Abstract 4058: Diverse patterns of biological response to phorbol esters and related protein kinaseC activators in LNCaP human prostate cancer cells

Abstract

All protein kinase C activators are not equivalent. Whereas phorbol 12-myristate 13-acetate (PMA) is the paradigmatic tumor promoter, bryostatin 1 or ingenol 3-angelate are in clinical trials as cancer chemotherapeutic agents. To better understand the structural features contributing to different biological outcomes, we tested a series of phorbol esters differing widely in hydrophobicity in two systems in which bryostatin 1 and PMA give very different responses. With U937 human leukemia cells, PMA inhibits cell growth and induces cell attachment, unlike bryostatin 1. We found that all the phorbol derivatives, like PMA, inhibited growth in a dose dependent manner. They likewise all induced attachment, a measure of differentiation, but several (sapintoxin D, phorbol 12, 13-dibenzoate, phorbol 12, 13-dihexanoate) differed in displaying a biphasic curve for this latter response. In the LNCaP cells, PMA inhibits growth and induces tumor necrosis factor alpha (TNF-alpha) secretion, whereas bryostatin 1 does not. We found that inhibition of growth in response to the phorbol esters ranged from full to partial (31-100 % of the PMA response) and the dose response curves ranged from monophasic to steeply biphasic. Similar divergent behavior was observed for induction of TNF-alpha secretion. For example, the secretion induced by phorbol 12, 13-dibenzoate and phorbol 12, 13-didecanoate was only 22% and 40% of the maximal response, respectively, and the response induced by sapintoxin D was very biphasic: 10 and 30 nM drug induced 56-59%, and 3000 and 10,000 nM induced only 8% and 5% of the maximal response. While none of the compounds induced as little response as did bryostatin 1, the results suggest that the difference was more quantitative in this system than qualitative. We conclude, moreover, that the hydrophobicity of the compounds was not the critical determinant of activity. Unlike the biological response, the translocation pattern of GFP-PKC delta did correlate with hydrophobicity, as the more lipophilic compounds PMA, octylindolactam V and phorbol 12, 13-didecanoate induced translocation to the plasma membrane, followed by translocation to internal membranes, while the more hydrophilic compounds translocated GFP-PKC delta mostly to the internal membranes. We thus conclude that the overall pattern of PKC delta translocation by itself cannot predict the different responses observed. Finally, Nano-pro technology, which can fingerprint the phosphorylation pattern of a protein, is revealing that the different phorbol esters induce divergent phosphorylation patterns of PKC delta, the isoform largely responsible for the phorbol ester induced apoptosis and TNF-alpha secretion in the LNCaP cells, and may provide a powerful tool for viewing the integrated outcome of the numerous elements impinging on this kinase following ligand interaction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4058. doi:10.1158/1538-7445.AM2011-4058