Synthesis and evaluation of novel boron-containing complexes of potential use for the selective treatment of malignant melanoma

Boron-containing complexes that have the potential to irreversibly accumulate in melanoma cells have been prepared by reaction of amino acids with 9-methoxy-9-borabicyclo[3.3.1]nonane. The ability of each complex to act as a substrate for tyrosinase has been probed by oximetry. Increased uptake of the lead candidate in a tyrosinase-rich cell line, compared with a tyrosinase-absent cell line, is reported, with results correlating well with that for a drug currently approved for BNCT.

A review of vertebroplasty for osteoporotic and malignant vertebral compression fractures

Vertebral compression fractures are a common clinical problem and the incidence of them will increase with the ageing population. Traditionally management has been conservative; however, there has been a growing trend towards vertebroplasty as an alternative therapy in patients with persisting severe pain. NICE produced guidance in 2003 recommending the procedure after 4 weeks of conservative management. Recent high-quality studies have been contradictory and there is currently a debate surrounding the role of the procedure with no agreement in the literature. We examine the evidence in both osteoporotic and malignant vertebral compression fractures; we also describe the benefits and side effects, alternative treatment options and the cost of the procedure. Finally, we recommend when vertebroplasty is most appropriately used based on the best available evidence.

Tyrosinase activated melanoma prodrugs

Metastatic malignant melanoma remains a highly aggressive form of skin cancer for which no reliable methods for treatment exist. Given the increasing incidence of this cancer, considerable attention has focused on the development of new and improved methods for tackling this disease. Within this article, methods for treating melanoma are reviewed and discussed with particular attention focusing on prodrugs that are activated by the tyrosinase enzyme. This enzyme is up-regulated and is of elevated activity within malignant melanomas compared with healthy melanocytes, providing an ideal in-situ tool for the activation of melanoma prodrugs. By way of background to the prodrug strategies discussed within this review, the causes of melanoma, the enzymology of tyrosinase, and the chemistry of the biosynthetic pathways associated with melanogenesis are presented. Aspects of the design, mode of action, and biological profiles of key prodrugs that are activated by tyrosinase, and that show potential for the treatment of melanoma, are then presented and compared.

Peptides generated ex vivo from serum proteins by tumor-specific exopeptidases are not useful biomarkers in ovarian cancer

BACKGROUND: The serum peptidome may be a valuable source of diagnostic cancer biomarkers. Previous mass spectrometry (MS) studies have suggested that groups of related peptides discriminatory for different cancer types are generated ex vivo from abundant serum proteins by tumor-specific exopeptidases. We tested 2 complementary serum profiling strategies to see if similar peptides could be found that discriminate ovarian cancer from benign cases and healthy controls. METHODS: We subjected identically collected and processed serum samples from healthy volunteers and patients to automated polypeptide extraction on octadecylsilane-coated magnetic beads and separately on ZipTips before MALDI-TOF MS profiling at 2 centers. The 2 platforms were compared and case control profiling data analyzed to find altered MS peak intensities. We tested models built from training datasets for both methods for their ability to classify a blinded test set. RESULTS: Both profiling platforms had CVs of approximately 15% and could be applied for high-throughput analysis of clinical samples. The 2 methods generated overlapping peptide profiles, with some differences in peak intensity in different mass regions. In cross-validation, models from training data gave diagnostic accuracies up to 87% for discriminating malignant ovarian cancer from healthy controls and up to 81% for discriminating malignant from benign samples. Diagnostic accuracies up to 71% (malignant vs healthy) and up to 65% (malignant vs benign) were obtained when the models were validated on the blinded test set. CONCLUSIONS: For ovarian cancer, altered MALDI-TOF MS peptide profiles alone cannot be used for accurate diagnoses.

