Investigating the mechanism of enhanced cytotoxicity of HPMA copolymer-Dox-AGM in breast cancer cells

Recently we have described an HPMA copolymer conjugate carrying both the aromatase inhibitor aminoglutethimide (AGM) and doxorubicin (Dox) as combination therapy. This showed markedly enhanced in vitro cytotoxicity compared to the HPMA copolymer-Dox (FCE28068), a conjugate that demonstrated activity in chemotherapy refractory breast cancer patients during early clinical trials. To better understand the superior activity of HPMA copolymer-Dox-AGM, here experiments were undertaken using MCF-7 and MCF-7ca (aromatase-transfected) breast cancer cell lines to: further probe the synergistic cytotoxic effects of AGM and Dox in free and conjugated form; to compare the endocytic properties of HPMA copolymer-Dox-AGM and HPMA copolymer-Dox (binding, rate and mechanism of cellular uptake); the rate of drug liberation by lysosomal thiol-dependant proteases (i.e. conjugate activation), and also, using immunocytochemistry, to compare their molecular mechanism of action. It was clearly shown that attachment of both drugs to the same polymer backbone was a requirement for enhanced cytotoxicity. FACS studies indicated both conjugates have a similar pattern of cell binding and endocytic uptake (at least partially via a cholesterol-dependent pathway), however, the pattern of enzyme-mediated drug liberation was distinctly different. Dox release from PK1 was linear with time, whereas the release of both Dox and AGM from HPMA copolymer-Dox-AGM was not, and the initial rate of AGM release was much faster than that seen for the anthracycline. Immunocytochemistry showed that both conjugates decreased the expression of ki67. However, this effect was more marked for HPMA copolymer-Dox-AGM and, moreover, only this conjugate decreased the expression of the anti-apoptotic protein bcl-2. In conclusion, the superior in vitro activity of HPMA copolymer-Dox-AGM cannot be attributed to differences in endocytic uptake, and it seems likely that the synergistic effect of Dox and AGM is due to the kinetics of intracellular drug liberation which leads to enhanced activity. (c) 2006 Elsevier B.V All rights reserved.

Non-promoting diterpene esters can induce Epstein-Barr Virus early antigen expression in the Raji cell line

A range of diterpene ester ligands with selective biological activity (e.g., irritant but not tumour promoting) were tested for their ability to induce Epstein-Barr virus (EBV) early antigen expression in the lymphoblastoid Raji cell line. All substituted compounds were found to be capable of inducing some antigen expression at nM−μM levels, including desacetyl-α-sapinine, a compound largely devoid of biological activity. The non-promoting, fluorescent compound, sapintoxin A, was virtually equipotent with promoting compounds. It was concluded that, although the assay has relevance to the specific condition of chronic diterpene ester exposure occurring in conjunction with high EBV infection rates, there was relatively poor correlation with mouse skin tumour promoting potential.

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

Postprandial lipoprotein lipase, insulin and gastric inhibitory polypeptide responses to test meals of different fatty acid composition: comparison of saturated, n-6 and n-3 polyunsaturated fatty acids

OBJECTIVE: The present study was carried out to investigate effects of meals, rich in either saturated fatty acids (SFA), or n-6 or n-3 fatty acids, on postprandial plasma lipid and hormone concentrations as well as post-heparin plasma lipoprotein lipase (LPL) activity. DESIGN: The study was a randomized single-blind study comparing responses to three test meals. SETTING: The volunteers attended the Clinical Investigation Unit of the Royal Surrey County Hospital on three separate occasions in order to consume the meals. SUBJECTS: Twelve male volunteers with an average age of 22.5 +/- 1.4 years (mean +/- SD), were selected from the University of Surrey student population; one subject dropped out of the study because he found the test meal unpalatable. INTERVENTIONS: Three meals were given in the early evening and postprandial responses were followed overnight for 11h. The oils used to prepare each of the three test meals were: a mixed oil rich in saturated fatty acids (SFA) which mimicked the fatty acid composition of the current UK diet, corn oil, rich in n-6 fatty acids and a fish oil concentrate (MaxEPA) rich in n-3 fatty acids. The oil under investigation (40 g) was incorporated into the test meals which were otherwise identical [208 g carbohydrates, 35 g protein, 5.65 MJ (1350 kcal) energy]. Postprandial plasma triacylglycerol (TAG), gastric inhibitory polypeptide (GIP), and insulin responses, as well as post-heparin LPL activity (measured at 12 h postprandially only) were investigated. RESULTS: Fatty acids of the n-3 series significantly reduced plasma TAG responses compared to the mixed oil meal (P < 0.05) and increased post-heparin LPL activity 15 min after the injection of heparin (P < 0.01). A biphasic response was observed in TAG, with peak responses occurring at 1 h and between 3-7 h postprandially. GIP and insulin showed similar responses to the three test meals and no significant differences were observed. CONCLUSION: We conclude that fish oils can decrease postprandial plasma TAG levels partly through an increase in post-heparin LPL activity, which however, is not due to increased GIP or insulin concentrations.

