Satindra Goswami

SKF83959 selectively regulates phosphatidylinositol-linked D1 dopamine receptors in rat brain

Previously a distinct D1‐like dopamine receptor (DAR) that selectively couples to phospholipase C/phosphatidylinositol (PLC/PI) was proposed. However, lack of a selective agonist has limited efforts aimed at characterizing this receptor. We characterized the in vitro and in vivo effects of SKF83959 in regulating PI metabolism. SKF83959 stimulates (EC50, 8 µm) phosphatidylinositol 4,5‐biphosphate hydrolysis in membranes of frontal cortex (FC) but not in membranes from PC12 cells expressing classical D1A DARs. Stimulation of FC PI metabolism was attenuated by the D1 antagonist, SCH23390, indicating that SKF83959 activates a D1‐like DAR. The PI‐linked DAR is located in hippocampus, cerebellum, striatum and FC. Most significantly, administration of SKF83959 induced accumulations of IP3 in striatum and hippocampus. In contrast to other D1 DAR agonists, SKF83959 did not increase cAMP production in brain or in D1A DAR‐expressing PC12 cell membranes. However, SKF83959 inhibited cAMP elevation elicited by the D1A DAR agonist, SKF81297, indicating that the compound is an antagonist of the classical D1A DAR. Lastly, we demonstrated that SKF83959 enhances [35S]guanosine 5′‐O‐(3‐thiotriphosphate) binding to membrane Gαq and Gαi proteins, suggesting that PI stimulation is mediated by activation of these guanine nucleotide‐binding regulatory proteins. Results indicate that SKF83959 is a selective agonist for the PI‐linked D1‐like DAR, providing a unique tool for investigating the functions of this brain D1 DAR subtype.

The role of the phosphatidyinositol-linked D1 dopamine receptor in the pharmacology of SKF83959.

SKF83959, previously described as an antagonist of the D1 dopamine receptor, has been shown to be a potent anti-parkinsonian agent. However, its mechanism of action is unknown. The present communication was designed to study the mechanism by which SKF83959 exerts its pharmacological effects. SKF83959 induced contralateral rotations in the unilateral 6-OHDA-lesioned rat model of Parkinsons disease (PD). The rotations were completely blocked by the D1 dopamine receptor antagonist, SCH23390. The response was not affected by the serotonin receptor antagonist, mesulergine and was transiently attenuated by alpha1 adrenergic or D2 dopamine receptor antagonists, prazosin or spiperone, respectively. Injection of 0.5 and 1 mg/kg SKF83959 elicited significant elevations in IP3 accumulation in lesioned as compared to intact striata. This effect was blocked by SCH23390 at a dose that completely obviated the rotational response to SKF83959, suggesting that activation of the PI-linked D1 dopamine receptor and the PLC/IP3 pathway may be the underlying mechanism for the rotational activity induced by SKF83959. The present data provide the first evidence that the PI-linked D1 dopamine receptor plays a role in regulating motor activity in striatum and that modulation of the D1 dopamine receptor/PLC/IP3 pathway may be a novel target in the discovery of drugs for the treatment of Parkinsons disease.

NO-releasing NSAIDs suppress NF-κB signaling in vitro and in vivo through S-nitrosylation

