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Abstract Locoregional failure (LRF) in patients with breast cancer post-surgery and post-irradiation is linked to a dismal prognosis. In a refined new model, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1/CD203a (ENPP1) to be closely associated with LRF. ENPP1hi circulating tumor cells (CTC) contribute to relapse by a self-seeding mechanism. This process requires the infiltration of polymorphonuclear myeloid-derived suppressor cells and neutrophil extracellular trap (NET) formation. Genetic and pharmacologic ENPP1 inhibition or NET blockade extends relapse-free survival. Furthermore, in combination with fractionated irradiation, ENPP1 abrogation obliterates LRF. Mechanistically, ENPP1-generated adenosinergic metabolites enhance haptoglobin (HP) expression. This inflammatory mediator elicits myeloid invasiveness and promotes NET formation. Accordingly, a significant increase in ENPP1 and NET formation is detected in relapsed human breast cancer tumors. Moreover, high ENPP1 or HP levels are associated with poor prognosis. These findings unveil the ENPP1/HP axis as an unanticipated mechanism exploited by tumor cells linking inflammation to immune remodeling favoring local relapse. Significance: CTC exploit the ENPP1/HP axis to promote local recurrence post-surgery and post-irradiation by subduing myeloid suppressor cells in breast tumors. Blocking this axis impairs tumor engraftment, impedes immunosuppression, and obliterates NET formation, unveiling new opportunities for therapeutic intervention to eradicate local relapse and ameliorate patient survival. This article is highlighted in the In This Issue feature, p. 1171

Locoregional failure (LRF) in patients with breast cancer post-surgery and post-irradiation is linked to a dismal prognosis. In a refined new model, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1/CD203a (ENPP1) to be closely associated with LRF. ENPP1hi circulating tumor cells (CTC) contribute to relapse by a self-seeding mechanism. This process requires the infiltration of polymorphonuclear myeloid-derived suppressor cells and neutrophil extracellular trap (NET) formation. Genetic and pharmacologic ENPP1 inhibition or NET blockade extends relapse-free survival. Furthermore, in combination with fractionated irradiation, ENPP1 abrogation obliterates LRF. Mechanistically, ENPP1-generated adenosinergic metabolites enhance haptoglobin (HP) expression. This inflammatory mediator elicits myeloid invasiveness and promotes NET formation. Accordingly, a significant increase in ENPP1 and NET formation is detected in relapsed human breast cancer tumors. Moreover, high ENPP1 or HP levels are associated with poor prognosis. These findings unveil the ENPP1/HP axis as an unanticipated mechanism exploited by tumor cells linking inflammation to immune remodeling favoring local relapse.Significance:CTC exploit the ENPP1/HP axis to promote local recurrence post-surgery and post-irradiation by subduing myeloid suppressor cells in breast tumors. Blocking this axis impairs tumor engraftment, impedes immunosuppression, and obliterates NET formation, unveiling new opportunities for therapeutic intervention to eradicate local relapse and ameliorate patient survival.This article is highlighted in the In This Issue feature, p. 1171

Supplementary Data from Tumor ENPP1 (CD203a)/Haptoglobin Axis Exploits Myeloid-Derived Suppressor Cells to Promote Post-Radiotherapy Local Recurrence in Breast Cancer

Supplementary Figure from Tumor ENPP1 (CD203a)/Haptoglobin Axis Exploits Myeloid-Derived Suppressor Cells to Promote Post-Radiotherapy Local Recurrence in Breast Cancer

The HLA system represents a central component of the antigen presentation machinery. As every patient possesses a defined set of HLA molecules, only certain antigens can be presented on the cell surface. Thus, studying HLA type-dependent antigen presentation can improve the understanding of variation in susceptibility to various diseases, including infectious diseases and cancer. In archival formalin-fixed paraffin-embedded (FFPE) tissue, the HLA type is difficult to analyze because of fragmentation of DNA, hindering the application of commonly used assays that rely on long DNA stretches. Addressing these difficulties, we present a refined approach for characterizing presence or absence of HLA-A*02, the most common HLA-A allele in the Caucasian population, in archival samples. We validated our genotyping strategy in a cohort of 90 samples with HLA status obtained by an NGS-based method. 90% (n = 81) of the samples could be analyzed with the approach. For all of them, the presence or absence of HLA-A*02 alleles was correctly determined with the method, demonstrating 100% sensitivity and specificity (95% CI: 91.40%–100% and 91.19%–100%). Furthermore, we provide an example of application in an independent cohort of 73 FFPE microsatellite-unstable (MSI) colorectal cancer samples. As MSI cancer cells encompass a high number of mutations in coding microsatellites, leading to the generation of highly immunogenic frameshift peptide antigens, they are ideally suited for studying relations between the mutational landscape of tumor cells and interindividual differences in the immune system, including the HLA genotype. Overall, our method can help to promote studying HLA type-dependency during the pathogenesis of a wide range of diseases, making archival and historic tissue samples accessible for identifying HLA-A*02 alleles. publishedVersion

