Exosomal microRNA in serum is a novel biomarker of recurrence in human colorectal cancer.

BACKGROUND: Functional microRNAs (miRNAs) in exosomes have been recognised as potential stable biomarkers in cancers. The aim of this study is to identify specific miRNAs in exosome as serum biomarkers for the early detection of recurrence in human colorectal cancer (CRC). METHODS: Serum samples were sequentially obtained from six patients with and without recurrent CRC. The miRNAs were purified from exosomes, and miRNA microarray analysis was performed. The miRNA expression profiles and copy number aberrations were explored using microarray and array CGH analyses in 124 CRC tissues. Then, we validated exosomal miRNAs in 2 serum sample sets (90 and 209 CRC patients) by quantitative real-time RT-PCR. RESULTS: Exosomal miR-17-92a cluster expression level in serum was correlated with the recurrence of CRC. Exosomal miR-19a expression levels in serum were significantly increased in patients with CRC as compared with healthy individuals with gene amplification. The CRC patients with high exosomal miR-19a expression showed poorer prognoses than the low expression group (P<0.001). CONCLUSIONS: Abundant expression of exosomal miR-19a in serum was identified as a prognostic biomarker for recurrence in CRC patients.

The AURKA/TPX2 axis drives colon tumorigenesis cooperatively with MYC.

BACKGROUND: The MYC oncogene has long been established as a central driver in many types of human cancers including colorectal cancer. However, the realization of MYC-targeting therapies remains elusive; as a result, synthetic lethal therapeutic approaches are alternatively being explored. A synthetic lethal therapeutic approach aims to kill MYC-driven tumors by targeting a certain co-regulator on the MYC pathway. PATIENTS AND METHODS: We analyzed copy number and expression profiles from 130 colorectal cancer tumors together with publicly available datasets to identify co-regulators on the MYC pathway. Candidates were functionally tested by in vitro assays using colorectal cancer and normal fibroblast cell lines. Additionally, survival analyses were carried out on another 159 colorectal cancer patients and public datasets. RESULTS: Our in silico screening identified two MYC co-regulator candidates, AURKA and TPX2, which are interacting mitotic regulators located on chromosome 20q. We found the two candidates showed frequent co-amplification with the MYC locus while expression levels of MYC and the two genes were positively correlated with those of MYC downstream target genes across multiple cancer types. In vitro, the aberrant expression of MYC, AURKA and TPX2 resulted in more aggressive anchorage-independent growth in normal fibroblast cells. Furthermore, knockdown of AURKA or TPX2, or treatment with an AURKA-specific inhibitor effectively suppressed the proliferation of MYC-expressing colorectal cancer cells. Additionally, combined high expression of MYC, AURKA and TPX2 proved to be a poor prognostic indicator of colorectal cancer patient survival. CONCLUSIONS: Through bioinformatic analyses and experiments, we proposed TPX2 and AURKA as novel co-regulators on the MYC pathway. Inhibiting the AURKA/TPX2 axis would be a novel synthetic lethal therapeutic approach for MYC-driven cancers.

Amplification of PVT-1 is involved in poor prognosis via apoptosis inhibition in colorectal cancers.

BACKGROUND: We previously conducted gene expression microarray analyses to identify novel indicators for colorectal cancer (CRC) metastasis and prognosis from which we identified PVT-1 as a candidate gene. PVT-1, which encodes a long noncoding RNA, mapped to chromosome 8q24 whose copy-number amplification is one of the most frequent events in a wide variety of malignant diseases. However, PVT-1 molecular mechanism of action remains unclear. METHODS: We conducted cell proliferation and invasion assays using colorectal cancer cell lines transfected with PVT-1siRNA or negative control siRNA. Gene expression microarray analyses on these cell lines were also carried out to investigate the molecular function of PVT-1. Further, we investigated the impact of PVT-1 expression on the prognosis of 164 colorectal cancer patients by qRT-PCR. RESULTS: CRC cells transfected with PVT-1 siRNA exhibited significant loss of their proliferation and invasion capabilities. In these cells, the TGF-ß signalling pathway and apoptotic signals were significantly activated. In addition, univariate and multivariate analysis revealed that PVT-1 expression level was an independent risk factor for overall survival of colorectal cancer patients. CONCLUSION: PVT-1, which maps to 8q24, generates antiapoptotic activity in CRC, and abnormal expression of PVT-1 was a prognostic indicator for CRC patients.

