Hypoxia–Induced Cytotoxic Drug Resistance in Osteosarcoma Is Independent of HIF-1Alpha
Citation: Adamski J, Price A, Dive C, Makin G (
Hypoxia-Induced Cytotoxic Drug Resistance in Osteosarcoma Is Independent of HIF-1Alpha
Jennifer Adamski 0
Andrew Price 0
Caroline Dive 0
Guy Makin 0
Dominique Heymann, Faculte de medecine de Nantes, France
0 1 Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research , Manchester , United Kingdom , 2 Institute of Cancer Sciences, Manchester Cancer Research Centre, Manchester Academic Health Sciences Centre, University of Manchester , Manchester , United Kingdom , 3 Department of Paediatric Oncology, Royal Manchester Children's Hospital , Manchester , United Kingdom
Survival rates from childhood cancer have improved dramatically in the last 40 years, such that over 80% of children are now cured. However in certain subgroups, including metastatic osteosarcoma, survival has remained stubbornly poor, despite dose intensive multi-agent chemotherapy regimens, and new therapeutic approaches are needed. Hypoxia is common in adult solid tumours and is associated with treatment resistance and poorer outcome. Hypoxia induces chemotherapy resistance in paediatric tumours including neuroblastoma, rhabdomyosarcoma and Ewing's sarcoma, in vitro, and this drug resistance is dependent on the oxygen-regulated transcription factor hypoxia inducible factor-1 (HIF-1). In this study the effects of hypoxia on the response of the osteosarcoma cell lines 791T, HOS and U2OS to the clinically relevant cytotoxics cisplatin, doxorubicin and etoposide were evaluated. Significant hypoxia-induced resistance to all three agents was seen in all three cell lines and hypoxia significantly reduced drug-induced apoptosis. Hypoxia also attenuated drug-induced activation of p53 in the p53 wild-type U2OS osteosarcoma cells. Drug resistance was not induced by HIF-1a stabilisation in normoxia by cobalt chloride nor reversed by the suppression of HIF-1a in hypoxia by shRNAi, siRNA, dominant negative HIF or inhibition with the small molecule NSC-134754, strongly suggesting that hypoxia-induced drug resistance in osteosarcoma cells is independent of HIF-1a. Inhibition of the phosphoinositide 3-kinase (PI3K) pathway using the inhibitor PI-103 did not reverse hypoxia-induced drug resistance, suggesting the hypoxic activation of Akt in osteosarcoma cells does not play a significant role in hypoxia-induced drug resistance. Targeting hypoxia is an exciting prospect to improve current anti-cancer therapy and combat drug resistance. Significant hypoxia-induced drug resistance in osteosarcoma cells highlights the potential importance of hypoxia as a target to reverse drug resistance in paediatric osteosarcoma. The novel finding of HIF-1a independent drug resistance suggests however other hypoxia related targets may be more relevant in paediatric osteosarcoma.
Osteosarcoma is the most common primary malignancy of bone
and occurs most frequently in late childhood and early adulthood.
 The introduction of dose intensive combination chemotherapy
has increased the overall survival for osteosarcoma patients to over
70%. [2,3] However in those with metastasis and in those who
relapse, prognosis remains poor with survival rates of only 20
30%. [4,5] There has been no improvement in the survival of
osteosarcoma patients in the last 20 years and therefore new
therapeutic options are urgently needed.
In vitro evidence of hypoxia-induced drug resistance exists for a
wide variety of cytotoxic agents in a wide variety of adult tumour
types.  Hypoxia is able to induce resistance to etoposide and
vincristine in neuroblastoma cells and doxorubicin, vincristine and
actinomycin-D in rhabdomyosarcoma and Ewings sarcoma cells.
[13,14] Markers of hypoxia including hypoxia-inducible factor-1
(HIF-1), vascular endothelial growth factor (VEGF) and carbonic
anhydrase IX (CA IX) can be detected in osteosarcomas 
and the presence of these markers correlates with poor patient
outcome, suggesting that hypoxia has an important role in
osteosarcoma. [15,17,18] The effect of hypoxia on drug response
in osteosarcoma has not been shown.
The main transcription factor responsible for the cellular
adaptation to hypoxia is HIF-1. HIF-1 is comprised of 2
subunits, a constitutionally expressed beta unit (HIF-1-b) and an
oxygen regulated alpha unit (HIF-1a or HIF-2a). [19,20] In the
presence of oxygen the alpha subunits are hydroxylated by
oxygen-dependant prolyl hydroxylases allowing binding to the
Von Hippel Lindau (VHL) protein and targeting for
ubiquitination and degradation. In hypoxia, hydroxylation does not occur
and the alpha subunits stabilise, dimerise with HIF-1b and
translocate to the nucleus where they regulate the transcription of
over 100 target genes, many of which are directly or indirectly
involved in drug resistance.  Known HIF-1 transcriptional
targets may induce drug resistance by affecting drug transport (eg.
increased p-glycoprotein ) or drug targets (eg. decreased
topiosomerase II ) or by changing the response to drugs, for
instance by modifying drug-induced apoptosis , reducing
druginduced senescence , or inducing autophagy in response to
morphologically apoptotic cells counted with a fluorescent microscope. Graphs represent the percentage of apoptotic cells in normoxia and hypoxia.
