Impact of Deoxycholic Acid on Oesophageal Adenocarcinoma Invasion: Effect on Matrix Metalloproteinases.

Bile acids (BAs) have been implicated in the development of oesophagitis, Barrett's oesophagus and oesophageal adenocarcinoma (OAC). However, whether BAs promote cancer invasiveness has not been elucidated. We evaluated the role of BAs, in particular deoxycholic acid (DCA), in OAC invasion. Migration and invasiveness in untreated and BA-treated oesophageal SKGT-4 cancer cells were evaluated. Activity and expression of different matrix metalloproteinases (MMPs) were determined by zymography, ELISA, PCR and Western blot. Finally, human OAC tissues were stained for MMP-10 by immunohistochemistry. It was found that SKGT-4 cells incubated with low concentrations of DCA had a significant increase in invasion. In addition, MMP-10 mRNA and protein expression were also increased in the presence of DCA. MMP-10 was found to be highly expressed both in-vitro and in-vivo in neoplastic OAC cells relative to non-neoplastic squamous epithelial cells. Our results show that DCA promotes OAC invasion and MMP-10 overexpression. This study will advance our understanding of the pathophysiological mechanisms involved in human OAC and shows promise for the development of new therapeutic strategies.

Cardioprotective effect of MMP-2-inhibitor-NO-donor hybrid against ischaemia/reperfusion injury.

Hypoxic injury of cardiovascular system is one of the most frequent complications following ischaemia. Heart injury arises from increased degradation of contractile proteins, such as myosin light chains (MLCs) and troponin I by matrix metalloproteinase 2 (MMP-2). The aim of the current research was to study the effects of 5-phenyloxyphenyl-5-aminoalkyl nitrate barbiturate (MMP-2-inhibitor-NO-donor hybrid) on hearts subjected to ischaemia/reperfusion (I/R) injury. Primary human cardiac myocytes and Wistar rat hearts perfused using Langendorff method have been used. Human cardiomyocytes or rat hearts were subjected to I/R in the presence or absence of tested hybrid. Haemodynamic parameters of heart function, markers of I/R injury, gene and protein expression of MMP-2, MMP-9, inducible form of NOS (iNOS), asymmetric dimethylarginine (ADMA), as well as MMP-2 activity were measured. Mechanical heart function, coronary flow (CF) and heart rate (HR) were decreased in hearts subjected to I/R Treatment of hearts with the hybrid (1-10 µmol/L) resulted in a concentration-dependent recovery of mechanical function, improved CF and HR. This improvement was associated with decreased tissue injury and reduction of synthesis and activity of MMP-2. Decreased activity of intracellular MMP-2 led to reduced degradation of MLC and improved myocyte contractility in a concentration-dependent manner. An infusion of a MMP-2-inhibitor-NO-donor hybrid into I/R hearts decreased the expression of iNOS and reduced the levels of ADMA. Thus, 5-phenyloxyphenyl-5-aminoalkyl nitrate barbiturate protects heart from I/R injury.

Novel Iron-Whey Protein Microspheres Protect Gut Epithelial Cells from Iron-Related Oxidative Stress and Damage and Improve Iron Absorption in Fasting Adults.

BACKGROUND: Iron food fortification and oral iron formulations are frequently limited by poor absorption, resulting in the widespread use of high-dose oral iron, which is poorly tolerated. METHODS: We evaluated novel iron-denatured whey protein (Iron-WP) microspheres on reactive oxygen species (ROS) and viability in gut epithelial (HT29) cells. We compared iron absorption from Iron-WP versus equimolar-dose (25 mg elemental iron) ferrous sulphate (FeSO4) in a prospective, randomised, cross-over study in fasting volunteers (n = 21 per group) dependent on relative iron depletion (a ferritin level ≤/>30 ng/mL). RESULTS: Iron-WP caused less ROS generation and better HT29 cell viability than equimolar FeSO4. Iron-WP also showed better absorption with a maximal 149 ± 39% increase in serum iron compared to 65 ± 14% for FeSO4 (p = 0.01). The response to both treatments was dependent on relative iron depletion, and multi-variable analysis showed that better absorption with Iron-WP was independent of baseline serum iron, ferritin, transferrin saturation, and haemoglobin in the overall group and in the sub-cohort with relative iron depletion at baseline (p < 0.01). CONCLUSIONS: Novel Iron-WP microspheres may protect gut epithelial cells and improve the absorption of iron versus FeSO4. Further evaluation of this approach to food fortification and supplementation with iron is warranted.

