A small molecule Inauhzin inhibits SIRT1 activity and suppresses tumour growth through activation of p53.
Bottom Line: Here, we report a novel small molecule Inauhzin (INZ) that effectively reactivates p53 by inhibiting SIRT1 activity, promotes p53-dependent apoptosis of human cancer cells without causing apparently genotoxic stress.Moreover, INZ stabilizes p53 by increasing p53 acetylation and preventing MDM2-mediated ubiquitylation of p53 in cells, though not directly in vitro.Hence, our study unearths INZ as a novel anti-cancer therapeutic candidate that inhibits SIRT1 activity and activates p53.
Affiliation: Department of Biochemistry & Molecular Biology and Cancer Center, Tulane University School of Medicine, Louisiana, LA, USA.Show MeSH
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Mentions: To elucidate possible cellular mechanisms underlying the protection of p53 by INZ from proteolysis in cells, we tested if this compound causes general genotoxicity to cells by conducting in vitro non-sequence-specific DNA-binding, in vivo immunofluorescence staining for H2AX Ser139 phosphorylation (γH2AX) and cellular p53 phosphorylation assays. We found that INZ is not genotoxic. First, it was a considerably poor DNA-binding agent in comparison with ActD, as the former hardly bound to DNA at 2 µM (Fig 4A), a dose that markedly induced p53 (Figs 1 and 2), while the latter bound to 50% of DNA molecules even at 0.3 µM (Fig 4A). Also, even though 2 µM INZ effectively induced p53 levels in cells compared to 10 µM Cisplatin (Cis), it did not appear to cause significant γH2AX focus formation, which is often used as a marker for DNA damage (Paull et al, 2000). As shown in Fig 4B and C and Fig S4 of Supporting Information, more than 80% of H460 cells treated with 10 µM Cis or 2 mM hydroxyurea (HU) were detected with more than 10 foci per nucleus, whereas, only less than 1.5% of H460 cells treated with 2 µM INZ contained such a high level of foci and ∼75% of INZ-treated cells were basically free of foci. Furthermore, the level of p53 phosphorylation at either serine 15 or serine 46 in INZ-treated H460 cells was not observed compared to the cells treated with Cis or Etoposide for 18 h (Fig 4D and E). Phosphorylation of p53 at serine 15 or serine 46 was previously shown to be responsive to severe DNA damage (Banin et al, 1998; Oda et al, 2000; Shieh et al, 1997). Finally, INZ did not activate AMPK (Fig 4F), which was also reported to activate p53 by phosphorylating serines 15 and 46 (Jones et al, 2005). All together, these results exclude the possibility that INZ might activate a kinase cascade that mediates p53 phosphorylation by causing DNA damage or activating AMPK.
Affiliation: Department of Biochemistry & Molecular Biology and Cancer Center, Tulane University School of Medicine, Louisiana, LA, USA.