Data are the mean 6 SEM of 3 independent experiments. * indicates p,0.05 and *** indicates p,0.001 determined by the 2-tailed student t-test. C,
72 hours after exposure to a 1 hour pulse of doxorubicin (0.14 mM), etoposide (0.8 mM) or cisplatin (6 mM) in normoxia or hypoxia U2OS cells were
stained with annexin V and 7-AAD and analysed by flow cytometry. Annexin V positive and/or 7-AAD positive cells were counted as apoptotic and
graphs represent the percentage of apoptotic cells and are the mean 6 SEM of 3 independent experiments.* indicates p,0.05 determined by the
2tailed student t-test. D, Protein from this experiment was immunoblotted for PARP, cleaved PARP, caspase-3 and cleaved caspase-3 The amount of
cleavage of PARP and caspase-3 was indicative of the amount of apoptosis occurring at that time point and was compared between normoxia and
drugs.  Hypoxia-induced drug resistance is dependent on
HIF1 in the majority of cases and inhibition of HIF-1 re-sensitises cells
to drug treatment in hypoxia. [6,811,13,2229] Thus in many
tumour types HIF-1 is a valid target to reverse hypoxia-induced
A number of other cellular pathways are differentially regulated
in hypoxia and may also contribute to hypoxia-induced drug
resistance. Wild-type p53 is inactivated in some tumour cells in
hypoxia, inducing resistance to p53-mediated apoptosis ,
and in some tumour types hypoxia-induced drug resistance occurs
only in cell lines with wild-type p53.  Activation of the
phosphoinositol-3-kinase (PI3K) pathway, nuclear factor kappa-B
(NFkB), cycloxygenase-2 (COX-2), activator protein-1 (AP-1),
cjun, Pim-1 and STAT-3 in hypoxia have all been found to induce
drug resistance, mainly by a reduction in drug-induced apoptosis.
[12,31,3439] Importantly, inhibiting this activation sensitises cells
to cytotoxic agents in hypoxia, and they are thus possible targets to
reverse hypoxia-induced drug resistance.
In this work we show for the first time that osteosarcoma cells
are resistant to the clinically relevant cytotoxics cisplatin,
doxorubicin and etoposide in hypoxia and that this resistance is
not dependent on HIF-1, or on an active PI3K pathway,
suggesting the need to investigate other hypoxia-related targets
in this tumour type.
Hypoxia Induces Drug Resistance in Osteosarcoma Cells
and Reduces Cytotoxic-induced Apoptosis
In all three osteosarcoma cell lines 24 hr exposure to 1%
oxygen, followed by 1 hr exposure to cytotoxic agent, and then
followed by a further 72 hrs exposure to 1% oxygen (hereafter
referred to as hypoxia), lead to significant resistance to cisplatin,
doxorubicin and etoposide (p,0.01 to p,0.001 2-way ANOVA)
in an SRB assay (Figure 1A). Returning the cells to 21% oxygen
after cytotoxic exposure did not induce drug resistance (data not
shown), as we have previously reported in neuroblastoma. 
Hypoxia-induced drug resistance was particularly pronounced in
U2OS cells, with a 439 fold increase in the IC50 for etoposide
(Table 1). The pattern of hypoxia-induced resistance to etoposide
and doxorubicin, with 791T cells showing the least and U2OS
cells the greatest, reflected the relative sensitivity of the three
osteosarcoma cell lines to these agents in normoxia; the IC50 for
etoposide in 791T cells in normoxia was 12.6 mM as compared to
0.8 mM in U2OS cells, whilst for doxorubicin the values were
0.7 mM and ,0.14 mM. However this was not the case with
cisplatin to which all three cell lines were similarly sensitive in
Hypoxia induces resistance to cytotoxic drugs by suppressing
apoptosis. [8,13,14,40] In U2OS cells there was a significant
reduction in apoptosis measured by morphological changes
(p,0.05001 2-tailed student t-test) (Figure 1B) and annexin V/
7-AAD positivity (p,0.050.01 2-tailed student t-test) (Figure 1C),
and a reduction in levels of cleaved caspase-3 and PARP on
western blotting (Figure 1D) in hypoxia compared to normoxia. A
significant reduction in apoptosis was also observed in HOS cells
exposed to all 3 drugs and in 791T cells exposed to cisplatin and
doxorubicin in at least 2 out of the 3 assays (data not shown). In
791T cells there was no significant difference in etoposide-induced
apoptosis between normoxia and hypoxia. 791T cells exposed to
etoposide have the least significant difference in response between
normoxia and hypoxia on SRB assay (p,0.01 2-way ANOVA
(Figure 1A)). Thus hypoxia-induced resistance to cytotoxic agents
in osteosarcoma cells is due to reduced drug-induced apoptosis.
