Pu-erh Tea Reduces Nitric Oxide Levels in Rats by Inhibiting Inducible Nitric Oxide Synthase Expression through Toll-Like Receptor 4

Pu-erh tea undergoes a unique fermentation process and contains theabrownins, polysaccharides and caffeine; although it is unclear about which component is associated with the down regulation of nitric oxide levels or how this process is mediated. To address this question we examined the effects of pu-erh tea on nitric oxide synthase (NOS) genes. Cohorts of rats were separately given four-week treatments of water as control, pu-erh tea, or the tea components: theabrownins, caffeine or polysaccharides. Five experimental groups were injected with lipopolysaccharides (LPS) to induce nitric oxide (NO) production, while the corresponding five control groups were injected with saline as a negative control. The serum and liver NO concentrations were examined and the NOS expression of both mRNA and protein was measured in liver. The results showed that the rats which were fed pu-erh tea or polysaccharides had lower levels of NO which corresponded with the down-regulation of inducible nitric oxide synthase (iNOS) expression. We further demonstrate that this effect is mediated through reduction of Toll-like receptor 4 (TLR4) signaling. Thus we find that the polysaccharide components in pu-erh tea reduce NO levels in an animal model by inhibiting the iNOS expression via signaling through TLR4.     

The Role of the BH4 Cofactor in Nitric Oxide Synthase Activity and Cancer Progression: Two Sides of the Same Coin

Cancer development is associated with abnormal proliferation, genetic instability, cell death resistance, metabolic reprogramming, immunity evasion, and metastasis. These alterations are triggered by genetic and epigenetic alterations in genes that control cell homeostasis. Increased reactive oxygen and nitrogen species (ROS, RNS) induced by different enzymes and reactions with distinct molecules contribute to malignant transformation and tumor progression by modifying DNA, proteins, and lipids, altering their activities. Nitric oxide synthase plays a central role in oncogenic signaling modulation and redox landscape. Overexpression of the three NOS isoforms has been found in innumerous types of cancer contributing to tumor growth and development. Although the main function of NOS is the production of nitric oxide (NO), it can be a source of ROS in some pathological conditions. Decreased tetrahydrobiopterin (BH4) cofactor availability is involved in NOS dysfunction, leading to ROS production and reduced levels of NO. The regulation of NOSs by BH4 in cancer is controversial since BH4 has been reported as a pro-tumoral or an antitumoral molecule. Therefore, in this review, the role of BH4 in the control of NOS activity and its involvement in the capabilities acquired along tumor progression of different cancers was described.     

The Roles of Nitric Oxide Synthase/Nitric Oxide Pathway in the Pathology of Vascular Dementia and Related Therapeutic Approaches

Vascular dementia (VaD) is the second most common form of dementia worldwide. It is caused by cerebrovascular disease, and patients often show severe impairments of advanced cognitive abilities. Nitric oxide synthase (NOS) and nitric oxide (NO) play vital roles in the pathogenesis of VaD. The functions of NO are determined by its concentration and bioavailability, which are regulated by NOS activity. The activities of different NOS subtypes in the brain are partitioned. Pathologically, endothelial NOS is inactivated, which causes insufficient NO production and aggravates oxidative stress before inducing cerebrovascular endothelial dysfunction, while neuronal NOS is overactive and can produce excessive NO to cause neurotoxicity. Meanwhile, inflammation stimulates the massive expression of inducible NOS, which also produces excessive NO and then induces neuroinflammation. The vicious circle of these kinds of damage having impacts on each other finally leads to VaD. This review summarizes the roles of the NOS/NO pathway in the pathology of VaD and also proposes some potential therapeutic methods that target this pathway in the hope of inspiring novel ideas for VaD therapeutic approaches.     

