Bacillamide D produced by Bacillus cereus from the mouse intestinal bacterial collection (miBC) is a potent cytotoxin in vitro.

The gut microbiota influences human health and the development of chronic diseases. However, our understanding of potentially protective or harmful microbe-host interactions at the molecular level is still in its infancy. To gain further insights into the hidden gut metabolome and its impact, we identified a cryptic non-ribosomal peptide BGC in the genome of Bacillus cereus DSM 28590 from the mouse intestine ( www.dsmz.de/miBC ), which was predicted to encode a thiazol(in)e substructure. Cloning and heterologous expression of this BGC revealed that it produces bacillamide D. In-depth functional evaluation showed potent cytotoxicity and inhibition of cell migration using the human cell lines HCT116 and HEK293, which was validated using primary mouse organoids. This work establishes the bacillamides as selective cytotoxins from a bacterial gut isolate that affect mammalian cells. Our targeted structure-function-predictive approach is demonstrated to be a streamlined method to discover deleterious gut microbial metabolites with potential effects on human health.

Toll-like receptor 3 expression in myeloid cells is essential for efficient regeneration after acute pancreatitis in mice.

Stringent regulation of the inflammatory response is crucial for normal tissue regeneration. Here, we analyzed the role of Toll-like receptor 3 (TLR3) in pancreatic regeneration after acute pancreatitis (AP). AP was induced by caerulein treatment in mice with global TLR3 deficiency (TLR3OFF ) or in mice re-expressing TLR3 exclusively in the myeloid cell lineage (TLR3Mye ). Compared to WT mice, TLR3OFF mice had a markedly increased formation of acinar-to-ductal metaplasia (ADM) that persisted until day 7 after initiation of AP. Pancreatic tissue of WT mice was completely regenerated after 5 days with no detectable ADM structures. The enhancing effect of TLR3-deficiency on ADM formation was closely linked with an increased and prolonged accumulation of macrophages in pancreata of TLR3OFF mice. Importantly, the phenotype of TLR3OFF mice was rescued in TLR3Mye mice, demonstrating the causative role of myeloid cell selective TLR3 signaling. Moreover, in vitro stimulation of macrophages through TLR3 initiated cell death by a caspase-8-associated mechanism. Therefore, these findings provide evidence that TLR3 signaling in myeloid cells is sufficient to limit inflammation and ADM formation and to promote regeneration after AP. Notably, resolution of inflammation after AP was associated with macrophage sensitivity to TLR3-mediated cell death.

Novel role for CRK adaptor proteins as essential components of SRC/FAK signaling for epithelial-mesenchymal transition and colorectal cancer aggressiveness.

Epithelial-mesenchymal transition (EMT) is a cell plasticity process required for metastasis and chemoresistance of carcinoma cells. We report a crucial role of the signal adaptor proteins CRK and CRKL in promoting EMT and tumor aggressiveness, as well as resistance against chemotherapy in colorectal and pancreatic carcinoma. Genetic loss of either CRKL or CRK partially counteracted EMT in three independent cancer cell lines. Strikingly, complete loss of the CRK family shifted cells strongly toward the epithelial phenotype. Cells exhibited greatly increased E-cadherin and grew as large, densely packed clusters, completely lacked invasiveness and the ability to undergo EMT induced by cytokines or genetic activation of SRC. Furthermore, CRK family-deficiency significantly reduced cell survival, proliferation and chemoresistance, as well as ERK1/2 phosphorylation and c-MYC protein levels. In accordance, MYC-target gene expression was identified as novel hallmark process positively regulated by CRK family proteins. Mechanistically, CRK proteins were identified as pivotal amplifiers of SRC/FAK signaling at focal adhesions, mediated through a novel positive feedback loop depending on RAP1. Expression of the CRK family and the EMT regulator ZEB1 was significantly correlated in samples from colorectal cancer patients, especially in invasive regions. Further, high expression of CRK family genes was significantly associated with reduced survival in locally advanced colorectal cancer, as well as in pan-cancer datasets from the TCGA project. Thus, CRK family adaptor proteins are promising therapeutic targets to counteract EMT, chemoresistance, metastasis formation and minimal residual disease. As proof of concept, CRK family-mediated oncogenic signaling was successfully inhibited by a peptide-based inhibitor.

BarrettNET-a prospective registry for risk estimation of patients with Barrett's esophagus to progress to adenocarcinoma.

