Apoptosis in radiation therapy: a double-edged sword
Radiation therapy achieves its therapeutic effects by inducing apoptosis and non-apoptotic cell death. The aim of this focused review is to highlight the aspects of the cell death pathways most relevant to conventional fractionated radiation therapy. I review reports on how our current understanding...
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Balcer-Kubiczek, E.K. 2018-06-19T18:44:40Z 2018-06-19T18:44:40Z 2012 Apoptosis in radiation therapy: a double-edged sword / E.K. Balcer-Kubiczek // Experimental Oncology. — 2012. — Т. 34, № 3. — С. 277-285. — Бібліогр.: 124 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/139044 Radiation therapy achieves its therapeutic effects by inducing apoptosis and non-apoptotic cell death. The aim of this focused review is to highlight the aspects of the cell death pathways most relevant to conventional fractionated radiation therapy. I review reports on how our current understanding of the molecular mechanisms of cell death may enable us to revise the four radiobiological principles (reoxygenation, repair of sublethal damage, redistribution of cells in the cell cycle, and repopulation of surviving cells) for radiation treatment with fractionated dose delivery. Apoptosis and non-apoptotic forms of cell death are not represented in the linear quadratic model, which is clinically used to calculate the effects of different total doses, dose per fraction and fraction number on reproductive cell death, a mode of cell death associated with lethal chromosome aberrations. Examples are provided to justify or not a reassessment of the role of apoptosis and non-apoptotic cell death in radiosensitivity, tumor cell proliferation and tumor microenvironment. As our understanding of apoptosis developed at the molecular level, so did our understanding of other forms of cell death, particularly autophagy and to a lesser extent, senescence. The linear quadratic model remains a guide for the treatment planner. The therapeutic clinical roles of apoptosis and non-apoptotic forms of cell death remain to be defined. Their relative importance will probably lie in tumor developmental history related to its type, size and stage. Radiobiological research should focus on the quantitative effects of dose and fractionation on the radiation induction of apoptotic and non-apoptotic types of cell death and the interplay among cell death pathways. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later”. I thank my colleagues, Dr. George H. Harrison and Dr. Juong Rhee, in the Department of Radiation Oncology, University of Maryland School of Medicine for reading the manuscript and providing valuable comments. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Reviews Apoptosis in radiation therapy: a double-edged sword Article published earlier |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Radiation therapy achieves its therapeutic effects by inducing apoptosis and non-apoptotic cell death. The aim of this focused review is to highlight the aspects of the cell death pathways most relevant to conventional fractionated radiation therapy. I review reports on how our current understanding of the molecular mechanisms of cell death may enable us to revise the four radiobiological principles (reoxygenation, repair of sublethal damage, redistribution of cells in the cell cycle, and repopulation of surviving cells) for radiation treatment with fractionated dose delivery. Apoptosis and non-apoptotic forms of cell death are not represented in the linear quadratic model, which is clinically used to calculate the effects of different total doses, dose per fraction and fraction number on reproductive cell death, a mode of cell death associated with lethal chromosome aberrations. Examples are provided to justify or not a reassessment of the role of apoptosis and non-apoptotic cell death in radiosensitivity, tumor cell proliferation and tumor microenvironment. As our understanding of apoptosis developed at the molecular level, so did our understanding of other forms of cell death, particularly autophagy and to a lesser extent, senescence. The linear quadratic model remains a guide for the treatment planner. The therapeutic clinical roles of apoptosis and non-apoptotic forms of cell death remain to be defined. Their relative importance will probably lie in tumor developmental history related to its type, size and stage. Radiobiological research should focus on the quantitative effects of dose and fractionation on the radiation induction of apoptotic and non-apoptotic types of cell death and the interplay among cell death pathways. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later”.
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Apoptosis in radiation therapy: a double-edged sword / E.K. Balcer-Kubiczek // Experimental Oncology. — 2012. — Т. 34, № 3. — С. 277-285. — Бібліогр.: 124 назв. — англ. |
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Experimental Oncology 34, 277–285, 2012 (September) 277
APOPTOSIS IN RADIATION THERAPY: A DOUBLE-EDGED SWORD
E.K. Balcer-Kubiczek
University of Maryland School of Medicine, Marlene and Stewart Greenebaum Cancer Center
Department of Radiation Oncology, Baltimore MD 21201, USA
Radiation therapy achieves its therapeutic effects by inducing apoptosis and non-apoptotic cell death. The aim of this focused review
is to highlight the aspects of the cell death pathways most relevant to conventional fractionated radiation therapy. I review reports
on how our current understanding of the molecular mechanisms of cell death may enable us to revise the four radiobiological principles
(reoxygenation, repair of sublethal damage, redistribution of cells in the cell cycle, and repopulation of surviving cells) for radiation
treatment with fractionated dose delivery. Apoptosis and non-apoptotic forms of cell death are not represented in the linear qua-
dratic model, which is clinically used to calculate the effects of different total doses, dose per fraction and fraction number on repro-
ductive cell death, a mode of cell death associated with lethal chromosome aberrations. Examples are provided to justify or not a reas-
sessment of the role of apoptosis and non-apoptotic cell death in radiosensitivity, tumor cell proliferation and tumor microenvironment.