The Discovery of Potentially Selective Human Neuronal Nitric Oxide Synthase (nNOS) Inhibitors: A Combination of Pharmacophore Modelling,CoMFA, Virtual Screening and Molecular Docking Studies

Neuronal nitric oxide synthase (nNOS) plays an important role in neurotransmission and smooth muscle relaxation. Selective inhibition of nNOS over its other isozymes is highly desirable for the treatment of neurodegenerative diseases to avoid undesirable effects. In this study, we present a workflow for the identification and prioritization of compounds as potentially selective human nNOS inhibitors. Three-dimensional pharmacophore models were constructed based on a set of known nNOS inhibitors. The pharmacophore models were evaluated by Pareto surface and CoMFA (Comparative Molecular Field Analysis) analyses. The best pharmacophore model, which included 7 pharmacophore features, was used as a search query in the SPECS database (SPECS^®, Delft, The Netherlands). The hit compounds were further filtered by scoring and docking. Ten hits were identified as potential selective nNOS inhibitors.     

Diabetes Mellitus Alters the Immuno-Expression of Neuronal Nitric Oxide Synthase in the Rat Pancreas

Nitric oxide is generated from nitric oxide synthase following hyperglycemia-induced oxidative stress during the course of diabetes mellitus (DM). We examined the temporal immuno-expression of neuronal nitric oxide synthase (nNOS) in the pancreas of diabetic and non-diabetic rats using immunohistochemical, immunofluorescence and western blot techniques 12 h, 24 h, 1 week, 2 weeks, 1, 8 and 15 months after induction of DM. nNOS co-localized with pancreatic beta cells but disappears 12 h after the onset of DM. In contrast, the nNOS content of pancreatic nerves increased significantly (p < 0.001) 24 h after the induction of DM, and decreased sharply thereafter. However, nNOS-positive ganglion cells were observed even 15 months post-diabetes. ROS increased by more than 100% two months after the onset of DM compared to non-diabetic control but was significantly (p < 0.000001) reduced at 9 months after the induction of DM. The pancreatic content of GSH increased significantly (p < 0.02) after 9 months of DM. Although, TBARS content was significantly (p < 0.009; p < 0.002) lower in aged (9 months) non-diabetic and DM rats, TBARS rate was markedly (p < 0.02) higher 9 months after the induction of DM when compared to younger age group. In conclusion, nNOS is present in pancreatic beta cell, but disappears 12 h after the onset of diabetes. In contrast, the tissue level of nNOS of pancreatic nerves increased in the first week of diabetes, followed by a sharp reduction. nNOS may play important roles in the metabolism of pancreatic beta cell.     

Trimethylamine N-Oxide (TMAO) Impairs Purinergic Induced Intracellular Calcium Increase and Nitric Oxide Release in Endothelial Cells

Trimethylamine N-oxide (TMAO) is a diet derived compound directly introduced through foodstuff, or endogenously synthesized from its precursors, primarily choline, L-carnitine, and ergothioneine. New evidence outlines high TMAO plasma concentrations in patients with overt cardiovascular disease, but its direct role in pathological development is still controversial. The purpose of the study was to evaluate the role of TMAO in affecting key intracellular factors involved in endothelial dysfunction development, such as reactive oxygen species, mitochondrial health, calcium balance, and nitric oxide release using bovine aortic endothelial cells (BAE-1). Cell viability and oxidative stress indicators were monitored after acute and prolonged TMAO treatment. The role of TMAO in interfering with the physiological purinergic vasodilatory mechanism after ATP stimulation was defined through measurements of the rise of intracellular calcium, nitric oxide release, and eNOS phosphorylation at Ser1179 (eNOS^Ser1179). TMAO was not cytotoxic for BAE-1 and it did not induce the rise of reactive oxygen species and impairment of mitochondrial membrane potential, either in the basal condition or in the presence of a stressor. In contrast, TMAO modified the purinergic response affecting intracellular ATP-induced calcium increase, nitric oxide release, and eNOS^Ser1179. Results obtained suggest a possible implication of TMAO in impairing the endothelial-dependent vasodilatory mechanism.     

