BRCA1 is an essential mediator of vinorelbine-induced apoptosis in mesothelioma

The rapeutic options for malignant pleural mesothelioma (MPM) are limited despite the increasing incidence globally. The vinca alkaloid vinorelbine exhibits clinical activity; however, to date, treatment optimization has not been achieved using biomarkers. BRCA1 regulates sensitivity to microtubule poisons; however, its role in regulating vinorelbine-induced apoptosis in mesothelioma is unknown. Here we demonstrate that BRCA1 plays an essential role in mediating vinorelbine-induced apoptosis, as evidenced by (1) the strong correlation between vinorelbine sensitivity and BRCA1 expression level; (2) induction of resistance to vinorelbine by BRCA1 using siRNA oligonucleotides; (3) dramatic down-regulation of BRCA1 following selection for vinorelbine resistance; and (4) the re-activation of vinorelbine-induced apoptosis following re-expression of BRCA1 in resistant cells. To determine whether loss of BRCA1 expression in mesothelioma was potentially relevant in vivo, BRCA1 immunohistochemistry was subsequently performed on 144 primary mesothelioma specimens. Loss of BRCA1 protein expression was identified in 38.9% of samples. Together, these data suggest that BRCA1 plays a critical role in mediating apoptosis by vinorelbine in mesothelioma, warranting its clinical evaluation as a predictive biomarker. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Advances in the systemic therapy of malignant pleural mesothelioma

Malignant pleural mesothelioma is an aggressive thoracic malignancy associated with exposure to asbestos, and its incidence is anticipated to increase during the first half of this century. Chemotherapy is the mainstay of treatment, yet sufficiently robust evidence to substantiate the current standard of care has emerged only in the past 5 years. This Review summarizes the evidence supporting the clinical activity of chemotherapy, discusses the use of end points for its assessment and examines the influence of clinical and biochemical prognostic factors on the natural history of malignant pleural mesothelioma. Early-phase clinical trials of second-line and novel agents are emerging from an increased understanding of mesothelioma cell biology. Coupled with high-quality translational research, such developments have real potential to improve the outlook of patients at a time of increasing incidence.

Safety and efficacy of the combination of trastuzumab with docetaxel for HER2-positive women with advanced breast cancer : a review of the existing clinical trials and results of the expanded access programme in the UK

Trastuzumab is a humanised monoclonal antibody against the extracellular domain of HER2 (human epidermal growth factor receptor-2) that is overexpressed in about 25% of human breast cancers. It has shown clinical benefit in HER2-positive breast cancer cases when used alone or in combination with chemotherapy. Trastuzumab increases the response rate to chemotherapy and prolongs survival when used in combination with taxanes. In this article, we review the clinical trials where trastuzumab has been administered together with docetaxel, and we present the results of the trastuzumab expanded access programme (EAP) in the UK. Combination of trastuzumab with docetaxel results in similar response rates and time-to-progression with the trastuzumab/paclitaxel combinations. The toxicity of the combination and the risk of heart failure are low. The clinical data for the docetaxel/trastuzumab combination indicate a favourable profile from both the efficacy and the safety point of view and confirm the feasibility and safety of trastuzumab administration both as monotherapy and in combination with docetaxel. © 2004 Blackwell Publishing Ltd.

Reversibility of liver failure secondary to metastatic breast cancer by vinorelbine and cisplatin chemotherapy

