Post-blast treatment with Nociceptin/Orphanin FQ peptide (NOP) receptor antagonist reduces brain injury-induced hypoxia and signaling proteins in vestibulomotor-related brain regions.

Mild traumatic brain injury (mTBI) diagnoses have increased due to aggressive sports and blast-related injuries, but the cellular mechanisms and pathology underlying mTBI are not completely understood. Previous reports indicate that Nociceptin Orphanin/FQ (N/OFQ), an endogenous neuropeptide, contributes to post-injury ischemia following mechanical brain injury, yet its specific role in cerebral hypoxia, vestibulomotor function and injury marker expression following blast-induced TBI is not known. This study is the first to identify a direct association of N/OFQ and its N/OFQ peptide (NOP) receptor with TBI-induced changes following a single 80psi head blast exposure in male rats. N/OFQ and NOP receptor expression increased in brain tissue and plasma following TBI, concurrent with vestibular dysfunction but preceding hypoxia and appearance of injury markers compared to sham rats. A single post-blast treatment with the NOP receptor antagonist, SB-612111, transiently improved acute vestibulomotor performance. It also prevented increases in markers of TBI-induced hypoxia, pro-apoptotic proteins and injury seen 8-10days post-blast. This study reveals an apparent role for the N/OFQ-NOP receptor system in blast TBI and suggests potential therapeutic utility of NOP receptor antagonists for mTBI.

Serum Monitoring and Phenotype Identification of Stage I Non-Small Cell Lung Cancer Patients.

A stage I non-small cell lung cancer (NSCLC) serum profiling platform is presented which is highly efficient and accurate. Test sensitivity (0.95) for stage I NSCLC is the highest reported so far. Test metrics are reported for discriminating stage I adenocarcinoma vs squamous cell carcinoma subtypes. Blinded analysis identified 23 out of 24 stage I NSCLC and control serum samples. Group-discriminating mass peaks were targeted for tandem mass spectrometry peptide/protein identification, and yielded a lung cancer phenotype. Bioinformatic analysis revealed a novel lymphocyte adhesion pathway involved with early-stage lung cancer.

Expression Profiling Identifies the Noncoding Processed Transcript of HNRNPU with Proliferative Properties in Pancreatic Ductal Adenocarcinoma.

A gene array was used to profile the expression of 22,875 long non-coding RNAs (lncRNAs) and a large number of protein coding genes in 47 specimens of pancreatic ductal adenocarcinoma (PDAC), adjacent benign pancreas and the pancreas from patients without pancreatic disease. Of the lncRNAs profiled, the expression of 126 were significantly increased and 260 were decreased in the tumors (p < 0.05, 2-fold). The expression of one lncRNA in particular, heterogeneous nuclear ribonucleoprotein U (HNRNPU) processed transcript (also known as ncRNA00201) was among the most significantly deregulated (increased four-fold) in the tumors compared to normal/adjacent benign tissues. Increased expression of HNRNPU processed transcript was associated with poor prognosis for patients with PDAC. The expression of HNRNPU processed transcript was increased in PDAC cell lines compared to noncancerous pancreatic cell lines. LNATM gapmer mediated inhibition of HNRNPU processed transcript reduced cell proliferation in Patu-T and PL45 pancreatic cancer cell lines. Reduced invasion and migration was reported upon HNRNPU processed transcript knockdown in Patu-T cells. Small interfering RNA (siRNA) knockdown of the HNRNPU protein coding gene correlated with a 55% reduction in the HNRNPU processed transcript expression and a corresponding reduction in proliferation of Patu-T and PL45 cells. However, gapmer inhibition of HNRNPU processed transcript did not affect HNRNPU mRNA levels. The lncRNA HNRNPU processed transcript expression is increased in both PDAC tissues and cell lines; knockdown of this lncRNA further reduces proliferation and invasion/migration of pancreatic carcinoma cells.

Globally increased ultraconserved noncoding RNA expression in pancreatic adenocarcinoma.