Radiotherapy is a mainstay cancer therapy whose antitumor effects partially depend on T cell responses. However, the role of Natural Killer (NK) cells in radiotherapy remains unclear. Here, using a reverse translational approach, we show a central role of NK cells in the radiation-induced immune response involving a CXCL8/IL-8–dependent mechanism. In a randomized controlled pancreatic cancer trial, CXCL8 increased under radiotherapy, and NK cell positively correlated with prolonged overall survival. Accordingly, NK cells preferentially infiltrated irradiated pancreatic tumors and exhibited CD56dim-like cytotoxic transcriptomic states. In experimental models, NF-κB and mTOR orchestrated radiation-induced CXCL8 secretion from tumor cells with senescence features causing directional migration of CD56dimNK cells, thus linking senescence-associated CXCL8 release to innate immune surveillance of human tumors. Moreover, combined high-dose radiotherapy and adoptive NK cell transfer improved tumor control over monotherapies in xenografted mice, suggesting NK cells combined with radiotherapy as a rational cancer treatment strategy.

Summary Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively). To define the heterogeneity of tumors and their associated microenvironments across subtypes, we sequenced 155,098 transcriptomes from 21 human biospecimens, including 54,523 SCLC transcriptomes. We observe greater tumor diversity in SCLC than lung adenocarcinoma, driven by canonical, intermediate, and admixed subtypes. We discover a PLCG2-high SCLC phenotype with stem-like, pro-metastatic features that recurs across subtypes and predicts worse overall survival. SCLC exhibits greater immune sequestration and less immune infiltration than lung adenocarcinoma, and SCLC-N shows less immune infiltrate and greater T cell dysfunction than SCLC-A. We identify a profibrotic, immunosuppressive monocyte/macrophage population in SCLC tumors that is particularly associated with the recurrent, PLCG2-high subpopulation.

IntroductionImmune checkpoint therapy (ICT) is associated with a distinct pattern of immune-related adverse events (irAEs) caused by inadvertently redirecting immune responses to healthy tissues. IrAEs can occur at any time; however, in most cases, they arise during the first 14 weeks of the beginning of immune checkpoint blockade. In many cases, immunotherapy must be discontinued due to irAEs. Early detection of irAEs triggers the temporary withholding of ICT or initiation of short-term immunosuppressive treatment, is crucial in preventing further aggravation of irAEs and enables safe re-exposure to ICT. This prospective study aims to evaluate the feasibility of an eHealth intervention for patients under immunotherapy (managing symptoms of immunotherapy, SOFIA). The SOFIA-App consists of two components: SOFIA-Monitoring, a tool to rate patient-reported outcomes (PROs) including irAEs, and SOFIA-Coaching, which provides important information about cancer-specific and immunotherapy-specific topics and the counselling services of the National Centre for Tumour Diseases (NCT) Heidelberg.Methods and analysisWe outlined a patient-level two-arm randomised controlled pilot trial of the intervention (SOFIA) versus no-SOFIA for patients with cancer beginning an immunotherapy, aged ≥18 years, recruited from the NCT, Heidelberg. Feasibility outcomes include: recruitment rate; drop-out rate; reasons for refusal and drop-out; willingness to be randomised, utilisation rate of SOFIA-Monitoring and utilisation time of SOFIA-Coaching, physicians utilisation rate of the PROs; feasibility of the proposed outcome measures and optimal sample size estimation. The clinical outcomes are measures of quality of life, psychosocial symptoms, self-efficacy, physician-patient communication and medical process data, which are assessed at the beginning of the intervention, postintervention and at 6-month follow-up.Ethics and disseminationThis trial protocol was approved by the Ethical Committee of Heidelberg University, Germany (Reference, S-581/2018).Trial registration numberWe registered the study in the German Clinical Trial Register (Reference: DRKS00021064). Findings will be disseminated broadly via peer-reviewed empirical journals, articles and conference presentations.

The role of radiotherapy in the treatment of pancreatic adenocarcinoma (PDAC) is controversial. In many other tumor types, radiotherapy is a mainstay therapy whose anti-tumor effects have partially been ascribed to T cell responses. The role of Natural Killer (NK) cells in radiotherapy tumor responses remains unclear. Here, we focused on the role of NK cells in radiotherapy of pancreatic cancer. In a randomized controlled pancreatic cancer trial, we found that CXCL8 serum levels increased under radiotherapy and NK cells positively correlated with prolonged overall patient survival. Accordingly, NK cells preferentially infiltrated irradiated tumors and exhibited CD56dim like cytotoxic transcriptomic states. In preclinical experimental systems in mice and tumor cells, NF-κB and mTOR signaling prompted radiation-induced CXCL8 secretion from senescent tumor cells causing directional migration of CD56dim NK cells into tumors. Thus, we link senescence-associated CXCL8 release to innate immune surveillance of human tumors. Moreover, combined radiotherapy and adoptive NK cell transfer improved tumor control over monotherapies in xenografted mice suggesting NK cells combined with radiotherapy as a rational cancer treatment strategy which may be integrated into PDAC treatment algorithms. Citation Format: Thomas Walle, Joscha A. Kraske, Boyu Liao, Carmen Timke, Benedicte Lenoir, Niels Halama, Peter E. Huber. CXCL8/Interleukin-8 orchestrates radiation-induced immune responses in pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB116.

Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively), which are associated with distinct therapeutic vulnerabilities. To define the heterogeneity of tumors and their associated microenvironments across subtypes, we sequenced 54,523 cellular transcriptomes from 21 human biospecimens. Our single-cell SCLC atlas reveals tumor diversity exceeding lung adenocarcinoma, driven by canonical, intermediate, and admixed subtypes. We discovered a PLCG2-high tumor cell population with stem-like, pro-metastatic features that recurs across subtypes and predicts worse overall survival, and manipulation of PLCG2 expression in cells confirms correlation with key metastatic markers. Treatment and subtype are associated with substantial phenotypic changes in the SCLC immune microenvironment, with greater T-cell dysfunction in SCLC-N than SCLC-A. Moreover, the recurrent, PLCG2-high subclone is associated with exhausted CD8+ T-cells and a pro-fibrotic, immunosuppressive monocyte/macrophage population, suggesting possible tumor-immune coordination to promote metastasis.

BackgroundMobile phone video call applications generally did not undergo testing in randomized controlled clinical trials prior to their implementation in patient care regarding the rate of successful patient visits and impact on the physician-patient relationship.MethodsThe NCT MOBILE trial was a monocentric open-label randomized controlled clinical trial of patients with solid tumors undergoing systemic cancer therapy with need of a follow-up visit with their consulting physician at outpatient clinics. 66 patients were 1:1 randomized to receive either a standard in-person follow-up visit at outpatient clinics or a video callviaa mobile phone application. The primary outcome was feasibility defined as the number of successful appointments at the first follow up visit. Secondary outcomes included success rate of further video calls, time spent by patient and physician, patient satisfaction, and quality of physician-patient relationship.FindingsSuccess rate of the first follow up visit in the intention-to-treat cohort was 87.8% for in-person visits and 78.7% for video calls (p=0.51, RR=0.88-1.43 95%CI). The most common reasons for failure were software incompatibility (12%) in the video call and no-show (6%) in the in-person visit arm. The success rate for further video visits was 91.6% (11 of 12 calls). Standardized patient questionnaires showed significantly decreased total time spent and less direct costs for patients (Δ95 to 246min 95%CI, Δ4.8 to 23.9€ 95%CI) and comparable time spent for physicians in the video call arm (Δ-6.4 to 5.4min, 95%CI). Doctor-patient relationship quality mean scores assessed by the validated standardized “questionnaire on quality of physician-patient interaction”(QQPPI) were higher in the video call arm (video call/in-person = 1.12 fold, p=0.02).InterpretationFollow-up visits with the tested mobile phone video call application were feasible but software compatibility should be critically evaluated.Trial registrationRetrospectively registered in the German Clinical Trials Register DRKS00015788, 26thOctober 2018

ESMO open 5(6), e000912 (2020). doi:10.1136/esmoopen-2020-000912, Published by BMJ, London

CD73hi macrophages are enriched in glioblastoma and may contribute to immunotherapy resistance.

Radiotherapy (RT) is currently used in more than 50% of cancer patients during the course of their disease in the curative, adjuvant or palliative setting. RT achieves good local control of tumor growth, conferring DNA damage and impacting tumor vasculature and the immune system. Formerly regarded as a merely immunosuppressive treatment, pre- and clinical observations indicate that the therapeutic effect of RT is partially immune mediated. In some instances, RT synergizes with immunotherapy (IT), through different mechanisms promoting an effective antitumor immune response. Cell death induced by RT is thought to be immunogenic and results in modulation of lymphocyte effector function in the tumor microenvironment promoting local control. Moreover, a systemic immune response can be elicited or modulated to exert effects outside the irradiation field (so called abscopal effects). In this review, we discuss the body of evidence related to RT and its immunogenic potential for the future design of novel combination therapies.

Dietary flavones have promising chemoprotective properties, in particular with regard to cancer, but problems with low oral bioavailability and sometimes unacceptable toxicity have made their use as protective additives to normal diets questionable. However, methylation of free phenolic hydroxyl groups leads to derivatives not susceptible to glucuronic acid or sulfate conjugation, resulting in increased metabolic stability. Methylation also leads to greatly improved transport through biological membranes, such as in intestinal absorption, and much increased oral bioavailability. Recent studies also indicate that methylation results in derivatives with increasing potency to kill cancer cells. They also show high potency towards inhibition of hormone-regulating enzymes, e.g., aromatase, important in the causation of breast cancer. Methylation of the flavones may also result in derivatives with diminished toxic side-effects and improved aqueous solubility. In conclusion, it appears that methylation of dietary flavones as well as of other food products may produce derivatives with much improved health effects.

