Induction of apoptosis by enediyne antibiotic calicheamicin ϑII proceeds through a caspase-mediated mitochondrial amplification loop in an entirely Bax-dependent manner

Abstract

Calicheamicin ϑII is a member of the enediyne class of antitumor antibiotics that bind to DNA and induce apoptosis. These compounds differ, however, from conventional anticancer drugs as they bind in a sequence-specific manner noncovalently to DNA and cause sequence-selective oxidation of deoxyriboses and bending of the DNA helix. Calicheamicin is clinically employed as immunoconjugate to antibodies directed against, for example, CD33 in the case of gemtuzumab ozogamicin. Here, we show by the use of the unconjugated drug that calicheamicin-induced apoptosis is independent from death-receptor/FADD-mediated signals. Moreover, calicheamicin triggers apoptosis in a p53-independent manner as shown by the use of p53 knockout cells. Cell death proceeds via activation of mitochondrial permeability transition, cytochrome c release and activation of caspase-9 and -3. The overexpression of Bcl-xL or Bcl-2 strongly inhibited calicheamicin-induced apoptosis. Knockout of Bax abrogated cell death after calicheamicin treatment. Thus, the activation of mitochondria and execution of cell death occur through a fully Bax-dependent mechanism. Interestingly, caspase inhibition by the pancaspase-inhibitor zVAD-fmk interfered with mitochondrial activation by calicheamicin. This places caspase activation upstream of the mitochondria and indicates that calicheamicin-triggered apoptosis is enhanced through death receptor-independent activation of the caspase cascade, that is, an amplification loop that is required for full activation of the mitochondrial pathway.

Introduction

In contrast to necrosis, apoptosis is a morphologically and biochemically defined form of cell death that plays a role under different physiological conditions, such as cell turnover, the immune response or embryonic development. In addition, the apoptotic program may be triggered by various, unphysiological death stimuli, for example, ionizing radiation (Belka et al., 2000, 2001), chemotherapeutic anticancer drugs (Engels et al., 2000; Wieder et al., 2001a) and by natural compounds (Wieder et al., 2001b). So far, three major pathways of apoptotic cell death have been defined: (i) the death receptor-mediated signalling cascade (Daniel et al., 2001, ii) the mitochondrial pathway involving the apoptosome (Daniel, 2000; Rudner et al., 2001), and (iii) the endoplasmic reticulum-dependent stress response (Belka and Budach, 2002; Rudner et al., 2002).

In previous studies, we demonstrated that inactivation of apoptotic pathways results in both apoptotic resistance in vitro and clinical resistance to anticancer treatment (Mrozek et al., 2003; Güner et al., 2003; Sturm et al., 2001; Sturm et al., 2003). One of the main regulatory steps of apoptotic cell death is controlled by the ratio of anti- and proapoptotic members of the Bcl-2 family of proteins function as a rheostat and determine the susceptibility to apoptosis (for reviews, see, Daniel, 2000; Daniel et al., 2003). For example, the overexpression of antiapoptotic Bcl-2 family members that can tilt the rheostat towards survival, thereby confering drug resistance, at least in some cellular tumor model systems (Rudner et al., 2001; Belka and Budach, 2002; Radetzki et al., 2002; von Haefen et al., 2003). On the other hand, forced expression of proapoptotic Bax or Bak or, for example, the BH3-only protein Nbk/Bik, is sufficient to increase the sensitivity of malignant cells to apoptosis and to overcome drug resistance (Hemmati et al., 2002; Radetzki et al., 2002; von Haefen et al., 2002). Interestingly, these in vitro data are in line with signalling analyses performed in primary carcinoma, lymphoma and leukemia cells. Upon activation of the mitochondria, cell death proceeds via the formation of the mitochondrial apoptosome and initiation of the caspase cascade. Consequently, loss of proapoptotic Bax results in the failure of cancer cells to activate caspase-3 in vivo (Prokop et al., 2000).

Calicheamicin is a naturally occurring hydrophobic enediyne antibiotic that was isolated in Micromonospora echinospora calichensis (Maiese et al., 1989). Calicheamicin and other naturally occurring enediynes exhibit significant activity against Gram-positive and -negative bacteria. In addition, calicheamicin exerts a potent antitumor activity and has been shown to induce apoptotic cell death in the picomolar range. Calicheamicin binds to the minor groove of the DNA helix, preferentially to the 3′ ends of oligopurine tracts, and causes sequence-selective oxidation of deoxyribose and bending of the DNA helix by an induced-fit mechanism of DNA target recognition (Salzberg and Dedon, 2000). In addition, calicheamicin and other naturally occurring enediyne antibiotics, including esperamicin, neocarcinostatin, kedarcidin and dynemicin constitute a unique class of reactive compounds, which can undergo aromatization to produce biradicals. Interestingly, phosphodiester bond breakage of DNA was shown to be dispensable for the apoptosis-inducing activity (Hiatt et al., 1994). Furthermore, prevention of apoptosis was observed in analogs that were electronically stabilized to impair aromatic rearrangements and generation of biradicals (Hiatt et al., 1994). Thus, apart from enforcing a conformational change in the DNA structure and the induction of DNA strand breaks, additional signalling events appear to be critically involved in the activation of apoptosis by calicheamicin and its analogs.

In clinical anticancer therapy, the enediyne calicheamicin is currently successfully employed as an immunoconjugate to antibodies serving as vehicles for the targeting of calicheamicin to tumor cells (Linenberger et al., 2001; van der Velden et al., 2001). So far, the mechanisms of apoptosis induction by these compounds remains enigmatic. Paradoxically, the induction of apoptotic cell death in PC12 cells by neocarcinostatin was shown to be potentiated in cells overexpressing the antiapoptotic factor Bcl-2 (Cortazzo and Schor, 1996; Schor et al., 1999). This phenomenon was suggested to occur in consequence of changes in the cellular redox state induced by Bcl-2 leading to enhanced activation of the prodrug neocarcinostatin.

