R R MNF is a unique bitopic agent with

(R,R′)-MNF is a unique bitopic agent with selective and potent β2AR agonistic and GPR55 inhibitory properties [10], [11] that are independently associated with the drug's anti-mitotic and anti-tumorigenic actions. In 1321N1, U118MG, and melanoma cell lines, (R,R′)-MNF activation of the β2AR decreases cell growth and motility [21], [22] while (R,R′)-MNF-associated antagonism of GPR55 produces anti-proliferative effects in HepG2 and PANC-1 k145 sale despite expression of β2ARs in these cells [9], [10]. Indeed, incubation of HepG2 and PANC-1 cells with β2AR agonists [other than (R,R′)-MNF] increases mitogenesis, and pretreatment with the selective β2AR antagonist ICI-118,551 fails to dampen the anti-mitogenic properties of (R,R′)-MNF [17]. C6 cells also express GPR55 and β2AR receptors [49]. Here, we demonstrate that (R,R′)-MNF significantly reduces internalization and intracellular accumulation of the GPR55 ligand T1117 and blocks the increase in ERK1/2 phosphorylation and cell motility elicited by GPR55 activation. Like (R,R′)-MNF, (R,R′)-Fen is a selective β2AR agonist but without anti-GPR55 activity. The reduction in ERK and AKT phosphorylation in (R,R′)-Fen-treated C6 cells (Fig. 4) led us to reexamine the relative contribution of β2AR agonism versus GPR55 antagonism to C6 cell proliferation and motility and to determine if the observed outcomes were the result of synergistic, additive or antagonistic effects. A dose-dependent attenuation in the phosphorylation of AKT and ERK1/2 was observed in C6 cells after incubation with either the selective [(R,R′)-Fen] or non-selective (ISO) β2AR agonist, and pre-incubation with 100nM ICI-118,551 dramatically decreased the potency of (R,R′)-Fen. The contribution of the AC/cAMP/PKA signaling cassette in mediating the response of β2AR agonists (Fig. 8) was independently validated upon cell treatment with forskolin, a direct activation of AC. Even though ICI-118,551-treated cells displayed a relatively modest increase in IC50 values for (R,R′)-MNF-mediated reduction in AKT and ERK1/2 phosphorylation, as compared to (R,R′)-Fen, the fact remains that β2AR antagonism had a negative impact on a subset of (R,R′)-MNF responses, including combatting C6 tumor cell proliferation. The potency of (R,R′)-Fen as a β2AR agonist is ~10-fold greater than (R,R′)-MNF [11], which may be reflected in the difference in the magnitude of the ICI-118,551 effect on (R,R′)-MNF and (R,R′)-Fen activity. The possibility exists, however, that the ability of (R,R′)-MNF to inhibit GPR55 activity has contributed to its relative refractoriness to ICI-118,551 (Fig. 8). Another potential explanation for the differential effect of ICI-118,551 is based upon the coupling of the β2-AR to both Gs and Gi proteins. (R,R′)-MNF, like most synthetic β2-AR agonists, couples to both Gs and Gi proteins [50]. ICI-118,551 has been shown to reduces Gs-dependent actions with either no effect on Gi coupling [51] or with some activation of Gi-dependent signaling [52]. Thus, siRNA-mediated β2-AR knock-down may result in different effects compared to pharmacological receptor blockade with ICI-118,551 due to the differences in G protein coupling pattern between (R,R′)-MNF and (R,R′)-Fen, as the latter is a Gs-selective β2-AR agonist [53]. It has been reported that the treatment of 1321N1 astrocytoma cells with (R,R′)-Fen increases β2AR-mediated accumulation of cAMP, which in turn activates PKA and ultimately leads to G1 cell-cycle arrest [21]. Similarly, induction of G1 cell-cycle arrest by cAMP analogs correlates with PKA activation in A172 glioma cells [54]. Increase in PKA activity has also been reported to be essential for the anti-tumorigenic effects of (R,R′)-MNF in melanoma cell lines [22]. In this study, we provide new evidence that showed a dose-dependent phosphorylation of filamin A and other PKA targets in response to (R,R′)-MNF by an increase in β2AR downstream signaling in C6 cells. Pharmacological inhibition of PKA blocked (R,R′)-MNF-dependent reduction in phosphorylation of AKT and, to a lesser extent, impeded the activating phosphorylation of ERK1/2, whereas in basal conditions, phospho-AKT level was refractory to PKA inhibition and phospho-ERK1/2 levels were significantly increased. These results are consistent with the data obtained after selective siRNA-mediated knockdown of β2AR, whereby marked increase in basal levels of phospho-AKT and phospho-ERK1/2 was accompanied by complete refractoriness to (R,R′)-MNF- and (R,R′)-Fen-mediated attenuation of AKT phosphorylation while minimally impacting the ability of (R,R′)-MNF to decrease ERK1/2 phosphorylation. These results indicate that basal β2AR activity exerts a tonic inhibition on AKT and ERK1/2 phosphorylation, which is intensified by β2AR agonists. Inhibition of GPR55 with CID-16020046 had also a negative impact on basal phospho-AKT and phospho-ERK1/2 levels, although to a lesser degree than (R,R′)-MNF (Fig. S5). We surmise that inhibition of constitutive GPR55 activity most probably contributed to the larger effect of (R,R′)-MNF on phospho-ERK1/2 levels in the β2AR siRNA-treated cells as compared to (R,R′)-Fen. Taken together, our findings support the notion of an (R,R′)-MNF-dependent increase in β2AR-AC-PKA signaling cascade and inhibition of GPR55 signal transduction, which attenuate pro-oncogenic signaling in C6 cells (Fig. 8).