Novel Epoxy-Tiglianes Stimulate Skin Keratinocyte Wound Healing Responses and Re-Epithelialization Via Protein Kinase C Activation

Epoxy-tiglianes are a novel class of diterpene esters. The prototype epoxy-tigliane, EBC-46 (tigilanol tiglate), possesses potent anti-cancer properties and is currently in clinical development as a local treatment for human and veterinary cutaneous tumors. EBC-46 rapidly destroys treated tumors and consistently promotes wound reepithelialization at sites of tumor destruction. However, the mechanisms underlying these keratinocyte wound healing responses are not completely understood. Here, we investigated the effects of EBC-46 and an analogue (EBC-211) at 1.51 nM-151 mu M concentrations, on wound healing responses in immortalized human skin keratinocytes (HaCaTs). Both EBC-46 and EBC-211 (1.51 nM-15.1 mu M) accelerated G0/G1-S and S-G2/M cell cycle transitions and HaCaT proliferation. EBC-46 (1.51-151 nM) and EBC-211 (1.51 nM-15.1 mu M) further induced significant HaCaT migration and scratch wound repopulation. Stimulated migration/wound repopulation responses were even induced by EBC-46 (1.51 nM) and EBC-211 (1.51-151 nM) with proliferation inhibitor, mitomycin C (1 mu M), suggesting that epoxy-tiglianes can promote migration and wound repopulation independently of proliferation. Expression profiling analyses showed that epoxy-tiglianes modulated keratin, DNA synthesis/replication, cell cycle/proliferation, motility/migration, differentiation, matrix metalloproteinase (MMP) and cytokine/chemokine gene expression, to facilitate enhanced responses. Although epoxy-tiglianes down-regulated established cytokine and chemokine agonists of keratinocyte proliferation and migration, enhanced HaCaT responses were demonstrated to be mediated via protein kinase C (PKC) phosphorylation and significantly abrogated by pan-PKC inhibitor, bisindolylmaleimide-1 (BIM-1, 1 mu M). By identifying how epoxytiglianes stimulate keratinocyte healing responses and re-epithelialization in treated skin, our findings support the further development of this class of small molecules as potential therapeutics for other clinical situations associated with impaired re-epithelialization, such as non-healing skin wounds.