Superoxide-dependent and -independent mitochondrial signaling during apoptosis in multiple myeloma cells

Abstract

Superoxide (O2) radicals have been linked to apoptosis. Here, we show that 2-methoxyestradiol (2ME2)-induced apoptosis in multiple myeloma (MM) cells is associated with O2 generation, whereas dexamethasone (Dex)-induced apoptosis occurs without concurrent increase in O2. In contrast, both these agents decrease mitochondrial transmembrane potential (Δψm). Treatment of MM cells with an antioxidant N-acetyl-L-cysteine blocks 2ME2, but not Dex-induced apoptosis as well as release of mitochondrial proteins cytochrome c (cyto c) and Smac. Taken together, these results demonstrate that there are at least two distinct apoptotic pathways: one dependent on O2, which is induced by 2ME2 and is associated with release of cyto c and Smac; and the other an independent of O2, which is triggered by Dex and associated with Smac release.

Main

Alterations in mitochondrial function during apoptosis trigger downstream caspase cascades via the release of cytochrome c (cyto c) and second mitochondria-derived activator of caspase (Smac) or DIABLO from mitochondria to cytosol (Liu et al., 1996; Du et al., 2000; Verhagen et al., 2000). The upstream mechanism(s) regulating the release of Smac and cyto c is unclear. Our prior studies showed that dexamethasone (Dex)-induced apoptosis in multiple myeloma (MM) cells involves release of Smac, but not cyto c (Chauhan et al., 1997); whereas 2-methoxyestradiol (2ME2)-induced apoptosis involves release of both cyto c and Smac, indicating differential upstream activator of Smac and cyto c release (Chauhan et al., 2002). Other studies have reported that the stress-induced changes in Δψm correlates with both an increase in Reactive oxygen species (ROS) and release of cyto c and Smac. The role of ROS, in particular, superoxide radical (O2), in mediating apoptosis in other systems is well established (Hockenbery et al., 1993); however, the relation between generation of O2 and the release of mitochondrial proteins remains controversial.

In the present study, we asked whether (1) 2ME2 or Dex-induced apoptosis involves alterations in the mitochondrial transmembrane potential (Δψm); (2) loss of Δψm is essential for generation of O2, and (3) O2 generation is an obligatory event for the release of mitochondrial proteins Smac and cyto c during 2ME2 or Dex-induced apoptosis.

We first determined whether 2ME2 or Dex induces apoptosis in MM cells. Dex-sensitive (MM.1S) and Dex-resistant (MM.1R) MM cells were treated with either 2ME2 (3 μ M) or Dex (5 μ M) for 48 h, and flow cytometric analysis was performed using dual fluorescence staining with DNA-binding fluorochrome Hoechst 33342 (HO) and propidium iodide (PI). As seen in Figure 1a, treatment of MM.1S cells with Dex induces apoptosis in MM.1S cells, but not in MM.1R cells (69±4%, P<0.005, n=3; 11+0.8%, P<0.004, n=3, respectively). In contrast, treatment with 2ME2 induces apoptosis in both MM.1S and MM.1R MM cells, (78±3.6%, P<0.005, n=3, and 66±3%, P<0.003, n=3, respectively) (Figure 1a), as in our prior studies (Chauhan et al., 2002). To further confirm 2ME2 or Dex-induced apoptosis, we examined poly (ADP-ribose) polymerase (PARP) cleavage, a signature event during apoptosis (Kaufmann et al., 1993). Total lysates from untreated and 2ME2- or Dex-treated MM.1S cells were subjected to immunoblot analysis with anti-PARP Abs (Pharmingen, Palo Alto, CA, USA). As seen in Figure 1b, both 2ME2 and Dex induce marked PARP cleavage in MM.1S cells, as evident by cleavage of 110 kDa full length PARP into 85 kDa fragment. The PARP cleavage is consistent with apoptosis and activation of proteases (Kaufmann et al., 1993). Moreover, 2ME2 induces PARP cleavage in Dex-resistant MM.1R cells (Figure 1b), consistent with 2ME2-induced apoptosis in these cells.

