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Introduction
Emerging evidence has shown that hematopoietic stem
cell transplantation can have tumor inhibitory effects in
patients with solid tumors, such as a case of regression of
unresectable, large pancreatic tumor and a case of
attenuation of multiple metastases of fibroblastic osteosarcoma
following non-myeloablative stem cell
transplantation[1_3]. Adult human
and murine hematopoietic stem cells and progenitor
cells display a tropism for intracerebral
gliomas in mice[4]. Likewise, experiments show that human neural stem cells
can inhibit mouse glioma
growth[5]. Mesenchymal stem cells (MSCs) have been identified as a kind of cell with
differentiation capacity along mesodermal lineages, migrate and
activate at a site of disease, and could be employed in
cell-replacement therapy and gene
therapy[6_8]. Human MSCs (hMSCs) can target microscopic tumors and contribute to
stem cell-based anticancer gene therapeutic approaches.
Animal studies have demonstrated that
gene-modified hMSC may serve as a platform for delivery of biological agents in
antitumors[9]. Human bone marrow-derived MSC may have
a tropism for brain tumors through the blood and increase
animal survival. Moreover, hMSC were recruited exclusively
into the brain glioma contrasted with the widespread
distribution of fibroblasts without tumor specificity
in vivo[10]. However, according to the recent studies, there are
discrepancies about the effect of standard MSCs on tumor cells. It
is described that unmodified MSCs are able to home to
several different tumor cells in mice and enhance their
growth[11]. Other studies suggest that unmodified hMSCs can inhibit
the growth of tumor cells, for example, the homing of hMSCs
to sites of Kaposi's sarcoma and potently inhibit tumor
growth in vivo, in the meantime downregulating the Akt
protein kinase of some types of tumor cells by cell_cell direct
contact[12]. The opposite of the results on the intrinsic
effects of MSC on tumor growth needs further examination.
Moreover, stem cells also show an antitumor effect in
metastatic cancers from breast, kidney, ovaries, prostate, and
pancreas[13]. Importantly, a study indicates that a product
containing stem cell differentiation stage factors inhibit
hepatocellular carcinoma growth in vivo
and in vitro[14]. In our previous study, we revealed that hMSCs are able to inhibit
the proliferation of breast cancer cells via the
Wnt/β-catenin pathway[15]. Taken together, a number of reports show that
stem cells display intrinsic antitumor effects, especially stem
cells home to tumorigenesis, indicating a
particular utility for tumor. However, the underlying molecular mechanism that
may be involved in multiple signaling pathways remains
unclear.
NF-κB is present broadly in the majority of cells and is a
key transcription factor, which is involved in cellular
proliferation, differentiation, carcinogenesis, and apoptosis.
The family of NF-κB transcription factors include
NF-κB1 (p105/p50), NF-κB2 (p100/p52), RelA (p65), RelB, and c-Rel.
The activation of NF-κB has been implicated in human
prostate cancer and tongue carcinoma
cells[16_18]. The upregulation of the
NF-κB signaling pathway in human thyroid cancer cells leads to apoptotic resistance and promotes invasive
ability, which is significantly inhibited by a specific
NF-κB inhibitor[19]. The inhibition of
NF-κB activation leads to the downregulation of gene products involved in cell survival,
proliferation, and invasion[20]. NF-κB significantly inhibits
cell growth and promotes cell death in human hepatocellular
cancer cells and ovarian cancer
cells[21_23]. Oncogenic H-Ras, an upstream activator of Akt, requires
NF-κB to suppress transformation-associated
apoptosis[24]. Deguelin, an Akt inhibitor, suppresses
NF-κB leading to the potentiation of apoptosis and the inhibition of cellular
invasion[25]. Embelin, an inhibitor of XIAP blocks the
NF-κB signaling pathway and leads to the suppression of
NF-κB-regulated anti-apoptotic and metastatic gene
expression[26]. Overall, the inhibition of
NF-κB activation provides convincing evidence for the critical role of antitumor therapy.