Costunolide Induces Apoptosis via the Reactive Oxygen Species and Protein Kinase B Pathway in Oral Cancer Cells

Oral cancer (OC) has been attracted research attention in recent years as result of its high morbidity and mortality. Costunolide (CTD) possesses potential anticancer and bioactive abilities that have been confirmed in several types of cancers. However, its effects on oral cancer remain unclear. This study investigated the potential anticancer ability and underlying mechanisms of CTD in OC in vivo and in vitro. Cell viability and anchorage-independent colony formation assays were performed to examine the antigrowth effects of CTD on OC cells; assessments for migration and invasion of OC cells were conducted by transwell; Cell cycle and apoptosis were investigated by flow cytometry and verified by immunoblotting. The results revealed that CTD suppressed the proliferation, migration and invasion of oral cancer cells effectively and induced cell cycle arrest and apoptosis; regarding the mechanism, CTD bound to AKT directly by binding assay and repressed AKT activities through kinase assay, which thereby downregulating the downstream of AKT. Furthermore, CTD remarkably promotes the generation of reactive oxygen species by flow cytometry assay, leading to cell apoptosis. Notably, CTD strongly suppresses cell-derived xenograft OC tumor growth in an in vivo mouse model. In conclusion, our results suggested that costunolide might prevent progression of OC and promise to be a novel AKT inhibitor.     

Chrysin Suppressed Inflammatory Responses and the Inducible Nitric Oxide Synthase Pathway after Spinal Cord Injury in Rats

Chrysin (CH), a natural plant flavonoid, has shown a variety of beneficial effects. Our present study was conducted to evaluate the therapeutic potential of CH three days after spinal cord injury (SCI) in rats and to probe the underlying neuroprotective mechanisms. SCI was induced using the modified weight-drop method in Wistar rats. Then, they were treated with saline or CH by doses of 30 and 100 mg/kg for 26 days. Neuronal function was assessed with the Basso Beattle Bresnahan locomotor rating scale (BBB). The water content of spinal cord was determined after traumatic SCI. The NF-κB p65 unit, TNF-α, IL-1β and IL-6 in serums, as well as the apoptotic marker, caspase-3, of spinal cord tissues were measured using commercial kits. The protein level and activity of inducible nitric oxide synthase (iNOS) were detected by western blot and a commercial kit, respectively. NO (nitric oxide) production was evaluated by the determination of nitrite concentration. The rats with SCI showed marked reductions in BBB scores, coupled with increases in the water content of spinal cord, the NF-κB p65 unit, TNF-α, IL-1β, IL-6, iNOS, NO production and caspase-3. However, a CH supplement dramatically promoted the recovery of neuronal function and suppressed the inflammatory factors, as well as the iNOS pathway in rats with SCI. Our findings disclose that CH improved neural function after SCI in rats, which might be linked with suppressing inflammation and the iNOS pathway.     

HCA (2-Hydroxy-Docosahexaenoic Acid) Induces Apoptosis and Endoplasmic Reticulum Stress in Pancreatic Cancer Cells

Pancreatic cancer has a high mortality rate due to its aggressive nature and high metastatic rate. When coupled to the difficulties in detecting this type of tumor early and the lack of effective treatments, this cancer is currently one of the most important clinical challenges in the field of oncology. Melitherapy is an innovative therapeutic approach that is based on modifying the composition and structure of cell membranes to treat different diseases, including cancers. In this context, 2-hydroxycervonic acid (HCA) is a melitherapeutic agent developed to combat pancreatic cancer cells, provoking the programmed cell death by apoptosis of these cells by inducing ER stress and triggering the production of ROS species. The efficacy of HCA was demonstrated in vivo, alone and in combination with gemcitabine, using a MIA PaCa-2 cell xenograft model of pancreatic cancer in which no apparent toxicity was evident. HCA is metabolized by α-oxidation to C21:5n-3 (heneicosapentaenoic acid), which in turn also showed anti-proliferative effect in these cells. Given the unmet clinical needs associated with pancreatic cancer, the data presented here suggest that the use of HCA merits further study as a potential therapy for this condition.     

