Chemical Inhibitors Targeting the Oncogenic m6A Modifying Proteins.

Epigenetics is brought to RNA, introducing a new dimension to gene expression regulation. Among numerous RNA modifications, N6-methyladenosine (m6A) is an abundant internal modification on eukaryote mRNA first identified in the 1970s. However, the significance of m6A modification in mRNA had been long neglected until the fat mass and obesity-associated (FTO) enzyme was identified as the first m6A demethylase almost 40 years later. The m6A modification influences nearly every step of RNA metabolism and thus broadly affects gene expression at multiple levels, playing a critical role in many biological processes, including cancer progression, metastasis, and immune evasion. The m6A level is dynamically regulated by RNA epigenetic machinery comprising methyltransferases such as methyltransferase-like protein 3 (METTL3), demethylases FTO and AlkB human homologue 5 (ALKBH5), and multiple reader proteins. The understanding of the biology of RNA epigenetics and its translational drug discovery is still in its infancy. It is essential to further develop chemical probes and lead compounds for an in-depth investigation into m6A biology and the translational discovery of anticancer drugs targeting m6A modifying oncogenic proteins.In this Account, we present our work on the development of chemical inhibitors to regulate m6A in mRNA by targeting the FTO demethylase, and the elucidation of their mode of action. We reported rhein to be the first substrate competitive FTO inhibitor. Due to rhein's poor selectivity, we identified meclofenamic acid (MA) that selectively inhibits FTO compared with ALKBH5. Based on the structural complex of MA bound with FTO, we designed MA analogs FB23-2 and Dac51, which exhibit significantly improved activities compared with MA. For example, FB23-2 is specific to FTO inhibition in vitro among over 400 other oncogenic proteins, including kinases, proteases, and DNA and histone epigenetic proteins. Mimicking FTO depletion, FB23-2 promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cells and inhibits the progression of primary cells in xenotransplanted mice. Dac51 treatment impairs the glycolytic activity of tumor cells and restores the function of CD8+ T cells, thereby inhibiting the growth of solid tumors in vivo. These FTO inhibitors were and will continue to be used as probes to promote biological studies of m6A modification and as lead compounds to target FTO in anticancer drug discovery.Toward the end, we also include a brief review of ALKBH5 demethylase inhibitors and METTL3 methyltransferase modulators. Collectively, these small-molecule modulators that selectively target RNA epigenetic proteins will promote in-depth studies on the regulation of gene expression and potentially accelerate anticancer target discovery.

Synergistic effect of a nonsteroidal anti-inflammatory drug in combination with topotecan on small cell lung cancer cells.

BACKGROUND: The topoisomerase I inhibitor topotecan (TPT) is used in the treatment of recurrent small cell lung cancer (SCLC). However, the drug has a limited success rate and causes distress to patients due to its side effects, such as hematologic toxicities, including anemia and thrombocytopenia. Due to these pharmacokinetic limitations and undesirable side effects of chemotherapeutic drugs, the development of combination therapies has gained popularity in SCLC. Meclofenamic acid (MA), a nonsteroidal anti-inflammatory drug, has demonstrated anticancer effects on various types of cancers through different mechanisms. This study aims to investigate the potential synergistic effects of MA and TPT on the small cell lung cancer cell line DMS114. METHODS AND RESULTS: To assess the cytotoxic and apoptotic effects of the combined treatment of MA and TPT, trypan blue exclusion assay, Annexin V, acridine orange/propidium iodide staining, western blot, and cell cycle analysis were conducted. The results demonstrated that the combination of MA and TPT elicited synergistic effects by enhancing toxicity in DMS114 cells (P < 0.01) without causing toxicity in healthy epithelial lung cells MRC5. The strongest synergistic effect was observed when the cells were treated with 60 µM MA and 10 nM TPT for 48 h (CI = 0,751; DRI = 10,871). CONCLUSION: This study, for the first time, furnishes compelling evidence that MA and TPT synergistically reduce cellular proliferation and induce apoptosis in SCLC cells. Combinations of these drugs holds promise as a potential therapeutic strategy to improve efficacy and reduce the side effects associated with TPT.