We previously found that Dkk-1 released from hMSCs
contributed to the depression of the proliferation of breast
cancer cells via the Wnt/β-catenin
pathway[15]. Moreover, in the present study, we investigated the role of
NF-κB in the depression of tumor cells mediated by hMSCs. Our finding
showed that NF-κB downregulation may be involved in the
inhibition of tumor cells mediated by hMSCs.
Material and methods
Cell culture The hMSCs designated Z3 were established
from human dermis tissues of a fetus aborted at 4 months'
gestation, and immortalized by the stable transfection of a
pGRN145 plasmid containing the cloned hTERT
gene[27]. The FACScan flow cytometer showed that hTERT(+) cells were
positive for CD29, CD44, CD105, and CD166, while CD31,
CD45, CD34, vWF, and HLA-DR were negative. Under
suitable conditions, hTERT(+) cells have the ability of multiple
lineage differentiation, including bone, fat, and nerve. The
hMSC-designated BMMS-03, derived from human fetal bone
marrow at 4 months, were established without
immortalization[28]. The FACScan flow cytometer showed that
BMMS-03 cells were positive for CD105 and CD166, while CD34 was
negative. For the experiments, Z3 and BMMS-03 were
cultured in Dulbecco's modified Eagle's medium with low
glucose and Iscove's modified Dulbecco's medium at 1:1
supplemented with 10% fetal calf serum (Gibco, USA), H7402/HepG2
human hepatoma cells and MCF-7 human breast cancer cells
(low metastasis potential)/LM-MCF-7 (high metastasis
potential, a subclone derived from lung metastasis MCF-7
cells) human breast cancer cells were cultured in RPMI-1640
containing 10% fetal calf serum[29]. The cells were incubated
in a humidified atmosphere with 5% CO2 at 37 °C.
Cell treatment Z3 or BMMS-03 cells were cultured as
described above 100% confluence. The supernatant derived
from the Z3 or BMMS-03 cultures was harvested and stored
at _80 °C until use as conditioned media for cell treatment.
Synchronized H7402/HepG2 human hepatoma cells or
MCF-7/LM-MCF-7 human breast cancer cells were pretreated with
a mixture of RPMI-1640 containing 10% fetal calf serum and
Z3- or BMMS-03-conditioned media (199:1, 49:1, 9:1, 4:1, or
1:1, namely 0.5%, 2.0%, 10%, 20%, or 50%) for 48_96 h,
respectively. During that time, the culture media were
replaced every 24 h. The treated tumor cells were examined by
the 5-bromodeoxyuridine (BrdU) incorporation assay, flow
cytometry assay, reporter gene assay, real-time PCR, and
Western blot analysis, respectively.
BrdU incorporation assay DNA synthesis or cell
proliferation was measured by the BrdU incorporation assay. BrdU
(Sigma, USA) was used to incorporate into DNA in place of
thymindine[30]. Briefly, the H7402 cells or MCF-7 cells were
seeded in a 96-well plate, respectively, and incubated with
treatment of 50% hMSC conditioned media for 48 h. BrdU
(10 µmol/L final concentration) was added and the cells were
re-incubated for an additional 4 h at 37 °C. The cells, which
incorporated BrdU into DNA, were detected using a
monoclonal antibody against BrdU (1:300 dilution, Neomarkers,
USA) at room temperature for 2 h. A goat anti-mouse
immunoglobulin G-labeled fluorescein-isothiocyanate (1:100
dilution; Sigma, USA) was used, as was propidium iodide
(Sigma, USA) nuclear counterstaining. The results were
expressed as the percentage of BrdU-positive cells over the
propidium iodide-positive cells.
Flow cytometry analysis Proliferation was assessed by
flow cytometry analysis. MCF-7 cells treated with 20% or
50% Z3- or BMMS-03-conditioned media as above for 48 h
were used for the flow cytometry analysis. The cells were
suspended in phosphate-buffered saline after being fixed
with ice-cold 70% alcohol and were stained with propidium
iodide (50 µg/mL). The proliferation index (PI) was
determined from the flow cytometry analysis data according to
the following formula:
PI=(G2/M+S)÷(G0/G1
+S+G2/M)×100%[31].