Arylquin 1 (Potent Par-4 Secretagogue) Inhibits Tumor Progression and Induces Apoptosis in Colon Cancer Cells

Colorectal cancer (CRC) is one of the most common gastrointestinal cancers worldwide. Current therapeutic strategies mainly involve surgery and chemoradiotherapy; however, novel antitumor compounds are needed to avoid drug resistance in CRC, as well as the severe side effects of current treatments. In this study, we investigated the anticancer effects and underlying mechanisms of Arylquin 1 in CRC. The MTT assay was used to detect the viability of SW620 and HCT116 cancer cells treated with Arylquin 1 in a dose-dependent manner in vitro. Further, wound-healing and transwell migration assays were used to evaluate the migration and invasion abilities of cultured cells, and Annexin V was used to detect apoptotic cells. Additionally, Western blot was used to identify the expression levels of N-cadherin, caspase-3, cyclin D1, p-extracellular signal-regulated kinase (ERK), p-c-JUN N-terminal kinase (JNK), and phospho-p38, related to key signaling proteins, after administration of Arylquin 1. Xenograft experiments further confirmed the effects of Arylquin 1 on CRC cells in vivo. Arylquin 1 exhibited a dose-dependent reduction in cell viability in cultured CRC cells. It also inhibited cell proliferation, migration, and invasion, and induced apoptosis. Mechanistic analysis demonstrated that Arylquin 1 increased phosphorylation levels of ERK, JNK, and p38. In a mouse xenograft model, Arylquin 1 treatment diminished the growth of colon tumors after injection of cultured cancer cells. Arylquin 1 may have potential anticancer effects and translational significance in the treatment of CRC.     

G Protein-Coupled Receptor 87 (GPR87) Promotes Cell Proliferation in Human Bladder Cancer Cells

G protein-coupled receptor 87 (GPR87) is a newly deorphanized member of the cell surface molecule G protein-coupled receptor family. GPR signaling was shown to play a role in promotion of cell growth and survival, metastasis, and drug resistance. The overexpression of GPR87 has also been reported in many malignant tumors including bladder cancer. The aim of the present study is to examine the effect of silencing GPR87 expression with a replication-deficient recombinant adenoviral vector expressing short hairpin RNA targeting GPR87 (Ad-shGPR87) and to explore the underlying molecular mechanisms in bladder cancer cells. Six GPR87-expressing human bladder cancer cells, HT1197, HT1376, J82, RT112, TCCSUP and UMUC3, were used. Infection with Ad-shGPR87 effectively downregulated the GPR87 expression, and significantly reduced the percentage of viable cells in 4 of 6 cell lines as detected by an MTT assay. Significant inhibition on cell proliferation with Ad-shGPR87 was observed in the wild-type p53 bladder cancer cell lines (HT1197, RT112, TCCSUP and UMUC3), but not in the mutant p53 cells (HT1376 and J82). As represented by a wild-type p53 RT112 cell, Ad-shGPR87 infection significantly enhanced p53 and p21 expression and caused caspase-dependent apoptosis. Furthermore, the treatment with Ad-shGPR87 exerted a significant antitumor effect against the GPR87-expressing RT112 xenografts. GPR87 appeared to be a promising target for gene therapy, and Ad-shGPR87 had strong antitumor effects, specifically anti-proliferative and pro-apoptotic effects, against GPR87-expressing human bladder cancer cells.     

Role of Nitric Oxide in Gene Expression Regulation during Cancer: Epigenetic Modifications and Non-Coding RNAs

Nitric oxide (NO) has been identified and described as a dual mediator in cancer according to dose-, time- and compartment-dependent NO generation. The present review addresses the different epigenetic mechanisms, such as histone modifications and non-coding RNAs (ncRNAs), miRNA and lncRNA, which regulate directly or indirectly nitric oxide synthase (NOS) expression and NO production, impacting all hallmarks of the oncogenic process. Among lncRNA, HEIH and UCA1 develop their oncogenic functions by inhibiting their target miRNAs and consequently reversing the inhibition of NOS and promoting tumor proliferation. The connection between miRNAs and NO is also involved in two important features in cancer, such as the tumor microenvironment that includes key cellular components such as tumor-associated macrophages (TAMs), cancer associated fibroblasts (CAFs) and cancer stem cells (CSCs).     