Proteomics analysis of meclofenamic acid-treated small cell lung carcinoma cells revealed changes in cellular energy metabolism for cancer cell survival.

Small cell lung carcinoma (SCLC) is a highly aggressive cancer with low survival rate. Although initial response to chemotherapy in SCLC patients is well-rated, the treatments applied after the disease relapses are not successful. Drug resistance is accepted to be one of the main reasons for this failure. Therefore, there is an urgent need for new treatment strategies for SCLC. Meclofenamic acid, a nonsteroidal anti-inflammatory drug, has been shown to have anticancer effects on various types of cancers via different mechanisms. The aim of this study was to investigate the alterations that meclofenamic acid caused on a SCLC cell line, DMS114 using the tools of proteomics namely two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF and nHPLC coupled to LC-MS/MS. Among the proteins identified by both methods, those showing significantly altered expression levels were evaluated using bioinformatics databases, PANTHER and STRING. The key altered metabolism upon meclofenamic acid treatment appeared to the cellular energy metabolism. Glycolysis was suppressed, whereas mitochondrial activity and oxidative phosphorylation were boosted. The cells underwent metabolic reprogramming to adapt into their new environment for survival. Metabolic reprogramming is known to cause drug resistance in several cancer types including SCLC. The identified differentially regulated proteins in here associated with energy metabolism hold value as the potential targets to overcome drug resistance in SCLC treatment.

TRPM4 inhibition by meclofenamate suppresses Ca2+-dependent triggered arrhythmias.

AIMS: Cardiac arrhythmias are a major factor in the occurrence of morbidity and sudden death in patients with cardiovascular disease. Disturbances of Ca2+ homeostasis in the heart contribute to the initiation and maintenance of cardiac arrhythmias. Extrasystolic increases in intracellular Ca2+ lead to delayed afterdepolarizations and triggered activity, which can result in heart rhythm abnormalities. It is being suggested that the Ca2+-activated nonselective cation channel TRPM4 is involved in the aetiology of triggered activity, but the exact contribution and in vivo significance are still unclear. METHODS AND RESULTS: In vitro electrophysiological and calcium imaging technique as well as in vivo intracardiac and telemetric electrocardiogram measurements in physiological and pathophysiological conditions were performed. In two distinct Ca2+-dependent proarrhythmic models, freely moving Trpm4-/- mice displayed a reduced burden of cardiac arrhythmias. Looking further into the specific contribution of TRPM4 to the cellular mechanism of arrhythmias, TRPM4 was found to contribute to a long-lasting Ca2+ overload-induced background current, thereby regulating cell excitability in Ca2+ overload conditions. To expand these results, a compound screening revealed meclofenamate as a potent antagonist of TRPM4. In line with the findings from Trpm4-/- mice, 10 µM meclofenamate inhibited the Ca2+ overload-induced background current in ventricular cardiomyocytes and 15 mg/kg meclofenamate suppressed catecholaminergic polymorphic ventricular tachycardia-associated arrhythmias in a TRPM4-dependent manner. CONCLUSION: The presented data establish that TRPM4 represents a novel target in the prevention and treatment of Ca2+-dependent triggered arrhythmias.

Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer.

Brain metastasis represents a substantial source of morbidity and mortality in various cancers, and is characterized by high resistance to chemotherapy. Here we define the role of the most abundant cell type in the brain, the astrocyte, in promoting brain metastasis. We show that human and mouse breast and lung cancer cells express protocadherin 7 (PCDH7), which promotes the assembly of carcinoma-astrocyte gap junctions composed of connexin 43 (Cx43). Once engaged with the astrocyte gap-junctional network, brain metastatic cancer cells use these channels to transfer the second messenger cGAMP to astrocytes, activating the STING pathway and production of inflammatory cytokines such as interferon-α (IFNα) and tumour necrosis factor (TNF). As paracrine signals, these factors activate the STAT1 and NF-κB pathways in brain metastatic cells, thereby supporting tumour growth and chemoresistance. The orally bioavailable modulators of gap junctions meclofenamate and tonabersat break this paracrine loop, and we provide proof-of-principle that these drugs could be used to treat established brain metastasis.

