Pterostilbene: A Novel Histone Deacetylase 1 Inhibitor (HDACi1) Demonstrating Efficacy in Multiple Myeloma.

Abstract 3844

Poster Board III-780

Pterostilbene (3,5-Dimethoxy-4'-hydroxy-trans-stilbene) is a key compound found predominantly in blueberries and grapes. Pterostilbene has been shown to exhibit potential anti-cancer characteristics, anti-hypercholesterolemia and anti-hypertriglyceridemia properties. However, the mechanism of anti-cancer effects has not been elucidated and this compound has not been previously evaluated in multiple myeloma (MM). In this study, we first examined the HDAC binding ability of pterostilbene in the RPMI8226 multiple myeloma (MM) cell line and 293 HEK cells by using a novel HDAC screening assay. Briefly, RPMI8226 cells or 293 HEK cells were lysed with M-PER supplemented with protease and phosphatase inhibitors. The lysates were diluted and incubated with pterostilbene in concentrations ranging from 1 to 200 μM or without drug. Proteolysis was performed using thermolysin and aliquots were removed every 5 minutes and western blot analysis completed using antibodies against different HDACs or GAPDH. The results showed that pterostilbene prevents specifically HDAC1 digestion by thermolysin in both RPMI8226 and 293 cells. Next, we examined H4 acetylation of lysine residues in RPMI8226 cells following treatment with pterostilbene using western blot analysis. Increases in histone acetylation in RPMI8226 cells following exposure to pterostilbene treatment occurred in a concentration-dependent manner. Specifically, 1 to10μM of the pterostilbene markedly induced histone acetylation in RPMI8226 cells within 24 hrs. We also analyzed pterostilbene's effect on MM tumor cell proliferation using the MTS assay and apoptosis with flow cytometric analysis following Annexin V staining, in cells from the RPMI8226 and MM1S MM cell lines and freshly obtained tumor cells from MM patients. Pterostilbene alone showed a 50% growth inhibition (IC₅₀) of cells from the RPMI8226 and MM1S lines as well as fresh bone marrow tumor cells from MM patients treated for 48 hours at approximately the same concentration (5 μM) as showing anti-MM effects in the MTS assay. Notably, the combination of pterostilbene and melphalan or the proteasome inhibitor bortezomib showed markedly increased inhibition of MM cell growth compared to treatment of the cells with the drugs alone. Since this effect may have resulted from decreased cell proliferation due to cell cycle arrest or increased cell death, we further determined apoptosis in cells from RPMI8226, U266, and MM1S cell lines as well as fresh tumor cells from MM patients following treatment with pterostilbene. Our data demonstrated that pterostilbene induced apoptotic cell death in a concentration dependent fashion. We also evaluated the combination of this compound with bortezomib (1-6 nM) and showed marked increases in apoptosis using this combination compared to either drug alone. These studies establish pterostilbene as a novel HDACi with potent anti-MM activity alone and also show its ability to enhance the anti-MM effects of chemotherapeutic agents and bortezomib. Currently, we are evaluating pterostilbene alone and in combination treatments using our SCID-hu murine models of human MM.