Strahlentherapie

und Onkologie Current Discussion

Combination of Ionising Irradiation and Hyperthermia

Activates Programmed Apoptotic and Necrotic Cell
Death Pathways in Human Colorectal Carcinoma Cells
Frederick Mantel1,*, Benjamin Frey1,*, Stefan Haslinger1, Petra Schildkopf1, Renate Sieber1, Oliver J. Ott1,
Barbara Lödermann1, Franz Rödel2, Rolf Sauer1, Rainer Fietkau1, Udo S. Gaipl1

Purpose: The malignancy of tumor cells can be attenuated by interfering with cell death pathways. Since hyperthermia (HT) is a very
potent radiosensitizer, the influence of HT (41.5 °C for 1 hour) alone and in combination with ionising irradiation (X-ray; 5 Gy or 10 Gy)
on the form of cell death as well as on the expression of proteins known to be major components in tumor cells’ apoptotic and necrotic
pathways were examined in colorectal tumor cells.
Material and Methods: The expression of proteins was analysed by western blot and the relative activity of caspases-3/7 by fluores-
cence-based assay. Colony formation was analysed using the clonogenic assay and cell death was determined with annexin V-FITC/prop-
idium iodide staining.
Results: Combining X-ray with HT led to similar activation of caspase-3/7 and p53 expression in comparison to irradiation only while
the amount of the pro-apoptotic proteins PUMA and Bax was increased in HCT15 and SW480 cells. HT alone or combinations with X-ray
further resulted in a temporarily increased level of the anti-apoptotic protein Bcl-2. Irradiation plus HT further led to an up-regulation of
IRF-5. The levels of RIP-1, a marker for programmed necrosis, increased in tumor cells which were treated with HT and/or X-ray. Combin-
ing 5 Gy irradiation with HT compared to irradiation resulted in a significantly increased number of necrotic tumor cells and in decreased
colony formation.
Conclusion: The combined treatment of colorectal tumor cells with X-ray and HT activates distinct tumor cell pathways and fosters the
early appearance of a necrotic tumor cell phenotype.

Key Words: Hyperthermia · Irradiation · Apoptosis · Necrosis · Colorectal cancer

Strahlenther Onkol 2010;186:587–99

DOI 10.1007/s00066-010-2154-x

Kombination von ionisierender Strahlung und Hyperthermie aktiviert programmierte apoptotische und nekrotische
Zelltodessignalwege in humanen kolorektalen Tumorzellen
Hintergrund: Die Malignität von Tumorzellen kann durch Eingriffe in Zelltodeswege abgemindert werden. Da Hyperthermie (HT) strahl-
ensensibilisierend wirkt haben wir in kolorektalen Tumorzelllinien den Einfluss von HT (41.5 °C für 1 Stunde) alleine oder in Kombination
mit ionisierender Strahlung (X-ray, 5 oder 10 Gy) auf Tumorzelltodesformen und auf die Expression von Proteinen, welche Hauptbe-
standteile von apoptotischen und nekrotischen Tumorzellsignalwegen sind, untersucht.
Material und Methodik: Die Expression der Proteine wurde mit Western-Blot-Technik und die relative Aktivität von Caspasen 3/7 mit
Fluoreszenz basierendem Ansatz bestimmt. Die Koloniebildungskapazität wurde mit klonogenem Assay und Zelltod mittels AnnexinV-
FITC/Propidiumjodid Färbung ermittelt.
Ergebnisse: Kombinationen von X-ray mit HT führten zu vergleichbaren Aktivierung von Caspase 3/7 und p53-Expression im Vergleich
zur alleinigen Bestrahlung, wohingegen die Menge der pro-apoptotischen Proteine PUMA und Bax in HCT15- und SW480-Zellen zunahm.
Alleinige HT Behandlung oder Kombinationen mit X-ray resultierten in einem vorübergehend erhöhten Level an anti-apoptotischem
Protein Bcl-2. Bestrahlungen plus HT führten weiterhin zu einer Hochregulation an IRF-5. Die Mengen an RIP-1, welches ein Marker für
programmierte Nekrose darstellt, waren nach Behandlung der Tumorzellen mit HT und/oder X-ray erhöht. Die Kombination von Bestrahl-
ung mit 5Gy mit HT im Vergleich zur alleinigen Bestrahlung resultierte in einer signifikant erhöhten Anzahl an nekrotischen Tumorzellen
und einer signifikant erniedrigten Koloniebildung.
Schlussfolgerung: Kombinationsbehandlungen von kolorektalen Tumorzellen mit X-ray und HT aktivieren distinkte Tumorzelltodessig-
nalwege und fördern das rasche Auftreten eines nekrotischen Tumorzellphänotyps.

Schlüsselwörter: Hyperthermie · Bestrahlung · Apoptose · Nekrose · kolorektales Karzinom

1
Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander University of Erlangen-Nürnberg, Germany,
2
Department of Radiotherapy and Oncology, University of Frankfurt, Germany.
* F. Mantel and B. Frey contributed equally to this work
Received: March 31, 2010; accepted: July 5, 2010
Published Online: November 8, 2010

Strahlenther Onkol 2010 · No. 11 © Urban & Vogel 587

Mantel F, et al. Cell Death Pathways Induced by X-ray and Heat

Introduction the existence of a molecular pathway mediating programmed

Hyperthermia (HT) has been established in the treatment of
malignant diseases as an adjuvant of already existing thera-
pies such as chemotherapy (CT) or radiotherapy (RT). HT,
being a safe cancer treatment modality, has been shown to
be an effective radiosensitizer [23]. The combination of ionis-
ing radiation (X-ray) and HT leads to significantly improved
tumor control in several malignomas, as recent randomized
clinical studies have demonstrated (summarised in [14]). An
emerging strategy to improve outcomes in rectal cancer is to
integrate innovative agents such as triple therapy into chemo-
radiation protocols. Biologically active, targeted agents like
VEGF inhibitors are discussed to improve the therapy out-
come [21]. Recent research also revealed that transmembrane
proteins, e.g. caveolin-1, are capable of modulating radiation-
induced cell death [4] and that specific activation of the im-
mune system contributes to cancer therapy success [3]. HT,
when used in combination with other therapies, may mediate
such anti-tumor immune responses [9]. We recently demon-
strated that HT given in addition to X-ray fosters the release
of the immune activating danger signal HMGB1 by colorectal
tumor cells [29, 30]. Like HMGB1, released heat shock pro-
teins induced by HT treatment have been shown to stimulate
professional antigen-presenting cells finally leading to specific
immune activation [31].
Apoptosis is defined as a programmed cell death mech-
anism and was first described by Kerr et al. [18]. Apoptotic
cells are immunological silent and are removed in a non- or
even anti-inflammatory manner [34]. Under certain circum-
stances, apoptosis is dispensable for radiation-induced clono-
genic death [38]. Recently, Hitomi and colleagues postulated
necrosis [13]. One particular type of programmed necrosis was
reported by Kroemer et al. and called necroptosis, which is
dependent on the upregulation of RIP-1, a serine/threonine
kinase associated with the death receptors [11]. To investigate
whether HT has an influence on this newly described path-
way we analyzed in addition to pro- and anti-apoptotic pro-
teins the expression of RIP-1 in colorectal SW480 and HCT15
cancer cells treated with X-ray and/or HT. Another form of
necrosis is so-called secondary necrosis. Secondarily necrotic
cells have undergone an apoptotic program for a certain time
before losing their membrane integrity [10]. The disruption of
the tumor cell membrane leads to the release of danger signals
and consecutively to immune activation [22].
In the present study we examined the form of tumor cell
death induction by X-ray plus HT and focussed not only on the
influence of HT alone but also in combination with X-ray on the
expression of proteins known to be major components in the
apoptotic and necrotic pathways of cells. Knowledge of the mo-
lecular mechanisms of radiation-induced cell death is essential to
adapt multimodal tumor therapies for each tumor entity [5].
The induction of apoptosis is accompanied with the ac-
tivation of caspases. Activated effector caspases lead to dis-
mantling of cellular structures, inactivation of anti-apoptotic
proteins, and to activation of destructive enzymes [1]. In this
study we examined the effect of HT plus X-ray on the activa-
tion of caspase-3 and -7, as two executioner caspases of either
intrinsic or extrinsic apoptosis, as well as on anti-apoptotic
proteins like Bcl-2. The expression of the tumor suppressor
protein p53 in colorectal SW480 and HCT15 tumor cells was
also investigated. SW480 cells, being a p53 double mutant cell