A fluorescent analogue of tauroursodeoxycholic acid reduces ER stress and is cytoprotective.

Tauroursodeoxycholic acid (TUDCA) is a cytoprotective ER stress inhibitor and chemical chaperone. It has therapeutic potential in a wide array of diseases but a specific macromolecular target or molecular mechanism of action remains obscure. This Letter describes an effective new synthetic approach to taurine conjugation of bile acids which we used to prepare 3α-dansyl TUDCA (4) as a probe for TUDCA actions. As a model of ER stress we used the hepatocarcinoma cell line HUH7 and stimulation with either deoxycholic acid (DCA, 200µM) or tunicamycin (5µg/ml) and measured levels of Bip/GRP78, ATF4, CHOP and XBP1s/XBP1u. Compound 4 was more effective than UDCA at inhibiting ER stress markers and had similar effects to TUDCA. In a model of cholestasis using the cytotoxic DCA to induce apoptosis, pretreatment with 4 prevented cell death similarly to TUDCA whereas the unconjugated clinically used UDCA had no effect. 3α-Dansyl TUDCA (4) appears to be a suitable reporter for TUDCA effects on ER stress and related cytoprotective activity.

Novel Selective Butyrylcholinesterase Inhibitors Incorporating Antioxidant Functionalities as Potential Bimodal Therapeutics for Alzheimer's Disease.

Isosorbide-2-carbamates-5-aryl esters are highly potent and very selective butyrylcholinesterase inhibitors. The objective of the present work was to address the hypothesis that the isosorbide-aryl-5-ester group could be replaced with an antioxidant functionality while maintaining inhibitor effects and selectivity. We successfully incorporated ferulic acid or lipoic acid groups producing potent selective inhibitors of butyrylcholinesterase (BuChE). The hybrid compounds were non-toxic to the murine hippocampal cell line HT-22 and lipoate esters were neuroprotective at 10 and 25 µM when the cells were challenged with glutamate (5 mM) in a similar manner to the positive control quercetin. The benzyl carbamate 7a was a potent inhibitor of BuChE (IC50 150 nM) and it was effective in reducing glutamate toxicity to neuronal cells at >5 µM. Representative compounds exhibited an antioxidant effect in the oxygen radical absorbance capacity assay as the lipoate 7d was not active, whereas the ferulate 8a showed a weak, but significant, activity with 0.635 ± 0.020 Trolox Equivalent.

Nitric oxide-matrix metaloproteinase-9 interactions: biological and pharmacological significance--NO and MMP-9 interactions.

Nitric oxide (NO) and matrix metalloproteinase 9 (MMP-9) levels are found to increase in inflammation states and in cancer, and their levels may be reciprocally modulated. Understanding interactions between NO and MMP-9 is of biological and pharmacological relevance and may prove crucial in designing new therapeutics. The reciprocal interaction between NO and MMP-9 have been studied for nearly twenty years but to our knowledge, are yet to be the subject of a review. This review provides a summary of published data regarding the complex and sometimes contradictory effects of NO on MMP-9. We also analyse molecular mechanisms modulating and mediating NO-MMP-9 interactions. Finally, a potential therapeutic relevance of these interactions is presented.

Matrix metalloproteinases in inflammatory bowel disease: an update.

