The existence of acute hypoxia events in tumours was shown a few years later, with the Keywords apoptosis; cancer; chemoresistance; endothelial cell; hypoxia-inducible factor-1; intermit
Trang 1Intermittent hypoxia is a key regulator of cancer cell and endothelial cell interplay in tumours
S Toffoli and C Michiels
Laboratory of Biochemistry and Cellular Biology (URBC), University of Namur – FUNDP, Belgium
Introduction
Hypoxia is increasingly perceived as one of the
tumour microenvironment features favouring tumour
cell survival, and also resistance to chemotherapy and
radiotherapy Hypoxia is defined as a decrease in
oxy-gen level within the tissue However, recent studies
have shown that the time frame within which this
decrease occurs and, more importantly, its duration
may vary greatly from one tumour to another, or even
from one area to another within the same tumour
These observations have led to the definition of two
kinds of hypoxia: chronic hypoxia and intermittent
hypoxia
Intermittent and chronic hypoxia in solid tumours
Chronic hypoxia in tumours, first described in 1955 [1,2], results from limitation of the diffusion of oxygen Oxygen diffuses to a distance of 100–150 lm from blood vessels in normal and malignant tissues At a greater distance, the oxygen tension becomes close to zero, and cells become hypoxic [1] In parallel with chronic hypoxia, it was suggested in 1979 that tran-sient hypoxia or intermittent hypoxia could also appear in tumours, due to the temporary ‘closure’ of blood vessels [3] The existence of acute hypoxia events
in tumours was shown a few years later, with the
Keywords
apoptosis; cancer; chemoresistance;
endothelial cell; hypoxia-inducible factor-1;
intermittent hypoxia; radioresistance;
reactive oxygen species; reoxygenation;
tumor cell
Correspondence
C Michiels, Laboratory of Biochemistry and
Cellular Biology (URBC), University of
Namur – FUNDP, 61 rue de Bruxelles, 5000
Namur, Belgium
Fax: +32 81 72 41 35
Tel: +32 81 72 41 31
E-mail: carine.michiels@fundp.ac.be
(Received 1 March 2008, accepted 9 April
2008)
doi:10.1111/j.1742-4658.2008.06454.x
Solid tumours are complex structures in which the interdependent relation-ship between tumour and endothelial cells modulates tumour development and metastasis dissemination The tumour microenvironment plays an important role in this cell interplay, and changes in its features have a major impact on tumour growth as well as on anticancer therapy respon-siveness Different studies have shown irregular blood flow in tumours, which is responsible for hypoxia and reoxygenation phases, also called intermittent hypoxia Intermittent hypoxia induces transient changes, the impact of which has been underestimated for a long time Recent in vitro and in vivo studies have shown that intermittent hypoxia could positively modulate tumour development, inducing tumour growth, angiogenic pro-cesses, chemoresistance, and radioresistance In this article, we review the effects of intermittent hypoxia on tumour and endothelial cells as well as its impacts on tumour development
Abbreviations
AP-1, activator protein-1; ARNT, aryl hydrocarbon receptor nuclear translocator; EPR, electron paramagnetic resonance; HIF-1, hypoxia-inducible factor-1; NF-jB, nuclear factor kappaB; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor.