Transcribed ultraconserved regions (T-UCRs) are a class of non-coding RNAs with 100% sequence conservation among human, rat and mouse genomes. T-UCRs are differentially expressed in several cancers, however their expression in pancreatic adenocarcinoma (PDAC) has not been studied. We used a qPCR array to profile all 481 T-UCRs in pancreatic cancer specimens, pancreatic cancer cell lines, during experimental pancreatic desmoplasia and in the pancreases of P48Cre/wt; KrasLSL-G12D/wt mice. Fourteen, 57 and 29% of the detectable T-UCRs were differentially expressed in the cell lines, human tumors and transgenic mouse pancreases, respectively. The vast majority of the differentially expressed T-UCRs had increased expression in the cancer. T-UCRs were monitored using an in vitro model of the desmoplastic reaction. Twenty-five % of the expressed T-UCRs were increased in the HPDE cells cultured on PANC-1 cellular matrix. UC.190, UC.233 and UC.270 were increased in all three human data sets. siRNA knockdown of each of these three T-UCRs reduced the proliferation of MIA PaCa-2 cells up to 60%. The expression pattern among many T-UCRs in the human and mouse pancreases closely correlated with one another, suggesting that groups of T-UCRs are co-activated in PDAC. Successful knockout of the transcription factor EGR1 in PANC-1 cells caused a reduction in the expression of a subset of T-UCRs suggesting that EGR1 may control T-UCR expression in PDAC. We report a global increase in expression of T-UCRs in both human and mouse PDAC. Commonalties in their expression pattern suggest a similar mechanism of transcriptional upregulation for T-UCRs in PDAC.

Blast Overpressure Waves Induce Transient Anxiety and Regional Changes in Cerebral Glucose Metabolism and Delayed Hyperarousal in Rats.

Physiological alterations, anxiety, and cognitive disorders are strongly associated with blast-induced traumatic brain injury (blast TBI), and are common symptoms in service personnel exposed to blasts. Since 2006, 25,000-30,000 new TBI cases are diagnosed annually in U.S. Service members; increasing evidence confirms that primary blast exposure causes diffuse axonal injury and is often accompanied by altered behavioral outcomes. Behavioral and acute metabolic effects resulting from blast to the head in the absence of thoracic contributions from the periphery were examined, following a single blast wave directed to the head of male Sprague-Dawley rats protected by a lead shield over the torso. An 80 psi head blast produced cognitive deficits that were detected in working memory. Blast TBI rats displayed increased anxiety as determined by elevated plus maze at day 9 post-blast compared to sham rats; blast TBI rats spent significantly more time than the sham controls in the closed arms (p < 0.05; n = 8-11). Interestingly, anxiety symptoms were absent at days 22 and 48 post-blast. Instead, blast TBI rats displayed increased rearing behavior at day 48 post-blast compared to sham rats. Blast TBI rats also exhibited suppressed acoustic startle responses, but similar pre-pulse inhibition at day 15 post-blast compared to sham rats. Acute physiological alterations in cerebral glucose metabolism were determined by positron emission tomography 1 and 9 days post-blast using (18)F-fluorodeoxyglucose ((18)F-FDG). Global glucose uptake in blast TBI rat brains increased at day 1 post-blast (p < 0.05; n = 4-6) and returned to sham levels by day 9. Our results indicate a transient increase in cerebral metabolism following a blast injury. Markers for reactive astrogliosis and neuronal damage were noted by immunoblotting motor cortex tissue from day 10 post-blast in blast TBI rats compared to sham controls (p < 0.05; n = 5-6).

Discriminating patients with early-stage pancreatic cancer or chronic pancreatitis using serum electrospray mass profiling.

Blood tests are needed to aid in the early detection of pancreatic ductal adenocarcinoma (PDAC), and monitoring pancreatitis development into malignancy especially in high risk patients. This study exhibits efforts and progress toward developing such blood tests, using electrospray-mass spectrometry (MS) serum profiling to distinguish patients with early-stage PDAC or pancreatitis from each other and from controls. Identification of significant serum mass peak differences between these individuals was performed using t tests and "leave one out" cross validation. Serum mass peak distributions of control individuals were distinguished from those of patients with chronic pancreatitis or early-stage PDAC with P values <10(-15), and patients with chronic pancreatitis were distinguished from those of patients with early-stage PDAC with a P value <10(-12). Sera from 12 out of 12 patients with PDAC stages I, IIA and IIB were blindly validated from controls. Tandem MS/MS identified a cancer phenotype with elements of PDAC involved in early-stage PDAC/control discrimination. These studies indicate electrospray-MS mass profiling can detect serum changes in patients with pancreatitis or early-stage pancreatic cancer. Such technology has the potential to aid in early detection of pancreatic cancer, biomarker development, and in monitoring development of pancreatitis into PDAC.

