Endothelin stimulates tyrosine phosphorylation of p125 and p130 in rat cerebral cortex

Stimulation of rat cerebral cortex with endothelin-1 (ET-1) caused an increase in the tyrosine phosphorylation of several proteins. Two of these phosphoproteins were identified by the immunoprecipitation assays as being the focal adhesion kinase p125^FAK and crk-associated substrate p130^Cas. This effect was time- and dose-dependent, with an EC₅₀ value of 3.9×10⁻⁸ M. In addition, the cerebral cortex ET receptor subtype involved in this action was determined by using BQ-123 and BQ-788, which are ET_A and ET_B receptor antagonists respectively. Our results indicate that the ET-1 effect on protein tyrosine phosphorylation occurred through ET_B receptors. The requirement for extracellular Ca²⁺ on ET-1 action was also studied. ET-1-stimulated tyrosine phosphorylation of both p125^FAK and p130^Cas was abolished in the absence of external Ca²⁺ or in the presence of nimodipine, a Ca²⁺ channel-blocker. These results suggest that the ET-1-stimulated protein tyrosine phosphorylation was secondary to Ca²⁺ influx through the dihydropyridine Ca²⁺-channel. In slices where protein kinase C was inhibited, ET-1-stimulated tyrosine phosphorylation of both proteins was reduced. These results indicate that ET-1 modulates the tyrosine phosphorylation of specific proteins, which may be involved in adhesion processes in the brain.     

Differential binding modes of the bromodomains of CREB-binding protein (CBP) and p300 with acetylated MyoD

We investigated whether blocking of monocyte chemoattractant-1 (MCP-1) function would inhibit recruitment of tumor-associated macrophages (TAMs) and prevent tumor angiogenesis and tumor growth of human malignant melanoma. B16-F1 melanoma cells were implanted onto the back of C57BL/6 mice (Day 0). At Day 7, a dominant negative MCP-1 mutant (7ND) gene was transfected in the thigh muscle to make overexpressed 7ND protein secreted into systemic circulation. 7ND treatment inhibited TAM recruitment and partially reduced tumor angiogenesis and tumor growth. Also, 7ND treatment attenuated inductions of tumor necrosis factor-α (TNFα), interleukin-1α (IL-1α), and vascular endothelial growth factor (VEGF) in the stroma and tumor. Melanoma cells expressed not only MCP-1 but also its receptor CCR2. Accordingly, it was suggested that MCP-1 would enhance tumor angiogenesis and early tumor growth in the early stages by inducing TNFα, IL-1α, and VEGF through TAM recruitment and probably the direct autocrine/paracrine effects on melanoma cells.     

A new in vitro wound model based on the co-culture of human dermal microvascular endothelial cells and human dermal fibroblasts

BACKGROUND INFORMATION: Different in vitro models, based on co-culturing techniques, can be used to investigate the behaviour of cell types, which are relevant for human wound and soft-tissue healing. Currently, no model exists to describe the behaviour of fibroblasts and microvascular endothelial cells under wound-specific conditions. In order to develop a suitable in vitro model, we characterized co-cultures comprising NHDFs (normal human dermal fibroblasts) and HDMECs (human dermal microvascular endothelial cells). The CCSWMA (co-culture scratch wound migration assay) developed was supported by direct visualization techniques in order to investigate a broad spectrum of cellular parameters, such as migration and proliferation activity, the differentiation of NHDFs into MFs (myofibroblasts) and the expression of endothelin-1 and ED-A-fibronectin (extra domain A fibronectin). The cellular response to hypoxia treatment, as one of the crucial conditions in wound healing, was monitored. RESULTS: The comparison of the HDMEC-NHDF co-culture with the respective mono-cultures revealed that HDMECs showed a lower proliferation activity when co-cultured, but their number was stable throughout a period of 48 h. NHDFs in co-culture were slightly slower at proliferating than in the mono-culture. The MF population was stable for 48 h in the co-culture, as well as in NHDF mono-culture. Co-cultures and HDMEC mono-cultures were characterized by a slower migration rate than NHDF mono-cultures. Hypoxia decreased both cell proliferation and migration in the mono-cultures, as well as in the co-cultures, indicating the general suitability of the assay. Exclusively, in co-cultures well-defined cell clusters comprising HDMECs and MFs formed at the edges of the in vitro wounds. CONCLUSIONS: On the basis of these results, the CCSWMA developed using co-cultures, including HDMECs, NHDFs and MFs, proved to be an effective tool to directly visualize cellular interaction. Therefore, it will serve in the future to evaluate the influence of wound-healing-related factors in vitro, as shown for hypoxia in the present study.     

