Rajib K. Paul

Negative Regulation of STAT3 Protein-mediated Cellular Respiration by SIRT1 Protein

In mammals, the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) is regulated by the deacetylase SIRT1. However, whether the newly described nongenomic actions of STAT3 toward mitochondrial oxidative phosphorylation are dependent on SIRT1 is unclear. In this study, Sirt1 gene knock-out murine embryonic fibroblast (MEF) cells were used to delineate the role of SIRT1 in the regulation of STAT3 mitochondrial function. Here, we show that STAT3 mRNA and protein levels and the accumulation of serine-phosphorylated STAT3 in mitochondria were increased significantly in Sirt1-KO cells as compared with wild-type MEFs. Various mitochondrial bioenergetic parameters, such as the oxygen consumption rate in cell cultures, enzyme activities of the electron transport chain complexes in isolated mitochondria, and production of ATP and lactate, indicated that Sirt1-KO cells exhibited higher mitochondrial respiration as compared with wild-type MEFs. Two independent approaches, including ectopic expression of SIRT1 and siRNA-mediated knockdown of STAT3, led to reduction in intracellular ATP levels and increased lactate production in Sirt1-KO cells that were approaching those of wild-type controls. Comparison of profiles of phospho-antibody array data indicated that the deletion of SirT1 was accompanied by constitutive activation of the pro-inflammatory NF-κB pathway, which is key for STAT3 induction and increased cellular respiration in Sirt1-KO cells. Thus, SIRT1 appears to be a functional regulator of NF-κB-dependent STAT3 expression that induces mitochondrial biogenesis. These results have implications for understanding the interplay between STAT3 and SIRT1 in pro-inflammatory conditions.

(R,S)-Ketamine Metabolites (R,S)-norketamine and (2S,6S)-hydroxynorketamine Increase the Mammalian Target of Rapamycin Function

Background:Subanesthetic doses of (R,S)-ketamine are used in the treatment of neuropathic pain and depression. In the rat, the antidepressant effects of (R,S)-ketamine are associated with increased activity and function of mammalian target of rapamycin (mTOR); however, (R,S)-ketamine is extensively metabolized and the contribution of its metabolites to increased mTOR signaling is unknown. Methods:Rats (n = 3 per time point) were given (R,S)-ketamine, (R,S)-norketamine, and (2S,6S)-hydroxynorketamine and their effect on the mTOR pathway determined after 20, 30, and 60 min. PC-12 pheochromocytoma cells (n = 3 per experiment) were treated with escalating concentrations of each compound and the impact on the mTOR pathway was determined. Results:The phosphorylation of mTOR and its downstream targets was significantly increased in rat prefrontal cortex tissue by more than ~2.5-, ~25-, and ~2-fold, respectively, in response to a 60-min postadministration of (R,S)-ketamine, (R,S)-norketamine, and (2S,6S)-hydroxynorketamine (P < 0.05, ANOVA analysis). In PC-12 pheochromocytoma cells, the test compounds activated the mTOR pathway in a concentration-dependent manner, which resulted in a significantly higher expression of serine racemase with ~2-fold increases at 0.05 nM (2S,6S)-hydroxynorketamine, 10 nM (R,S)-norketamine, and 1,000 nM (R,S)-ketamine. The potency of the effect reflected antagonistic activity of the test compounds at the &agr;7-nicotinic acetylcholine receptor. Conclusions:The data demonstrate that (R,S)-norketamine and (2S,6S)-hydroxynorketamine have potent pharmacological activity both in vitro and in vivo and contribute to the molecular effects produced by subanesthetic doses of (R,S)-ketamine. The results suggest that the determination of the mechanisms underlying the antidepressant and analgesic effects of (R,S)-ketamine requires a full study of the parent compound and its metabolites.

