Different Pathways Mediate Cytochrome c Release After Photodynamic Therapy with Hypericin

Abstract In this study we show that overexpression of Bcl-2 in PC60R1R2 cells reveals a caspase-dependent mechanism of cytochrome c release following photodynamic therapy (PDT) with hypericin. Bcl-2 overexpression remarkably delayed cytochrome c release, procaspase-3 activation and poly(adenosine diphosphate-ribose)polymerase cleavage during PDT-induced apoptosis while it did not protect against PDT-induced necrosis. PDT-treated cells showed a reduction in the mitochondrial membrane potential which occurred with similar kinetics in PC60R1R2 and PC60R1R2/Bcl-2 cells, and was affected neither by the permeability transition pore inhibitor cyclosporin A nor by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk). Hypericin-induced mitochondrial depolarization coincided with cytochrome c release in PC60R1R2 cells while it precedes massive cytochrome c efflux in PC60R1R2/Bcl-2 cells. Preincubation of PC60R1R2 cells with zVAD-fmk or cyclosporin A did not prevent the mitochondrial efflux of cytochrome c, and caspase inhibition only partially protected the cells from PDT-induced apoptosis. In contrast, in PC60R1R2/Bcl-2 cells cytochrome c release and apoptosis were suppressed by addition of zVAD-fmk or cyclosporin A. These observations suggest that the progression of the PDT-induced apoptotic process in Bcl-2–overexpressing cells involves a caspase-dependent feed-forward amplification loop for the release of cytochrome c.

[1]  Z. Oltvai,et al.  Bcl-2 functions in an antioxidant pathway to prevent apoptosis , 1993, Cell.

[2]  P. D. de Witte,et al.  Photodynamic therapy efficacy and tissue distribution of hypericin in a mouse P388 lymphoma tumor model. , 2000, Cancer letters.

[3]  T. Tsuruo,et al.  Involvement of Bcl-2 cleavage in the acceleration of VP-16-induced U937 cell apoptosis. , 1998, Biochemical and biophysical research communications.

[4]  C. Hadjur,et al.  Photodynamically induced cytotoxicity of hypericin dye on human fibroblast cell line MRC5. , 1995, Journal of photochemistry and photobiology. B, Biology.

[5]  G. Kroemer,et al.  Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis , 1996, FEBS letters.

[6]  P. Vandenabeele,et al.  Hypericin‐induced photosensitization of HeLa cells leads to apoptosis or necrosis , 1998, FEBS letters.

[7]  John C Reed,et al.  Bcl-2 family proteins , 1998, Oncogene.

[8]  G. Kroemer,et al.  The thiol crosslinking agent diamide overcomes the apoptosis-inhibitory effect of Bcl-2 by enforcing mitochondrial permeability transition , 1998, Oncogene.

[9]  John Calvin Reed,et al.  Bcl-2 family proteins and mitochondria. , 1998, Biochimica et biophysica acta.

[10]  P. Vandenabeele,et al.  The Activation of the c-Jun N-terminal Kinase and p38 Mitogen-activated Protein Kinase Signaling Pathways Protects HeLa Cells from Apoptosis Following Photodynamic Therapy with Hypericin* , 1999, The Journal of Biological Chemistry.

[11]  W. Fiers,et al.  Both TNF receptors are required for TNF-mediated induction of apoptosis in PC60 cells. , 1995, Journal of immunology.

[12]  B. Mignotte,et al.  Mitochondria and apoptosis. , 1998, European journal of biochemistry.

[13]  B. McManus,et al.  Bcl-2 overexpression blocks caspase activation and downstream apoptotic events instigated by photodynamic therapy , 1999, British Journal of Cancer.

[14]  B. McManus,et al.  Early release of mitochondrial cytochrome c and expression of mitochondrial epitope 7A6 with a porphyrin-derived photosensitizer: Bcl-2 and Bcl-xL overexpression do not prevent early mitochondrial events but still depress caspase activity. , 1999, Laboratory investigation; a journal of technical methods and pathology.

[15]  N. Oleinick,et al.  The photobiology of photodynamic therapy: cellular targets and mechanisms. , 1998, Radiation research.

[16]  M. Agarwal,et al.  The Induction of Partial Resistance to Photodynamic Therapy by the Protooncogene BCL‐2 , 1996, Photochemistry and photobiology.

[17]  Pierre-Yves Turpin,et al.  Hypericin-Induced Cell Photosensitization Involves an Intracellular pH Decrease , 1996 .

[18]  B. Zhivotovsky,et al.  Cleavage of Bcl-2 is an early event in chemotherapy-induced apoptosis of human myeloid leukemia cells , 1999, Leukemia.

[19]  D. Bredesen,et al.  Expression of bcl‐2 inhibits necrotic neural cell death , 1995, Journal of neuroscience research.

[20]  Xiaodong Wang,et al.  Bid, a Bcl2 Interacting Protein, Mediates Cytochrome c Release from Mitochondria in Response to Activation of Cell Surface Death Receptors , 1998, Cell.

[21]  Luca Scorrano,et al.  Mitochondria and cell death. Mechanistic aspects and methodological issues. , 1999, European journal of biochemistry.

[22]  G. Núñez,et al.  Role of cytochrome c and dATP/ATP hydrolysis in Apaf‐1‐mediated caspase‐9 activation and apoptosis , 1999, The EMBO journal.

