Prognostic stratification of patients with advanced renal cell carcinoma treated with sunitinib: comparison with the Memorial Sloan-Kettering prognostic factors model

<p>Abstract</p> <p>Background</p> <p>The treatment paradigm in advanced renal cell carcinoma (RCC) has changed in the recent years. Sunitinib has been established as a new standard for first-line therapy. We studied the prognostic significance of baseline characteristics and we compared the risk stratification with the established Memorial Sloan Kettering Cancer Center (MSKCC) model.</p> <p>Methods</p> <p>This is a retrospective analysis of patients treated in six Greek Oncology Units of HECOG. Inclusion criteria were: advanced renal cell carcinoma not amenable to surgery and treatment with Sunitinib. Previous cytokine therapy but no targeted agents were allowed. Overall survival (OS) was the major end point. Significance of prognostic factors was evaluated with multivariate cox regression analysis. A model was developed to stratify patients according to risk.</p> <p>Results</p> <p>One hundred and nine patients were included. Median follow up has been 15.8 months and median OS 17.1 months (95% CI: 13.7-20.6). Time from diagnosis to the start of Sunitinib (<= 12 months vs. >12 months, p = 0.001), number of metastatic sites (1 vs. >1, p = 0.003) and performance status (PS) (<= 1 vs >1, p = 0.001) were independently associated with OS. Stratification in two risk groups ("low" risk: 0 or 1 risk factors; "high" risk: 2 or 3 risk factors) resulted in distinctly different OS (median not reached [NR] vs. 10.8 [95% confidence interval (CI): 8.3-13.3], p < 0.001). The application of the MSKCC risk criteria resulted in stratification into 3 groups (low and intermediate and poor risk) with distinctly different prognosis underlying its validity. Nevertheless, MSKCC model did not show an improved prognostic performance over the model developed by this analysis.</p> <p>Conclusions</p> <p>Studies on risk stratification of patients with advanced RCC treated with targeted therapies are warranted. Our results suggest that a simpler than the MSKCC model can be developed. Such models should be further validated.</p

Synthetic, enzyme kinetic, and protein crystallographic studies of C-[béta]-D-glucopyranosyl pyrroles and imidazoles reveal and explain low nanomolar inhibition of human liver glycogen phosphorylase

LBK

The role of CXC-chemokine receptor CXCR2 and suppressor of cytokine signaling-3 (SOCS-3) in renal cell carcinoma

BACKGROUND: Chemokine receptor signaling pathways are implicated in the pathobiology of renal cell carcinoma (RCC). However, the clinical relevance of CXCR2 receptor, mediating the effects of all angiogenic chemokines, remains unclear. SOCS (suppressor of cytokine signaling)-3 is a negative regulator of cytokine-driven responses, contributing to interferon-α resistance commonly used to treat advanced RCC with limited information regarding its expression in RCC. METHODS: In this study, CXCR2 and SOCS-3 were immunohistochemically investigated in 118 RCC cases in relation to interleukin (IL)-6 and (IL)-8, their downstream transducer phosphorylated (p-)STAT-3, and VEGF expression, being further correlated with microvascular characteristics, clinicopathological features and survival. In 30 cases relationships with hypoxia-inducible factors, i.e. HIF-1a, p53 and NF-κΒ (p65/RelA) were also examined. Validation of immunohistochemistry and further investigation of downstream transducers, p-JAK2 and p-c-Jun were evaluated by Western immunoblotting in 5 cases. RESULTS: Both CXCR2 and IL-8 were expressed by the neoplastic cells their levels being interrelated. CXCR2 strongly correlated with the levels of HIF-1a, p53 and p65/RelA in the neoplastic cells. Although SOCS-3 was simultaneously expressed with p-STAT-3, its levels tended to show an inverse relationship with p-JAK-2 and p-c-Jun in Western blots and were positively correlated with HIF-1a, p53 and p65/p65/RelA expression. Neither CXCR2 nor SOCS-3 correlated with the extent of microvascular network. IL-8 and CXCR2 expression was associated with high grade, advanced stage and the presence/number of metastases but only CXCR2 adversely affected survival in univariate analysis. Elevated SOCS-3 expression was associated with progression, the presence/number of metastasis and shortened survival in both univariate and multivariate analysis. CONCLUSIONS: Our findings implicate SOCS-3 overexpression in RCC metastasis and biologic aggressiveness advocating its therapeutic targeting. IL-8/CXCR2 signaling also contributes to the metastatic phenotype of RCC cells but appears of lesser prognostic utility. Both CXCR2 and SOCS-3 appear to be related to transcription factors induced under hypoxia

