Apoptosis
DOI 10.1007/s10495-015-1168-3
ORIGINAL PAPER
Hyperthermia: an effective strategy to induce apoptosis in cancer
cells
Kanwal Ahmed1 • Yoshiaki Tabuchi2 • Takashi Kondo1
 Springer Science+Business Media New York 2015
Abstract Heat has been used as a medicinal and healing
modality throughout human history. The combination of
hyperthermia (HT) with radiation and anticancer agents has
been used clinically and has shown positive results to a
certain extent. However, the clinical results of HT treat-
ment alone have been only partially satisfactory. Cell death
following HT treatment is a function of both temperature
and treatment duration. HT induces cancer cell death
through apoptosis; the degree of apoptosis and the apop-
totic pathway vary in different cancer cell types. HT-in-
duced reactive oxygen species production are responsible
for apoptosis in various cell types. However, the underlying
mechanism of signal transduction and the genes related to
this process still need to be elucidated. In this review, we
summarize the molecular mechanism of apoptosis induced
by HT, enhancement of heat-induced apoptosis, and the
genetic network involved in HT-induced apoptosis.
Keywords Hyperthermia  Apoptosis  ROS  Genetic
network
& Takashi Kondo
kondot@med.u-toyama.ac.jp
1
Department of Radiological Sciences, Graduate School of
Medicine and Pharmaceutical Sciences, University of
Toyama, Toyama 930-0194, Japan
2 plays essential roles in the development and maintenance
Division of Molecular Genetic Research, Life Science
Research Center, University of Toyama, Toyama 930-0194,
Japan
Introduction
The idea of treating cancer cells using hyperthermia (HT)
dates back at least 5000 years. The main reason for the
relative lack of interest in HT in modern cancer research is
that the past technologies could not deliver effective and
homogeneous heating to all sites, particularly deep-seeded
tumors. Moreover, research and implementation have been
hampered by the lack of noninvasive temperature mea-
surement techniques. The recent development of new
techniques (e.g., nanotechnology, computer modeling, and
noninvasive thermometry) to control and directly apply
heat has stimulated interest in HT again. Generally, there is
no intrinsic difference in HT sensitivity between normal
and tumor cells [1]. Nevertheless, a selective in vivo tumor
killing effect is achieved at temperatures between 40 and
44 C, which is related to a characteristic difference in
physiology between normal and tumor cells. The archi-
tecture of the vasculature in solid tumors is complex and
includes hypoxic and low-pH regions [2–4] which are not
found in normal tissues under undisturbed conditions.
These environmental factors make tumor cells more sen-
sitive to HT. The effectiveness of HT treatment depends on
temperature and treatment duration [5]. Most normal tis-
sues are not damaged by treatment for 1 h at a temperature
of up to 44 C [6]. Only nerve tissues appear more sensi-
tive [7]. Promising results from recent clinical trials indi-
cate the effectiveness of HT treatment as an adjunct to
radiotherapy or chemotherapy in treating numerous cancers
[8].
HT is known to induce apoptosis in cancer cells.
Apoptosis is a tightly regulated cell suicide program that
of tissue homeostasis by eliminating unnecessary and
harmful cells [9]. Impairment of this native defense
123