As our understanding of apoptosis developed at the molecular level, so did our understanding of other forms of cell death, particu-
larly autophagy and to a lesser extent, senescence. The linear quadratic model remains a guide for the treatment planner. The thera-
peutic clinical roles of apoptosis and non-apoptotic forms of cell death remain to be defined. Their relative importance will probably
lie in tumor developmental history related to its type, size and stage. Radiobiological research should focus on the quantitative effects
of dose and fractionation on the radiation induction of apoptotic and non-apoptotic types of cell death and the interplay among cell
death pathways. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later”.
Key Words: apoptosis, non-apoptotic cell death, radiation therapy, tumor response, normal tissue response.
INTRODUCTION
Ionizing radiation has been used clinically for the
treatment of a wide range of human cancers for more
than 100 years [1]. Radiotherapy reduces the risk
of cancer recurrence, promotes tumor control, and
improves survival [2]. At least 50% of all cancer pa-
tients will receive radiotherapy at some stage during
the course of their illness. It is currently used to treat
localized solid tumors, such as cancers of the lung,
colon/rectum, larynx, thyroid, brain/CNS, breast,
prostate, or cervix, and can also be used to treat leu-
kemia and lymphoma [2, 3]. The aim of radiotherapy
is to destroy cancer cells with ionizing radiation while
limiting the damage to nearby healthy tissue. This
aim has been facilitated by innovations in technology
and engineering, followed by the computer revolution
applied to treatment planning, and the recent develop-
ment of sophisticated irradiation techniques, including
proton and intensity-modulated radiotherapy [4].
Current practice of fractionated radiotherapy,
where tumors are irradiated multiple times, usually 30,
with small doses per fraction, usually 1.8–2 Gy, over
several weeks has its roots in decades of clinical obser-
vations and radiobiological research. Collectively, the
data indicate that the biologic effects of a given dose
depend strongly on the details of how this dose is de-
livered over time. Fractionation of the radiation dose
produces, in most cases, better tumor control while
reducing the level of normal tissue damage compared
to a single dose. The underlying interpretation has
been encapsulated in the four Rs of radiation therapy:
repair of radiation-induced damage between fractions,
redistribution of cells in the cell cycle, repopulation
of the tumor during the treatment period by surviving
tumor cells and reoxygenation of hypoxic cells [5].
A fifth R, radiosensitivity expressing a genetic charac-
teristic of cells, has been proposed as a major factor
determining radiotherapy individual outcome [6].
There are at least eight forms of cell death that may
account for cell killing both normal and tumor tissues
[7, 8]. Of those, cell death modalities most relevant
to this discussion are apoptosis [9], autophagy [10]
(a history of autophagy reviewed in ref. [11]) and the
loss of clonogenic survival [12]. Both apoptosis and
autophagy, and their crosstalk are important in under-
standing clonogenic radiosensitivity in vitro and in vivo
[13, 17–19]. The loss of clonogenic survival is the form
of cell death quantified by the clonogenic assay, the
experimental technique for assessing the fraction
of cells dying (or more precisely, of cells surviving) [7,
12]. In the context of radiation biology/oncology cell
death is equated with any process that leads to the loss
of the proliferative capacity of the cells. Cells that are
able to form colonies from a single cell are considered
to have survived the treatment. The clonogenic in vi-
tro data generally, but not always, agree with tumor
response in vivo [20–22]. Irradiated cells may also
die by mitotic catastrophe, senescence and necrosis
[8]. Mitotic catastrophe might not a bona fide form
Received: June 8, 2012.
