Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

<p>Abstract</p> <p>Background</p> <p>Along with high affinity binding of epibatidine (<it>K</it>_d1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (<it>K</it>_d2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [³H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites.</p> <p>Results</p> <p>Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [³H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [³H]epibatidine binding after adding a large concentration of cold competitor. Fourth, nonspecific binding of a heterologous competitor changed estimates of high and low inhibition constants but did not change the ratio of those estimates.</p> <p>Conclusions</p> <p>Investigating the low affinity site of α4β2 nAChR with equilibrium binding when ligand depletion and nonspecific binding are present likely needs special attention to experimental design and data interpretation beyond fitting total binding data. Manipulation of maximum ligand and receptor concentrations and intentionally increasing ligand depletion are potentially helpful approaches.</p

Targeting RNS/caveolin-1/MMP signaling cascades to protect against cerebral ischemia-reperfusion injuries: potential application for drug discovery

Reactive nitrogen species (RNS) play important roles in mediating cerebral ischemia-reperfusion injury. RNS activate multiple signaling pathways and participate in different cellular events in cerebral ischemia-reperfusion injury. Recent studies have indicated that caveolin-1 and matrix metalloproteinase (MMP) are important signaling molecules in the pathological process of ischemic brain injury. During cerebral ischemia-reperfusion, the production of nitric oxide (NO) and peroxynitrite (ONOO-), two representative RNS, down-regulates the expression of caveolin-1 (Cav-1) and, in turn, further activates nitric oxide synthase (NOS) to promote RNS generation. The increased RNS further induce MMP activation and mediate disruption of the blood-brain barrier (BBB), aggravating the brain damage in cerebral ischemia-reperfusion injury. Therefore, the feedback interaction among RNS/Cav-1/MMPs provides an amplified mechanism for aggravating ischemic brain damage during cerebral ischemia-reperfusion injury. Targeting the RNS/Cav-1/MMP pathway could be a promising therapeutic strategy for protecting against cerebral ischemia-reperfusion injury. In this mini-review article, we highlight the important role of the RNS/Cav-1/MMP signaling cascades in ischemic stroke injury and review the current progress of studies seeking therapeutic compounds targeting the RNS/Cav-1/MMP signaling cascades to attenuate cerebral ischemia-reperfusion injury. Several representative natural compounds, including calycosin-7-O-β-D-glucoside, baicalin, Momordica charantia polysaccharide (MCP), chlorogenic acid, lutein and lycopene, have shown potential for targeting the RNS/Cav-1/MMP signaling pathway to protect the brain in ischemic stroke. Therefore, the RNS/Cav-1/MMP pathway is an important therapeutic target in ischemic stroke treatment.published_or_final_versio

EMBLICA EXTRACT INDUCES TYPE I PRO-COLLAGEN SYNTHESIS AND INHIBITS COLLAGENASE ACTIVITY IN MOUSE FIBROBLASTS

Decrease of type I collagen generation as well as the increase of its degradation via matrix metalloproteinases (MMPs; e.g. gelatinases and collagenases) are considered as a major cause of skin aging. A potent natural antioxidant exblica extract from emblica fruits (Phyllanthus emblica) was herein evaluated for collagen promoting activity in mouse fibroblasts. We demonstrated that the level of intracellular type I pro-collagen increased following emblica extract treatment in concentration-dependent manner. Emblica extract at the concentration of 100 µg/mL exhibited the maximum effect on type I pro-collagen; approximately 7 fold-induction versus non-treated control determined by Western blot analysis. Moreover, our result indicated that emblica extract affected the MMPs function. Percentage of collagenase inhibition exceeded forty at the concentration of 100 µg/mL of emblica extract. Taken together, emblica extract has a promising anti-aging effect attributed to its positive effect on type I collagen formation as well as the protection against collagen degradation

