Ischemic preconditioning protects the brain against injury via inhibiting CaMKII-nNOS signaling pathway.

Although studies have shown that cerebral ischemic preconditioning (IPC) can ameliorate ischemia/reperfusion (I/R) induced brain damage, but its precise mechanisms remain unknown. Therefore, the aim of this study was to investigate the neuroprotective mechanisms of IPC against ischemic brain damage induced by cerebral I/R and to explore whether the Calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated up-regulation of nNOS ser847-phosphorylation signaling pathway contributed to the protection provided by IPC. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The rats were pretreated with 3 min of IPC alone or KN62 (selective antagonist of CaMKII) treatment before IPC, after reperfusion for 3 days, 6 min ischemia was induced. Cresyl violet staining was used to examine the survival of hippocampal CA1 pyramidal neurons. Immunoblotting was performed to measure the phosphorylation of CaMKII, nNOS, c-Jun and the expression of FasL. Immunoprecipitation was used to examine the binding between PSD95 and nNOS. The results showed that IPC could significantly protect neurons against cerebral I/R injury, furthermore, the combination of PSD95 and nNOS was increased, coinstantaneously the phosphorylation of CaMKII and nNOS (ser847) were up-regulated, however the activation of c-Jun and FasL were reduced. Conversely, KN62 treatment before IPC reversed all these effects of IPC. Taken together, the results suggest that IPC could diminish ischemic brain injury through CaMKII-mediated up-regulation of nNOS ser847-phosphorylation signaling pathway.

Rap2B promotes migration and invasion of human suprarenal epithelioma.

The aim of our study was to elucidate the role of Rap2B in the development of human suprarenal epithelioma and to investigate the effect of Rap2B on suprarenal epithelioma cells migration and invasion. We use tissue microarray and immunohistochemistry to evaluate Rap2B staining in 75 suprarenal epithelioma tissues and 75 tumor-adjacent normal renal tissues. And the expression of Rap2B protein in human suprarenal epithelioma cells and tissues was detected by western blot simultaneously. The role of Rap2B in suprarenal epithelioma cells migration and invasion was detected by using wound healing assay, cell migration assay, and matrigel invasion assay. After that, we performed western blot analysis and gelatin zymography to detect MMP-2 protein expression and enzyme activity. Our research showed that Rap2B expression was increased in tumor tissues compared with tumor-adjacent normal renal tissues. But no correlation was found between Rap2B expression and clinicopathological parameters. In addition, we found that Rap2B promoted the cell migration and invasion abilities, and Rap2B increased MMP-2 expression and enzyme activity in suprarenal epithelioma cells. Our data indicated that Rap2B expression is significantly increased in human suprarenal epithelioma and Rap2B can promote the cell migration and invasion abilities, which may provide a new target for the treatment of suprarenal epithelioma.

Expression, purification, and characterization of RGD-mda-7, a His-tagged mda-7/IL-24 mutant protein.

RGD peptide (Arg-Gly-Asp tripeptide) binds to integrin αVß(3) and αVß(5), which is selectively expressed in tumor neovasculature and on the surface of some tumor cells. Some studies showed that coupling the RGD peptides to anticancer drugs yielded compounds with increased efficiency against tumors and lowered toxicity to normal tissues. The melanoma differentiation-associated gene-7/interleukin-24 gene (mda-7/IL-24) is a novel tumor-suppressor/cytokine gene that exhibits potent tumor-suppressive activity without damaging normal cells. To enhance the antitumor effect, we inserted a glycine residue into the wild type (mda-7/IL-24) between (164)Arg and (165)Asp to form a RGD peptide, named RGD-mda-7, then expressed RGD-mda-7 in Escherichia coli. Herein, we describe the expression and purification of RGD-mda-7. We detected the characterizations of immunostimulatory activity, tumor targeting, potent cytopathic effect, and apoptosis inducing exploited by RGD-mda-7 in tumor cells, and also compared these characterizations with wtmda-7/IL-24. The data showed that RGD-mda-7 had more potent tumor targeting and apoptosis-inducing effects than wtmda-7/IL-24.

nNOS downregulation attenuates neuronal apoptosis by inhibiting nNOS-GluR6 interaction and GluR6 nitrosylation in cerebral ischemic reperfusion.

Glutamate receptor 6 (GluR6) is well documented to play a pivotal role in ischemic brain injury, which is mediated by the GluR6·PSD95·MLK3 signaling module and subsequent c-Jun N-terminal kinase (JNK) activation. Our recent studies show that GluR6 is S-nitrosylated in the early stages of ischemia-reperfusion. NO (Nitric Oxide) is mainly generated from neuronal nitric oxide synthase (nNOS) in cerebral neurons during the early stages of reperfusion. Here, the effect of nNOS downregulation on GluR6 S-nitrosylation and GluR6-mediated signaling was investigated in cerebral ischemia and reperfusion. Administration of nNOS oligonucleotides confirmed that GluR6 nitrosylation is induced by nNOS-derived endogenous NO and further activates the GluR6·PSD95·MLK3 signaling module and JNK signaling pathway. Moreover, this study revealed for the first time that nNOS can bind with GluR6 during ischemic reperfusion, and PSD95 is involved in this interaction. In summary, our results suggest that nNOS binds with GluR6 via PSD95 and then produces endogenous NO to S-nitrosylate GluR6 in cerebral ischemia-reperfusion, which provides a new approach for stroke therapy.

