A preliminary study of pamidronic acid downregulation of angiogenic factors IGF-1/PECAM-1 expression in circulating level in bone metastatic breast cancer patients
OncoTargets and Therapy
a preliminary study of pamidronic acid downregulation of angiogenic factors igF-1/ PecaM-1 expression in circulating level in bone metastatic breast cancer patients
Zeng Wang 1 2 3 4
Meiqin Yuan 0 1 2 4
Xiaojia Wang 0 1 2 4
0 Department of c hemotherapy center, Zhejiang cancer hospital
1 Zhejiang Key l ab of Diagnosis & Treatment Technology on Thoracic Oncology
2 guonong Yang
3 Department of Pharmacy
4 ling Ya chen
5 Department of Pharmacy, integrated chinese and Western Medicine hospital of Zhejiang Province , hangzhou, Zhejiang, People's republic of china
8 1 0 2 - l u J - 2 1 n o 7 0 2 . 6 4 . 9 5 . 7 3 y b / m o c . s s re .
pamidronic acid; insulin-like growth factor-1; platelet endothelial cell adhesion
open access to scientific and medical research
O r i g i n a l r e s e a r c h
Bone tissue is one of the most common sites in malignant tumor distant metastasis.
It is particularly common in patients with breast cancer; the incidence rate of bone
metastasis in metastatic breast cancer patients is ~65%–75%, among which the
proportion of bone metastasis as the first symptom is 27%–50%, including bone pain,
pathological fracture, hypercalcemia, spinal cord, or nerve root compression syndromes.1
Bisphosphonates (BPs) are well established for the treatment and prevention of the
skeletal-related events of breast cancer patients with bone metastases. BPs have been
shown to reduce and delay skeletal-related events as well as to improve patients’ quality
of life. Recent studies have shown that BPs might also act as a potential inhibitor of
tumor angiogenesis by affecting the vascular endothelial growth factor (VEGF).2–4
Insulin-like growth factor-1 (IGF-1) is known to stimulate neovascularization by
inducing factors, such as VEGF.5,6 Activation of IGF-1 receptor (IGF-1R)-mediating
signaling in breast cancer cells promotes cell proliferation, invasion, and migration,
suggesting that IGF-1/IGF-1R plays an important role in tumor angiogenesis.
Moreover, platelet endothelial cell adhesion molecule-1
(PECAM-1/CD31), a cell adhesion molecule with
proangiogenic and proinflammatory activity, plays an
important role in the process of angiogenesis. It is reported
that there is a correlation between microvessel density and
VEGF expression in many cancers.7,8
Santini et al9 proved that pamidronic acid (PA) could
downregulate circulating VEGF concentrations in vivo. His
research team also showed that zoledronic acid, a potent
third-generation BP, induced greater suppression of VEGF
concentrations in cancer patients with metastatic bone
disease.10 Despite evidence on the fall of VEGF concentration
after administration of BP in treatment-naïve patients,
however, there is no literature about the effects of BP treatment
on serum (IGF-1and PECAM-1) expression.
Therefore, the purpose of this study was to investigate
the effect of PA treatment on serum IGF-1 and PECAM-1
of patients with breast cancer who had progressive osseous
Eligibility criteria included stage IV bone metastatic female
breast cancer patients, 18 years of age or older; a
performance status not .2 on the Eastern Cooperative Oncology
Group scale; a life expectancy of .3 months; and adequate
organ function, including neutrophil count .1.5×109/L,
platelet count .100×109/L, normal hepatic and renal
function (Cr #265 μmol/L, CrCl $30 mL/min), electrolytes
within the normal range, and no acute or chronic infections
or inflammatory diseases. Moreover, a negative pregnancy
test was necessary for females in childbearing age. Diagnosis
of bone metastases had to include the use of X-ray, CT scan,
or MRI. No antitumor therapy for these patients was
permitted within 28 days before PA administration.
Patients were excluded from study participation for any
of the following reasons: history of other malignancy
(unless .5 years disease-free) excluding completely
resected nonmelanoma skin cancer; active or uncontrolled
infection; concurrent active dental problems, including
infection of the teeth (maxillary or mandibular), dental
trauma, or a concurrent or prior diagnosis of osteonecrosis
of the jaw; recent (within 6 weeks) or planned dental or jaw
surgery; history of uncontrolled or symptomatic angina,
arrhythmias, or congestive heart failure; previous
treatment with any BPs within 1 month before study initiation;
known hypersensitivity to BPs; history of treatment with
calcitonin, gallium nitrate, or mithramycin within 28 days
before study; or pregnancy or lactation in potentially
The study was approved by the medical ethics committee
of Zhejiang Cancer Hospital, Hangzhou. Written informed
consent was obtained from all patients prior to enrollment.
