Osteopontin Upregulates the Expression of Glucose Transporters in Osteosarcoma Cells
Citation: Hsieh I-S, Yang R-S, Fu W-M (
Osteopontin Upregulates the Expression of Glucose Transporters in Osteosarcoma Cells
I-Shan Hsieh 0
Rong-Sen Yang 0
Wen-Mei Fu 0
Effie C. Tsilibary, National Center for Scientific Research Demokritos, Greece
0 1 Department of Pharmacology, College of Medicine, National Taiwan University , Taipei, Taiwan , 2 Department of Orthopedic Surgery, National Taiwan University Hospital , Taipei , Taiwan
Osteosarcoma is the most common primary malignancy of bone. Even after the traditional standard surgical therapy, metastasis still occurs in a high percentage of patients. Glucose is an important source of metabolic energy for tumor proliferation and survival. Tumors usually overexpress glucose transporters, especially hypoxia-responsive glucose transporter 1 and glucose transporter 3. Osteopontin, hypoxia-responsive glucose transporter 1, and glucose transporter 3 are overexpressed in many types of tumors and have been linked to tumorigenesis and metastasis. In this study, we investigated the regulation of glucose transporters by osteopontin in osteosarcoma. We observed that both glucose transporters and osteopontin were upregulated in hypoxic human osteosarcoma cells. Endogenously released osteopontin regulated the expression of glucose transporter 1 and glucose transporter 3 in osteosarcoma and enhanced glucose uptake into cells via the avb3 integrin. Knockdown of osteopontin induced cell death in 20% of osteosarcoma cells. Phloretin, a glucose transporter inhibitor, also caused cell death by treatment alone. The phloretin-induced cell death was significantly enhanced in osteopontin knockdown osteosarcoma cells. Combination of a low dose of phloretin and chemotherapeutic drugs, such as daunomycin, 5-Fu, etoposide, and methotrexate, exhibited synergistic cytotoxic effects in three osteosarcoma cell lines. Inhibition of glucose transporters markedly potentiated the apoptotic sensitivity of chemotherapeutic drugs in osteosarcoma. These results indicate that the combination of a low dose of a glucose transporter inhibitor with cytotoxic drugs may be beneficial for treating osteosarcoma patients.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This study was financially supported by Ministry of Science and Technology (NSC 101-2325-B-002-070). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Osteosarcoma is the most common type of bone cancer in
teenagers and is highly metastatic [1,2]. Surgery and
chemotherapy are the standard treatment options for high-grade
osteosarcoma. However, approximately 20% of patients have lung
metastases at initial diagnosis and 40% patients experience
metastasis at a later stage. The 5-year survival rate for
osteosarcoma patients with metastases is 20%, compared with
65% for patients with localized disease .
Glucose is a source of metabolic energy that maintains tumor
cells ability to proliferate and survive. Glucose transporters
(GLUTs) move glucose into the cytosol to fuel aerobic glycolysis,
also known as the Warburg effect . GLUT1 and GLUT3 are
class I glucose transporters that possess a high affinity for glucose
and are hypoxia-responsive. Hypoxia is an important factor during
tumor progression. Under hypoxic conditions, HIF-1
(hypoxiainducible factor 1) regulates the expression of numerous genes,
such as VEGF (vascular endothelial growth factor), iNOS, EPO,
LDHA (lactate dehydrogenase A), PDK1 (pyruvate dehydrogenase
kinase 1), GLUT1, and GLUT3 . Expression of GLUT1 and
GLUT3 is regulated by developmental stage and metabolic state.
Upregulation of GLUT1 and GLUT3 are reported to be
associated with poor prognosis in breast cancer .
Overexpression of GLUT1 also corresponds with poor survival in non-small
cell lung cancer  and tumor aggressiveness in transitional cell
carcinoma of the bladder . Many cancers overexpress GLUTs
because of the energy requirement associated with uncontrolled
proliferation and metastasis ; however, few studies examine
the relationship between osteosarcoma progression and GLUTs.
Osteopontin (OPN) is a noncollagenous bone matrix protein
that earned its name from its discovery in osteoblasts [12,13].