Transfection and reporter gene
assay The activity of NF-κB was determined by transfecting the cells with a target
gene-dependent reporter plasmid and measuring luciferase
activity. The pGL-NF-κB reporter plasmid containing the
NF-κB-dependent luciferase reporter gene (provided by Dr
Chuan-shu Huang form New York University, New York,
USA) and the pRL-TK plasmid containing the renilla luciferase
gene as an internal control were cotransfected into
MCF-7/LM-MCF-7 breast cancer cells and H7402/HepG2 human
hepatoma cells in triplicate by using Lipofectamine 2000
(Invitrogen, Carlsbad, California, USA) according to the
manufacturer's recommendation, when treated with
conditioned media of Z3 cells or BMMS-03 cells.The cells were
harvested 48 h after transfection, and the activities of target
gene luciferase and renilla luciferase were determined with
the dual luciferase reporter assay kit (Promega, San Luis
Obispo, CA, USA). Luciferase and renilla luciferase
luminiscence were measured by using a luminometer. All of
the data shown in this study were obtained from at least
three independent experiments.
Quantitative real-time PCR Quantitative real-time PCR
was performed to examine the expression level of
NF-κB in treated cells and untreated cells by a LightCycler PCR
analyzer (Roche, Mannheim, Germany) using the one-step
RT-PCR system. Relative quantitation was performed using the
LightCycler fast start DNA master SYBR green I kit (Roche,
Germany) to detect PCR products in real time with the
LightCycler. The oligonucleotide primers for PCR were based
on published mRNA sequences as follows: human NF-κB2
sense primer, 5'-CATGGAGAGTTGCTACAACC-3'; human
NF-κB2 antisense primer, 5´-TCTCTGCTTAGGCTGTTCCA-3';
human GAPDH sense primer,
5'-TGTTGCCATCAATGACCCCTT-3'; and GAPDH antisense primer,
5'-CTCCACGACGTACTCAGCG-3'. After denaturation at 95 °C for 10 min, PCR was
performed for 45 cycles (10 s at 95 °C, 5 s at 60 °C, and 15 s at
72 °C). Each experiment was repeated 5 times independently.
All amplified products were sequence verified. Control
reactions were performed in the absence of reverse transcriptase
and were negative. The target gene transcripts relative to
the housekeeping gene GAPDH were quantified, and the
results were expressed as a relative value compared with
GAPDH.
Western blot analysis The expression levels of
NF-κB and the phosphorylation of inhibitor
κBα(p-IκBα) were detected in H7402 cells and MCF-7 cells by Western blot
analysis. After treatment with 10% conditioned media of Z3
or BMMS-03 cells for 96 h, the MCF-7 cells and H7402 cells
were lysed in lysis buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2%
SDS, 5% 2-mercaptoethanol, and 10% glycerol) at 4 °C for 20
min. The lysate was centrifuged at 10
000×g at 4 °C for 20 min. The protein concentrations of the supernatant were
determined with a Bio-Rad protein assay kit (Bio-Rad,
Hercules, CA, USA). The proteins in the cell lysate were
separated on a 12% SDS-PAGE gel and transferred to a
nitrocellulose filter, then incubated with an antibody against
NF-κB at a dilution of 1:400 (NeoMarkers, USA) or an antibody
against p-IκBα at a dilution of 1:300 (Santa Cruz, CA, USA)
for 2 h at room temperature, respectively, and re-incubated
with a peroxidase-conjugated secondary antibody for 1 h at
room temperature. The proteins of NF-κB and
p-IκBα were detected by the enhanced chemiluminescence system (Sigma,
USA).
Statistical analysis The analysis of data was carried out
with Student's t-test.
Results
Depression of the proliferation of tumor cells treated
with conditioned media from hMSC We investigated the
effect of hMSCs on tumor cells by using conditioned media
from hMSC culture. The proliferation of H7402 and MCF-7
cells treated with 20% or 50% conditioned media from Z3 cell
or BMMS-03 cell cultures were assessed by the BrdU
incorporation assay and flow cytometry analysis. The BrdU
incorporation assay indicated that the positive cells in the S
phase of the treated group were significantly lower than that
in the control group (Figure 1A). The average positive rate
of BrdU staining was 38.2% in the control group of H7402
cells; however, the average positive rate of BrdU staining was
29.2% in the H7402 cells inoculated with 50% Z3 conditioned
media (P<0.01 vs control), and was 30.1% in the H7402 cells
inoculated with 50% BMMS-03-conditioned media
(P<0.01 vs control). A similar result was obtained in the MCF-7 cells.
