The discovery of indolone GW5074 during a comprehensive search for non-polyamine-based polyamine transport inhibitors.

The native polyamines putrescine, spermidine, and spermine are essential for cell development and proliferation. Polyamine levels are often increased in cancer tissues and polyamine depletion is a validated anticancer strategy. Cancer cell growth can be inhibited by the polyamine biosynthesis inhibitor difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthesis pathway. Unfortunately, cells treated with DFMO often replenish their polyamine pools by importing polyamines from their environment. Several polyamine-based molecules have been developed to work as polyamine transport inhibitors (PTIs) and have been successfully used in combination with DFMO in several cancer models. Here, we present the first comprehensive search for potential non-polyamine based PTIs that work in human pancreatic cancer cells in vitro. After identifying and testing five different categories of compounds, we have identified the c-RAF inhibitor, GW5074, as a novel non-polyamine based PTI. GW5074 inhibited the uptake of all three native polyamines and a fluorescent-polyamine probe into human pancreatic cancer cells. GW5074 significantly reduced pancreatic cancer cell growth in vitro when treated in combination with DFMO and a rescuing dose of spermidine. Moreover, GW5074 alone reduced tumor growth when tested in a murine pancreatic cancer mouse model in vivo. In summary, GW5074 is a novel non-polyamine-based PTI that potentiates the anticancer activity of DFMO in pancreatic cancers.

Neuronal Aquaporin 1 Inhibits Amyloidogenesis by Suppressing the Interaction Between Beta-Secretase and Amyloid Precursor Protein.

The accumulation of amyloid-ß (Aß) is a characteristic event in the pathogenesis of Alzheimer's disease (AD). Aquaporin 1 (AQP1) is a membrane water channel protein belonging to the AQP family. AQP1 levels are elevated in the cerebral cortex during the early stages of AD, but the role of AQP1 in AD pathogenesis is unclear. We first determined the expression and distribution of AQP1 in brain tissue samples of AD patients and two AD mouse models (3xTg-AD and 5xFAD). AQP1 accumulation was observed in vulnerable neurons in the cerebral cortex of AD patients, and in neurons affected by the Aß or tau pathology in the 3xTg-AD and 5xFAD mice. AQP1 levels increased in neurons as aging progressed in the AD mouse models. Stress stimuli increased AQP1 in primary cortical neurons. In response to cellular stress, AQP1 appeared to translocate to endocytic compartments of ß- and γ-secretase activities. Ectopic expression of AQP1 in human neuroblastoma cells overexpressing amyloid precussir protein (APP) with the Swedish mutations reduced ß-secretase (BACE1)-mediated cleavage of APP and reduced Aß production without altering the nonamyloidogenic pathway. Conversely, knockdown of AQP1 enhanced BACE1 activity and Aß production. Immunoprecipitation experiments showed that AQP1 decreased the association of BACE1 with APP. Analysis of a human database showed that the amount of Aß decreases as the expression of AQP1 increases. These results suggest that the upregulation of AQP1 is an adaptive response of neurons to stress that reduces Aß production by inhibiting the binding between BACE1 and APP.

Reduced skeletal muscle secreted frizzled-related protein 3 is associated with inflammation and insulin resistance.

OBJECTIVE: To investigate the role of secreted frizzled-related protein 3 (Sfrp3) in insulin sensitivity (ISi) and ß-cell function in humans across a spectrum of glucose homeostasis. METHODS: Subjects included those with normal glucose homeostasis (NGT; n = 18), prediabetes (PD; n = 11), or type 2 diabetes (T2D; n=12). Serum and skeletal muscle (SkM) Sfrp3 levels were measured by ELISA and qPCR, respectively, and insulin signaling pathway was assessed by Western blot. IS and ß-cell function were assessed by indices derived from frequently sampled intravenous glucose tolerance test. RESULTS: SkM Sfrp3 mRNA levels were significantly reduced in PD and T2D versus NGT. Similarly, serum Sfrp3 levels tended to be decreased in PD and T2D versus NGT. SkM Sfrp3 mRNA levels correlated negatively with circulating proinflammatory cytokines (IL-6, IFN-γ) and positively with IS. In vitro-differentiated myotubes from lean insulin-sensitive subjects treated with either lipopolysaccharide (LPS) or recombinant IL-6 demonstrated a dose-dependent reduction in Sfrp3 gene expression. Treatment of myotubes with recombinant Sfrp3 restored LPS- and IL-6-induced attenuation of insulin-stimulated Akt phosphorylation. CONCLUSIONS: Inflammation-induced reduction in SkM Sfrp3 expression may contribute to insulin resistance, and this effect may be prevented by addition of exogenous Sfrp3. Thus, Sfrp3 may be a novel target for insulin sensitization.