Overexpression of regucalcin enhances its nuclear localization and suppresses L-type Ca2+ channel and calcium-sensing receptor mRNA expressions in cloned normal rat kidney proximal tubular epithelial NRK52E cells

The effect of regucalcin (RC), a regulatory protein in intracellular signaling pathway, on the gene expression of various mineral ion transport-related proteins was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing RC. NRK52E cells (wild-type) and stable RC/pCXN2 transfectant were cultured for 72 h in medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured 24-72 h in a medium containing either vehicle, aldosterone (10(-8) or 10(-7) M), or parathyroid hormone (PTH) (1-34) (10(-8) or 10(-7) M) without BS. RC was markedly localized in the nucleus of transfectants. Overexpression of RC caused a significant increase in rat outer medullary K(+) channel (ROMK) mRNA expression, while it caused a remarkable decrease in L-type Ca(2+) channel and calcium-sensing receptor (CaR) mRNA expressions. Overexpression of RC did not have an effect on epithelial sodium channel (ENaC), Na, K-ATPase (alpha-subunit), Type II Na-Pi cotransporter (NaPi-IIa), angiotensinogen, Na(+)-Ca(2+) exchanger, and glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expressions. Hormonal effect on gene expression, moreover, was examined. Culture with aldosterone (10(-8) or 10(-7) M) caused a significant increase in ENaC, Na, K-ATPase, and ROMK mRNA expressions in the wild-type cells. Those increases were weakened in the transfectants. Culture with PTH (10(-8) or 10(-7) M) significantly decreased NaPi-IIa mRNA expression in the wild-type cells. This effect was not altered in the transfectants. PTH significantly decreased angiotensinogen mRNA expression in the wild-type cells and the transfectants, while aldosterone had no effect. Culture with PTH (10(-8) or 10(-7) M) caused a significant decrease in L-type Ca(2+) channel and CaR mRNA expressions in the wild-type cells, while the hormone significantly increased Na(+)-Ca(2+) exchanger mRNA expression. The effects of PTH on L-type Ca(2+) channel, CaR, and Na(+)-Ca(2+) exchanger mRNA expressions were also seen in the transfectants. This study demonstrates that overexpression of RC caused a remarkable increase in its nuclear localization, and that it has suppressive effects on the gene expression of L-type Ca(2+) channel or CaR, which regulates intracellular Ca(2+) signaling, among various regulator proteins for mineral ions in NRK52E cells.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Global DNA promoter methylation in frontal cortex of alcoholics and controls

LOX-1 (Lectin-like oxidized low-density lipoprotein receptor-1) is the primary endothelial receptor of oxidized LDL (oxLDL). Both in vitro and in vivo experiments have shown this protein to be important in the initiation of atherosclerosis and to be up-regulated by pro-atherogenic factors. Recently, it has been demonstrated that Olr1, the gene encoding Lox-1, is important for tumor growth and for maintaining the transformed state in different cancer cell lines, suggesting that it acts in a molecular pathway connecting cancer and atherosclerosis. Both diseases in humans are characterized by uncontrolled regulation of cellular growth and differentiation.We present evidence that Olr1 is expressed during mouse embryogenesis in developmental stages (from 7.5 to 9.5 dpc) in which cardiogenesis occurs. In addition, we identify two novel Olr1 isoform (hereafter referred to as D3D5Olr1 and D2D5Olr1) whose spatio-temporal expression pattern overlaps with Olr1 in vivo. In vitro, D3D5Olr1 localizes to the cell surface membrane as Olr1, in contrast with D2D5Olr1; these data suggest that D2D5Olr1 isoform translates a receptor that does not reach the plasma membrane. Accordingly, in silico transmembrane protein topology prediction analyses, show that D2D5Olr1 does not contain any transmembrane region. Finally, both isoforms can activate the same genetic pathways underlying Olr1 expression, such as, hypoxia and inflammation, even if with a different efficiency.All these data suggest a new functional involvement of Olr1, and probably of its spliceforms, in murine cardiogenesis and angiogenesis.     

