S150 Effects of putative senotherapies, fisetin and navitoclax, on senescent small airway fibroblasts in COPD

<h3>Background</h3> Chronic obstructive pulmonary disease (COPD) is associated with accelerated lung ageing and cellular senescence. Small airway fibroblasts (SAF) are senescent in COPD and may be implicated in small airways disease. Senescent cells are in a permanent state of cell cycle arrest with increased expression of cell cycle inhibitors p16^Ink4a and p21^Cip1. Senotherapeutics can remove or alter the phenotype of senescent cells. We hypothesised that two senotherapeutics, fisetin and navitoclax, could reduce the senescence markers, p16^Ink4a and p21^Cip1, associated with senescent SAF in COPD. <h3>Objective</h3> Investigate effects of fisetin and navitoclax on p16^Ink4a and p21^Cip1 expression in COPD SAF. <h3>Methods</h3> SAF were isolated from lung resection tissue of COPD patients (n=6) and non-smokers (n=8). Cells were cultured with fisetin (1–100 µM) and navitoclax (0.1–10 M). Cellular senescence was induced using 300 MH₂O₂. Expression of senescence markers, p16^Ink4a and p21^Cip1, was measured using western blots. Cell viability was assessed using MTT assays. <h3>Results</h3> p21^Cip1, but not p16^Ink4a, was significantly increased in COPD SAF compared to non-smoker SAF (p=0.02). Cell viability was decreased in COPD SAF treated with navitoclax by 60.2% (p=0.11, n=4). COPD SAF treated with 100 M navitoclax showed a concentration-dependent decrease in expression of p16^INK4 by 83.3% (p=0.13, n=4) and p21^Cip1 by 86.4% (p=0.13, n=4), which was not seen at any concentration of fisetin. Next, H₂O₂ was used to induce senescence further in COPD SAF. Treatment of COPD SAF with 300 M H₂O₂increased the expression of senescence marker p21^Cip1 by 101.5% (p=0.58, n=7). In this model, the highest concentrations of navitoclax, but not fisetin, significantly reduced cell viability in senescence-induced COPD SAF (by 83.7 ± 2.1%, p=0.038). Both fisetin and navitoclax significantly reduced expression of p16^Ink4a (by 77.1± 8.7%, p=0.024 and 99.8 ± 0.2%, p=0.0082, respectively) and p21^Cip1(by 88.6 ± 2.5%, p=0.024 and 95.4 ± 4.5%, p=0.018, respectively) in senescence-induced COPD SAF. Similar results were seen in senescence-induced non-smoker SAF. <h3>Conclusion</h3> Fisetin and navitoclax both reduce p16^Ink4a and p21^Cip1 expression in senescent COPD SAF. Further work is needed to investigate if these drugs alter other markers of senescence or induce apoptosis.