Dimitrijevic, Sasa M; (1995) Interaction of tigliane and daphnane diterpenoid esters with protein kinase C isozymes in vitro. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Different biological effects elicited by different phorbol esters in mammalian cells are thought to be due mainly to their differential interaction with, and modification of, the cellular pool of PKC isozymes. However, in most investigations only TPA, as the most potent tumor-promoting phorbol ester, has been used. Inspite of observations that other phorbol esters exert more specific biological effects in vivo, relatively few studies have been reported so far on their interaction with individual PKC isozymes in vitro or in vivo. In vivo interaction of different phorbol esters with PKC involves penetration of the phorbol ester molecule into the inner layer of the cellular membrane and binding to the regulatory domain of PKC followed by redistribution of the PKC pool from the cytosolic to the membrane bound fraction. It is thought that PKC redistribution ("translocation") is followed by an activation step and phosphorylation of substrate(s). Tigliane (TPA, PdBu, DOPP, DOPPA and Sap A) and daphnane (Thy A and Rx) diterpenoids of the phorbol ester group, were investigated for their ability to interact with purified recombinant protein kinase C (PKC) isozymes. Representative compounds of distinct biological activity were chosen in an attempt to establish a correlation between their in vivo effects and their ability to interact with individual PKC isozymes. Utilising PKC isozymes α, β1, β2, ỿ, δ, ε, and ζ purified from a baculovirus/Sf9 insect cell expression system and a phosphatidylserine / Triton X-100 mixed micellar system as an in vitro cell membrane model, binding and activation of PKC isozymes by seven different phorbol esters was studied. Binding affinity and activation potency of individual compounds were found to correlate well with high tumor promoting activity of TPA and PdBu on one side and with the non-promoting action of DOPPA and Rx. However, the non-promoters DOPP and Sap A and a second stage tumor promoter Thy A were effective agonists of PKC isozymes. To study the ability of some PEs to induce association of PKC isozymes with cellular membranes (i.e. "translocation"), a membrane fraction obtained from HL-60 cells was used, in order to approach in vivo conditions. Although the ability of the investigated PEs to induce "translocation" of PKC isozymes corresponded to their ability to induce PKC activation, the ability of micromolar Ca2+ concentrations to induce membrane association of n-PKCs, ε and δ, was not in agreement with our activation results and current theory of Ca2+ independency of the n-PKC isozymes. These results suggested that specific biological effects of different phorbol esters could not solely be explained through differences in their interaction with PKC isozymes in vitro. It is possible that an intracellular component, absent in an artificial system, is responsible for modulation of phorbol ester effects in vivo. Additionally a daphnane diterpene and a second stage tumor promoter mezerein, was isolated from previously uninvestigated Daphne blagayana, a plant indegenous to the Balkan. For the first time, detailed one and two dimensional NMR (1H, 13C, COSY and NOESY) experiments were conducted to confirm the previously determined structure of mezerein. Computer assisted molecular modelling and structure analysis enabled determination of molecular minimum free energy and interatomic distances of the pharmacophore's functional groups. These values were similar to those obtained for a highly potent tumor promoter TPA. As an activator of individual PKC isozymes in vitro, mezerein appeared to be different from TPA. Mezerein was relatively less potent (when compared with TPA) as an activator of the novel PKC isozymes δ and ε. This suggested that differences in biological activity of mezerein and TPA could be, in part, due to differences in their ability to activate the PKC isozymes δ and ε.

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