Arsenic Trioxide (As₂O₃) Mediated Cell Death in Hodgkin Lymphoma (HL) and Non-Hodgkin Lymphoma (NHL): Investigation of Reactive Oxygen Species (ROS), Caspase, and MAP Kinase Signaling Pathways.

The cytotoxic activity of As₂O₃ in leukemia and myeloma has been shown to be mediated in part by ROS. We determined the mechanism of cytotoxicity of As₂O₃ in HL and NHL cell lines. Ramos, HF-1, SUDHL4 (NHL lines), and L428 (HL line) cells were cultured with increasing clinically relevant As₂O₃ concentrations (1.0mm-10mm) at 24-72 hours with and without the oxidizing agent, buthionine sulfoxime (BSO, 100mm), and with and without caspase inhibitors Z-VAD-FMK and Z-IETD-FMK. Apoptosis was measured by Annexin-V/propidium iodide (PI) staining and detected by flow cytometry (FACS). ROS was measured by oxidation of 2′7′ dichlorofluorescein diacetate (H₂DCFDA) to Dichlorofluorescein (DCF) and analyzed by FACS. Immunoblots were performed for bcl-2, PARP, cleaved caspases 3, 8, 9, and mitogen activated protein kinase (MAPK) pathways (ERK, JNK, and p38 activation). As₂O₃ alone at 10mM resulted in dose- and time-dependent apoptosis in all cell lines (HL and NHL) with >75% annexin⁺/PI⁺ between 48 and 72 hours. In Ramos and L428 cells, a combination of As₂O₃ (2 mM) and BSO 100mM resulted in >80% annexinV⁺/PI⁺, while either agent alone resulted in <15% apoptosis (p=0.001). A four-fold increase in ROS was seen in all four cell lines with As₂O₃/BSO, but not with As₂O₃ alone. Both ROS production and apoptosis induced by As₂O₃/BSO and As₂O₃ alone were reversible with the anti-oxidant N-acetylcysteine (NAC) (10mM). Furthermore, As₂O₃/BSO induced PARP cleavage and caspase-3 activation in all NHL cell lines, but not in L428. In Ramos, Z-VAD-FMK and Z-IETD-FMK blocked As₂O₃-induced apoptosis (suggesting a caspase-dependent mechanism), but had no effect on As₂O₃/BSO-induced cell death (caspase-independent). To investigate the signaling pathways involved, we performed western blot analysis for phospho-p38 (p-p38), phospho-JNK and phospho-ERK (p-ERK) in Ramos and L428 cells following As₂O₃ alone and As₂O₃/BSO. We found up-regulation of p-p38 in Ramos cells, while in L428, p-ERK was activated following As₂O₃ and As₂O₃/BSO. Inhibitors of p-38 in Ramos cells resulted in up-regulation of ERK. In summary, As₂O₃ induced dose- and time-dependent apoptosis in HL and NHL cell lines was significantly enhanced with the addition of BSO, and inhibited by NAC, suggesting that apoptosis was in part ROS-dependent. In Ramos, As₂O₃ alone resulted in caspase-dependent apoptosis, while addition of BSO resulted in caspase-independent apoptosis. As₂O₃ alone and combined with BSO resulted in activation of MAPK pathways in HL and NHL lines. These data provide a basis and a mechanism for As₂O₃-induced cell death in HL and NHL and have therapeutic implications.