Correspondence: Fax: 410-706-6666
E-mail: ekubicze@umaryland.edu
Abbreviations used: GFP — green fluorescent protein; HIF-1 —
hypoxia-inducible factor-1; LC3 — light chain 3 of the microtubule-
associated protein 1; a mammalian homolog of autophagy-related
protein 8 (Atg8); LQ — the linear quadratic model or formula;
NCCD — Nomenclature Committee on Cell Death; PARP-1 —
poly(adenine diphosphate-ribose) polymerase-1; TFF1 — trefoil
factor 1 (pS2); TFF3 — trefoil factor 3 (ITF); TGF β — transforming
growth factor β; TUNNEL — terminal deoxynucleotidyl transferase-
mediated deoxyuridine triphosphate (dUTP) nick end labeling.
Exp Oncol 2012
34, 3, 277–285
INVITED REVIEW
278 Experimental Oncology 34, 277–285, 2012 (September)
of cell death, because cells that experience mitotic
catastrophe eventually die by apoptosis or senesce
[8, 23]. Senescent cells are considered to be dead
reproductively and usually not contributing to radiation
response. There is a renewed interest in stress-in-
duced senescence because of a possible relationship
between autophagy and senescence in treated tumor
cells and the involvement of these two death modali-
ties in tumor dormancy and disease recurrence [24]
(reviewed in ref. [25]). In contrast to off-target effects
of diffused cytotoxins, radiation-induced “bystander
death” is an unclassified and poorly understood type
of cell death, perhaps relevant to risk estimation at low
radiation dose levels but not effects of typical thera-
peutic dose levels [26].
Prior reviews dating back more than two decades
have addressed the role of apoptosis in determining
radiation response [13, 14, 27–31]. The purpose of this
review is present a few examples from currently ac-
tive research with tumors and tumor cells of primarily
non-hematological origin that justify a reassessment
of the role of apoptosis and non-apoptotic cell death
in radiation sensitivity, tumor cell proliferation and
tumor microenvironment.
RADIATION SURVIVAL CURVES AND
MECHANISMS OF CELL DEATH
Cell death following irradiation is the stochastic
effect or “chance effect” which a consequence of the
random, statistical nature of damage. Thus, the magni-
tude of an effect is independent of dose, but the prob-
ability of an effect increases with dose. This stochastic
nature of cell killing is easily inferred from the shape
of survival curves, that is, plots of the logarithm of the
percentage of surviving (clonogenic) cells as a func-
tion of the dose [12]. The initial portion of survival
curve in the low (therapeutic) dose range (<3–4 Gy)
can be conveniently described by the linear-quadratic
(LQ) formula, which enables radiation biologists and
treatment planners to calculate cell killing by different
total doses, size of dose fraction, dose rate and frac-
tion number [32].
Implicit in the LQ formula is the assumption that
radiation produces two different types of damage:
non-repairable damage described by the linear com-
ponent (~ dose) and repairable damage described
by the quadratic component (~ dose2). Non-repairable
damage is synonymous with intrinsic radiosensitiv-
ity, because the linear component is invariant with
respect to dose delivery variables, such as dose rate
or fractionation, but modifiable by genetic back-
ground. Well-known examples of genes involved
in genetic control of intrinsic radiosensitivity are genes
involved in cell-cycle progression and DNA repair
(http://sciencepark.mdanderson.org/ labs/wood/dna).
There have been attempts in the 1990s to correlate
the incidence of apoptosis with clonogenic cell survival
and to factor apoptosis in the LQ formula [14, 15, 27–
31]. Several authors proposed that radiation-induced
apoptosis contributes only to the linear component
of the LQ formula, that is, to intrinsic radiosensitivity
of a cell [33–37]. This was primarily based on obser-
vations that dose response curves for both apoptosis
incidence and non-repairable damage are a linear
function of the dose and that apoptosis incidence
is independent of how the dose was distributed in time.
It has been now recognized that the above-men-
tioned direct correlation between sensitivity to the
induction of apoptosis and loss of clonogenicity ex-
ists only in a limited number of tissues, including
thymocytes, spermatogonia, hair-follicle cells, stem
cells of the small intestine and bone marrow, and tis-
sues in developing embryos as well tumors arising
from these tissues [35, 36, 38–43]. In contrast, other
studies with solid tumor models in vitro and in vivo
generally failed to find an association between sensi-
tivity to apoptosis and sensitivity to therapeutic agents
including ionizing radiation. For results underlying this
conclusion in experimental settings, see refs. [13–15,
27, 28]. Similar results were obtained in clinical set-
tings. The majority of studies found no or negative
association between high apoptosis incidence and
survival and/or recurrence in cervical, bladder and
head and neck patients treated with radiotherapy [44]
(and references therein). In addition, a retrospective
study of 2739 colorectal cancer patients treated with
chemo- and radiotherapy showed no association
between apoptosis resistance and treatment failure.