Hypoxia-induced Drug Resistance in Osteosarcoma Cells
is not Dependent on HIF-1
Hypoxia-induced drug resistance is usually dependent on
1. Hypoxia rapidly stabilised HIF-1a and HIF-2a in all three
osteosarcoma cell lines and the corresponding up-regulation of CA
95% confidence interval IC50 (mM)
Figure 2. Regulation of HIF-1 expression in osteosarcoma cells. A, Western blots showing the time course of HIF-1a and HIF-2a stabilisation
and protein levels of the HIF-1 transcriptional target carbonic anhydrase IX (CA IX) in 791T, HOS and U2OS cells after exposure to hypoxia. GAPDH is a
loading control. Data are representative of 3 independent experiments. B, Graphs show 2(2DDCT) where CT is the cross-threshold and represents the
change in Glut-1 mRNA expression with time in 791T, HOS and U2OS cells in hypoxia relative to normoxia, where 1 would be equivalent expression in
normoxia and hypoxia and greater than 1 represents an increase in hypoxia relative to normoxia. Data are the mean 6 SEM of 2 independent
experiments. C, VEGF-A levels in the supernatant of HOS and U2OS cells detected by enzyme-linked immunosorbent assay after 24 hours in normoxia
or hypoxia, normalised to cell number. Data are the mean 6 SEM of 3 independent experiments. * indicates p,0.05 as determined by the 2-tailed
cisplatin (0150 mM), doxorubicin (02.5 mM) or etoposide (050 mM) for 1 hour. An SRB assay was performed 72 hours after treatment. B, Western
blotting performed on cell lysates from cells simultaneously maintained in hypoxia shows reduced expression of HIF-1a and CA IX, indicating
suppressed transcriptional activity, throughout the experiment. D, Stable 791T clones expressing shRNAi to HIF-1a (C24) and firefly luciferase as a
control (L3) were similarly processed and treated with doxorubicin (048 mM) or etoposide (0180 mM) for 1 hour. C, Whole cell lysates from cells
simultaneously plated were harvested at 24 hours (24H) (at treatment) and 96 hours (96 H) of hypoxia (the experiment end). Western blotting for
HIF1a and CA IX shows suppression of HIF-1a expression and transcriptional activity. E, 791T cells were transiently transfected with siRNA to HIF-1a or a
non-targeting control (NT). 8 hours after transfection the hypoxic arm was transferred to hypoxia and after 16 hours cells were treated with cisplatin
for 1 hour (0150 mM). 72 hours after treatment cells were assessed by SRB assay. F, Western blotting on cell lysates collected from cells
simultaneously transfected after 24 hours (24 H) and 96 hours (96 H) of hypoxia shows suppression of HIF-1a and CA IX expression. All graphs show
the mean absorbance relative to the untreated controls against log drug concentration and are the mean of 3 independent experiments 6 SEM. The
difference in the drug response of the shRNAi clones and the siRNA transfected cells between hypoxia and normoxia remains highly significant in all
cases despite HIF-1a suppression (p,0.001, 2-way ANOVA). Western blots are representative of 3 independent experiments. GAPDH and actin were
IX protein levels (Figure 2A), Glut-1 mRNA levels (Figure 2B) and
levels of secreted VEGF-A (Figure 2C), indicates that it is
transcriptionally active in these cell lines. HIF-1 was stabilised in
normoxia by exposing cells to cobalt chloride at either 50 mM
(791T) or 25 mM (HOS and U2OS) for 24 hrs. Functional activity
of cobalt chloride stabilised HIF-1 was confirmed by an increase in
protein levels of CA IX (Figure 3A). Despite this activation of the
HIF-1 pathway in normoxia, 24 hr cobalt chloride treatment did
not induce drug resistance (Figure 3B), suggesting that
transcriptionally active HIF-1 is not sufficient for hypoxia-induced drug
resistance in osteosarcoma cells. To further investigate the role of
HIF-1 in hypoxia-induced drug resistance in HOS and 791T cells,
stable clones were generated in which HIF-1a was suppressed by
short-hairpin RNA interference (shRNAi). Significant resistance to
cisplatin, doxorubicin and etoposide remained in hypoxia
compared to normoxia (Figure 4A), despite a reduction in
HIF1a protein levels sufficient to prevent transcription of CA IX
(Figure 4B), and there was no significant difference in
hypoxiainduced resistance to cisplatin, etoposide and doxorubicin between
the HIF-1a and the luciferase repressed cells, in which HIF-1a
levels and function were normal (Figure 4A, 4B). Similarly 791T
HIF-1a shRNAi cells remained significantly resistant to
doxorubicin and etoposide in hypoxia compared to the luciferase shRNAi
control (Figure 4D), despite significantly reduced HIF-1a protein
levels and suppressed HIF-1 function (Figure 4C). Both these
results were verified in second HIF-1a suppressed clones (data not
shown). In 791T cells highly significant (p,0.001, 2-way ANOVA)
resistance to cisplatin in hypoxia remains after transient
transfection of HIF-1a short interfering RNA (siRNA) (Figure 4E), despite
reduction in protein levels of HIF-1a and CA IX (Figure 4F). Thus
in HOS and 791T cells, suppression of HIF-1a sufficient to inhibit
