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.

Abstract

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.