Antitumour prodrug development using cytochrome P450 (CYP) mediated activation

An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme, or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed, including identification of prodrugs activated by tumour-specific polymorphic CYPs, use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxic tumour regions. The last approach is best exemplified by AQ4N, a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite, AQ4 (T50/80 K-m = 26.7 mu M, V-max = 0.43 mu M/mg protein/min; RIF-1 K-m = 33.5 mu M, V-max = 0.42 mu M/mg protein/min) via AQM, a mono-N-oxide intermediate (T50/80 K-m = 37.5 mu M; V-max = 1.4 mu M/mg protein/min; RIF-1 K-m = 37.5 mu M; V-max = 1.2 mu M/mg protein/min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3-fold over a 12 h period, after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely, cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air, despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4, but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (K-m = 4 mu M, V-max = 3.5 pmol/mg protein/min) and normal kidney (K-m = 4 mu M, V-max = 4.0 pmol/mg protein/min), both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species, the process requires low oxygen conditions. Hence, AQ4N metabolism should be restricted to hypoxic tumour cells. The isoform selectivity of AQ4N reduction, in addition to its air sensitivity, indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active-site-bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.

Retinal pericytes control expression of nitric oxide synthase and endothelin-1 in microvascular endothelial cells

Pericytes are known to communicate with endothelial cells by direct contact and by releasing cytokines such as TGF-beta. There is also strong evidence that pericytes act as regulators of endothelial cell proliferation and differentiation. We have investigated the effect of pericyte-conditioned medium (PCM) on proliferation of human microvascular endothelial cells in vitro, together with the expression of the vasoregulatory molecules, constitutive and inducible nitric oxide synthases (ecNOS and iNOS), and endothelin-1 (ET-1). Expression was measured at the mRNA level using semiquantitative RT-PCR for all three genes and at the protein level for ecNOS and iNOS using Western blotting. Growth curves for HMECs showed that PCM inhibits proliferation, eventually leading to cell death. Exposure to PCM repressed iNOS mRNA expression fivefold after 6 h. A similar, though delayed, reduction in protein levels was observed. ecNOS mRNA was slightly induced at 6 h, though there was no significant change in ecNOS protein. By contrast, ET-1 mRNA was induced 2.3-fold after 6 h exposure to PCM. We conclude that pericytes release a soluble factor or factors that are potent inhibitors of endothelial cell growth and promote vasoconstriction by up-regulating endothelin-1 and down-regulating iNOS. (C) 2000 Academic Press.

Male morph predicts investment in larval immune function in the dung beetle, Onthophagus taurus

Investment in immunity is costly, so that resource-based trade-offs between immunity and sexually selected ornaments might be expected. The amount of resources that an individual can invest in each trait will be limited by the total resources available to them. It would therefore be informative to investigate how investment in immune function changes during growth or production of the sexual trait as resources are diverted to it. Using the dung beetle, Onthophagus taurus, which displays both sexual and male dimorphism in horn size, we examined changes in one measure of immune function, phenoloxidase (PO) activity, in the hemolymph of larvae prior to and during horn growth. We found that PO levels differed between small- and large-horned males throughout the final instar prior to the point where investment in horn growth was taking place. PO levels in females were intermediate to the 2 male morphs. These differences could not be accounted for by differences in condition, measured as hemolymph protein levels and weight. We suggest that the observed differences might be associated with sex- and morph-specific variation in juvenile hormone levels.