Many flavonoids have been shown to possess prooxidant properties, capable of causing oxidative stress, especially at larger doses. Here, we examined the potential cell toxicity caused by exposure to the hydroxylated flavones chrysin, apigenin, luteolin and quercetin in comparison to the methylated flavones 5,7-dimethoxyflavone and 3',4'-dimethoxyflavone in normal Rainbow trout hepatocytes. The hydroxylated flavones, especially chrysin, demonstrated cell toxicity and inhibition of DNA synthesis at very low (2 microM) concentrations. The cytotoxicity of chrysin may partially be due to its metabolism by myeloperoxidase, which was shown to be present in these normal trout liver cells (164pmol/(min mg protein)). In contrast, methylated flavones showed no significant metabolism by myeloperoxidase and no signs of toxicity, even at much higher concentrations. These results may be useful for further investigations of cytotoxicity of dietary flavonoids.

Dietary flavonoids and other polyphenols show great potential as cancer chemopreventive agents in cell culture studies. This does not translate well into in vivo activity, because of extensive conjugative metabolism of these compounds in the intestine and liver. This paper presents a review of a flavonoid subclass in which all hydroxyl groups are capped by methylation. This results in dramatically increased metabolic stability and membrane transport in the intestine/liver, thus improving oral bioavailability. The methoxyflavones also show increased cancer chemopreventive properties. At the cancer initiation stage, bioactivation of polyaromatic hydrocarbon carcinogens and binding to DNA are markedly diminished through effects on CYP1A1/1B1 transcription but also through direct interactions with the proteins. At the cancer promotion stage, the proliferation of cancer cells, but not normal cells, is inhibited with greater potency than with the unmethylated flavones. Limited mechanistic experiments, such as of effects on cell cycle regulation, indicate that the mechanisms of methoxyflavone activities are unique, including aromatase inhibition. The cancer preventive effects and mechanisms of the polymethoxyflavones, such as tangeretin and nobiletin, are discussed in comparison. It is concluded that the methoxyflavones have properties that may make them particularly useful as cancer chemopreventive agents.

Ellagic acid (EA), a polyphenol present in berries, has been demonstrated to prevent oesophageal and colon cancer in animals. To better understand the site-specificity of these effects, we studied the accumulation and transport of [14C]EA in rat aerodigestive epithelial cells in-vivo and in cultured human cells. When [14C]EA was administered to rats by gavage, a high content of EA was found in the oesophagus and small intestine at 0.5h after oral administration and in the colon at 12h, with very low amounts in plasma and peripheral tissues. Studies in human intestinal Caco-2 and human oesophageal HET-1A cells found very limited transcellular transport (Caco-2) of EA but high accumulation (Caco-2 and HET-1A) in the cells. In more detailed studies in the Caco-2 cells, accumulation of EA displayed ATP- and Na+-dependency. Multiple interventions permitted the exclusion of a number of transporters as mediators of this uptake. A dramatically reduced transport of EA at low pH (5.5) compared with high pH (7.4) suggested an important role for the negative charge of EA. This was supported by the organic anion transport inhibitors 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and bromosulfophthalein. The latter produced as much as 78% inhibition at the 100 μm concentration. Finally, Caco-2 cells were shown to express organic anion transporter 4 (OAT4) mRNA, as was the human large intestine. EA appears to be accumulated along the aerodigestive tract using OATlike transporters, one of which might be OAT4.

S-adenosyl-l-methionine (SAMe) is an endogenous molecule that is known to be protective against hepatotoxic injury. Although oral SAMe appears to be absorbed across the intestinal mucosa, its systemic bioavailability is low. The reason for this is unknown. Using the Caco-2 cell culture model for enterocyte absorption, we determined the mode by which SAMe is transported across this cell monolayer. We also determined the extent it is taken up by both Caco-2 cells and hepatocytes. In Caco-2 cells transport was observed in both apical to basolateral and basolateral to apical directions. The apparent permeability coefficients (Papp) appeared to be concentration independent and were similar in both directions (0.7 times 10−6 and 0.6 times 10−6 cms−1, respectively), i.e. identical to that of the paracellular transport marker mannitol (0.9 times 10−6 and 0.7 times 10−6 cms−1). This mode of transport was supported by a four-fold increase in the Papp for SAMe transport in Ca++-free buffer. Cellular uptake of SAMe was examined in both Caco-2 cells and cultured rat hepatocytes. Uptake by hepatocytes was not saturable in a concentration range of 0.001–100 μm. Accumulation by both cell types was very low, with a cell:medium ratio at equilibrium of only 0.2–0.5. This low cell accumulation supports the finding of paracellular transport as the only mode of cell membrane transport. Increased hepatocellular protection for SAMe may be accomplished by converting SAMe to a more lipid-soluble prodrug.

