A Combination of Herbal Extracts Targets Prostate Cancer by Upregulating AMPK thru the Tumor Suppressor Protein LKB1
Authors: MacDonald Amber , Donohoe Dallas , Bettaieb Ahmed , Han Anna , Zhao Yi , Whelan Jay 
Published: June 2021

BackgroundZyflamend (ZYF), a select blend of 10 herbal extracts (holy basil, turmeric, ginger, green tea, rosemary, hu zhang, barberry, oregano, Baikal skullcap, and Chinese goldthread), effectively inhibits tumor growth in vivo using a preclinical model of castrate‐resistant prostate cancer (CR‐PCa). These effects were mediated by activating multiple anti‐tumorigenic pathways, many of which are downstream of 5′ adenosine monophosphate‐activated protein kinase (AMPK), a tumor suppressor and master energy sensor of the cell. We show that phosphorylation and activation of AMPK (pAMPK) by ZYF inhibits mTORC1 signaling, fatty acid synthesis (via inhibition of FAS, ACC, SREBP‐1C) and glucose oxidation, and increases mitochondrial beta‐oxidation. The importance of its anti‐cancer effects in PCa has been bolstered by several case studies from MD Anderson Cancer Center demonstrating clinical benefits using ZYF, and another AMPK activator, metformin in clinically unresponsive forms of the disease. Currently, two predominant upstream kinases are known to phosphorylate and activate AMPK at Thr172: liver kinase B1 (LKB1) and calcium/calmodulin‐dependent protein kinase kinase β (CaMKKβ). LKB1 is a known tumor suppressor protein whereby inactivation is associated with a variety of cancers, including PCa. CaMKKβ responds to a rise of intracellular calcium levels, but its activation of AMPK has been reported to promote the growth of advanced PCa via interaction with androgen receptor signaling, a pathway that ZYF also down regulates.ObjectiveTo interrogate the upstream molecular mechanisms responsible for ZYF‐mediated activation of AMPK in CWR22Rv1 cells, a CR‐PCa cell line.MethodsThe roles of LKB1 and CaMKKβ in AMPK activation will be investigated by targeting upstream mediators of both kinases ±ZYF, and using pharmacologic and molecular techniques to regulate their activities in CWR22Rv1 cells. Confirmation of the involvement of LKB1 in this process will be determined using CaMKKβ and AMPK containing, but LKB1 null, HeLa cells.ResultsZYF significantly reduced ATP levels, which promotes phosphorylation of AMPK by upstream kinases. ZYF increases pAMPK, the phosphorylation and activation of LKB1 (Ser428) and its upstream activator PKCϚ (Thr410). In the presence of chemical and molecular inhibitors of LKB1 (radicicol & siLKB1), ZYF failed to rescue their effects, suggesting an LKB1‐dependent mechanism. In LKB1‐null HeLa cells, ZYF failed to increase pAMPK, results recapitulated by AICAR, an LKB1‐dependent activator of AMPK. Effects of ZYF appear to be independent of CaMKKβ because inhibition of this enzyme (STO‐609, EGTA, BAPTA‐AM) failed to prevent the phosphorylation of AMPK by ZYF. Furthermore, ZYF increases the phosphorylation of CaMKKβ at Ser511, a site regulated by death‐associated protein kinase (DAPK). Inhibition of DAPK, in the presence of ZYF, decreases phosphorylation of CaMKKβ at Ser511.ConclusionsThese results suggest that ZYF's activation of AMPK is mediated by LKB1, possibly via activation of PKCϚ, but independent of CaMKKβ. Moreover, ZYF inhibits CaMKKβ activity by a mechanism involving DAPK's phosphorylation of CaMKKβ at Ser511. Importantly, this demonstrates that “foods” can have a place in treating cancer.Support or Funding InformationThis work was supported by the USDA National Institute of Food and Agriculture, Hatch project 1002398.