Cystathionine Gamma-Lyase Regulates TNF-α-Mediated Injury Response in Human Colonic Epithelial Cells and Colonoids.

Cystathionine gamma-lyase (CSE) and TNF-α are now recognized as key regulators of intestinal homeostasis, inflammation, and wound healing. In colonic epithelial cells, both molecules have been shown to influence a variety of biological processes, but the specific interactions between intracellular signaling pathways regulated by CSE and TNF-α are poorly understood. In the present study, we investigated these interactions in normal colonocytes and an organoid model of the healthy human colon using CSE-specific pharmacological inhibitors and siRNA-mediated transient gene silencing in analytical and functional assays in vitro. We demonstrated that CSE and TNF-α mutually regulated each other's functions in colonic epithelial cells. TNF-α treatment stimulated CSE activity within minutes and upregulated CSE expression after 24 h, increasing endogenous CSE-derived H2S production. In turn, CSE activity promoted TNF-α-induced NF-ĸB and ERK1/2 activation but did not affect the p38 MAPK signaling pathway. Inhibition of CSE activity completely abolished the TNF-α-induced increase in transepithelial permeability and wound healing. Our data suggest that CSE activity may be essential for effective TNF-α-mediated intestinal injury response. Furthermore, CSE regulation of TNF-α-controlled intracellular signaling pathways could provide new therapeutic targets in diseases of the colon associated with impaired epithelial wound healing.

Deletion of cystathionine-γ-lyase in bone marrow-derived cells promotes colitis-associated carcinogenesis.

Ulcerative colitis (UC) is characterized by widespread relapsing inflammation of the colonic mucosa. Colitis-associated cancer (CAC) is one of the most serious complications of a prolonged history of UC. Hydrogen sulfide (H2S) has emerged as an important physiological mediator of gastrointestinal homeostasis, limiting mucosal inflammation and promoting tissue healing in response to injury. Inhibition of cystathionine-γ-lyase (CSE)-dependent H2S production in animal models of UC has been shown to exacerbate colitis and delay tissue repair. It is unknown whether CSE plays a role in CAC, or the downregulation of CSE expression and/or activity promotes CAC development. In humans, we observed a significant decrease in CSE expression in colonic biopsies from patients with UC. Using the dextran sodium sulfate (DSS) model of epithelium injury-induced colitis and global CSE KO mouse strain, we demonstrated that CSE is critical in limiting mucosal inflammation and stimulating epithelial cell proliferation in response to injury. In vitro studies showed that CSE activity stimulates epithelial cell proliferation, basal and cytokine-stimulated cell migration, as well as cytokine regulation of transepithelial permeability. In the azoxymethane (AOM)/DSS model of CAC, the loss of CSE expression accelerated both the development and progression of CAC. The increased tumor multiplicity and severity of CAC observed in CSE-KO mice were associated with reduced levels of mucosal IL-10 expression and increased levels of IL-6. Restoring CSE expression in bone marrow (BM) cells of CSE-KO mice through reciprocal BM transplantation raised mucosal IL-10 expression, decreased IL-6 level, and reduced the number of aberrant crypt foci and tumors in AOM/DSS-treated mice. These studies demonstrate that CSE expression in BM cells plays a critical role in suppressing CAC in mice. Furthermore, the data suggest that the inhibitory effects of CSE on the development of CAC are due, in part, to the modulation of mucosal pro-and anti-inflammatory cytokine expression.

Developing future translational scientists through authentic learning and assessments.

