Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpression of Bcl-x(L) or Bcl-2.
Apoptosis is required for proper tissue homeostasis. Defects in apoptosis signaling pathways, thus, contribute to carcinogenesis and chemoresistance. A major goal in chemotherapy is, therefore, to find cytotoxic agents that restore the ability of tumor cells to undergo apoptosis. We show here that the sesquiterpene lactone helenalin (10-50 microM) induces apoptosis in leukemia Jurkat T cells even if they lack the CD95 death receptor or overexpress the antiapoptotic proteins Bcl-x(L) or Bcl-2. Activated peripheral blood mononuclear cells, however, are not affected (10-50 microM helenalin). Helenalin led to a time-dependent (0-24 h) cleavage of the specific caspase-3-like substrate Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin as well as to the proteolytic processing of procaspase-3 and -8. Caspase activation was a necessary requirement for apoptosis because the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk, 50 microM) completely abrogated helenalin-induced DNA fragmentation as well as phosphatidylserin translocation. Although the initiator caspase-8 was activated, the helenalin-induced signaling pathway did not require the CD95 death receptor as shown using cells without or with an antibody (ZB4)-blocked CD95 receptor. Helenalin also did not induce CD95 or CD95-ligand expression. On the other hand, helenalin was found to induce the release of cytochrome c from mitochondria that was not inhibited by the caspase inhibitor zVAD-fmk, which indicated that cytochrome c release precedes caspase activation. Cytochrome c release was accompanied by dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), which was partly inhibited by zVAD-fmk, which suggests that caspases are involved in loss of DeltaPsi(m). Most importantly, overexpression of the mitochondria protecting proteins Bcl-x(L) or Bcl-2 failed to confer resistance to helenalin-induced apoptosis, although the data presented here suggest that helenalin induces a mitochondria-dependent pathway. Thus, helenalin is a promising experimental cytotoxic agent that possibly points to new strategies to overcome apoptosis resistance attributable to overexpression of antiapoptotic Bcl-2 proteins.
Dirsch
V M
VM
Department of Pharmacy, Center of Drug Research, University of Munich, Butenandtstrasse 5-13, D-81377 Munich, Germany. Verena.Dirsch@cup.uni-muenchen.de
Stuppner
H
H
Vollmar
A M
AM
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D004789:Enzyme Activation; D004790:Enzyme Induction / Q000187:drug effects; D053222:Fas Ligand Protein; D006801:Humans; D019169:Jurkat Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D020782:Matrix Metalloproteinases / Q000096:biosynthesis; D008562:Membrane Glycoproteins / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000502:physiology; D012717:Sesquiterpenes / Q000494:pharmacology*; D045789:Sesquiterpenes, Guaiane; D051020:bcl-X Protein; D019014:fas Receptor / Q000502:physiology*
D000972:Antineoplastic Agents, Phytogenic; C494811:BCL2L1 protein, human; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012717:Sesquiterpenes; D045789:Sesquiterpenes, Guaiane; D051020:bcl-X Protein; D019014:fas Receptor; C001329:helenalin; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D020782:Matrix Metalloproteinases