EDITORIALS

Arsenic Trioxide, a Novel Mitochondriotoxic Anticancer Agent?

Guido Kroemer, Hugues de Thé

Affiliations of authors: G. Kroemer, Centre National de la Recherche Scientifique (CNRS), EST1984, Villejuif, France; H. de Thé, CNRS, UPR9051, Laboratoire associé No. 11 Comité de Paris de la Ligue contre le Cancer, Laboratoire associé à l'Université de Paris VII, Hôpital St. Louis, Paris, France.

Correspondence to: Guido Kroemer, M.D., Ph.D., Centre National de la Recherche Scientifique, Unité Propre de Recherche 420, Génétique Moléculaire et Biologie du Développement, 19, rue Guy Môquet, BP8, 94801 Villejuif Cedex, France (e-mail: kroemer{at}infobiogen.fr).

During the last few years, it has become increasingly clear that mitochondria play a major rate-limiting role in apoptosis (1-3). In general terms, the apoptotic process can be subdivided into three phases: the initiation phase, the decision/effector phase, and the degradation phase. During the heterogeneous initiation phase, which is essentially premitochondrial, specific pro-apoptotic signal transduction pathways or nonspecific damage pathways are activated. These pathways converge on the mitochondria during the decision/effector phase, where they trigger progressive permeabilization of mitochondrial membranes, mostly as a result of the action of the permeability transition pore complex (PTPC). Thus, the mitochondrion (or to be more precise, the PTPC, which interacts with the Bcl-2/Bax complex) "decides" the cell's fate and determines the point of no return of the process (4,5). The morphologic and biochemical features of apoptosis become manifest during the postmitochondrial degradation phase, in which soluble intermembrane proteins (SIMPs) released from mitochondria play an active role: AIF (apoptosis-inducing factor) translocates to the nucleus, where it induces large-scale DNA fragmentation (6); cytochrome c triggers the activation of pro-caspase-9 (7); and pro-caspases 2, 3, and 9 intervene in a cascade of proteolytic destruction (8,9). Inhibition of SIMPs does not prevent cell death as such, although it may cause a shift from apoptotic to non-apoptotic cell death (2).

The goal of chemotherapy is to kill tumor cells, mostly by inducing apoptosis. What then is the practical implication of PTPC and SIMPs? Some gross alterations in mitochondrial function, such as a dissipation of the mitochondrial inner transmembrane potential ({Delta}{Psi}m) or the release of SIMPs (via the outer membrane), are near-to-general features of apoptosis (1-3). The mere detection of mitochondrial changes thus does not distinguish between two fundamentally different possibilities, namely, that a chemotherapeutic agent acts directly on mitochondria or rather that it compromises mitochondrial function in an indirect fashion, by activating extramitochondrial pro-apoptotic signal transduction or damage pathways. To discriminate between these possibilities, it is necessary to perform experiments in which the chemotherapeutic agent is added to purified mitochondria in vitro and local effects (activation of the PTPC and release of SIMPs) are studied. One particularly interesting possibility is to combine different components of the cell (e.g., nuclei, cytosols, and mitochondria) in vitro to study the minimum requirements for caspase activation or induction of nuclear apoptosis induced by anticancer agents. With this approach, it has become clear that a number of experimental drugs act primarily on mitochondria to induce apoptosis. This applies to lonidamine (an agent used in phase II studies of breast cancer therapy) (10), betulinic acid (an agent that selectively kills neuroectodermal cells) (11,12), thiol-cross-linking agents such as diamide (13), and ligands of the peripheral benzodiazepine receptor (14) including photo-activable porphyrin derivatives (15). In contrast, classical chemotherapeutic agents such as etoposide, doxorubicin, or cisplatin have no direct mitochondrial effects (11). Such agents act by inducing p53, by activating ceramide, by stimulating the CD95/CD95L pathway, and/or by inducing major shifts in redox potentials. Each of these alterations then can affect mitochondria in an indirect fashion, p53 by affecting the redox balance (see below) (16,17), ceramide by formation of ganglioside GD3 and/or influencing the function of BAD (which interacts with Bcl-2) (18), CD95 by apical caspases (which act on mitochondria) (19), and redox imbalances by effects on redox-sensitive sites within the PTPC (20). The level at which a chemotherapeutic agent acts is likely to have some practical impact because, on theoretical grounds, agents that directly affect mitochondria should bypass any resistance due to interruption of the indirect death-inducing pathways (e.g., mutations of p53 and loss of CD95). Whether this is the case for arsenic trioxide awaits further confirmation.