The Interprofessional Research Design course uses authentic learning pedagogy to bring together students from different education tracks (PhD, MD, MD/PhD training) to engage in interprofessional collaborative skills toward completion of a capstone project, a National Health Institutes (NIH) R21-style grant proposal. The course, underpinned by principles of team science, begins with a leadership training workshop to introduce students to effective leadership and teamwork strategies for interprofessional team environments. We used several assessments during the course to monitor leadership and team dynamics. We analyzed three assessments (leadership self-efficacy testing, iterative team contracts and reflective essays) to better understand students' learning experiences. Self-efficacy testing was administered before leadership training (pre) and at the end of the course (post-then); scores were analyzed using a repeated repeated measures ANOVA. Iterative team contracts were analyzed qualitatively using both deductive and descriptive methods. Reflective essays were analyzed using a general inductive approach. Nine teams of 32 students (23 MD; 9 PhD) participated in the class over 2017-2018. Self-efficacy testing using post-then timing to control for response shift showed a statistically significant increase in self-efficacy across all measures. Deductive qualitative analysis of iterative contracting showed evidence of team processes which support successful team performance; and descriptive analysis mapped productive behaviors. In reflective essays, seven of the nine teams collectively described their experiences positively; e.g., themes included empathizing with group members, sophisticated communication and collaborative workflow/styles. For negative experiences, themes were related to basic communication, poor integration and the theory-practice gap of leadership training. These findings demonstrate the usefulness of an authentic learning pedagogy focused on teaching the practice of team science.

Efficacy of Novel Aminooxyacetic Acid Prodrugs in Colon Cancer Models: Towards Clinical Translation of the Cystathionine ß-Synthase Inhibition Concept.

Upregulation of hydrogen sulfide (H2S) biosynthesis, at least in part related to the upregulation of cystathionine ß-synthetase (CBS) in cancer cells, serves as a tumor-promoting factor and has emerged as a possible molecular target for antitumor drug development. To facilitate future clinical translation, we have synthesized a variety of novel CBS-targeting, esterase-cleavable prodrugs based on the structure of the prototypical CBS inhibitor aminooxyacetic acid (AOAA). The pharmacological properties of these compounds were evaluated in cell-free assays with recombinant human CBS protein, the human colon cancer cell line HCT116, and in vivo using various tumor-bearing mice models. The prodrug YD0251 (the isopropyl ester derivative of AOAA) was selected for detailed characterization. YD0251 exhibits improved antiproliferative efficacy in cell culture models when compared to AOAA. It is up to 18 times more potent than AOAA at suppressing HCT116 tumor growth in vivo and is effective when administered to tumor-bearing mice either via subcutaneous injection or oral gavage. Patient-derived xenografts (PDTXs) with higher levels of CBS protein grew significantly larger than tumors with lower levels, and YD0251 treatment inhibited the growth of PDTXs with elevated CBS, whereas it had no significant effect on PDTXs with low CBS protein levels. The toxicity of YD0251 was assessed in mice subjected to subchronic administration of supratherapeutic doses the inhibitor; no significant alteration in circulating markers of organ injury or histopathological alterations were noted, up to 60 mg/kg/day × 5 days. In preparation to a future theranostic concept (to match CBS inhibitor therapy to high-CBS expressors), we identified a potential plasma marker of CBS-expressing tumors. Colon cancer cells produced significant levels of lanthionine, a rare metabolic intermediate of CBS-mediated H2S biosynthesis; forced expression of CBS into non-transformed epithelial cells increased lanthionine biogenesis in vitro and in vivo (measured in the urine of tumor-bearing mice). These current results may be useful to facilitate the translation of a CBS inhibition-based antitumor concept into the clinical space.

Successful Lumpectomy in a Patient With Multicentric Breast Cancer.

When there is extensive breast cancer, patients typically undergo mastectomy. However, lumpectomy may still be performed for patients who are motivated to avoid a mastectomy and understand the risk for positive margins requiring second surgery in unique cases. This report details the surgical management and clinical reasoning behind lumpectomy for a multicentric breast cancer spanning 5 cm. The lumpectomy was a success with negative margins on final pathology.

3-Mercaptopyruvate sulfurtransferase supports endothelial cell angiogenesis and bioenergetics.