While it is well accepted that enediyne antibiotics are very efficient inducers of apoptosis in the picomolar range (Nicolaou et al., 1993, 1994b), the molecular mechanisms of cell death induction and execution are far from being clear. The aim of the present study was, therefore, to unravel the proapoptotic signalling pathways induced by synthetic calicheamicin ϑII, an analog of the naturally occurring calicheamicin.

Results

Dose-dependent induction of apoptosis by calicheamicin ϑII

Depending on their concentration, many cytostatic substances cause necrotic, membrane damaging as well as apoptotic cell death (Wieder et al., 1998; Friedrich et al., 2001). We therefore determined the unspecific, cytotoxic effect of calicheamicin ϑII (further designated as calicheamicin) in BJAB cells by the determination of LDH release into the culture medium. As shown in Figure 1a, calicheamicin did not significantly reduce viability of BJAB cells after 24 h of incubation at concentrations 100 pM, thereby indicating that a necrosis-like mechanism, for example, through direct membrane damage, does not play a role for its death-inducing potency. After 48 h, however, 25% of the cells showed LDH release, which can be attributed to secondary necrosis that follows apoptosis in vitro. Calicheamicin potently induced DNA fragmentation in up to 60% of the cells. Apoptosis induction was concentration-dependent with a half-maximum concentration of 10 pM (Figure 1b), and DNA fragmentation was already observed after 24 h (Figure 1c). The percentage of cells showing loss of membrane integrity at 48 h (Figure 1a) corresponds well to the number of cells showing DNA fragmentation at 24 h (Figure 1c). Thus, DNA fragmentation precedes membrane damage induced in calicheamicin-induced cells. This indicates that calicheamicin induces apoptotic cell death in BJAB cells and this is followed by secondary necrosis.

Figure 1
figure1

Cytotoxic versus proapoptotic effects of calicheamicin. (a) BJAB cells were treated with different concentrations of calicheamicin for 24 h (open circles) or 48 h (closed circles). Then, viability was determined by the LDH release assay. Values are given as % of control±s.d. (n=3). (b) Dose response: BJAB cells were treated with different concentrations of calicheamicin. After 72 h of incubation, DNA fragmentation was measured by flow cytometric analysis by assessing the cellular DNA content. Values are given as percentages of cells with hypodiploid DNA±s.d. (n=3). (c) Time course: BJAB cells were treated with 100 pM calicheamicin. After different times of incubation, DNA fragmentation was measured by flow cytometric analysis of cellular DNA content. Values are given as percentages of cells with hypodiploid DNA±s.d. (n=3)

Calicheamicin-induced apoptosis occurs independently of CD95/Fas signalling

To investigate the involvement of CD95/Fas or other death receptor systems in calicheamicin-induced cell death, we employed BJAB cells overexpressing a dominant-negative FADD (FADD-dn) mutant. Both mock and FADD-dn transfectants express endogenous FADD (27 kDa), while the FADD-dn transfectants also show a lower band representing the truncated FADD-dn (Wieder et al., 2001a). To provide a positive control for the biological activity of the FADD-dn, we functionally characterized the FADD-dn-overexpressing BJAB cells. Vector-transfected BJAB cells were very sensitive towards challenge with recombinant human soluble Fas ligand (CD95 ligand, CD178): apoptosis as determined by phosphatidylserine exposure on the outer membrane (as determined by Annexin-V–FITC binding to propidium iodide (PI) -negative cells) reached approximately 45% and DNA fragmentation 65% after Fas ligand exposure. In contrast, BJAB/FADD-dn cells were completely resistant against this death stimulus (Figure 2a and c). In the presence of Fas ligand only 10% of BJAB/FADD-dn cells died, thereby clearly demonstrating the efficiency of FADD-dn in these cells. In contrast, neither phosphatidylserine exposure (Figure 2b) nor DNA fragmentation (Figure 2d) were significantly influenced by FADD-dn overexpression when the cells were treated with 1–100 pM calicheamicin. CD95/Fas-independent cell death was also observed in another experimental approach: treatment of BJAB cells in the presence of an antagonistic anti-CD95/Fas antibody did neither impair calicheamicin-induced phosphatidylserine exposure (Figure 3b) nor DNA fragmentation (Figure 3d). The efficiency of the blocking anti-CD95/Fas antibody was demonstrated by blocking the activation of the CD95/Fas pathway by an agonistic anti-CD95/Fas antibody employed as CD95/Fas receptor agonist. Phosphatidylserine exposure on the outer plasma membrane layer (Figure 3a) and DNA fragmentation (Figure 3c) induced upon treatment with agonistic anti-CD95/Fas were inhibited by approximately 73%. Thus, we conclude that calicheamicin-induced apoptosis in BJAB cells proceeds independently of CD95/Fas and FADD signalling.

Figure 2
figure2

Overexpression of dominant-negative FADD in BJAB cells does not impair calicheamicin-induced phosphatidylserine exposure and DNA fragmentation. Vector- or FADD-dn-transfected BJAB cells were either left untreated (control) or incubated with soluble recombinant human Fas ligand for 24 h. Then, phosphatidylserine exposure (a) and DNA fragmentation (c) were measured by flow cytometric analysis. Additionally, vector- or FADD-dn-transfected BJAB cells were treated with different concentrations of calicheamicin for 48 h. Then, phosphatidylserine exposure (b) and DNA fragmentation (d) were measured likewise. Values of phosphatidylserine exposure and DNA fragmentation are given as percentages of annexin-V-positive/ PI-negative cells±s.d. (n=3) and as percentages of cells with hypodiploid DNA±s.d. (n=3), respectively

