Sulforaphane-enriched extracts from glucoraphanin-rich broccoli exert antimicrobial activity against gut pathogens in vitro and innovative cooking methods increase in vivo intestinal delivery of sulforaphane.

PURPOSE: Studies on broccoli (Brassica oleracea var. italica) indicate beneficial effects against a range of chronic diseases, commonly attributed to their bioactive phytochemicals. Sulforaphane, the bioactive form of glucoraphanin, is formed by the action of the indigenous enzyme myrosinase. This study explored the role that digestion and cooking practices play in bioactivity and bioavailability, especially the rarely considered dose delivered to the colon. METHODS: The antimicrobial activity of sulforaphane extracts from raw, cooked broccoli and cooked broccoli plus mustard seeds (as a source myrosinase) was assessed. The persistence of broccoli phytochemicals in the upper gastrointestinal tract was analysed in the ileal fluid of 11 ileostomates fed, in a cross-over design, broccoli soup prepared with and without mustard seeds. RESULTS: The raw broccoli had no antimicrobial activity, except against Bacillus cereus, but cooked broccoli (with and without mustard seeds) showed considerable antimicrobial activity against various tested pathogens. The recovery of sulforaphane in ileal fluids post soup consumption was < 1% but the addition of mustard seeds increased colon-available sulforaphane sixfold. However, when sulforaphane was extracted from the ileal fluid with the highest sulforaphane content and tested against Escherichia coli K12, no inhibitory effects were observed. Analysis of glucosinolates composition in ileal fluids revealed noticeable inter-individual differences, with six "responding" participants showing increases in glucosinolates after broccoli soup consumption. CONCLUSIONS: Sulforaphane-rich broccoli extracts caused potent antimicrobial effects in vitro, and the consumption of sulforaphane-enriched broccoli soup may inhibit bacterial growth in the stomach and upper small intestine, but not in the terminal ileum or the colon.

Antioxidant and antigenotoxic activities of Ginkgo biloba L. leaf extract are retained after in vitro gastrointestinal digestive conditions.

PURPOSE: The recognized biological properties of Ginkgo biloba extracts potentiate their utilization as an ingredient for functional foods. However, the digestive conditions can affect the chemical composition of the extracts and consequently their biological properties, which can lead to food safety problems. Thus, the impact of in vitro-simulated upper gastrointestinal tract digestion on the chemical composition and bioactivity of Ginkgo biloba leaf extract (GBE) was evaluated. METHODS: Physicochemical conditions of human digestion were simulated in vitro, and its impact on the chemical composition of GBE was investigated by electrospray ionization-mass spectrometry. The persistence of bioactivity was investigated by subjecting GBE and the in vitro digested extract (DGBE) to the same methodology. Antioxidant properties were assessed using 2',7'-dichlorofluorescein diacetate to measure the intracellular oxidation of Schizosaccharomyces pombe cells pre-incubated with GBE or DGBE and exposed to H2O2. Antigenotoxicity was tested by comet assay in HT-29 colon cancer cells pre-incubated with GBE or DGBE and challenged with H2O2. RESULTS: The chemical analysis revealed a considerable change in chemical composition upon digestion. Pre-incubation with GBE or DGBE attenuated the H2O2-imposed intracellular oxidation in wild-type S. pombe cells, unlike the oxidative stress response-affected mutants sty1 and pap1, and decreased H2O2-induced DNA damage in HT-29 cells. The extracts did not induce toxicity in these eukaryotic models. CONCLUSION: The chemical composition of GBE was affected by in vitro digestion, but the antioxidant and antigenotoxic activities persisted. Therefore, G. biloba extract may be suitable for use as food additive and contribute to a healthy colon.

Development and Validation of an Algorithm to Determine Spontaneous versus Provider-Initiated Preterm Birth in US Vital Records.