ATP13A3 and caveolin-1 as potential biomarkers for difluoromethylornithine-based therapies in pancreatic cancers.

The purpose of this paper was to better understand the role of polyamine transport in pancreatic cancers.This paper identifies potential biomarkers for assessing the relative tumor commitment to polyamine biosynthesis or transport. Cell lines with low polyamine import activity and low ATP13A3 protein levels appear committed to polyamine biosynthesis and required high concentrations of the polyamine biosynthesis inhibitor, difluoromethylornithine (DFMO) to inhibit their growth (e.g., AsPC-1 and Capan 1). In contrast, cell lines with high polyamine import activity and high ATP13A3 protein expression (e.g., L3.6pl) demonstrated a commitment to polyamine transport and required lower DFMO concentrations to inhibit their growth. Pancreatic cancer cell lines which were most sensitive to DFMO also gave the highest EC50 values for the polyamine transport inhibitors (PTIs) tested indicating that more PTI was needed to inhibit the active polyamine transport systems of these cell lines. Most significant is that the combination therapy of DFMO+PTI was efficacious against both cell types with the PTI showing low efficacy in cell lines with low polyamine transport activity and high efficacy in cell lines with high polyamine transport activity. High ATP13A3 protein expression and moderate to low Cav-1 protein expression was shown to be predictive of tumors which effectively escape DFMO via polyamine import. In summary, this report demonstrates for the first time the role of ATP13A3 in polyamine transport and its use as a potential biomarker along with Cav-1 to select tumors most susceptible to DFMO. These findings may help stratify patients in the ongoing clinical trials with DFMO-based therapies and help predict tumor response.

Proteomics analyses of subcutaneous adipocytes reveal novel abnormalities in human insulin resistance.

OBJECTIVE: To provide a more global view of adipocyte changes in human insulin resistance by proteomics analyses. METHODS: Baseline biopsies of abdominal subcutaneous adipose tissue were obtained from 23 subjects without diabetes. Euglycemic clamps were used to divide subjects into an insulin-resistant group (IR, N = 10) and an insulin-sensitive (IS, N = 13) group, which were of similar age and gender but unequal adiposity (greater in IR). Proteins of isolated adipocytes were quantified by mass spectrometry using normalized spectral abundance factors. RESULTS: Of 1,245 proteins assigned, 30 were detected in at least 12 of the 23 subjects that differed significantly in abundance ≥1.5-fold between IR and IS. IR displayed a pattern of increased cytoskeletal proteins and decreased mitochondrial proteins and FABP4 and FABP5. In subgroup analyses of adiposity-matched subjects, several of these changes were less pronounced in IR, but the abundance of proteins related to lipid metabolism and the unfolded/misfolded protein response were significantly and unfavorably altered. CONCLUSIONS: These results confirm lower abundance of mitochondrial proteins and suggest increased cytoskeletal proteins and decreased FABP4 and FABP5 in subcutaneous adipocytes of typical IR individuals. Changes in proteins related to lipid metabolism and the unfolded/misfolded protein may discriminate IR and IS individuals of equal adiposity.

Polyamine transport inhibitors: design, synthesis, and combination therapies with difluoromethylornithine.

