Affiliations of authors: P. Costantini, E. Jacotot, G. Kroemer, Centre National de la Recherche Scientifique, Institut Gustave Roussy, Villejuif, France; D. Decaudin, Institut Curie, Département d'Hématologie, Paris, France.
Correspondence to: Guido Kroemer, M.D., Ph.D., 19 rue Guy Môquet, B.P. 8, F-94801 Villejuif, France (e-mail: kroemer{at}infobiogen.fr).
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ABSTRACT |
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INTRODUCTION |
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CELL BIOLOGY OF APOPTOSIS |
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The understanding of apoptosis has recently been facilitated by the development of cell-free systems. Instead of considering the cell as a black box, subcellular fractions (e.g., mitochondria, nuclei, and cytosols) are mixed together with the aim to reconstitute the apoptosis phenomenon by recapitulating the essential steps of the process in vitro (19,2231). With the use of this approach, we have demonstrated that apoptosis of mammalian cells develops in several steps (see Fig. 1). Schematically, it appears that proapoptotic second messengers, whose nature depends on the apoptosis-inducing agent, accumulate in the cytosol during the initiation phase. These agents then induce mitochondrial membrane permeabilization, allowing cells to enter the decision phase. The apoptotic changes of mitochondria consist in a
m loss, transient swelling of the mitochondrial matrix, mechanical rupture of the outer membrane and/or its nonspecific permeabilization by giant proteinpermeant pores, and release of soluble intermembrane proteins (SIMPs) through the outer membrane (5,6,10,11,1921,32). Once the mitochondrial membrane barrier function is lost, several factors, e.g., the metabolic consequences at the bioenergetic level, the loss of redox homeostasis, and the perturbation of ion homeostasis, contribute to cell death. The activation of proteases (caspases) and nucleases by SIMPs is necessary for the acquisition of apoptotic morphology (4,19,2231,33). This latter phase corresponds to the degradation step, beyond the point of no return of the apoptotic process. Different SIMPs provide a molecular link between mitochondrial membrane permeabilization and the activation of catabolic hydrolases: cytochrome c (a heme protein that participates in caspase activation) (34), certain procaspases (in particular, procaspases 2 and 9, which, in some cell types, are selectively enriched in mitochondria) (25,35), and AIF (19,22,24). AIF is a nuclear-encoded intermembrane flavoprotein that translocates to the nucleus where it induces the caspase-independent peripheral chromatin condensation and the degradation of DNA into 50-kilobase-pair fragments (24).
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MOLECULAR MECHANISMS OF MITOCHONDRIAL MEMBRANE PERMEABILIZATION |
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Is the PTPC (and its components) the only mechanism by which mitochondrial membranes are permeabilized? The answer is not clear. Although apoptosis is almost universally accompanied by a loss of the m and although PTPC inhibitors (CsA, BA, and/or proteins of the Bcl-2 family) frequently inhibit the mitochondrial and postmitochondrial manifestations of apoptosis, it cannot be excluded that additional mechanisms exist. Thus, proapoptotic members of the Bcl-2 family, such as Bax or Bid, may cause outer-membrane permeabilization without inducing an immediate
m dissipation (37,38). Apoptosis without a complete
m loss has also been reported to occur in some cell lines, such as HL60 (21). Because Bax can kill yeast cells lacking VDAC expression, at least in some experimental settings (59), it is possible that Bax (and other proapoptotic members of the Bcl-2 family?) may act in an autonomous fashion, i.e., by forming giant channels and/or acting on mitochondrial structures other than the PTPC.
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NEOPLASIA AND CHEMOTHERAPY: ROLE OF MITOCHONDRIA |
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The gene coding for ANT2 (one of the three ANT isoenzymes), whose expression is normally repressed in quiescent cells, is transcribed in dedifferentiated, proliferating tumor cells (60,61). Three other putative PTPC components (Fig. 2, C), i.e., PBR, the PBR-associated protein Prax-1, and mitochondrial creatine kinase, are also overexpressed in some tumors (52,6265). Intriguingly, mitochondrial creatine kinase can confer apoptosis resistance (and inhibition of the PTPC) in the presence of creatine (52,64). Moreover, overexpression of PBR confers a relative resistance to oxidative stress (66). More important, the expression of functional Bax is frequently reduced in cancer cells, either at the transcriptional level or because of loss-of-function mutations (6771), and Bcl-2 or its antiapoptotic homologues are overexpressed in a large percentage of neoplasias (7275). This strengthens the hypothesis that the composition and/or the control of the PTPC can be altered in tumors.
