World Trade Center exposure, post-traumatic stress disorder, and subjective cognitive concerns in a cohort of rescue/recovery workers.

OBJECTIVE: To determine whether World Trade Center (WTC)-exposure intensity and post-traumatic stress disorder (PTSD) are associated with subjective cognitive change in rescue/recovery workers. METHOD: The population included 7875 rescue/recovery workers who completed a subjective cognition measure, the Cognitive Function Instrument (CFI), between 3/1/2018 and 2/28/2019 during routine monitoring, indicating whether they had experienced cognitive and functional difficulties in the past year. Higher scores indicated greater self-perceived cognitive change. Probable PTSD, depression, and alcohol abuse were evaluated by validated mental health screeners. Logistic regression assessed the associations of WTC exposure and current PTSD with top-quartile (≥2) CFI score, and of early post-9/11 PTSD with top-quartile CFI in a subpopulation (N = 6440). Models included demographics, smoking, depression, and alcohol abuse as covariates. RESULTS: Mean age at CFI completion was 56.7 ± 7.7 (range: 36-81). Participants with high-intensity WTC exposure had an increased likelihood of top-quartile CFI score (odds ratio[OR] vs. low exposure: 1.32, 95%CI: 1.07-1.64), controlling for covariates. Current and early PTSD were both associated with top-quartile CFI (OR: 3.25, 95%CI: 2.53-4.19 and OR: 1.56, 95%CI: 1.26-1.93) respectively. CONCLUSIONS: High-intensity WTC exposure was associated with self-reported cognitive change 17 years later in rescue/recovery workers, as was PTSD. Highly WTC-exposed subgroups may benefit from additional cognitive evaluation and monitoring of cognition over time.

Quantifying Argonaute 2 (Ago2) expression to stratify breast cancer.

BACKGROUND: Argonaute-2 (Ago2) is an essential component of microRNA biogenesis implicated in tumourigenesis. However Ago2 expression and localisation in breast cancer remains undetermined. The aim was to define Ago2 expression (mRNA and protein) and localisation in breast cancer, and investigate associations with clinicopathological details. METHODS: Ago2 protein was stained in breast cancer cell lines and tissue microarrays (TMAs), with intensity and localization assessed. Staining intensity was correlated with clinicopathological details. Using independent databases, Ago2 mRNA expression and gene alterations in breast cancer were investigated. RESULTS: In the breast cancer TMAs, 4 distinct staining intensities were observed (Negative, Weak, Moderate, Strong), with 64.2% of samples stained weak or negatively for Ago2 protein. An association was found between strong Ago2 staining and, the Her2 positive or basal subtypes, and between Ago2 intensity and receptor status (Estrogen or Progesterone). In tumours Ago2 mRNA expression correlated with reduced relapse free survival. Conversely, Ago2 mRNA was expressed significantly lower in SK-BR-3 (HER2 positive) and BT-20 (Basal/Triple negative) cell lines. Interestingly, high levels of Ago2 gene amplification (10-27%) were observed in breast cancer across multiple patient datasets. Importantly, knowledge of Ago2 expression improves predictions of breast cancer subtype by 20%, ER status by 15.7% and PR status by 17.5%. CONCLUSIONS: Quantification of Ago2 improves the stratification of breast cancer and suggests a differential role for Ago2 in breast cancer subtypes, based on levels and cellular localisation. Further investigation of the mechanisms affecting Ago2 dysregulation will reveal insights into the molecular differences underpinning breast cancer subtypes.

Use of an engineered honey to eradicate preformed biofilms of important wound pathogens: an in vitro study.