The druggability of the ATP binding site of glycogen phosphorylase kinase probed by coumarin analogues

Glycogen phosphorylase kinase (PhK) converts by phosphorylation, the inactive glycogen phosphorylase (GPb) into active GPa in the glycogenolytic pathway. It is a complex enzyme comprising of the catalytic (γ) and three regulatory subunits (α, β, δ) forming a hexadecamer with stoichiometry (αβγδ)4. Several studies have indicated PhK as a promising target for the development of antihyperglycemics as its inhibition blocks glycogenolysis in liver and a potential therapeutic target for cancer against pathological angiogenesis and tumor progression. The identification of compounds that inhibit the kinase through their direct binding to its catalytic site is an effective approach to identify bioactive molecules of therapeutic significance. Towards this, the structure of the N-terminal kinase domain (residues 1–298) of the catalytic γ subunit of PhK (PhKγtrnc) has been determined by X-ray crystallography while staurosporine and indirubin analogues have been characterized as potent inhibitors targeting the ATP binding site. In this study, a series of 38 synthetic analogues of naturally occurring coumarins were screened for inhibition of PhKγtrnc, in vitro, using a photometric assay. The IC50 values of the two most potent compounds were determined for PhKγtrnc and the pharmacologically relevant target, human liver isoform (PHKG2A). Their cellular efficacy and toxicity in HepG2 cells were further assessed ex vivo. Docking experiments and the structural comparison with previously described inhibitors reveal the binding mode of the coumarin scaffold at a no hinge region of the ATP site of PhK and the role of a conserved β3-Lys in binding. The experimental findings provide structural insights with implications to the kinase targeting and drug design

Metabolic acidosis may be as protective as hypercapnic acidosis in an ex-vivo model of severe ventilator-induced lung injury: a pilot study

<p>Abstract</p> <p>Background</p> <p>There is mounting experimental evidence that hypercapnic acidosis protects against lung injury. However, it is unclear if acidosis <it>per se </it>rather than hypercapnia is responsible for this beneficial effect. Therefore, we sought to evaluate the effects of hypercapnic (respiratory) versus normocapnic (metabolic) acidosis in an ex vivo model of ventilator-induced lung injury (VILI).</p> <p>Methods</p> <p>Sixty New Zealand white rabbit ventilated and perfused heart-lung preparations were used. Six study groups were evaluated. Respiratory acidosis (RA), metabolic acidosis (MA) and normocapnic-normoxic (Control - C) groups were randomized into high and low peak inspiratory pressures, respectively. Each preparation was ventilated for 1 hour according to a standardized ventilation protocol. Lung injury was evaluated by means of pulmonary edema formation (weight gain), changes in ultrafiltration coefficient, mean pulmonary artery pressure changes as well as histological alterations.</p> <p>Results</p> <p>HPC group gained significantly greater weight than HPMA, HPRA and all three LP groups (P = 0.024), while no difference was observed between HPMA and HPRA groups regarding weight gain. Neither group differ on ultrafiltration coefficient. HPMA group experienced greater increase in the mean pulmonary artery pressure at 20 min (P = 0.0276) and 40 min (P = 0.0012) compared with all other groups. Histology scores were significantly greater in HP vs. LP groups (p < 0.001).</p> <p>Conclusions</p> <p>In our experimental VILI model both metabolic acidosis and hypercapnic acidosis attenuated VILI-induced pulmonary edema implying a mechanism other than possible synergistic effects of acidosis with CO2 for VILI attenuation.</p

A multidisciplinary study of 3-(β-d-glucopyranosyl)-5-substituted-1,2,4-triazole derivatives as glycogen phosphorylase inhibitors: Computation, synthesis, crystallography and kinetics reveal new potent inhibitors

