Ionising Irradiation and Hyperthermia

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Combination of Ionising Irradiation and HyperthermiaActivates Programmed Apoptotic and Necrotic CellDeath Pathways in Human Colorectal Carcinoma CellsFrederick Mantel1,*, Benjamin Frey1,*, Stefan Haslinger1, Petra Schildkopf 1, 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– 99DOI 10.1007/s00066-010-2154-x

Kombination von ionisierender Strahlung und Hyperthermie aktiviert programmierte apoptotische und nekrotischeZelltodessignalwege 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 Vergleichzur 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

Current Discussion

Strahlentherapieund Onkologie

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, 2010Published Online: November 8, 2010

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



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

588 Strahlenther Onkol 2010

the existence of a molecular pathway mediating programmednecrosis [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

IntroductionHyperthermia (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

1.0

0.1

0.01

0.001

0.0001colony formatio

n fraction

0.00001

**

**

w/o

a b

HT 5 Gy 5 Gy+ HT

10 Gy 10 Gy+ HT

1.0

0.1

0.01

0.001

0.0001colony formatio

n fraction

0.00001

w/o HT 5 Gy 5 Gy+ HT

10 Gy 10 Gy+ HT

SW480 HCT15

Figure 1. Colony formation of SW480 and HCT15 colorectal tumor cells after treatment with ionising irradiation and/or hyperthermia. The time intervalbetween 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.




589Strahlenther Onkol 2010

line, have lost one copy of chromosome 17p and thus the p53gene 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 Baxas 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 leadsto 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

db

35

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apoptotic cells [%]

necrotic cells [%]

*

**

**

**

**

**

w/o HT 5Gy 5Gy

+ HT

10Gy 10Gy

+ HT

w/o HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

c

HCT15

HCT15

48h

48h

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0

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apoptotic cells [%]

necrotic cells [%]

**

**

**

*

*

*

**

w/o HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

w/o HT 5Gy 5Gy

+ HT

10Gy 10Gy

+ HT

48h

a

SW480

SW48048h

Figure 2. Cell death of SW480 and HCT15 colorectal tumor cells after treatment with ionising irradiation and/or hyperthermia. Two days aftertreatment 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 stainedwith 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 einerKombination 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, welchejeweils in Duplikaten durchgeführt wurden. *p < 0.05, **p < 0.01.




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 – averagehalf-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 tumorcells. 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]. PIis 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-

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relative activity of caspase 3/7

relative activity of caspase 3/7

0h 8h 24h 32h 48h 72h 0h 8h 24h 32h 48h 72h

SW480 HCT15

HT

5Gy

5Gy + HT

10Gy

10Gy + HT

HT

5Gy

5Gy + HT

10Gy

10Gy + HT

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 afluorescence-based caspase activity assay. The relative caspase activity was calculated by the quotient of the activity value of the treated samplesand the activity value of the negative control (untreated cells). Both values were first corrected by subtraction of the blank value (medium withoutcells). 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- oderKombinationsbehandlung wurde mit Fluoreszenz basierendem Caspase Aktivitätstest analysiert. Die relative Caspaseaktivität wurde mit demQuotienten 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.

590 Strahlenther Onkol 2010 · No. 11




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 Figures4–9 and have been corrected with the actin-control expres-

sion. The indicated protein contents were set in relation to the

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p53 (53 kDa) content

(densitometric value)

w/oa c

b d

actin actin

p53 p53

24h 24h

48h 48h

p53

SW480 HCT15

SW480 HCT15

p53

actin actin

HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

w/o HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

w/o HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

w/o HT 5Gy 5Gy+ HT

10 Gy 10 Gy+ HT

p53 (53 kDa) content


p53 (53 kDa

) content


p53 (53 kDa

) content


Figure 4. Expression of p53 in colorectal tumor cells after treatment with ionising irradiation and/or hyperthermia. The levels of the protein p53in 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. Theimmunoblot bands as well as the densitometric protein expression value corrected with the actin loading control are displayed. The expressionof 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 p53Protein 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 repräsentativeDaten von drei voneinander unabhängigen Experimenten. Gy: Gray; HT: Hyperthermie (41.5 °C für 1 Stunde); w/o: unbehandelte Kontrolle.