Significance of Multiple Bioactivation Pathways for Meclofenamate as Revealed through Modeling and Reaction Kinetics.

Meclofenamate is a nonsteroidal anti-inflammatory drug used in the treatment of mild-to-moderate pain yet poses a rare risk of hepatotoxicity through an unknown mechanism. Nonsteroidal anti-inflammatory drug (NSAID) bioactivation is a common molecular initiating event for hepatotoxicity. Thus, we hypothesized a similar mechanism for meclofenamate and leveraged computational and experimental approaches to identify and characterize its bioactivation. Analyses employing our XenoNet model indicated possible pathways to meclofenamate bioactivation into 19 reactive metabolites subsequently trapped into glutathione adducts. We describe the first reported bioactivation kinetics for meclofenamate and relative importance of those pathways using human liver microsomes. The findings validated only four of the many bioactivation pathways predicted by modeling. For experimental studies, dansyl glutathione was a critical trap for reactive quinone metabolites and provided a way to characterize adduct structures by mass spectrometry and quantitate yields during reactions. Of the four quinone adducts, we were able to characterize structures for three of them. Based on kinetics, the most efficient bioactivation pathway led to the monohydroxy para-quinone-imine followed by the dechloro-ortho-quinone-imine. Two very inefficient pathways led to the dihydroxy ortho-quinone and a likely multiply adducted quinone. When taken together, bioactivation pathways for meclofenamate accounted for approximately 13% of total metabolism. In sum, XenoNet facilitated prediction of reactive metabolite structures, whereas quantitative experimental studies provided a tractable approach to validate actual bioactivation pathways for meclofenamate. Our results provide a foundation for assessing reactive metabolite load more accurately for future comparative studies with other NSAIDs and drugs in general. SIGNIFICANCE STATEMENT: Meclofenamate bioactivation may initiate hepatotoxicity, yet common risk assessment approaches are often cumbersome and inefficient and yield qualitative insights that do not scale relative bioactivation risks. We developed and applied innovative computational modeling and quantitative kinetics to identify and validate meclofenamate bioactivation pathways and relevance as a function of time and concentration. This strategy yielded novel insights on meclofenamate bioactivation and provides a tractable approach to more accurately and efficiently assess other drug bioactivations and correlate risks to toxicological outcomes.

Differential Contribution of Gap Junctions to the Membrane Properties of ON- and OFF-Bipolar Cells of the Rat Retina.

Gap junctions are ubiquitous within the retina, but in general, it remains to be determined whether gap junction coupling between specific cell types is sufficiently strong to mediate functionally relevant coupling via electrical synapses. From ultrastructural, tracer coupling and immunolabeling studies, there is clear evidence for gap junctions between cone bipolar cells, but it is not known if these gap junctions function as electrical synapses. Here, using whole-cell voltage-clamp recording in rat (male and female) retinal slices, we investigated whether the gap junctions of bipolar cells make a measurable contribution to the membrane properties of these cells. We measured the input resistance (RN) of bipolar cells before and after applying meclofenamic acid (MFA) to block gap junctions. In the presence of MFA, RN of ON-cone bipolar cells displayed a clear increase, paralleled by block of the electrical coupling between these cells and AII amacrine cells in recordings of coupled cell pairs. For OFF-cone and rod bipolar cells, RN did not increase in the presence of MFA. The results for rod bipolar cells are consistent with the lack of gap junctions in these cells. However, for OFF-cone bipolar cells, our results suggest that the morphologically identified gap junctions between these cells do not support a junctional conductance that is sufficient to mediate effective electrical coupling. Instead, these junctions might play a role in chemical and/or metabolic coupling between subcellular compartments.

Meclofenamic acid promotes cisplatin-induced acute kidney injury by inhibiting fat mass and obesity-associated protein-mediated m6A abrogation in RNA.

