melitten and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

We investigated the antitumoral efficacy, endocrine consequences, and molecular mechanisms underlying cell death induced by the Hecate-chorionic gonadotropin (CG)beta conjugate, a fusion protein of a 23-amino acid lytic peptide Hecate with a 15-amino acid (81-95) fragment of the human CGbeta chain. Transgenic (TG) mice expressing the inhibin alpha-subunit promoter (inhalpha)/Simian Virus 40 T-antigen (Tag) transgene, developing luteinizing hormone (LH) receptor (R) expressing Leydig and granulosa cell tumors, and wild-type control littermates were treated either with vehicle, Hecate, or Hecate-CGbeta conjugate for 3 weeks. Hecate-CGbeta conjugate treatment reduced the testicular and ovarian tumor burden (P < .05), whereas a concomitant increase (testis; P < .05) or no change (ovary) in tumor volumes occured with Hectate treatment. A drop in serum progesterone, produced by the tumors, and an increase in LH levels occured in Hecate-CGbeta treated mice, in comparison with vehicle and Hecate groups, providing further support for the positive treatment response. Hecate-CGbeta conjugate induced a rapid and cell-specific membrane permeabilization of LHR-expressing cells in vitro, suggesting a necrotic mode of cell death without activation of apoptosis. These results prove the principle that the Hecate-CGbeta conjugate provides a novel specific lead into gonadal somatic cell cancer therapy by targeted destruction of LHR-expressing tumor cells.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blotting, Northern; Caspase 3; Caspases; Cell Death; Cell Line, Tumor; Cell Separation; Chorionic Gonadotropin, beta Subunit, Human; Disease Models, Animal; Enzyme Activation; Female; Flow Cytometry; Granulosa Cell Tumor; Humans; Leydig Cell Tumor; Male; Melitten; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence; Necrosis; Ovarian Neoplasms; Progesterone; Promoter Regions, Genetic; Protein Structure, Tertiary; Receptors, LH; Recombinant Fusion Proteins; Testicular Neoplasms; Time Factors

Opsonization of apoptotic cells with complement proteins contributes to their clearance by phagocytes. Little is known about the lytic effects of complement on apoptotic cells. Sensitivity of cells treated with anti-Fas antibody (Jurkat cells), staurosporine or etoposide (Raji cells) to lysis by complement was examined. As shown here, early apoptotic cells are more sensitive to lysis by antibody and complement than control cells. More complement C3 and C9 bound to apoptotic than to control cells, even though antibody binding was similar. Enhanced killing and C3/C9 deposition were blocked by benzyloxy-Val-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor. Complement-mediated lysis of early apoptotic cells was also prevented by inhibitors of caspases 6, 8, 9 or 10. In contrast, caspase inhibitors had no effect on the lysis of non-apoptotic Jurkat and Raji cells. Early apoptotic Jurkat cells were also more sensitive to lysis by the pore formers streptolysin O and melittin. Sensitivity of Jurkat Bcl-2 transfectants to lysis by complement was analyzed. Enhanced Bcl-2 expression was associated with reduced C3 deposition and lower sensitivity to complement-mediated lysis. These results demonstrate that at an early stage in apoptosis, following caspase activation, cells become sensitive to necrotic-type death by complement and other pore formers. Furthermore, they suggest that Bcl-2 is actively protecting Jurkat cells from complement-mediated lysis.

Topics: Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Apoptosis; Bacterial Proteins; Caspase Inhibitors; Caspases; Complement C3; Complement C9; Enzyme Inhibitors; Etoposide; Flow Cytometry; Genes, bcl-2; Humans; Jurkat Cells; Melitten; Staurosporine; Streptolysins; Transfection