Risk stratification in patients with Barrett's esophagus (BE) to prevent the development of esophageal adenocarcinoma (EAC) is an unsolved task. The incidence of EAC and BE is increasing and patients are still at unknown risk. BarrettNET is an ongoing multicenter prospective cohort study initiated to identify and validate molecular and clinical biomarkers that allow a more personalized surveillance strategy for patients with BE. For BarrettNET participants are recruited in 20 study centers throughout Germany, to be followed for progression to dysplasia (low-grade dysplasia or high-grade dysplasia) or EAC for >10 years. The study instruments comprise self-administered epidemiological information (containing data on demographics, lifestyle factors, and health), as well as biological specimens, i.e., blood-based samples, esophageal tissue biopsies, and feces and saliva samples. In follow-up visits according to the individual surveillance plan of the participants, sample collection is repeated. The standardized collection and processing of the specimen guarantee the highest sample quality. Via a mobile accessible database, the documentation of inclusion, epidemiological data, and pathological disease status are recorded subsequently. Currently the BarrettNET registry includes 560 participants (23.1% women and 76.9% men, aged 22-92 years) with a median follow-up of 951 days. Both the design and the size of BarrettNET offer the advantage of answering research questions regarding potential causes of disease progression from BE to EAC. Here all the integrated methods and materials of BarrettNET are presented and reviewed to introduce this valuable German registry.

BH3 mimetic ABT-737 and a proteasome inhibitor synergistically kill melanomas through Noxa-dependent apoptosis.

The Bcl-2 family is important in modulating sensitivity to anticancer drugs in many cancers, including melanomas. The BH3 mimetic ABT-737 is a potent small molecule inhibitor of the anti-apoptotic proteins Bcl-2/Bcl-X(L)/Bcl-w. In this report, we examined whether ABT-737 is effective in killing melanoma cells in combination with the proteasome inhibitor MG-132, and further evaluated the mechanisms of action. Viability, morphological, and Annexin V apoptosis assays showed that ABT-737 alone exhibited little cytotoxicity, yet it displayed strong synergistic lethality when combined with MG-132. In addition, the detection of Bax/Bak activation indicated that the combination treatment synergistically induced mitochondria-mediated apoptosis. Furthermore, mechanistic analysis revealed that this combination treatment induced expression of the pro-apoptotic protein Noxa- and caspase-dependent degradation of the anti-apoptotic protein, Mcl-1. Finally, siRNA-mediated inhibition of Mcl-1 expression significantly increased sensitivity to ABT-737 in these cells, and knocking down Noxa expression protected the cells from cytotoxicity induced by the combination treatment. These findings demonstrate that ABT-737 combined with MG-132 synergistically induced Noxa-dependent mitochondrial-mediated apoptosis. In summary, this study indicates promising therapeutic potential of targeting anti-apoptotic Bcl-2 family members in treating melanoma, and it validates rational molecular approaches that target anti-apoptotic defenses when developing cancer treatments.

Active N-Ras and B-Raf inhibit anoikis by downregulating Bim expression in melanocytic cells.

B-Raf and N-Ras proteins are often activated in melanoma, yet their roles in producing inherent survival signals are not fully understood. In this study, we investigated how N-RAS(Q61K) and B-RAF(V600E) contribute to melanoma's resistance to apoptosis induced by detachment from the extracellular matrix (anoikis). We found that expression of constitutively active N-RAS(Q61K) and B-RAF(V600E) downregulated the proapoptotic Bim protein in an immortalized melanocyte cell line. Bim is one of the main proapoptotic mediators of anoikis. Western blot analysis showed that detachment increased Bim expression in melanocytes, and Annexin V staining indicated that detachment induced cell death significantly in melanocytes. Blocking Bim expression by using RNAi vectors or by expressing N-RAS(Q61K) significantly inhibited anoikis in melanocytes. In summary, this report indicates that N-RAS(Q61K) and B-RAF(V600E) contribute to melanoma's resistance to apoptosis in part by downregulating Bim expression, suggesting that Bim is a possible treatment target for overriding melanoma's inherent defenses against cell death.

Prostatic fluid concentrations of isoflavonoids in soy consumers are sufficient to inhibit growth of benign and malignant prostatic epithelial cells in vitro.