However, high apoptosis in a subset of rectal patients
correlated less recurrences and/or survival [45] (and
references therein).
There have been several explanations of experi-
mental and clinical results described above. As pro-
posed by Brown and colleagues [30, 44], the time
period over which apoptosis occurs following irra-
diation could be different in different cell types. Cells
such as thymocytes, lymphocytes, lymphoblasts and
stem cells undergo apoptosis shortly after treatment
(peaking usually at 3–4 h post-irradiation) and prior
to the first division after treatment (also termed an “in-
terphase cell death”) [35, 38].In these cells apoptosis
incidence generally correlates with clonogenic cell
killing. In contrast, this early apoptosis does not occur
in epithelial cells and tumors of epithelial or mesenchy-
mal origin. In these cells, apoptosis occurs much later
and subsequent to mitosis (also termed a “postmitotic
cell death”) [28–31, 34, 39]. Late apoptosis does not
correlate with clonogenic cell killing and usually does
not occur at therapeutic dose levels (<3–4 Gy).
Although the genetic mechanisms of X-ray ac-
tion on cells were far from understood in the early
days of radiation research, scientists realized that the
clonogenic assay might not take into account all the
consequences of irradiation. For example, they noted
that the number of cells in the colonies produced
by surviving cells depends on the dose they received:
the larger dose, the larger proportion of small colo-
nies that frequently include morphologically-altered
cells such as giant and senescent cells [12, 46–53].
Hurwitz and Tolmach [50] and Thompson and Suit
Experimental Oncology 34, 277–285, 2012 (September) 279
[51] investigated the fate of irradiated cells of differ-
ent origins using simple imaging tools available in the
1960s. By this approach, they were able to record the
division history of specific, initially single cells; that is,
whether they divided between observations, produced
morphologically normal or altered daughter cells,
or disappeared (following their detaching from the
substratum). These observations furnished evidence
that even “killed” cells can carry out a limited number
of divisions and that the average number of divisions
of which non-surviving cells are capable before the on-
set of death varies with a dose and cell type. The elimi-
nation of “killed” cells from the colony was attributed
to lysis of detached and rounded cells. It is now known
that detachment from a solid substrate is one of early
morphological features of apoptosis and that apop-
totic cells are typically engulfed by surrounding cells,
and therefore disappear. The anchorage-dependent
mode of cell death by apoptosis induced by abnormal
detachment from the extracellular matrix is termed
anoikis (from a Greek word meaning “homelessness”)
[7, 8]. A well-designed study of early and late death
processes in irradiated cells [15] and real-time imag-
ing studies in the late 1990s using advanced imaging
technologies have refined and extended results on fate
of irradiated cells [54–56].
REPOPULATION AND MECHANISMS
OF CELL DEATH
Repopulation of tumors, during and after radia-
tion treatment, is considered one of the main reasons
for the failure of conventional fractionated radiation
therapy, because tumor cell division between frac-
tions may in part compensate for the cell death pro-
duced by each fraction [57–60]. The cell population
kinetics have been studied experimentally in several
animal models as well as be retrospectively by ana-
lyzing clinical data [57, 61–63 (reviewed in ref. [58]).
It is important to present examples of results from
these studies in order to place current research in the
proper context. Denekamp [63] and Withers et al.
[57] showed that tumor repopulation is not evident
at the beginning of the treatment and that the process
becomes clinically apparent 3–5 weeks after the start
of the treatment. This implies that for treatment times
shorter than 3–5 weeks tumor proliferation had little
effect. Following the lag phase, accelerated repopu-
lation takes place; the term “accelerated repopula-
tion” describes more rapid multiplication surviving
clonogens after irradiation than before [57, 61–64].
For treatments longer than 5 weeks, the effect of re-
population is eguivalent to a loss of tumor radiation
dose of 0.6–1.3 Gy/day [57, 63, 64].