mechanism promotes aberrant cellular proliferation and
accumulation of genetic defects, which ultimately result in
tumorigenesis [9]. The regulation of apoptosis at several
levels is essential for maintaining the delicate balance
between cell survival and death signaling required to pre-
vent disease [9]. Caspases have major and central roles in
the apoptotic mechanism. The term caspases is derived
from cysteine-dependent aspartate-specific proteases.
There are three pathways to the activation of caspases: the
intrinsic or mitochondrial pathway, the extrinsic or death
receptor pathway, and the recently described, but less
understood, intrinsic endoplasmic reticulum (ER) pathway
[10]. Although heat shock has been examined for decades,
studies are still in progress to determine the precise
mechanism of HT-induced apoptosis. In this review, we
outline the effects of HT at the cellular and molecular
levels.
Cellular changes induced by HT
HT induces many changes in cells. For example, an
increase in temperature causes protein unfolding and
aggregation. HT also alters the internal organization of
cells including disruption of the cytoskeleton, fragmenta-
tion of the Golgi system and ER, and a decrease in the
number of mitochondria and lysosomes [11]. HT also
affects nuclear processes and the cell membrane [12]. The
key targets of HT in cells are briefly discussed below.
Cytoskeletal alteration
The cytoskeleton, in its intact form, is responsible for
maintaining cell structure and anchoring many organelles.
The cytoskeleton participates in various activities including
cell movement, cell division, distribution of a number of
intracellular membranous organelles, macromolecule
transport, and regulation of protein synthesis [13]. Partic-
ularly in eukaryotes, one of the major impairments
observed in response to stress conditions are defects of the
cytoskeleton [12]. The effects of heat shock on the
cytoskeleton vary depending on the strength and duration
of the applied HT as well as biological factors such as cell
type [14]. The three major cytoskeletal systems are not
necessarily affected simultaneously during heat stress.
Sometimes one or two are affected while the remaining
system is not rearranged [15]. Following HT treatment, the
microtubules slightly retract from the cell periphery.
Cytoplasmic bundles of microfilaments are disassembled
with the accumulation of actin aggregates and the inter-
mediate filaments become concentrated and form a tight
ring around the nucleus [16]. Along with disruption of the
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Apoptosis
cytoskeleton, loss of the correct organelle localization and
breakdown of intracellular transport processes have also
been reported [12].
Cellular proteins
HT induces unfolding and aggregation of proteins due to
interactions of the exposed hydrophobic groups. Misfolded
and aggregated proteins not only lose their biological
activity but may also disturb protein homeostasis, damage
membranes, and induce apoptosis [17]. Heat shock proteins
(HSPs) are a family of major cell regulatory proteins. They
function as molecular chaperones in regulating cellular
homeostasis. HSPs prevent the formation of misfolded
protein structures both under normal conditions and during
exposure to stresses such as high temperature [18]. How-
ever, a massive stress can exhaust the protein folding
capacity of HSPs resulting in protein misfolding and
aggregation. This chaperone overload leads to gross dis-
turbances in cellular physiology [19] and may lead to cell
death [20].
Cell cycle
Synchronized cell cultures exhibit variations in their
susceptibility to heat in accordance with the phase of the
cell cycle. Phosphoinositide 3-kinase-related kinases
(PIKK) family members such as ataxia-telangiectasia
mutated (ATM) and ATM-and Rad 3-related (ATR)
kinases play critical roles in early signal transduction
through cell cycle check points [21]. ATM activates
checkpoint kinase 2 (Chk2) and ATR activates checkpoint
kinase 1(Chk1). Chk1 and Chk2 are serine/threonine
kinases that are important for cell-cycle checkpoint acti-
vation following induction of DNA damage [22]. One of
the earliest signs of heat stress is the rapid phosphoryla-
tion of ATR at serine 428 and Chk1 at serine 345 [22].
Heat-induced activation of ATR-Chk1 signaling arrests
cell cycle progression at the G2/M phase [22]. Heat pro-
duces chromosomal lesions that trigger cell death if they
are not removed before the onset of mitosis. The rapid
stimulation of ATR at temperatures above 40 C is an
indicator of DNA damage [19]. Although ATM activation
is delayed at temperatures above 40 C, the ATM-Chk2
pathway is mainly not involved in HT-induced apoptosis
[23]. Our study also revealed that the ATR-Chk1 pathway
rather than the ATM-Chk2 pathway plays a predominant
role in apoptosis inhibition under HT [22]. Inhibition of
the ATR-Chk1 pathway abrogates G2/M checkpoint
activation and promotes caspase-3 cleavage to induce
apoptosis under HT [22].
Apoptosis
DNA
Although the effect of HT on higher eukaryotes has been
extensively studied, little is known about its effects on
DNA integrity and replication. Reports in the literature of
HT-induced double strand breaks (DSBs) are controversial.
However, in 2012 it was reported that HT induces DSBs in
G1 and G2 cells that were marked by cH2AX. In contrast,
HT does not induce DSBs in S-phase cells but instead
