Cross Talk between Receptor Guanylyl Cyclase C and c-src Tyrosine Kinase Regulates Colon Cancer Cell Cytostasis

Increased activation of c-src seen in colorectal cancer is an indicator of a poor clinical prognosis, suggesting that identification of downstream effectors of c-src may lead to new avenues of therapy. Guanylyl cyclase C (GC-C) is a receptor for the gastrointestinal hormones guanylin and uroguanylin and the bacterial heat-stable enterotoxin. Though activation of GC-C by its ligands elevates intracellular cyclic GMP (cGMP) levels and inhibits cell proliferation, its persistent expression in colorectal carcinomas and occult metastases makes it a marker for malignancy. We show here that GC-C is a substrate for inhibitory phosphorylation by c-src, resulting in reduced ligand-mediated cGMP production. Consequently, active c-src in colonic cells can overcome GC-C-mediated control of the cell cycle. Furthermore, docking of the c-src SH2 domain to phosphorylated GC-C results in colocalization and further activation of c-src. We therefore propose a novel feed-forward mechanism of activation of c-src that is induced by cross talk between a receptor GC and a tyrosine kinase. Our findings have important implications in understanding the molecular mechanisms involved in the progression and treatment of colorectal cancer.

Inhibition of Tyrosine Phosphorylation of Sperm Flagellar Proteins, Outer Dense Fiber Protein-2 and Tektin-2, Is Associated With Impaired Motility During Capacitation of Hamster Spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

Tyrphostin-A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoa

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.

miR-219-5p inhibits receptor tyrosine kinase pathway by targeting EGFR in glioblastoma

Glioblastoma is one of the common types of primary brain tumors with a median survival of 12-15 months. The receptor tyrosine kinase (RTK) pathway is known to be deregulated in 88% of the patients with glioblastoma. 45% of GBM patients show amplifications and activating mutations in EGFR gene leading to the upregulation of the pathway. In the present study, we demonstrate that a brain specific miRNA, miR-219-5p, repressed EGFR by directly binding to its 3'-UTR. The expression of miR-219-5p was downregulated in glioblastoma and the overexpression of miR-219-5p in glioma cell lines inhibited the proliferation, anchorage independent growth and migration. In addition, miR-219-5p inhibited MAPK and PI3K pathways in glioma cell lines in concordance with its ability to target EGFR. The inhibitory effect of miR-219-5p on MAPK and PI3K pathways and glioma cell migration could be rescued by the overexpression of wild type EGFR and vIII mutant of EGFR (both lacking 3'-UTR and thus being insensitive to miR-219-5p) suggesting that the inhibitory effects of miR-219-5p were indeed because of its ability to target EGFR. We also found significant negative correlation between miR-219-5p levels and total as well as phosphorylated forms of EGFR in glioblastoma patient samples. This indicated that the downregulation of miR-219-5p in glioblastoma patients contribute to the increased activity of the RTK pathway by the upregulation of EGFR. Thus, we have identified and characterized miR-219-5p as the RTK regulating novel tumor suppressor miRNA in glioblastoma.

Efficacious Anticancer Drug Delivery Mediated by a pH-Sensitive Self-Assembly of a Conserved Tripeptide Derived from Tyrosine Kinase NGF Receptor

We present herein a short tripeptide sequence (Lys-Phe-Gly or KFG) that is situated in the juxtamembrane region of the tyrosine kinase nerve growth factor (Trk NGF) receptors. KFG self-assembles in water and shows a reversible and concentration-dependent switching of nanostructures from nanospheres (vesicles) to nanotubes, as evidenced by dynamic light scattering, transmission electron microscopy, and atomic force microscopy. The morphology change was associated with a transition in the secondary structure. The tripeptide vesicles have inner aqueous compartments and are stable at pH7.4 but rupture rapidly at pH approximate to 6. The pH-sensitive response of the vesicles was exploited for the delivery of a chemotherapeutic anticancer drug, doxorubicin, which resulted in enhanced cytotoxicity for both drug-sensitive and drug-resistant cells. Efficient intracellular release of the drug was confirmed by fluorescence-activated cell sorting analysis, fluorescence microscopy, and confocal microscopy.

