RepSox improves viability and regulates gene expression in rhesus monkey-pig interspecies cloned embryos.

OBJECTIVE: To investigate the effect of the small molecule, RepSox, on the expression of developmentally important genes and the pre-implantation development of rhesus monkey-pig interspecies somatic cell nuclear transfer (iSCNT) embryos. RESULTS: Rhesus monkey cells expressing the monomeric red fluorescent protein 1 which have a normal (42) chromosome complement, were used as donor cells to generate iSCNT embryos. RepSox increased the expression levels of the pluripotency-related genes, Oct4 and Nanog (p < 0.05), but not of Sox2 compared with untreated embryos at the 2-4-cell stage. Expression of the anti-apoptotic gene, Bcl2, and the pro-apoptotic gene Bax was also affected at the 2-4-cell stage. RepSox treatment also increased the immunostaining intensity of Oct4 at the blastocyst stage (p < 0.05). Although the blastocyst developmental rate was higher in the group treated with 25 µM RepSox for 24 h than in the untreated control group (2.4 vs. 1.2%, p > 0.05), this was not significant. CONCLUSION: RepSox can improve the developmental potential of rhesus monkey-pig iSCNT embryos by regulating the expression of pluripotency-related genes.

Influence of nano-carrier architecture on in vitro siRNA delivery performance and in vivo biodistribution: polyplexes vs micelleplexes.

Micelle-based siRNA carriers ("micelleplexes") were prepared from the A-B-C triblock copolymer poly(ethylene glycol)-poly(n-butyl acrylate)-poly(2-(dimethylamino)ethyl methacrylate) (PEG-PnBA-PDMAEMA), and their in vitro performance and in vivo biodistribution properties were compared with the benchmark PEGylated and basic polycation systems PEG-PDMAEMA and PDMAEMA, respectively. The micelle architecture, incorporating increased PEG shielding and a larger particle size (∼50 nm) than polycation-based complexes (polyplexes; ∼10 nm), enhances siRNA delivery performance in two important aspects: in vitro gene silencing efficiency and in vivo tumor accumulation. The in vitro gene silencing efficiency of the micelleplexes (24% in HeLa cells) was significantly better than the statistically insignificant levels observed for PDMAEMA and PEG-PDMAEMA polyplexes under identical conditions. This enhancement is linked to the different mechanisms by which micelleplexes are internalized (i.e., caveolar, etc.) compared to PDMAEMA and PEG-PDMAEMA polyplexes. Folate-functionalization significantly improved micelleplex uptake but had negligible influence on gene-silencing efficiency, suggesting that this parameter is not limited by cellular internalization. In vivo biodistribution analysis revealed that siRNA delivered by micelleplexes was more effectively accumulated and retained in tumor tissues than that delivered by PEGylated polyplexes. Overall, the micelle particle size and architecture appear to improve in vitro and in vivo delivery characteristics without significantly changing other properties, such as cytotoxicity and resistance to enzymes and dissociation. The self-assembled nature of micelleplexes is expected to enable incorporation of imaging modalities inside the hydrophobic micelle core, thus combining therapeutic and diagnostic capabilities. The findings from the present study suggest that the micelleplex-type carrier architecture is a useful platform for potential theranostic and tumor-targeting applications.

Modest anti-cancer activity of a bile acid acylated heparin derivative in a PC14PE6 induced orthotopic lung cancer model.

PURPOSE: A novel chemically modified heparin derivative, heparin-deoxycholic acid nano-particles, has lower anticoagulant activity, and was recently reported to have significant anti-tumor effects on squamous head and neck cancer cells. Therefore, the aim of this study was to evaluate the anti-tumor effects of heparin-deoxycholic acid nano-particles in a human lung adenocarcinoma cell line. MATERIALS AND METHODS: An orthotopic lung cancer model in 16 mice was developed using intra-thoracic injections of 0.5x10(6) PC14PE6 cells. Ten days after inoculation, the mice were divided into two groups. PBS and Heparin-DOCA particles were injected once a day every 3 days in the tail vein, for a total of 5 injections. The body weight and survival of each mouse were monitored and the tumor size in the lung was measured by SPECT-CT before and after heparin-DOCA nano-particle treatment. RESULTS: IThe HD particles had no significant cytotoxicity when the PC9 cells were treated in vitro. There was no statistical difference in tumor size, body weight and survival between the HD treated and control groups in vivo. Furthermore, there was no difference in the amount of CD31 between tumor tissues in the two study groups. CONCLUSION: HD synthesized with unfractionated heparin had no apparent inhibitory effects on tumor growth in a PC14PE6 cell induced orthotopic lung cancer mouse model. The HD particles did not significantly inhibit tumor-induced angiogenesis at the tumor sites.

Clinicopathological implications of EpCAM expression in adenocarcinoma of the lung.

BACKGROUND: The frequency of epithelial cell adhesion molecule (EpCAM) expression was investigated in non-small cell lung cancer (NSCLC) cells and human tissues, and its clinicopathological significance in adenocarcinoma of the lung was evaluated. MATERIALS AND METHODS: EpCAM expression was analysed by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry in human NSCLC cells. EpCAM protein expression was evaluated in 234 adenocarcinoma tissues using immunohistochemistry. RESULTS: A high expression level of EpCAM was observed in human NSCLC cells by flow cytometry and RT-PCR. EpCAM overexpression was detected in 120/234 (51.3%) surgically resected adenocarcinoma tissues. EpCAM overexpression occurred significantly more frequently in adenocarcinoma than in bronchioloalveolar carcinoma (p=0.02). The overall survival did not differ significantly between EpCAM-overexpressing and EpCAM-negative patients (p=0.40). CONCLUSION: These findings suggest EpCAM plays a role in the carcinogenesis of adenocarcinoma of the lung and might provide a promising molecule for targeted therapy in NSCLC.