arrests and decelerates the progression of the replication
fork in a temperature-dependent manner. This response
also induces the phosphorylation of H2AX at the sites of
replication. The formation of cH2AX at or close to repli-
cation forks prevents DNA strands from totally collapsing,
which would result in the formation of DSBs. H2AX might
be phosphorylated both in a DSB-dependent and DSB-in-
dependent manner. cH2AX formation is mediated by dif-
ferent PIKKs: ATM triggers the DSB-associated
phosphorylation, whereas DNA-dependent protein kinase
triggers the replication arrest/delay-associated phosphory-
lation [24].
Pro-apoptotic signal transduction induced by HT
The response of cells to HT depends on the magnitude and
duration of HT treatment. HT triggers several signaling
pathways, some facilitate cell survival including the
upregulation of HSPs, and some initiate the cell death
program. There is significant interest in understanding the
underlying mechanisms that mediate HT-induced apopto-
sis. Most data suggest that HT kills cells through activation
of the intrinsic apoptotic pathway. Caspase-2 has been
shown to play an important role in cell death induced by
microtubule disruption and heat stress [25–28]. Several
recent reports indicate that caspase-2 forms a complex with
its adaptor protein RAIDD immediately after HT treatment,
which in turn activates caspase-2 and cleaves Bid [25, 27,
28]. tBid then stimulates mitochondrial outer membrane
potential (MOMP), cytochrome c release, and formation of
an Apaf-1-caspase-9 apoptosome [25, 28]. It has also been
reported that heat itself induces conformational changes in
Bax and/or Bak that render them more susceptible to
activation by tBid [29]. Another study revealed that this
canonical pathway may not fully account for the cell death
induced by heat shock. In some instances, caspase-2 and
Bid are not essential for cell death or serve as an amplifi-
cation loop to promote MOMP and caspase activation
downstream of the apoptosome [30]. Recently, it has been
discovered that another BH3-only family member, Bim,
plays a significant role in mediating cell death indepen-
dently of the caspase-2-Bid pathway [31]. Bim induces
apoptosis following HT treatment through a Bax/Bak-de-
pendent pathway consistent with its established role as a
direct activator of Bax and Bak [32]. Whether Bim also
induces Bax/Bak-independent activation of caspase-3 in
the absence of MOMP remains unclear. As to how Bim is
activated following HT treatment, it is worth noting that
heat stress disrupts intermediary, actin, and tubulin net-
works [12] as well as Bim. Bim associates with the LC8
chain of the dynein motor complex and is liberated in
response to cytoskeletal damage [33]. Moreover, HT is a
strong activator of c-Jun N-terminal kinases (JNKs) [34,
35]. Phosphorylation of Bim by JNK enhances its
proapoptotic activity [35].
In addition to the intrinsic pathway, HT is also reported to
affect the extrinsic apoptotic pathway by activating cell
surface death receptors, which transmit apoptotic signals
initiated by specific ligands such as Fas ligands, tumor
necrosis factor-related apoptosis-inducing ligand (TRAIL)
and TNF-a. Stress-induced signaling was previously shown
to directly affect Fas-mediated cell death. Indeed, studies of
Ipr mouse lymphoid cells have revealed that both c-irradi-
ation-induced apoptosis and HT-induced apoptosis are par-
tially dependent on Fas signaling [36]. Signaling from Fas
can either directly activate downstream effector caspases or
trigger the mitochondrial apoptosis amplification loop. This
signaling pathway involves caspase-8-mediated activation
of the proapoptotic Bcl-2 family member Bid followed by
the release of cytochrome c, which binds to the apoptosome
complex formed by Apaf-1 and caspase-9 [37]. HT at 44 C
for 20 min was reported to increase the expression level of
Fas and to simultaneously activate caspase-8 and caspase-3
in U937 cells [38]. In Jurkat cells, mild HT at 42 C for
30 min did not induce any increase in the surface expression
level of Fas whereas caspase-8 was activated, suggesting that
heat stress affects Fas signaling downstream of the Fas
receptor [37]. With these reports we can say that in addition
to the duration and intensity of heat stress, the signaling
effect of HT is also dependent on the cell type. TRAIL is
another ligand involved in the extrinsic apoptosis pathway; it
is well known to play a critical role as an inducer of apoptosis
in various cancer cells in vitro and has been shown to limit
tumor growth efficiently in vivo with minimal damage to
normal tissues [39, 40]. The apoptotic signal of TRAIL is
transduced by binding to the death receptors TRAILR1
(DR4) and TRAILR2 (DR5), which are members of the TNF
receptor superfamily. These receptors are expressed more
frequently on the surface of tumor cells than on the surface of
normal cells and thus induce the extrinsic apoptotic signal to
target cancer cells [41]. TRAIL binding to the death recep-
tors DR4 and DR5 results in conformational changes that
expose the binding surface of the Fas-associated death
domain, an adaptor protein. The adaptor molecule recruits
the initiators caspases-8 and -10 to promote the formation of
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the death-inducing signaling complex. Activated caspases-8
and -10 directly activate caspase-3 or activated caspase-8
merges with the mitochondrial apoptosis pathway similarly
