Abstract 3195: Transient duration of action is the primary mechanism responsible for the unique biology of bryostatin 1

Abstract

Abstract Bryostatin 1 (bryo 1) is a natural compound tested in several clinical trials as an anticancer agent and in preclinical studies for treatment of Alzheimer disease. Despite the clinical interest, the mechanisms responsible for its unique behavior are not fully understood. Bryo 1 is a protein kinase C activator that paradoxically antagonizes many but not all phorbol ester responses. To obtain a broader view of the differences in response to bryo 1 and to the phorbol ester PMA, we have compared their effects on global RNA expression as well as on the levels of important regulatory proteins in two cellular systems (LNCaP and U937 cell lines) where bryo 1 has different biology than PMA. In LNCaP cells, where bryo 1 does not induce secretion of TNF alpha or cause apoptosis, bryo 1 fails to induce translocation of PKC alpha, delta, epsilon and pPKD1 to the nuclear enriched fraction or the phosphorylation of PKCdelta at Y311. For many other early responses detected at 60 min (activation of MEK/ERK pathway and of several transcription factors including NFKB, AP1, EGR1) bryo 1 acts similarly to PMA. In contrast, bryo 1 fails to induce the late responses detected at 6 hrs (phosphorylation of p65, translocation of cRel and RelB) and induces early termination of the induced responses both at the protein and RNA levels as detected for a selected panel of genes. The early termination of the induced responses by bryo 1 can be detected as well in U937 cells, where bryo 1 fails to induce differentiation and apoptosis, unlike PMA. Microarray analysis of LNCaP and U937 cells treated for 6 and 8 hrs, respectively, revealed significant, extensive changes in gene expression compared to control but did not identify genes uniquely responsive to bryo 1. Typically, the effect of bryo 1 was smaller than that of PMA. qPCR analysis of about 3 dozen genes spanning the breadth of the microarray response patterns indicated a single mechanism - genes were similarly activated by PMA and bryo 1 early on but the effect of bryo 1 was transient. Further microarray analysis was performed on LNCaP and U937 samples treated for 60 min and 6 hrs to address the following questions: 1) Are there genes that are induced selectively by bryo 1 at 1 hr? 2) Does transient response to bryo 1, which was only shown for selected genes by qPCR but was inferred to explain the extensive reduced expression at 6 hr in response to bryo 1 compared to PMA, actually explain the reduced expression? While some genes are selectively induced by bryo 1 treatment, the altered levels of expression are limited, suggesting that it does not reflect a unique site of action. Looking at genes showing a reduced level of expression at 6 hr in response to bryo 1 compared to PMA, we confirm that transiency of action is the primary mechanism. These insights provide a guide for evaluation of second generation bryostatin analogs and a focus on the biochemical basis for the transiency of bryo 1 effect. Citation Format: Noemi Kedei, Aleksandra M. Michalowski, Peter M. Blumberg. Transient duration of action is the primary mechanism responsible for the unique biology of bryostatin 1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3195. doi:10.1158/1538-7445.AM2014-3195