Matrix metalloproteinases (MMPs) are known to be upregulated in inflammatory bowel disease (IBD) and other inflammatory conditions, but while their involvement is clear, their role in many settings has yet to be determined. Studies of the involvement of MMPs in IBD since 2006 have revealed an array of immune and stromal cells which release the proteases in response to inflammatory cytokines and growth factors. Through digestion of the extracellular matrix and cleavage of bioactive proteins, a huge diversity of roles have been revealed for the MMPs in IBD, where they have been shown to regulate epithelial barrier function, immune response, angiogenesis, fibrosis, and wound healing. For this reason, MMPs have been recognised as potential biomarkers for disease activity in IBD and inhibition remains a huge area of interest. This review describes new roles of MMPs in the pathophysiology of IBD and suggests future directions for the development of treatment strategies in this condition.

Mechanisms of platelet-stimulated colon cancer invasion: role of clusterin and thrombospondin 1 in regulation of the P38MAPK-MMP-9 pathway.

Platelets have been implicated in colon cancer metastasis and prognosis but the underlying molecular mechanisms remain unclear. We evaluated the role of the different mitogen-activated protein kinase (MAPK) pathways in platelet-stimulated matrix metalloproteinase-9 (MMP-9) generation and colon cancer invasion. In addition, proteins released during platelet-tumour cell interactions were studied. For this purpose, interactions of Caco-2 and HT29 cells with platelets were studied using scanning electron microscopy, aggregometry, flow cytometry and cell invasion chambers. Quantitative PCR and zymography were used to study MMP-9 gene expression and activity, respectively, whereas western blot was used to study p38MAPK. Finally, the origin of proteins during platelet-cancer cell interactions was investigated using stable isotope labelling by amino acids in cell culture (SILAC)-based proteomics. We found that platelets promoted p38MAPK phosphorylation and MMP-9 up-regulation in both cell lines, with the subsequent cell-invasion-promoting effects. Pharmacological inhibition of p38MAPK led to a significant down-regulation of MMP-9 and colon cancer cell invasiveness. Also, p38MAPK-small interfering RNA abolished the induction of platelet-stimulated MMP-9. SILAC experiments demonstrated that thrombospondin 1 (TSP1) was released mainly from platelets and clusterin by both platelets and cancer cells. Finally, inhibition of TSP1 and clusterin abolished p38MAPK phosphorylation, MMP-9 activity and platelet-stimulated colon cancer invasion. Our results indicate that platelet-secreted TSP1 and clusterin promote the signal regulation of MMP-9 in platelet-induced colonic cancer invasion via a P38MAPK-regulated pathway. These findings are relevant to the development of therapeutic approaches to preventing and reducing tumour cell metastasis induced by colon adenocarcinoma.

New highly toxic bile acids derived from deoxycholic acid, chenodeoxycholic acid and lithocholic acid.

We have prepared a new panel of 23 BA derivatives of DCA, chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) in order to study the effect of dual substitution with 3-azido and 24-amidation, features individually associated with cytotoxicity in our previous work. The effect of the compounds on cell viability of HT-1080 and Caco-2 was studied using the 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Compounds with high potency towards reduction of cell viability were further studied using flow cytometry in order to understand the mechanism of cell death. Several compounds were identified with low micromolar IC50 values for reducing cell viability in the Caco-2 and HT1080 cell lines, making them among the most potent BA apoptotic agents reported to date. There was no evidence of relationship between overall hydrophobicity and cytotoxicity supporting the idea that cell death induction by BAs may be structure-specific. Compounds derived from DCA caused cell death through apoptosis. There was some evidence of selectivity between the two cell lines studied which may be due to differing expression of CD95/FAS. The more toxic compounds increased ROS production in Caco-2 cells, and co-incubation with the antioxidant N-acetyl cysteine blunted pro-apoptotic effects. The properties these compounds suggest that there may be specific mechanism(s) mediating BA induced cell death. Compound 8 could be useful for investigating this phenomenon.

Ursodeoxycholic acid attenuates colonic epithelial secretory function.

Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl(-) secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl(-) secretory responses to the Ca(2+) and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n = 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na(+)/K(+)-ATPase activity and basolateral K(+) channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg(-1)) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA (50-200 µm) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea.