591Strahlenther Onkol 2010 · No. 11




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 backgroundfluorescence. The latter was subtracted from the values ob-

tained of the samples.

b d

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w/o

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PUMA PUMA

24h 24h

48h 48h

PUMA

SW480 HCT15

SW480 HCT15

PUMA

actin actin

HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

w/o HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

w/o HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

w/o HT 5Gy 5Gy+ HT

10Gy 10Gy+ 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. Theimmunoblot 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 anPUMA Protein in SW480 und HCT15 kolorektalen Tumorzellen 24 Stunden (a, c) oder 48 Stunden (b, d) nach Behandlung mit X-ray und/oder HT wurdenmit Western Blot-Technik analysiert. Die Immunoblot-Banden sowie der densitometrisch ermittelte Protein Expressionslevel, welcher mit den Wertender Aktin-Ladekontrolle korrigiert wurde, sind dargestellt. Die Expression von PUMA der unbehandelten Probe wurde auf 1 gesetzt. Die Abbildung zeigtrepräsentative Daten von drei voneinander unabhängigen Experimenten. Gy: Gray; HT: Hyperthermie (41.5 °C für 1 Stunde); w/o: unbehandelte Kontrolle.





Statistical Analyses

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 (**).

Results

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.

a c

b d

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w/o

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bax bax

24h 24h

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SW480 HCT15

SW480 HCT15

bax

actin actin

HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

w/o HT 5Gy 5Gy

+ HT

10Gy 10Gy+ HT

w/o HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

w/o HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

Bax (23 kDa)

content


Bax (23 kDa)

content


Bax (23 kDa) content


Figure 6. Expression of Bax in colorectal tumor cells after treatment with ionising irradiation and/or hyperthermia. The levels of the protein Baxin 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. Theimmunoblot bands as well as the densitometric protein expression value corrected with the actin loading control are displayed. The expressionof 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 mitWestern Blot-Technik analysiert. Die Immunoblot-Banden sowie der densitometrisch ermittelte Protein Expressionslevel, welcher mit den Werten derAktin-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.





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.

2.5

2.0

1.5

1.0

0.5

0

2.5

2.0

1.5

1.0

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0

2.5

3.0

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1.5

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0

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3.0

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1.0

0.5

0

Bcl-2 (28 kDa) content


w/o

a c

b d

actin actin

bcl-2 bcl-2

24h 24h

48h 48h

bcl-2

SW480 HCT15

SW480 HCT15

bcl-2

actin actin

HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

w/o HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

w/o HT 5 Gy 5Gy+ HT

10Gy 10Gy+ HT

w/o HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

Bcl-2 (28 kDa) con

tent

(densitometric va

lue)

Bcl-2 (28 kDa) con

tent


Bcl-2 (28 kDa) content


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. Theimmunoblot 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 anBcl-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 mitWestern Blot-Technik analysiert. Die Immunoblot-Banden sowie der densitometrisch ermittelte Protein Expressionslevel, welcher mit den Werten derAktin-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 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 activityrose 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 reacheda maximum after 72 hours. Cells treated with X-ray plus HT

reached similar activity levels of caspase-3/7, 48 hours and

1.5

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4

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0

3

4

2

1

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IRF-5 (60 kDa) content


w/o

a c

b d

actin actin

IRF-5 IRF-5

24h 24h

48h 48h

IRF-5

SW480 HCT15

SW480 HCT15

IRF-5

actin actin

HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

w/o HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

w/o HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT

w/o HT 5Gy 5Gy+ HT

10Gy 10Gy+ HT


(densitometr

ic value)

IRF-5 (60 kDa

) content




Figure 8. Expression of IRF-5 in colorectal tumor cells after treatment with ionising irradiation and/or hyperthermia. The levels of the protein IRF-5in 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. Theimmunoblot 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 mitWestern Blot-Technik analysiert. Die Immunoblot-Banden sowie der densitometrisch ermittelte Protein Expressionslevel, welcher mit den Werten derAktin-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.





