A Breast Cancer Stem Active Cobalt(III)-Cyclam Complex Containing Flufenamic Acid with Immunogenic Potential.

The cytotoxic and immunogenic-activating properties of a cobalt(III)-cyclam complex bearing the non-steroidal anti-inflammatory drug, flufenamic acid is reported within the context of anti-cancer stem cell (CSC) drug discovery. The cobalt(III)-cyclam complex 1 displays sub-micromolar potency towards breast CSCs grown in monolayers, 24-fold and 31-fold greater than salinomycin (an established anti-breast CSC agent) and cisplatin (an anticancer metallopharmaceutical), respectively. Strikingly, the cobalt(III)-cyclam complex 1 is 69-fold and 50-fold more potent than salinomycin and cisplatin towards three-dimensionally cultured breast CSC mammospheres. Mechanistic studies reveal that 1 induces DNA damage, inhibits cyclooxygenase-2 expression, and prompts caspase-dependent apoptosis. Breast CSCs treated with 1 exhibit damage-associated molecular patterns characteristic of immunogenic cell death and are phagocytosed by macrophages. As far as we are aware, 1 is the first cobalt complex of any oxidation state or geometry to display both cytotoxic and immunogenic-activating effects on breast CSCs.

Development of LM-41 and AF-2112, two flufenamic acid-derived TEAD inhibitors obtained through the replacement of the trifluoromethyl group by aryl rings.

The Hippo pathway regulates organ size and tissue homeostasis by controlling cell proliferation and apoptosis. The YAP-TEAD transcription factor, the downstream effector of the Hippo pathway, regulates the expression of genes such as CTGF, Cyr61, Axl and NF2. Aberrant Hippo activity has been identified in multiple types of cancers. Flufenamic acid (FA) was reported to bind in a liphophilic TEAD palmitic acid (PA) pocket, leading to reduction of the expression of Axl and NF2. Here, we show that the replacement of the trifluoromethyl moiety in FA by aromatic groups, directly connected to the scaffold or separated by a linker, leads to compounds with better affinity to TEAD. Co-crystallization studies show that these compounds bind similarly to FA, but deeper within the PA pocket. Our studies identified LM-41 and AF-2112 as two TEAD binders that strongly reduce the expression of CTGF, Cyr61, Axl and NF2. LM-41 gave the strongest reduction of migration of human MDA-MB-231 breast cancer cells.

Identification and validation of fusidic acid and flufenamic acid as inhibitors of SARS-CoV-2 replication using DrugSolver CavitomiX.

In this work, we present DrugSolver CavitomiX, a novel computational pipeline for drug repurposing and identifying ligands and inhibitors of target enzymes. The pipeline is based on cavity point clouds representing physico-chemical properties of the cavity induced solely by the protein. To test the pipeline's ability to identify inhibitors, we chose enzymes essential for SARS-CoV-2 replication as a test system. The active-site cavities of the viral enzymes main protease (Mpro) and papain-like protease (Plpro), as well as of the human transmembrane serine protease 2 (TMPRSS2), were selected as target cavities. Using active-site point-cloud comparisons, it was possible to identify two compounds-flufenamic acid and fusidic acid-which show strong inhibition of viral replication. The complexes from which fusidic acid and flufenamic acid were derived would not have been identified using classical sequence- and structure-based methods as they show very little structural (TM-score: 0.1 and 0.09, respectively) and very low sequence (~ 5%) identity to Mpro and TMPRSS2, respectively. Furthermore, a cavity-based off-target screening was performed using acetylcholinesterase (AChE) as an example. Using cavity comparisons, the human carboxylesterase was successfully identified, which is a described off-target for AChE inhibitors.

A Single High Dose of Flufenamic Acid in Rats does not Reduce the Damage Associated with the Rat Lithium-Pilocarpine Model of Status Epilepticus but Leads to Deleterious Outcomes.

