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

Background Chronic obstructive pulmonary disease (COPD) is associated with accelerated lung ageing and cellular senescence(definition). 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 p16Ink4a and p21Cip1. Senotherapeutics can remove or alter the phenotype of senescent cells. We hypothesised that two senotherapeutics, fisetin and navitoclax, could reduce the senescence markers, p16Ink4a and p21Cip1, associated with senescent SAF in COPD. Objective Investigate effects of fisetin and navitoclax on p16Ink4a and p21Cip1 expression in COPD SAF. Methods 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, p16Ink4a and p21Cip1, was measured using western blots. Cell viability was assessed using MTT assays. Results p21Cip1, but not p16Ink4a, 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 p16INK4 by 83.3% (p=0.13, n=4) and p21Cip1 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 p21Cip1 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 p16Ink4a (by 77.1± 8.7%, p=0.024 and 99.8 ± 0.2%, p=0.0082, respectively) and p21Cip1(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. Conclusion Fisetin and navitoclax both reduce p16Ink4a and p21Cip1 expression in senescent COPD SAF. Further work is needed to investigate if these drugs alter other markers of senescence or induce apoptosis.