Evaluation of protein kinase C-activating effect of different type of Euphorbia diterpene phorbol esters in human platelets

Phorbol esters are the tetracyclic diterpenoids derived from Croton tiglium and from other plants of the family Euphorbiaceae. Phorbol esters, such as phorbol 12-myristate 13-acetate, are known as tumor promoters and many biological activities of the compounds are mediated through their direct activation of protein kinase C (PKC). On the other hand, some kinds of phorbol Euphorbia diterpene esters, such as ingenol 3-angelate and prostratin, can activate PKC without tumor-promoting activities. Moreover, ingenol 3-angelate is able to induce primary necrosis in dysplastic keratinocytes, an effect that may be mediated by activation of a non-classic type of PKC- PKCδ. Recently, ingenol 3-angelate has been approved for the topical treatment of actinic keratosis. Therefore, finding novel phorbol diterpene esters from natural sources seems to be an important task. Human platelets contain 5 isoforms of PKC, including α, β, δ, θ, and ζ, and activation of PKC results in platelet aggregation. Therefore, in the present study, platelets were used to evaluate PKC-activating activities of five phorbol diterpene esters, which were isolated from European Euphorbia species. We found that 20-deoxyingenol 3-angelate (1) and 12-deoxy-16-hydroxyphorbol-20-acetate-6-angelate-13-isobutyrate (2) induced significant platelet aggregation accompanied by induction of phosphorylation of PKC substrates in platelets. In contrast, two jatrophanes (3, 4), and a myrsinol-type diterpene (5) neither induced platelet aggregation nor PKC activation. PKD, a substrate of PKCδ, was phosphorylated in response to 1 and 2, indicating that these two compounds are able to activate PKCδ. Our results suggest that 1 and 2 are PKC activator, but their selectivity to different isoforms of PKC remains to be determined.