Higher EpCAM-Positive Extracellular Vesicle Concentration in Ascites Is Associated with Shorter Progression-Free Survival of Patients with Advanced High-Grade Serous Carcinoma.

Platinum-resistant high-grade serous carcinoma (HGSC) is an incurable disease, so biomarkers that could help with timely treatment adjustments and personalized approach are extensively being sought. Tumor-derived extracellular vesicles (EVs) that can be isolated from ascites and blood of HGSC patients are such promising biomarkers. Epithelial cell adhesion molecule (EpCAM) expression is upregulated in most epithelium-derived tumors; however, studies on prognostic value of EpCAM overexpression in ovarian carcinoma have shown contradictory results. The aim of our study was to evaluate the potential of total and EpCAM-positive EVs as prognostic and predictive biomarkers for advanced HGSC. Flow cytometry was used to determine the concentration of total and EpCAM-positive EVs in paired pretreatment ascites and plasma samples of 37 patients with advanced HGSC who underwent different first-line therapy. We found that higher EpCAM-positive EVs concentration in ascites is associated with shorter progression-free survival (PFS) regardless of treatment strategy. We also found a strong correlation of EpCAM-positive EVs concentration between ascites and plasma. Our findings indicate that EpCAM-positive EVs in ascites of patients with advanced HGSC have the potential to serve as prognostic biomarkers for predicting early recurrence and thereby likelihood of more aggressive tumor biology and development of chemoresistance.

Characteristics of Extracellular Vesicles from a High-Grade Serous Ovarian Cancer Cell Line Derived from a Platinum-Resistant Patient as a Potential Tool for Aiding the Prediction of Responses to Chemotherapy.

Platinum-resistant high-grade serous ovarian cancer (HGSOC) is invariably a fatal disease. A central goal of ovarian cancer research is therefore to develop new strategies to overcome platinum resistance. Treatment is thus moving towards personalized therapy. However, validated molecular biomarkers that predict patients' risk of developing platinum resistance are still lacking. Extracellular vesicles (EVs) are promising candidate biomarkers. EpCAM-specific EVs are largely unexplored biomarkers for predicting chemoresistance. Using transmission electron microscopy, nanoparticle tracking analysis and flow cytometry, we compared the characteristics of EVs released from a cell line derived from a clinically confirmed cisplatin-resistant patient (OAW28) and EVs released from two cell lines from tumors sensitive to platinum-based chemotherapy (PEO1 and OAW42). We demonstrated that EVs released from the HGSOC cell line of chemoresistant patients exhibited greater size heterogeneity, a larger proportion of medium/large (>200 nm) Evs and a higher number of released EpCAM-positive EVs of different sizes, although the expression of EpCAM was predominant in EVs larger than 400 nm. We also found a strong positive correlation between the concentration of EpCAM-positive EVs and the expression of cellular EpCAM. These results may contribute to the prediction of platinum resistance in the future, although they should first be validated in clinical samples.

Application of Dynamic and Static Light Scattering for Size and Shape Characterization of Small Extracellular Nanoparticles in Plasma and Ascites of Ovarian Cancer Patients.

In parallel to medical treatment of ovarian cancer, methods for the early detection of cancer tumors are being sought. In this contribution, the use of non-invasive static (SLS) and dynamic light scattering (DLS) for the characterization of extracellular nanoparticles (ENPs) in body fluids of advanced serous ovarian cancer (OC) and benign gynecological pathology (BP) patients is demonstrated and critically evaluated. Samples of plasma and ascites (OC patients) or plasma, peritoneal fluid, and peritoneal washing (BP patients) were analyzed. The hydrodynamic radius (Rh) and the radius of gyration (Rg) of ENPs were calculated from the angular dependency of LS intensity for two ENP subpopulations. Rh and Rg of the predominant ENP population of OC patients were in the range 20-30 nm (diameter 40-60 nm). In thawed samples, larger particles (Rh mostly above 100 nm) were detected as well. The shape parameter ρ of both particle populations was around 1, which is typical for spherical particles with mass concentrated on the rim, as in vesicles. The Rh and Rg of ENPs in BP patients were larger than in OC patients, with ρ ≈ 1.1-2, implying a more elongated/distorted shape. These results show that SLS and DLS are promising methods for the analysis of morphological features of ENPs and have the potential to discriminate between OC and BP patients. However, further development of the methodology is required.

The contribution of copper efflux transporters ATP7A and ATP7B to chemoresistance and personalized medicine in ovarian cancer.