Paraben esters: review of recent studies of endocrine toxicity, absorption, esterase and human exposure, and discussion of potential human health risks

This toxicology update reviews research over the past four years since publication in 2004 of the first measurement of intact esters of p-hydroxybenzoic acid (parabens) in human breast cancer tissues, and the suggestion that their presence in the human body might originate from topical application of bodycare cosmetics. The presence of intact paraben esters in human body tissues has now been confirmed by independent measurements in human urine, and the ability of parabens to penetrate human skin intact without breakdown by esterases and to be absorbed systemically has been demonstrated through studies not only in vitro but also in vivo using healthy human subjects. Using a wide variety of assay systems in vitro and in vivo, the oestrogen agonist properties of parabens together with their common metabolite (p-hydroxybenzoic acid) have been extensively documented, and, in addition, the parabens have now also been shown to possess androgen antagonist activity, to act as inhibitors of sulfotransferase enzymes and to possess genotoxic activity. With the continued use of parabens in the majority of bodycare cosmetics, there is a need to carry out detailed evaluation of the potential for parabens, together with other oestrogenic and genotoxic co-formulants of bodycare cosmetics, to increase female breast cancer incidence, to interfere with male reproductive functions and to influence development of malignant melanoma which has also recently been shown to be influenced by oestrogenic stimulation. Copyright (C) 2008 John Wiley & Sons, Ltd.

MARCKS functions as a novel growth/tumour suppressor in cells of melanocyte origin

Protein kinase C (PKC) plays a pivotal role in modulating the growth of melanocytic cells in culture. We have shown previously that a major physiological substrate of PKC, the 80 kDa myristoylated alanine-rich C-kinase substrate (MARCKS), can be phosphorylated in quiescent, non-tumorigenic melanocytes exposed transiently to a biologically active phorbol ester, but cannot be phosphorylated in phorbol ester-treated, syngeneic malignant melanoma cells. Despite its ubiquitous distribution, the function of MARCKS in cell growth and transformation remains to be demonstrated clearly. We report here that MARCKS mRNA and protein levels are down-regulated significantly in the spontaneously derived murine B16 melanoma cell line compared with syngeneic normal Mel-ab melanocytes. In contrast, the tumourigenic v-Ha-ras-transfonned melan-ocytic line, LTR Ras 2, showed a high basal level of MARCKS phosphorylation which was not enhanced by treatment of cells with phorbol ester. Furthermore, protein levels of MARCKS in LTR Ras 2 cells were similar to those expressed in Mel-ab melanocytes. However, in four out of six murine tumour cell lines investigated, levels of MARCKS protein were barely detectable. Transfection of B16 cells with a plasmid containing the MARCKS cDNA in the sense orientation produced two neomycin-resistant clones displaying reduced proliferative capacity and decreased anchorage-independent growth compared with control cells. In contrast, transfection with the antisense MARCKS construct produced many colonies which displayed enhanced growth and transforming potential compared with control cells. Thus, MARCKS appears to act as a novel growth suppressor in the spontaneous transformation of cells of melanocyte origin and may play a more general role in the tumour progression of other carcinomas.

Highly accurate detection of ovarian cancer using CA125 but limited improvement with serum matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling

Objectives: Our objective was to test the performance of CA125 in classifying serum samples from a cohort of malignant and benign ovarian cancers and age-matched healthy controls and to assess whether combining information from matrix-assisted laser desorption/ionization (MALDI) time-of-flight profiling could improve diagnostic performance. Materials and Methods: Serum samples from women with ovarian neoplasms and healthy volunteers were subjected to CA125 assay and MALDI time-of-flight mass spectrometry (MS) profiling. Models were built from training data sets using discriminatory MALDI MS peaks in combination with CA125 values and tested their ability to classify blinded test samples. These were compared with models using CA125 threshold levels from 193 patients with ovarian cancer, 290 with benign neoplasm, and 2236 postmenopausal healthy controls. Results: Using a CA125 cutoff of 30 U/mL, an overall sensitivity of 94.8% (96.6% specificity) was obtained when comparing malignancies versus healthy postmenopausal controls, whereas a cutoff of 65 U/mL provided a sensitivity of 83.9% (99.6% specificity). High classification accuracies were obtained for early-stage cancers (93.5% sensitivity). Reasons for high accuracies include recruitment bias, restriction to postmenopausal women, and inclusion of only primary invasive epithelial ovarian cancer cases. The combination of MS profiling information with CA125 did not significantly improve the specificity/accuracy compared with classifications on the basis of CA125 alone. Conclusions: We report unexpectedly good performance of serum CA125 using threshold classification in discriminating healthy controls and women with benign masses from those with invasive ovarian cancer. This highlights the dependence of diagnostic tests on the characteristics of the study population and the crucial need for authors to provide sufficient relevant details to allow comparison. Our study also shows that MS profiling information adds little to diagnostic accuracy. This finding is in contrast with other reports and shows the limitations of serum MS profiling for biomarker discovery and as a diagnostic tool