OBJECTIVE: We previously reported on the ability of SurgihoneyRO (SHRO), an engineered honey, to prevent biofilm formation in vitro, but data were lacking regarding the activity against preformed biofilms. This study aims to assess whether SHRO has any antibacterial activity against mature, preformed biofilms and whether there is any evidence to support the observed clinical effectiveness when SHRO has been used anecdotally on acute and chronic wounds where biofilm is most likely present. METHOD: We tested the in vitro antibacterial activity of SHRO against the mature biofilms of 16 clinically relevant wound pathogens, in terms of impacts on biofilm seeding and biofilm biomass. The honey was serially double diluted from 1:3 down to 1:6144, and the lowest dilution achieving a statistically significant reduction in biomass of ≥50%, compared with untreated controls, was recorded. RESULTS: All 16 bacterial isolates were susceptible to SHRO, with reduced biofilm seeding observed for all, and percentage reductions ranging from 58% (ACI_C59) to 94.3% (MDR_B) for the strongest concentration of honey (1:3). Furthermore at this concentration, biofilm seeding of the test biofilm was reduced by 80-94.3% (when compared with the positive control) for 12/16 isolates. We additionally demonstrated that SHRO has antibiofilm impacts, with the 24 hour exposure resulting in disruption of the biofilm, reduced seeding and reduced biomass. CONCLUSION: SHRO is effective at reducing seeding of preformed biofilms of clinically important wound pathogens in vitro, and also has antibiofilm activity. This supports the anecdotal clinical data for antibiofilm efficacy, and supports the use of SHRO as a promising topical wound care agent.

In vitro activity of an engineered honey, medical-grade honeys, and antimicrobial wound dressings against biofilm-producing clinical bacterial isolates.

OBJECTIVE: Honey is recognised to be a good topical wound care agent owing to a broad-spectrum of antimicrobial activity combined with healing properties. Surgihoney RO (SH1) is a product based on honey that is engineered to produce enhanced reactive oxygen species (ROS) and has been reported to be highly antimicrobial. The objective was to investigate the ability of the engineered honey and its comparators to prevent biofilm formation in vitro. METHOD: We tested the ability of three medical-grade honeys SH1, Activon manuka honey (MH) and Medihoney manuka honey (Med), alongside five antimicrobial dressings (AMDs) to prevent the formation of biofilms by 16 isolates. Honeys were serially double diluted from 1:3 down to 1:6144 and the lowest dilution achieving a statistically significant reduction in biomass of at least 50%, compared with untreated controls, was recorded. RESULTS: Although all the honeys were antibacterial and were able to prevent the formation of biofilms, SH1 was the most potent, with efficacy at lower dilutions than the medical honeys for five isolates, and equivalent dilutions for a further six. Additionally, SH1 was superior in antibacterial potency to three commercially available AMDs that contain honey. CONCLUSION: SH1 is effective at preventing bioflms from forming and is superior to medical honeys and AMDs in in vitro tests. DECLARATION OF INTEREST: Surgihoney RO was provided free of charge for testing by Matoke Holdings, UK and the hospital pharmacy provided the other honeys and dressings. This paper presents independent research funded by the National Institute for Health Research (NIHR). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.

Hot and not-so-hot females: reproductive state and thermal preferences of female Arizona Bark Scorpions (Centruroides sculpturatus).

For ectotherms, environmental temperatures influence numerous life history characteristics, and the body temperatures (Tb ) selected by individuals can affect offspring fitness and parental survival. Reproductive trade-offs may therefore ensue for gravid females, because temperatures conducive to embryonic development may compromise females' body condition. We tested whether reproduction influenced thermoregulation in female Arizona Bark Scorpions (Centruroides sculpturatus). We predicted that gravid females select higher Tb and thermoregulate more precisely than nonreproductive females. Gravid C. sculpturatus gain body mass throughout gestation, which exposes larger portions of their pleural membrane, possibly increasing their rates of transcuticular water loss in arid environments. Accordingly, we tested whether gravid C. sculpturatus lose water faster than nonreproductive females. We determined the preferred Tb of female scorpions in a thermal gradient and measured water loss rates using flow-through respirometry. Gravid females preferred significantly higher Tb than nonreproductive females, suggesting that gravid C. sculpturatus alter their thermoregulatory behaviour to promote offspring fitness. However, all scorpions thermoregulated with equal precision, perhaps because arid conditions create selective pressure on all females to thermoregulate effectively. Gravid females lost water faster than nonreproductive animals, indicating that greater exposure of the pleural membrane during gestation enhances the desiccation risk of reproductive females. Our findings suggest that gravid C. sculpturatus experience a trade-off, whereby selection of higher Tb and increased mass during gestation increase females' susceptibility to water loss, and thus their mortality risk. Elucidating the mechanisms that influence thermal preferences may reveal how reproductive trade-offs shape the life history of ectotherms in arid environments.