OPN interacts with cells through many different integrins,
including avb1, avb3, and avb5, via the GRGDS. OPN also
binds to the CD44 receptor on the cell membrane to regulate
cytokine production, cell trafficking, cell proliferation, migration,
and cell survival [14,15]. OPN expression is associated with the
progression of several cancers, including breast, ovarian, prostate,
renal, oral, colorectal, pancreatic, liver, lung, skin, and thyroid
cancers, glioblastoma, and sarcomas. The interaction of OPN with
various receptors, including several integrins and CD44, induces
the activation of signal transduction pathways leading to cell
migration and invasion. The level of OPN is also related to tumor
stage and is a biomarker for cancer progression and prognosis in
many cancers. The upregulation of OPN levels concomitant with
cancer type-specific markers aids in early detection of many
malignancies . VEGF, THBS3 (thrombospondin 3),
osteocalcin, osteonectin, VS38c, and S100 are specific markers
for osteosarcoma . Higher levels of these markers are detected
in the peripheral blood of osteosarcoma patients .
Overexpression of OPN also occurs in many osteosarcoma samples .
Although osteopontin has multiple physiological functions,
including the attachment of osteogenic cells to the bone matrix,
control of mineralization, coupling of bone formation, and
resorption , however, the role of OPN in osteosarcoma is
still not clear. In this study, we found that GLUTs and OPN
increased during hypoxic conditions in osteosarcoma. OPN
upregulated GLUT1 and GLUT3 expression via avb3 integrin
and the AKT, JNK, and p38 pathways in osteosarcoma cells.
Knockdown of OPN increased cell death in osteosarcoma cell
lines. Chemotherapeutic drugs in combination with a low dose of
glucose transporter inhibitor exerted synergistic cytotoxic action.
Taken together, these data suggest a new therapeutic strategy for
Materials and Methods
The human osteosarcoma cell lines MG63, U-2OS, and 143B
were purchased from the American Type Culture Collection
(Rockville, MD). MG63 cells were cultured with DMEM (Gibco;
Grand Island, NY), U-2OS cells were cultured with RPMI 1640,
and 143B cells were cultured with MEM. All cell cultures were
supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan,
UT) and maintained at 37uC in a humidified atmosphere with 5%
An OPN-shRNA (short-hairpin RNA) conjugated to the
pLKO.1 vector containing a puromycin-resistant region was
provided by the National RNAi Core Facility at the Institute of
Molecular Biology/Genomic Research Center in Taipei in
Taiwan. The sequences as shown below:
shRNA plasmids and TurboFect Transfection Reagent
(#R0531; Thermo Scientific) were premixed with Opti-MEM I
(Gibco, Grand Island, NY) separately for 5 min, mixed with each
other for 25 min, and then applied to MG63 and U-2OS cell
cultures. The control shRNA (empty vector; ev) was used as a
negative control. For transient transfection, cells were transfected
with two different OPN-shRNA plasmids for 24 h.
After washing with cold phosphate-buffered saline (PBS), cells
were lysed with 50 ml radioimmunoprecipitation assay buffer
[RIPA; 50 mM HEPES, 150 mM NaCl, 4 mM EDTA, 10 mM
Na4P2O7, 100 mM NaF, 2 mM Na3VO4, 1% Triton X-100,
0.25% sodium deoxycholate, 50 mM 4-(2-aminoethyl)-benzene
sulfonylfluoride, 50 mg ml21 leupeptin, 20 mg ml21 aprotinin,
pH 7.4] on ice for 30 min. Following centrifugation of lysates at
14,500 r.p.m. for 1 h, the supernatant was isolated and used for
western blotting. Protein concentration was measured using a
BCA assay kit (Pierce, Rockford, IL) with bovine serum albumin as
a standard. Equal concentrations of protein were separated on 8%
sodium dodecyl sulfate-polyacrylamide (SDS) gels and transferred
to nitrocellulose membranes (Millipore, Bedford, MA, USA). The
membranes were incubated for 1 h with 5% dry skim milk in PBS
buffer to block nonspecific binding and then incubated overnight
at 4uC with the following primary antibodies: rabbit
anti-GLUT1, 2, 3, and 4 (1:1,000; Millipore, Billerica, MA), anti-OPN
(1:1,000; Abcam Inc., Cambridge, MA), and mouse anti-b-actin
(1:10,000; Santa Cruz Biotechnology, Dallas, TX). After washing
with phosphate buffered saline Tween (PBST), the membranes
were then incubated with mouse anti-rabbit or goat anti-mouse
peroxidase-conjugated secondary antibody (1:1,000; Santa Cruz
Figure 1. Hypoxia increases osteopontin expression in human osteosarcoma cells. MG63 osteosarcoma cells were treated with the
chemical hypoxic agent CoCl2 (100 mM). Osteopontin (OPN) mRNA (6 h) (A) and protein (24 h) (B) levels were increased by CoCl2 treatment. Data are
presented as the mean 6 S.E.M. (n = 3), *p#0.05, as compared with the control (con).
