Cannabinoid Receptor Activation Correlates with the Proapoptotic Action of the β₂-Adrenergic Agonist (R,R′)-4-Methoxy-1-Naphthylfenoterol in HepG2 Hepatocarcinoma Cells

Abstract

Inhibition of cell proliferation by fenoterol and fenoterol derivatives in 1321N1 astrocytoma cells is consistent with ␤ 2 -adrenergic receptor (␤ 2 -AR) stimulation. However, the events that result in fenoterol-mediated control of cell proliferation in other cell types are not clear. Here, we compare the effect of the ␤ 2 -AR agonists (R,RЈ)-fenoterol (Fen) and (R,RЈ)-4-methoxy-1-naphthylfenoterol (MNF) on signaling and cell proliferation in HepG2 hepatocarcinoma cells by using Western blotting and [ 3 H]thymidine incorporation assays. Despite the expression of ␤ 2 -AR, no cAMP accumulation was observed when cells were stimulated with isoproterenol or Fen, although the treatment elicited both mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt activation. Unexpectedly, isoproterenol and Fen promoted HepG2 cell growth, but MNF reduced proliferation together with increased apoptosis. The mitogenic responses of Fen were attenuated by 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118,551), a ␤ 2 -AR antagonist, whereas those of MNF were unaffected. Because of the coexpression of ␤ 2 -AR and cannabinoid receptors (CBRs) and their impact on HepG2 cell proliferation, these G␣ i /G␣ o -linked receptors may be implicated in MNF signaling. Cell treatment with (R)-(ϩ)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a synthetic agonist of CB 1 R and CB 2 R, led to growth inhibition, whereas inverse agonists of these receptors blocked MNF mitogenic responses without affecting Fen signaling. MNF responses were sensitive to pertussis toxin. The ␤ 2 -ARdeficient U87MG cells were refractory to Fen, but responsive to the antiproliferative actions of MNF and WIN 55,212-2. The data indicate that the presence of the naphthyl moiety in MNF results in functional coupling to the CBR pathway, providing one of the first examples of a dually acting ␤ 2 -AR-CBR ligand.

Selective GPR55 antagonism reduces chemoresistance in cancer cells

“…The effect of MNF (0-10 μM) on [ 3 H]-thymidine incorporation into PANC-1, MDA-MB-231, and MCF-7 cells was investigated following previously described procedures [20, 21]. The effect of MNF on doxorubicin cytotoxicity in PANC-1 cells was measured using [ 3 H]-thymidine incorporation.…”

“…We have also demonstrated that treatment of these cell lines with ( R,R’ )-4’-methoxy-1-naphthylfenoterol (MNF) inhibits cellular uptake of Tocrifluor 1117 in a concentration-dependent manner and has a negative impact on GPR55 signaling, as evidenced by the significant impairment in ligand-inducible changes in p-ERK levels, cell morphology, and migration using scratch wound healing assay [21]. In addition, incubation of HepG2 cells with MNF decreased signaling through the MEK/ERK and PI3K/AKT pathways to produce cell cycle arrest and apoptosis [20]. …”

Activation of β-adrenergic receptor promotes cellular proliferation in human glioblastoma

“…However, all three subtypes of β-ARs appear to be detectable in U87-MG tumors in vivo, indicating that β-ARs expression processes may take place and may be functional during tumor development (24). Additionally, (R,R')-4-methoxy-1-naphthylfenoterol, a selective β 2 -AR agonist, was identified to inhibit cellular proliferation of U87-MG cells (28). All these data suggest that β-ARs may also be functional, by an unknown mechanism.…”

“…As aforementioned, obtaining β-AR expression in U87-MG tumors in vivo would imply that β-AR may facilitate tumor formation by promoting cellular proliferation. Another potential probability is that this cell type-specific divergence on cellular proliferation may be due to cross-talk between β-ARs and other GPCR-linked signaling cascades including gamma-aminobutyric acid B receptors (GABA B R) (37), α 2 -AR (38), bradykinin B 2 receptor (39), oxytocin (40), and cannabinoid receptors (28). For example, the ISO-induced signaling cascade, cellular proliferation and cellular migration in pancreatic ductal adenocarcinoma cells may be potently inhibited following stimulation of GABA B R signaling (37).…”

Identification and Validation of Targets for Cancer Immunotherapy: From the Bench-to-Bedside

“…MTT assay is very convenient, however some considerations need to be assessed to avoid false positives such as cell densities, correct culture medium, filtration of media to remove precipitate, optimisation of MTT concentrations and incubation times [69]. ]-thymidine and subsequently the incorporated radioactivity is measured, following washes to remove unincorporated radioactivity, using a Scintillation Counter [70]. As [ 3 H]-thymidine is radioactive an analogue called bromodeoxyuridine (BrdU) was developed to replace it in assays.…”