Ovarian cancer has the highest mortality rate among all gynecologic cancers, with most patients presenting with advanced stage tumors. About a third of patients do not respond to primary platinum-based chemotherapy treatment, and over time up to 80 % of others develop chemoresistance, rendering recurrent disease incurable. Moreover, according to latest EMSO-ESGO (European Society for Medical Oncology - European Society for Gynecological Oncology) consensus conference manuscript on ovarian cancer, there are currently no validated molecular predictive biomarkers for platinum resistance. Recent studies suggest that the copper efflux transporters ATP7A and ATP7B play an important role in platinum resistance. In addition, by exploring their role in mediating resistance, new pathways of platinum resistance emerge, such as lysosomal storage disorders, which might be explored in the future as a new target to circumvent platinum resistance. This review outlines a challenging clinical hurdle in ovarian cancer therapy due to platinum resistance, links between the essential trace element copper and cytotoxic platinum-based medicines, and enigmatic mechanisms of ATP7A and ATP7B mediating platinum resistance. It then presents clinical studies showing a significant association of ATP7A and ATP7B with response to cisplatin/carboplatin and prognosis. Based on the results of in vitro assays, disease-relevant animal models, and clinical studies to date, it may be concluded that APT7A and ATP7B deserve further development as predictive markers of platinum resistance in ovarian cancer. Both transporters could play a particularly important role in early estimation of therapy response to identify platinum-resistant tumors and to adjust the treatment of ovarian cancer patients accordingly.

Toxicological properties of Δ9-tetrahydrocannabinol and cannabidiol.

Cannabis sativa L. contains more than 100 phytocannabinoids that can interact with cannabinoid receptors CB1 and CB2. None of the cannabinoid receptor ligands is entirely CB1- or CB2-specific. The effects of cannabinoids therefore differ not just because of different potency at cannabinoid receptors but also because they can interact with other non-CB1 and non-CB2 targets, such as TRPV1, GPR55, and GPR119. The most studied phytocannabinoid is Δ9-tetrahydrocannabinol (THC). THC is a partial agonist at both cannabinoid receptors, but its psychotomimetic effect is produced primarily via activation of the CB1 receptor, which is strongly expressed in the central nervous system, with the noteworthy exception of the brain stem. Although acute cognitive and other effects of THC are well known, the risk of irreversible neuropsychological effects of THC needs further research to elucidate the association. Unlike THC, phytocannabinoid cannabidiol (CBD) does not appear to have psychotomimetic effects but may interact with some of the effects of THC if taken concomitantly. CBD administered orally has recently undergone well-controlled clinical trials to assess its safety in the treatment of paediatric epilepsy syndromes. Their findings point to increased transaminase levels as a safety issue that calls for postmarketing surveillance for liver toxicity. The aim of this review is to summarise what is known about acute and chronic toxicological effects of both compounds and address the gaps in knowledge about the safety of exogenous cannabinoids that are still open.

Potential of osteopontin in the management of epithelial ovarian cancer.

Background Osteopontin (sOPN) is a promising blood tumour marker for detecting epithelial ovarian cancer (EOC). However, other clinical uses of sOPN as a tumour marker in EOC are still lacking. Since sOPN concentrations in serum are not associated with those in ascites, we compared clinical value of sOPN concentrations in the two body fluids. Patients and methods The study included 31 women with advanced EOC and 34 women with benign gynaecological pathology. In the EOC group, serum for sOPN analysis was obtained preoperatively, after primary debulking surgery and after chemotherapy. In the control group, serum was obtained before and after surgery. Ascites and peritoneal fluid were obtained during surgery. sOPN concentrations were determined by flow cytometry bead-based assay. Results The sensitivity and specificity of sOPN in detecting EOC was 91.2% and 90.3% (cut-off = 47.4 ng/ml) in serum, and 96.8% and 100% (cut-off = 529.5 ng/ml) in ascites. Kaplan-Meier analysis showed a significant association between higher serum sOPN concentration and overall survival (p = 0.018) or progression free survival (p = 0.008). Higher ascites sOPN concentrations were associated with suboptimally debulked tumour and unresectable disease. Higher serum sOPN concentrations were associated with refractory disease or incomplete response to platinum-based chemotherapy. Conclusions The study showed that ascites sOPN level mirrors present disease and is superior to serum level for diagnostic purposes and surgical planning, although the end result of treatment is the response of the whole body in fighting the disease. The preoperative sOPN concentration in serum thus better reflects disease outcome.

Control values of ovarian cancer tumor markers and standardisation of a protocol for sampling peritoneal fluid and performing washing during laparoscopy.

