Recombinant cathepsin S propeptide attenuates cell invasion by inhibition of cathepsin L-like proteases in tumor microenvironment

Human cathepsin L along with cathepsin S, K, and V are collectively known as cathepsin L-like proteases due to their high homology. The overexpression and aberrant activity of each of these proteases has been implicated in tumorigenesis. These proteases contain propeptide domains that can potently inhibit both their cognate protease and other proteases within the cathepsin L-like subfamily. In this investigation, we have produced the cathepsin S propeptide recombinantly and have shown that it is a potent inhibitor of the peptidolytic, elastinolytic, and gelatinolytic activities of the cathepsin L-like proteases. In addition, we show that this peptide is capable of significantly attenuating tumor cell invasion in a panel of human cancer cell lines. Furthermore, fusion of an IgG Fc-domain to the COOH terminus of the propeptide resulted in a chimeric protein with significantly enhanced ability to block tumor cell invasion. This Fc fusion protein exhibited enhanced stability in cell-based assays in comparison with the unmodified propeptide species. This approach for the combined inhibition of the cathepsin L-like proteases may prove useful for the further study in cancer and other conditions where their aberrant activity has been implicated. Furthermore, this strategy for simultaneous inhibition of multiple cysteine cathepsins may represent the basis for novel therapeutics to attenuate tumorigenesis.

A randomised interventional trial of ω-3-polyunsaturated fatty acids on endothelial function and disease activity in systemic lupus erythematosus

Objective: To determine the clinical effect of dietary supplementation with low-dose ?-3-polyunsaturated fatty acids on disease activity and endothelial function in patients with systemic lupus erythematosus. Methods: A 24-week randomised double-blind placebo-controlled parallel trial of the effect of 3 g of ?-3-polyunsaturated fatty acids on 60 patients with systemic lupus erythematosus was performed. Serial measurements of disease activity using the revised Systemic Lupus Activity Measure (SLAM-R) and British Isles Lupus Assessment Group index of disease activity for systemic lupus erythematosus (BILAG), endothelial function using flow-mediated dilation (FMD) of the brachial artery, oxidative stress using platelet 8-isoprostanes and analysis of platelet membrane fatty acids were taken at baseline, 12 and 24 weeks. Results: In the fish oil group there was a significant improvement at 24 weeks in SLAM-R (from 9.4 (SD 3.0) to 6.3 (2.5), p

Agelastatin A: a novel inhibitor of osteopontin-mediated adhesion, invasion, and colony formation

Effective inhibitors of osteopontin (OPN)-mediated neoplastic transformation and metastasis are still lacking. (-)-Agelastatin A is a naturally occurring oroidin alkaloid with powerful antitumor effects that, in many cases, are superior to cisplatin in vitro. In this regard, past comparative assaying of the two agents against a range of human tumor cell lines has revealed that typically (-)-agelastatin A is 1.5 to 16 times more potent than cisplatin at inhibiting cell growth, its effects being most pronounced against human bladder, skin, colon, and breast carcinomas. In this study, we have investigated the effects of (-)-agelastatin A on OPN-mediated malignant transformation using mammary epithelial cell lines. Treatment with (-)-agelastatin A inhibited OPN protein expression and enhanced expression of the cellular OPN inhibitor, Tcf-4. (-)-Agelastatin A treatment also reduced beta-catenin protein expression and reduced anchorage-independent growth, adhesion, and invasion in R37 OPN pBK-CMV and C9 cell lines. Similar effects were observed in MDA-MB-231 and MDA-MB-435s human breast cancer cell lines exposed to (-)-agelastatin A. Suppression of Tcf-4 by RNA interference (short interfering RNA) induced malignant/invasive transformation in parental benign Rama 37 cells; significantly, these events were reversed by treatment with (-)-agelastatin A. Our study reveals, for the very first time, that (-)-agelastatin A down-regulates beta-catenin expression while simultaneously up-regulating Tcf-4 and that these combined effects cause repression of OPN and inhibition of OPN-mediated malignant cell invasion, adhesion, and colony formation in vitro. We have also shown that (-)-agelastatin A inhibits cancer cell proliferation by causing cells to accumulate in the G(2) phase of cell cycle.

BRCA1: mechanisms of inactivation and implications for management of patients.

The BRCA1 gene was cloned in 1994 as one of the genes that conferred genetic predisposition to early-onset breast and ovarian cancer. Since then, a genetic test for identification of high-risk individuals has been developed. Despite being implicated in many important cellular pathways, including DNA repair and regulation of transcription, the exact mechanism by which inactivation of BRCA1 might lead to malignant transformation of cells remains unknown. We examine the mechanisms that underlie inactivation of BRCA1 and assess how they affect management of patients, in terms of both primary and secondary cancer prevention strategies. Furthermore, we look at the potential usefulness of BRCA1 as a prognostic tool and as a predictive marker of response to different classes of drugs. Finally, throughout this review, we draw links between the functional consequences of BRCA1 inactivation, in terms of key cellular signalling pathways, and how they might explain specific clinical observations in individuals who carry mutations in the gene.