Figure 3
figure3

Antagonistic anti-Fas/CD95 antibody does not impair calicheamicin-induced phosphatidylserine exposure and DNA fragmentation. BJAB cells were either left untreated (control) or incubated with an agonistic anti-CD95/Fas antibody in the presence or absence of 5 μg/ml blocking, antagonistic anti-Fas/CD95 antibody as indicated in the figure. After 24 h of incubation, phosphatidylserine exposure (a) and DNA fragmentation (c) were measured by flow cytometry. Additionally, BJAB cells were treated with 10 pM calicheamicin in the presence or absence of 5 μg/ml antagonistic anti-Fas/CD95 antibody as indicated in the figure. After 48 h of incubation, phosphatidylserine exposure (b) and DNA fragmentation (d) were measured. Values of phosphatidylserine exposure and DNA fragmentation are given as percentages of annexin-V-positive/PI-negative cells±s.d. (n=3) and as percentages of cells with hypodiploid DNA±s.d. (n=3), respectively

Calicheamicin-induced apoptosis is mediated by the loss of mitochondrial membrane potential, cytochrome c release and caspase-9 and -3 processing

To determine the involvement of the mitochondrial pathway, we measured mitochondrial activation after treatment of BJAB cells with calicheamicin. Exposure to calicheamicin resulted in a dramatic loss of the mitochondrial membrane potential as determined by the cationic dye JC-1 (Figure 4a). After 24 h of incubation, activation of the mitochondria was observed in 58% of the population, thereby clearly preceding DNA fragmentation that only reached 15% at this time point (Figure 4b).

Figure 4
figure4

Calicheamicin-induced mitochondrial permeability transition precedes DNA fragmentation. BJAB cells were incubated with 100 pM calicheamicin. After different times of incubation, mitochondrial permeability transition (a) and DNA fragmentation (b) were measured in parallel by flow cytometric analysis on the single cell level. Values of mitochondrial permeability transition and DNA fragmentation are given as percentages of cells with low ΔΨm±s.d. (n=3) and as percentages of cells with hypodiploid DNA±s.d. (n=3), respectively. Mitochondrial permeability transition and DNA fragmentation of the medium controls (time 0 h) did not change over time

To gain further insight into calicheamicin-induced cell death, we investigated cytochrome c release and consecutive processing of caspase-9 and -3 by means of Western blot analysis. Treatment of BJAB cells led to the release of mitochondrial cytochrome c into the cytosol after incubation with 10–100 pM calicheamicin for 48 h (Figure 5a). Cytochrome c release was paralleled by procaspase-9 processing and appearance of the the 37 kDa cleavage product of procaspase-9 at 48 and 72 h. (Figure 5b). Weak caspase-3 processing to the active p17 subunit was detectable as early as 48 h and then reached a maximum at 72 h. Thus, cytochrome c release and procaspase-9 processing precede caspase-3 activation.

Figure 5
figure5

Calicheamicin induces cytochrome c release and caspase-3 activation. BJAB cells were incubated with different concentrations of calicheamicin or with 1 μg/ml epirubicin as positive control. After 48 or 72 h of incubation, cytosolic extracts were prepared and 30 μg of cytosolic protein were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and subjected to Western blot analysis. Immunoblots developed with anticytochrome c antibody (a) or anticaspase-3 or -9 antibody (b) are shown. Arrows indicate the positions of cytochrome c, procaspase-3 and -9, the 17 kDa active subunit of caspase-3 (p17) and the 37 kDa cleavage product of procaspase-9. The experiment was repeated twice and similar results were obtained

To further investigate the functional role of caspases in the calicheamicin-mediated death signalling pathway, we employed the broad-spectrum caspase inhibitor zVAD-fmk (von Haefen et al., 2002). Interestingly, mitochondrial permeability transition after treatment of BJAB cells with calicheamicin was inhibited by zVAD-fmk in a concentration-dependent manner (Figure 6a). Likewise, calicheamicin-induced DNA fragmentation was inhibited by zVAD-fmk (Figure 6b). Inhibition of both apoptotic hallmarks reached about 50%. We therefore conclude that caspase activation after calicheamicin treatment plays a functional role in death signalling upstream of the mitochondria as well as in the execution of cell death downstream of the mitochondria. A similar mechanism was observed previously during paclitaxel (taxol)-induced apoptosis that relies on a caspase-mediated amplification loop for full activation of the mitochondrial apoptosis pathway (von Haefen et al., 2003).

Figure 6
figure6

Calicheamicin-induced mitochondrial permeability transition and DNA fragmentation are inhibited by the broad-spectrum caspase inhibitor zVAD-fmk. (a) BJAB cells were incubated in the presence of 100 pM calicheamicin. Some cultures were preincubated for 2 h with 10, 20 or 40 μ M zVAD-fmk or 0.2% DMSO (0 μ M zVAD-fmk) as control. After 48 h of incubation, the percentages of cells showing low ΔΨm were determined by flow cytometric analysis. Inhibition of the calicheamicin-induced effects is given in % of control±s.d. (n=3). In these experiments, the percentage of cells with low ΔΨm after calicheamicin treatment was 54%. (b) BJAB cells were incubated in the presence of 100 pM calicheamicin. Some cultures were preincubated for 2 h with 10, 20 or 40 μ M zVAD-fmk or 0.2% DMSO (0 μ M zVAD-fmk) as control. After 48 h of incubation, the percentages of cells showing hypoploid DNA were determined by flow cytometric analysis. Inhibition of the calicheamicin-induced effects is given in % of control±s.d. (n=3). In these experiments, the percentage of cells with hypodiploid DNA after calicheamicin treatment was 36%

Role of antiapoptotic Bcl-2 and Bcl-xL and proapoptotic Bax on calicheamicin-induced mitochondrial permeability transition and DNA fragmentation

Mitochondrial apoptosis is controlled by anti- and proapoptotic members of the Bcl-2 protein family (for review, see, Martinou and Green, 2001). Indeed, the overexpression of Bcl-2 in Jurkat cells completely blocked calicheamicin-induced loss of mitochondrial membrane potential (Figure 7b) and DNA fragmentation (Figure 7d). In these experiments, we employed epirubicin as a positive control since we had shown previously that epirubicin treatment led to hierarchical activation of the mitochondria followed by the activation of caspase-3 and DNA fragmentation (Wieder et al., 2001a). As expected, epirubicin-induced the loss of mitochondrial membrane potential as well as DNA fragmentation were strongly inhibited in Bcl-2-overexpressing Jurkat cells (Figure 7a and c).

