Synergistic Interaction of the Histone Deacetylase Inhibitor SAHA with the Proteasome Inhibitor Bortezomib in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is an incurable B cell lymphoma and novel treatment strategies are urgently needed. Proteasome inhibitors, e.g. bortezomib, act by targeting the catalytic 20S core of the proteasome and induce apoptosis in tumor cells. Among other mechanisms, they lead to cytoplasmic accumulation of the IκBa protein, resulting in a reduced NF-κB activity. Histone deacetylase inhibitors (HDAI), e.g. SAHA, promote histone acetylation, chromatin uncoiling, and transcription of a variety of genes. Previous studies have indicated that HDAIs also interfere with NF-κB signaling. Since NF-κB is constitutively activated MCL cells and plays a major role in a variety of cellular processes, we hypothesized synergist effects of bortezomib and HDAI in MCL cells. Human mantle cell lymphoma cell lines (JeKo-1 and Granta-519) were exposed to bortezomib and/ or SAHA for 4 to 48 hours. Cell viability and apoptosis were quantified by the MTT and annexin-V assay, respectively. The effect of the combination of both agents was analyzed using the median effect method of Chou and Talalay. Reactive oxygen species (ROS) were quantified by the fluorophore H₂DCFDA. The functional role of ROS generation was assessed using the free radical scavenger N-acetyl-l-cysteine (LNAC). In addition, activated caspases, proteasome- and NF-κB activity were quantified. After 48 hours of incubation, IC50 of SAHA and bortezomib were noted at 0.8μM and 7.7nm in JeKo-1 cells and at 3.9μM and 5.7nm in Granta-519 cells, respectively. Combined incubation resulted in synergistic cytotoxic effects, as indicated by CI values <1. This was accompanied by an increase of caspase-3, -8 and -9 activity. In addition, coexposure of bortezomib and SAHA led to an enhanced ROS generation (5.4 fold in Jeko-1 and 9.2 fold in Granta-519 as compared to untreated controls), while the agents alone only modestly induced reactive oxygen species. LNAC could block the ROS generation and reduced the apoptosis significantly. As expected, bortezomib reduced the NF-κB activity. Interestingly, exposure to SAHA led to an increase of NF-κB activity after 4 hours and a decrease after 24 hours. The combination of both drugs resulted in a decrease of NF-κB activity. Moreover, combined treatment with bortezomib and SAHA resulted in a marked reduction of proteasome activity that was more pronounced than the proteasome inhibition by the single agents. In conclusion, this is the first report giving evidence that SAHA and bortezomib synergistically induce apoptosis in mantle cell lymphoma cells through the generation of reactive oxygen species and disruption of the NF-κB pathway. Our data provide a framework for clinical studies with the combination of both agents in patients with mantle cell lymphoma.