The Histone Deacetylase Inhibitor Vorinostat (SAHA) Induces Apoptosis and Cell Cycle Arrest in Hodgkin Lymphoma (HL) Cell Lines by Altering Several Survival Signaling Pathways and Synergizes with Doxorubicin, Gemcitabine and Bortezomib.

Several epigenetic changes have been recently implicated in the pathogenesis of several human malignancies, including HL. This pathway is currently explored for cancer therapy using hypomethylating agents and histone deacetylase (HDAC) inhibitors. The purpose of this study was to examine the potential clinical value for HDAC inhibition in HL. Therefore, we examined the single agent activity of vorinostat (suberoylanilide hydroxamic acid, SAHA) in a panel of three well defined HL-derived cell lines (HD-LM2, L-428, and KM-H2). Furthermore, we determined the molecular mechanisms of vorinostst activity in these cell lines. Vorinostat showed antiproliferative effect in all HL cell lines in a time- and dose-dependent manner, as determined with the MTS assay. The most sensitive cell line was the HD-LM2 with an IC50 of 2 micromolar, but antiproliferative activity was observed at concentrations between 0.1 and 0.5 micromolar. This antiproliferative effect was due to cell cycle arrest at the G2/M phase, as determined by Propidium Iodide DNA staining and FACS analysis. This effect was most prominent in the HD-LM2 cells, as the G2M fraction increased by 6 fold within 24 hours of incubation with 10 micromolar of vorinostat, and by 4 fold when 5 micromolar was used. Cell cycle arrest was followed with an increase in Annexin-V binding indicative of induction of apoptosis. This apoptotic effect was associated with activation of the caspase pathway, which resulted in caspase 3 and PARP cleavage. Furthermore, the antiproliferative activity was associated with de-phosphorylation of three important survival signals that are known to promote HL survival: STAT6, Akt and ERK. To determine the potential synergy of vorinostat with conventional chemotherapy and modern targeted small molecules, we treated HL cell lines with the combination of vorinostat and doxorubicin, gemcitabine, and the proteasome inhibitor bortezomib. In all the combinations SAHA significantly enhanced the effect of the other drugs, as determined with the MTS assay and the calculation of the Combination Index. Moreover, the combination of vorinostat and bortezomib arrested the cells in G2/M phase more efficiently and showed increased rate of apoptosis than each drug alone. In contrast, there was no added benefit when vorinostat was combined with the heat shock protein-90 inhibitor 17-AAG or with the mTOR inhibitor CCI-779. Our data indicate that HDAC inhibition by vorinostat may have a clinical value in HL, especially when combined with bortezomib or with chemotherapy.