The role of RNA methylation on the sixth N atom of adenylate (m6A) in acute kidney injury (AKI) is unknown. FTO (fat mass and obesity-associated protein) reverses the m6A modification in cisplatin-induced AKI. Here, we aimed to determine FTO's role in AKI. We induced AKI in c57BL/6 mice by intraperitoneal cisplatin injection and treated the animal with vehicle or an FTO inhibitor meclofenamic acid (MA) for 3 days. Moreover, as an in vitro model, human kidney proximal tubular cells (HK2 cells) were treated with cisplatin. We found that the cisplatin treatment reduces FTO expression and increases m6A levels in vivo and in vitro MA aggravated renal damage and increased apoptosis in cisplatin-treated kidneys, phenotypes that were correlated with reduced FTO expression and increased m6A levels. Moreover, MA promoted apoptosis in cisplatin-treated HK2 cells, which was correlated with the reduced FTO expression and increased m6A in HK2 cells. FTO protein overexpression reduced m6A levels and inhibited apoptosis in cisplatin-treated HK2 cells and also blocked the MA-induced increase in m6A levels and apoptosis rates. In agreement, overexpression of the m6A-generating methyltransferase-like 3 and 14 (METTL3 and METTL14) or siRNA-mediated FTO knockdown promoted apoptosis and enhanced m6A levels in cisplatin-treated HK2 cells. MA increased p53 mRNA and protein levels in AKI both in vitro and in vivo, and FTO overexpression reduced p53 expression and reversed the MA-induced p53 increase in AKI. In conclusion, reduced renal FTO expression in cisplatin-induced AKI increases RNA m6A levels and aggravates renal damages.

Gap junctional coupling between retinal amacrine and ganglion cells underlies coherent activity integral to global object perception.

Coherent spike activity occurs between widely separated retinal ganglion cells (RGCs) in response to a large, contiguous object, but not to disjointed objects. Since the large spatial separation between the RGCs precludes common excitatory inputs from bipolar cells, the mechanism underlying this long-range coherence remains unclear. Here, we show that electrical coupling between RGCs and polyaxonal amacrine cells in mouse retina forms the synaptic mechanism responsible for long-range coherent activity in the retina. Pharmacological blockade of gap junctions or genetic ablation of connexin 36 (Cx36) subunits eliminates the long-range correlated spiking between RGCs. Moreover, we find that blockade of gap junctions or ablation of Cx36 significantly reduces the ability of mice to discriminate large, global objects from small, disjointed stimuli. Our results indicate that synchronous activity of RGCs, derived from electrical coupling with amacrine cells, encodes information critical to global object perception.

Preferential electrical coupling regulates neocortical lineage-dependent microcircuit assembly.

Radial glial cells are the primary neural progenitor cells in the developing neocortex. Consecutive asymmetric divisions of individual radial glial progenitor cells produce a number of sister excitatory neurons that migrate along the elongated radial glial fibre, resulting in the formation of ontogenetic columns. Moreover, sister excitatory neurons in ontogenetic columns preferentially develop specific chemical synapses with each other rather than with nearby non-siblings. Although these findings provide crucial insight into the emergence of functional columns in the neocortex, little is known about the basis of this lineage-dependent assembly of excitatory neuron microcircuits at single-cell resolution. Here we show that transient electrical coupling between radially aligned sister excitatory neurons regulates the subsequent formation of specific chemical synapses in the neocortex. Multiple-electrode whole-cell recordings showed that sister excitatory neurons preferentially form strong electrical coupling with each other rather than with adjacent non-sister excitatory neurons during early postnatal stages. This preferential coupling allows selective electrical communication between sister excitatory neurons, promoting their action potential generation and synchronous firing. Interestingly, although this electrical communication largely disappears before the appearance of chemical synapses, blockade of the electrical communication impairs the subsequent formation of specific chemical synapses between sister excitatory neurons in ontogenetic columns. These results suggest a strong link between lineage-dependent transient electrical coupling and the assembly of precise excitatory neuron microcircuits in the neocortex.

Electrical synapses connect a network of gonadotropin releasing hormone neurons in a cichlid fish.

