Potent efficacy of Stachybotrys microspora triprenyl phenol-7, a small molecule having anti-inflammatory and antioxidant activities, in a mouse model of acute kidney injury.

Acute kidney injury (AKI) increases the risk of chronic kidney disease (CKD), complicates existing CKD, and can lead to the end-stage renal disease. However, there are no approved effective therapeutics for AKI. Recent studies have suggested that inflammation and oxidative stress are the primary causes of AKI. We previously reported the potential anti-inflammatory and antioxidant activities of Stachybotrys microspora triprenyl phenol-7 (SMTP-7). The aim of the present study was to evaluate the efficacy of SMTP-7 in AKI model mice. AKI was induced in mice by ischemia of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after the removal of right kidney. The efficacy of SMTP-7 was determined by measuring the renal function using urine and serum samples and morphological assessment. For deciphering the mechanism of action of SMTP-7, inflammatory cytokines and oxidative stress in kidney were detected. SMTP-7 (0.01, 0.1, 1, 10 mg/kg) dose-dependently improved the renal function. In addition, it improved the damage to renal tubules and exhibited anti-inflammatory and antioxidant activities in the kidney of AKI mice. These results indicate the potential of SMTP-7 as a medicinal compound for the treatment of AKI.

Screening Reveals Sterol Derivatives with Pro-Differentiation, Pro-Survival, or Potent Cytotoxic Effects on Oligodendrocyte Progenitor Cells.

Inducing the formation of new oligodendrocytes from oligodendrocyte progenitor cells (OPCs) represents a potential approach to repairing the loss of myelin observed in multiple sclerosis and other diseases. Recently, we demonstrated that accumulation of specific cholesterol precursors, 8,9-unsaturated sterols, is a dominant mechanism by which dozens of small molecules enhance oligodendrocyte formation. Here, we evaluated a library of 56 sterols and steroids to evaluate whether other classes of bioactive sterol derivatives may also influence mouse oligodendrocyte precursor cell (OPC) differentiation or survival. From this library, we identified U-73343 as a potent enhancer of oligodendrocyte formation that induces 8,9-unsaturated sterol accumulation by inhibition of the cholesterol biosynthesis enzyme sterol 14-reductase. In contrast, we found that mouse OPCs are remarkably vulnerable to treatment with the glycosterol OSW-1, an oxysterol-binding protein (OSBP) modulator that induces Golgi stress and OPC death in the low picomolar range. A subsequent small-molecule suppressor screen identified mTOR signaling as a key effector pathway mediating OSW-1's cytotoxic effects in mouse OPCs. Finally, evaluation of a panel of ER and Golgi stress-inducing small molecules revealed that mouse OPCs are highly sensitive to these perturbations, more so than closely related neural progenitor cells. Together, these studies highlight the wide-ranging influence of sterols and steroids on OPC cell fate, with 8,9-unsaturated sterols positively enhancing differentiation to oligodendrocytes and OSW-1 able to induce lethal Golgi stress with remarkable potency.

N-Ethyl-2-Pyrrolidinone-Substituted Flavan-3-Ols with Anti-inflammatory Activity in Lipopolysaccharide-Stimulated Macrophages Are Storage-Related Marker Compounds for Green Tea.

Fresh green tea (GT) is commonly considered to have better sensory flavor and higher commercial value than long-term-stored GT; however, the chemical variations during storage are unclear. In this study, the chemical profiles of stored GT were surveyed among time-series samples from 0 to 19 months using a nontargeted metabolomics method. Seven N-ethyl-2-pyrrolidinone-substituted flavan-3-ols (EPSFs) increased from 0.022 ± 0.019 to 3.212 ± 0.057 mg/g within 19 months (correlation coefficients with storage duration ranging from 0.936 to 0.965), and they were the most significantly increased compounds among the 127 identified compounds. Two representative EPSFs (R-EGCG-cThea and S-EGCG-cThea) possess potential anti-inflammatory properties by suppressing the expression, phosphorylation, and nuclear translocation of nuclear factor kappa-B (NF-κB) p65 in lipopolysaccharide-stimulated macrophages based on western blotting and immunofluorescence results. In conclusion, EPSFs were found to be marker compounds for stored GT and showed potential anti-inflammatory activity by regulating the NF-κB signaling pathway.

