G protein-membrane interactions I: Gαi1 myristoyl and palmitoyl modifications in protein-lipid interactions and its implications in membrane microdomain localization.

G proteins are fundamental elements in signal transduction involved in key cell responses, and their interactions with cell membrane lipids are critical events whose nature is not fully understood. Here, we have studied how the presence of myristic and palmitic acid moieties affects the interaction of the Gαi1 protein with model and biological membranes. For this purpose, we quantified the binding of purified Gαi1 protein and Gαi1 protein acylation mutants to model membranes, with lipid compositions that resemble different membrane microdomains. We observed that myristic and palmitic acids not only act as membrane anchors but also regulate Gαi1 subunit interaction with lipids characteristics of certain membrane microdomains. Thus, when the Gαi1 subunit contains both fatty acids it prefers raft-like lamellar membranes, with a high sphingomyelin and cholesterol content and little phosphatidylserine and phosphatidylethanolamine. By contrast, the myristoylated and non-palmitoylated Gαi1 subunit prefers other types of ordered lipid microdomains with higher phosphatidylserine content. These results in part explain the mobility of Gαi1 protein upon reversible palmitoylation to meet one or another type of signaling protein partner. These results also serve as an example of how membrane lipid alterations can change membrane signaling or how membrane lipid therapy can regulate the cell's physiology.

[Review of medical or combined treatment of local or locally advanced oesophageal cancer]. / A lokális vagy helyileg elõrehaladott nyelõcsõrák gyógyszeres és kombinált kezeléseinek áttekintése.

More than 800 oesophageal tumours are diagnosed each year in Hungary. The disease is characterized by high mortality. The curative treatment traditionally surgical, although the study results of recent decades pointed out that patient outcome can be improved with the proper application of radio- and chemotherapy. The diverse study designs, the low number of recruited patients and the sometimes conflicting results make the determination of optimal treatment difficult. The aim of this work is to facilitate the choice of treatment modality with the best outcome, especially with the view of medical oncologists. The treatment remains surgery for very early tumours (up to pT1b) and palliative therapy for tumours with metastasis. In other cases additional therapy, such as chemotherapy, radiotherapy, or their combinations, and targeted therapies, may result in improved survival. There are data mostly for neoadjuvant therapy because patients after surgery are rarely candidates for adjuvant therapy. Neoadjuvant chemotherapy may improve survival over surgery alone, but this improvement is more pronounced and supported by more evidence for neoadjuvant chemoradiotherapy (CRT). Certain results suggest that in selected cases after neoadjuvant CRT omission of surgery might not compromise survival, but the routine omission of surgery is not advised. However, the agent given concomitantly to radiotherapy may have importance. Besides cisplatin and fluorouracil other platinum derivatives (carboplatin, oxaliplatin) and taxanes (docetaxel, paclitaxel) can be used without compromising survival but with more favourable toxicity profile.

Membrane Assays to Characterize Interaction of Drugs with ABCB1.

ATP-binding cassette sub-family B member 1 (ABCB1) [P-glycoprotein (P-gp), multidrug resistance protein 1 (MDR1)] can affect the pharmacokinetics, safety, and efficacy of drugs making it important to identify compounds that interact with ABCB1. The ATPase assay and vesicular transport (VT) assay are membrane based assays that can be used to measure the interaction of compounds with ABCB1 at a lower cost and higher throughput compared to cellular-based assays and therefore can be used earlier in the drug development process. To that end, we tested compounds previously identified as ABCB1 substrates and inhibitors for interaction with ABCB1 using the ATPase and VT assays. All compounds tested interacted with ABCB1 in both the ATPase and VT assays. All compounds previously identified as ABCB1 substrates activated ABCB1-mediated ATPase activity in the ATPase assay. All compounds previously identified as ABCB1 inhibitors inhibited the ABCB1-mediated transport in the VT assay. Interestingly, six of the ten compounds previously identified as ABCB1 inhibitors activated the basal ATPase activity in activation assays suggesting that the compounds are substrates of ABCB1 but can inhibit ABCB1 in inhibition assays. Importantly, for ATPase activators the EC50 of activation correlated with the IC50 values from the VT assay showing that interactions of compounds with ABCB1 can be measured with similar levels of potency in either assay. For ATPase nonactivators the IC50 values from the ATPase inhibition and VT inhibition assay showed correlation. These results demonstrate the utility of membrane assays as tools to detect and rank order drug-transporter interactions.

