Uptake and metabolism of ß-apo-8'-carotenal, ß-apo-10'-carotenal, and ß-apo-13-carotenone in Caco-2 cells.

ß-Apocarotenoids are eccentric cleavage products of carotenoids formed by chemical and enzymatic oxidations. They occur in foods containing carotenoids and thus might be directly absorbed from the diet. However, there is limited information about their intestinal absorption. The present research examined the kinetics of uptake and metabolism of ß-apocarotenoids. Caco-2 cells were grown on 6-well plastic plates until a differentiated cell monolayer was achieved. ß-Apocarotenoids were prepared in Tween 40 micelles, delivered to differentiated cells in serum-free medium, and incubated at 37°C for up to 8 h. There was rapid uptake of ß-apo-8'-carotenal into cells, and ß-apo-8'-carotenal was largely converted to ß-apo-8'-carotenoic acid and a minor metabolite that we identified as 5,6-epoxy-ß-apo-8'-carotenol. There was also rapid uptake of ß-apo-10'-carotenal into cells, and ß-apo-10'-carotenal was converted into a major metabolite identified as 5,6-epoxy-ß-apo-10'-carotenol and a minor metabolite that is likely a dihydro-ß-apo-10'-carotenol. Finally, there was rapid cellular uptake of ß-apo-13-carotenone, and this compound was extensively degraded. These results suggest that dietary ß-apocarotenals are extensively metabolized in intestinal cells via pathways similar to the metabolism of retinal. Thus, they are likely not absorbed directly from the diet.

Apocarotenoids: Emerging Roles in Mammals.

Apocarotenoids are cleavage products of C40 isoprenoid pigments, named carotenoids, synthesized exclusively by plants and microorganisms. The colors of flowers and fruits and the photosynthetic process are examples of the biological properties conferred by carotenoids to these organisms. Mammals do not synthesize carotenoids but obtain them from foods of plant origin. Apocarotenoids are generated upon enzymatic and nonenzymatic cleavage of the parent compounds both in plants and in the tissues of mammals that have ingested carotenoid-containing foods. The best-characterized apocarotenoids are retinoids (vitamin A and its derivatives), generated upon central oxidative cleavage of provitamin A carotenoids, mainly ß-carotene. In addition to the well-known biological actions of vitamin A, it is becoming apparent that nonretinoid apocarotenoids also have the potential to regulate a broad spectrum of critical cellular functions, thus influencing mammalian health. This review discusses the current knowledge about the generation and biological activities of nonretinoid apocarotenoids in mammals.

Analysis of Electronic Cigarette-Related Injury Presenting to U.S. Emergency Departments, 2008-2017.

BACKGROUND: Several types of injuries associated with electronic cigarette malfunction have been reported in the literature since their introduction to the U.S. market in 2007. The traumatic consequences of electronic nicotine delivery system (ENDS) malfunction remain an under-researched topic. OBJECTIVES: Using information from a national database of emergency department (ED) visits, we sought to characterize the nature and frequency of ENDS injuries over a 10-year study period. METHODS: Archived information from the National Electronic Injury Surveillance System was accessed for the years 2008 to 2017. Incidents related to ENDS-related trauma were manually identified. Data extracted included patient demographics, injury type and location, and patient disposition. RESULTS: A total of 49 incidents were recorded during the years 2008 to 2017, including 18 cases in 2017, 25 cases in 2016, five cases in 2015, and one case in 2013. There were no identified ED visits for an e-cigarette-related burn or explosion prior to 2013. Using statistical weights, the estimated annual national incidence is 835 cases. Most of the injuries were thermal burns. The primary location of injury was in the lower extremity, followed by the upper extremity and hand. CONCLUSIONS: Our study demonstrates a significant increase in the number of ENDS-related injuries over the study period, particularly in males under the age of 45 years. This rise mirrors the growth of the ENDS market and this trend can be expected to continue. As the use of ENDS is expected to increase, physicians should become familiar with the nature of associated injuries.

The sound field of a rotating dipole in a plug flow.