Identification of antibiotics for use in selection of the chytrid fungi Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans.

Global amphibian populations are being decimated by chytridiomycosis, a deadly skin infection caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). Although ongoing efforts are attempting to limit the spread of these infections, targeted treatments are necessary to manage the disease. Currently, no tools for genetic manipulation are available to identify and test specific drug targets in these fungi. To facilitate the development of genetic tools in Bd and Bsal, we have tested five commonly used antibiotics with available resistance genes: Hygromycin, Blasticidin, Puromycin, Zeocin, and Neomycin. We have identified effective concentrations of each for selection in both liquid culture and on solid media. These concentrations are within the range of concentrations used for selecting genetically modified cells from a variety of other eukaryotic species.

Discovery of BMS-986235/LAR-1219: A Potent Formyl Peptide Receptor 2 (FPR2) Selective Agonist for the Prevention of Heart Failure.

Formyl peptide receptor 2 (FPR2) agonists can stimulate resolution of inflammation and may have utility for treatment of diseases caused by chronic inflammation, including heart failure. We report the discovery of a potent and selective FPR2 agonist and its evaluation in a mouse heart failure model. A simple linear urea with moderate agonist activity served as the starting point for optimization. Introduction of a pyrrolidinone core accessed a rigid conformation that produced potent FPR2 and FPR1 agonists. Optimization of lactam substituents led to the discovery of the FPR2 selective agonist 13c, BMS-986235/LAR-1219. In cellular assays 13c inhibited neutrophil chemotaxis and stimulated macrophage phagocytosis, key end points to promote resolution of inflammation. Cardiac structure and functional improvements were observed in a mouse heart failure model following treatment with BMS-986235/LAR-1219.

HSP70 Inhibition Leads to the Activation of Proteasomal System under Mild Hyperthermia Conditions in Young and Senescent Fibroblasts.

Aging has been characterized with the accumulation of oxidized proteins, as a consequence of progressive decline in proteostasis capacity. Among others, proteasomal system is an efficient protein turnover complex to avoid aggregation of oxidized proteins. Heat shock protein 70 (HSP70) is another critical player that is involved in some key processes including the correct folding of misfolded proteins and targeting aggregated proteins to the proteasome for rapid degradation. The aim of this study was to determine the role of proteasomal system and heat shock proteins to maintain proteome balance during replicative senescence in mild hyperthermia conditions. Our results demonstrated that HSP40/70 machinery is induced by mild hyperthermia conditions independent from senescence conditions. Since HSP70 is largely responsible for the rapidly inducible cell protection following hyperthermia, the activation of "heat shock response" resulted in the elevation of HSP40/70 expressions as well as the proteasome activity. Interestingly, when HSP70 expression was inhibited, increased proteasomal activation was shown to be responsive to mild hyperthermia. Since HSP70 is involved in various stress-related pathways such as oxidative and endoplasmic reticulum stress, depletion of HSP70 expression may induce proteasomal degradation to maintain proteome balance of the cell. Thus, our data suggests that in mild heat stress conditions, molecular chaperone HSP70 plays an important role to avoid protein oxidation and aggregation; however, activities of proteasomal system are induced when HSP70 expression is depleted.

Natural pseurotins and analogs thereof inhibit activation of B-cells and differentiation into the plasma cells.

BACKGROUND: The frequency of allergic diseases is constantly rising. Dysregulated production of isotype E immunoglobulins is one of the key factors behind allergic reactions and its modulation is therefore an important target for pharmacological intervention. Natural products of the pseurotin family were reported to be inhibitors of IgE production in B-cells. Mechanistic details underlying these effects are however not well understood. PURPOSE: In the present study, we synthesized new analogs of natural pseurotins and extensively investigated their inhibitory effects on activation, proliferation and differentiation of B-cells, as well as on the production of IgE. STUDY DESIGN: Effects of two natural pseurotins (pseurotins A and D) and a collection of fully synthetic pseurotin analogs were studied on mouse B-cells stimulated by the combination of IL-4 and E. coli lipopolysaccharide. The IgE production was determined along with cell viability and cell proliferation. The phosphorylation of selected members of the STAT transcription factor family was subsequently investigated. Finally, the in vivo effect of pseurotin D on the ovalbumin-induced delayed type hypersensitivity response was tested in mice. RESULTS: We discovered that several fully synthetic pseurotin analogs were able to decrease the production of IgE in stimulated B-cells with potency comparable to that of pseurotins A and D. We found that the two natural pseurotins and the active synthetic analogs inhibited the phosphorylation of STAT3, STAT5 and STAT6 proteins in stimulated B-cells, resulting in the inhibition of B-cell proliferation and differentiation into the plasma cells. In vivo, pseurotin D decreased ovalbumin-induced foot pad edema. CONCLUSION: Our results advance the current mechanistic understanding of the pseurotin-induced inhibition of IgE production in B-cells by linking the effect to STAT signaling, and associated modulation of B-cell proliferation and differentiation. Together with our finding that structurally simpler pseurotin analogs were able to reproduce the effects of natural pseurotins, the presented work has implications for the future research on these secondary metabolites in the context of allergic diseases.

