Otsuki Tai-ichiro, Kanno Takeshi, Fujita Yumiko, Tabata Chiharu, Fukuoka Kazuya, Nakano Takashi, Gotoh Akinobu, Nishizaki Tomoyuki
Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, Nishinomiya, Japan.
Cell Physiol Biochem. 2012;30(1):210-20. doi: 10.1159/000339058. Epub 2012 Jun 19.
BACKGROUND/AIMS: A(3) adenosine receptor mediates apoptosis in cancer cells via diverse signaling pathways. The present study examined A(3) adenosine receptor-mediated apoptosis in Lu-65 cells, a human giant cell lung carcinoma cell line.
MTT assay, TUNEL staining, real-time RT-PCR, Western blotting, and assay of caspase-3, -8, and -9 activities were carried out in Lu-65 cells, and A(3) adenosine receptor or p53 was knocked-down by transfecting each siRNA into cells.
Extracellular adenosine induces Lu-65 cell apoptosis in a concentration (0.01-10 mM)-dependent manner, and the effect was inhibited by the A(3) adenosine receptor inhibitor MRS1191 or by knocking-down A(3) adenosine receptor or p53. Like adenosine, the A(3) adenosine receptor agonist 2-Cl-IB-MECA also induced Lu-65 cell apoptosis. Adenosine upregulated expression of p53 and Noxa mRNAs and activated caspase-3 and -9, but not caspase-8. Those adenosine effects were still inhibited by knocking-down A(3) adenosine receptor or p53.
The results of the present study show that adenosine upregulates p53 expression via A(3) adenosine receptor, to promote p53-dependent Noxa gene transcription, causing activation of caspase-9 and the effector caspase-3 to induce Lu-65 cell apoptosis.
背景/目的：A(3)腺苷受体通过多种信号通路介导癌细胞凋亡。本研究检测了A(3)腺苷受体介导的人巨细胞肺癌细胞系Lu-65细胞凋亡情况。
对Lu-65细胞进行MTT检测、TUNEL染色、实时逆转录聚合酶链反应、蛋白质印迹法以及半胱天冬酶-3、-8和-9活性检测，并通过将每种小干扰RNA转染到细胞中来敲低A(3)腺苷受体或p53。
细胞外腺苷以浓度（0.01 - 10 mM）依赖性方式诱导Lu-65细胞凋亡，且该效应被A(3)腺苷受体抑制剂MRS1191或敲低A(3)腺苷受体或p53所抑制。与腺苷一样，A(3)腺苷受体激动剂2-Cl-IB-MECA也诱导Lu-65细胞凋亡。腺苷上调p53和Noxa mRNA的表达并激活半胱天冬酶-3和-9，但不激活半胱天冬酶-8。敲低A(3)腺苷受体或p53仍可抑制那些腺苷效应。
本研究结果表明，腺苷通过A(3)腺苷受体上调p53表达，促进p53依赖的Noxa基因转录，导致半胱天冬酶-9和效应半胱天冬酶-3激活，从而诱导Lu-65细胞凋亡。