PROTECTIVE EFFECT OF A PHYLLANTHUS EMBLICA EXTRACT AGAINST CISPLATIN-INDUCED APOPTOSIS IN DERMAL PAPILLA FIBROBLASTS

Cisplatin is a widely prescribed anticancer agent that causes hair loss in patients. Since the dermal papilla (DP) fibroblasts are known as a key mediator in controlling hair growth and loss, protection against cisplatin-induced cell damage on these cells may lead to a new strategy for hair loss protection in chemotherapy patients. We herein reported that cisplatin induced DP cell death in a concentration-dependent manner through apoptosis mechanism, which was further found to be related to the intracellular reactive oxygen species (ROS) generation. Therefore, extract from emblica fruits (Phyllanthus emblica), a known natural antioxidant, was examined for its protective effect on cisplatin-induced DP fibroblast cell death. Our results indicated that emblica extract attenuated the intracellular ROS induction following cisplatin treatment and subsequently protected against cisplatin-induced DP fibroblast apoptotic cell death. Co-treatment of emblica extract (250-500 µg/mL) and cisplatin 100 µmol/L (LD50) markedly increased the DP fibroblast relative cell viability to the same level as the non-treated control

CURCUMIN INDUCES ANOIKIS IN H460 NON-SMALL LUNG CANCER CELLS THROUGH SUPEROXIDE ANION INDUCTION

Anoikis or apoptosis triggered by loss of cell anchorage plays an important role in prevention of tumor cell metastasis. During metastasis process, anticancer drugs are not able to completely eliminate cancer cells that suspended in the blood stream; therefore, the use of natural safety compounds to enhance apoptosis of these suspended cells may improve the therapy. Curcumin (diferuloylmethane), a major active component in turmeric, Curcuma longa, has been shown to inhibit neoplastic initiation, promotion and progression of a wide variety of tumor cells. However, the effect of curcumin on anoikis process has not been well characterized. We herein demonstrated that 10 µM of curcumin triggered 1.2-folds anoikis of detached lung carcinoma H460 cells compared with controlled cells. Cell viability was detected by XTT assay and anoikis cells were indicated by annexin V staning assay. Mechanism of curcumin induced anoikis was associated with its ability to generate intracellular reactive oxygen species (ROS). Curcumin caused 1.6-folds induction of intracellular ROS level as detected by DCF-DA and flow cytometry. Our inhibitory study revealed that superoxide anion generated by curcumin was a principle ROS responsible for anoikis induction in these cells. Thus, our results showed that curcumin induced anoikis in detached-H460 cells resulted from increasing of intracellular ROS

SENSITIZING EFFECT OF CURCUMIN ON CISPLATIN-INDUCED APOPTOSIS INVOLVES SUPEROXIDEANION INDUCTION AND BCL-2 DEGRADATION

The possibility of using curcumin as a chemotherapeutic sensitizing agent has been intensively demonstrated in some cancers. However, the effect of curcumin on non-small cell lung cancer (NSCLC), one of the most resistant cancers, is largely unknown. The aim of this study was to investigate the sensitizing effect of curcumin on cisplatin-induced apoptosis in NSCLC cells. Curcumin was shown to induce intracellular superoxide anion generation, down-regulate anti-apoptotic Bcl-2 protein, and subsequently sensitize NSCLC H460 cells to cisplatin-induced apoptosis. Amplification and overexpression of bcl-2 protein has been implicated in chemotherapeutic resistance in many cancers and overexpression of this protein strongly rendered H-460 cells resistant to cisplatin-induced apoptosis. The present study showed that co-treatment of the cells with curcumin and cisplatin resulted in increased apoptosis and reversal of Bcl-2-mediated cisplatin resistance. The mechanism by which curcumin down-regulates Bcl-2 and sensitizes cells to cisplatin-induced apoptosis involves proteasomal degradation of Bcl-2, since a specific proteasome inhibitor lactacystin reversed effect of curcumin on bcl-2 level. These findings indicate a novel pathway for curcumin regulation of Bcl-2, which benefits the development of a cisplatin sensitizing agent