Arsenic trioxide has recently become a therapeutic agent of choice for the treatment of acute promyelocytic leukemia (APL). The report by Zhu et al. (21) in this issue of the Journal shows that arsenic trioxide induces (as do most other chemotherapeutic agents) an early {Delta}{Psi}m collapse, thus extending the observation that arsenite (the trivalent inorganic salt formed by arsenic trioxide) causes the release of cytochrome c from mitochondria to the cytosol (22). Cell-free systems of apoptosis reveal that arsenite requires mitochondria to induce nuclear apoptosis in vitro (23). Moreover, arsenite acts on isolated mitochondria to induce the opening of PTPC, presumably by acting as a thiol-oxidizing agent (20). In accordance with this idea, intracellular glutathione levels determine the dose of arsenite required to kill cells (21,24). Arsenite also acts on purified PTPC reconstituted in liposomes in vitro (23). In such a system, recombinant Bcl-2 prevents the opening of PTPC, which is in agreement with the fact that transfection-enforced overexpression of Bcl-2 protects cells against the pro-apoptotic effect of arsenite (23).

If these data suggest that arsenic trioxide can induce apoptosis via a direct mitochondrial effect leading to caspase activation, they do not rule out that this pleiotropic agent may have additional effects that also contribute to induction of cell death. Indeed, arsenic may interfere with a staggering variety of cellular processes, e.g., cell cycle progression, DNA repair, ubiquitination, tubulin polymerization, nitric oxide synthesis, and oncogene expression or activation. In view of this large number of potential cellular targets, issues such as dose, cell type, or cellular environment become critical. From a therapeutic perspective, differences in arsenic sensitivity should exist between tumor cells and normal cells. Some reports have highlighted clear differences in arsenic sensitivity of some cell lines compared with others. For example, human T-cell lymphotropic virus type I-infected cells, myeloma cells, and transformed lymphocytes are exquisitely sensitive to arsenic (25,26). However, results observed in cell lines should be interpreted with caution. Indeed, cellular levels of glutathione and selenium (both of which potentially titrate intracellular arsenic) are major determinants of arsenic-triggered cell death (21,24,27). The redox state of cell lines in culture is likely to differ from that of cells in vivo. In that sense, very little is known about the consequences of in vivo exposure to micromolar arsenic concentrations, with the notable exception of APL, in which arsenic trioxide induces dramatic clinical remissions. Although nonleukemic cells show some toxicity, the selective disappearance of leukemic cells in vivo implies their greater sensitivity to the drug.

The mechanism by which eradication of the leukemic clones is achieved in vivo is still unclear. In vitro studies in cell lines initially suggested the mechanism to be apoptosis, while lower doses were later found to induce terminal differentiation (27-30). In contrast to the observations in established APL cell lines, primary APL blasts are relatively resistant to rapid apoptosis induction by arsenite when cultured ex vivo. In patients or animal models, apoptosis and differentiation seem to coexist in the few cases analyzed, but their respective contribution to clinical remissions is elusive (30,31). At the molecular level, arsenic was reported to target the leukemia-specific PML/RAR{alpha} (i.e., promyelocytic leukemia protein/retinoic acid receptor {alpha}) fusion protein for degradation (21,30,32,33). Since the PML/RAR{alpha} fusion protein may antagonize both differentiation and apoptosis, its degradation could trigger these two events, yet, in some settings, PML/RAR{alpha} degradation is observed in the absence of apoptosis (27). Arsenic has also been reported to alter the intranuclear localization of PML protein, to enhance its pro-apoptotic properties, and finally to induce its degradation (32,34,35). However, mouse cells lacking the PML (or PML/RAR{alpha}) genes were shown to undergo normal cell death in response to arsenic (36), a finding conflicting with the results of other reports (35,37). Finally, prolonged exposure to arsenic can induce the degradation of RAR{alpha} (27), which points to a possible effect of this drug on the retinoic acid (RA) response. In support of this possibility, arsenic sensitizes some APL cells to RA-triggered differentiation (27). Moreover, arsenic and RA cooperate to induce tumor regression and prolong survival in an animal model of APL (31). This observation suggests that, especially when used at relatively low doses (in the low µM range), arsenite may have effects that can be dissociated from its acute mitochondriotoxicity.