Trang 2demonstration that intermittent hypoxia resulted from
transient changes in blood flow [1,4,5] Histological
analysis of tumour blood vessels showed that
struc-tural abnormalities were responsible for this irregular
blood flow Indeed, tumour blood vessels are often
tor-tuous and dilated, with excessive branching and
numerous dead ends [6] Moreover, compression of
these vessels by tumour cells, associated with the
immaturity of the tumour vascular network, which is
characterized by an absence of or a loose association
with mural cells, pericytes and vascular smooth muscle
cells, could also play a role in the heterogeneity of the
blood flow [7–9]
The blood flow stop periodicity, depending on the
architectural complexity and maturation level of the
tumour vascular network, is very variable from one
tumour to another, and also within the same tumour
[10,11] Therefore, a precise duration for blood flow
interruption in tumours cannot be given However,
studies of murine and human tumours have shown
that the blood flow fluctuations observed in these
tumours could vary from several minutes to more
than 1 h in duration [10–16] These blood flow
irregu-larities in tumours can be demonstrated by different
methods Direct real-time measurement in vivo of
tumour blood flow fluctuations can be performed by
the use of microprobes that are directly implanted in
tumours Different microprobe systems can be used
to study the blood flow fluctuations One of the most
used microprobes is the Eppendorf polarographic
needle electrode, which allows measurement of the
oxygen partial pressure (po2) within tissues [12,17]
Polarographic oxygen microelectrode functioning is
based on reduction of oxygen at the surface of a
cathode by applying a negative voltage between the
cathode and the anode The reduction current
mea-sured with this kind of electrode is proportional to
the number of oxygen molecules being reduced, and
diminishes when blood flow is decreased or
inter-rupted [18,19] Other microprobes, such as the
Oxy-Lite laser Doppler probe, allow monitoring of tumour
blood perfusion [14] These probes illuminate the
tissue under observation with single-frequency light
from optical fibres coupled to a sensor Mobile red
blood cells scatter the monochromatic light and
gen-erate a signal that is proportional to the mean
eryth-rocyte velocity multiplied by the number of moving
erythrocytes within the sampling volume [20,21] This
signal decreases when the blood flow diminishes or
stops, and vice versa However, the spatial resolution
of these techniques is low, and the use of
polaro-graphic or laser Doppler microprobes is restricted
to easily accessible tumours [22] For less accessible
neoplasms, the direct real-time measurement in vivo of oxygen tension is performed by the use of imaging techniques [18,22,23], most of which are based on magnetic resonance Blood oxygen level-dependent magnetic resonance imaging and electron paramag-netic resonance (EPR) oxymetry are examples of such techniques [22–24] Blood flow modifications observed with blood oxygen level-dependent magnetic reso-nance imaging are based on the oxygenation status of endogenous haemoglobin This becomes paramagnetic when it is deoxygenated, and it is then detectable by magnetic resonance imaging Changes in blood flow modify the blood concentration of paramagnetic deoxyhaemoglobin and hence induce variations in the magnetic resonance signal [22,23,25,26] On the other hand, EPR oxymetry is based on the broadening of the resonance spectrum of a paramagnetic material
by oxygen [27] Modifications in the EPR signal are directly correlated with the oxygen concentration, which is linked to the blood flow [18] One injection
of a paramagnetic agent, such as India ink or char-coal, directly into a tumour is sufficient to allow repeated measurements to be performed over a rela-tively long period [18,24] Indirect measurements
in vivo of tumour po2 fluctuation can also be per-formed by the use of a double hypoxia marker tech-nique [11,28–30] 2-Nitroimidazoles (e.g misonidazole, EF5, CCI-103F, and pimonidazole) are commonly used as hypoxia markers These molecules are reduced by cellular nitroreductases at po2 levels below
10 mmHg to intermediates that covalently bind to cellular macromolecules [31–33] Hypoxic markers are administered in vivo separately at different times according to a pre-established timing schedule Tumour areas stained only by one marker show tran-sient changes in hypoxia during the time interval between the injections of the two hypoxia markers [22,29] Reduced 2-nitroimidazoles can be detected
