Abstract
Bisindolylmaleimide VIII (Bis VIII) has been previously shown to enhance Fas-mediated apoptosis through a protein kinase C-independent mechanism. In the present study, we examined the effect of Bis VIII on apoptosis induced by DR5 (TRAIL-R2), using an agonistic anti-human DR5 monoclonal antibody, TRA-8. Our results demonstrated that Bis VIII was able to enhance the apoptosis-inducing activity of TRA-8 both in vitro and in vivo. The combination of TRA-8 and Bis VIII led to a synergistic and sustained activation of the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase, which was mediated by MAPK kinase 4 and was caspase-8-dependent. The mitochondrial pathway is involved in the synergistic induction of apoptosis by Bis VIII and TRA-8. Bis VIII alone induced the loss of mitochondrial membrane potential in a caspase-independent fashion without subsequent release of cytochrome c. However, in the presence of Bis VIII, TRA-8 induced more profound loss of mitochondrial membrane potential and release of cytochrome c. These results suggest that the enhanced and persistent activation of the JNK/p38 and the decreased mitochondrial membrane potential play a crucial role in synergistic induction of the death receptor-mediated apoptosis by Bis VIII. The unique ability of Bis VIII to enhance DR5-mediated apoptosis signal transduction discloses a potential utility of this compound in combination with anti-DR5 antibody in cancer therapy.
Highlights
Bisindolylmaleimide VIII (Bis VIII) has been previously shown to enhance Fas-mediated apoptosis through a protein kinase C-independent mechanism
Bis VIII Facilitates DR5-mediated Apoptosis and Enhances Tumoricidal Activity of TRA-8 —To determine whether Bis VIII has a similar apoptosis-enhancing effect on DR5-mediated apoptosis to that on Fas-mediated apoptosis as previously shown [28], we examined cell death of a human astrocytoma cell line (1321N1) mediated by TRA-8 in the absence or presence of Bis VIII. 1321N1 cells are relatively insensitive to DR5-mediated apoptosis as a short term treatment with TRA8-induced moderate cell death in a dose-dependent manner with no more than 30% of cells being killed by a high concentration of TRA-8 (1000 ng/ml) within a 12-h incubation (Fig. 1A)
To determine whether caspase-8 is required for the activation of the MKK4/Jun Nterminal kinase (JNK)/p38 pathway by DR5 signaling, we examined the effect of caspase inhibitors on cell death and the kinase activation induced by TRA-8 and Bis VIII
Summary
Reagents—An agonistic anti-human DR5 monoclonal antibody, TRA-8, was prepared as described [25]. Bis VIII and SB203580 were purchased from Alexis Biochemicals (San Diego, CA). The various caspase inhibitors of fluoromethyl ketone-derivatized peptides, such as Z-VAD-FMK, Z-DEVD-FMK, Z-IETD-FMK, and Z-LEHD-FMK, were purchased from R & D Systems, Inc. Carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone and N,N,-dihexyl-2-(4-fluorophenyl)-indole-3-acetamide were from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA), and anisomycin and chelerythrine. Cells were cultured with the indicated concentration of TRA-8 and/or Bis VIII overnight. Cell viability was determined using the ATPLite assay with the medium control taken to represent 100% of viability
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