SW480 HCT15

1.0 1.0
**

0.1 0.1 **
colony formation fraction
colony formation fraction

0.01 0.01

0.001 0.001

0.0001 0.0001

0.00001 0.00001
w/o HT 5 Gy 5 Gy 10 Gy 10 Gy w/o HT 5 Gy 5 Gy 10 Gy 10 Gy
+ HT + HT + HT + HT
a b

Figure 1. Colony formation of SW480 and HCT15 colorectal tumor cells after treatment with ionising irradiation and/or hyperthermia. The time interval
between irradiation and application of HT was 4 hours. The data are obtained from two independent experiments, each performed in duplicates. *p < 0.05,
**p < 0.01. Gy: Gray; HT: hyperthermia (41.5 °C for 1 h); w/o: untreated control.
Abbildung 1. Koloniebildung von SW480 und HCT15 kolorektalen Tumorzellen nach Behandlung mit ionisierender Bestrahlung und/oder Hyper-
thermie. Das Zeitintervall zwischen Bestrahlung und Hyperthermiebehandlung betrug 4 Stunden. Die Daten stammen aus zwei voneinander un-
abhängig Experimenten, welche jeweils in Duplikaten durchgeführt wurden. *p < 0.05, **p < 0.01. Gy: Gray; HT: Hyperthermie (41.5 °C für 1 Stunde);
w/o: unbehandelte Kontrolle.

588 Strahlenther Onkol 2010

Mantel F, et al. Cell Death Pathways Induced by X-ray and Heat

48h SW480 48h HCT15

35 35

30 30

apoptotic cells [%]

**
apoptotic cells [%]

25 ** 25

20 20

15 15
** **
10 10

5 5

0 0
w/o HT 5 Gy 5 Gy 10 Gy 10 Gy w/o HT 5 Gy 5 Gy 10 Gy 10 Gy
+ HT + HT + HT + HT
a c
48h SW480 48h HCT15
** **

* 35 **
**
35
30
30 *
*
25
necrotic cells [%]
necrotic cells [%]

25
20
20
* 15
15
10 10 **

5 5

0 0
w/o HT 5 Gy 5 Gy 10 Gy 10 Gy w/o HT 5 Gy 5 Gy 10 Gy 10 Gy
b + HT + HT d + HT + HT

Figure 2. Cell death of SW480 and HCT15 colorectal tumor cells after treatment with ionising irradiation and/or hyperthermia. Two days after
treatment of colorectal tumor cells with ionising irradiation (5 Gy or 10 Gy), HT (41.5 °C for 1 h), or a combination of both, the cells were stained
with AxV-FITC/PI, and cell death was analysed by flow cytometry. The time interval between irradiation and HT was 4 hours. The percentage of
apoptotic and necrotic tumor cells 48 hours after treatment is displayed in (a, c) and (b, d) respectively. The data are obtained from four indepen-
dent experiments, each performed in duplicate. *p < 0.05, **p < 0.01.
Abbildung 2. Zelltod von SW480 und HCT15 kolorektalen Tumorzellen nach Behandlung mit ionisierender Bestrahlung und/oder Hyperthermie.
Zwei Tage nach Behandlung von kolorektalen Tumorzellen mit ionisierender Bestrahlung (5 Gy oder 10 Gy), HT (41.5 °C für 1 Stunde), oder einer
Kombination aus beiden wurden die Zellen mit AxV-FITC/PI gefärbt und der Zelltod mittels durchflusszytometrischer Analyse bestimmt. Das Zei-
tintervall zwischen Bestrahlung und Hyperthermiebehandlung betrug 4 Stunden. Der Prozentsatz an apoptotischen und nekrotischen Tumorzel-
len 48 Stunden nach Behandlung ist in (a, c) bzw. (b, d) dargestellt. Die Daten stammen aus vier voneinander unabhängig Experimenten, welche
jeweils in Duplikaten durchgeführt wurden. *p < 0.05, **p < 0.01.

line, have lost one copy of chromosome 17p and thus the p53
gene for this allele. The remaining p53 allele is also mutated.
However, the tumor cells retain the proficiency for some p53
functions [27]. At least 16 genes have been identified to medi-
ate cell death by p53 [35]. Among them, we analyzed the ex-
pression of PUMA, the p53 up-regulated modulator of apop-
tosis. PUMA belongs to the BH3-only proteins of the Bcl-2
protein family as it shows homology to the BH3 (Bcl-2 homol-
ogy region) domain. It is a highly conserved protein among
different species and has been shown to be very effective in
the induction of apoptosis [37].
The balance between pro- and anti-apoptotic proteins de-
cides over the cell’s determination for apoptosis or survival [7,
8]. We determined the expression of the effector protein Bax
as a pro-apoptotic Bcl-2 family member and the expression of
the anti-apoptotic Bcl-2. In addition, whether treatment with
irradiation alone or with a combination of X-ray and HT leads
to differences in RIP-1 (see above) or IRF-5 expression lev-
els was also studied. Interferon regulatory factors (IRF) are
a family of transcription factors known for their role in host
immune response to pathogens and oncogenesis [32]. IRF-5
seems to be critical for the induction of apoptosis in response
to DNA damage [16]. Signalling through IRF-5 sensitizes p53-
deficient tumors to cell death [17] and displays a promising
target for colorectal cancer therapeutics [15].
Materials and Methods
Cell Culture
Human colorectal adenocarcinoma SW480 cells and human
colorectal adenocarcinoma HCT15 cells were grown in Dul-
becco’s modified Eagle’s medium (DMEM; PAN-Biotech
GmbH, Aidenbach, Germany) supplemented with 10% fetal

