Preferential Inhibitory Effect Research Articles

Exploring the effects of extracranial injections of botulinum toxin type A on activation and sensitization of central trigeminovascular neurons by cortical spreading depression in male and female rats.

OnabotulinumtoxinA (onabotA), is assumed to achieve its therapeutic effect in migraine through blocking activation of unmyelinated meningeal nociceptors and their downstream communications with central dura-sensitive trigeminovascular neurons in the spinal trigeminal nucleus (SPV). The present study investigated the mechanism of action of onabotA by assessing its effect on activation and sensitization of dura-sensitive neurons in the SPV by cortical spreading depression (CSD). It is a follow up to our recent study on onabotA effects on activation and sensitization of peripheral trigeminovascular neurons. In anesthetized male and female rats, single-unit recordings were used to assess effects of extracranial injections of onabotA (five injections, one unit each, diluted in 5 μl of saline were made along the lambdoid (two injection sites) and sagittal (two injection sites) suture) vs. vehicle on CSD-induced activation and sensitization of high-threshold (HT) and wide-dynamic range (WDR) dura-sensitive neurons in the SPV. Single cell analysis of onabotA pretreatment effects on CSD-induced activation and sensitization of central trigeminovascular neurons in the SPV revealed the ability of this neurotoxin to prevent activation and sensitization of WDR neurons (13/20 (65%) vs. 4/16 (25%) activated neurons in the control vs. treated groups, p = 0.022, Fisher's exact). By contrast, onabotA pretreatment effects on CSD-induced activation and sensitization of HT neurons had no effect on their activation (12/18 (67%) vs. 4/7 (36%) activated neurons in the control vs. treated groups, p = 0.14, Fisher's exact). Regarding sensitization, we found that onabotA pretreatment prevented the enhanced responses to mechanical stimulation of the skin (i.e. responses reflecting central sensitization) in both WDR and HT neurons. In control but not treated WDR neurons, responses to brush (p = 0.004 vs. p = 0.007), pressure (p = 0.002 vs. p = 0.79) and pinch (p = 0.007 vs. 0.79) increased significantly two hours after CSD. Similarly, in control but not treated HT neurons, responses to brush (p = 0.002 vs. p = 0.79), pressure (p = 0.002 vs. p = 0.72) and pinch (p = 0.0006 vs. p = 0.28) increased significantly two hours after CSD. Unexpectedly, onabotA pretreatment prevented the enhanced responses of both WDR and HT neurons to mechanical stimulation of the dura (commonly reflecting peripheral sensitization). In control vs. treated WDR and HT neurons, responses to dural stimulation were enhanced in 70 vs. 25% (p = 0.017) and 78 vs. 27% (p = 0.017), respectively. The ability of onabotA to prevent activation and sensitization of WDR neurons is attributed to its preferential inhibitory effects on unmyelinated C-fibers. The inability of onabotA to prevent activation of HT neurons is attributed to its less extensive inhibitory effects on the thinly myelinated Aδ-fibers. These findings provide further pre-clinical evidence about differences and potentially complementary mechanisms of action of onabotA and calcitonin gene-related peptide-signaling neutralizing drugs.

Novel insight into atogepant mechanisms of action in migraine prevention.

Recently, we showed that while atogepant-a small-molecule calcitonin gene-related peptide (CGRP) receptor antagonist-does not fully prevent activation of meningeal nociceptors, it significantly reduces a cortical spreading depression (CSD)-induced early response probability in C fibres and late response probability in Aδ fibres. The current study investigates atogepant effect on CSD-induced activation and sensitization of high threshold (HT) and wide dynamic range (WDR) central dura-sensitive trigeminovascular neurons. In anaesthetized male rats, single-unit recordings were used to assess effects of atogepant (5 mg/kg) versus vehicle on CSD-induced activation and sensitization of HT and WDR trigeminovascular neurons. Single cell analysis of atogepant pretreatment effects on CSD-induced activation and sensitization of central trigeminovascular neurons in the spinal trigeminal nucleus revealed the ability of this small molecule CGRP receptor antagonist to prevent activation and sensitization of nearly all HT neurons (8/10 versus 1/10 activated neurons in the control versus treated groups, P = 0.005). In contrast, atogepant pretreatment effects on CSD-induced activation and sensitization of WDR neurons revealed an overall inability to prevent their activation (7/10 versus 5/10 activated neurons in the control versus treated groups, P = 0.64). Unexpectedly however, in spite of atogepant's inability to prevent activation of WDR neurons, it prevented their sensitization (as reflected their responses to mechanical stimulation of the facial receptive field before and after the CSD). Atogepant' ability to prevent activation and sensitization of HT neurons is attributed to its preferential inhibitory effects on thinly myelinated Aδ fibres. Atogepant's inability to prevent activation of WDR neurons is attributed to its lesser inhibitory effects on the unmyelinated C fibres. Molecular and physiological processes that govern neuronal activation versus sensitization can explain how reduction in CGRP-mediated slow but not glutamate-mediated fast synaptic transmission between central branches of meningeal nociceptors and nociceptive neurons in the spinal trigeminal nucleus can prevent their sensitization but not activation.