Although accepted as a process, the mechanisms
behind accelerated repopulation and its onset are
topics still debated in the literature. One of the pos-
sible mechanisms responsible for tumor repopulation
is accelerated cancer stem cell division [65, 66]. The
cancer stem cell hypothesis proposed that a subset
of tumor cells is able to maintain and propagate tumor
[67–70]. The term “tumor stem cells” was first used
by Makino in 1959 [67] to describe rare tumor cells
that are more resistant to chemotherapy than the bulk
of tumor cells. The current view is that cancer stem
cells originate either from malignant transformation
of a normal somatic stem cell or a progenitor (non-
stem) cell [65, 66, 68–70]. The possibility of intercon-
version of tumor stem and non-stem cells and what are
key factors in influencing this plasticity are a matter
of debate [66, 69–73]. Mechanisms of accelerated
repopulation based on the cancer stem cell hypothesis
have been proposed by Dörr [74] and more recently
revisited by Marcu et al. [75] and Pajonk et al. [66]. The
latter study suggested that radiation damage might
recruit quiescent cancer stem cells into the proliferat-
ing pool [66]. Other likely mechanisms, named by Dörr
[74] “the three As of repopulation”, include accelera-
tion of stem cell division, abortive division and asym-
metrical loss in stem cell division. Accelerated stem
cell division implies a treatment-induced shortening
of the cancer stem cell cycle time. Marcu et al. [75]
modeled post-irradiation accelerated repopulation
assuming different cell cycle durations. The authors
concluded that accelerated cancer stem cell division
is the least likely mechanism responsible for tumor re-
population because it would require a shortening of the
cancer stem cell cycle to about 1 h, which is biologi-
cally implausible. The third hypothetical mechanism,
the loss of asymmetrical division (resulting in two stem
cells, instead of one stem and one differentiated cell)
remains untested.
An alternative mechanism of tumor repopulation
considers non-stem, senescent tumor cells [24, 25,
76]. Cellular senescence could be activated as a part
of an adaptive stress response [24, 76–78]. Recent
studies demonstrated that the pro-survival function
of autophagy (protective autophagy) is required for the
efficient execution of the stress-induced senescence
program [78–80]. Accordingly, protective autophagy
helps stressed tumor cell survive in a setting of in-
creased metabolic demands, mitigate damage and
promote recovery of normal functions; alternatively,
autophagy helps achieve cellular remodeling asso-
ciated with senescence by degradation of specific
cellular components [78, 81]. Independent regulation
of apoptosis and autophagy observed in some cellular
settings; in this scenario, inhibiting one death pathway
results in activating expressing the other pathway [11,
16, 17]. Crosstalk between the two death pathways was
also reported; under this alternative scenario, apop-
tosis depends on prior autophagy [79, 80]. Gewirtz
[25] described a model whereby the functional con-
sequences of protective autophagy and senescence
depend on the nature and quantity of the cellular dam-
age. When the damage is extensive such as following
a large single dose of radiation (e.g. 20 Gy), autophagy
and senescence might be insufficient to maintain cells
in a protective state and the majority of irradiated cells
die. In contrast, when irradiation is delivered over time,
such as during fractionated therapy (typically 6 weeks),
280 Experimental Oncology 34, 277–285, 2012 (September)
the cells experience progressive but moderate radia-
tion damage after each fraction (about 2 Gy). There
are several reports showing that during intra-fraction
intervals (typically 24 h), cells are not able to repair
completely DNA damage before the application of the
next radiation dose induces new DNA damage [13, 15,
82–84]. In two studies [82, 83], accumulation of DNA
double strand breaks did not trigger apoptosis in vivo
and in vitro and diverted a fraction of cells into cell cycle
exit [82]. The authors hypothesized that a growth arrest
phenotype may precede senescence [82].
From the standpoint of radiation therapy concerns,
a limitation of the studies by Řezáčová et al. [82] and
Rűbe et al. [83] are that the only times points examined
were during fractionated irradiation (up to 5 d in both
studies). In contrast, Li et al. [85] studied DNA damage
over an extended period of up to 21 d post-irradiation;
the authors showed two distinct phases of DNA dou-
ble-strand break induction, an acute phase peaking
at 3–5 h during first 24-h post X-irradiation, and a post-
acute phase lasting peaking at 5 d during 1–21 d post
X-irradiation. In addition, they reported activation
of both apoptotic and non-apoptotic pathways in sur-
vivors during the second wave of DNA double-strand
break induction [85]. These results are consistent with
observations from several laboratories [13–17, 22, 23,
41, 46, 47, 51–53], collectively termed “lethal sector-
ing” [86], which describes the induction of protective
and death subroutines in individual survivors. There
is no direct evidence that the impairment of autophagy
facilitates escape from senescence and reentry of cells
to the cell cycle [25]. However, it must be noted that
the detailed analysis of patterns of growth of irradiated
experimental tumors led Frindel et al. [62] to suggest
that a proportion of cells exhibiting a growth arrest phe-
notype “are in a reversible state and can be stimulated
to re-enter division”.
