Proteasome Inhibition Results in TRAIL Sensitization of Primary Keratinocytes by Removing the Resistance-Mediating Block of Effector Caspase Maturation

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts potent cytotoxic activity against transformed keratinocytes, whereas primary keratinocytes are relatively resistant. In several cell types, inhibition of the proteasome sensitizes for TRAIL-induced apoptosis by interference with NF-B activation. Here we describe a novel intracellular mechanism of TRAIL resistance in primary cells and how this resistance is removed by proteasome inhibitors independent of NF-B in primary human keratinocytes. This sensitization was not mediated at the receptor-proximal level of TRAIL DISC formation or caspase 8 activation but further downstream. Activation of caspase 3 was critical, as it only occurred when mitochondrial apoptotic pathways were activated, as reflected by Smac/DIABLO, HtrA2, and cytochrome c release. Smac/DIABLO and HtrA2 are needed to release the X-linked inhibitor-of-apoptosis protein (XIAP)-mediated block of full caspase 3 maturation. XIAP can effectively block caspase 3 maturation and, intriguingly, is highly expressed in primary but not in transformed keratinocytes. Ectopic XIAP expression in transformed keratinocytes resulted in increased resistance to TRAIL. Our data suggest that breaking of this resistance via proteasome inhibitors, which are potential anticancer drugs, may sensitize certain primary cells to TRAIL-induced apoptosis and could thereby complicate the clinical applicability of a combination of TRAIL receptor agonists with proteasome inhibitors.

NF-κB-independent sensitization of glioblastoma cells for TRAIL-induced apoptosis by proteasome inhibition

“…Proteasome inhibitors have previously been reported to enhance TRAIL-induced apoptosis in a variety of cancers, for example glioblastoma (Kasuga et al, 2004;Kim et al, 2004;Yin et al, 2005) and prostate, colon or bladder cancer (Johnson et al, 2003), as well as primary kerationocytes (Leverkus et al, 2003). Although some potential mediators of this sensitization effect have been indicated, for example p21 (Lashinger et al, 2005), c-FLIP (Sayers et al, 2003), Bik and Bim (Nikrad et al, 2005), whereas others have been excluded, for instance Bcl-2 (Nencioni et al, 2005), Bax (He et al, 2004) and Bcl-xL (Johnson et al, 2003), the specific contribution of NF-kB to this phenomenon remained unclear.…”

Prospective strategies to enforce selectively cell death in cancer cells

“…In combination with inhibitors of MEK, AKT or NF-kB, chemotherapy will induce apoptosis in cancer cells (Figure 2c). As a limitation, certain normal cells utilize the same survival pathways for protection from chemotherapy (Leverkus et al, 2003). It may be expected that apoptosis in normal cells will limit this approach.…”

“…TNF, FasL, TRAIL (activators of capsase-8) potentiate DNA-damaging drugs, which induce mitochondrial release of inhibitors of IAP to reactivate caspase-3. This, in turn, sensitizes cells to TRAIL (Leverkus et al, 2003). The proteasome inhibitor PS-341 sensitizes neoplastic cells to TRAILmediated apoptosis by reducing levels of c-FLIP (Sayers et al, 2003).…”

“…It should be kept in mind, however, that synergistic drug combinations might be synergistically toxic to a patient due to synergy in normal cells. For example, proteasome inhibition results in TRAIL sensitization of normal keratinocytes by removing the resistance-mediating block of caspases (Leverkus et al, 2003). To ensure cancer cell death, drug combinations should potentiate apoptosis in cancer cells selectively.…”

Regulation of apoptosis proteins in cancer cells by ubiquitin

“…Leverkus et al (2003) showed that proteasome inhibitors enhance TRAIL-mediated apoptosis of primary keratinocytes by enhancing the release of proapoptotic molecules such as SMAC/DIABLO and cytochrome c from mitochondria (Figure 3). It was found that TRAIL could not induce apoptosis effectively in Bax-deficient cells, apparently due to the formation of p20/p12 caspase-3 but not the fully active p17/p12 dimer.…”