Pharmacological Profile of the Novel Antiepileptic Drug Candidate Padsevonil: Characterization in Rodent Seizure and Epilepsy Models.

The antiepileptic drug (AED) candidate, (4R)-4-(2-chloro-2,2-difluoroethyl)-1-{[2-(methoxymethyl)-6-(trifluoromethyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl]methyl}pyrrolidin-2-one (padsevonil), is the first in a novel class of drugs that bind to synaptic vesicle protein 2 (SV2) proteins and the GABAA receptor benzodiazepine site, allowing for pre- and postsynaptic activity, respectively. In acute seizure models, padsevonil provided potent, dose-dependent protection against seizures induced by administration of pilocarpine or 11-deoxycortisol, and those induced acoustically or through 6 Hz stimulation; it was less potent in the pentylenetetrazol, bicuculline, and maximal electroshock models. Padsevonil displayed dose-dependent protective effects in chronic epilepsy models, including the intrahippocampal kainate and Genetic Absence Epilepsy Rats from Strasbourg models, which represent human mesial temporal lobe and absence epilepsy, respectively. In the amygdala kindling model, which is predictive of efficacy against focal to bilateral tonic-clonic seizures, padsevonil provided significant protection in kindled rodents; in mice specifically, it was the most potent AED compared with nine others with different mechanisms of action. Its therapeutic index was also the highest, potentially translating into a favorable efficacy and tolerability profile in humans. Importantly, in contrast to diazepam, tolerance to padsevonil's antiseizure effects was not observed in the pentylenetetrazol-induced clonic seizure threshold test. Further results in the 6 Hz model showed that padsevonil provided significantly greater protection than the combination of diazepam with either 2S-(2-oxo-1-pyrrolidinyl)butanamide (levetiracetam) or 2S-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl] butanamide (brivaracetam), both selective SV2A ligands. This observation suggests that padsevonil's unique mechanism of action confers antiseizure properties beyond the combination of compounds targeting SV2A and the benzodiazepine site. Overall, padsevonil displayed robust efficacy across validated seizure and epilepsy models, including those considered to represent drug-resistant epilepsy. SIGNIFICANCE STATEMENT: Padsevonil, a first-in-class antiepileptic drug candidate, targets SV2 proteins and the benzodiazepine site of GABAA receptors. It demonstrated robust efficacy across a broad range of rodent seizure and epilepsy models, several representing drug-resistant epilepsy. Furthermore, in one rodent model, its efficacy extended beyond the combination of drugs interacting separately with SV2 or the benzodiazepine site. Padsevonil displayed a high therapeutic index, potentially translating into a favorable safety profile in humans; tolerance to antiseizure effects was not observed.

Dahuang Zhechong Pill suppresses colorectal cancer liver metastasis via ameliorating exosomal CCL2 primed pre-metastatic niche.