Tyrosine 308 Is Necessary for Ligand-directed Gs Protein-biased Signaling of β2-Adrenoceptor

Background: Ligand-specific receptor signaling is often referred to as functional selectivity or biased agonism. Results: Single amino acid substitution on β2-adrenoreceptor (Y308F) converts a ligand-specific signaling from Gs-biased to promiscuous Gs and Gi dual signaling. Conclusion: Specific ligand-receptor interaction results in receptor conformation(s) sufficient to convey biased signaling. Significance: Our work reveals a molecular mechanism for biased agonism. Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, β2-adrenoceptor (β2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of β2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of β2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi coupling. We have previously shown that although most β2-AR agonists cause both Gs and Gi activation, (R,R′)-fenoterol preferentially activates β2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on β2-AR essential for Gs-biased signaling. Following stimulation with a β2-AR-Gs-biased agonist (R,R′)-4′-aminofenoterol, the Gi disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type β2-AR. Interestingly, Y308F substitution on β2-AR enabled (R,R′)-4′-aminofenoterol to activate Gi and to produce these responses in a pertussis toxin-sensitive manner without altering β2-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of β2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of β2-AR stabilize receptor conformations favoring the receptor-Gs protein coupling and subsequently result in Gs-biased agonism.

Capillary electrophoresis–laser-induced fluorescence (CE-LIF) assay for measurement of intracellular d-serine and serine racemase activity

An enantioselective capillary electrophoresis-laser-induced fluorescence (CE-LIF) method for the analysis of D-serine (D-Ser) in cellular matrices has been developed. The assay involves derivatization with FITC followed by CE-LIF using 0.5 mM hydroxyl propyl-β-cyclodextrin in borate buffer [80 mM, pH 9.3]. The method was able to resolve D-Ser and L-Ser with an enantioselectivity (α) of 1.03 and a resolution (R(s)) of 1.37. Linearity was established from 0.25 to 100.00 μM. The assay was also able to enantioselectively resolve 6 additional amino acid racemates. The method was applied to the determination of intracellular D-Ser concentrations in PC-12, C6, 1312N1, and HepG2 cell lines. This method was used to determine the concentration-dependent increases in D-Ser and associated EC₅₀ values produced by L-Ser and the concentration-dependent decreases in d-Ser and associated IC₅₀ values produced by glycine, a competitive inhibitor of serine racemase (SR). Western blot analysis determined that the PC-12 and C6 cell lines contained monomeric and dimeric forms of SR while the 1321N1 and HepG2 cells contained only the monomeric form. Although the SR dimer has been identified as the active form of the enzyme, all four of the tested cell lines expressed enzymatically active SR.

Nicotinic acetylcholine receptor antagonists alter the function and expression of serine racemase in PC-12 and 1321N1 cells.

Western blot analysis demonstrated that PC-12 cells express monomeric and dimeric forms of serine racemase (m-SR, d-SR) and that 1321N1 cells express m-SR. Quantitative RT-PCR and functional studies demonstrated that PC-12 cells express homomeric and heteromeric forms of nicotinic acetylcholine receptors (nAChR) while 1321N1 cells primarily express the α7-nAChR subtype. The effect of nAChR agonists and antagonists on SR activity and expression was examined by following concentration-dependent changes in intracellular d-Ser levels and SR protein expression. Incubation with (S)-nicotine increased d-Ser levels, which were attenuated by the α7-nAChR antagonist methyllycaconitine (MLA). Treatment of PC-12 cells with mecamylamine (MEC) produced a bimodal reduction of d-Ser reflecting MEC inhibition of homomeric and heteromeric nAChRs, while a unimodal curve was observed with 1321N1 cells, reflecting predominant expression of α7-nAChR. The nAChR subtype selectivity was probed using α7-nAChR selective inhibitors MLA and (R,S)-dehydronorketamine and α3β4-nAChR specific inhibitor AT-1001. The compounds reduced d-Ser in PC-12 cells, but only MLA and (R,S)-dehydronorketamine were effective in 1321N1 cells. Incubation of PC-12 and 1321N1 cells with (S)-nicotine, MEC and AT-1001 did not affect m-SR or d-SR expression, while MLA and (R,S)-dehydronorketamine increased m-SR expression but not SR mRNA levels. Treatment with cycloheximide indicated that increased m-SR was due to de novo protein synthesis associated with phospho-active forms of ERK1/2, MARCKS, Akt and rapamycin-sensitive mTOR. This effect was attenuated by treatment with the pharmacological inhibitors U0126, LY294002 and rapamycin, which selectively block the activation of ERK1/2, Akt and mTOR, respectively, and siRNAs directed against ERK1/2, Akt and mTOR. We propose that nAChR-associated changes in Ca(2+) flux affect SR activity, but not expression, and that MLA and (R,S)-dehydronorketamine bind to allosteric sites on the α7-nAChR and promote multiple signaling cascades that converge at mTOR to increase m-SR levels.