Tissue microarray study for classification of breast tumors

Clinical and pathological heterogeneity of breast cancer hinders selection of appropriate treatment for individual cases. Molecular profiling at gene or protein levels may elucidate the biological variance of tumors and provide a new classification system that correlates better with biological, clinical and prognostic parameters. We studied the immunohistochemical profile of a panel of seven important biomarkers using tumor tissue arrays. The tumor samples were then classified with a monothetic (binary variables) clustering algorithm. Two distinct groups of tumors are characterized by the estrogen receptor (ER) status and tumor grade (p = 0.0026). Four biomarkers, c-erbB2, Cox-2, p53 and VEGF, were significantly overexpressed in tumors with the ER-negative (ER-) phenotype. Eight subsets of tumors were further identified according to the expression status of VEGF, c-erbB2 and p53. The malignant potential of the ER-/VEGF+ subgroup was associated with the strong correlations of Cox-2 and c-erb132 with VEGF. Our results indicate that this molecular classification system, based on the statistical analysis of immunohistochemical profiling, is a useful approach for tumor grouping. Some of these subgroups have a relative genetic homogeneity that may allow further study of specific genetically-controlled metabolic pathways. This approach may hold great promise in rationalizing the application of different therapeutic strategies for different subgroups of breast tumors. (C) 2003 Elsevier Inc. All rights reserved.

Regulation of cyclin D1 by the BRCA1-BARD1 complex

Background BRCA1 and cyclin D₁ are both essential for normal breast development and mutation or aberration of their expression is associated with breast cancer [1,2]. Cyclin D₁ is best known as a G₁ cyclin where it regulates the G₁ to S phase transition by acting as a rate-limiting subunit of CDK4/6 kinase activity. More recently, however, Stacey has demonstrated that cyclin D₁ levels in G₂/M determine whether a cell continues to proliferate or exits the cell cycle [3]. The majority of BRCA1 in the cell is bound to BARD1 through their N-terminal RING domains. Heterodimerization is essential for the stability and correct localization of the complex and confers ubiquitin ligase activity to BRCA1. The importance of the ligase activity of BRCA1 to breast cancer development is inferred from the fact that N-terminal diseaseassociated mutations are proposed to reduce ligase activity [4]. Methods Protein–protein interactions were demonstrated using yeast-two-hybrid and coimmunoprecipitation. Protein levels were altered through overexpression, siRNA and antisense technology. The effect of proteasome inhibitors and cycloheximide treatment was also examined. Results We initially identified cyclin D₁ as a binding partner of BARD1 in a yeast-two-hybrid screen and defined the minimal binding region as the N-terminus of BARD1. This interaction was confirmed in vivo by coimmunoprecipitation. The N-terminus of BARD1 also binds BRCA1 and imparts ubiquitin ligase activity to the complex. Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Traditionally polyubiquitin chains linked through lysine 48 target proteins for degradation by the 26 S proteasome. We have demonstrated that cyclin D₁ protein levels are inversely related to BRCA1 and BARD1 levels in several model systems. Furthermore, regulation of cyclin D1 levels occurs through a post-transcriptional mechanism and requires the ligase activity of BRCA1. Interestingly, this phenomenon is cell-cycle regulated, occurring in G₂/M. Conclusion We propose that cyclin D₁ is a potential substrate for BRCA1 ubiquitination and that this targets cyclin D₁ for proteasomal-mediated degradation. Future work will focus on ascertaining the functional consequence of cyclin D₁ regulation by the BRCA1–BARD1 complex; in particular, the impact of BRCA1, mediated through regulation of cyclin D₁, on the proliferation versus differentiation decision.

PARP inhibition induces BAX/BAK-independent synthetic lethality of BRCA1-deficient non-small cell lung cancer