L1 cell adhesion molecule as a predictor for recurrence in pulmonary carcinoids and large-cell neuroendocrine tumors.

Pulmonary neuroendocrine tumors are a distinct subset of neoplasms with indolent to aggressive behavior. This study was conducted to evaluate the prognostic role of L1 cell adhesion molecule (L1CAM) in pulmonary neuroendocrine tumors. We retrospectively analyzed L1 expression in 55 cases of completely resected carcinoids and large-cell neuroendocrine carcinomas, by the immunohistochemistry with monoclonal antibody A10-A3 against human L1. L1 immunoreactivity was detected in 34 (61.8%) of 55 specimens. There was a significant correlation between L1 expression and the World Health Organization classification of this tumor (Spearman rank correlation, rho=0.60, p<0.001). With median follow-up of 52.0 months, the 5-year survival rate for patients with low expression of L1 (<20% of tumor cells stained) was significantly better compared with those with high expression of L1 (82.6% vs. 43.7%, p=0.005). L1 was also a significant independent predictor of disease-free survival, and patients with high L1 expression have a higher risk for recurrence compared with those with low L1 expression (hazard ratio, 3.0; 95% confidence interval, 1.2-8.3; p=0.034). L1 expression is significantly associated with aggressiveness and further studies with larger samples are needed to validate potential prognostic value for pulmonary neuroendocrine tumors.

Nanoparticulate carrier containing water-insoluble iodinated oil as a multifunctional contrast agent for computed tomography imaging.

Contrast-enhanced computed tomography (CT) imaging is a valuable and routine strategy for the clinical diagnosis of various diseases. However, all current CT contrast agents are liquids, so they flow through the blood vessels and disappear very quickly by extravasation. If it were possible to make a blood-compatible particulate contrast agent, we could highlight a particular tissue by either passive or active targeting. In this work, Pluronic F127 and a naturally iodinated compound, Lipiodol, were used to form radiopaque nanoreservoir structures. The resultant nanoparticles have a stable structure at high concentrations, sufficient X-ray absorption, a safety profile similar to or better than that of Iopromide, and a longer circulation time than commercial iodinated preparations. The utility of the resultant radiopaque nanoparticles as a contrast agent was tested using micro-SPECT/CT imaging in vivo. Together with the very good solubility of hydrophobic drugs (e.g., Taxol) in Lipiodol, these results suggest the possibility that these particulate structures and their bioconjugates could become functional CT contrast agents that could deliver therapeutic agents to a particular tissue.

Mouse orthotopic lung cancer model induced by PC14PE6.

PURPOSE: This study was undertaken to investigate in detail the xenograft mouse orthotopic lung cancer model induced by PC14PE6 adenocarcinoma cells. MATERIALS AND METHODS: Three cell doses (0.5x10(6); 1x10(6); 2x10(6)) of PC14PE6 cells were injected into the lungs of male BALB/c nude mice by the intrathoracic injection method. The lung and other organs, including brain, liver, spleen, kidney, muscle, adrenal gland, and lymph node on knee, were removed and stained with H/E to detect the presence of tumor cells. RESULTS: The reliable tumorigenicity time in the PC14PE6 adenocarcinoma cell-inoculated BALB/c nude mouse was 10 days after intrathoracic injection. The average life span of the three groups after inoculation was 14 days in the 2x10(6) cells inoculum group; 25 days in the 1x10(6) cells inoculum group; and 32 days in the 0.5x10(6) cells inoculum group. The PC14PE6 adenocarcinoma cells induced orthotopic lung cancer limited within the thorax. CONCLUSIONS: This orthotopic lung cancer model is an efficient cancer model with easy inoculation methods, rapid and high tumorigenicity, and simple monitoring methods for metastasis.

Endogenous somatostatin inhibits interaction of insulin and cholecystokinin on exocrine secretion of isolated, perfused rat pancreas.

INTRODUCTION: Although somatostatin inhibits pancreatic exocrine secretion, the inhibitory mechanism of endogenous somatostatin is not clearly understood. AIM: To investigate the effect of endogenous somatostatin on the interaction between endogenous insulin and exogenous cholecystokinin (CCK) in exocrine secretion of the totally isolated, perfused rat pancreas. METHODOLOGY: Endogenous releases of somatostatin and insulin were induced by 18 mM glucose. Streptozotocin (75 mg/kg) or cysteamine (300 mg/kg) was injected into rats 24 hours before the experiment to deplete insulin or somatostatin in the pancreas. RESULTS: Glucose (18 mM) enhanced CCK (10 pM)-stimulated secretions of fluid and amylase in the normal pancreas, which was further elevated by a somatostatin antagonist. Exogenous insulin (100 nM) also enhanced CCK-stimulated secretions in the streptozotocin-treated pancreas, which was also markedly increased by the somatostatin antagonist. The glucose (18 mM)-enhanced CCK-stimulated secretions were much higher in the cysteamine-treated pancreas than in the normal pancreas, which was dose-dependently reduced by exogenous somatostatin (30, 100 pM). However, endogenous or exogenous somatostatin did not modify the pancreatic responses to CCK alone. CONCLUSION: Endogenous somatostatin inhibits the interaction of endogenous insulin and CCK on pancreatic exocrine secretion in the rat rather than reducing the action of CCK alone or endogenous release of insulin.