Abstract 5: The effects of SIRT1 inhibitors nicotinamide and Ex-527 on lymphoma cells

Abstract

Abstract SIRT1 is a well-known lysine-deacetylase, having both histone and non-histone proteins as molecular targets. SIRT1 may function as a tumor promoter as well as a tumor suppressor in a context-dependent manner. We previously demonstrated that the treatment with resveratrol at concentrations able to induce apoptosis leads to SIRT1 down-regulation in lymphoma cells. Here we look for SIRT1 inhibition in order to investigate the effects on proliferation potential, apoptosis induction and targets modulation at a molecular level. The aim is to study whether SIRT1 inhibition affects cell cycle and proliferation capability of lymphoma cells. Experimental procedures. Diffuse large-B cell lymphoma derived cell line Toledo; human lymphoblastoid cell line GGB #7; SIRT1 inhibitors: nicotinamide (NAM) and Ex-527; cell cycle analysis; caspase-3 activity fluorimetric assay; genotoxicity assay gamma-H2AX; fluorescence-activated cell sorting intracellular staining for acetylated-H4K16 histone. New data. We found that the calculated IC50s for SIRT1 inhibition is 50mM for NAM and 343μM for Ex-527. The treatment with either NAM or Ex-527 leads to cell cycle arrest in both Toledo and GGB#7 cells, although in a different fashion. Namely, NAM causes an S-phase accumulation of Toledo while it leads GGB #7 to G0/G1 phase arrest with a concomitant decrease in S-phase already after 10mM. In both cell lines NAM 50mM induces apoptosis as demonstrated by caspase-3 activation and sub-G1 peak appearance in the cell cycle profiles. Ex-527 causes an accumulation in the S phase of Toledo cells and a decrease of G2/M in GGB#7 cells. No induction of apoptosis at the IC50 concentration was observed in both cell lines. Genotoxicity of the above mentioned treatments was investigated through the quantitation of gamma-H2AX histone released by treated cells. No genotoxic effects have been observed upon the treatments with these two inhibitors even at the concentration where NAM induces apoptosis. In order to correlate the observed changes with SIRT1 inhibition, we studied the acetylated H4K16 histone, a direct target of SIRT1 deacetylation. Our preliminary data indicate that NAM treatment increases the percentage of acetylated H4K16. Conclusions. Lymphoma and lymphoblastoid cells are affected by SIRT1 inhibition. NAM and Ex-527 cause cell cycle arrest and growth inhibition. Only NAM 50mM induces apoptosis without genotoxic effects. Overall, our data suggest the direct involvement of SIRT1 during the observed cell cycle arrest and growth inhibition mediated by these inhibitors. Citation Format: Manuela Guardi, Mariaelena Pistoni, Raffaele Frazzi. The effects of SIRT1 inhibitors nicotinamide and Ex-527 on lymphoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5. doi:10.1158/1538-7445.AM2017-5