Strahlenther Onkol 2010 589

Mantel F, et al. Cell Death Pathways Induced by X-ray and Heat

SW480 HCT15
40 40

35 HT 35 HT
relative activity of caspase 3/7

relative activity of caspase 3/7

30 5 Gy 5 Gy
30
5 Gy + HT 5 Gy + HT
25 10 Gy 25 10 Gy

20 10 Gy + HT 10 Gy + HT
20

15 15

10 10

5 5

0 0
a 0h 8h 24 h 32 h 48 h 72 h b 0h 8h 24 h 32 h 48 h 72 h

Figure 3. Activation of caspase-3/7 in colorectal tumor cells after treatment with ionising irradiation and/or hyperthermia. The activation of cas-
pase-3 and -7 in SW480 (a) or HCT15 (b) colorectal tumor cells 0, 8, 24, 32, 48 and 72 hours after single or combined treatment were analysed using a
fluorescence-based caspase activity assay. The relative caspase activity was calculated by the quotient of the activity value of the treated samples
and the activity value of the negative control (untreated cells). Both values were first corrected by subtraction of the blank value (medium without
cells). One representative set of experiments out of two is displayed. Gy: Gray; HT: hyperthermia (41.5 °C for 1 h); w/o: untreated control.
Abbildung 3. Aktivierung von Caspasen3/7 in kolorektalen Tumorzellen nach Behandlung mit ionisierender Bestrahlung und/oder Hyperther-
mie. Die Aktivierung von Caspase-3 und -7 in SW480 (a) und HCT15 (b) kolorektalen Tumorzellen 0, 8, 24, 32, 48 und 72 Stunden nach Einzel- oder
Kombinationsbehandlung wurde mit Fluoreszenz basierendem Caspase Aktivitätstest analysiert. Die relative Caspaseaktivität wurde mit dem
Quotienten des Aktivitätswertes der behandelten Probe und dem Aktivitätswert der unbehandelten Probe berechnet. Beide Werte wurden zu-
nächst durch Subtraktion des Leerwertes (Medium ohne Zellen) bereinigt. Ein repräsentatives Set von zwei Experimenten ist dargestellt. Gy: Gray;
HT: Hyperthermie (41.5 °C für 1 Stunde); w/o: unbehandelte Kontrolle.

bovine serum (FBS; Biochrom AG, Berlin, Germany), 1% so-
dium pyruvate, 2 mM glutamine, 100 U/ml penicillin, and 100
µg/ml streptomycin at 37 °C in 5% CO2 and 90% humidity.
The cell line SW480 was obtained from the American Type
Culture Collection (ATCC; Wesel, Germany) and the HCT15
cells from the German Collection of Microorganisms and Cell
Cultures (DSMZ; Braunschweig, Germany).
Irradiation and Hyperthermia Treatment
A GE Inspection Technologies X-ray generator (Hürth, Ger-
many) was used for ionising irradiation (X-ray) of the cells,
which were irradiated with a single dose of 5 Gy – average
half-weekly dose in tumor therapy – (120 kV, 21.5 mA for 0.7
min) or a cumulative weekly dose of 10 Gy (120 kV, 22.7 mA
for 1.3 min). For hyperthermia treatment, cells were heated in
a HT chamber (constructed by our physicists) for 1 hour at a
constant temperature of 41.5 °C. The temperature variations
which cells were exposed to were less than 0.2 °C. For cells
treated with a combination of X-ray and HT, the time interval
between the two treatments was 4 hours. The cells were stored
at 37 °C during this time interval.
Determination of Colony Formation
The clonogenic assay was performed on single-cell suspension
of exponentially growing SW480 or HCT15 colorectal tumor
cells. Cells were counted, plated in growth medium into Pe-
tri dishes, and were irradiated 12 hours after plating. After
14 days, the tumor cells were stained with methylene blue for
0.5 hour. Colonies greater than 50 cells were counted using an
automatic colony analyzing machine.
Induction and Detection of Cell Death
Flow cytometry was used to detect death of colorectal tumor
cells after X-ray and/or HT treatment. To distinguish apop-
totic from primary and secondary necrotic cells, the exposure
of phosphatidylserine (PS) by apoptotic and necrotic cells
was analyzed by binding of FITC-labelled annexin V (AxV-
FITC), and necrosis was differed from apoptosis by co-stain-
ing with propidium iodide (PI) as described previously [19]. PI
is able to penetrate into cells which have lost their membrane
integrity and intercalates DNA. Analyses by flow cytometry
were performed with an EPICS XL MCL (Coulter, Fullerton,
CA, USA) apparatus.
Western Blot Analysis
The intracellular amounts of p53, PUMA, Bcl-2, Bax, IRF-
5 and RIP-1 were analyzed by western blot 24 and 48 hours
after treatment. The cells were washed in ice-cold PBS and
suspended with RIPA buffer containing protease inhibitors.
After 30 minutes incubation on ice, the samples were centri-
fuged at 13000 rpm for 8 minutes at 4 °C. The supernatants
were collected, and loading buffer was added in a ratio of 1:6.
The samples were then denatured at 100 °C for 10 minutes.
Then, 30 µg protein samples were run on a 10% or 12% SDS-

590 Strahlenther Onkol 2010 · No. 11

Mantel F, et al. Cell Death Pathways Induced by X-ray and Heat

24h SW480 24h HCT15

p53 p53
actin actin

4 5

(densitometric value)
(densitometric value)

p53 (53 kDa) content

p53 (53 kDa) content

3
2
2

1
1

0 0
w/o HT 5 Gy 5 Gy 10 Gy 10 Gy w/o HT 5 Gy 5 Gy 10 Gy 10 Gy
a + HT + HT c + HT + HT

SW480 HCT15
48h 48h
p53
p53
actin actin
4 5

4
(densitometric value)
(densitometric value)

p53 (53 kDa) content

p53 (53 kDa) content

3
2
2

1
1

0 0
w/o HT 5 Gy 5 Gy 10 Gy 10 Gy w/o HT 5 Gy 5 Gy 10 Gy 10 Gy
b + HT + HT d + HT + HT

Figure 4. Expression of p53 in colorectal tumor cells after treatment with ionising irradiation and/or hyperthermia. The levels of the protein p53
in SW480 and HCT15 colorectal tumor cells 24 (a, c) or 48 hours (b, d) after treatment with X-ray and/or HT were analysed by western blot. The
immunoblot bands as well as the densitometric protein expression value corrected with the actin loading control are displayed. The expression
of p53 in untreated cells was set to 1. The figure shows representative data from three independent experiments. Gy: Gray; HT: hyperthermia
(41.5 °C for 1 h); w/o: untreated control.
Abbildung 4. Expression von p53 in kolorektalen Tumorzellen nach Behandlung mit ionisierender Bestrahlung und/oder Hyperthermie. Die Menge an p53
Protein in SW480 und HCT15 kolorektalen Tumorzellen 24 Stunden (a, c) oder 48 Stunden (b, d) nach Behandlung mit X-ray und/oder HT wurden mit Wes-
tern Blot-Technik analysiert. Die Immunoblot-Banden sowie der densitometrisch ermittelte Protein Expressionslevel, welcher mit den Werten der Aktin-
Ladekontrolle korrigiert wurde, sind dargestellt. Die Expression von p53 der unbehandelten Probe wurde auf 1 gesetzt. Die Abbildung zeigt repräsentative
Daten von drei voneinander unabhängigen Experimenten. Gy: Gray; HT: Hyperthermie (41.5 °C für 1 Stunde); w/o: unbehandelte Kontrolle.