Polymer-drug conjugates: towards a novel approach for the treatment of endrocine-related cancer

The last decade has seen successful clinical application of polymer–protein conjugates (e.g. Oncaspar, Neulasta) and promising results in clinical trials with polymer–anticancer drug conjugates. This, together with the realisation that nanomedicines may play an important future role in cancer diagnosis and treatment, has increased interest in this emerging field. More than 10 anticancer conjugates have now entered clinical development. Phase I/II clinical trials involving N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (PK1; FCE28068) showed a four- to fivefold reduction in anthracycline-related toxicity, and, despite cumulative doses up to 1680 mg/m2 (doxorubicin equivalent), no cardiotoxicity was observed. Antitumour activity in chemotherapy-resistant/refractory patients (including breast cancer) was also seen at doxorubicin doses of 80–320 mg/m2, consistent with tumour targeting by the enhanced permeability (EPR) effect. Hints, preclinical and clinical, that polymer anthracycline conjugation can bypass multidrug resistance (MDR) reinforce our hope that polymer drugs will prove useful in improving treatment of endocrine-related cancers. These promising early clinical results open the possibility of using the water-soluble polymers as platforms for delivery of a cocktail of pendant drugs. In particular, we have recently described the first conjugates to combine endocrine therapy and chemotherapy. Their markedly enhanced in vitro activity encourages further development of such novel, polymer-based combination therapies. This review briefly describes the current status of polymer therapeutics as anticancer agents, and discusses the opportunities for design of second-generation, polymer-based combination therapy, including the cocktail of agents that will be needed to treat resistant metastatic cancer.

The impact of date palm fruits and their component polyphenols, on gut microbial ecology, bacterial metabolites and colon cancer cell proliferation

The fruit of the date palm (Phoenix dactylifera L.) is a rich source of dietary fibre and polyphenols. We have investigated gut bacterial changes induced by the whole date fruit extract (digested date extract; DDE) and its polyphenol-rich extract (date polyphenol extract; DPE) using faecal, pH-controlled, mixed batch cultures mimicking the distal part of the human large intestine, and utilising an array of microbial group-specific 16S rRNA oligonucleotide probes. Fluorescence microscopic enumeration indicated that there was a significant increase in the growth of bifidobacteria in response to both treatments, whilst whole dates also increased bacteroides at 24 h and the total bacterial counts at later fermentation time points when compared with DPE alone. Bacterial metabolism of whole date fruit led to the production of SCFA, with acetate significantly increasing following bacterial incubation with DDE. In addition, the production of flavonoid aglycones (myricetin, luteolin, quercetin and apigenin) and the anthocyanidin petunidin in less than 1 h was also observed. Lastly, the potential of DDE, DPE and metabolites to inhibit Caco-2 cell growth was investigated, indicating that both were capable of potentially acting as antiproliferative agents in vitro, following a 48 h exposure. This potential to inhibit growth was reduced following fermentation. Together these data suggest that consumption of date fruits may enhance colon health by increasing beneficial bacterial growth and inhibiting the proliferation of colon cancer cells. This is an early suggestion that date intake by humans may aid in the maintenance of bowel health and even the reduction of colorectal cancer development.

