A_3 Adenosine Receptor Mediates Apoptosis in 5637 Human Bladder Cancer Cells by G_q Protein/PKC-Dependent AIF Upregulation

摘要

Background/Aims: A_3 adenosine receptor mediates apoptosis in a variety of cancer cells via diverse signaling pathways. The present study was conducted to assess A_3 adenosine receptor-mediated apoptosis in human bladder cancer cell lines and to understand the underlying mechanism. Methods: Human bladder cancer cell lines such as 253J, 5637, KK-47, TCCSUP, T24, and UMUC-3 cells were cultured. The siRNA to silence the A_3 adenosine receptor-targeted gene was constructed and transfected into cells. MTT assay, TUNEL staining, Western blotting, and real-time RT-PCR were carried out. Results: For all the investigated cell types adenosine induced apoptosis in a concentration (0.01-10 mM)- and treatment time (24-48 h)-dependent manner. Adenosine-induced 5637 cell death was significantly inhibited by the A3 adenosine receptor inhibitor MRS1191 or knocking-down A_3 adenosine receptor, and the A_3 adenosine receptor agonist 2-Cl-IB-MECA mimicked the adenosine effect. The adenosine effect was prevented by GF109203X, an inhibitor of protein kinase C (PKC), but it was not affected by forskolin, an activator of adenylate cyclase. Adenosine-induced 5637 cell death, alternatively, was not inhibited by the pan-caspase inhibitor Z-VAD. Adenosine upregulated expression of apoptosis-inducing factor (AIF), that is suppressed by knocking-down A_3 adenosine receptor, and accumulated AIF in the nucleus. Conclusion: The results of the present study show that adenosine induces 5637 cell apoptosis by upregulating AIF expression via an A_3 adenosine receptor-mediated G_q protein/PKC pathway.