Figure 2. Hypoxia increases the expression of glucose transporters in human osteosarcoma cells. (A) The mRNA levels of glucose
transporter (GLUT) 1, 2, 3, 4, 6, 8, 10, and 12 were evaluated using quantitative PCR. After treatment with CoCl2 (100 mM, 6 h), GLUT 1, 2, and 3 mRNA
levels were increased. CoCl2 (100 mM, 24 h) also increased GLUT1 (B) and GLUT3 (C) protein levels in MG63 cells. Data are presented as the mean 6
S.E.M. (n = 3), *p#0.05, compared with the control group (con).
Biotechnology, Dallas, TX) for 1 h. The blots were visualized by
enhanced chemiluminescence (ECL; Millipore, Billerica, MA)
using a UVP imaging system (UVP, Upland, CA). For
normalization purposes, each blot was also probed with mouse
anti-bactin (1:10,000; Santa Cruz Biotechnology, Dallas, TX).
Quantitative Real-Time PCR
Total RNA was extracted using a TRIzol kit (MDBio, Inc.,
Taipei, Taiwan). 2 mg RNA was used for reverse transcription that
was performed with a commercial kit (Invitrogen, Carlsbad, CA).
Quantitative real-time PCR was performed using a TaqMan/
SYBR Master Mix (Thermo Scientific) and analyzed with a model
StepOne plus System (Applied Biosystems; Foster City, CA). After
pre-incubation at 50uC for 2 min and 95uC for 10 min, the PCR
was performed at 40 cycles of 95uC for 15 sec and 60uC for 1 min.
The threshold was set above the non-template control background
and within the linear phase of target gene amplification to
calculate the cycle number at which the transcript was detected
(denoted as CT). The cDNA was amplified with gene specific
primers as shown below:
Forward: CCAGC TGCCA TTGCC GTT
Figure 3. Osteopontin increases GLUT1 and GLUT3 expression in osteosarcoma cell lines. OPN (24 h) increased GLUT1 (A) and GLUT3 (B)
protein levels in a concentration-dependent manner in MG63 osteosarcoma cells. OPN (10 ng/ml, 24 h) also increased GLUT1 and GLUT3 protein
expression in U-2OS (C) and 143B (D) osteosarcoma cells. Data are presented as the mean 6 S.E.M. (n = 4), *p#0.05, as compared with the control
Cell viability assay
Cell viability was assessed by MTT [3-(4,5-dimethyl
thiazol-2yl)-2,5-diphenyl tetrazolium bromide] (Sigma-Aldrich, St. Louis,
MO) assay. The culture medium was aspirated 24 h after drug
treatment and MTT (0.5 mg/ml) was added to each well. The
MTT was removed after 30 min and the cells were lysed using
100 ml dimethylsulfoxide (DMSO). The absorbance was measured
at 550 nm and 630 nm using a microplate reader (Bio-Tek,
Glucose uptake assay
Glass coverslips were coated with poly-D-lysine for 1 h at room
temperature and then rinsed with sterile d.d. H2O (3 times/
5 min). Cells were seeded onto coverslips for 24 h. They were then
treated with OPN (100 ng/ml) for 24 h followed by
2-deoxy-Dglucose (2-NBDG) for 30 min at 37uC. After uptake of 2-NBDG,
the cells were put on ice and fixed with 4% paraformaldehyde in
PBS for 15 min at 4uC. Images were obtained from a fluorescent
microscope using an excitation wavelength of 485 nm and an
emission wavelength of 535 nm (model SP5 TCS; Leica,
Figure 4. Osteopontin increases glucose uptake in MG63 osteosarcoma cells. 2-NBDG, a fluorescent D-glucose analog, was used as an
indicator of glucose uptake. Note that treatment with OPN (100 ng/ml) for 24 h enhanced 2-NBDG uptake into MG63 cells, as shown by confocal
microscopy (A) and flow cytometric analysis (B).