BACKGROUND: Determining whether initiation of preterm birth was spontaneous, or through labour induction or caesarean without labour or membrane rupture is critical in surveillance and aetiological research on preterm birth, although this information is not explicitly included on the US Birth Certificate. Algorithms combining several fields from birth certificates have been developed to infer the initiating event, but none has been validated against manual review of original obstetric records. Our objective was to develop a birth certificate-based algorithm to determine initiation of preterm birth and validate it by manual review of original records. METHODS: We developed an algorithm from the 2003 US Standard Birth Certificate to determine spontaneous vs. indicated preterm birth. The algorithm was first tested on obstetrical records from 80 preterm (<37 weeks) births in Columbus OH (2006-12) abstracted by an obstetrics research nurse and reviewed by an obstetrician-gynecologist. Onset of delivery was spontaneous if the initiating event was premature rupture of membranes (PROM) or contractions, or indicated if the initiating event was induction or caesarean without labour or PROM. The algorithm was validated in an independent sample of 100 preterm births from four hospitals. RESULTS: Codes for tocolysis, fetal intolerance of labour, and anaesthesia during labour did not predict labour and were dropped. The final algorithm correctly classified 73/80 cases, kappa = 0.83. In the validation, 86/100 cases were correctly classified. The kappa statistic was 0.68 (0.52, 0.83); predictive values for spontaneous and indicated onset were 85% (75%, 92%) and 89% (71%, 98%). CONCLUSIONS: The algorithm distinguished spontaneous from indicated preterm birth, using birth certificates, with good accuracy.

Bcl-xL Is a Key Mediator of Apoptosis Following KRASG12C Inhibition in KRASG12C-mutant Colorectal Cancer.

Novel covalent inhibitors of KRASG12C have shown limited response rates in patients with KRASG12C-mutant (MT) colorectal cancer. Thus, novel KRASG12C inhibitor combination strategies that can achieve deep and durable responses are needed. Small-molecule KRASG12C inhibitors AZ'1569 and AZ'8037 were used. To identify novel candidate combination strategies for AZ'1569, we performed RNA sequencing, siRNA, and high-throughput drug screening. Top hits were validated in a panel of KRASG12CMT colorectal cancer cells and in vivo. AZ'1569-resistant colorectal cancer cells were generated and characterized. We found that response to AZ'1569 was heterogeneous across the KRASG12CMT models. AZ'1569 was ineffective at inducing apoptosis when used as a single agent or combined with chemotherapy or agents targeting the EGFR/KRAS/AKT axis. Using a systems biology approach, we identified the antiapoptotic BH3-family member BCL2L1/Bcl-xL as a top hit mediating resistance to AZ'1569. Further analyses identified acute increases in the proapoptotic protein BIM following AZ'1569 treatment. ABT-263 (navitoclax), a pharmacologic Bcl-2 family inhibitor that blocks the ability of Bcl-xL to bind and inhibit BIM, led to dramatic and universal apoptosis when combined with AZ'1569. Furthermore, this combination also resulted in dramatically attenuated tumor growth in KRASG12CMT xenografts. Finally, AZ'1569-resistant cells showed amplification of KRASG12C, EphA2/c-MET activation, increased proinflammatory chemokine profile and cross-resistance to several targeted agents. Importantly, KRAS amplification and AZ'1569 resistance were reversible upon drug withdrawal, arguing strongly for the use of drug holidays in the case of KRAS amplification. Taken together, combinatorial targeting of Bcl-xL and KRASG12C is highly effective, suggesting a novel therapeutic strategy for patients with KRASG12CMT colorectal cancer.

Cancer-Associated SF3B1 Mutations Confer a BRCA-Like Cellular Phenotype and Synthetic Lethality to PARP Inhibitors.

Mutations in SF3B1 have been identified across several cancer types. This key spliceosome component promotes the efficient mRNA splicing of thousands of genes including those with crucial roles in the cellular response to DNA damage. Here, we demonstrate that depletion of SF3B1 specifically compromises homologous recombination (HR) and is epistatic with loss of BRCA1. More importantly, the most prevalent cancer-associated mutation in SF3B1, K700E, also affects HR efficiency and as a consequence, increases the cellular sensitivity to ionizing radiation and a variety of chemotherapeutic agents, including PARP inhibitors. In addition, the SF3B1 K700E mutation induced unscheduled R-loop formation, replication fork stalling, increased fork degradation, and defective replication fork restart. Taken together, these data suggest that tumor-associated mutations in SF3B1 induce a BRCA-like cellular phenotype that confers synthetic lethality to DNA-damaging agents and PARP inhibitors, which can be exploited therapeutically. SIGNIFICANCE: The cancer-associated SF3B1K700E mutation induces DNA damage via generation of genotoxic R-loops and stalled replication forks, defective homologous recombination, and increased replication fork degradation, which can be targeted with PARP inhibitors.

Ex Vivo Fecal Fermentation of Human Ileal Fluid Collected After Wild Strawberry Consumption Modulates Human Microbiome Community Structure and Metabolic Output and Protects Against DNA Damage in Colonic Epithelial Cells.

