Phosphatidylinositol 3-Kinase/AKT Pathway Inhibition by Doxazosin Promotes Glioblastoma Cells Death,Upregulation of p53 and Triggers Low Neurotoxicity

Glioblastoma is the most frequent and malignant brain tumor. Treatment includes chemotherapy with temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide, as well as the human glioblastoma cell line U138-MG. We investigated doxazosin’s (an antihypertensive drug) activity against glioblastoma cells (C6 and U138-MG) and its neurotoxicity on primary astrocytes and organoptypic hippocampal cultures. For this study, the following methods were used: citotoxicity assays, flow cytometry, western-blotting and confocal microscopy. We showed that doxazosin induces cell death on C6 and U138-MG cells. We observed that doxazosin’s effects on the PI3K/Akt pathway were similar as LY294002 (PI3K specific inhibitor). In glioblastoma cells treated with doxasozin, Akt levels were greatly reduced. Upon examination of activities of proteins downstream of Akt we observed upregulation of GSK-3β and p53. This led to cell proliferation inhibition, cell death induction via caspase-3 activation and cell cycle arrest at G0/G1 phase in glioblastoma cells. We used in this study Lapatinib, a tyrosine kinase inhibitor, as a comparison with doxazosin because they present similar chemical structure. We also tested the neurocitotoxicity of doxazosin in primary astrocytes and organotypic cultures and observed that doxazosin induced cell death on a small percentage of non-tumor cells. Aggressiveness of glioblastoma tumors and dismal prognosis require development of new treatment agents. This includes less toxic drugs, more selective towards tumor cells, causing less damage to the patient. Therefore, our results confirm the potential of doxazosin as an attractive therapeutic antiglioma agent.     

TFPI1 Mediates Resistance to Doxorubicin in Breast Cancer Cells by Inducing a Hypoxic-Like Response

Thrombin and hypoxia are important players in breast cancer progression. Breast cancers often develop drug resistance, but mechanisms linking thrombin and hypoxia to drug resistance remain unresolved. Our studies using Doxorubicin (DOX) resistant MCF7 breast cancer cells reveals a mechanism linking DOX exposure with hypoxic induction of DOX resistance. Global expression changes between parental and DOX resistant MCF7 cells were examined. Westerns, Northerns and immunocytochemistry were used to validate drug resistance and differentially expressed genes. A cluster of genes involved in the anticoagulation pathway, with Tissue Factor Pathway Inhibitor 1 (TFPI1) the top hit, was identified. Plasmids overexpressing TFPI1 were utilized, and 1% O₂ was used to test the effects of hypoxia on drug resistance. Lastly, microarray datasets from patients with drug resistant breast tumors were interrogated for TFPI1 expression levels. TFPI1 protein levels were found elevated in 3 additional DOX resistant cells lines, from humans and rats, indicating evolutionarily conservation of the effect. Elevated TFPI1 in DOX resistant cells was active, as thrombin protein levels were coincidentally low. We observed elevated HIF1α protein in DOX resistant cells, and in cells with forced expression of TFPI1, suggesting TFPI1 induces HIF1α. TFPI1 also induced c-MYC, c-SRC, and HDAC2 protein, as well as DOX resistance in parental cells. Growth of cells in 1% O₂ induced elevated HIF1α, BCRP and MDR-1 protein, and these cells were resistant to DOX. Our in vitro results were consistent with in vivo patient datasets, as tumors harboring increased BCRP and MDR-1 expression also had increased TFPI1 expression. Our observations are clinically relevant indicating that DOX treatment induces an anticoagulation cascade, leading to inhibition of thrombin and the expression of HIF1α. This in turn activates a pathway leading to drug resistance.     

Thrombin induces macrophage migration inhibitory factor release and upregulation in urothelium: a possible contribution to bladder inflammation

Purpose

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine expressed by urothelial cells that mediates bladder inflammation. We investigated the effect of stimulation with thrombin, a Protease Activated Receptor-1 (PAR1) agonist, on MIF release and MIF mRNA upregulation in urothelial cells.