Initiating and regulating vertebrate reproduction requires pulsatile release of gonadotropin-releasing hormone (GnRH1) from the hypothalamus. Coordinated GnRH1 release, not simply elevated absolute levels, effects the release of pituitary gonadotropins that drive steroid production in the gonads. However, the mechanisms underlying synchronization of GnRH1 neurons are unknown. Control of synchronicity by gap junctions between GnRH1 neurons has been proposed but not previously found. We recorded simultaneously from pairs of transgenically labeled GnRH1 neurons in adult male Astatotilapia burtoni cichlid fish. We report that GnRH1 neurons are strongly and uniformly interconnected by electrical synapses that can drive spiking in connected cells and can be reversibly blocked by meclofenamic acid. Our results suggest that electrical synapses could promote coordinated spike firing in a cellular assemblage of GnRH1 neurons to produce the pulsatile output necessary for activation of the pituitary and reproduction.

Meclofenamic acid improves the signal to noise ratio for visual responses produced by ectopic expression of human rod opsin.

PURPOSE: Retinal dystrophy through outer photoreceptor cell death affects 1 in 2,500 people worldwide with severe impairment of vision in advanced stages of the disease. Optogenetic strategies to restore visual function to animal models of retinal degeneration by introducing photopigments to neurons spared degeneration in the inner retina have been explored, with variable degrees of success. It has recently been shown that the non-steroidal anti-inflammatory and non-selective gap-junction blocker meclofenamic acid (MFA) can enhance the visual responses produced by an optogenetic actuator (channelrhodopsin) expressed in retinal ganglion cells (RGCs) in the degenerate retina. Here, we set out to determine whether MFA could also enhance photoreception by another optogenetic strategy in which ectopic human rod opsin is expressed in ON bipolar cells. METHODS: We used in vitro multielectrode array (MEA) recordings to characterize the light responses of RGCs in the rd1 mouse model of advanced retinal degeneration following intravitreal injection of an adenoassociated virus (AAV2) driving the expression of human rod opsin under a minimal grm6 promoter active in ON bipolar cells. RESULTS: We found treated retinas were light responsive over five decades of irradiance (from 1011 to 1015 photons/cm2/s) with individual RGCs covering up to four decades. Application of MFA reduced the spontaneous firing rate of the visually responsive neurons under light- and dark-adapted conditions. The change in the firing rate produced by the 2 s light pulses was increased across all intensities following MFA treatment, and there was a concomitant increase in the signal to noise ratio for the visual response. Restored light responses were abolished by agents inhibiting glutamatergic or gamma-aminobutyric acid (GABA)ergic signaling in the MFA-treated preparation. CONCLUSIONS: These results confirm the potential of MFA to inhibit spontaneous activity and enhance the signal to noise ratio of visual responses in optogenetic therapies to restore sight.

Meclofenamic acid selectively inhibits FTO demethylation of m6A over ALKBH5.

Two human demethylases, the fat mass and obesity-associated (FTO) enzyme and ALKBH5, oxidatively demethylate abundant N(6)-methyladenosine (m(6)A) residues in mRNA. Achieving a method for selective inhibition of FTO over ALKBH5 remains a challenge, however. Here, we have identified meclofenamic acid (MA) as a highly selective inhibitor of FTO. MA is a non-steroidal, anti-inflammatory drug that mechanistic studies indicate competes with FTO binding for the m(6)A-containing nucleic acid. The structure of FTO/MA has revealed much about the inhibitory function of FTO. Our newfound understanding, revealed herein, of the part of the nucleotide recognition lid (NRL) in FTO, for example, has helped elucidate the principles behind the selectivity of FTO over ALKBH5. Treatment of HeLa cells with the ethyl ester form of MA (MA2) has led to elevated levels of m(6)A modification in mRNA. Our collective results highlight the development of functional probes of the FTO enzyme that will (i) enable future biological studies and (ii) pave the way for the rational design of potent and specific inhibitors of FTO for use in medicine.

Different effects of the nonsteroidal anti-inflammatory drugs meclofenamate sodium and naproxen sodium on proteasome activity in cardiac cells.