BACKGROUND AND PURPOSE: During angiogenesis, quiescent endothelial cells (ECs) are activated by various stimuli to form new blood vessels from pre-existing ones in physiological and pathological conditions. Many research groups have shown that hydrogen sulfide (H2 S), the newest member of the gasotransmitter family, acts as a proangiogenic factor. To date, very little is known about the regulatory role of 3-mercaptopyruvate sulfurtransferase (3-MST), an important H2 S-producing enzyme in ECs. The aim of our study was to explore the potential role of 3-MST in human EC bioenergetics, metabolism, and angiogenesis. EXPERIMENTAL APPROACH: To assess in vitro angiogenic responses, we used EA.hy926 human vascular ECs subjected to shRNA-mediated 3-MST attenuation and pharmacological inhibition of proliferation, migration, and tube-like network formation. To evaluate bioenergetic parameters, cell respiration, glycolysis, glucose uptake, and mitochondrial/glycolytic ATP production were measured. Finally, global metabolomic profiling was performed to determine the level of 669 metabolic compounds. KEY RESULTS: 3-MST-attenuated ECs subjected to shRNA or pharmacological inhibition of 3-MST significantly reduced EC proliferation, migration, and tube-like network formation. 3-MST silencing also suppressed VEGF-induced EC migration. From bioenergetic and metabolic standpoints, 3-MST attenuation decreased mitochondrial respiration and mitochondrial ATP production, increased glucose uptake, and perturbed the entire EC metabolome. CONCLUSION AND IMPLICATIONS: 3-MST regulates bioenergetics and morphological angiogenic functions in human ECs. The data presented in the current report support the view that 3-MST pathway may be a potential candidate for therapeutic modulation of angiogenesis. LINKED ARTICLES: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.

Apigenin Decreases Acinar Cell Damage in Pancreatitis.

OBJECTIVE: Chronic pancreatitis is the consequence of multiple episodes of recurrent acute pancreatitis (RAP). We hypothesized that apigenin can minimize the sequelae of RAP by limiting acinar cells' proinflammatory signaling pathways. METHODS: AR42J acinar cells were treated in vitro with transforming growth factor ß (TGF-ß), apigenin, and other inhibitors. Dual luciferase reporter assay measured parathyroid hormone-related protein (PTHrP) promoter activity. MAPK/ERK pathway activity was assessed by immunoblotting and in vivo by immunohistochemistry with a cerulein-induced RAP mouse model. Nuclear factor κ B nuclear localization was analyzed in vitro in cells stimulated with tumor necrosis factor α. Primary acini were isolated and treated with cerulein; interleukin 6 messenger RNA was measured comparing PTHrP wild-type and knockout mice. RESULTS: Apigenin and PD98059 each downregulated TGF-ß stimulation of PTHrP P3 promoter activity. In a RAP mouse model, apigenin reduced pERK nuclear localization in acinar cells and preserved acinar cell architecture. Apigenin suppressed tumor necrosis factor α-mediated signaling by decreasing nuclear factor κ B nuclear localization and decreased interleukin 6 messenger RNA levels via a PTHrP-dependent mechanism. CONCLUSIONS: Apigenin reduced inflammatory responses in experimental models of RAP. The mechanisms mediating the actions of apigenin, in part, are owing to attenuation of PTHrP and TGF-ß proinflammatory signaling.

Operative complications and economic outcomes of cholecystectomy for acute cholecystitis.

BACKGROUND: Recent management of acute cholecystitis favors same admission (SA) or emergent cholecystectomy based on overall shorter hospital stay and therefore cost savings. We adopted the practice of SA cholecystectomy for the treatment of acute cholecystitis at our tertiary care center and wanted to evaluate the economic benefit of this practice. We hypothesized that the existence of complications, particularly among patients with a higher degree of disease severity, during SA cholecystectomy could negate the cost savings. AIM: To compare complication rates and hospital costs between SA vs delayed cholecystectomy among patients admitted emergently for acute cholecystitis. METHODS: Under an IRB-approved protocol, complications and charges for were obtained for SA, later after conservative management (Delayed), or elective cholecystectomies over an 8.5-year period. Patients were identified using the acute care surgery registry and billing database. Data was retrieved via EMR, operative logs, and Revenue Cycle Operations. The severity of acute cholecystitis was graded according to the Tokyo Guidelines. TG18 categorizes acute cholecystitis by Grades 1, 2, and 3 representing mild, moderate, and severe, respectively. Comparisons were analyzed with χ 2, Fisher's exact test, ANOVA, t-tests, and logistic regression; significance was set at P < 0.05. RESULTS: Four hundred eighty-six (87.7%) underwent a SA while 68 patients (12.3%) received Delayed cholecystectomy. Complication rates were increased after SA compared to Delayed cholecystectomy (18.5% vs 4.4%, P = 0.004). The complication rates of patients undergoing delayed cholecystectomy was similar to the rate for elective cholecystectomy (7.4%, P = 0.35). Mortality rates were 0.6% vs 0% for SA vs Delayed. Patients with moderate disease (Tokyo 2) suffered more complications among SA while none who were delayed experienced a complication (16.1% vs 0.0%, P < 0.001). Total hospital charges for SA cholecystectomy were increased compared to a Delayed approach ($44500 ± $59000 vs $35300 ± $16700, P = 0.019). The relative risk of developing a complication was 4.2x [95% confidence interval (CI): 1.4-12.9] in the SA vs Delayed groups. Among eight patients (95%CI: 5.0-12.3) with acute cholecystitis undergoing SA cholecystectomy, one patient will suffer a complication. CONCLUSION: Patients with Tokyo Grade 2 acute cholecystitis had more complications and increased hospital charges when undergoing SA cholecystectomy. This data supports a selective approach to SA cholecystectomy for acute cholecystitis.