NO-NSAIDs are promising anticancer drugs, comprising an NSAID, an NO-releasing moiety, and a spacer linking them. Although the effect of NO-NSAIDs on a wide variety of signaling and other cellular mechanisms has been deciphered, a key question remains unanswered, that being the role of NO to the overall biological effect of these agents. It has been shown that NO can directly modify sulfhydryl residues of proteins through S-nitrosylation and induce apoptosis. We studied 3 NO-NSAIDs having a different NSAID, spacer, and NO-releasing moiety. In vitro: aspirin, NO-ASA, naproxen, and NO-naproxen inhibited HT-29 human colon cancer cell growth, the IC(50)s being >5000, 192±6, 2800±210 and 95±5μM at 24h, respectively. NO-Aspirin and NO-naproxen reduced NF-κB protein levels, and activated caspase-3 enzyme in a dose- and time-dependent manner. Based on the biotin switch assay, NO-ASA and NO-naproxen S-nitrosylated NF-κB p65 in a time-dependent manner. Pretreatment of the cells with carboxy-PTIO, abrogated the S-nitrosylation of NF-κB p65. In vivo: rats treated with NO-ASA, NONO-ASA, and NO-naproxen showed S-nitrosylation of NF-κB p65 in the stomach tissue, increases in plasma TNF-α, and reductions in mucosal PGE(2) levels. These data provide a mechanistic role for NO and a rational for the chemopreventive effects of NO-NSAIDs.

JS-K; a nitric oxide-releasing prodrug, modulates β-catenin/TCF signaling in leukemic Jurkat cells: Evidence of an S-nitrosylated mechanism

β-Catenin is a central player of the Wnt signaling pathway that regulates cell-cell adhesion and may promote leukemia cell proliferation. We examined whether JS-K, an NO-donating prodrug, modulates the Wnt/β-catenin/TCF-4 signaling pathway in Jurkat T-Acute Lymphoblastic Leukemia cells. JS-K inhibited Jurkat T cell growth in a concentration and time-dependent manner. The IC(50)s for cell growth inhibition were 14±0.7 and 9±1.2μM at 24 and 48h, respectively. Treatment of the cells with JS-K for 24h, caused a dose-dependent increase in apoptosis from 16±3.3% at 10μM to 74.8±2% at 100μM and a decrease in proliferation. This growth inhibition was also due, in part, to alterations in the different phases of the cell cycle. JS-K exhibited a dose-dependent cytotoxicity as measured by LDH release at 24h. However, between 2 and 8h, LDH release was less than 20% for any indicated JS-K concentration. The β-catenin/TCF-4 transcriptional inhibitory activity was reduced by 32±8, 63±5, and 93±2% at 2, 10, and 25μM JS-K, respectively, based on luciferase reporter assays. JS-K reduced nuclear β-catenin and cyclin D1 protein levels, but cytosolic β-catenin expression did not change. Based on a time-course assay of S-nitrosylation of proteins by a biotin switch assay, S-nitrsolyation of nuclear β-catenin was determined to precede its degradation. A comparison of the S-nitrosylated nuclear β-catenin to the total nuclear β-catenin showed that β-catenin protein levels were degraded at 24h, while S-nitrosylation of β-catenin occurred earlier at 0-6h. The NO scavenger PTIO abrogated the JS-K mediated degradation of β-catenin demonstrating the need for NO.

Positional Isomers of Aspirin Are Equally Potent in Inhibiting Colon Cancer Cell Growth: Differences in Mode of Cyclooxygenase Inhibition

We compared the differential effects of positional isomers of acetylsalicylic acid (o-ASA, m-ASA, and p-ASA) on cyclooxygenase (COX) inhibition, gastric prostaglandin E2 (PGE2), malondialdehyde, tumor necrosis factor-alpha (TNF-α) levels, superoxide dismutase (SOD) activity, human adenocarcinoma colon cancer cell growth inhibition, cell proliferation, apoptosis, and cell-cycle progression. We also evaluated the gastric toxicity exerted by ASA isomers. All ASA isomers inhibit COX enzymes, but only the o-ASA exerted an irreversible inhibitory profile. We did not observe a significant difference between ASA isomers in their ability to decrease the in vivo synthesis of PGE2 and SOD activity. Furthermore, all isomers increased the levels of gastric and TNF-α when administered orally at equimolar doses. We observed a dose-dependent cell growth inhibitory effect; the order of potency was p-ASA > m-ASA ≈ o-ASA. There was a dose-dependent decrease in cell proliferation and an increase in apoptosis, with a concomitant Go/G1 arrest. The ulcerogenic profile of the three ASA isomers showed a significant difference between o-ASA (aspirin) and its two positional isomers when administered orally at equimolar doses (1 mmol/kg); the ulcer index (UI) for o-ASA indicated extensive mucosal injury (UI = 38), whereas m-ASA and p-ASA produced a significantly decreased toxic response (UI = 12 and 8, respectively) under the same experimental conditions. These results suggest that the three positional isomers of ASA exert practically the same biologic profile in vitro and in vivo but showed different safety profiles. The mechanism of gastric ulcer formation exerted by aspirin and its two isomers warrants a more detailed and thorough investigation.

