Advanced aqueous-phase phosphoramidation reactions for effectively synthesizing peptide-oligonucleotide conjugates trafficked into a human cell line.

Peptide-oligonucleotide conjugates (POCs) have held promise as effective therapeutic agents in treating microbial infections and human genetic diseases including cancers. In clinical applications, POCs are especially useful to circumvent cellular delivery and specificity problems of oligonucleotides. We previously reported that nucleic acid phosphoramidation reactions performed in aqueous solutions have the potential for facile POC synthesis. Here, we carried out further studies to significantly improve aqueous-phase two-step phosphoramidation reaction yield. Optimized reactions were employed to effectively synthesize POCs for delivery into human A549 cells. We achieved optimization of aqueous-phase two-step phosphoramidation reaction and improved reaction yield by (1) determining appropriate co-solutes and co-solute concentrations to acquire higher reaction yields, (2) exploring a different nucleophilicity of imidazole and its derivatives to stabilize essential nucleic acid phosphorimidazolide intermediates prior to POC formation, and (3) enhancing POC synthesis by increasing reactant nucleophilicity. The advanced two-step phosphoramidation reaction was exploited to effectively conjugate a well-studied cell penetrating peptide, the Tat(48-57) peptide, with oligonucleotides, bridged by either no linkers or a disulfide-containing linker, to have the corresponding POC yields of 47-75%. Phosphoramidation-synthesized POCs showed no cytotoxicity to human A549 cells at studied POC concentrations after 24 h inoculation and were successfully trafficked into the human A549 cell line as demonstrated by flow cytometry, fluorescent microscopy, and confocal laser scanning microscopy study. The current report provides insight into aqueous-phase phosphoramidation reactions, the knowledge of which was used to develop effective strategies for synthesizing POCs with crucial applications including therapeutic agents for medicine.

Schwann Cell Migration Induced by Earthworm Extract via Activation of PAs and MMP2/9 Mediated through ERK1/2 and p38.

The earthworm, which has stasis removal and wound-healing functions, is a widely used Chinese herbal medicine in China. Schwann cell migration is critical for the regeneration of injured nerves. Schwann cells provide an essentially supportive activity for neuron regeneration. However, the molecular migration mechanisms induced by earthworms in Schwann cells remain unclear. Here, we investigate the roles of MAPK (ERK1/2, JNK and p38) pathways for earthworm-induced matrix-degrading proteolytic enzyme (PAs and MMP2/9) production in Schwann cells. Moreover, earthworm induced phosphorylation of ERK1/2 and p38, but not JNK, activate the downstream signaling expression of PAs and MMPs in a time-dependent manner. Earthworm-stimulated ERK1/2 and p38 phosphorylation was attenuated by pretreatment with U0126 and SB203580, resulting in migration and uPA-related signal pathway inhibition. The results were confirmed using small interfering ERK1/2 and p38 RNA. These results demonstrated that earthworms can stimulate Schwann cell migration and up-regulate PAs and MMP2/9 expression mediated through the MAPK pathways, ERK1/2 and p38. Taken together, our data suggests the MAPKs (ERK1/2, p38)-, PAs (uPA, tPA)-, MMP (MMP2, MMP9) signaling pathway of Schwann cells regulated by earthworms might play a major role in Schwann cell migration and nerve regeneration.

RSC96 Schwann Cell Proliferation and Survival Induced by Dilong through PI3K/Akt Signaling Mediated by IGF-I.

Schwann cell proliferation is critical for the regeneration of injured nerves. Dilongs are widely used in Chinese herbal medicine to remove stasis and stimulate wound-healing functions. Exactly how this Chinese herbal medicine promotes tissue survival remains unclear. The aim of the present study was to investigate the molecular mechanisms by which Dilong promote neuron regeneration. Our results show that treatment with extract of Dilong induces the phosphorylation of the insulin-like growth factor-I (IGF-I)-mediated phosphatidylinositol 3-kinase/serine-threonine kinase (PI3K/Akt) pathway, and activates protein expression of cell nuclear antigen (PCNA) in a time-dependent manner. Cell cycle analysis showed that G(1) transits into the S phase in 12-16 h, and S transits into the G(2) phase 20 h after exposure to earthworm extract. Strong expression of cyclin D1, cyclin E and cyclin A occurs in a time-dependent manner. Small interfering RNA (siRNA)-mediated knockdown of PI3K significantly reduced PI3K protein expression levels, resulting in Bcl(2) survival factor reduction and a marked blockage of G(1) to S transition in proliferating cells. These results demonstrate that Dilong promotes the proliferation and survival of RSC96 cells via IGF-I signaling. The mechanism is mainly dependent on the PI3K protein.

Plasma matrix metalloproteinase-9 level is better than serum matrix metalloproteinase-9 level to predict gastric cancer evolution.

PURPOSE: Matrix metalloproteinase-9 (MMP-9) in blood is a promising new tumor marker. The aims of the present study are to compare the usefulness of plasma and serum MMP-9 levels for predicting gastric cancer development, invasion, and survival. EXPERIMENTAL DESIGN: In this nested case-control study, 114 gastric cancer patients and 87 healthy controls were enrolled. MMP-9 levels and activities were quantitatively measured by ELISA assay and zymography. The results were compared with the occurrence, clinicopathologic features, and outcomes of gastric cancer patients. The follow-up time for all patients was at least 5 years. RESULTS: Serum MMP-9 levels were significantly higher than plasma MMP-9 levels. Both plasma and serum MMP-9 levels correlated significantly with active MMP-9 identified by zymography (P = 0.002 and P = 0.048, respectively). Plasma MMP-9 level was significantly elevated in gastric cancer patients when compared with control subjects (P < 0.001). Serum MMP-9 levels did not differ between the groups. Receiver-operator characteristics analysis showed the values of sensitivity (82.5%) and specificity (65.5%) at the maximum accuracy for plasma MMP-9 at >or=60 ng/mL (P < 0.001). Elevated plasma MMP-9 correlated significantly with lymph node metastasis [odds ratio (OR), 3.43; P = 0.019], lymphatic invasion (OR, 7.58; P = 0.009), and venous invasion (OR, 4.14; P = 0.033). Patients with elevated plasma MMP-9 levels had poorer survival rates than those with normal plasma MMP-9 levels (P = 0.038). Serum MMP-9 level did not correlate well with gastric cancer-invasive phenotypes or survival. CONCLUSION: Our results suggest plasma MMP-9 level is a better marker than serum MMP-9 level for predicting gastric cancer development and progression.