Differential Activation by Daphnetoxin and Mezerein of PKC-Isotypes α, βI, δ and ζ

Abstract

Daphnetoxin, a mezerein derivative, was isolated from the stem bark of Daphne gnidium. Mezerein is a PKC activator that exhibits antileukemic properties. However, daphnetoxin and its analogue 12-hydroxydaphnetoxin were described as being devoid of this effect. In the present study daphnetoxin and mezerein were compared as PKC activators on classical (α and βI), novel (δ) and atypical (ζ) isoforms, using an alternative in vivo yeast phenotypic assay. The aim was to clarify if daphnetoxin is a PKC activator and if the differences between the antiproliferative effect of mezerein and of its analogue daphnetoxin may be ascribed to differences on their potency or selectivity as PKC activators. Yeast samples expressing each of the mammalian PKC isoforms tested were incubated with daphnetoxin or mezerein. Growth inhibition caused by these drugs was assumed to be due to PKC activation since it did not occur when expression was not induced. Mezerein inhibited the growth of yeast expressing PKCα (IC₅₀ = 1190 ± 237 nM; n = 20), PKCβI (IC₅₀ = 908 ± 46 nM; n = 20), and PKCδ (IC₅₀ = 141 ± 25 nM; n = 20) but not of yeast expressing PKCζ. Daphnetoxin also inhibited the growth of yeast expressing isoforms α, βI and δ, being more potent than mezerein on PKCα (IC₅₀ = 536 ± 183 nM; n = 20; P < 0.05), as potent as mezerein on PKCβI (IC₅₀ = 902 ± 129 nM; n = 20) and less potent than mezerein upon PKCδ (IC₅₀ = 3370 ± 492 nM; n = 20; P < 0.05). These results show that daphnetoxin is a potent PKC activator but with a selectivity different from that of mezerein. It is suggested that the lack of antileukemic and antiproliferative effects of daphnetoxin may be due to its lower potency to activate PKCδ.

Key words

Daphne gnidium L. - Thymelaeaceae - daphnetoxin - mezerein - PKC isoforms - yeast phenotypic assay

References

• 1 Felhauer M, Hecker E. Screening of Thymelaeaceae species for irritant, cocarcinogenic and antineoplastic activity.  Planta Medica. 1986;  52 553-4
• 2 Kupchan S M, Baxter R L. Mezerein: antileukemic principle isolated from Daphne mezereum L.  Science. 1975;  187 652-3
• 3 Jeffrey A M, Liskamp R MJ. Computer-assisted molecular modeling of tumor promoters: rationale for the activity of phorbol esters, teleocidin B, and aplysiatoxin.  Proceedings of the National Academy of Sciences USA. 1986;  83 241-5
• 4 Slaga T J, Fischer S M, Nelson K, Gleason G L. Studies on the mechanism of skin tumor promotion: evidence for several stages in promotion.  Proceedings of the National Academy of Sciences USA. 1980;  77 3659-63
• 5 Miyake R, Tanaka Y, Tsuda T, Kaibushi K, Kikkawa U, Nishizuka Y. Activation of protein kinase C by non-phorbol tumor promoter, mezerein.  Biochemical and Biophysical Research Communications. 1984;  121 649-56
• 6 Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C.  Science. 1992;  258 607-14
• 7 Blumberg P M. Complexities of the protein kinase C pathway.  Molecular Carcinogenesis. 1991;  4 339-44
• 8 Ronlán A, Wickberg B. The structure of mezerein, a major toxic principle of Daphne mezereum L.  Tetrahedron Letters. 1970;  49 4261-4
• 9 Ulubelen A, Terem B, Tuzlaci E. Coumarins and flavonoids from Daphne gnidioides .  Journal of Natural Products. 1986;  49 692-4
• 10 Shieh H L, Hansen H, Zhu J, Riedel H. Differential protein kinase C ligand regulation detected in vivo by a phenotypic yeast assay.  Molecular Carcinogenesis. 1995;  12 166-76
• 11 Stout G H, Balkenhol W G, Poling M, Hickernell G L. The isolation and structure of daphnetoxin, the poisonous principle of Daphne species.  Journal of the American Chemical Society. 1970;  92 1070-1
• 12 Ito H, Yasuki F, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations.  Journal of Bacteriology. 1983;  153 163-8
• 13 Gailly P, Gong M C, Somlyo A V, Somlyo A P. Possible role of atypical protein kinase C activated by arachidonic acid in Ca²⁺ sensitization of rabbit smooth muscle.  Journal of Physiology. 1997;  500 95-109
• 14 Adolf W, Dossajt S F, Seip E H, Hecker E. Skin irritant diterpene orthoesters of daphnane type from Peddiea africana and P. volkensii .  Phytochemistry. 1985;  24 2047-9
• 15 Nyborg J, La Cour T. X-ray diffraction study of molecular structure and conformation of mezerein.  Nature. 1975;  257 824-5
• 16 Kupchan S M, Sweeny J G, Baxter R L, Murae T, Zimmerly V A, Sickles BR. Gnididin, gniditrin, and gnidicin, novel potent antileukemic diterpenoid esters from Gnidia lamprantha .  Journal of the American Chemical Society. 1975;  97 672-3
• 17 Geiges D, Meyer T, Marte B, Vanek M, Weissgerber G, Stabel S. et al . Activation of protein kinase C subtypes α, γ, δ, ε, ζ and η by tumor-promoting and nontumor-promoting agents.  Biochemical Pharmacology. 1997;  53 865-75
• 18 Fujii T, García-Bermejo M L, Bernabó J L, Caamaño J, Ohba M, Kuroki T. et al . Involvement of protein kinase Cδ (PKCδ) in phorbol ester-induced apoptosis in LNCaP prostate cancer cells.  The Journal of Biological Chemistry. 2000;  275 7574-82