The objective of this study was to examine the ability of dietary polyphenols to inhibit cytochrome P450 (CYP) 1A1 expression and activity and benzo[a]pyrene (BaP) DNA binding, with the main emphasis on prevention of chemical-induced hepatic carcinogenesis. For this purpose we used Hep G2 cells, a good model of the normal human hepatocyte for CYP1A1 cell signaling. First, when these cells were exposed to a low concentration (1 microM) of BaP, DNA binding occurred, which dramatically increased after 6 h of treatment. BaP also dramatically induced CYP1A1 activity, protein expression and mRNA levels, the likely reason for the marked increase in DNA binding. Second, we screened 25 polyphenols with highly varying chemical structures for maximum ability to inhibit CYP1A1 activity in the Hep G2 cells. Highly varying responses were obtained, ranging from a 10-fold induction by some polyphenols to almost complete inhibition, in particular by 5,7-dimethoxyflavone (DMF), a flavonoid found in some tropical plants. Third, we examined the ability of DMF to inhibit DNA binding of BaP and the mechanisms involved. DMF (2-20 microM) inhibited BaP-induced DNA binding. DMF also inhibited BaP-induced CYP1A1 activity, CYP1A1 protein expression and mRNA levels. Moreover, DMF directly inhibited the catalytic activity of recombinant CYP1A1 with an IC50 of 0.8 microM. In conclusion, DMF was a highly potent inhibitor of BaP-induced DNA binding and CYP1A1 protein expression and activity in the Hep G2 cells. These properties may make DMF an effective chemoprotectant in chemical-induced liver cancer.

Increasing epidemiological evidence supports the view that dietary flavonoids have protective roles in oral diseases, including cancer. However, the dietary forms of flavonoids, the flavonoid glycosides, must first be hydrolyzed to the aglycones, which is thought to occur mainly in the intestine. In the present study we tested whether this hydrolytic activity occurs in the oral cavity. Saliva was collected from human subjects, incubated with flavonoid glycosides, and analyzed for aglycone formation by HPLC. When quercetin 4'-glucoside or genistein 7-glucoside was incubated with human saliva, hydrolysis to quercetin and genistein, respectively, was detected within minutes. Studies of additional flavonoid glycosides demonstrated that glucose conjugates were rapidly hydrolyzed, but not conjugates with other sugars, i.e., rutin, quercitrin, and naringin. In a limited study of 17 subjects, the interindividual variability in the hydrolysis of genistein 7-glucoside was >20-fold. This supports the contention that salivary hydrolysis of certain flavonoid glucosides may be important in some individuals but not in others. Support for a bacterial contribution to this hydrolysis was obtained from the inhibitory effect of antibacterials in vivo and in vitro and from experiments with subcultured oral bacterial colonies. However, cytosol isolated from oral epithelial cells was also capable of effective hydrolysis. Dietary flavonoid glucosides may thus be hydrolyzed in the oral cavity by both bacteria and shedded epithelial cells to deliver the biologically active aglycones at the surface of the epithelial cells. The aglycones quercetin and genistein both potently inhibited proliferation of oral cancer cells. The large interindividual variability in this hydrolytic activity may be a factor that should be taken into consideration in future studies.

Resveratrol is a dietary constituent suggested to have protective effects against cancer as well as cardiovascular disease. The purpose of the study was to learn whether this agent could be absorbed in man and enter the systemic circulation. This was examined by measuring transport and metabolism of resveratrol (5–40 μM) by the human intestinal epithelial cell line Caco-2 cultured in Transwells. Transport across the Caco-2 monolayer occurred in a direction-independent manner with Papp values of ≅ 7 times 10−6 cm s−1, much higher than for the paracellular transport marker mannitol (≅ 0.4 times 10−6 cms−1), suggesting efficient absorption in-vivo. At the highest resveratrol concentration, the absorption increased, possibly due to saturation of metabolism. In sharp contrast to previous findings in the rat, the metabolism of resveratrol in Caco-2 cells involved mainly sulfation and, to a minor extent, glucuronidation. At low resveratrol concentrations, most of the sulfate conjugate was exported to the apical side, presumably by MRP2, which is well expressed in these cells. At high concentrations, there was a shift towards the basolateral side, possibly involving MRP3, which was recently shown also to be expressed in Caco-2 cells. These results indicate that absorption of resveratrol in-vivo may be high but with limited bioavailability due to efficient sulfate conjugation. Extensive accumulation of resveratrol in the Caco-2 cells, demonstrated in additional experiments, suggests enterocytes as a major target site for this cancer preventive agent.