Impact of SHMT1 polymorphism on the clinical outcome of patients with metastatic colorectal cancer treated with first-line FOLFIRI+bevacizumab.

The impact of thymidylate synthase (TYMS), methylenetetrahydrofolate reductase (MTHFR), and serine hydroxymethyltransferase 1 (SHMT1) gene polymorphisms and that of dihydropyrimidine dehydrogenase (DPD) enzyme activity, serum total homocysteine level, and estimated serum creatinine clearance on first-line 5-fluorouracil, leucovorin, irinotecan, and bevacizumab (FOLFIRI+bevacizumab) regimen efficacy in metastatic colorectal cancer patients was investigated. DNA was extracted from peripheral blood mononuclear cells. Genotyping was performed for TYMS 5'UTR variable number tandem repeat, TYMS 3'UTR ins/del, MTHFR C677T, and SHMT1 C1420T polymorphisms. The DPD activity of peripheral blood mononuclear cells was also determined. The univariate and multivariate analyses demonstrated that the SHMT1 1420T allele was associated with better response (P=0.025) and longer progression-free survival (PFS) (P=0.00004) and overall survival (OS) (P=0.034). Grade ≥2 hypertension was also an independent prognostic factor of longer progression-free survival and OS. Bevacizumab-related hypertension might be a predictive marker of treatment efficacy (P=0.0002 for OS) in the case of wild (CC) SHMT1 1420 genotypes only.

Mouse Bsep ATPase assay: a nonradioactive tool for assessment of the cholestatic potential of drugs.

The mouse ortholog of the human bile salt export pump (BSEP) transporter was expressed in a baculovirus-infected insect cell (Sf9) system to study the effect of membrane cholesterol content on the transporter function. The transport activity of cholesterol-loaded mouse Bsep-HAM-Sf9 vesicles was determined in a vesicular transport assay with taurochenodeoxycholate (TCDC), a known BSEP substrate. Mouse Bsep transports TCDC at a high rate that can be sensitively detected in the ATPase assay. Cholesterol upload of the Sf9 membrane potentiates both TCDC transport and TCDC-stimulated ATPase activities. Inhibitory effect of BSEP interactors on probe substrate transport was tested in both vesicular transport and ATPase assays using cholesterol-loaded membrane vesicles. A good rank order correlation was found between IC(50) values measured in TCDC-stimulated mBsep ATPase assay and in the human BSEP vesicular transport assay utilizing taurocholate (TC) as probe substrate. This upgraded form of the mouse Bsep-HAM ATPase assay is a user friendly, sensitive, nonradioactive method for early high-throughput screening of drugs with BSEP-related cholestatic potential. It may complement the human BSEP-mediated taurocholate vesicular transport inhibition assay.

Effect of membrane cholesterol on BSEP/Bsep activity: species specificity studies for substrates and inhibitors.

The efflux transporter responsible for the canalicular elimination of bile salts from the hepatocytes is the bile salt export pump (BSEP, ABCB11). Absence or inhibition of this transporter leads to bile salt retention in the hepatocyte and in turn can lead to cholestatic liver disease. We expressed the BSEP/Bsep protein from three species (human, rat, and mouse) in a baculovirus-infected Sf9 system. Vesicles prepared from these cells were used to evaluate bile salt transport of four conjugated bile salts. Because the Sf9 system contains less membrane cholesterol than the liver canalicular membrane, the effect of added cholesterol on the kinetics of BSEP/Bsep-mediated bile salt transport was also investigated. Cholesterol treatment increased the V(max) values in all the species, with the most pronounced effect observed in the rat transporter. In contrast, K(m) values, with the exception of glycochenodeoxycholate, remained largely unchanged. The species-specific bile salt transport inhibition potential of three compounds known to cause clinical cholestasis was investigated in vesicles containing BSEP/Bsep. Troglitazone and glibenclamide inhibited the BSEP/Bsep-mediated transport of different bile salts with similar affinities, whereas the potential of cyclosporine A to inhibit bile salt transport showed species- and bile salt-specific variations. In conclusion, the cholesterol-loaded Sf9 vesicles overexpressing BSEP/Bsep seem to be a useful system for the identification of potential cholestatic compounds and can also be used for the investigation of species specificity. We observed greater differences in IC(50) values for inhibitors than in K(m) values for substrates between species.