An alternative model has been proposed by Meyn
and colleagues [87]. The authors evaluated single
dose- and fractionation protocols in experimental
tumors and showed that compared to a single dose,
fractionated radiation is a more efficient inducer
of apoptosis; in fact, a proportion of apoptotic cells
was directly correlated with the number of fraction and
inversely correlated with tumor growth rates in each
radiation protocol. In addition, the authors concluded
that the balance between apoptotic death and cell divi-
sion of survivors after each dose fraction might result
in the lag period before the onset of repopulation.
A provocative study by Huang and colleagues [88]
provided yet another mechanism of tumor repopula-
tion. They reported that under radiation therapy, dy-
ing cells in the tumor mass support the proliferation
of other live tumor cells. This work demonstrated that
the activation of a key player in apoptotic cell death,
caspase-3, in damaged cells is responsible for synthe-
sis and efflux of prostaglandin E2 .How prostaglandin
E2 stimulate the growth of tumor cells is controversial,
because as recently noted both extracellular and in-
tracellular prostaglandin E2 participates in a receptor-
or Bax-mediated apoptotic death, respectively [89].
Connell and Weichselbaum [90] and Lauber et al. [91]
critically addressed the relevance of work by Huang
et al. [88] to radiation therapy. Just to highlight one
point, Huang et al. used one or two large X-ray doses
(6–12 Gy) in their experiments [88]. These doses are
in the range of doses only used in specialized radiation
procedures (for example, proton therapy or stereo-
tactic body radiotherapy) that employ 1–5 fractions
delivered over a short period, at most 2 weeks [92].
Because compensatory repopulation starts 3–4 weeks
after initiation of radiation therapy, repopulation is not
a factor in such types of radiation treatment. In ad-
dition, it has been shown that apoptosis-inducing
drugs (for example, taxanes or PARP-inhibitors) given
prior to radiation therapy significantly reduce tumor
growth and volume, compared to radiation therapy
alone [23, 93, 94], whereas the opposite effect would
be expected based on the study by Huang et al. [88].
MICROENVIRONMENT AND MECHANISMS
OF CELL DEATH
A solid tumor is a complex system composed
of a mass of proliferating tumor cells, a blood vessel
network, lymphatic vessels, and a variety of non-
tumor cells and molecules all of which contribute
to the local microenvironment. The importance of the
tumor-specific milieu was recognized more than
120 years ago by Paget who described the concept
of “seed and soil” to explain site-specific metastatic
dissemination [95]; he concluded that “although the
best work in pathology of cancer is done by those who
are studying the nature of the seed” (cancer cell), the
“observations of the properties of the soil” (optimal
milieu for tumor growth) “may also be useful” [95].
It has been recognized for more than 40 years that
interactions between the tumor cell and components
of its microenvironment shape and determine the
malignancy phenotype. However, how this complex
and intertwined tumor system responds to radiation
therapy is still poorly understood.
The underlying differences between the physiol-
ogy of normal and tumor tissues stem from the tumor
vasculature [96]. Structurally, tumor vessels are often
dilated and leaky. A heterogeneous zonal variability
of blood supply within a tumor correlates spatially
with metabolic activity and oxygen supply [97]. It has
been recently proposed that the tumor vasculature
can arise from proliferation of endothelial cells from
local, pre-existing vessels (angiogenesis) or by colo-
nization of circulating endothelial and other specific
pro-angiogenic cells, mainly myeloid bone marrow-
derived cells (vasculogenesis) [98]. Which of the two
mechanisms prevails in radiation therapy is a topic still
debated in the literature [65, 66, 96–98]. While a more
comprehensive discussion of mechanisms of tumor
vascularization in naпve and radiation treated tumors
is outside the scope of this review, it needs to be men-
tioned that Kozin et al. [99] recently reviewed single-
dose effects (12–50 Gy) on a population of endothelial
Experimental Oncology 34, 277–285, 2012 (September) 281
cells and blood perfusion in preclinical models [99];
the authors concluded that a body of experimental evi-
dence supports endothelial cell-based angiogenesis
rather than an alternative mechanism of vasculogen-
esis proposed by Kioi et al. [98].