Prenatal cocaine exposure uncouples mGluR1 from Homer1 and Gq Proteins.

Cocaine exposure during gestation causes protracted neurobehavioral changes consistent with a compromised glutamatergic system. Although cocaine profoundly disrupts glutamatergic neurotransmission and in utero cocaine exposure negatively affects metabotropic glutamate receptor-type 1 (mGluR1) activity, the effect of prenatal cocaine exposure on mGluR1 signaling and the underlying mechanism responsible for the prenatal cocaine effect remain elusive. Using brains of the 21-day-old (P21) prenatal cocaine-exposed rats, we show that prenatal cocaine exposure uncouples mGluR1s from their associated synaptic anchoring protein, Homer1 and signal transducer, Gq/11 proteins leading to markedly reduced mGluR1-mediated phosphoinositide hydrolysis in frontal cortex (FCX) and hippocampus. This prenatal cocaine-induced effect is the result of a sustained protein kinase C (PKC)-mediated phosphorylation of mGluR1 on the serine residues. In support, phosphatase treatment of prenatal cocaine-exposed tissues restores whereas PKC-mediated phosphorylation of saline-treated synaptic membrane attenuates mGluR1 coupling to both Gq/11 and Homer1. Expression of mGluR1, Homer1 or Gα proteins was not altered by prenatal cocaine exposure. Collectively, these data indicate that prenatal cocaine exposure triggers PKC-mediated hyper-phosphorylation of the mGluR1 leading to uncoupling of mGluR1 from its signaling components. Hence, blockade of excessive PKC activation may alleviate abnormalities in mGluR1 signaling and restores mGluR1-regulated brain functions in prenatal cocaine-exposed brains.

Abstract 5560: All three positional isomers of acetyl salicylic acids are equally potent in inhibiting colon cancer cell growth: Differences in mode of COX inhibition