ETHNOPHARMACOLOGICAL RELEVANCE: Dahuang Zhechong Pill (DZP) is a classical formula from "Synopsis of Prescriptions of the Golden Chamber". It has been used for treatment of abdominal masses (including tumorous diseases) for thousands of years. AIM OF THE STUDY: Our previous work showed that DZP suppresses CCl-4 induced hepatic fibrosis by downregulating the expression of interleukin-13. We aimed to test if DZP suppresses the metastasis of colorectal cancer (CRC) by ameliorating the fibrosis status of the future metastatic organ. MATERIALS AND METHODS: Liver metastasis was observed by injection of MC38-EGFP cells with stably expressing enhanced green fuorescence protein beneath the splenic capsule of C57BL/6J mice. MC38-EGFP-derived exosomes were analyzed by Label-free comparative proteomics. mRNA expression was determined by Quantitative PCR. Protein expression was determined by immunohistochemistry, immunofuorescence and Western blot. Collagen deposition was determined by Masson staining. All data were statistically analyzed using SPSS. RESULTS: DZP drastically reduced the metastatic tumor number and fluorescence intensity in a splenic liver metastasis model. It also lowered the expression of mature TGF-ß1 and decreased the fibronectin contents & collagen deposition. Exosome proteomics showed that the upregualted CC chemokine ligand-2 (CCL2) was repressed by DZP treatment. Importantly, DZP markedly lowered the expression of CCL2 and its receptor CCR2 in the liver. Exosomal CCL2 activated macrophage recruitment and shifted the M1/M2 paradigm to a M2 phenotype. DZP reduced the macrophage infiltration and attenuated the M2 polarizaion in tumor-bearing mice liver. It decreased the F4/80 positive areas and specifically reduced the ratio of CCR2+ positive macrophage. Anti-fibrosis and inhibition of CCR2 suppress the growth and metastasis of CRC. CONCLUSIONS: DZP inhibits the liver metastasis of CRC by suppressing CCL2 mediated M2-skewing paradigm and ameliorating the pro-fibrotic microenvironment.

3-Hydroxy-2-Pyrrolidinone as a Potential Bidentate Ligand for in Vivo Chelation of Uranyl with Low Cytotoxicity and Moderate Decorporation Efficacy: A Solution Thermodynamics, Structural Chemistry, and in Vivo Uranyl Removal Survey.

Uranium poses a threat for severe renal and bone damage in vivo. With the rapid development of nuclear industry, it is more urgent than ever to search for potential in vivo uranium chelators. In this work, 3-hydroxy-2-pyrrolidinone (HPD) is investigated as a new potential uranium decorporation ligand. The potentiometric titration measurements were carried out, and the stability constants were determined to be log ß110 = 10.5(7), log ß120 = 20.7(9), and log ß130 = 28.2(4). The species distribution diagram shows that nearly all uranyl is complexed by HPD at pH 7.4 under the defined condition. A single crystal of uranyl and HPD complexes, [(UO2)3O(H2O)3(C4H6NO2)3]·NO3·12H2O (uranyl-HPD), was obtained via an evaporation method. The overall structure of uranyl-HPD is a trimer that consists of three uranyl units and three HPD ligands. The uranyl unit is equatorially coordinated by three oxygen atoms from two HPD agents, one coordinated water molecule, and one µ3-O atom that is shared by three uranyl units. The results of the cytotoxicity assay indicate that the ligand is less toxic than the chelators used clinically (i.e., DTPA-ZnNa3 and 3-hydroxy-1,2-dimethyl-4(1 H)-pyridone (DFP)). The results of the uranium removal assay using the NRK-52E cell show that it could reduce as much as 58% of the uranium content at the cellular level. Furthermore, the in vivo uranium decorporation assays demonstrate that HPD can remove 52% of uranium deposited in the kidney but shows poor uranium removal efficacy in the bone.

The evaluation of an immunoperoxidase assay applicable in antiviral drug screening.

Bovine viral diarrhea virus (BVDV) fall into cytopathic (CP) and noncytopathic (NCP) biotypes, based on their ability to kill cultured cells. NCP-BVDV can not be titrated by conventional means as used for CP-BVDV, which has impeded the identification of antiviral drugs targeting NCP-BVDV virus strains. In this study, the application of an immunoperoxidase assay in the screening of antiviral drugs was tested using two known BVDV inhibitors, ribavirin and ammonium chloride (NH4Cl). Phospholipase C inhibitor U73122 was identified to affect BVDV infection by using this immunoperoxidase assay. In addition, the results of immunoperoxidase assay were validated by real-time PCR. Taken together, the immunoperoxidase assay is a useful and versatile method suitable for antiviral drug screening targeting NCP-BVDV.

New Bis-Alkenoic Acid Derivatives from a Marine-Derived Fungus Fusarium solani H915.

