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 (alpha and beta I), novel (delta) and atypical (zeta) 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 PKC alpha (IC(50) = 1190 +/- 237 nM; n = 20), PKC beta I (IC(50) = 908 +/- 46 nM; n = 20), and PKC delta (IC(50) = 141 +/- 25 nM; n = 20) but not of yeast expressing PKC zeta. Daphnetoxin also inhibited the growth of yeast expressing isoforms alpha, beta I and delta, being more potent than mezerein on PKC alpha (IC(50) = 536 +/- 183 nM; n = 20; P < 0.05), as potent as mezerein on PKC beta I (IC(50) = 902 +/- 129 nM; n = 20) and less potent than mezerein upon PKC delta (IC(50) = 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 PKC delta.

Differential Activation of Protein Kinase C Isoforms by Euxanthone, Revealed by anIn VivoYeast Phenotypic Assay

“…Drugs were dissolved in DMSO and the final concentration in the yeast cultures never exceeded 0.1 % (v/v). Yeast transformation was performed as described for PKC-a, -bI, -d and -z [6]. YEplac181, encoding the cDNA for the mouse PKC-h (kindly offered by Dr. Nigel Goode, Royal Veterinary College, London, UK) was also used.…”

“…YEplac181, encoding the cDNA for the mouse PKC-h (kindly offered by Dr. Nigel Goode, Royal Veterinary College, London, UK) was also used. Cell lysis, immunoblotting and the yeast phenotypic assay was carried out basically as described previously [6].…”

“…The yeast phenotypic assay is an alternative in vivo assay based on the growth inhibition (reflecting an increase in the cell doubling time) of transformed yeast cells, which is proportional to the degree of activation of the mammalian PKC isoform expressed [6]. Because only a mammalian PKC isoform is functionally expressed separately and effects not mediated by this protein can easily be detected by measuring growth in the absence of isoform expression, it is a convenient method to elucidate the selectivity of potential PKC activators.…”

An Update on the Other Telomerase Inhibitors: Non-G-Quadruplex Interactive Agent, Non-Antisense, Non-Reverse Transcriptase Telomerase Inhibitors

“…As was seen in the glioma cells, no loss in telomerase activity was seen upon cellular differentiation [55]. Mezerein acts indirectly on telomerase activity as a protein kinase C activator [59](see below). …”

New insights into cancer‐related proteins provided by the yeast model

“…The yeast PKC phenotypic assay was also extensively used in the characterization of the potency and selectivity of known PKC activators [73] and inhibitors [74,75], as well as in the screening for new potent and selective PKC pharmacological modulators of individual mammalian PKC isoforms [74,[76][77][78]. More recently, using yeast cells expressing PKCa, bI, d, e or f, a new potent and selective activator of nPKCd and e was discovered [79].…”