Proteasome inhibitor PS-341 blocks cell growth and induces apoptosis in anaplastic large cell lymphoma cell lines

Blockade of the proteasome degradation pathway has emerged as a novel strategy to induce apoptosis in several tumor models, and PS-341 (Velcade) is among the newest compounds that show proteasome inhibitor activity that strictly correlates with antitumor effect. In this study, we investigated the antiproliferative activity of PS-341 in Anaplastic Large Cell Lymphomas (ALCLs), a high grade non-Hodgkin lymphoma characterized by the presence of specific chromosomal translocations involving the anaplastic lymphoma kinase (ALK) gene. Here we report that when administered for increasing time intervals PS-341 potently inhibited the growth of both ALK-positive (KARPAS, SUDHL1, SR786) and -negative (FE-PD) ALCL cell lines, irrespective of ALK-fusion proteins status and activity. The IC50s, as assayed by MTT following 24 h exposure, were 4.5-6 nM for SUDHL1, SR786 cells and the ALK-negative cell line FE-PD, whereas 17.8 nM for KARPAS. These cells resulted somehow more resistant to drug treatment, as time-dependent cleavage of PARP protein observed between 8 and 12 hours in SR786, SUDHL1 and FE-PD with 20 nM PS341, occurred only at higher concentrations (100 nM) in KARPAS cells. Because of the PS-341 pro-apoptotic activity, we assessed the activation of apoptotic caspase proteases and found that effector caspase-3 was strongly induced between 4 and 8 hours in ALCL lymphoma cells, and preceded the activation of caspase-8 and -9. Indeed, the caspase-8 inhibitor IETD-CHO prevented the formation of the active form of caspase-8 in the presence of PS-341, while it failed to block the procaspase-3 processing and PARP cleavage under the same experimental conditions. In contrast, the pan-caspase inhibitor ZVAD-fmk and the specific caspase-3 peptide inhibitor DEVD-CHO prevented both caspase-3 activation and PARP cleavage in all the cell lines. As expected, activation of caspases and mitochondrial cytochrome-c release was delayed in KARPAS cells, as well as up-regulation of the two cell-cycle inhibitors p27Kip and p21WAF. In contrast, the anti-apoptotic protein Mcl-1 was overexpressed in KARPAS cells and only slightly downregulated in the presence of PS-341, despite the potent inhibition of proteasome activity (90%) measured by fluorometric assay. Together, these data demonstrate the efficacy of PS-341 at inducing apoptosis in ALCL lymphoma cells, and suggest that induction of apoptosis may depend not solely on the inhibition of the 20S Proteasome, but also on the specific molecular profile of the individual cell line