the transcriptional activity of HIF-1 does not prevent hypoxia
from inducing significant resistance to cisplatin, doxorubicin and
etoposide. HIF-1 function in U2OS cells was inhibited by transient
transfection of a dominant negative HIF vector (DN-HIF)
expressing a truncated HIF-1a which lacks the trans-activation
domain.  Despite functional inhibition of HIF-1 (Figure 5A),
significant resistance to cisplatin, doxorubicin and etoposide
remained in hypoxia with no observable difference in drug
response between the DN transfected cells and the empty vector
controls (Figure 5B). Finally the small molecule NSC134754, an
inhibitor of both HIF-1a and HIF-2a, was used.  NSC134754
reduced HIF-1a protein levels in U2OS cells in hypoxia, and
reduced levels of CA IX (Figure 6A). Significant hypoxia-induced
resistance to cisplatin, doxorubicin and etoposide remained despite
this functional inhibition of HIF-1 (Figure 6B). The failure of
HIF1 inhibition, by a range of methods, to significantly impact on the
resistance to cisplatin, doxorubicin and etoposide induced by
hypoxia in any of the 3 osteosarcoma cells, suggests strongly that
Hypoxia Activates the PI3K/Akt Pathway in Osteosarcoma
Cells However Inhibition of this Pathway does not Affect
Hypoxia-induced Drug Resistance
Activation of PI3K and Akt prevents apoptosis and induces
drug resistance in both normoxia and hypoxia and PI3K
inhibition is able to reverse this resistance. [12,4244] In both
U2OS and 791T cells hypoxia increased protein levels of pS473
Akt, indicating activation of the PI3K pathway in these cells in
hypoxia (Figure 7A). In U2OS and 791T cells protein levels of
PTEN, a negative regulator of PI3K, were reduced in hypoxia.
HOS cells have an aberrantly activated PI3K pathway with strong
expression of PTEN, Akt and pS473 Akt in both normoxia and
hypoxia (data not shown). However, despite inhibition of PI3K
activation by the small molecule inhibitor PI-103, shown by
reduced pS473 Akt levels (Figure 7B), significant hypoxia-induced
drug resistance remains, regardless of the scheduling of PI3K
inhibition relative to cytotoxic exposure (Figure 7C).
Phosphorylation of p53 at Serine 15 in Response to Drug
Exposure is Reduced in Hypoxia in Osteosarcoma Cells
Cisplatin, etoposide and doxorubicin exert their cytotoxic effect
through the activation of p53 and the initiation of apoptosis.
[45,46] Oncogenically transformed cells undergo p53 dependent
apoptosis in hypoxia, therefore hypoxia selects for cells which are
deficient in p53.  In p53 wild type cells, suppression of p53
activity protects against cytotoxic-induced apoptosis. [30,32,33,48]
The inactivation of p53 in hypoxia is both HIF-1 dependent
[33,49], and HIF-1 independent.  In p53 wild type U2OS cells
p53 protein was readily detectable in untreated cells, suggesting
protein stabilisation. However phosphorylation of p53 protein on
serine 15, an indication of p53 activation, was only detected after
exposure to cytotoxic drugs (Figure 8A). Phosphorylation of p53
on serine 15 after exposure to cisplatin, etoposide and
doxorubicin, was reduced in hypoxia compared to normoxia (Figure 8A),
correlating with reduction in the protein levels of the known p53
transcriptional targets p21 and NOXA, suggesting that this
reduction in p53 phosphorylation leads to a reduction in the
transcriptional activity of p53 in hypoxia. To investigate whether
p53 inactivation in hypoxia was dependent upon functional
HIF1, U2OS cells were transiently transfected with the DN-HIF
vector. Despite functional inhibition of HIF-1 (Figure 8B)
reduction in p53 phosphorylation on serine 15 and p21 protein levels
after etoposide exposure in hypoxia were not altered (Figure 8C).
This suggests reduced p53 activation in hypoxia in U2OS cells is
not dependent on functional HIF-1. p53 inactivation may thus be
contributing to reduced cytotoxic drug-induced apoptosis in
hypoxia in U2OS cells.
Hypoxia-induced drug-resistance has been observed in vitro in
rhabdomyosarcoma, Ewings sarcoma and neuroblastoma, [13,14]
although this is not a universal phenomenon, and hypoxic
sensitisation has also been reported. Cytotoxic drug resistance in
hypoxia can vary between tumour type and with drug used.
[13,50,51] Evidence exists of the importance of hypoxia in
osteosarcoma, but the effect of hypoxia on the response of
osteosarcoma cells to clinically relevant cytotoxic drugs has not
Highly significant resistance to etoposide, cisplatin and
doxorubicin in hypoxia was seen in all 3 osteosarcoma cell lines,
consistent with previous data showing hypoxia-induced resistance
to cisplatin, doxorubicin and etoposide in a range of different
tumour types. [68,10,11,13,14,23,25,27,29,31,35,36,38,48,52]
Drug-induced apoptosis was reduced in HOS and U2OS cells
exposed to cisplatin, doxorubicin and etoposide and in 791T cells
exposed to cisplatin and doxorubicin, suggesting reduced apoptosis
as the underlying mechanism for hypoxia-induced drug resistance.
