Katarina Černe

Cell-Penetrating Mimics of Agonist-Activated G-Protein Coupled Receptors

Cell-penetrating peptides have proven themselves as valuable vectors for intracellular delivery. Relatively little is known about the frequency of cell-penetrating sequences in native proteins and their functional role. By computational comparison of peptide sequences, we recently predicted that intracellular loops of G-protein coupled receptors (GPCR) have high probability for occurrence of cell-penetrating motifs. Since the loops are also receptor and G-protein interaction sites, we postulated that the short cell-penetrating peptides, derived from GPCR, when applied extracellularly can pass the membrane and modulate G-protein activity similarly to parent receptor proteins. Two model systems were analyzed as proofs of the principle. A peptide based on the C-terminal intracellular sequence of the rat angiotensin receptor (AT1AR) is shown to internalize into live cells and elicit blood vessel contraction even in the presence of AT1AR antagonist Sar1-Thr8-angiotensin II. The peptide interacts with the same selectivity towards G-protein subtypes as agonist-activated AT1AR and blockade of phospholipase C abolishes its effect. Another cell-penetrating peptide, G53-2 derived from human glucagon-like peptide receptor (GLP-1R) is shown to induce insulin release from isolated pancreatic islets. The mechanism was again found to be shared with the original GLP-1R, namely G11-mediated inositol 1,4,5-triphosphate release pathway. These data reveal a novel possibility to mimic the effects of signalling transmembrane proteins by application of shorter peptide fragments.

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.

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

Abstract 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.

Expression of organic cation transporter 3 (SLC22A3) and plasma membrane monoamine transporter (SLC29A4) in human umbilical vein endothelial cells and their relevance for histamine uptake

Background Increased plasma histamine levels lead to pathological events. Endothelial cells actively participate in histamine clearance by promoting its uptake via yet unidentified carriers, thus limiting histamine effects. The organic cation transporter 3 (OCT3) and plasma membrane monoamine transporter (PMAT) are the two most prominent transporters for endogenous monoamines. OCT3 and PMAT show Na/K-ATPase independency. Both are highly sensitive to inhibition by the isocyanine compound, decynium-22. However, OCT3 is highly sensitive to corticosteron, whereas PMAT is not. We showed in the past that decynium-22 inhibits histamine uptake in cultured human umbilical vascular endothelial cells (HUVEC). In the present study we identified the expression of OCT3 and PMAT in freshly isolated and cultured HUVEC as well as some characteristics of histamine uptake in cultured HUVEC such as ouabain and corticosteron sensitivity.

Soluble osteopontin concentrations in serum and ascites of women with advanced serous ovarian cancer

Background Despite advances in surgery and combination chemotherapy, ovarian cancer is first in terms of death rates of gynaecological malignancies. More than 90% of ovarian cancers arise from surface epithelium and the serous histological subtype is the most commonly diagnosed epithelial ovarian carcinoma. Extensive seeding of the peritoneal cavity by tumour cells is often associated with ascites, particularly in advanced, high-grade serous carcinomas. Currently CA-125 is the most widely used biomarker in the evaluation and management of women with epithelial ovarian cancer. However, in approximately 15% of these patients CA-125 is not indicative of disease status or progression. Therefore, an alternative tumour marker would be useful. Osteopontin is a secreted, integrin-binding glykophosphoprotein which is overexpressed in ovarian cancer cells and thus may serve as a serum biomarker. By combining the data from blood and fluid from the proximity of the tumour we might be more likely to discover a protein biomarker secreted from the tumour rather than deriving from another part of the body.

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

Abstract 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.

Chapter Eight - Implications of Microvesicle and Cell Surface Protein Shedding for Biomarker Studies, Cancerogenesis, and Therapeutic Target Discovery in Ovarian Cancer

Abstract Ovarian cancer is one of the most aggressive and lethal cancers in women. There is currently no accurate noninvasive diagnostic test for ovarian cancer; most patients are diagnosed at an advanced stage, presenting with peritoneal metastasis and ascites. Although ovarian cancer patients often respond initially to chemotherapy, recurrence is almost always accompanied by chemoresistance. The identification of proteins that are abundantly and predominantly expressed in ovarian cancer is therefore relevant to diagnosis, tumor imaging, and targeted therapies. Proteins with high secretion rates or proteins that are extensively cleaved from the cell surface, specifically from tumor cells, are a potential source of circulating biomarkers for ovarian cancer. The process of surface protein shedding as observed in ascites-derived tumor cells from ovarian cancer patients is also relevant to tumor invasion and metastasis, as exemplified by the role of cadherin. Additionally, proteins, as well as lipids, DNA, and RNA, can reach the blood in microvesicles that are shed from tumor cells. A roughly fivefold increase in their blood concentration is reported in ovarian cancer patients. Tumor-derived vesicles are fully equipped to facilitate the escape of tumor cells from immune surveillance. At the same time, they are involved in the establishment of an optimal environment for metastatic tumor cells. On the basis of the role of tumor microvesicles in tumor progression, another door has been opened to a new ovarian cancer therapy. This review addresses the relevance of secretion and shedding of microvesicles and membrane surface protein from tumor cells for biomarker discovery, cancerogenesis, and new therapies in ovarian cancer.