Proteasome inhibitors sensitize colon carcinoma cells to TRAIL-induced apoptosis via enhanced release of Smac/DIABLO from the mitochondria

Ist Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary.
Pathology & Oncology Research (Impact Factor: 1.86). 02/2006; 12(3):133-42. DOI: 10.1007/BF02893359
Source: PubMed


The synergistic interaction between proteasome inhibitors and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising approach to induce cell death in tumor cells. However, the molecular and biochemical mechanisms of this synergism have been proven to be cell type specific. We therefore focused our investigation on TRAIL-resistant colon carcinoma cells in this study. DNA fragmentation, mitochondrial membrane depolarization and increased caspase-3-like enzyme activity was exclusively induced only by combined treatment with proteasome inhibitors (epoxomicin, MG132, bortezomib/PS-341) and TRAIL. The expression level of anti-apoptotic proteins (XIAP, survivin, Bcl-2, Bcl-XL), regulated by NF-kappaB transcription factor, was not effected by any of these treatments. TRAIL alone induced only partial activation of caspase-3 (p20), while the combination of TRAIL and proteasome inhibition led to the full proteolytic activation of caspase-3 (p17). Only the combination treatment induced marked membrane depolarization and the release of cytochrome c, HtrA2/Omi and Smac/DIABLO. Apoptosis-inducing factor (AIF) was not released in any of these conditions. These results are consistent with a model where the full activation of caspase-3 by caspase-8 is dependent on the release of Smac/DIABLO in response to the combined treatment. This molecular mechanism, independent of the inhibition NF-kappaB activity, may provide rationale for the combination treatment of colon carcinomas with proteasome inhibitors and recombinant TRAIL or agonistic antibody of TRAIL receptors.

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    • "It was noted that the Smac protein released from the mitochondria was more abundant than the Cyto c protein. This notion is in agreement with the findings in other types of cancer cells treated with PIs [19] [20]. A recent observation in our laboratory also showed that mitochondrial release of the Smac protein happens much earlier than that of the Cyto c protein during MG132-induced apoptosis of lung cancer cells, supporting the view that the Smac protein is more critical than the Cyto c protein in initiating PI-induced apoptosis [21]. "
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    ABSTRACT: Malignant pleural mesothelioma (MPM) is an aggressive malignancy tightly associated with asbestos exposure. The increasing incidence of MPM and its resistance to all therapeutic modalities necessitate an urgent development of new treatments for MPM. Proteasome inhibitors (PIs) have emerged as promising agents for treating human cancers that are refractory to current chemotherapies. In this study, we characterized MG132, a commonly used PI, for its proapoptotic and anti-invasion activities in NCI-H2452 and NCI-H2052 human thoracic MPM cell lines to determine the therapeutic effect of PIs on MPM. We found that as low as 0.5 microM MG132 caused a significant apoptosis in both cell lines as evidenced by DNA damage, cleavage of poly ADP-ribose polymerase and caspases 3, 7, and 9, and mitochondrial release of Smac/DIABLO and Cytochrome c. Mitochondrial caspase activation was found to be the underlying mechanism of the MG132-induced apoptosis. Mcl-1, among the Bcl-2 and IAP (inhibitor of apoptosis protein) antiapoptotic family proteins tested, was proved to be a major inhibitor of the MG132-induced apoptosis in MPM cells. Meanwhile, subapoptotic doses of MG132 inhibited the invasion of both MPM cell lines through reducing Rac1 activity. These observations demonstrate that MG132 possesses proapoptotic and anti-invasion activities in human MPM cells, therefore encouraging further investigations on the value of PIs for treating MPM.
    Translational oncology 10/2008; 1(3):129-40. DOI:10.1593/tlo.08133 · 2.88 Impact Factor
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    • "Sensitization of tumour cells to TRAIL-induced apoptosis by bortezomib has been ascribed to enhanced surface expression of TRAIL receptors (Johnson et al., 2003; Liu et al., 2007; Voortman et al., 2007), enhanced release of SMAC/Diablo (Johnson et al., 2003; Leverkus et al., 2003; Nagy et al., 2006), reduction of c-FLIP levels (Sayers et al., 2003; Koschny et al., 2007a) or Bik accumulation (Nikrad et al., 2005; Zhu et al., 2005). We did find an increased expression of DR4 and 5 in SW620 cells and of DR5 in SW480 cells under the influence of bortezomib (not shown). "
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    ABSTRACT: A pair of isogenic colon carcinoma cells, SW480 and 620, was used to investigate the mechanisms of acquired tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistance during tumour progression. Whereas primary tumour SW480 cells are sensitive to TRAIL-induced apoptosis, metastatic SW620 cells are resistant. The apoptotic signalling activated by TRAIL in SW480 cells is a type II pathway. We show that in SW620 cells, although caspase-8 is recruited and activated at the death-inducing-signalling complex and Bid is cleaved, this does not lead to caspase-9 activation. Comparison of Bcl-2, Bcl-xL and Mcl-1 levels in both cell lines showed no difference. In SW620 cells transfected with a tBid-GFP construct, tBid-GFP was correctly localized to the mitochondria. Thus, the resistance of SW620 cells is at the level of the mitochondria that can withstand large amounts of tBid. Although caspase-3 was directly cleaved by caspase-8 in SW620 cells to yield the p20 fragment, no further autocatalytic maturation into the p17 fragment was observed. We show that, in contrast to SW480 cells, the SW620 cell line expresses high amounts of X-linked inhibitor of apoptosis (XIAP). Downregulation of XIAP with bortezomib or small-interfering RNA was sufficient to restore the sensitivity of SW620 cells to TRAIL-induced apoptosis in the absence of SMAC/Diablo or cytochrome c release from the mitochondria. Thus, SW620 cells have developed a dual resistance to TRAIL-induced apoptosis: a block at the level of the mitochondria and, after a conversion to a type I pathway, an increased expression of XIAP which inhibits this pathway.
    Oncogene 07/2008; 27(46):6012-22. DOI:10.1038/onc.2008.197 · 8.46 Impact Factor
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