Hypoxia-induced resistance to cisplatin-induced apoptosis has
been previously reported in a number of tumour cell types
[25,35,39,48,52,53] as has hypoxia-induced resistance to
doxorubicin [14,27,35] and etoposide. [7,10,13,27,36,53] Although 791T
cells showed highly significant (p = 0.0012) resistance to etoposide
in hypoxia (Figure 1) they consistently showed an equivalent
degree of etoposide-induced apoptosis in normoxia and hypoxia,
suggesting that other resistance mechanisms may be active in these
HIF-1 is the major factor in hypoxia-induced drug resistance.
Cytotoxic drug resistance in hypoxia is dependent upon functional
HIF-1 in a number of different tumour cell types. [6,8
11,13,14,2229] HIF-1 function is important for resistance to
multiple cytotoxic agents including etoposide, doxorubicin and
cisplatin. In a wide range of different tumour types
hypoxiainduced drug resistance can be reversed by HIF-1 inhibition.
[6,8,10,11,13,14,22,23,25,27,29] However, in the osteosarcoma
cell lines HOS, U2OS and 791T stabilisation of HIF-1a in
normoxia, with activation of the HIF-1 pathway, failed to induce
drug resistance (Figure 3), suggesting that HIF-1 activation is not
sufficient for cytotoxic drug resistance in these cells. Furthermore
targeting HIF-1 in hypoxia, with several different approaches,
dominant negative HIF-1, shRNAi, siRNA and the small molecule
inhibitor NSC-134754, failed to reverse drug resistance in
hypoxia, despite very clear evidence of functional inhibition of
the HIF-1 pathway (Figures 46). This data suggests strongly that
hypoxia-induced drug resistance is independent of HIF-1 in these
osteosarcoma cells. HIF-1 independent mechanisms of drug
resistance in hypoxia are under-investigated and rarely reported.
However changes in apoptotic proteins can be independent of
HIF-1 [8,14,54] and in several cell types hypoxia-induced drug
resistance is only partially reversed by HIF-1 inhibition suggesting
the existence of HIF-1 independent mechanisms of drug
resistance. [9,38] HIF-1 null renal proximal tubular cells remain
resistant to cisplatin in hypoxia , and in pancreatic carcinoma
cells hypoxia-induced resistance to cisplatin, doxorubicin and
gemcitabine-induced apoptosis is dependent on the survival kinase
Pim-1, the induction of which in hypoxia is independent of HIF-1.
 Inhibition of PI3K, COX-2, NFkB, STAT-3 and AP-1 can
reverse resistance to cytotoxic drugs in hypoxia, implying a role for
these pathways in hypoxia-induced drug resistance. [12,31,3439]
However the degree to which they are dependent on functional
HIF-1 is often uncertain. Previous publications reporting the
HIF1 independence of hypoxia-induced drug resistance have been
contradicted subsequently by the demonstration of HIF-1
dependence, accounted for by an initial failure to adequately suppress
HIF-1. Thus in HepG2 hepatoma cells initial experiments
resulting in 50% reduction of HIF-1 activity suggested that
resistance to etoposide-induced apoptosis in hypoxia did not
depend upon HIF-1, but subsequent experiments achieving 95%
reduction in HIF-1 activity showed very clear dependence on
functional HIF-1. [29,36] In HOS and 791T osteosarcoma cells
functional inhibition of HIF-1, as measured by the up-regulation
of protein levels of CA IX, was achieved through shRNAi
(Figure 4B and 4C). In 791T cells complete loss of HIF-1a protein
and functional inhibition, as measured by CA IX, was also
achieved with transient transfection of siRNA (Figure 4F). In both
cell lines this loss of functional HIF-1 did not reverse
hypoxiainduced drug resistance. In U2OS cells transient transfection of
dominant negative HIF-1a, which significantly reduced the
transcription of both CA IX and GLUT-1 in hypoxia, did not
alter hypoxia-induced drug resistance to all 3 drugs (Figure 5).
Finally, despite inhibition of HIF-1 function by NSC-134754 in
U2OS cells, hypoxia-induced resistance to all 3 cytotoxics
persisted (Figure 6). Thus in these three osteosarcoma cell lines,
four different methods of inhibition of HIF-1 function failed to
have any effect upon hypoxia-induced drug resistance, providing
strong evidence for the HIF-1 independence of this phenomenon.
Consistent with previous reports of increased activation of Akt
in hypoxia, hypoxia leads to phosphorylation of Akt at serine 473
in 791T and U2OS osteosarcoma cells (Figure 7A). 
Phosphorylation of Akt correlated with reduced levels of PTEN,
also previously reported, [31,55] and consistent with the normal
regulation of this pathway.  Activation of the PI3K pathway is
a recognised cause of cytotoxic resistance in hypoxia, protecting
against both drug-induced and serum withdrawal-induced
apoptosis. [12,42,44,5759] Mechanisms include inhibition of GSK-3
activity [43,60,61] and activation of NFkB.  Activation of the
PI3K pathway may also stabilise and activate HIF-1  and
inactivate p53,  both of which are known to reduce apoptosis
in hypoxia. However, although Akt is activated in hypoxia in 791T
and U2OS osteosarcoma cells, inhibition of PI3K with PI-103 is
not able to re-sensitise cells to cytotoxic agents, regardless of
scheduling (Figure 7B), suggesting it does not contribute
significantly to hypoxia-induced drug resistance in these cells. This
differs from the situation in lung cancer, pancreatic cancer and
phaeochromocytoma cells in which hypoxic resistance to
cytotoxic-induced apoptosis is reversed by PI3K inhibition [12,42,44].