BACKGROUND: Epilepsy is one of the most common neurologic diseases, and around 30% of all epilepsies, particularly the temporal lobe epilepsy (TLE), are highly refractory to current pharmacological treatments. Abnormal synchronic neuronal activity, brain glucose metabolism alterations, neurodegeneration and neuroinflammation are features of epilepsy. Further, neuroinflammation has been shown to contribute to dysregulation of neuronal excitability and the progression of epileptogenesis. Flufenamic acid (FLU), a non-steroidal anti-inflammatory drug, is also characterized by its wide properties as a dose-dependent ion channel modulator. In this context, in vitro studies have shown that it abolishes seizure-like events in neocortical slices stimulated with a gamma-aminobutyric acid A (GABAA) receptor blocker. However, little is known about its effects in animal models. Thus, our goal was to assess the efficacy and safety of a relatively high dose of FLU in the lithium-pilocarpine rat model of status epilepticus (SE). This animal model reproduces many behavioral and neurobiological features of TLE such as short-term brain hypometabolism, severe hippocampal neurodegeneration and inflammation reflected by a marked reactive astrogliosis. METHODS: FLU (100 mg/kg, i.p.) was administered to adult male rats, 150 min before SE induced by pilocarpine. Three days after the SE, brain glucose metabolism was assessed by 2-deoxy-2-[18F]-fluoro-D-glucose ([18F]FDG) positron emission tomography (PET). Markers of hippocampal integrity, neurodegeneration and reactive astrogliosis were also evaluated. RESULTS: FLU neither prevented the occurrence of the SE nor affected brain glucose hypometabolism as assessed by [18F]FDG PET. Regarding the neurohistochemical studies, FLU neither prevented neuronal damage nor hippocampal reactive astrogliosis. On the contrary, FLU increased the mortality rate and negatively affected body weight in the rats that survived the SE. CONCLUSIONS: Our results do not support an acute anticonvulsant effect of a single dose of FLU. Besides, FLU did not show short-term neuroprotective or anti-inflammatory effects in the rat lithium-pilocarpine model of SE. Moreover, at the dose administered, FLU resulted in deleterious effects.

Topical Application of Butyl Flufenamate Ointment Promotes Cranial Defect Healing in Mice by Inducing BMP2 Secretion in Skin Mesenchymal Stem Cells.

Bone defects and fractures heal slowly compared with injuries to other tissues, creating a heavy burden for patients, their families, and society. Alongside conventional treatment methods for fractures and bone defects, adjuvant therapies play an important but underappreciated role. In a previous study, we found that systemic administration of flufenamic acid promoted osteogenesis in vivo, but its side effects limited the application of our findings. In the present study, we assess the effects of external butyl flufenamate ointment on the healing of cranial defects in mice. We found that application of butyl flufenamate ointment on the surface of the skin accelerated the healing of cranial defects in mice by promoting BMP2 secretion from mouse-skin mesenchymal stem-cells. These findings indicate that butyl flufenamate ointment has potential therapeutic value for treating superficial fractures or bone defects while avoiding the toxicity and side effects of systemic medication, representing a safe and convenient adjuvant therapy to promote healing of superficial bone defects and fractures.

Flufenamic acid improves survival and neurologic outcome after successful cardiopulmonary resuscitation in mice.

BACKGROUND: Brain injury is the main cause of high mortality and disability after successful cardiopulmonary resuscitation (CPR) from sudden cardiac arrest (CA). The transient receptor potential M4 (TRPM4) channel is a novel target for ameliorating blood-brain barrier (BBB) disruption and neuroinflammation. Herein, we tested whether flufenamic acid (FFA), which is reported to block TRPM4 with high potency, could confer neuroprotection against brain injury secondary to CA/CPR and whether its action was exerted by blocking the TRPM4 channel. METHODS: Wild-type (WT) and Trpm4 knockout (Trpm4-/-) mice subjected to 10-min CA/CPR were randomized to receive FFA or vehicle once daily. Post-CA/CPR brain injuries including neurologic deficits, survival rate, histological damage, edema formation, BBB destabilization and neuroinflammation were assessed. RESULTS: In WT mice subjected to CA/CPR, FFA was effective in improving survival and neurologic outcome, reducing neuropathological injuries, attenuating brain edema, lessening the leakage of IgG and Evans blue dye, restoring tight junction protein expression and promoting microglia/macrophages from the pro-inflammatory subtype toward the anti-inflammatory subtype. In comparison to WT mice, Trpm4-/- mice exhibited less neurologic deficiency, milder histological impairment, more BBB integrity and more anti-inflammatory microglia/macrophage polarization. As expected, FFA did not provide a benefit of superposition compared with vehicle in the Trpm4-/- mice after CA/CPR. CONCLUSIONS: FFA mitigates BBB breach and modifies the functional status of microglia/macrophages, thereby improving survival and neurologic deficits following CA/CPR. The neuroprotective effects occur at least partially by interfering with the TRPM4 channel in the neurovascular unit. These results indicate the significant clinical potential of FFA to improve the prognosis for CA victims who are successfully resuscitated.