Integration of leadership training for graduate and medical students engaged in translational biomedical research: Examining self-efficacy and self-insight.

INTRODUCTION: Formal training in team leadership is not taught in biomedical research graduate training programs or medical schools. METHODS: We piloted a Leadership Training Workshop for graduate biomedical and medical students enrolled in our Interprofessional Research Design Course. RESULTS: The Kane-Baltes self-efficacy survey demonstrated improved leadership skills (median scores pretraining and post-training were 71 and 76.6; paired t-test, p=0.04). CONCLUSIONS: Most students demonstrated significant improvement in self-awareness pertaining to their own innate leadership styles.

Upregulation of Cystathionine-ß-Synthase in Colonic Epithelia Reprograms Metabolism and Promotes Carcinogenesis.

The trans-sulfuration enzyme cystathionine-ß-synthase (CBS) and its product hydrogen sulfide (H2S) are aberrantly upregulated in colorectal cancers, where they contribute to tumor growth and progression by both autocrine and paracrine mechanisms. However, it is unknown whether the CBS/H2S axis plays a role in colorectal carcinogenesis. Here, we report upregulation of CBS in human biopsies of precancerous adenomatous polyps and show that forced upregulation of CBS in an adenoma-like colonic epithelial cell line is sufficient to induce metabolic and gene expression profiles characteristic of colorectal cancer cells. Differentially expressed metabolites (65 increased and 20 decreased) clustered into the glycolytic pathway, nucleotide sugars, intermediates of the pentose phosphate pathway, and lipogenesis, including primarily phospholipids, sphingolipids, and bile acids. CBS upregulation induced broad changes in the NCM356 cell transcriptome with over 350 differentially expressed genes. These genes overlapped significantly with gene sets related to glycolysis, hypoxia, and a colon cancer cell phenotype, including genes regulated by NF-κB, KRAS, p53, and Wnt signaling, genes downregulated after E-cadherin knockdown, and genes related to increased extracellular matrix, cell adhesion, and epithelial-to-mesenchymal transition. The CBS-induced switch to an anabolic metabolism was associated with increased NCM356 cell bioenergetics, proliferation, invasion through Matrigel, resistance to anoikis, and CBS-dependent tumorigenesis in immunocompromised mice. Genetic ablation of CBS in CBS heterozygous mice (CBS+/- ) reduced the number of mutagen-induced aberrant colonic crypt foci. Taken together, these results establish that activation of the CBS/H2S axis promotes colon carcinogenesis. Cancer Res; 77(21); 5741-54. ©2017 AACR.

Consensus Molecular Subtypes of Colorectal Cancer and their Clinical Implications.