SCOPE: Wild strawberries (Fragaria vesca) are richer in (poly)phenols than common commercial strawberry varieties, e.g., Fragaria × ananassa. (Poly)phenols and their microbiota-derived metabolites are hypothesized to exert bioactivity within the human gut mucosa. To address this, the effects of wild strawberries are investigated with respect to their bioactivity and microbiota-modulating capacity using both in vitro and ex vivo approaches. METHODS AND RESULTS: Ileal fluids collected pre- (0h) and post-consumption (8h) of 225 g wild strawberries by ileostomates (n = 5) and also in vitro digested strawberry varieties (Fragaria vesca and Fragaria × ananassa Duchesne) supernatants are collected. Subsequent fermentation of these supernatants using an in vitro batch culture proximal colon model reveals significant treatment-specific changes in microbiome community structure in terms of alpha but not beta diversity at 24 h. Nutri-kinetic analysis reveals a significant increase in the concentration of gut microbiota catabolites, including 3-(4hydroxyphenyl)propionic acid, 3-(3-hydroxyphenyl)propanoic acid, and benzoic acid. Furthermore, post-berry ileal fermentates (24 h) significantly (p < 0.01) decrease DNA damage (% Tail DNA, COMET assay) in both HT29 cells (∼45%) and CCD 841 CoN cells (∼25%) compared to untreated controls. CONCLUSIONS: Post berry consumption fermentates exhibit increased overall levels of (poly)phenolic metabolites, which retains their bioactivity, reducing DNA damage in colonocytes.

Ex vivo fecal fermentation of human ileal fluid collected after raspberry consumption modifies (poly)phenolics and modulates genoprotective effects in colonic epithelial cells.

Diets rich in fruit and vegetables are associated with a decreased incidence of colorectal cancer (CRC) due, in part, to the bioactive (poly)phenolic components and their microbiota-mediated metabolites. This study investigated how such compounds, derived from ingested raspberries in the gastrointestinal tract, may exert protective effects by reducing DNA damage. Ileal fluids collected pre- and post-consumption of 300 g of raspberries by ileostomists (n = 11) were subjected to 24 h ex vivo fermentation with fecal inoculum to simulate interaction with colonic microbiota. The impact of fermentation on (poly)phenolics in ileal fluid was determined and the bioactivity of ileal fluids pre- and post fermentation investigated. (Poly)phenolic compounds including sanguiin H-6, sanguiin H-10 and cyanidin-3-O-sophoroside decreased significantly during fermentation while, in contrast, microbial catabolites, including 3-(3'-hydroxyphenyl)propanoic acid, 3-hydroxybenzoic acid and benzoic acid increased significantly. The post-raspberry ileal fermentate from 9 of the 11 ileostomates significantly decreased DNA damage (~30%) in the CCD 841 CoN normal cell line using an oxidative challenge COMET assay. The raspberry ileal fermentates also modulated gene expression of the nuclear factor 2-antioxidant responsive element (Nrf2-ARE) pathway involved in oxidative stress cytoprotection, namely Nrf2, NAD(P)H dehydrogenase, quinone-1 and heme oxygenase-1. Four of the phenolic catabolites were assessed individually, each significantly reducing DNA damage from an oxidative challenge over a physiologically relevant 10-100 µM range. They also induced a differential pattern of expression of key genes in the Nrf2-ARE pathway in CCD 841 CoN cells. The study indicates that the colon-available raspberry (poly)phenols and their microbial-derived catabolites may play a role in protection against CRC in vivo.

Clinical Positioning of the IAP Antagonist Tolinapant (ASTX660) in Colorectal Cancer.

Inhibitors of apoptosis proteins (IAPs) are intracellular proteins, with important roles in regulating cell death, inflammation, and immunity. Here, we examined the clinical and therapeutic relevance of IAPs in colorectal cancer. We found that elevated expression of cIAP1 and cIAP2 (but not XIAP) significantly correlated with poor prognosis in patients with microsatellite stable (MSS) stage III colorectal cancer treated with 5-fluorouracil (5FU)-based adjuvant chemotherapy, suggesting their involvement in promoting chemoresistance. A novel IAP antagonist tolinapant (ASTX660) potently and rapidly downregulated cIAP1 in colorectal cancer models, demonstrating its robust on-target efficacy. In cells co-cultured with TNFα to mimic an inflammatory tumor microenvironment, tolinapant induced caspase-8-dependent apoptosis in colorectal cancer cell line models; however, the extent of apoptosis was limited because of inhibition by the caspase-8 paralogs FLIP and, unexpectedly, caspase-10. Importantly, tolinapant-induced apoptosis was augmented by FOLFOX in human colorectal cancer and murine organoid models in vitro and in vivo, due (at least in part) to FOLFOX-induced downregulation of class I histone deacetylases (HDAC), leading to acetylation of the FLIP-binding partner Ku70 and downregulation of FLIP. Moreover, the effects of FOLFOX could be phenocopied using the clinically relevant class I HDAC inhibitor, entinostat, which also induced acetylation of Ku70 and FLIP downregulation. Further analyses revealed that caspase-8 knockout RIPK3-positive colorectal cancer models were sensitive to tolinapant-induced necroptosis, an effect that could be exploited in caspase-8-proficient models using the clinically relevant caspase inhibitor emricasan. Our study provides evidence for immediate clinical exploration of tolinapant in combination with FOLFOX in poor prognosis MSS colorectal cancer with elevated cIAP1/2 expression.

