Requirement of Apoptotic Protease-Activating Factor-1 for Bortezomib-Induced Apoptosis but Not for Fas-Mediated Apoptosis in Human Leukemic Cells

Abstract

Bortezomib is a highly selective inhibitor of the 26S proteasome and has been approved for clinical use in the treatment of relapsing and refractory multiple myeloma and mantle cell lymphoma. Clinical trials are also underway to assess the role of bortezomib in several other human malignancies, including leukemia. However, the mechanism(s) by which bortezomib acts remain to be fully understood. Here, we studied the molecular requirements of bortezomib-induced apoptosis using the human T-cell leukemic Jurkat cells stably transfected with or without shRNA against apoptotic protease-activating factor-1 (Apaf-1). The Apaf-1-deficient Jurkat T cells were resistant to bortezomibinduced apoptosis, as assessed by caspase-3 activity, poly(ADPribose) polymerase cleavage, phosphatidylserine externalization, and hypodiploid DNA content. In contrast, Apaf-1-deficient cells were sensitive to Fas-induced apoptosis. Bortezomib induced an upregulation of the pro-apoptotic protein Noxa, loss of mitochondrial transmembrane potential, and release of cytochrome c in cells expressing or not expressing Apaf-1. Transient silencing of Apaf-1 expression in RPMI 8402 T-cell leukemic cells also diminished bortezomib-induced apoptosis. Fas-associated death domain (FADD)-deficient Jurkat cells were resistant to Fas-mediated apoptosis yet remained sensitive to bortezomib. Our results show that bortezomib induces apoptosis by regulating pathways that are mechanistically different from those activated upon death receptor ligation. Furthermore, in silico analyses of public transcriptomics databases indicated elevated Apaf-1 expression in several hematologic malignancies, including acute lymphoblastic and myeloid leukemia. We also noted variable Apaf-1 expression in a panel of samples from patients with acute lymphoblastic leukemia. Our results suggest that the expression of Apaf-1 may be predictive of the response to proteasome inhibition.

Expression Profiles of the Individual Genes Corresponding to the Genes Generated by Cytotoxicity Experiments with Bortezomib in Multiple Myeloma

“…For instance, the LP-1 cell line was found to be resistant to bortezomib in our present study (Figure 3). A previous study suggested that the expression of Apaf-1 might be predictive of the response to proteasome inhibition [29]. Based on our present results, patients with MM mimicking the molecular profile/behavior of LP-1 at any clinical evaluation point during the long-term clinical course of MM will be candidates for therapeutic regimens other than bortezomib.…”

Copper oxide nanoparticles trigger macrophage cell death with misfolding of Cu/Zn superoxide dismutase 1 (SOD1)

“…7 a, b). We also performed western blot for polyubiquitinated proteins as described previously [ 45 ], and we could detect an accumulation of polyubiquitinated proteins in cells exposed to a BMD 20 dose (25 µg/mL) of CuO NPs (Fig. 6 c).…”

Recent advances and novel treatment paradigms in acute lymphocytic leukemia

“…Their mechanism of action involves inhibition of the proteasome which affects critical pathways for cellular survival through nuclear factor Kβ signaling, Jun N-terminal kinase (JNK) pathway, histone deacetylase action and suppression of anti-apoptotic proteins [Mujtaba and Dou, 2011; Dou and Zonder, 2014]. Other studies using cell lines derived from T-ALL indicated that expression of Apaf-1 has an important role in bortezomib-induced apoptosis [Ottosson-Wadlund et al 2013]. The Nalm-6 cell line [Adachi et al 2006] recapitulates aspects of B-ALL biology and in vitro experiments using bortezomib highlighted an array of antileukemic mechanisms.…”