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 PUMAAll treatments did not result in significant higher levels of PU-

MA in the SW480 and HCT15 tumor cells 24 hours after appli-

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

2.0

1.5

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0

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RIP-1 (74 kDa) content


w/o

a c

b d

actin actin

RIP-1

24h 24h

48h 48h

RIP-1

SW480 HCT15

SW480 HCT15

RIP-1

actin actin

HT 5 Gy 5 Gy+ HT

10 Gy 10 Gy+ HT


10 Gy 10 Gy+ HT


10 Gy 10 Gy+ HT


10 Gy 10 Gy+ HT

RIP-1


(densitometri

c value)





Figure 9. Expression of RIP-1 in colorectal tumor cells after treatment with ionising irradiation and/or hyperthermia. The levels of the protein RIP-1in 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-1in untreated cells was set to 1. The figure shows representative data from three independent experiments.Gy: Gray; HT: hyperthermia (41.5 °C for1 h); w/o: untreated control.

Abbildung 9. Expression von RIP-1 in kolorektalen Tumorzellen nach Behandlung mit ionisierender Bestrahlung und/oder Hyperthermie. Die Menge anRIP-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 mitWestern Blot-Technik analysiert. Die Immunoblot-Banden sowie der densitometrisch ermittelte Protein Expressionslevel, welcher mit den Werten derAktin-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.





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-2One 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 ledto 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 similarexpression of RIP-1 in comparison to irradiation or HT alone.

DiscussionPreoperative 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 incombination 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





permeabilization (MOMP). This means that PUMA binds

anti-apoptotic Bcl-2 proteins that sequester direct activator

BH3-only proteins, like Bid, leading to the release of Bid from

Bcl-2. Bid then directly activates Bax leading to MOMP [8].

Higher amounts of the pro-apoptotic Bax were also observed

after combined treatment of colorectal tumor cells with irra-

diation and HT (Figure 6). The amount of the anti-apoptotic

Bcl-2 was primarily increased after 24 hours in cells treated

with the combination of X-ray plus HT (Figure 7). However,

after 48 hours, the combinatory treatments resulted in slightly

lower Bcl-2 expression levels in SW480 and HCT15 cells in

comparison to irradiation only (Figure 7b and d). A novelaranorosin derivative, K050, could be a potent therapeutic

agent against Bcl-2-overexpressing human malignancies [25].

Taken together, we have shown thatthe pro-apoptotic Bcl-2 family members

Bax and PUMA are up-regulated within

cells treated with a combination of irra-

diation and HT, and the anti-apoptotic

Bcl-2 is, after an initial up-regulation,

finally down-regulated. The cells tend

towards cell death as the pro-apoptotic

proteins outbalance Bcl-2 [8].

IRF-5 is a transcription factor

known to be critical in the induction of

apoptosis after DNA damage. It sensi-

tises tumor cells to cell death whereas

silencing of IRF-5 desensitizes tumor

cells [16]. It was shown for the first time

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

show increased amounts of IRF-5 after

X-ray plus HT treatment (Figure 8b).

In contrast to apoptosis, necrosis is

been known to be a non-physiological

event. In addition, a programmed form

of necrosis, so-called necroptosis, has

recently been reported [13, 20]. The lat-

ter is dependent on the serine/threonine

kinase RIP-1 which is associated with

the death receptors. RIP-1 promotes

the mitochondrial permeability transi-

tion caused by the permeability transi-

tion pore complex (PTPC). Therefore

it induces the accumulation of prone-

crotic ceramides or binds to the adenine

nucleotide translocase, another compo-

nent of the PTPC [12]. This study illus-

trates for the first time that in colorec-

tal tumor cells the expression levels of RIP-1 are increased after application of

X-ray alone, HT alone or in combination of both. This sug-

gests that necroptosis can be induced by HT. Figure 10 gives

a schematic overview of some of the apoptosis- and necro-

sis-regulating proteins examined in our study that are up- or

down-regulated in colorectal tumor cells after treatment with

X-ray plus HT.