The effect of OPN on the cellular uptake of 2-NBDG was also
measured by flow cytometry. Briefly, 56105 cells were incubated
in 6-well plates for 24 h. The cells were treated with OPN
(100 ng/ml) at 37uC for 24 h. The cells were then detached by
trypsin and 2-NBDG was added and incubated at 37uC for
another 0.5 h. The cells were collected and washed twice with
icecold PBS buffer. Finally, the cells were resuspended in cold PBS
buffer for flow cytometric analysis. The relative values of 2-NBDG
staining intensity were obtained by dividing the fluorescence
intensity of each measurement by that of control cells.
Values are expressed as the mean 6 S.E.M from at least three
experiments. Results were analyzed with one-way analysis of
variance (ANOVA), followed by Students t-test. Significance was
defined as p,0.05.
Hypoxia increases osteopontin expression in human
Hypoxia is a major regulator of tumor development and
aggressiveness . Osteopontin levels are also known to be
upregulated in a variety of cancers. A dose of 100 mM cobalt
chloride (CoCl2), a hypoxia-mimetic agent that induces HIF1a
expression, stabilization, and activation, was used to mimic the
hypoxia seen during tumor development. We observed that
osteopontin mRNA (6 h, Figure 1A) and protein (24 h, Figure 1B)
levels were markedly increased in MG63 human osteosarcoma
cells after treatment with CoCl2, indicating that osteopontin may
play a role in osteosarcoma progression.
Hypoxia increases the expression of glucose transporters
in human osteosarcoma cells
Glucose transporters are another common regulator of tumor
growth [5,24]. Tumor cells turn on the hypoxia-inducible
transcription factor oxygen-sensing system and regulate the
downstream genes, such as VEGF, iNOS, EPO, GLUT1, and
GLUT3, to adapt to hypoxia and increase tissue oxygenation
[7,2527]. In MG63 human osteosarcoma cells, the mRNA levels
of GLUT1, GLUT2, and GLUT3, but not GLUT 4, 6, 8, 10, or
12, were increased after a 6-h treatment of CoCl2 (100 mM)
(Figure 2A). Because GLUT1 and GLUT3 possess a high affinity
for glucose, we then measured the protein expression of GLUT1
and GLUT3. We observed that protein levels of GLUT1 and
GLUT3 were upregulated after treatment of CoCl2 (100 mM,
24 h) (Figures 2B and 2C).
Osteopontin increases GLUT1 and GLUT3 expression in
Because osteopontin is one of the hypoxia-inducible genes and a
cancer progression marker, we performed further investigations of
the effect of OPN on the regulation of glucose transporters. We
observed that treatment with OPN for 24 h increased GLUT1
(Figure 3A) and GLUT3 (Figure 3B) protein expression in a
concentration-dependent manner in MG63 osteosarcoma cells.
GLUT1 and GLUT3 are also upregulated by OPN (10 ng/ml)
treatment in two other osteosarcoma cell lines, U-2OS (Figure 3C)
and 143B (Figure 3D). These results demonstrate that OPN can
regulate glucose transporter expression in osteosarcoma.