Pentoxifylline-stimulated capacitation and acrosome reaction in hamster spermatozoa: involvement of intracellular signalling molecules

We investigated the role of cAMP/cGMP, protein kinases and intracellular calcium ( [Ca2+](i)) in pentoxifylline-stimulated hamster sperm capacitation and the acrosome reaction (AR) in vitro. Treatment with pentoxifylline [0.45 mM) initially increased sperm cAMP values 2.8-fold, compared with untreated controls (396 +/- 9.2 versus 141 +/- 6.0 fmoles/10(6) spermatozoa; mean +/- SEM, n = 6) after 15 min, although by 3 h, cAMP values were similar (503-531 fmoles/10(6) spermatozoal, cGMP values (similar to 27 fmoles/10(6) spermatozoa) were the same in treated and control spermatozoa. Both sperm capacitation and the AR, determined from the absence of an acrosomal cap, were stimulated by pentoxifylline; these were almost completely inhibited by a Cl-/HCO(3)(-)antiporter inhibitor (4,4-diisothiocyanato-stilbene-2,2 disulphonic acid; 1 mM) defined from the degree of sperm motility and by a protein kinase A inhibitor (H89; 10 mu M) A protein kinase G inhibitor (staurosporine, 1 nM) did not affect pentoxifylline-stimulated capacitation but inhibited the AR by 50%. A protein tyrosine kinase inhibitor (tyrphostin A-47, 0.1 mM) had no effect on either pentoxifylline-stimulated capacitation or AR, A phospholipase A(2) inhibitor (aristolochic acid, 0.4 mM) markedly inhibited the pentoxifylline-stimulated AR but not capacitation. When intracellular sperm calcium [Ca2+](i) was measured using fura-2-AM, there was an early rise 271 nM at 0.5 hi in pentoxifylline(-treated spermatozoa; this appeared to be due to intracellular mobilization rather than to uptake. In the absence of extracellular Ca2+, sperm motility was maintained in the presence of pentoxifylline, but capacitation did not occur; spermatozoa exhibited a low level of hyperactivated motility and had a poor rate of AR(20.5 +/- 2.3%). These results suggest that: (i) the pentoxifylline-stimulated early onset of sperm capacitation may be mediated by an early rise in cAMP and [Ca2+/-](i) and involves protein kinase A activity; and (ii) pentoxifylline-stimulated AR may require phospholipase A;A(2) and protein kinase C activity.

Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate

Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [P-32] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by Western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [P-32] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC alpha and epsilon, the major isotypes of PKC in BPAECs, by TPA (100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [P-32] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.

Essential role of PI3-kinase pathway in p53-mediated transcription: Implications in cancer chemotherapy

The PI3-kinase pathway is the target of inactivation in achieving better cancer chemotherapy. Here, we report that p53-mediated transcription is inhibited by pharmacological inhibitors and a dominant-negative mutant of PI3-kinase, and this inhibition was relieved by a constitutively active mutant of PI3-kinase. Akt/PKB and mTOR, the downstream effectors of PI3-kinase, were also found to be essential. LY294002 (PI3-kinase inhibitor) pre-treatment altered the post-translational modifications and the sub-cellular localization of p53. Although LY294002 increased the chemosensitivity of cells to low concentrations of adriamycin (adriamycin-low), it protected the cells from cytotoxicity induced by high concentrations of adriamycin (adriamycin-high) in a p53-dependent manner. Further, we found that LY294002 completely abolished the activation of p53 target genes (particularly pro-apoptotic) under adriamycin-high conditions, whereas it only marginally repressed the p53 target genes under adriamycin-low conditions; in fact, it further activated the transcription of NOXA, HRK, APAF1 and CASP5 genes. Thus, the differential effect of PI3-kinase on p53 functions seems to be responsible for the differential regulation of DNA damage-induced cytotoxicity and cell death by PI3-kinase. Our finding becomes relevant in the light of ongoing combination chemotherapy trials with the PI3-kinase pathway inhibitors and underscores the importance of p53 status in the careful formulation of combination chemotherapies. Oncogene (2010) 29, 3605-3618; doi: 10.1038/onc.2010.123; published online 26 April 2010