by immunohistochemistry or immunofluorescence staining after the tumour resection Using radio-labelled 2-nitroimidazoles (e.g [18 F]fluoromisonida-zole), transiently hypoxic areas can also be detected
in vivo by positron emission tomography, which is based on the detection of electromagnetic radiation emitted indirectly by the positron-emitting radio-isotope [22,34] Modifications in blood flow are shown by performing scans after each hypoxia mar-ker injection [18,22,23,35] The use of these techniques and their combination allow a better understanding
of spatial and temporal changes in hypoxia in solid tumours, and also allow the linkage of these changes with other tumour microenvironmental parameters [18,22,23,35]
Trang 3Hypoxia-inducible factor-1 (HIF)
a-subunit stabilization and HIF-1
activation under intermittent hypoxia
Hypoxia induces numerous changes in gene expression
in normal and tumour cells [36] This adaptive response
to hypoxia is orchestrated by a family of transcription
factors induced by hypoxia The most important and
best-studied member of this family is hypoxia-inducible
factor-1 (HIF-1) HIF-1 is a heterodimeric transcription
factor composed of the HIF-1a (120 kDa) and aryl
hydrocarbon receptor nuclear translocator (ARNT,
94 kDa; also called HIF-1b) subunits These two
subun-its belong to the Per-ARNT-Sim basic–helix–loop helix
family [37,38] HIF-1a and ARNT are constitutively
expressed [39], but the formation of HIF-1 transcription
factor in the nucleus depends on HIF-1a stabilization,
which is principally O2-dependent [40] Under
nor-moxia, HIF-1a is hydroxylated on proline 402 in the
N-terminal domain and proline 564 in the C-terminal
domain by prolyl-4-hydroxylases [41] These
hydroxyla-tions allow the binding of von Hippel–Lindau tumour
suppressor protein on the oxygen-dependent
degrada-tion (ODD) domain of HIF-1a [42] von Hippel–Lindau
tumour suppressor protein acts as the substrate
recogni-tion protein of the E3 ubiquitin ligase complex [43], and
induces the ubiquitination of HIF-1a on its N-terminal
and C-terminal domains (amino acids 390–417 and
549–582, respectively) [41] This ubiquitination targets
HIF-1a for proteasomal degradation On the other
hand, under hypoxic conditions, the prolyl hydroxylase
activity decreases and the degradation pathway
described above is interrupted [44] HIF-1a therefore
rapidly accumulates and translocates into the nucleus,
where, after dimerization with ARNT, it induces the
transcription of target genes involved, notably, in
glycolysis (e.g the glyceraldehyde-3-phosphate
dehydro-genase gene) and angiogenesis [e.g the vascular
endo-thelial growth factor (VEGF) gene] [45], thus allowing
cells to adapt to hypoxia [46]
The stabilization of HIF-1a and activation of HIF-1
have been widely studied under chronic hypoxia The
new interest in intermittent hypoxia in recent years has
led us to consider again this point: can the succession
of short hypoxia and reoxygenation phases, typical
of intermittent hypoxia, also stabilize HIF-1a and
activate HIF-1?
In the absence of oxygen, HIF-1a is rapidly
stabi-lized, and short, intermittent hypoxia periods can be
sufficient to induce HIF-1 Indeed, Yuan et al showed,
in vitro, HIF-1a stabilization during intermittent
hypoxia (cycles of 30 s of hypoxia followed by 4 min
of reoxygenation) [47] This increase in HIF-1a
abun-dance was dependent on the number of intermittent hypoxia cycles The kinetics used by Yuan et al undoubtedly demonstrate that short hypoxia–reoxy-genation cycles can induce HIF-1a stabilization How-ever, considering these kinetics, the increase in abundance of HIF-1a during intermittent hypoxia cycles could be due to an accumulation of HIF-1a sub-unit during each cycle, and not to an increase in its stabilization Indeed, although HIF-1a may be extre-mely rapidly degraded when cells are reoxygenated, its degradation after 4 min of reoxygenation was not assayed by Yuan et al Furthermore, Berra et al showed that HIF-1a could still be detected after 5 min
of reoxygenation in HeLa cells incubated for 1 h or or
8 h under hypoxia They showed that the half-life of HIF-1a is inversely proportional to the duration of hypoxic stress [48], suggesting that long hypoxia peri-ods could decrease HIF-1a stability Other recent stud-ies have also shown an increase in HIF-1a abundance
in the course of intermittent hypoxia cycles, using longer cycles of 1 h of hypoxia followed by 30 min of reoxygenation [49,50] The times used in these studies allowed the demonstration of complete HIF-1a degra-dation after each cycle of 30 min of reoxygenation, showing that HIF-1a had not accumulated in the course of intermittent hypoxia cycles, and therefore that it is its stabilization that is increased in these conditions [50]