Decreased Expression and Uncoupling of Endothelial Nitric Oxide Synthase in the Cerebral Cortex of Rats with Thioacetamide-Induced Acute Liver Failure

Acute liver failure (ALF) is associated with deregulated nitric oxide (NO) signaling in the brain, which is one of the key molecular abnormalities leading to the neuropsychiatric disorder called hepatic encephalopathy (HE). This study focuses on the effect of ALF on the relatively unexplored endothelial NOS isoform (eNOS). The cerebral prefrontal cortices of rats with thioacetamide (TAA)-induced ALF showed decreased eNOS expression, which resulted in an overall reduction of NOS activity. ALF also decreased the content of the NOS cofactor, tetrahydro-L-biopterin (BH4), and evoked eNOS uncoupling (reduction of the eNOS dimer/monomer ratio). The addition of the NO precursor L-arginine in the absence of BH4 potentiated ROS accumulation, whereas nonspecific NOS inhibitor L-NAME or EDTA attenuated ROS increase. The ALF-induced decrease of eNOS content and its uncoupling concurred with, and was likely causally related to, both increased brain content of reactive oxidative species (ROS) and decreased cerebral cortical blood flow (CBF) in the same model.     

Echinacoside Induces Apoptosis in Human SW480 Colorectal Cancer Cells by Induction of Oxidative DNA Damages

Echinacoside is a natural compound with potent reactive oxygen species (ROS)-scavenging and anti-oxidative bioactivities, which protect cells from oxidative damages. As cancer cells are often under intense oxidative stress, we therefore tested if Echinacoside treatment would promote cancer development. Surprisingly, we found that Echinacoside significantly inhibited the growth and proliferation of a panel of cancer cell lines. Treatment of the human SW480 cancer cells with Echinacoside resulted in marked apoptosis and cell cycle arrest, together with a significant increase in active caspase 3 and cleaved PARP, and upregulation of the G1/S-CDK blocker CDKN1B (p21). Interestingly, immunocytochemistry examination of drug-treated cancer cells revealed that Echinacoside caused a significant increase of intracellular oxidized guanine, 8-oxoG, and dramatic upregulation of the double-strand DNA break (DSB)-binding protein 53BP1, suggesting that Echinacoside induced cell cycle arrest and apoptosis in SW480 cancer cells via induction of oxidative DNA damages. These results establish Echinacoside as a novel chemical scaffold for development of anticancer drugs.     

Anti-Oncogenic gem-Dihydroperoxides Induce Apoptosis in Cancer Cells by Trapping Reactive Oxygen Species

Organic gem-dihydroperoxides (DHPs) and their derived peroxides have attracted a great deal of attention as potential anti-cancer agents. However, the precise mechanism of their inhibitory effect on tumors is unknown. To determine the mechanism of the inhibitory effects of DHPs, we examined the effects of DHPs on leukemia K562 cells. As a result, certain DHPs used in this study exhibited growth-inhibitory activity according to a clear structure-activity relationship. The most potent DHP, 12AC3O, induced apoptosis in K562 cells, but not in peripheral blood monocytes (PBMCs) or fibroblast cells. 12AC3O induced apoptosis through the intrinsic mitochondrial pathway and thereafter through the extrinsic pathway. The activity of the former pathway was partly attenuated by a JNK inhibitor. Interestingly, 12AC3O induced apoptosis by trapping a large amount of ROS, leading to an extremely lower intracellular ROS level compared with that in the cells in the steady-state condition. These results suggest that an appropriate level of intracellular ROS was necessary for the maintenance of cancer cell growth. DHPs may have a potential to be a novel anti-cancer agent with minimum adverse effects on normal cells.     

Crassolide Induces G2/M Cell Cycle Arrest,Apoptosis, and Autophagy in Human Lung Cancer Cells via ROS-Mediated ER Stress Pathways

