Glutathione depletion enhances arsenic trioxide‐induced apoptosis in lymphoma cells through mitochondrial‐independent mechanisms (2010)

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 μmol/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 μmol/l) or ATO (2 μmol/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). Arsenic trioxide (ATO)-induced apoptosis in lymphoma cells with or without buthionine sulfoximine (BSO). (A) Ramos, HF1, and SUDHL4 cells were incubated with increasing concentrations of ATO (10–100 μmol/l) for 18 h. Percentage apoptosis was measured by annexinV/propidium iodide (PI) staining and analyzed by flow cytometry. Dose-dependent apoptosis was seen in all cell lines, with effective dose for 50% apoptosis (ED50) between 10–20 μmol/l for Ramos and HF-1 and 20–50 μmol/l for SUDHL4. P values represent respective concentrations compared with control. (B) Ramos, HF1, and SUDHL4 cells were treated with 2 μmol/l ATO, 100 μmol/l BSO alone, or ATO/BSO combined for 48 h. The percentage of apoptotic cells was determined by AnnexinV/PI staining and analyzed by flow cytometry as described. P values compare ATO + BSO versus ATO and BSO alone. In all three cell lines, there was >75% apoptosis when BSO was added to ATO. P values represent respective concentrations compared with control and single agent alone. (C) ROS was measured in Ramos, HF1, and SUDHL4 cells. Cells were incubated with ATO alone (10 μmol/l) or ATO 2 μmol/l combined with BSO 100 μmol/l for 16 h. Increase in ROS was measured by staining with H2DCF-DA and analyzed by flow cytometry. Peak shift to the right denotes an increase in ROS production. P value for level of ROS production was <0·05 for control versus ATO/BSO and for ATO/BSO versus ATO+BSO+NAC for all three cell lines. (D) N-acetylcysteine (NAC) attenuated ATO- and ATO/BSO-induced apoptosis. Ramos, HF1, and SUDHL4 cells were pretreated with NAC 10 mmol/l for 4 h followed by incubation with ATO 10 μmol/l or ATO 2 μmol/l and BSO 100 μmol/l, and in combination with NAC for 48 h. Percentage apoptosis was determined as previously described. P values compared ATO+NAC versus ATO and ATO+BSO+NAC versus ATO+BSO. *P < 0·05, **P < 0·01, and ***P < 0·001. (E) Ramos cells were pre-incubated with 1000 units of catalase following treatment with the indicated concentrations of ATO or ATO/BSO for 48 h. Apoptosis was measured by annexinV/PI staining. (F) Peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of three CLL patients and from (G) two patients with follicular lymphoma as described in the text. Cells were incubated with indicated concentrations of ATO or ATO/BSO for 24 h (all CLL cases), 48 h (follicular lymphoma Patient #1), and 72 h (follicular lymphoma Patient #2). Percentage apoptosis was determined as described before. P values compare ATO versus control. *P < 0·05, **P < 0·01, and ***P < 0·001. 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 μmol/l of ATO alone. Interestingly, significantly less cell death was seen compared with the same concentrations (5–10 μmol/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 (Δψm), suggesting an alternative cell death pathway. ATO, but not ATO/BSO-induced apoptosis, was mediated by Bax translocation, mitochondrial depolarization, and caspase activation. (A) Ramos cells were treated with ATO (2 μmol/l or 10 μmol/l) alone or combined with 100 μmol/l BSO for the indicated periods of time. Mitochondrial and cytosolic fractions were separated as described in the text and analyzed by Western blotting for pro-apoptotic Bax and cytochrome c proteins. Mn-SOD and actin were used as internal controls for mitochondrial and cytosolic extracts, respectively. There was release of cytochrome c from the mitochondria to the cytosol over time with ATO alone, with translocation of Bax to the mitochondria (left panel), while in the presence of BSO, there was no change in mitochondrial Bax or cytosolic cytochrome c, suggesting an alternate cell death pathway. (B) Apoptosis was measured in wild type and Bax−/−/Bak−/− MEFs. Cells were plated the night before the treatment. Cells were treated with the indicated concentrations of ATO and ATO/BSO for 24 h followed by annexinV/PI staining and flow cytometry. (C) SUDHL4 cells were treated with ATO or ATO/BSO for 16 h followed by incubation with 200 nmol/l mitotracker red and 1 μg/ml Hoechst stain for 30 min at 37°C followed by washing with Hanks Balanced Salt Solution and fixed in 3·7% formaldehyde. Fixed cells were used for confocal microscopy. Original magnification ×40. (D) Mitochondrial membrane potential was measured in Ramos and SUDHL4 cells. Cells were treated with indicated concentrations of ATO versus ATO/BSO for 16 h, followed by staining with Tetramethylrhodamine (TMRE). Cells were analyzed by flow cytometry. 