Figure 7
figure7

Overexpression of Bcl-2 blocks calicheamicin-induced mitochondrial permeability transition and DNA fragmentation. Vector- or Bcl-2-transfected Jurkat cells were either left untreated (control), or incubated with 1 μg/ml epirubicin (Epi) for 48 h. Then, mitochondrial permeability transition (a) and DNA fragmentation (c) were measured by flow cytometric analysis. Additionally, vector- or Bcl-2-transfected Jurkat cells were treated with different concentrations of calicheamicin for 48 h. Then, mitochondrial permeability transition (b) and DNA fragmentation (d) were measured by flow cytometry. Values of mitochondrial permeability transition and DNA fragmentation are given as percentages of cells with low ΔΨm±s.d. (n=3) and as percentages of cells with hypodiploid DNA±s.d. (n=3), respectively

These data were confirmed in BJAB cells overexpressing Bcl-xL (Figure 8). Although the efficiency of Bcl-xL was slightly lower in BJAB B cells as compared with Bcl-2 overexpression in the Jurkat T cells, calicheamicin-induced mitochondrial activation and DNA fragmentation were inhibited by 40–65% by Bcl-xL (Figure 8b and d). Epirubicin was again used as a positive control to check the cellular system functionally. In Bcl-xL-overexpressing BJAB cells, epirubicin-induced loss of mitochondrial membrane potential and DNA fragmention were reduced by 67 and 78%, respectively (Figure 8a and c).

Figure 8
figure8

Overexpression of Bcl-xL in BJAB cells inhibits calicheamicin-induced mitochondrial permeability transition and DNA fragmentation. Vector- or Bcl-xL-transfected BJAB cells were either left untreated (control), or incubated with 1 μg/ml epirubicin (Epi) for 48 h. Then, mitochondrial permeability transition (a) and DNA fragmentation (c) were measured by flow cytometric analysis by the use of the cationic dye JC-1. Additionally, vector- or Bcl-xL-transfected BJAB cells were treated with different concentrations of calicheamicin for 48 h. Then, mitochondrial permeability transition (b) and DNA fragmentation (d) were determined. Values of mitochondrial permeability transition and DNA fragmentation are given as percentages of cells with low ΔΨm±s.d. (n=3) and as percentages of cells with hypodiploid DNA (n=3), respectively

In recent studies, evidence was provided that Bax is a key player in different forms of apoptosis initiated via the mitochondrial pathway (Martinou and Green, 2001; Roucou and Martinou, 2001; Smaili et al., 2001; Bosanquet et al., 2002; Hemmati et al., 2002; von Haefen et al., 2002). To this end, we investigated the influence of Bax knockout on calicheamicin-mediated cell death in human colorectal cancer HCT116 cells (Figure 9). Bax-proficient HCT116 wild-type cells were susceptible to treatment with calicheamicin. At picomolar concentrations of calicheamicin, mitochondrial activation and DNA fragmentation were observed in 40 and 28% of the cell population, respectively. In contrast, HCT116 Bax knockout cells cells showed only marginal mitochondrial activation of 13% (Figure 9b) and DNA fragmentation of 9% (Figure 9d). Bax dependency was also observed in the case of epirubicin-induced apoptosis. This demonstrates both the Bax dependency of anthracycline-induced apoptosis and the efficiency of the Bax knockout in our experimental system (Figure 9a and c). Notably, HCT116 cells express significant levels of Bak (Gillissen et al., 2003). As Bak has been shown in a variety of reports to be a key mediator of mitochondrial apoptosis, our present data indicate that calicheamicin operates in a Bak-independent but entirely Bax-dependent manner.

Figure 9
figure9

Bax knockout in HCT116 cells abrogates calicheamicin-induced mitochondrial permeability transition and DNA fragmentation. HCT116 wild-type or HCT116 Bax knockout cells were either left untreated (control), or incubated with 1 μg/ml epirubicin (Epi) for 48 h. Then, mitochondrial permeability transition (a) and DNA fragmentation (c) were measured by flow cytometric analysis. Additionally, HCT116 wild type or HCT116 Bax −/− cells were incubated in the presence of different concentrations of calicheamicin for 48 h. Then, mitochondrial permeability transition (b) and DNA fragmentation (d) were measured by flow cytometry. Values of mitochondrial permeability transition and DNA fragmentation are given as percentages of cells with low ΔΨm±s.d. (n=3) and as percentages of cells with hypodiploid DNA±s.d. (n=3), respectively

Given the central role of Bax and to gain a more complete picture of how calicheamicin induces the activation of the mitochondrial pathway, we studied the role of p53, a transcriptional activator of the Bax gene. HCT116 p53 k.o. cells (Bunz et al., 1998) were compared with HCT116 control cells that carry two wild-type p53 alleles (HCT116 p53 wt). The induction of apoptosis was determined by flow cytometric analysis of genomic DNA fragmentation on the single cell level. Apoptosis occurred to an identical extent in the p53-deficient HCT116 p53 k.o. cells (49%) as compared with the HCT116 p53 wt cells (48%) (Figure 10a). In contrast, the anthracyclin drug epirubicin-induced apoptotic cell death in a clearly p53-dependent manner (Figure 10b). The HCT116 p53 wt cells exhibited an apoptosis rate of 59%, whereas only 27% of the p53 k.o. cells underwent apoptotic DNA fragmentation upon exposure to epirubicin. This corroborates that mechanisms of calicheamicin-induced cell death differ from that induced by the majority of conventional anticancer drugs.

