Heterogeneous role of caspase-8 in fenretinide-induced apoptosis in epithelial ovarian carcinoma cell lines.

The mechanism of action of fenretinide, a synthetic retinoid currently undergoing testing as a chemopreventive and chemotherapeutic agent, is incompletely understood. In the present study, fenretinide caused apoptotic changes, including DNA fragmentation and cleavage of caspase substrates, in six low-passage ovarian cancer cell lines. However, the caspase activation pathway used by this agent varied. Transient transfection of cDNA-encoding cytokine response modifier A (CrmA), a caspase-8 inhibitor, diminished fenretinide-induced death in OV177 cells. Likewise, IETD(OMe)-fluoromethylketone (fmk) inhibited fenretinide-induced apoptosis by >80% in OV177 or OV266 cells and by approximately 50% in OV17, OV167, or OV207 cells. Further analysis demonstrated that inhibition of Fas ligand, tumor necrosis factor-alpha, or TRAIL signaling with blocking reagents did not affect fenretinide-induced apoptosis, raising the possibility that fenretinide activates caspase-8 in a death receptor-independent manner. In contrast, CrmA transfection or IETD(OMe)-fmk treatment did not inhibit fenretinide-induced apoptosis in OV202 cells. These divergent behaviors did not correlate with increased levels of procaspase-10, which is relatively resistant to CrmA and IETD(OMe)-fmk, nor with the expression of procaspase-8 and -9, apoptotic protease activating factor-1, or cellular FLICE-like inhibitory protein. Similarly, fenretinide treatment increased ceramide levels equally in cells that do (OV177) and do not (OV202) rely on caspase-8 to initiate apoptosis. These results indicate that synthetic retinoids can use caspase-8 as an initiating caspase, but they also indicate unexpected heterogeneity in caspase activation pathways among closely related cell lines.

Temporal aspects of onion-induced antiplatelet activity.

Organosulfur compounds in onion extracts are formed following the lysis of the S-alk(en)yl-L-cysteine sulfoxides by alliinase. These compounds inhibit the aggregation of human blood platelets and offer the potential for positive cardiovascular health benefits. An experiment was designed to examine temporal and temperature effects on onion-induced antiplatelet activity. Platelet aggregation is induced by various agonists, including ADP, collagen, and thrombin. Unexpectedly, all freshly-juiced onion extracts (ca. 5 minutes post-juicing) appeared to exhibit both an agonist-free aggregation peak (AFP) and a platelet inhibitory peak (PIP) characteristic of inhibition of platelet aggregation. The AFP was minimal by 30 minutes and dissipated in all treatments by 120 minutes, while the PIP increased as onion extracts aged and did not change after 30 minutes at 25 degrees C. This finding confirms the observation that the in vitro platelet inhibitory activity of onion organosulfur compounds is time dependent. Freshly-prepared onion extracts were incubated with the ADP scavenger enzyme apyrase (E.C. 3.6.1.5). AFPs were abolished in apyrase-treated extracts, suggesting that this response may have been due to free ADP in onion extracts, although an amount of ADP required to generate such a response would be unexpected in onion extracts. In addition, platelet aggregates were not observed in the AFP, suggesting this response may be associated with changes in light transmission through platelet rich plasma that are not associated with platelet aggregation. Artifacts of analysis are, therefore, possible when assessing onion-induced antiplatelet activity with freshly-juiced extracts. Temporal formation of platelet-inhibiting organosulfur compounds should be taken into account during both in vitro and in vivo assessment of onion-induced antiplatelet activity.

Synthetic Smac/DIABLO peptides enhance the effects of chemotherapeutic agents by binding XIAP and cIAP1 in situ.

Inhibitor of apoptosis proteins (IAPs) interact with and inhibit caspases-3, -7, and -9. This interaction can be inhibited by Smac/DIABLO, a polypeptide released from mitochondria upon initiation of the apoptotic signaling process. Here we demonstrate that the first 4-8 N-terminal amino acids of Smac/DIABLO fused to the Drosophila antennapaedia penetratin sequence, a carrier peptide, enhance the induction of apoptosis and long term antiproliferative effects of diverse antineoplastic agents including paclitaxel, etoposide, 7-ethyl-10-hydroxycamptothecin (SN-38), and doxorubicin in MCF-7 breast cancer cells. Similar effects were observed in additional breast cancer and immortalized cholangiocyte cell lines. Further analysis demonstrated that the Smac-penetratin fusion peptide crossed the cellular membrane, bound XIAP and cIAP1, displaced caspase-3 from cytoplasmic aggregates, and enhanced drug-induced caspase action in situ. These studies demonstrate that inhibition of IAP proteins can modulate the efficacy of antineoplastic agents.