Purpose: The development of liver metastases from breast cancer is associated with a very poor prognosis, estimated at 4 months median survival. Since treatment with many chemotherapeutic agents is relatively contraindicated, we assessed the safety, tolerability and potential efficacy of combination chemotherapy with vinorelbine and cisplatin (ViP). Method: Pilot study in 11 patients with histologically confirmed breast carcinoma, radiological evidence of liver metastases and serum bilirubin greater than 1.5 times the upper limit of normal. Patients received up to six cycles of cisplatin (75 mg/m 2) every 21 days and vinorelbine (20 mg/m 2) on days 1 and 8 of every 21-day cycle. Measurement of liver lesions was performed on CT scan every 8 weeks into treatment. Results: The most frequently reported adverse event was myelosuppression. Other adverse effects included nausea, vomiting and mild neurotoxicity. Two patients died after one treatment with ViP, one of whom suffered an intracerebral haemorrhage that was possibly treatment-related. Improvement in liver function tests was observed in 10 patients, and mean time to normalization of bilirubin levels was 36 days. Partial responses were documented radiologically in 7 out of 11 patients treated. Median overall survival from trial entry was 6.5 months (range 11-364 days), with one patient alive 13 months from trial entry. Conclusion: Normalization of liver function is possible with ViP treatment of metastatic breast cancer, offering the potential to prolong survival. Phase II clinical trials of this regimen in this patient group should include measurement of quality of life in order to assess risk versus benefit.

Isolation, structure determination and cytotoxicity studies of tryptophan alkaloids from an Australian marine sponge Hyrtios sp

Mass-guided fractionation of the MeOH extract from a specimen of the Australian marine sponge Hyrtios sp. resulted in the isolation of two new tryptophan alkaloids, 6-oxofascaplysin (2), and secofascaplysic acid (3), in addition to the known metabolites fascaplysin (1) and reticulatate (4). The structures of all molecules were determined following NMR and MS data analysis. Structural ambiguities in 2 were addressed through comparison of experimental and DFT-generated theoretical NMR spectral values. Compounds 1–4 were evaluated for their cytotoxicity against a prostate cancer cell line (LNCaP) and were shown to display IC50 values ranging from 0.54 to 44.9 μM.

Identification of eusynstyelamide B as a potent cell cycle inhibitor following the generation and screening of an ascidian-derived extract library using a real time cell analyzer

Ascidians are marine invertebrates that have been a source of numerous cytotoxic compounds. Of the first six marine-derived drugs that made anticancer clinical trials, three originated from ascidian specimens. In order to identify new anti-neoplastic compounds, an ascidian extract library (143 samples) was generated and screened in MDA-MB-231 breast cancer cells using a real-time cell analyzer (RTCA). This resulted in 143 time-dependent cell response profiles (TCRP), which are read-outs of changes to the growth rate, morphology, and adhesive characteristics of the cell culture. Twenty-one extracts affected the TCRP of MDA-MB-231 cells and were further investigated regarding toxicity and specificity, as well as their effects on cell morphology and cell cycle. The results of these studies were used to prioritize extracts for bioassay-guided fractionation, which led to the isolation of the previously identified marine natural product, eusynstyelamide B (1). This bis-indole alkaloid was shown to display an IC50 of 5 μM in MDA-MB-231 cells. Moreover, 1 caused a strong cell cycle arrest in G2/M and induced apoptosis after 72 h treatment, making this molecule an attractive candidate for further mechanism of action studies.

Feeding sorghum ergot (Claviceps africana) to sows before farrowing inhibits milk production

Objective: To assess the impact of feeding different amounts of sorghum ergot to sows before farrowing. Design: Fifty-one pregnant sows from a continually farrowing piggery were sequentially inducted into the experiment each week in groups of four to seven, as they approached within 14 days of farrowing. Diets containing sorghum ergot sclerotia within the range of 0 (control) up to 1.5% w/w (1.5% ergot provided 7 mg alkaloids/kg, including 6 mg dihydroergosine/kg) were randomly allocated and individually fed to sows. Ergot concentrations were varied with each subsequent group until an acceptable level of tolerance was achieved. Diets with ergot were replaced with control diets after farrowing. Post-farrowing milk production was assessed by direct palpation and observation of udders, and by piglet responses and growth. Blood samples were taken from sows on three days each week, for prolactin estimation. Results: Three sows fed 1.5% ergot for 6 to 10 days preceding farrowing produced no milk, and 87% of their piglets died despite supplementary feeding of natural and artificial colostrums, milk replacer, and attempts to foster them onto normally lactating sows. Ergot inclusions of 0.6% to 1.2% caused lesser problems in milk release and neo-natal piglet mortality. Of 23 sows fed either 0.3% or 0.6% ergot, lactation of only two first-litter sows were affected. Ergot caused pronounced reductions in blood prolactin, and first-litter sows had lower plasma prolactin than multiparous sows, increasing their susceptibility to ergot. Conclusion: Sorghum ergot should not exceed 0.3% (1 mg alkaloid/kg) in diets of multiparous sows fed before farrowing, and should be limited to 0.1 % for primiparous sows, or avoided completely.

