Immune Netw.  2016 Jun;16(3):183-188. 10.4110/in.2016.16.3.183.

Induction of Nuclear Enlargement and Senescence by Sirtuin Inhibitors in Glioblastoma Cells

• Yoon KB
• Park KR
• Kim SY
• Han SY

• KMID: 2299792
• DOI: http://doi.org/10.4110/in.2016.16.3.183

Abstract

Sirtuin family members with lysine deacetylase activity are known to play an important role in anti-aging and longevity. Cellular senescence is one of the hallmarks of aging, and downregulation of sirtuin is reported to induce premature senescence. In this study, we investigated the effects of small-molecule sirtuin inhibitors on cellular senescence. Various small molecules such as tenovin-1 and EX527 were employed for direct sirtuin activity inhibition. U251, SNB-75, and U87MG glioblastoma cells treated with sirtuin inhibitors exhibited phenotypes with nuclear enlargement. Furthermore, treatment of rat primary astrocytes with tenovin-1 also increased the size of the nucleus. The activity of senescence-associated β-galactosidase, a marker of cellular senescence, was induced by tenovin-1 and EX527 treatment in U87MG glioblastoma cells. Consistent with the senescent phenotype, treatment with tenovin-1 increased p53 expression in U87MG cells. This study demonstrated the senescence-inducing effect of sirtuin inhibitors, which are potentially useful tools for senescence research.

Keyword

Sirtuin; Senescence; Tenovin-1; EX527

MeSH Terms

Aging*
Animals
Astrocytes
Cell Aging
Down-Regulation
Glioblastoma*
Humans
Longevity
Lysine
Phenotype
Rats
Lysine

Figure

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  Figure 1 Effect of sirtuin inhibitors on nuclear size in glioblastoma cells. U251 and SNB-75 glioblastoma cells were treated with DMSO (control), 5 µM tenovin-1, or 5 µM EX527 for 48 h, as indicated. The cells were stained with DAPI and the size of the nucleus was measured as described in the Materials and Methods section.

• 

  Figure 2 Effect of sirtuin inhibitors on nuclear size in rat primary astrocytes. (A) Rat primary astrocytes were treated with DMSO (control) and 5 µM tenovin-1 for 48 h, as indicated. The cells were stained with DAPI and the number of cells with a nucleus larger than 12.8 µm (the average nuclear size in the DMSO-treated group) was determined (** indicates a p-value<0.01). (B) Rat primary astrocytes were treated with DMSO (control), 0.1 µg/mL LPS, 5 µM tenovin-1, or LPS and 5 µM tenovin-1 for 48 h. The cells were stained with DAPI and the number of cells with a nucleus larger than 12.8 µm was determined (** indicates a p-value < 0.01).

• 

  Figure 3 Effect of sirtuin inhibitors on the senescence markers. (A) U87MG glioblastoma cells and (B) SF295 cells were treated with DMSO (control), 5 µM tenovin-1, and 5 µM EX527 for 24 h. Senescence-associated β-galactosidase (SA-β-gal) activity was then measured according to the manufacturer's protocol. (C) U87MG cells were treated with tenovin-1 with the indicated concentration. Cell lysates were then subjected to western blotting using antibodies against acetylated tubulin (Ac-tubulin), p-53, and β-actin.

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