Evasion of apoptosis contributes to both tumourigenesis and drug resistance in non-small cell lung carcinoma (NSCLC). The pro-apoptotic BCL-2 family proteins BAX and BAK are critical regulators of mitochondrial apoptosis. New strategies for targeting NSCLC in a mitochondria-independent manner should bypass this common mechanism of apoptosis block. BRCA1 mutation frequency in lung cancer is low; however, decreased BRCA1 mRNA and protein expression levels have been reported in a significant proportion of lung adenocarcinomas. BRCA1 mutation/deficiency confers a defect in homologous recombination DNA repair that has been exploited by synthetic lethality through inhibition of PARP (PARPi) in breast and ovarian cells; however, it is not known whether this same synthetic lethal mechanism exists in NSCLC cells. Additionally, it is unknown whether the mitochondrial apoptotic pathway is required for BRCA1/PARPi-mediated synthetic lethality. Here we demonstrate that silencing of BRCA1 expression by RNA interference sensitizes NSCLC cells to PARP inhibition. Importantly, this sensitivity was not attenuated in cells harbouring mitochondrial apoptosis block induced by co-depletion of BAX and BAK. Furthermore, we demonstrate that BRCA1 inhibition cannot override platinum resistance, which is often mediated by loss of mitochondrial apoptosis signalling, but can still sensitize to PARP inhibition. Finally we demonstrate the existence of a BRCA1-deficient subgroup (11-19%) of NSCLC patients by analysing BRCA1 protein levels using immunohistochemistry in two independent primary NSCLC cohorts. Taken together, the existence of BRCA1-immunodeficient NSCLC suggests that this molecular subgroup could be effectively targeted by PARP inhibitors in the clinic and that PARP inhibitors could be used for the treatment of BRCA1-immunodeficient, platinum-resistant tumours. Copyright (C) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Spin-orbit effects on photoionization-produced alignment of the Ar+ 3p(4)(P-3)4p levels

We have determined photoionization spectra of Ar with excitation of the 3p(4)(P-3)4p states emphasizing the effects of two different classes of Ar+ spin-orbit interactions. The spin-orbit splitting of each Ar+ state adequately describes the resonant excitation of the quartet states of Ar+, and gives Ar photoionization cross sections with excitation of the 3p4(3P)4p P-2(3/2)o and P-4(5/2)o levels of Ar+ in sufficiently good agreement with experiment to identify the observed resonances and to estimate the excitation strengths. In addition, we demonstrate the importance of spin-orbit induced mixing of different Ar+ LS-coupled states for observables such as the alignment of the 3p(4)(P-3)4p P-4(5/2)o level and the excitation of Rydberg series converging to the 3p(4)(P-3)4p S-2(o) and S-4(o) thresholds.

Diabetes downregulates large-conductance Ca2+-activated potassium beta 1 channel subunit in retinal arteriolar smooth muscle

Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca(2+) release events (Ca(2+)-sparks), trigger the activation of large-conductance Ca(2+)-activated K(+)(BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca(2+)](i) release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca(2+)-sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BKalpha subunit was unchanged. The Ca(2+)-sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BKbeta1 subunit confers Ca(2+)-sensitivity to BK channel complexes and both transcript and protein levels for BKbeta1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BKbeta1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension.

Changes in lymphocyte subsets, interleukin 2, and soluble interleukin 2 receptor in old and very old age

In this study, the changes in some of the cellular components of the immune system and the activity of the cytokine interleukin 2, important for immune activation and lymphocyte proliferation, were measured in a large cross-sectional study of all age groups including octogenarian and nonagenarian subjects. In 206 apparently well community-living subjects, the absolute lymphocyte count and T and B cell numbers fell a little in old and very old subjects. Within the T cell compartment, helper/inducer CD4+ T cells, together with their subsets identified as 'naive' (CD4+/CD45RA+) and 'memory' (CD4+/CD45RO+) cells, also showed a decline with increased age. The suppressor/cytotoxic CD8+ subset showed no age-related change. The levels of the cytokine interleukin 2 were very low in octogenarian and nonagenarian subjects, while the soluble interleukin 2 receptor levels increased with increasing age. The interleukin 2 levels were associated with number and percentage of the 'memory' (CD4+/CD45RO+) subset of T cells which mediates the host response to previously met antigens. Since the interleukin 2 values were very low in the oldest groups and were associated with a reduced 'memory' (CD4+/CD45RO+) compartment, this suggests a possible mechanism of why the very elderly subject is more susceptible to morbidity and mortality from infectious or other agents.

