Inhibition of Polyamine Catabolism Reduces Cellular Senescence

Abstract

The aging of the global population has necessitated the identification of effective anti-aging technologies based on scientific evidence. Polyamines (putrescine, spermidine, and spermine) are essential for cell growth and function. Age-related reductions in polyamine levels have been shown to be associated with reduced cognitive and physical functions. We have previously found that the expression of spermine oxidase (SMOX) increases with age; however, the relationship between SMOX expression and cellular senescence remains unclear. Therefore, we investigated the relationship between increased SMOX expression and cellular senescence using human-liver-derived HepG2 cells. Intracellular spermine levels decreased and spermidine levels increased with the serial passaging of cells (aged cells), and aged cells showed increased expression of SMOX. The levels of acrolein-conjugated protein, which is produced during spermine degradation, also increases. Senescence-associated β-gal activity was increased in aged cells, and the increase was suppressed by MDL72527, an inhibitor of acetylpolyamine oxidase (AcPAO) and SMOX, both of which are enzymes that catalyze polyamine degradation. DNA damage accumulated in aged cells and MDL72527 reduced DNA damage. These results suggest that the SMOX-mediated degradation of spermine plays an important role in cellular senescence. Our results demonstrate that cellular senescence can be controlled by inhibiting spermine degradation using a polyamine-catabolizing enzyme inhibitor.

Keywords: acrolein; aging; polyamine metabolism; senescence; spermine oxidase.

Figure 1

Effect of aging and MDL72527 on polyamine levels. HepG2 cells cultured in the absence (open column) or presence (filled column) of 20 µM MDL72527 for 3 days (young) and 3 months with serial passage (aged). Polyamine levels were measured as described in the Materials and Methods section. Spermidine (A) and spermine (B) levels were calculated as nmol/mg protein and expressed as mean ± standard deviation (SD) of triplicate determinations. The dots indicate individual data points. * p < 0.05, *** p < 0.005.

Figure 2

Effect of aging on the protein levels of polyamine-metabolizing enzymes and senescence-related proteins. (A) Polyamine-metabolizing pathway. AdoMet; S-adenosylmethionine, dcAdoMet; decarboxylated S-adenosylmethionine, N¹-AcSPD; N¹-acetylspermidine, N¹-AcSPM; N¹-acetylspermine. (B) Cells were cultured in the absence (none) or presence (MDL) of 20 µM MDL72527 for 3 days (young) and serially passaged for 3 months (aged). Western blotting was performed as described in the Materials and Methods. The full-length blots are shown in Supplementary Figure S1. (C) Bands were quantified as described in Materials and Methods, normalized to actin and expressed as relative amount to untreated young cells. Data are presented as the mean + SD of triplicate determinations. * p < 0.05, *** p < 0.005.

Figure 3

Effect of aging and MDL72527 on senescence-associated β-gal activity. Cells were cultured in the absence (open column) or presence (filled column) of 20 µM MDL72527 for 3 days (young) or 3 months with serial passaging (aged) and seeded on a black 96-well plate. Senescence-associated β-gal activity was measured as described in the Materials and Methods. Data are presented as the mean ± SD of triplicate measurements. Dots indicate individual measurements. ns, not significant; *** p < 0.005.

Figure 4

Effect of aging and MDL72527 on DNA damage. (A) Cells were cultured in the absence (none) or presence (MDL) of 20 µM MDL72527 for 3 days (young) and serially passaged for 3 months (aged). Cells were seeded on coverslips, cultured for 1 d, and fixed. DNA damage was visualized as described in the Materials and Methods section. Red fluorescence indicates phospho-γH2AX, and blue fluorescence indicates nuclei. Bar = 20 μm. (B) Red fluorescence intensity in cells was measured as described in the Materials and Methods section and shown as mean + SD of at least 10 cells from three independent staining. Open columns and filled columns indicate none and MDL, respectively. ** p < 0.01, *** p < 0.005.