Viewpoint survey of mental health service users' experiences of discrimination in England 2008-2012.

PURPOSE: Research suggests levels of discrimination among mental health service users in England are high, but fell over the course of the first phase of the Time to Change programme to reduce stigma and discrimination (2008-2011). The aim of this study was to determine changes in discrimination levels, both overall and by the area of life in which discrimination is experienced, since Time to Change began and over the first year of its second phase (2011-2012). METHOD: Separate samples of mental health service users were interviewed annually from 2008 to 2012 using the Discrimination and Stigma Scale. In 2011 and 2012, social capital was also measured using the Resource Generator-UK. RESULTS: Sample percentages of participants reporting the experience of discrimination in one or more life areas for years 2008-2012 were 91.4, 86.5, 86.2, 87.9 and 91.0 %, respectively. A multivariable logistic regression model was performed to test for significant differences by study year, weighted to match the study population and adjusted for employment status and diagnosis as potential confounding factors. The odds of reporting discrimination in one or more life areas were significantly lower as compared to 2008 for all subsequent years except for 2012 (0.76, 95 % CI 0.49-1.19). However, a weighted multiple regression model provided evidence of decreased mean overall discrimination in 2012 as compared to 2008 (mean decrease -7.57, 95 % CI -11.1 to -4.0, p < 0.001). The weighted mean number of social resources was 13.5 in 2012 as compared to 14.0 in 2011 (mean difference -0.60, 95 % CI -1.25 to 0.06). CONCLUSIONS: While the overall level of discrimination across the life areas studied has fallen over 2008-2012, there is no evidence that more people using mental health services experience no discrimination. We suggest that the pattern suggesting a recent rise in discrimination following an earlier reduction may be linked to economic austerity. Further, the welfare benefits system has become an increasing source of discriminatory experience.

[Detection of Burkholderia cepacia complex strains in the University Hospital Olomouc]. / Záchyt kmenu Burkholderia cepacia komplex ve Fakultní nemocnici Olomouc.

BACKGROUND: The aim was to assess the epidemiology of Burkholderia cepacia complex strains isolated at the Department of Microbiology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, determine the most frequent strains and confirm or rule out potential clonal spread of the strains. MATERIAL AND METHODS: Over a period of eight months, all strains classified as Burkholderia cepacia complex were collected. Susceptibility to selected antimicrobial agents was determined and adequate molecular genetic methods were used to assess their genetic relationship. RESULTS: A total of 52 isolates were tested, with the most frequent (88.5 %) being genomovar II (Burkholderia multivorans). More than 46 % of them were genetically related; 58.3 % of them were detected in intensive care units. All isolates were highly resistant to antimicrobial agents. In four cases, deaths associated with Burkholderia multivorans infection were reported. CONCLUSION: It may be assumed that genetically related strains of Burkholderia multivorans spread from the hospital setting. As yet, the source of infection has not been determined and further investigations are needed.

The impact of the World Trade Center attack on FDNY firefighter retirement, disabilities, and pension benefits.

BACKGROUND: Our goal was to examine the effect of the World Trade Center (WTC) attack and subsequent New York City Fire Department (FDNY) rescue/recovery activities on firefighter retirements. We also analyzed the financial impact associated with the increased number and proportion of service-connected "accidental" disability retirements on the FDNY pension system. METHODS: A total of 7,763 firefighters retired between 9/11/1994 and 9/10/2008. We compared the total number of retirements and the number and proportion of accidental disability retirements 7 years before and 7 years after the WTC attack. We categorized WTC-related accidental disability retirements by medical cause and worked with the New York City Office of the Actuary to approximate the financial impact by cause. RESULTS: In the 7 years before 9/11 there were 3,261 retirements, 48% (1,571) of which were accidental disability retirements. In the 7 years after 9/11, there were 4,502 retirements, 66% (2,970) were accidental disability retirements, of which 47% (1,402) were associated with WTC-related injuries or illnesses. After 9/11, the increase in accidental disability retirements was, for the most part, due to respiratory-related illnesses. Additional increases were attributed to psychological-related illnesses and musculoskeletal injuries incurred at the WTC site. Pension benefits associated with WTC-related accidental disability retirements have produced an increased financial burden of over $826 million on the FDNY pension system. CONCLUSIONS: The WTC attacks affected the health of the FDNY workforce resulting in more post-9/11 retirements than expected, and a larger proportion of these retirees with accidental disability pensions.