Figure 10
figure10

p53 knockout in HCT116 cells does not affect calicheamicin-induced DNA fragmentation. HCT116 wild-type or HCT116 p53 knockout cells were incubated in the presence of different concentrations of calicheamicin for 48 h (a). Then, genomic DNA fragmentation was measured by flow cytometry. The number of cells undergoing DNA fragmentation is given as percentages of cells with hypodiploid DNA±s.d. (n=3), respectively. Alternatively, cells were incubated with 1 μg/ml epirubicin (Epi) for 48 h (b)

Discussion

Calicheamicin is a naturally occurring enediyne antibiotic with high antileukemic potency and represents the clinically most promising candidate of this class of antitumor agents (Thorson et al., 2000). The tremendous potency of calicheamicin or its synthetic ϑII analog against leukemic cells is, however, accompanied by cytotoxic side effects. Therefore, calicheamicin is employed as an immunoconjugate to focus its cell killing activity to cancer cells. Conjugates to CD33 that is expressed on more than 90% of myeloid leukemic blasts are currently evaluated in therapy of acute myeloid leukemias (reviewed in Nabhan and Tallman, 2002). In this vein treatment of a patient with an anti-CD33 calicheamicin immunoconjugate (gemtuzumab ozogamicin, CMA-676, Mylotarg) led to molecular remission of Philadelphia/bcr-abl-positive acute myeloid leukemia (de Vetten et al., 2000). Meanwhile, gemtuzumab ozogamicin was successfully applied in phase I and phase II trials in patients with acute myeloid leukemias (Sievers et al., 1999; van der Velden et al., 2001).

Although calicheamicin has already found its way to the clinic as immunoconjugate, the molecular mechanisms leading to apoptosis in cancer cells remained enigmatic. This is surprising in view of the impressive apoptosis-inducing potency of this class of antitumor agents. In the present study, we therefore employed the synthetic and highly effective calicheamicin ϑII to investigate apoptosis signalling by this drug. Here, we show that calicheamicin triggers the mitochondrial pathway of apoptosis. A model for calicheamicin-induced apoptosis induction is provided in Figure 11.

Figure 11
figure11

Signalling model of calicheamicin-induced apoptosis. Calicheamicin binds to the minor groove of the DNA helix, preferentially to the 3′ ends of oligopurine tracts and causes sequence-selective oxidation of deoxyribose and bending of the DNA helix by an induced-fit mechanism of DNA target recognition. In addition, calicheamicin forms biradicals that induce DNA strand breaks and additional, so far undefined damage signals. These events trigger a nuclear stress signal that ultimately initiates the mitochondrial apoptosis signalling cascade through death receptor- and FADD-independent mechanisms. The activation of mitochondria occurs through an entirely Bax-dependent mechanism that can be inhibited by either Bcl-2 or Bcl-xL. Mitochondrial activation for apoptosis results in release of cytochrome c into the cytosol, processing of procaspase-9 and subsequent activation of procaspase-3. Notably, inhibition of caspase activities by the pancaspase-inhibitor zVAD-fmk partially interferes with mitochondrial membrane potential breakdown. This suggests that caspase processing might be initiated upstream of the mitochondria. Alternatively, caspases might mediate an autoamplification loop in calicheamicin-induced apoptosis that is required for the full activation of the mitochondrial pathway as described earlier for paclitaxel-induced cell death. Casp: caspase

In contrast to a previous finding suggesting a paradoxically enhanced apoptosis induction in cells overexpressing Bcl-2, both Bcl-2 and Bcl-xL strongly inhibited calicheamicin-induced cell death in the present study. Moreover, calicheamicin induced apoptosis in an entirely Bax-dependent manner: the knockout of proapoptotic Bax in HCT116 cells almost completely abrogated apoptosis induction by calicheamicin. Interestingly, the activation of the mitochondria occurred in a caspase-dependent manner. The pancaspase inhibitor zVAD-fmk not only interfered with cell death execution and inhibited apoptotic DNA fragmentation, but also inhibited mitochondrial activation by calicheamicin. Nevertheless, calicheamicin induced apoptosis in a death-receptor-independent manner. Thus, death receptor-induced apoptosis that is known to trigger caspase activation upstream of the mitochondria (Belka et al., 2000; Daniel, 2000; Daniel et al., 2001), could be ruled out as cause for the mitochondrial activation in the present case. This places caspase activation upstream of the mitochondria and may indicate a unique property of endiyene compounds. Calicheamicin induces apoptotic DNA fragmentation without affecting membrane integrity, thereby ruling out unspecific toxic effects, that can be observed at high concentrations of other hydrophobic substances, for example, ceramides (Wieder et al., 1997) or alkylphosphocholines (Wieder et al., 1998). Apoptotic hallmarks, such as phosphatidylserine exposure and DNA fragmentation, were already observed at very low concentrations of calicheamicin ranging from 10 to 100 pM. Neither overexpression of a dominant-negative mutant of the adaptor protein FADD nor blocking of CD95/Fas by an antagonistic antibody significantly inhibited calicheamicin-induced apoptosis. Thus, apoptosis signalling after calicheamicin treatment proceeds in a CD95/Fas-independent manner through the mitochondrial pathway.

