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Intracellular signal pathway of heat-induced apoptosis in human lung cancer cell lines

人肺癌细胞系热诱导凋亡的胞内信号通路

基本信息

批准号：
12671334
负责人：
OTSUKI Yoshinori
金额：
$ 0.9万
依托单位：
Osaka Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

This study was designed to detect heat-induced apoptosis in non-small human lung cancer cells with two different histological types and to elucidate its signaling. We analyzed the chronological changes in intracellular Ca^<2+> concentrations ([Ca^<2+>]_i) during heat-induced apoptosis in human lung cancer cell lines LK-2 (squamous cell carcinoma) and LU65A Gage cell carcinoma). In LK-2 cells, increased [Ca^<2+>]_i levels were maintained at levels between 250-350 nM 9 h after heat-shock. Treatment with BAPTA, an intracellular Ca^<2+> chelator, prior to heat-shock decreased the fequency of heat-induced apoptosis in LK-2, while thapsigargin, a selective endoplasmic reticulum Ca^-ATPase inhibitor did not change the number of apoptotic cells, regardless of the presence or absence of Ca^<2+>-supplemented medium. In LU65A cells, treatment with BAPTA or thapsigargin did not alter the apoptotic rates. Western blotting demonstrated that although expression of Bax and Bcl-2 were not changed by heat-shock, P53 expession was elevated in LK-2, but not LU65A cells. Immunohistochemistry showed that p53 was localized predominantly in the cytoplasmic of LK-2 cells, suggesting that p53 protein is not functional in LK-2. Heat-shock also elevated caspases 3, 8 and 9 activity in both cell lines. We conclude that a temporal increase in [Ca^<2+>]_i is the important initiating factor in hyperthermia-induced apoptosis in LK-2 cells and that, in these two lung cancer cell lines, apoptosis may occur through 'cross-talk' between P53-independent mitochondrial and death receptor pathways.

本研究旨在检测两种不同组织学类型的非小细胞肺癌细胞中热诱导的凋亡，并阐明其信号转导。我们分析了人肺癌细胞株LK-2（鳞状细胞癌）和LU 65 A（Gage细胞癌）在热诱导凋亡过程中细胞内Ca^<2 +>浓度（[Ca^<2+]_i）的时间变化。在LK-2细胞中，在热休克后9小时，增加的[Ca^<2+>]_i水平维持在250-350 nM之间的水平。在热休克前用BAPTA（一种细胞内Ca^2+螯合剂）处理LK-2可降低热诱导凋亡的频率，而毒胡萝卜素（一种选择性内质网Ca^-ATP酶抑制剂）则不改变凋亡细胞的数量，无论是否存在补充Ca^2+的培养基。在LU 65 A细胞中，用BAPTA或毒胡萝卜素处理不改变凋亡率。Western blotting结果显示，热休克处理后LK-2细胞Bax和Bcl-2蛋白表达无明显变化，但P53蛋白表达明显升高，而LU 65 A细胞则无明显变化。免疫组化显示p53主要定位于LK-2细胞的胞质中，表明p53蛋白在LK-2中没有功能。热休克也提高了这两种细胞系中的半胱天冬酶3、8和9的活性。我们的结论是，[Ca^<2+>]_i的时间增加是高血压诱导的LK-2细胞凋亡的重要起始因素，在这两种肺癌细胞系中，凋亡可能通过P53非依赖性线粒体和死亡受体途径之间的“串扰”发生。