Fatty acid compositions of inositol and choline phospholipids of breast tumours and normal breast tissue

The fatty acid compositions of the -choline and -inositol phospholipids of breast tumours of women undergoing surgery for treatment of breast disease (malignant n = 12; benign n = 10) and normal breast tissue of women undergoing breast reduction surgery (n = 6) were determined. The fatty acid compositions of erythrocyte phospholipids were also determined in the same subjects and in an additional number of normal healthy volunteers (n = 16). Levels of oleic acid were lower in both phospholipid fractions of erythrocytes of women with breast disease and in the phosphatidylcholine fraction of breast tumours compared with normal breast tissue. Significantly higher levels of linoleic acid were found in erythrocytes of tumour-bearing subjects and a similar trend was evident in the phosphatidylcholine fraction of tumour compared with normal breast tissues. Conversely, lower levels of two of the products of linoleic acid chain elongation and desaturation, dihomogamma-linolenic and arachidonic acids, were found in the erythrocyte phospholipids of tumour-bearing subjects and in the choline phospholipids of breast tumour tissues. These data suggest that in women with breast disease, there may be inhibition of 6-desaturase, and enhanced activity of 9-desaturase, enzymes which play an important role in determining membrane phospholipid fatty acid composition. This pattern of altered fatty acid composition characteristic of erythrocyte phospholipids of tumour-bearing subjects and phosphatidylcholine of breast tumour tissue was less evident in the case of the breast tumour phosphatidylinositol in which differences other than those described were seen.

Plasma lipids and prolactin in patients with breast cancer

In a comparative study of pre- and postmenopausal women with benign and malignant breast disease, a number of differences were observed in circulating plasma prolactin and lipid concentrations. Plasma lipids, phospholipids, triglycerides, cholesterol and free fatty acids were all higher in blood obtained from breast cancer patients prior to surgery. HDL-Cholesterol levels were significantly lower in these patients. These differences remained when the patient groups were sub-divided according to menopausal status. Plasma prolactin concentrations were also found to be higher in cancer compared with non-cancer patients, this effect being more marked in premenopausal than in postmenopausal patients. Premenopausal patients with invasive or poorly differentiated disease had significantly higher prolactin levels than those with non-invasive disease. No correlations were found between plasma prolactin and any of the lipid fractions.

Eight-plex iTRAQ analysis of variant metastatic human prostate cancer cells identifies candidate biomarkers of progression: an exploratory study.

BACKGROUND: Due to the heterogeneity in the biological behavior of prostate cancer, biomarkers that can reliably distinguish indolent from aggressive disease are urgently needed to inform treatment choices. METHODS: We employed 8-plex isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to profile the proteomes of two distinct panels of isogenic prostate cancer cells with varying growth and metastatic potentials, in order to identify novel biomarkers associated with progression. The LNCaP, LNCaP-Pro5, and LNCaP-LN3 panel of cells represent a model of androgen-responsive prostate cancer, while the PC-3, PC-3M, and PC-3M-LN4 panel represent a model of androgen-insensitive disease. RESULTS: Of the 245 unique proteins identified and quantified (>or=95% confidence; >or=2 peptides/protein), 17 showed significant differential expression (>or=+/-1.5), in at least one of the variant LNCaP cells relative to parental cells. Similarly, comparisons within the PC-3 panel identified 45 proteins to show significant differential expression in at least one of the variant PC-3 cells compared with parental cells. Differential expression of selected candidates was verified by Western blotting or immunocytochemistry, and corresponding mRNA expression was determined by quantitative real-time PCR (qRT-PCR). Immunostaining of prostate tissue microarrays for ERp5, one of the candidates identified, showed a significant higher immunoexpression in pre-malignant lesions compared with non-malignant epithelium (P < 0.0001, Mann-Whitney U-test), and in high Gleason grade (4-5) versus low grade (2-3) cancers (P < 0.05). CONCLUSIONS: Our study provides proof of principle for the application of an 8-plex iTRAQ approach to uncover clinically relevant candidate biomarkers for prostate cancer progression.