MMP inhibition by barbiturate homodimers.

We have studied some homodimeric compounds derived from 5-homopiperazine substituted pyrimidine triones (barbiturates) with linkers in the range 2-20 carbon atoms. The compounds were designed to be capable of resisting absorption, to be stable in the gut and to maintain inhibitory potency against gelatinases and related function. The compounds were then assessed for inhibitory potency against a panel of MMPs (1, 2, 8, 9 and 13). The dimer compounds had similar potency and selectivity to the homopiperazine barbiturate monomer class. At 100 nM, selected dimers significantly inhibited cancer cell invasion in a matrigel assay using Caco-2 cells stimulated by hepatic growth factor. Finally, selected dimers showed adequate stability in simulated intestinal fluid to suggest the capacity to transit to the colon.

Synthesis and biological evaluation of Foscan® bile acid conjugates to target esophageal cancer cells.

Porphyrins and chlorins such as Foscan® have a natural proclivity to accumulate in cancer cells. This trait has made them good candidates for photosensitizers and as imaging agents in phototherapy. In order to improve on cellular selectivity to lower post-treatment photosensitivity bile acid porphyrin bioconjugates have been prepared and investigated in esophageal cancer cells. Bile acids which are known to selectively bind to, or be readily taken up by cancer cells were chosen as targeting moieties. Synthesis of the conjugates was achieved via selective nucleophilic monofunctionalization of 5,10,15,20-tetrahydroxyphenylporphyrins with propargyl bromide followed by Cu(I) mediated cycloaddition with bile acid azides in good yields. The compounds were readily taken up by esophageal cancer cells but showed no PDT activity.

A nitrophenyl-based prodrug type for colorectal targeting of prednisolone, budesonide and celecoxib.

Celecoxib is a COX-2 inhibitor drug that can be used to reduce the risk of colorectal adenocarcinoma. Glucocorticoids are used in the treatment of inflammatory bowel disease. A limitation to the use of both drug types is that they undergo absorption from the intestinal tract with serious side effects. The prodrug systems introduced here involve forming a nitro-substituted acylsulfonamide group in the case of celecoxib and a nitro-substituted 21-ester for the glucocorticoids. Drug release is triggered by the nitro reductase action of the colonic microflora, liberating a cyclization competent species. The release of the active parent drugs was evaluated in vitro using Clostridium perfringens and epithelial transport through Caco-2 monolayer evaluation was carried out to estimate the absorption properties of the prodrugs compared to the parental drugs.

Structure requirements for anaerobe processing of azo compounds: implications for prodrug design.

This Letter generalizes the metabolism of the azo class of compounds by Clostridium perfringens, an anaerobe found in the human colon. A recently reported 5-aminosalicylic acid-based prednisolone prodrug was shown to release the drug when incubated with the bacteria, while the para-aminobenzoic acid (PABA) based analogue did not. Instead, it showed a new HPLC peak with a relatively close retention time to the parent which was identified by LCMS as the partially reduced hydrazine product. This Letter investigates azoreduction across a panel of substrates with varying degrees of electronic and steric similarity to the PABA-based compound. Azo compounds with an electron donating group on the azo-containing aromatic ring showed immediate disproportionation to their parent amines without any detection of hydrazine intermediates by HPLC. Compounds containing only electron withdrawing groups are partially and reversibly reduced to produce a stable detectable hydrazine. They do not disproportionate to their parent amines, but regenerate the parent azo compound. This incomplete reduction is relevant to the design of azo-based prodrugs and the toxicology of azo-based dyes.

Azo-reductase activated budesodine prodrugs for colon targeting.