Gabapentin and (S)-pregabalin decrease intracellular D-Serine concentrations in PC-12 cells

The effects of gabapentin (GBP) and (S)-pregabalin (PGB) on the intracellular concentrations of d-serine and the expression of serine racemase (SR) in PC-12 cells were determined. Intracellular d-serine concentrations were determined using an enantioselective capillary electrophoresis assay with laser-induced fluorescence detection. Increasing concentrations of GBP, 0.1-20μM, produced a significant decrease in d-serine concentration relative to control, 22.9±6.7% at 20μM (*p<0.05), with an IC(50) value of 3.40±0.29μM. Increasing concentrations of PGB, 0.1-10μM, produced a significant decrease in d-serine concentration relative to control, 25.3±7.6% at 10μM (*p<0.05), with an IC(50) value of 3.38±0.21μM. The compounds had no effect on the expression of monomeric-SR or dimeric-SR as determined by Western blotting. The results suggest that incubation of PC-12 cells with GBP and PGB reduced the basal activity of SR, which is most likely a result of the decreased Ca(2+) flux produced via interaction of the drugs with the α(2)-δ subunit of voltage-gated calcium channels. d-Serine is a co-agonist of the N-methyl d-aspartate receptor (NMDAR) and reduced d-serine concentrations have been associated with reduced NMDAR activity. Thus, GBP and PGB may act as indirect antagonists of NMDAR, a mechanism that may contribute to the clinical effects of the drugs in neuropathic pain.

Cannabinoid receptor activation correlates with the proapoptotic action of the β2-adrenergic agonist (R,R')-4-methoxy-1-naphthylfenoterol in HepG2 hepatocarcinoma cells.

Inhibition of cell proliferation by fenoterol and fenoterol derivatives in 1321N1 astrocytoma cells is consistent with β2-adrenergic receptor (β2-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 β2-AR agonists (R,R′)-fenoterol (Fen) and (R,R′)-4-methoxy-1-naphthylfenoterol (MNF) on signaling and cell proliferation in HepG2 hepatocarcinoma cells by using Western blotting and [3H]thymidine incorporation assays. Despite the expression of β2-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 β2-AR antagonist, whereas those of MNF were unaffected. Because of the coexpression of β2-AR and cannabinoid receptors (CBRs) and their impact on HepG2 cell proliferation, these Gαi/Gαo-linked receptors may be implicated in MNF signaling. Cell treatment with (R)-(+)-[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 CB1R and CB2R, 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 β2-AR-deficient 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 β2-AR-CBR ligand.

Antitumor activity of (R,R’)‐4‐methoxy‐1‐naphthylfenoterol in a rat C6 glioma xenograft model in the mouse

