Concurrent regulation of LKB1 and CaMKK2 in the activation of AMPK in castrate-resistant prostate cancer by a well-defined polyherbal mixture with anticancer properties

Abstract

BackgroundZyflamend, a blend of herbal extracts, effectively inhibits tumor growth using preclinical models of castrate-resistant prostate cancer mediated in part by 5′-adenosine monophosphate-activated protein kinase (AMPK), a master energy sensor of the cell. Clinically, treatment with Zyflamend and/or metformin (activators of AMPK) had benefits in castrate-resistant prostate cancer patients who no longer responded to treatment. Two predominant upstream kinases are known to activate AMPK: liver kinase B1 (LKB1), a tumor suppressor, and calcium-calmodulin kinase kinase-2 (CaMKK2), a tumor promotor over-expressed in many cancers. The objective was to interrogate how Zyflamend activates AMPK by determining the roles of LKB1 and CaMKK2.MethodsAMPK activation was determined in CWR22Rv1 cells treated with a variety of inhibitors of LKB1 and CaMKK2 in the presence and absence of Zyflamend, and in LKB1-null HeLa cells that constitutively express CaMKK2, following transfection with wild type LKB1 or catalytically-dead mutants. Upstream regulation by Zyflamend of LKB1 and CaMKK2 was investigated targeting protein kinase C-zeta (PKCζ) and death-associated protein kinase (DAPK), respectively.ResultsZyflamend’s activation of AMPK appears to be LKB1 dependent, while simultaneously inhibiting CaMKK2 activity. Zyflamend failed to rescue the activation of AMPK in the presence of pharmacological and molecular inhibitors of LKB1, an effect not observed in the presence of inhibitors of CaMKK2. Using LKB1-null and catalytically-dead LKB1-transfected HeLa cells that constitutively express CaMKK2, ionomycin (activator of CaMKK2) increased phosphorylation of AMPK, but Zyflamend only had an effect in cells transfected with wild type LKB1. Zyflamend appears to inhibit CaMKK2 by DAPK-mediated phosphorylation of CaMKK2 at Ser511, an effect prevented by a DAPK inhibitor. Alternatively, Zyflamend mediates LKB1 activation via increased phosphorylation of PKCζ, where it induced translocation of PKCζ and LKB1 to their respective active compartments in HeLa cells following treatment. Altering the catalytic activity of LKB1 did not alter this translocation.DiscussionZyflamend’s activation of AMPK is mediated by LKB1, possibly via PKCζ, but independent of CaMKK2 by a mechanism that appears to involve DAPK.ConclusionsTherefore, this is the first evidence that natural products simultaneously and antithetically regulate upstream kinases, known to be involved in cancer, via the activation of AMPK.Electronic supplementary materialThe online version of this article (10.1186/s12906-018-2255-0) contains supplementary material, which is available to authorized users.

Sanghuangporus sanghuang Mycelium Prevents Paracetamol-Induced Hepatotoxicity through Regulating the MAPK/NF-κB, Keap1/Nrf2/HO-1, TLR4/PI3K/Akt, and CaMKKβ/LKB1/AMPK Pathways and Suppressing Oxidative Stress and Inflammation

“…In addition, AMPK activation prevents inflammation through the IKK/NF-κB signaling pathway [49]. CaMKKβ, an AMPK-activating kinase, may exert anti-inflammatory effects and reduce inflammatory responses to paracetamol stimulation [50]. LKB1 is a key upstream kinase and critical downstream molecule of AMPK and is essential for its activation [51].…”

In Vitro Effect and Mechanism of Action of Ergot Alkaloid Dihydroergocristine in Chemoresistant Prostate Cancer Cells

“…In prostate cells, CaMKK2 appeared to be the dominant kinase of AMPK, a master regulator of cellular homeostasis and tumor suppressor, by directly phosphorylating AMPK at Thr172 and activating AMPK (68,69). Phosphorylation of CaMKK2 at Ser155 by deathassociated protein kinase (DAPK) inhibits CaMKK2 activity (70,71). Considering the effects of dopaminergic signaling on intracellular calcium levels and that the activation of CaMKK2 is dependent on calcium-mediated calmodulin (14), we investigated whether the CaMKK2-AMPK axis is responsive to DHECS treatment in PCa cells.…”

Zyflamend induces apoptosis in pancreatic cancer cells via modulation of the JNK pathway

“…Additionally, Zyflamend has shown the capability to interact with a variety of integral cellular signaling pathways beyond COX. These signaling pathways and mechanisms of interaction include: AMP-activated protein kinase (AMPK) [ 19 , 20 ], nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [ 21 , 22 ], mammalian target of rapamycin (mTORC1) [ 20 ], apoptosis, cell growth [ 13 , 23 ], endoplasmic reticulum (ER) stress [ 22 , 24 , 25 ] and finally autophagy [ 14 ]. While these studies show that Zyflamend could exhibit profound potential in the therapeutic application, more research is required to elucidate the molecular basis underlying its anti-cancer effects.…”