Wenhu Duan

Differential Roles of NMDA Receptor Subtypes in Ischemic Neuronal Cell Death and Ischemic Tolerance

Background and Purpose— Activation of NMDA subtypes of glutamate receptors is implicated in cell damage induced by ischemia as well as for the establishment of ischemic tolerance after ischemic preconditioning in animal models. We investigated the contributions of NR2A- and NR2B-containing NMDA receptors to ischemic cell death and ischemic tolerance in a rat model of transient global ischemia. Methods— Transient global ischemia was produced in rats by 4-vessel occlusion. Neuronal injury was analyzed by Fluoro-Jade B and Nissl staining. Phosphorylation of CREB was detected by Western blotting and immunohistochemistry. In situ hybridization and reverse transcriptase–polymerase chain reaction were used to evaluate the mRNA level of cpg15 and bdnf. Results— NR2A subtype-specific antagonist NVP-AAM077 enhanced neuronal death after transient global ischemia and abolished the induction of ischemic tolerance. In contrast, NR2B subtype-specific antagonist ifenprodil attenuated ischemic cell death and enhanced preconditioning-induced neuroprotection. Furthermore, selectively blocking NR2A-, but not NR2B-, containing NMDA receptors inhibited ischemia-induced phosphorylation of CREB and the subsequent upregulation of CREB target genes such as cpg15 and bdnf. Conclusions— We found that NR2A- and NR2B-containing NMDA receptor subtypes play differential roles in ischemic neuronal death and ischemic tolerance, suggesting attractive new strategies for the development of drugs for patients with stroke.

Differential Roles of NR2A- and NR2B-Containing NMDA Receptors in Activity-Dependent Brain-Derived Neurotrophic Factor Gene Regulation and Limbic Epileptogenesis

Fleeting activation of NMDA receptors (NMDARs) induces long-term modification of synaptic connections and refinement of neuronal circuits, which may underlie learning and memory and contribute to pathogenesis of a diversity of neurological diseases, including epilepsy. Here, we found that NR2A and NR2B subunit-containing NMDARs were coupled to distinct intracellular signaling, resulting in differential BDNF expression and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. Selective activation of NR2A-containing NMDARs increased BDNF gene expression. Activation of NR2B-containing NMDARs led to ERK1/2 phosphorylation. Furthermore, selectively blocking NR2A-containing NMDARs impaired epileptogenesis and the development of mossy fiber sprouting in the kindling and pilocarpine rat models of limbic epilepsy, whereas inhibiting NR2B-containing NMDARs had no effects in epileptogenesis and mossy fiber sprouting. Interestingly, blocking either NR2A- or NR2B-containing NMDARs decreased status epilepticus-induced neuronal cell death. The specific requirement of NR2A and its downstream signaling for epileptogenesis implicates attractive new targets for the development of drugs that prevent epilepsy in patients with brain injury.

Gambogic acid inhibits the catalytic activity of human topoisomerase IIα by binding to its ATPase domain

This study is intended to characterize the cellular target of gambogic acid (GA), a natural product isolated from the gamboge resin of Garcinia hurburyi tree, which possesses potent in vitro and in vivo antitumor activities. The antiproliferative activity of GA was further confirmed here in a panel of human tumor cells and multidrug-resistant cells. We found that GA significantly inhibited the catalytic activity of topoisomerase (Topo) II and, to a comparatively less extent, of Topo I, without trapping and stabilizing covalent topoisomerase-DNA cleavage complexes. Down-regulation of Topo IIα but not Topo I and Topo IIβ, reduced GA-induced apoptosis and the phosphorylation of c-Jun, and restored cell proliferation upon GA treatment. Moreover, GA antagonized etoposide-induced DNA damage and abrogated the antiproliferative activity of etoposide, whereas it did not affect camptothecin-induced DNA damage. By dissecting the actions of GA on the individual steps of Topo IIα catalytic cycle, we found that GA inhibited DNA cleavage and ATP hydrolysis. Moreover, GA directly bound to the ATPase domain of Topo IIα, and may share common binding sites with ATP. The results reported here show that GA exerts its antiproliferative effect by inhibiting the catalytic activity Topo IIα. They also indicate that GA inhibits Topo IIα-mediated DNA cleavage and modulate the activity of Topo II poisons, which provide rationale for further clinical evaluation of GA. [Mol Cancer Ther 2007;6(9):2429–40]

Simple cyclohexanediamine-derived primary amine thiourea catalyzed highly enantioselective conjugate addition of nitroalkanes to enones.

A highly enantioselective conjugate addition of nitroalkanes to enones has been developed. The process is efficiently catalyzed by a simple chiral cyclohexanediamine-derived primary amine thiourea with a broad substrate scope.

SH-7, a new synthesized shikonin derivative, exerting its potent antitumor activities as a topoisomerase inhibitor