PAGE and blotted to PVDF membranes (Millipore, Billerica,
MA, USA). The membranes were blocked for at least 30 min-
utes with 5% non-fat dried milk in TBST and washed three
times with TBST for 10 minutes. They were incubated with
the primary antibody overnight at 4 °C. The following primary
antibodies were used: anti-p53 (dilution 1:2,000, Cell Signal-
ing), anti-PUMA (dilution 1:1,000, Cell Signaling), anti-Bcl-2
(dilution 1:200, Santa Cruz Biotechnology), anti-Bax (1:200,
Santa Cruz Biotechnology), anti-IRF-5 (dilution 1:1,000,
Cell Signaling) and anti-RIP-1 (dilution 1:200, Santa Cruz
Biotechnology). After washing three times with TBST for
10 minutes, the membranes were incubated with horse perox-
idase-conjugated rabbit anti-mouse (1:10,000 dilution), goat
anti-rabbit (1:20,000 dilution) or donkey anti-goat (1:7,500 di-
lution) secondary antibody, respectively, soluted in 5% milk
in TBST for 1 hour. After washing, the membranes were incu-
bated with ECL for 1 minute and then visualised using Amer-
sham Hyperfilm ECL (GE Healthcare Limited, UK).
The densitometric values of the protein expressions ob-
tained by western blot analyses are displayed in the Figures
4–9 and have been corrected with the actin-control expres-
sion. The indicated protein contents were set in relation to the

Strahlenther Onkol 2010 · No. 11 591

Mantel F, et al. Cell Death Pathways Induced by X-ray and Heat

24h SW480 24h HCT15

PUMA PUMA

actin actin

1.5 4
PUMA (18 kDa) content

PUMA (18 kDa) content

(densitometric value)

(densitometric value)
3
1.0

0.5
1

0 0
w/o HT 5 Gy 5 Gy 10 Gy 10 Gy w/o HT 5 Gy 5 Gy 10 Gy 10 Gy
+ HT + HT + HT + HT
a c

48h SW480 48h HCT15

PUMA PUMA

actin actin

1.5 4
PUMA (18 kDa) content
PUMA (18 kDa) content

(densitometric value)
(densitometric value)

3
1.0

0.5
1

0 0
w/o HT 5 Gy 5 Gy 10 Gy 10 Gy w/o HT 5 Gy 5 Gy 10 Gy 10 Gy
b + HT + HT d + HT + HT

Figure 5. Expression of PUMA in colorectal tumor cells after treatment with ionising irradiation and/or hyperthermia. The levels of the protein PU-
MA in SW480 and HCT15 colorectal tumor cells 24 (a, c) or 48 hours (b, d) after treatment with X-ray and/or HT were analysed by western blot. The
immunoblot bands as well as the densitometric protein expression value corrected with the actin loading control are displayed. The expression of
PUMA in untreated cells was set to 1. The figure shows representative data from three independent experiments. Gy: Gray; HT: hyperthermia (41.5
°C for 1 h); w/o: untreated control.
Abbildung 5. Expression von PUMA in kolorektalen Tumorzellen nach Behandlung mit ionisierender Bestrahlung und/oder Hyperthermie. Die Menge an
PUMA Protein in SW480 und HCT15 kolorektalen Tumorzellen 24 Stunden (a, c) oder 48 Stunden (b, d) nach Behandlung mit X-ray und/oder HT wurden
mit Western Blot-Technik analysiert. Die Immunoblot-Banden sowie der densitometrisch ermittelte Protein Expressionslevel, welcher mit den Werten
der Aktin-Ladekontrolle korrigiert wurde, sind dargestellt. Die Expression von PUMA der unbehandelten Probe wurde auf 1 gesetzt. Die Abbildung zeigt
repräsentative Daten von drei voneinander unabhängigen Experimenten. Gy: Gray; HT: Hyperthermie (41.5 °C für 1 Stunde); w/o: unbehandelte Kontrolle.

expression of the proteins in untreated cells. The basal expres-
sion level in untreated was set to 1.
Analysis of Caspase-3 and -7 Activities
The activities of caspase-3 and -7 were analyzed 0, 8, 24, 32, 48
and 72 hours after the treatments using a fluorescence-based
caspase activity assay (Apo-ONE Homogeneous Caspase-
3/7 Assay, Promega, Madison, WI, USA). Briefly, an equal
amount of cells were given into a 96-well plate in duplicate,
and the assay reagent was added in a ratio of 1:1. The contents
were mixed using a plate shaker at 250 rpm for 30 seconds.
The fluorescence emission was measured using a fluorescence
plate reader (HTS 7000 Bio Assay Reader, Perkin Elmer)
with an excitation filter of 485 nm and an emission filter of 535
nm. Not only blanks containing assay reagent but also culture
medium without cells were used to measure the background
fluorescence. The latter was subtracted from the values ob-
tained of the samples.

592 Strahlenther Onkol 2010 · No. 11

Mantel F, et al. Cell Death Pathways Induced by X-ray and Heat

24h SW480 24h HCT15

bax bax

actin actin

2.5 2.5

2.0 2.0
(densitometric value)

(densitometric value)
Bax (23 kDa) content

Bax (23 kDa) content

1.5 1.5

1.0 1.0

0.5 0.5

0 0
w/o HT 5 Gy 5 Gy 10 Gy 10 Gy w/o HT 5 Gy 5 Gy 10 Gy 10 Gy
a + HT + HT c + HT
+ HT

48h SW480 48h HCT15

bax bax

actin actin

2.5 2.5

2.0 2.0
(densitometric value)
(densitometric value)

Bax (23 kDa) content

Bax (23 kDa) content

1.5 1.5

1.0 1.0

0.5 0.5

0 0
w/o HT 5 Gy 5 Gy 10 Gy 10 Gy w/o HT 5 Gy 5 Gy 10 Gy 10 Gy
b + HT + HT d + HT + HT

Figure 6. Expression of Bax in colorectal tumor cells after treatment with ionising irradiation and/or hyperthermia. The levels of the protein Bax
in SW480 and HCT15 colorectal tumor cells 24 (a, c) or 48 hours (b, d) after treatment with X-ray and/or HT were analysed by western blot. The
immunoblot bands as well as the densitometric protein expression value corrected with the actin loading control are displayed. The expression
of Bax in untreated cells was set to 1. The figure shows representative data from three independent experiments. Gy: Gray; HT: hyperthermia (41.5
°C for 1 h); w/o: untreated control.
Abbildung 6. Expression von Bax in kolorektalen Tumorzellen nach Behandlung mit ionisierender Bestrahlung und/oder Hyperthermie. Die Menge an
Bax-Protein in SW480 und HCT15 kolorektalen Tumorzellen 24 Stunden (a, c) oder 48 Stunden (b, d) nach Behandlung mit X-ray und/oder HT wurden mit
Western Blot-Technik analysiert. Die Immunoblot-Banden sowie der densitometrisch ermittelte Protein Expressionslevel, welcher mit den Werten der
Aktin-Ladekontrolle korrigiert wurde, sind dargestellt. Die Expression von Bax der unbehandelten Probe wurde auf 1 gesetzt. Die Abbildung zeigt reprä-
sentative Daten von drei voneinander unabhängigen Experimenten. Gy: Gray; HT: Hyperthermie (41.5 °C für 1 Stunde); w/o: unbehandelte Kontrolle.

Statistical Analyses Results

Representative western blot data of three independent exper-
iments are displayed. The cell death data are obtained from
four independent experiments each performed in duplicate.
The colony formation experiments were repeated at least
twice. Statistical analyses were performed using the Student’s
t-test. A p value < 0.05 was considered as significant (*) and a
p value < 0.01 as highly significant (**).
X-ray (5 Gy) Combined with Hyperthermia Reduces
Colony Formation of Colorectal Tumor Cells
In both SW480 and HCT15 colorectal tumor cells HT highly
significantly enhanced radiosensitivity after 5 Gy irradiation
(Figure 1a and b). Since after irradiation with 10 Gy only very
few clonogenic tumor cells were spared, the application of HT
after X-ray only slightly, but not significantly, further reduced
the colony formation of SW480 (Figure 1a) and HCT15 (Fig-
ure 1b) tumor cells.