The development of polyamine transport inhibitors (PTIs), in combination with the polyamine biosynthesis inhibitor difluoromethylornithine (DFMO), provides a method to target cancers with high polyamine requirements. The DFMO+PTI combination therapy results in sustained intracellular polyamine depletion and cell death. A series of substituted benzene derivatives were evaluated for their ability to inhibit the import of spermidine in DFMO-treated Chinese hamster ovary (CHO) and L3.6pl human pancreatic cancer cells. Several design features were discovered which strongly influenced PTI potency, sensitivity to amine oxidases, and cytotoxicity. These included changes in (a) the number of polyamine chains appended to the ring system, (b) the polyamine sequence, (c) the attachment linkage of the polyamine to the aryl core, and (d) the presence of a terminal N-methyl group. Of the series tested, the optimal design was N(1),N(1'),N(1″)-(benzene-1,3,5-triyltris(methylene))tris(N(4)-(4-(methylamino)butyl)butane-1,4-diamine, 6b, which contained three N-methylhomospermidine motifs. This PTI exhibited decreased sensitivity to amine oxidases and low toxicity as well as high potency (EC50 = 1.4 µM) in inhibiting the uptake of spermidine (1 µM) in DFMO-treated L3.6pl human pancreatic cancer cells.

A DESD-box helicase functions in salinity stress tolerance by improving photosynthesis and antioxidant machinery in rice (Oryza sativa L. cv. PB1).

The exact mechanism of helicase-mediated salinity tolerance is not yet understood. We have isolated a DESD-box containing cDNA from Pisum sativum (Pea) and named it as PDH45. It is a unique member of DEAD-box helicase family; containing DESD instead of DEAD/H. PDH45 overexpression driven by constitutive cauliflower mosaic virus-35S promoter in rice transgenic [Oryza sativa L. cv. Pusa Basmati 1 (PB1)] plants confers salinity tolerance by improving the photosynthesis and antioxidant machinery. The Na(+) ion concentration and oxidative stress parameters in leaves of the NaCl (0, 100 or 200 mM) treated PDH45 overexpressing T1 transgenic lines were lower as compared to wild type (WT) rice plants under similar conditions. The 200 mM NaCl significantly reduced the leaf area, plant dry mass, net photosynthetic rate (PN), stomatal conductance (gs), intercellular CO2 (Ci), chlorophyll (Chl) content in WT plants as compared to the transgenics. The T1 transgenics exhibited higher glutathione (GSH) and ascorbate (AsA) contents under salinity stress. The activities of antioxidant enzymes viz. superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) were significantly higher in transgenics; suggesting the existence of an efficient antioxidant defence system to cope with salinity induced-oxidative damage. Yeast two-hybrid assay indicated that the PDH45 protein interacts with Cu/Zn SOD, adenosine-5'-phosphosulfate-kinase, cysteine proteinase and eIF(4G), thus confirming the involvement of ROS scavenging machinery in the transgenic plants to provide salt tolerance. Furthermore, the T2 transgenics were also able to grow, flower, and set viable seeds under continuous salinity stress of 200 mM NaCl. This study provides insights into the mechanism of PDH45 mediated salinity stress tolerance by controlling the generation of stress induced reactive oxygen species (ROS) and also by protecting the photosynthetic machinery through a strengthened antioxidant system.

Effect of acetazolamide on aquaporin-1 and fluid flow in cultured choroid plexus.

Acetazolamide (AZA), used in treatment of early or infantile hydrocephalus, is effective in some cases, while its effect on the choroid plexus (CP) remains ill-defined. The drug reversibly inhibits aquaporin-4 (AQP4), the most ubiquitous "water pore" in the brain, and perhaps modulation of AQP1 (located apically on CP cells) by AZA may reduce cerebrospinal fluid (CSF) production. We sought to elucidate the effect of AZA on AQP1 and fluid flow in CP cell cultures.CP tissue culture from 10-day Sprague-Dawley rats and a TRCSF-B cell line were grown on Transwell permeable supports and treated with 100 µM AZA. Fluid assays to assess direction and extent of fluid flow, and AQP1 expression patterns by immunoblot, Immuncytochemistry (ICC), and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were performed.Immunoblots and ICC analyses showed a decrease in AQP1 protein shortly after AZA treatment (lowest at 12 h), with transient AQP1 reduction mediated by mRNA expression (lowest at 6 h). Transwell fluid assays indicated a fluid shift at 2 h, before significant changes in AQP1 mRNA or protein levels.Timing of AZA effect on AQP1 suggests the drug alters protein transcription, while affecting fluid flow by a concomitant method. It is plausible that other mechanisms account for these phenomena, as the processes may occur independently.