3-(β-d-Glucopyranosyl)-5-substituted-1,2,4-triazoles have been revealed as an effective scaffold for the development of potent glycogen phosphorylase (GP) inhibitors but with the potency very sensitive to the nature of the alkyl/aryl 5-substituent (Kun et al., Eur. J. Med. Chem. 2014, 76, 567). For a training set of these ligands, quantum mechanics-polarized ligand docking (QM-PLD) demonstrated good potential to identify larger differences in potencies (predictive index PI = 0.82) and potent inhibitors with K 's < 10 μM (AU-ROC = 0.86). Accordingly, in silico screening of 2335 new analogues exploiting the ZINC docking database was performed and nine predicted candidates selected for synthesis. The compounds were prepared in O-perbenzoylated forms by either ring transformation of 5-β-d-glucopyranosyl tetrazole by N-benzyl-arenecarboximidoyl chlorides, ring closure of C-(β-d-glucopyranosyl)formamidrazone with aroyl chlorides, or that of N-(β-d-glucopyranosylcarbonyl)arenethiocarboxamides by hydrazine, followed by deprotections. Kinetics experiments against rabbit muscle GPb (rmGPb) and human liver GPa (hlGPa) revealed five compounds as potent low μM inhibitors with three of these on the submicromolar range for rmGPa. X-ray crystallographic analysis sourced the potency to a combination of favorable interactions from the 1,2,4-triazole and suitable aryl substituents in the GP catalytic site. The compounds also revealed promising calculated pharmacokinetic profiles. [Abstract copyright: Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Biochemical studies of enzymes in glycogen metabolism