Aims To describe the oral disposition of the dietary flavonoid chrysin in healthy volunteers.

Flavonoids, dietary components in vegetables, fruits and beverages, may protect against coronary heart disease, stroke and cancer. However, the bioavailability of these compounds is questionable. A previous study in ileostomy patients of the most abundant flavonoid, quercetin, suggested a 52% absorption of its major dietary forms, monoglucoside (QMG) and diglucoside (QDG), from an onion meal. However, this was based on indirect measurements after acid hydrolysis. Because human intestinal Caco-2 cell monolayers showed minimal absorption of the glucosides, we repeated the study in ileostomy patients, using molecularly specific analytical methodology for the intact glucosides and quercetin. The onion meal had high concentrations of both QMG and QDG with only trace amounts of quercetin. The intake of QMG and QDG in four patients ranged from 10.9 to 51.6 mg. No QMG or QDG was detected in the ileostomy fluid. In contrast, the amounts of the aglycone quercetin were substantial, 2.9-11.3 mg. This corresponded to 19.5-35.2% of total quercetin glucosides ingested, implying absorption of 64.5-80.7%. These findings suggest a different interpretation than that from the previous study, i.e., that both QMG and QDG are efficiently hydrolyzed in the small intestine by beta-glucosidases to quercetin, most of which is then absorbed.

Cooked-food mutagens formed when frying meat have been suggested to contribute to the etiology of colon, breast and prostate cancer. The most prevalent of these mutagens is 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which after absorption is bioactivated by both phase I and phase II enzymes. Although available data suggest absorption of PhIP in humans, the extent and mechanism of absorption are unknown. In the present study we examined the transport of [(3)H]PhIP through the human Caco-2 intestinal epithelial cell monolayer, a well-accepted model of human intestinal absorption. The influx, or absorption, was extensive and linear for 2 h and up to a PhIP concentration of 5 microM. Still, the basolateral to apical efflux [apparent permeability coefficient (P(app)) 54.2 +/- 0.7x10(-6) cm/s, mean +/- SEM, n = 24] was 3.6 times greater than the apical to basolateral influx (P(app) 15.1 +/- 0.6x10(-6) cm/s, n = 21, P < 0.0001). Equilibrium exchange experiments demonstrated the efflux to be a true active process. Preincubations with verapamil, an inhibitor of P-glycoprotein-mediated transport, or MK-571, an inhibitor of multidrug resistance-associated protein-mediated transport, stimulated influx and reduced efflux of PhIP, suggesting that PhIP is a substrate for both of these transporters. These findings should be considered when determining exposure to the cooked food mutagens.

Although the bioflavonoids, a large group of polyphenolic natural products, exert chemopreventive effects in cardiovascular disease and cancer, there is little information about the disposition of these dietary components in man. The objective of this study was to investigate the plasma-protein binding of the most abundant bioflavonoid, quercetin, using 14C-labelled quercetin. An ultracentrifugation assay (170 000 g for 16 h at 20°C) was shown to sediment plasma proteins. Binding of quercetin to normal plasma was extensive (99.1 ± 0.5%, mean ± s.d., n = 5). The unbound fraction varied as much as 6-fold, 0.3–1.8%, between subjects. This high binding was independent of quercetin concentration over the range 1.5–15 μM (0.5–5 μg mL−1). Human serum albumin was the primary protein responsible for the binding of quercetin in plasma (99.4 ± 0.1%). Binding by α1-acid glycoprotein (39.2 ± 0.5%) and very-low-density lipoproteins (< 0.5% of total quercetin) did not make substantial contributions to overall plasma binding. The equilibrium association constant for the binding of quercetin to serum albumin was 267 ± 33 times 103 M−1 (n=15). Thermodynamic data for the binding of quercetin to serum albumin indicated spontaneous, endothermic association. Displacement studies suggested that in man the ‘IIA’ subdomain binding site of human serum albumin was the primary binding site for quercetin. Association of quercetin with erythrocytes was significantly (P < 0.001) reduced by plasma protein binding. These data indicate poor cellular availability of quercetin because of its extensive binding to plasma proteins.

We present a transformation, called mirroring, that enables pipelining in semi-systolic and systolic linear arrays. Mir-roring doubles the throughput of semi-systolic arrays and increases the throughput of systolic arrays by one third.

In shared-memory multiprocessors, combining networks serve to eliminate hot spots due to concurrent access to the same memory location. Examples are the NYU Ultracomputer, the IBM RP3 and the Fluent Machine. We present a problem that occurs when trying to implement the Fluent Machine's network nodes with network chips that do not know their position within the network. We formulate the problem mathematically and present two solutions. The first solution requires some additional hardware around nodes that can be put outside network chips. The second solution requires a minor modification of the routing algorithm but no additional hardware is needed.