BACKGROUND: The differential intestinal metabolism of the soy isoflavones is likely to influence the ability of soy to prevent prostate cancer. While daidzein, genistein, and equol have direct antiproliferative effects on prostatic epithelial cells in vitro, there are no such data for the isoflavone glycitein, or seven metabolites: O-desmethylangolensin (ODMA), 6-hydroxyODMA (6H-ODMA), dihydrodaidzein (DHD), cis-4-hydroxyequol (C4HE), 3'-hydroxydaidzein (3HD), 6-hydroxydaidzein (6HD), and 8-hydroxydaidzein (8HD). In the current study, the in vitro activities of these compounds were elucidated, and the active ranges of concentrations were compared to that found in Caucasian prostatic fluid (PF) and plasma samples. METHODS: The effects of isoflavonoids on cell growth, cell cycle distribution, and apoptosis (active Caspase 3) were examined on benign prostatic epithelial cells (PrEC), and the prostate cancer cell line LNCaP. RESULTS: PF concentrations of genistein, equol, and daidzein (but not ODMA or DHD) were often within the ranges that reduce PrEC growth in vitro. Profound differences in sensitivities were observed with LNCaP. The hydroxydaidzeins, C4HE, and 6H-ODMA had significant inhibitory effects at 10(-5)M on PrEC growth (but not LNCaP). Glycitein had significant effects on both. Reductions in cell growth were typically associated with both changes in cell cycle distribution and Caspase 3 activation. When five isoflavonoids were used in combination at concentrations present in PF samples, synergistic effects were observed. CONCLUSION: The profound differences in sensitivities of prostatic epithelial cells to these compounds along with their synergistic effects suggest that multiple metabolites in vivo may be optimal for preventing prostate cancer.

Molecular characterization of human prostate carcinoma cell lines.

BACKGROUND: This study presents a comprehensive survey and characterization of available prostate carcinoma cell lines, most of which have been widely used but are incompletely characterized. METHODS: A total of 21 cell lines were investigated, including three "classical" (DU 145, LNCaP, and PC-3) and 18 "non-classical" lines (1013L, 22Rv1, ALVA-55, ALVA-101, ARCaP, CWR-R1, DuCaP, DuPro-1, LAPC-4, MDA PCa 1, MDA PCa 2a, MDA PCa 2b, NCI-H660, PC-346C, PC-93, PSK-1, UM-SCP-1, and VCaP). Cytogenetics, DNA profiling, expression of basal, luminal, and neuroendocrine differentiation markers, and mutation analyses of the TP53 and androgen receptor (AR) genes were performed. RESULTS: Based on cytogenetics and DNA profiling analyses, out of the 18 "non-classical" lines, six were confirmed to be unique, eight (in four pairs) were confirmed to be related in origin, and four lines were identified as cross-contaminants. Of this latter group, PC-93 was found to be a derivative of HeLa, whereas DuPro-1, ALVA-55, and ALVA-101 were derivatives of PC-3. The 17 genuine prostate cell lines expressed keratin 8 (K8) and K18. Nine showed AR expression, of which five harbored mutations in the AR gene. Prostate-specific antigen and DD3 were exclusively detected in AR expressing cell lines. Seven lines expressed the basal cell marker K5, three of these lines showed co-expression of AR. CONCLUSIONS: This study defines a collection of 17 genuine prostate carcinoma cell lines. This collection, although small, constitutes a variety of different types and stages of prostate cancer, while it also partly reflects the heterogeneous nature of this malignancy.

Soy isoflavonoid equol modulates the growth of benign and malignant prostatic epithelial cells in vitro.

BACKGROUND: The dietary consumption of high levels of soy has been linked to reduced risks for prostate cancer (PC) in Asians and vegetarians. In vitro studies have focused on the two most abundant isoflavones in soy, genistein and daidzein. However, daidzein is differentially metabolized by gut microflora in humans, yielding compounds with very different bioactivities and half-lives. Asians are significantly more likely to produce the metabolite equol than Caucasians, suggesting its role in the prevention of PC. We hypothesize that equol is a bioactive metabolite that exerts direct antiproliferative effects on prostatic epithelial cells. METHODS: Benign and malignant prostatic epithelial cells were treated in vitro with equol, genistein, and daidzein by using the range of concentrations found in the prostatic fluids of Asians consuming soy. Growth and cell cycle distribution were analyzed over time. RESULTS: After 9 days of treatment, equol inhibited growth of benign human prostatic epithelial cells (PrEC) by 37% at 10(-6) M and 80% at 10(-5) M. Although genistein also had profound effects, daidzein appeared only one tenth as potent as equol. Equol and daidzein caused an accumulation of cells in G0/G1, whereas genistein arrested cells in G2/M. The isoflavonoids demonstrated differential effects on the established PC cell lines 22Rv1, LNCaP, LAPC-4, PC-3, and DU 145. PC-3 cells showed the greatest resistance. CONCLUSION: Equol is a biologically active metabolite of daidzein that has potent antiproliferative effects on benign and malignant prostatic epithelial cells at concentrations that can be obtained naturally through dietary soy consumption.