Type 2 diabetes mellitus (T2D) is a serious threat to global health constituting a ma-jor socioeconomic problem. Currently, T2D drugs have many adverse side effects, including severe hypoglycemia. Glycogen metabolism is controlled by a number of enzymes in a complex and dynamic manner. Glycogen phosphorylase (GP) and gly-cogen phosphorylase kinase (PhK) are two enzymes that play a critical role in glyco-gen metabolism and in glucose homeostasis. In the framework of this PhD thesis, within the overarching goal to discover new therapeutic agents for T2D, studies on the structure-activity relationship of these two enzymes were performed. GP is a val-idated target for the discovery of new antihyperglycaemic agents for patients with T2D. In the first part, the inhibitory effect of polyphenolic extracts from 19 samples from byproducts of the industrial juicing process of pomegranate (Punica Granatum) and 23 samples from plants of the Rosaceae family was assessed. The most potent inhibitory extracts showed IC50 values below 10 μg / mL. Employing the affinity crys-tallography method, the most bioactive molecules of the extracts were determined. For the pomegranate samples, the most bioactive molecules were ellagic and chlorogenic acid (found bound at the inhibition and active site of the enzyme, re-spectively). For the Rosaceae samples, the most bioactive molecules were glucose, gallic, ellagic and chlorogenic acid found bound at the active, inhibitory, and the new allosteric site, respectively. The binding of chlorogenic acid to GP has not been ob-served thus far, and structural analysis of the GP-chlorogenic acid complex in the crystal revealed the structural basis of its inhibitory activity. Recent studies have highlighted the usefulness of plant extracts in the production of bio-functional food products and the results of this study constitute an important step towards the design of new biofunctional products for T2D patients. Phk phosphorylates the inactive phosphorylase b converting it to the active phosphorylase a. The enzyme is one of the most complex protein kinases, consisting of four subunits (α, β, γ and δ) with heterodimer stoichiometry (αβγδ)4 and a molecular weight of 1.3 MDa. In the sec-ond part of the dissertation, biochemical and biophysical studies of PhK subunits γ and α were performed. Expression studies of human γ truncated subunit of PhK were performed in E. coli strains and then the protein was purified by liquid chroma-tography methods and crystallization tests were performed. The α subunit of the human muscle PhK was also studied. Initially, gene optimization studies were per-formed to express it in the E.coli bacterial system. The amplified gene was inserted into the pETM-11 vector and to enhance its solubility, the molecular chaperone Trig-ger Factor was used. This was followed by chromatographic purification, and bio-physical studies to determine the oligomerization state (DLS), as well as experiments to determine the folding and its secondary structure elements (CD), and final the sample was analyzed for its homogeneity and purity through negative stain EM.Ο Σακχαρώδης διαβήτης τύπου 2 (ΣΔ2) είναι μια σοβαρή απειλή για την παγκόσμια υγεία, ενώ αποτελεί ένα τεράστιο κοινωνικό και οικονομικό πρόβλημα για τις αναπτυγμένες και αναπτυσσόμενες χώρες. Τα φάρμακα που σχετίζονται με τη θεραπεία της διαταραχής συνδέονται με πολλές αρνητικές παρενέργειες μεταξύ των οποίων και ο κίνδυνος της υπογλυκαιμίας. Ο μεταβολισμός του γλυκογόνου ελέγχεται από ένα πλήθος ενζύμων με πολύπλοκο και δυναμικό τρόπο. Η φωσφορυλάση του γλυκογόνου (GP) και η κινάση της φωσφορυλάσης του γλυκογόνου (PhK) είναι δύο ένζυμα με κρίσιμο ρόλο στο μεταβολισμό του γλυκογόνου και στη ρύθμιση των επιπέδων γλυκόζης στο αίμα. Στο πλαίσιο της διδακτορικής διατριβής μελετήθηκε η σχέση δομής-δράσης των δύο αυτών ενζύμων με απώτερο στόχο την εκμετάλλευσης της παραγόμενης γνώσης στην ανακάλυψη νέων θεραπευτικών μέσων για το ΣΔ2. Η GP αποτελεί έναν επικυρωμένο στόχο για την ανακάλυψη αντιυπεργλυκαιμικών παραγόντων για ασθενείς με ΣΔ2. Στο πρώτο μέρος της διατριβής αξιολογήθηκε η ανασταλτική δράση στη GP, πολυφαινολικών εκχυλισμάτων 19 παραπροϊόντων βιομηχανικής χυμοποίησης ροδιού (Punica Granatum) και 23 φυτών της οικογένειας Rosaceae. Τα πλέον ισχυρά ανασταλτικά εκχυλίσματα εμφάνισαν τιμές IC50 χαμηλότερες από 10 μg/mL. Με τη μέθοδο της κρυσταλλογραφίας συγγένειας προσδιορίστηκαν τα πλέον βιοδραστικά μόρια των εκχυλισμάτων. Για τα εκχυλίσματα από τα παραπροϊόντα βιομηχανικής χυμοποίησης ροδιού τα πλέον βιοδραστικά μόρια ήταν το ελλαγικό και ο χλωρογενικό οξύ που βρέθηκαν συνδεδεμένα στο κέντρο αναστολής και στο καταλυτικό κέντρο του ενζύμου, αντίστοιχα. Για τα εκχυλίσματα των Rosaceae τα πλέον βιοδραστικά μόρια ήταν η γλυκόζη, το ελλαγικό, το γαλλικό και το χλωρογενικό οξύ τα οποία βρέθηκαν συνδεδεμένα στο καταλυτικό κέντρο, στο κέντρο αναστολής και στο νέο αλλοστερικό κέντρο, αντίστοιχα. Η σύνδεση του χλωρογενικού οξέος στην GP παρατηρήθηκε για πρώτη φορά και η δομική ανάλυση του συμπλόκου GP-χλωρογενικού οξέος στον κρύσταλλο, απεκάλυψε την δομική βάση της ανασταλτικής του δραστικότητας. Πρόσφατες μελέτες έχουν αναδείξει τη χρησιμότητα φυτικών εκχυλισμάτων στην παραγωγή βιολειτουργικών προϊόντων δια-τροφής και τα αποτελέσματα των μελετών της παρούσας διατριβής αποτελούν ένα σημαντικό βήμα για το σχεδιασμό νεών βιολειτουργικών προϊόντων για ασθενείς με ΣΔ2. Η PhK ενεργοποιεί τη φωσφορυλάση b και τη μετατρέπει σε φωσφορυλάση a. Το ένζυμο είναι μία από τις πιο πολύπλοκες πρωτεϊνικές κινάσες, αποτελούμενη από τέσσερις υπομονάδες (α, β, γ και δ) με στοιχειομετρία δεκαεξαμερούς (αβγδ)4 και μοριακό βάρος 1,3 MDa. Στο δεύτερο μέρος της διατριβής, πραγματοποιήθηκαν βιοχημικές και βιοφυσικές μελέτες των υπομονάδων γ και α της PhK. Διεξήχθησαν μελέτες έκφρασης της κολοβωμένης ανθρώπινης ηπατικής υπομονάδας γ της PhK, σε βακτηριακά στελέχη E. coli και στη συνέχεια απομόνωση της μέσω χρωματογραφικού καθαρισμού. Ακολούθησαν δοκιμές κρυστάλλωσης της πρωτεΐνης. Η δεύτερη υπομονάδα που μελετήθηκε ήταν η α από την ανθρώπινη μυϊκή PhK. Αρχικά έγιναν μελέτες βελτιστοποίησης του γονιδίου με σκοπό την έκφρασή της στο βακτηριακό σύστημα E.coli. Το γονίδιο που ενισχύθηκε εισάχθηκε στο φορέα pETM-11. Για την επιτυχή έκφρασή της στο διαλυτό κλάσμα, χρησιμοποιήθηκε ο μοριακός συνοδός Trigger Factor. Στη συνέχεια ακολούθησε χρωματογραφικός καθαρισμός της, και βιοφυσικές μελέτες με σκοπό τον προσδιορισμό της κατάστασης ολιγομερισμού (πειράματα DLS), πειράματα προσδιορισμού του βαθμού αναδίπλωσης και ποσοτικοποίησης των στοιχείων της δευτεροταγούς δομής της (πειράματα CD), ενώ τέλος έγινε ανάλυση της ομοιογένειας και της καθαρότητας του δείγματος μέσω πειραμάτων αρνητικής χρώσης ΕΜ (negative stain EM)

