Anti-cancer effect of 131I-nimotuzumab on immune deficiency mouse bearing human larynx cancer

JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017

ANTI-CANCER EFFECTS OF 131I-NIMOTUZUMAB ON IMMUNE
DEFICIENCY MOUSE BEARING HUMAN LARYNX CANCER
Nguyen Thi Kim Huong*; Ho Anh Son**; Nguyen Thi Thu***
Vo Thi Cam Hoa***; Pham Huy Quyen*; Nguyen Linh Toan**
SUMMARY
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Objectives: To assess the anti-cancer effects of
I-nimotuzumab on immune deficient
mouse bearing human laryngeal cancer. Subjects and methods: Three groups of mice bearing
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human laryngeal cancers were injected via the tail vein by I-nimotuzumab, nimotuzumab or
0.9% NaCl, separately. Tumor volume was measured every week. The average survival time
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and survival rate was calculated for mice in each group. Results:
I-nimotuzumab could
reducing the tumor volume with extending the average life span and increase the survival rate
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for the mice. Conclusions:
I-nimotuzumab had the highly anti-cancer effects on immune
deficient mice bearing human laryngeal cancer.
* Keywords: Larynx cancer:

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I-nimotuzumab; Nude mouse.

INTRODUCTION
Laryngeal cancer is the second common

malignancy in head and neck cancer.
Larynx cancer accounts for 2% of all cancer
cases [2]. In Vietnam, larynx cancer ranks
common second after nasopharyngeal
cancer in head and neck cancer and
ranks fourth of all types of cancers. This
disease is common in men (80%), usually
in ages 40 - 60 years, but the current trend
is that more young people are affected
with growing burden on society and health
system [1]. Epidermal growth factor receptor
(EGFR) is often overexpressed in larynx
cancer with over 90% in head and neck
cancer. EGFR signaling plays an important
role in the development, progression,
invasion, metastasis and angiogenesis in

head and neck cancer. In addition,
overexpression of EGFR has a poor
prognosis of overall survival in patients in
Eastern and Western countries. In particular,
EGFR expression level is considered to
be a predictor of laryngeal squamous cell
carcinoma [3].
Currently treatment for head neck cancer
and larynx cancer is the still the major
challenge due to its inadequate response
to conventional treatments such as
chemotherapy or radiotherapy. Targeted
therapy is one of the promising cancer

treatments and has superior advantages
over conventional method. This method
however is that focuses only destroying
cancer cells, and in addition, targeted
therapies have limited adverse effects on

* Haiphong Medical-Pharmaceutical University
** Vietnam Military Medical University
*** Nuclear Research Institute
Corresponding author: Ho Anh Son ()
Date received: 03/10/2017
Date accepted: 20/11/2017

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JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017
healthy organs and on the overall condition
of the cancer patients. Using radiolabeled
antibody, or radioimmunotherapy is an
emerging method which attracting research
attention during the past half century.
There have been some successes in this
field that have been applied to the clinical
treatment of cancer patients. However,
this is a new approach that has not yet
been investigated in Vietnam.
In this study, we used EGFR-specific
monoclonal antibody, nimotuzumab in
combination with 131I to form 131I-nimotuzumab

complex, and evaluated the antitumor activity
of this complex in immunocompromised
mice xenografted with the human laryngeal
cancer line Hep2. This is the initial basis
for the development of this complex
application for laryngeal cancer treatment
in the future.
SUBJECTS AND METHODS
1. Subjects.
The immune-deficient BALB/c nude mice
are imported from Charlie-River Company,
USA. The mouse was bred in clean room
conditions with the air is filtered and have
positive pressure. Room temperature is
maintained at 25 ± 20C, humidity 55 ± 5%,
light is automatically turned on at 7:00 am,
off at 7:00 pm. Chaw (Zeigler, USA) and
drinking water be sterilized before use.
Each mouse cage is mounted on a selfventilated rack and filters through the
membrane ensuring good isolation from
pathogens.

2. Materials.
* 131

I-nimotuzumab:

Nimotuzumab monoclonal antibody
combines with 131I to form 131I-nimotuzumab
complex. The technical procedure was

carried out at Vietnam Nuclear Research
Institute (NRI), with radiation concentrations
of 100 - 200 mCi/mL.
* Human laryngeal cancer line:
Human laryngeal cancer cell line Hep2
were purchased from ATCC (American
Type Culture Collection, P.O. Box 1549,
Manassas, VA 20108, USA).
3. Methods.
* Xenograft experiments:
Hep2 cells (1 x 106) were subcutaneously
injected in a volume of 100 µL into the left
and right thigh of thirty immune-deficient
BALB/c nude mice. All animals were
housed in individually ventilated cages,
after the tumor appears, and has a
diameter of approximately 30 mm, then
the mice were randomly divided into three
groups:
- 131I-nimotuzumab treatment group
(n = 10): mice were intravenous ịnjected a
single dose of 1 mCi/ml of 31I-nimotuzumab
through tail veins.
- Nimotuzumab treatment group (n = 10):
mice were intravenous injected a single
dose of 1 mCi/ml of nimotuzumab through
tail veins.
- Control group (n = 10): mice were
intravenous ịnjected with 0.9% NaCl through
tail veins.