Connexin hemichannel inhibition ameliorates epidermal pathology in a mouse model of keratitis ichthyosis deafness syndrome.

Mutations in five different genes encoding connexin channels cause eleven clinically defined human skin diseases. Keratitis ichthyosis deafness (KID) syndrome is caused by point mutations in the GJB2 gene encoding Connexin 26 (Cx26) which result in aberrant activation of connexin hemichannels. KID syndrome has no cure and is associated with bilateral hearing loss, blinding keratitis, palmoplantar keratoderma, ichthyosiform erythroderma and a high incidence of childhood mortality. Here, we have tested whether a topically applied hemichhanel inhibitor (flufenamic acid, FFA) could ameliorate the skin pathology associated with KID syndrome in a transgenic mouse model expressing the lethal Cx26-G45E mutation. We found that FFA blocked the hemichannel activity of Cx26-G45E in vitro, and substantially reduced epidermal pathology in vivo, compared to untreated, or vehicle treated control animals. FFA did not reduce the expression of mutant connexin hemichannel protein, and cessation of FFA treatment allowed disease progression to continue. These results suggested that aberrant hemichannel activity is a major driver of skin disease in KID syndrome, and that the inhibition of mutant hemichannel activity could provide an attractive target to develop novel therapeutic interventions to treat this incurable disease.

Development of LM98, a Small-Molecule TEAD Inhibitor Derived from Flufenamic Acid.

The YAP-TEAD transcriptional complex is responsible for the expression of genes that regulate cancer cell growth and proliferation. Dysregulation of the Hippo pathway due to overexpression of TEAD has been reported in a wide range of cancers. Inhibition of TEAD represses the expression of associated genes, demonstrating the value of this transcription factor for the development of novel anti-cancer therapies. We report herein the design, synthesis and biological evaluation of LM98, a flufenamic acid analogue. LM98 shows strong affinity to TEAD, inhibits its autopalmitoylation and reduces the YAP-TEAD transcriptional activity. Binding of LM98 to TEAD was supported by 19 F-NMR studies while co-crystallization experiments confirmed that LM98 is anchored within the palmitic acid pocket of TEAD. LM98 reduces the expression of CTGF and Cyr61, inhibits MDA-MB-231 breast cancer cell migration and arrests cell cycling in the S phase during cell division.

Pro-IL-1ß Is an Early Prognostic Indicator of Severe Donor Lung Injury During Ex Vivo Lung Perfusion.

BACKGROUND: Ex vivo lung perfusion (EVLP) is used to evaluate and recondition extended criteria donor lungs for transplantation. Interleukin-1ß (IL-1ß) has been identified as a prognostic indicator of nonrecovery during EVLP. This may be an effect of inflammasome activation or cellular necrosis following donation and graft preservation. Delineating the mechanism of IL-1ß release is required. METHODS: The inactive intracellular precursor molecule, pro-IL-1ß, was characterized along with the pro-IL-1ß processing enzyme, caspase-1, in the perfusate of n = 20 human lungs that had undergone EVLP (n = 10 lungs that failed to recover and were discarded versus n = 10 lungs that reconditioned and were transplanted). In an experimental porcine model, n = 8 lungs underwent EVLP and were randomized to receive either a specific NLRP3 inflammasome inhibitor or control. RESULTS: Significant increases in pro-IL-1ß and caspase-1 were observed in the perfusate from human lungs that did not recondition during EVLP compared with those that successfully reconditioned and were used for transplantation. Within the porcine EVLP, NLRP3 inflammasome inhibition reduced IL-1ß within the perfusate compared with controls, but this had no impact on lung function, hemodynamics, or inflammation. CONCLUSIONS: Our data suggest that pro-IL-1ß is passively released following cellular necrosis of the donor lung.