Investigating the prevalence and causes of prescribing errors in general practice: the PRACtICe Study

Aim: To determine the prevalence and nature of prescribing errors in general practice; to explore the causes, and to identify defences against error. Methods: 1) Systematic reviews; 2) Retrospective review of unique medication items prescribed over a 12 month period to a 2% sample of patients from 15 general practices in England; 3) Interviews with 34 prescribers regarding 70 potential errors; 15 root cause analyses, and six focus groups involving 46 primary health care team members Results: The study involved examination of 6,048 unique prescription items for 1,777 patients. Prescribing or monitoring errors were detected for one in eight patients, involving around one in 20 of all prescription items. The vast majority of the errors were of mild to moderate severity, with one in 550 items being associated with a severe error. The following factors were associated with increased risk of prescribing or monitoring errors: male gender, age less than 15 years or greater than 64 years, number of unique medication items prescribed, and being prescribed preparations in the following therapeutic areas: cardiovascular, infections, malignant disease and immunosuppression, musculoskeletal, eye, ENT and skin. Prescribing or monitoring errors were not associated with the grade of GP or whether prescriptions were issued as acute or repeat items. A wide range of underlying causes of error were identified relating to the prescriber, patient, the team, the working environment, the task, the computer system and the primary/secondary care interface. Many defences against error were also identified, including strategies employed by individual prescribers and primary care teams, and making best use of health information technology. Conclusion: Prescribing errors in general practices are common, although severe errors are unusual. Many factors increase the risk of error. Strategies for reducing the prevalence of error should focus on GP training, continuing professional development for GPs, clinical governance, effective use of clinical computer systems, and improving safety systems within general practices and at the interface with secondary care.

MALDI MS profiling of post-DRE urine samples highlights the potential of b-Microseminoprotein as a marker for prostatic diseases

BACKGROUND. To use spectra acquired by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) from pre- and post-digital rectal examination (DRE) urine samples to search for discriminating peaks that can adequately distinguish between benign and malignant prostate conditions, and identify the peaks’ underlying biomolecules. METHODS. Twenty-five participants with prostate cancer (PCa) and 27 participants with a variety of benign prostatic conditions as confirmed by a 10-core tissue biopsy were included. Pre- and post-DRE urine samples were prepared for MALDI MS profiling using an automated clean-up procedure. Following mass spectra collection and processing, peak mass and intensity were extracted and subjected to statistical analysis to identify peaks capable of distinguishing between benign and cancer. Logistic regression was used to combine markers to create a sensitive and specific test. RESULTS. A peak at m/z 10,760 was identified as b-microseminoprotein (b-MSMB) and found to be statistically lower in urine from PCa participants using the peak’s average areas. By combining serum prostate-specific antigen (PSA) levels with MALDI MS-measured b-MSMB levels, optimum threshold values obtained from Receiver Operator characteristics curves gave an increased sensitivity of 96% at a specificity of 26%. CONCLUSIONS. These results demonstrate that with a simple sample clean-up followed by MALDI MS profiling, significant differences of MSMB abundance were found in post-DRE urine samples. In combination with PSA serum levels, obtained from a classic clinical assay led to high classification accuracy for PCa in the studied sample set. Our results need to be validated in a larger multicenter prospective randomized clinical trial.

GTP binding controls complex formation by the human ROCO protein MASL1

The human ROCO proteins are a family of multi-domain proteins sharing a conserved ROC-COR supra-domain. The family has four members: leu- cine-rich repeat kinase 1 (LRRK1), leucine-rich repeat kinase 2 (LRRK2), death-associated protein kinase 1 (DAPK1) and malignant fibrous histiocy- toma amplified sequences with leucine-rich tandem repeats 1 (MASL1). Previous studies of LRRK1/2 and DAPK1 have shown that the ROC (Ras of complex proteins) domain can bind and hydrolyse GTP, but the cellular consequences of this activity are still unclear. Here, the first biochemical characterization of MASL1 and the impact of GTP binding on MASL1 complex formation are reported. The results demonstrate that MASL1, similar to other ROCO proteins, can bind guanosine nucleotides via its ROC domain. Furthermore, MASL1 exists in two distinct cellular com- plexes associated with heat shock protein 60, and the formation of a low molecular weight pool of MASL1 is modulated by GTP binding. Finally, loss of GTP enhances MASL1 toxicity in cells. Taken together, these data point to a central role for the ROC/GTPase domain of MASL1 in the reg- ulation of its cellular function.