Materials and Methods

MIF and PAR1 expression was examined in normal human immortalized urothelial cells (UROtsa) using real-time RT-PCR, Western blotting and dual immunostaining. MIF and PAR1 immunostaining was also examined in rat urothelium. The effect of thrombin stimulation (100 nM) on urothelial MIF release was examined in UROtsa cells (in vitro) and in rats (in vivo). UROtsa cells were stimulated with thrombin, culture media were collected at different time points and MIF amounts were determined by ELISA. Pentobarbital anesthetized rats received intravesical saline (control), thrombin, or thrombin +2% lidocaine (to block nerve activity) for 1 hr, intraluminal fluid was collected and MIF amounts determined by ELISA. Bladder or UROtsa MIF mRNA was measured using real time RT-PCR.

Results

UROtsa cells constitutively express MIF and PAR1 and immunostaining for both was observed in these cells and in the basal and intermediate layers of rat urothelium. Thrombin stimulation of urothelial cells resulted in a concentration- and time-dependent increase in MIF release both in vitro (UROtsa; 2.8-fold increase at 1 hr) and in vivo (rat; 4.5-fold) while heat-inactivated thrombin had no effect. In rats, thrombin-induced MIF release was reduced but not abolished by intravesical lidocaine treatment. Thrombin also upregulated MIF mRNA in UROtsa cells (3.3-fold increase) and in the rat bladder (2-fold increase) where the effect was reduced (1.4-fold) by lidocaine treatment.

Conclusions

Urothelial cells express both MIF and PAR1. Activation of urothelial PAR1 receptors, either by locally generated thrombin or proteases present in the urine, may mediate bladder inflammation by inducing urothelial MIF release and upregulating urothelial MIF expression.     

Effects of the blockade of high voltage-activated calcium channels on in vitro pineal melatonin synthesis

The presence of high voltage-activated calcium channels in the rat pineal gland is well known. However, their role in pineal metabolism is not completely understood and is even controversial. Better to understand this matter, we investigated the effects of L-, N- or P/Q-type calcium channel blockers (nifedipine, omega-conotoxin GVIA, omega-agatoxin IVA, respectively) on melatonin content and arylalkylamine-N-acetyltransferase activity of denervated rat pineal glands kept for 48 h in culture and stimulated with norepinephrine. Melatonin was measured by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase activity was quantified by radiometric assay. Pre-incubation with any of these high voltage-activated calcium channel blockers reduced the melatonin production induced by norepinephrine although arylalkylamine-N-acetyltransferase activity was reduced only by the N-type calcium channel antagonist, omega-conotoxin GVIA. The results indicate that calcium influx through L-, N- or P/Q-type of high voltage-activated calcium channels is necessary for the full expression of the metabolic process leading to melatonin synthesis in the rat pineal glands. However, the mechanisms involved in this process are different for the L- or P/Q- and N-type calcium channels.     

Biological, nutritional, and histochemical basis for improving an artificial diet for Bracon hebetor Say (Hymenoptera: Braconidae)

The biology of Bracon hebetor Say (Hymenoptera: Braconidae) reared on fifth instars of Anagasta kuehniella (Zeller) (Lepidoptera: Pyralidae) (natural diet) and in vitro (artificial diet) was evaluated. Data on the number of instars, development time and food intake were collected, and histochemical tests were conducted to detect proteins and lipids in the parasitoid's digestive tract. The data disclosed differences that can help to improve artificial rearing of B. hebetor. B. hebetor had three instars in both diets, but the developmental time on the artificial diets was prolonged due to the increase in larval and pupal development times. Larvae grew faster on the natural host and required a lower food intake (2.7 microl) as compared to that required by the larvae feeding on the artificial diet (3.8 microl). Analysis of diet protein content and host hemolymph and the observations on the parasitoid larvae gut content indicated altogether the artificial diets requires the addition of others sources of proteins and lipids to improve the overall nutrition quality of the in vitro rearing system for this ectoparasitoid.     