Fusarium solani H915 is a fungus derived from mangrove sediments. From its ethyl acetate extract, a new alkenoic acid, fusaridioic acid A (1), three new bis-alkenoic acid esters, namely, fusariumester A1 (2), A2 (3) and B (4), together with three known compounds (5⁻7), were isolated. The structures of the new compounds were comprehensively characterized by high resolution electrospray ionization-mass spectrometry (HR-ESI-MS), 1D and 2D nuclear magnetic resonance (NMR). Additionally, the antifungal activities against tea pathogenic fungi Pestalotiopsis theae and Colletotrichum gloeosporioides were studied. The new compound, 4, containing a ß-lactone ring, exhibited moderate inhibitory activity against P. theae, with an MIC of 50 µg/disc. Hymeglusin (6), a typical ß-lactone antibiotic and a terpenoid alkaloid, equisetin (7), exhibited potent inhibitory activities against both fungal species. The isolated compounds were evaluated for their effects on zebrafish embryo development. Equisetin clearly imparted toxic effect on zebrafish even at low concentrations. However, none of the alkenoic acid derivatives exhibited significant toxicity to zebrafish eggs, embryos, or larvae. Thus, the ß-lactone containing alkenoic acid derivatives from F. solani H915 are low in toxicity and are potent antifungal agents against tea pathogenic fungi.

Flavoalkaloids with a Pyrrolidinone Ring from Chinese Ancient Cultivated Tea Xi-Gui.

Chinese Xi-Gui tea is one ancient cultivated variety of Camellia sinensis var. assamica. At present, it is used for producing expensive and elite tea in China. Five new flavoalkaloids, (-)-6-(5''' S)- N-ethyl-2-pyrrolidinone-epicatechin-3- O-gallate (ester-type catechins pyrrolidinone E, etc-pyrrolidinone E, 1), (-)-6-(5''' R)- N-ethyl-2-pyrrolidinone-epicatechin-3- O-gallate (etc-pyrrolidinone F, 2) (-)-8-(5''' S)- N-ethyl-2-pyrrolidinone-epicatechin-3- O-gallate (etc-pyrrolidinone G, 3a), (-)-8-(5''' S)- N-ethyl-2-pyrrolidinone-catechin-3- O-gallate (etc-pyrrolidinone I, 4a), (-)-8-(5''' R)- N-ethyl-2-pyrrolidinone-catechin-3- O-gallate (etc-pyrrolidinone J, 4b), and one new naturally occurring natural product (-)-8-(5''' R)- N-ethyl-2-pyrrolidinone-epicatechin-3- O-gallate (etc-pyrrolidinone H, 3b) together with the known flavoalkaloids etc-pyrrolidinones A-D (5, 6, 7a, and 7b) were detected and isolated from Xi-Gui green tea. Their structures were identified by comprehensive NMR spectroscopic analyses. Absolute configurations of 1-3 were established by comparison of the CD analyses with epicatechin-3- O-gallate (ECG). Compounds 1-4 were evaluated for their protection against high glucose induced cell senescence on human umbilical vein endothelia cells (HUVECs) and showed significant protection effects ( p < 0.01) at both 1.0 and 10 µM. A discussion on the possible evolution of tea plants divergent from related food plants on the basis of phytochemical view is also provided.

C-8 N-Ethyl-2-pyrrolidinone-Substituted Flavan-3-ols from the Leaves of Camellia sinensis var. pubilimba.

Camellia sinensis var. pubilimba, one variety of the genus Camellia sect. Thea (Theaceae), has been used for producing green tea mainly by the local people of its growing areas of Guangxi province, China. Forty compounds, including eight C-8 N-ethyl-2-pyrrolidinone-substituted flavan-3-ols (1-8) and their substituted unit N-ethyl-5-hydroxy-2-pyrrolidinone (9), four flavan-3-ol monomers (10-13) and one dimer (14), nine flavonoids (15-23), three hydrolyzable tannins (24-26), two lignans (27-28), 11 simple phenolics (29-39), and caffeine (40), were first isolated and identified from the leaves. Their structures were determined by detailed spectroscopic analysis and comparison with the literature data and authentic samples. Both 1 and 4 were obtained as a mixture of the N-ethyl-2-pyrrolidinone C-5 enantiomers (1a and 1b and 4a and 4b), respectively, while the resolution of another three pairs of enantiomers (2 and 3, 5 and 6, and 7 and 8) was achieved. Among them, 1b is a new compound whose NMR data together with its enantiomer (1a) were reported for the first time, while 2 and 3 are two new natural products. Most of the isolates exhibited significant antioxidant activities, stronger than ascorbic acid and trolox, while parts of the isolates, particularly C-8 N-ethyl-2-pyrrolidinone-substituted flavan-3-ols, showed obvious inhibitory effects on acetylcholinesterase (AChE). The results indicated that C. sinensis var. pubilimba is a valuable plant resource for tea production.

