Retinoid-related molecules require caspase 9 for the effective release of Smac and the rapid induction of apoptosis

Abstract

Certain retinoid-related molecules (RRMs) with agonist or antagonist activities have been described to induce apoptosis in a variety of cancer cell lines and show promise for the treatment of cancer. Similar to other chemotherapeutic drugs, these retinoid analogs have been suggested to induce apoptosis through the intrinsic pathway, which requires the release of cytochrome c from the mitochondria for the effective activation of caspase 9. Expression of a catalytically inactive form of caspase 9, which functions as a dominant negative mutant, inhibits the induction of DEVDase activity and nuclear fragmentation by selective RRMs. Whereas the RRMs could induce the release of cytochrome c in the absence of caspase 9 activity, the later is necessary for the effective release of Smac/Diablo from the mitochondria. Furthermore, overexpression of Bcl-2 or Bcl-X L also inhibits RRM-induced apoptosis. We demonstrate that activation of caspase 2 by the agonist MX2870-1 requires caspase 9 activity and is inhibited by Bcl-2 overexpression. In contrast, the antagonist MX781 induces cleavage of procaspase 2 upstream of mitochondria and independently of caspase 9. Thus, two retinoid analogs with unique characteristics activate two distinct apical caspases (2 or 9) to initiate apoptosis. In addition to caspase-mediated cell death, sustained exposure to the RRMs can also lead to loss of cell viability in cells lacking caspase 9 activity or in cells stimulated in the presence of the caspase inhibitor Z-VADfmk. Moreover, MX2870-1 and MX781 produce cell cycle arrest independently of caspase activity and the retinoid receptors.

Apoptotic volume decrease as a geometric determinant for cell dismantling into apoptotic bodies

“…However, inhibition of caspase 9 (and downstream executioner caspase 3) prevents Fas-induced apoptotic body release but not blebbing. 25 However, in the case of retinoids, the apoptotic signal upstream of mitochondria is caspase-independent. 26 That is why the pan caspase inhibitor Z-VAD-fmk cannot block retinoid-induced blebbing, but prevents the executioner caspase-dependent apoptotic body release.…”

“…However, inhibition of caspase 9 by stably overexpressing a catalytically inactive mutant of this caspase prevents apoptotic body release but only partially inhibits membrane blebbing induced by all these stimuli. 25 In addition, the pan caspase inhibitor N-benzyloxycarbonyl-valine-alanineaspartic acid-fluoromethylketone (Z-VAD-fmk) completely inhibits blebbing and apoptotic body release induced by Fas stimulation, but only the apoptotic body release induced by retinoids.…”

“…The release of bleb vesicles is a protease-dependent process, in which caspases seem to have a central role. 24,25 It has been shown that caspase inhibition prevents the release of apoptotic bodies induced by extrinsic and intrinsic pathway activators, but not their formation by blebbing. 25 Membrane blebbing induced by retinoids (activators of the intrinsic pathway) and Fas stimulation (extrinsic pathway) is completely abolished by stable overexpression of B-cell lymphoma 2 (Bcl-2).…”

“…24,25 It has been shown that caspase inhibition prevents the release of apoptotic bodies induced by extrinsic and intrinsic pathway activators, but not their formation by blebbing. 25 Membrane blebbing induced by retinoids (activators of the intrinsic pathway) and Fas stimulation (extrinsic pathway) is completely abolished by stable overexpression of B-cell lymphoma 2 (Bcl-2). However, inhibition of caspase 9 by stably overexpressing a catalytically inactive mutant of this caspase prevents apoptotic body release but only partially inhibits membrane blebbing induced by all these stimuli.…”

A novel retinoid-related molecule inhibits pancreatic cancer cell proliferation by a retinoid receptor independent mechanism via suppression of cell cycle regulatory protein function and induction of caspase-associated apoptosis

“…Other RRMs have been described and, in general, they show enhanced efficacy with fewer side effects. They frequently kill cells via an apoptotic mechanism (Bayon et al, 2003;Garattini et al, 2004a, b;Lopez-Hernandez et al, 2004;Zuco et al, 2004).…”