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The response to tumor-initiating inflammatory and genetic insults can vary among morphologically indistinguishable cells, suggesting as yet uncharacterized roles for epigenetic plasticity during early neoplasia. To investigate the origins and impact of such plasticity, we performed single-cell analyses on normal, inflamed, premalignant, and malignant tissues in autochthonous models of pancreatic cancer. We reproducibly identified heterogeneous cell states that are primed for diverse, late-emerging neoplastic fates and linked these to chromatin remodeling at cell-cell communication loci. Using an inference approach, we revealed signaling gene modules and tissue-level cross-talk, including a neoplasia-driving feedback loop between discrete epithelial and immune cell populations that was functionally validated in mice. Our results uncover a neoplasia-specific tissue-remodeling program that may be exploited for pancreatic cancer interception.

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

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

Factor analysis can drive biological discovery by decomposing single-cell gene expression data into a minimal set of gene programs that correspond to processes executed by cells in a sample. However, matrix factorization methods are prone to technical artifacts and poor factor interpretability. We have developed Spectra, an algorithm that identifies user-provided gene programs, modifies them to dataset context as needed, and detects novel programs that together best explain expression covariation. Spectra overcomes the dominance of cell-type signals by modeling cell-type-specific programs, and can characterize interpretable cell states along a continuum. We show that it outperforms existing approaches in challenging tumor immune contexts; Spectra finds factors that change under immune checkpoint therapy, disentangles the highly correlated features of CD8+T-cell tumor reactivity and exhaustion, finds a novel program that explains continuous macrophage state changes under therapy, and identifies cell-type-specific immune metabolic programs.

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

The response to tumor-initiating inflammatory and genetic insults can vary amongst morphologically indistinguishable cells, suggesting yet uncharacterized roles for epigenetic plasticity during early neoplasia. To investigate the origins and impact of such plasticity, we perform single-cell analyses on normal, inflamed, pre-malignant and malignant tissues in autochthonous models of pancreatic cancer. We reproducibly identify heterogeneous cell-states that are primed for diverse late-emerging neoplastic fates and link these to chromatin remodeling at cell-cell communication loci. Using a new inference approach, we reveal signaling gene modules and tissue-level crosstalk, including a neoplasia-driving feedback loop between discrete epithelial and immune cell populations that we validate by genetic perturbation in mice. Our results uncover a neoplasia-specific tissue remodeling program that may be exploited for pancreas cancer interception.One-Sentence SummarySingle-cell analysis reveals that enhanced epigenetic plasticity drives pro-neoplastic crosstalk in early pancreatic cancer.

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.

Patients with cancer frequently receive immune-checkpoint inhibitors (ICIs), which may modulate immune responses to COVID-19 vaccines. Recently, cytokine release syndrome (CRS) was observed in a patient with cancer who received BTN162b2 vaccination under ICI treatment. Here, we analyzed adverse events and serum cytokines in patients with 23 different tumors undergoing (n = 64) or not undergoing (n = 26) COVID-19 vaccination under ICI therapy in a prospectively planned German single-center cohort study (n = 220). We did not observe clinically relevant CRS (≥grade 2) after vaccination (95% CI 0-5.6%; Common Terminology of Adverse Events v.5.0) in this small cohort. Within 4 weeks after vaccination, serious adverse events occurred in eight patients (12.5% 95% CI 5.6-23%): six patients were hospitalized due to events common under cancer therapy including immune related adverse events and two patients died due to conditions present before vaccination. Despite absence of CRS symptoms, a set of pairwise-correlated CRS-associated cytokines, including CXCL8 and interleukin-6 was1.5-fold upregulated in 40% (95% CI 23.9-57.9%) of patients after vaccination. Hence, elevated cytokine levels are common and not sufficient to establish CRS diagnosis.

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.

Evaluation of the in vitro human liver microsome and hepatocyte metabolism of ketotifen demonstrated that norketotifen (NK) is the major demethylated hepatic metabolite of ketotifen. It is here reported that NK is completely devoid of the severe and dose-limiting sedative effects of ketotifen. Thus, while ketotifen is clinically dose-limited to 1 mg, bid, there are no dose-limiting sedative effects elicited by NK, even after the highest single-dose (16 mg) or after repeat-doses (8 mg × 7 days) in humans or after the highest doses given to dogs in repeat-dose toxicological studies (40 mg/kg × 14 days). In addition, NK-but not ketotifen-was found to express potent and dose-dependent inhibition of the release of the pro-inflammatory cytokine TNFα from activated human buffy coat preparations. Thus, when used as an anti-inflammatory drug, ketotifen is the sedating prodrug which is converted to NK a nonsedating metabolite with anti-inflammatory activity.

