第３回 国際癌治療増感研究協会
国際研究奨励賞

＜研究テーマ＞

Molecular cellular mechanisms of the nitroxide(TEMPO)-derived sensitization of hyperthermia-induced apoptosis

趙　　慶　利（Qing-Li ZHAO)　氏

富山医科薬科大学医学部放射線基礎医学教室（中国）

＜Brief Career History＞

I finished my doctor of medicine degree at Harbin Medical University in 1985. After graduation I worked in the university hospital's radiology department for 8 years until year 1993. For two years I went on research training at Kobe University, after which I started my graduate studies in the same university under the radiological sciences department. After finishing my doctoral degree (PhD) I started my work as research associate at the radiological sciences department of Toyama Medical and Pharmaceutical University and continuously work here up to the present. 　　　　　　　

＜Title of Study＞

Molecular cellular mechanisms of the nitroxide (TEMPO)-derived sensitization of hyperthermia-induced apoptosis

＜Purpose＞

Hyperthermia is known to induce apoptosis of a variety of normal and cancer cells. Nitroxides have been shown to possess the SOD-mimic anti-oxidant and protective activity against superoxide and other reactive oxygen species (ROS) in culture cells and animals. Unexpectedly, however, results of our ongoing research suggest that appropriate concentrations of a nitoroxide TEMPO potentiate strongly hyperthermia-induced apoptosis of U937 lymphoblastic leukemia cells in culture. Since my finding is novel based on the literature search, the main purpose is to delineate the molecular and cellular mechanisms by which Tempo enhances heat-induced apoptosis in a model cell system, and to contribute to opening of a new field of hyperthermic oncotherapy.

＜Methods＞

(1) Quantitative induction of U937 cell apoptosis by treatments with either TEMPO (1~10 mM at 37｡C for 10-30 min), 44｡C heating for 10-30 min, or Tempo-plus-heating (Annexin V-FITC/propidium iodide, DNA fragmentation). (2) Detection of Activation of the c-Jun-terminal kinase (JNK) pathway and the effects of JNK inhibitors on the induced apoptosis (3) Detection of mitochondrial changes: Cytochrome c release from mitochondria, mitochondrial membrane potential (DYm), Western blot analysis of pro- and anti-apoptotic Bcl-2 family proteins, and mitochondrial ROS production, changes in ATP levels. (4) Distinction the apoptogenic mechanism by the heat/TEMPO-induced death signaling to cytochrome c/caspase cascade or by the heat/TEMPO-induced mitochondrial dysfunction.

＜Expected Result＞

A 10 min treatment with 1-5 mM TEMPO at 37｡C or suboptimal heating (44｡C/10 min) alone had no effect on the apoptosis and its related events in U937 cells. However, the combined treatment with 5 mM TEMPO and 44｡C for 10 min elicited ~90% apoptosis in 6 h, as did the 44｡C/30 min heating. The enhanced apoptosis was time-dependent, through a mitochondrial dysfunction such as progressive increase in low-DY, decrease of superoxide, release of cytochrome c without altered cellular expression of Bcl-2, Bcl-XL and Bax in the whole cell lysates. In early time after the combined treatment, JNK1 was activated moderately. Thus, these results indicate a new finding that Tempo sensitizes greatly the heat-induced apoptosis through a unique mitochondrial mechanism.

＜Study Plan in the Future＞

To make clear the molecular mechanism in the above model system, I further study (i) why cytochrome c is released without altered expression of Bcl-2 proteins that regulate cytochrome c release and apoptosis by searching the targeting of activated JNK or BH3-only proapoptotic Bcl-2 family proteins to mitochondria, (ii) why antioxidant Tempo, when combined with suboptimal heating (44｡C/10 min) and optimal heating (44｡C/30 min), causes apoptotic events such as the mitochondrial respiratory and ATP-generating dysfunction that are intimately related with apoptosis and necrosis . These work-out will establish a new field for hyperthermia oncotherapy.

＜連絡先＞

Qing-Li ZHAO　　　　　　　　　　　　　　　
Toyama Medical and Pharmaceutical University
2630 Sugitani, Toyama 930-0194 Japan
TEL: 076-434-7267
FAX: 076-434-5190