Original Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2007.00310.x |
Soluble Fms-like Tyrosine
Kinase-1 expression inhibits the growth of multiple
myeloma in nude mice
Junru LIU, Juan LI, Chang SU,
Beihui HUANG, and Shaokai LUO*
Department
of Hematology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou
510080, China
Received: February
7, 2007
Accepted: April 3,
2007
This work was
supported by a grant from the Sci-Tech Program Foundation of Guangdong Province
(No. 2004B30301003)
*Corresponding
author: Tel, 86-20-87755766-8831; Fax, 86-20-87750632; E-mail,
[email protected]
Abstract Angiogenesis is an essential factor in the growth and progression
of hematological malignancies including multiple myeloma (MM). Vascular
endothelial growth factor and its receptors have been shown to be targets for
treating tumors. This study explores the effect of adenovirus-mediated delivery
of soluble vascular endothelial growth factor receptor Fms-like tyrosine kinase-1
(sFLT-1) on the growth of MM cell line KM3 in nude mice. sFLT-1 cDNA was
amplified by reverse transcription-polymerase chain reaction from human
umbilical vein endothelial cells and was used as a transgene to construct an
adenoviral vector carrying sFLT-1 (ADV-sFLT). Cell proliferation and
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays were
carried out to evaluate the effect of ADV-sFLT on human umbilical vein
endothelial cells and KM3 cells in vitro. Eighteen female BALB/c nude
mice were inoculated subcutaneously with KM3 cells, and they were randomly
divided into three groups and injected intravenously with ADV-sFLT, ADV-LacZ,
or phosphate-buffered saline (PBS). The volume of KM3 xenografts was measured
twice a week. Three weeks after the initial treatment, the volume of MM
xenografts in the mice treated with ADV-sFLT, ADV-LacZ, or PBS was 770.3228.73
mm3,
1983.3643.72 mm3,
and 2042.0582.31 mm3, respectively (P<0.01,
ADV-sFLT versus ADV-LacZ or PBS). The value of microvessel density was
29.176.85, 79.177.35, and 78.838.54 in the tumors treated with ADV-sFLT,
ADV-LacZ, and PBS, respectively (P<0.01, ADV-sFLT versus ADV-LacZ or
PBS). This study suggested that the adenovirus-mediated sFLT-1 gene
greatly inhibits MM-derived tumor growth and angiogenesis in mouse xenograft,
and might serve as a new therapy for MM.
Keywords multiple myeloma; gene therapy;
adenovirus; vascular endothelial growth factor; receptor
Multiple myeloma (MM) accounts for 1% of all malignancies and 10% of
malignant hematological neoplasms. Despite the use of conventional and
high-dose chemotherapy, it remains incurable [1]. Novel approaches need to be
found to improve the treatment efficiency of MM. Drugs such as thalidomide
achieve responses in patients with refractory and relapsed MM based on its
antiangiogenic activity [2–4]. The bone marrow microenvironment has become a target to treat
MM.
Angiogenesis is associated with the growth, progression and
metastasis of most solid tumors [5]. As a result, decreased angiogenesis with
novel antiangiogenic agents appears to be a promising and exciting therapeutic
approach for cancer. Further studies have also suggested that angiogenesis
plays a very important role in hematological malignancies including MM and there
is a strong correlation between microvessel density (MVD) and disease
progression and poor prognosis of MM [6–10]. Vascular endothelial
growth factor (VEGF) is a potent angiogenic factor and exerts its biological
functions by interacting with its receptors such as Fms-like tyrosine kinase-1
(FLT-1) and fetal liver kinase-1 (Flk-1)/kinase insert domain-containing
receptor (KDR) [11]. It has been reported that the patients of MM overexpress
VEGF and have high levels of serum VEGF correlated with poor prognosis [12].
Importantly, FLT-1 protein but not Flk-1 is expressed in MM cell lines and MM
patient cells [13]. It has been found that VEGF binds to FLT-1 with an affinity
7–10
times higher than to KDR. These data provided the framework for targeting VEGF
and FLT-1 in the novel therapeutics of MM. Several anti-VEGF drugs such as
GW654652 and PTK787 have been shown to have anti-MM activity in vitro
[14,15].