to Fas signaling [42]. Previously, the synergistic effect of HT
(42–43 C) and TRAIL was observed to be cytotoxic to CX-
1 human colorectal cancer cells through the activation of
caspases-8, -9 and -3 and cytochrome c release from mito-
chondria [43]. TNF-a is another death receptor ligand that
plays a role in the extrinsic apoptotic pathway. It acts by
binding to the two TNF receptors (TNFR), TNFR1 and
TNFR2. It has been reported that in U937 macrophages after
HT treatment at 42 C for 30 min, TNF-a binds to the
TNFR1 receptor and mediates the downregulation of heat
shock factor 1 induction, which decreases cell survival and
induces apoptosis through the activation of caspase-8 [44].
Another study showed that 15 min HT treatment of mice at
42 C for 7 days initiates TNF-a-mediated hepatotoxicity,
whereas 15 min treatment at 42 C HT for 1 day prevents
hepatic injury [45]. Repeated HT treatments rapidly down-
regulate the levels of the caspase-8 inhibitory protein, called
the FLICE inhibitory protein (FLIP), which activates cas-
pase-8, and thereby enhances TNFR-mediated apoptosis
[45] (Fig. 1).
Furthermore, HT is also reported to trigger ER stress
due to the accumulation of unfolded or misfolded proteins
in ER which triggers cell death [46, 47]. Elevation of
cytosolic calcium level and activation of glucose-related
protein (GRP) 78 and GRP 94 are HT-induced ER stress
markers [48]. Change in cytosolic calcium level is a critical
mediator of HT-induced cell apoptosis [38, 46, 48, 49].
Role of intracellular ROS in HT-induced apoptosis
Reactive oxygen species (ROS) are derived from the
metabolism of oxygen as the by-product of cell respiration
and are continuously produced in all aerobic organisms.
Oxidative stress occurs as a consequence of an imbalance
between ROS production and the available neutralizing
antioxidants. ROS, including superoxide, hydrogen perox-
ide (H2O2), and hydroxyl radicals (OH.), play pivotal roles
in both physiological and pathological processes [50]. HT
is one of the factors responsible for ROS production in
various cell types [34, 35, 49, 51, 52].
The superoxide anion (O-• 2 ) is the precursor of most
ROS and a mediator in oxidative chain reactions [53]. HT
decreases superoxide dismutase 1 (SOD-1) mRNA levels,
cytoplasmic SOD protein levels, and enzyme activity,
leading to the enhancement of ROS generation [54]. O-• 2 work A, the core is IL1B, which is associated with bio-
can dismute to produce H2O2 either spontaneously or
through a reaction catalyzed by SOD. In addition, O-•
2 may
react with other radicals including NO. The product per-
oxynitrite (ONOO -) is also a very powerful oxidant [55].
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Apoptosis
When we used SOD/catalase mimetic platinum nanoparti-
cles in a study of the role of ROS after HT treatment,
significant protection against apoptosis was noted [56].
Enhanced ROS production in mitochondria during heat
stress leads to nonspecific modifications of lipids, proteins,
and nucleic acids, resulting in bioenergetic dysfunction
[57]. Mitochondrial membrane constituents are particularly
susceptible to oxidative damage by ROS [57]. Because
intracellular oxidative stress plays an important role in the
modulation of apoptosis, we have studied the combination
of HT with various chemicals that produce organic free
radicals, superoxide, hydrogen peroxide, and nitric oxide
intracellularly and with stable free radicals (Table 1).
These chemicals act as heat sensitizers. An ideal sensitizer
would be nontoxic at basal temperature but would become
toxic at hyperthermic temperatures. HT also produces
synergistic effects when combined with radiation or cyto-
toxic drugs at low temperatures (40.5–43 C) in vitro and
in vivo [65, 66]. Interestingly, we also observed the syn-
ergistic effects of mild HT (41 C for 20 min) and a non-
toxic dose of an antimetastatic 16-membered macrolide
compound arising from the immediate generation of ROS
in U937 cells [52]. In addition, another study by us showed
the synergistic killing of human osteosarcoma (HOS) cells
in the presence of b-lapachone (a quinine-containing
compound) and mild HT (42 C for 60 min) [67]. Long-
lasting elevation of NQO1 (NADPH: quinone oxidore-
ductase) levels in HOS cells in the presence of mild HT
plays an important role in the synergistic effects [67].
Genes and gene network related to HT-induced
apoptosis
HT induces complex cellular responses and changes in
signal transduction. As a result, overall responses are dif-
ficult to determine by conventional methods. The appro-
priate tools for elucidating HT-induced responses are DNA
microarrays and bioinformatics systems. Heat stress affects
numerous genes and biological functions in a wide variety
of cell types under apoptotic and non-apoptotic conditions.
Our group previously compared differences in gene
expression patterns between cells under apoptotic and non-
apoptotic conditions using a microarray system and dif-
ferent cell lines [68, 69]. Through the use of an ingenuity
pathways analysis tool, we identified two significant gene
networks in U937 cells shown in Fig. 2a (non-apoptotic
condition) and Fig. 2b (apoptotic condition). In gene net-
logical activities such as cellular compromise responses as
well as cellular function and maintenance. This network
also contains HSPs including Hsp70 (HSPA1A), Hsp60
(HSPD1), and Hsp27 (HSPB1). Induction of HSPs is a
Apoptosis