The colorectal cancer (CRC) Subtyping Consortium has unified six independent molecular classification systems, based on gene expression data, into a single consensus system with four distinct groups, known as the Consensus Molecular Subtypes (CMS); clinical implications are discussed in this review. This article is based on a literature review relevant to the CMS of CRC indexed in PubMed (US National Library of Medicine) as well as the authors' own published data. The CMS were determined and correlated with epigenomic, transcriptomic, microenvironmental, genetic, prognostic and clinical characteristics. The CMS1 subtype is immunogenic and hypermutated. CMS2 tumors are activated by the WNT-ß-catenin pathway and have the highest overall survival. CMS3 feature a metabolic cancer phenotype and CMS4 cancers have the worst survival and have a strong stromal gene signature. The Consensus Molecular Subtypes of CRC may better inform clinicians of prognosis, therapeutic response, and potential novel therapeutic strategies.

Patient-derived Xenografts from Colorectal Carcinoma: A Temporal and Hierarchical Study of Murine Stromal Cell Replacement.

BACKGROUND/AIM: Patient-derived xenografting (PDX) of human colorectal cancer (CRC) is the preferred experimental model to study tumor response to therapeutic agents. Gradually, human stromal cells are replaced by mouse stromal cells; however, the exact timing of the replacement of human with murine stromal cells in human CRC xenograft has not been fully elucidated. We hypothesize that orthologous murine transcripts functionally substitutes for the loss due to replacement of human stromal genes. MATERIALS AND METHODS: Human CRC were implanted in athymic nude mice in replicates and followed-up over time. Using next-generation sequencing, we determined the temporal kinetics of human stromal cell replacement with the orthologous murine transcripts. RESULTS: CRC cell-induced re-organization of the normal, quiescent murine stromal cells into a protumorigenic phenotype supporting human CRC growth occurs at initial implantation. CONCLUSION: Murine cell replacement occurs in a time- and size-dependent manner.

How does inflammation drive mutagenesis in colorectal cancer?

Colorectal cancer (CRC) is a major health challenge worldwide. Factors thought to be important in CRC etiology include diet, microbiome, exercise, obesity, a history of colon inflammation and family history. Interventions, including the use of non-steroidal anti-Inflammatory drugs (NSAIDs) and anti-inflammatory agents, have been shown to decrease incidence in some settings. However, our current understanding of the mechanistic details that drive CRC are insufficient to sort out the complex and interacting factors responsible for cancer-initiating events. It has been known for some time that the development of CRC involves mutations in key genes such as p53 and APC, and the sequence in which these mutations occur can determine tumor presentation. Observed recurrent mutations are dominated by C to T transitions at CpG sites, implicating the deamination of 5-methylcytosine (5mC) as a key initiating event in cancer-driving mutations. While it has been widely assumed that inflammation-mediated oxidation drives mutations in CRC, oxidative damage to DNA induces primarily G to T transversions, not C to T transitions. In this review, we discuss this unresolved conundrum, and specifically, we elucidate how the known nucleotide excision repair (NER) and base excision repair (BER) pathways, which are partially redundant and potentially competing, might provide a critical link between oxidative DNA damage and C to T mutations. Studies using recently developed next-generation DNA sequencing technologies have revealed the genetic heterogeneity in human tissues including tumors, as well as the presence of DNA damage. The capacity to follow DNA damage, repair and mutagenesis in human tissues using these emerging technologies could provide a mechanistic basis for understanding the role of oxidative damage in CRC tumor initiation. The application of these technologies could identify mechanism-based biomarkers useful in earlier diagnosis and aid in the development of cancer prevention strategies.

Screening of a composite library of clinically used drugs and well-characterized pharmacological compounds for cystathionine ß-synthase inhibition identifies benserazide as a drug potentially suitable for repurposing for the experimental therapy of colon cancer.