Oncolytic potential of a novel KGFR tyrosine kinase inhibitor using a KGFR-selective breast cancer xenograft model.

BACKGROUND: Keratinocyte growth factor (KGF)/KGF receptor (KGFR) signaling produces a rapid increase in the progression of breast cancer. Molecular modeling was used to create a group of KGFR-selective kinase inhibitors (TKI). Compound L-27 is a potent and selective KGFR TKI. The present study examined the oncolytic potential of L-27 using a breast cancer xenograft model. MATERIALS AND METHODS: An orthotopic xenograft model was developed with KGF-transfected MCF-7 cells to examine the influence of L-27 upon KGFR-mediated tumor progression. RESULTS: L-27 was found to produce a dose-related reduction in the growth and metastasis of mouse xenograft tumors. Furthermore, L-27 treatment did not produce any signs of gross toxicity. CONCLUSION: L-27 was found to reduce the growth and metastasis of MCF-7 tumor xenografts with elevated expression of KGF. Thus, KGFR TKI may provide a new therapeutic approach for the treatment of breast and other types of cancer.

Distinguishing patients with stage I lung cancer versus control individuals using serum mass profiling.

Serum mass profiling can discern physiological changes associated with specific disease states and their progression. Sera (86 total) from control individuals and patients with stage I nonsmall cell lung cancer or benign small pulmonary nodules were discriminated retrospectively by serum changes discerned by mass profiling. Control individuals were distinguished from patients with Stage I lung cancer or benign nodules with test sensitivities of 89% and 83%. Lung cancer patients versus those with benign nodules were distinguished with 80% sensitivity. This study exhibits progress toward a minimally-invasive aid in early detection of lung cancer and monitoring small pulmonary nodules for malignancy.

Brain injury: neuro-inflammation, cognitive deficit, and magnetic resonance imaging in a model of blast induced traumatic brain injury.

Blast wave-induced traumatic injury from terrorist explosive devices can occur at any time in either military or civilian environments. To date, little work has focused on the central nervous system response to a non-penetrating blast injury. We have evaluated the effect of a single 80-psi blast-overpressure wave in a rat model. Histological and immunochemical studies showed an early inflammatory response, tissue damage and the initiation of apoptosis. With regard to inflammation, polymorphonuclear leukocytes and lymphocytes infiltrated brain parenchyma within 1 h post-blast. Glial-fibrillary protein, cyclo-oxygenase-2ir, interleukin-1ß and tumor necrosis factor were present by 1 h and remained detectable at three weeks post-injury. High mobility group box-1 protein was detectable at three weeks. With regard to tissue damage, S100ß and 4-hydroxynonenal were present at 1 h and remained detectable at three weeks. Amyloid precursor protein was detectable at three weeks. As for apoptosis, Cleaved Caspase-3 was detectable at three weeks. Morris water maze assessment of cognitive function showed that blast injured animals required significantly more time to reach the platform on day 1 of training and traveled a greater distance to get to the platform on days 1 and 2. Blast-injured animals showed a significant increase in swimming speed (p<0.001), increased total distance traveled (p<0.001) and increased number of entries into the previous quadrant that had contained the escape platform (p<0.05). Magnetic resonance imaging showed hyperintense regions in the somatosensory area within 1 h. T2 relaxation times and apparent diffusion coefficients show increasing trends in both somatosensory and cortical regions. These data indicate an early and lasting response of brain tissue to non-penetrating blast over-pressure injury. This early inflammatory response is indicative of a mild traumatic brain injury. There is evidence of early hippocampal dysfunction.

Silica-based branched hollow microfibers as a biomimetic extracellular matrix for promoting tumor cell growth in vitro and in vivo.