Paired related homoeobox 1, a new EMT inducer, is involved in metastasis and poor prognosis in colorectal cancer.

BACKGROUND: Paired related homoeobox 1 (PRRX1) has been identified as a new epithelial-mesenchymal transition (EMT) inducer in breast cancer. However, the function of PRRX1 in colorectal cancer (CRC) has not been elucidated. METHODS: We utilised ectopic PRRX1-expressing cell lines to analyse the function of PRRX1 in CRC. The clinical significance of PRRX1 was also examined on three independent CRC case sets. RESULTS: PRRX1 induced EMT and the stem-like phenotype in CRC cells. In contrast to studies of breast cancer, abundant expression of PRRX1 was significantly associated with metastasis and poor prognosis in CRC. CONCLUSION: PRRX1 is an indicator of metastasis and poor prognosis in CRC cases. Further investigation is required to uncover the signalling network regulating PRRX1.

Increased lipophilicity and subsequent cell partitioning decrease passive transcellular diffusion of novel, highly lipophilic antioxidants.

Oxidative stress is considered a cause or propagator of acute and chronic disorders of the central nervous system. Novel 2, 4-diamino-pyrrolo[2,3-d]pyrimidines are potent inhibitors of iron-dependent lipid peroxidation, are cytoprotective in cell culture models of oxidative injury, and are neuroprotective in brain injury and ischemia models. The selection of lead candidates from this series required that they reach target cells deep within brain tissue in efficacious amounts after oral dosing. A homologous series of 26 highly lipophilic pyrrolopyrimidines was examined using cultured cell monolayers to understand the structure-permeability relationship and to use this information to predict brain penetration and residence time. Pyrrolopyrimidines were shown to be a more permeable structural class of membrane-interactive antioxidants where transepithelial permeability was inversely related to lipophilicity or to cell partitioning. Pyrrole substitutions influence cell partitioning where bulky hydrophobic groups increased partitioning and decreased permeability and smaller hydrophobic groups and more hydrophilic groups, especially those capable of weak hydrogen bonding, decreased partitioning, and increased permeability. Transmonolayer diffusion for these membrane-interactive antioxidants was limited mostly by desorption from the receiver-side membrane into the buffer. Thus, in this case, these in vitro cell monolayer models do not adequately mimic the in vivo situation by underestimating in vivo bioavailability of highly lipophilic compounds unless acceptors, such as serum proteins, are added to the receiving buffer.

Novel, highly lipophilic antioxidants readily diffuse across the blood-brain barrier and access intracellular sites.

In an accompanying article, an in vitro assay for permeability predicts that membrane-protective, antioxidant 2,4-diamino-pyrrolo[2, 3-d]pyrimidines should have improved blood-brain barrier (BBB) permeation over previously described lipophilic antioxidants. Using a first-pass extraction method and brain/plasma quantification, we show here that two of the pyrrolopyrimidines, one of which is markedly less permeable, readily partition into rat brain. The efficiency of extraction was dependent on serum protein binding, and in situ efflux confirms the in vitro data showing that PNU-87663 is retained in brain longer than PNU-89843. By exploiting inherent fluorescence properties of PNU-87663, its distribution within brain and within cells in culture was demonstrated using confocal scanning laser microscopy. PNU-87663 rapidly partitioned into the cell membrane and equilibrates with cytoplasmic compartments via passive diffusion. Although partitioning of PNU-87663 favors intracytoplasmic lipid storage droplets, the compound was readily exchangeable as shown by efflux of compound from cells to buffer when protein was present. The results demonstrated that pyrrolopyrimidines were well suited for quickly accessing target cells within the central nervous system as well as in other target tissues.

Expression of a P-selectin ligand in zona pellucida of porcine oocytes and P-selectin on acrosomal membrane of porcine sperm cells. Potential implications for their involvement in sperm-egg interactions.