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

8509 Background: 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. The emerging consensus on SCLC subtypes has led to new questions, such as whether subtypes are associated with different disease stages, metastatic potential, or immune microenvironments; whether there is plasticity between subtypes; and whether novel SCLC phenotypes exist. Single cell RNA sequencing (scRNA-seq) offers a unique opportunity to address these questions by dissecting intratumoral transcriptional heterogeneity and the surrounding tumor microenvironment (TME). However, efforts to apply this technology to human SCLC tumors have been limited, as these tumors are infrequently resected. Methods: We have optimized protocols to process both surgical resections and biopsies to construct the first single-cell atlas of SCLC patient tumors (N = 21), with comparative lung adenocarcinoma (LUAD) and normal lung data. We leverage computational methods including diffusion maps and non-negative matrix factorization to perform a deep annotation of SCLC phenotypes and the surrounding immune TME. We perform validation experiments using flow cytometry, Vectra, and immunohistochemistry in independent SCLC cohorts, as well as genetic manipulation in preclinical SCLC models. Results: Our data reveals substantial transcriptional heterogeneity in SCLC both within and across tumors and confirms a pro-metastatic gene program in SCLC-N subtype characterized by epithelial-mesenchymal transformation and axonogenesis. Beyond known subtypes, we discover a PLCG2-high tumor cell population with stem-like, pro-metastatic features that recurs across subtypes and predicts significantly worse overall survival. 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. Conclusions: This atlas of SCLC illustrates how canonical subtypes and a novel PLCG2-high recurrent tumor subclone enlist diverse gene programs to create tumor heterogeneity and facilitate metastasis in a profoundly immunosuppressed TME. Our dataset provides further insight into tumor and immune biology in SCLC at single-cell resolution, with potential implications for design of novel targeted therapies and immunotherapeutic approaches.

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.

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

This paper reviews our current understanding of the absorption, bioavailability, and metabolism of resveratrol, with an emphasis on humans. The oral absorption of resveratrol in humans is about 75% and is thought to occur mainly by transepithelial diffusion. Extensive metabolism in the intestine and liver results in an oral bioavailability considerably less than 1%. Dose escalation and repeated dose administration of resveratrol does not appear to alter this significantly. Metabolic studies, both in plasma and in urine, have revealed major metabolites to be glucuronides and sulfates of resveratrol. However, reduced dihydroresveratrol conjugates, in addition to highly polar unknown products, may account for as much as 50% of an oral resveratrol dose. Although major sites of metabolism include the intestine and liver (as expected), colonic bacterial metabolism may be more important than previously thought. Deconjugation enzymes such as β-glucuronidase and sulfatase, as well as specific tissue accumulation of resveratrol, may enhance resveratrol efficacy at target sites. Resveratrol analogs, such as methylated derivatives with improved bioavailability, may be important in future research.

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 have many biological properties that could make them useful as chemopreventive agents. However, very poor oral bioavailability makes them largely ineffective in vivo. The low bioavailability is mainly due to highly efficient glucuronic acid and sulfate conjugation of these mono- or polyhydroxylated agents in the intestinal/hepatic barrier. This review describes how the methyl capping of all free hydroxyl groups of flavones results in dramatically increased metabolic stability, as the metabolism is shifted to less efficient CYP-mediated oxidation. This was demonstrated best by using the human liver S9 fraction with an appropriate selection of cofactors. In addition, the intestinal transport of flavones was much improved through methylation, as shown in Caco-2 cell Transwell experiments. In vivo in the rat, oral administration of one methylated flavone resulted in high bioavailability and tissue distribution with no detectable levels of its unmethylated analogue. In addition to increased metabolic stability, methylation resulted in markedly increased inhibition of cancer cell proliferation. Thus, methylation appears to be a simple and effective way of increasing both metabolic resistance and transport of the flavonoids and, most important, some of their major biological activities.

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.

