Date of Award
Master of Science
Dallas Donohoe, Michael McEntee
Zyflamend, a select blend of 10 herbal extracts, effectively inhibits tumor growth using preclinical models of castrate-resistant prostate cancer (CR-PCa), mediated in part by activating AMPK, a master energy sensor of the cell. Currently, two predominant upstream kinases are known to phosphorylate/activate AMPK at Thr172: LKB1, a known tumor suppressor, and CaMKK2, a tumor promotor over-expressed in a number of cancers. Thus, the overall objective was to interrogate how Zyflamend activates AMPK and determine the roles of LKB1 and CaMKK2 in this activation, by targeting upstream mediators of both kinases ± Zyflamend using pharmacologic and molecular techniques in CWR22Rv1 (CR-PCa cell line) and HeLa (LKB1-null cell line) cells. Zyflamend-mediated activation of AMPK appears to be LKB1 dependent, while coordinately and negatively regulating CaMKK2 activity. Zyflamend failed to rescue the activation of AMPK in the presence of chemical and molecular inhibitors of LKB1 (radicicol & siLKB1), an effect not observed in the presence of inhibitors of CaMKK2 (STO-609, EGTA) in CWR22Rv1 cells. Using LKB1-null and catalytically dead LKB1-transfected HeLa cells that constitutively express CaMKK2, ionomycin (activator of CaMKK2) increased phosphorylation of AMPK, but Zyflamend had no effect. Zyflamend appears to inhibit CaMKK2 by DAPK-mediated phosphorylation at Ser511, an effect prevented by a DAPK inhibitor. Alternatively, Zyflamend increased pAMPK only in HeLa cells transfected with wild type LKB1. Zyflamend increased phosphorylation of PKCζ [Zeta], a known activator of LKB1, and inhibition of PKCζ reduced LKB1 phosphorylation. Using various constructs of HeLa cells, nuclear and cytosolic localization of LKB1 and PKCζ, respectively, were reversed following Zyflamend treatment, consistent with phosphorylation of AMPK by LKB1 in the cytosol. These results suggest that Zyflamend’s activation of AMPK is mediated by LKB1, possibly via activation of PKCζ, but independent of CaMKK2. Moreover, Zyflamend inhibits CaMKK2 activity by a mechanism involving DAPK phosphorylation of CaMKK2 at Ser511.
MacDonald, Amber Frances, "AMPK Activation by Zyflamend: A novel pathway regulating metabolism and growth in prostate cancer. " Master's Thesis, University of Tennessee, 2017.