Multiprobabilistic prediction in early medical diagnoses

This paper describes the methodology of providing multiprobability predictions for proteomic mass spectrometry data. The methodology is based on a newly developed machine learning framework called Venn machines. Is allows to output a valid probability interval. The methodology is designed for mass spectrometry data. For demonstrative purposes, we applied this methodology to MALDI-TOF data sets in order to predict the diagnosis of heart disease and early diagnoses of ovarian cancer and breast cancer. The experiments showed that probability intervals are narrow, that is, the output of the multiprobability predictor is similar to a single probability distribution. In addition, probability intervals produced for heart disease and ovarian cancer data were more accurate than the output of corresponding probability predictor. When Venn machines were forced to make point predictions, the accuracy of such predictions is for the most data better than the accuracy of the underlying algorithm that outputs single probability distribution of a label. Application of this methodology to MALDI-TOF data sets empirically demonstrates the validity. The accuracy of the proposed method on ovarian cancer data rises from 66.7 % 11 months in advance of the moment of diagnosis to up to 90.2 % at the moment of diagnosis. The same approach has been applied to heart disease data without time dependency, although the achieved accuracy was not as high (up to 69.9 %). The methodology allowed us to confirm mass spectrometry peaks previously identified as carrying statistically significant information for discrimination between controls and cases.

Comparison of biological behavior between early-stage adenocarcinoma and squamous cell carcinoma of the uterine cervix

Objectives: (1) To compare the anatomopathological variables and recurrence rates in patients with early-stage adenocarcinoma (AC) and squamous cell carcinoma (SCC) of the uterine cervix; (2) to identify the independent risk factors for recurrence. Study design: This historical cohort study assessed 238 patients with carcinoma of the uterine cervix (113 and IIA), who underwent radical hysterectomy with pelvic lymph node dissection between 1980 and 1999. Comparison of category variables between the two histological types was carried out using the Pearson`s X-2 test or Fisher exact test. Disease-free survival rates for AC and SCC were calculated using the Kaplan-Meier method and the curves were compared using the log-rank test. The Cox proportional hazards model was used to identify the independent risk factors for recurrence. Results: There were 35 cases of AC (14.7%) and 203 of SCC (85.3%). AC presented lower histological grade than did SCC (grade 1: 68.6% versus 9.4%; p < 0.001), lower rate of lymphovascular space involvement (25.7% versus 53.7%; p = 0.002), lower rate of invasion into the middle or deep thirds of the uterine cervix (40.0% versus 80.8%; p < 0.001) and lower rate of lymph node metastasis (2.9% versus 16.3%; p = 0.036). Although the recurrence rate was lower for AC than for SCC (11.4% versus 15.8%), this difference was not statistically significant (p = 0.509). Multivariate analysis identified three independent risk factors for recurrence: presence of metastases in the pelvic lymph nodes, invasion of the deep third of the uterine cervix and absence of or slight inflammatory reaction in the cervix. When these variables were adjusted for the histological type and radiotherapy status, they remained in the model as independent risk factors. Conclusion: The AC group showed less aggressive histological behavior than did the SCC group, but no difference in the disease-free survival rates was noted. (C) 2006 Elsevier Ireland Ltd. All rights reserved.

Early Changes in Gene Expression Induced by Tobacco Smoke: Evidence for the Importance of Estrogen within Lung Tissue

Lung cancer is the leading cause of cancer deaths in the United States, surpassing breast cancer as the primary cause of cancer-related mortality in women. The goal of the present study was to identify early molecular changes in the lung induced by exposure to tobacco smoke and thus identify potential targets for chemoprevention. Female A/J mice were exposed to either tobacco smoke or HEPA-filtered air via a whole-body exposure chamber (6 h/d, 5 d/wk for 3, 8, and 20 weeks). Gene expression profiles of lung tissue from control and smoke-exposed animals were established using a 15K cDNA microarray. Cytochrome P450 1b1, a phase I enzyme involved in both the metabolism of xenobiotics and the 4-hydroxylation of 17 beta-estradiol (E(2)), was modulated to the greatest extent following smoke exposure. A panel of 10 genes were found to be differentially expressed in control and smoke-exposed lung tissues at 3, 8, and 20 weeks (P < 0.001). The interaction network of these differentially expressed genes revealed new pathways modulated by short-term smoke exposure, including estrogen metabolism. In addition, E(2) was detected within murine lung tissue by gas chromatography-coupled mass spectrometry and immunohistochemistry. Identification of the early molecular events that contribute to lung tumor formation is anticipated to lead to the development of promising targeted chemopreventive therapies. In conclusion, the presence of E2 within lung tissue when combined with the modulation of cytochrome P450 1b1 and other estrogen metabolism genes by tobacco smoke provides novel insight into a possible role for estrogens in lung cancer. Cancer Prev Res; 3(6); 707-17. (C) 2010 AACR.