TRAIL FasL Extrinsic pathway

DR4/DR5 TNFα
Fas
TNFreceptors HT
Death
domain
Intrinsic pathway
DD DD
(DD)

FADD FADD TRADD Procaspase-2

Procaspase-8 FADD
Procaspase-2 JNK
Caspase-8 FLIP RAIDD
p-JNK
tBid Caspase-2
ER stress
Bid Bim
Caspase10 Bid tBid
Ca2+-dependent
enzymes Bax
Bak
Cytochrome c
[Ca2+]i
Apaf-1/ caspase-9
Cytochrome c
Caspase-3
Apaf-1/ caspase-9

Caspase-3
DNA and nuclear fragmentation
Cell death (Apoptosis)

Fig. 1 Mechanism of apoptosis induced by hyperthermia (HT).
Extrinsic pathway: Activated by cell surface death receptors. Ligands
such as Fas L, TNF-a and tumor necrosis factor-related apoptosis-
inducing ligand (TRAIL) activate death receptors. Caspase-8 plays
the main role in the extrinsic apoptotic pathway. These receptors
activate caspase-8 followed by cleavage of Bid into tBid, MOMP
stimulation, cytochrome c release, and formation of the Apaf-
1/caspase-9 complex, which in turn activates effector caspase-3
inducing apoptosis. TRAIL and Fas L also directly activate caspase-3.
HT downregulates the levels of the FLICE inhibitory protein (FLIP),
which activates caspase-8 and enhances TNFa-mediated apoptosis.
Intrinsic pathway: Heat shock induces the formation of the RIADD-
caspase-2 complex, which activates caspase-2 followed by cleavage
of Bid into tBid and stimulation of the mitochondrial apoptotic
pathway. Another pro-apoptotic BH3-only protein, Bim, plays an
important role in HT-induced apoptosis through the Bax/Bak-
dependent pathway. HT also induces ER stress, which increases
intracellular calcium ion concentration ([Ca2?]i) as the mediator of
apoptosis

Table 1 Heat-sensitizing compounds

Drug Cell line Concentration/C Target for apoptosis Reference

2, 20 -azobis (2, 4-dimethylvaleronitrile) (free radical initiator) U937 5 mM/44 C (10 min) Mitochondria [48]
a-phenyl-tert-buthyl nitrone (neuroprotector) U937 10 mM/44 C (10 min) Mitochondria/Fas/ [49]
Tempo (2,2,6,6 tetramethyl-piperidine-N-oxyl) U937 10 mM/43 C (30 min) Mitochondria [51]
Sanazole (hypoxicradiosensitizer) U937 10 mM/44 C (20 min) Fas/Caspase8 [58]
Verapamil (Ca2? channel blocker) U937 100 lM/42–44 C (30 min) Mitochondria [59]
Lidocaine (local anesthetic) U937 1 mM/44 C (10 min) Mitochondria [60]
6-Formylpterin (intracellular hydrogen peroxide generator) U937 300 lM/44 C (20 min) Mitochondria [61]
Furan-fused tetracyclic compounds (antiviral agent) U937 20 lM/44 C (20 min) Mitochondria [62]
Sodium butyrate HCT116 1 mM/44 C (60 min) Mitochondria [63]
Benzocycloalkene derivatives U937 20 lM/44 C (20 min) Mitochondria/Fas [38]
Docosahexaenoic acid (DHA) U937 20 lM/44 C (10 min) Mitochondria 64]
Withaferin A Hela cells 0.5–1 lM/44 C (30 min) Mitochondria [34]

response to heat and these proteins block apoptosis by network A. Another study by us also revealed the upreg-
interfering with caspase activation [70]. Furthermore, ulation of cytoprotective molecules such as HSPs and
HMOX1, an anti-apoptotic molecule is also present in gene BAG3 in oral squamous cell carcinoma cells. BAG3, a