The use of nonsteroidal anti-inflammatory drugs (NSAIDs) like meclofenamate sodium (MS), used to reduce pain, has been associated with an increased risk of cardiovascular disease (CVD). Naproxen (NAP), another NSAID, is not associated with increased risk of CVD. The molecular mechanism(s) by which NSAIDs induce CVD is unknown. We investigated the effects of MS and NAP on protein homeostasis and cardiotoxicity in rat cardiac H9c2 cells and murine neonatal cardiomyocytes. MS, but not NAP, significantly inhibited proteasome activity and reduced cardiac cell viability at pharmacological levels found in humans. Although proteasome subunit gene and protein expression were unaffected by NSAIDs, MS treated cell lysates showed higher 20S proteasome content, while purified proteasomes from MS treated cells had lower proteasome activity and higher levels of oxidized subunits than proteasomes from control cells. Addition of exogenous proteasome to MS treated cells improved cell viability. Both MS and NAP increased ROS production, but the rate of ROS production was greater in MS than in NAP treated cells. The ROS production is likely from mitochondria, as MS inhibited mitochondrial Complexes I and III, major sources of ROS, while NAP inhibited Complex I. MS also impaired mitochondrial membrane potential while NAP did not. Antioxidants were able to prevent the reduced cell viability caused by MS treatment. These results suggest that NSAIDs induce cardiotoxicity by a ROS dependent mechanism involving mitochondrial and proteasome dysfunction and may explain why some NSAIDs should not be given to patients for long periods.

An Analysis of Responses to Defibrotide in the Pulmonary Vascular Bed of the Cat.

Defibrotide is a polydisperse mixture of single-stranded oligonucleotides with many pharmacologic properties and multiple actions on the vascular endothelium. Responses to defibrotide and other vasodepressor agents were evaluated in the pulmonary vascular bed of the cat under conditions of controlled pulmonary blood flow and constant left atrial pressure. Lobar arterial pressure was increased to a high steady level with the thromboxane A2 analog U-46619. Under increased-tone conditions, defibrotide caused dose-dependent decreases in lobar arterial pressure without altering systemic arterial and left atrial pressures. Responses to defibrotide were significantly attenuated after the administration of the cyclooxygenase inhibitor sodium meclofenamate. Responses to defibrotide were also significantly attenuated after the administration of both the adenosine 1 and 2 receptor antagonists 8-cyclopentyl-1,3-dimethylxanthine and 8-(3-chlorostyryl)caffeine. Responses to defibrotide were not altered after the administration of the vascular selective adenosine triphosphate-sensitive potassium channel blocker U-37883A, or after the administration of the nitric oxide synthase inhibitor L-N-(1-iminoethyl)-ornithine. These data show that defibrotide has significant vasodepressor activity in the pulmonary vascular bed of the cat. They also suggest that pulmonary vasodilator responses to defibrotide are partially dependent on both the activation of the cyclooxygenase enzyme and adenosine 1 and 2 receptor pathways and independent of the activation of adenosine triphosphate-sensitive potassium channels or the synthesis of nitric oxide in the pulmonary vascular bed of the cat.

Renal Effects of Cyclooxygenase Inhibition When Nitric Oxide Synthesis Is Reduced and Angiotensin II Levels Are Enhanced.

The involvement of both cyclooxygenase (COX) isoforms in regulating renal function is well known but their interactions with other regulatory mechanisms, such as angiotensin II (Ang II) and nitric oxide (NO), are not well defined. This study has evaluated the relative contribution of both COX isoforms in regulating renal function when NO synthesis is reduced with and without a simultaneous increment in Ang II levels. The renal responses to a nonselective (meclofenamate) or a selective COX2 (nimesulide) inhibitor were examined in dogs pretreated with L-NAME with or without an intrarenal Ang II infusion. Meclofenamate induced a greater (P < 0.05) renal vasoconstriction than nimesulide in dogs pretreated with L-NAME. This vasoconstriction seems to be Ang II-dependent because it was reduced (P < 0.05) by captopril administration. Meclofenamate also induced a greater (P < 0.05) renal vasoconstriction than that elicited by nimesulide in dogs with reduced NO synthesis and elevated Ang II levels. The renal vasoconstriction induced by nimesulide but not that elicited by meclofenamate in dogs pretreated with L-NAME and Ang II, decreased (P < 0.05) during an extracellular volume expansion. These results demonstrate that the nonselective COX inhibition induces a greater renal vasoconstriction than that elicited by the selective COX2 inhibition when NO synthesis is reduced, and when NO synthesis is reduced and Ang II levels are elevated.

