Bortezomib-Induced Apoptosis in Cutaneous T-Cell Lymphoma and Adult T-Cell Leukemia/Lymphoma Cells Partially Depends on up-Regulation of Noxa, Functional Repression of Mcl-1, and Down-Regulation of C-Flip and XIAP.

Bortezomib, a proteasome inhibitor, which was originally developed as an inhibitor of NF-kappaB pathways, is currently used for the treatment of multiple myeloma (MM) and mantle cell lymphoma (MCL). The mechanisms of action of this anti-tumor agent have been studied by several investigators. As bortezomib has been reported effective for a fraction of peripheral T-cell lymphomas including adult T-cell leukemia/lymphoma (ATLL) and cutaneous T-cell lymphoma (CTCL) at clinically achievable concentrations, it is expected to be applicable for these refractory T-cell malignancies. In this study, we have explored the underlying mechanisms of bortezomib-induced apoptosis in cell lines established from CTCL and ATLL together with those from MM by particularly focusing on the level of mitochondrial membrane injury. It was because the apoptosis induced by bortezomib in CTCL and ATLL cells accompanied the activation of caspase-3, -8, and -9, and the disturbance of mitochondrial membrane potential preceded the process of bortezomib-induced apoptosis when detected by DiIC1 and Annexin V staining. In 6 MM lines including KMS-12-PE, 2 CTCL lines including Hut78, and 7 ATLL lines including ATN1 and MT4, anti-apoptotic factors such as c-Flip and XIAP were down-regulated after exposure to bortezomib, probably via inhibition of NF-kappaB signaling. This reduction of c-Flip and XIAP was also confirmed in the primary tumor cells derived from the patients with ATLL after exposure to 20nM bortezomib. These findings indicate that the activation of both intrinsic and extrinsic apoptosis pathways led to activation of caspases via reduced expression of negative regulators of apoptosis. Among the members of the BH3-only family protein, up-regulation of Noxa was consistently seen at both the transcriptional and protein levels in a p53-independent and c-Myc-independent manner after exposure to bortezomib, whereas the expression of other BH3-only family proteins showed no consistent changes. Of anti-apoptotic Bcl-2 family proteins, accumulation of Mcl-1 and its cleaved form were observed, but no altered expression of other Bcl-2 family members was seen. Specific repression of Noxa by small interfering RNA partially rescued CTCL and ATLL cells from bortezomib–induced apoptosis, suggesting a crucial role of Noxa in bortezomib-induced apoptosis in these cell types as well as in MM cells. Immunoprecipitation assays indicated time-dependent binding of Noxa and Mcl-1 in all cell types, suggesting that functional repression of Mcl-1 led to the loss of mitochondrial outer membrane potential. Similar results showing transcriptional and translational up-regulation of Noxa followed by increased amount of the Mcl-1/Noxa complexes after exposure to 20nM bortezomib were also obtained in primary tumor cells derived from patients with ATLL. Taken together, we conclude that bortezomib-induced apoptosis in ATLL/CTCL cells at least partly depends on up-regulation of Noxa and functional repression of Mcl-1 in addition to the reduced amount of c-Flip and XIAP proteins, although the mechanisms leading to the transcriptional activation of Noxa after exposure to bortezomib remains to be clarified.