[Changes in renal function during bisphosphonate treatment of breast cancer patients]. / A biszfoszfonát-kezelés során észlelheto vesefunkció-változás vizsgálata emlorákos betegeinknél.

Renal function aberrations during bisphosphonate treatment is a well-known phenomenon. In our retrospective study we examined renal functions of 97 breast cancer patients with bone metastasis during their first year of bisphosphonate treatment i.e. (1) frequency of initial renal function alterations; (2) frequency of decreasing renal function during bisphosphonate treatment; (3) the connection between the laboratory findings and the renal function parameters measured at the beginning of bisphosphonate treatment. At the beginning of bisphosphonate treatment we found a surprisingly high rate (26.80%) of decreased creatinine clearance calculated by the Cockcroft-Gault formula. Decreased creatinine clearance at least once during bisphosphonate treatment has been found in 32.99% of the patients, and in 13.4% of the patients with decreased renal function parameters before bisphosphonates it remained decreased during the one-year period. Expected normal renal function is prognosticated by the renal function parameters and serum calcium level measured before starting bisphosphonate treatment. However, we could not demonstrate any connection between decreasing renal function and either the route of administration or the generation or type of bisphosphonates or the previous use of platinum compounds. Our analysis confirms the necessity of monitoring renal function before and during bisphosphonate treatment, and it is advisable to calculate the creatinine clearance in the upper quarter of the normal range of creatinine levels. In case of decreased renal function, change to a less nephrotoxic bisphosphonate or discontinuing the treatment is suggested. While our results are at variance with the published literature, the above-mentioned questions should be examined in a prospective trial.

Schizotypal Traits and the Dark Triad From an Ecological Perspective: A Nonclinical Sample Study.

The Dark Triad is a collection of socially aversive personality traits, namely subclinical psychopathy, Machiavellianism, and subclinical narcissism. These deviant traits, however, contribute to the success of individuals with dark personality traits. Therefore, Dark Triad traits can be conceived as pseudopathologies. Schizotypal traits have also been studies from the perspective of behavioral adaptations. In this study, we investigated whether schizotypal traits were associated with the Dark Triad traits and how schizotypal symptoms can be considered as parts of dark interpersonal strategies that contribute to the individual success of people with dark personality traits. A sample of 277 university students (198 females and 79 males; Mage = 20.64; SDage = 2.15) were recruited to fill out the Short Dark Triad and the Schizotypal Personality Questionnaire-Brief Revised. Statistical analyses revealed that Machiavellianism was positively associated with restricted emotional and social life. Narcissism was negatively associated with interpersonal problems. Psychopathy was positively associated with distorted perceptions/cognitions and disorganization. Results of the study are discussed within a behavioral ecology framework. This perspective emphasizes the adaptive values connected to schizotypal personality traits. We further discuss how these adaptive traits fit into strategies of individuals with Dark Triad traits, and how these schizotypal traits might restrict or further promote their individual success.

Predictive Value of Early Skin Rash in Cetuximab-Based Therapy of Advanced Biliary Tract Cancer.

Randomized trials in advanced biliary tract cancer (BTC) did not show benefit of cetuximab addition over chemotherapy. This is probably due to the lack of predictive biomarkers. The aim of this study was to explore possible predictive factors. Between 2009 and 2014, 57 patients were treated in 3-week cycles with cetuximab (250 mg/m2/week, loading dose: 400 mg/m2), gemcitabine (1000 mg/m2 on day 1 and 8), and capecitabine (1300 mg/m2/day on days 1-14). The objective response rate (ORR), progression-free (PFS) and overall survival (OS) and the adverse events (AEs) were evaluated. An exploratory analysis was performed to find possible predictive factors on clinicopathological characteristics, routine laboratory parameters and early AEs, which occurred within 2 months from the beginning of treatment. The ORR was 21%. The median PFS and OS were 34 (95% CI: 24-40) and 54 (43-67) weeks, respectively. The most frequent AEs were skin toxicities. In univariate analysis performance status, previous stent implantation, thrombocyte count at the start of therapy, early neutropenia and skin rash statistically significantly influenced the ORR, PFS and/or OS. In multivariate Cox regression analysis only normal thrombocyte count at treatment start and early acneiform rash were independent markers of longer survival. In patients showing early skin rash compared to the others the median PFS was 39 vs. 13 weeks and the median OS was 67 vs. 26 weeks, respectively. It is suggested that early skin rash can be used as a biomarker to select patients who would benefit from the treatment with cetuximab plus chemotherapy.