Use of granulated sugar therapy in the management of sloughy or necrotic wounds: a pilot study.

OBJECTIVE: To determine the in vitro antimicrobial efficacy of three types of sugar and conduct a pilot clinical study with a view to developing a protocol for a randomised controlled trial (RCT). METHOD: In the in vitro studies three types of granulated sugar (Demerara, granulated beet sugar and granulated cane sugar) were tested to determine their minimum inhibitory concentrations (MICs) against 18 Gram-negative and Gram-positive bacteria in a micro-titre broth dilution assay; growth inhibition of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa in different concentrations of sugar (0.38-25%) was also tested over 12-hours in an agar diffusion assay. The pilot clinical study selected patients from a vascular surgical ward and a vascular outpatient department. All had acute or chronic exuding wounds, some of which were infected. White granulated sugar was applied to the wounds. The following parameters were assessed: surface area; wound characteristics including pain, malodour, appearance (slough/granulation); exudate level; pain level and bacterial load. Patients with diabetes had their blood sugar levels checked daily. All patients completed a short health questionnaire at the start and end of the study. Staff completed a satisfaction questionnaire at the end of the study. The study period was 21 days. RESULTS: In vitro tests demonstrated that sugar inhibits bacterial growth. All three types of sugars had MICs ranging from 6-25% in the bacterial strains tested. The diffusion tests showed that strains were able to grow well in low concentrations of sugar but were completely inhibited in higher concentrations. The two granulated sugars were found to be slightly more effective than Demerara sugar, so the latter was excluded from the clinical pilot study. Twenty-two patients (20 inpatients and two outpatients) with sloughy or necrotic wounds were recruited into the clinical study. Two patients had MRSA and two had Staphylococcus colonisation at baseline. Blood sugar levels remained stable in the seven patients with insulin-dependent diabetes mellitus. All wounds were clean/debrided in a mean of 11.13 days. Pain and malodour reduced markedly. Patient and staff surveys revealed overwhelming support for the sugar therapy. CONCLUSION: The pilot study achieved its aim of developing a protocol for a RCT. Preliminary data suggest that sugar is an effective wound cleansing and is safe to use in patients with insulin-dependent diabetes. In vitro studies demonstrate that sugar inhibits bacterial growth. CONFLICT OF INTEREST: None.

Emerging peptide nanomedicine to regenerate tissues and organs.

Peptide nanostructures containing bioactive signals offer exciting novel therapies of broad potential impact in regenerative medicine. These nanostructures can be designed through self-assembly strategies and supramolecular chemistry, and have the potential to combine bioactivity for multiple targets with biocompatibility. It is also possible to multiplex their functions by using them to deliver proteins, nucleic acids, drugs and cells. In this review, we illustrate progress made in this new field by our group and others using peptide-based nanotechnology. Specifically, we highlight the use of self-assembling peptide amphiphiles towards applications in the regeneration of the central nervous system, vasculature and hard tissue along with the transplant of islets and the controlled release of nitric oxide to prevent neointimal hyperplasia. Also, we discuss other self-assembling oligopeptide technology and the progress made with these materials towards the development of potential therapies.

Lipid analysis of Neochloris oleoabundans by liquid state NMR.

