SMAD3 phosphorylation inhibitor, tomentosenol A arrests cell cycle progression and induces apoptotic signalling in cultured fibroblasts: implications for anti-scarring activity

Hypertrophic scars are characterised by excessive fibroblast proliferation, myofibroblast activation, aberrant extracellular matrix deposition and reduced apoptosis. Current anti-scar therapies have variable efficacy and are limited by recurrence, tissue damage and pain, so new strategies are needed. In this study, we examined the efficacy of tomentosenol A, a meroterpenoid compound isolated from Australian stingless bee propolis, to intervene in the development of the cellular hallmarks of scar tissue. The effects of tomentosenol A were compared to two structural analogues (4S)- and (4R)-ficifolidione, using cultured HEK293 cells expressing a SMAD binding element/secreted embryonic alkaline phosphatase reporter and cultured normal adult human dermal fibroblasts. Cell cycle progression was assessed by CDK2 (Tyr15) and Histone H3 (Ser10) phosphorylation, apoptotic signalling by caspase-3/7 activity, and fibroblast wound closure by live-cell scratch-wound assay. The findings revealed that all compounds inhibited TGF-β1-stimulated fibroblast activation, as evidenced by prevention of α-smooth muscle actin assembly in cytoskeletal microfilaments, but only tomentosenol A prevented TGF-β1-stimulated SMAD signalling and fibronectin secretion. Tomentosenol A stimulated G1/S transition and arrested cells at G2/M; (4S)-ficifolidione was without effect on cell cycle progression while (4R)-ficifolidione arrested cells at G1/S. Only tomentosenol A stimulated caspase-3/7 activity, and this effect was attenuated by TGF-β1. Tomentosenol A also concentration-dependently slowed fibroblast wound closure. The findings establish tomentosenol A as a lead compound to test for anti-scarring activity. The structure in the vicinity of the free syncarpic acid conjugated with a pinene moiety appears critical to the anti-scarring potential of tomentosenol A.