Investigation of urinary volatile organic metabolites as potential cancer biomarkers by solid-phase microextraction in combination with gas chromatography-mass spectrometry

BACKGROUND: Non-invasive diagnostic strategies aimed at identifying biomarkers of cancer are of great interest for early cancer detection. Urine is potentially a rich source of volatile organic metabolites (VOMs) that can be used as potential cancer biomarkers. Our aim was to develop a generally reliable, rapid, sensitive, and robust analytical method for screening large numbers of urine samples, resulting in a broad spectrum of native VOMs, as a tool to evaluate the potential of these metabolites in the early diagnosis of cancer. METHODS: To investigate urinary volatile metabolites as potential cancer biomarkers, urine samples from 33 cancer patients (oncological group: 14 leukaemia, 12 colorectal and 7 lymphoma) and 21 healthy (control group, cancer-free) individuals were qualitatively and quantitatively analysed. Dynamic solid-phase microextraction in headspace mode (dHS-SPME) using a carboxenpolydimethylsiloxane (CAR/PDMS) sorbent in combination with GC-qMS-based metabolomics was applied to isolate and identify the volatile metabolites. This method provides a potential non-invasive method for early cancer diagnosis as a first approach. To fulfil this objective, three important dHS-SPME experimental parameters that influence extraction efficiency (fibre coating, extraction time and temperature of sampling) were optimised using a univariate optimisation design. The highest extraction efficiency was obtained when sampling was performed at 501C for 60min using samples with high ionic strengths (17% sodium chloride, wv 1) and under agitation. RESULTS: A total of 82 volatile metabolites belonging to distinct chemical classes were identified in the control and oncological groups. Benzene derivatives, terpenoids and phenols were the most common classes for the oncological group, whereas ketones and sulphur compounds were the main classes that were isolated from the urine headspace of healthy subjects. The results demonstrate that compound concentrations were dramatically different between cancer patients and healthy volunteers. The positive rates of 16 patients among the 82 identified were found to be statistically different (Po0.05). A significant increase in the peak area of 2-methyl3-phenyl-2-propenal, p-cymene, anisole, 4-methyl-phenol and 1,2-dihydro-1,1,6-trimethyl-naphthalene in cancer patients was observed. On average, statistically significant lower abundances of dimethyl disulphide were found in cancer patients. CONCLUSIONS: Gas chromatographic peak areas were submitted to multivariate analysis (principal component analysis and supervised linear discriminant analysis) to visualise clusters within cases and to detect the volatile metabolites that are able to differentiate cancer patients from healthy individuals. Very good discrimination within cancer groups and between cancer and control groups was achieved.

Solid phase microextraction, mass spectrometry and metabolomic approaches for detection of potential urinary cancer biomarkers: a powerful strategy for breast cancer diagnosis

A sensitive assay to identify volatile organic metabolites (VOMs) as biomarkers that can accurately diagnose the onset of breast cancer using non-invasively collected clinical specimens is ideal for early detection. Therefore the aim of this study was to establish the urinary metabolomic profile of breast cancer patients and healthy individuals (control group) and to explore the VOMs as potential biomarkers in breast cancer diagnosis at early stage. Solid-phase microextraction (SPME) using CAR/PDMS sorbent combined with gas chromatography–mass spectrometry was applied to obtain metabolomic information patterns of 26 breast cancer patients and 21 healthy individuals (controls). A total of seventy-nine VOMs, belonging to distinct chemical classes, were detected and identified in control and breast cancer groups. Ketones and sulfur compounds were the chemical classes with highest contribution for both groups. Results showed that excretion values of 6 VOMs among the total of 79 detected were found to be statistically different (p < 0.05). A significant increase in the peak area of (−)-4-carene, 3-heptanone, 1,2,4-trimethylbenzene, 2-methoxythiophene and phenol, in VOMs of cancer patients relatively to controls was observed. Statiscally significant lower abundances of dimethyl disulfide were found in cancer patients. Bioanalytical data were submitted to multivariate statistics [principal component analysis (PCA)], in order to visualize clusters of cases and to detect the VOMs that are able to differentiate cancer patients from healthy individuals. Very good discrimination within breast cancer and control groups was achieved. Nevertheless, a deep study using a larger number of patients must be carried out to confirm the results.