Cystathionine-ß-synthase (CBS) has been recently identified as a drug target for several forms of cancer. Currently no potent and selective CBS inhibitors are available. Using a composite collection of 8871 clinically used drugs and well-annotated pharmacological compounds (including the LOPAC library, the FDA Approved Drug Library, the NIH Clinical Collection, the New Prestwick Chemical Library, the US Drug Collection, the International Drug Collection, the 'Killer Plates' collection and a small custom collection of PLP-dependent enzyme inhibitors), we conducted an in vitro screen in order to identify inhibitors for CBS using a primary 7-azido-4-methylcoumarin (AzMc) screen to detect CBS-derived hydrogen sulfide (H2S) production. Initial hits were subjected to counterscreens using the methylene blue assay (a secondary assay to measure H2S production) and were assessed for their ability to quench the H2S signal produced by the H2S donor compound GYY4137. Four compounds, hexachlorophene, tannic acid, aurintricarboxylic acid and benserazide showed concentration-dependent CBS inhibitory actions without scavenging H2S released from GYY4137, identifying them as direct CBS inhibitors. Hexachlorophene (IC50: ∼60µM), tannic acid (IC50: ∼40µM) and benserazide (IC50: ∼30µM) were less potent CBS inhibitors than the two reference compounds AOAA (IC50: ∼3µM) and NSC67078 (IC50: ∼1µM), while aurintricarboxylic acid (IC50: ∼3µM) was equipotent with AOAA. The second reference compound NSC67078 not only inhibited the CBS-induced AzMC fluorescence signal (IC50: ∼1µM), but also inhibited with the GYY4137-induced AzMC fluorescence signal with (IC50 of ∼6µM) indicative of scavenging/non-specific effects. Hexachlorophene (IC50: ∼6µM), tannic acid (IC50: ∼20µM), benserazide (IC50: ∼20µM), and NSC67078 (IC50: ∼0.3µM) inhibited HCT116 colon cancer cells proliferation with greater potency than AOAA (IC50: ∼300µM). In contrast, although a CBS inhibitor in the cell-free assay, aurintricarboxylic acid failed to inhibit HCT116 proliferation at lower concentrations, and stimulated cell proliferation at 300µM. Copper-containing compounds present in the libraries, were also found to be potent inhibitors of recombinant CBS; however this activity was due to the CBS inhibitory effect of copper ions themselves. However, copper ions, up to 300µM, did not inhibit HCT116 cell proliferation. Benserazide was only a weak inhibitor of the activity of the other H2S-generating enzymes CSE and 3-MST activity (16% and 35% inhibition at 100µM, respectively) in vitro. Benserazide suppressed HCT116 mitochondrial function and inhibited proliferation of the high CBS-expressing colon cancer cell line HT29, but not the low CBS-expressing line, LoVo. The major benserazide metabolite 2,3,4-trihydroxybenzylhydrazine also inhibited CBS activity and suppressed HCT116 cell proliferation in vitro. In an in vivo study of nude mice bearing human colon cancer cell xenografts, benserazide (50mg/kg/days.q.) prevented tumor growth. In silico docking simulations showed that benserazide binds in the active site of the enzyme and reacts with the PLP cofactor by forming reversible but kinetically stable Schiff base-like adducts with the formyl moiety of pyridoxal. We conclude that benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo. Further pharmacokinetic, pharmacodynamic and preclinical animal studies are necessary to evaluate the potential of repurposing benserazide for the treatment of colorectal cancers.

Cystathionine-beta-synthase inhibition for colon cancer: Enhancement of the efficacy of aminooxyacetic acid via the prodrug approach.

Colon cancer cells contain high levels of cystathionine-beta-synthase (CBS). Its product, hydrogen sulfide (H2S) promotes the growth and proliferation of colorectal tumor cells. In order to improve the antitumor efficacy of the prototypical CBS inhibitor aminooxyacetic acid (AOAA), we have designed and synthesized YD0171, a methyl ester derivative of AOAA. The antiproliferative effect of YD0171 exceeded the antiproliferative potency of AOAA in HCT116 human colon cancer cells. The esterase inhibitor paraoxon prevented the cellular inhibition of CBS activity by YD0171. YD0171 suppressed mitochondrial respiration and glycolytic function and induced G0/G1 arrest, but did not induce tumor cell apoptosis or necrosis. Metabolomic analysis in HCT116 cells showed that YD0171 affects multiple pathways of cell metabolism. The efficacy of YD0171 as an inhibitor of tumor growth was also tested in nude mice bearing subcutaneous HCT116 cancer cell xenografts. Animals were treated via subcutaneous injection of vehicle, AOAA (1, 3 or 9 mg/kg/day) or YD0171 (0.1, 0.5 or 1 mg/kg/day) for 3 weeks. Tumor growth was significantly reduced by 9 mg/kg/day AOAA, but not at the lower doses. YD0171 was more potent: tumor volume was significantly inhibited at 0.5 and 1 mg/kg/day. Thus, the in vivo efficacy of YD0171 is 9-times higher than that of AOAA. YD0171 (1 mg/kg/day) attenuated tumor growth and metastasis formation in the intracecal HCT116 tumor model. YD0171 (3 mg/kg/day) also reduced tumor growth in patient-derived tumor xenograft (PDTX) bearing athymic mice. YD0171 (3 mg/kg/day) induced the regression of established HCT116 tumors in vivo. A 5-day safety study in mice demonstrated that YD0171 at 20 mg/kg/day (given in two divided doses) does not increase plasma markers of organ injury, nor does it induce histological alterations in the liver or kidney. YD0171 caused a slight elevation in plasma homocysteine levels. In conclusion, the prodrug approach improves the pharmacological profile of AOAA; YD0171 represents a prototype for CBS inhibitory anticancer prodrugs. By targeting colorectal cancer bioenergetics, an emerging important hallmark of cancer, the approach exemplified herein may offer direct translational opportunities.