One novel method of inhibiting the angiogenic action of VEGF is to
use soluble FLT-1 (sFLT-1), a potent and selective inhibitor of VEGF. sFLT-1 is
a known, endogenously expressed, alternatively spliced form of the FLT-1
receptor [16]. It is devoid of the FLT-1 transmembrane domain and the entire
intracellular tyrosine kinase-containing region. sFLT-1 binds to VEGF with the
same affinity and specificity as that of the full-length receptor, but this
binding does not initiate signal transduction [17,18]. In addition, the mutant
consisting of the first three ectodomains of FLT-1 is reported to be able to
bind to VEGF with affinity similar to that of sFLT-1, indicating the sFLT-1(13)
could be sufficient as a potent inhibitor of VEGF mediating angiogenesis [17].
sFLT-1 inhibits the angiogenic action of VEGF in two ways. First, by
sequestering VEGF, thus making it unavailable for angiogenic action; and
second, by heterodimerizing with the extracellular ligand binding region of the
membrane spanning FLT-1 and Flk-1/KDR receptors, thereby blocking the
phosphorylation and activation of downstream signal transduction pathways for
endothelial cell proliferation [16,18].
Several studies have shown that adenovirus-mediated or
plasmid-mediated gene transfer of sFLT-1 inhibited tumor angiogenesis and
growth both in vitro and in vivo [19–22]. Almost all of these
studies focused on solid tumor therapy.
In this study, we investigated the effects of sFLT-1 gene therapy on
the hematological tumor MM in nude mice. We constructed the sFLT-1 gene that
codes the 13 immunoglobulin (Ig)-like domains and established an adenoviral
vector expressing sFLT-1. We used an adenoviral vector for the intravenous
delivery of sFLT-1 and showed its therapeutic effect in a murine model system
of human MM.
Materials and Methods
Cell culture
KM3 human MM cell line was kindly presented by Dr. Jian Hou (Second Military Medical University,
Shanghai, China). KM3 and human embryonic kidney cell line 293 from Amercian
Type Culture Collection (ATCC, Manassas, USA) were maintained in complete
Dulbecco’s modified Eagles’s medium (DMEM) supplemented with 10% fetal bovine
serum (Gibco BRL, Garlsbad, USA), 1% penicillin and streptomycin. Human
umbilical vein endothelial cells (HUVECs) were obtained from ATCC and grown in
DMEM containing 10% fetal bovine serum supplemented with 75 mg/ml endothelial
cell growth supplement (ECGS) (Sigma-Aldrich, St. Louis, USA). All of the cells
were grown at 37 ºC in a humidified atmosphere of 5% CO2.
Cloning of the 1–3 Ig-like domains of FLT-1
Total cellular RNA was isolated from HUVECs with Trizol reagent (Invitrogen,
Carlsbad, USA) according to the manufacturer’s instructions. The isolated RNA
was applied as a template for the first strand cDNA synthesis by reverse
transcription (RT) and cDNA used as a template for polymerase chain reaction
(PCR). The upstream sense primer (5‘-TTGGTACCCATGGTCAGCTACTGGGACA-3‘,
nt 250–268, KpnI), and the downstream antisense primer (5‘-GGAAGCTTCCTCAATGCACTGAGGTGTT-3‘
nt 1230–1213, HindIII) were designed to amplify the coding sequence
for the 13 Ig-like domains of FLT-1 according to the sequence information from
GenBank (accession no. X51602)
[17,19]. The amplified product was subcloned directly into the PMD-18T easy
cloning vector (TaKaRa, Dalian, China) following the manufacturer’s
instructions. The resultant clone, T-sFLT, was confirmed by DNA sequencing and
restriction digestion analysis (KpnI and HindIII).
Construction of recombinant
adenoviral vector expressing sFLT-1
T-sFLT was digested with KpnI and HindIII restriction
enzymes and ligated to the pshuttle-CMV
vector (Stratagene, Glen Burnie, USA). After sequence verification, the
resultant plasmid was named pShuttle-sFLT. This shuttle vector was then
linearized with PmeI and used for homologous DNA recombination with
AdEasy adenoviral vector (Stratagene) containing the adenovirus genome in the Escherichia
coli strain BJ5183. The plasmid containing the genome of Ad-sFLT was
obtained as a result of this recombination and designated pAd-sFLT. Insertion
of the pShuttle-sFLT in the plasmid was confirmed by PCR analysis and
restriction mapping with PacI digestion.
Recombinant adenovirus ADV-sFLT expressing sFLT-1 was generated by
transfection of 293 cells with PacI-digested pAd-sFLT and confirmed by
PCR analysis. The virus titer expressed in p.f.u./ml was determined by plaque
formation assay using 293 cells. The control virus ADV-LacZ was constructed
similarly.