A novel scaffold composed of loosely branched hollow silica microfibers that has been proven to be highly biocompatible is proposed for the 3D culture of cancer cells. The MCF-7 cancer cells can grow and proliferate freely inside the scaffold in the form of multicellular spheroids. MCF-7 cancer cells cultured on the current 3D silica scaffold retained significantly more oncological characters than those cultured on the conventional 2D substrate and can serve as in vitro tumor model for studying cancer treatment.

Utility of nuclear magnetic resonance spectroscopy for pancreatic cancer studies.

OBJECTIVES: The aims of this study were (1) to determine nuclear magnetic resonance spectroscopic characteristics and metabolite profiles of serum samples from patients with pancreatic cancer compared with noncancerous control samples and (2) to ascertain if the accuracy of metabolite identification by 1D spectra can be improved upon by confirmation of spin-system assignment using more sophisticated experiments. METHODS: Nuclear magnetic resonance spectra, including 1D, total correlation spectroscopy, and heteronuclear multiple/single quantum coherence, were obtained from serum samples from patients with pancreatic cancer and control subjects and used to determine serum levels of a range of metabolites. RESULTS: The data show that total choline (P = 0.03), taurine (P = 0.03), and glucose plus triglycerides (P = 0.01) are significantly higher in cancer versus control samples. Also detected were species that could not be individually identified and that were designated UCM (unresolved complex matter). Levels of UCM are significantly higher in subjects with cancer, being almost double those of control samples. CONCLUSIONS: Although metabolites such as lactate, taurine, glucose, choline, and triglycerides can be determined from 1D spectra, accuracy is improved by confirmation of spin-system assignment with total correlation spectroscopy and heteronuclear multiple/single quantum coherence spectral analysis. In addition, we introduce a new metric, UCM, which is at higher concentrations in cancer compared with control samples.

Serum profiling to distinguish early- and late-stage ovarian cancer patients from disease-free individuals.

Sera mass spectrometry (MS) peak differences were analyzed from 35 ovarian cancer patients and 16 disease-free individuals. "Leave one out" cross validation was used to assign "% cancer peaks" in control and ovarian cancer sera samples. Sera MS discriminated stage I/II and stage III/V ovarian cancer patients versus controls with ROC curve area values of 0.82 and 0.92. Test sensitivities for ovarian cancer stage I/II and III/V were 80% and 93% respectively. These results indicate that MS is useful for distinguishing sera from early-stage ovarian cancer patients, and has potential as a test for early detection of this disease.

Distinguishing non-small cell lung adenocarcinoma patients from squamous cell carcinoma patients and control individuals using serum profiling.

Goals of this study were to analyze the ability of mass spectrometry serum profiling to distinguish non-small cell lung adenocarcinoma from squamous cell carcinoma patients and healthy controls. Sera were obtained from 19 adenocarcinoma patients, 24 squamous cell carcinoma patients, and 21 controls. Identifications of significant mass-to-charge ratio (m/z) peak differences between these groups were performed using t-tests. A "leave one out" cross-validation procedure yielded discriminatory lung adenocarcinoma versus squamous cell carcinoma p and ROC curve values of <.0001 and 0.92, respectively. Test sensitivity and specificity were 84% and 79%, respectively. This approach could aid in lung cancer diagnosis and sub-typing.

Serum discrimination of early-stage lung cancer patients using electrospray-ionization mass spectrometry.

The goal of this study was to evaluate the usefulness of electrospray ionization-mass spectrometry (ESI-MS) technology to distinguish sera of early-stage lung cancer patients from control individuals. ESI-MS m/z (mass divided by charge) data were generated from sera of 43 non-small cell lung cancer patients (pathological stages I and II) and 21 control individuals. Identifications of m/z peak area significances between cancer and control ESI-MS sera spectra were performed using t-tests. A "leave one out" cross validation procedure, which mimics blinded sera analysis and corrects for "over-fitting" of data, yielded discriminatory cancer versus control distribution p value and ROC curve area value of <0.001 and 0.87, respectively. Analysis without the "leave one out" cross validation procedure yielded a ROC curve area of 0.99 for discrimination of sera from lung cancer patients versus control individuals. Predictive value measurements revealed overall test efficiency and sensitivity for distinguishing sera from lung cancer patients from controls (using "leave one out" cross validation) of 80% and 84%, respectively. ESI-MS serum analysis between control individuals and lung cancer patients who smoked or did not smoke had p values in ranges indicating that smoking effects are not pronounced in our analysis. These studies indicate that ESI-MS analyses of sera from early stage non-small cell lung cancer patients were helpful in distinguishing these patients from control individuals.