Increase of glucose uptake by OPN in osteosarcoma cells
Because of the fact that OPN can upregulate the expression of
glucose transporters in osteosarcoma cells, we further examined
the effect of OPN on glucose uptake in MG63 cells. 2-NBDG, a
fluorescent D-glucose analog for direct measurement of glucose
uptake, was used to examine the effect of OPN on glucose uptake.
Immunofluorescence showed that 2-NBDG uptake was increased
following treatment with OPN (100 ng/ml, 24 h) (Figure 4A).
Flow cytometric analysis showed that the fluorescence of 2-NBDG
was right-shifted by treatment with OPN (100 ng/ml, 24 h)
(Figure 4B). These results indicate that exogenous OPN can
further increase glucose uptake into hypoxic osteosarcoma cells.
with empty vector (ev) (Figure 5A). CoCl2 treatment (100 mM,
6 h) upregulated GLUT1 (Figure 5B) and GLUT3 (Figure 5C)
mRNA expression in MG63 cells, which was antagonized by OPN
shRNA transfection, indicating that endogenously released OPN is
involved in hypoxia-induced GLUT1 and GLUT3 expression.
In addition, OPN knockdown for 48 h decreased cell viability in
both MG63 (Figure 6A) and U-2OS (Figure 6B) osteosarcoma
cells. Treatment with phloretin (500 mM), a glucose transporter
inhibitor, for 24 h also decreased cell viability in MG63
(Figure 6A) and U-2OS (Figure 6B) osteosarcoma cells. The
apoptotic effect of phloretin was further enhanced in OPN
knockdown cells. These results indicate that endogenously released
OPN plays an important role in regulating GLUTs expression and
cell survival in osteosarcoma cells.
The avb3 integrin and MAPK pathways are involved in
osteopontin-induced glucose transporter upregulation in
Osteopontin, a secreted adhesive glycoprotein with a functional
RGD cell-binding domain, interacts primarily with the avb3
integrin. As shown in Figure 7A, the OPN-induced increase of
GLUT1 and GLUT3 protein expression was significantly
antagonized by a avb3 monoclonal antibody (2 mg/ml) and
Knockdown of osteopontin decreases the expression of
glucose transporters and cell viability in osteosarcoma
The role of endogenously released OPN was investigated by
OPN knockdown in osteosarcoma cells using shRNA transfection.
Transfection with two sequences of OPN-specific shRNA (sh1 and
sh2) for 24 h downregulated OPN protein expression compared
PF573228 (focal adhesion kinase (FAK) inhibitor, 5 mM) in MG63
cells, indicating that OPN increased GLUT1 and GLUT3
expression via avb3 integrin and caused the activation of the
downstream protein kinase FAK. OPN-induced increase of
GLUT1 and GLUT3 protein expression was also markedly
inhibited by the PI3K inhibitor LY294002 (20 mM), the JNK
inhibitor SP600125 (20 mM), and the p38 inhibitor SB203580
(20 mM), whereas the ERK inhibitor PD98059 (20 mM) did not
affect the OPN-induced expression of GLUT1 and GLUT3
(Figure 7B). It was also found that OPN time-dependently
phosphorylated phosphoinositide-3 kinase (PI3K/AKT),
Junamino-terminal kinase (JNK), and the p38 pathway, which were
antagonized by an avb3 integrin monoclonal antibody
(Figure 7C). These results indicate that several kinases, including
PI3K/AKT, JNK, and p38, are involved in the regulation of
glucose transporters by OPN via avb3 integrin.
The synergistic cytotoxic effect of chemotherapy drugs in
combination with a GLUT inhibitor in osteosarcoma cells
Phloretin is a competitive inhibitor of glucose transporters that
has potent antioxidant activity, as well as anti-proliferative and
apoptotic effects in cancer cells, such as hepatocellular carcinoma
(HepG2) , colon cancer (HT-29) , melanoma (B16) ,
and breast cancer (MCF10A). Here we used a low concentration
of phloretin (100 mM) in combination with chemotherapeutic
drugs, such as daunomyacin (1 mM), 5-Fu (10 mM), etoposide
(10 mM), and methotrexate (10 mM), in the osteosarcoma cell lines
MG63, U-2OS, and 143B. Treatment with phloretin (100 mM),
daunomyacin (1 mM), 5-Fu (10 mM), etoposide (10 mM), or
methotrexate (10 mM) alone for 24 h caused only 20% cell death.
