Modulation of heat‐induced cell death in PC‐3 prostate cancer cells by the antioxidant inhibitor diethyldithiocarbamate

Abstract

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). Materials and methods PC-3 cells were treated with thermotherapy at 42, 43 or 44uC 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 44uC for 60 min. Results Cell survival was significantly lower after heating cells at 43uC for 60, 90 and 120 min and at 44uC for all periods tested (P<0.05). At 43uC apoptosis increased with the duration of heating and was similarly enhanced after heating at 44uC for 30 min. Necrosis was not increased by heating at 42 or 43uC, but was markedly enhanced after heating at 44uC with both the duration of heating and with time after heating. Significant increases in DCF production were induced by heating at 43uC for 60, 90 and 120 min (P<0.05) and at 44uC 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 43uC, with significant differences among cells heated for 60, 90 and 120 min (P<0.05). After heating at 44uC, 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 43uC for 90 and 120 min (P<0.05) and at 44uC for 30 and 60 min (P<0.05 and <0.025, respectively). Hsp 70 levels decreased after heating at 44uC 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.

Measurement and mathematical modeling of thermally induced injury and heat shock protein expression kinetics in normal and cancerous prostate cells

“…At lower temperatures, the rates and magnitudes of all HSP expression diminished proportionally. Although our study focused on higher heating temperatures (44 À60 C) than prior studies, our results correspond closely to previous studies where Hsp70 expression was elevated in PC3 cell monolayer following mild heat shock (43 C) [68,69]. Very few papers exist that investigate thermally induced Hsp27 or Hsp60 expression in PC3 cells.…”

Selective hyperthermia using magnetoliposomes to target cervical lymph node metastasis in a rabbit tongue tumor model

“…Moriyama et al found from their in vitro study using PC-3 prostate cancer cells that heating at 43°C increased apoptosis proportionally to the duration of heating; heating for up to 60 min at 44°C increased both apoptosis and necrosis, while longer heating at 44°C mainly increased necrosis. 23) As shown in Figs. 4 and 5, our quantitative assay of apoptosis and necrosis 24 h after hyperthermia at 43-44.5°C for approximately 30 min revealed that both apoptosis and necrosis were induced.…”

Regulation of the multidrug resistance transporter P‐glycoprotein in multicellular prostate tumor spheroids by hyperthermia and reactive oxygen species

“…In this respect, it has been demonstrated that in large tumor spheroids overexpressing P-glycoprotein low levels of ROS downregulated P-glycoprotein, whereas in small tumor spheroids, which are devoid of an MDR phenotype, oxidative stress induced upregulation of P-glycoprotein. 16,17 Heat incubation-induced oxidative stress has been recently reported for human T lymphocytes, 51 rat cardiomyocytes, 52 BC-8 macrophages 36, PC-3 prostate cancer cells 53 and HL-60 cells 24 ; however the source of ROS has not yet been identified. In our study hyperthermia treatment of tumor spheroids resulted in a robust increase of ROS presumably through the activity of an NADPH-oxidase, which has recently been demonstrated by us to be highly expressed in small tumor spheroids but downregulated with the induction of cell quiescence and MDR in large tumor spheroids.…”