HIF-1a stabilization does not always translate into HIF-1 activity One can therefore ask whether hypoxia periods interrupted by reoxygenation periods can be sufficient to induce the transcription of HIF-1 target genes HIF-1a degradation after each reoxygenation makes HIF-1 inactive In these circumstances, HIF-1 can only be transcriptionally active during the hypoxia phases, which can be short Reporter assays showed a significant gradual increase in hypoxia response element (HRE) promoter activity in PC12 cells incubated under intermittent hypoxia, in the course of hypoxia–reoxy-genation cycles [47] Interestingly, with the same incu-bation time, HIF-1 transcriptional activity observed under intermittent hypoxia was almost equal to HIF-1 transcriptional activity observed under chronic hypoxia [50] Moreover, with the same incubation time under hypoxia (duration of reoxygenation under intermittent hypoxia was not considered in this case), Yuan et al showed higher HIF-1 transcriptional activity in com-parison to chronic hypoxia [47]
Although intermittent hypoxia can induce, like chronic hypoxia, HIF-1a stabilization as well as HIF-1 transcriptional activity, some differences can be seen between these two kinds of hypoxia It was shown in PC12 cells and EAhy926 endothelial cells that, under
Trang 4transient hypoxia, extracellular signal-related
kinase 1⁄ 2 mitogen-activated protein kinases and
phos-phoinositide-3-kinase are not required for HIF-1
sta-bilization and transcriptional activity [47,50], whereas
the inhibition of these kinases under chronic hypoxia
impaired HIF-1 target gene expression [51,52] On the
other hand, at least in endothelial cells, protein
kina-se A is involved in HIF-1a phosphorylation under
intermittent hypoxia but not under chronic hypoxia,
and protein kinase A inhibition decreased the
tran-scription of HIF-1 target genes [50] These results
sug-gest that the pathways regulating HIF-1 activity under
chronic or intermittent hypoxia are different Figure 1
shows a brief comparison of HIF-1a stabilization and
HIF-1 activation under intermittent hypoxia and
chronic hypoxia
Tumour resistance induced by
intermittent hypoxia
The effects of chronic hypoxia have been extensively
studied, and it has been clearly demonstrated that
chronic hypoxia protects tumour cells from apoptosis
induced by radiotherapy and chemotherapy [53–60]
Recent studies have shown that intermittent hypoxia
could also protect tumour cells from anticancer
treat-ments
Martinive et al showed, in vivo, a decrease in
tumour cell apoptosis in transplantable liver tumour
implanted in mice subjected to cycles of intermittent
hypoxia before irradiation (10 Gy) with respect to mice
kept under normoxia [49] This inhibition of apoptosis
under transient hypoxia was also observed in vitro in
FsaII fibrocarcinoma cells and B16 melanoma cells [49] Moreover, Dong & Wang demonstrated the possi-bility of death-resistant cell selection by the repetition
of hypoxia episodes [61] Such selected cells were shown to be resistant to cell death induced by different types of molecules, such as azide, cisplatin and stauro-sporine [61]
Transient hypoxia could also render tumours more invasive Cairns et al observed a highly significant increase in the number of lung micrometastases in KHT tumour-bearing mice exposed to 12 cycles per day (for 8–15 days) of 10 min of hypoxia followed by
10 min of reoxygenation, in comparison to control mice Interestingly, no increase in lung micrometastasis was observed in mice exposed to chronic hypoxia [15], suggesting again that intermittent hypoxia has different effects from chronic hypoxia
In addition, it was shown by Durand & Aquino-Parsons that blood flow decreases could transiently arrest the division of tumour cells in S-phase [62] These cells are the main targets of chemotherapy, and the arrest of their cell cycle during S-phase reduces considerably their sensitivity to antiproliferative drugs, but it also implies a more rapid initiation of tumour cell repopulation when the blood flow restarts [62] More generally, the transient cessation of tumour blood flow reduces tumour cell exposure to the most highly diffusible anticancer agents, but also reduces their sensitivity to radiotherapy because of the decrease
in oxygen supply during tumour irradiation [57,62] The protection against antitumour treatment can also
be linked to a particular phenotype acquired by the cells in the course of intermittent hypoxia phases An
Fig 1 Effects of intermittent hypoxia and chronic hypoxia on HIF-1a stabilization and HIF-1 target gene transcription.