This study is an evaluation of liquid state NMR as a tool for analyzing the lipid composition of algal cultures used for biodiesel production. To demonstrate the viability of this approach, (13)C NMR was used to analyze the lipid composition of intact cells of the algal species, Neochloris oleoabundans (UTEX #1185). Two cultures were used in this study. One culture was "healthy" and grown in conventional media, whereas the other culture was "nitrogen-starved" and grown in media that lacked nitrate. Triglyceride was determined to be present in both cultures by comparing the algal NMR spectra with published chemical shifts for a wide range of lipids and with a spectrum obtained from a triglyceride standard (glyceryl trioleate). In addition, it is shown that (1) the signal-to-noise ratio of the approximately 29.5 ppm methylene peak is indicative of the lipid content and (2) the nitrogen-starved culture contained a greater lipid content than the healthy culture, as expected. Furthermore, the nitrogen-starved culture produced spectra that primarily contained the characteristic peaks of triglyceride (at approximately 61.8 and approximately 68.9 ppm), whereas the healthy culture produced spectra that contained several additional peaks in the glycerol region, likely resulting from the presence of monoglyceride and diglyceride. Finally, potential interferences are evaluated (including the analysis of phospholipids via (31)P NMR) to assess the specificity of the acquired spectra to triglyceride. These results indicate that NMR is a useful diagnostic tool for selectively identifying lipids in algae, with particular relevance to biodiesel production.

Psychoneuroimmunological outcomes and quality of life.

According to the American Psychological Association the field of psychoneuroimmunology comprises a rapidly expanding body of knowledge that strongly suggests a reciprocal interaction between mind (cognition) and body. This body of knowledge indicates that cognitive events have somatic consequences that impact directly on the immune system and disease processes, which in turn lead to maladaptive cognitive processes. Disease sufferers' thought processes and perceived quality of life may thus have a direct bearing on their somatic functioning and disease process, and interventions at this level may have a material impact on their prognosis.

Quantifying Tip60 (Kat5) stratifies breast cancer.

Breast cancer is stratified into four distinct clinical subtypes, using three key biomarkers (Her2/Neu gene status, Estrogen and Progesterone receptor status). However, each subtype is a heterogeneous group, displaying significant variation in survival rates and treatment response. New biomarkers are required to provide more precise stratification of breast cancer cohorts to inform personalised treatment options/predict outcomes. Tip60 is a member of the MYST sub-family of histone acetyltransferases (HATs), and is directly involved in genome maintenance, gene regulation and DNA damage response/repair pathways (key chemotherapeutic influencing mechanisms). We aimed to determine if quantifying Tip60 staining patterns improved breast cancer stratification. We defined Tip60 protein in vivo, quantifying location (cytoplasmic, nuclear), percent of cells and staining intensity in a breast cancer tissue microarray (n = 337). A significant association of specific Tip60 staining patterns with breast cancer subtype, ER or PR status and Tumour grade was found. Importantly, low Tip60 mRNA expression correlated with poor overall survival and relapse free survival. We found Tip60 is a biomarker able to stratify breast cancer patients, and low Tip60 expression is a significant risk factor indicating a higher chance of disease reoccurrence. This work highlights Tip60 regulation as a key factor influencing the development of breast cancer.

Metallothionein induction and deinduction in human prostatic carcinoma cells: relationship with resistance and sensitivity to adriamycin.

Human prostate carcinoma cell line DU-145 was used to examine the relationship between the intracellular levels of cysteine-rich metallothionein (MT) and the sensitivity or resistance of cells to Adriamycin (ADR). The basis for the poor response of human prostate carcinomas to ADR was studied. Cadmium-resistant (Cdr) cells, capable of growth in 10(-5) M cadmium, were derived from DU-145 cadmium-sensitive (Cds) cells, by exposure to increasing concentrations of cadmium. The relative rates of MT synthesis were measured by L-[35S]cysteine incorporation and MT separation by high-performance liquid chromatography. Cdr cells, continuously exposed to cadmium, show a steady-state rate of MT synthesis (designated as control = 100%) which is 3.5 times the basal rate in Cds cells (29%). Dose-response curves, using clonal and cell count assays, show that the dose levels required to produce inhibition of growth to 50% and 90% of control, respectively, of ADR for Cdr cells (19.00 and 132.0 ng/ml) are 1.5 to 1.7 times those for Cds cells (12.5 and 77.5 ng/ml). In the absence of cadmium, deinduction of MT occurs with MT synthesis declining, after 70 and 118 h, to 29% and 19% of control. Correspondingly, in such deinduced cells (Cdr minus cadmium), the 50% inhibitory doses of ADR in clonal and growth assays are 3.5 and 4.8 ng/ml, respectively. Thus, deinduced cells are 3 and 4 times more sensitive to ADR than Cds and Cdr cells. This increased sensitivity is explained by the rapid and marked inhibition of MT synthesis upon exposure to ADR, even in the presence of cadmium, so that after 6 and 10 h in the presence of 10 ng/ml of ADR, the rates drop to 62% and 19% of control. On the basis of these results, we propose that: (a) the increased levels of MT increase the resistance of Cdr cells to ADR and that this may be partly responsible for the poor response of prostate carcinomas to ADR; (b) MT deinduction results in increased sensitivity to ADR; and (c) ADR inhibits MT synthesis. Thus, it is suggested that a treatment regimen consisting of ADR exposure followed by a second exposure, during increased ADR sensitivity, may be effective for growth inhibition of slow-growing prostatic carcinomas.