Taken together, both apoptotic and necrotic cell death

programs were activated in colorectal tumor cells treated with

X-ray plus HT. Because a major death form of colorectal tu-

mor cells 48 hours after a combinatory treatment with irradia-

tion and HT was necrosis, we assume that treated tumor cells

undergo secondary necrosis shortly after starting the apop-totic program. Fostering distinct tumor cell pathways and the

release of immune-activating danger signals, e.g. extracel-

X-ray plus HT

down-regulation

blocking

activation

Bax

tumor cell death

PUMA

RIP-1

IRF-5

Bcl-2

caspase3/7 p53#

a c t i v a t i o n

via mitochondrial permeability transition

Figure 10. Apoptosis and necrosis regulating proteins in colorectal tumor cells after treatmentwith ionising irradiation and/or hyperthermia. A schematic overview and possible interactionsof proteins that are involved in the apoptotic and necrotic cell death program of colorectal tu-mor cells after treatment with X-ray and/or HT is given. Up-regulation of PUMA and Bax maylead to caspase-3/7 activation. Bcl-2, after an initial up-regulation, is finally down-regulatedwhen the activity of caspases-3/7 increases (48 hours after treatment). The up-regulation of p53may lead to activation of Bax and to blocking of Bcl-2. IRF-5 may indirectly activate caspases-3/7while RIP-1 leads to cell death by promoting the mitochondrial permeability transition. #p53 ismutated in SW480 and HCT15 cells.

Abbildung 10. Apoptose und Nekrose regulierende Proteine in kolorektalen Tumorzellen nachBehandlung mit ionisierender Bestrahlung und/oder Hyperthermie. Ein schematischer Über-blick und mögliche Interaktionen von Proteinen, welche an apoptotischen und nekrotischenZelltodesprogrammen in kolorektalen Tumorzellen nach Behandlung mit ionisierender Be-strahlung und/oder Hyperthermie beteiligt sind, sind dargestellt. Die Hochregulierung vonPUMA und Bax können zur Aktivierung von Caspasen3/7 führen. Bcl-2 wird nach einer anfäng-lichen Hochregulation schließlich herunterreguliert, wenn die Aktivität an Caspasen3/7 steigt(48 Stunden nach Kombinationsbehandlung). Die Hochregulierung von p53 kann zur Aktivie-rung von Bax und zur Blockade von Bcl-2 führen. IRF-5 kann indirekt durch Caspasen3/7 aktiviertwerden wohingegen RIP-1 zum Zelltod führt indem die Ausbildung der mitochondrialen Perme-abilitäts-Transitions-Pore gefördert wird. #p53 ist mutiert in SW480 und HCT15 Zellen.





lular heat-shock proteins [24] and HMGB1 [29], contributeto 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.

AcknowledgementsThis work was supported by the ELAN Fond (ST-08.06.30.1) of the Friedrich-Alexander University of Erlangen-Nuremberg, by theEuropean Commissions (NOTE (TPA4 FP6)), and by the GermanResearch Foundation (Graduate School of the SFB 643).

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Address for CorrespondencePriv.-Doz. Dr. Udo S. Gaipl, PhDDepartment of Radiation OncologyRadiation ImmunobiologyUniversity Hospital ErlangenFriedrich-Alexander University of Erlangen-NürnbergUniversitätsstr. 2791054 ErlangenGermanyPhone: (+49) 9131-85-32311, Fax -39335e-mail: [email protected]


Ionising Irradiation and Hyperthermia

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