The trial is registered through the Hospital Pharmaceutical
Research Fund Project of Zhejiang Provincial Pharmaceutical
Association, number 2011ZYY05.
According to the reduction in IGF-1 circulating levels, it
was defined as modified when at least a 20% reduction
was recorded 2 days after PA administration, otherwise as
Treatment and sample collection
PA was administered on an outpatient basis.
All patients received 45 mg of PA infusion in 100 mL of
0.9% saline intravenously over a period of 3 hours for 2 days.
Peripheral venous blood samples (5 mL) were collected
prior to PA infusion and 48 hours after infusion. After
collection, the blood samples were kept at room temperature
for 30 minutes to allow clotting, and then were immediately
centrifuged at 3,000× g for 5 minutes. Serum samples were
aliquoted and stored at −80°C until detection.
Serum levels of IGF-1 and PECAM-1 were assayed using
a solid-phase sandwich enzyme-linked immunosorbent
assay (Quantikine Immunoassays; R&D Systems, Inc.,
Minneapolis, MN, USA). The assay was performed according
to the manufacturer’s instructions. Each serum sample was
analyzed in duplicate.
The follow-up period was defined as the time from
diagnosis to the last visit/observation or death. Progression-free
survival was defined as the time between the date of the
time from randomization until patient’s tumor progression
or death. The log-rank test was used to estimate and
compare survival. All statistical analyses were carried out on an
intention-to-treat basis using the SPSS 15.0 software package
(SPSS Inc., Chicago, IL, USA).
As the angiogenic cytokine levels were not normally
distributed, data were summarized by median and range.
The nonparametric test (Wilcoxon test) was used to assess
differences among groups.
Table 1 summarizes the characteristics of patients
evaluated in this study. The number of advanced breast cancer
patients enrolled was ten, with a median age of 41 years
(range 33–60). At baseline, ten patients were treated with
pamidronic for 2 days. Nine patients (90.0%) had ductal
breast carcinoma, and only three patients (30%) were
postmenopausal. Eight patients (80.0%) had hormone
receptorpositive breast cancer, and the other two patients (20.0%) had
hormone receptor-negative breast cancer. No patients were
ever-smokers and two patients (20%) had drinking history.
Most of the patients (90%) had a reproductive history. Two
patients (20%) had diabetes mellitus, and four (40%) patients
had other underlying disease (mainly hypertension, cardiac
disease, and hepatitis).
Further, three (30%) patients had adjuvant radiotherapy,
five (50%) patients had adjuvant chemotherapy, and five
(50%) patients had adjuvant hormonotherapy. In five patients,
bone was the only site of metastatic disease, whereas three
patients had lung metastases and three patients had liver
metastases. Advanced metastases: at the time of initial visits,
before treatment, there were metastatic lesions. Recurrent
metastases: metastasis occurred after treatment.
serum angiogenic cytokine levels analysis
The number of patients who had a reduction in the
circulating IGF-1 and PECAM-1 levels after PA infusion is
summarized in Table 2. The mean ± standard deviation baseline
IGF-1 and PECAM-1 concentration for the ten patients was
1,370.0±523.3 and 5,478.7±2,655.2 pg/mL, respectively. The
median IGF-1 and PECAM-1 values showed a decrease of
22.6% and 30.0%, respectively, 2 days after the first infusion
of PA, and these reductions were both statistically significant
Patient characteristics according to
Reduction in IGF-1 circulating levels was defined as .20%
decrease recorded 2 days after PA administration. Ten
patients were separated into two groups: six patients (60%)
in whom there occurred at least 20% reduction in IGF-1
serum levels after PA infusion for 2 days were classified as
the reduced IGF-1 group, and the other four patients (40%)
were entered in the unmodified IGF-1 group.
All the patient characteristics according to IGF-1
modifications are summarized in Table 3. It is suggested that
compared to IGF-1 unmodified patients, IGF-1 modified
patients might benefit from the adjuvant hormonotherapy.
Further multivariate analysis confirmed that adjuvant
hormonotherapy history is a favorable factor contributing to
IGF-1 modifications (Table 4).
The impact of IGF-1 modification on patients’
By the follow-up time of March 10, 2016, two patients
had died. Both patients belonged to the IGF-1
unmodified group. Though the mortality between IGF-1 modified
group and IGF-1 unmodified group had no statistical
significance (P=0.053), it still had a certain value for treatment
Median value (maximum, minimum)
number of patients
Median age (year)
Breast cancer histotype
Metastases other than
bone locations (patients)
no other locations
Abbreviations: BMi, body mass index; igF-1, insulin-like growth factor-1; er,
estrogen receptor; Pr, progesterone receptor; her-2, human epithelial growth
consideration. Further, the progression-free survival of
subsequent first-line chemotherapy treatment in IGF-1 modified
patients was longer than that of IGF-1 unmodified patients
(P=0.009), indicating that the prognosis of patients in IGF-1
unmodified group was relatively poor (Figure 1).