The average positive rate of BrdU staining was 46.3% in the
control group of MCF-7 cells; however, the average positive
rate of BrdU staining was 33.7% in the cells inoculated with
50% Z3 conditioned media (P<0.05
vs control), and was 37.2% in the cells inoculated with 50% BMMS-03-conditioned
media (P<0.01 vs control). Furthermore, the flow cytometry
analysis was carried out in MCF-7 cells treated with
conditioned media from hMSCs at 2 concentrations of 20% and
50% for 48 h. The flow cytometry analysis showed that the
PI decreased in the treated cells compared with the control.
The PI was 37.33% or 36.85% in MCF-7 cells treated with 20%
Z3- or BMMS-03- conditioned media, and 30.49% or
32.60% treated with 50% conditioned media from Z3 or
BMMS-03, but the PI was 42.18% in the control cells
(P<0.01; Figure 1B). The data indicate that the conditioned media from hMSCs
are able to inhibit the proliferation of hepatoma cells and
breast cancer cells.
NF-κB downregulation may involve the depression of
tumor cells mediated by conditioned media from
hMSCs Next, we sought to investigate the molecular mechanisms
underlying the inhibitory effect of hMSCs on tumor cells.
We addressed whether or not NF-κB was involved in the
depression of tumor cells mediated by conditioned media
from hMSCs. First, we examined the transcriptional activity
of NF-κB in tumor cells after the treatment with conditioned
media from hMSCs. The NF-κB-dependent luciferase
plasmid was transfected into MCF-7/LM-MCF-7 breast cancer
cells and H7402/HepG2 hepatoma cells in triplicate when
treated with 10% conditioned media from Z3 cells. It was
found that the activity of NF-κB-dependent luciferase was
downregulated (Figure 2A). A similar result was obtained in
MCF-7 human breast cells employing 10% conditioned
media from BMMS-03 cells (Figure 2B). The treatment of
MCF-7 cells with conditioned media from Z3 cells of 0.5%, 2.0%, or
10.0% significantly resulted in the depression of
NF-κB in a dose-dependent fashion (Figure 2C), suggesting that the
NF-κB-mediated transactivation of luciferase activity in
tumor cells was downregulated by conditioned media from
hMSCs.
Second, we examined the expression of NF-κB at the
mRNA and protein levels. The NF-κB2 and GAPDH mRNA
levels were determined by quantitative real-time PCR. The
PCR products for NF-κB2 and GAPDH were 131 and 202 bp,
respectively. The results showed that the expression level
of NF-κB2 mRNA was downregulated in H7402 cells treated
with 10% Z3 conditioned media (Figure 3). The Western blot
analysis revealed that the expression levels of
NF-κB were downregulated in H7402 and MCF-7 cells treated with 10%
conditioned media from Z3 or BMMS-03 stem cells, and the
downregulation of NF-κB in treated cells was accompanied
by low p-IκBα (Figure 4). Taken together, our data indicate
that some soluble factors in the conditioned media from Z3
and BMMS-03 cells were responsible for the suppression of
hepatoma cells and breast cancer cells, in which
NF-κB downregulation may involve in the inhibition.
Discussion
With the development of study in stem cell biology, more
and more attention has been paid to the research of the
relationship between stem cells and tumor cells. However, to
date, the molecular mechanism of the inhibitory effect of
stem cells on the phenotype of tumor cells remains unclear.
Stem cells share several characteristics of cancer cells,
including loss of contact inhibition and immortality.