Activation of multiple receptors stimulates extracellular vesicle release from trophoblast cells.

Reports of the stimulated release of extracellular vesicles (EVs) are few, and the mechanisms incompletely understood. To our knowledge, the possibility that the activation of any one of the multitudes of G-protein-coupled receptors (GPCRs) expressed by a single cell-type might increase EV release has not been explored. Recently, we identified the expression of cholecystokinin (CCK), gastrin, gastrin/cholecystokinin types A and/or B receptors (CCKAR and/or -BR), and the bitter taste receptor, TAS2R14 in the human and mouse placenta. specifically, trophoblast. These GPCR(s) were also expressed in four different human trophoblast cell lines. The current objective was to employ two of these cell lines-JAR choriocarcinoma cells and HTR-8/SVneo cells derived from first-trimester human villous trophoblast-to investigate whether CCK, TAS2R14 agonists, and other GPCR ligands would each augment EV release. EVs were isolated from the cell-culture medium by filtration and ultracentrifugation. The preparations were enriched in small EVs (<200 nm) as determined by syntenin western blot before and after sucrose gradient purification, phycoerythrin (PE)-ADAM10 antibody labeling, and electron microscopy. Activation of TAS2R14, CCKBR, cholinergic muscarinic 1 & 3, and angiotensin II receptors, each increased EV release by 4.91-, 2.79-, 1.87-, and 3.11-fold, respectively (all p < .05 versus vehicle controls), without significantly changing EV diameter. A progressive increase of EV concentration in conditioned medium was observed over 24 hr consistent with the release of preformed EVs and de novo biogenesis. Compared to receptor-mediated stimulation, EV release by the calcium ionophore, A23187, was less robust (1.63-fold, p = .08). Diphenhydramine, a TAS2R14 agonist, enhanced EV release in JAR cells at a concentration 10-fold below that required to increase intracellular calcium. CCK activation of HTR-8/SVneo cells, which did not raise intracellular calcium, increased EV release by 2.06-fold (p < .05). Taken together, these results suggested that other signaling pathways may underlie receptor-stimulated EV release besides, or in addition to, calcium. To our knowledge, the finding that the activation of multiple GPCRs can stimulate EV release from a single cell-type is unprecedented and engenders a novel thesis that each receptor may orchestrate intercellular communication through the release of EVs containing a subset of unique cargo, thus mobilizing a specific integrated physiological response by a network of neighboring and distant cells.

Electrospinning of PVA-carboxymethyl cellulose nanofibers for flufenamic acid drug delivery.

A prominent medical application of nanotechnology is represented in drug delivery. In this work, carboxymethyl cellulose (CMC) and poly(vinyl alcohol) (PVA) were used for producing CMC/PVA aqueous-based nanofibers loaded with flufenamic acid (FFA) as a drug containing amine groups. The CMC/PVA solutions with 90/10, 80/20, 70/30, 60/40 and 50/50 ratios were considered for electrospinning. Two integration methods were studied for loading FFA on the nanofibers during the electrospinning process. The characterization techniques of SEM, AFM, fluorescence microscopy and FT-IR spectroscopy were used to study the produced nanofibers, indicating a uniform distribution of FFA throughout the samples. The resulting nanofibers were formed in a diameter range of 176-285 nm and exhibited a 5 h degradation time in the PBS buffer solution. A standard diagram of drug loading was obtained for the samples. The drug release pattern was examined using a dialysis tube method. UV-visible spectroscopy revealed a time-dependent drug release behavior in CMC/PVA/FFA nanofibers where a sharp release occurred over the first 20 min. However, a prolonged release time of 10 h was achieved using a cross-linker (EDC).

