Apaf-1/Cytochrome c-independent and Smac-dependent Induction of Apoptosis in Multiple Myeloma (MM) Cells*

Smac, a second mitochondria-derived activator of caspases, promotes caspase activation in the cytochrome c(cyto-c)/Apaf-1/caspase-9 pathway. Here, we show that treatment of multiple myeloma (MM) cells with dexamethasone (Dex) triggers the release of Smac from mitochondria to cytosol and activates caspase-9 without concurrent release of cyto-c and Apaf-1 oligomerization. Smac binds to XIAP (an inhibitor of apoptosis protein) and thereby, at least in part, eliminates its inhibitory effect on caspase-9. Interleukin-6, a growth factor for MM, blocks Dex-induced apoptosis and prevents release of Smac. Taken together, these findings demonstrate that Smac plays a functional role in mediating Dex-induced caspase-9 activation and apoptosis in MM cells.

The cellular response to diverse classes of stress inducers includes growth arrest and activation of apoptosis. Apoptosis is triggered through a controlled program that is associated with distinctive morphological changes, including membrane blebbing, cytoplasmic and nuclear condensation, chromatin aggregation, and formation of apoptotic bodies (1). The induction of apoptosis involves a cascade of initiator and effector caspases that are activated sequentially (2,3). Caspases, a family of cysteine proteases with aspartate substrate specificity, are present in cells as catalytically inactive zymogens (2). Effector caspases, such as caspase-3, are activated by initiator caspases, such as caspase-9. Once activated, the effector caspases induce proteolytic cleavage of various cellular targets, inducing poly-(ADP-ribose) polymerase (4,5), DNA-dependent protein kinase, protein kinase C-␦, and other substrates (6), ultimately leading to cell death.
One of the major caspase cascades is triggered by the release of mitochondrial apoptogenic protein, cytochrome c (cyto-c) (11)(12)(13). Cytosolic cyto-c binds to the CED-4 homolog Apaf-1 and induces caspase-9-dependent activation of caspase-3 (14 -17). Recent studies have identified another important regulator of apoptosis, Smac (second mitochondria-derived activator of caspase) or DIABLO, which is released from mitochondria into the cytosol during apoptosis (18 -20) and functions by eliminating inhibitory effects of IAPs on caspases (20,21).
Our prior study demonstrated that dexamethasone (Dex)induced apoptosis is independent of cyto-c release and associated with caspase-3 activation (22,23). In the present study, we examined the upstream signaling leading to caspase-3 activation. The results demonstrate that Dex-induced apoptosis in MM cells is mediated by Smac, which activates caspase-9 by binding to and inhibiting XIAP. Interleukin-6 (IL-6), a growth factor for MM, blocks Dex-induced release of Smac and apoptosis. Taken together, this study provides evidence for an Apaf-1-cyto-c-independent pathway mediating caspase-9 activation via Smac. Moreover, these findings also demonstrate a functional role of Smac in IL-6-mediated block during Dex-induced apoptosis.
Caspase Activity Assays-Caspase-9 activation was performed using LEHD-pNA as a substrate, as per the manufacturer's instructions (colorimetric assay kit, Biovision, Palo Alto, CA). MM.1S MM cells were also treated with Dex (10 M) in the presence or absence of caspase-9 inhibitor LEHD-FMK (5 M) for 24 h and then analyzed for apoptosis.
Quantification of Apoptosis-Flow cytometric analyses: dual fluorescence staining with DNA-binding fluorochromes Hoechst 33342 (HO) and propidium iodide (PI) was used to quantitate the percentage of apoptotic (HOϩPIϪ) cells using flow cytometry (The Vantage, Becton Dickinson), as described previously (24). DNA fragmentation assays were also performed as described previously (24) RESULTS AND DISCUSSION To determine whether Dex-induced apoptosis in MM cells is associated with the release of Smac, MM.1S MM cells were treated with Dex for various times, and cytosolic and mitochondrial extracts were analyzed for the levels of Smac. The results demonstrate that Dex treatment is associated with an increase in Smac levels in the cytosol at 24 and 48 h, with a concomitant decrease in mitochondrial Smac levels (Fig. 1A). Dex-induced increase in cytosolic Smac and a corresponding decrease in mitochondrial Smac levels were specific, because no change was observed in the levels of tubulin protein and mitochondrial matrix protein, Hsp60, respectively (Fig. 1A). Similar results were obtained when patient MM cells were exposed to Dex (Fig.  1B). These results suggest that Dex-induced apoptosis is accompanied by accumulation of Smac in the cytosol.