An analytical far field solution for a rotating point dipole source in a plug flow is derived. The shear layer of the jet is modelled as an infinitely thin cylindrical vortex sheet and the far field integral is calculated by the stationary phase method. Four numerical tests are performed to validate the derived solution as well as to assess the effects of sound refraction from the shear layer. First, the calculated results using the derived formulations are compared with the known solution for a rotating dipole in a uniform flow to validate the present model in this fundamental test case. After that, the effects of sound refraction for different rotating dipole sources in the plug flow are assessed. Then the refraction effects on different frequency components of the signal at the observer position, as well as the effects of the motion of the source and of the type of source are considered. Finally, the effect of different sound speeds and densities outside and inside the plug flow is investigated. The solution obtained may be of particular interest for propeller and rotor noise measurements in open jet anechoic wind tunnels.

Synthesis of apo-13- and apo-15-lycopenoids, cleavage products of lycopene that are retinoic acid antagonists.

Consumption of the tomato carotenoid, lycopene, has been associated with favorable health benefits. Some of lycopene's biological activity may be due to metabolites resulting from cleavage of the lycopene molecule. Because of their structural similarity to the retinoic acid receptor (RAR) antagonist, ß-apo-13-carotenone, the "first half" putative oxidative cleavage products of the symmetrical lycopene have been synthesized. All transformations proceed in moderate to good yield and some with high stereochemical integrity allowing ready access to these otherwise difficult to obtain terpenoids. In particular, the methods described allow ready access to the trans isomers of citral (geranial) and pseudoionone, important flavor and fragrance compounds that are not readily available isomerically pure and are building blocks for many of the longer apolycopenoids. In addition, all of the apo-11, apo-13, and apo-15 lycopenals/lycopenones/lycopenoic acids have been prepared. These compounds have been evaluated for their effect on RAR-induced genes in cultured hepatoma cells and, much like ß-apo-13-carotenone, the comparable apo-13-lycopenone and the apo-15-lycopenal behave as RAR antagonists. Furthermore, molecular modeling studies demonstrate that the apo-13-lycopenone efficiently docked into the ligand binding site of RARα. Finally, isothermal titration calorimetry studies reveal that apo-13-lycopenone acts as an antagonist of RAR by inhibiting coactivator recruitment to the receptor.

Substrate Specificity of Purified Recombinant Chicken ß-Carotene 9',10'-Oxygenase (BCO2).

Provitamin A carotenoids are oxidatively cleaved by ß-carotene 15,15'-dioxygenase (BCO1) at the central 15-15' double bond to form retinal (vitamin A aldehyde). Another carotenoid oxygenase, ß-carotene 9',10'-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9'-10' bond to yield an ionone and an apo-10'-carotenoid. Previously published substrate specificity studies of BCO2 were conducted using crude lysates from bacteria or insect cells expressing recombinant BCO2. Our attempts to obtain active recombinant human BCO2 expressed in Escherichia coli were unsuccessful. We have expressed recombinant chicken BCO2 in the strain E. coli BL21-Gold (DE3) and purified the enzyme by cobalt ion affinity chromatography. Like BCO1, purified recombinant chicken BCO2 catalyzes the oxidative cleavage of the provitamin A carotenoids ß-carotene, α-carotene, and ß-cryptoxanthin. Its catalytic activity with ß-carotene as substrate is at least 10-fold lower than that of BCO1. In further contrast to BCO1, purified recombinant chicken BCO2 also catalyzes the oxidative cleavage of 9-cis-ß-carotene and the non-provitamin A carotenoids zeaxanthin and lutein, and is inactive with all-trans-lycopene and ß-apocarotenoids. Apo-10'-carotenoids were detected as enzymatic products by HPLC, and the identities were confirmed by LC-MS. Small amounts of 3-hydroxy-ß-apo-8'-carotenal were also consistently detected in BCO2-ß-cryptoxanthin reaction mixtures. With the exception of this activity with ß-cryptoxanthin, BCO2 cleaves specifically at the 9'-10' bond to produce apo-10'-carotenoids. BCO2 has been shown to function in preventing the excessive accumulation of carotenoids, and its broad substrate specificity is consistent with this.