Dynamic changes in mitogen-activated protein kinase (MAPK) activities in the corpus luteum of the bonnet monkey (Macaca radiata) during development, induced luteolysis, and simulated early pregnancy: A role for p38 MAPK in the regulation of luteal function

Changes in MAPK activities were examined in the corpus luteum (CL) during luteolysis and pregnancy, employing GnRH antagonist (Cetrorelix)-induced luteolysis, stages of CL, and hCG treatment to mimic early pregnancy as model systems in the bonnet monkey. We hypothesized that MAPKs could serve to phosphorylate critical phosphoproteins to regulate luteal function. Analysis of several indices for structural (caspase-3 activity and DNA fragmentation) and functional (progesterone and steroidogenic acute regulatory protein expression) changes in the CL revealed that the decreased luteal function observed during Cetrorelix treatment and late luteal phase was associated with increased caspase-3 activity and DNA fragmentation. As expected, human chorionic gonadotropin treatment dramatically increased luteal function, but the indices for structural changes were only partially attenuated. All three MAPKs appeared to be constitutively active in the mid-luteal-phase CL, and activities of ERK-1/2 and p38-MAPK (p38), but not Jun N-terminal kinase (JNK)-1/2, decreased significantly (P < 0.05) within 12 - 24 h after Cetrorelix treatment. During the late luteal phase, in contrast to decreased ERK-1/2 and p38 activities, JNK-1/2 activities increased significantly (P < 0.05). Although human chorionic gonadotropin treatment increased ERK-1/2 and p38 activities, it decreased JNK-1/2 activities. The activation status of p38 was correlated with the phosphorylation status of an upstream activator, MAPK kinase-3/6 and the expression of MAPK activated protein kinase-3, a downstream target. Intraluteal administration of p38 kinase inhibitor (SB203580), but not MAPK kinase-1/2 inhibitor (PD98059), decreased the luteal function. Together, these data suggest an important role for p38 in the regulation of CL function in primates.

Genome-wide analysis and experimentation of plant serine/threonine/tyrosine-specific protein kinases

Protein tyrosine phosphorylation plays an important role in cell growth, development and oncogenesis. No classical protein tyrosine kinase has hitherto been cloned from plants. Does protein tyrosine kinase exist in plants? To address this, we have performed a genomic survey of protein tyrosine kinase motifs in plants using the delineated tyrosine phosphorylation motifs from the animal system. The Arabidopsis thaliana genome encodes 57 different protein kinases that have tyrosine kinase motifs. Animal non-receptor tyrosine kinases, SRC, ABL, LYN, FES, SEK, KIN and RAS have structural relationship with putative plant tyrosine kinases. In an extended analysis, animal receptor and non-receptor kinases, Raf and Ras kinases, mixed lineage kinases and plant serine/threonine/tyrosine (STY) protein kinases, form a well-supported group sharing a common origin within the superfamily of STY kinases. We report that plants lack bona fide tyrosine kinases, which raise an intriguing possibility that tyrosine phosphorylation is carried out by dual-specificity STY protein kinases in plants. The distribution pattern of STY protein kinase families on Arabidopsis chromosomes indicates that this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. Genome-wide analysis is supported by the functional expression and characterization of At2g24360 and phosphoproteomics of Arabidopsis. Evidence for tyrosine phosphorylated proteins is provided by alkaline hydrolysis, anti-phosphotyrosine immunoblotting, phosphoamino acid analysis and peptide mass fingerprinting. These results report the first comprehensive survey of genome-wide and tyrosine phosphoproteome analysis of plant STY protein kinases.

