SAHA (suberoylanilide hydroxamic acid) is an inhibitor of class I and II histone deacetylases. This agent has been shown to induce hyperacetylation of histones leading to the altered expression of genes such as induction of p21CIPI/WAFI and p27KIP1, resulting in cell cycle arrest and apoptosis. Bortezomib is an inhibitor of the 26S proteasome, an enzyme complex that plays an essential role in the ubiquitin-mediated degradation of many proteins including cell-cycle regulatory proteins such as p16, p21, p27, and the tumor suppressor, p53. Similar to SAHA, inhibitors of the 26S proteasome can induce cell-cycle arrest and apoptosis in vitro and in vivo. Our hypothesis was the combination of these two agents would result in synergistic antitumor effects. HT-29 and HCT-116 colon carcinoma cells were exposed to SAHA (0.5, 1, 2 uM), bortezomib (0.1, 0.5, 1 uM), or both agents (SAHA: 0.5, 1, 2; bortezomib: 0.1, 0.5, 1 uM) concurrently for 24 hours. MTT assays coupled to dose effect analysis (isobologram) demonstrated a striking anti-proliferative synergy between the compounds at all doses tested. Subsequent analyses were performed using 0.1 uM of bortezomib and 0.5 uM SAHA after a 24-hour concurrent exposure. The induction of apoptosis in both cell lines was more than additive in the combination, as reflected by an increase in the amount of PARP cleavage and a higher percentage of apoptotic and necrotic cells via a flow cytometry-based assay. Cell cycle analysis demonstrated a dramatic increase in the proportion of cells in the G2/M phase that was associated with an induction of the cyclin-dependent kinase inhibitor, p21. Flow cytometric analysis using phospho-specific antibodies showed a reduction in the levels of the pro-survival pathway proteins AKT and pAKT as well as reduced levels of ERK and pERK. IkB, a known target for proteasomal degradation, was also measured in the cytosol. Interestingly, in both cell lines, IkB was degraded in an apparent proteasome-independent fashion, supporting data from published work by others that in HT29 cells, IkB degradation may actually be stimulated by proteasome inhibitors. These results suggest that combined targeting of cell-cycle regulatory and survival pathways by SAHA and bortezomib results in an apoptotic response that may reflect the net response of molecular events dictating cell fate. Further studies to elucidate these pathways are ongoing in vitro and in vivo.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]