ß-Apo-10'-carotenoids Modulate Placental Microsomal Triglyceride Transfer Protein Expression and Function to Optimize Transport of Intact ß-Carotene to the Embryo.

ß-Carotene is an important source of vitamin A for the mammalian embryo, which depends on its adequate supply to achieve proper organogenesis. In mammalian tissues, ß-carotene 15,15'-oxygenase (BCO1) converts ß-carotene to retinaldehyde, which is then oxidized to retinoic acid, the biologically active form of vitamin A that acts as a transcription factor ligand to regulate gene expression. ß-Carotene can also be cleaved by ß-carotene 9',10'-oxygenase (BCO2) to form ß-apo-10'-carotenal, a precursor of retinoic acid and a transcriptional regulator per se The mammalian embryo obtains ß-carotene from the maternal circulation. However, the molecular mechanisms that enable its transfer across the maternal-fetal barrier are not understood. Given that ß-carotene is transported in the adult bloodstream by lipoproteins and that the placenta acquires, assembles, and secretes lipoproteins, we hypothesized that the aforementioned process requires placental lipoprotein biosynthesis. Here we show that ß-carotene availability regulates transcription and activity of placental microsomal triglyceride transfer protein as well as expression of placental apolipoprotein B, two key players in lipoprotein biosynthesis. We also show that ß-apo-10'-carotenal mediates the transcriptional regulation of microsomal triglyceride transfer protein via hepatic nuclear factor 4α and chicken ovalbumin upstream promoter transcription factor I/II. Our data provide the first in vivo evidence of the transcriptional regulatory activity of ß-apocarotenoids and identify microsomal triglyceride transfer protein and its transcription factors as the targets of their action. This study demonstrates that ß-carotene induces a feed-forward mechanism in the placenta to enhance the assimilation of ß-carotene for proper embryogenesis.

Lymphocyte density determined by computational pathology validated as a predictor of response to neoadjuvant chemotherapy in breast cancer: secondary analysis of the ARTemis trial.

BACKGROUND: We have previously shown lymphocyte density, measured using computational pathology, is associated with pathological complete response (pCR) in breast cancer. The clinical validity of this finding in independent studies, among patients receiving different chemotherapy, is unknown. PATIENTS AND METHODS: The ARTemis trial randomly assigned 800 women with early stage breast cancer between May 2009 and January 2013 to three cycles of docetaxel, followed by three cycles of fluorouracil, epirubicin and cyclophosphamide once every 21 days with or without four cycles of bevacizumab. The primary endpoint was pCR (absence of invasive cancer in the breast and lymph nodes). We quantified lymphocyte density within haematoxylin and eosin (H&E) whole slide images using our previously described computational pathology approach: for every detected lymphocyte the average distance to the nearest 50 lymphocytes was calculated and the density derived from this statistic. We analyzed both pre-treatment biopsies and post-treatment surgical samples of the tumour bed. RESULTS: Of the 781 patients originally included in the primary endpoint analysis of the trial, 609 (78%) were included for baseline lymphocyte density analyses and a subset of 383 (49% of 781) for analyses of change in lymphocyte density. The main reason for loss of patients was the availability of digitized whole slide images. Pre-treatment lymphocyte density modelled as a continuous variable was associated with pCR on univariate analysis (odds ratio [OR], 2.92; 95% CI, 1.78-4.85; P < 0.001) and after adjustment for clinical covariates (OR, 2.13; 95% CI, 1.24-3.67; P = 0.006). Increased pre- to post-treatment lymphocyte density showed an independent inverse association with pCR (adjusted OR, 0.1; 95% CI, 0.033-0.31; P < 0.001). CONCLUSIONS: Lymphocyte density in pre-treatment biopsies was validated as an independent predictor of pCR in breast cancer. Computational pathology is emerging as a viable and objective means of identifying predictive biomarkers for cancer patients. CLINICALTRIALS.GOV: NCT01093235.