Histological changes caused by meclofenamic acid in androgen-independent prostate cancer tumors: evaluation in a mouse model.

Meclofenamic acid is a nonsteroidal anti-inflammatory drug that has shown therapeutic potential for different types of cancers, including androgen-independent prostate neoplasms. The antitumor effect of diverse nonsteroidal anti-inflammatory drugs has been shown to be accompanied by histological and molecular changes that are responsible for this beneficial effect. The objective of the present work was to analyze the histological changes caused by meclofenamic acid in androgen-independent prostate cancer. Tumors were created in a nude mouse model using PC3 cancerous human cells. Meclofenamic acid (10 mg/kg/day; experimental group, n=5) or saline solution (control group, n=5) was administered intraperitoneally for twenty days. Histological analysis was then carried out on the tumors, describing changes in the cellular architecture, fibrosis, and quantification of cellular proliferation and tumor vasculature. Meclofenamic acid causes histological changes that indicate less tumor aggression (less hypercellularity, fewer atypical mitoses, and fewer nuclear polymorphisms), an increase in fibrosis, and reduced cellular proliferation and tumor vascularity. Further studies are needed to evaluate the molecular changes that cause the beneficial and therapeutic effects of meclofenamic acid in androgen-independent prostate cancer.

Block of gap junctions eliminates aberrant activity and restores light responses during retinal degeneration.

Retinal degeneration leads to progressive photoreceptor cell death, resulting in vision loss. Subsequently, inner retinal neurons develop aberrant synaptic activity, compounding visual impairment. In retinal ganglion cells, light responses driven by surviving photoreceptors are obscured by elevated levels of aberrant spiking activity. Here, we demonstrate in rd10 mice that targeting disruptive neuronal circuitry with a gap junction antagonist can significantly reduce excessive spiking. This treatment increases the sensitivity of the degenerated retina to light stimuli driven by residual photoreceptors. Additionally, this enhances signal transmission from inner retinal neurons to ganglion cells, potentially allowing the retinal network to preserve the fidelity of signals either from prosthetic electronic devices, or from cells optogenetically modified to transduce light. Thus, targeting maladaptive changes to the retina allows for treatments to use existing neuronal tissue to restore light sensitivity, and to augment existing strategies to replace lost photoreceptors.

Acidosis prevents and alkalosis augments endothelium-dependent contractions in mouse arteries.

Changes in pH modulate the responsiveness of vascular smooth muscle cells to vasoconstrictor stimuli, but their effect on endothelium-dependent responses is unknown. Therefore, the influence of moderate changes in pH on responses to endothelium-dependent and -independent agonists was determined in aortae and carotid arteries of 15- to 26-week-old male C57BL/6N mice. Isolated rings were suspended in Halpern-Mulvany myographs for isometric tension recording. The preparations were exposed to either acidic (pH 7), control (pH 7.4) or alkaline (pH 7.8) modified Krebs-Ringer bicarbonate buffer solutions and their contractions and relaxations compared. Endothelium-dependent relaxations to acetylcholine (in the presence of meclofenamate or of the thromboxane-prostanoid (TP) receptor antagonist S18886) were comparable at the three pH values tested in contracted aortic or carotid arterial rings. Endothelium-dependent contractions of quiescent carotid arteries were reduced in acidosis and potentiated in alkalosis compared to control; these effects were reversible. The carotid arteries produced equal amounts of 6-keto prostaglandin F1α and thromboxane B2 at the different pH values tested. Contractions to the full TP receptor agonist U46619 were similar in the three milieus, but after inducing partial TP receptor blockade (with low concentrations of the TP receptor antagonist S18886) they were depressed in acidosis compared to alkalosis. Prostacyclin as a partial TP receptor activator also induced weaker contractions at low than at high pH, whereas its vasodilator effect was not affected. These findings demonstrate that changes in pH modulate endothelium-dependent contractions in mouse arteries primarily by altering the sensitivity of TP receptors of vascular smooth muscle to endothelium-derived contracting factors.