In vitro characterization of anti-inflammatory activities of 3RS, 7R, 11R-phytanic acid.

The aim of the research described here was to investigate the in vitro immunomodulatory effects of 3RS, 7R, 11R-phytanic acid (3RS-PHY) from the perspective of efficacy against autoimmune diseases. 3RS-PHY is a milk component with strong agonist activity at the peroxisome proliferator activated receptor (PPAR). As PPAR is a therapeutic target for several human diseases, 3RS-PHY intake may have possible health benefits. Recently, we chemically synthesized a preparation of 3RS-PHY and demonstrated that 3RS-PHY inhibited T-cell production of interferon (IFN)-γ. However, the overall immunomodulatory effects were not evaluated. In this study, mouse splenocytes, purified T-cells and B-cells were stimulated by mitogens and incubated with 3RS-PHY, followed by evaluation of cytokine and antibody production. A macrophage-like cell line J774.1 was also incubated with 3RS-PHY to evaluate nitric oxide production. 3RS-PHY decreased mRNA levels not only of IFN-γ but also of interleukin (IL)-2, IL-10 and IL-17A in splenocytes and similar effects were confirmed at the protein level. In addition, 3RS-PHY had a direct action on T-cells with preferential inhibitory effects on Th1 and Th17 cytokines such as IFN-γ and IL-17A. Furthermore, 3RS-PHY suppressed antibody secretion by B-cells and nitric oxide production by J774.1 almost completely, indicating that 3RS-PHY is a bioactive fatty acid with anti-inflammatory properties. These findings encourage further investigations, including in vivo experiments, to evaluate whether 3RS-PHY actually shows the potential to prevent autoimmune diseases, and provide basic information to produce milk and dairy products with an increased 3RS-PHY concentration.

Reciprocal positive regulation between BRD4 and YAP in GNAQ-mutant uveal melanoma cells confers sensitivity to BET inhibitors

Uveal melanoma (UM) is the most common intraocular cancer in adults. UMs are usually initiated by a mutation in GNAQ or GNA11 (encoding Gq or G11, respectively), unlike cutaneous melanomas (CMs), which usually carry a BRAF or NRAS mutation. Currently, there are no clinically effective targeted therapies for UM carrying Gq/11 mutations. Here, we identified a causal link between Gq activating mutations and hypersensitivity to bromodomain and extra-terminal (BET) inhibitors. BET inhibitors transcriptionally repress YAP via BRD4 regardless of Gq mutation status, independently of Hippo core components LATS1/2. In contrast, YAP/TAZ downregulation reduces BRD4 transcription exclusively in Gq-mutant cells and LATS1/2 double knockout cells, both of which are featured by constitutively active YAP/TAZ. The transcriptional interdependency between BRD4 and YAP identified in Gq-mutated cells is responsible for the preferential inhibitory effect of BET inhibitors on the growth and dissemination of Gq-mutated UM cells compared to BRAF-mutated CM cells in both culture cells and animal models. Our findings suggest BRD4 as a viable therapeutic target for Gq-driven UMs that are addicted to unrestrained YAP function.

Progerin mislocalizes myocardin-related transcription factor in Hutchinson-Guilford Progeria syndrome.