Disease-free and overall survival at 3.5 years for neoadjuvant bevacizumab added to docetaxel followed by fluorouracil, epirubicin and cyclophosphamide, for women with HER2 negative early breast cancer: ARTemis Trial.

BACKGROUND: The ARTemis trial previously reported that addition of neoadjuvant bevacizumab (Bev) to docetaxel (D) followed by fluorouracil, epirubicin and cyclophosphamide (D-FEC) in HER2 negative breast cancer improved the pathological complete response (pCR) rate. We present disease-free survival (DFS) and overall survival (OS) with central pathology review. PATIENTS AND METHODS: Patients were randomized to 3 cycles of D followed by 3 cycles of FEC (D-FEC), ±4 cycles of Bev (Bev + D-FEC). DFS and OS were analyzed by treatment and by central pathology reviewed pCR and Residual Cancer Burden (RCB) class. RESULTS: A total of 800 patients were randomized [median follow-up 3.5 years (IQR 3.2-4.4)]. DFS and OS were similar across treatment arms [DFS hazard ratio (HR)=1.18 (95% CI 0.89-1.57), P = 0.25; OS HR = 1.26 (95% CI 0.90-1.76), P = 0.19). Both local pathology report review and central histopathology review confirmed a significant improvement in DFS and OS for patients who achieved a pCR [DFS HR = 0.38 (95% CI 0.23-0.63), P < 0.001; OS HR = 0.43 (95% CI 0.24-0.75), P = 0.003]. However, significant heterogeneity was observed (P = 0.02); larger improvements in DFS were obtained with a pCR achieved with D-FEC than a pCR achieved with Bev + D-FEC. As RCB class increased, significantly worse DFS and OS was observed (P for trend <0.0001), which effect was most marked in the ER negative group. CONCLUSIONS: The addition of short course neoadjuvant Bev to standard chemotherapy did not demonstrate a DFS or OS benefit. Achieving a pCR with D-FEC is associated with improved DFS and OS but not when pCR is achieved with Bev + D-FEC. At the present time therefore, Bev is not recommended in early breast cancer. CLINICALTRIALS.GOV NUMBER: NCT01093235.

Carotenoids and Retinoids: Nomenclature, Chemistry, and Analysis.

Carotenoids are polyenes synthesized in plants and certain microorganisms and are pigments used by plants and animals in various physiological processes. Some of the over 600 known carotenoids are capable of metabolic conversion to the essential nutrient vitamin A (retinol) in higher animals. Vitamin A also gives rise to a number of other metabolites which, along with their analogs, are known as retinoids. To facilitate discussion about these important molecules, a nomenclature is required to identify specific substances. The generally accepted rules for naming these important molecules have been agreed to by various Commissions of the International Union of Pure and Applied Chemistry and International Union of Biochemistry. These naming conventions are explained along with comparisons to more systematic naming rules that apply for these organic chemicals. Identification of the carotenoids and retinoids has been advanced by their chemical syntheses, and here, both classical and modern methods for synthesis of these molecules, as well as their analogs, are described. Because of their importance in biological systems, sensitive methods for the detection and quantification of these compounds from various sources have been essential. Early analyses that relied on liquid adsorption and partition chromatography have given way to high-performance liquid chromatography (HPLC) coupled with various detection methods. The development of HPLC coupled to mass spectrometry, particularly LC/MS-MS with Multiple Reaction Monitoring, has resulted in the greatest sensitivity and specificity in these analyses.

Mechanisms of selective delivery of xanthophylls to retinal pigment epithelial cells by human lipoproteins.