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens.

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, α-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Gremlin is a key pro-fibrogenic factor in chronic pancreatitis.

UNLABELLED: The current study aims to identify the pro-fibrogenic role of Gremlin, an endogenous antagonist of bone morphogenetic proteins (BMPs) in chronic pancreatitis (CP). CP is a highly debilitating disease characterized by progressive pancreatic inflammation and fibrosis that ultimately leads to exocrine and endocrine dysfunction. While transforming growth factor (TGF)-ß is a known key pro-fibrogenic factor in CP, the TGF-ß superfamily member BMPs exert an anti-fibrogenic function in CP as reported by our group recently. To investigate how BMP signaling is regulated in CP by BMP antagonists, the mouse CP model induced by cerulein was used. During CP induction, TGF-ß1 messenger RNA (mRNA) increased 156-fold in 2 weeks, a BMP antagonist Gremlin 1 (Grem1) mRNA levels increased 145-fold at 3 weeks, and increases in Grem1 protein levels correlated with increases in collagen deposition. Increased Grem1 was also observed in human CP pancreata compared to normal. Grem1 knockout in Grem1 (+/-) mice revealed a 33.2 % reduction in pancreatic fibrosis in CP compared to wild-type littermates. In vitro in isolated pancreatic stellate cells, TGF-ß induced Grem1 expression. Addition of the recombinant mouse Grem1 protein blocked BMP2-induced Smad1/5 phosphorylation and abolished BMP2's suppression effects on TGF-ß-induced collagen expression. Evidences presented herein demonstrate that Grem1, induced by TGF-ß, is pro-fibrogenic by antagonizing BMP activity in CP. KEY MESSAGES: • Gremlin is upregulated in human chronic pancreatitis and a mouse CP model in vivo. • Deficiency of Grem1 in mice attenuates pancreatic fibrosis under CP induction in vivo. • TGF-ß induces Gremlin mRNA and protein expression in pancreatic stellate cells in vitro. • Gremlin blocks BMP2 signaling and function in pancreatic stellate cells in vitro. • This study discloses a pro-fibrogenic role of Gremlin by antagonizing BMP activity in chronic pancreatitis.

Apigenin inhibits pancreatic stellate cell activity in pancreatitis.