Enzymatic sulfation of chiral phenolic ethanolamine drugs, e.g. β-agonists, has been shown to be stereoselective in humans. The reaction appears to be specific for the monoamine (M) form of the phenolsulfotransferases (PSTs). In further studies of the stereochemistry of this reaction, we have found the hepatoblastoma-derived cell line Hep G2 to be an excellent human model. These cells contain the M form PST in quantities exceeding those of human liver by about 4-fold. Thus, sulfate conjugates of the β-agonist drugs can easily be synthesized for subsequent structural and enzyme kinetic studies. Although less abundant, the phenol (P) form PST as well as dehydroepiandrosterone sulfotransferase are also expressed in the Hep G2 cells.

BackgroundResveratrol is a natural compound suggested to have beneficial health effects. However, people are consuming resveratrol for this reason without having the adequate scientific evidence for its effects in humans. Therefore, scientific valid recommendations concerning the human intake of resveratrol based on available published scientific data are necessary. Such recommendations were formulated after the Resveratrol 2010 conference, held in September 2010 in Helsingør, Denmark.MethodologyLiterature search in databases as PUBMED and ISI Web of Science in combination with manual search was used to answer the following five questions: (1)Can resveratrol be recommended in the prevention or treatment of human diseases?; (2)Are there observed "side effects" caused by the intake of resveratrol in humans?; (3)What is the relevant dose of resveratrol?; (4)What valid data are available regarding an effect in various species of experimental animals?; (5)Which relevant (overall) mechanisms of action of resveratrol have been documented?Conclusions/significanceThe overall conclusion is that the published evidence is not sufficiently strong to justify a recommendation for the administration of resveratrol to humans, beyond the dose which can be obtained from dietary sources. On the other hand, animal data are promising in prevention of various cancer types, coronary heart diseases and diabetes which strongly indicate the need for human clinical trials. Finally, we suggest directions for future research in resveratrol regarding its mechanism of action and its safety and toxicology in human subjects.

Metabolism of propranolol by the human hepatoma cell line Hep G2 was studied. Although metabolism qualitatively was similar to that in-vivo, the P450-mediated N-desisopropylation clearly predominated. Pretreatment of cells with 3-methylcholanth-rene increased the activity of this pathway 14-fold, whereas phenobarbitone had no effect. This is similar to the pathway-selective inductive response observed for cigarette smoking in-vivo. As in-vivo, secondary metabolism of N-desisopropylpropranolol was extensive. This could, however, be completely blocked by 0·1 μm clorgyline, a potent MAO type A inhibitor. As in human liver microsomes, the stereochemistry of propranolol metabolism demonstrated a preference for the R(+)-enantiomer. These observations emphasize the usefulness of the Hep G2 cell line as a model of man.

Dietary polyphenols, including flavonoids, have been implied to have cancer preventive properties. Suggested mechanisms include inhibition of carcinogen-activating cytochrome P450 (CYP) transcription and activities. These studies have focused mainly on CYP1A1. However, CYP1B1 has recently been shown to be of particular importance in smoking-induced oral and oesophageal cancer. Previous observations in our laboratory demonstrated that methoxylated flavonoids may be effective inhibitors of CYP1A1 transcription and activity as well as being orally bioavailable. In this study, an initial screening of 19 methoxylated flavones, using the ethoxyresorufin de-ethylation assay in human oral squamous cell carcinoma SCC-9 cells pretreated with 1μM benzo[a]pyrene, identified six strongly inhibitory compounds for further studies. The effect of these flavones on CYP1B1 mRNA expression was measured with quantitative branched DNA methodology. Four of the compounds −3′,4′-dimethoxyflavone and 5,7,4′-trimethoxyflavone and, in particular, 7,3′-dimethoxyflavone and 7,4′-dimethoxyflavone — were potent inhibitors of CYP1B1 mRNA expression. Two of the more common unmethylated polyphenols — curcumin and quercetin — were also potent inhibitors. Whereas most unmethylated polyphenols, such as curcumin and quercetin, have very poor bioavailability, the high metabolic stability of the methoxylated flavones studied here suggests that these CYP1B1 inhibitors may also be effective in-vivo.

Previous studies have shown chrysin, 7-hydroxyflavone and 7,4′-dihydroxyflavone to be the most potent flavonoid inhibitors of aromatase. However, very poor oral bioavailability is a major limitation for the successful use of dietary flavonoids as chemopreventive agents. We have recently shown that methylated flavones, including 5,7-dimethoxyflavone, 7-methoxyflavone and 7,4′-dimethoxyflavone, are much more resistant to metabolism than their unmethylated analogs and have much higher intestinal absorption. In this study, we examined these fully methylated flavones as potential aromatase inhibitors for the prevention and/or treatment of hormone-dependent cancers. Whereas 5,7-dimethoxyflavone had poor effect compared to its unmethylated analog chrysin, 7-methoxyflavone and 7,4′-dimethoxyflavone were almost equipotent to their unmethylated analogs with IC 50 values of 2–9 μM. Thus, some fully methylated flavones appear to have great potential as cancer chemopreventive/chemotherapeutic agents.