[In vitro methods suitable for the prediction of drug and ABC transporter, especially ABCG2 interactions]. / ABC transzporterek-különös tekintettel az ABCG2-re--és gyógyszerjelölt molekulák kölcsönhatásának predikciójára alkalmas in vitro módszerek.

ABCG2, a transporter of the ATP-binding cassette family, is known to play a prominent role in the absorption, distribution, metabolism, and excretion of xenobiotics. Drug-transporter interactions are commonly screened by in vitro systems using transfected insect and/or human cell lines.

Interaction of ivermectin with multidrug resistance proteins (MRP1, 2 and 3).

Ivermectin is a potent antiparasitic drug from macrocyclic lactone (ML) family, which interacts with the ABC multidrug transporter P-glycoprotein (Pgp). We studied the interactions of ivermectin with the multidrug resistance proteins (MRPs) by combining cellular and subcellular approaches. The inhibition by ivermectin of substrate transport was measured in A549 cells (calcein or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, BCECF) and in HL60-MRP1 (calcein). Ivermectin induced calcein and BCECF retention in A549 cells (IC(50) at 1 and 2.5microM, respectively) and inhibited calcein efflux in HL60-MRP1 (IC(50)=3.8microM). The action of ivermectin on the transporters ATPase activity was followed on membranes from Sf9 cells overexpressing human Pgp, MRP1, 2 or 3. Ivermectin inhibited the Pgp, MRP1, 2 and 3 ATPase activities after stimulation by their respective activators. Ivermectin showed a rather good affinity for MRPs, mainly MRP1, in the micromolar range, although it was lower than that for Pgp. The transport of BODIPY-ivermectin was followed in cells overexpressing selectively Pgp or MRP1. In both cell lines, inhibition of the transporter activity induced intracellular retention of BODIPY-ivermectin. Our data revealed the specific interaction of ivermectin with MRP proteins, and its transport by MRP1. Although Pgp has been considered until now as the sole active transporter for this drug, the MRPs should be taken into account for the transport of ivermectin across cell membrane, modulating its disposition in addition to Pgp. This could be of importance for optimizing clinical efficacy of ML-based antiparasitic treatments. This offers fair perspectives for the use of ivermectin or non-toxic derivatives as multidrug resistance-reversing agents.

[Assessment of suicidal behaviour in general practice]. / Az öngyilkos viselkedés gyakorisága és jellegzetességei a családorvosi gyakorlatban.