Assessment of a multiplexing high throughput immunochemical SPR biosensor in measuring multiple proteins on a single biosensor chip

Multiplexed immunochemical detection platforms offer the potential to decrease labour demands, increase sample throughput and decrease overall time to result. A prototype four channel multiplexed high throughput surface plasmon resonance biosensor was previously developed, for the detection of food related contaminants. A study focused on determining the instruments performance characteristics was undertaken. This was followed by the development of a multiplexed assay for four high molecular weight proteins. The protein levels were simultaneously evaluated in serum samples of 10-week-old veal calves (n = 24) using multiple sample preparation methods. Each of the biosensor's four channels were shown to be independent of one another and produced multiplexed within run repeatability (n = 6) ranging from 2.0 to 6.7%CV, for the four tested proteins, whilst between run reproducibility (n = 4) ranged from 1.5 to 8.9%CV. Four calibration curves were successfully constructed before serum sample preparation was optimised for each protein. Multiplexed concentration analysis was successfully performed on four channels revealing that each proteins concentration was consistent across the twenty-four tested animals. Signal reproducibility (n > 19) on a further long term study revealed coefficient of variation ranging from 1.1% to 7.3% and showed that the multiplexed assay was stable for at least 480 cycles. These findings indicate that the performance characteristics fall within the range of previously published data for singleplex optical biosensors and that the multiplexing biosensor is fit-for-purpose for simultaneous concentration analysis in many different types of applications such as the multiplexed detection of markers of growth-promoter abuse and multiplexed detection of residues of concern in food safety. © 2013 Elsevier B.V.

Activation of protease calpain by oxidized and glycated LDL increases the degradation of endothelial nitric oxide synthase

Oxidation and glycation of low-density lipoprotein (LDL) promote vascular injury in diabetes; however, the mechanisms underlying this effect remain poorly defined. The present study was conducted to determine the effects of 'heavily oxidized' glycated LDL (HOG-LDL) on endothelial nitric oxide synthase (eNOS) function. Exposure of bovine aortic endothelial cells with HOG-LDL reduced eNOS protein levels in a concentration- and time-dependent manner, without altering eNOS mRNA levels. Reduced eNOS protein levels were accompanied by an increase in intracellular Ca(2+), augmented production of reactive oxygen species (ROS) and induction of Ca(2+)-dependent calpain activity. Neither eNOS reduction nor any of these other effects were observed in cells exposed to native LDL. Reduction of intracellular Ca(2+) levels abolished eNOS reduction by HOG-LDL, as did pharmacological or genetic through calcium channel blockers or calcium chelator BAPTA or inhibition of NAD(P)H oxidase (with apocynin) or inhibition of calpain (calpain 1-specific siRNA). Consistent with these results, HOG-LDL impaired acetylcholine-induced endothelium-dependent vasorelaxation of isolated mouse aortas, and pharmacological inhibition of calpain prevented this effect. HOG-LDL may impair endothelial function by inducing calpain-mediated eNOS degradation in a ROS- and Ca(2+)-dependent manner.

Simvastatin decreases the level of heparin-binding protein in patients with acute lung injury

Heparin-binding protein is released by neutrophils during inflammation and disrupts the integrity of the alveolar and capillary endothelial barrier implicated in the development of acute lung injury and systemic organ failure. We sought to investigate whether oral administration of simvastatin to patients with acute lung injury reduces plasma heparin-binding protein levels and improves intensive care unit outcome.

A highly potent and specific MET therapeutic protein antagonist with both ligand-dependent and ligand-independent activity

Activation of the MET oncogenic pathway has been implicated in the development of aggressive cancers that are difficult to treat with current chemotherapies. This has led to an increased interest in developing novel therapies that target the MET pathway. However, most existing drug modalities are confounded by their inability to specifically target and/or antagonize this pathway. Anticalins, a novel class of monovalent small biologics, are hypothesized to be "fit for purpose" for developing highly specific and potent antagonists of cancer pathways. Here, we describe a monovalent full MET antagonist, PRS-110, displaying efficacy in both ligand-dependent and ligand-independent cancer models. PRS-110 specifically binds to MET with high affinity and blocks hepatocyte growth factor (HGF) interaction. Phosphorylation assays show that PRS-110 efficiently inhibits HGF-mediated signaling of MET receptor and has no agonistic activity. Confocal microscopy shows that PRS-110 results in the trafficking of MET to late endosomal/lysosomal compartments in the absence of HGF. In vivo administration of PRS-110 resulted in significant, dose-dependent tumor growth inhibition in ligand-dependent (U87-MG) and ligand-independent (Caki-1) xenograft models. Analysis of MET protein levels on xenograft biopsy samples show a significant reduction in total MET following therapy with PRS-110 supporting its ligand-independent mechanism of action. Taken together, these data indicate that the MET inhibitor PRS-110 has potentially broad anticancer activity that warrants evaluation in patients.

