Proton induces apoptosis of hypoxic tumor cells by the p53-dependent and p38/JNK MAPK signaling pathways.
Tumor hypoxia is a main obstacle for radiation therapy. To investigate whether exposure to a proton beam can overcome radioresistance in hypoxic tumor cells, three kinds of cancer cells, Lewis lung carcinoma (LLC) cells, hepatoma HepG2 and Molt-4 leukemia cells, were treated with a proton beam (35 MeV, 1, 2, 5, 10 Gy) in the presence or absence of hypoxia. Cell death rates were determined 72 h after irradiation. Hypoxic cells exposed to the proton beam underwent a typical apoptotic program, showing condensed nuclei, fragmented DNA ladders, and poly-ADP-ribose polymerase (PARP) cleavage. Fluorescence-activated cell sorter analysis revealed a significant increase in Annexin-V-positive cells. Cells treated with the proton beam and hypoxia displayed increased expression of p53, p21 and Bax, but decreased levels of phospho-Rb, Bcl-2 and XIAP, as well as activated caspase-9 and -3. The proton beam with hypoxia induced cell death in wild-type HCT116 cells, but not in a p53 knockout cell line, demonstrating a requirement for p53. As reactive oxygen species (ROS) were also significantly increased, apoptosis could also be abolished by treatment with the anti-oxidant N-acetyl cysteine (NAC). P38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) were activated by the treatment, and their respective DN mutants restored the cell death induced by either proton therapy alone or with hypoxia. In conclusion, proton beam treatment did not differently regulate cancer cell apoptosis either in normoxic or hypoxic conditions via a p53-dependent mechanism and by the activation of p38/JNK MAPK pathways through ROS.
Lee
Kheun Byeol
KB
School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Korea.
Kim
Kye-Ryung
KR
Huh
Tae-Lin
TL
Lee
You Mie
YM
D000818:Animals; D017209:Apoptosis / Q000528:radiation effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D018827:Carcinoma, Lewis Lung; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000528:radiation effects; D015687:Cell Hypoxia; D004307:Dose-Response Relationship, Radiation; D005720:Gamma Rays; D045325:HCT116 Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D051379:Mice; D009369:Neoplasms / Q000201:enzymology / Q000473:pathology*; D018384:Oxidative Stress / Q000528:radiation effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000528:radiation effects*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D017382:Reactive Oxygen Species; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020169:Caspases