The composition of potentially bioactive triterpenoid glycosides in red raspberry is influenced by tissue, extraction procedure and genotype.

The beneficial effects of consumption of berry fruits on a range of chronic diseases has been attributed (at least in part) to the presence of unique phytochemicals. Recently, we identified novel ursolic acid-based triterpenoid glycosides (TTPNs) in raspberry fruit and demonstrated their survival in human ileal fluids after feeding which confirmed their colon-availability in vivo. In this paper, in vitro digestion studies demonstrated that certain TTPNs were stable under gastrointestinal conditions and confirmed that these components may have been responsible for bioactivity noted in previous studies. Sequential extractions of raspberry puree, isolated seeds and unseeded puree showed that certain TTPN components (e.g. peak T1 m/z 679, and T2 m/z 1358) had different extractabilities in water/solvent mixes and were differentially associated with the seeds. Purified seed TTPNs (mainly T1 and T2) were shown to be anti-genotoxic in HT29 and CCD841 cell based in vitro colonocyte models. Further work confirmed that the seeds contained a wider range of TTPN-like components which were also differentially extractable in water/solvent mixes. This differential extractability could influence the TTPN composition and potential bioactivity of the extracts. There was considerable variation in total content of TTPNs (∼3-fold) and TTPN composition across 13 Rubus genotypes. Thus, TTPNs are likely to be present in raspberry juices and common extracts used for bioactivity studies and substantial variation exists in both content and composition due to genetics, tissue source or extraction conditions, which may all affect observed bioactivity.

Novel colon-available triterpenoids identified in raspberry fruits exhibit antigenotoxic activities in vitro.

SCOPE: Ileostomy studies provide a unique insight into digestion of food, allowing identification of physiologically relevant dietary phytochemicals and their metabolites important to gut health. We previously reported the consistent increase of components in ileal fluids of ileostomates after consumption of raspberries with use of nontargeted LC-MSn techniques and data deconvolution software highlighting two major unknown components (m/z 355 and 679). METHODS AND RESULTS: In-depth LC-MSn analyses suggested that the ileal m/z 355 components were p-coumaroyl glucarates. These compounds have not been identified previously and were confirmed in raspberry extracts after partial purification. The major ileal component with m/z 679 was a glycoside with an aglycone of m/z 517 and was present as two peaks in extracts of whole puree, unseeded puree, and isolated seeds. These components were purified using Sephadex LH20 and C18 SPE units and identified as major, novel raspberry triterpenoid glycosides. This triterpenoid-enriched fraction (100 nM) protected against H2 O2 -induced DNA damage in both colon cancer and normal cell lines and altered expression of cytoprotective genes. CONCLUSION: The presence of these novel raspberry triterpenoid components in ileal fluids indicates that they would be colon-available in vivo, so confirmation of their anticancer bioactivities is of key physiological relevance.

In vitro and in vivo models of colorectal cancer: antigenotoxic activity of berries.

The etiology of colorectal cancer (CRC), a common cause of cancer-related mortality globally, has strong associations with diet. There is considerable epidemiological evidence that fruits and vegetables are associated with reduced risk of CRC. This paper reviews the extensive evidence, both from in vitro studies and animal models, that components of berry fruits can modulate biomarkers of DNA damage and that these effects may be potentially chemoprotective, given the likely role that oxidative damage plays in mutation rate and cancer risk. Human intervention trials with berries are generally consistent in indicating a capacity to significantly decrease oxidative damage to DNA, but represent limited evidence for anticarcinogenicity, relying as they do on surrogate risk markers. To understand the effects of berry consumption on colorectal cancer risk, future studies will need to be well controlled, with defined berry extracts, using suitable and clinically relevant end points and considering the importance of the gut microbiota.