Vasomodulatory effect of novel peroxovanadate compounds on rat aorta: Role of rho kinase and nitric oxide/cGMP pathway

The present study was undertaken to assess the role of reactive oxygen species (ROS) in rat aortic ring vasoreactivity and integrity by using various peroxovanadate (pV) compounds. All the pV compounds (1 nM-300 mu M) used in the present study exerted concentration-dependent contractions on endothelium intact rat aortic rings. All compounds with an exception of DPV-asparagine (DPV-asn) significantly altered vascular integrity as shown by diminished KCl responses. Phenylephrine (PE)-mediated contractions (3 nM-300 mu M) were unaltered in the presence of these compounds. Acetylcholine (Ach)-mediated relaxation in PE (1 mu M) pre-contracted rings was significantly reduced in presence of diperoxovanadate (DPV), poly (sodium styrene sulfonate-co-maleate)-pV (PSS-CoM-pV) and poly (sodium styrene 4-sulfonate)-pV (PSS-pV). However, no significant change in Ach-mediated responses was observed in the presence of poly (acrylate)-pV (PM-pV) and DPV-asn. DPV-asn was thus chosen to further elucidate mechanism involved in peroxide mediated modulation of vasoreactivity. DPV-asn (30 nM-300 mu M) exerted significantly more stable contractions, that was found to be catalase (100 U/ml) resistant in comparison with H(2)O(2) (30 nM-300 mu M) in endothelium intact aortic rings. These contractile responses were found to be dependent on extracellular Ca(2+) and were significantly inhibited in presence of ROS scavenger N-acetylcysteine (100 mu M). Intracellular calcium chelation by BAPTA-AM (10 mu M) had no significant effect on DPV-asn (30 nM-300 mu M) mediated contraction. Pretreatment of aortic rings by rho-kinase inhibitor Y-27632 (10 mu M) significantly inhibited DPV-asn-mediated vasoconstriction indicating role of voltage-dependent Ca(2+) influx and downstream activation of rho-kinase. The small initial relaxant effect obtained on addition of DPV-asn (30 nM-1 mu M) in PE (1 mu M) pre-contracted endothelium intact rings, was prevented in the presence of guanylate cyclase inhibitor, methylene blue (10 mu M) and/or nitric oxide synthase (NOS) inhibitor, L-NAME (100 mu M) suggesting involvement of nitric oxide and cGMP. DPV-asn, like H(2)O(2), exerted a response of vasoconstriction in normal arteries and vasodilation at low concentrations (30 nM-1 mu M) in PE-pre contracted rings with overlapping mechanisms. These findings suggest usefulness of DPV-asn having low toxicity, in exploring the peroxide-mediated effects on various vascular beds. The present study also convincingly demonstrates role of H(2)O(2) in the modulation of vasoreactivity by using stable peroxide DPV-asn and warrants future studies on peroxide mediated signaling from a newer perspective. (C) 2011 Published by Elsevier Ltd.

Synthesis, ABTS-Radical Scavenging Activity, and Antiproliferative and Molecular Docking Studies of Novel Pyrrolo1,2-a]quinoline Derivatives

A new 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)-radical scavenging and antiproliferative agents of pyrrolo1,2-a]quinoline derivatives have been synthesized. An efficient method for the synthesis of 14 novel diversified pyrrolo1,2-a]quinoline derivatives has been described using 4-(1,3-dioxolan-2-yl)quinoline and different phenacyl bromides in acetone and followed by reacting with different acetylenes in dimethylformamide/K2CO3. The structure of the newly synthesized compounds was determined by infrared, H-1 NMR, C-13 NMR, mass spectrometry, and elemental analysis. The in vitro antioxidant activity revealed that among all the tested compounds 5n exhibited maximum scavenging activity with ABTS. Compound 5b has showed good antiproliferative activity as an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase.