3-chlorophenylhydrazone (CCCP; Invitrogen, Carlsbad, CA, USA) was used as a positive control. (E) Mitochondrial membrane potential was measured in wild type and Bax−/−/Bak−/− MEFs. Cells were plated the night before the treatment and treated with the indicated concentrations of ATO and ATO/BSO for 6 h and stained with TMRE followed by flow cytometry. Unless indicated, p values represent comparison with control. (F) Ramos, HF1, and SUDHL4 cells were treated for 16 h with increasing concentrations of ATO or (G) using BSO (100 μmol/l) in combination with lower dose ATO (2 μmol/l) for 16 h. Western blots of whole cell extracts were performed to detect caspase, PARP, and BID proteins. GAPDH was used as internal control. (H) Western blot of cleaved caspase 7 was measured in cells treated with ATO or ATO/BSO with and without NAC. (I) Ramos, SUDHL4, and HF1 cells were pre-treated with the pan-caspase inhibitor, Z-VAD-FMK (50 μmol/l) for 4 h, followed by treatment with ATO 10 μmol/l or ATO 2 μmol/l combined with BSO 100 μmol/l for 48 h. Percentage apoptosis was determined by flow cytometry after staining the cells with annexin V/PI. Abbreviations: H, hours; MEFs, mouse embryonic fibroblasts; Mito-Bax, mitochondrial-related Bax. *P < 0·05, **P < 0·01, and ***P < 0·001, unless otherwise indicated. To further examine the role of Bax translocation and Δψm, we used immortalized wild type and Bax−/−Bak−/− mouse embryonic fibroblasts (MEFs). Treatment of wild type MEFs with ATO alone resulted in enhanced apoptosis compared with Bax−/−Bak−/− MEFs, while ATO/BSO induced similar apoptosis in wild type and Bax−/−Bak−/− 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 Δψm loss, as indicated by the loss of red stain in Hoechst-stained blue cells (Fig 2C). The loss of Δψm was also determined by tetramethylrhodamine (TMRE) staining (Fig 2D). Treatment of cells with ATO/BSO did not show significant Δψm change, indicating mitochondrial-independent cell death in these cells. Moreover, compared with changes in Δψm in wild-type MEFs and the Bax−/−Bak−/− double knockout MEFs, there was a substantial difference in Δψm in wild type and Bax−/−Bak−/− 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 Δψm 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 μmol/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). However, increase in cleaved caspase 7 (Fig 2H) and PARP cleavage was observed following treatment of each of the three cell lines with both ATO and ATO/BSO (Fig 2H). To further test the dependence of the caspases in ATO–related apoptosis (± BSO), cell lines were pre-treated with the pan-caspase inhibitor, Z-VAD-FMK. Z-VAD–FMK effectively blocked apoptosis with ATO alone in all cell lines, while much less inhibition was seen in ATO/BSO-treated cells (Fig 2I). The present study found that ATO alone induced apoptosis in NHL cell lines through ROS, Bax, and caspase-dependent pathways, although relatively high in vitro concentrations of ATO were required for effect. Addition of BSO to ATO resulted in highly synergistic cell death with apoptosis that occurred through a Bax- and caspase-independent pathway. Furthermore, we discovered critical differences between primary lymphoma/leukaemia cells and in vitro cell lines. BSO did not enhance ATO-related cell death in primary cells, which was likely due to higher basal levels of intracellular GSH in cell lines compared with primary cells. Continued investigation of arsenic-based therapy, including new arsenical compounds that are able to overcome known mechanisms of ATO resistance (e.g. GSH-redox mechanisms) (Diaz et al, 2008; Tsimberidou et al, 2009) and through novel methods of arsenic delivery (Chen et al, 2009), for the treatment of lymphoma is warranted. We would like to thank members of Flow Cytometry and cell imaging facility of Northwestern University. Supported in part from grants from the National Cancer Institute (AME, K23 CA109613-A1 and LCP, R01CA121192) and a Merit review grant from the Department of Veterans Affairs.