In vitro deacetylation assays

Assays were carried out using purified components in the Fluor de Lys Fluorescent Assay Systems. Relevant FdL substrates were used at 7 mM and NAD+ was used at 1 mM. Tenovin-6 was solubilized in DMSO with the final DMSO concentration in the reaction being less than 0.25%. For SirT1 and HDAC8, 1 unit of enzyme was used for each reaction, and for SirT2 and SirT3, 5 units were used for each reaction. Reactions were carried out at 37 ℃ for 1 hr.

Cell lines

CLL cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20 ℃ for several months.

Reaction Conditions

1, 5 or 10 μM; 8 or 24 hrs

Applications

After 24-hr culture, Tenovin-6 showed a significant dose-dependent cytotoxic effect to CLL cells. In CLL cells treated with Tenovin-6 (10 μM) for 8 hrs, the metabolic activity reduced substantially, which was similar to the effects of Fludarabine (3 μM).

Animal models

Female SCID mice bearing ARN8 melanomas

Dosage form

50 mg/kg/day; i.p.

Applications

In female SCID mice bearing ARN8 melanomas, Tenovin-6 significantly reduced tumor growth (day 6, p = 0.045; day 11, p = 0.0179; days 13 and 15, p = 0.0247).

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

Tenovin-6 is an analog of tenovin-1 with more water-soluble and it inhibits the protein deacetylase activities of SIRT1, SIRT2 and SIRT3 with IC50 value of 21 μM, 10 μM, and 67 μM, respectively [1].
It has been identified that　the cytotoxic effects of　tenovin-6 may occur through the dysregulation of autophagy rather　than induction of apoptosis in chronic lymphocytic leukemia cells. Sir2p homologs could be targets　for tenovin-6 in mammalian cells.
Tenovin-6 decreases purified human SirT1 peptide deacetylase activity in vitro with an IC50 of 21 mM and human SirT2 activity with an IC50 of 10 mM. Inhibition of SirT3 by tenovin-6 in this assay was significantly lower with an IC50 of 67 mM. And the activity of HDAC8 is poorly inhibited by tenovin-6 with an IC50 above the highest concentration of 90 mM. In xenograft models, tenovins induce apoptosis in malignant cell lines, containing those derived from lympho-reticular neoplasia and decrease human tumor growth. Anti-leukaemic properties of Sirtuin inhibitors have also been demonstrated in recent pre-clinical studies on Tenovin in chronic myeloid leukaemia and Nicotinamide in CLL15 associated with increased p53-pathway function [1, 2].
Tenovin-6 has potent antitumor activity against human gastric cancer cells via DR5 up-regulation. Interestingly, tenovin-6 induced apoptosis in the cell lines, those with wild-type TP53, mutant-type and null versions, which were accompanied by up regulation of death receptor 5. In the KatoIII cell line ( TP53-null), death receptor 5 silencing markedly attenuated tenovin-6-induced apoptosis, indicating that the pivotal mechanism behind its antitumor effects is based on activation of the death receptor signal pathway. Tenovin-6 combined with docetaxel or SN-38 exerted a slight to moderate synergistic cytotoxicity against gastric cancer cells [3].
References:
[1]. Lain S, Hollick JJ, Campbell J, et al. Discovery, in vivo activity, and mechanism of action of a small-molecule p53 activator. Cancer Cell, 2008, 13(5): 454-463.
[2]. MacCallum SF, Groves MJ, James J, et al. Dysregulation of autophagy in chronic lymphocytic leukemia with the small-molecule Sirtuin inhibitor Tenovin-6. Scientific Reports, 2013, 3: 1275.
[3]. Hirai S, Endo S, Saito R, et al. Antitumor Effects Of A Sirtuin Inhibitor, Tenovin-6, Against Gastric Cancer Cells Via Death Receptor 5 Up-Regulation. PLoS ONE, 2014, 9(7): e102831.