The selectin family of cell adhesion molecules mediates initial leukocyte adhesion to vascular endothelial cells at sites of inflammation. O-glycan structural similarities between oligosaccharides from human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and from zona pellucida glycoproteins of porcine oocytes indicate the possible existence of a P-selectin ligand in the zona pellucida. Here, using biochemical as well as morphological approaches, we demonstrate that a P-selectin ligand is expressed in the porcine zona pellucida. In addition, a search for a specific receptor for this ligand leads to the identification of P-selectin on the acrosomal membrane of porcine sperm cells. In vitro binding of porcine acrosome-reacted sperm cells to oocytes was found to be Ca2+ dependent and inhibitable with either P-selectin, P-selectin receptor-globulin, or leukocyte adhesion blocking antibodies against P-selectin and PSGL-1. Moreover, porcine sperm cells were found to be capable of binding to human promyeloid cell line HL-60. Taken together, our findings implicate a potential role for the oocyte P-selectin ligand and the sperm P-selectin in porcine sperm-egg interactions.

Pyrrolopyrimidines: novel brain-penetrating antioxidants with neuroprotective activity in brain injury and ischemia models.

A novel group of antioxidant compounds, the pyrrolopyrimidines, has been discovered recently. Many of these possess significantly improved oral bioavailability (56-70% in rats), increased efficacy and potency in protecting cultured neurons against iron-induced lipid peroxidative injury and as much as a 5-fold increase in brain uptake compared with the 21-aminosteroid antioxidant compound, tirilazad mesylate (U-74006F), described earlier. They appear to quench lipid peroxidation reactions by electron-donating and/or radical-trapping mechanisms. Several compounds in the series, such as U-101033E and U-104067F, demonstrate greater ability than tirilazad to protect the hippocampal CA1 region in the gerbil transient (5-min) forebrain ischemia model. Delaying treatment until 4 hr after the ischemic insult still results in significant CA1 neuronal protection. U-101033E is still effective in salvaging a portion of the CA1 neuronal population when the ischemic duration is extended to 10 min. In addition, U-101033E has been found to be protective in the context of focal cerebral ischemia, reducing infarct size in the mouse permanent middle cerebral artery occlusion model, in contrast to tirilazad which is minimally effective. These results suggest that antioxidant compounds with improved brain parenchymal penetration are better able to limit certain types of ischemic brain damage than those which are localized in the cerebral microvasculature. However, the activity of U-101033E in improving early post-traumatic recovery in mice subjected to severe concussive head injury is similar to that of tirilazad. Last, the oral bioavailability of many pyrrolopyrimidines suggests that they may be useful for certain chronic neurodegenerative disorders in which lipid peroxidation plays a role.

Intestinal absorption of sodium dodecyl sulfate in the rodent: evidence for paracellular absorption.

Experiments were performed to define the mechanism of intestinal absorption of dodecyl sulfate (DS), an amphipathic organic anion whose chemical structure resembles that of dodecanoate, a C12 fatty acid anion. With jejunal segments perfused in single-pass fashion in the anesthetized rat, steady-state absorption of DS was concentration dependent, with the apparent permeability constant (P(app)) ranging from 4 to 22 x 10(-5) cm/s. When DS concentration was held constant and net water absorption was induced by decreasing perfusate osmolality, DS absorption increased in direct proportion to water absorption, suggesting absorption by solvent drag via the paracellular route. However, DS absorption continued even when water secretion was induced by a hypertonic perfusate. Consequently, for all experiments, DS absorption could be empirically described as the sum of two terms: 1) absorption in the absence of water absorption (P(app) = 5.6 x 10(-5) cm/s) and 2) absorption induced by water movement [(delta P(app)/delta water absorption) = 0.2 x 10(-5) cm x s(-1) x microl segment(-1) x min(-1)]. In a polarized epithelial monolayer of renal epithelial cells (Madin-Darby canine kidney cells), DS was absorbed predominantly by a paracellular pathway, as the absorption rate increased threefold when paracellular junction pore size was increased by the addition of cytochalasin D. The calculated apparent radius was 2.9 A, indicating that the cross section of the molecule, not its length, determined the rate of absorption. It is concluded that absorption of DS in the intact animal occurs slowly and mostly via the paracellular route, because the fixed negative charge on the molecule retards rapid passive entry into the enterocyte, as occurs with protonated fatty acids. That absorption of DS persisted despite net water secretion suggests a low level of transcellular absorption across the jejunal enterocyte also occurs.

Analytical and numerical techniques for the evaluation of free radical damage in cultured cells using scanning laser microscopy.