[23]  G. Cortopassi,et al.  Induction of the mitochondrial permeability transition causes release of the apoptogenic factor cytochrome c. , 1998, Free radical biology & medicine.

[24]  P. Song,et al.  HYPERICIN AND ITS PHOTODYNAMIC ACTION , 1986, Photochemistry and photobiology.

[25]  C. Borner,et al.  Alphaviruses induce apoptosis in Bcl‐2‐overexpressing cells: evidence for a caspase‐mediated, proteolytic inactivation of Bcl‐2 , 1998, The EMBO journal.

[26]  M. Collins,et al.  Induction of apoptosis by valinomycin: mitochondrial permeability transition causes intracellular acidification , 1998, Cell Death and Differentiation.

[27]  W. Merlevede,et al.  Photodynamic treatment of basal cell carcinoma and squamous cell carcinoma with hypericin. , 1998, Anticancer research.

[28]  Kevin K. W Wang,et al.  Calpain and caspase: can you tell the difference? , 2000, Trends in Neurosciences.

[29]  D. Kessel,et al.  Enhanced apoptotic response to photodynamic therapy after bcl-2 transfection. , 1999, Cancer research.

[30]  M. V. Heiden,et al.  Bcl-xL Regulates the Membrane Potential and Volume Homeostasis of Mitochondria , 1997, Cell.

[31]  P. Agostinis,et al.  Antitumour activity of photosensitized hypericin on A431 cell xenografts. , 1996, Anticancer research.

[32]  G. C. Miller,et al.  PHOTOACTIVATION OF HYPERICIN GENERATES SINGLET OXYGEN IN MITOCHONDRIA AND INHIBITS SUCCINOXIDASE , 1992, Photochemistry and photobiology.

[33]  D. Meruelo,et al.  The chemical and biological properties of hypericin—a compound with a broad spectrum of biological activities , 1995, Medicinal research reviews.

[34]  Y. Tsujimoto,et al.  Retardation of chemical hypoxia-induced necrotic cell death by Bcl-2 and ICE inhibitors: possible involvement of common mediators in apoptotic and necrotic signal transductions. , 1996, Oncogene.

[35]  Dean P. Jones,et al.  Prevention of Apoptosis by Bcl-2: Release of Cytochrome c from Mitochondria Blocked , 1997, Science.

[36]  B. Dutrillaux,et al.  Light-induced photoactivation of hypericin affects the energy metabolism of human glioma cells by inhibiting hexokinase bound to mitochondria. , 1998, Cancer research.

[37]  D. Meruelo,et al.  Therapeutic agents with dramatic antiretroviral activity and little toxicity at effective doses: aromatic polycyclic diones hypericin and pseudohypericin. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[38]  Wei Zhao,et al.  Hypericin‐Induced Phototoxicity in Cultured Fibroblasts and Swine Erythrocytes , 1996, Photochemistry and photobiology.

[39]  H. Falk,et al.  A Convenient Semisynthetic Route to Hypericin. , 1993 .

[40]  W. Merlevede,et al.  Differential cytotoxic effects induced after photosensitization by hypericin. , 1997, Journal of photochemistry and photobiology. B, Biology.

[41]  P. Petit,et al.  Over‐expression of Bcl‐2 does not protect cells from hypericin photo‐induced mitochondrial membrane depolarization, but delays subsequent events in the apoptotic pathway , 1999, FEBS letters.

[42]  G. Kroemer,et al.  Caspases disrupt mitochondrial membrane barrier function , 1998, FEBS letters.

[43]  D. Bredesen,et al.  Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. , 1993, Science.

[44]  P. Vandenabeele,et al.  A Caspase-activated Factor (CAF) Induces Mitochondrial Membrane Depolarization and Cytochrome c Release by a Nonproteolytic Mechanism , 1998, The Journal of experimental medicine.

[45]  Ruedi Aebersold,et al.  Molecular characterization of mitochondrial apoptosis-inducing factor , 1999, Nature.

[46]  A. Almasan,et al.  Distinct stages of cytochrome c release from mitochondria: evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis , 2000, Cell Death and Differentiation.

[47]  G. Salvesen,et al.  Target Protease Specificity of the Viral Serpin CrmA , 1997, The Journal of Biological Chemistry.

[48]  L. Baert,et al.  Fluorescence detection of flat bladder carcinoma in situ after intravesical instillation of hypericin. , 2000, The Journal of urology.

[49]  T. Obsil,et al.  The effect of hypericin and hypocrellin-A on lipid membranes and membrane potential of 3T3 fibroblasts. , 1999, Biochimica et biophysica acta.

[50]  A. Andreoni,et al.  LASER PHOTOSENSITIZATION OF CELLS BY HYPERICIN , 1994, Photochemistry and photobiology.

[51]  D. Green,et al.  The Release of Cytochrome c from Mitochondria: A Primary Site for Bcl-2 Regulation of Apoptosis , 1997, Science.

[52]  R. Pardini,et al.  Time-course of hypericin phototoxicity and effect on mitochondrial energies in EMT6 mouse mammary carcinoma cells. , 1998, Free radical biology & medicine.

[53]  S. Srinivasula,et al.  Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade , 1997, Cell.