Crassolide, a cembranoid diterpene extracted from the soft coral Lobophytum crissum, has been proven to possess antioxidant and immunomodulatory properties. In the present study, we assessed the anticancer effects of crassolide on human H460 non-small-cell lung cancer (NSCLC) cells. We found that crassolide exerted cytotoxic effects on H460 cancer cells in vitro, inducing G2/M phase arrest and apoptosis. In addition, in H460 cells exposed to crassolide, the expression of the autophagy-related proteins LC3-II and beclin was increased, while the expression of p62 was decreased. Moreover, inhibiting autophagy with chloroquine (CQ) suppressed the crassolide-induced G2/M arrest and apoptosis of H460 cells. Moreover, we also found that crassolide induced endoplasmic reticulum (ER) stress in lung cancer cells by increasing the expression of ER stress marker proteins and that the crassolide-induced G2/M arrest, apoptosis, and autophagy were markedly attenuated by the ER stress inhibitor 4-phenylbutyric acid (4-PBA). Furthermore, we found that crassolide promoted reactive oxygen species (ROS) production by H460 cells and that the ROS inhibitor N-acetylcysteine (NAC) decreased the crassolide-induced ER stress, G2/M arrest, apoptosis, and autophagy. In conclusion, our findings show that crassolide inhibits NSCLC cell malignant biological behaviors for the first time, suggesting that this effect may be mechanistically achieved by inducing G2/M arrest, apoptosis, and autophagy through ROS accumulation, which activates the ER stress pathway. As a result of our findings, we now have a better understanding of the molecular mechanism underlying the anticancer effect of crassolide, and we believe crassolide might be a candidate for targeted cancer therapy.     

Low-Dose Tacrolimus Promotes the Migration and Invasion and Nitric Oxide Production in the Human-Derived First Trimester Extravillous Trophoblast Cells In Vitro

Placentation is one of the most important determinants for a successful pregnancy, and this is dependent on the process of trophoblast migration and invasion. Progesterone receptors (PGR) are critical effectors of progesterone (P4) signaling that is required for trophoblast migration and invasion conducive to a successful gestation. In immune complicated pregnancies, evidence has shown that abnormal placentation occurs because of aberrant expression of PGR. Therapeutic intervention with tacrolimus (FK506) was able to restore PGR expression and improve pregnancy outcomes in immune-complicated gestations; however, the exact mode of action of tacrolimus in assisting placentation is not clear. Here, we attempt to uncover the mode of action of tacrolimus by examining its effects on trophoblast invasion and migration in the human-derived extravillous trophoblast (EVT) cell line, the HTR-8/SVneo cells. Using a variety of functional assays, we demonstrated that low-dose tacrolimus (10 ng/mL) was sufficient to significantly (p < 0.001) stimulate the migration and invasion of the HTR-8/SVneo cells, inducing their cytosolic/nuclear progesterone receptor expression and activation, and modulating their Nitric Oxide (NO) production. Moreover, tacrolimus abrogated the suppressive effect of the NOS inhibitor N^ω- Nitro-L-Arginine Methyl Ester (L-NAME) on these vital processes critically involved in the establishment of human pregnancy. Collectively, our data suggest an immune-independent mode of action of tacrolimus in positively influencing placentation in complicated gestations, at least in part, through promoting the migration and invasion of the first trimester extravillous trophoblast cells by modulating their NO production and activating their cytosolic/nuclear progesterone-receptors. To our knowledge, this is the first report to show that the mode of action of tacrolimus as a monotherapy for implantation failure is plausibly PGR-dependent.     

Inhibition of the FGF/FGFR System Induces Apoptosis in Lung Cancer Cells via c-Myc Downregulation and Oxidative Stress

Lung cancer represents an extremely diffused neoplastic disorder with different histological/molecular features. Among the different lung tumors, non-small-cell lung cancer (NSCLC) is the most represented histotype, characterized by various molecular markers, including the expression/overexpression of the fibroblast growth factor receptor-1 (FGFR1). Thus, FGF/FGFR blockade by tyrosine kinase inhibitors (TKi) or FGF-ligand inhibitors may represent a promising therapeutic approach in lung cancers. In this study we demonstrate the potential therapeutic benefit of targeting the FGF/FGFR system in FGF-dependent lung tumor cells using FGF trapping (NSC12) or TKi (erdafitinib) approaches. The results show that inhibition of FGF/FGFR by NSC12 or erdafitinib induces apoptosis in FGF-dependent human squamous cell carcinoma NCI-H1581 and NCI-H520 cells. Induction of oxidative stress is the main mechanism responsible for the therapeutic/pro-apoptotic effect exerted by both NSC12 and erdafitinib, with apoptosis being abolished by antioxidant treatments. Finally, reduction of c-Myc protein levels appears to strictly determine the onset of oxidative stress and the therapeutic response to FGF/FGFR inhibition, indicating c-Myc as a key downstream effector of FGF/FGFR signaling in FGF-dependent lung cancers.