A recent study in human hepatic preparations demonstrated that a methylated flavone, 5,7-dimethoxyflavone (DMF), abundant in certain plants, such as the leaves of Piper caninum, has high metabolic ...

Polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (BaP) mainly induce lung cancer in humans, but induce liver cancer in fishes. The chemoprevention of cancers through inhibition of molecular events via phytochemicals is a potentially beneficial area of research, and has been carried out in human cell cultures in the past. Carcinogenesis initiation events are thought to occur in similar ways in fish and humans. Our study investigated the feasibility of using cultured rainbow trout CRL-2301 liver cells as a model for BaP-induced carcinogenesis and its prevention by dietary phytochemicals. Treatment with 1 microM BaP resulted in extensive time-dependent covalent binding to cellular DNA and marked cytochrome P450 (CYP) 1A induction, for both about a 20-fold increase, which is similar to what has been observed in cultured human cells. A surprisingly high expression of epoxide hydrolase (EH) activity in these cells likely contributed substantially to the bioactivation of BaP. Two methoxylated flavones and the stilbene resveratrol were effective inhibitors of both the BaP-DNA binding and CYP 1A induction, in particular 5,7-dimethoxyflavone (5,7-DMF), supporting a role for these dietary compounds as cancer chemopreventive agents. Unlike in human liver or bronchial cells, the main mechanism of inhibition of BaP-induced CYP 1A activity in trout liver cells appears to be direct competition at the protein level. Different cellular responses in any particular model used can be expected and the effect of cell context on the biological responses to xenobiotics, including carcinogens as well as polyphenols, must be considered. The trout CRL-2301 cells' sensitivity to BaP treatment is a clear advantage when contemplating a model system for studies of PAH-induced carcinogenesis and cancer chemoprevention. However, extrapolation to human organs should be done cautiously.

Dietary modulation of carcinogenesis-related pathwaysDietary item or component studied: 5,7- dimethoxyflavone (5,7-DMF) and 30,40-dimethoxyflavone (30,40-DMF)Pathways studied: BaP- DNA binding by inhibition of CYP1B1/1A1 activity and/or protein expressionStudy type (in vitro, animals, humans): in vitroImpact on pathway (including dose-response): 5,7-DMF: BaP-DNA binding was markedly inhibited and the BaP-induced part of the CYP1B1 mRNA expression (P

Poor oral bioavailability has been a major limitation for the successful use of dietary flavonoids as cancer chemopreventive agents. In this study, we examined fully methylated flavones as promising improved agents. In the human oral SCC-9 cancer cells, 5,7-dimethoxyflavone and 5,7,4'-trimethoxyflavone were both 10 times more potent inhibitors of cell proliferation (IC(50) values 5-8 microM) than the corresponding unmethylated analogs chrysin and apigenin. Flow cytometry indicated that both methylated flavones arrested the SCC-9 cells in the G1 phase with a concomitant decrease in the S phase, dramatically different from the unmethylated analogs, which promoted G2/M phase arrest. Both methylated compounds inhibited the proliferation of two other cancer cell lines with very little effect on two immortalized normal cell lines. Examination of additional flavone structures indicated that methylated flavones in general have antiproliferative properties. Finally, we demonstrated that 5,7-dimethoxyflavone, in contrast to its unmethylated analog chrysin, was well absorbed and had high oral bioavailability as well as tissue accumulation in vivo in the rat. Thus, fully methylated flavones appear to have great potential as cancer chemopreventive/chemotherapeutic agents, in particular in oral cancer.

The metabolic stability of two potential cancer chemopreventive flavones, i.e. 5,7-dimethoxyflavone (5,7-DMF) and 3',4'-dimethoxyflavone (3',4'-DMF), compared with the non-methylated flavone galangin (3,5,7-trihydroxyflavone), was investigated in human hepatic preparations. Galangin, as expected, was extensively metabolized mainly by glucuronidation in human liver S9 fractions in the presence of appropriate co-factors. In contrast, 5,7-DMF and 3',4'-DMF were metabolically highly stable with only a small fraction of 3',4'-DMF undergoing oxidation. Consistent with the S9 fraction results, galangin was almost completely depleted after 2-h incubations in freshly plated hepatocytes. The hepatocytes also showed some metabolism of 3',4'-DMF, but virtually none of 5,7-DMF. In human liver microsomes, 5,7-DMF was more metabolically stable than 3',4'-DMF. The observations present a new strategy for examining the metabolic stability of dietary flavonoids and suggest that methylated flavonoids may have a high oral bioavailability compared with their non-methylated forms, which will make them more likely to be useful as cancer chemoprotectants.