Strahlenther Onkol 2010 · No. 11 593

Mantel F, et al. Cell Death Pathways Induced by X-ray and Heat

24h SW480 24h HCT15

bcl-2 bcl-2

actin actin

2.5 3.0

2.0 2.5
Bcl-2 (28 kDa) content

Bcl-2 (28 kDa) content

(densitometric value)

(densitometric value)
2.0
1.5
1.5
1.0
1.0
0.5
0.5

0 0
w/o HT 5 Gy 5 Gy 10 Gy 10 Gy w/o HT 5 Gy 5 Gy 10 Gy 10 Gy
a + HT + HT c + HT + HT

48h SW480 48h HCT15

bcl-2 bcl-2

actin actin

2.5 3.0

2.5
Bcl-2 (28 kDa) content

2.0
Bcl-2 (28 kDa) content

(densitometric value)
(densitometric value)

2.0
1.5
1.5
1.0
1.0
0.5 0.5

0 0
w/o HT 5 Gy 5 Gy 10 Gy 10 Gy w/o HT 5 Gy 5 Gy 10 Gy 10 Gy
b + HT + HT d + HT + HT

Figure 7. Expression of Bcl-2 in colorectal tumor cells after treatment with ionising irradiation and/or hyperthermia. The levels of the protein Bcl-
2 in SW480 and HCT15 colorectal tumor cells 24 (a, c) or 48 hours (b, d) after treatment with X-ray and/or HT were analysed by western blot. The
immunoblot bands as well as the densitometric protein expression value corrected with the actin loading control are displayed. The expression of
Bcl-2 in untreated cells was set to 1. The figure shows representative data from three independent experiments. Gy: Gray; HT: hyperthermia (41.5
°C for 1 h); w/o: untreated control.
Abbildung 7. Expression von Bcl-2 in kolorektalen Tumorzellen nach Behandlung mit ionisierender Bestrahlung und/oder Hyperthermie. Die Menge an
Bcl-2 Protein in SW480 und HCT15 kolorektalen Tumorzellen 24 Stunden (a, c) oder 48 Stunden (b, d) nach Behandlung mit X-ray und/oder HT wurden mit
Western Blot-Technik analysiert. Die Immunoblot-Banden sowie der densitometrisch ermittelte Protein Expressionslevel, welcher mit den Werten der
Aktin-Ladekontrolle korrigiert wurde, sind dargestellt. Die Expression von Bcl-2 der unbehandelten Probe wurde auf 1 gesetzt. Die Abbildung zeigt reprä-
sentative Daten von drei voneinander unabhängigen Experimenten. Gy: Gray; HT: Hyperthermie (41.5 °C für 1 Stunde); w/o: unbehandelte Kontrolle.

X-ray Combined with Hyperthermia Induces Predomi-
nantly Necrosis in Colorectal Tumor Cells
Colorectal SW480 and HCT 15 tumor cells behaved similar
in regards to cell death induction (Figure 2). The dying tumor
cells became secondarily necrotic instead of continuing their
apoptotic program 2 days after treatment of SW480 (Figure 2a
and b) or HCT15 (Fig. 2c and d) with X-ray and/or HT; necro-
sis was the prominent form of cell death. For this reason, fur-
ther cell pathway analyses focussed on the time points 24 and
48 hours after treatment. In comparison to untreated cells or
cells treated with HT only, X-ray alone led to a highly signifi-
cant increase of necrotic cells after 48 hours, which could be
further significantly increased when combing 5 Gy irradiation
with HT. In addition, HT alone also resulted in a significantly
increased number of necrotic tumor cells. Irradiation with a
single dose of 10 Gy had similar effects on necrosis when com-
pared to irradiation plus HT. Addition of HT to 5 Gy irradi-
ated cells produced a statistically significant increase in necro-
sis but did not produce a statistically significant change in the
percentage of apoptotic cells.

594 Strahlenther Onkol 2010 · No. 11

Mantel F, et al. Cell Death Pathways Induced by X-ray and Heat

24h SW480 24h HCT15

IRF-5 IRF-5
actin actin

1.5 4

IRF-5 (60 kDa) content

IRF-5 (60 kDa) content

(densitometric value)
(densitometric value)

3
1.0

0.5
1

0 0
w/o HT 5 Gy 5 Gy 10 Gy 10 Gy w/o HT 5 Gy 5 Gy 10 Gy 10 Gy
+ HT + HT + HT + HT
a c
48h SW480 48h HCT15

IRF-5 IRF-5

actin actin

1.5 4
IRF-5 (60 kDa) content
IRF-5 (60 kDa) content

(densitometric value)
(densitometric value)

3
1.0

0.5
1

0 0
w/o HT 5 Gy 5 Gy 10 Gy 10 Gy w/o HT 5 Gy 5 Gy 10 Gy 10 Gy
b + HT + HT d + HT + HT

Figure 8. Expression of IRF-5 in colorectal tumor cells after treatment with ionising irradiation and/or hyperthermia. The levels of the protein IRF-5
in SW480 and HCT15 colorectal tumor cells 24 (a, c) or 48 hours (b, d) after treatment with X-ray and/or HT were analysed by western blot. The
immunoblot bands as well as the densitometric protein expression value corrected with the actin loading control are displayed. The expression of
IRF-5 in untreated cells was set to 1. The figure shows representative data from three independent experiments. Gy: Gray; HT: hyperthermia (41.5
°C for 1 h); w/o: untreated control.
Abbildung 8. Expression von IRF-5 in kolorektalen Tumorzellen nach Behandlung mit ionisierender Bestrahlung und/oder Hyperthermie. Die Menge an
IRF-5 Protein in SW480 und HCT15 kolorektalen Tumorzellen 24 Stunden (a, c) oder 48 Stunden (b, d) nach Behandlung mit X-ray und/oder HT wurden mit
Western Blot-Technik analysiert. Die Immunoblot-Banden sowie der densitometrisch ermittelte Protein Expressionslevel, welcher mit den Werten der
Aktin-Ladekontrolle korrigiert wurde, sind dargestellt. Die Expression von IRF-5 der unbehandelten Probe wurde auf 1 gesetzt. Die Abbildung zeigt reprä-
sentative Daten von drei voneinander unabhängigen Experimenten. Gy: Gray; HT: Hyperthermie (41.5 °C für 1 Stunde); w/o: unbehandelte Kontrolle.

X-ray Combined with HT Leads to Similar Activation of
Caspase-3 and -7 Compared to Irradiation Only
To determine the activation of caspase-3 and -7 in colorectal
SW480 and HCT15 carcinoma cells treated with irradiation
and/or HT a fluorescence-based caspase activity assay 0, 8, 24,
32, 48 and 72 hours after the respective treatments was per-
formed (Figure 3a and b). After 8 hours, the different treat-
ments did not lead to an increase in caspase activity compared
to untreated cells. After 24 hours, the relative caspase activity
rose showing the highest values for cells treated with X-ray
or X-ray plus HT. The increase in caspase activity continued
and was most pronounced 48 hours after treatment for the ir-
radiated-only cells when comparing 5 Gy with 5 Gy plus HT.
In the case of irradiation with 10 Gy, the activity for caspase-
3/7 was further increased compared to 5 Gy irradiation and
similar in tumor cells treated with 10 Gy or 10 Gy plus HT. Af-
ter 48 hours, the level of caspase activity reached a plateau in
SW480 cells which were irradiated or treated with irradiation
plus HT. In the case of HCT15 cells, the caspase-3/7 activity
further increased, especially with the 10 Gy dose and reached
a maximum after 72 hours. Cells treated with X-ray plus HT
reached similar activity levels of caspase-3/7, 48 hours and

Strahlenther Onkol 2010 · No. 11 595

Mantel F, et al. Cell Death Pathways Induced by X-ray and Heat

24h SW480 24h HCT15

RIP-1 RIP-1
actin actin

2.0 2.0

RIP-1 (74 kDa) content

(densitometric value)
RIP-1 (74 kDa) content
(densitometric value)