Effect of kosmotropicity of ionic liquids on the enzyme stability in aqueous solutions

This paper examined the effect of several pyridinium and imidazolium-based ionic liquids (ILs) on the protease stability in aqueous solutions. In general, the enzyme was found quite active at low concentrations of hydrophilic ILs. In aqueous environment, the enzyme was stabilized by the kosmotropic anions (such as CF3COO- and CH3COO-) and chaotropic cations (such as [BuPy]+ and [EMIM]+), but was destabilized by chaotropic anions (such as tosylate and BF4-) and kosmotropic cations (such as [BMIM]+).     

Protection status as determinant of carbon stock drivers in Cerrado sensu stricto

天然植被在全球碳循环和碳储存中扮演着重要角色。巴西大草原塞拉多保护区(Cerrado)因自身固有特性被认为是一个碳汇。本研究的目的是评估具有不同保护状况的三个地区,控制区(法定保护区)、保护区(PA)和非保护区(Non-PA)地上生物量与生物多样性关系的变化。这三个被研究的地区都位于巴西米纳斯吉拉斯州(Minas Gerais)北部。根据森林清查资料,该研究对地上碳储量进行了估算,并测量了每个地区生物多样性指标的三个维度：功能性状优势度、分类学多样性和功能多样性。对物种的以下功能性状进行了评价：木材密度、最大直径和种子大小。通过建立广义线性模型,评估了碳储量、群落加权平均值、物种丰富度和多样性以及功能多样性指数在不同地区间的差异。 研究结果表明,未受保护的地区碳储量、物种丰富度、物种多样性、功能丰富度和功能分散度均较低,而保护区和非保护区群落加权平均值最大直径和种子大小均低于法定保护区。广义线性模型结果表明,碳储量与物种和功能丰富度指数在同一地区内和不同地区间存在相关性,因此,物种丰富度可以作为功能丰富度和碳储量的替代指标。物种丰富度和群落加权平均值最大直径对碳储量有正向影响,功能分散度对碳储量有负向影响。功能丰富度、物种多样性和群落加权平均值种子大小出现在最佳模型中,但对碳储量没有显著的直接影响。因此,我们的结论是,在缺乏保护的巴西塞拉多地区会降低物种丰富度和碳储量。     

Trade-offs between ontogenetic changes and food consumption in Brazilian silverside Atherinella brasiliensis from two tropical estuaries

Salinity variation in estuarine environments influences the distribution of fish species as well as the availability of food resources to be used by them. This study examines the effect of the range of salinity on the trade-off between growth and feeding intensity of Atherinella brasiliensis from two tropical estuaries (positive and hypersaline). To investigate the effects of salinity, we hypothesized that hypersalinity negatively affects foraging intensity, consumption and prey selection by the Brazilian silverside, leading to differences in body condition. Sampling was carried out using the beach seine method in two areas of the estuaries (upper and lower zone) during rainy and dry periods. A total of 2549 stomachs (1124 for the positive estuary and 1425 for the hypersaline estuary) were examined, and the results indicated a dissimilarity of 92.7% of the diet between environments. In the positive estuary, there was more predation on Calanoida, Gastropoda, Hymenoptera, Ceratopogonidae larvae and Decapoda larvae, while Alga and plant-material characterized the diet in the hypersaline estuary. Significant correlations between the volume of food and salinity were observed in both estuaries. The vacuity index indicated that hypersaline environments presented higher contributions of semifull stomachs, indicating an intense consumption of algae. On the other hand,in the positive estuary, these values were less intense, but the stomachs were always with animal items. The variation found for both environments reinforces the effect of salinity on the physiological mechanism of the populations once the higher proportions of filled stomachs in the hypersaline environment indicate the need for constant and high ingestion of prey to guarantee the pronounced energy expenditure with osmoregulation.