(R,R’)‐4‐methoxy‐1‐naphthylfenoterol (MNF) inhibits cancer cell proliferation in vitro through cell‐type specific modulation of β2‐adrenergic receptor and/or cannabinoid receptor function. Here, we report an investigation into antitumor activity of MNF in rat C6 glioma cells. The potent antiproliferative action of MNF in these cells (IC50 of ~1 nmol/L) was refractory to pharmacological inhibition of β2‐adrenergic receptor while a synthetic inverse agonist of cannabinoid receptor 1 significantly blocked MNF activity. The antitumor activity of MNF was then assessed in a C6 glioblastoma xenograft model in mice. Three days after subcutaneous implantation of C6 cells into the lower flank of nude mice, these animals were subjected to i.p. injections of saline or MNF (2 mg/kg) for 19 days and tumor volumes were measured over the course of the experiment. Gene expression analysis, quantitative RT‐PCR and immunoblot assays were performed on the tumors after treatment. Significant reduction in mean tumor volumes was observed in mice receiving MNF when compared with the saline‐treated group. We identified clusters in expression of genes involved in cellular proliferation, as well as molecular markers for glioblastoma that were significantly downregulated in tumors of MNF‐treated mice as compared to saline‐injected controls. The efficacy of MNF against C6 glioma cell proliferation in vivo and in vitro was accompanied by marked reduction in the expression of cell cycle regulator proteins. This study is the first demonstration of MNF‐dependent chemoprevention of a glioblastoma xenograft model and may offer a potential mechanism for its anticancer action in vivo.

In vitro caloric restriction induces protective genes and functional rejuvenation in senescent SAMP8 astrocytes

Astrocytes are key cells in brain aging, helping neurons to undertake healthy aging or otherwise letting them enter into a spiral of neurodegeneration. We aimed to characterize astrocytes cultured from senescence‐accelerated prone 8 (SAMP8) mice, a mouse model of brain pathological aging, along with the effects of caloric restriction, the most effective rejuvenating treatment known so far. Analysis of the transcriptomic profiles of SAMP8 astrocytes cultured in control conditions and treated with caloric restriction serum was performed using mRNA microarrays. A decrease in mitochondrial and ribosome mRNA, which was restored by caloric restriction, confirmed the age‐related profile of SAMP8 astrocytes and the benefits of caloric restriction. An amelioration of antioxidant and neurodegeneration‐related pathways confirmed the brain benefits of caloric restriction. Studies of oxidative stress and mitochondrial function demonstrated a reduction of oxidative damage and partial improvement of mitochondria after caloric restriction. In summary, caloric restriction showed a significant tendency to normalize pathologically aged astrocytes through the activation of pathways that are protective against the age‐related deterioration of brain physiology.

Abstract 4535: Inhibition of cell proliferation by (R,R')-4′-methoxy-1-naphthylfenoterol in breast cancer cell lines

(R,R9)-4′-methoxy-1-naphthylfenoterol (MNF) is a bitopic ligand that acts as an agonist of the β2-adrenergic receptor and an antagonist of the orphan G-protein-coupled receptor GPR55. Activation of GPR55 is pro-oncogenic through cannabinoid-related signal transduction and MNF treatment inhibits proliferation of different tumor cell lines, representing cancers of the liver, pancreas and brain. Of significance, MNF exerts antitumor activity in a xenograft glioma model in nude mice. In the current study, the anti-proliferative response mediated by MNF has been examined in human breast cancer cell lines. In this study, MCF-7 and MDA-MB-231 cells were screened for the presence of epidermal growth factor receptor (EGFR) and GPR55 proteins by Western blotting. The EGFR in such cancer cells plays a key role in the control of growth and proliferation. When compared to MCF-7 cells, MDA-MB-231 cells contained high levels of EGFR and GPR55 and showed exquisite sensitivity to MNF as evidenced by the significant reduction in constitutive phosphorylation of the extracellular signal-regulated kinase (ERK1/2), with an IC50 of 0.08 nM. [3H]-Thymidine incorporation assay was performed and revealed that MNF elicited a 2-fold higher growth inhibition in MDA-MB-231 cells as compared to MCF-7 cells (50% vs. 24.9% inhibition at 10 µM of MNF; p Citation Format: Rajib K. Paul, Artur Wnorowski, Michel Bernier, Irving W. Wainer. Inhibition of cell proliferation by (R,R9)-4′-methoxy-1-naphthylfenoterol in breast cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4535. doi:10.1158/1538-7445.AM2014-4535