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Helenalin Triggers a CD95 Death Receptor-independent Apoptosis That Is Not Affected by Overexpression of Bcl-x_L or Bcl-2¹

Department of Pharmacy, Center of Drug Research, University of Munich, D-81377 Munich, Germany [V. M. D., A. M. V.], and Institute of Pharmacy, University of Innsbruck, A-6020 Innsbruck, Austria [H. S.]

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 µM) 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 µM 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 µM) 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 (_m), which was partly inhibited by zVAD-fmk, which suggests that caspases are involved in loss of _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.

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