Generation of a non-small cell lung cancer transcriptome microarray

Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. At present no reliable biomarkers are available to guide the management of this condition. Microarray technology may allow appropriate biomarkers to be identified but present platforms are lacking disease focus and are thus likely to miss potentially vital information contained in patient tissue samples.

Cholinergic-induced Ca2+ signaling in interstitial cells of Cajal from the guinea pig bladder.

Acetylcholine released from parasympathetic excitatory nerves activates contraction in detrusor smooth muscle. Immunohistochemical labeling of guinea pig detrusor with anti-c-Kit and anti-VAChT demonstrated a close structural relationship between interstitial cells of Cajal (ICC) and cholinergic nerves. The ability of guinea pig bladder detrusor ICC to respond to the acetylcholine analog, carbachol, was investigated in enzymatically dissociated cells, loaded with the Ca(2+) indicator fluo 4AM. ICC fired Ca(2+) transients in response to stimulation by carbachol (1/10 microM). Their pharmacology was consistent with carbachol-induced contractions in strips of detrusor which were inhibited by 4-DAMP (1 microM), an M(3) receptor antagonist, but not by the M(2) receptor antagonist methoctramine (1 microM). The source of Ca(2+) underlying the carbachol transients in isolated ICC was investigated using agents to interfere with influx or release from intracellular stores. Nifedipine (1 microM) or Ni(2+) (30-100 microM) to block Ca(2+) channels or the removal of external Ca(2+) reduced the amplitude of the carbachol transients. Application of ryanodine (30 microM) or tetracaine (100 microM) abolished the transients. The phospholipase C inhibitor, U-73122 (2.5 microM), significantly reduced the responses. 2-Aminoethoxydiethylborate (30 microM) caused a significant reduction and Xestospongin C (1 microM) was more effective, almost abolishing the responses. Intact in situ preparations of guinea pig bladder loaded with a Ca(2+) indicator showed distinctively different patterns of spontaneous Ca(2+) events in smooth muscle cells and ICC. Both cell types responded to carbachol by an increase in frequency of these events. In conclusion, guinea pig bladder detrusor ICC, both as isolated cells and within whole tissue preparations, respond to cholinergic stimulation by firing Ca(2+) transients. PMID: 18171995 [PubMed - indexed for MEDLINE]

Cell-specific processing of chromogranin A in endocrine cells of the rat stomach

The rat stomach is rich in endocrine cells. The acid-producing (oxyntic) mucosa contains ECL cells, A-like cells, and somatostatin (D) cells, and the antrum harbours gastrin (G) cells, enterochromaffin (EC) cells and D cells. Although chromogranin A (CgA) occurs in all these cells, its processing appears to differ from one cell type to another. Eleven antisera generated to different regions of rat CgA, two antisera generated to a human (h) CgA sequences, and one to a bovine Ib) CgA sequence, respectively, were employed together with antisera directed towards cell-specific markers such as gastrin (G cells), serotonin (EC cells), histidine decarboxylsae (ECL cells) and somatostatin (D cells) to characterize the expression of CgA and CgA-derived peptides in the various endocrine cell populations of the rat stomach. In the oxyntic mucosa, antisera raised against CgA(291-319) and CGA(316-321) immunostained D cells exclusively, whereas antisera raised against bCgA(82-91) and CgA(121-128) immunostained A-like cells and D cells. Antisera raised against CgA(318-349) and CgA(437-448) immunostained ECL cells and A-like cells, but not D cells. In the antrum, antisera against CgA(291-319) immunostained D cells, and antisera against CgA(351-356) immunostained G cells. Our observations suggest that each individual endocrine cell type in the rat stomach generates a unique mixture of CgA-derived peptides, probably reflecting cell-specific differences in the post-translational processing of CgA and its peptide products. A panel of antisera that recognize specific domains of CgA may help to identify individual endocrine cell populations.

Thermogravimetric evaluation of perennial ryegrass (Lolium perenne) for the prediction of in vitro dry matter digestibility

Thermogravimetry (TG) can be used for assessing the compositional differences in grasses that relate to dry matter digestibility (DMD) determined by pepsin-cellulase assay. This investigation developed regression models for predicting DMD of herbage grass during one growing season using TG results. The calibration samples were obtained from a field trial of eight cultivars and two breeding lines. The harvested materials from five cuts were analysed by TG to identify differences in the combustion patterns within the range of 30-600 degrees C. The discrete results including weight loss, peak height, area, temperature, widths and residue of three decomposition peaks were regressed against the measured DMD values of the calibration samples. Similarly, continuous weight loss results of the same samples were also utilised to generate DMD models. The r(2) for validation of the discrete and the best continuous models were 0.90 and 0.95, respectively, and the two calibrations were validated using independent samples from 24 plots from a trial carried out in 2004. The standard error for prediction of the 24 samples by the discrete model (4.14%) was higher than that by the continuous model (2.98%). This study has shown that DMD of grass could be predicted from the TG results. The benefit of thermal analysis is the ability to detect and show changes in composition of cell wall fractions of grasses during different cuts in a year.

