Glutathione depletion enhances arsenic trioxide-induced apoptosis in lymphoma cells through mitochondrial-independent mechanisms.

Arsenic trioxide (ATO) is an effective therapeutic agent for acute promyelocytic leukaemia (APL) (Evens et al, 2004). In APL, ATO induces differentiation at low concentrations, while inducing apoptosis at higher concentrations (Miller et al, 2002). In addition, ATO-induced apoptosis in APL is mediated through the mitochondrial apoptotic pathway, resulting in part from the production of reactive oxygen species (ROS), such as hydrogen peroxide (Dai et al, 1999; Yi et al, 2002). High intracellular levels of glutathione (GSH) confer resistance to ATO partly through the detoxification of ROS. Compounds that promote ROS and/or deplete protective metabolites such as GSH may sensitize tumour cells to oxidative cytolysis. Buthionine sulfoximine (BSO), a selective inhibitor of gamma glutamylcysteine synthetase, is known to effectively deplete cellular GSH (Gartenhaus et al, 2002; Davison et al, 2003). We evaluated herein the cytotoxic activity and cell death pathways induced by ATO alone and combined with BSO in non-Hodgkin lymphoma (NHL) cell lines and primary lymphoproliferative cells. With ATO 10 lmol/l, approximately 15–30% apoptosis was seen in Ramos, HF1, and SUDHL4 cell lines (Fig 1A). NHL cell lines were subsequently treated with BSO (100 lmol/l) or ATO (2 lmol/l) alone or in combination. Minimal apoptosis was seen with BSO or ATO alone at these concentrations, while BSO combined with ATO was highly synergistic, inducing over 75% apoptosis in all cell lines (Fig 1B). We next measured ROS prior to and after ATO and/or BSO. ATO alone induced minimal ROS, while ATO/BSO combined resulted in pronounced ROS production. To determine ROS-dependence, cells were co-incubated with the antioxidant, N-acetylcysteine (NAC). Pretreatment with NAC significantly reduced ROS levels in cells treated with ATO/BSO (Fig 1C). Furthermore, NAC blocked ATO/BSO-induced apoptosis as well as ATO alone (Fig 1D). Catalase did not inhibit ATO-induced apoptosis, while ATO/BSO-induced apoptosis was significantly reduced (Fig 1E). These data suggest that ATO-induced apoptosis is attributed primarily through the depletion of GSH, while ATO/BSO induced apoptosis is more prominently ROS-mediated. Primary chronic lymphocytic leukaemia (CLL) and follicular lymphoma cells were treated with increasing concentrations of ATO ± BSO (Fig 1F,G). Apoptosis in primary CLL cells was approximately 75% with 5 lmol/l of ATO alone. Interestingly, significantly less cell death was seen compared with the same concentrations (5–10 lmol/l ATO) in NHL cell lines (Fig 1A). Further, the addition of BSO to ATO in primary CLL or follicular lymphoma cells did not enhance apoptosis compared with ATO alone. We hypothesized that low intracellular GSH content might explain the higher sensitivity of primary cells to ATO alone and the lower sensitivity to ATO/BSO. GSH levels in CLL cells were 4–5 fold lower compared with levels seen in Ramos or HF1 cells (data not shown). We investigated whether ATO-induced apoptosis was dependent on Bax translocation. As shown in Fig 2A, ATO alone redistributed Bax from the cytosol to the mitochondria (evident at 6 hours) with release of cytochrome C in Ramos cells. Of note, treatment of cells with combined ATO/BSO did not affect Bax translocation or change in mitochondrial membrane potential (Dwm), suggesting an alternative cell death pathway. To further examine the role of Bax translocation and Dwm, we used immortalized wild type and BaxBak mouse embryonic fibroblasts (MEFs). Treatment of wild type MEFs with ATO alone resulted in enhanced apoptosis compared with BaxBak MEFs, while ATO/BSO induced similar apoptosis in wild type and BaxBak MEFs (Fig 2B). Further, cells stained with mitotracker red and Hoechst followed by confocal microscopy showed that treatment of cells with ATO alone resulted in Dwm loss, as indicated by the loss of red stain in Hoechst-stained blue cells (Fig 2C). The loss of Dwm was also determined by tetramethylrhodamine (TMRE) staining (Fig 2D). Treatment of cells with ATO/BSO did not show significant Dwm change, indicating mitochondrial-independent cell death in these cells. Moreover, compared with changes in Dwm in wild-type MEFs and the Bax Bak double knockout MEFs, there was a substantial difference in Dwm in wild type and BaxBak double knockout MEFs following ATO treatment, while ATO/BSO did not show any difference (Fig 2E). Altogether, these studies implicate the dependence of Bax and loss of Dwm in NHL cells treated with ATO alone, but not with ATO/BSO. With ATO alone, there was increased caspase 8, 9 and caspase 3 cleavage in all lymphoma cell lines at 5–10 lmol/l (Fig 2F). ATO also induced PARP and BID activation. These findings suggest involvement of both cell death pathways using ATO alone, although with more prominent activation of the intrinsic cascade. In contrast, minimal activation of the intrinsic cascade was observed with ATO/BSO (Fig 2G). correspondence