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Aspirin, 2-(acetyloxy)-benzoic acid or acetyl salicylic acid (o-ASA) is the prototypic chemopreventive agent. The mode of action of o-ASA is irreversible inhibition of cyclo-oxygenases (COX-1 and COX-2). o-ASA has potentially serious gastrointestinal side effects ranging from dyspepsia to gastrointestinal bleeding, obstruction, and perforation. There are 2 other positional isomers of acetyl salicylic acid, 3-(acetyloxy)-benzoic acid (m-ASA) and 4-(acetyloxy)-benzoic acid (p-ASA). In this study we did a head-to-head comparison between these 3 structural isomers in terms of their ulcerogenic properties, mode of COX inhibition, effects on colon cancer growth and cell kinetics. Methods: Cell line: HT-29 human colon adenocarcimoa; Cell growth: MTT; Cell cycle phase distribution: Flow cytometry; Apoptosis: subdiploid (sub-G/G1) peak in DNA content histograms; Proliferation: PCNA. COX-1, -2 enzyme activity: colorimetric kit; Enzyme inhibition reversibility: dialysis. In vivo: 6 hrs after drug administration rats were euthanized, stomachs removed, cut along the greater curvature, rinsed with water, and observed (with magnifying lenses) to count the numbers and measure the lengths (in mm) of all hemorrhagic lesions (ulcer index). Tissue samples were immediately frozen in liquid nitrogen for PGE2, SOD, and MDA determination. Results: All 3 positional isomers inhibited the growth of HT-29 cells with IC50s >3 mM. At 1 mM and 3 mM, the 3 positional isomers showed similar but dose-dependent: (i) increases in apoptosis; (ii) reductions in proliferation; (iii) increases in G/G1 phase, decreases in S and G2/M phases, suggesting a G/G1 block. COX-1 inhibition before and after dialysis with 1 mM concentrations were: o-ASA 72 ± 3% and 72 ± 4%; m-ASA 54 ± 2% and 38 ± 2%; p-ASA 48 ± 2% and 29 ± 1%. COX-2 inhibition before and after dialysis with 3 mM concentrations were: o-ASA 83 ± 2% and 81 ± 2%; m-ASA 80 ± 2% and 56 ± 3%; p-ASA 80 ± 3% and 38 ± 2%. For indomethacine (positive control) at 1 µM the corresponding values were: COX-1 69 ± 2% and 24 ± 1%; COX-2 67 ± 2% and 32 ± 2%. In the stomachs all 3 positional isomers reduced PGE2 and SOD levels and increased MDA to the same extent. Ulcer index in o-ASA treated animals was 38 whereas in m-ASA and p-ASA treated animals it was 12 and 8 respectively. However, in the stomachs of the m-ASA and p-ASA treated animals there were significant numbers of micro-hemorrhagic lesions (m-ASA = 45, p-ASA = 37) representing pre-ulcers. Conclusion: m-ASA and p-ASA are reversible inhibitors of COX-1 and COX-2 whereas o-ASA in an irreversible inhibitor. Gastric side effects were qualitatively and quantitatively different possibly because o-ASA inhibits COX-1 to a greater extent than m-ASA or p-ASA. All 3 positional isomers had similar effects on HT-29 cell kinetics. m-ASA and p-ASA may be alternatives to traditional ASA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5560. doi:10.1158/1538-7445.AM2011-5560

Abstract 1487: Nitric oxide-releasing NSAIDs suppress NF-κB signaling and increase caspase-3 enzyme activity in vitro and in vivo through S-nitrosylation

Introduction: Nitric oxide-releasing nonsteroidal anti-inflammatory drugs (NO-NSAIDs) consisting of a conventional NSAID to which an NO releasing moiety -ONO2 is covalently attached through a spacer have emerged as a new class of pharmaceutical agents. The released NO has proapoptotic effects which may cause DNA damage, leading to p53 activation, proteasome inhibition, and/or cytochrome c release from mitochondria, resulting from activation of the mitochondrial permeability transition pore or damage to mitochondrial membrane phospholipids. NO can directly modify sulfhydryl residues of proteins through S-nitrosylation, which has emerged as an important posttranslational protein modification based on prototypic redox mechanisms in signal transduction. The transcription factor, NF-κB is constitutively expressed in many tumors and characterizes all inflammatory responses. As apoptosis is a dominant anti-tumor pathway for chemotherapy-induced cell death, NF-κB may also be involved in resistance to cancer chemotherapy. Caspase-3 activation is intimately involved in apoptosis. In this study we evaluated the actions of meta-NO-aspirin (m-NO-ASA) and NO-naproxen which inhibit cell growth and induce apoptosis on NF-κB expression and caspase-3 activity and determined whether these agents S-nitrosylate these proteins. Methods: m-NO-ASA and NO-naproxen synthesized by us. Cell line: HT-29 human colon cancer cells. Growth inhibition: MTT. Animal studies: Rats (5 per group) were gavaged with ASA (180 mg/kg) or naproxen (40 mg/kg) or equimolar amounts of NO-ASA or NO-naproxen and killed 6h later after which their stomachs were rapidly removed, gently washed, and frozen in liquid nitrogen. Caspase-3 activity: commercial kit. Protein S-nitrosylation: Biotin switch assay (Jaffrey et al, Nature Cell Biology, 2001, 3:193-197); NF-κB, caspase-3, TNF-α, and biotinylated protein levels: immunoblotting. We also used the NO-donor SNAP as a positive control. Results: In vitro: m-NO-ASA and NO-naproxen inhibited the growth of HT29 colon cancer cells with IC50s of 187 ± 10 µM and 97 ± 5 µM at 24 hr respectively. NF-κB expression was reduced, however, caspase-3 enzyme activity and protein levels were increased by these agents in a dose- and time-dependent manner. NO-NSAID treatment for 24 hr at 0.5 x IC50, IC50 and 2 x IC50 caused an increase in both the total S-nitrosylated proteins and S-nitrosylated NF-κB (p-65 protein) and caspase-3 expression. These effects were reversed by carboxy-PTIO (an NO scavenger). In vivo: NO-NSAIDs caused an increase in total S-nitrosylated proteins and an increase in S-nitrosylated NF-κB, and TNF-α protein levels. Conclusions: These data strongly suggest S-nitrosylation as a potential molecular mechanism for the observed effects of NO-NSAIDs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1487.

