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Arsenic trioxide (ATO) is an effective therapeutic agent for acute promyelocytic leukemia (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 in part through the detoxification of ROS. Compounds that promote ROS and/or deplete protective metabolites such as GSH are able to sensitize tumor cells to oxidative cytolysis. Buthionine sulfoximine (BSO), a selective inhibitor of gamma glutamylcysteine synthetase, is known to effectively deplete cellular GSH (Davison, et al 2003, Gartenhaus, et al 2002). We evaluated herein the cytotoxic activity and cell death pathways induced by ATO alone and combined with BSO in non-Hodgkin’s lymphoma (NHL) cell lines and primary lymphoproliferative cells.
With ATO 10µM, approximately 15–30% apoptosis was seen in Ramos, HF1, and SUDHL4 cell lines (Figure 1A). NHL cell lines were subsequently treated with BSO (100µM) or ATO (2µM) alone or in combination. Minimal apoptosis was seen with BSO or ATO alone, while BSO combined with ATO was highly synergistic inducing over 75% apoptosis in all cell lines (Figure 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, we co-incubated cells with the antioxidant, N-acetylcysteine (NAC). Pretreatment with NAC significantly reduced ROS levels in cells treated with ATO/BSO (Figure 1C). Furthermore, NAC blocked ATO/BSO-induced apoptosis as well as ATO alone (Figures 1D). Catalase did not inhibit ATO-induced apoptosis, while ATO/BSO-induced apoptosis was significantly reduced (Figure 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 leukemia (CLL) and follicular lymphoma cells were treated with increasing concentrations of ATO +/− BSO (Figure 1F and 1G). Apoptosis in primary CLL cells was approximately 75% with 5µM of ATO alone. Interestingly, significantly less cell death was seen compared with the same concentrations (5–10µM ATO) in NHL cell lines (Figure 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. We found that 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 is dependent on Bax translocation. As shown in Figure 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.
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 (Figure 2B). Further, we stained cells with mitotracker red and Hoechst followed by confocal microscopy. Treatment of cells with ATO alone resulted in Δψm loss as indicated by the loss of red stain in Hoechst-stained blue cells (Figure 2C). We also determined the loss of Δψm by tetramethylrhodamine (TMRE) staining (Figure 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 (Figure 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 9 and caspase 3 cleavage in all lymphoma cell lines at 5–10µM (Figure 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 (Figure 2G). However, increase in cleaved caspase 7 (Figure 5C) and PARP cleavage was observed following treatment of each of the three cell lines with both ATO and ATO/BSO (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 (Figure 2I).
In the present study, we 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/leukemia 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.