The pro-tumorigenic effects of metabolic alterations in glioblastoma including brain tumor initiating cells

De-regulated cellular energetics is an emerging hallmark of cancer with alterations to glycolysis, oxidative phosphorylation, the pentose phosphate pathway, lipid oxidation and synthesis and amino acid metabolism. Understanding and targeting of metabolic reprogramming in cancers may yield new treatment options, but metabolic heterogeneity and plasticity complicate this strategy. One highly heterogeneous cancer for which current treatments ultimately fail is the deadly brain tumor glioblastoma. Therapeutic resistance, within glioblastoma and other solid tumors, is thought to be linked to subsets of tumor initiating cells, also known as cancer stem cells. Recent profiling of glioblastoma and brain tumor initiating cells reveals changes in metabolism, as compiled here, that may be more broadly applicable. We will summarize the profound role for metabolism in tumor progression and therapeutic resistance and discuss current approaches to target glioma metabolism to improve standard of care.     

Induction of the intrinsic apoptosis pathway in insulin-secreting cells is dependent on oxidative damage of mitochondria but independent of caspase-12 activation

Pro-inflammatory cytokine-mediated beta cell apoptosis is activated through multiple signaling pathways involving mitochondria and endoplasmic reticulum. Activation of organelle-specific caspases has been implicated in the progression and execution of cell death. This study was therefore performed to elucidate the effects of pro-inflammatory cytokines on a possible cross-talk between the compartment-specific caspases 9 and 12 and their differential contribution to beta cell apoptosis. Moreover, the occurrence of ROS-mediated mitochondrial damage in response to beta cell toxic cytokines has been quantified. ER-specific caspase-12 was strongly activated in response to pro-inflammatory cytokines; however, its inhibition did not abolish cytokine-induced mitochondrial caspase-9 activation and loss of cell viability. In addition, there was a significant induction of oxidative mitochondrial DNA damage and elevated cardiolipin peroxidation in insulin-producing RINm5F cells and rat islet cells. Overexpression of the H₂O₂ detoxifying enzyme catalase effectively reduced the observed cytokine-induced oxidative damage of mitochondrial structures. Taken together, the results strongly indicate that mitochondrial caspase-9 is not a downstream substrate of ER-specific caspase-12 and that pro-inflammatory cytokines cause apoptotic beta cell death through activation of caspase-9 primarily by hydroxyl radical-mediated mitochondrial damage.     

Sorafenib enhances proteasome inhibitor-mediated cytotoxicity via inhibition of unfolded protein response and keratin phosphorylation

Hepatocellular carcinoma (HCC) is highly resistant to conventional systemic therapies and prognosis for advanced HCC patients remains poor. Recent studies of the molecular mechanisms responsible for tumor initiation and progression have identified several potential molecular targets in HCC. Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC. It acts by inhibiting the serine/threonine kinases and the receptor type tyrosine kinases. In preclinical experiments sorafenib had anti-proliferative activity in hepatoma cells and it reduced tumor angiogenesis and increased apoptosis. Here, we demonstrate for the first time that the cytotoxic mechanisms of sorafenib include its inhibitory effects on protein ubiquitination, unfolded protein response (UPR) and keratin phosphorylation in response to endoplasmic reticulum (ER) stress. Moreover, we show that combined treatment with sorafenib and proteasome inhibitors (PIs) synergistically induced a marked increase in cell death in hepatoma- and hepatocyte-derived cells. These observations may open the way to potentially interesting treatment combinations that may augment the effect of sorafenib, possibly including drugs that promote ER stress. Because sorafenib blocked the cellular defense mechanisms against hepatotoxic injury not only in hepatoma cells but also in hepatocyte-derived cells, we must be careful to avoid severe liver injury.