Cancer cells withstand an adverse microenvironment (hypoxia, acidosis, hypoglycemia, and shortage of growth factors) by virtue of metabolic adaptation. Solid tumors are characterized by a resistance to hypoxia coupled to an increased anaerobic glycolysis that is not influenced by the oxygen concentration (Warburg effect) (7678). The Warburg effect is still not fully understood. In this context, it may be intriguing that the hypoxia-inducible transcription factor, in conjunction with mutant p53 protein, accounts for the overexpression of the glycolytic enzyme hexokinase II (79), which is associated with VDAC in normal brain and in a variety of tumors (but not in other normal tissues) (80). For instance, mitochondria from hepatoma or hepatocellular carcinoma do bind hexokinase II, but normal liver cell mitochondria do not (78). Whether this or additional alterations in the composition of the PTPC may explain the Warburg effect remains an open question.
Chemotherapy aims at the specific eradication of cancer cells, mostly through the induction of apoptosis. What is the practical implication of the mitochondrial control of apoptosis? As mentioned above, mitochondrial membrane permeabilization is a near-to-general feature of apoptosis (2,4,81). The mere detection of such mitochondrial alterations thus does not distinguish between two fundamentally different options: the direct action of a chemotherapeutic drug on mitochondria or the indirect perturbation of mitochondrial function through the activation of proapoptotic signal transduction pathways and/or damage of extramitochondrial structures. To distinguish between these possibilities, experiments have to be performed in which the effects of the anticancer drugs on isolated mitochondria (step 2 in Fig. 1) or on purified PTPC (Fig. 3
) are evaluated. One particularly interesting possibility consists in combining different cellular components (i.e., nuclei, cytosols, mitochondria, etc.) in a cell-free system to study the minimum requirement for the activation of caspases or the induction of nuclear apoptosis by anticancer agents. Several conventional anticancer agents, such as etoposide, doxorubicin, or cisplatin, have no direct effect on mitochondria (82). Instead, they activate signal transduction pathways that are also involved in physiologic apoptosis. In the following sections, we will discuss which of the endogenous and xenobiotic agents have a direct effect on mitochondria.
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PROAPOPTOTIC SIGNAL-TRANSDUCING MOLECULES ACTING ON MITOCHONDRIA |
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Redox metabolism.
An enhanced generation of reactive oxygen species is not always the result of cellular damage; it also can result from overexpression of the proapoptotic antioncogene p53 (83) or from treatment of cells with the second messenger ceramide (84). Changes in cellular redox potentials due to an enhanced generation of reactive oxygen species (or a decrease in their detoxification), depletion of nonoxidized glutathione, or depletion of NADPH suffice to induce or to facilitate PTPC opening (53,85,86). Peroxynitrite (which is formed by the reaction of nitric oxide with superoxide anion) is also a potent PTPC trigger (87,88) (Fig. 4). The mitochondrial megachannel possesses several redox-sensitive sites, one that is modulated by NADPH and another that is in equilibrium with mitochondrial matrix glutathione (89). This latter site is likely to be Cys56 of the ANT, based on the observation that oxidation of this thiol (which is exposed to the matrix) suffices to convert ANT into a large nonspecific pore (90).
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Lipid messengers. Ceramide is generated in cells exposed to several apoptosis-inducing stimuli, including signaling via the Fas/Apo-1/CD95 receptor or the tumor necrosis factor receptor, nonspecific stress, or cytotoxic drugs (92). When added to cells, ceramide induces mitochondrial membrane permeabilization (23,93,94) but does not induce PTPC opening in isolated mitochondria (23). To induce apoptosis, ceramide must be converted into ganglioside GD3 in the Golgi apparatus (94). GD3 then translocates to mitochondria and causes PTPC opening in a direct fashion, since this has been demonstrated both in isolated mitochondria and in intact cells (95). In addition to GD3, the fatty acid palmitate (which interacts with ANT) (96) and the lipid oxidation product 4-hydroxyhexenal (97) can induce PTPC opening when they are added to purified mitochondria.