Figure 5. Osteosarcoma cells treated with the small molecule inhibitor of HIF-1a NSC134754 remain resistant in hypoxia. A, U2OS
cells were treated with 20 mM NSC-134754 for 24 hours in hypoxia prior to exposure to a range of concentrations of cisplatin (0300 mM), doxorubicin
(0100 mM) or etoposide (04000 mM) for 1 hour. Untreated controls were exposed to the same concentration ranges of cisplatin, doxorubicin and
etoposide in normoxia and hypoxia. 72 hours after treatment cells were fixed and a SRB assay performed Graphs show the mean absorbance relative
to the untreated controls (no chemotherapy agent) and are the average of 3 independent experiments 6 SEM. B, Simultaneously plated cells treated
with NSC134754 and incubated in hypoxia for 24 hours (time of treatment) or 96 hours (end of experiment) were harvested for whole cell lysates and
western blotting performed for HIF-1a and CA IX. Western blots are representative 3 independent experiments with GAPDH used as a loading
control. The difference between the response to cytotoxics in normoxia and hypoxia remains highly significant despite treatment with NSC134754
(p,0.001 2-way ANOVA).
Figure 6. Cobalt Chloride stabilises and transcriptionally activates HIF-1a in normoxia but does not induce resistance. B, 24 hours
after plating osteosarcoma cells were treated with cobalt chloride (791T 50 mM; HOS 25 mM; U2OS 25 mM) for 24 hours before treatment with a range
of concentrations of cisplatin (791T 050 mM; HOS 025 mM; U2OS 0200 mM), doxorubicin (791T 016 mM; HOS 05 mM; U2OS 040 mM) or
etoposide (791T 050 mM; HOS 050 mM; U2OS 01000 mM). Following a one hour drug exposure cells were incubated with or without cobalt
chloride for a further 72 hours before fixing and performing a sulphorhodamine-B assay. Graphs show the mean absorbance relative to the untreated
controls (UnT) against the log of the drug concentrations and are the average of 3 independent experiments 6 SEM. A, Whole cell lysates of cells
treated with the above doses of cobalt chloride for the length of the experiment (96 hours) were harvested for western blotting to determine HIF-1a
stabilisation and expression of downstream target CA IX. The western blots are representative of 3 independent experiments with GAPDH as a
Modification of p53 in hypoxia is well reported  and its
inactivation leads to a reduction in drug-induced apoptosis. [30
33,48] Furthermore HIF-1 mediated inactivation of p53 in
normoxia also induces chemoresistance. [37,49] It has been
suggested that, although U2OS cells have wild type p53, the
pathway is non-functional because of mdm2 over-expression. 
However, when U2OS cells are exposed to cytotoxic agents
phosphorylation of p53 leads to the disassociation of p53 from
mdm2, activation of down-stream targets and p53dependent
apoptosis. [66,67] Increased p21 protein levels after cytotoxic drug
exposure in our experiments imply an active downstream pathway
in U2OS cells consistent with this data (Figure 8A). p53
inactivation in hypoxia (Figure 8A), may contribute to the reduced
drug-induced apoptosis in hypoxia seen in U2OS osteosarcoma
cells, as in HepG2 hepatoma cells (Sermius 2008). p53 inactivation
in U2OS cells is not dependent on functional HIF-1 (Figure 8B
and C), and this is consistent with the contribution of p53
inactivation to hypoxia-induced drug resistance. HOS cells are
known to have non-functioning mutated p53, and although the
p53 status of 791T cells is not described, we have not observed a
p53-regulated response to DNA damaging agents. However
significant hypoxia-induced drug resistance was observed in both
these cell lines, although the degree of resistance was significantly
greater in U2OS cells. Thus, although it may contribute to
hypoxia-induced drug resistance in U2OS cells, p53 inactivation
cannot be the only cause of hypoxia-induced resistance in
osteosarcoma cells. Potential alternative drug resistance
mechanisms including activation of NFkB , c-jun  and p-ERK
1/2  have all been reported as contributing to
hypoxiainduced drug resistance in cancer cells with inactive p53 pathways.
In conclusion the significant hypoxia-induced drug resistance in
these three osteosarcoma cell lines suggests that hypoxia is a
potential target in osteosarcoma. However the failure of HIF-1
inhibition to reverse drug resistance in hypoxia suggests that
alternative approaches are needed. p53 inactivation in hypoxia
may contribute to drug resistance in osteosarcoma cells with a
functioning p53 pathway but cannot be the cause of drug
resistance in all osteosarcoma cells. Further work is needed to
identify a targetable pathway on which hypoxia-induced drug
resistance in osteosarcoma is dependent.
