December 1, 1998Journal of Biological Chemistry374 citationsOpen Access

Activation of Mitochondria and Release of Mitochondrial Apoptogenic Factors by Betulinic Acid

SFSimone Fulda CSCarsten Scaffidi SSSantos A. Susín

Key Points

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Abstract

Different classes of anticancer drugs may trigger apoptosis by acting on different subcellular targets and by activating distinct signaling pathways. Here, we report that betulinic acid (BetA) is a prototype cytotoxic agent that triggers apoptosis by a direct effect on mitochondria. In isolated mitochondria, BetA directly induces loss of transmembrane potential independent of a benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone-inhibitable caspase. This is inhibited by bongkrekic acid, an agent that stabilizes the permeability transition pore complex. Mitochondria undergoing BetA-induced permeability transition mediate cleavage of caspase-8 (FLICE/MACH/Mch5) and caspase-3 (CPP32/Yama) in a cell-free system. Soluble factors such as cytochrome c or apoptosis-inducing factor released from BetA-treated mitochondria are sufficient for cleavage of caspases and nuclear fragmentation. Addition of cytochromec to cytosolic extracts results in cleavage of caspase-3, but not of caspase-8. However, supernatants of mitochondria, which have undergone permeability transition, and partially purified apoptosis-inducing factor activate both caspase-8 and caspase-3 in cytosolic extracts and suffice to activate recombinant caspase-8. These findings show that induction of mitochondrial permeability transition alone is sufficient to trigger the full apoptosis program and that some cytotoxic drugs such as BetA may induce apoptosis via a direct effect on mitochondria. Different classes of anticancer drugs may trigger apoptosis by acting on different subcellular targets and by activating distinct signaling pathways. Here, we report that betulinic acid (BetA) is a prototype cytotoxic agent that triggers apoptosis by a direct effect on mitochondria. In isolated mitochondria, BetA directly induces loss of transmembrane potential independent of a benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone-inhibitable caspase. This is inhibited by bongkrekic acid, an agent that stabilizes the permeability transition pore complex. Mitochondria undergoing BetA-induced permeability transition mediate cleavage of caspase-8 (FLICE/MACH/Mch5) and caspase-3 (CPP32/Yama) in a cell-free system. Soluble factors such as cytochrome c or apoptosis-inducing factor released from BetA-treated mitochondria are sufficient for cleavage of caspases and nuclear fragmentation. Addition of cytochromec to cytosolic extracts results in cleavage of caspase-3, but not of caspase-8. However, supernatants of mitochondria, which have undergone permeability transition, and partially purified apoptosis-inducing factor activate both caspase-8 and caspase-3 in cytosolic extracts and suffice to activate recombinant caspase-8. These findings show that induction of mitochondrial permeability transition alone is sufficient to trigger the full apoptosis program and that some cytotoxic drugs such as BetA may induce apoptosis via a direct effect on mitochondria. permeability transition apoptosis-inducing factor betulinic acid fluorescence-activated cell sorting benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone bongkrekic acid phosphate-buffered saline polyacrylamide gel electrophoresis bovine serum albumin poly(ADP-ribose) polymerase monoclonal antibody 1,4-piperazinediethanesulfonic acid mitochondrial transmembrane potential. Anticancer agents with different modes of action have been reported to trigger apoptosis in chemosensitive cells (1Fisher D.E. Cell. 1994; 78: 539-542Abstract Full Text PDF PubMed Scopus (1368) Google Scholar). Alterations of mitochondrial functions such as permeability transition (PT)1 have been found to play a major role in the apoptotic process including cell death induced by chemotherapeutic agents (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar, 3Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 5-37Crossref Scopus (587) Google Scholar, 4Marchetti P. Castedo M. Susin S.A. Zamzami N. Hirsch T. Macho A. Haeffner A. Hirsch T. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1155-1160Crossref PubMed Scopus (779) Google Scholar, 5Zamzami N. Susin S.A. Marchetti P. Hirsch T. Gomez-Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1544Crossref PubMed Scopus (1262) Google Scholar, 6Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar, 7Decaudin D. Geley S. Hirsch T. Castedo M. Marchetti P. Macho A. Kofler R. Kroemer G. Cancer Res. 1997; 57: 62-67PubMed Google Scholar). Mitochondria undergoing PT release apoptogenic proteins such as cytochrome c or apoptosis-inducing factor (AIF) from the mitochondrial intermembrane space into the cytosol, where they can activate caspases and endonucleases (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar, 3Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 5-37Crossref Scopus (587) Google Scholar, 6Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar, 8Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4256) Google Scholar, 9Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4384) Google Scholar, 10Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4435) Google Scholar, 11Li P. Nijhawan D. Budihardjo I. Srinivasula S. Ahmad M. Alnemri A.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6183) Google Scholar). However, activated caspases can also induce PT, probably via a direct effect on the PT pore complex (3Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 5-37Crossref Scopus (587) Google Scholar, 12Marzo I. Brenner C. Zamzami N. Susin S.A. Beutner G. Brdiczka D. Remy R. Xie Z.-H. Reed J.C. Kroemer G. J. Exp. Med. 1998; 187: 1261-1271Crossref PubMed Scopus (614) Google Scholar). These findings suggest that caspases can act upstream and downstream of mitochondria. Mitochondrial function during apoptosis is controlled by the Bcl-2 family of proteins localized to intracellular membranes including the mitochondrial membrane (13Kroemer G. Nat. Med. 1997; 3: 614-620Crossref PubMed Scopus (1706) Google Scholar). Overexpression of the anti-apoptotic molecules Bcl-2 and Bcl-XL has been found to confer resistance to anticancer treatment (14Kim C.N. Wang X. Huang Y. Ibrado A.M. Liu L. Fang G. Bhalla K. Cancer Res. 1997; 57: 3115-3120PubMed Google Scholar, 15Dole M. Nuñez G. Merchant A.K. Maybaum J. Rode C.K. Bloch C.A. Castle V.P. Cancer Res. 1994; 54: 3253-3259PubMed Google Scholar, 16Dole M.G. Jasty R. Cooper M.J. Thompson C.B. Nuñez G. Castle V.P. Cancer Res. 1995; 55: 2576-2582PubMed Google Scholar). Bcl-2 and Bcl-XL may inhibit apoptosis through the capacity to prevent PT and/or to stabilize the barrier function of the outer mitochondrial membrane (5Zamzami N. Susin S.A. Marchetti P. Hirsch T. Gomez-Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1544Crossref PubMed Scopus (1262) Google Scholar, 6Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar, 13Kroemer G. Nat. Med. 1997; 3: 614-620Crossref PubMed Scopus (1706) Google Scholar, 17Vander Heiden M.G. Chandel N.S. Williamson E.K. Schumacker P.T. Thompson C.B. Cell. 1997; 91: 627-637Abstract Full Text Full Text PDF PubMed Scopus (1233) Google Scholar, 18Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO J. 1998; 17: 37-49Crossref PubMed Scopus (1105) Google Scholar).Cytotoxic drugs such as doxorubicin can activate apoptosis pathways by such as the C. I. Nat. Med. 1996; PubMed Scopus Google Scholar, S. C. I. Cancer Res. 1997; 57: Google Scholar, S. M. C. J. 1998; PubMed Scopus Google Scholar, S. C. T. 1998; PubMed Scopus Google Scholar, M. I. C. S. K. 1997; PubMed Google Scholar, C. S. 1997; Scopus Google Scholar, J. Cancer 1997; PubMed Scopus Google Scholar, M. S. J. 1997; PubMed Scopus Google Scholar). caspase-8 (FLICE/MACH/Mch5) is in of a downstream including caspase-3 (CPP32/Yama) A. I. M. G. C. M. S. J. J. Full Text PDF PubMed Google Scholar, C. S. P. Science. PubMed Scopus Google Scholar, 1996; 3: Google Scholar, S. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. A.M. 1996; 3: Google Scholar, M. A.M. K. C. M. R. M. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A.M. K. S. J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, S. I. M. M. EMBO J. 1995; PubMed Scopus Google Scholar, C. M. EMBO J. 1997; PubMed Scopus Google Scholar). of the and of treatment with cytotoxic drugs such as doxorubicin have been in a of and of by has been found to inhibit cell death C. I. Nat. Med. 1996; PubMed Scopus Google Scholar, S. C. I. Cancer Res. 1997; 57: Google Scholar, S. M. C. J. 1998; PubMed Scopus Google Scholar, S. C. T. 1998; PubMed Scopus Google Scholar, M. I. C. S. K. 1997; PubMed Google Scholar, C. S. 1997; Scopus Google Scholar, J. Cancer 1997; PubMed Scopus Google Scholar, M. S. J. 1997; PubMed Scopus Google acid (BetA) is a anticancer with for E. Nat. Med. 1995; PubMed Scopus Google Scholar, S. C. M. C. M. Nuñez G. Cancer Res. 1997; 57: Google Scholar). found that BetA-induced apoptosis from anticancer agents such as doxorubicin S. C. M. C. M. Nuñez G. Cancer Res. 1997; 57: Google Scholar). BetA-induced apoptosis is not with of such as and not of mitochondrial function including loss of mitochondrial permeability transition of apoptosis such as of the and nuclear S. C. M. C. M. Nuñez G. Cancer Res. 1997; 57: Google Scholar). This that BetA may have a direct effect on mitochondria. BetA directly activate mitochondria and the of the apoptosis drugs have been reported to act by apoptosis in cells (1Fisher D.E. Cell. 1994; 78: 539-542Abstract Full Text PDF PubMed Scopus (1368) Google Scholar). of apoptosis by anticancer drugs or of death of mitochondrial and of the death molecules of apoptosis J. Cancer 1997; PubMed Scopus Google Scholar). the cell death may and drugs may the have not been in Here, we report that of anticancer agents by BetA may act by directly mitochondria, in downstream of a cell-free we found that BetA directly PT in isolated mitochondria, and induction of PT to the in of PT by of Bcl-2 or Bcl-XL or by the of apoptosis in cells and in a cell-free such as of of cleavage of and nuclear fragmentation. In to cytotoxic drugs such as or not induce mitochondrial in isolated mitochondria, that mitochondrial PT, which in cells during apoptosis by S. C. I. Cancer Res. 1997; 57: Google the of a of pathways or BetA cells S. C. M. C. M. Nuñez G. Cancer Res. 1997; 57: Google Scholar). to BetA induced mitochondrial in but not in cell not However, BetA-treated mitochondria isolated from the cell mitochondrial PT and nuclear to mitochondria from the of BetA for may by and/or of the to the mitochondrial by in mitochondria BetA-treated of different caspases and nuclear found in cells with mitochondrial and BetA-induced loss of not inhibited by the This that downstream of mitochondria and that of mitochondria is sufficient to trigger downstream to In in loss of inhibited by that of mitochondria is by upstream S. and Cancer Res. BetA to a of cytotoxic agents apoptosis-inducing effect is by of on BetA-induced apoptosis a of the of different caspases and mitochondrial death the caspase-8 which in full of the cleavage of and of mitochondrial PT C. S. F. C. A. EMBO J. 1998; 17: PubMed Scopus Google Scholar, C. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, A. F. A. L. R. J.C. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Thompson C.B. S. A. 1997; PubMed Scopus Google Scholar). of caspase-8 in cells in which mitochondrial PT and of downstream caspases are by of Bcl-2 or Bcl-XL (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar). In in BetA-treated of both caspase-8 and caspase-3 to mitochondrial proteins such as cytochromec and released from mitochondria permeability transition have been to directly induce cleavage of caspases (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 5-37Crossref Scopus (587) Google Scholar, 6Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar). mitochondrial PT and apoptosis cleavage of caspase-8 and caspase-3 independent of in BetA-treated caspase-8 activated by mitochondria undergoing PT in the of signaling complex released from mitochondria may mediate caspase-8 cleavage BetA treatment partially purified recombinant caspase-8. In cytochromec not induce of induced caspase-3 that the of caspase-8 downstream of mitochondria from that of to for partially purified has been to induce and we found of with isolated mitochondria. However, may of cytosolic that may mediate of downstream targets such as or X. C. Wang X. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, M. K. A. S. 1997; Scopus Google classes of anticancer drugs may the apoptotic distinct they induce a that the of a of findings may have for of apoptosis of mitochondrial PT, as by cytotoxic drugs such as may sufficient for induction of apoptosis in cells and may the for upstream drugs cells that have a in upstream apoptosis pathways. In that cells with a in the C. S. 1997; Scopus Google which to to chemotherapeutic are to BetA-induced cell death S. C. M. C. M. Nuñez G. Cancer Res. 1997; 57: Google Scholar). findings may to and a of cytotoxic agents with direct mitochondrial that some of of Anticancer agents with different modes of action have been reported to trigger apoptosis in chemosensitive cells (1Fisher D.E. Cell. 1994; 78: 539-542Abstract Full Text PDF PubMed Scopus (1368) Google Scholar). Alterations of mitochondrial functions such as permeability transition (PT)1 have been found to play a major role in the apoptotic process including cell death induced by chemotherapeutic agents (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar, 3Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 5-37Crossref Scopus (587) Google Scholar, 4Marchetti P. Castedo M. Susin S.A. Zamzami N. Hirsch T. Macho A. Haeffner A. Hirsch T. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1155-1160Crossref PubMed Scopus (779) Google Scholar, 5Zamzami N. Susin S.A. Marchetti P. Hirsch T. Gomez-Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1544Crossref PubMed Scopus (1262) Google Scholar, 6Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar, 7Decaudin D. Geley S. Hirsch T. Castedo M. Marchetti P. Macho A. Kofler R. Kroemer G. Cancer Res. 1997; 57: 62-67PubMed Google Scholar). Mitochondria undergoing PT release apoptogenic proteins such as cytochrome c or apoptosis-inducing factor (AIF) from the mitochondrial intermembrane space into the cytosol, where they can activate caspases and endonucleases (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar, 3Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 5-37Crossref Scopus (587) Google Scholar, 6Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar, 8Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4256) Google Scholar, 9Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4384) Google Scholar, 10Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4435) Google Scholar, 11Li P. Nijhawan D. Budihardjo I. Srinivasula S. Ahmad M. Alnemri A.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6183) Google Scholar). However, activated caspases can also induce PT, probably via a direct effect on the PT pore complex (3Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 5-37Crossref Scopus (587) Google Scholar, 12Marzo I. Brenner C. Zamzami N. Susin S.A. Beutner G. Brdiczka D. Remy R. Xie Z.-H. Reed J.C. Kroemer G. J. Exp. Med. 1998; 187: 1261-1271Crossref PubMed Scopus (614) Google Scholar). These findings suggest that caspases can act upstream and downstream of mitochondria. Mitochondrial function during apoptosis is controlled by the Bcl-2 family of proteins localized to intracellular membranes including the mitochondrial membrane (13Kroemer G. Nat. Med. 1997; 3: 614-620Crossref PubMed Scopus (1706) Google Scholar). Overexpression of the anti-apoptotic molecules Bcl-2 and Bcl-XL has been found to confer resistance to anticancer treatment (14Kim C.N. Wang X. Huang Y. Ibrado A.M. Liu L. Fang G. Bhalla K. Cancer Res. 1997; 57: 3115-3120PubMed Google Scholar, 15Dole M. Nuñez G. Merchant A.K. Maybaum J. Rode C.K. Bloch C.A. Castle V.P. Cancer Res. 1994; 54: 3253-3259PubMed Google Scholar, 16Dole M.G. Jasty R. Cooper M.J. Thompson C.B. Nuñez G. Castle V.P. Cancer Res. 1995; 55: 2576-2582PubMed Google Scholar). Bcl-2 and Bcl-XL may inhibit apoptosis through the capacity to prevent PT and/or to stabilize the barrier function of the outer mitochondrial membrane (5Zamzami N. Susin S.A. Marchetti P. Hirsch T. Gomez-Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1544Crossref PubMed Scopus (1262) Google Scholar, 6Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar, 13Kroemer G. Nat. Med. 1997; 3: 614-620Crossref PubMed Scopus (1706) Google Scholar, 17Vander Heiden M.G. Chandel N.S. Williamson E.K. Schumacker P.T. Thompson C.B. Cell. 1997; 91: 627-637Abstract Full Text Full Text PDF PubMed Scopus (1233) Google Scholar, 18Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO J. 1998; 17: 37-49Crossref PubMed Scopus (1105) Google Scholar). drugs such as doxorubicin can activate apoptosis pathways by such as the C. I. Nat. Med. 1996; PubMed Scopus Google Scholar, S. C. I. Cancer Res. 1997; 57: Google Scholar, S. M. C. J. 1998; PubMed Scopus Google Scholar, S. C. T. 1998; PubMed Scopus Google Scholar, M. I. C. S. K. 1997; PubMed Google Scholar, C. S. 1997; Scopus Google Scholar, J. Cancer 1997; PubMed Scopus Google Scholar, M. S. J. 1997; PubMed Scopus Google Scholar). caspase-8 (FLICE/MACH/Mch5) is in of a downstream including caspase-3 (CPP32/Yama) A. I. M. G. C. M. S. J. J. Full Text PDF PubMed Google Scholar, C. S. P. Science. PubMed Scopus Google Scholar, 1996; 3: Google Scholar, S. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. A.M. 1996; 3: Google Scholar, M. A.M. K. C. M. R. M. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A.M. K. S. J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, S. I. M. M. EMBO J. 1995; PubMed Scopus Google Scholar, C. M. EMBO J. 1997; PubMed Scopus Google Scholar). of the and of treatment with cytotoxic drugs such as doxorubicin have been in a of and of by has been found to inhibit cell death C. I. Nat. Med. 1996; PubMed Scopus Google Scholar, S. C. I. Cancer Res. 1997; 57: Google Scholar, S. M. C. J. 1998; PubMed Scopus Google Scholar, S. C. T. 1998; PubMed Scopus Google Scholar, M. I. C. S. K. 1997; PubMed Google Scholar, C. S. 1997; Scopus Google Scholar, J. Cancer 1997; PubMed Scopus Google Scholar, M. S. J. 1997; PubMed Scopus Google Scholar). acid (BetA) is a anticancer with for E. Nat. Med. 1995; PubMed Scopus Google Scholar, S. C. M. C. M. Nuñez G. Cancer Res. 1997; 57: Google Scholar). found that BetA-induced apoptosis from anticancer agents such as doxorubicin S. C. M. C. M. Nuñez G. Cancer Res. 1997; 57: Google Scholar). BetA-induced apoptosis is not with of such as and not of mitochondrial function including loss of mitochondrial permeability transition of apoptosis such as of the and nuclear S. C. M. C. M. Nuñez G. Cancer Res. 1997; 57: Google Scholar). This that BetA may have a direct effect on mitochondria. BetA directly activate mitochondria and the of the apoptosis drugs have been reported to act by apoptosis in cells (1Fisher D.E. Cell. 1994; 78: 539-542Abstract Full Text PDF PubMed Scopus (1368) Google Scholar). of apoptosis by anticancer drugs or of death of mitochondrial and of the death molecules of apoptosis J. Cancer 1997; PubMed Scopus Google Scholar). the cell death may and drugs may the have not been in Here, we report that of anticancer agents by BetA may act by directly mitochondria, in downstream of a cell-free we found that BetA directly PT in isolated mitochondria, and induction of PT to the in of PT by of Bcl-2 or Bcl-XL or by the of apoptosis in cells and in a cell-free such as of of cleavage of and nuclear fragmentation. In to cytotoxic drugs such as or not induce mitochondrial in isolated mitochondria, that mitochondrial PT, which in cells during apoptosis by S. C. I. Cancer Res. 1997; 57: Google the of a of pathways or BetA cells S. C. M. C. M. Nuñez G. Cancer Res. 1997; 57: Google Scholar). to BetA induced mitochondrial in but not in cell not However, BetA-treated mitochondria isolated from the cell mitochondrial PT and nuclear to mitochondria from the of BetA for may by and/or of the to the mitochondrial by in mitochondria BetA-treated of different caspases and nuclear found in cells with mitochondrial and BetA-induced loss of not inhibited by the This that downstream of mitochondria and that of mitochondria is sufficient to trigger downstream to In in loss of inhibited by that of mitochondria is by upstream S. and Cancer Res. BetA to a of cytotoxic agents apoptosis-inducing effect is by of on BetA-induced apoptosis a of the of different caspases and mitochondrial death the caspase-8 which in full of the cleavage of and of mitochondrial PT C. S. F. C. A. EMBO J. 1998; 17: PubMed Scopus Google Scholar, C. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, A. F. A. L. R. J.C. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Thompson C.B. S. A. 1997; PubMed Scopus Google Scholar). of caspase-8 in cells in which mitochondrial PT and of downstream caspases are by of Bcl-2 or Bcl-XL (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar). In in BetA-treated of both caspase-8 and caspase-3 to mitochondrial proteins such as cytochromec and released from mitochondria permeability transition have been to directly induce cleavage of caspases (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 5-37Crossref Scopus (587) Google Scholar, 6Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar). mitochondrial PT and apoptosis cleavage of caspase-8 and caspase-3 independent of in BetA-treated caspase-8 activated by mitochondria undergoing PT in the of signaling complex released from mitochondria may mediate caspase-8 cleavage BetA treatment partially purified recombinant caspase-8. In cytochromec not induce of induced caspase-3 that the of caspase-8 downstream of mitochondria from that of to for partially purified has been to induce and we found of with isolated mitochondria. However, may of cytosolic that may mediate of downstream targets such as or X. C. Wang X. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, M. K. A. S. 1997; Scopus Google classes of anticancer drugs may the apoptotic distinct they induce a that the of a of findings may have for of apoptosis of mitochondrial PT, as by cytotoxic drugs such as may sufficient for induction of apoptosis in cells and may the for upstream drugs cells that have a in upstream apoptosis pathways. In that cells with a in the C. S. 1997; Scopus Google which to to chemotherapeutic are to BetA-induced cell death S. C. M. C. M. Nuñez G. Cancer Res. 1997; 57: Google Scholar). findings may to and a of cytotoxic agents with direct mitochondrial that some of of drugs have been reported to act by apoptosis in cells (1Fisher D.E. Cell. 1994; 78: 539-542Abstract Full Text PDF PubMed Scopus (1368) Google Scholar). of apoptosis by anticancer drugs or of death of mitochondrial and of the death molecules of apoptosis J. Cancer 1997; PubMed Scopus Google Scholar). the cell death may and drugs may the have not been in Here, we report that of anticancer agents by BetA may act by directly mitochondria, in downstream of mitochondria. a cell-free we found that BetA directly PT in isolated mitochondria, and induction of PT to the in of PT by of Bcl-2 or Bcl-XL or by the of apoptosis in cells and in a cell-free such as of of cleavage of and nuclear fragmentation. In to cytotoxic drugs such as or not induce mitochondrial in isolated mitochondria, that mitochondrial PT, which in cells during apoptosis by S. C. I. Cancer Res. 1997; 57: Google the of a of pathways or BetA cells S. C. M. C. M. Nuñez G. Cancer Res. 1997; 57: Google Scholar). to BetA induced mitochondrial in but not in cell not However, BetA-treated mitochondria isolated from the cell mitochondrial PT and nuclear to mitochondria from the of BetA for may by and/or of the to the mitochondrial by in mitochondria In BetA-treated of different caspases and nuclear found in cells with mitochondrial and BetA-induced loss of not inhibited by the This that downstream of mitochondria and that of mitochondria is sufficient to trigger downstream to In in loss of inhibited by that of mitochondria is by upstream S. and Cancer Res. BetA to a of cytotoxic agents apoptosis-inducing effect is by of mitochondria. on BetA-induced apoptosis a of the of different caspases and mitochondrial death the caspase-8 which in full of the cleavage of and of mitochondrial PT C. S. F. C. A. EMBO J. 1998; 17: PubMed Scopus Google Scholar, C. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, A. F. A. L. R. J.C. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Thompson C.B. S. A. 1997; PubMed Scopus Google Scholar). of caspase-8 in cells in which mitochondrial PT and of downstream caspases are by of Bcl-2 or Bcl-XL (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar). In in BetA-treated of both caspase-8 and caspase-3 to mitochondrial proteins such as cytochromec and released from mitochondria permeability transition have been to directly induce cleavage of caspases (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 5-37Crossref Scopus (587) Google Scholar, 6Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. 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