The xanthophylls, lutein and zeaxanthin, are dietary carotenoids that selectively accumulate in the macula of the eye providing protection against age-related macular degeneration. To reach the macula, carotenoids cross the retinal pigment epithelium (RPE). Xanthophylls and ß-carotene mostly associate with HDL and LDL, respectively. HDL binds to cells via a scavenger receptor class B1 (SR-B1)-dependent mechanism, while LDL binds via the LDL receptor. Using an in-vitro, human RPE cell model (ARPE-19), we studied the mechanisms of carotenoid uptake into the RPE by evaluating kinetics of cell uptake when delivered in serum or isolated LDL or HDL. For lutein and ß-carotene, LDL delivery resulted in the highest rates and extents of uptake. In contrast, HDL was more effective in delivering zeaxanthin and meso-zeaxanthin leading to the highest rates and extents of uptake of all four carotenoids. Inhibitors of SR-B1 suppressed zeaxanthin delivery via HDL. Results show a selective HDL-mediated uptake of zeaxanthin and meso-zeaxanthin via SR-B1 and a LDL-mediated uptake of lutein. This demonstrates a plausible mechanism for the selective accumulation of zeaxanthin greater than lutein and xanthophylls over ß-carotene in the retina. We found no evidence of xanthophyll metabolism to apocarotenoids or lutein conversion to meso-zeaxanthin.

ß-Apo-13-carotenone regulates retinoid X receptor transcriptional activity through tetramerization of the receptor.

Retinoid X receptor (RXRα) is activated by 9-cis-retinoic acid (9cRA) and regulates transcription as a homodimer or as a heterodimer with other nuclear receptors. We have previously demonstrated that ß-apo-13-carotenone, an eccentric cleavage product of ß-carotene, antagonizes the activation of RXRα by 9cRA in mammalian cells overexpressing this receptor. However, the molecular mechanism of ß-apo-13-carotenone's modulation on the transcriptional activity of RXRα is not understood and is the subject of this report. We performed transactivation assays using full-length RXRα and reporter gene constructs (RXRE-Luc) transfected into COS-7 cells, and luciferase activity was examined. ß-Apo-13-carotenone was compared with the RXRα antagonist UVI3003. The results showed that both ß-apo-13-carotenone and UVI3003 shifted the dose-dependent RXRα activation by 9cRA. In contrast, the results of assays using a hybrid Gal4-DBD:RXRαLBD receptor reporter cell assay that detects 9cRA-induced coactivator binding to the ligand binding domain demonstrated that UVI3003 significantly inhibited 9cRA-induced coactivator binding to RXRαLBD, but ß-apo-13-carotenone did not. However, both ß-apo-13-carotenone and UVI3003 inhibited 9-cRA induction of caspase 9 gene expression in the mammary carcinoma cell line MCF-7. To resolve this apparent contradiction, we investigated the effect of ß-apo-13-carotenone on the oligomeric state of purified recombinant RXRαLBD. ß-Apo-13-carotenone induces tetramerization of the RXRαLBD, although UVI3003 had no effect on the oligomeric state. These observations suggest that ß-apo-13-carotenone regulates RXRα transcriptional activity by inducing the formation of the "transcriptionally silent" RXRα tetramer.

The human enzyme that converts dietary provitamin A carotenoids to vitamin A is a dioxygenase.

ß-Carotene 15-15'-oxygenase (BCO1) catalyzes the oxidative cleavage of dietary provitamin A carotenoids to retinal (vitamin A aldehyde). Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase, incorporating oxygen from O2 and H2O into its cleavage products. This was based on a study that used conditions that favored oxygen exchange with water. We incubated purified recombinant human BCO1 and ß-carotene in either (16)O2-H2(18)O or (18)O2-H2(16)O medium for 15 min at 37 °C, and the relative amounts of (18)O-retinal and (16)O-retinal were measured by liquid chromatography-tandem mass spectrometry. At least 79% of the retinal produced by the reaction has the same oxygen isotope as the O2 gas used. Together with the data from (18)O-retinal-H2(16)O and (16)O-retinal-H2(18)O incubations to account for nonenzymatic oxygen exchange, our results show that BCO1 incorporates only oxygen from O2 into retinal. Thus, BCO1 is a dioxygenase.

PD-L1 protein expression in breast cancer is rare, enriched in basal-like tumours and associated with infiltrating lymphocytes.