However, the combination of phloretin with these
chemotherapeutic drugs markedly increased cell death (.50%) in all three
osteosarcoma cell lines (Figure 8A). Representative images of
phloretin in combination with chemotherapeutic drugs are shown
in Figure 8B. These results indicate that the addition of a low dose
of glucose transporter inhibitor with chemotherapeutic drugs can
enhance cytotoxicity in osteosarcoma cells.
In this study we demonstrate that both osteopontin and glucose
transporters are crucial factors in osteosarcoma. Endogenously
released OPN regulated GLUTs expression in hypoxia, which was
reversed by knockdown of OPN. Inhibition of the function of
OPN and GLUTs induced cell death in osteosarcoma cell lines.
Combination of a GLUT inhibitor and chemotherapy drugs
exerted a synergistic apoptotic effect in osteosarcoma.
Cobalt chloride (CoCl2), a hypoxia-mimetic agent, has been
demonstrated to inhibit the prolyl hydroxylase domain-containing
Figure 8. The cytotoxic effect of chemotherapeutic drugs is enhanced by combination with a glucose transporter inhibitor. (A)
Treatment of MG63 cells with phloretin (100 mM), daunomycin (1 mM), 5-Fu (10 mM), etoposide 10 mM, or methotrexate (10 mM) alone for 24 h
induced a low level of cell death. However, the combination of phloretin with chemotherapeutic drugs (daunomycin, 5-Fu, etoposide, and
methotrexate) markedly increased cell death in three osteosarcoma cell lines: MG63, U-2OS, and 143B. Representative photographs are shown in
panel B. Data are presented as the mean 6 S.E.M. (n = 4). *p#0.05, compared with the control (con), #p#0.05, compared with the respective
treatment of the chemotherapeutic drug alone.
enzymes (PHDs) , which plays a key role in oxygen-dependent
degradation of the transcription factor, to activate
hypoxiamediated signaling by stabilizing hypoxia-inducible transcription
factor-1a (HIF-1a) . Here we used CoCl2 to mimic the
hypoxic condition, an inevitable cellular stress experienced during
tumor progression and solid tumor development [35,36]. During
hypoxia, HIF-1a degradation is inhibited and its activity is
increased. Increase of HIF-1a activity is mediated through the
PI3K-Akt and MAPK signaling pathways [37,38]. OPN is a
boneassociated extracellular matrix protein that is produced by
numerous cell types, such as osteoblasts, osteoclasts, T
lymphocytes, NK cells, and epithelial cells. OPN influences normal
physiological processes, including bone resorption, wound healing,
tissue remodeling, and vascularization . OPN has also been
shown to be involved in all stages of cancer progression; for
instance, tumor invasion, angiogenesis, and metastasis .
Here, we found that CoCl2-induced hypoxia upregulated OPN
mRNA and protein expression in osteosarcoma cells. The
overexpression of GLUTs is requisite for cell proliferation, like
that seen in cancer, in order to increase the energy supply. We also
demonstrated that GLUTs can be upregulated by both CoCl2 and
Because GLUTs were upregulated by OPN, glucose uptake was
evaluated using a fluorescent D-glucose analog, 2-NBDG, in
MG63 osteosarcoma cells with confocal microscopy and flow
cytometry [43,44]. The intracellular fluorescence intensity of
2NBDG was enhanced by treatment with OPN in MG63,
indicating that OPN increases nutrient availability to
osteosarcoma cells. This effect was mediated by avb3 integrin, FAK
phosphorylation, and the AKT, JNK, and p38 MAPK pathways.
Knockdown of OPN expression was performed in the
osteosarcoma cell lines MG63 and U-2OS using two different
shRNA plasmids. Cell survival was decreased by approximately
20% in OPN knockdown cells. Hypoxia-induced expression of
GLUTs was also inhibited by OPN knockdown. These results
indicate that endogenously released OPN can regulate GLUTs
expression, glucose uptake, and the survival of osteosarcoma cells.