Smac is known to promote caspase activation in the cyto-c/ Apaf-1/caspase-9 pathway; therefore, we next examined the release of cyto-c triggered by Dex or IR in MM.1S MM cells. The cytosolic extracts from Dex-or IR-treated cells were subjected to immunoblot analyses with anti-cyto-c and anti-Smac. As in our previous findings (22), treatment of MM.1S MM cells with Dex did not induce release of cyto-c in the cytosol (Fig. 1C,  upper panel); in contrast, ␥-radiation (IR) stimulated the release of cyto-c (Fig. 1C, upper panel), demonstrating that the release cyto-c is functional in these cells. To assay for Smac release these immunoblots were then stripped and reprobed with anti-Smac. As seen in Fig. 1C, both Dex and IR induced release of Smac in the cytosol. Furthermore, low to undetectable cytosolic Smac or cyto-c levels were observed in the untreated cells. Reprobing the immunoblots with anti-tubulin confirms equal protein loading (Fig. 1C).
Since Dex-induced apoptosis is associated with Smac release, but not cyto-c release, we next determined whether Apaf-1 oligomerization is required for Smac-related signaling. For these experiments, we utilized the same MM.1S MM cells model (22) to determine whether IR or Dex induce Apaf-1 oligomerization. Cells were transiently cotransfected with FLAG-Apaf-1 and T7-Apaf-1 or empty vector and treated with Dex or IR. Cell lysates were incubated with dATP. As shown in  (Fig. 1D, lane 3). The finding that IR induces Apaf-1 oligomerization in MM.1S MM cells indicates that the Apaf-1 oligomerization system is functionally intact and served as a positive control. Taken together, these findings suggest that Dex-induced apoptosis in MM cells is mediated by Smac and is independent of cyto-c/Apaf-1 mechanism.
To examine whether Dex-induced Smac release and apoptosis are associated with processing of caspase-9, the cytosolic extracts from Dex-treated cells were subjected to immunoblot analysis with anti-caspase-9. The results demonstrate that treatment of MM.1S cells with Dex induces proteolytic cleavage of procaspase-9 into 37-and 35-kDa fragments ( Fig. 2A, upper  panel). Reprobing the immunoblot with anti-tubulin confirms equal protein loading ( Fig. 2A, lower panel). We next assayed for catalytic activity of caspase-9 using LEHD-pNA conjugated substrate in a colorimetric protease assays (26). Incubation of cytosolic extracts from Dex-treated MM.1S cells with LEHD-pNA was associated with efficient cleavage of LEHD-pNA (Fig.  2B). Although LEHD-pNA may be cleaved by caspases other than caspase-9, our study indicates that activated caspase-8 in MM.1S MM cells does not cleave this substrate (Fig. 2B), further supporting its specificity for caspase-9. Taken together, these findings demonstrate that treatment of MM.1S cells with Dex is associated with activation of caspase-9.
We next asked whether caspase-9 activation is an obligatory event during Dex-induced apoptosis. MM.1S MM cells were cultured with Dex in the presence or absence of caspase-9 tetrapeptide inhibitor LEHD-FMK for 24 h and then assayed for proteolytic cleavage of caspase-9 and caspase-3. LEHD-FMK abrogates Dex-induced cleavage of both caspase-9 and caspase-3 (Fig. 2C, left and right panel). In contrast, LEHD-FMK did not inhibit anti-Fas-induced caspase-8 or caspase-8mediated caspase-3 cleavage in MM.1S MM cells (data not shown), further indicating the selectivity of LEHD-FMK for caspase-9. We next determined whether blocking caspase-9 activation affects Dex-induced apoptosis, MM.1S MM cells were cultured with Dex in the presence or absence of caspase-9 inhibitor LEHD-FMK for 24 h and then assayed for apoptosis using flow cytometric analysis with PI and HO dual staining to determine the percentage for PIϪ and HOϩ apoptotic cells. Dex-induced apoptosis (51 ϩ 3% apoptotic cells (n ϭ 3)) was significantly inhibited in cells pretreated with caspase-9 inhibitor (27 ϩ 2% apoptotic cells (n ϭ 3)) (Fig. 2D). Other studies have demonstrated that caspase-9 proteolytically cleaves and activates procaspase-3 (14). In that context, our previous studies have shown that Dex triggers caspase-3 activation in MM.1S MM cells (22,23). Taken together, these results suggest that Dex induces sequential activation of Smac 3 caspase-9 3 caspase-3 and is independent of cyto-c⅐Apaf-1 apoptosome complex formation.
