In vitro evaluation of senolytics targeting BCL2 family members to selectively deplete senescent primary human alveolar and small airway-derived epithelial cells

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and ultimately fatal lung disease. Aging is a significant risk factor for patients with IPF, making the understanding of aging mechanisms in the lung crucial for addressing the causes of IPF. One hallmark characteristic of aging is cellular senescence, which is observed to be higher in the fibrotic lung compared to age-matched healthy individuals. This study was designed to identify a suitable in vitro model for studying senescence in primary alveolar and small airway epithelial cells as well as lung-related epithelial cell lines. Initial testing of different profibrotic and senescence-inducing compounds on an epithelial lung cell line (A549) revealed that bleomycin treatment resulted in the most significant and robust increase in senescence-associated markers. These markers included gene expression of CDKN1A/CDKN2A, p21 protein expression, and beta-galactosidase staining. Following these results, bleomycin was also used in subsequent experiments involving primary cells. This study then assessed senolytic compounds targeting anti-apoptotic proteins of the BCL2 family for their potential to induce apoptosis specifically in senescent cells. Although apoptosis increased in a dose-dependent manner, no selectivity for senescent cells was observed. The compounds tested either had no effect or showed similar increases in cytotoxic/apoptotic signals for both healthy and senescent cells. Further research is required to identify alternative targets that can selectively eliminate senescent cells, potentially serving as alternative therapeutic targets.