The sizes of the tumors were measured
once per week using microcaliper from week 1
to week 8 after injection. In addition,
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JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017
mortality of mice was monitored daily and
the number of days was recorded for
each mouse in the treatment and control
groups throughout the follow-up period.
Life tables were used to calculate the
survival rates and average survival time.
* Ethics statement:
All animal care and experimental protocols
were approved by the animal ethics
committee of the Vietnam Military Medical

Universtiy under the guidelines of the
Animals (Scientific Procedures) Act 1986.
* Statistics:
Data was analyzed using the ANOVA
tests or chi-square test where appropriate.
Values are expressed as means ± SD.
The statistical package for the Social
Sciences version 21.0 (SPSS, Chicago, IL)
was used for all the analyses. p values of
< 0.05 were considered statistically significant.

RESULTS

1. Treatment with131I-nimotuzumab reduces the volume of tumors.
To investigage the effect of 131I-nimotuzumab in vivo, we measured tumor size in
xenograft mice model barering human laryngeal cancer cell line Hep2 after treatment
with 131I-nimotuzumab (nimotuzumab and NaCl 0.9% as control) at different times
after injection.

Figure 1: Volume of the tumor after one week of treatment.
A) The table compares the average volume of tumors of mice of different treatment
groups; there was no statistically significant difference between the groups of mice. B)
Histogram showing the average tumor volume of the study groups.
Results showed that at one week after treatment, the volume of tumors of groups of
mice was similar.
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JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017

*

Figure 2: Volume of the tumor after two weeks of treatment.
A) The table compares the average volume of tumors of mice of different treatment
groups; in that group of mice treated with 131I-nimotuzumab had significantly smaller
tumor volume than control group, with * = p < 0.05, 131I-nimotuzumab vs. control; and
no significant difference between the other groups. B) Histogram shows the average
tumor volume of the study groups.
However, by the second week after injection, the tumor volume in the 131I-nimotuzumab
treated mice group was significantly lower than the control group (p < 0.05). While the
tumor volume of the group treated with nimotuzumab was smaller than the control group,
but there was no significant difference between the two groups, also no difference
between the groups of 131I-nimotuzumab and nimotuzumab treatment.

*

Figure 3: Volume of the tumor after eight weeks of treatment.
A) The table compares the average volume of tumors of mice in different treatment
groups; the group of mice treated with 131I-nimotuzumab had significantly smaller tumor
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JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017
volume than control groups, with * = p < 0.05, 131I-nimotuzumab vs. control; and no
significant difference between the other groups. B) Histogram shows the average tumor
volume of the study groups.

*

*

*

**

**

**

**

Figure 4: Volume of tumors in different groups of mice during eight
weeks of treatment.

The mice treated with

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I-nimotuzumab had a significantly smaller tumor volume

than the control group at most of the time of the survey, from the 2nd week of treatment,
with * = p < 0.05 ; ** = p < 0.01,

131

I-nimotuzumab vs. control; and no significant difference

between the other groups.
Follow-up at subsequent times showed that the

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I-nimotuzumab treatment group

had significantly smaller tumor volumes than the control group at eight weeks
(with p < 0.05 - 0.01). While there was no statistically significant difference between the
other groups at that time.
The results show that

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I-nimotuzumab complex had a significant effect in inhibiting

the development of human larynx cancer when compared to the control group, in addition

the effect of

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I-nimotuzumab is stronger than nimotuzumab when compared with the

control group. Thus, the efficacy of anti-cancer increased with the combination of
nimotuzumab versus using nimotuzumab alone.
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131

I with


JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017
2. Treatment with 131I-nimotuzumab increased average survival time and the
survival rates of mice.

