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Cancer cells are more sensitive to oxidative stress due to higher levels of reactive oxygen species. Therefore, the ability of anti-cancer agent combretastatin A-4 (CA-4) and triazole analogues to induce reactive oxygen species may be important for selectivity against cancer cells. The purpose of the present study was to investigate the structural requirements for reactive oxygen species production by CA-4 and the triazole analogues Ana-2, Ana-3 and Ana-4. Ana-2 and Ana-3 mimic the cis configuration in CA-4; Ana-3 lacks the phenolic hydroxyl group, while Ana-4 mimics a trans configuration. The rat pheochromocytoma cancer cell line PC12 was used as model system. CA-4 and Ana-2 were highly toxic; Ana-3 was less toxic, whereas Ana-4 was non-toxic. The probe dihydroethidium detected reactive oxygen species production from CA-4, Ana-2, and Ana-3. CA-4 and Ana-2 also induced oxidation of the reactive oxygen species probe dihydrorhodamine and activation of caspase-3. Thus, the phenolic hydroxyl group in CA-4 and Ana-2 was necessary for dihydrorhodamine oxidation, caspase-3 activation, and increased cytotoxicity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Caspase-3 is known as the key executioner caspase, activated in both the intrinsic and extrinsic apoptotic pathway, and an effector far downstream in the apoptotic cascade. Procaspase-activating compound-1 (PAC-1) and 1541 were launched as direct activators of procaspase-3 to caspase-3, and anticipated to be promising therapeutic agents for the treatment of cancer. PAC-1 has recently been evaluated in a phase I preclinical trial. However, little is known about the effect of these substances in cells. Activation of caspase-3 in whole cells may be more complicated than thought, as it is likely that this key protease is tightly regulated both in development and apoptosis. In this study, we investigated the effect of epidermal growth factor (EGF) on PAC-1-induced caspase-3 activity and cell death. We show that EGF can block caspase-3 activity generated by PAC-1, and protect both PC12 cells and primary cerebellar granule neurons against PAC-1-induced death. Similar results were obtained with 1541. Both substances reduced cellular p-ERK levels. Crosstalk between caspase-3 and growth factor signaling pathways may present a challenge for the use of such caspase-3-activating substances in cancer therapy, since aberrant growth factor signaling is frequently seen in malignant cells. This study adds important knowledge about cellular effects of procaspase-3 activators like PAC-1 and 1541. Effects mediated by these substances may also contribute to the understanding of caspase signaling in cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibited both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy., (2010 Elsevier Inc. All rights reserved.)