DCAMKL-1 regulates epithelial-mesenchymal transition in human pancreatic cells through a miR-200a-dependent mechanism.

Pancreatic cancer is an exceptionally aggressive disease in great need of more effective therapeutic options. Epithelial-mesenchymal transition (EMT) plays a key role in cancer invasion and metastasis, and there is a gain of stem cell properties during EMT. Here we report increased expression of the putative pancreatic stem cell marker DCAMKL-1 in an established KRAS transgenic mouse model of pancreatic cancer and in human pancreatic adenocarcinoma. Colocalization of DCAMKL-1 with vimentin, a marker of mesenchymal lineage, along with 14-3-3 σ was observed within premalignant PanIN lesions that arise in the mouse model. siRNA-mediated knockdown of DCAMKL-1 in human pancreatic cancer cells induced microRNA miR-200a, an EMT inhibitor, along with downregulation of EMT-associated transcription factors ZEB1, ZEB2, Snail, Slug, and Twist. Furthermore, DCAMKL-1 knockdown resulted in downregulation of c-Myc and KRAS through a let-7a microRNA-dependent mechanism, and downregulation of Notch-1 through a miR-144 microRNA-dependent mechanism. These findings illustrate direct regulatory links between DCAMKL-1, microRNAs, and EMT in pancreatic cancer. Moreover, they demonstrate a functional role for DCAMKL-1 in pancreatic cancer. Together, our results rationalize DCAMKL-1 as a therapeutic target for eradicating pancreatic cancers.

miR-132 and miR-212 are increased in pancreatic cancer and target the retinoblastoma tumor suppressor.

Numerous microRNAs (miRNAs) are reported as differentially expressed in cancer, however the consequence of miRNA deregulation in cancer is unknown for many miRNAs. We report that two miRNAs located on chromosome 17p13, miR-132 and miR-212, are over-expressed in pancreatic adenocarcinoma (PDAC) tissues. Both miRNAs are predicted to target the retinoblastoma tumor suppressor, Rb1. Validation of this interaction was confirmed by luciferase reporter assay and western blot in a pancreatic cancer cell line transfected with pre-miR-212 and pre-miR-132 oligos. Cell proliferation was enhanced in Panc-1 cells transfected with pre-miR-132/-212 oligos. Conversely, antisense oligos to miR-132/-212 reduced cell proliferation and caused a G(2)/M cell cycle arrest. The mRNA of a number of E2F transcriptional targets were increased in cells over expressing miR-132/-212. Exposing Panc-1 cells to the ß2 adrenergic receptor agonist, terbutaline, increased the miR-132 and miR-212 expression by 2- to 4-fold. We report that over-expression of miR-132 and miR-212 result in reduced pRb protein in pancreatic cancer cells and that the increase in cell proliferation from over-expression of these miRNAs is likely due to increased expression of several E2F target genes. The ß2 adrenergic pathway may play an important role in this novel mechanism.

Distinguishing early-stage pancreatic cancer patients from disease-free individuals using serum profiling.

This study evaluated the usefulness of electrospray mass spectrometry to distinguish sera of early-stage pancreatic cancer patients from disease-free individuals. Sera peak data were generated from 33 pancreatic cancer patients and 30 disease-free individuals. A "leave one out" cross-validation procedure discriminated stage I/II pancreatic cancer versus disease-free sera with a p value <.001 and a receiver-operator characteristic curve area value of 0.85. Predictive values for cancer stage I/II test efficiency, specificity, and sensitivity were 78%, 77%, and 79%, respectively. These studies indicate that electrospray mass spectrometry is useful for distinguishing sera of early-stage pancreatic cancer patients from disease-free individuals.

Molecular MRI assessment of vascular endothelial growth factor receptor-2 in rat C6 gliomas.