Impaired activities of antioxidant enzymes elicit endothelial dysfunction in spontaneous hypertensive rats despite enhanced vascular nitric oxide generation

Objective: Enhanced oxidative stress is involved in mediating the endothelial dysfunction associated with hypertension. The aim of this study was to investigate the relative contributions of pro-oxidant and anti-oxidant enzymes to the pathogenesis of endothelial dysfunction in genetic hypertension. Methods: Dilator responses to endothelium-dependent and endothelium-independent agents such as acetylcholine (ACh) and sodium nitroprusside were measured in the thoracic aortas of 28-week-old spontaneously hypertensive rats (SHR) and their matched normotensive counterparts, Wistar Kyoto rats (WKY). The activity and expression (mRNA and protein levels) of endothelial nitric oxide synthase (eNOS), p22-phox, a membrane-bound component of NAD(P)H oxidase, and antioxidant enzymes, namely, superoxide dismutases (CuZn- and Mn-SOD), catalase and glutathione peroxidase (GPx), were also investigated in aortic rings. Results: Relaxant responses to ACh were attenuated in phenylephrine-precontracted SHR aortic rings, despite a 2-fold increase in eNOS expression and activity. Although the activity and/or expression of SODs, NAD(P)H oxidase (p22-phox) and GPx were elevated in SHR aorta, catalase activity and expression remained unchanged compared to WKY. Pretreatment of SHR aortic rings with the inhibitor of xanthine oxidase, allopurinol, and the inhibitor of cyclooxygenase, indomethacin, significantly potentiated ACh-induced relaxation. Pretreatment of SHR rings with catalase and Tiron, a superoxide anion (O) scavenger, increased the relaxant responses to the levels observed in WKY rings whereas pyrogallol, a O -generator, abolished relaxant responses to ACh. Conclusion: These data demonstrate that dysregulation of several enzymes, resulting in oxidative stress, contributes to the pathogenesis of endothelial dysfunction in SHR and indicate that the antioxidant enzyme catalase is of particular importance in the reversal of this defect. © 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Aflatoxin exposure is inversely associated with IGF1 and IGFBP3 levels in vitro and in Kenyan schoolchildren

SCOPE: This study explores the relationship between aflatoxin and the insulin-like growth factor (IGF) axis and its potential effect on child growth.

METHODS AND RESULTS: One hundred and ninety-nine Kenyan schoolchildren were studied for aflatoxin-albumin adduct (AF-alb), IGF1 and IGF-binding protein-3 (IGFBP3) levels using ELISA. AF-alb was inversely associated with IGF1 and IGFBP3 (p < 0.05). Both IGF1 and IGFBP3 were significantly associated with child height and weight (p < 0.01). Children in the highest tertile of AF-alb exposure (>198.5 pg/mg) were shorter than children in the lowest tertile (<74.5 pg/mg), after adjusting for confounders (p = 0.043). Path analysis suggested that IGF1 levels explained ∼16% of the impact of aflatoxin exposure on child height (p = 0.052). To further investigate this putative mechanistic pathway, HHL-16 liver cells (where HHL-16 is human hepatocyte line 16 cells) were treated with aflatoxin B1 (0.5, 5 and 20 μg/mL for 24-48 h). IGF1 and IGFBP3 gene expression measured by quantitative PCR and protein in culture media showed a significant down-regulation of IGF genes and reduced IGF protein levels.

CONCLUSION: Aflatoxin treatment resulted in a significant decrease in IGF gene and protein expression in vitro. IGF protein levels were also lower in children with the highest levels of AFB-alb adducts. The data suggest that aflatoxin-induced changes in IGF protein levels could contribute to growth impairment where aflatoxin exposure is high.