Hutchinson–Guilford Progeria syndrome (HGPS) is a rare genetic disease of premature aging and early death due to cardiovascular disease. The arteries of HGPS children and mice are pathologically stiff, and HGPS mice also display reduced arterial contractility. We recently showed that reduced contractility is an early event in HGPS and linked to an aberrantly low expression of smooth muscle myosin heavy chain (smMHC). Here, we have explored the basis for reduced smMHC abundance and asked whether it is a direct effect of progerin expression or a longer-term adaptive response. Myh11, the gene encoding for smMHC, is regulated by myocardin-related transcription factors (MRTFs), and we show that HGPS aortas have a reduced MRTF signature. Additionally, smooth muscle cells (SMCs) isolated from HGPS mice display reduced MRTF nuclear localization. Acute progerin expression in WT SMCs phenocopied both the decrease in MRTF nuclear localization and expression of Myh11 seen in HGPS. Interestingly, RNA-mediated depletion of MRTF-A in WT SMCs reproduced the preferential inhibitory effect of progerin on Myh11 mRNA relative to Acta2 mRNA. Our results show that progerin expression acutely disrupts MRTF localization to the nucleus and suggest that the consequent decrease in nuclear coactivator activity can help to explain the reduction in smMHC abundance and SMC contractility seen in HGPS.

Peripheral and central mechanisms of migraine provide rationale for combination therapy

The purpose of this study was to determine the effects of BoNT-A/atogepant (ATO) combination treatment on activation/sensitization of high-threshold (HT) and wide-dynamic range (WDR) trigeminovascular neurons in the spinal trigeminal nucleus (STN) by CSD. ATO is a small molecule CGRP receptor antagonist. (A) Individual analysis: in the control group, CSD activated 80% of HT and 70% of WDR neurons whereas in the treatment group it activated 10% of HT (p = 0.001, c2 = 9.8) and 0% of WDR (p = 0.001, c2 = 10.7) neurons. Similarly, in the control group, CSD sensitized 80% of HT and 60% of WDR neurons whereas in the treatment group it sensitized 0% of HT and 5% of WDR neurons. (B) Group analysis: statistical analyses of all neurons revealed that in the control group, spontaneous activity increased significantly in both HT (c2 = 6.4, p = 0.039) and WDR (c2 = 7.8, p = 0.004) neurons, whereas in the treatment group it remained unchanged in the HT (c2 = 1.4, p = 0.35) and decreased, rather than increased, significantly (c2 = 8.6, p = 0.003) in the WDR neurons. A similar pattern was seen in responses to mechanical stimulation of the dura and skin. Given BoNT-A preferential inhibitory effects on unmyelinated C- but not thinly myelinated Ad-fibers, and fremanezumab preferential inhibitory effects on Ad- but not C-fibers, it is reasonable to propose that the robust inhibition of activation and sensitization of the HT and WDR trigeminovascular neurons by the BoNT-A/ATO combination treatment was achieved through a dual blockade of both classes of meningeal nociceptors.

METTL3-mediated m6A methylation negatively modulates autophagy to support porcine blastocyst development‡.

N6-methyladenosine (m6A) catalyzed by METTL3 regulates the maternal-to-zygotic transition in zebrafish and mice. However, the role and mechanism of METTL3-mediated m6A methylation in blastocyst development remains unclear. Here, we show that METTL3-mediated m6A methylation sustains porcine blastocyst development via negatively modulating autophagy. We found that reduced m6A levels triggered by METTL3 knockdown caused embryonic arrest during morula-blastocyst transition and developmental defects in trophectoderm cells. Intriguingly, overexpression of METTL3 in early embryos resulted in increased m6A levels and these embryos phenocopied METTL3 knockdown embryos. Mechanistically, METTL3 knockdown or overexpression resulted in a significant increase or decrease in expression of ATG5 (a key regulator of autophagy) and LC3 (an autophagy marker) in blastocysts, respectively. m6A modification of ATG5 mRNA mainly occurs at 3'UTR, and METTL3 knockdown enhanced ATG5 mRNA stability, suggesting that METTL3 negatively regulated autophagy in an m6A dependent manner. Furthermore, single-cell qPCR revealed that METTL3 knockdown only increased expression of LC3 and ATG5 in trophectoderm cells, indicating preferential inhibitory effects of METTL3 on autophagy activity in the trophectoderm lineage. Importantly, autophagy restoration by 3MA (an autophagy inhibitor) treatment partially rescued developmental defects of METTL3 knockdown blastocysts. Taken together, these results demonstrate that METTL3-mediated m6A methylation negatively modulates autophagy to support blastocyst development.