The early systemic and gastrointestinal oxygenation effects of hemorrhagic shock resuscitation with hypertonic saline and hypertonic saline 6% dextran-70: A comparative study in dogs

The smaller volemic state from hypertonic (7.5%) saline (HS) solution administration in hemorrhagic shock can determine lesser systemic oxygen delivery and tissue oxygenation than conventional plasma expanders. In a model of hemorrhagic shock in dogs, we studied the systemic and gastrointestinal oxygenation effects of HS and hyperoncotic (6%) dextran-70 in combination with HS (HSD) solutions in comparison with lactated Ringer's (LR) and (6%) hydroxyethyl starch (HES) solutions. Forty-eight mongrel dogs were anesthetized, mechanically ventilated, and subjected to splenectomy. A gastric air tonometer was placed. in the stomach for intramucosal gastric CO2 (Pgco(2)) determination and for the calculation of intramucosal. pH (pHi):[pHi = pHa - log(Pgco(2)/Paco(2))].The dogs were hemorrhaged (42% of blood volume) to hold mean arterial blood pressure at 40-50 mm Hg over 30 min and were then resuscitated with LR (n = 12) in a 3:1 relation to removed blood volume; HS (n = 12), 6 mL / kg; HSD (n = 12), 6 mL / kg; and HES (mean molecular weight, 200 kDa; degree of substitution, 0.5) (n = 12) in a 1:1 relation to the removed blood volume. Hemodynamic, systemic, and gastric oxygenation variables were measured at baseline, after 30 min of hemorrhage, and 5, 60, and 120 min after intravascular fluid resuscitation. After fluid resuscitation, HS showed significantly lower arterial pH and mixed venous Po-2 and higher systemic oxygen uptake index and systemic oxygenation extraction than LR and HES (P < 0.05), whereas HSD showed significantly lower arterial pH than LR and HES (P < 0.05). Only HS and HSD did not return arterial pH and pHi to control levels (P < 0.05). In conclusion, all solutions improved systemic and gastrointestinal oxygenation after hemorrhagic shock in dogs. However, the HS solution showed the worst response in comparison to LR and HES solutions in relation to systemic oxygenation, whereas HSD showed intermediate values. HS and HSD solutions did not return regional oxygenation to control values.

Folic acid supplementation during early hepatocarcinogenesis: cellular and molecular effects

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Familial cancer: depressed NK-cell cytotoxicity in healthy and cancer affected members

Este estudo se reporta às funções de células natural killer (NK), como adesão, lise e citotoxicidade e de subpopulações de células T em uma família com alta prevalência de pacientes com câncer e que apresentaram: glioblastoma, leucemia mielóide crônica, osteoblastoma, melanoma e carcinomas gástrico, pancreático e cólon retal. Quinze membros dessa família foram estudados, sendo 13 sadios, acompanhados por 5 anos e dois com câncer: glioblastoma e leucemia mielóide crônica. Duas pessoas sadias, no momento da avaliação, desenvolveram posteriormente osteoblastoma mandibular ou melanoma maligno. Como controle, foram avaliados 19 indivíduos saudáveis de faixa etária equivalente. A determinação de linfócitos T CD3+ e de suas subpopulações CD4+ e CD8+ foi realizada empregando-se anticorpos monoclonais e a atividade citotóxica de células NK, avaliada pelo teste de single-cell contra células alvo da linhagem eritroleucêmica K562. Os resultados mostraram que as percentagens de células T totais (CD3+), da subpopulação CD4+ e da relação CD4/CD8 foram significativamente menores nos indivíduos da família estudada em comparação aos valores observados no grupo controle. em todos os membros dessa família a percentagem de formação de conjugados entre células NK-células alvo foi inferior ao valor mínimo observado nos controles. Essa alteração poderia estar relacionada a defeito na expressão de moléculas de adesão, presentes na membrana de células NK, como provável causa das alterações funcionais dessas células. A herança dos mecanismos determinantes desta deficiência pode ser um fator de risco, com valor prognóstico para o desenvolvimento de cancer.

DNA viruses in human cancer: An integrated overview on fundamental mechanisms of viral carcinogenesis

The first experimental data suggesting that neoplasm development in animals might be influenced by infectious agents were published in the early 1900s. However, conclusive evidence that DNA viruses play a role in the pathogenesis of some human cancers only emerged in the 1950s, when Epstein-Barr virus (EBV) was discovered within Burkitt lymphoma cells. Besides EBV, other DNA viruses consistently associated with human cancers are the hepatitis B virus (HBV), human papillomavirus (HPV), and Kaposi sarcoma herpesvirus (KSHV). Although each virus has unique features, it is becoming clearer that all these oncogenic agents target multiple cellular pathways to support malignant transformation and tumor development. (c) 2006 Elsevier B.V.. All rights reserved.