Methoxylated flavones were recently shown to be promising cancer chemopreventive agents. Their high metabolic stability compared with the hydroxylated analogs was shown in our laboratory using the human hepatic S9 fraction with cofactors for glucuronidation, sulfation, and oxidation. In the present study, the resistance of methoxylated flavones toward oxidative metabolism was investigated with human liver microsomes and recombinant cytochrome P450 (P450) isoforms. Among 15 methoxylated flavones investigated, the two partially methylated compounds, tectochrysin and kaempferide, were among the most susceptible to microsomal oxidation (Cl(int) 283 and 82 ml/min/kg). Of the fully methylated compounds, 5,7-dimethoxyflavone and 5-methoxyflavone were the most stable (Cl(int) 13 and 18 ml/min/kg, respectively), whereas 4'-methoxyflavone, 3'-methoxyflavone, 5,4'-dimethoxyflavone, and 7,3'-dimethoxyflavone were the least stable (Cl(int) 161, 140, 119, and 92 ml/min/kg, respectively), emphasizing the importance of the positions of the methoxy substituents in the flavone ring system. Among the five P450 isoforms tested, CYP1A1 showed the highest rate of metabolism of fully methylated compounds, followed by CYP1A2 and CYP3A4. CYP2C9 and CYP2D6 gave minimal disappearance of the parent compound. Finally, in incubations with hepatic S9 fraction with cofactors for oxidation and both conjugation reactions, partially methylated flavones, as expected, were much less metabolically stable than fully methylated flavones, confirming that oxidative demethylation is the rate-limiting metabolic reaction for fully methylated flavones only. In summary, the rate of oxidative metabolism of methoxylated flavones, mainly involving CYP1A1 and CYP1A2, varied widely, even between compounds with very similar structures.

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.

Oral cancer, originating from smoking-induced lesions of the basal cells in the complex stratified oral epithelium, is difficult to treat. Early detection of premalignant lesions, e.g., leukoplakia, has suggested the possibility of chemopreventive measures, such as topical application of antimutagenic/antiproliferative dietary or pharmaceutical agents. As an extension of a study in human oral epithelial cell monolayers, we determined the carcinogen, i.e., benzo[a]pyrene (BaP), transport, bioactivation, and DNA binding in a bioengineered human gingival epithelial tissue construct and the chemopreventive effects of dietary polyphenols. Short-term experiments showed that both types of compounds can traverse this tissue as well as be effectively taken up by the tissue. The model cigarette smoke carcinogen BaP very slowly, but to a great extent, accumulated in the tissue with maximal uptake at 24 h. Such exposure clearly resulted in DNA binding of BaP by the tissue. This DNA binding was associated with BaP-induced CYP1B1 as well as CYP1A1 expression, as evidenced by mRNA measurements. Cotreatment of the oral tissue with dietary polyphenols, including resveratrol and quercetin, and BaP, resulted in significant inhibition of the BaP-DNA binding. Using fluorescence microscopy as well as simultaneous autoradiography, we also demonstrated that quercetin indeed penetrates the entire stratified tissue layer, but that quercetin was also oxidized within the cells. Thus, this bioengineered oral tissue construct opens up improved ways of understanding and preventing/treating smoking-induced oral cancer.

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.

Quercetin is an abundant flavonoid in the human diet with numerous biological activities, which may contribute to the prevention of human disease but also may be potentially harmful. Quercetin is oxidized in cells to products capable of covalently binding to cellular proteins, a process that may be important for its biological activities. In the present study, using radiolabeled drug and quantifying the products after electrophoretic separation, proteins to which oxidized quercetin is binding irreversibly were identified. The binding of quercetin to human serum albumin (HSA) in human blood and the effect of stimulation of neutrophilic myeloperoxidase on this binding were also measured. The in vitro binding of quercetin to eight proteins in the presence of catalytic amounts of horseradish peroxidase and hydrogen peroxide was highly selective for HSA. For all proteins the binding was dramatically decreased by reduced L-glutathione. In the blood samples, the release of neutrophilic myeloperoxidase by phorbol ester caused a 3-fold increase in the binding of quercetin to HSA. This study shows that quercetin in the presence of peroxidase/hydrogen peroxide covalently links to proteins with a particularly high affinity for HSA and that this also may occur in vivo after exposure to quercetin. This provides further insights into the complex behavior of this major dietary flavonoid.