A New Insight into Meloxicam: Assessment of Antioxidant and Anti-Glycating Activity in In Vitro Studies.

Meloxicam is a non-steroidal anti-inflammatory drug, which has a preferential inhibitory effect to cyclooxyganase-2 (COX-2). Although the drug inhibits prostaglandin synthesis, the exact mechanism of meloxicam is still unknown. This is the first study to assess the effect of meloxicam on protein glyco-oxidation as well as antioxidant activity. For this purpose, we used an in vitro model of oxidized bovine serum albumin (BSA). Glucose, fructose, ribose, glyoxal and methylglyoxal were used as glycating agents, while chloramine T was used as an oxidant. We evaluated the antioxidant properties of albumin (2,2-di-phenyl-1-picrylhydrazyl radical scavenging capacity, total antioxidant capacity and ferric reducing antioxidant power), the intensity of protein glycation (Amadori products, advanced glycation end products) and glyco-oxidation (dityrosine, kynurenine, N-formylkynurenine, tryptophan and amyloid-β) as well as the content of protein oxidation products (advanced oxidation protein products, carbonyl groups and thiol groups). We have demonstrated that meloxicam enhances the antioxidant properties of albumin and prevents the protein oxidation and glycation under the influence of various factors such as sugars, aldehydes and oxidants. Importantly, the antioxidant and anti-glycating activity is similar to that of routinely used antioxidants such as captopril, Trolox, reduced glutathione and lipoic acid as well as protein glycation inhibitors (aminoguanidine). Pleiotropic action of meloxicam may increase the effectiveness of anti-inflammatory treatment in diseases with oxidative stress etiology.

Different phytotoxic effect of Lolium multiflorum Lam. leaves against Echinochloa oryzoides (Ard.) Fritsch and Oryza sativa L.

Rice cultivation, particularly prone to weed issues, requires practices able to effectively control them, however reducing the use of herbicides, responsible for damage to human health and ecosystem sustainability. Alternative strategies for weed management can be based on plant-plant interaction phenomena. In this context, a group of organic farmers has developed a pragmatic approach for weed containment using Lolium multiflorum Lam. as a cover crop before rice. The present study aimed to confirm the farmer field observations reporting a preferential inhibitory effect of L. multiflorum on Echinochloa oryzoides (Ard.) Fritsch, one of the most yield-damaging rice weed, compared with Oryza sativa L. The study showed that L. multiflorum was able to significantly reduce the seed germination of E. oryzoides. It was found to be more susceptible than O. sativa both to the effect of the aqueous extract and powder of L. multiflorum leaves (23-79% vs. 3-57% and 26-100% vs. 23-31%, respectively). In addition, the leaf extract was able to affect E. oryzoides growth starting from 20% concentration both in relation to the root and shoot length while O. sativa exhibited differences compared with the control only under the influence of extract 50%. The L. multiflorum leaf characterization by NMR and UPLC-HR-MS analyses led to the identification of 35 compounds including several polyphenols, glycosyl flavonoids and glycosyl terpenoids, as well as different amino acids and organic acids. Some of them (e.g. protocatechuic and gallic acids) are already known as allelochemicals confirming that L. multiflorum is a source of plant growth inhibitors.

The Preferential Inhibitory Effect of Olmesartan, a New Angiotensin II Type 1 Antagonist, on Sympathetic Nerve Terminals in Isolated Canine Splenic Artery