Antibacterial and antibiofilm effects of flufenamic acid against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are one of the most serious surgery complications, and their prevention is of utmost importance. Flufenamic acid is a non-steroid anti-inflammatory drug approved for clinical use to relieve inflammation and pain in rheumatoid arthritis patients. In this study, we explored the antibacterial efficacy of flufenamic acid and the mechanisms underlying this effect. By using minimal inhibitory concentration (MIC), time-kill, resistance induction assays, and the antibiotic synergy test, we demonstrated that flufenamic acid inhibited the growth of methicillin-resistant staphylococci and did not induce resistance when it was used at the MIC. Furthermore, flufenamic acid acted synergistically with the beta-lactam antibiotic oxacillin and did not show significant toxicity toward mammalian cells. The biofilm inhibition assay revealed that flufenamic acid could prevent biofilm formation on medical implants and destroy the ultrastructure of the bacterial cell wall. RNA sequencing and quantitative RT-PCR indicated that flufenamic acid inhibited the expression of genes associated with peptidoglycan biosynthesis, beta-lactam resistance, quorum sensing, and biofilm formation. Furthermore, flufenamic acid efficiently ameliorated a local infection caused by MRSA in mice. In conclusion, flufenamic acid may be a potent therapeutic compound against MRSA infections and a promising candidate for antimicrobial coating of implants and surgical devices.

Two isostructural Co(II) flufenamato and niflumato complexes with bathocuproine: Analogues with a different cytotoxic activity.

Two novel Co(II) fenamato complexes containing bathocuproine (bcp), namely [Co(bcp)(flu)2] (1) and [Co(bcp)(nif)2] (2) (flu = flufenamato, nif = niflumato) were synthesized and characterized by elemental analysis, single-crystal X-ray structure analysis as well as absorption and fluorescence spectroscopy. Investigation of their molecular structure revealed that both complexes are isostructural and form analogous complex molecules, with a Co(II) atom hexacoordinated by two nitrogen atoms of bcp and four oxygen atoms of two chelate bonded flu (1) and nif (2) ligands in a distorted octahedral arrangement. Surprisingly, the results of cytotoxicity experiments on four cancer cell lines (HeLa, HT-29, PC-3 and MCF-7) have revealed that despite similar structure of the complexes, the nif complex exhibits significantly higher activity, being the most effective against the PC-3 cell line (IC50 (MTT) = 6.11 ±â€¯1.95 µM). Further studies performed on PC-3 cell line have shown that the mechanism of the cytotoxic action of nif complex (2) might involve activation of autophagic processes and apoptosis, while for its flu analogue (1) apoptosis was detected.

Flufenamic acid alleviates sepsis-induced lung injury by up-regulating CBR1.

Investigate the effect of flufenamic acid (FFA) on lung injury of sepsis rats. Rat sepsis model was established using cecal ligation and puncture (CLP). The pathomorphology of lung tissue was detected by Hematoxylin-eosin (H&E) staining. The expression levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and high mobility group box-1 (HMGB-1) in serum and TNF-α, IL-6, malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) in lung tissues. The viability of RLE-6TN cells was detected by CCK-8 assay. The expression of carbonyl reductase 1 (CBR1) in RLE-6TN cells was analyzed by Western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. The inflammatory response was obviously enhanced in CLP-constructed sepsis rats and alleviated by FFA treatment. Sepsis induced the increase of W/D ratio, promoted the levels of TNF-α, IL-6, HMGBR1, and MDA and inhibited the levels of SOD and GSH. FFA could effectively alleviate the sepsis-induced lung injury. The viability of RLE-6TN cells induced by LPS was improved with the treatment of FFA. CBR1 expression in LPS-induced RLE-6TN cells was decreased and FFA could up-regulate the CBR1 expression. In addition, LPS-induced lung injury promoted the inflammatory response in lung tissues, increased the W/D ratio and levels of TNF-α, IL-6, HMGBR1, and MDA while inhibited the levels of SOD and GSH. FFA could effectively improve the LPS-induced lung injury while the effect of FFA on LPS-induced lung injury was alleviated by CBR1 interference. FFA may alleviate sepsis-induced lung injury by up-regulating CBR1.

Stimulatory Effects of Soluplus® on Flufenamic Acid ß-Cyclodextrin Supramolecular Complex: Physicochemical Characterization and Pre-clinical Anti-inflammatory Assessment.