Western blot analysis for
sFLT-1
Conditioned media and cell lysates were generated by infecting KM3 cells
at a multiplicity of infection (MOI) of 100 of either ADV-sFLT or ADV-LacZ for
72 h. Cell lysates and conditioned media were electrophoretically separated on
a 12% sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. The
gel was then transferred to polyvinylidene difluoride membranes (Millipore,
Billerica, USA), probed by rabbit polyclonal antibody FLT (Santa Cruz
Biotechnology, Santa Cruz, USA), then reacted with horseradish
peroxidase-labeled anti-rabbit mouse antibody. Detection of the bands was
carried out using an enhanced chemiluminescent system (Cell Signaling
Technology, Danvers, USA).
Verification of expression of
sFLT-1 by enzyme-linked immunosorbent assay (ELISA)
Expression of sFLT-1 was verified with the ELISA method using a
commercial kit (VEGFR1, referred to in this study as sFLT-1; Boster, Wuhan,
China) according to the instructions provided by the manufacturer. KM3 cells
were planted in a 6-well plate at a density of 3´105 cells/well and infected with ADV-sFLT or
ADV-LacZ at a MOI of 100. The supernatants were taken at 12, 24 and 48 h and
subjected to ELISA detection. Briefly, the 96-microwell plate provided in the
kit was coated with mouse monoclonal anti-sFLT-1 antibody and washed with wash
buffer. Cell supernatant samples were diluted 1:50 by sample dilutant, and 100
ml of diluted sample was added to the microwell plate in duplicate. Similarly,
sFLT-1 standards ranging from 0.03 to 2 ng/ml were added to the microwell
plates in duplicate. After washing four times with wash buffer, 200 ml of
biotin-conjugated polyclonal antibody against sFLT-1 was added and incubated at
room temperature for 2 h. After washing the wells again three times,
tetramethylbenzidine substrate solution was added and incubated at room
temperature for 30 min. The enzymatic reaction was stopped by adding 100 ml of
stop solution to the wells, and absorbance was determined by spectrophotometric
readings at 450 nm. The sFLT-1 concentration in the cell supernatant samples
was calculated based on the standard curve.
HUVEC proliferation inhibition
assay
Conditioned media was obtained from KM3
cells infected with ADV-sFLT or ADV-LacZ. HUVECs were grown in 12-well plates
at 37 ºC in DMEM. At 80% confluence, DMEM was removed, the cell layers were
washed with PBS, and 1.0 ml of 5´conditioned
medium was added. After 15 min, purified recombinant human VEGF165 (R&D
Systems, Minneapolis, USA) at 10 ng/ml was added to each well and incubated at
37 ºC. After 72 h, the cells were trypsinized, and the number of viable cells
was counted using a Trypan blue assay.
KM3 proliferation inhibition
assay
The inhibitory effect of ADV-sFLT on KM3 cell growth was assessed
using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), which
was cleaved by viable cells to yield a dark blue formazan product. KM3 cells
were infected with ADV-sFLT or ADV-LacZ at an MOI of 100 for 2 h, then the
infection media was removed. Serum-free DMEM medium with or without VEGF165 (10
ng/ml) was added. The cells were incubated for 1, 2, 3 and 4 d and a cell count
was carried out daily by MTT assay.
In vivo animal studies
To test the effect of ADV-sFLT on the growth of MM in vivo,
4-week-old BALB/c female nude mice were divided into three groups with six mice
in each group. All experimental procedures conformed with institutional
guidelines for the care and use of laboratory animals in Sun Yat-sen University
(Guangzhou, China), and to the NIH Guide for Care and Use of Laboratory Animals
(revised 1985). All mice were inoculated with 3´107 KM3 cells subcutaneously on day 0. The mice
were then given 1´109 p.f.u.
of ADV-sFLT, ADV-LacZ or PBS, injected into the tail vein on day 1. Animal
weight and tumor measurements were obtained twice a week. Tumor volume was
calculated using the formula V=L´W2/2 (L, length; W, width). Animals were killed at the end of day 21
or when the one-dimensional tumor diameter exceeded 2.0 cm. To evaluate the
expression of sFLT-1 in the plasma of treated mice, plasmas were obtained when
the mice were killed, then subjected to ELISA detection as described above.
Immunohistochemical analysis
for microvessel formation
Angiogenesis was measured by estimating the MVD. The tumor specimens
of mice were harvested under anesthesia and fixed immediately in formalin. To
analyze the microvessel formation in tumors, antigen retrieval was carried out
by boiling the sections for 10 min in 0.01 M citrate buffer in a microwave
oven. Tumor sections of five micrometers were then treated with 3% goat serum
for 1 h at 25 ºC to reduce nonspecific staining followed by 1 h incubation at
37 ºC with goat anti-mouse CD31 polyclonal antibody (Boster) at a concentration
of 1:100. The regions of highest MVD (“hot spot” regions) were
scanned at low magnification (100´) and
counted at higher magnification (200´). MVD
was expressed as the average number of vessels in three hot spots.
