Benzylguanine Induces Cellular Senescence and Decreases Relaxation of Vascular Smooth Muscle in the Rat Aorta

Cellular senescence (CS) is defined as a condition of permanent cessation of growth, accompanied by distinct morphological and physiological alterations. This phenomenon is closely associated with DNA damage. Recently, our department identified that inhibition of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) by O-6-benzylguanine (O6-BG) induces CS in vascular smooth muscle cells (VSMCs). Consequently, we have resolved to validate these findings in an animal model. The objective of this study was to examine the impact of O6-BG on CS in vascular smooth muscle and to assess its influence on the relaxation response.

Wistar rats were administered O6-BG by gavage with a dose of 12 mg/kg (two doses every 24 hours). After 12 weeks, animals were sacrificed, the aorta was removed and cleaned, and samples were prepared for Western blotting. Small aortic rings were the isolated for vascular response measurement.

Senescent markers p27Kip1 and p21 Cipl were significantly upregulated after O6- BG treatment, which confirms that MGMT inhibition leads to CS. We did not observe any statistically significant changes in the relative expression of the proteins p53 or ERK1/2. This means that the expression of cyclin-dependent kinase inhibitors is regulated independently of the cell cycle regulator p53 and the ERK1/2 branch of the MAPK signaling pathway. Furthermore, we observed significantly decreased relaxation response of aortic smooth muscle to sodium nitroprusside (SNP), which confirms known adverse effects of CS on the vascular system.

To conclude, we found that treatment with O6-BG triggered an increase in the relative expression of p27, p21, and PCNA compared to healthy aortic tissues, acting as mediators and markers of senescence. Our findings provide new insights into the signaling of CS in a model of MGMT inhibition.