Potent antitumor effect elicited by RGD-mda-7, an mda-7/IL-24 mutant, via targeting the integrin receptor of tumor cells.

The melanoma differentiation-associated gene-7/interleukin-24 gene (mda-7/IL-24) is a novel tumor-suppressor/cytokine gene that exhibits potent tumor-suppressive activity without damaging normal cells. To enhance the antitumor effect, an mda-7/IL-24 mutant, RGD-mda-7, which includes the cell adhesive sequence 164Arg-165Gly-166Asp (RGD motif), was constructed and evaluated for bioactivity. RGD peptide binds to integrins α(V)ß(3) and α(V)ß(5), which are selectively expressed in tumor neovasculature and in the surface of some tumor cells. The wtmda-7/IL-24 and RGD-mda-7 were expressed in Escherichia coli and then purified and renatured. The immunostimulatory activity of RGD-mda-7 was assayed by stimulating peripheral blood mononuclear cells. The results suggested that the abilities of RGD-mda-7 to induce IL-6, TNF-α, and IFN-γ production were higher than wtmda-7/IL-24. Tumor targeting of RGD-mda-7 was assayed using cell adhesion experiments. The antitumor effect of the purified RGD-mda-7 on cell proliferation in vitro was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake, cell apoptosis by staining with fluorescent probes of FITC-annexin V and DAPI, and caspase-3 expression and activity. The in vitro results showed that RGD-mda-7 inhibited the proliferation of multiple tumor cell lines (Hela, ACHN, HepG2, and A549). Staining with fluorescent probes of FITC-annexin V and DAPI indicated that RGD-mda-7 could induce apoptosis more effectively in four tumor cell lines than wtmda-7/IL-24, but has no effect on normal cell line NHLF. Western blotting showed that treatment of tumor cells with RGD-mda-7 could activate apoptotic pathway by cleavage of caspase-3 as same as wtmda-7/IL-24. Further, RGD-mda-7 group showed a higher cleaved level of caspase-3, but not in NHLF cells. These results demonstrate that RGD-MDA-7 possesses more potent antitumor effects than wtmda-7/IL-24 and therefore merits further investigation in preclinical and clinical studies.

A critical role of Sp1 transcription factor in regulating the human Ki-67 gene expression.

Ki-67 plays a crucial role in cell proliferation as well as maintenance or regulation of cell division. The mechanism governing the Ki-67 gene expression remains unknown. Thus, we cloned the core promoter of the human Ki-67 gene and further investigated its transcriptional regulation. The putative Sp1 binding sites were confirmed by electrophoretic mobility shift assay together with an anti-Sp1 antibody-mediated supershift assay. Deletion mutagenesis and firefly luciferase reporter gene assay demonstrated the essential contribution of Sp1 on transcriptional activation of the Ki-67 gene. In this study, we first confirm that there are three Sp1 binding sites in the Ki-67 core promoter. Two Sp1 sites (one at position -159 to -145 nt and the other at position -14 to +12 nt) are mainly involved in transcriptional regulation of the Ki-67 gene. Overexpression of Sp1 can enhance the Ki-67 promoter activity. However, down-regulation of Sp1 expression using siRNA-Sp1 and mithramycin effectively inhibits the Ki-67 gene transcription. Our results suggest that Sp1 is essential for basal promoter activity of the human Ki-67 gene. Inhibition of the Ki-67 transcriptional activity through abolishment of Sp1 may provide the useful prospect for gene therapy.

Oncolytic-adenovirus-expressed RNA interference for cancer therapy.

IMPORTANCE OF THE FIELD: RNA interference (RNAi) has generated considerable excitement for its potential cancer therapeutic applications. Because of the difficulties in delivering a large amount of siRNA to cancer cells and the short half-life of siRNA, it is important to choose an efficient delivery system for transduction of siRNA into target cells. Oncolytic adenovirus offers a better platform by virtue of its high transfection efficiency and selective replication in cancer cells. AREAS COVERED IN THIS REVIEW: This review focuses on the synergism between oncolytic adenovirus and siRNA antitumor responses, and discusses recent progresses in oncolytic-adenovirus-expressed siRNA. WHAT THE READER WILL GAIN: siRNA-expressing oncolytic adenovirus can generate a significantly enhanced antitumor effect through gene knockdown and viral oncolysis. TAKE HOME MESSAGE: Due to its potency and target specificity, using siRNA delivery by oncolytic adenovirus has generated much excitement and will open new avenues for treatment of human cancer.