Combination of ionising irradiation and hyperthermia activates programmed apoptotic and necrotic cell death pathways in human colorectal carcinoma cells.
OBJECTIVE
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.


METHODS
The expression of proteins was analysed by western blot and the relative activity of caspases-3/7 by fluorescence- based assay. Colony formation was analysed using the clonogenic assay and cell death was determined with annexin V-FITC/propidium 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. Combining 5 Gy irradiation with HT compared to irradiation resulted in a significantly increased number of necrotic tumor cells and in decreased colony formation.


CONCLUSIONS
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.
Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen, Germany.
Frederick
F
Mantel
Benjamin
B
Frey
Stefan
S
Haslinger
Petra
P
Schildkopf
Renate
R
Sieber
Oliver J
OJ
Ott
Barbara
B
Lödermann
Franz
F
Rödel
Rolf
R
Sauer
Rainer
R
Fietkau
Udo S
US
Gaipl
D017209:Apoptosis / Q000528:radiation effects*; D051017:Apoptosis Regulatory Proteins / Q000032:analysis; D015153:Blotting, Western; D053148:Caspase 3 / Q000032:analysis; D053179:Caspase 7 / Q000032:analysis; D016923:Cell Death / Q000528:radiation effects*; D015179:Colorectal Neoplasms / Q000473:pathology*; D003131:Combined Modality Therapy; D004307:Dose-Response Relationship, Radiation; D004789:Enzyme Activation / Q000528:radiation effects; D006801:Humans; D006979:Hyperthermia, Induced; D009336:Necrosis; D014411:Neoplastic Stem Cells; D011518:Proto-Oncogene Proteins / Q000032:analysis; D051560:Proto-Oncogene Proteins c-bcl-6 / Q000032:analysis; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000032:analysis; D051028:bcl-2-Associated X Protein / Q000032:analysis
D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; C438378:BBC3 protein, human; D011518:Proto-Oncogene Proteins; D051560:Proto-Oncogene Proteins c-bcl-6; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D053148:Caspase 3; D053179:Caspase 7