Human LS174T Research Articles

A32 CATHELICIDIN REGULATES GOBLET CELL MUCUS SECRETION DURING CITROBACTER RODENTIUM-INDUCED COLITIS

Abstract Background Colonic goblet cells by secreting Muc2 mucin and specific proteins is critical for physically entrapping and expelling invading enteropathogens. Thus, is not surprising that Muc2-/- littermates exhibit increased susceptibility to attaching/effacing Citrobacter rodentium colonization. The colonic epithelium also secretes small cathelicidin peptide, which potentially interacts intimately with goblet cells and was presumed to accumulate within the sterile inner mucus layer as a simple antimicrobial peptide defense. Aims To determine the effects of cathelicidin on mucin secretion in goblet cells during C. rodentium-induced colitis and the impact on the mucus barrier defense. Methods We used cathelicidin-deficient (Camp-/-) mice, mouse colonoids and human colonic LS174T like-goblet epithelial cells to elucidate the mechanisms by which cathelicidin regulates goblet cell secretions. Results Camp -/- littermates infected with C. rodentium displayed increased fecal shedding and epithelial colonization. Camp-/- littermates at the peak of C. rodentium infection (7 dpi) showed a deficient mucin layer with fewer Alcian blue/PAS filled goblet cells and a reduction in fucose (UEA-1+) and N-acetylglucosamine (WGA+) glycoproteins. By transmission electron microscopy (TEM), goblet cells in Camp-/- colons were swollen and retained a large number of mucus granules during C. rodentium infection. C. rodentium infected Camp-/- littermates showed impaired reactive oxygen species (ROS) production and a transcriptomic profiling associated with decreased ROS biosynthesis and an increase in ROS negative regulators. In mucin producing LS174T colonic epithelial cells, human cathelicidin LL-37 promptly induced the secretion of goblet cell-associated TFF3 and RELMβ, via a ROS-dependent mechanism. Conclusions These findings revealed that mice lacking cathelicidin (Camp-/-) were more susceptible to C. rodentium colonization caused by defective goblet cell mucus and mucin-associated protein secretion via a ROS-dependent mechanism. Importantly, cathelicidin regulated mucus secretion revealing a non microbicidal action of this peptide with homeostatic properties on the colonic mucus barrier, critical in excluding luminal microbiota away from the epithelia to clear bacterial infections and restore gut homeostasis. Funding Agencies NSERC

Flagellin is essential for initial attachment to mucosal surfaces by Clostridioides difficile.

Mucins are glycoproteins which can be found in host cell membranes and as a gelatinous surface formed from secreted mucins. Mucosal surfaces in mammals form a barrier to invasive microbes, particularly bacteria, but are a point of attachment for others. Clostridioides difficile is an anaerobic bacterium, which colonizes the mammalian gastrointestinal (GI) tract and is a common cause of acute GI inflammation leading to a variety of negative outcomes. Although C. difficile toxicity stems from secreted toxins, colonization is a prerequisite for C. difficile disease. While C. difficile is known to associate with the mucous layer and underlying epithelium, the mechanisms underlying these interactions that facilitate colonization are less well understood. To understand the molecular mechanisms by which C. difficile interacts with mucins, we used ex vivo mucosal surfaces to test the ability of C. difficile to bind to mucins from different mammalian tissues. We found significant differences in C. difficile adhesion based upon the source of mucins, with highest levels of binding observed to mucins purified from the human colonic adenocarcinoma line LS174T and lowest levels of binding to porcine gastric mucin. We also observed defects in adhesion by mutants deficient in flagella but not type IV pili. These results imply that interactions between host mucins and C. difficile flagella facilitate the initial host attachment of C. difficile to host cells and secreted mucus. Importance Clostridioides difficile is one of the leading causes of hospital-acquired infections worldwide and presents challenges in treatment due to recurrent gastrointestinal disease after treatment with antimicrobials. The mechanisms by which C. difficile colonizes the gut represent a key gap in knowledge, including its association with host cells and mucosa. Our results show the importance of flagellin for specific adhesion to mucosal hydrogels and can help to explain prior observations of adhesive defects in flagellin and pilin mutants.

Synthesis and Evaluation of ePSMA-DM1: A New Theranostic Small-Molecule Drug Conjugate (T-SMDC) for Prostate Cancer.