Pyrrolizidine Alkaloids in Crotalaria Taxa from Northern Australia: Risk to Grazing Livestock.

Crotalaria species containing hepatotoxic pyrrolizidine alkaloids grow widely in pastures in northern Australia and have sporadically poisoned grazing livestock. The diverse Crotalaria taxa present in these pastures include varieties, subspecies, and chemotypes not previously chemically examined. This paper reports the pyrrolizidine alkaloid composition and content of 24 Crotalaria taxa from this region and assesses the risk of poisoning in livestock consuming them. Alkaloids present in C. goreensis, C. aridicola subsp. densifolia, and C. medicaginea var. neglecta lack the esterified 1,2-unsaturated functionality required for pyrrole adduct formation, and these taxa are not hepatotoxic. Taxa with high levels of hepatotoxic alkaloids, abundance, and biomass pose the greatest risk to livestock health, particularly C. novae-hollandiae subsp. novae-hollandiae, C. ramosissima, C. retusa var. retusa, and C. crispata. Other species containing moderate alkaloid levels, C. spectabilis and C. mitchellii, also pose significant risk when locally abundant.

Risks from plants containing pyrrolizidine alkaloids for livestock and meat quality in Northern Australia.

This chapter describes poisoning associated with consumption of pyrrolizidine alkaloid (PA)-containing plants (Crotalaria spp., Heliotropium spp. and Senecio spp.) by cattle and horses in rangelands of northern Australia, as well as the risks for meat quality of PA residues and potential health hazards to consumers.

Indole oxygenase from the leaves of Tecoma stans

A new indole oxygenase from the leaves of Tecoma stans was isolated and purified to near homogeneity. The purified enzyme system catalyses the conversion of indole to anthranilic acid. It is optimally active at pH 5.2 and at 30°C. Oxygen (2 mol) is consumed and anthranilic acid (1 mol) is formed for every mole of indole oxidized. Neither sulfhydryl reagents nor sulfhydryl compounds inhibited the enzyme activity. The oxygenase also attacks, apart from indole, 5-hydroxyindole, 5-bromoindole and 5-methylindole. It is not inhibited by copper specific chelators or non-heme iron specific chelators. Atebrin did not inhibit the enzyme activity suggesting that it is not a flavoprotein, unlike other indole oxygenases and indole oxidases. Dialysis resulted in complete loss of enzyme activity. The inactive enzyme could not be reactivated by addition of various cofactors.

Regulation of oxidative-stress responsive genes by arecoline in human keratinocytes

Background and Objective: Arecoline, an arecanut alkaloid present in the saliva of betel quid chewers, has been implicated in the pathogenesis of a variety of inflammatory oral diseases, including oral submucous fibrosis and periodontitis. To understand the molecular b asis of arecoline action in epithelial changes associated with these diseases, we investigated the effects of arecoline on human keratinocytes with respect to cell growth regulation and the expression of stress-responsive genes.Material and Methods:Human keratinocyte cells (of the HaCaT cell line) were treated with arecoline, following which cell viability was assessed using the Trypan Blue dye-exclusion assay, cell growth and proliferation were analyzed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and 5-bromo-2-deoxyuridine incorporation assays, cell cycle arrest and generation of reactive oxygen species were examined using flow cytometry, and gene expression changes were investigated using the reverse transcription-polymerase chain reaction technique. The role of oxidative stress, muscarinic acetylcholine receptor and mitogen-activated protein kinase (MAPK) pathways were studied using specific inhibitors. Western blot analysis was performed to study p38 MAPK activation.Results:Arecoline induced the generation of reactive oxygen species and cell cycle arrest at the G1/G0 phase in HaCaT cells without affecting the expression of p21/Cip1. Arecoline-induced epithelial cell death at higher concentrations was caused by oxidative trauma without eliciting apoptosis. Sublethal concentrations of arecoline upregulated the expression of the following stress-responsive genes: heme oxygenase-1; ferritin light chain; glucose-6-phosphate dehydrogenase; glutamate-cysteine ligase catalytic subunit; and glutathione reductase.Additionally, there was a dose-dependent induction of interleukin-1alfa mRNA by arecoline via oxidative stress and p38 MAPK activation. Conclusion:our data highlight the role of oxidative stress in arecoline-mediated cell death, gene regulation and inflammatory processes in human keratinocytes.