Scanning laser microscopy (SLM) was used to develop an assay to visualize the generation of intracellular reactive oxygen species (ROS) and to evaluate the effect of the lipophilic antioxidant U-87,663 on ROS formation. Cultured N18 neuroglioma cells were challenged by extracellular addition of cumene hydroperoxide, and subsequent intracellular generation of ROS was characterized by measuring the fluorescence intensity of the ROS indicator 2',7'-dichlorofluorescein (DCF). The kinetics of the reaction between ROS and the indicator DCF, or the antioxidant U-87,663, can be most accurately assessed if results from individual cell clusters are analyzed independently. It is possible and necessary to account for the these experimental and analytical properties in order to characterize the properties of the antioxidant activity precisely. We determined that the temporal increase in DCF fluorescence was consistent with the reaction of DCF with free radicals generated from cumene hydroperoxide, as was the loss of fluorescence from U-87,663. The rate constants for the free radical reactions revealed that ROS reaction with DCF is 10 times faster than with U-87,663. These differences in reaction rates combined with differences in the cellular distribution of the ROS indicator DCF, the antioxidant U-87,663, and the bulk of the ROS prevented detection of any protection of U-87,663 may offer.

Neuroprotective efficacy of microvascularly-localized versus brain-penetrating antioxidants.

The 21-aminosteroid (lazaroid) tirilazad mesylate has been demonstrated to be a potent inhibitor of lipid peroxidation and to reduce traumatic and ischemic damage in a number of experimental models. Currently, tirilazad is being actively investigated in phase III clinical trials in head and spinal cord injury, ischemic stroke and subarachnoid hemorrhage. This compound acts in large part to protect the microvascular endothelium and consequently to maintain normal blood-brain barrier (BBB) permeability and cerebral blood flow autoregulatory mechanisms. However, due to its limited penetration into brain parenchyma, tirilazad has generally failed to affect delayed neuronal damage to the selectively vulnerable hippocampal CA1 and striatal regions. Recently, we have discovered a new group of antioxidant compounds, the pyrrolopyrimidines, which possess significantly improved ability to penetrate the BBB and gain direct access to neural tissue. Several compounds in the series, such as U-101033E, have demonstrated greater ability to protect the CA1 region in the gerbil transient forebrain ischemia model with a post-ischemic therapeutic window of at least four hours. In addition, U-101033E has been found to reduce infarct size in the mouse permanent middle cerebral artery occlusion model in contrast to tirilazad which is minimally effective. These results suggest that antioxidant compounds with improved brain parenchymal penetration are better able to limit certain types of ischemic brain damage compared to those which are localized in the cerebral microvasculature. On the other hand, microvascularly-localized agents like tirilazad appear to have better ability to limit BBB damage.

Expression of a widely distributed, novel sulfated membrane glycoprotein by bovine endothelia and certain transporting epithelia.

We generated monoclonal antibodies (MAbs) against cultured bovine brain microvessel endothelial cells (BMEC) for use as probes to study membrane protein traffic and polarity. One MAb recognized a heterogeneous family of acidic sulfoglycoproteins called gp4A4 with molecular weights of 50-65 KD and 85 KD. Gp4A4 is a long-lived integral membrane protein which resides mostly at the plasma membrane, and a portion appears to be in equilibrium with an intracellular pool via endocytosis. Gp4A4 is expressed by many endothelial cells, except for fenestrated capillaries in choroid plexus, and specific epithelial cells in bile duct, kidney, and choroid plexus. A comparison of indirect immunoperoxidase and immunofluorescence detection using semi-thin cryosections gave contrasting results on the apparent distribution of gp4A4 on the apical and basolateral membranes of cerebral endothelia and choroid plexus epithelia. Immunogold labeling of ultra-thin cryosections showed that gp4A4 was expressed by the apical and basolateral membrane domains of BMEC and choroid plexus epithelia. This was consistent with the results using indirect immunofluorescence microscopy. On an average, gp4A4 expression by cerebral endothelia was not asymmetric and was considerably variable between capillaries. These results emphasize the need to compare several different techniques in assessing polarized expression of cell surface antigens in vivo.

Transcellular permeability of chlorpromazine demonstrating the roles of protein binding and membrane partitioning.