Modulation of Pacemaker Potentials in Murine Small Intestinal Interstitial Cells of Cajal by Gamisoyo-San, a Traditional Chinese Herbal Medicine.

BACKGROUND: The Gamisoyo-san (GSS) has been used for -improving the gastrointestinal (GI) symptoms. The purpose of this study was to investigate the effects of GSS, a traditional Chinese herbal medicine, on the pacemaker potentials of mouse small intestinal interstitial cells of Cajal (ICCs). METHODS: ICCs from the small intestines were dissociated and cultured. Whole-cell patch-clamp configuration was used to record pacemaker potentials and membrane currents. RESULTS: GSS depolarized ICC pacemaker potentials in a dose-dependent manner. Pretreatment with 4-diphenylacetoxypiperidinium iodide completely inhibited GSS-induced pacemaker potential depolarizations. Intracellular GDP-ß-S inhibited GSS-induced effects, and in the presence of U-73122, GSS-induced effects were inhibited. Also, GSS in the presence of a Ca2+-free solution or thapsigargin did not depolarize pacemaker potentials. However, in the presence of calphostin C, GSS slightly depolarized pacemaker potentials. Furthermore, GSS inhibited both transient receptor potential melastatin7 and Ca2+-activated Cl- channel (anoctamin1) currents. CONCLUSION: GSS depolarized pacemaker potentials of ICCs via G protein and muscarinic M3 receptor signaling pathways and through internal or external Ca2+-, phospholipase C-, and protein kinase C-dependent and transient receptor potential melastatin 7-, and anoctamin 1-independent pathways. The study shows that GSS may regulate GI tract motility, suggesting that GSS could be a basis for developing novel prokinetic agents for treating GI motility dysfunctions.

Equisetin as potential quorum sensing inhibitor of Pseudomonas aeruginosa.

OBJECTIVE: To screen for the quorum-sensing (QS) inhibitors from marine-derived fungi and evaluate their anti-QS properties in Pseudomonas aeruginosa. RESULTS: QS inhibitory activity was found in secondary metabolites of a marine fungus Fusarium sp. Z10 using P. aeruginosa QSIS-lasI biosensor. The major active compound of this fungus was isolated by HPLC and identified as equisetin. Subinhibitory concentration of equisetin could inhibit the formation of biofilm, swarming motility, and the production of virulence factors in P. aeruginosa. The inhibition of las, PQS, and rhl system by equisetin were determined using Escherichia coli MG4/pKDT17, E.coli pEAL08-2, and E.coli pDSY, respectively. Real-time RT-PCR assays showed that equisetin could downregulate the mRNA expression of QS-related genes. CONCLUSIONS: Equisetin proved its potential as an inhibitor against P. aeruginosa QS system and might also serve as precursor compound in development of novel therapeutics for infectious diseases by optimal design of structures.

Berberine activates bitter taste responses of enteroendocrine STC-1 cells.

Glucagon-like peptide-1 (GLP-1) is involved in the regulation of insulin secretion and glucose homeostasis. GLP-1 release is stimulated when berberine interacts with a novel G protein family (TAS2Rs) in enteroendocrine cells. In this study, we used STC-1 cells and examined a marked increase in Ca2+ in response to various bitter compounds. Ca2+ responses to traditional Chinese medicine extracts, including berberine, phellodendrine and coptisine, in STC-1 cells were suppressed by the phospholipase C (PLC) inhibitor U-73122, suggesting the involvement of bitter taste receptors in changing the physiological status of enteroendocrine cells in a PLC-dependent manner. STC-1 cells showed berberine-up-regulated preproglucagon (GLP-1 precursor) mRNA and GLP-1 secretion. A QPCR analysis demonstrated that TAS2R38, a subtype of the bitter taste receptor, was associated with GLP-1 secretion. Berberine-mediated GLP-1 secretion was attenuated in response to small interfering RNA silencing of TAS2R38. The current studies demonstrated that Gα-gustducin co-localized with GLP-1 and Tas2r106 in the STC-1 cells. We further utilized inhibitors of PLC and TRPM5, which are known to participate in taste signal transduction, to investigate the underlying pathways mediated in berberine-induced GLP-1 secretion. Berberine-induced GLP-1 release from enteroendocrine cells is modulated in a PLC-dependent manner through a process involving the activation of bitter taste receptors. Together, our data demonstrated a berberine-mediated GLP-1 secretion pathway in mouse enteroendocrine cells that could be of therapeutic relevance to hyperglycemia and the role of bitter taste receptors in the function of the small intestine.

Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic ß-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity.

Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP7, is at a high level in pancreatic ß-cells and is important for insulin exocytosis. To better understand IP7 regulation in ß-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP7. Although the inhibitors have diverse targets, they all perturbed IP7 levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP7 levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high Km for ATP makes IP7 synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP7 concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wortmannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (U73122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP7 indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic ß-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP7's role in cellular signal transduction is likely to have been underestimated.

Molecularly Targeted Cancer Combination Therapy with Near-Infrared Photoimmunotherapy and Near-Infrared Photorelease with Duocarmycin-Antibody Conjugate.

Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that uses an antibody-photoabsorber conjugate (APC). However, the effect of NIR-PIT can be enhanced when combined with other therapies. NIR photocaging groups, based on the heptamethine cyanine scaffold, have been developed to release bioactive molecules near targets after exposure to light. Here, we investigated the combination of NIR-PIT using panitumumab-IR700 (pan-IR700) and the NIR-releasing compound, CyEt-panitumumab-duocarmycin (CyEt-Pan-Duo). Both pan-IR700 and CyEt-Pan-Duo showed specific binding to the EGFR-expressing MDAMB468 cell line in vitro In in vivo studies, additional injection of CyEt-Pan-Duo immediately after NIR light exposure resulted in high tumor accumulation and high tumor-background ratio. To evaluate the effects of combination therapy in vivo, tumor-bearing mice were separated into 4 groups: (i) control, (ii NIR-PIT, (iii) NIR-release, (iv) combination of NIR-PIT and NIR-release. Tumor growth was significantly inhibited in all treatment groups compared with the control group (P < 0.05), and significantly prolonged survival was achieved (P < 0.05 vs. control). The greatest therapeutic effect was shown with NIR-PIT and NIR-release combination therapy. In conclusion, combination therapy of NIR-PIT and NIR-release enhanced the therapeutic effects compared with either NIR-PIT or NIR-release therapy alone. Mol Cancer Ther; 17(3); 661-70. ©2017 AACR.

Emulsion-Based Intradermal Delivery of Melittin in Rats.

Bee venom (BV) has long been used as a traditional medicine. The aim of the present study was to formulate a BV emulsion with good rheological properties for dermal application and investigate the effect of formulation on the permeation of melittin through dermatomed rat skin. A formulated emulsion containing 1% (w/v) BV was prepared. The emulsion was compared with distilled water (DW) and 25% (w/v) N-methyl-2-pyrrolidone (NMP) in DW. Permeation of melittin from aqueous solution through the dermatomed murine skin was evaluated using the Franz diffusion cells. Samples of receptor cells withdrawn at pre-determined time intervals were measured for melittin amount. After the permeation study, the same skin was used for melittin extraction. In addition, a known amount of melittin (5 µg/mL) was added to stratum corneum, epidermis, and dermis of the rat skin, and the amount of melittin was measured at pre-determined time points. The measurement of melittin from all samples was done with HPLC-MS/MS. No melittin was detected in the receptor phase at all time points in emulsion, DW, or NMP groups. When the amount of melittin was further analyzed in stratum corneum, epidermis, and dermis from the permeation study, melittin was still not detected. In an additional experiment, the amount of melittin added to all skin matrices was corrected against the amount of melittin recovered. While the total amount of melittin was retained in the stratum corneum, less than 10% of melittin remained in epidermis and dermis within 15 and 30 min, respectively. Skin microporation with BV emulsion facilitates the penetration of melittin across the stratum corneum into epidermis and dermis, where emulsified melittin could have been metabolized by locally-occurring enzymes.