BACKGROUND: Expression of programmed death ligand 1 (PD-L1) in solid tumours has been shown to predict whether patients are likely to respond to anti-PD-L1 therapies. To estimate the therapeutic potential of PD-L1 inhibition in breast cancer, we evaluated the prevalence and significance of PD-L1 protein expression in a large collection of breast tumours. PATIENTS AND METHODS: Correlations between CD274 (PD-L1) copy number, transcript and protein levels were evaluated in tumours from 418 patients recruited to the METABRIC genomic study. Immunohistochemistry was used to detect PD-L1 protein in breast tumours in tissue microarrays from 5763 patients recruited to the SEARCH population-based study (N = 4079) and the NEAT randomised, controlled trial (N = 1684). RESULTS: PD-L1 protein data was available for 3916 of the possible 5763 tumours from the SEARCH and NEAT studies. PD-L1 expression by immune cells was observed in 6% (235/3916) of tumours and expression by tumour cells was observed in just 1.7% (66/3916). PD-L1 was most frequently expressed in basal-like tumours. This was observed both where tumours were subtyped by combined copy number and expression profiling [39% (17/44) of IntClust 10 i.e. basal-like tumours were PD-L1 immune cell positive; P < 0.001] and where a surrogate IHC-based classifier was used [19% (56/302) of basal-like tumours were PD-L1 immune cell positive; P < 0.001]. Moreover, CD274 (PD-L1) amplification was observed in five tumours of which four were IntClust 10. Expression of PD-L1 by either tumour cells or infiltrating immune cells was positively correlated with infiltration by both cytotoxic and regulatory T cells (P < 0.001). There was a nominally significant association between PD-L1 and improved disease-specific survival (hazard ratio 0.53, 95% confidence interval 0.26-1.07; P = 0.08) in ER-negative disease. CONCLUSIONS: Expression of PD-L1 is rare in breast cancer, markedly enriched in basal-like tumours and is correlated with infiltrating lymphocytes. PD-L1 inhibition may benefit the 19% of patients with basal-like tumours in which the protein is expressed. NEAT CLINICALTRIALSGOV: NCT00003577.

Cellular localization of ß-carotene 15,15' oxygenase-1 (BCO1) and ß-carotene 9',10' oxygenase-2 (BCO2) in rat liver and intestine.

The intestine and liver are crucial organs for vitamin A uptake and storage. Liver accounts for 70% of total body retinoid stores. Vitamin A deficiency (VAD) is a major micronutrient deficiency around the world. The provitamin A carotenoid, ß-carotene, is a significant source of vitamin A in the diet. ß-Carotene 15,15' oxygenase-1 (BCO1) and ß-carotene 9',10' oxygenase-2 (BCO2) are the two known carotenoid cleavage enzymes in humans. BCO1 and BCO2 are highly expressed in liver and intestine. Hepatocytes and hepatic stellate cells are two main cell types involved in the hepatic metabolism of retinoids. Stellate-like cells in the intestine also show ability to store vitamin A. Liver is also known to accumulate carotenoids, however, their uptake, retention and metabolism in specific liver and intestinal cell types is still unknown. Hence, we studied the cellular and subcellular expression and localization of BCO1 and BCO2 proteins in rat liver and intestine. We demonstrate that both BCO1 and BCO2 proteins are localized in hepatocytes and mucosal epithelium. We also show that BCO1 is also highly expressed in hepatic stellate cells (HSC) and portal endothelial cells in liver. At the subcellular level in liver, BCO1 is found in cytosol, while BCO2 is found in mitochondria. In intestine, immunohistochemistry showed strong BCO1 immunoreactivity in the duodenum, particularly in Brunner's glands. Both BCO1 and BCO2 showed diffuse presence along epithelia with strong immunoreactivity in endothelial cells and in certain epithelial cells which warrant further investigation as possible intestinal retinoid storage cells.

Actions of ß-apo-carotenoids in differentiating cells: differential effects in P19 cells and 3T3-L1 adipocytes.

