Helenalin triggers a CD95 death receptor-independent apoptosis that is notaffected by overexpression of Bcl-X-L or Bcl-2

Apoptosis is required for proper tissue homeostasis. Defects in apoptosis s ignaling pathways, thus, contribute to carcinogenesis and chemoresistance. A major goal in chemotherapy is, therefore, to find cytotoxic agents that r estore the ability of tumor cells to undergo apoptosis. We show here that t he sesquiterpene lactone helenalin (10-50 muM) induces apoptosis in leukemi a Jurkat T cells even if they lack the CD95 death receptor or overexpress t he antiapoptotic proteins Bcl-x(L), or Bcl-2. Activated peripheral blood mo nonuclear cells, however, are not affected (10-50 muM helenalin). Helenalin led to a time-dependent (0-24 h) cleavage of the specific caspase-3-like s ubstrate Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin as well as to th e proteolytic processing of procaspase-3 and -8. Caspase activation was a n ecessary requirement for apoptosis because the broad-spectrum caspase inhib itor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk, 50 mu M) completely abrogated helenalin-induced DNA fragmentation as well as phos phatidylserin translocation. Although the initiator caspase-8 was activated , the helenalin-induced signaling pathway did not require the CD95 death re ceptor 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 f rom mitochondria that was not inhibited by the caspase inhibitor zVAD-fink, which indicated that cytochrome c release precedes caspase activation. Cyt ochrome c release was accompanied by dissipation of the mitochondrial trans membrane potential (Delta Psi (m)), which was partly inhibited by zVAD-fmk, which suggests that caspases are involved in loss of Delta Psi (m). Most i mportantly, overexpression of the mitochondria protecting proteins Bcl-x(L) or Bcl-2 failed to confer resistance to helenalin-induced apoptosis, altho ugh the data presented here suggest that helenalin induces a mitochondria-d ependent pathway. Thus, helenalin is a promising experimental cytotoxic age nt that possibly points to new strategies to overcome apoptosis resistance attributable to overexpression of antiapoptotic Bcl-2 proteins.