Evidence for altered Numb isoform levels in Alzheimer's disease patients and a triple transgenic mouse model.

The cell fate determinant Numb exists in four alternatively spliced variants that differ in the length of their PTB (phosphotyrosine-binding domain, either lacking or containing an 11 amino acid insertion) and PRR (proline-rich region, either lacking or containing a 48 amino acid insertion). We previously reported that Numb switches from isoforms containing the PTB insertion to isoforms lacking this insertion in neural cultures subjected to stress induced by trophic factor withdrawal. The switch in Numb isoforms enhances the generation of amyloid-ß peptide (Aß), the principle component of senile plaques in Alzheimer's disease (AD). Here we examine the expression of the Numb isoforms in brains from AD patients and triple transgenic (3xTg) AD mice. We found that levels of the Numb isoforms lacking the PTB insertion are significantly elevated in the parietal cortex but not in the cerebellum of AD patients when compared to control subjects. Levels of Numb isoforms lacking the PTB insertion were also elevated in the cortex but not cerebellum of 12 month-old 3xTg AD mice with Aß deposits compared to younger 3xTg-AD mice and to non-transgenic mice. Exposure of cultured neurons to Aß resulted in an increase in the levels of Numb isoforms lacking the PTB domain, consistent with a role for Aß in the aberrant expression of Numb in vulnerable brain regions of AD patients and mice. Collectively, the data show that altered expression of Numb isoforms in vulnerable neurons occurs during AD pathogenesis and suggest a role for Numb in the disease process.

Expression of aquaporin 1 and 4 in a congenital hydrocephalus rat model.

BACKGROUND: Hydrocephalus occurs because of an imbalance of bulk fluid flow in the brain, and aquaporins (AQPs) play pivotal roles in cerebral water movement as essential mediators during edema and fluid accumulation. AQP1 is a water channel found in the choroid plexus (CP), and AQP4 is expressed at the brain-CSF interfaces and astrocytic end feet; excessive fluid accumulation may involve expression of changes in these AQPs during various stages of hydrocephalus. OBJECTIVE: To determine the alterations of CP AQP1 expression in congenital hydrocephalus; detect hydrocephalus-induced AQP1 expression in the cortical parenchyma, ependyma, and pia mater of hydrocephalic animals; and evaluate AQP4 expression in congenital hydrocephalus through progressive stages of the condition. METHODS: We evaluated differential expression of AQPs 1 and 4 in the congenital hydrocephalus Texas rat at postnatal days 5, 10, and 26 in isolated CP and cortex by enzyme-linked immunosorbent assay, Western blot, quantitative reverse transcriptase polymerase chain reaction, and immunohistochemistry. RESULTS: The CP exhibited a 34% decrease in AQP1 expression in young hydrocephalic pups (postnatal days 5 and 10), which became normal (postnatal day 26) just before death. With advancing hydrocephalus, expression of AQPs 1 and 4 increased at the brain-CSF interfaces; AQP1 was localized to the endothelium of cortical capillaries with increased AQP4 expression in surrounding astrocytes end feet. AQP1 expression level was increased in the pia mater, with prominent AQP4 expression in the subpial layers. Subependymal capillaries expressed AQP1 in the endothelium, with increasing AQP4 expression in surrounding astrocytes. Hydrocephalic animals (postnatal day 26) had significant nonendothelial (CD34) AQP1 expression in the septal nucleus of the basal forebrain, an area affected by increased intracranial pressure. CONCLUSION: Biphasic AQP1 expression in the CP with increased AQPs 1 and 4 at the brain-fluid interfaces may indicate compensatory mechanisms to regulate choroidal cerebrospinal fluid secretion and increase parenchymal fluid absorption in the high-pressure hydrocephalic condition.