Budesodine is a synthetic glurocorticoid that undergoes substantial first pass metabolism, limiting systemic exposure. Its use in treatment of inflammatory bowel disease would benefit from a targeting strategy that could lead to a local topical effect, improving safety and increasing anti-inflammatory efficacy. A two-step prodrug strategy involving azoreduction/cyclization that we developed previously for prednisolone is here applied with some variations to budesonide. The budesodine prodrugs were tested using an in vitro azoreductase assay simulating human colonic microflora. The kinetics of amino steroid ester cyclization and its pH dependence was also evaluated. The stability of the prodrugs systems in simulated human duodenal and gastric fluid was evaluated to determine the likelihood of intact intestinal transit. The propionic acid derived prodrug 3 undergoes rapid activation by Clostridium perfingens and its putative reduction product cyclizes with acceptable rapidity when synthesized independently. These properties of 3 suggest that it has potential in management of ulcerative colitis.

New fluorescent bile acids: synthesis, chemical characterization, and disastereoselective uptake by Caco-2 cells of 3-deoxy 3-NBD-amino deoxycholic and ursodeoxycholic acid.

Deoxycholic acid (DCA), a secondary bile acid (BA), and ursodeoxycholic acid (UDCA), a tertiary BA, cause opposing effects in vivo and in cell suspensions. Fluorescent analogues of DCA and UDCA could help investigate important questions about their cellular interactions and distribution. We have prepared a set of isomeric 3α- and 3ß-amino analogues of UDCA and DCA and derivatised these with the discrete fluorophore, 4-nitrobenzo-2-oxa-1,3-diazol (NBD), forming the corresponding four fluorescent adducts. These absorb in the range 465-470 nm and fluoresce at approx. 535 nm. In order to determine the ability of the new fluorescent bile acids to mimic the parents, their uptake was studied using monolayers of Caco-2 cells, which are known to express multiple proteins of the organic anion-transporting peptide (OATP) subfamily of transporters. Cellular uptake was monitored over time at 4 and 37°C to distinguish between passive and active transport. All four BA analogues were taken up but in a strikingly stereo- and structure-specific manner, suggesting highly discriminatory interactions with transporter protein(s). The α-analogues of DCA and to a lesser extent UDCA were actively transported, whereas the ß-analogues were not. The active transport process was saturable, with Michaelis-Menten constants for 3α-NBD DCA (5) being K(m)=42.27±12.98 µM and V(max)=2.8 ± 0.4 nmol/(mg protein*min) and for 3α-NBD UDCA (3) K(m)=28.20 ± 7.45 µM and V(max)=1.8 ± 0.2 nmol/(mg protein*min). These fluorescent bile acids are promising agents for investigating questions of bile acid biology and for detection of bile acids and related organic anion transport processes.

A double prodrug system for colon targeting of benzenesulfonamide COX-2 inhibitors.

The design, synthesis and delivery potential of a new type of benzenesulfonamide cyclo-oxygenase-2 (COX-2) inhibitor prodrug is investigated using celecoxib. The approach involves a double prodrug that is activated first by azoreductases and then by cyclization triggering drug release. We studied the intramolecular aminolysis of the acylsulfonamide. The cyclization was surprisingly rapid at physiological pH and very fast at pH 5. The prodrug is activated specifically under conditions found in the colon but highly stable in the presence of human and rodent intestinal extracts. Finally, the prototype with celecoxib was transported much more slowly in the Caco-2 transepithelial model than the parent. The design therefore shows significant promise for the site specific delivery of benzenesulfonamide COX-2 inhibitors to the colon.

N-substituted homopiperazine barbiturates as gelatinase inhibitors.

Matrix metalloproteinases are implicated in a wide range of pathophysiological processes and potent selective inhibitors for these enzymes continue to be eagerly sought. 5,5-Disubstituted barbiturates hold promise as inhibitor types being stable in vivo and relatively selective for the gelatinases (MMP-2 and MMP-9). In this paper we describe the synthesis of 5-piperazine and -homopiperazine substituted barbiturates. The activity of these compounds as gelatinase inhibitors was evaluated using supernatants from 12-O-tetradecanoylphorbol-13-acetate (PMA)-stimulated HT-1080 cells as well as using recombinant human MMPs. N-Acyl homopiperazine compounds were found to be potent inhibitors of the gelatinases (range in nM) and generally more potent than the corresponding piperazine analogues. The panel of N-acyl homopiperazines was enlarged in order to exploit differences between the gelatinases at the S2' site in order to design MMP-2- or MMP-9-selective inhibitors. Compounds in this group exhibited single digit nano-molar potency and some selectivity between the two enzymes. Representative potent compounds were effective inhibitors of cancer cell migration.