Trang 5example of such acquired resistance is described by
Dong & Wang [61]: upregulation of Bcl-XL has been
observed in immortalized rat kidney epithelial cells
exposed to repeated periods of hypoxia It was shown
in these cells that Bcl-XL could directly interact with
the proapoptotic molecule Bax at the mitochondrial
level, impeding Bax oligomerization and cytochrome c
release, and hence preventing cell apoptosis [61]
Genetic instability due to abnormal DNA
meta-bolism linked to impaired activity of enzymes such as
topoisomerases, helicases and ligases is often observed
in hypoxic tumours [63] Strand breaks, translocations,
transversions and other chromosomal rearrangements
observed in these conditions can also be responsible
for tumour resistance Reynolds et al showed that
hypoxia could induce a 3–4-fold elevation in mutation
frequency, and higher levels of mutagenesis were
observed in cells exposed multiple times to hypoxia
[63], suggesting that exposure of cells to transient
hypoxia could also induce resistance to antitumour
treatments by this mechanism Moreover, oxidative
injuries generated by reoxygenation in the course of
intermittent hypoxia phases can also be responsible for
DNA damage through an increase in 8-oxoguanine,
which has been shown to miscode for A and lead to
C:G to A:T transversions [64]
Reactive oxygen species (ROS) generated during the
reoxygenation periods can also play an important role,
modifying gene expression through the regulation of
the activity of some transcription factors, such as
activator protein-1 (AP-1) or nuclear factor kappaB
(NF-jB)
AP-1 is known to play a pivotal role in
tumorigen-esis, regulating the expression and function of cell
cycle regulators such cyclin D1, p53, p21, p19, and
p16 Moreover, its activity was shown to increase in
multiple human tumour types, and its inhibition can
block tumour promotion, transformation, progression,
and invasion [65] AP-1 activation was shown in
PC12 cells under intermittent hypoxia, and was
clearly associated with ROS production and, more
particularly, with superoxide (O2Æ–) anion generation
[66] Furthermore, it was shown that AP-1 activation
involved c-fos, the activation of which persisted for
several hours after the intermittent hypoxia ‘stimulus’
[66] Deregulation of c-fos and c-jun proteins can
induce transformation in vivo [67], and c-fos
upregu-lation was shown in tumour formation and, more
particularly, in liver tumour development In this kind
of tumour, AP-1 and c-fos were shown to be able to
downregulate tumour suppressor genes and favour
angiogenesis and tumour invasiveness [68] Therefore,
AP-1 activation under intermittent hypoxia, associated
with c-fos upregulation, could promote tumour devel-opment
NF-jB can also be activated by ROS [69] ROS pro-duction during the reoxygenation periods [70] might also be able to activate NF-jB Ryan et al showed in HeLa cells and bovine aortic endothelial cells that transient hypoxia activated NF-jB in a number of hypoxia–reoxygenation cycles in an ROS-dependent manner [71] Despite the potential production of ROS during reoxygenation concomitant with NF-jB activa-tion, these authors suggested that NF-jB activation under intermittent hypoxia was not linked to ROS production, because no decrease in NF-jB activation
in the presence of the ROS scavenger N-acetyl-l-cyste-ine was observed [71] However, inhibition of NF-jB activation by N-acetyl-l-cysteine has been shown to occur not through ROS-dependent mechanisms, but rather through inhibition of tumour necrosis factor-stimulated signal transduction by lowering tumour necrosis factor receptor affinity [72,73] or through inhi-bition of its DNA-binding activity [74] Therefore, involvement of ROS in NF-jB activation under inter-mittent hypoxia cannot be completely excluded It has
to be noted that NF-jB activation by ROS is extre-mely cell type-dependent Beyond the question of the regulation mechanisms of NF-jB, its activation under intermittent hypoxia remains a critical point, because NF-jB plays an important role in tumour development through its ability to induce the transcription of genes coding for apoptosis inhibitor factors (cIAPs, Bcl-XL, FLICE), proproliferation molecules (interleukin-2, G1 cyclins), proangiogenic factors (VEGF, interleukin-8), and enzymes that lead to extracellular matrix degra-dation (matrix metalloproteases) [75–78] In addition, NF-jB activation was reported as an early event in malignant transformation in vitro [79], and continuous activation of NF-jB was also shown in many kinds of solid tumours [80] Conversely, NF-jB inhibition impairs tumour development NF-jB inhibition in prostate cancer in mice led to a marked reduction in the growth of tumour, demonstrating again the impor-tant role played by this transcription factor in tumour development [80]