Osteonectin promotes prostate cancer cell migration and invasion: a possible mechanism for metastasis to bone.

The mechanism underlying the "organ-specific" metastasis of prostate cancer cells to the bone is still poorly understood. It is not clear whether the cells only invade the bone and proliferate there or whether they invade many tissues but survive mainly in the bone ("seed and soil"). Extracts from various organs were used as chemoattractants in the in vitro chemotaxis and invasion assays. Results show that, in comparison with extracts of other tissues, bone extracts promote a 2- to 4-fold increase in chemotaxis by human prostate epithelial cells and a 4-fold increase in the invasive ability of human prostate carcinoma cells. The purified active factor from bone and from marrow stromal-cell-conditioned medium is a low glycosylated osteonectin that specifically promotes the invasive ability of bone-metastasizing prostate (and breast) cancer cells but not that of non-bone-metastasizing tumor cells. It does not stimulate the growth of prostate cancer cells in vitro or in vivo. Because osteonectin specifically enhances matrix metalloprotease activity in prostate and breast cancer cells (and not in other tumor cell types), we conclude that prostate cancer cell metastasis to the bone is, in part, mediated by the ability of osteonectin to promote migration, protease activity, and invasion.

Ornithine as a possible marker of cancer.

The nonprotein amino acid ornithine is the major source of polyamines in mammalian physiological systems. Increased urinary polyamine levels have been demonstrated in humans with varied types of cancers. The metabolism of DL-[1-14C]ornithine monohydrochloride in rats with either Walker 256 carcinoma or chemically induced methylcholanthrene tumors was studied. Following the i.p. injection of 3 muCi[14C]ornithine per 100 g body weight, the decarboxylation of ornithine-yielding 14CO2 was monitored by utilizing the vibrating reed electrometer-ionization chamber model of Davidson and Schwabe. Tumor-bearing animals showed significant increases in ornithine metabolism as compared to controls; for Walker 256 the tumor-bearing animal to control ratio rose from 1.16 to 1.78, for methylcholanthrene implants it rose from 1.19 to 1.82, and for methylcholanthrene paintings it rose from 1.00 to 2.20. With tumor regression ornithine levels of metabolism in the tumor-bearing animals returned to base line or nearly base-line levels. These results encourage us in our attempt to develop ornithine as a biological marker of cancer.

Cadmium-induced malignant transformation of human prostate epithelial cells.

Prostate cancer has become epidemic, and environmental factors such as cadmium may be partly responsible. This study reports malignant transformation of the nontumorigenic human prostatic epithelial cell line RWPE-1 by in vitro cadmium exposure. The cadmium-transformed cells exhibited a loss of contact inhibition in vitro and rapidly formed highly invasive and occasionally metastatic adenocarcinomas upon inoculation into mice. The transformed cells also showed increased secretion of MMP-2 and MMP-9, a phenomenon observed in human prostate tumors and linked to aggressive behavior. Cadmium-induced malignant transformation of human prostate epithelial cells strongly fortifies the evidence for a potential role of cadmium in prostate cancer.

Malignant transformation of human prostate epithelial cells by N-nitroso-N-methylurea.