BACKGROUND: Determination of the tumor marker concentration in peritoneal fluid (PF) may help to assess its potential to detect small concentration changes between benign ovarian pathology and early stage ovarian cancer. Peritoneal washing, which can also be obtained when PF is absent, is already included in the International Federation of Gynecology and Obstetrics (FIGO) staging classification for ovarian cancer but sampling has not yet been standardized. Since our aim was to evaluate the relationship between marker concentration in PF and washing, standardization of the sampling protocol was a prerequisite to ensure reliable results. METHODS: Thirty-three women with non-malignant pathology of the reproductive organs were included in the study. We used three promising tumor markers for evaluation of the marker concentration in local fluid: osteopontin (sOPN), splice variant 6 of sCD44 (sCD44-v6) and vascular cell adhesion molecule-1 (sVCAM-1). After aspiration of PF, washing of the uterus, ovaries and pelvic peritoneum was performed with saline solution. Patients were divided into two groups based on the solution volume: A-20 ml and B-50 ml. To determine the efficiency of washing in relation to solution volume, washing was repeated three times. Concentrations of markers in samples were determined using flow cytometry. RESULTS: Mean concentrations of markers were significantly higher (P <0.001) in PF than in the first washing. We demonstrated a significant positive correlation between marker concentrations in PF and first washing (sOPN: r = 0.447, P = 0.048; sCD44-v6: r = 0.660, P = 0.002; sVCAM-1: r = 0.526, P = 0.017). When using a smaller solution volume for washing, significantly higher (sVCAM-1: 2.5-fold, P = 0.021; sOPN: 3-fold, P = 0.024) or equal (sCD44-v6) mean concentrations of tumor markers were obtained. CONCLUSIONS: Our work demonstrates for the first time that concentrations of sOPN, sCD44-v6 and sVCAM-1 in PF correlate with peritoneal washing in women with non-malignant pathology of the reproductive organs. This indicates that, for selected tumor markers, washing can replace PF when PF is absent. A standardized protocol for sampling PF and performing washing during laparoscopy was established.

Circulating serum sVCAM-1 concentration in advanced ovarian cancer patients: correlation with concentration in ascites.

BACKGROUND: Vascular cell adhesion molecule-1 (VCAM-1) is associated with ovarian cancer progression but the origin of its soluble form (sVCAM-1) in serum is not well investigated. The purpose of this study was to elucidate whether the concentration of sVCAM-1 in serum correlates with the concentration in ascites, that represents local tumour environment, and with systemic inflammation, various clinicopathological characteristics, and patient outcome. PATIENTS AND METHODS: Thirty-six patients with advanced ovarian cancer were included in the study. Serum for sVCAM-1 analysis was obtained prior to surgery. Ascites samples were collected at the beginning of the operation. Clinical data were collected from patients' medical records. sVCAM-1 in samples was analysed by flow cytometric bead-based assay. The mean follow-up period was 11 months (range 0-23) from the time of surgery. RESULTS: Serum sVCAM-1 concentrations are positively correlated to ascites sVCAM-1 concentrations. There was a weakly positive correlation of serum sVCAM-1 with tumour size and no correlation with inflammatory tumour markers, FIGO stage or grade. Higher concentrations of sVCAM-1 were associated with poor disease outcome (death from ovarian cancer) in almost all cases before chemotherapy was started. CONCLUSIONS: This is the first study demonstrating that serum concentrations of sVCAM-1 in advanced ovarian cancer patients correlate with sVCAM-1 concentrations in ascites, thus expressing the biologic potential of malignant disease to metastasis, rather than systemic inflammation. Higher serum and ascites sVCAM-1 concentrations might have predictive potential for different biologic behaviour.

Analysis of cytotoxicity of melittin on adherent culture of human endothelial cells reveals advantage of fluorescence microscopy over flow cytometry and haemocytometer assay.

Melittin, from the honeybee venom, is a membrane active protein, whose cytotoxicity to human endothelial cells has not been described yet. In this work, we studied its time-dependent cytotoxicity on human umbilical vein endothelial cells (HUVECs). Since HUVECs grow in culture as adherent cells, suspension of cells is required before measuring cytotoxicity with a haemocytometer or flow cytometry. Therefore, we also tried to discover whether the result of cytotoxicity tests of melittin is influenced by the preparation of the cell suspension. For this purpose, we compared the results of haemocytometer-based trypan blue assay and flow cytometry using 7-aminoactinomycin D (7-AAD) with results of fluorescence microscopy using 7-AAD and 4', 6-diamidino-2-phenylindole (DAPI). Melittin over 60 min exposure evoked a rapid decline in the survival of HUVEC. After 60 min exposure to melittin, the phase contrast microscopy demonstrated massive necrosis in the remaining attached cells. Fluorescence microscopy detected both viable and non-viable cells in adequate proportions at all exposure times, whereas haemocytometer-based assay and flow cytometry highly underestimated the percentage of non-viable cells or even failed to detect any dead cells. Our data clearly indicate that the induction of large-scale damage to adherent endothelial cells by melittin results in a loss of the majority of necrotic cells during sample preparation for flow cytometry or a haemocytometer-based assay. In the case of adherent cell culture, therefore, fluorescence microscopy was shown to be a more appropriate method for quantitative analysis of cell death caused by a fast-acting cytolytic toxin such as melittin.