Transcellular permeability of the neuroleptic-anesthetic chlorpromazine (CPZ) was examined using a cell type (MDCK) that forms a confluent monolayer of polarized cells resulting in distinct apical (AP) and basolateral (BL) membrane domains separated by tight junctions. Because CPZ is membrane interactive, transmonolayer flux was analyzed as two kinetic events: cell uptake from the AP donor solution and efflux into the BL side receiver. Using the rate of cell uptake in the presence of different concentrations of BSA, an intrinsic cell partition coefficient of 3700 +/- 130 and an operational dissociation binding constant of 0.4 +/- 0.05 mM were calculated. In contrast to uptake, efflux of CPZ from either the AP or the BL side of the cell monolayer was approximately 10(4)-fold slower and was dependent upon the avidity of CPZ for the protein acceptor in the receiver solution. These results emphasized the importance of simultaneously measuring disappearance of a lipophilic molecule from the donor solution and its appearance in the receiver and demonstrated how interactions with proteins on either side of the cellular barrier influence permeability. Appearance kinetics showed that the composition of the receiving environment is critical to model a particular in vivo situation and implied that the intrinsic permeability of membrane-interactive molecules in vitro does not necessarily predict penetration beyond the initial cellular barrier in vivo.

Use of a biophysical-kinetic model to understand the roles of protein binding and membrane partitioning on passive diffusion of highly lipophilic molecules across cellular barriers.

The novel antioxidants U-78517F and U-74006F, or lazaroids, are highly lipophilic organic molecules with poor brain uptake. To understand this paradoxical behavior better, continuous monolayers of Madin-Darby canine kidney (MDCK) epithelial cells with distinct apical (AP) and basolateral (BL) plasma membrane domains grown on polycarbonate membrane filters and plastic were used to examine the mechanism of transcellular diffusion. Independent kinetic experiments were used to quantify AP to BL flux, efflux from the AP and BL membranes and AP membrane partitioning as functions of bovine serum albumin (BSA) concentration. Fluxes were appropriately reduced to permeability coefficients (Pe) for the membrane, aqueous boundary layer (ABL) and filter, BSA-drug binding constants, and effective (Ke) and intrinsic (Kintr) membrane partition coefficients in the absence of metabolism. Both Pe and Ke decreased exponentially with increased BSA concentration and a concomitant decrease in free drug concentration. Uptake was ABL-controlled under the conditions used and its Pe was 1,000-fold faster than that for efflux due to a large Kintr. Therefore, diffusion across the cellular barrier was limited kinetically by the equilibrium between protein-bound drug and free drug partitioned into the cell membrane and the rate-limiting desorption of drug from the cell membrane into the aqueous receiver. This suggests that brain uptake of these lipophilic antioxidants is limited by interactions with plasma proteins and, possibly, by unfavorable partitioning from the endothelium into the underlying tissue. The present biophysical kinetic model is proposed as generally useful in studying the penetrative ability of other membrane interacting molecules.

Permeability of bovine brain microvessel endothelial cells in vitro: barrier tightening by a factor released from astroglioma cells.

It has been shown both in vivo and in culture that astrocytes communicate with brain microvessel endothelial cells (BMECs) to induce many of the blood-brain barrier characteristics attributed to these unique cells. However, the results using cultured cells are conflicting as to whether this communication is dependent upon cell-cell contact. In this study we used primary cultures of bovine BMECs grown as monolayers on polycarbonate filters to study the formation of the barrier in vitro and examine its modulation by rat C6 glioma cells. Effects were examined by treating postconfluent BMEC monolayers with medium conditioned continually by C6 cells from the basolateral side to mimic the in vivo orientation. Cell monolayer integrity was assessed using electrical resistance and by measuring diffusion of uncharged molecules. BMEC monolayers form a functionally polarized and leaky barrier, with maximal resistance of 160 omega . cm2 and significant flux of molecules of molecular weight less than 350 Da. Treatment with rat or human astroglioma cells rather than pericytoma cells or transformed fibroblasts results in a concentration-dependent 200-440% increase in electrical resistance and a coincident 50% decrease in permeability to sucrose and dextran (70 kDa). The decrease in passive diffusion is most likely due to a change in tight junctions and not to transcellular vesicular traffic. The findings support that astroglioma cells release one or more signals that are required for cultured BMECs to express a "differentiated" phenotype associated with a tighter barrier, increased gamma-glutamyl transpeptidase activity, and decreased pinocytic activity. The relative ease and quickness of this culture system makes it amenable to studies on cell-cell interaction and regulation of barrier maintenance.