Expression of hyoscyamine 6 beta-hydroxylase in the root pericycle cells and accumulation of its product scopolamine in leaf and stem tissues of Datura metel L.

Hyoscyamine 60-hydroxylase (H6H: EC 1.14.11.11), a key enzyme at the terminal step of tropane alkaloid biosynthesis, converts hyoscyamine to scopolamine. The accumulation of scopolamine in different organs, in particular the aerial parts for storage, is subject to the expression of hyoscyamine 6-phydroxylase as well as its transport from the site of synthesis. To understand the molecular basis of this regulation, we have analyzed, in parallel, the relative levels of hyoscyamine and scopolamine, and the accumulation of H6H (both protein and transcript) in leaves, stems and roots of D. metel. The root, stem and leaf tissues all contain about 0.51-0.65 mg g(-1) dry weight of scopolamine. Hyoscyamine content was extremely low in leaf and stem tissues and was about 0.28 mg g(-1) dry weight in the root tissue. H6H protein and its transcript were found only in roots but not in the aerial parts viz. stems and leaves. The immunolocalization studies performed on leaf, stem, root as well as hairy root tissues showed that H6H was present only in the pericycle cells of young lateral and hairy roots. These studies suggest that the conversion of hyoscyamine to scopolamine takes place in the root pericycle cells, and the alkaloid biosynthesized in the roots gets translocated to the aerial parts in D. metel. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

An alpha-aminomethyl carbanion equivalent via a novel Barbier reaction: (1H-naphtho[1,8-de]-1,2,3-triazin-2-yl)methyl anion

A novel sonication-promoted Barbier reaction putatively generated the titled species from the corresponding naphthotriazinylmethyl chloride and magnesium in THF: its formal addition to a variety of carbonyl compounds in situ occurred in excellent yields. Subsequent catalytic hydrogenolysis of the triazine moiety demasked the amine, thus defining a route to various phenylethylamines (including the alkaloid 'mescaline'), or ethanolamines (in two cases), in excellent overall yields. (C) 2000 Elsevier Science Ltd. All rights reserved.

Cyclin dependent protein kinases as drug targets – potential in cardiovascular diseases

Complications of atherosclerosis such as myocardial infarction and stroke are the primary cause of death in Western societies. The development of atherosclerotic lesions is a complex process, including endothelial cell dysfunction, inflammation, extracellular matrix alteration and vascular smooth muscle cell (VSMC) proliferation and migration. Various cell cycle regulatory proteins control VSMC proliferation. Protein kinases called cyclin dependent kinases (CDKs) play a major role in regulation of cell cycle progression. At specific phases of the cell cycle, CDKs pair with cyclins to become catalytically active and phosphorylate numerous substrates contributing to cell cycle progression. CDKs are also regulated by cyclin dependent kinase inhibitors, activating and inhibitory phosphorylation, proteolysis and transcription factors. This tight regulation of cell cycle is essential; thus its deregulation is connected to the development of cancer and other proliferative disorders such as atherosclerosis and restenosis as well as neurodegenerative diseases. Proteins of the cell cycle provide potential and attractive targets for drug development. Consequently, various low molecular weight CDK inhibitors have been identified and are in clinical development. Tylophorine is a phenanthroindolizidine alkaloid, which has been shown to inhibit the growth of several human cancer cell lines. It was used in Ayurvedic medicine to treat inflammatory disorders. The aim of this study was to investigate the effect of tylophorine on human umbilical vein smooth muscle cell (HUVSMC) proliferation, cell cycle progression and the expression of various cell cycle regulatory proteins in order to confirm the findings made with tylophorine in rat cells. We used several methods to determine our hypothesis, including cell proliferation assay, western blot and flow cytometric cell cycle distribution analysis. We demonstrated by cell proliferation assay that tylophorine inhibits HUVSMC proliferation dose-dependently with an IC50 value of 164 nM ± 50. Western blot analysis was used to determine the effect of tylophorine on expression of cell cycle regulatory proteins. Tylophorine downregulates cyclin D1 and p21 expression levels. The results of tylophorine’s effect on phosphorylation sites of p53 were not consistent. More sensitive methods are required in order to completely determine this effect. We used flow cytometric cell cycle analysis to investigate whether tylophorine interferes with cell cycle progression and arrests cells in a specific cell cycle phase. Tylophorine was shown to induce the accumulation of asynchronized HUVSMCs in S phase. Tylophorine has a significant effect on cell cycle, but its role as cell cycle regulator in treatment of vascular proliferative diseases and cancer requires more experiments in vitro and in vivo.