Cytosolic calcium. Ca2+ ions are among the most efficient triggers of the PTPC. At supraphysiologic doses (>>10 µM), Ca2+ suffices to induce permeability transition (PT); at lower doses, it facilitates the induction of PT by other stimuli (53,85). Increases in free Ca2+ concentrations are also important comediators of apoptotic cell death. It is unknown as to what extent Ca2+ triggers cell death via direct mitochondrial effects. However, it appears that Bcl-2 overexpression enhances the tolerance of mitochondria to Ca2+ (98).
Proapoptotic members of the Bcl-2 family. On induction of apoptosis, several proapoptotic Bcl-2 family members can translocate from the cytosol (Bax, Bid, and Bad) or from microtubuli (Bim) to mitochondria, where they incorporate into the outer membrane and may undergo a conformational change (36,99,100). The mechanism of Bax translocation is unclear (99), but Bid translocation may involve its cleavage by caspase 8 (101), and Bad translocation may involve its dephosphorylation, causing its release from cytosolic 14-3-3 protein (102). Bax and Bak cause mitochondrial membrane permeabilization in a way that is inhibited by the PTPC inhibitors CsA and BA (39,41,42), although this conclusion has been contested (37). Proapoptotic signaling may also lead to the inactivation of antiapoptotic members of the Bcl-2 family. Inactivation of Bcl-2 is achieved by chemotherapeutic agents (such as paclitaxel), which act on microtubule assembly and cause its hyperphosphorylation (103) and, simultaneously, favor opening of the PT pore (104). In addition, Bcl-2 can be cleaved by caspase 3 in a reaction that yields a proapoptotic product (105).
Caspases. Ligation of some receptors can lead to a rapid proteolytic activation of caspases within seconds or minutes. This was first documented for the Fas/Apo-1/CD95 receptor, which, on interaction with the Fas/Apo-1/CD95 ligand, recruits caspase 8 into the receptor complex and causes its activation. Caspases can act on members of the Bcl-2 family (e.g., caspase 8 cleaves Bid, caspase 1 cleaves Bcl-XL, and caspase 3 cleaves Bcl-2; see above), thereby activating proapoptotic members of the Bcl-2 family (Bid) or inactivating antiapoptotic members (Bcl-2 and Bcl-XL). In the Fas/Apo-1/CD95-triggered pathway, mitochondrial membrane permeabilization (mediated by Bid or ceramide/GD3) may be a prerequisite of cell death ("type 2 cells") but may be dispensable when caspase 8 activates other caspases in a direct fashion ("type 1 cells") (106,107).
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CHEMOTHERAPEUTIC AGENTS ACTING DIRECTLY ON MITOCHONDRIA |
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Arsenite (the trivalent inorganic salt formed by arsenic trioxide) recently has become a therapeutic agent of choice for the treatment of acute promyelocytic leukemia (APL) (116). Moreover, human T-cell leukemia/lymphotropic virus type I (HTLV-I)-infected cells, myeloma cells, and transformed lymphocytes are extremely sensitive to arsenic (117,118). Cell-free systems of apoptosis have revealed that arsenite requires mitochondria to induce nuclear apoptosis in vitro (58). Moreover, arsenite acts on isolated mitochondria to induce PTPC opening (89). Since arsenite toxicity is modulated by the reduced glutathione content (119), it may be speculated that it acts as a thiol-oxidizing agent (89). However, arsenite does not cause oxidation of Cys56 of ANT as other thiol-reactive agents do (90). Arsenite acts on the purified PTPC reconstituted into liposomes in vitro (58). In such a system, recombinant Bcl-2 prevents PTPC opening. This parallels the observation that transfection-mediated overexpression of Bcl-2 protects cells against the proapoptotic effect of arsenite (58).
CD437 (6[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid) is a new synthetic retinoid acid receptor (RAR
) agonist inducing apoptosis of human breast, lung, cervical, and ovarian carcinomas, melanoma, prostate cancer cells, neuroblastoma, and APL. Several mechanisms of induction of the cell-death process have been reported: activation of AP-1 complex (120); increase of p53, p21, and Bax (121); decrease of Bcl-XL (122); cell-cycle arrest; and activation of caspase 3 and caspase 7. Although it was tacitly assumed that CD437 acts via RAR
, CD437 can also kill RAR
-negative cell lines (123) and cytoplasts (i.e., cells without a nucleus) (124). Thus, CD437-dependent apoptosis does not require activation of RAR
or that of any other nuclear RAR. Moreover, in intact cells CD437-dependent caspase activation is preceded by the release of cytochrome c from mitochondria, and this release is not affected by the caspase inhibitor Z-VAD-fmk (124). CD437 also causes membrane permeabilization when added to purified mitochondria, and this effect is prevented by the PTPC inhibitors CsA and BA. Since CD437-mediated cell killing is suppressed by CsA and BA (124), it appears plausible that CD437 exerts its cytotoxic effects via the PTPC.