Immunohistochemical and autoradiographic findings suggest that minoxidil is not localized in specific cells of vibrissa, pelage, or scalp follicles.

Immunohistochemistry with a minoxidil antibody suggested that minoxidil-immunoreactivity is associated with the root sheaths, laterally orientated differentiating matrix cells, and dividing epithelial cells of cultured vibrissa follicles of pigmented and albino neonatal mice. The dermal papilla and connective tissue sheath were devoid of minoxidil-immunoreactivity. To verify that minoxodil-immunoreactivity in the follicles was specific, immunostaining was conducted with dissected whisker pads, formalin-fixed "dead" follicles, and sections of spleen, liver and kidney (non-haired organs) cultured with minoxidil. Microscopic examination revealed minoxidil-immunoreactivity in all of these tissues. Follicles and whisker pads cultured with minoxidil, then washed for one h in media were devoid of minoxidil-immunoreactivity. These data suggest that minoxidil-immunoreactivity in cultured vibrissa follicles is probably non-specific. Sections of skin from C3H and CF1 mice which were topically dosed with minoxidil (in vivo) showed no minoxidil-immunoreactivity. Autoradiography demonstrated that tritiated minoxidil was bound in vivo and in vitro only to melanin granules in pigmented follicles of rodent and human tissue. This is probably non-specific binding since melanin is known to accumulate several chemically and pharmacologically unrelated drugs. It is reasonable to conclude that, under the conditions of these experiments, minoxidil is not specifically localized in any cells of whisker, pelage or, scalp follicles.

Ciglitazone, a hypoglycemic agent: early effects on the pancreatic islets of ob/ob mice.

Chronic administration of ciglitazone (5-4[1-methyl-cyclohexylmethoxy)-benzyl]-thiazolidine-2,4 dione) decreased both plasma glucose and insulin concentrations in ob/ob mice. When given as an admixture to the feed, blood glucose levels were reduced as early as 12 hours after initiation of treatment. Concomitant with the decrease in circulating insulin, there was an increased hormone content in the beta-cells as judged by RIA and aldehyde-fuchsin staining. Acute oral dosing with ciglitazone produced a 41% reduction in circulating insulin at a time when glucose concentrations were as yet unaffected. Ciglitazone also inhibited glucose-stimulated insulin secretion in vitro. The results suggest that the hypoglycemic agent, ciglitazone, may reduce plasma glucose and insulin concentrations at least partially as the result of independent mechanisms.

Analysis of pancreatic islet cells and hormone content in the spontaneously diabetic KKAy mouse by morphometry, immunocytochemistry and radioimmunoassay.

The splenic pancreas of 165 day old diabetic KKAy and age-matched nondiabetic C57BL/6 mice was examined by morphometry and immunocytochemistry at the light microscopic level and by radioimmunoassay to evaluate the morphology, surface area, endocrine cell composition and hormone content of the pancreatic islets. The insulin cells of the diabetic mice were severely degranulated and many of the glucagon, somatostatin and pancreatic polypeptide cells were displaced from the mantle to the core of the islet tissue where the non-insulin cells appeared to lose their continuity. The topography of some of the islets of KKAy mice was further deranged by acinar cells among the endocrine tissue. Morphometric analysis revealed that the surface area of the islets of KKAy mice was significantly expanded in comparison with that of C57BL/6 mice. The volume and numerical percents of the insulin cells were significantly increased whereas those of the glucagon and somatostatin cells were decreased in the KKAy mice. Since only the mean absolute number of insulin cells was elevated in the diabetic mice, the alteration in the relative proportions of the non-insulin cells and hypertrophy of the islets seemed to be a manifestation of insulin cell hyperplasia. Pancreatic insulin and somatostatin contents were markedly diminished in the islets of KKAy compared with those of C57BL/6 mice. These results demonstrate that the microscopic anatomy, endocrine cell populations and hormone content of the pancreatic islets are deranged in the KKAy mouse with severe hyperinsulinemia and hyperglycemia.