123
 Apoptosis

Fig. 2 Genetic changes with or PP

without HT treatment were A B
examined by microarray CLU A,PD,PP,RB,T,TR
PP A,E,T PD
analysis using the ingenuity PD
pathway analysis tool. Gene JUN
HSPB1 PP
networks a (under anti-
E,PP
JUND E
PP
apoptotic conditions) and A,E,PP E,P A,P,PP A,E,P,PP,
b (under apoptotic conditions) PP
TR RB,T ID2
E,L,LO,RB,T,TR
were identified. The networks HMOX1 HSPD1 A E
are shown graphically as nodes GADD45A MAPK8 A,E,P,RB
PP
E,RB E,LO
and edges (i.e., the biological IL1B PP A,PP
relationships between nodes). E,I,L A,PP A,L
The nodes and edges are shown
A,E,I,L,LO,M,PD
E,LO
PP BCL2L11
E A,L,PP,RB PP,TR
using various shapes and labels FAS
that represent the functional CDKN1A HSPA1A A,L,PP
A

class of genes and the nature of PP CASP8 CASP3 PP

E A,L,PP A
the relationship between the I A A,L,PP L
nodes PD L,M,P,PD
BAG3 A,PP
PP,RE,TR A
DUSP1 PP
PMAIP1
CASP1 A
L CASP7

A, activation/deactivation cytokine A B binding only

E, expression enzyme A B acts on
I, inhibition kinase A B inhibits
L, proteolysis others A B inhibits and acts on
LO, localization peptidase direct interaction
M, biochemical modification phosphatase indirect interaction
P, phosphorylation/dephosphorylation transcription regulator
PD, protein-DNA binding transmembrane receptor
PP, protein-protein binding upregulation
RE, reaction
T, transcription activation

TR, translocation

cochaperone of Hsp70, is a stress-inducible protein and Conclusions

demonstrates a cytoprotective property against various
stresses, including heat stress [71]. Induction of HSPs and/
or BAG3 is a common response to heat with or without cell
death, as shown in several microarray studies [72–76].
Interestingly, gene network B, which is derived from
apoptosis-specific transcripts, contains caspases (CASP1,
CASP3, CASP7, and CASP8), Fas, MAPK8, and JUN,
which are primarily associated with the molecular func-
tion of cell death, including apoptosis. It is well known
that the MAPK8 (JNK) pathway is involved in heat-in-
duced apoptosis [34, 35]. The PMAIP1 (Noxa) and
BCL2L11 (Bim) proteins present in gene network B are
also pro-apoptotic proteins. In many microarray studies,
heat stress was also observed to induce the expression of
cell-death-associated genes, such as those encoding acti-
vating transcription factor 3, dual specificity phosphatase
1, and growth arrest and DNA-damage-inducible b [72,
74, 75].
HT treatment has evolved into an increasingly viable
therapy for many tumors. It is preferred to use HT in
combination with radiotherapy or chemotherapy. HT cau-
ses protein aggregation and arrests cell cycle progression at
the G2/M phase. It induces apoptosis through extrinsic and
intrinsic pathways. HT acts as a sensitizing agent in com-
bination with radiotherapy and chemotherapy. Depending
on the applied techniques and duration of treatment, the
side effects of HT include pain, unpleasant sensations,
burns, neuropathies, and blood coagulation [77]. HT ther-
apy is currently under study in many clinical trials, par-
ticularly in Europe, Japan, and the US. The goal of these
trials is to improve and better understand this promising
technique [78]. Future areas of challenge and opportunity
for HT include the improvement of the understanding of
thermal biology, improvement of technologies for delivery
and monitoring of HT treatments in patients, successful

123
Apoptosis
high-quality clinical trials, and combination of HT with
emerging cancer therapies [78].
Acknowledgments This work was supported by Japan Society for
the Promotion of Science Grant-in-Aid for Challenging Exploratory
Research (No. 26670551).
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