Phloretin, a natural product, exists in the fruit trees in glucosidic
form, namely phloridzin. Both of phloretin and phloridizin are
present in apple and pulp . Phloretin amounts to 12.5 mg/mL
in apple juice and 219 mg/mL in carrot juice. Phloretin has a
marked effect on the survival of colon cancer cells at
concentrations as low as 50 mmol/L . It has been demonstrated that
phloretin, isoliquiritigenin and other hydroxylated chalcones had
cytotoxic activity by inducing collapse of mitochondrial membrane
potential and increasing oxygen uptake . Phloretin (the
aglucon of phlorizin) is reported to induce human liver cancer
cell apoptosis and exert significant anti-tumor effects in HepG2
xenograft animal model by administering phloretin (10 mg/kg)
intraperitoneally . Although phlorizin makes the tumor cells
impermeable to glucose, it also makes all other cells all over the
body impermeable to glucose cells. However, phlorizin derivatives
can sensitize the cancer cells for treatment with heat and other
modalities in patients . Here we used phloretin in
osteosarcoma. Inhibition of GLUTs by 500 mM phloretin induced
approximately 50% cell death in osteosarcoma cell lines. OPN
knockdown enhanced the cell death induced by phloretin to 80%,
indicating that phloretin-induced cell death was more sensitive in
OPN knockdown osteosarcoma cells. This apoptosis enhancing
effect also appeared at low dose of phloretin (100 mM, 6 h) in both
MG63 (Figure S1A) and U-2OS (Figure S1B) osteosarcoma cells,
indicating that OPN knockdown sensitized the tumor cells to
phloretin-induced apoptosis. Both glucose and OPN are important
to osteosarcoma cell survival. Glucose uptake is related to cancer
cell survival and drug sensitivity. For instance, the decrease in
glucose uptake at an early time point after a high dose of cisplatin
reflects cisplatin chemosensitivity in ovarian cancer cells .
Phloretin also sensitizes cancer cells to daunorubicin and
overcomes drug resistance in hypoxia . Chemotherapy is used
in the treatment of osteosarcoma. As shown above, osteosarcoma
is sensitive to the glucose transporter inhibitor phloretin.
Moreover, combination of a low dose of glucose transporter
inhibitor with chemotherapy drugs markedly enhanced cell death
in osteosarcoma cell lines. These results indicate that a
combination therapy of a low dose of a GLUTs inhibitor and a cytotoxic
drug may offer a new therapeutic option to osteosarcoma patients.
In conclusion, endogenously released OPN is important for the
regulation of GLUT1 and GLUT3 expression in osteosarcoma.
Inhibition of glucose uptake by a transporter inhibitor can induce
cell death in osteosarcoma. Furthermore, the combination of a low
dose of phloretin with chemotherapeutic drugs markedly
enhanced cell death in osteosarcoma cells.
Figure S1 Low dose of phloretin enhances cell death of
osteosarcoma in osteopontin knockdown cells.
Knockdown of OPN expression (24 h) by transient transfection of
OPNshRNA (shOPN1 and shOPN2) induced approximately 10% cell
death in MG63 (A) and U-2OS (B) cells. Inhibition of glucose
transporter activity by phloretin (100 mM, 6 h) caused cell death in
the empty vector (ev) group. The cytotoxic effect of phloretin was
enhanced by OPN knockdown in both MG63 (A) and U-2OS (B)
cells after short duration of 6 h treatment. Cell viability was
measured by MTT assay. Data are presented as the mean 6
S.E.M. (n = 4). *p#0.05, compared with control group (con); #p#
0.05, compared with respective vehicle-treated group.
Conceived and designed the experiments: WMF RSY. Performed the
experiments: ISH. Analyzed the data: ISH. Contributed reagents/
materials/analysis tools: ISH. Contributed to the writing of the manuscript:
ISH. Preparation of the first draft of manuscript: ISH. Interpretation of the
results and draft revision: WMF. Scientific guidance and draft revision:
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