HIF-1 activation in tumours is often associated with
a poor prognosis It allows the tumour cells to survive
in the absence of oxygen, regulating their cell metabo-lism and inducing the production of prosurvival mole-cules, but also inducing the formation of new blood vessels, favouring metastasis [81] HIF-1 activation is always associated with hypoxia, but it was shown that the production of ROS under normoxia was also able
to stabilize HIF-1a subunit and contribute to HIF-1 activation [82] The production of ROS during the
Trang 6reoxygenation periods under intermittent hypoxia
could then also influence HIF-1 activity However,
HIF-1a subunit degradation is always observed during
reoxygenation after incubation under chronic or
tran-sient hypoxia Therefore, HIF-1 activation during
reoxygenation after a hypoxia period should be
impaired in this case Paradoxically, it was shown that
reoxygenation could stimulate HIF-1 signalling
Increases in the translation of HIF-1 target genes and
HRE–green fluorescent protein construction transcripts
were observed after reoxygenation, despite the
com-plete degradation of HIF-1a [83,84] This peculiar
observation was explained by Moeller et al., who
showed that reoxygenation could enhance downstream
HIF-1 signalling by depolymerizing stress granules
They showed that a pool of HIF-1-regulated
tran-scripts were kept untranslated in the course of hypoxia
in stress granules that were depolymerized during
reox-ygenation, allowing the rapid translation of
seques-trated transcripts under normoxia [83] Interestingly,
Moeller et al also observed stress granule formation in
tumour cells under hypoxia as well as their
degrada-tion during reoxygenadegrada-tion Hence, they suggested that
this post-transcriptional regulation process could help
cancer cells to recover from a hypoxic shock and
pre-pare the cells for a future insult This mechanism, the
regulation of which could involve ROS, as suggested
again by Moeller et al., could also explain, at least in
part, the cancer cell resistance to anticancer treatment
observed under intermittent hypoxia It would be
inter-esting to investigate the involvement of stress granules
in the gradual increase in the abundance of HIF-1a
observed after each hypoxia step in the course of
hypoxia–reoxygenation cycles
Effects of intermittent hypoxia on
tumour vasculature
Tumour blood vessel formation is essential for tumour
development As well as comprising a tumour cell
dis-semination pathway in the body, tumour blood vessels
supply to cancer cells the oxygen and nutrients
essen-tial for their survival and proliferation In the absence
of angiogenesis and new blood vessel formation,
tumour growth is restricted, and the tumour size
remains ‘microscopic’, generally not increasing beyond
0.5 mm, even in the case of a highly proliferative
tumour, in which cell division is balanced by cell
apop-tosis induced by unfavourable survival conditions [85–
88] In these circumstances, in situ tumours can remain
dormant ‘indefinitely’ in the absence of angiogenesis
[89] Indeed, antiangiogenic treatments were shown to
be able to impair or slow down tumour development
and to reduce the volume of some solid tumours [90– 92] One of the main targets of these treatments com-prises the endothelial cells One endothelial cell can control the survival of approximately 50–100 tumour cells [93] Therefore, the destruction of a few endothe-lial cells may induce the death of a large number of tumour cells Moreover, it was shown that endothelial cell suppression could also mediate apoptosis in drug-resistant tumour cells [94,95] The role played by the tumour vascular network is thus critical in the devel-opment of a tumour, and therefore the effects of the tumour microenvironment on the cells constituting this network, i.e the endothelial cells, must also be consid-ered Indeed, the tumour environment induces faster endothelial cell proliferation than in normal tissue, and the turnover of endothelial cells in tumours was estimated to be 20–2000 times faster [96] Moreover, significant differences have been shown in the tran-scriptome of tumour endothelial cells in comparison to endothelium in surrounding normal tissue [97–99] In addition, tumour cells can favour endothelial cell survival within tumours by the production of VEGF, and particularly after irradiation [100] As described previously in this review, intermittent hypoxia influ-ences tumour cell behaviour Transient hypoxia also affects endothelial cells It was shown in vivo that intermittent hypoxia had a proangiogenic effect An increase in capillary density in mouse brains was observed after the repetition of cycles of 4 min of hypoxia followed by 4 min of reoxygenation for