One group of investigators proposed that the
response of tumors to irradiation is affected by the
sensitivity of tumor endothelial cells [100, 101]. Gar-
cia-Barros et al. reported that the tumor-associated
endothelial cells undergo massive and rapid ceramide-
mediated apoptosis within few hours after irradiation
leading to indirect tumor cell death. There is no in-
dependent confirmation of these results, as noted
by Kozin et al. [99]. Indeed, numerous other studies
reported negligible radiation effects on vessel struc-
ture and function during a few weeks post-irradiation
(see Table I in ref. [99] and references therein). Ogawa
et al. [102] attributed findings of Garcia-Barros et al.
to unusual tumor-host relationships in the tumor model
they used in [100, 101]. However, note that because
of the short experimental time frame, apoptosis of tu-
mor cells that would have occurred at later time points
cannot be ruled out. As discussed, apoptosis is not
a major contributor of long-term tumor response post-
irradiation. An obvious alternative mechanism, not
considered by Garcia-Barros et al. [100, 101], is direct
tumor cell killing by radiation; this can be assessed
using the conventional in vivo clonogenic assay.
One of consequences of disorganized architecture
of tumor vessels is a heterogeneous variation of oxy-
gen within the cell mass ranging from and hypoxic
(<0.5% to 1.5% O2) to normoxic (>1.5% O2) with me-
dian values much lower than normal. The histological
studies of human bronchial carcinoma by Thomlinson
and Gray were among the first to provide a mechanism
for spatially heterogeneous distribution of oxygen
concentration in tumors [103]. They postulated that
because of their rapid growth, tumor cells are progres-
sively pushed away from vessels beyond the effective
diffusion distance (of about 150 μm) thus become hy-
poxic and eventually necrotic. With minor refinements,
this basic mechanism has been validated in cancers
of other organs, including the ovary, esophagus, and
head and neck [104]. Glucose and nutrient distribu-
tions are thought to follow similar patterns to that
of oxygen. Consequently, the viable regions of tumor
are characterized by variability of oxygen and glucose
content in space. In addition, the efficient efflux of hy-
drogen ions from tumor cells combined with inefficient
buffering capacity of tumor interstitial fluid generates
extracellular acidosis.
Hypoxia is detrimental to successful radiation ther-
apy because hypoxic cells are typically 2.5 to 3 times
radioresistant than normoxic cells (as measured
by the clonogenic assay) [104]. Hypoxia is detrimental
to chemotherapy because anticancer drugs might not
reach the target cells distant from blood vessels [105]
and because hypoxia up-regulates genes involved
in multidrug resistance [106]. Finally, hypoxia com-
promises curability by cancer surgery, because the
low oxygen environment promotes survival of tumor
cells with a more aggressive phenotype, including
diminished pro-death mechanisms (for example,
apoptosis), enhanced pro-survival mechanisms (for
example, switching aerobic to anaerobic energy
production or activating protective autophagy and/
or senescence) [104–106]. Finally, hypoxia results
in a limited response to the presence of cancer cells
by the immune system. Thus, hypoxia in solid tumors
has a negative impact on the ability of current cancer
treatment modalities to control solid tumors.
Landmark studies in the early 1990s demonstrated
the operation of a specific oxygen-sensing process
controlled by hypoxia-inducible factor-1 (HIF-1)
in tumor cells [107]. As a transcription factor, HIF-1 up-
regulates more than more than 100 genes coding
proteins essential for glucose and iron metabolism,
mobility, proliferation, cell survival, immune surveil-
lance, angiogenesis and drug resistance [107].
Together, the consequences of HIF-1 are directed
toward maintaining energy production and survival
of the tumor in a hostile microenvironment.
Modulation of radiation-induced death pathways
by factors associated with the tumor microenvironment
(hypoxia, energy depletion and acidosis) is far from
completely understood. Several lines of evidence in-
dicate that apoptosis and autophagy co-exist in tumor
cells and can be activated as independent pathways,
but they are also interconnected processes. For ex-
ample, both irradiation and hypoxia up-regulate au-
tophagic death and inhibit apoptotic death. However,
contributions of these processes to the overall survival
depend on the relative magnitude of cellular stresses
as well as the cellular context [108–110].
Finally, there is increasing evidence that radio-
therapy leads to significant alterations in the tumor
microenvironment through the induction of soluble
signals (including regulatory proteins, growth fac-
tors, cytokines and chemokines) [111, 112].The
most significant among them are survival-regulatory
proteins including epidermal growth factor [112],
pro-inflammatory cytokines [113], fibroblastic growth
factor [114], transforming growth factors α (TGF-α)
and β (TGF-β) [79] and trefoil factors 1 (TFF1 and
3 (TFF3) [115]. Unlike fibroblast growth factor, and
epithelial growth factors and TGF-α , which are early
radiation-induced events, activation of TGF-β in tumor
cells is a biphasic event with the second wave of the
induction beginning 1 d post-irradiation and persisting
for up 2–3 d post-irradiation [116]; the late induction
phase may be associated the irradiation-induced
oxidative stress [111]. The late extracellular induction
of TGF-β has a tissue-wide, broad spectrum of cellular
consequences including growth arrest, differentiation,
migration, invasion, angiogenesis, evasion of the im-
mune system, and apoptosis [117]. In addition, the
TGF-β induction in fractionated radiation may lead
to de novo interactions between microenvironmental
factors and tumor cells, and between different micro-
environment factors with each dose delivery, thus per-
282 Experimental Oncology 34, 277–285, 2012 (September)
petuating its bioactivity during radiation therapy [111].