Figure 1
figure1

2ME2 and Dex induce apoptosis in MM cells. Human MM.1S (Dex-sensitive) and MM.1R (Dex-resistant) MM cells (Chauhan et al., 1997) were grown in RPMI-1640 media supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. Cells were treated with 5 μ M Dex or 3 μ M 2ME2 (Sigma Chemical Co, St Louis, MO, USA). (a) MM.1S and MM.1R MM cells were treated with either 2ME2 or Dex for 48 h, and analyzed for apoptosis by flow cytometric analysis using dual fluorescence staining with DNA-binding fluorochrome HO and PI. PI-negative and HO-positive stained cells represent apoptotic cells. Results are mean±s.d. of three independent experiments (P<0.005). (b) MM.1S and MM.1R cells were treated with 2ME2 (3 μ M) or Dex (5 μ M) for 48 h. Total cell lysates were separated by 7.5% SDS–PAGE and analysed by immunoblotting (IB) with anti-PARP Abs. Blots are representative of three independent experiments with similar results. FL, full length; CF, cleaved fragment. (c) Effects of 2ME2 and Dex on mitochondrial membrane potential (Δψm). Serum-starved MM.1S cells were treated with either 2ME2 (3 μ M) (filled square) or Dex (5 μ M) (filled circle) for the indicated times, incubated with CMXRos for the last 20 min, and analysed by flow cytometry, as described previously (Wang et al., 1999). Briefly, 2ME2 or Dex-treated MM cells were stained with lipophilic cationic dye CMXRos (Mitotracker Red) (Molecular Probes, Eugene, OR, USA) in PBS for 20 min at at 37°C and analysed by flow cytometry to assay for alterations in Δψm (Poot and Pierce, 1999). Results are mean±s.d. of three independent experiments (P<0.003). (D) Effects of 2ME2 and Dex on O2. Serum-starved MM.1S were treated with 2ME2 (3 μ M) or Dex (5 μ M) for 6, 12, and 24h; harvested; and stained with membrane permeable dye HE, as previously described (Rothe and Valet, 1990). Superoxide anions oxidize HE to fluorescent ethidium permitting analysis by flow cytometry (The Vantage, Becton Dickinson, FACScan using excitation at 480 nm and emission at 630 nm). Cells were also treated with H2O2, as a positive control for generation of O2. Results are mean±s.d. of three independent experiments (P<0.005)

Figure 2
figure2

Effect of antioxidant NAC on 2ME2 or Dex-induced apoptosis and generation of O2. (a) MM.1S cells were treated with 2ME2 (3 μ M) or Dex (5 μ M) in the presence or absence of NAC (10 μ M) for 48 h, and analysed for apoptosis by flow cytometric analyses (The Vantage, Becton Dickinson) using PI and HO staining, as previously described (Chauhan et al., 2002). Apoptotic cells were PI-HO+ cells (P<0.003, n=3). (b) Effect of NAC on 2ME2-induced O2 production. MM.1S were treated with 2ME2 (3 μ M) in the presence or absence of NAC (10 μ M) for 12 h, incubated with superoxide sensitive dye HE for the last 15 min, and analysed by flow cytometry (P<0.005, n=3). Cells were also treated with Dex as a negative control. (c) Effect of NAC on 2ME2- and Dex-induced caspase-9 activation. MM.1S cells were treated with 2ME2 (3 μ M) or Dex (5 μ M) in the presence or absence of NAC (10 μ M) for 24 h and harvested. Cytosolic extracts were assayed for protease activity using LEHD-pNA as substrate as per manufacturer's instructions (colorimetric assay kit, Biovision, Palo Alto, CA, USA). Results are representative of three independent experiments (mean±s.d., n=3). (d) Effect of NAC on 2ME2- and Dex-induced caspase-3 cleavage. MM.1S cells were treated with 2ME2 (3 μ M) or Dex (5 μ M) in the presence or absence of NAC (10 μ M) for 24 h and harvested. Cytosolic extracts were separated by 12.5% SDS–PAGE and analysed by IB with anti-caspase-3 Ab. Blots shown are representative of three independent experiments with similar results

Mitochondria play a critical role in apoptosis induction during stress (Bossy-Wetzel and Green, 1999). We therefore next examined whether treatment of MM.1S cells affected mitochondrial membrane potential (Δψm). MM.1S cells were treated with 2ME2 (3 μ M) or Dex (5 μ M) for 6, 12 and 24 h; stained with CMXRos; and analysed by flow cytometry as previously described (Wang et al., 1999). As seen in Figure 1c, both 2ME2 and Dex trigger a significant decrease in Δψm in MM.1S cells, as measured by an increase in CMXRos negative cells (P<0.003 for 2ME2 and P<0.005 for Dex, n=3). Similar decreases in Δψm were observed in 2ME2-treated Dex-resistant MM.1R cells (data not shown).