ß-Apo-carotenoids, including ß-apo-13-carotenone and ß-apo-14'-carotenal, are potent retinoic acid receptor (RAR) antagonists in transactivation assays. We asked how these influence RAR-dependent processes in living cells. Initially, we explored the effects of ß-apo-13-carotenone and ß-apo-14'-carotenal on P19 cells, a mouse embryonal carcinoma cell line that differentiates into neurons when treated with all-trans-retinoic acid. Treatment of P19 cells with either compound failed to block all-trans-retinoic acid induced differentiation. Liquid chromatography tandem mass spectrometry studies, however, established that neither of these ß-apo-carotenoids accumulates in P19 cells. All-trans-retinoic acid accumulated to high levels in P19 cells. This suggests that the uptake and metabolism of ß-apo-carotenoids by some cells does not involve the same processes used for retinoids and that these may be cell type specific. We also investigated the effects of two ß-apo-carotenoids on 3T3-L1 adipocyte marker gene expression during adipocyte differentiation. Treatment of 3T3-L1 adipocytes with either ß-apo-13-carotenone or ß-apo-10'-carotenoic acid, which lacks RAR antagonist activity, stimulated adipocyte marker gene expression. Neither blocked the inhibitory effects of a relatively large dose of exogenous all-trans-retinoic acid on adipocyte differentiation. Our data suggest that in addition to acting as transcriptional antagonists, some ß-apo-carotenoids act through other mechanisms to influence 3T3-L1 adipocyte differentiation.

Substrate specificity of purified recombinant human ß-carotene 15,15'-oxygenase (BCO1).

Humans cannot synthesize vitamin A and thus must obtain it from their diet. ß-Carotene 15,15'-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15-15' double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (kcat/Km). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of ß-carotene with a Vmax = 197.2 nmol retinal/mg BCO1 × h, Km = 17.2 µM and catalytic efficiency kcat/Km = 6098 M(-1) min(-1). The enzyme also catalyzed the oxidative cleavage of α-carotene, ß-cryptoxanthin, and ß-apo-8'-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of ß-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of ß-carotene. The shorter ß-apocarotenals (ß-apo-10'-carotenal, ß-apo-12'-carotenal, ß-apo-14'-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-ß-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.

Anthracyclines and taxanes in the neo/adjuvant treatment of breast cancer: does the sequence matter?

BACKGROUND: In early breast cancer, adjuvant chemotherapy decreases the risks of recurrence and breast cancer mortality, and neoadjuvant treatment leads to equivalent long-term outcomes. A large number of clinical trials have attempted to refine systemic therapeutic strategies in early breast cancer, but little attention has been paid to the sequence of anthracyclines and taxanes. Based on preclinical observations, there is limited rationale to administer the taxane before the anthracycline. METHODS: We searched PubMed, the American Society of Clinical Oncology website, and clinicaltrials.gov with the goal of identifying published or ongoing studies that aimed at comparing reverse sequences of anthracyclines and taxanes. Given the nature and the small number of studies identified, we did not attempt to quantitatively pool the study results. RESULTS: We retrieved seven studies in the adjuvant setting and eight in the neoadjuvant setting: 10 randomized trials (only 2 were phase IIII), 3 retrospective studies, and 2 ongoing phase II trials. A total of nearly 5000 patients were included in such studies. None of the clinical trials has shown disadvantages in terms of efficacy or toxicity for sequences in which the taxane was administered first. In the neoadjuvant setting, studies have collectively shown similar or increased pathological complete response rates for sequences in which the taxane was administered first. CONCLUSION: Given the available information, there seems to be sufficient evidence to suggest that a taxane followed by an anthracycline is a sequence option that can be incorporated into daily clinical practice.

A randomised, placebo-controlled trial of weekly paclitaxel and saracatinib (AZD0530) in platinum-resistant ovarian, fallopian tube or primary peritoneal cancer†.