Effects of olmesartan (RNH-6270: (5-methyl-2-oxo-1,3-dioxolen-4-yl)methoxy-4-(1-hydroxy-1-methylethyl)-2-propyl{4-[2-(tetrazol-5-yl)-phenyl]phenyl}methylimidazol-5-carboxylase, an active form of olmesartan medoxomil (CS-866)) was investigated in isolated, perfused canine splenic arterial preparations. Neither exogenous noradrenaline- nor ATP-induced vasoconstrictor responses were modified by treatment with the used concentrations of olmesartan (1 – 100 nM). A high concentration of 10 nM angiotensin II caused a potentiation of either noradrenaline- and ATP-induced constrictions, although 1 nM angiotensin II did not induce any potentiating effects for these responses. These potentiations were inhibited by olmesartan in a concentration-related manner. Periarterial nerve electrical stimulation (PNS) readily induced a biphasic constriction consisting of an initial P2X purinoceptor-mediated vasoconstriction followed by a prolonged mainly α1-adrenoceptor-mediated response. PNS-induced 1st and 2nd peaked responses were significantly inhibited by olmesartan in a concentration-related manner. With a low concentration of 1 nM angiotensin II, which did not induce any vascular effects by itself, PNS-induced responses were markedly enhanced. The enhanced responses were inhibited by olmesartan. It is concluded that endogenous angiotensin II exerts its stimulating action on the releases of ATP and noradrenaline from the periarterial sympathetic nerve terminal, and olmesartan has an inhibitory property on angiotensin II-induced potentiation of endogenous ATP- and noradrenaline-induced responses.

Sulfonate and sulfamate derivatives possessing benzofuran or benzothiophene nucleus as potent carbonic anhydrase II/IX/XII inhibitors

In the current work, we report the discovery of new sulfonate and sulfamate derivatives of benzofuran- and benzothiophene as potent inhibitors of human carbonic anhydrases (hCAs) II, IX and XII. A set of derivatives, 1a–t, having different substituents on the fused benzofuran and benzothiophene rings (R = alkyl, cyclohexyl, aryl, NH2, NHMe, or NMe2) was designed and synthesized. Most of the derivatives exhibited higher potency than acetazolamide as inhibitors of the purified hCAII, IX and XII isoforms. The most potent inhibitors for hCAII, hCAIX and hCAXII were 1g, 1b and 1d with an IC50 ± SEM values of 0.14 ± 0.03, 0.13 ± 0.03 and 0.17 ± 0.06 µM, respectively. In addition, compounds 1d and 1n exerted preferential inhibitory effect against hCAXII isozyme with good potencies. Some selected compounds were docked within the active pocket of these isozymes and binding of the molecules revealed that sulfonate and sulfamate rings were located towards the active cavity and compounds coordinated to zinc ions.

Oral Administration of Meloxicam Suppresses Low-Dose Endotoxin Challenge–Induced Pain in Thoroughbred Horses

Nonsteroidal anti-inflammatory drugs such as flunixin meglumine have been used to treat signs of systemic inflammatory conditions, but it is also known to have the side effect to small intestine mucosa. It may be considered to be due to inhibition of both cyclooxygenase (COX)-1 and COX-2. On the other hand, meloxicam is widely used in equine clinical practice and an effective nonsteroidal anti-inflammatory drug with the preferential inhibitory effect on COX-2. However, it has not yet been evaluated in equine systemic inflammation. The aim of this study was to evaluate the effect of meloxicam administered 60 minutes prior lipopolysaccharide (LPS)-induced inflammatory response in five Thoroughbred horses using a crossover test. Clinical parameters including body temperature, heart rate, respiratory rate, behavioral pain score, and hoof wall surface temperature were recorded, and plasma tumor necrosis factor-alpha, cortisol, and leukocyte counts were measured at various times before and after LPS infusion for 420 minutes. At time points 60, 90 (P < .01), 120, and 180 (P < .05) minutes, pain scores were significantly lower in meloxicam-treated horses. There was no significant difference in other parameters. In the present study, we revealed the analgesic effect of meloxicam using an equine low-dose endotoxin model.

2-Deoxy-D-Glucose inhibits aggressive triple-negative breast cancer cells by targeting glycolysis and the cancer stem cell phenotype