Stress-induced switch in Numb isoforms enhances Notch-dependent expression of subtype-specific transient receptor potential channel.

The Notch signaling pathway plays an essential role in the regulation of cell specification by controlling differentiation, proliferation, and apoptosis. Numb is an intrinsic regulator of the Notch pathway and exists in four alternative splice variants that differ in the length of their phosphotyrosine-binding domain (PTB) and proline-rich region domains. The physiological relevance of the existence of the Numb splice variants and their exact regulation are still poorly understood. We previously reported that Numb switches from isoforms containing the insertion in PTB to isoforms lacking this insertion in neuronal cells subjected to trophic factor withdrawal (TFW). The functional relevance of the TFW-induced switch in Numb isoforms is not known. Here we provide evidence that the TFW-induced switch in Numb isoforms regulates Notch signaling strength and Notch target gene expression. PC12 cells stably overexpressing Numb isoforms lacking the PTB insertion exhibited higher basal Notch activity and Notch-dependent transcription of the transient receptor potential channel 6 (TRPC6) when compared with those overexpressing Numb isoforms with the PTB insertion. The differential regulation of TRPC6 expression is correlated with perturbed calcium signaling and increased neuronal vulnerability to TFW-induced death. Pharmacological inhibition of the Notch pathway or knockdown of TRPC6 function ameliorates the adverse effects caused by the TFW-induced switch in Numb isoforms. Taken together, our results indicate that Notch and Numb interaction may influence the sensitivity of neuronal cells to injurious stimuli by modulating calcium-dependent apoptotic signaling cascades.

Receptor channel TRPC6 is a key mediator of Notch-driven glioblastoma growth and invasiveness.

Glioblastoma multiforme (GBM) is the most frequent and incurable type of brain tumor of adults. Hypoxia has been shown to direct GBM toward a more aggressive and malignant state. Here we show that hypoxia increases Notch1 activation, which in turn induces the expression of transient receptor potential 6 (TRPC6) in primary samples and cell lines derived from GBM. TRPC6 is required for the development of the aggressive phenotype because knockdown of TRPC6 expression inhibits glioma growth, invasion, and angiogenesis. Functionally, TRPC6 causes a sustained elevation of intracellular calcium that is coupled to the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Pharmacologic inhibition of the calcineurin-NFAT pathway substantially reduces the development of the malignant GBM phenotypes under hypoxia. Clinically, expression of TRPC6 was elevated in GBM specimens in comparison with normal tissues. Collectively, our studies indicate that TRPC6 is a key mediator of tumor growth of GBM in vitro and in vivo and that TRPC6 may be a promising therapeutic target in the treatment of human GBM.

Evidence for reduced lymphatic CSF absorption in the H-Tx rat hydrocephalus model.

BACKGROUND: There is mounting evidence that spinal fluid absorption takes place not only at the arachnoid villi, but also at several extracranial sites, which might serve as a reserve mechanism for, or be primarily involved in the absorption of CSF in hydrocephalus. METHODS: We compared the nasal lymphatic pathway in congenital Hydrocephalus-Texas (H-Tx) rats in unaffected and affected hydrocephalic (HC) siblings with that of control Sprague Dawley (SD) rat pups. The animals were examined after immediate post mortem injection of Evan's blue dye into the cisterna magna at 6 and 10 days of age. The specimens were evaluated for amount of dye penetration into the nasal passages. RESULTS: We found more dye visualization in the olfactory regions of control SD (14/16 at P6, 14/16 at P10) and unaffected H-Tx (13/17 at P6, 13/16 at P10) compared with HC animals (0/14 at P6, 3/15 at P10). This difference was more pronounced at 10 days of age. The dye was not visualized in the cervical lymph nodes or venous channels in these acute experiments. CONCLUSION: The results of this study suggest that nasal lymphatic cerebrospinal fluid absorption is reduced in the H-Tx rat hydrocephalus model.