Modulation of heat-induced cell death in PC-3 prostate cancer cells by the antioxidant inhibitor diethyldithiocarbamate.
OBJECTIVE
To examine the relationships between the form of cell death (apoptosis or necrosis), reactive oxygen species (ROS) generation, superoxide dismutase (SOD) activity and the level of heat-shock protein 70 (hsp 70) expression after thermotherapy of PC-3 prostate cancer cells; also assessed were the tumoricidal effects of combined treatment with both heat and the antioxidant inhibitor diethyldithiocarbamate (DDC).


METHODS
PC-3 cells were treated with thermotherapy at 42, 43 or 44 degrees C for 30, 60, 90 or 120 min. Cell proliferation, ROS generation, SOD activity and cellular hsp 70 level were determined using tetrazolium-based cytotoxicity, fluorescent dichlorofluorescein (DCF) and nitroblue tetrazolium assays, Western blot analysis and flow cytometry, respectively. The apoptotic and necrotic cells were determined by staining with propidium iodide and fluorescein isothiocyanate-labelled annexin V. These variable were also measured after combined treatment of PC-3 cells with 1 mmol/L DDC and thermotherapy at 43 or 44 degrees C for 60 min.


RESULTS
Cell survival was significantly lower after heating cells at 43 degrees C for 60, 90 and 120 min and at 44 degrees C for all periods tested (P<0.05). At 43 degrees C apoptosis increased with the duration of heating and was similarly enhanced after heating at 44 degrees C for 30 min. Necrosis was not increased by heating at 42 or 43 degrees C, but was markedly enhanced after heating at 44 degrees C with both the duration of heating and with time after heating. Significant increases in DCF production were induced by heating at 43 degrees C for 60, 90 and 120 min (P<0.05) and at 44 degrees C at all times (P<0.010-0.005). There was a significant correlation between the level of ROS generation and necrosis (P<0.001) but no correlation between the ROS level and apoptosis. SOD activity increased in cells after heating at 43 degrees C, with significant differences among cells heated for 60, 90 and 120 min (P<0.05). After heating at 44 degrees C, SOD activity was maximal in cells heated for 30 min (P<0.005), by 30 min and then decreased with time after heating. There were significant increases in hsp 70 level in cells heated at 43 degrees C for 90 and 120 min (P<0.05) and at 44 degrees C for 30 and 60 min (P<0.05 and <0.025, respectively). Hsp 70 levels decreased after heating at 44 degrees C for 90 and 120 min. The combination of DDC and heating significantly increased ROS generation and the percentage of cell death, and decreased SOD activity (P<0.05).


CONCLUSIONS
These findings show a qualitative change in the form of cell death induced by thermotherapy of PC-3 cells, which changed from apoptosis to necrosis according to the degree and duration of heating. Mild thermotherapy induced marginally low occurrence of apoptosis of PC-3 cells and DDC may represent a useful future strategy for the treatment of prostate carcinoma.
Moriyama-Gonda
N
N
Department of Urology, Shimane Medical University, Izumo, Japan. gonada-n@shimane-med.ac.jp
Igawa
M
M
Shiina
H
H
Urakami
S
S
Shigeno
K
K
Terashima
M
M
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D000975:Antioxidants / Q000378:metabolism*; D017209:Apoptosis; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival; D003131:Combined Modality Therapy; D004050:Ditiocarb / Q000494:pharmacology* / Q000627:therapeutic use*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D006979:Hyperthermia, Induced / Q000379:methods*; D008297:Male; D009336:Necrosis; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D017382:Reactive Oxygen Species / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D000975:Antioxidants; D018840:HSP70 Heat-Shock Proteins; D017382:Reactive Oxygen Species; D004050:Ditiocarb