Secondary (iso)BAs cooperate with endogenous ligands to activate FXR under physiological and pathological conditions.

IsoBAs, stereoisomers of primary and secondary BAs, are found in feces and plasma of human individuals. BA signaling via the nuclear receptor FXR is crucial for regulation of hepatic and intestinal physiology/pathophysiology. AIM: Investigate the ability of BA-stereoisomers to bind and modulate FXR under physiological/pathological conditions. METHODS: Expression-profiling, luciferase-assays, fluorescence-based coactivator-association assays, administration of (iso)-BAs to WT and cholestatic mice. RESULTS: Compared to CDCA/isoCDCA, administration of DCA/isoDCA, UDCA/isoUDCA only slightly increased mRNA expression of FXR target genes; the induction was more evident looking at pre-mRNAs. Notably, almost 50% of isoBAs were metabolized to 3-oxo-BAs within 4 h in cell-based assays, making it difficult to study their actions. FRET-based real-time monitoring of FXR activity revealed that isoCDCA>CDCA stimulated FXR, and isoDCA and isoUDCA allowed fully activated FXR to be re-stimulated by a second dose of GW4064. In vivo co-administration of a single dose of isoBAs followed by GW4064 cooperatively activated FXR, as did feeding of UDCA in a background of endogenous FXR ligands. However, in animals with biliary obstruction and concomitant loss of intestinal BAs, UDCA was unable to increase intestinal Fgf15. In contrast, mice with an impaired enterohepatic circulation of BAs (Asbt-/-, Ostα-/-), administration of UDCA was still able to induce ileal Fgf15 and repress hepatic BA-synthesis, arguing that UDCA is only effective in the presence of endogenous FXR ligands. CONCLUSION: Secondary (iso)BAs cooperatively activate FXR in the presence of endogenous BAs, which is important to consider in diseases linked to disturbances in BA enterohepatic cycling.

Bile acid toxicity structure-activity relationships: correlations between cell viability and lipophilicity in a panel of new and known bile acids using an oesophageal cell line (HET-1A).

The molecular mechanisms and interactions underlying bile acid cytotoxicity are important to understand for intestinal and hepatic disease treatment and prevention and the design of bile acid-based therapeutics. Bile acid lipophilicity is believed to be an important cytotoxicity determinant but the relationship is not well characterized. In this study we prepared new azido and other lipophilic BAs and altogether assembled a panel of 37 BAs with good dispersion in lipophilicity as reflected in RPTLC RMw. The MTT cell viability assay was used to assess cytotoxicity over 24 h in the HET-1A cell line (oesophageal). RMw values inversely correlated with cell viability for the whole set (r2=0.6) but this became more significant when non-acid compounds were excluded (r2=0.82, n=29). The association in more homologous subgroups was stronger still (r2>0.96). None of the polar compounds were cytotoxic at 500 microM, however, not all lipophilic BAs were cytotoxic. Notably, apart from the UDCA primary amide, lipophilic neutral derivatives of UDCA were not cytotoxic. Finally, CDCA, DCA and LagoDCA were prominent outliers being more toxic than predicted by RMw. In a hepatic carcinoma line, lipophilicity did not correlate with toxicity except for the common naturally occurring bile acids and their conjugates. There were other significant differences in toxicity between the two cell lines that suggest a possible basis for selective cytotoxicity. The study shows: (i) azido substitution in BAs imparts lipophilicity and toxicity depending on orientation and ionizability; (ii) there is an inverse correlation between RMw and toxicity that has good predictive value in homologous sets; (iii) lipophilicity is a necessary but apparently not sufficient characteristic for BA cytocidal activity to which it appears to be indirectly related.