Small-molecule drug conjugates (SMDCs) are compounds in which a therapeutic payload is conjugated to a targeting vector, for specific delivery to the tumor site. This promising approach can be translated to the treatment of prostate cancer by selecting a targeting vector which binds to the prostate-specific membrane antigen (PSMA). Moreover, the addition of a bifunctional chelator to the molecule allows for the use of both diagnostic and therapeutic radionuclides. In this way, the distribution of the SMDC in the body can be monitored, and combination therapy regimes can be implemented. We combined a glutamate-urea-lysine vector to the cytotoxic agent DM1 and a DOTA chelator via an optimized linker to obtain the theranostic SMDC (T-SMDC) ePSMA-DM1. ePSMA-DM1 retained a high binding affinity to PSMA and demonstrated PSMA-specific uptake in cells. Glutathione stability assays showed that the half-life of the T-SMDC in a reducing environment was 2 h, and full drug release was obtained after 6 h. Moreover, 100 nM of ePSMA-DM1 reduced the cell viability of the human PSMA-positive LS174T cells by >85% after 72 h of incubation, which was comparable to a 10-fold higher dose of free DM1. [111In]In-ePSMA-DM1 and [177Lu]Lu-ePSMA-DM1 were both obtained in high radiochemical yields and purities (>95%), with >90% stability in PBS and >80% stability in mouse serum for up to 24 h post incubation at 37 °C. SPECT/CT imaging studies allowed for a faint tumor visualization of [111In]In-ePSMA-DM1 at 1 h p.i., and the ex vivo biodistribution showed tumor uptake (2.39 ± 0.29% ID/g) at 1 h p.i., with the compound retained in the tumor for up to 24 h. Therefore, ePSMA-DM1 is a promising T-SMDC candidate for prostate cancer, and the data obtained so far warrant further investigations, such as therapeutic experiments, after further optimization.

Proof of concept of improved fluorescence-guided surgery of colon cancer liver metastasis using color-coded imaging of a tumor-labeling fluorescent antibody and indocyanine green restricted to the adjacent liver segment

BackgroundIndocyanine green has been used for fluorescence-guided surgery of liver metastasis and labeling of liver segments. However, indocyanine green is nonspecific, and indocyanine green labeling does not always clearly outline tumor margins. In addition, it is difficult to distinguish between a tumor and its adjacent liver segment colored with indocyanine green alone. In the present study, we performed fluorescence-guided surgery in an orthotopic colon-cancer liver metastasis mouse model by labeling the metastatic liver tumor with an anti-carcinoembryonic antigen fluorescent antibody and with indocyanine green restricted to the adjacent liver segment. MethodsA liver metastasis model was established with human LS174T colon cancer tumor fragments. To label the tumor, mice received SGM-101, an anti-carcinoembryonic antigen antibody conjugated to a near-infrared fluorophore (700 nm), currently in clinical trials, 3 days before surgery. Indocyanine green (800 nm) was injected after ligation of the tumor-bearing Glissonean pedicle with fluorescence labeling restricted to the liver segment adjacent to the tumor. Bright-light surgery and fluorescence-guided surgery were performed to resect the liver metastasis. To assess recurrence, mice underwent necropsy 3 weeks after surgery and the tumor was weighed. ResultsFluorescence-guided anatomic left lateral lobectomy and fluorescence-guided partial liver resection were both performed with color-coded double labeled imaging. Tumor weight 3 weeks after surgery was significantly lower with fluorescence-guided surgery compared to bright-light surgery (38 ± 57 mg vs 836 ± 668 mg, P = .011) for partial liver resection. ConclusionThe present study provides a proof-of-concept that color-coded and double labeling of the tumor and adjacent liver segment has the potential to improve liver metastasectomy.

Abstract 5426: Down-regulation of cancer metabolism enhances radiosensitivity by impairing the heat shock response

Abstract Therapy (radiation) resistance can, inter alia, be caused by an increased lactate dehydrogenase (LDH) activity, elevated concentrations of the oncometabolite lactate and an overexpression of anti-apoptotic heat shock proteins (HSPs). In this study we aimed to break radioresistance by targeting the cancer metabolism. We could show, for the first time, that the lactate metabolism and heat shock response are co-regulated in two cancer cell systems (murine B16F10 melanoma cells, human LS174T colorectal adenocarcinoma cells). A CRISPR/Cas9 induced double knockout of LDHA/B (LDH−/−) as well as a pharmacological inhibition of LDH by Oxamate or GNE-140 reduces tumor growth, ROS production and synthesis of different HSPs, including Hsp90, Hsp70 and Hsp27. Moreover, the membrane Hsp70 density was significantly reduced on tumor cells after inhibition of the LDH activity as a consequence of a down-regulated lipid metabolism affecting the Hsp70-anchoring, tumor-specific glycosphingolipid Gb3. Our results demonstrate that influencing cancer metabolism increases radiation sensitivity by an impairment of the stress response. Since presently available LDH inhibitors have a low stability and high toxicity in vivo, we are currently investigating more potent LDH inhibitors with a beneficial safety profile. In summary, targeting the lactate metabolism provides a promising strategy to improve radiosensitivity by impairing the stress response. Citation Format: Melissa Schwab, Katharina Thunborg, Omid Azimzadeh, Christine von Toerne, Maxim Shevtsov, Masa Zdralevic, Jacques Pouyssegur, Kathrin Renner, Marina Kreutz, Peter Vaupel, Gabriele Multhoff. Down-regulation of cancer metabolism enhances radiosensitivity by impairing the heat shock response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5426.

