the five groups. Animals receiving
saline or PEG-Hb alone have much more proliferative tumor cells with
average PCNA scores over 4, while cisplatin treatment (groups 2, 3, and 4)
can significantly down-regulate the expression of PCNA, especially in group
4, which received cisplatin in combination with a higher dose (0.6 g/kg) of
PEG-Hb. On the other hand, many more apoptotic cells could be observed in
the tumors harvested from cisplatin or cisplatin conbined with PEG-HB
Table 2. Pimonidazole binding scores in cervical cancer xenografts
Group 1 Group 2 Group 3 Group 4 Group 5
Scores 2.8190.86 2.9390.79 1.9390.71 0.8290.55
'
1.2790.71
'
PB0.01 as compared with group1 and group 2.
Table 3. PCNA scores and apoptosis index in tumor sections
Group 1 Group 2 Group 3 Group 4 Group 5
PCNA Scores 4.1490.24 2.4390.28 2.2290.34 1.5490.12* 4.0490.21
Apoptosis Index
(%)
4.1291.24 32.593.28* 36.694.62* 45.498.72* 6.0491.28
*PB0.01 as compared with Group 1 and Group 5. PCNA, Proliferating Cell Nuclear
Antigen.
PEG-Hb Can Sensitize Cisplatins Efficacy in Cervical Cancer Model 557
treatment groups (PB0.01). 45.4 percent of nuclei are apoptotic in the case of
cisplatin conbined with PEG-Hb treatment in group 4 (PB0.001).
DISCUSSION
Cervical cancer is a major health issue worldwide. Each year, approximately
500,000 new cases of cervical cancer are diagnosed, and more than 270,000
deaths result from this disease [22]. In the United States (US), in 2007, an
estimated 11,150 cases of cervical cancer are expected to be diagnosed, and
every 2.5 h a woman will die of cervical cancer in the US [14]. The treatment
of cervical cancer can include surgery, radiation therapy, chemoradiation
therapy, or chemotherapy. However, more and more evidence revealed that
hypoxia is a common phenomenon in cervical cancers [2, 11, 12], which
endows cervical carcinomas significantly more malignant, metastatic, radio-
and chemoresistant. To date, a variety of strategies designed to improve tumor
oxygenation to overcome resistance to radiation and chemotherapy has been
investigated. These efforts, such as hyperoxic gas inhalation [16], VEGF
blockade [1, 36], mild hyperthermia [7], respiratory inhibition [27], and Ras
inhibition [4, 6, 25] have had only limited success. With the encouraging
development of artificial oxygen carriers in recent years, hemoglobin-based
oxygen carriers (HBOCs), which initially developed as alternatives for blood
transfusion, might be biochemically tailored for specific clinical indications
such as cancer chemo- and radiotherapy sensitization [38].
In fact, although Raabe et al. demonstated that low-dose (0.3 g/kg)
application of HBOC-201 (a glutaraldehyde-polymerized bovine hemoglobin
solution; Biopure, Cambridge, MA) did not improve the response of the
rhabdomyosarcoma R1H of the rat to fractionated irradiation [23] since the
application of low dose hemoglobin solution HBOC-201 (0.3 g/kg) alone
failed to improve tumor tissue or healthy skeletal muscle oxygenation [9],
several experimental tumor studies have demonstrated that administration of
ultrapurified polymerized bovine hemoglobin solution could result in
decreased tumor hypoxia [24, 33, 35] and increased both irradiation [24, 28,
30, 32] and chemotherapeutic response [3032, 34]. However, several studies
have shown that these earlier generations of hemoglobin derivatives induce
several undesired effects, including vasoconstriction, hypertension, renal
toxicity, and platelet aggregation [5, 37]. Recent development in the field of
artificial oxygen carriers based on the modification of hemoglobin have led to
the formulation of polyethylene glycol (PEG)-conjugated Hb compounds,
which was shown to be free of hypertension and was also shown to release
oxygen in an artificial capillary in a manner virtually identical to native RBCs
[37]. Some of these kinds of products could also increase tumor oxygen
558 M. Yu et al.
content [18, 20, 29] and improve the effectiveness of radio- and chemotherapy
in rodent tumor models [18, 29].
In our present study, the evaluated PEG-Hb, which is also derived from
PEG-conjugated hemoglobin, can effectively increase the oxygen-carrying
capacity of blood and thus improve tumor oxygenation. Cisplatin is one of the
most important chemotherapeutic drugs in clinical use. The US National
Cancer Institute alert in February 1999 stated that concomitant cisplatin-based
chemotherapy and radiotherapy should be considered for all patients with
cervical cancer [10] and it is believed to kill cells through interaction with
DNA, mainly by formation of various DNA adducts, which lead to initiation
of apoptosis [8]. There is evidence that cisplatin was less effective under
hypoxic conditions [17]. Thus, in this study, we also investigated the
chemotherapy sensitizing effect of PEG-Hb in vivo. We found that the
reduction of tumor volume as well as pro-apoptotic (TUNEL-positive) and
anti-proliferative effects in HeLa nude mice xenograft with co-administration
of cisplatin and higher dose of PEG-Hb are more pronounced than that
produced by cisplatin alone. In addition, no manifestation of the side effects
(body weight drop and mortality) associated with the PEG-Hb is observed.
In conclusion, the results of this study suggest that the evaluated PEG-Hb
are useful in enhancing the oxygenation of tumor tissue, resulting in more
therapeutic response of cisplatin. Application of this type of oxygen carrier
may provide a means of increasing the effectiveness of certain chemother-
apeutic agents such as cisplatin. However, the tailoring of optimal dosage/
schedule is still to be further optimized.
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This paper was first published online on iFirst on 9 December 2008.
PEG-Hb Can Sensitize Cisplatins Efficacy in Cervical Cancer Model 561