Mitochondrial apoptosis is the most prominent intrinsic apoptosis signalling pathway (for reviews, see, Kroemer and Reed, 2000; Martinou and Green, 2001). It is triggered by various cytotoxic drugs such as taxol or epirubicin (Wieder et al., 2001a; von Haefen et al., 2003), natural compounds such as piceatannol (Wieder et al., 2001b), truncated ceramides (von Haefen et al., 2002) or ionizing radiation (Belka et al., 2000). In a first series of experiments, we demonstrated that challenge with calicheamicin led to a dramatic decrease of the mitochondrial membrane potential, which was observed in approximately 80% of BJAB cells. Mitochondrial permeability transition preceded DNA fragmentation, thereby indicating that mitochondrial activation plays an important role in calicheamicin signalling. This assumption was substantiated by measurements showing that calicheamicin-induced mitochondrial activation was accompanied by cytochrome c release into the cytosol and cleavage of caspase-9 that preceded processing of procaspase-3 to the active 17 kDa subunit. Both cytochrome c release and procaspase-9 processing are generally accepted as essential parts of mitochondrial signalling and execution (Li et al., 1997; Liu et al., 1996; Raisova et al., 2000; von Haefen et al., 2002). This is well in line with our finding that calicheamicin-induced cell death can be inhibited by Bcl-2 and Bcl-xL, as these proteins are key regulators of mitochondrial cytochrome c release. Specifically, employing two independent cellular systems we did not observe the previously suggested paradoxical enhancement of calicheamicin-induced apoptosis in Bcl-2-overexpressing PC12 cells (Cortazzo and Schor, 1996). This indicates that calicheamicin-induced apoptosis critically depends on the activation of the mitochondrial pathway of apoptosis. This model is further supported by the fact that loss of Bax impaired calicheamicin-induced apoptosis. Bax directly mediates the release of cytochrome c and has been implicated in the formation of cytochrome c releasing channels in the outer mitochondrial membrane or the opening of VDAC pores (Tsujimoto and Shimizu, 2000; von Ahsen et al., 2000; van Loo et al., 2002). The effect of Bax loss or Bcl-2/Bcl-xL overexpression was even more pronounced when apoptotic DNA fragmentation was investigated as compared with ΔΨm breakdown. This indicates that loss of ΔΨm can occur in the absence of apoptosis execution.

In a recent review, Martinou and Green have put forward two models for breaking the mitochondrial barrier: (i) after a death signal BH3-only proteins (where BH3 stands for Bcl-2 homology domain 3) activate members of the Bax subfamily leading to their insertion in the outer mitochondrial membrane, where they form large channels and (ii) death stimuli activate a pathway involving classical permeability transition pore openers such as Ca2+. The latter pathway was suggested to operate independently of members of the Bax subfamily of Bcl-2 homologs (Martinou and Green, 2001). In this context, we found that HCT116 Bax knockout cells were insensitive towards calicheamicin, whereas HCT116 wild-type cells died by apoptosis after challenge with the drug. This is worth noting since these cells express significant amounts of Bak (Gillissen et al., 2003). Thus, Bak is not sufficient to mediate sensitivity for caliceamicin in this model. This indicates that calicheamicin triggers cell death preferentially via Bax and not via Bak, that is, in an entirely Bax-dependent manner.

Interestingly, cell death induction by calicheamicin was independent from p53. Both p53 wild-type and p53 knockout HCT116 cells underwent apoptosis to an identical extent when exposed to calicheamicin. This was surprising in view of the established role of p53 as direct transcriptional activator of Bax and indirect transcriptional activator of the Bak gene. Apart from pointing out the potential usefulness of this compound in the treatment of p53 mutated cancers, this finding supports the notion of calicheamicin differing from conventional anticancer drugs.

Caspases are the main executioners of apoptotic cell death (Cohen, 1997). Furthermore, evidence has been provided that caspases are also involved in intracellular signalling pathways (for review, see, Salvesen and Dixit, 1997). For example, a caspase-3-driven mitochondrial feedback loop has been proposed during cytotoxic drug- and UV radiation-induced apoptosis (Slee et al., 2000). Moreover, a caspase-3- and -8-driven feedback amplification loop was recently shown to be required for full release of cytochrome c during paclitaxel-induced apoptosis (von Haefen et al., 2003). Interestingly, the broad-spectrum caspase inhibitor zVAD-fmk almost completely inhibited mitochondrial activation and DNA fragmentation upon calicheamicin ϑII treatment. Thus, calicheamicin-induced mitochondrial permeability transition proceeds in a caspase-dependent manner. The necessity of caspases for mitochondrial activation distinguishes calicheamicin-mediated from, for example, ceramide-induced cell death. In the case of ceramide, it has been recently demonstrated that signalling events upstream of mitochondria are caspase independent, whereas the execution of cell death downstream of mitochondria proceeds in a caspase-dependent manner (Utz and Anderson, 2000; Daniel et al., 2001; Ameisen, 2002; Distelhorst, 2002; Koonin and Aravind, 2002).

This caspase dependency of mitochondrial apoptosis is of special interest, since caspases are generally considered to act as initiators and executioners that are activated either downstream of mitochondrial apoptosis (Engels et al., 2000; Wieder et al., 2001a) or link mitochondria to the the death receptor pathway (Belka et al., 2000, 2001). Even while caspases act upstream of the mitochondria in the latter case, we could clearly exclude death receptor initiated signalling events in calicheamicin-induced apoptosis. Thus, caspases appear to be initiators of calicheamicin-induced mitochondrial activation and execution of apoptosis in a Bax-dependent manner.

The mechanisms of caspase activation by calicheamicin remain, however, to be elucidated. Caspase activation taking place upstream of the mitochondria has recently been described as potential link between nuclear stress, for example by the topoisomerase II inhibitor etoposide, and the mitochondrial apoptosis pathway (Lassus et al., 2002; Robertson et al., 2002). This may indicate that the key steps of caspase activation are initiated upstream of the mitochondria and are then utilized to amplify the mitochondrial cell death pathway in a Bcl-2-dependent manner (Marsden et al., 2002). Nevertheless, zVAD-fmk is a rather weak inhibitor of caspase-2 and this indirectly suggests that caspase-2 is not the key effector in this setting.

