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

本课题研究了V-ATP酶抑制剂如巴弗洛霉素、伴卡那霉素和灵菌菌素促进神经突起生长（NOG）、细胞分化、生长停滞和凋亡的机制，以及V-ATP酶的质子转运机制，发现：（1）V-ATP酶抑制剂如巴弗洛霉素以新的RNA、蛋白质合成和丝氨酸/苏氨酸激酶依赖方式诱导细胞凋亡和NOG;和K-252 a-或A-激酶-独立的方式，但它们彼此不同：细胞凋亡诱导与NOG诱导的不同之处在于其对酪氨酸磷酸酶、花生四烯酸级联反应或钙调蛋白激酶的不敏感性。（2）灵菌红素也能诱导细胞凋亡和NOG，但它们不是由其H^+/Cl^-共转运活性诱导的，（3）NH_4Cl不诱导细胞凋亡或NOG，细胞内外pH差不诱导细胞凋亡或NOG。（4）成功地合成了具有V-ATP酶抑制活性的康卡那霉素A光亲和探针。(5)灵菌红素是一种H^+/Cl^-共转运体，能使ATP酶如F-、V-、P-ATP酶和电子传递活性解偶联，其中灵菌红素抑制质子传递，但不抑制ATP水解（或电子传递）活性。灵菌红素还能强烈地可逆地解偶联（H^+/K^+）ATP酶依赖的猪胃粘膜质子转位。(6)我们成功地克隆了嗜热栖热菌（Thermus termophilus）的V-ATP酶基因，并对其质子泵依赖的ATP合成和操纵子结构进行了研究。

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).