Mediation of Anti-Keloid Effects of mTOR Inhibitors by Autophagy-Independent Machinery

Abstract

Abstract Objective: Blocking mechanistic target of rapamycin (mTOR) activation with mTOR inhibitors has promising therapeutic potential for keloids. However, the precise mechanism of mTOR inhibitors remains unclear. This study was aimed to investigate the role of autophagy machinery in the anti-keloid effects of mTOR inhibitors. Methods: We first validated the biological effects induced by the mTOR inhibitors rapamycin (100 nmol/L) and KU-0063794 (5 μmol/L) on the proliferation, apoptosis, migration, and collagen synthesis of keloid fibroblasts (KFs) derived from Han Chinese persons through a Cell Counting Kit-8 assay, 5-Bromo-2’-deoxyuridine incorporation, Annexin V/propidium iodide staining, migration, and western blotting. To explore whether autophagy machinery is involved in the anti-keloid effects of mTOR inhibitors, we first blocked the autophagy activation induced by rapamycin and KU-0063794 with a pharmacological autophagy inhibitor (wortmannin) or by silencing the key autophagy gene (ATG5), and we then re-evaluated these biological effects on KFs. Results: Blocking mTOR activation with either rapamycin or KU-0063794 completely inhibited proliferation, migration, and collagen synthesis of primary KFs but did not affect apoptosis. Incubating KFs with the autophagy inhibitor wortmannin or performing ATG5 silencing abrogated the subsequent activation of autophagic activity induced by rapamycin (rapamycin + E-64d + pepstatin vs. rapamycin + wortmannin + E-64d + pepstatin: 1.88 ± 0.38 vs. 1.02 ± 0.35, F = 6.86, P = 0.013), (non-sense control + rapamycin vs. ATG5 small interfering RNA + rapamycin: 1.46 ± 0.18 vs. 0.75 ± 0.20, respectively; F = 7.68, P = 0.01) or KU-0063794 (KU-0063794 + E-64d + pepstatin vs. KU-0063794 + wortmannin + E-64d + pepstatin: 1.65 ± 0.35 vs. 0.76 ± 0.17, F = 10.01, P = 0.004), (NC + KU-0063794 vs. ATG5 small interfering RNA + KU-0063794: 1.59 ± 0.50 vs. 0.77 ± 0.09, F = 5.93, P = 0.02) as evidenced by decreased accumulation of LC3-II. However, blockage of autophagy induction in mTOR inhibitor-treated KFs with both methods did not disturb their anti-keloid effects, such as inhibition of cell viability, cell migration, and collagen synthesis (P > 0.05 each). Conclusion: Blocking mTOR activation with the mTOR inhibitors rapamycin and KU-0063794 showed anti-keloid effects in KFs. Restoration of autophagy inhibition by mTOR inhibitors does not contribute to their anti-keloid effects.