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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 p53 level and activity as well as p53-dependent apoptosisA. Cells were treated with 2 µM INZ for the indicated time and harvested for IB. * Indicates residual signals of p53.B-C. H460 cells were treated with 2 µM INZ and harvested for real-time PCR. Values represent mean ± SD (n = 3).D-E. Induction of apoptosis by 2 µM INZ analysed by FACS. The apoptotic cells, identified by sub-G1 DNA content, were presented in the M1 population. Quantification of apoptosis of H1299 and H460 cells was shown in (E). The results shown are representative of three-independent experiments. Values represent mean ± SD (n = 3), **p < 0.01.F-G. INZ induces p53-dependent senescence. Senescence-associated β-galactosidase staining was performed in cultured cells for 6 days in the presence of 2 µM INZ or 10 µM Nutlin-3. β-galactosidase activity was measured by the absorbance of 5,5′-dibromo-4,4′-dichloro-indigo at 650 nm generated by the β-galactosidase staining. Values represent mean ± SD (n = 3), **p < 0.01. Representative photomicrographs of the cells by β-galactosidase staining were shown in (G).
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fig02: INZ induces p53 level and activity as well as p53-dependent apoptosisA. Cells were treated with 2 µM INZ for the indicated time and harvested for IB. * Indicates residual signals of p53.B-C. H460 cells were treated with 2 µM INZ and harvested for real-time PCR. Values represent mean ± SD (n = 3).D-E. Induction of apoptosis by 2 µM INZ analysed by FACS. The apoptotic cells, identified by sub-G1 DNA content, were presented in the M1 population. Quantification of apoptosis of H1299 and H460 cells was shown in (E). The results shown are representative of three-independent experiments. Values represent mean ± SD (n = 3), **p < 0.01.F-G. INZ induces p53-dependent senescence. Senescence-associated β-galactosidase staining was performed in cultured cells for 6 days in the presence of 2 µM INZ or 10 µM Nutlin-3. β-galactosidase activity was measured by the absorbance of 5,5′-dibromo-4,4′-dichloro-indigo at 650 nm generated by the β-galactosidase staining. Values represent mean ± SD (n = 3), **p < 0.01. Representative photomicrographs of the cells by β-galactosidase staining were shown in (G).

Mentions: To further characterize the effect of INZ on p53 cellular functions, we performed a set of time course experiments using the same aforementioned approaches with 2 µM INZ, as this concentration was sufficient to significantly induce p53 level and activity (Fig 1D). We found that INZ induced p53 level in a time-dependent manner as early as 6 h post-treatment in both p53-containing H460 and HCT116 cells (Fig 2A). Correspondingly, three of p53 targets, MDM2, p21 and Puma, were also induced in a time-dependent manner in p53-containing H460 and HCT116, but not in p53- H1299 and HCT116, cells (Fig 2A). Interestingly, Puma was induced earlier (3–6 h) and cleaved PARP was detected later on (∼12 h; Fig 2A). Apparently, cleaved PARP was p53-dependent as it was not detected in p53- cells (Fig 2A). The induction of p53 targets was clearly at the transcriptional level as p21 mRNA and miR34a, but not p53 mRNA and miR24, were highly induced in H460 cells (Fig 2B and C).


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 p53 level and activity as well as p53-dependent apoptosisA. Cells were treated with 2 µM INZ for the indicated time and harvested for IB. * Indicates residual signals of p53.B-C. H460 cells were treated with 2 µM INZ and harvested for real-time PCR. Values represent mean ± SD (n = 3).D-E. Induction of apoptosis by 2 µM INZ analysed by FACS. The apoptotic cells, identified by sub-G1 DNA content, were presented in the M1 population. Quantification of apoptosis of H1299 and H460 cells was shown in (E). The results shown are representative of three-independent experiments. Values represent mean ± SD (n = 3), **p < 0.01.F-G. INZ induces p53-dependent senescence. Senescence-associated β-galactosidase staining was performed in cultured cells for 6 days in the presence of 2 µM INZ or 10 µM Nutlin-3. β-galactosidase activity was measured by the absorbance of 5,5′-dibromo-4,4′-dichloro-indigo at 650 nm generated by the β-galactosidase staining. Values represent mean ± SD (n = 3), **p < 0.01. Representative photomicrographs of the cells by β-galactosidase staining were shown in (G).
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fig02: INZ induces p53 level and activity as well as p53-dependent apoptosisA. Cells were treated with 2 µM INZ for the indicated time and harvested for IB. * Indicates residual signals of p53.B-C. H460 cells were treated with 2 µM INZ and harvested for real-time PCR. Values represent mean ± SD (n = 3).D-E. Induction of apoptosis by 2 µM INZ analysed by FACS. The apoptotic cells, identified by sub-G1 DNA content, were presented in the M1 population. Quantification of apoptosis of H1299 and H460 cells was shown in (E). The results shown are representative of three-independent experiments. Values represent mean ± SD (n = 3), **p < 0.01.F-G. INZ induces p53-dependent senescence. Senescence-associated β-galactosidase staining was performed in cultured cells for 6 days in the presence of 2 µM INZ or 10 µM Nutlin-3. β-galactosidase activity was measured by the absorbance of 5,5′-dibromo-4,4′-dichloro-indigo at 650 nm generated by the β-galactosidase staining. Values represent mean ± SD (n = 3), **p < 0.01. Representative photomicrographs of the cells by β-galactosidase staining were shown in (G).
Mentions: To further characterize the effect of INZ on p53 cellular functions, we performed a set of time course experiments using the same aforementioned approaches with 2 µM INZ, as this concentration was sufficient to significantly induce p53 level and activity (Fig 1D). We found that INZ induced p53 level in a time-dependent manner as early as 6 h post-treatment in both p53-containing H460 and HCT116 cells (Fig 2A). Correspondingly, three of p53 targets, MDM2, p21 and Puma, were also induced in a time-dependent manner in p53-containing H460 and HCT116, but not in p53- H1299 and HCT116, cells (Fig 2A). Interestingly, Puma was induced earlier (3–6 h) and cleaved PARP was detected later on (∼12 h; Fig 2A). Apparently, cleaved PARP was p53-dependent as it was not detected in p53- cells (Fig 2A). The induction of p53 targets was clearly at the transcriptional level as p21 mRNA and miR34a, but not p53 mRNA and miR24, were highly induced in H460 cells (Fig 2B and C).

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