Betulinic acid, a pentacyclic triterpene, is a novel experimental anticancer drug. It possesses an antitumoral activity in vitro and in vivo in melanoma, neuroectodermal tumors, and glioma cell lines. Fulda et al. (82) have shown that betulinic acid induces apoptosis via direct mitochondrial alterations. All of these effects have been observed in intact cells and in cell-free systems. When added to isolated mitochondria, betulinic acid directly induces loss of m in a way that is not affected by the caspase inhibitor Z-VAD-fmk and yet is inhibited by BA. In a cell-free system comprising mitochondria, cytosols, and purified nuclei, mitochondria undergoing betulinic acid-induced permeability transition mediate cytosolic caspase activation (caspase 8 and caspase 3) and nuclear fragmentation via the liberation of soluble factors, such as cytochrome c or AIF (125). Bcl-2 and Bcl-XL block all mitochondrial and cellular manifestations of apoptosis induced by betulinic acid, as does BA, an inhibitor of the PTPC (125).
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DRUG DESIGN: PROAPOPTOTIC PEPTIDES TARGETED TO MITOCHONDRIA |
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The data discussed above suggest that lethal peptides may be targeted to mitochondria and more specifically, at least in the case of Vpr, to the PTPC. Ellerby et al. (130) recently have fused the mitochondriotoxic (KLAKLAK)2 motif to a targeting peptide that interacts with endothelial cells. Such a fusion peptide is internalized and induces mitochondrial membrane permeabilization in angiogenic endothelial cells and kills MDA-MD-435 breast cancer xenografts transplanted into nude mice. Similarly, a recombinant chimeric protein containing interleukin 2 (IL-2) protein fused to Bax selectively binds to and kills IL-2 receptor-bearing cells in vitro (134). Thus, specific cytotoxic agents that target surface receptors, translocate into the cytoplasm, and induce apoptosis via mitochondrial membrane permeabilization might be useful in treating cancer.
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TWO STRATEGIES TO OVERCOME BCL-2-MEDIATED INHIBITION OF APOPTOSIS AT THE MITOCHONDRIAL LEVEL |
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A second strategy to overcome Bcl-2-mediated cytoprotection consists in the use of ligands of the PBR (135,136), an outer mitochondrial membrane protein that physically interacts with VDAC and ANT (50). Ligands of the mitochondrial benzodiazepin receptor, such as PK11195, can overcome the apoptosis resistance of Bcl-2-overexpressing cells in response to diverse stimuli, including glucocorticoids (136), the topoisomerase inhibitor etoposide (136), LND (56), or arsenite (58). PK11195 also abolishes the resistance of Bcl-2-overexpressing mitochondria to the induction of PTPC opening by the ANT ligand atractyloside in vitro (136). Protoporphyrin IX, a PBR ligand, can also override Bcl-2-mediated cytoprotection (135). Verteporfin, a porphyrin-derived photosensitizer, similarly causes mitochondrial membrane permeabilization irrespective of Bcl-2 or Bcl-XL overexpression (137). Diazepam, also a ligand of the mitochondrial peripheral benzodiazepine receptor, and LND have synergistic effects in the treatment of nude mice bearing human glioblastoma (138).
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PERSPECTIVES |
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On theoretic grounds, selective eradication of transformed cells by use of mitochondrion-specific agents should be effective. One strategy is to target a toxic agent to selected cell types on the basis of the specific expression of surface receptors. Another, yet to be developed, strategy would aim at exploiting differences in the composition or regulation of the PTPC between normal and tumor cells. Future research will tell to which extent cell targeting (by use of retroviral or adenoviral vectors, use of integrin-specific domains, etc.) and/or targeting of tumor-specific alterations in the PTPC will prove to be useful in cancer therapy.
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NOTES |
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Manuscript received June 23, 1999; revised April 5, 2000; accepted May 1, 2000.
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