The results of this study suggest that PA treatment could
suppress circulating serum IGF-1 and PECAM-1 concentrations,
and the prognosis of patients in IGF-1 unmodified group
was relatively poor; moreover, previous hormonotherapy
may improve patients’ responses to BP therapy in IGF-1
modification. PA, a kind of nitrogen-containing BP that
inhibits the activity of farnesyl diphosphate synthase, is a
key enzyme in the mevalonate pathway. Apart from
antiresorptive activity, PA has been reported to suppress tumor
angiogenesis in animal models.11 These findings, together
with reports that PA modified circulating angiogenic
factors in metastatic cancer patients, suggest its potential role
in antitumor angiogenesis.9
Numerous studies have suggested that
IGF-1/IGF-1Rmediated activation of downstream signaling pathways
plays an important part in progression of breast cancer.12–14
Tang et al15 demonstrated that PA and clodronate functionally
inhibited both in vitro and in vivo tumor angiogenesis
induced by IGF-1-stimulated MCF-7 cells.
As we know, tumor angiogenesis is affected by the
activity of multiple, rather than single, angiogenesis
PECAM-1 is a molecule expressed on all cells within
the vascular compartment; apart from exhibiting adhesive
properties, PECAM-1 is also an efficient signaling molecule
and is now known to have diverse roles in vascular biology,
including roles in angiogenesis, platelet function,
thrombosis, and so on.17 Xu18 suggest that PECAM-1, VEGF, and
microvessel density play roles in oncogenesis and
progression of renal cell carcinoma. There is a significant correlation
among abundant PECAM-1, VEGF, and microvessel density
in renal cell carcinoma, and PECAM-1 and VEGF may be
important targets for antiangiogenesis therapy in patients
with renal cell carcinoma.
In our study, the results suggested that PA might have
potential antiangiogenic properties and exhibits adhesive
property in bone metastatic breast cancer by decreasing the
expression of IGF-1 and PECAM-1 concentrations.
Furthermore, we found that the serum concentrations of
IGF-1 in breast cancer patients who received adjuvant
hormonotherapy were lower than other breast cancer patients.
Since the introduction of the molecular subtypes of breast
carcinoma in 2000, breast cancer has been classified based on
gene expression profiling into five subtypes, including:
luminal A, luminal B, Her-2, triple negative basal-like, and triple
negative without basal-like markers.19 About 80% of breast
cancers present an estrogen receptor and/or progesterone
receptor expression.20 Numerous studies nowadays report that
estrogens cooperate with the IGF system at multiple levels
both in physiology and disease. More and more studies have
focused on this bidirectional cross-talk in cancers. Notably,
cancer cells show frequent deregulation of the IGF system
with overexpression of IGF-1R, but frequent upregulation
of the classical estrogen receptor.21,22 Above all, the potential
effect of PA on IGF-1 in hormone receptor-positive breast
cancer patients deserves more attention.
In conclusion, the present data reveal that the
antiangiogenic effect of PA might act through a double blocking
mechanism by downregulation of IGF-1 and PECAM-1
expression. Moreover, the prognosis of patients in IGF-1
unmodified group was relatively poor. However, there are
some limitations in this investigation. First, the detection
points after PA treatment, of this study are not sufficient,
and the compliance management of patients who participated
in the study needs to be strengthened. Second, the number
of patients included was small since the number of patients
who met the inclusion criteria was small. Third, this is a
single-center investigation. Thus, future clinical trials with
large population, and more detection points, in multicenters
should be designed to evaluate the efficacy of PA, as well as
other BPs, used as an antiangiogenic drug in bone metastatic
breast cancer patients.
This work was sponsored by 1022 Talent Training Program of
Zhejiang Cancer Hospital, Hospital Pharmaceutical Research
Fund Project of Zhejiang Provincial Pharmaceutical
Association (2011ZYY05), General Research Program of Medical
Health in Zhejiang Province (No 2014KYB039), Scientific
Research Fund Project of Integrated Chinese and Western
Medicine Institute in Zhejiang Province (2014LYK021),
Science and Technology in Zhejiang Province Chinese
Medicine Program (2012ZA101, 2015ZA148, 2016ZA038),
Hangzhou City Science and Technology Project Planning
Guide (Social Development) (20130733Q15), Hangzhou
City Health Science and Technology Project (2013A43), and
Public Welfare Technology Application Studies Program of
The authors report no conflicts of interest in this work.
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