The challenge of LC/MS/MS multi-mycotoxin analysis - Heracles battling the Hydra?

Mycotoxins are secondary metabolites of filamentous fungi. They pose a health risk to humans and animals due to their harmful biological properties and common occurrence in food and feed. Liquid chromatography/mass spectrometry (LC/MS) has gained popularity in the trace analysis of food contaminants. In this study, the applicability of the technique was evaluated in multi-residue methods of mycotoxins aiming at simultaneous detection of chemically diverse compounds. Methods were developed for rapid determination of toxins produced by fungal genera of Aspergillus, Fusarium, Penicillium and Claviceps from cheese, cereal based agar matrices and grains. Analytes were extracted from these matrices with organic solvents. Minimal sample clean-up was carried out before the analysis of the mycotoxins with reversed phase LC coupled to tandem MS (MS/MS). The methods were validated and applied for investigating mycotoxins in cheese and ergot alkaloid occurrence in Finnish grains. Additionally, the toxin production of two Fusarium species predominant in northern Europe was studied. Nine mycotoxins could be determined from cheese with the method developed. The limits of quantification (LOQ) allowed the quantification at concentrations varying from 0.6 to 5.0 µg/kg. The recoveries ranged between 96 and 143 %, and the within-day repeatability (as relative standard deviation, RSDr) between 2.3 and 12.1 %. Roquefortine C and mycophenolic acid could be detected at levels of 300 up to 12000 µg/kg in the mould cheese samples analysed. A total of 29 or 31 toxins could be analysed with the method developed for agar matrices and grains, with the LOQs ranging overall from 0.1 to 1250 µg/kg. The recoveries ranged generally between 44 and 139 %, and the RSDr between 2.0 and 38 %. Type-A trichothecenes and beauvericin were determined from the cereal based agar and grain cultures of F. sporotrichioides and F. langsethiae. T-2 toxin was the main metabolite, the average levels reaching 22000 µg/kg in the grain cultures after 28 days of incubation. The method developed for ten ergot alkaloids from grains allowed their quantification at levels varying from 0.01 to 10 µg/kg. The recoveries ranged from 51 to 139 %, and the RSDr from 0.6 to 13.9 %. Ergot alkaloids were measured in barley and rye at average levels of 59 and 720 µg/kg, respectively. The two most prevalent alkaloids were ergocornine and ergocristine. The LC/MS methods developed enabled rapid detection of mycotoxins in such applications where several toxins co-occurred. Generally, the performance of the methods was good, allowing reliable analysis of the mycotoxins of interest with sufficiently low quantification limits. However, the variation in validation results highlighted the challenges related to optimising this type of multi-residue methods. New data was obtained about the occurrence of mycotoxins in mould cheeses and of ergot alkaloids in Finnish grains. In addition, the study revealed the high mycotoxin-producing potential of two common fungi in Finnish crops. The information can be useful when risks related to fungal and mycotoxin contamination will be assessed.