Statistical analysis
Data were reported as the mean±SE of measurement. P<0.05
was considered statistically significant in all of the analyses. Analysis of
significance of difference among multiple groups was carried out by anova.
The statistical analyses were carried out with spss
software, version 12.0 for Windows (SPSS, Chicago, USA).
Results
Cloning and validation of
sFLT-1
The sFLT-1 was obtained by PCR amplification from HUVECs and was
subcloned into PMD-18T easy vector [Fig. 1(A)]. Restriction digestion
mapping and DNA sequencing of this vector confirmed the presence of sFLT-1 [Fig.
1(B)].
Construction of recombinant
adenovirus expressing sFLT-1
As a first step of constructing an adenovirus, the sFLT-1 was
subcloned into the pShuttle-CMV vector, generating pShuttle-sFLT. Recombinant
adenovirus pAd-sFLT was then generated through co-transfecting the
pShuttle-sFLT with an E1A/B-deleted adenoviral backbone vector. Presence of
sFLT-1 in the recombinant adenovirus was confirmed by PCR (980 bp).
Restriction digestion of pAd-sFLT with PacI resulted in 3 kb and
30 kb fragments. The 3 kb fragment indicated the presence of pShuttle-sFLT (Fig.
2).
Expression of sFLT-1
To determine the expression/secretion of sFLT-1 in vitro, KM3
cells were infected with 100 MOI of ADV-sFLT. This conditioned media and cell
lysate were subjected to Western blot analysis. The results showed that sFLT-1
was present in conditioned media and cell lysate from ADV-sFLT but not
ADV-LacZ, confirming the adenovirus-mediated secretion of sFLT-1. The sFLT-1
identified by western blot was
approximately 37 kda [Fig.
3(A)]. The expression of sFLT-1 was further subjected to ELISA
verification. The results showed that the KM3 cells infected with ADV-sFLT efficiently
secreted sFLT-1 [Fig. 3(B)].
Inhibition of HUVEC
proliferation by ADV-sFLT
The concentrated conditioned media obtained from ADV-sFLT, ADV-LacZ
infected cells and uninfected cells was applied to the HUVECs grown in 12-well
plates, followed by stimulation with VEGF of 10 ng/ml. The cell count after 72
h incubation was (9.62±1.32)´104,
(32.37±2.64)´104 and
(33.00±2.55)´104 in
ADV-sFLT, ADV-LacZ and PBS, respectively (P<0.01). Conditioned media
from ADV-sFLT cells inhibited HUVEC proliferation by 70% and 71% compared with
conditioned media from ADV-LacZ and PBS, respectively [Fig. 4(A)].
Inhibition of KM3
proliferation by ADV-sFLT in vitro
MTT proliferation test showed that there was no significant difference
among the KM3 cells treated with ADV-sFLT, ADV-LacZ or PBS (P>0.05)
in the presence or absence of VEGF. The results showed that ADV-sFLT can not
inhibit the proliferation of KM3 cells directly in vitro [Fig. 4(B)].
Effect of ADV-sFLT on MM growth
in vivo
At day 21, the mean size of tumors in the ADV-sFLT, ADV-LacZ and PBS
groups reached approximately 770.32±28.73 mm3, 1983.36±43.72 mm3,
and 2042.05±82.31 mm3, respectively. The growth of tumors treated with
ADV-sFLT therapeutic viruses was inhibited by 157% and 165% compared with the
ADV-LacZ and PBS groups, respectively (P<0.01). These results
suggested that sFLT-1 gene therapy was effective in suppressing the growth of
xenografted MM (Fig. 5). Plasma levels of sFLT-1 in ADV-sFLT, ADV-LacZ
and PBS were determined by ELISA. The average sFLT-1 concentration level in the
ADV-sFLT, ADV-LacZ and PBS groups was 9.291.19 ng/ml, 0.940.17 ng/ml and
0.760.16 ng/ml, respectively. The expression level of sFLT-1 in ADV-sFLT was
almost 10-fold higher than the ADV-LacZ and PBS groups, which were almost
negligible (Fig. 6).