2 weeks [101] Moreover, in vitro, an increase in endo-thelial cell migration and formation of tubes was also reported under intermittent hypoxia [49] Therefore, transient hypoxia could increase angiogenic processes also in tumours Furthermore, it was observed that endothelial cells become, like tumour cells, radio-resistant after an intermittent hypoxia preconditioning
In vitro, an increase in the survival of endothelial cells was observed after irradiation (2 Gy) when intermit-tent hypoxia preconditioning was performed [49] This protective effect of intermittent hypoxia against radiotherapy on endothelial cells was shown to be HIF-1-dependent Indeed, a decrease in endothelial cell survival after a low level of irradiation (2 Gy) on cells previously incubated under intermittent hypoxia was shown when HIF-1a was silenced by small interfering RNA [49] Endothelial cell radioprotection through the repetition of hypoxia–reoxygenation cycles was also observed in vivo Terminal dUTP nick-end labelling assays showed a decrease in the number of apoptotic cells in the vasculature of transplantable liver tumour borne by mice when rodents were submitted to three cycles of 1 h of hypoxia followed by 30 min of
Trang 7reoxygenation before tumour irradiation (10 Gy) [49].
Interestingly, chronic hypoxia incubation before the
irradiation did not protect endothelial cells against
apoptosis: in contrast to intermittent hypoxia, it even
drastically increased cell apoptosis [49] Moeller et al
also showed in vivo a radioprotective effect of
hypoxia⁄ reoxygenation in endothelial cells after
irradi-ation [83] They suggested that this radioprotection
was induced by the secretion of endothelial
cell-radio-protective cytokines by tumour cells after
reoxy-genation They showed that tumour cell-conditioned medium recovered after incubation under hypoxia fol-lowed by reoxygenation was more radioprotective for endothelial cells than conditioned medium from tumour cells incubated under normoxia, normoxia with radiation, or hypoxia without reoxygenation [83] Moreover, it was shown that this endothelial cell radioprotection mediated by tumour cells after hypoxia–reoxygenation was also HIF-1-dependent Indeed, no significant endothelial cell radioprotective
Fig 2 Schematic representation of the
effects of intermittent hypoxia on cancer
cells and endothelial cells within a tumour.
Fig 3 Schematic representation of the
effects of HIF-1 activation under intermittent
hypoxia on cancer cells and endothelial cells
within a tumour.
Trang 8effect was observed when conditioned medium was
taken from HIF-1-incompetent tumour cells [83]
Therefore, these results suggest that intermittent
hypoxia protects endothelial cells in a direct manner
by acting directly on the endothelial cell phenotype, as
observed by Martinive et al in vitro [49], and also by
indirect pathways involving secreted molecules released
from tumour cells, as suggested by Moeller et al [83]
Conclusion
Until now, most attention has been paid to chronic
hypoxia However, during the last few years, a new
concept has arisen, showing first that changes in po2
level are not always sustained in tumours but that they
can be transient, and second that intermittent hypoxia
can exert effects that are different from those induced
by chronic hypoxia Both tumour cells and endothelial
cells are affected by intermittent hypoxia, which can be
perceived as the consequence of different stresses
resulting from repeated combinations of hypoxia and
reoxygenation periods, which may induce different cell
responses In contrast, chronic hypoxia causes a
pro-longed and unique modification of the cell
environ-ment Figures 2 and 3 schematically summarize the
effects of intermittent hypoxia The major conclusion
drawn from these observations is the intricate interplay
between tumour cells and endothelial cells, each
favouring the survival of the other This delicate ballet
has to be understood in detail in order to allow the
design of new therapies targeting these processes
Acknowledgements
Se´bastien Toffoli is recipient of a FNRS-Te´le´vie grant
Carine Michiels is research director of FNRS (Fonds
National de la Recherche Scientifique, Belgium) This
article presents results of the Belgian Programme on
Interuniversity Poles of Attraction initiated by the
Belgian State, Prime Minister’s Office, Science Policy
Programming The responsibility is assumed by its
authors
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