Importantly, stem cells and tumor cells have similar signaling pathways
that regulate self-renewal and differentiation, such as Wnt,
BMP, mitogen-activated protein kinase (MAPK), and Notch
pathways, which determine the diverse developmental fates
of cells[32_34]. The microenvironment around stem cells plays
an essential role in preventing carcinogenesis by providing
primarily inhibitory signals for both proliferation and
differentiation[35]. Understanding the signaling circuitry involved
in cancer cells may provide an insight into the molecular
mechanisms of tumorigenesis. Some researchers have
observed that hMSCs are able to home to the sites of tumorigenesis,
and the tropism of MSC may possibly be suitable for cancer
therapy.
In the present study, we treated human hepatoma cells
and human breast cancer cells with conditioned media from
Z3 or BMMS-03 hMSCs. The BrdU incorporation assay and
flow cytometry assay demonstrated that the proliferation
ability of H7402 and MCF-7 cells was inhibited significantly
with treatment of 20% or 50% conditioned media from Z3 or
BMMS-03 hMSCs. These findings strongly suggest that
some soluble factors in the conditioned media from Z3 or
BMMS-03 cells are able to inhibit tumor cells. We
hypothesize that the interaction of tumor cells and hMSCs is
influenced by ligand-dependent extracellular signaling, in which
NF-κB may be involved in the signalings. Therefore, we
investigated the regulation of NF-κB at the levels of
transcription, mRNA, protein and activity regulation. The
results demonstrated that the transcriptional activity of
NF-κB was downregulated in H7402/HepG2 human hepatoma
cells and MCF-7/LM-MCF-7 human breast cancer cells after
treatment with 10% conditioned media from Z3 or BMMS-03
cells by measuring luciferase activity. Real-time PCR and the
Western blot analysis showed the downregulation of
NF-κB at the mRNA and protein levels. In the resting state, the
activity of NF-κB is sequestered in the cytoplasm as an
inactive precursor complex with inhibitory
κB (IκB). Members of the IkB family include IκBα,
IκBβ, IκBγ, IκBε, and Bcl-3. The biological activity of
NF-κB is tightly regulated by IκB specific to
IκBα[36]. It appears that the newly synthesized
protein IκB rapidly re-associates with the newly released
NF-κB, thereby markedly reducing the amount of
NF-κB translocated into the nucleus for the activation of cytokine genes.
The activity of the NF-κB transcription factor is regulated by
p-IκB through multiple intracellular signal transduction
pathways. Inflammatory cytokines, such as tumor necrosis
factor (TNF), activate the IκB kinase (IKK) complex to
phosphorylate the conserved N-terminal region of
IκB proteins. Additionally, protein kinase C directly regulates the
phosphorylation of IκB, which results in the release of
NF-κB and the appearance of NF-κB DNA-binding
activity[37]. Mitogen-activated/extracellular response kinase kinase 1 has been
found to participate in IKK activation in response to
TNF-b in 293 cells[38]. Once IκB is phosphorylated and degraded,
active NF-κB is released and translocates into the nucleus
to enhance the expression of genes. Further study was
performed to determine the implications of this phenomenon
with regard to p-IκB. Our results indicated that the
downregulation of NF-κB and p-IκBα in treated cells was
cooperatively regulated in H7402 and MCF-7 cells. A
number of reports showed that NF-κB is regulated by the
intracellular signal transduction cascades. A variety of active
factors regulate the activity of NF-κB, such as
TNF-α/β, interferonβ/γ, interleukin (IL)-1α, IL-1β, IL-2,
TGF-β, and macrophage colony-stimulating factor. However, the
factors in the conditioned media released from hMSCs that are
responsible for the downregulation of NF-κB in tumor cells
remain unclear.
Taken together, our study illustrates that conditioned
media of hMSCs inhibits the proliferation of hepatoma cells
and breast cancer cells, which may be related to multiple
signaling pathways, such as the downregulation of either
Wnt/β-catenin[18] and/or NF-κB, and/or others. The
mechanism needs to be further investigated. The application of
hMSCs may be used in the therapeutic strategy of cancers.
Acknowledgements
We thank Dr Chuan-shu HUANG (from Nelson Institute
of Environmental Medicine, New York University School of
Medicine, New York, USA) for providing the
pGL-NF-κB and pRL-TK reporter plasmid.
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