BACKGROUND: Chronic pancreatitis (CP) is characterized by recurrent pancreatic injury, resulting in inflammation, necrosis, and fibrosis. There are currently no drugs limiting pancreatic fibrosis associated with CP, and there is a definite need to fill this void in patient care. MATERIALS AND METHODS: Pancreatitis was induced in C57/BL6 mice using supraphysiologic doses of cerulein, and apigenin treatment (once daily, 50 µg per mouse by oral gavage) was initiated 1 wk into the recurrent acute pancreatitis (RAP) protocol. Pancreata were harvested after 4 wk of RAP. Immunostaining with fibronectin antibody was used to quantify the extent of pancreatic fibrosis. To assess how apigenin may decrease organ fibrosis, we evaluated the effect of apigenin on the proliferation and apoptosis of human pancreatic stellate cells (PSCs) in vitro. Finally, we assessed apigenin's effect on the gene expression in PSCs stimulated with parathyroid hormone-related protein, a profibrotic and proinflammatory mediator of pancreatitis, using reverse transcription-polymerase chain reaction. RESULTS: After 4 wk of RAP, apigenin significantly reduced the fibrotic response to injury while preserving acinar units. Apigenin inhibited viability and induced apoptosis of PSCs in a time- and dose-dependent manner. Finally, apigenin reduced parathyroid hormone-related protein-stimulated increases in the PSC messenger RNA expression levels of extracellular matrix proteins collagen 1A1 and fibronectin, proliferating cell nuclear antigen, transforming growth factor-beta, and interleukin-6. CONCLUSIONS: These in vivo and in vitro studies provide novel insights regarding apigenin's mechanism(s) of action in reducing the severity of RAP. Additional preclinical testing of apigenin analogs is warranted to develop a therapeutic agent for patients at risk for CP.

Pharmacological inhibition and genetic knockdown of exchange protein directly activated by cAMP 1 reduce pancreatic cancer metastasis in vivo.

cAMP plays a critical role in regulating migration of various cancers. This role is context dependent and is determined by which of the two main cAMP sensors is at play: cAMP-dependent protein kinase or exchange protein directly activated by cAMP (EPAC). Recently, we have shown that the cAMP sensor protein EPAC1 promotes invasion/migration of pancreatic ductal adenocarcinoma (PDA) in vitro. In this study, we investigated the role of EPAC1 in invasion and metastasis of PDA in vivo, and evaluated the therapeutic potential of EPAC inhibitors as antimetastasis agents for this neoplasm. We employed an orthotopic metastatic mouse model in which the PDA cells MIA PaCa-2 were injected into the pancreas of athymic nude mice, and their local and distant spread was monitored by in vivo imaging and histologic evaluation of the number of metastatic foci in the liver. Either genetic suppression of EPAC1 or its pharmacologic inhibition with 3-(5-tert-butyl-isoxazol-3-yl)-2-[(3-chloro-phenyl)-hydrazono]-3-oxo-propionitrile, an EPAC-specific antagonist recently identified in our laboratory, decreased invasion and metastasis of the PDA cells. Mechanistically, EPAC1 promotes activation and trafficking of integrin ß1, which plays an essential role in PDA migration and metastasis. Our data show that EPAC1 facilitates metastasis of PDA cells and EPAC1 might be a potential novel therapeutic target for developing antimetastasis agents for PDA.

The therapeutic potential of cystathionine ß-synthetase/hydrogen sulfide inhibition in cancer.

SIGNIFICANCE: Cancer represents a major socioeconomic problem; there is a significant need for novel therapeutic approaches targeting tumor-specific pathways. RECENT ADVANCES: In colorectal and ovarian cancers, an increase in the intratumor production of hydrogen sulfide (H2S) from cystathionine ß-synthase (CBS) plays an important role in promoting the cellular bioenergetics, proliferation, and migration of cancer cells. It also stimulates peritumor angiogenesis inhibition or genetic silencing of CBS exerts antitumor effects both in vitro and in vivo, and potentiates the antitumor efficacy of anticancer therapeutics. CRITICAL ISSUES: Recently published studies are reviewed, implicating CBS overexpression and H2S overproduction in tumor cells as a tumor-growth promoting "bioenergetic fuel" and "survival factor," followed by an overview of the experimental evidence demonstrating the anticancer effect of CBS inhibition. Next, the current state of the art of pharmacological CBS inhibitors is reviewed, with special reference to the complex pharmacological actions of aminooxyacetic acid. Finally, new experimental evidence is presented to reconcile a controversy in the literature regarding the effects of H2S donor on cancer cell proliferation and survival. FUTURE DIRECTIONS: From a basic science standpoint, future directions in the field include the delineation of the molecular mechanism of CBS up-regulation of cancer cells and the delineation of the interactions of H2S with other intracellular pathways of cancer cell metabolism and proliferation. From the translational science standpoint, future directions include the translation of the recently emerging roles of H2S in cancer into human diagnostic and therapeutic approaches.