Most anticancer agents have been clinically used before their modes of action have been understood, and arsenic is no exception to this rule. The future will tell to which extent progress in fundamental understanding will invert this order and boost rational drug design. Irrespective of the molecular details, it appears that arsenic constitutes a welcome addition to the clinician's armamentarium for the chemotherapy of leukemia.

REFERENCES

1 Kroemer G, Petit PX, Zamzami N, Vayssiere JL, Mignotte B. The biochemistry of programmed cell death. FASEB J 1995;9:1277-87.[Abstract/Free Full Text]

2 Kroemer G, Dallaporta B, Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 1998;60:619-42.[Medline]

3 Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281:1309-12.[Abstract/Free Full Text]

4 Marzo I, Brenner C, Zamzami N, Susin SA, Beutner G, Brdiczka D, et al. The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins. J Exp Med 1998;187:1261-71.[Abstract/Free Full Text]

5 Marzo I, Brenner C, Zamzami N, Jurgensmeier JM, Susin SA, Vieira HL, et al. Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 1998;281:2027-31.[Abstract/Free Full Text]

6 Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, et al. Molecular characterization of mitochondrial apoptosis-inducing factor (AIF).Nature 1999;397:441-6.[Medline]

7 Liu XS, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 1996;86:147-57.[Medline]

8 Mancini M, Nicholson DW, Roy S, Thornberry NA, Peterson EP, Casciola-Rosen LA, et al. The caspase-3 precursor has a cytosolic and mitochondrial distribution: implications for apoptotic signaling. J Cell Biol 1998;140:1485-95.[Abstract/Free Full Text]

9 Susin SA, Lorenzo HK, Zamzami N, Marzo I, Brenner C, Larochette N, et al. Mitochondrial release of caspase-2 and -9 during the apoptotic process. J Exp Med 1999;189:381-94.[Abstract/Free Full Text]

10 Ravagnan L, Marzo I, Costantini P, Susin SA, Zamzami N, Petit PX, et al. Lonidamine triggers apoptosis via a direct, Bcl-2-inhibited effect on the mitochondrial permeability transition pore. Oncogene. In press 1999.

11 Fulda S, Susin SA, Kroemer G, Debatin KM. Molecular ordering of apoptosis induced by anticancer drugs in neuroblastoma cells. Cancer Res 1998;58:4453-60.[Abstract]

12 Fulda S, Scaffidi C, Susin SA, Krammer PH, Kroemer G, Peter ME, et al. Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid. J Biol Chem 1998;273:33942-8.[Abstract/Free Full Text]

13 Zamzami N, Marzo I, Susin SA, Brenner C, Larochette N, Marchetti P, et al. The thiol-crosslinking agent diamide overcomes the apoptosis-inhibitory effect of Bcl-2 by enforcing mitochondrial permeability transition. Oncogene 1998;16:1055-63.[Medline]

14 Hirsch T, Decaudin D, Susin SA, Marchetti P, Larochette N, Resche-Rignon M, et al. PK11195, a ligand of the mitochondrial benzodiazepine receptor, facilitates the induction of apoptosis and reverses Bcl-2-mediated cytoprotection. Exp Cell Res 1998;241:426-34.[Medline]

15 Verma A, Facchina SL, Hirsch DJ, Song SY, Dillahey LF, Williams JR, et al. Photodynamic tumor therapy: mitochondrial benzodiazepine receptors as a therapeutic target. Mol Med 1998;4:40-5.[Medline]

16 Marchetti P, Castedo M, Susin SA, Zamzami N, Hirsch T, Macho A, et al. Mitochondrial permeability transition is a central coordinating event of apoptosis. J Exp Med 1996;184:1155-60.[Abstract]

17 Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B. A model for p53-induced apoptosis. Nature 1997;389:300-5.[Medline]

18 Basu S, Bayoumy S, Zhang Y, Lozano J, Kolesnick R. BAD enables ceramide to signal apoptosis via Ras and Raf-1. J Biol Chem 1998;273:30419-26.[Abstract/Free Full Text]

19 Susin SA, Zamzami N, Castedo M, Daugas E, Wang HG, Geley S, et al. The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis. J Exp Med 1997;186:25-37.[Abstract/Free Full Text]

20 Costantini P, Chernyak BV, Petronilli V, Bernardi P. Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites. J Biol Chem 1996;271:6746-51.[Abstract/Free Full Text]

21 Zhu XH, Shen YL, Jing Y, Cai X, Jia PM, Huang Y, et al. Apoptosis and growth inhibition in malignant lymphocytes after treatment with arsenic trioxide at clinically achievable concentrations. J Natl Cancer Inst 1999;91:772-8.[Abstract/Free Full Text]

22 Chen YC, Lin-Shiau SY, Lin JK. Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis. J Cell Physiol 1998;177:324-33.[Medline]

23 Larochette N, Decaudin D, Jacotot E, Brenner C, Marzo I, Susin SA, et al. Arsenite induces apoptosis via a direct effect on the mitochondrial permeability transition pore. Exp Cell Res. In press 1999.