Trefoil factors, TFF1 and TFF3, represent a distinct
class of tumor suppressor genes, whose downstream
functions in irradiated cells remain yet to be elucidated.
However, recent studies shed some light on the nature
of the cellular and molecular events targeted by TFF
signaling [118–122]. Together, these results indicate
that the secreted TFF1 and TFF3 proteins have anti-
apoptotic, anti-inflammatory and, paradoxically, anti-
proliferative effects on the tumor and its microenviron-
ment. Whether and to what degree, the action of TFF
proteins might counterbalance the effects of growth
factors and other soluble proteins remains unknown.
However, clinical relevance of TFF1 and TFF3 to radia-
tion therapy can be established based on two effects.
First, both genes are activated in a p53-independent
fashion [123]; p53 is the most frequently mutated gene
in human cancers [124]. Secondly, both genes display
the unique coordinate, delayed and persistent expres-
sion pattern in irradiated cells [115, 121, 123]. Thus,
trefoil factors might exert long-lasting protective ef-
fects on normal tissues outside the radiation treatment
volume; examples include the salivary gland, heart,
lung, colon, small intestine and prostate, because
these normal tissues are unavoidably irradiated in the
course radiotherapy of head and neck, breast, lung
and prostate cancers.
CONCLUDING THOUGHTS
Despite the enormous importance of the discovery
of molecularly controlled death pathways, the con-
tribution of apoptosis, autophagy and senescence
to radiation induced cell death as measured long-term
in solid tumors (by clonogenic assays in vitro and
in vivo) remains unclear.
One reason might be the frequent use of the
apoptosis-necrosis paradigm or, more recently, the au-
tophagy-senescence-necrosis paradigm to describe
total cell killing death following irradiation. As noted
in this review and previously by others (notably by Steel
[27], Brown and Attardi [30]) most of such studies as-
sessed radiation apoptotic and non-apoptotic effects
at an early fixed time after a single large dose (usually
~10 Gy). Thus, future radiobiological research should
focus on the quantitative (rather than qualitative) ef-
fects of dose, fractionation and time on the induction
of apoptotic and non-apoptotic types of cell death.
At present, the published data are too fragmentary
even to conclude whether or not there is a dose thresh-
old for the induction of different modes of cell death.
The second observation is the often-imprecise and
confusing classification of cell death in the literature. For
example, the term “apoptosis” is frequently misapplied
in the context of cell death by radiation. The Nomencla-
ture Committee on Cell Death (NCCD) published the
guidelines in 2008 and 2012 on the use of cell death
terminology [7, 8], but those are usually not followed
[125]. The NCCD reports emphasized the importance
of the biochemical features rather than the common-
place reliance on morphological features. As discussed
by Bucur et al. [125], the same techniques used to de-
tect apoptosis can also detect necrosis (examples in-
clude microscopic observations of DNA fragmentation,
TUNEL and Annexin V staining). Popular autophagy
detection methods that rely on solely the redistribution
of GFP-LC3 fusion proteins into vesicular structures are
not considered sufficient for diagnosis [8].
Thirdly, because of tumor heterogeneity in a single
patient and phenotypic variations among patients
undergoing radiation therapy for the same clini-
cally defined disease, it would be important to assess
whether and how the different death types within the
tumor (and among patients) might evolve in the course
of treatment. Radiation affects multiple facets of tumor
cell physiology. Consequently, it could be expected
that different cell death mechanisms are not mutually
exclusive but rather operate in side-by-side or, con-
versely, overlap albeit to a variable degree and several
characteristics might be displayed at the same time and
most likely in a dose-dependent manner. The crosstalk
between pro-survival and pro-death pathways and the
activation of yet unknown backup pathways add to the
complexity of how the cell eventually dies.
ACKNOWLEDGMENT
I thank my colleagues, Dr. George H. Harrison
and Dr. Juong Rhee, in the Department of Radiation
Oncology, University of Maryland School of Medicine
for reading the manuscript and providing valuable
comments.
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