BACKGROUND: We investigated whether the Src inhibitor saracatinib (AZD0530) improved efficacy of weekly paclitaxel in platinum-resistant ovarian cancer. PATIENTS AND METHODS: Patients with platinum-resistant ovarian, fallopian tube or primary peritoneal cancer were randomised 2 : 1 to receive 8-week cycles of weekly paclitaxel (wPxl; 80 mg/m(2)/week ×6 with 2-week break) plus saracatinib (S; 175 mg o.d.) or placebo (P) continuously, starting 1 week before wPxl, until disease progression. Patients were stratified by taxane-free interval (<6 versus ≥6 months/no prior taxane). The primary end point was progression-free survival (PFS) rate at 6 months. Secondary end points included overall survival (OS) and response rate (RR). RESULTS: A total of 107 patients, median age 63 years, were randomised. Forty-three (40%) had received >2 lines of prior chemotherapy. The 6-month PFS rate was 29% (wPxl + S) versus 34% (wPxl + P) (P = 0.582). Median PFS was 4.7 versus 5.3 months (hazard ratio 1.00, 95% confidence interval 0.65-1.54; P = 0.99). RR (complete + partial) was 29% (wPxl + S) versus 43% (wPxl + P), P value = 0.158. Grade 3/4 adverse events were 36% versus 31% (P = 0.624); the most frequent G3/4 toxicities were vomiting (5.8% saracatinib versus 8.6% placebo), abdominal pain (5.8% versus 0%) and diarrhoea (4.3% versus 5.7%). Febrile neutropenia was more common in the saracatinib arm (4.3%) than placebo (0%). Response, PFS and OS were all significantly (P < 0.05) better in patients with taxane interval ≥6 months/no prior taxane (n = 85) than those <6 months (n = 22), regardless of randomisation. CONCLUSIONS: Saracatinib does not improve activity of weekly paclitaxel in platinum-resistant ovarian cancer. Taxane-free interval of ≥6 months/no prior taxane was associated with better outcome in both groups. TRIALS REGISTRATION: Clinicaltrials.gov NCT01196741; ISRCTN 32163062.

Association between CD8+ T-cell infiltration and breast cancer survival in 12,439 patients.

BACKGROUND: T-cell infiltration in estrogen receptor (ER)-negative breast tumours has been associated with longer survival. To investigate this association and the potential of tumour T-cell infiltration as a prognostic and predictive marker, we have conducted the largest study of T cells in breast cancer to date. PATIENTS AND METHODS: Four studies totalling 12 439 patients were used for this work. Cytotoxic (CD8+) and regulatory (forkhead box protein 3, FOXP3+) T cells were quantified using immunohistochemistry (IHC). IHC for CD8 was conducted using available material from all four studies (8978 samples) and for FOXP3 from three studies (5239 samples)-multiple imputation was used to resolve missing data from the remaining patients. Cox regression was used to test for associations with breast cancer-specific survival. RESULTS: In ER-negative tumours [triple-negative breast cancer and human epidermal growth factor receptor 2 (human epidermal growth factor receptor 2 (HER2) positive)], presence of CD8+ T cells within the tumour was associated with a 28% [95% confidence interval (CI) 16% to 38%] reduction in the hazard of breast cancer-specific mortality, and CD8+ T cells within the stroma with a 21% (95% CI 7% to 33%) reduction in hazard. In ER-positive HER2-positive tumours, CD8+ T cells within the tumour were associated with a 27% (95% CI 4% to 44%) reduction in hazard. In ER-negative disease, there was evidence for greater benefit from anthracyclines in the National Epirubicin Adjuvant Trial in patients with CD8+ tumours [hazard ratio (HR) = 0.54; 95% CI 0.37-0.79] versus CD8-negative tumours (HR = 0.87; 95% CI 0.55-1.38). The difference in effect between these subgroups was significant when limited to cases with complete data (P heterogeneity = 0.04) and approached significance in imputed data (P heterogeneity = 0.1). CONCLUSIONS: The presence of CD8+ T cells in breast cancer is associated with a significant reduction in the relative risk of death from disease in both the ER-negative [supplementary Figure S1, available at Annals of Oncology online] and the ER-positive HER2-positive subtypes. Tumour lymphocytic infiltration may improve risk stratification in breast cancer patients classified into these subtypes. NEAT ClinicalTrials.gov: NCT00003577.