1.5 1.5

1.0 1.0

0.5 0.5

0 0
w/o HT 5 Gy 5 Gy 10 Gy 10 Gy w/o HT 5 Gy 5 Gy 10 Gy 10 Gy
+ HT + HT + HT + HT
a c
48h SW480 48h HCT15
RIP-1 RIP-1
actin actin

2.0 2.0
RIP-1 (74 kDa) content
(densitometric value)

RIP-1 (74 kDa) content

(densitometric value)

1.5 1.5

1.0 1.0

0.5 0.5

0 0
w/o HT 5 Gy 5 Gy 10 Gy 10 Gy w/o HT 5 Gy 5 Gy 10 Gy 10 Gy
b + HT + HT d + HT + HT

Figure 9. Expression of RIP-1 in colorectal tumor cells after treatment with ionising irradiation and/or hyperthermia. The levels of the protein RIP-1
in SW480 and HCT15 colorectal tumor cells 24 (a, c) or 48 h (b, d) after treatment with X-ray and/or HT were analysed by western blot. The immu-
noblot bands as well as the densitometric protein expression value corrected with the actin loading control are displayed. The expression of RIP-1
in untreated cells was set to 1. The figure shows representative data from three independent experiments. Gy: Gray; HT: hyperthermia (41.5 °C for
1 h); w/o: untreated control.
Abbildung 9. Expression von RIP-1 in kolorektalen Tumorzellen nach Behandlung mit ionisierender Bestrahlung und/oder Hyperthermie. Die Menge an
RIP-1 Protein in SW480 und HCT15 kolorektalen Tumorzellen 24 Stunden (a, c) oder 48 Stunden (b, d) nach Behandlung mit X-ray und/oder HT wurden mit
Western Blot-Technik analysiert. Die Immunoblot-Banden sowie der densitometrisch ermittelte Protein Expressionslevel, welcher mit den Werten der
Aktin-Ladekontrolle korrigiert wurde, sind dargestellt. Die Expression von RIP-1 der unbehandelten Probe wurde auf 1 gesetzt. Die Abbildung zeigt reprä-
sentative Daten von drei voneinander unabhängigen Experimenten. Gy: Gray; HT: Hyperthermie (41.5 C für 1 Stunde); w/o: unbehandelte Kontrolle.

72 hours after treatment compared to irradiated only cells. HT
only led to no detectable caspase-3/7 activity at all time points
investigated.
X-ray Alone or Combined with HT Increases p53
Expression in Colorectal Tumor Cells
The expression levels of p53 in SW480 or HCT15 cells 24 hours
(Figure 4a and c) and 48 hours (Figure 4b and d) after treat-
ment are displayed. Colorectal tumor cells treated with irradia-
tion only or treated with a combination of irradiation and HT
expressed more p53 than untreated cells or cells treated with
HT only 48 hours after the applications. HCT15 cells already
increased the p53 expression 24 hours after the respective
treatments (Figure 4c). HT only led to a slight increase in p53
expression compared to untreated cells (Figure 4b and d).
Hyperthermia and Combinations with X-ray Increases
Expression of PUMA
All treatments did not result in significant higher levels of PU-
MA in the SW480 and HCT15 tumor cells 24 hours after appli-

596 Strahlenther Onkol 2010 · No. 11

Mantel F, et al. Cell Death Pathways Induced by X-ray and Heat
cation (Figure 5a and c). The expression of PUMA was gener-
ally lower in SW480 cells. 48 hours after treatment, a higher
level of PUMA was observed when HT was given alone or in
combinations with X-ray in both SW480 and HCT15 tumor
cells (Figure 5b and d).
Hyperthermia and Combinations with X-ray Increase the
Expression of the Pro-apoptotic Protein Bax
Combinatory treatments of SW480 or HCT15 tumor cells with
irradiation and hyperthermia or HT alone caused a higher ex-
pression of the pro-apoptotic protein Bax as early as 48 hours
after treatment (Figure 6). This effect was more pronounced
in SW480 cells, especially for the combinatory treatment with
5 Gy or with HT only (Figure 6b). However, a similar tenden-
cy was observed for HCT15 cells (Figure 6d).
Hyperthermia and Combinations with X-ray Modulate
the Expression of the Anti-apoptotic Protein Bcl-2
One day after treatment of SW480 colorectal tumor cells, Bcl-
2 was up-regulated in cells that had been treated with irradia-
tion plus HT or HT only compared to irradiated and untreated
cells (Figure 7a). In HCT15 cells, all treatments also led to an
increased expression of Bcl-2 after 24 hours (Figure 7c). How-
ever, waiting for another 24 hours, the expression of Bcl-2 re-
turned to basal levels in SW480 and HCT15 cells (Figure 7b
and d).
Irradiation Plus Hyperthermia Leads to Up-regulation of
IRF-5
IRF-5 sensitizes tumor cells for apoptosis. Figure 8 displays
the level of IRF-5 in SW480 or HCT15 cells 24 and 48 hours
after treatment. The expression levels of IRF-5 did not differ
under all conditions 24 hours after treatment in SW480 cells
(Figure 8a). In HCT15 cells, the amount of IRF-5 was signifi-
cantly increased when applying HT, X-ray or combinations of
both. After 48 hours, the combination of X-ray and HT re-
sulted in a slightly higher level of IRF-5 in SW480 cells in com-
parison to single treatments (Figure 8b). The total amount of
IRF-5 decreased in HCT15 cells; however, all treatments led
to an increased expression of IRF-5 in comparison to untreat-
ed cells (Figure 8d).
Hyperthermia, X-ray, and Combinations of Both Increase
Expression of RIP-1 in Colorectal Tumor Cells
To analyse whether treatment with X-ray and/or HT causes the
activation of the necroptotic pathway, the expression of RIP-
1 in colorectal SW480 and HCT15 tumor cells was examined.
After treatment, no changes were observed in both SW480 and
HCT15 cells after 24 hours (Figure 9a and c). However, after
48 hours the levels of RIP-1 increased in colorectal tumor cells
which were treated with HT and/or X-ray (Figure 9b and d).
A combination of irradiation and hyperthermia led to similar
expression of RIP-1 in comparison to irradiation or HT alone.
Strahlenther Onkol 2010 · No. 11
Discussion
Preoperative chemoradiotherapy, as compared with postop-
erative chemoradiotherapy, improves local control of colorec-
tal cancer [28]. Hyperthermia has been established in the
treatment of malignant diseases in combination with irradia-
tion. HT given in addition to RT significantly improved the
number of complete responses and significant regressions in
patients with locally advanced carcinoma of the rectum and
significantly improved the 5-year survival rates [6]. Preclini-
cal studies proved a synergy of heat with X-ray and also with
modulated electromagnetic field in killing tumor cells [2]. In
recent years, knowledge about the immune biological mode
of action of HT is increasing. Radiosensitivity of tumor cells is
often determined by the clonogenic assay. The latter defines
the “surviving fraction” of cells treated with different radia-
tion doses. We showed that HT given in addition to X-ray (5
Gy) significantly reduces the colony formation of SW480 and
HCT15 cells, respectively (Figure 1). The strength of this as-
say is that it provides information about the percentage of tu-
mor cell colonies that are not mitotically active. However, it
cannot give information about the viability of the irradiated
tumor cells or the type of cell death occurring since the cells
could also be senescent [20]. Therefore, AxV-FITC/PI stain-
ing was applied to examine cell death of colorectal tumor cells
after X-ray and/or HT treatment. Secondary necrotic cells re-
sult from apoptotic cells that have lost their membrane integ-
rity while undergoing apoptosis [10]. It was shown here that
48 hours after treatment with X-ray plus HT, colorectal tumor
cells display higher necrotic rates compared to apoptotic rates
(Figure 2). HT was more potent in sensitizing tumor cells for
necrosis when a lower dose of X-ray was applied. One could
speculate that this may be a basis for the higher efficacy of HT
when applied together with a lower total dose of RT in for
example already pre-irradiated areas [33].
We conclude that the dying colorectal SW480 and HCT15
cells have proceeded rather quickly into secondary necro-
sis instead of continuing their apoptotic program. The latter
was present, since prominent apoptotic proteins displayed an
increased expression (Fig 4–6). We showed here that HT in
combination with X-ray fosters immune activating necrotic
cell death forms and leads to activation of apoptotic and ne-
crotic programs in tumor cells. Treatment with irradiation or
combinations of X-ray with HT lead to a higher level of p53
and high activation levels of caspase-3/7 in colorectal carci-
noma cells.
PUMA and Bax contribute to the activation of caspase-
3/7. It was shown in this study that 48 hours after application,
HT given in combination with irradiation leads to a higher ex-
pression level of PUMA in comparison to irradiation alone.
Yu et al. [36] demonstrated with gene knockout (KO) experi-
ments the necessity of PUMA for apoptosis induced by p53,
hypoxia and DNA-damaging agents in human colorectal can-
cer cells. PUMA functions as a de-repressor of direct activa-
tors of Bax or Bak leading to mitochondrial outer membrane
597
Mantel F, et al. Cell Death Pathways Induced by X-ray and Heat