Molecular and kinetic characterization of histamine transport into adult rat cultured astrocytes.

Astrocytes have a key role in the clearance and inactivation of histamine in the adult central nervous system, but transporters which mediate histamine uptake into astrocytes have not been fully characterized. We therefore investigated the kinetic and molecular characteristics of histamine uptake into cultured adult rat astrocytes. [(3)H]-histamine was taken up by astrocytes in a temperature-, time- and concentration-dependent manner and was inhibited up to 60-70% by 1mM ouabain or by substitution of NaCl with choline chloride. Specific [(3)H]-histamine uptake, determined as the difference between transport at 37 and 4°C, displayed saturation kinetics with the apparent Michaelis-Menten constant (K(m)) of 141 and 101µM and the apparent maximal uptake rate (V(max)) of 22.5 and 17.8pmol/min/mg protein, as estimated from the Woolf and the Eadie-Hofstee plots, respectively. Since our data suggested the presence of a carrier-operated histamine uptake system, we assessed the possible involvement of the organic cation transporters (OCT) 1, 2 and 3, which have been previously described to play a role in histamine transport in the central nervous system. Low level mRNA expression of all OCT isoforms was detected, but in contrast to rat brain cortex homogenate, where OCT3 was the most prominently expressed OCT isoform, OCT2 mRNA was the predominant OCT species in cultured astrocytes. However, OCT inhibitors corticosterone and decynium 22 (D22) had no effect or only modestly reduced [(3)H]-histamine uptake. Thus, our data indicate that adult rat astrocytes possess an efficient high-capacity, low-affinity carrier-operated histamine uptake system, which does not seem to involve OCTs.

Mechanisms of changes in coronary arterial tone induced by bee venom toxins.

Our study elucidates some mechanisms of contractions or relaxations of isolated porcine left anterior descending coronary artery (LAD) induced by two peptides from the honeybee venom, melittin and apamin. Contractions or relaxations were measured on relaxed or precontracted arteries, respectively. Melittin at lower concentrations (0.1-10 microg/ml) induced transient relaxation, and contraction at higher concentrations (>or=7 microg/ml). The removing of the endothelium diminished the melittin-induced relaxation but did not affect the maximal contraction. The inhibition of prostaglandin and nitric oxide (NO) synthesis (by indomethacin and by N-omega-Nitro-l-arginine, respectively) and the use of K(+) channel inhibitors (apamin and charybdotoxin) showed that melittin evoked relaxation via an endothelium-dependent mechanism (NO production), and by activation of charybdotoxin-sensitive K(+) channels of smooth muscle. Apamin alone did not affect contraction or relaxation, but the inhibition of NO and prostanoid production revealed the involvement of apamin-sensitive K(+) channels of smooth muscle in melittin-induced relaxation. Our data show that melittin and apamin could affect contractility of porcine LAD at concentrations similar to those encountered in multiple honeybee stings in humans. Melittin could directly affect contractility of porcine LAD, whereas apamin acts as a modulator of the relaxant response to melittin.

Lacidipine decreases the honeybee venom-induced vasoconstriction of the isolated porcine coronary artery.

The venom of the honeybee Apis mellifera induces cardiovascular dysfunction. As its effects on coronary arteries have not yet been described, we studied the effects of the whole honeybee venom (non-volatile part) in the isolated porcine left anterior descending coronary artery (LAD) and the influence of L-type Ca2+ channel blocker, lacidipine, upon the venom effects in LAD. The venom produced concentration dependent contractions (7 - 70 µg/ml) of the porcine LAD; maximal effect of the venom was approximately the same as the effect of 30 mM KCl. Lacidipine concentration dependently (0.1-10 µM) and significantly (P ≤ 0.05) decreased the venom-induced vasoconstriction. The results indicate the involvement of L-type Ca2+ channels in coronary contraction, induced by bee venom.