Involvement of Synthesis and Phosphorylation of Nuclear Protein Factors That Bind to the Positivecis-Acting Element in the Transcriptional Activation of the CYP2B1/B2 Gene by Phenobarbitonein Vivo

The synthesis and phosphorylation of protein factor(s) that bind to the positivecis-acting element (−69 to −98 nt) of the CYP2B1/B2 gene have been examinedin vivoin the rat. Treatment of rats with cycloheximide, a protein synthetic inhibitor, suppresses basal as well as phenobarbitone-induced levels of CYP2B1/B2 mRNA and its run-on transcription. Under these conditions, complex formation of the nuclear extract with the positive element is also inhibited, as judged by gel shift assays. Treatment of rats with 2-aminopurine, a general protein kinase inhibitor, blocks the phenobarbitone-mediated increase in CYP2B1/B2 mRNA, cell-free transcription of a minigene construct containing the positive element, pP450e179DNA, and binding of nuclear proteins to the positive element. Treatment of rats with okadaic acid, a protein phosphatase inhibitor, mimics the effects of phenobarbitone, but only partially. Thus, both phenobarbitone and okadaic acid individually enhance binding of the nuclear protein(s) to the positive element, cell-free transcription of the minigene construct, and phosphorylation of the not, vert, similar26- and 94-kDa proteins binding to the positive element. But unlike phenobarbitone, okadaic acid is not an inducer of CYP2B1/B2 mRNA or its run-on transcription. Thus, phenobarbitone-responsive positive element interactions constitute only a minimal requirement, and okadaic acid is perhaps not able to bring about the total requirement for activation of CYP2B1/B2 gene transcription that should include interaction between the minimal promoter and further upstream elements. An intriguing feature is the antagonistic effect of okadaic acid on phenobarbitone-mediated effects on CYP2B1/B2 mRNA levels, cell-free and run-on transcription, and nuclear protein binding to the positive element. The reason for this antagonism is not clear. It is concluded that phenobarbitone treatment enhancesin vivothe synthesis and phosphorylation of protein factors binding to the positive element and these constitute a minimal requirement for the transcriptional activation of the CYP2B1/B2 gene.

Defining the pathway to insulin-like growth factor system targeting in cancer

The insulin-like growth factors (IGEs; IGF-1 and IGF-2) play central roles in cell growth, differentiation, survival, transformation and metastasis. The biologic effects of the IGFs are mediated by the IGF-1 receptor (IGF-1R), a receptor tyrosine kinase with homology to the insulin receptor (IR). Dysregulation of the ICE system is well recognized as a key contributor to the progression of multiple cancers, with IGF-1R activation increasing the tumorigenic potential of breast, prostate, lung, colon and head and neck squamous cell carcinoma (HNSCC). Despite this relationship, targeting the IGF-1R has only recently undergone development as a molecular cancer therapeutic. As it has taken hold, we are witnessing a robust increase and interest in targeting the inhibition of IGF-1R signaling. This is accentuated by the list of over 30 drugs, including monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs) that are under evaluation as single agents or in combination therapies 1]. The ICE-binding proteins (IGFBPs) represent the third component of the ICE system consisting of a class of six soluble secretory proteins. They represent a unique class of naturally occurring ICE-antagonists that bind to and sequester IGF-1 and IGF-2, inhibiting their access to the IGF-1R. Due to their dual targeting of the IGFs without affecting insulin action, the IGFBPs are an untapped ``third'' class of IGF-1R inhibitors. in this commentary, we highlight some of the significant aspects of and prospects for targeting the IGF-1R and describe what the future may hold. (C) 2010 Elsevier Inc. All rights reserved.

H2O2 has a role in cellular regulation

H2O2, in addition to producing highly reactive molecules through hydroxyl radicals or peroxidase action, can exert a number of direct effects on cells, organelles and enzymes. The stimulations include glucose transport, glucose incorporation into glycogen, HMP shunt pathway, lipid synthesis, release of calcium from mitochondria and of arachidonate from phospholipids, poly ADP ribosylation, and insulin receptor tyrosine kinase and pyruvate dehydrogenase activities. The inactivations include glycolysis, lipolysis, reacylation of lysophospholipids, ATP synthesis, superoxide dismutase and protein kinase C. Damages to DNA and proteoglycan and general cytotoxicity possibly through oxygen radicals were also observed. A whole new range of effects will be opened by the finding that H2O2 can act as a signal transducer in oxidative stress by oxidizing a dithiol protein to disulphide form which then activates transcription of the stress inducible genes. Many of these direct effects seem to be obtained by dithiol-disulphide modification of proteins and their active sites, as part of adaptive responses in oxidative stress.