Since loss of Δψm is associated with O2 production (Zamzami et al., 1995; Huang et al., 2000), we next determined whether 2ME2 or Dex also affected O2 generation. MM.1S cells were treated with 2ME2 or Dex for 6, 12 and 24h; stained with dihydroethidium (HE); and analysed by flow cytometry. As seen in Figure 1d, 2ME2, but not Dex, induces generation of O2 in these cells. As a control, treatment of cells with H2O2 results in an increase in O2. To confirm whether higher doses of Dex affected O2 levels, MM.1S cells were treated with 10 μ M of Dex. No increases in O2 were observed in response to Dex (data not shown), suggesting that O2 production is not a prerequisite for Dex-induced apoptosis. Additionally, 2ME2- or Dex-treated MM.1S cells were also analysed for alterations in H2O2 levels using 2′-7′-Dichlorodihydrofluorescein diacetate (DCF-DA). No alterations in H2O2 levels were observed in response to Dex, whereas treatment with 2ME2 decreased the H2O2 levels (data not shown). These findings are consistent with a prior study showing a decrease in H2O2 and an increase in O2 in response to 2ME2 in ML-1 cells (Huang et al., 2000). Taken together, these findings demonstrate that (1) 2ME2-induced apoptosis is associated with a decrease in Δψm and an increase in O2, and (2) Dex-triggered apoptosis involves a decrease in Δψm, without a concurrent increase in O2.

Multiple prior studies have reported that mitochondria are a major site for the generation of O2 via the electron transport chain (ETC) (Curtin et al., 2002). To determine whether blockade of mitochondrial ETC affect 2ME2-induced generation of O2, MM.1S cells were exposed to a specific inhibitor of ETC (complex-I, NADH-ubiquinone oxidoreductase) rotenone for 1 h prior to treatment with 2ME2; and then analysed for O2 levels. The results demonstrated a marked inhibition (73±5.1% decrease; P<0.05, n=3) in 2ME2-induced O2 production in cells incubated with rotenone compared to the cells treated with 2ME2 alone. These findings suggest that 2ME2 trigger O2 generation via mitochondrial ETC.

Previous studies have shown that generation of O2 mediates apoptosis in response to various stress agents (Dussmann et al., 2003; Li et al., 2003). Conversely, the scavenging of UV-generated H2O2 by N-acetyl-1-cysteine (NAC) (Sigma Chemicals, St Louis, MO, USA) inhibited UV-induced apoptosis (Ding et al., 2002). Furthermore, NAC blocked the methylglyoxal-induced apoptosis in Jurkat cells (Du et al., 2001). To determine whether generation of O2 is an obligatory event during 2ME2-induced apoptosis, MM.1S cells were treated with 2ME2 for 12, 24 and 48 h in the presence or absence of NAC (10 μ M), and analysed for apoptosis by PI/HO staining. As seen in Figure 2a, cotreatment of MM.1S MM cells with NAC for 48 h significantly (P<0.003, n=3) blocked 2ME2, but not Dex-induced apoptosis in these cells. The blockade in 2ME2-induced apoptosis by NAC was detectable as early as 12 h, and maximally at 48 h. Moreover, 2ME2-induced generation of O2 is markedly reduced in the presence of NAC, as determined by HE staining (Figure 2b). To provide specificity for NAC in blocking O2 mediated signaling, we used another radical scavenger pyrrolidine dithiocarbomate (PDTC). Treatment of MM.1S cells with 25 μ M of PDTC prevented 2ME2-induced apoptosis, whereas apoptosis triggered by Dex remained unaffected (data not shown). Additionally, MM.1S cells were also cotreated with 2ME2 and a specific inhibitor of O2 Tiron, and analysed for apoptosis. The results demonstrated that Tiron significantly blocked 2ME2-induced apoptosis (78±4.1% apoptotic cells in 2ME2-treated cells versus 26±3.2% apoptotic cells in Tiron+2ME2-treated cells). These findings further confirm a role of O2 in mediating 2ME2-induced apoptosis in MM cells.

Our previous study showed that 2ME2- and Dex-induced apoptosis is associated with caspase-9 and -3 activation. We next determined whether NAC affects Dex- or 2ME2-induced catalytic activity of caspase-9 using LEHD-pNA conjugated substrate in a colorimetric protease assay (Colorimetric assay kit, Biovision, Palo Alto, CA, USA), as previously described (Chauhan et al., 2001). MM.1S cells were treated with 2ME2- or Dex for 12 h and cytosolic extracts were incubated with LEHD-pNA. As seen in Figure 2c, incubation of cytosolic extracts from 2ME2-or Dex-treated MM.1S cells with LEHD-pNA was associated with efficient cleavage of LEHD-pNA, whereas cotreatment with NAC abrogated 2ME2, but not Dex -induced cleavage of LEHD-pNA. Similar results were obtained after 24 or 48 h exposure of MM.1S cells to either 2ME2 or Dex (data not shown). Furthermore, 2ME2-triggered caspase-3 cleavage was also blocked by NAC, whereas Dex-induced caspase-3 cleavage remained unaffected in the presence of NAC (Figure 2d).