We report the malignant transformation of adult human prostate epithelial cells after multiple exposures to the chemical carcinogen N-nitroso-N-methylurea. Such transformants showed morphological alterations and anchorage-independent growth in soft agar and induced carcinomas when transplanted into nude mice. No p53 or ras mutations were observed. Stepwise chromosomal changes in the progression to tumorigenicity were observed. Loss of the p arms of chromosome 8 (p10>pter) and chromosome 10 (p10>pter) and gain of the q arm of chromosome 8 (q10>qter) were only observed in the tumor outgrows. These findings provide the first evidence of malignant transformation of human prostate epithelial cells exposed to a chemical carcinogen.

Retinoic acid modulates extracellular urokinase-type plasminogen activator activity in DU-145 human prostatic carcinoma cells.

Effects of all-trans retinoic acid (RA) on the net enzymatic activity of secreted, extracellular urokinase-type plasminogen activator (u-PA) in DU-145 human prostatic carcinoma cells were examined to assess the potential use of retinoids in human prostate cancer prevention and treatment. u-PA is associated with tumor progression involving invasion and metastasis. Based on a chromogenic substrate assay, results show that DU-145 cells secrete five times more u-PA than normal human prostatic epithelium. DU-145 cells were treated with 0.1 to 10 micrometer RA for 48 h. This short treatment of cells with RA did not inhibit growth. After a 48-h treatment of cultures with RA, serum-free conditioned medium was analyzed for u-PA activity by SDS-PAGE zymography. Two major bands of u-PA with Mr of approximately 54,000 (high molecular weight u-PA) and approximately 33,000 (low molecular weight u-PA) were detected. Plasminogen-dependent catalytic activity of these bands could be specifically inhibited with antibody to u-PA, confirming that these bands represent u-PA. A 48-h treatment with 1.0 micrometer RA reduced u-PA activity in conditioned medium to 51.6% of control. A 50% reduction in free u-PA antigen level, as compared to control, was further demonstrated at 1.0 micrometer RA by Western blot analysis and densitometry. These results show that RA can decrease the net extracellular urokinase activity produced by prostatic carcinoma cells. It is proposed that these effects of RA may have important implications not only in the chemoprevention of prostate cancer, by inhibition of promotion of prostatic intraepithelial neoplasia to invasive carcinoma, but also in tumor progression during invasion and metastasis, by decreasing extracellular matrix degradation, as shown in our accompanying article (M. M. Webber and A. Waghray, Clin. Cancer Res., 1: 755-761, 1995).

Urokinase-mediated extracellular matrix degradation by human prostatic carcinoma cells and its inhibition by retinoic acid.

Both normal and malignant prostatic epithelial cells in culture secrete urokinase-type plasminogen activator (u-PA) into the culture medium. u-PA has been shown to have a direct association with invasive and metastatic potential of many types of cancers. We propose that prostate cancer has the intrinsic ability to invade and metastasize because of its inherent ability to secrete the serine protease u-PA. We further propose that in prostate cancer, u-PA is the key enzyme which occupies a place at the apex of the proteolytic cascade and initiates the degradative process. Subsequently, collagenases are recruited after activation of procolla-genases by another serine protease plasmin formed by the activation of plasminogen by u-PA. Extracellular proteolysis involving plasmin can cause massive degradation of the extracellular matrix. We show that u-PA alone can use fibronectin as a substrate and degrade it, but u-PA alone did not degrade laminin. Serum-free conditioned medium from DU-145 human prostatic carcinoma cells has the ability to degrade both fibronectin and laminin. However, treatment of cultures with 1 microM all-trans retinoic acid (RA) for 48 h reduced the ability of serum-free conditioned medium to cause u-PA-mediated degradation of fibronectin and laminin. Thus, RA had a protective effect on these extracellular matrix glycoproteins. Treatment of cells with RA also decreased their ability to invade Matrigel in the in vitro invasion assay in a dose-dependent manner. RA at the 0.5, 1, and 10 microM level reduced invasion to 65.7%, 46.7%, and 34.3% of control, respectively. RA reduced extracellular proteolysis and thus inhibited extracellular matrix degradation and invasion. These results may also explain one mechanism by which retinoids inhibit invasion and metastasis in vitro and in vivo. These studies have important translational value in the chemoprevention of progression of prostatic intraepithelial neoplasia to invasive carcinoma.