In vitro activity: Salermide is a potent inhibitor of Sirt1 (Sirtuin 1) and Sirt2 (Sirtuin 2). It can cause strong cancer-specific apoptotic cell death. Sirt1 and Sirt2 belong to the family of NAD+ (nicotinamide adenine dinucleotide-positive)-dependent class III histone deacetylases and are involved in regulating lifespan. As cancer is a disease of ageing, targeting Sirtuins is emerging as a promising antitumour strategy. Salermide is a reverse amide with a strong in vitro inhibitory effect on Sirt1 and Sirt2. Salermide was well tolerated by mice at concentrations up to 100 muM and prompted tumour-specific cell death in a wide range of human cancer cell lines. The antitumour activity of Salermide was primarily due to a massive induction of apoptosis. This was independent of global tubulin and K16H4 acetylation, which ruled out a putative Sirt2-mediated apoptotic pathway and suggested an in vivo mechanism of action through Sirt1. Consistently with this, RNA interference-mediated knockdown of Sirt1, but not Sirt2, induced apoptosis in cancer cells. Although p53 has been reported to be a target of Sirt1, genetic p53 knockdowns showed that the Sirt1-dependent proapoptotic effect of Salermide is p53-independent. Taken together,these results suggest Salermide's promise as an anticancer drug and provide evidence for the molecular mechanism through which Sirt1 is involved in human tumorigenesis.

Kinase Assay: Recombinant His-tagged human Sirt1 and Sirt2 were assayed for deacetylase activity using the HDAC fluorescent activity assay (BIOMOL, Plymouth, PA, USA). Reactions were carried out at 37 °C for 60 min. Results are expressed as the mean and standard deviation of four independent experiments.

Cell Assay: Cell lines (SW480, MDA-MB-231, MOLT4, KG1A, K562 and Raji) were obtained from the American Type Culture Collection (VA, USA). Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described earlier. IC50 index was calculated using four Salermide concentrations (25, 50, 75 and 100 μM) for 24 h. The percentage of apoptotic cells was determined with the FACSCalibur (BD, Heidelberg, Germany) apparatus The Annexin V FACS analysis was carried out according to the manufacturer's protocol (Annexin V-FITC, BD-Pharmingen, San Diego, CA, USA).