Effect of ADV-sFLT on tumor
microvessel formation
To determine whether sFLT-1 gene therapy inhibited intratumoral vessel
formation, we assessed the MVD in the tumor nodules resected from ADV-sFLT and
control mice using endothelial cell-specific CD31 immunostaining. The mean
number of microvessels in the ADV-sFLT, ADV-LacZ and PBS groups was 29.17±6.85,
79.17±7.35 and 78.83±8.54, respectively (P<0.01). As shown in Fig.
7, the density of CD31+ vascular endothelial cells was
dramatically reduced in tumor nodules resected from mice treated with
ADV-sFLT, suggesting a significant antiangiogenic effect of sFLT-1 gene therapy
against experimental MM.
Discussion
Antiangiogenic therapy shows promise as a strategy for cancer
treatment because most tumor growth is dependent on the formation of new blood
vessels. Several approaches have been tested recently for their antitumor
effectiveness in delivering sFLT-1 gene therapy in vivo [19–22]. Mahasreshti
et al. reported that adenovirus-mediated sFLT-1 significantly inhibited
the proliferation of ovarian tumor [20]. In addition, sFLT-1 resulted in a
significant increase in the survival times of mice compared with control mice.
In another study, Ye et al. observed that systemic delivery of the
sFLT-1 gene mediated by mammalian cells could be effective in inhibiting tumor
angiogenesis and growth [19]. These data showed that sFLT-1 gene therapy was
practical and effective. Nearly all of these studies focused on solid tumors.
Preclinical studies show that VEGF directly triggers MM cell growth, migration
and survival, increases osteoclast activity and chemotaxis, and inhibits
dendritic cell maturation [23]. Thus, VEGF and its receptors could be important
targets for the development of new drugs and present an alternative method for
the control of MM.
In this study, we applied adenovirus-mediated sFLT-1 gene therapy to
a MM mouse model. We successfully constructed ADV-sFLT expressing the 13
Ig-like domains of sFLT-1, as verified by Western blot and ELISA assay. Results
presented in this report showed that the transfer of the sFLT-1 gene
effectively inhibited the growth of human MM tumors in immunodeficient mice. We
observed the direct effect of ADV-sFLT on proliferation of KM3 cells in the
presence/absence of VEGF. Unfortunately, we could not find any relationship between
the proliferation of KM3 cells and ADV-sFLT. This result showed that ADV-sFLT
exerted its anti-MM effect without affecting the tumor cells proliferation
directly. To determine whether the inhibitory effect of ADV-sFLT on tumor
growth was associated with the suppression of tumor angiogenesis, we examined
the distribution of the endothelial cell-specific antigen CD31 in tumor
section. As expected, the MVD of tumor was decreased in the ADV-sFLT group
compared with the control. This result showed that ADV-sFLT exerted its
biological effect mainly through inhibiting the angiogenesis of tumor without
affecting the growth of tumor itself. According to our study, sFLT-1 can also
inhibit the growth of hematological tumor. Systemic delivery of ADV-sFLT could
be effective in inhibiting MM growth through decreasing the microvessel density
of tumors.
Recombinant adenoviral vectors are effective tools for gene delivery
because of their superiority of gene transfer efficacy in vivo in a wide
spectrum of both dividing and non-dividing cell types. Potential side-effects
of adenoviruses have been reported, such as liver toxicity and animal death
[24,25]. However, no visible toxicity such as weight loss, change in animal
behavior, decreased food or water intake, or premature mortality, was found in
animals injected with ADV-sFLT in our study. Perhaps the time we observed was
not long enough to find its side-effects.
Recently, several anti-VEGF targeted therapies have been used to
inhibit proliferation of MM. Some artificially synthesized drugs such as
GW654652 and PTK787/ZK222584, which are small-molecular tyrosine kinase
inhibitors inhibiting VEGF receptors, can inhibit the proliferation of MM
cells line and patients MM cell in vitro [14,15]. Another VEGF receptor
tyrosine kinase inhibitor, pazopanib, can also inhibit growth and migration of
MM cells in vitro and in vivo [26]. All of these drugs belong to
non-native inhibitors and have certain toxicities to the human body. However,
sFLT-1 is a known potent and selective endogenous inhibitor of VEGF-mediated
angiogenesis. Theoretically it has no side-effects. Comparison of sFLT-1 with
other synthesized drugs will be carried out in further research.
In conclusion, ADV-sFLT gene therapy targeted to bone marrow could
effectively suppress the development of MM without any side-effects in a mouse
xenograft model. The mechanism of its anti-MM is based on its antiangiogenic
effect. This method might be more practical for clinical applications. It is
further confirmation that sFLT-1 could serve as a new approach for the
treatment of MM.
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