Given the interaction of calicheamicin and other enediyne compounds with the DNA, we favor the concept of a nuclear stress signal that initiates apoptosis. In this line, there is recent evidence that caspase activation could potentially be initiated out of the cell cycle by direct interaction with cell cycle regulating proteins: caspase-3 was shown to interact with p21 physically (Suzuki et al., 1999) or cyclin D3 (Mendelsohn et al., 2002). Another possibility how caspase activation may take place upstream of the mitochondria could be a mitochondrial amplification loop (Slee et al., 2000; von Haefen et al., 2003). There, caspase-3 and -8 activation is initiated downstream of the mitochondria but then serves as secondary amplifier to achieve optimal mitochondrial activation, possibly via a Bid-dependent mechanism. In the case of calicheamicin, the exact signaling requirements and caspases involved in this loop are under current investigation.

In summary, the data presented in this study provide experimental evidence how calicheamicin mediates apoptosis in a p53-independent manner through an entirely Bax-dependent activation of the mitochondrial pathway. These insights into cell death pathways after calicheamicin treatment not only help to understand the impressive potency of this anticancer compound, but also serve as basis for the currently undertaken efforts to elucidate the upstream events in enediyne-induced apoptosis initiation.

Materials and methods

Materials

Polyclonal rabbit anti-human caspase-3 (developed against human recombinant protein) and monoclonal mouse anti-human cytochrome c (clone 7H8. 2C12) antibodies were from Pharmingen (Hamburg, Germany) and were used at 1 : 2000 and 1 : 1000, respectively. Polyclonal goat anti-human caspase-9 was from R&D Systems GmbH (Wiesbaden-Nordenstadt, Germany) and was used at 1 : 1000. Secondary anti-rabbit, anti-goat and anti-mouse horseradish peroxidase (HRP)-conjugated antibodies were from Promega (Mannheim, Germany) or Southern Biotechnology Associates (Birmingham, AL, USA) and were used at 1 : 5000. Monoclonal, antagonistic anti-CD95/Fas antibody (clone SM1/23) was from Bender Med Systems (Vienna, Austria) and was used at a concentration of 5 μg/ml. RNase A was from Roth (Karlsruhe, Germany). The broad-spectrum caspase inhibitor zVAD-fmk (in which z stands for benzyloxycarbonyl and fmk for fluoromethyl ketone) was from Calbiochem-Novabiochem GmbH (Bad Soden, Germany) and was dissolved in dimethyl sulfoxide (DMSO) to give a 20 mM stock solution. According to the manufacturer, this inhibitor was synthesized as a methyl ester to enhance cell permeability. DMSO (0.2%) (vehicle) was added to controls. This treatment did not induce significant apoptotic DNA fragmentation (data not shown). Epirubicin was purchased from Pharmacia Upjohn (Erlangen, Germany). Calicheamicin ϑII was prepared by total synthesis in 49 steps as described previously (Nicolaou et al., 1994a) and was dissolved in DMSO to give a 0.1 μ M stock solution. Calicheamicin was then further diluted into growth medium to achieve picomolar concentrations.

Cell culture

Control vector- and FADD-dn-transfected BJAB cells stably expressing a dominant-negative FADD mutant lacking the N-terminal death effector domain have been described earlier (Wieder et al., 2001a). Bcl-xL-transfected BJAB cells were a kind gift of S Fulda (University of Ulm, Germany) and have been characterized in a previous study (Fulda et al., 2001). Control vector- and Bcl-2-transfected Jurkat cells (Belka et al., 2000) stably overexpressing the antiapoptotic protein Bcl-2 were a kind gift of K Schulze-Osthoff (University of Münster, Germany). Cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 0.56 g/l L-glutamine, 100 000 U/l penicillin and 0.1 g/l streptomycin. Media and culture reagents were from Life Technologies GmbH (Karlsruhe, Germany). BJAB and Jurkat cells were subcultured every 3–4 days by dilution of the cells to a concentration of 1 × 105 cells/ml. Human colorectal cancer HCT116 wild-type cells, HCT116 p53 knockout (Bunz et al., 1998) and HCT116 Bax knockout cells (Zhang et al., 2000) were a kind gift of Bert Vogelstein (Johns Hopkins University School of Medicine, Baltimore, MD, USA). Confluent HCT116 cells were subcultured every 4–5 days after detaching the cells with 0.1% trypsin, 0.02% ethylenediamine tetraacetic acid (EDTA) in phosphate-buffered saline (PBS). HCT116 cell clones were cultured under the same conditions as described for the BJAB and Jurkat cells above.

Measurement of cell death

Cytotoxicity of calicheamicin was measured by the release of lactate dehydrogenase (LDH) as described previously (Friedrich et al., 2001). After incubation with different concentrations of calicheamicin for 24 and 48 h, LDH activity released by BJAB cells was measured in the cell culture supernatants using the Cytotoxicity Detection Kit from Boehringer-Mannheim (Mannheim, Germany). The supernatants were centrifuged at 300 g for 5 min. Cell-free supernatants (20 μl) were diluted with 80 μl PBS and 100 μl reaction mixture containing 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT), sodium lactate, NAD+ and diaphorase were added. Then, time-dependent formation of the reaction product was quantified photometrically at 490 nm. The maximum amount of LDH activity released by the cells was determined by lysis of the cells using 0.1% Triton X-100 in culture medium and was set as 100% cell death.

Measurement of CD95/Fas-mediated cell death

For determination of CD95/Fas-mediated cell death, 1 × 105 BJAB cells/ml were treated with either recombinant human soluble Fas ligand (rhs Super-Fas ligand; Alexis, Grünberg, Germany) at 0.1 μg/ml or with an agonistic, that is, crosslinking, anti-CD95/Fas antibody (clone anti-APO-1 IgG3) at 1 μg/ml for 24 h (Dhein et al., 1992). Then, cell death was assessed by measurement of phosphatidylserine exposure or by measurement of DNA fragmentation as described below. In some experiments, a blocking anti-CD95/Fas antibody (clone SM1/23) was added at 5 μg/ml.