Due to limited availability of pharmacological therapies, triple-negative breast cancer (TNBC) is the subtype with worst outcome. We hypothesised that 2-Deoxy-D-Glucose (2-DG), a glucose analogue, may hold potential as a therapy for particularly aggressive TNBC. We investigated 2-DG’s effects on TNBC cell line variants, Hs578T parental cells and their isogenic more aggressive Hs578Ts(i)8 variant, using migration, invasion and anoikis assays. We assessed their bioenergetics by Seahorse. We evaluated metabolic alterations using a Seahorse XF Analyzer, citrate synthase assay, immunoblotting and flow cytometry. We assessed the cancer stem cell (CSC) phenotype of the variants and 2-DG’s effects on CSCs. 2-DG significantly inhibited migration and invasion of Hs578Ts(i)8 versus Hs578T and significantly decreased their ability to resist anoikis. Investigating 2-DG’s preferential inhibitory effect on the more aggressive cells, we found Hs578Ts(i)8 also had significantly decreased oxidative phosphorylation and increased glycolysis compared to Hs578T. This is likely due to mitochondrial dysfunction in Hs578Ts(i)8, shown by their significantly decreased mitochondrial membrane potential. Furthermore, Hs578Ts(i)8 had a significantly increased proportion of cells with CSC phenotype, which was significantly decreased by 2-DG. 2-DG may have benefit as a therapy for TNBC with a particularly aggressive phenotype, by targeting increased glycolysis. Studies of more cell lines and patients’ specimens are warranted.

Antibiotic anisomycin selectively targets leukemia cell lines and patient samples through suppressing Wnt/β-catenin signaling

Chronic myeloid leukemia (CML) responds well to BCR-ABL tyrosine kinase inhibitors (TKI), such as imatinib and dasatinib. However, these inhibitors have been less effective as single agents in the blast phase-CML. In this work, we show that anisomycin, a clinically available drug, targets CML cells at all stages of development and enhances BCR-ABL TKIs’ efficacy. Anisomycin at nanomolar concentration inhibits proliferation and induces apoptosis in a panel of CML cell lines in a dose-dependent manner. It induces apoptosis CD34 stem/progenitor cells isolated from patients with blast phase CML. Using colony formation and serial replating assays, we further show that anisomycin inhibits CML CD34 cell differentiation, proliferation and self-renewal. Additionally, anisomycin is less effective in normal bone marrow (NBM) CD34 cells, suggesting the selective anti-leukemia activity of anisomycin. Combination of anisomycin with imatinib or dasatinib achieves significantly better efficacy than TKI alone in leukemia cell lines and patient samples while sparing normal counterparts. Mechanistically, we demonstrate that p38 MAPK/JNK activation is not required for anti-leukemia activities of anisomycin. Instead, anisomycin displays preferential inhibitory effects to Wnt/β-catenin-mediated signaling in CML. Our work provides the preclinical evidence on the potent efficacy of anisomycin in leukemia and its mechanisms of action. Our work suggests that anisomycin is a potential drug to overcome resistance to BCR-ABL TKI treatment in blast phase CML.

Preferential Disruption of Prefrontal GABAergic Function by Nanomolar Concentrations of the α7nACh Negative Modulator Kynurenic Acid.

Increased concentrations of kynurenic acid (KYNA) in the prefrontal cortex (PFC) are thought to contribute to the development of cognitive deficits observed in schizophrenia. Although this view is consistent with preclinical studies showing a negative impact of prefrontal KYNA elevation on executive function, the mechanism underlying such a disruption remains unclear. Here, we measured changes in local field potential (LFP) responses to ventral hippocampal stimulation in vivo and conducted whole-cell patch-clamp recordings in brain slices to reveal how nanomolar concentrations of KYNA alter synaptic transmission in the PFC of male adult rats. Our data show that prefrontal infusions of KYNA attenuated the inhibitory component of PFC LFP responses, a disruption that resulted from local blockade of α7-nicotinic acetylcholine receptors (α7nAChR). At the cellular level, we found that the inhibitory action exerted by KYNA in the PFC occurred primarily at local GABAergic synapses through an α7nAChR-dependent presynaptic mechanism. As a result, the excitatory-inhibitory ratio of synaptic transmission becomes imbalanced in a manner that correlates highly with the level of GABAergic suppression by KYNA. Finally, prefrontal infusion of a GABAAR positive allosteric modulator was sufficient to overcome the disrupting effect of KYNA and normalized the pattern of LFP inhibition in the PFC. Thus, the preferential inhibitory effect of KYNA on prefrontal GABAergic transmission could contribute to the onset of cognitive deficits observed in schizophrenia because proper GABAergic control of PFC output is one key mechanism for supporting such cortical functions.SIGNIFICANCE STATEMENT Brain kynurenic acid (KYNA) is an astrocyte-derived metabolite and its abnormal elevation in the prefrontal cortex (PFC) is thought to impair cognitive functions in individuals with schizophrenia. However, the mechanism underlying the disrupting effect of KYNA remains unclear. Here we found that KYNA biases the excitatory-inhibitory balance of prefrontal synaptic activity toward a state of disinhibition. Such disruption emerges as a result of a preferential suppression of local GABAergic transmission by KYNA via presynaptic inhibition of α7-nicotinic acetylcholine receptor signaling. Therefore, the degree of GABAergic dysregulation in the PFC could be a clinically relevant contributing factor for the onset of cognitive deficits resulting from abnormal increases of cortical KYNA.