AIM: To assess the prevalence of suicidal behavior (wish to die, suicidal thoughts, suicide attempts) and to determine the characteristics of suicide attempters in primary care, including screening for major mental disorders. METHOD: A Hungarian urban general practitioner's district with 1248 inhabitants was screened for suicidal behavior as well as for major mental disorders. All the patients (n=382) who visited their general practitioner within a two-week period were asked to participate. 277 patients completed the Prime-MD questionnaire, an easy-to-use diagnostic instrument developed for general practitioners to recognize the most common psychiatric disorders, like depressive (major depressive disorder, minor depressive disorder), anxiety (panic disorder, generalized anxiety disorder), somatoform, eating and alcohol related disorders. Detailed data about suicidal thoughts and attempts were also collected by the structured questions of MINI-Plus diagnostic interview. RESULTS: Prevalence of suicide attempts in primary care was 2.9%. 9% of the patients had either suicidal thoughts or suicide attempts in the previous month. Suicidal patients were more ready to use psychotropic drugs, they assessed their health status more poorly, and had more mental symptoms than the control group (non-suicidal patients). 60% of suicidal patients and 11.5% of the investigated population had a current depressive episode. Beside depressive symptoms, anxiety disorders and alcohol problems were also more common among suicidal patients. The rate of previous psychiatric treatments was also higher in suicidal patients, who generally visited their general practitioners less frequently than non-suicidal patients. According to multivariate logistic regression, suicidal patients are more ready to take antidepressants, they tend to have more previous psychiatric treatments and suicidal attempts, and they visit their general practitioners less frequently and have a current depressive episode. CONCLUSION: Suicidal behavior and mental disorders are frequent in primary care. Since almost every tenth patient visiting their general practitioner has suicidal thoughts or depressive or anxiety disorder, the recognition of suicide risk and mental disorders is very important in primary care. As for preventing suicides, the diagnosing and treating of mental disorders -especially affective disorders- are very important for general practitioners. In addition to pharmacotherapy, psychotherapies are also important in treating patients in crisis situations, or with suicidal thoughts or depressive disorder. The modified Prime-MD questionnaire can be an effective, easy-to-use method in the hand of the general practitioners to identify suicidal risk and to recognize the most common mental disorders in the average population.

Synergistic effect of Avemar on proinflammatory cytokine production and Ras-mediated cell activation.

Macrophages activated by lipopolysaccharide and/or phorbol esters exhibited high sensitivity to Avemar, a fermented wheat germ extract. Avemar synergized with lipopolysaccharide and PMA in the induction of the transcription of cytokine genes and release of inflammatory cytokines. At higher concentrations the preparation had a significant negative effect on the proliferation and survival of activated myeloid cell types. Avemar treatment induced the synthesis of ICAM-1 and synergized with the ICAM-inducing effect of TNF, but had no effect on VCAM-1 expression on microvascular endothelial cells. The effect of Avemar on signaling pathways, which are involved in cell activation was studied on HeLa cells as a model system. Avemar treatment increased the activity of stress kinases in a concentration-dependent way, resulting in the activation of AP-1 transcription factor. NF-kappa B-sensitive reporters were also activated by Avemar; in contrast, no effect of the preparation was observed on PKA-sensitive signaling pathways.

Tumor cells expressing membrane-bound tumor necrosis factor activate macrophages and have a compromised growth in immunosuppressed and immunodeficient mice.

Tumor necrosis factor (TNF)-alpha producing tumors as vaccines were demonstrated to induce a therapeutic anti-tumor immune response, but their clinical use is limited by the toxicity of soluble TNF. We investigated the growth characteristics and immunomodulatory properties of HeLa cells producing an uncleavable transmembrane form of TNF (preTNF). The growth of the transformed tumors was compromised in both immunosuppressed and severe combined immunodeficient mice; no signs of TNF toxicity were detected. Macrophages co-cultured with the transformed cells showed increased phagocytosis and cytokine production, indicating that activated macrophages may be the mediators of the anti-tumor effect. preTNF producing tumor cells are promising safe anti-tumor vaccine candidates.

[Effect of imatinib treatment of gastrointestinal stromal tumors]. / Az imatinibkezelés hatása gastrointestinalis stroma eredetú daganatokban.

INTRODUCTION: Advanced malignant gastrointestinal stromal tumours are practically resistant to further radio- or chemotherapy. These tumours are characterized by the presence of C-KIT (a transmembrane tyrosin kinase) mutation which can be specified by CD117 expression. Imatinib (2-fenilaminopirimidine) is a selective inhibitor of the mutated C-KIT. AIM: The purpose of our study was to determine the potential antitumour effect of imatinib in patients with gastrointestinal stroma tumour patients. MATERIALS AND METHODS: An open, non-randomized trial was performed involving 38 patients each of which had received/metastatic disease associated with CD117 positivity. Consecutively daily doses of 400-600 mg imatinib was administered orally to the patients. The evaluation was carried out on 37 patients in a form of an interim analysis. RESULTS: After a 3-18 months observation period 1 complete, 19 partial remissions and 10 static diseases could be registered (78%), in association of only grade 1-2 toxicity. CONCLUSIONS: The imatinib treatment improved the quality of life of the patients with gastrointestinal stroma tumour and their life expectancy became considerably prolonged. Further follow-up of the patients as well as design of a prospective, randomized trial on a larger patient material is urgently needed.