Angiogenesis is essential to tumour progression and a precise evaluation of angiogenesis is important for tumour early diagnosis and treatment. The quantitative and dynamic in vivo assessment of tumour angiogenesis can be achieved by molecular magnetic resonance imaging (mMRI). Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are the main regulatory systems in angiogenesis and have been used as hot targets for radionuclide-based molecular imaging. However, little research has been accomplished in targeting VEGF/VEGFRs by mMRI. In our study, we aimed to assess the expression of VEGFR2 in C6 gliomas by using a specific molecular probe with mMRI. The differential uptake of the probe conjugated to anti-VEGFR2 monoclonal antibody, shown by varied increases in T(1) signal intensity during a 2 hr period, demonstrated the heterogeneous expression of VEGFR2 in different tumour regions. Microscopic fluorescence imaging, obtained for the biotin group in the probe with streptavidin-Cy3, along with staining for cellular VEGFR2 levels, laminin and CD45, confirmed the differential distribution of the probe which targeted VEGFR2 on endothelial cells. The angiogenesis process was also assessed using magnetic resonance angiography, which quantified tumour blood volume and provided a macroscopic view and a dynamic change of the correlation between tumour vasculature and VEGFR2 expression. Together these results suggest mMRI can be very useful in assessing and characterizing the expression of specific angiogenic markers in vivo and help evaluate angiogenesis associated with tumour progression.

Influence of novel KGFR tyrosine kinase inhibitors on KGF-mediated proliferation of breast cancer.

BACKGROUND: Keratinocyte growth factor (KGF) acts at the KGF receptor (KGFR) to produce a rapid stimulation of breast cancer cell proliferation and motility which is mediated via the Erk signaling pathway. Enhancement of KGF/KGFR signal transduction may be an early step in the metastatic progression of breast cancer. Receptor modeling of KGFR was used to identify selective KGFR tyrosine kinase (TK) inhibitor molecules that have the potential to bind selectively to the KGFR. The present study evaluated the biological activity of 57 of these KGFR TK inhibitor compounds on breast cancer cells. MATERIALS AND METHODS: These compounds were tested for their ability to inhibit KGF-mediated breast cancer cell proliferation in MCF-7 breast cancer cells. Furthermore, the effects of the most effective proliferation inhibitors were examined on Erk signaling and on the relative density of cell membrane KGFR. RESULTS: It was observed that 27 of the 57 compounds tested produced a 20% or greater reduction in KGF-mediated proliferation; while five compounds produced greater than 50% inhibition. In addition, the most potent inhibitors also reduced Erk signaling and cell membrane density of the KGFR. CONCLUSION: The compounds examined appear to be selective KGFR inhibitors which inhibit KGF-mediated activity and reduce the expression of KGFR on cancer cells. These results may lead to the development of a novel class of anticancer agents for the prevention of metastatic cancer progression.

Identification of a novel putative pancreatic stem/progenitor cell marker DCAMKL-1 in normal mouse pancreas.

Stem cells are critical in maintaining adult homeostasis and have been proposed to be the origin of many solid tumors, including pancreatic cancer. Here we demonstrate the expression patterns of the putative intestinal stem cell marker DCAMKL-1 in the pancreas of uninjured C57BL/6 mice compared with other pancreatic stem/progenitor cell markers. We then determined the viability of isolated pancreatic stem/progenitor cells in isotransplantation assays following DCAMKL-1 antibody-based cell sorting. Sorted cells were grown in suspension culture and injected into the flanks of athymic nude mice. Here we report that DCAMKL-1 is expressed in the main pancreatic duct epithelia and islets, but not within acinar cells. Coexpression was observed with somatostatin, NGN3, and nestin, but not glucagon or insulin. Isolated DCAMKL-1+ cells formed spheroids in suspension culture and induced nodule formation in isotransplantation assays. Analysis of nodules demonstrated markers of early pancreatic development (PDX-1), glandular epithelium (cytokeratin-14 and Ep-CAM), and isletlike structures (somatostatin and secretin). These data taken together suggest that DCAMKL-1 is a novel putative stem/progenitor marker, can be used to isolate normal pancreatic stem/progenitors, and potentially regenerates pancreatic tissues. This may represent a novel tool for regenerative medicine and a target for anti-stem cell-based therapeutics in pancreatic cancer.