Getting to the root of neuronal signalling in plant-parasitic nematodes using RNA interference

A variety of genes expressed in preparasitic second-stage juveniles (J2) of plant-parasitic nematodes appear to be vulnerable to RNA interference (RNAi) in vitro by coupling double-stranded (ds)RNA soaking with the artificial stimulation of pharyngeal pumping. Also, there is mounting evidence that the in planta generation of nematode-specific double-stranded RNAs (dsRNAs) has real utility in the control of these pests. Although neuronally-expressed genes in Caenorhabditis elegans are commonly refractory to RNAi, we have discovered that neuronally-expressed genes in plant-parasitic nematodes are highly susceptible to RNAi and that silencing can be induced by simple soaking procedures without the need for pharyngeal stimulation. Since most front-line anthelmintics that are used for the control of nematode parasites of animals and humans act to disrupt neuromuscular coordination, we argue that intercellular signalling processes associated with neurons have much appeal as targets for transgenic plant-based control strategies for plant-parasitic nematodes. FMRFamide-like peptides (FLPs) are a large family of neuropeptides which are intimately associated with neuromuscular regulation, and our studies on flp gene function in plant-parasitic nematodes have revealed that their expression is central to coordinated locomotory activities. We propose that the high level of conservation in nervous systems across nematodes coupled with the RNAi-susceptibility of neuronally-expressed genes in plant-parasitic nematodes provides a valuable research tool which could be used to interrogate neuronal signalling processes in nematodes.

Interleukin-8 signaling promotes androgen-independent proliferation of prostate cancer cells via induction of androgen receptor expression and activation

The aim of our study was to assess the importance of the CXC chemokine and interleukin (IL)-8 in promoting the transition of prostate cancer (CaP) to the androgen-independent state. Stimulation of the androgen-dependent cell lines, LNCaP and 22Rv1, with exogenous recombinant human interleukin-8 (rh-IL-8) increased androgen receptor (AR) gene expression at the messenger RNA (mRNA) and protein level, assessed by quantitative polymerase chain reaction and immunoblotting, respectively. Using an androgen response element-luciferase construct, we demonstrated that rh-IL-8 treatment also resulted in increased AR transcriptional activity in both these cell lines, and a subsequent upregulation of prostate-specific antigen and cyclin-dependent kinase 2 mRNA transcript levels in LNCaP cells. Blockade of CXC chemokine receptor-2 signaling using a small molecule antagonist (AZ10397767) attenuated the IL-8-induced increases in AR expression and transcriptional activity. Furthermore, in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, coadministration of AZ10397767 reduced the viability of LNCaP and 22Rv1 cells exposed to bicalutamide. Our data show that IL-8 signaling increases AR expression and promotes ligand-independent activation of this receptor in two androgen-dependent cell lines, describing two mechanisms by which this chemokine may assist in promoting the transition of CaP to the androgen-independent state. In addition, our data show that IL-8-promoted regulation of the AR attenuates the effectiveness of the AR antagonist bicalutamide in reducing CaP cell viability.

Substitution of pseudokinase domain residue Val-617 by large non-polar amino acids causes activation of JAK2

Explaining the uniqueness of the acquired somatic JAK2 V617F mutation, which is present in more than 95% of polycythemia vera patients, has been a challenge. The V617F mutation in the pseudokinase domain of JAK2 renders the unmutated kinase domain constitutively active. We have performed random mutagenesis at position 617 of JAK2 and tested each of the 20 possible amino acids for ability to induce constitutive signaling in Ba/F3 cells expressing the erythropoietin receptor. Four JAK2 mutants, V617W, V617M, V617I, and V617L, were able to induce cytokine independence and constitutive downstream signaling. Only V617W induced a level of constitutive activation comparable with V617F. Also, only V617W stabilized tyrosine-phosphorylated suppressor of cytokine signaling 3 ( SOCS3), a mechanism by which JAK2 V617F overcomes inhibition by SOCS3. The V617W mutant induced a myeloproliferative disease in mice, mainly characterized by erythrocytosis and megakaryocytic proliferation. Although JAK2 V617W would predictably be pathogenic in humans, the substitution of the Val codon, GTC, by TTG, the codon for Trp, would require three base pair changes, and thus it is unlikely to occur. We discuss how the predicted conformations of the activated JAK2 mutants can lead to better screening assays for novel small molecule inhibitors.