The present study demonstrates the solubility and dissolution of flufenamic acid (FLF)/ß-cyclodextrin (ß-CD)/Soluplus® supramolecular ternary inclusion complex. The binary and ternary inclusion complexes were prepared using solvent evaporation and the microwave irradiation method. The prepared inclusion complexes were evaluated for physicochemical characterization and anti-inflammatory activity using a murine paw edema mol. The phase solubility studies demonstrated 4.59-fold and 17.54-fold enhancements in FLF solubility with ß-CD alone and ß-CD:Soluplus® combination compared with pure FLF, respectively. The in vitro drug release results revealed a significant improvement (P < 0.05) in the release pattern compared with pure FLF. Maximum release was found with flufenamic acid binary and ternary complexes prepared using the microwave irradiation method, i.e., 75.23 ± 3.12% and 95.36 ± 3.23% in 60 min, respectively. The physicochemical characterization results showed complex formation and conversion of the crystalline form of FLF to an amorphous form. The SEM study revealed the presence of a more agglomerated and amorphous structure of the solid particles, which confirmed the formation of complexes. The anti-inflammatory effect of the complex was higher than pure FLF. Therefore, the FLF:ß-CD:Soluplus® inclusion complex may be a very valuable formulation with improved solubility, dissolution, and anti-inflammatory effect.

Flufenamic acid inhibits osteoclast formation and bone resorption and act against estrogen-dependent bone loss in mice.

Postmenopausal osteoporosis is one of the most common types of osteoporosis resulting from estrogen deficiency in elderly women. Nonsteroidal anti-inflammatory drugs (NSAIDs) are important drugs for pain relief in patients with osteoporosis. In this study, we report for the first time that flufenamic acid, a clinically approved and widely used NSAID, not only has analgesic properties but also shows a significant effect in terms of preventing postmenopausal osteoporosis. Quantitative RT-PCR analysis showed that treatment with flufenamic acid significantly downregulated the genes associated with osteoclast differentiation. Meanwhile, RNA-sequencing and western blot analyses suggested that flufenamic acid could inhibit the bone resorption by suppressing the phosphorylation of MAPK pathways. Moreover, an ovariectomy (OVX)-induced bone-loss mouse model indicated that flufenamic acid might be a potent drug for preventing osteoporotic fractures, as verified by micro-CT scanning and histological analysis. Therefore, this study proposes an attractive and potent drug with analgesic properties for the prevention of postmenopausal osteoporosis.

Inhibition of medaka ovulation by gap junction blockers due to its disrupting effect on the transcriptional process of LH-induced Mmp15 expression.

Using medaka, we found that in vitro follicle ovulation, but not germinal vesicle breakdown, was inhibited by three gap junction blockers, carbenoxolone, mefloquine, and flufenamic acid. The blockers specifically inhibited follicular expression of matrix metalloproteinase-15 mRNA and the protein (mmp15/Mmp15), a protease indispensable for medaka ovulation, indicating that gap junctional communication may be required for successful ovulation and mmp15/Mmp15 expression. Further experiments using carbenoxolone as the representative of the gap junction blockers showed that expression of nuclear progestin receptor (Pgr), a transcription factor required for mmp15 expression, was not affected by carbenoxolone treatment, but the formation of phosphorylated Pgr was considerably suppressed. Carbenoxolone treatment caused a decrease in the Pgr binding to the promoter region of mmp15. mRNA expression of cyclin-dependent protein kinase-9 (cdk9) and cyclin I (ccni), whose translation products are demonstrated to be involved in Pgr phosphorylation in the medaka ovulating follicles, was suppressed by carbenoxolone treatment. Transcripts of connexin 34.5 (cx34.5) and connexin 35.4 (cx35.4) were dominantly expressed in the follicle cells of ovulating follicles. The results indicate that gap junctional communication plays an important role in medaka ovulation.

Low concentration flufenamic acid enhances osteogenic differentiation of mesenchymal stem cells and suppresses bone loss by inhibition of the NF-κB signaling pathway.

