Abstract
Ingenol-3–angelate (I3A) is a non-tumor promoting phorbol ester-like compound identified in the sap of Euphoria peplus. Similar to tumor promoting phorbol esters, I3A is a diacylglycerol (DAG) analogue that binds with high affinity to the C1 domains of PKCs, recruits PKCs to cellular membranes and promotes enzyme activation. Numerous anti-cancer activities have been attributed to I3A and ascribed to I3A’s effects on PKCs. We show here that I3A also binds to and activates members of the RasGRP family of Ras activators leading to robust elevation of Ras-GTP and engagement of the Raf-Mek-Erk kinase cascade. In response to I3A, recombinant proteins consisting of GFP fused separately to full-length RasGRP1 and RasGRP3 were rapidly recruited to cell membranes, consistent with direct binding of the compound to RasGRP’s C1 domain. In the case of RasGRP3, IA3 treatment led to positive regulatory phosphorylation on T133 and activation of the candidate regulatory kinase PKCδ. I3A treatment of select B non-Hodgkin’s lymphoma cell lines resulted in quantitative and qualitative changes in Bcl-2 family member proteins and induction of apoptosis, as previously demonstrated with the DAG analogue bryostatin 1 and its synthetic analogue pico. Our results offer further insights into the anticancer properties of I3A, support the idea that RasGRPs represent potential cancer therapeutic targets along with PKC, and expand the known range of ligands for RasGRP regulation.
Highlights
Diacylglycerol (DAG) is a potent second messenger that is generated in cells in response to membrane receptor stimulation of phospholipid metabolism
Compared to PKCa, RasGRP1 and RasGRP3 bound I3A with approximately similar affinities, indicating that there was no selectivity between these two families of targets, at least in vitro
I3A Activates RasGRPs in Select B-non-Hodgkin’s lymphoma (B-NHL) Cell Lines We previously showed that DAG analogues such as bryostatin 1 and the synthetic analogue pico could activate RasGRPs in BNHL cell lines that have been characterized as sensitive to induction of apoptosis by these same compounds [25,26]
Summary
Diacylglycerol (DAG) is a potent second messenger that is generated in cells in response to membrane receptor stimulation of phospholipid metabolism. DAG and DAG analogues such as PMA (phorbol 12-myristate 13-acetate) bind conventional and novel forms of protein kinase C (PKC) through a conserved domain called C1. This process contributes to PKC membrane localization and enzyme activation. Prolonged exposure to DAG analogues can negatively impact PKC activity through induced enzyme degradation. Some DAG analogues, such as PMA, are potent tumor promoters. Other DAG analogues, such as prostratin and bryostatin 1, are non-tumor promoters or may inhibit tumor promotion. Medicinal DAG analogues such as bryostatin 1 exert a variety of anti-cancer cell and immune modulatory effects.
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