Histone acetyltransferase inhibitor CPTH6 preferentially targets lung cancer stem-like cells.

Cancer stem cells (CSCs) play an important role in tumor initiation, progression, therapeutic failure and tumor relapse. In this study, we evaluated the efficacy of the thiazole derivative 3-methylcyclopentylidene-[4-(4′-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6), a novel pCAF and Gcn5 histone acetyltransferase inhibitor, as a small molecule that preferentially targets lung cancer stem-like cells (LCSCs) derived from non-small cell lung cancer (NSCLC) patients. Notably, although CPTH6 inhibits the growth of both LCSC and NSCLC cell lines, LCSCs exhibit greater growth inhibition than established NSCLC cells. Growth inhibitory effect of CPTH6 in LCSC lines is primarily due to apoptosis induction. Of note, differentiated progeny of LCSC lines is more resistant to CPTH6 in terms of loss of cell viability and reduction of protein acetylation, when compared to their undifferentiated counterparts. Interestingly, in LCSC lines CPTH6 treatment is also associated with a reduction of stemness markers. By using different HAT inhibitors we provide clear evidence that inhibition of HAT confers a strong preferential inhibitory effect on cell viability of undifferentiated LCSC lines when compared to their differentiated progeny. In vivo, CPTH6 is able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Strikingly, the ability of CPTH6 to inhibit tubulin acetylation is also confirmed in vivo. Overall, our studies propose histone acetyltransferase inhibition as an attractive target for cancer therapy of NSCLC.

Identification of β-glucosidase 1 as a biomarker and its high expression in hepatocellular carcinoma is associated with resistance to chemotherapy drugs

Context and objective: Long-term prognosis of hepatocellular carcinoma (HCC) patients is challenging, and novel biomarkers are needed to predict patient risk and serve as potential therapeutic target.Results: We found β-glucosidase 1 is significantly overexpressed and activated in primary HCC tissue and multiple HCC cell lines. β-Glucosidase 1 expression is associated with predicting prognosis of HCC patients under chemotherapy. Silencing β-glucosidase 1 inhibits growth and survival of HCC cells, with preferential inhibitory effects on high β-glucosidase 1-expressing cells. Combination of chemo drug with β-glucosidase 1 inhibitor sensitized HCC cells to chemotherapy.Conclusion: Our data support β-glucosidase 1 as a HCC biomarker due to its prognosis significance.

Therapeutic potential of mTOR inhibitors for targeting cancer stem cells.

The mammalian target of rapamycin (mTOR) pathway is aberrantly activated in many cancer types. As the intricate network of regulatory mechanisms controlling mTOR activity is uncovered, more refined drugs are designed and tested in clinical trials. While first generation mTOR inhibitors have failed to show clinical efficacy due partly to the feedback relief of oncogenetic circuits, newly developed inhibitors show greater promise as anti-cancer agents. An effective drug must defeat the cancer stem cells (CSCs) while sparing the normal stem cells. Due to its opposing role on normal and malignant stem cells, mTOR lends itself very well as a therapeutic target. Indeed, a preferential inhibitory effect on CSCs has already been shown for some mTOR inhibitors. These results provide a compelling rationale for the clinical development of mTOR-targeted therapies.