24 Dai J, Weinberg RS, Waxman S, Jing Y. Malignant cells can be sensitized to undergo growth inhibition and apoptosis by arsenic trioxide through modulation of the glutathione redox system. Blood 1999;93:268-77.[Abstract/Free Full Text]

25 Bazarbachi A, El-Sabban ME, Nasr R, Quignon F, Awaraji C, Kersual J, et al. Arsenic trioxide and interferon-alpha synergize to induce cell cycle arrest and apoptosis in human T-cell lymphotropic virus type I-transformed cells. Blood 1999;93:278-83.[Abstract/Free Full Text]

26 Rousselot P, Labaume S, Marolleau JP, Larghero J, Noguera MH, Brouet JC, et al. Arsenic trioxide and melarsoprol induce apoptosis in plasma cell lines and plasma cells from myeloma patients. Cancer Res 1999;59:1041-8.[Abstract/Free Full Text]

27 Gianni M, Koken MH, Chelbi-Alix MK, Benoit G, Lanotte M, Chen Z, et al. Combined arsenic and retinoic acid treatment enhances differentiation and apoptosis in arsenic-resistant NB4 cells. Blood 1998;91:4300-10.[Abstract/Free Full Text]

28 Kizaki M, Muto A, Kinjo K, Ueno H, Ikeda Y. Application of heavy metal and cytokine for differentiation-inducing therapy in acute promyelocytic leukemia [letter]. J Natl Cancer Inst 1998;90:1906-7.[Free Full Text]

29 Chen GQ, Zhu J, Shi XG, Ni JH, Zhong HJ, Si GY, et al. In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of bcl-2 expression and modulation of PML-RAR alpha/PML proteins. Blood 1996;88:1052-61.[Abstract/Free Full Text]

30 Chen GQ, Shi XG, Tang W, Xiong SM, Zhu J, Cai X, et al. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): I. As2O3 exerts dose-dependent dual effects on APL cells. Blood 1997;89:3345-53.[Abstract/Free Full Text]

31 Lallemand-Breitenbach V, Guillemin MC, Janin A, Daniel MT, Degos L, Kogan S, et al. Retinoic acid and arsenic synergize to eradicate leukemic cells in a mouse model of acute promyelocytic leukemia. J Exp Med. In press 1999.

32 Muller S, Matunis MJ, Dejean A. Conjugation with the ubiquitin-related modifier SUMO-1 regulates the partitioning of PML within the nucleus. EMBO J 1998;17:61-70.[Abstract/Free Full Text]

33 Shao W, Fanelli M, Ferrara FF, Riccioni R, Rosenauer A, Davison K, et al. Arsenic trioxide as an inducer of apoptosis and loss of PML/RAR alpha protein in acute promyelocytic leukemia cells. J Natl Cancer Inst 1998;90:124-33.[Abstract/Free Full Text]

34 Zhu J, Koken MH, Quignon F, Chelbi-Alix MK, Degos L, Wang ZY, et al. Arsenic-induced PML targeting onto nuclear bodies: implications for the treatment of acute promyelocytic leukemia. Proc Natl Acad Sci U S A 1997;94:3978-83.[Abstract/Free Full Text]

35 Quignon F, De Bels F, Koken M, Feunteun J, Ameisen JC, de The H. PML induces a novel caspase-independent death process. Nat Genet 1998;20:259-65.[Medline]

36 Wang ZG, Rivi R, Delva L, Konig A, Scheinberg DA, Gambacorti-Passerini C, et al. Arsenic trioxide and melarsoprol induce programmed cell death in myeloid leukemia cell lines and function in a PML and PML-RAR alpha independent fashion. Blood 1998;92:1497-504.[Abstract/Free Full Text]

37 Pucetti E, Sterndorf T, Hoelzer D, Will H, Ottman O, Ruthardt M. PIC-1 SUMO-1 modified PML/RARa is directly involved in arsenic trioxide-induced apoptosis in acute promyelocytic leukemia. Blood 1998;92 Suppl 1:212a.


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