Successful treatment of solitary bone metastasis of non-small cell lung cancer with bevacizumab and hyperthermia.

Non-small cell lung cancer (NSCLC) represents 85 % of all malignant lung cancers. In metastatic disease the principle goal of palliative therapy is to prolong survival with least toxicity and best patients' quality of life. Bevacizumab (BEV) has been approved as first line treatment in combination with platinum based chemotherapy and maintenance therapy in NSCLC. BEV can be added safely to several chemotherapeutic agents, however there is no data on coadministration with thermotherapy. Even in localized disease no robust evidence exists about the beneficial effect of loco-regional thermotherapy on overall survival, but it might be used successfully in symptom palliation. In this article a successful co-administration of BEV and hyperthermia is reported in a patient with monolocalized bone metastasis from previously operated NSCLC. This case suggests that electrohyperthermia can probably be incorporated in palliative therapy added not only to radiotherapy or chemotherapy but also to anti-angiogenic BEV treatment.

The expressions of ABCC4 and ABCG2 xenobiotic transporters in human keratinocytes are proliferation-related.

Xenobiotic transporters of the ATP-binding cassette (ABC) protein superfamily play important roles in maintaining the biochemical barrier of various tissues, but their precise functions in the skin are not yet known. Screening of the expressions of the known xenobiotic transporter genes in two in vitro keratinocyte differentiation models revealed that the ABCC4 and ABCG2 transporters are highly expressed in proliferating keratinocytes, their expressions decreasing along with differentiation. Abrogation of the ABCC4 and ABCG2 protein functions by siRNA-mediated silencing and chemical inhibition did not affect the proliferation of HaCaT cells. In contrast, disruption of the ABCG2 function had no effect on normal human epidermal keratinocyte proliferation, while the inhibition of ABCC-type transporters by probenecid resulted in a striking decrease in the proliferation of the cells. These results indicate that, besides their possible therapy-modulating effects, xenobiotic transporters may contribute significantly to other keratinocyte functions, such as cell proliferation.

Specific inhibition of the ABCG2 transporter could improve the efficacy of photodynamic therapy.

Photodynamic therapy is based on the selective accumulation of a photosensitizer in tumors, followed by destruction of the target tissue by a light source. Protoporphyrin IX, a well-known photosensitizer, was recently reported as an endogenous substrate for the multidrug transporter ABCG2. We investigated the role of ABCG2 protein in the porphyrin extrusion ability of keratinocytes, with regard to the impact of the specific inhibition of ABCG2 by a non-toxic fumitremorgin C analog, Ko-134, on photodynamic therapy efficacy. We studied the level of porphyrin accumulation in response to delta-aminolevulinic acid pretreatment in proliferating and highly differentiated HaCaT keratinocytes. An in vitro model of photodynamic therapy on HaCaT cells was established with a therapeutically approved narrow-bandwidth red-light source. The porphyrin extrusion ability of HaCaT cells proved to correlate with their ABCG2 expression which was higher in proliferating cells than in differentiated cells. Moreover, the specific inhibition of ABCG2 by Ko-134 enhanced the sensitivity of keratinocytes to photodynamic therapy in vitro. These results suggest that ABCG2 may serve as a target molecule via which to improve the photodynamic therapy of skin lesions: its inhibition by the non-toxic Ko-134 is a promising therapeutic modality.

Ivermectin interacts with human ABCG2.

Ivermectin is an antiparasitic drug frequently administered to humans. It has a limited brain exposure that is attributed to the efflux activity of ABCB1/Abcb1. ABCG2/Abcg2 is also a major transporter present in most pharmacologically important barriers. However, interaction of ivermectin with Abcg2 shows species specificity and in many studies was confounded by the masking effect of ABCB1/Abcb1. In this study using cellular and membrane assays we show that ivermectin displays a high-affinity interaction with human ABCG2 with IC(50) values in the 1-1.5 µM range. This interaction may have implications in human ABCG2-mediated drug-drug interactions of ivermectin.