We next determined the mechanism of Smac-mediated Dexinduced caspase-9 activation. Two potential mechanisms for capsase-9 activation have been suggested (18). First, the release of cyto-c leads to Apaf-1 oligomerization, which then activates caspase-9; second, Smac interacts with IAPs (inhibitors of apoptosis protein), such as X-chromosome-linked IAP (XIAP), and eliminates the inhibitory effects of IAPs on caspase-9 (27). Since Dex-induced apoptosis is not associated with cyto-c release or Apaf-1 multimerization, we asked whether XIAP interacts with Smac during Dex-induced apoptosis. MM.1S MM cells were transiently transfected with Myc-XIAP and treated with Dex for 24 h. Cytosolic extracts were subjected to immunoprecipitation with anti-Myc and immunoblotting with anti-caspase-9, anti-Smac, or anti-XIAP. As  shown in Fig. 2E, Dex treatment induces an interaction between XIAP and Smac. Importantly, Dex treatment also leads to dissociation of XIAP from caspase-9 (Fig. 2E). Equivalent levels of transfected XIAP protein were confirmed by reprobing the filters with anti-XIAP (Fig. 2E). These findings are in concert with other studies demonstrating that Smac promotes caspase activity of initiator caspase-9 by binding to and inhibiting IAPs (7,20,21).
To explore the functional significance of Smac release during Dex-induced apoptosis, we utilized IL-6, a known inhibitor of Dex-triggered apoptosis in MM cells. IL-6 abrogates Dex-induced apoptosis in MM.1S MM cells (Fig. 3A, Refs. 23 and 28), as evidenced by DNA fragmentation assay. To determine whether IL-6 affects Dex-induced release of Smac, MM.1S MM cells were treated with Dex in the presence or absence of IL-6 (100 ng), and cytosolic extracts were analyzed for Smac levels. The Dex-induced increase in cytosolic Smac was significantly abrogated in the cells pretreated with IL-6 ( Fig. 3B). This IL-6-mediated block in the Dex-induced Smac levels was specific, because no change was observed in the levels of tubulin (Fig. 3B). Additional evidence for supporting the role of Smac during Dex-induced signaling was obtained by utilizing Dexresistant (MM.1R) MM cells. MM.1R cells were treated with Dex or IR for 48 h, and cytosolic extracts were analyzed for Smac levels. As shown in Fig. 3C, IR, but not Dex, induces Smac release. The finding that IR induces release of Smac from mitochondria to cytosol in MM.1R MM cells indicates that the Smac release system is functionally intact and served as a positive control. The observation that IL-6 prevents Dex-induced Smac release and apoptosis, coupled with the lack of Smac release and apoptosis by Dex in MM.1R (Dex-resistant) cells, supports a role for Smac in mediating Dex-induced apoptosis and is consistent with the known resistance to Dex treatment in MM patients with advanced disease and high serum levels of IL-6 (28,29).
Collectively, the present study demonstrates that Dex-induced apoptosis in MM cells is associated with Smac release and caspase-9 activation, without concurrent release of cyto-c and Apaf-1 oligomerization. In contrast, treatment of Dexresistant cells with Dex fails to induce release of Smac and apoptosis. Furthermore, IL-6 blocks Dex-induced apoptosis in MM cells and inhibits Smac release, thereby conferring Dexresistance. Taken together, these findings provide evidence for a Smac-dependent activation of caspase-9 and apoptosis, independent of Apaf-1/cyto-c (Fig. 3D), and suggest therapeutic strategies based upon targeting both Smac and XIAP.