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A small molecule Inauhzin inhibits SIRT1 activity and suppresses tumour growth through activation of p53.

Zhang Q, Zeng SX, Zhang Y, Zhang Y, Ding D, Ye Q, Meroueh SO, Lu H - EMBO Mol Med (2012)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology and Cancer Center, Tulane University School of Medicine, Louisiana, LA, USA.

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INZ induces acetylation of p53, but not tubulin, in cells, which is affected by knockdown of SIRT1A-D. Cells were treated with INZ, Etoposide or TSA as indicated. Total levels of p53 and acetylated p53 at lysine 382 were assessed by IB (70 µg of total proteins was used per lane; true to all panels in this figure).E. H460 cells were plated in 6-well plates 18 h prior to infection with SIRT1 shRNA or control GFP shRNA. To increase shRNA knockdown efficiency, cells were infected again 24 h later. At 24 h after second infection, cells were treated with Etoposide for 6 h followed by addition of INZ for 12 h.F-G. Cells infected with shGFP or shSIRT1 adenovirus in (E) were seeded at 3000 cells per well in 96-well culture plates and incubated overnight at 37C. Various concentrations of INZ (G) or INZ combined with 2 µM Etopside (F) were added into the plates. Cell growth inhibition was measured using WST cell growth assays. IC50 values are represented as mean ± standard deviation (n = 3). ** Indicates p < 0.01.
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fig05: INZ induces acetylation of p53, but not tubulin, in cells, which is affected by knockdown of SIRT1A-D. Cells were treated with INZ, Etoposide or TSA as indicated. Total levels of p53 and acetylated p53 at lysine 382 were assessed by IB (70 µg of total proteins was used per lane; true to all panels in this figure).E. H460 cells were plated in 6-well plates 18 h prior to infection with SIRT1 shRNA or control GFP shRNA. To increase shRNA knockdown efficiency, cells were infected again 24 h later. At 24 h after second infection, cells were treated with Etoposide for 6 h followed by addition of INZ for 12 h.F-G. Cells infected with shGFP or shSIRT1 adenovirus in (E) were seeded at 3000 cells per well in 96-well culture plates and incubated overnight at 37C. Various concentrations of INZ (G) or INZ combined with 2 µM Etopside (F) were added into the plates. Cell growth inhibition was measured using WST cell growth assays. IC50 values are represented as mean ± standard deviation (n = 3). ** Indicates p < 0.01.

Mentions: Previous studies have demonstrated that p53 is also modulated by reversible acetylation, which is inverse to ubiquitylation (Li et al, 2002) because the two post-translational modifications occur at similar lysine residues within p53. Hence, we tested whether INZ would influence p53 acetylation in cells. Indeed, at 2 µM it induced p53 acetylation at lysine 382 as detected by anti-acetylated K382 antibodies, which correlated well with the increment of p53 levels (Fig 5A) and more markedly than did Etoposide at 10 µM (Fig 5B and C). Interestingly, INZ induced acetylation of p53 in H460 cells, but not tubulin in constrast with trichostatin A (TSA), which induced acetylation of tubulin (Fig 5D) by inhibiting the activity of the HDAC family, such as HDAC1 and HDAC2 (Finnin et al, 1999).


A small molecule Inauhzin inhibits SIRT1 activity and suppresses tumour growth through activation of p53.

Zhang Q, Zeng SX, Zhang Y, Zhang Y, Ding D, Ye Q, Meroueh SO, Lu H - EMBO Mol Med (2012)

INZ induces acetylation of p53, but not tubulin, in cells, which is affected by knockdown of SIRT1A-D. Cells were treated with INZ, Etoposide or TSA as indicated. Total levels of p53 and acetylated p53 at lysine 382 were assessed by IB (70 µg of total proteins was used per lane; true to all panels in this figure).E. H460 cells were plated in 6-well plates 18 h prior to infection with SIRT1 shRNA or control GFP shRNA. To increase shRNA knockdown efficiency, cells were infected again 24 h later. At 24 h after second infection, cells were treated with Etoposide for 6 h followed by addition of INZ for 12 h.F-G. Cells infected with shGFP or shSIRT1 adenovirus in (E) were seeded at 3000 cells per well in 96-well culture plates and incubated overnight at 37C. Various concentrations of INZ (G) or INZ combined with 2 µM Etopside (F) were added into the plates. Cell growth inhibition was measured using WST cell growth assays. IC50 values are represented as mean ± standard deviation (n = 3). ** Indicates p < 0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376857&req=5

fig05: INZ induces acetylation of p53, but not tubulin, in cells, which is affected by knockdown of SIRT1A-D. Cells were treated with INZ, Etoposide or TSA as indicated. Total levels of p53 and acetylated p53 at lysine 382 were assessed by IB (70 µg of total proteins was used per lane; true to all panels in this figure).E. H460 cells were plated in 6-well plates 18 h prior to infection with SIRT1 shRNA or control GFP shRNA. To increase shRNA knockdown efficiency, cells were infected again 24 h later. At 24 h after second infection, cells were treated with Etoposide for 6 h followed by addition of INZ for 12 h.F-G. Cells infected with shGFP or shSIRT1 adenovirus in (E) were seeded at 3000 cells per well in 96-well culture plates and incubated overnight at 37C. Various concentrations of INZ (G) or INZ combined with 2 µM Etopside (F) were added into the plates. Cell growth inhibition was measured using WST cell growth assays. IC50 values are represented as mean ± standard deviation (n = 3). ** Indicates p < 0.01.
Mentions: Previous studies have demonstrated that p53 is also modulated by reversible acetylation, which is inverse to ubiquitylation (Li et al, 2002) because the two post-translational modifications occur at similar lysine residues within p53. Hence, we tested whether INZ would influence p53 acetylation in cells. Indeed, at 2 µM it induced p53 acetylation at lysine 382 as detected by anti-acetylated K382 antibodies, which correlated well with the increment of p53 levels (Fig 5A) and more markedly than did Etoposide at 10 µM (Fig 5B and C). Interestingly, INZ induced acetylation of p53 in H460 cells, but not tubulin in constrast with trichostatin A (TSA), which induced acetylation of tubulin (Fig 5D) by inhibiting the activity of the HDAC family, such as HDAC1 and HDAC2 (Finnin et al, 1999).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology and Cancer Center, Tulane University School of Medicine, Louisiana, LA, USA.

Show MeSH
Related in: MedlinePlus