Arsenic Trioxide-Induced Apoptosis and Its Enhancement by Buthionine Sulfoximine in Hepatocellular Carcinoma Cell Lines

We treated four hepatocellular carcinoma cell lines, HLE, HLF, HuH7, and HepG2 with ATO and demonstrated that arsenic trioxide (ATO) at low doses (1–3 μM) induced a concentration-dependent suppression of cell growth in HLE, HLF, and HuH7. HLE cells underwent apoptosis at 2 μM ATO, which was executed by the activation of caspase-3 through the mitochondrial pathway mediated by caspase-8 activation and Bid truncation. When these cell lines were exposed to ATO in combination with l- S,R-buthionine sulfoximine (BSO) which inhibits GSH synthesis, a synergistic growth suppression was induced, even in HepG2 showing a lower sensitivity to ATO than other cell lines tested. The intracellular GSH levels after the treatment with ATO plus BSO were considerably decreased in HLE cells compared with those after the treatment with ATO or BSO alone. The production of reactive oxygen species (ROS) which was examined by 2′,7′-dichlorodihydrofluorescein diacetate, increased significantly after the treatment with ATO plus BSO in HLE cells. These findings indicate that ATO at low concentrations induces growth inhibition and apoptosis, and furthermore that the ATO-BSO combination treatment enhances apoptosis through increased production of ROS in hepatocellular carcinoma cells.