Osteosarcoma cell lines U2OS (ATCC), HOS (ATCC) and
791T (Paterson Institute for Cancer Research Cell Bank) were
maintained in GIBCO RPMI medium with 10% FCS in 95% air
and 5% CO2 at 37uC. All cell lines were authenticated by CRUK
in July 2010 using STR profiling.
For hypoxia experiments, cells were incubated and treated in an
InVivo2 Hypoxia workstation 400 (Ruskin Technology Limited)
flushed with 1% O2, 5% CO2, and 94% N2 (subsequently referred
to as hypoxia).
Analysis of Cell Population Growth by SRB Assay
Drug response was assessed using the sulphorhodamine-B (SRB)
assay. After 24 hours pre-incubation in normoxia or hypoxia cells
in log phase were exposed to etoposide (Sigma-Aldrich E1383),
cisplatin (Sigma-Aldrich 479306) or doxorubicin (Sigma-Aldrich
D1515) for a period of 1 hour then further incubated in normoxia
or hypoxia for 72 hour before processing as previously described.
 IC50 values were calculated on GraphPad Prism5 software
using the Hill equation and represent 50% of the drugs maximal
Protein Detection by Western Blotting
Cells were harvested for western blotting as described. 
Primary antibodies were applied overnight in PBST or 15% milk
in PBST: Actin (1:1000; Sigma A4700), Akt (1:1000; Cell
Signalling 9272), CA IX (1:1000; Bayer), Cleaved caspase-3
(1:100; Cell Signalling 9661), GapDH (1:2500; Sigma G9545),
HIF-1a (1:1000; BD Transduction Laboratories 610958), HIF-2a
(ep190b) (1:500; Novus Biologicals NB 100132H), NOXA
(1:1000; Imgenex IMG-349A), p53 (1:1000; Santa Cruz
Biotechnology (Do-1) sc-126), PARP (1:1000; Cell Signalling 9542),
Phospho-Akt (Ser 473) (1:1000; Cell Signalling (193H12) 4058),
Phosphop53 (Ser15) (1:1000; Cell Signalling 92840), PTEN
(1:1000; BD Pharmingen 559600), PUMA (1:1000; Sigma
(bbc3C-Terminal) P4618), WAF1 (P21) (1:500; Oncogene
OP64100UG). Secondary antibodies were either goat anti-mouse
horseradish peroxidase or goat anti-rabbit horseradish peroxidase
(DAKO P0447 and P0448).
VEGF was determined by the Quantikine Human VEGF-A
Immunoassay (R&D Systems). Cells were allowed to adhere for 24
hours, the medium replaced and flasks incubated in hypoxia or
normoxia for a further 24 hours. The supernatant was then
removed and analysed for VEGF-A levels in pg/ml. VEGF levels
were normalised to cell number.
Total RNA was isolated using the Qiagen RNeasy Kit. Reverse
transcription was performed using the TaqMan Reverse
Transcription Reagent Kit (Applied Biosystems) according to the
manufacturers guidelines. Glut-1 and CA IX were amplified using
the following primer sequences (shown 59 to 39): Glut-1
GGTTGTGCCATACTCATGACC (left primer),
CAGATAGGACATCCAGGGTAGC (right primer); CA IX
CCTTTGCCAGAGTTGACGAG (left primer),
GCAACTGCTCATAGGCACTG (right primer) and universal probes 67 and 25 for
Glut1 and CA IX respectively (Roche). Succinate dehydrogenate
complex A (SDHA), L14, L32 and beta-2-microglobulin (B2M)
were housekeeper genes. RT-PCR was performed with 5ng
template cDNA using TaqMan Master Mix and an ABI Prism
7900 HT sequence detection system (Applied Biosystems).
Crossthreshold (CT) values were calculated using the 2.1 software (ABI).
The 2(2DDCT) was calculated to represent the fold change of the
target gene mRNA in hypoxia compared to normoxia .
Cells were harvested 48 or 72 hours following drug exposure.
For Annexin-V/7-AAD staining trypsinised cells were stained in
96 wells plate to identify apoptotic cells. Data were collected on
BD FACSArrayTM and analysed by FlowJo software. The
percentage of apoptotic cells was calculated by combining all the
annexin-V positive cells or 7-AAD positive cells. Western blotting
was performed for cleaved caspase 3 and cleaved PARP.
Morphological changes of apoptosis were assessed 48 hours after
drug exposure. Cell pellets were fixed in 10% formalin
(SigmaAldrich) and re-suspended in ProLong Gold antifade with DAPI
(Molecular Probe). Apoptotic nuclear morphology was quantified
using an Olympus BX51 UV fluorescence microscope, counting 3
full fields or at least 300 cells.
Induction of HIF-1a in Normoxia using Cobalt Chloride
Cells were exposed to 24 hours cobalt chloride (50 mM 791T
cells, 25 mM HOS and U2OS cells) before exposure to a range of
concentrations of cisplatin or etoposide for 1 hour. An SRB assay
was performed after 72 hours. Cells simultaneously plated and
treated were lysed after 96 hours of cobalt chloride exposure and
protein levels of HIF-1a and CA IX assessed by western blotting.
