The activation of AKT and ERK

The activation of AKT and ERK1/2 results in the stabilization and increase in the co-transcriptional function of β-catenin [31], [32], [33]. Here, (R,R′)-MNF was found to reduce the levels of phospho-active AKT and ERK, which, in turn, may regulate the abundance and signaling potential of β-catenin. Knockdown of β2AR abrogated (R,R′)-MNF-mediated increase in β-catenin phosphorylation on Ser-33, while treatment of C6 cells with the GPR55 agonist, O-1602, significantly increased β-catenin stability through reduction in phospho-β-catenin (Ser-33) levels. This effect was blocked by (R,R′)-MNF most likely through inhibition of ERK signaling. Cell treatment with (R,R′)-MNF dose-dependently reduced C6 cell motility in a β2AR-independent fashion whereas the negative action of (R,R′)-Fen on cell motility was thwarted by ICI-118,551 pretreatment. The ability of (R,R′)-MNF at impeding wound closure elicited by GPR55 agonists, such as O-1602, AM251 and LPI, is consistent with earlier reports showing a link between cell motility and GPR55 signaling [12], [13]. It appears that both β2AR activation and GPR55 inhibition play a role in the attenuation of C6 cell motility by (R,R′)-MNF. U87MG cells do not express functional β2ARs and are refractory to (R,R′)-Fen [21], yet (R,R′)-MNF exerts anti-mitogenic activity [17] that correlates with inhibition of GPR55 activity [10]. Here, (R,R′)-MNF but not ISO (β2AR agonist) dose-dependently reduced phospho-ERK1/2 levels under basal conditions and after cell treatment with GPR55 agonists. These results are reminiscent of those collected from C6 cells and demonstrate the conserved anti-tumorigenic effects of (R,R′)-MNF in glioma cell lines. The fact that (R,R′)-MNF administration has significantly retarded glioma tumor growth in U87MG xenograft mouse model indicates that the drug should be active in malignant primary protease inhibitor cocktail receptor tumors and brain tumor cell lines that do not express functional β2ARs. Interconnections of the β2AR- and GPR55-mediated signaling pathways may enhance the expression and activation of a number of signaling molecules, transcriptional activators, and effector proteins that modulate tumor cell survival and progression. Based on the results presented here, it is reasonable to assume that the dual anti-proliferative properties of (R,R′)-MNF could be used as monotherapy and combination therapy to combat malignant primary brain tumors and brain tumor cell lines harboring β2ARs [55], [56] and/or overexpressing GPR55 that is often associated with aggressive glioblastomas [12]. To conclude, the bitopic function of (R,R′)-MNF works in concert to reduce pro-oncogenic signaling and inhibit tumor growth and metastasis.
Disclosure of potential conflicts of interest Drs. Bernier and Wainer are listed as co-inventors on a patent for the use of fenoterol and fenoterol analogues, including (R,R′)-MNF, in the treatment of glioblastomas and astrocytomas (US20130005799 A1). Drs. Paul, Bernier and Wainer are listed as co-inventors on a patent for the use of fenoterol analogues for regulating cannabinoid receptor activity-related disorders and diseases (WO2013177418 A1). Drs. Paul, Bernier and Wainer have assigned theirs rights in the patents to the U.S. government but will receive a percentage of any royalties that may be received by the government. Dr. Wainer is currently Chief Scientific Officer at Mitchell Woods Pharmaceuticals, which has licensed the use of (R,R′)-MNF from the U.S. government for use in the treatment of pancreatic, brain and liver cancers.
Author contributions
Acknowledgments We thank Nagendra Singh from National Institute on Aging for performing thymidine incorporation experiments. We also thank Maciej Maj and Izabela Grzesiuk for excellent technical assistance. This work was supported by funds from the Intramural Research Program of the National Institute on Aging/NIH, the Foundation for Polish Science (TEAM 2009-4/5 programme), the internal research fund for young researchers of the Medical University of Lublin (MNsd 65), and the Polpharma Scientific Foundation (scholarship to A.W.).