To determine whether inhibition of caspases affect 2ME2-induced O2 generation, MM.1S cells were treated with pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk). No signififcant alterations in 2ME2-induced O2 generation were detected; however, z-VAD-fmk significant decreased 2ME2-induced apoptosis (data not shown). These results clearly suggest that O2 generation occurs upstream of caspase activation in response to 2ME2. Together, these findings suggest that 2ME2-induced apoptosis and associated caspase-9>caspase-3 activation is, at least in part, mediated by generation of O2, whereas Dex-induced apoptosis and associated caspase signaling is independent of O2.

Release of mitochondrial apoptogenic proteins cyto c and Smac/DIABLO induce activation of caspase-9 and -3 (Liu et al., 1996; Du et al., 2000). Having shown that scavenging of O2 by NAC blocks 2ME2, but not Dex -induced apoptosis and associated activation of caspase-9 and -3, we next asked whether release of cyto c or Smac is similarly affected by NAC. MM.1S cells were treated with 2ME2 in the presence or absence of NAC (10 μ M); cytosolic extracts were then prepared and subjected to immunoblot analysis with anti-Smac or anti-cyto c Abs. As seen in Figure 3a and b (upper panels), cotreatment of MM.1S cells with NAC significantly decreases (5–6-fold decrease) 2ME2-induced release of both Smac and cyto c. In contrast, Dex-induced release of Smac is not blocked by NAC (Figure 3c, upper panel). Reprobing the immunoblots with anti-SHP2 (Src homology-2 domain containing protein tyrosine phosphatase) Abs (Santa Cruz Biotech, Santa Cruz, CA, USA) confirms equal protein loading (Figure 3a–c lower panels). These findings show that O2 generation is associated with cyto c- or Smac-mediated apoptotic signaling triggered in response to 2ME2, but not Dex, in MM cells.

Figure 3
figure3

Effect of NAC on 2ME2 or Dex-induced release of Smac or cyto c. (a–c) MM.1S cells were treated with 2ME2 (3 μM) or Dex (5 μ M) in the presence or absence of NAC and harvested at 24 h. Cells were then washed twice with PBS, and the pellet was suspended in cold buffer A (20 mM HEPES, pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 10 μg/ml leupeptin and aprotinin and pepstatin A) containing 250 nM sucrose. After homogenization using a Dounce homogenizer, cytosolic extracts were isolated, separated by 12.5% SDS–PAGE, and analysed by IB with anti-Smac (a and c, upper panel) or anti-cyto c (b, upper panel) Abs. As a control for equal loading of proteins, filters were also reprobed with anti-SHP2 Ab (a, b and c, lower panel). The blots were developed by enhanced chemiluminesence (ECL), using the manufacturer's protocol (Amersham). Blots shown are representative of three independent experiments with similar results. (d) Schematic representation of 2ME2 and Dex-induced effects on mitochondrial signaling. Two distinct apoptotic signaling pathways were observed; one induced by Dex (dash-lined arrows), which is independent of O2 and associated with the release of Smac; and a second induced by 2ME2 (dark-lined arrows), which is associated with an increase in O2 and release of both Smac and cyto c. Both 2ME2 and Dex reduce Δψm

Collectively, our findings have an important biologic and therapeutic application. We provide evidence for at least two distinct apoptotic signaling pathways; one induced by Dex, which is independent of O2 and associated with the release of Smac; and a second induced by 2ME2, which is associated with an increase in O2 and release of both Smac and cyto c (Figure 3d, schema). Importantly, both 2ME2 and Dex reduce Δψm. The ROS have been linked to the release of Smac or cyto c from mitochondria to cytosol during apoptosis (Bossy-Wetzel and Green, 1999; Matsuyama and Reed, 2000; Distelhorst, 2002), and our results clearly demonstrate that accumulation of Smac in cytosol during Dex-induced apoptosis occurs independent of an increase in O2. In contrast, 2ME2-induced apoptosis and release of both Smac and cyto c requires generation of O2.

Our findings also provide the framework for novel targeted therapeutics based upon drug-induced signaling cascades. For example, Smac agonists or ectopic overexpression of Smac peptide may enhance drug-induced cytotoxicity, as recently reported (Guo et al., 2002; Ng et al., 2002). The observation that inhibition of SOD induces apoptosis of MM cells further suggests that the combination of SOD inhibitor (2ME2) with modalities that produce free radicals may enhance anti-MM activity.

Abbreviations

MM:

multiple myeloma

O2:

superoxide

cyto c:

cytochrome c

Smac:

second mitochondrial activator of caspase

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Acknowledgements

This investigation was supported by NIH Grants 50947 and CA 78373, a Doris Duke Distinguished Clinical Research Scientist Award (KCA), The Myeloma Research Fund, and The Cure Myeloma Fund.

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Correspondence to Kenneth C Anderson.

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Keywords

  • multiple myeloma
  • apoptosis
  • superoxide
  • cytochrome c
  • Smac (second mitochondrial activator of caspases)
  • 2-methoxyestradiol
  • dexamethasone

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