Search for cell-death inducing antitumor agents based on the anti-tumor activity of V-ATPase inhibitors

在本课题中，我们研究了V-ATPase的抑制剂，如巴非霉素、刀豆素和灵芝菌素对轴突生长、细胞分化、生长抑制和细胞凋亡的作用机制，以及V-ATPase的质子转运机制。我们发现：(1)巴非霉素等V-ATPase抑制剂在新的RNA、蛋白质合成、丝氨酸/苏氨酸激酶依赖的甘露聚糖酶和K-252A或A-激酶非依赖性途径中诱导细胞凋亡和NOG，但它们不同于NOG诱导的不同于NOG-诱导的是对酪氨酸磷酸酶的敏感性，(2)细胞的凋亡和NOG也被灵芝菌素诱导，但不是由其H~+/Cl~-传递活性所诱导的；(3)细胞内外的pH差不能诱导细胞的凋亡或NOG，因为它们不是被NH_4CL诱导的。(4)我们成功地合成了具有V-ATPase抑制活性的刀豆素A光亲和探针。(5)原球菌素是一种H~(++)/C~(2+)转运体，具有解偶联的ATPase，如F-、V-、P-ATPase和电子传递活性，其中抑制质子传递，但不具有ATP水解(或电子传递)活性。猪胃粘膜的质子转运也强烈且可逆地解偶联(H^+/K^+)ATPase。(6)成功地克隆了真细菌(Thermus Term Ophilus)V-ATPase的质子泵依赖性的ATP合成，并测定了其操纵子结构。

In this project, we have investigated the mechanism of incuction of neurite outgrowth (NOG), cell differentiation, growth inhibiton and apoptosis by inhibitors against V-ATPases, such as bafilomycin, concanamycins and prodigiosins, and the mechanism of proton transport in V-ATPases.We found that (1) V-ATPase inhibitors like bafilomycin induced apoptosis and NOG in new RNA-, protein-syntheses, and serine/threonine kinase-dependent mannaer, and K-252a- or A-kinase-independent manner, but they are different from each other : apoptosis-induction is different from NOG-induction in its insensitivitiy in tyrosin-phosphatase, arachidonate-cascade or Calmodulin-kinase independent manner, (2) apoptosis and NOG are also induced by prodigiosins but they are not induced by its H^+/Cl^- symporting activities, and (3) apoptosis or NOG is not induced by pH-differences between inside and outside of cells, because they are not induced by NH_4Cl, (4) We have succeeded in the synsesis of concanamycin A-photoaffinity probes active in V-ATPase-inhibition. (5) Prodigiosins are H^+/Cl^- symporters that uncoupled ATPases like F-, V-, P-ATPase and electron transport activity, in which prodigiosins inhibited proton-transport but no ATP hydrolysis (or electron transport) activiteis. Prodigiosins also strongly and reversibly uncoupled (H^+/K^+) ATPase-dependent proton-translocation from hog gastric mucosa. (6) We have succeeded in the cloning, proton-pump dependent ATP synthesis, and determation of operon structure of V-ATPase from a eubacterium (Thermus termophilus).