Measurement of phosphatidylserine exposure

Cell death was determined by staining cells with annexin-V–FITC and counterstaining with PI. During apoptosis, the phospholipid phosphatidylserine is exposed to the outer leaflet of the plasma membrane (Fadok et al., 2001; Schlegel and Williamson, 2001). Annexin-V–FITC then binds to phosphatidylserine leading to an increase of the fluorescence. On the other hand, PI is excluded from cells with intact membranes. PI positivity is therefore a sign of cell necrosis, whereas cells which are annexin-V–FITC positive but PI negative are generally defined as apoptotic (Vermes et al., 1995). For the Annexin-V/PI assay, 1 × 105 cells were washed twice with ice-cold PBS and then resuspended in binding buffer (10 mM N-(2-hydroxyethyl)piperazin-N′-3(propansulfonicacid) (HEPES)/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) at a concentration of 1 × 106 cells/ml. Next, 5 μl of Annexin-V–FITC (BD Pharmingen, Heidelberg, Germany) and 10 μl of 50 μg/ml PI (Sigma-Aldrich, Taufkirchen, Germany) were added to the cells. Analyses were performed on an FACScan (Becton Dickinson, Heidelberg, Germany) using the CellQuest analysis software.

Measurement of DNA fragmentation

DNA fragmentation was measured essentially as described (Daniel et al., 1999; Essmann et al., 2000). After treatment with different concentrations of calicheamicin, cells were detached by trypsination and collected by centrifugation at 300 g for 5 min. Cells were first washed with complete cell culture medium to stop tryptic digestion and then with PBS at 4°C. Cells were fixed in PBS/0.74% (v/v) formaldehyde on ice for 30 min, pelleted, incubated with ethanol/PBS (2 : 1, v/v) for 15 min, pelleted and resuspended in PBS containing 40 μg/ml RNase A. RNA was digested for 30 min at 37°C. Cells were pelleted again and finally resuspended in PBS containing 50 μg/ml PI. Nuclear DNA fragmentation was quantified by flow cytometric determination of hypodiploid DNA. Data were collected and analysed using an FACScan (Becton Dickinson; Heidelberg, Germany) equipped with the CELLQuest software. Data are given in % hypoploidy (subG1), which reflects the number of apoptotic cells.

Measurement of the mitochondrial permeability transition

After incubation with different concentrations of calicheamicin ϑII, mitochondrial permeability transition was determined by staining the cells with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanin iodide (JC-1; Molecular Probes, Leiden, The Netherlands) exactly as described elsewhere (Wieder et al., 2001a). Mitochondrial permeability transition was then quantified by flow cytometric determination of cells with decreased fluorescence, that is, with mitochondria displaying a lower membrane potential. Data were collected and analysed using an FACScan (Becton Dickinson) equipped with the CELLQuest software. Data are given in % cells with low ΔΨm, which reflects the number of cells undergoing mitochondrial apoptosis.

Determination of cytochrome c release and processing of caspase-3

Cytosolic extracts were prepared according to a method described previously (Raisova et al., 2001; von Haefen et al., 2002). After incubation of BJAB cells in medium supplemented with different concentrations of calicheamicin, cells were harvested in PBS, equilibrated in hypotonic buffer (20 mM HEPES (pH 7.4), 10 mM KCl, 2 mM MgCl2, 1 mM EDTA) and centrifuged at 300 g for 5 min. The resulting cell pellets were then dissolved in hypotonic buffer containing phenylmethyl sulfonylfluoride (final concentration 0.1 mM) and incubated on ice for 15 min. Cells were homogenized by passing the cells through a syringe (G 20) approximately 20 times. The membranes were pelleted by twofold centrifugation at 10 000 g, 4°C for 10 min and the supernatant of the second centrifugation was used as cytosolic extract. Protein concentration was determined using the bicinchoninic acid assay from Pierce (Rockford, IL, USA). Then, Western blot analyses with 30 μg, cytosolic protein were performed as described below and conducted with anticytochrome c antibodies or anticaspase-3/-9 antibodies.

Western blot analysis

Cytosolic protein (30 μg) were loaded per lane and were separated by SDS–PAGE. Then, blotting of proteins onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany) was performed exactly as described (Wieder et al., 2001a). After blotting, the membrane was blocked for 1 h in PBST (PBS, 0.05% Tween-20) containing 3% nonfat dry milk and incubated with primary antibody for 1 h. After the membrane had been washed three times in PBST, secondary antibody in PBST was applied for 1 h. Finally, the membrane was washed in PBST again and the ECL enhanced chemiluminescence system from Amersham Buchler (Braunschweig, Germany) was used to visualize the protein bands in question.

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Acknowledgements

This work was supported by grants from the Deutsche José Carreras Leukämie-Stiftung, the Verein zur Förderung der Tagesklinik and the Deutsche Krebshilfe. We thank B Vogelstein (Johns Hopkins University School of Medicine, Baltimore, MD, USA) for kindly providing HCT116 wild-type, p53 −/− and Bax −/− cells and the congeneic controls, S Fulda (University of Ulm, Germany) for kindly providing BJAB cells overexpressing Bcl-xL and K Schulze-Osthoff (University of Düsseldorf, Germany) for kindly providing Jurkat cells overexpressing Bcl-2. The excellent technical assistance of A Richter is gratefully acknowledged.

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Correspondence to Peter T Daniel.

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Prokop, A., Wrasidlo, W., Lode, H. et al. Induction of apoptosis by enediyne antibiotic calicheamicin ϑII proceeds through a caspase-mediated mitochondrial amplification loop in an entirely Bax-dependent manner. Oncogene 22, 9107–9120 (2003). https://doi.org/10.1038/sj.onc.1207196

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Keywords

  • calicheamicin
  • apoptosis
  • caspase-3
  • Bax
  • Bcl-2
  • cytochrome c
  • mitochondria
  • BJAB
  • Jurkat
  • HCT116

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