The Effect of Alpha-Blocker Treatment on Bladder Hypoxia Inducible Factor-1 Alpha Regulation during Lower Urinary Tract Obstruction

Aims: To determine whether alpha 1-blocker treatment, in chronic bladder outlet obstruction (BOO), influences bladder tissue ischemia. Materials and Methods: This prospective study included 60 patients with BOO, of which 40 were under alpha 1-blocker medication and 20 without treatment. Patients underwent transurethral resection of the prostate (TURF) or suprapubic prostatectomy (SPP). Ten patients with non-muscle invasive bladder cancer underwent transurethral resection of the bladder tumor and served as the control group. Tissue specimens were immunohistochemically stained for hypoxia inducible factor-1 alpha (HIF-1 alpha). Results: Bladder tissue from obstructed subjects showed high immunoreactivity to HIF-1 alpha. The specimens from the control group, showed no or weak, mainly cytoplasmic immunoreactivity to HIF-1 alpha. Patients under alpha -blocker treatment did not differ in the number of HIF-1 alpha positive cells compared to subjects with no treatment (median number 86.8 [20-150] and 88.6 [0-175], respectively) (p &gt; 0.05). The lowest bladder pressure at which H1F-1 alpha was up regulated, was detected at detnisor pressure Qmax (PdetQmax) = 60 cm H2O. Conclusions: Treatment with alpha-blockers in obstructed patients considered as non-responders, does not result in HIF-1 alpha down regulation, thus bladder continues to be under chronic stress

The effect of alpha-blocker treatment on bladder hypoxia inducible factor-1 alpha regulation during lower urinary tract obstruction

AIMS: To determine whether &#945;1-blocker treatment, in chronic bladder outlet obstruction (BOO), influences bladder tissue ischemia. MATERIALS AND METHODS: This prospective study included 60 patients with BOO, of which 40 were under &#945;1-blocker medication and 20 without treatment. Patients underwent transurethral resection of the prostate (TURP) or suprapubic prostatectomy (SPP). Ten patients with non-muscle invasive bladder cancer underwent transurethral resection of the bladder tumor and served as the control group. Tissue specimens were immunohistochemically stained for hypoxia inducible factor-1&#945; (HIF-1&#945;). RESULTS: Bladder tissue from obstructed subjects showed high immunoreactivity to HIF-1&#945;. The specimens from the control group, showed no or weak, mainly cytoplasmic immunoreactivity to HIF-1&#945;. Patients under &#945; -blocker treatment did not differ in the number of HIF-1&#945; positive cells compared to subjects with no treatment (median number 86.8 [20-150] and 88.6 [0-175], respectively) (p > 0.05). The lowest bladder pressure at which HIF-1&#945; was up regulated, was detected at detrusor pressure Qmax (PdetQmax) = 60 cm H2O. CONCLUSIONS: Treatment with &#945;-blockers in obstructed patients considered as non-responders, does not result in HIF-1&#945; down regulation, thus bladder continues to be under chronic stress