1‐(1,4‐dihydro‐5,8‐dihydroxy‐1,4‐dioxonaphthalen‐2‐yl)‐4‐methylpent‐3‐enylfuran‐2‐caroxylate (SH‐7), a new naphthoquinone compound, derived from shikonin, exhibited obvious inhibitory actions on topoisomerase II (Topo II) and topoisomerase I (Topo I), which were stronger than its mother compound shikonin. Notably, the SH‐7s inhibitory potency on Topo II was much stronger than that on Topo I. In addition, SH‐7 significantly stabilized Topo II‐DNA cleavable complex and elevated the expression of phosphorylated‐H2AX. The in vitro cell‐based investigation demonstrated that SH‐7 displayed wide cytotoxicity in diversified cancer cell lines with the mean IC50 value of 7.75 μM. One important finding is SH‐7 displayed significant cytotoxicity in the 3 MDR cell lines, with an average IC50 value nearly equivalent to that of the corresponding parental cell lines. The average resistance factor (RF) of SH‐7 was 1.74, which was much lower than those of reference drugs VP‐16 (RF 145.92), ADR (RF 105.97) and VCR (RF 197.39). Further studies illustrated that SH‐7 had the marked apoptosis‐inducing function on leukemia HL‐60 cells, which was validated to be of mitochondria‐dependence. The in vivo experiments showed that SH‐7 had inhibitory effects on S‐180 sarcoma implanted to mice, SMMC‐7721, BEL‐7402 human hepatocellular carcinoma and PC‐3 human prostate cancer implanted to nude mice. Taken together, these results suggest that SH‐7 induces DSBs as a Topo II inhibitor, which was crucial to activate the apoptotic process, and subsequently accounts for its both in vitro and in vivo antitumor activities. The well‐defined Topo II inhibitory activity, antitumor effects particularly with its obvious anti‐MDR action, better solubility and less toxicity make SH‐7 as a potential antitumor drug candidate for further research and development.

A Simple Primary Amine Thiourea Catalyzed Highly Enantioselective Conjugate Addition of α,α-Disubstituted Aldehydes to Maleimides

Conjugate addition reactions are powerful and versatile processes for the formation of C C bonds in organic synthesis as a result of a wide variety of substances that can serve as electrophiles and nucleophiles and, consequently, a diverse array of products can be generated. Notably, in recent years, organocatalytic, enantioselective conjugate reactions have been subject to intensive investigations. The general observation is that enals, enones, and nitroolefins are often used as Michael acceptors. In sharp contrast, the use of maleimides 2, for C C bond formation remains elusive, despite the broad synthetic and biological utilities of chiral a-branched succinimides 3 (Scheme 1). To the best

Shikonin derivatives: synthesis and inhibition of human telomerase.

We synthesized DL-shikonin, shikonin, alkanin, and their cyclo-derivatives and acyl-derivatives. These compounds have low cytotoxicity, as well as inhibitory activity against the telomerase enzyme, except cyclo-derivatives.

Caspase-8 preferentially senses the apoptosis-inducing action of NG-18, a Gambogic acid derivative, in human leukemia HL-60 cells.

Gambogic acid (GA) is the major active ingredient of gamboge secreted from a Chinese traditional medicine Garcinia hanburryi possessing potent anti-tumor activity. N-(2-ethoxyethyl)gambogamide (NG-18), a derivative of GA, also efficiently inhibits proliferation of cultured human tumor cells. The inhibition effect of NG-18 is associated with its ability to induce apoptosis. In the present study, NG-18 markedly induced leukemia HL-60 cells apoptosis, and the extrinsic and intrinsic apoptosis pathways were activated almost at the same time. NG-18-induced tumor cell apoptosis was associated with up-regulation of pro-apoptotic Bcl-2 family member Bax, and down-regulation of anti-apoptotic protein Bcl-2. The NG-18-induced apoptosis was blocked completely by a pan-caspase inhibitor Z-VAD-FMK, indicating that caspases were functionally and actively involved in this process. The specific inhibition of caspase-8 activity using Z-IETD-FMK significantly blocked NG-18-induced apoptosis. In contrast, inhibition of other initiator caspases, caspase-2 or -9, using Z-VDVAD-FMK or Z-LEHD-FMK respectively had no effect on NG-18-induced apoptosis. Altogether, our data demonstrated that NG-18-induced apoptosis was dependent on caspases and caspase-8 acted as a key executor in the event.

Synthesis and biological activity of novel shikonin analogues

A series of shikonin analogues with side chain variants have been synthesized and evaluated for antitumor activity. These novel analogues show a broad spectrum of in vitro cytotoxicity against various cancer cell lines. Additionally, some analogues were also found to have the ability to decrease the expression level of HIF-1alpha in breast cancer cells MDA-MB-231 under hypoxia. The features of these analogues suggest their potential in cancer therapy.

GA3, a new gambogic acid derivative, exhibits potent antitumor activities in vitro via apoptosis-involved mechanisms

AbstractAim:Gambogic acid (GA) is the major active ingredient of gamboge, which is secreted from a Chinese traditional medicine, Garcinia hanburyi, which possesses potent antitumor activity. GA3, a new GA derivative, has been shown to possess better water solubility than GA. The aim of the present study was to examine the antitumor activity of GA3 and the mechanism underlying it.Methods:The growth inhibition of cancer cell lines induced by GA3 was assessed using the SRB assay. DAPI staining, flow cytometry, a DNA fragment assay, and Western blot analysis were used to study the apoptotic mechanisms of GA3.Results:GA3 displayed wide cytotoxicity in diversified human cancer cell lines with a mean IC50 value of 2.15 μmol/L. GA3 was also effective against multidrug resistant cells, with an average resistance factor (RF) that was much lower than that of the reference drug, doxorubicin. Mechanistic studies revealed that GA3-induced apoptosis in HL-60 cells proceeded via both extrinsic and intrinsic pathways, with caspase-8 functioning upstream of caspase-9. In addition, GA3-driven apoptotic events were associated with up-regulation of Bax, down-regulation of Bcl-2 and cleavage of Bid. Moreover, GA3 triggered cytochrome c release from the mitochondria, in particular bypassing the involvement of the mitochondrial membrane potential.Conclusion:Better solubility and a potential anti-MDR activity, combined with a comparable antitumor efficacy, make GA3 a potential drug candidate in cancer therapy that deserves further investigation.