Taken together, we have shown that

X-ray plus HT
the pro-apoptotic Bcl-2 family members
Bax and PUMA are up-regulated within
via mitochondrial permeability transition cells treated with a combination of irra-
diation and HT, and the anti-apoptotic
Bcl-2 is, after an initial up-regulation,
PUMA Bax activ finally down-regulated. The cells tend
ation
towards cell death as the pro-apoptotic
activation
proteins outbalance Bcl-2 [8].
RIP-1
IRF-5 is a transcription factor
caspase3/7 # known to be critical in the induction of
p53
apoptosis after DNA damage. It sensi-
IRF-5
tises tumor cells to cell death whereas
down-regulation
silencing of IRF-5 desensitizes tumor
cells [16]. It was shown for the first time
Bcl-2
blocking
in this study that IRF-5 is also modulat-
ed by X-ray and/or HT. In HCT15 cells,
the amount of IRF-5 was significantly
increased when applying HT, X-ray
or combinations of both while SW480
tumor cell death show increased amounts of IRF-5 after
X-ray plus HT treatment (Figure 8b).
Figure 10. Apoptosis and necrosis regulating proteins in colorectal tumor cells after treatment In contrast to apoptosis, necrosis is
with ionising irradiation and/or hyperthermia. A schematic overview and possible interactions
of proteins that are involved in the apoptotic and necrotic cell death program of colorectal tu-
been known to be a non-physiological
mor cells after treatment with X-ray and/or HT is given. Up-regulation of PUMA and Bax may event. In addition, a programmed form
lead to caspase-3/7 activation. Bcl-2, after an initial up-regulation, is finally down-regulated of necrosis, so-called necroptosis, has
when the activity of caspases-3/7 increases (48 hours after treatment). The up-regulation of p53 recently been reported [13, 20]. The lat-
may lead to activation of Bax and to blocking of Bcl-2. IRF-5 may indirectly activate caspases-3/7 ter is dependent on the serine/threonine
while RIP-1 leads to cell death by promoting the mitochondrial permeability transition. #p53 is
mutated in SW480 and HCT15 cells. kinase RIP-1 which is associated with
the death receptors. RIP-1 promotes
Abbildung 10. Apoptose und Nekrose regulierende Proteine in kolorektalen Tumorzellen nach
Behandlung mit ionisierender Bestrahlung und/oder Hyperthermie. Ein schematischer Über- the mitochondrial permeability transi-
blick und mögliche Interaktionen von Proteinen, welche an apoptotischen und nekrotischen tion caused by the permeability transi-
Zelltodesprogrammen in kolorektalen Tumorzellen nach Behandlung mit ionisierender Be- tion pore complex (PTPC). Therefore
strahlung und/oder Hyperthermie beteiligt sind, sind dargestellt. Die Hochregulierung von it induces the accumulation of prone-
PUMA und Bax können zur Aktivierung von Caspasen3/7 führen. Bcl-2 wird nach einer anfäng-
crotic ceramides or binds to the adenine
lichen Hochregulation schließlich herunterreguliert, wenn die Aktivität an Caspasen3/7 steigt
(48 Stunden nach Kombinationsbehandlung). Die Hochregulierung von p53 kann zur Aktivie- nucleotide translocase, another compo-
rung von Bax und zur Blockade von Bcl-2 führen. IRF-5 kann indirekt durch Caspasen3/7 aktiviert nent of the PTPC [12]. This study illus-
werden wohingegen RIP-1 zum Zelltod führt indem die Ausbildung der mitochondrialen Perme- trates for the first time that in colorec-
abilitäts-Transitions-Pore gefördert wird. #p53 ist mutiert in SW480 und HCT15 Zellen. tal tumor cells the expression levels of
RIP-1 are increased after application of
permeabilization (MOMP). This means that PUMA binds X-ray alone, HT alone or in combination of both. This sug-
anti-apoptotic Bcl-2 proteins that sequester direct activator gests that necroptosis can be induced by HT. Figure 10 gives
BH3-only proteins, like Bid, leading to the release of Bid from a schematic overview of some of the apoptosis- and necro-
Bcl-2. Bid then directly activates Bax leading to MOMP [8]. sis-regulating proteins examined in our study that are up- or
Higher amounts of the pro-apoptotic Bax were also observed down-regulated in colorectal tumor cells after treatment with
after combined treatment of colorectal tumor cells with irra- X-ray plus HT.
diation and HT (Figure 6). The amount of the anti-apoptotic Taken together, both apoptotic and necrotic cell death
Bcl-2 was primarily increased after 24 hours in cells treated programs were activated in colorectal tumor cells treated with
with the combination of X-ray plus HT (Figure 7). However, X-ray plus HT. Because a major death form of colorectal tu-
after 48 hours, the combinatory treatments resulted in slightly mor cells 48 hours after a combinatory treatment with irradia-
lower Bcl-2 expression levels in SW480 and HCT15 cells in tion and HT was necrosis, we assume that treated tumor cells
comparison to irradiation only (Figure 7b and d). A novel undergo secondary necrosis shortly after starting the apop-
aranorosin derivative, K050, could be a potent therapeutic totic program. Fostering distinct tumor cell pathways and the
agent against Bcl-2-overexpressing human malignancies [25]. release of immune-activating danger signals, e.g. extracel-