Abstract B86: JS‐K, a nitric oxide‐donating prodrug, modulates β‐catenin/TCF signaling in leukemic Jurkat cells through S‐nitrosylation

Introduction: Leukemia is the most common form of cancer in children and acute lymphoblastic leukemia (ALL) is the primary cause of cancer‐related mortality. The deregulation of theWnt signaling cascade and its components has been implicated in T‐cell ALL as well as in B‐cell chronic lymphocytic leukemia (CLL). The protein β‐catenin has a central role in Wnt signaling pathway and may promote leukemia cell proliferation and differentiation. β‐catenin is expressed in T‐ ALL cells, tumor lines of hematopoietic origin and primary leukemia cells but is undetectable in normal peripheral blood T cells. Among the leukemic cell lines, β‐catenin is expressed in high levels in Jurkat T‐cells. JS‐K [O2‐(2,4‐dinitrophenyl)1‐[(4‐ethoxycarbonyl)piperazin‐1yl]diazen‐1‐ium‐1,2‐diolate] is a prodrug of the diazeniumdiolate class that releases NO through a reaction catalyzed by GST utilizing glutathione. This agent has been shown to inhibit the growth of various cancer cell lines. This report highlights the effects of JSK on the growth properties of human Jurkat T‐acute lymphoblastic leukemia cells, the β‐catenin signaling pathway, and protein S‐nitrosylation. Methods: JS‐K: synthesized by us. Cell growth: MTT; cell cycle phase distribution: flow cytometry; apoptosis: subdiploid (sub‐G0/G1) peak in DNA content histograms. Transient transfection: luciferase reporter constructs TOPflash or FOPflash. Luciferase activity: kit per manufacturer9s instructions. Protein S‐nitrosylation: Biotin switch assay (Jaffrey et al, Nature Cell Biology, 2001, 3, 193–197); β‐catenin and biotinylated protein levels: immunoblotting. Results: JS‐K inhibited the growth of Jurkat cells, IC50s of 12 ± 3 µM and 7 ± 1 µM at 24 h and 48 hr, respectively. This effect was due, in part, to a dose‐dependent induction of apoptosis and alterations in the distribution of the cells in the cell cycle, arrest was in G0/G1. JS‐K dose‐dependently inhibited β‐catenin/TCF‐4 signaling by reporter assays, and reduced β‐catenin levels in the nucleus and cytoplasm. JS‐K treatment for 24 hr at 0.5 × IC50, 1 × IC50 and 2 × IC50 caused an increase in total S‐nitrosylated proteins and also caused a reduction in S‐nitrosylated β‐catenin expression. These effects were reversed by carboxy‐PTIO (an NO scavenger). Conclusions: These findings establish a strong inhibitory effect of JS‐K in human Jurkat T‐acute lymphoblastic leukemia cells, an effect that is through inhibition of Wnt/β‐catenin/TCF‐4 signaling and through induction of apoptosis modulated by protein S‐nitrosylation. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B86.