Combination therapy of prostate cancer with HPMA copolymer conjugates containing PI3K/mTOR inhibitor and docetaxel

Combination therapies have been investigated to address the current challenges of anti-cancer therapeutics. In particular, a novel paradigm of combination therapy targeting both cancer stem/progenitor cells and bulk tumor cells is promising to improve the long-term therapeutic benefit against prostate cancer. Among the therapeutic agents with anti-CSC activities, the PI3K/mTOR inhibitors exhibit preferential inhibitory effect on prostate cancer stem/progenitor cells and potent cytotoxicity against bulk tumor cells. The combination of PI3K/mTOR inhibitor and traditional chemotherapy docetaxel may show superior therapeutic effect over single drug treatment. Aiming to further improve the combinational anti-tumor and anti-CSC effect, we developed the combination therapy containing two HPMA copolymer–drug conjugates, incorporated with PI3K/mTOR inhibitor GDC-0980 (P-(GDC-0980)) and docetaxel (P-DTX), respectively. The anti-tumor and anti-CSC effects of the single and combination therapy were investigated in vitro and on PC-3 prostate cancer xenografts in nude mice. Our evaluations showed that P-(GDC-0980) suppressed CD133+ prostate stem/progenitor cell growth even at the low dose which does not cause significant growth inhibition in bulk tumor cells. The combination therapy exhibited effective anti-CSC effect as well as enhanced anti-bulk tumor effect in vitro. Among all the single and combination dosing regimens of free drugs and conjugates, the macromolecular combination therapy showed significantly prolonged mice survival in vivo.

Abstract 236: Pharmacological and genetic inhibition of FAK attenuates cancer stem cell function in vitro and in vivo.

Abstract Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that orchestrates cell signaling through integrins and growth factor receptors. FAK has been implicated in multiple steps of carcinogenesis including tumor initiation, growth and metastasis. Amplification and overexpression of FAK have been observed in multiple aggressive human cancers including breast and ovarian. VS-4718 is a potent and selective FAK kinase inhibitor that was previously shown by us to exhibit preferential inhibitory activities on breast cancer stem cells. We have further extended our investigation of the role of FAK on cancer stem cells to other solid tumors and report here that pharmacological attenuation of FAK activity by VS-4718 or RNAi-mediated depletion of FAK exhibits preferential inhibitory effects on cancer stem cells. To determine if FAK plays a role in the biology of cancer stem cells, we depleted FAK expression in breast, ovarian and mesothelioma cancer cell lines by RNAi. Our results indicated that shRNA-mediated knock-down of FAK inhibits tumorsphere formation in vitro. In parallel, VS-4718 was evaluated in a multitude of cancer stem cell assays both in vitro and in vivo. Pre-treatment of SUM159 cells with VS-4718 in matrigel reduced the percentage of ALDEFLUOR+ cancer stem cells and side population (SP). Similar effects were observed in ovarian cancer cell lines OVCAR-8 and OVCAR-5 where VS-4718 inhibited cancer stem cells as measured by multiple CSC assays. In an analogous fashion, VS-4718 also reduced the proportion of the ALDEFLUOR+ cells in H2052 human mesothelioma cells. In direct contrast, standard-of-care agents paclitaxel, carboplatin or pemetrexed increased the percentage of cancer stem cells, suggesting these agents do not effectively target cancer stem cells. Importantly combination of VS-4718 with standard-of-care agents attenuated chemotherapy-induced increases in the percentage of cancer stem cells in vitro in all three cancer models. The in vivo effect of VS-4718 on cancer stem cells was evaluated in SUM159 and MDA-MB-231 human triple negative breast cancer xenograft models. Following systemic administration, VS-4718 significantly reduced the proportion of cancer stem cells in tumors as evidenced by decreases in the percentage of ALDEFLUOR+ cells and tumorsphere-forming efficiency relative to vehicle-treated tumors and significantly abrogated tumor-initiating capabilities of cancer cells in a limiting dilution re-implantation assay. In summary, our results indicate the importance of FAK in the self-renewal of cancer stem cells in vitro and in vivo, and support the clinical development of FAK inhibitors to target cancer stem cells for the treatment of multiple cancers. Citation Format: Vihren Kolev, Quentin Wright, Christian Vidal, Irina Shapiro, Mahesh Pavdal, Mitchell Keegan, Qunli Xu, Jonathan Pachter. Pharmacological and genetic inhibition of FAK attenuates cancer stem cell function in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 236. doi:10.1158/1538-7445.AM2013-236