BACKGROUND: As the representative of fenamic acids, an important group of NSAIDs, flufenamic acid (FFA) has been used for anti-inflammation and analgesia in the clinic. Recently, researches have focused on the role of some members of NSAIDs in promoting osteogenesis. However, little attention has been paid to the subgroup of fenamic acids, and it remains unclear whether FFA and other fenamic acids could regulate mesenchymal stem cells' (MSCs) lineage commitment and bone regeneration. METHODS: Here we treated two kinds of human MSCs with FFA at different concentrations in vitro and examined the effect of FFA on osteogenic differentiation of human MSCs. This was followed by heterotopic bone formation assay in nude mice. In addition, ovariectomized and aged mice were used as osteoporotic models to test the effect of FFA on osteoporosis. Besides, activators and inhibitor of nuclear factor-κB (NF-κB) signaling pathway and western blot were used to clarify the mechanism of the promoting effect of low concentration FFA on osteogenesis. RESULTS: Our results indicated that low concentrations of FFA could significantly enhance osteogenic differentiation of human MSCs in vitro, as well as in vivo. In addition, FFA treatment suppressed bone loss in ovariectomized and aged mice. Mechanistically, FFA at low concentrations promoted osteogenesis differentiation of human MSCs by inhibition of the NF-κB signaling pathway. CONCLUSIONS: Collectively, our study suggested that low concentration FFA could be used in bone tissue engineering or osteoporosis by promoting osteogenic differentiation of human MSCs.

Redox-cycling and intercalating properties of novel mixed copper(II) complexes with non-steroidal anti-inflammatory drugs tolfenamic, mefenamic and flufenamic acids and phenanthroline functionality: Structure, SOD-mimetic activity, interaction with albumin, DNA damage study and anticancer activity.

Copper(II) complexes containing non-steroidal anti-inflammatory drugs (NSAIDs) have been the subject of many research papers and reviews. Here we report the synthesis, spectroscopic study and biological activity of novel mixed copper(II) complexes with NSAIDs: tolfenamic (tolf), mefenamic (mef) and flufenamic (fluf) acids and phenanthroline (phen): [Cu(tolf-O,O')2(phen)] (1), [Cu(mef-O,O')2(phen)] (2), [Cu(fluf-O,O')2(phen)] (3). Complexes were characterized by X-ray analysis and EPR spectroscopy. Complexes 1-3 are monomeric, six-coordinate and crystallize in a monoclinic space group. Interaction of Cu(II) complexes with DNA was studied by means of absorption titrations, viscosity measurements and gel electrophoresis. The relative ability of the complexes to cleave DNA even in the absence of hydrogen peroxide is in the order 3 > 2 > 1. Application of the reactive oxygen species (ROS) scavengers, L-histidine, DMSO and SOD confirmed that singlet oxygen, hydroxyl radicals (Fenton reaction) and superoxide radical were formed, respectively. Thus, in addition to mechanism of intercalation, redox-cycling mechanism which in turn lead to the formation of ROS contribute to DNA damage. Cu(II) complexes exhibit excellent SOD-mimetic activity in the order 3~1 > 2. The fluorescence spectroscopy revealed that albumin may act as a targeted drug delivery vehicle for Cu(II) complexes (K~106). The anticancer activities of complexes 1-3 were investigated using an MTS assay (reduction of the tetrazolium compound) against three cancer cell lines (HT-29 human colon adenocarcinoma, HeLa and T-47D breast cancer cells) and mesenchymal stromal cells (MSC). The most promising compound, from the viewpoint of its NSAID biological activity is 3, due to the presence of the three fluorine atoms participating in the formation of weak hydrogen-bonds at the DNA surface.

Gap junction mediated signaling between satellite glia and neurons in trigeminal ganglia.

Peripheral sensory ganglia contain the somata of neurons mediating mechanical, thermal, and painful sensations from somatic, visceral, and oro-facial organs. Each neuronal cell body is closely surrounded by satellite glial cells (SGCs) that have properties and functions similar to those of central astrocytes, including expression of gap junction proteins and functional dye coupling. As shown in other pain models, after systemic pain induction by intra-peritoneal injection of lipopolysaccharide, dye coupling among SGCs in intact trigeminal ganglion was enhanced. Moreover, neuron-neuron and neuron-SGC coupling was also detected. To verify the presence of gap junction-mediated coupling between SGCs and sensory neurons, we performed dual whole cell patch clamp recordings from both freshly isolated and short term cultured cell pairs dissociated from mouse trigeminal ganglia. Bidirectional gap junction mediated electrical responses were frequently recorded between SGCs, between neurons and between neurons and SGCs. Polarization of SGC altered neuronal excitability, providing evidence that gap junction-mediated interactions between neurons and glia within sensory ganglia may contribute to integration of peripheral sensory responses, and to the modulation and coordinaton of neuronal activity.