598 Strahlenther Onkol 2010 · No. 11

Mantel F, et al. Cell Death Pathways Induced by X-ray and Heat
lular heat-shock proteins [24] and HMGB1 [29], contribute
to the development of specific anti-tumor immunity. The
latter could be achieved by distinct combinatory treatments
with radiotherapy, chemotherapy, HT, and further immune-
modulating agents [26]. Therefore, the analyses of molecules
involved in cell death pathways could be important predictors
for the success of combinatory cancer therapies.
Acknowledgements
This work was supported by the ELAN Fond (ST-08.06.30.1) of
the Friedrich-Alexander University of Erlangen-Nuremberg, by the
European Commissions (NOTE (TPA4 FP6)), and by the German
Research Foundation (Graduate School of the SFB 643).
References
1. Adrain C, and Martin SJ. The mitochondrial apoptosome: a killer unleashed
by the cytochrome seas. Trends Biochem Sci 2001;26:390–7.
2. Andocs G, Renner H, Balogh L, et al. Strong synergy of heat and modu-
lated electromagnetic field in tumor cell killing. Strahlenther Onkol
2009;185:120–6.
3. Apetoh L, Ghiringhelli F, Tesniere A, et al. Toll-like receptor 4-dependent
contribution of the immune system to anticancer chemotherapy and ra-
diotherapy. Nat Med 2007;13:1050–9.
4. Barzan D, Maier P, Zeller WJ, et al. Overexpression of caveolin-1 in lympho-
blastoid TK6 cells enhances proliferation after irradiation with clinically
relevant doses. Strahlenther Onkol 2010;186:99–106.
5. Belka C, Betsch A, Marini P, et al. [Death inducing ligands in combination
with ionizing radiation: objective and current knowledge]. Strahlenther
Onkol 2003;179:141–51.
6. Berdov BA, and Menteshashvili GZ. Thermoradiotherapy of patients with
locally advanced carcinoma of the rectum. Int J Hyperthermia 1990;6:
881–90.
7. Budach W. [Bcl-XL and bcl-2 expression in squamous epithelial carcinomas
of the head-neck]. Strahlenther Onkol 1999;175:533–4.
8. Chipuk JE, and Green DR. How do BCL-2 proteins induce mitochondrial
outer membrane permeabilization? Trends Cell Biol 2008;18:157–64.
9. Dayanc BE, Beachy SH, Ostberg JR, et al. Dissecting the role of hyperther-
mia in natural killer cell mediated anti-tumor responses. Int J Hyperthermia
2008;24:41–56.
10. Gaipl US, Kuenkele S, Voll RE, et al. Complement binding is an early feature
of necrotic and a rather late event during apoptotic cell death. Cell Death
Differ 2001;8:327–34.
11. Galluzzi L, and Kroemer G. Necroptosis: a specialized pathway of pro-
grammed necrosis. Cell 2008;135:1161–3.
12. Golstein P, and Kroemer G. Cell death by necrosis: towards a molecular
definition. Trends Biochem Sci 2007;32:37–43.
13. Hitomi J, Christofferson DE, Ng A, et al. Identification of a molecular sig-
naling network that regulates a cellular necrotic cell death pathway. Cell
2008;135:1311–23.
14. Horsman MR, and Overgaard J. Hyperthermia: a potent enhancer of radio-
therapy. Clin Oncol (R Coll Radiol) 2007;19:418–26.
15. Hu G, and Barnes BJ. Interferon regulatory factor-5-regulated pathways
as a target for colorectal cancer therapeutics. Expert Rev Anticancer Ther
2006;6:775–84.
16. Hu G, and Barnes BJ. IRF-5 is a mediator of the death receptor-induced
apoptotic signaling pathway. J Biol Chem 2009;284:2767–77.
17. Hu G, Mancl ME, and Barnes BJ. Signaling through IFN regulatory factor-5
sensitizes p53-deficient tumors to DNA damage-induced apoptosis and cell
death. Cancer Res 2005;65:7403–12.
18. Kerr JF, Wyllie AH, and Currie AR. Apoptosis: a basic biological phenomenon
with wide-ranging implications in tissue kinetics. Br J Cancer 1972;26:
239–57.
Strahlenther Onkol 2010 · No. 11
19. Koopman G, Reutelingsperger CP, Kuijten GA, et al. Annexin V for flow cy-
tometric detection of phosphatidylserine expression on B cells undergoing
apoptosis. Blood 1994;84:1415–20.
20. Kroemer G, Galluzzi L, Vandenabeele P, et al. Classification of cell death:
recommendations of the Nomenclature Committee on Cell Death 2009. Cell
Death Differ 2009;16:3–11.
21. Marquardt F, Rodel F, Capalbo G, et al. Molecular targeted treatment and
radiation therapy for rectal cancer. Strahlenther Onkol 2009;185:371–378.
22. Matzinger P. The danger model: a renewed sense of self. Science
2002;296:301–5.
23. Molls M. Hyperthermia–the actual role in radiation oncology and future
prospects. Part I. Strahlenther Onkol 1992;168:183–190.
24. Multhoff G. Activation of natural killer cells by heat shock protein 70. Int J
Hyperthermia 2009;25:169–75.
25. Nakashima T, Tanaka R, Yamashita Y, et al. Aranorosin and a novel deriva-
tive inhibit the anti-apoptotic functions regulated by Bcl-2. Biochem Bio-
phys Res Commun 2008;377:1085–90.
26. Niyazi M, Marini P, Daniel PT, et al. Efficacy of a triple treatment with irradi-
ation, agonistic TRAIL receptor antibodies and EGFR blockade. Strahlenther
Onkol 2009;185:8–18.
27. Rochette PJ, Bastien N, Lavoie J, et al. SW480, a p53 double-mutant cell line
retains proficiency for some p53 functions. J Mol Biol 2005;352:44–57.
28. Sauer R, Becker H, Hohenberger W, et al. Preoperative versus postoperative
chemoradiotherapy for rectal cancer. N Engl J Med 2004;351:1731–40.
29. Schildkopf P, Frey B, Mantel F, et al. Application of hyperthermia in ad-
dition to ionizing irradiation fosters necrotic cell death and HMGB1 re-
lease of colorectal tumor cells. Biochem Biophys Res Commun 2010;391:
1014–20.
30. Schildkopf P, Holmer R, Sieber R, et al. Hyperthermia in combination with
X-irradiation induces inflammatory forms of cell death. Autoimmunity
2009;42:311–3.
31. Sherman M, and Multhoff G. Heat shock proteins in cancer. Ann N Y Acad Sci
2007;1113:192–201.
32. Taniguchi T, Ogasawara K, Takaoka A, et al. IRF family of transcription fac-
tors as regulators of host defense. Annu Rev Immunol 2001;19:623–55.
33. Vernon CC, Hand JW, Field SB, et al. Radiotherapy with or without hyper-
thermia in the treatment of superficial localized breast cancer: results
from five randomized controlled trials. International Collaborative Hyper-
thermia Group. Int J Radiat Oncol Biol Phys 1996;35:731–44.
34. Voll RE, Herrmann M, Roth EA, et al. Immunosuppressive effects of apop-
totic cells. Nature 1997;390:350–1.
35. Vousden KH, and Lu X. Live or let die: the cell’s response to p53. Nat Rev
Cancer 2002;2:594–604.
36. Yu J, Wang Z, Kinzler KW, et al. PUMA mediates the apoptotic response
to p53 in colorectal cancer cells. Proc Natl Acad Sci U S A 2003;100:
1931–6.
37. Yu J, and Zhang L. No PUMA, no death: implications for p53-dependent
apoptosis. Cancer Cell 2003;4:248–9.
38. Zhang P, Castedo M, Tao Y, et al. Caspase independence of radio-induced
cell death. Oncogene 2006;25:7758–70.
Address for Correspondence
Priv.-Doz. Dr. Udo S. Gaipl, PhD
Department of Radiation Oncology
Radiation Immunobiology
University Hospital Erlangen
Friedrich-Alexander University of Erlangen-Nürnberg
Universitätsstr. 27
91054 Erlangen
Germany
Phone: (+49) 9131-85-32311, Fax -39335
e-mail: udo.gaipl@uk-erlangen.de
599