Figure 5: The average survival time and the survival rates of mice treated with
131
I-nimotuzumab, nimotuzumab and control group.
A) The average survival time analysis of the three groups of mice, in which the
I-nimotuzumab treatment group had a significantly higher survival time than the
control group, with p < 0.05; and there was no difference in which between the treatment
group with nimotuzumab. Use the chi square test. B) Histogram showing everage
survival time and survival rates of mice treated with 131I-nimotuzumab, nimotuzumab,
and control group.
131

The effect of 131I-nimotuzumab was also
assessed through the average survival
time and survival rate when compared on
the mice treated with nimotuzumab and
control group. Results showed that the
mice treated with 131I-nimotuzumab, the
average survival time was 15.829 weeks,
significantly higher than of control group,
with average survival time was 14.222 weeks
(p = 0.033), and trend to higher than the
mice treated with nimotuzumab had aveage
survival time was 15.657 even the difference
was not statistic significant. At the same
time, the survival rate of the treated group
by 131I-nimotuzumab was 0.9, which was

higher than the control group with the
survival rate was 0.5, and was the same
with the mice treated with nimotuzumab.
The results showed that 131I-nimotuzumab
treament increased average survival time
and the survival rates of mice bearing
laryngeal cancer compared to control group.
DISCUSSION
In this study we investiaged the
combination of 131I with nimotuzumab, an
anti-EGFR monoclonal antibody and
assessed the anti-cancer effects of this
complex in immunodeficiency mice bearing
human laryngeal cancer. Results showed

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JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017
that 131I-nimotuzumab complex was higher
effective against larynx cancer than
nimotuzumab alone when compared to
control group. Treatment with 131Inimotuzumab reduced the volume of
tumors, also increased the average
survival time and the survival rate of mice
compared to the using nimotuzumab
group alone and control group. Thus, our
findings suggest that the combination of
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I and nimotuzumab increases the effective
treatment of human larynx cancer on
animal model.
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I and nimotuzumab itself has certain
effects on the treatment of head and neck
cancer and larynx cancer in particular, but
the effect is still limited [4] and usually is
combined with other treatment methods to
improve the efficiency [5]. In our study,
although the mice treated with nimotuzumab
had a smaller tumor volume than the
control group at most of the time of study,
however, the difference was not significant.
This suggests that using nimotuzumab

alone is not so effective in treating
laryngeal cancer, which is consistent with
some previous studies. In this study we
conducted a combination of nimotuzumab
with 131I to exploit the advantages of these
substances. Among EGFR-specific
monoclonal antibodies (cetuximab, gefitinib,
erlotinib, panitumumab and nimotuzumab),
the nimotuzumab had the distinct properties
and advantages than the other antibody
types. Nimotuzumab binds sustainable
and stable to the receptor via double bonds
so it can bind to cells with moderate to
high levels of EGFR expression. Due to
nimotuzumab has a low affinity with the
healthy cells with low EGFR expression,
150

so it avoids dose-dependent toxicity limitation.
Unlike other anti-EGFR antibodies with
typical skin toxicity, in clinical trials with
nimotuzumab, 90% of patients showed
no evidence of severe skin toxicity [6, 7].
Among the radioactive isotopes used
for radiotherapy, 131 I has outstanding
advantages such as: the half-life of 131I is
eight days, which is in accordance with
the half-life of the antibody, thus, it will
prolong the duration of treatment. In addition,
131

I emission  ray, which allows imaging
to monitor the absorption, distribution,
metabolism and excretion of radioactive
medications. It also emisses the shorter 
for effectively treat tumors even small
ones. Easy renal clearance of 131I helps to
limit the accumulation of iodine in the
bone [8]. Moreover, accumulation of clinical
trials have demonstrated the efficacy of
131
I in the treatment of hematopoietic
and solid tumors. 131I also has an advantages
such as: inexpensive, can be used to locate
the tumor and use it for therapeutics, and
has a long history of successfully treating
malignant tumors. In addition, 131I emits
the powerful -rays as an additional element
that supports the emission of  particles in
radioimmunoassay, as  particles generate
large amounts of energy efficiency to destroy
the cancer cells [9, 10]. Accordingly, the
superior efficacy of the 131I-nimotuzumab
complex in the treatment of larynx cancer
on animal model is probably due to the
combination of the advantages of each
constituent.
The combination of 131I and nimotuzumab
has proven to be the primary efficacy
in treatment of laryngeal cancer.
Thiscombination combination is also

JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017
innovative point in this study and opens
new prospects for the treatment of laryngeal
cancer and other tumors that overexpress
the EGFR. The results of this study are
an important basis for further research
into the application of 131I-nimotuzumab
complex in the treatment of larynx cancer
in clinical trials.
CONCLUSIONS
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I-nimotuzumab complex is effective
in treatment of immune deficiency mouse
model bearing human larynx cancer.
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I-nimotuzumab treatment animal showed
the reduction the tumor volume. It increased
survival time and survival rates of mice.
The therapeutic efficacy of 131I-nimotuzumab
was higher than using nimotuzumab.
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