The Molecular Mechanisms Underlying Adenosine-induced Apoptosis In BEL-7404 Liver Cancer Cells

[Background]Adenosine, chemically a purine nucleoside that is abundantly present inside and outside cells, exhibits a variety of modulatory actions in our whole body, especially in the cardiovascular and central nervous systems. It is reported to suppress cell growth through apoptosis in various cancer cells via diverse extrinsic and intrinsic signaling pathways, which made it a potential molecular target for anti-cancer drug design.Mitochondria, the major sites for ATP generation and Reactive Oxygen Species (ROS) production, are crucial for energy production, intermediary metabolism and oxidative phosphorylation. Accumulating evidence has shown that mitochondria are the most important sensors for apoptosis.Our previous studies showed that extracellular adenosine induces Caspase-dependent apoptosis in Huh-7 and HepG2 human liver cancer cells by activating Caspase-8/9/3. Also, adenosine induces Caspase-independent apoptosis in Huh-7 cells via up-regulating AMID expression. And it is reported that some chemotherapeutic drugs induce apoptosis of liver cancer cells through ROS generation. However, it remains unknown whether adenosine induces cell apoptosis via the mitochondrial pathway in human liver cancer cells and what role do ROS play in the process of adenosine-induced apoptosis.[Aims]To examine whether adenosine inhibits the growth of liver cancer cells via the mitochondria-dependent apoptotic pathway and investigate the role of ROS in the adenosine-induced apoptosis, with the aim of further elucidating the new mechanisms underlying adenosine-induced apoptosis in tumor cells.[Methods]1. The effects of adenosine on cell viability of 7404 cells were identified by the CCK-8 assay and crystal violet staining.2. Cell cycle profile of the cells was examined by PI staining and FACS analysis.3. Adenosine-induced apoptosis was determined by means of Annexin V-FITC/PI double staining.4. To understand molecular mechanism underlying the apoptosis, the activation of Caspase and Bcl-2 family proteins, the effects of adenosine on mitochondrial membrane potential (MMP) and mitochondria-related protein release were examined using Western blot, JC-1 dye staining and subcellular fractionation techniques.5. The effect of adenosine on intracellular ROS levels was evaluated by flow cytometry analysis.6. Cells were treated with adenosine in the absence or presence of NAC and subjected to ROS detection, apoptosis assay, Western blot analysis of expression of Cytochrome C and JC-1 assay.[Results]1. Extracellular adenosine significantly reduced 7404 cell viability in a dose-and time-dependent manner.2. Adenosine induced G2/M cell cycle arrest and Sub-G1 phase occurrence in 7404 cells.3. Adenosine induced apoptosis in 7404 cells, predominantly late stages of apoptosis.4. Adenosine induced accumulation of Bak, decreased Bcl-xL and Mcl-1, subsequently caused the mitochondrial dysfunction and the release of DIABLO, Cytochrome C and AIF from mitochondria to cytoplasm in the cells, responsible for Caspase-9 activation, eventually induced apoptosis in BEL-7404 liver cancer cells.5. Adenosine significantly increased ROS production in a time-and dose-dependent manner.6. ROS inhibitor, NAC could block adenosine-induced MMP loss, Cytochrome C release and cell apoptosis.[Conclusions]Our study demonstrated that adenosine inhibits BEL-7404 cell growth by inducing cell apoptosis via both the mitochondrial and death receptor pathways. Moreover, our study first observed that adenosine increases ROS production in tumor cells and revealed the involvement of ROS-mediated mitochondrial dysfunction in adenosine-induced apoptosis, which provide additional evidence that adenosine could act as an effective anticancer agent for targeted cancer therapy.