Short Hairpin RNA Interference for HIF-1a
The HIF-1a target sequence GTCTCGAGATGCAGCCAGA
 was incorporated into p-Silencer 2.1-U6 Hygro (Ambion
(AM5760)). This plasmid was stably transfected into cells by
electroporation at 1050uF, 260 V. After hygromycin selection
(100 mg/ml for 791T and U2OS cells and 20 mg/ml for HOS
cells), single clones were screened for HIF-1a protein expression in
hypoxia. All clones were maintained in RPMI containing 10%
FCS and hygromycin (100 mg/ml for 791T cells, 5 mg/ml for
HOS cells and 25 mg/ml for U2OS cells).
For control the firefly luciferase target sequence
CTTACGCTGAGTACTTCGA replaced the HIF-1a target sequence.
Transfection and selection were as above. Single clones were transfected
with the expression vector pBactin-IRES-GFP-ff-Luc using
FuGENE HD transfection reagent as per the manufacturers
instructions (Roche 04709705001). 24 hours after transfection cells
were sorted on the BD FACSVantageTM SE Cell Sorter (BD
Biosciences) and cells positive for GFP retained. GFP positive were
incubated in medium containing streptomycin and penicillin
(50units/ml, penicillin-streptomycin liquid, Invitrogen 15070-063,
diluted 1:100) for 24 hours and then subjected to a luciferase assay
as per the manufacturers instructions (Promega E1500).
Luminescence was measured on the FLUOstar OPTIMA microplate
reader and normalised to cell number. Clones were selected for
significant reduction in luciferase expression. HIF-1a protein levels
in hypoxia in the luciferase shRNAi clones did not differ from the
Small Interfering RNA Interference for HIF-1a
siRNAs targeted to HIF-1a and non-targeting (NT) control
siRNA were from Dharmacon SMARTpool (Thermo-Scientific
L004018-00 and D-001810-01-20). Cells were transfected with
siRNA at 25 nM using the DharmaFECT 2 siRNA transfection
reagent (Thermo-Scientific T-2002) according to the
manufacturers instructions. After 24 hours siRNA was replaced by full growth
medium. 68 hours after transfection cells were incubated in
hypoxia until drug exposure 32 hours after transfection (after 24
hours in hypoxia). Simultaneously plated and transfected cells
were harvested for HIF-1a and CA IX protein detection by
HIF-1 Inhibition by Dominant Negative HIF (DN HIF)
Both the pEF IRES-P HIF-no TAD EGFP plasmid (dominant
negative HIF (DN HIF)) [6,28] and the pEF IRES-P EGFP empty
vector (EV) control were kindly donated by Dr Kaye Williams,
University of Manchester. DN-HIF or EV plasmids were
transiently transfected into U2OS osteosarcoma cells using
FuGENE HD transfection reagent as per manufacturers
instructions. 24 hours after transfection cells were seeded for SRB assay.
Cells from the same pool were simultaneously seeded for
quantification of Glut-1 and CA IX mRNA by qPCR.
HIF-1 Inhibition by NSC-134754
Cells were treated with 20 mM NSC 134754  (National
Cancer Institute, Bethesda) for 24 hours in normoxia or hypoxia
before exposure to cytotoxic for 1 hour. The concentration of
20 mM NSC-134754 was maintained throughout. Identical plates
without NSC 134754 treatment were used as controls. After 72
hours an SRB assay was performed. Protein levels of HIF-1 and
CA IX were determined in simultaneously plated cells by western
Activation of Akt and PI3K Inhibition
Protein levels of total Akt, Akt pS473, and PTEN were assessed
after 48 hours incubation in normoxia and hypoxia by western
blotting. After a 24 hour incubation period in normoxia or
hypoxia cells were treated with 1 mM PI-103 (Calbiochem 528100)
followed by cytotoxic for 1 hour. After 72 hours an SRB assay was
performed. The concentration of 1 mM PI-103 was maintained
throughout. Cells simultaneously plated and treated with and
without PI-103 were harvested at the end of the experiment (after
96 hours of PI-103 treatment) for western blotting.
p53 Activation in Response to Cytotoxic Treatment and
the Influence of HIF-1
24 hours after exposure to SRB IC50 doses of cytotoxic for
1 hour lysates were assessed for total p53, p53 pS15, p21(WAF1),
PUMA and NOXA by western blotting. U2OS cells were
transiently transfected with the DN HIF or EV plasmids. 24
hours after transfection cells were pre-incubated in normoxia or
hypoxia for 24 hours then exposed to 1 mM etoposide. 24 hours
later protein levels were assessed by western blotting.
Simultaneously transfected and plated cells were maintained in normoxia
or hypoxia and harvested at 24 hours for qPCR for CA IX and
Statistical significance of differences was assessed using 2-tailed
students t test or 2-way ANOVA, a p value of less than 0.05
considered significant. Experiments show the average of 3
independent experiments unless otherwise stated and western
blots are representative of 3 independent experiments. Error bars
Conceived and designed the experiments: JA AP CD GM. Performed the
experiments: JA AP. Analyzed the data: JA GM. Wrote the paper: JA GM.
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