Co(III), Ni(II) and Cu(II) complexes with a tetradentate Schiff base ligand: synthesis, characterization, electrochemical behavior, binding assessment and in vitro cytotoxicity

Two new Schiff base cobalt(III) ([Co(LH)Cl2], 1) and nickel(II) ([Ni(LH)ClO4], 2) complexes with a diimine-dioxime ligand, (4,9-diaza-3,10-diethyl-3,9-dodecadiene-2,11-dione bis oxime (LH2 )), were synthesized and characterized. The compounds were obtained in MeOH from corresponding metal salts and LH2 in molar ratio 1:1 and further characterized by mass spectrometry, IR spectroscopy, electrochemistry, and elemental analysis. Previously reported copper(II) analog ([Cu2(LH)2]·(ClO4)2, 3) was joined to 1 and 2, and the three metal analogs, 1–3, were further investigated in terms of their electrochemical behavior. Binding studies of the complexes with deoxyribonucleic acid (DNA) and human serum albumin (HSA) were carried out using both spectrophotometric and electrochemical methods. All three complexes exhibit binding affinity towards the DNA chain through intercalative interactions. The binding reaction with HSA showed for 1 and 3 decrease in the peak current obtained in the case of complexes before the addition of HSA, while the Ni complex–HSA possesses the same electroactivity as starting complex. The cytotoxicity of LH2 as well as its metal complexes, and cisplatin were evaluated on CT-26 mouse colon carcinoma and human LS174T cancer cell lines employing MTT assay. The copper(II) complex exhibited very promising anticancer activity compared with cisplatin.

Structural and Chemical Alterations to the Intestinal Mucus Barrier during Giardia duodenalis Infection

Background During the course of infection with the protozoan parasite Giardia duodenalis, both mucus production and structure of the mucus layers have been shown to be altered throughout the large and small intestines. This potentially has significant impacts on mucosal barrier function, predisposing a host to mucosal inflammation, which contributes both to Giardia pathogenesis and to development of post-infectious complications. We have attempted to characterize the mechanisms by which Giardia modulates mucus production and secretion by intestinal goblet cells, as well as chemical alterations to mucin proteins that occur in response to Giardia infection. Several effects have been found to be dependent on Giardia cysteine protease activity, suggesting a potential mechanism involving protease activated receptors. Methods The human colonic epithelial cell line LS174T was infected with Giardia trophozoites (isolates NF, WB, S2, and GSM). Prior to infection, trophozoites were treated with E64, a broad-spectrum cysteine protease inhibitor, and LS174T were treated with a pepducin PAR2 antagonist, BAPTA, a calcium chelator, or U0126, an ERK1/2 inhibitor. MUC2 mucin gene expression was assessed using quantitative PCR (qPCR). Wild type (WT) and PAR2 deficient (PAR2 −/−) mice were infected with Giardia trophozoites. The colonic mucus layer was stained using WGA and qPCR was performed for Muc2 and Muc5ac in the small and large intestines. Expression of glycosyltransferase enzymes was assessed using qPCR. Results Giardia NF, S2, and WB, but not GSM, upregulate MUC2 expression in LS174T cells. This increase was attenuated by inhibition of Giardia cysteine proteases and by antagonism of PAR2 or inhibition of calcium release or ERK1/2 signaling in LS174T cells. Giardia infected WT mice show increased Muc2 and Muc5ac expression in the colon, and increased Muc5ac but decreased Muc2 expression in the jejunum. These changes were not seen in PAR2−/− mice. Both WT infected and PAR2−/− non-infected mice showed thinning of the colonic mucus layer compared to WT controls. There was some recovery in thickness in PAR2−/− infected mice. Expression of the fucosyltransferase Fut2 was increased in the jejunums of WT mice, while the sulfotransferase Chst4 was decreased. Expression of the glycan core synthase C2GnT1 was increased in the WT jejunum, while C1GalT1 expression was slightly decreased in the jejunum and colon. Conclusions Giardia-induced alterations to mucin gene expression in vitro and in vivo are dependent on PAR2 signaling. Canonical PAR2 signaling, characterized by calcium release and ERK1/2 pathway activation, contribute to altered expression of the MUC2 mucin in colonic goblet cell cultures. Expression of several glycosyltransferases, responsible for the addition of glycan groups to intestinal mucins, are altered during Giardia infection in vivo, suggesting mucosal alterations may involve chemical modifications of mucin proteins.

The Relative Indirect Anthelmintic Effect of Caprine Milk on Mucins Gene Expression in Vitro Using IL-22 Treated LS174T Cells Model of Helminth Infection

Mucus secretion by intestinal goblet cells constitutes an important mechanism in TH2 response following helminth infection mediated by the key cytokine IL-22. This indirect mechanism rather than directly attacking the parasite is important in preventing helminth attachment hence promoting helminth expulsion from the intestinal tract. We hypothesized that natural products having an anthelmintic activity like caprine milk may exert similar response. Using human intestinal LS174T cells treated with IL-22 to simulate helminth infection, we tested whether or not the co-treatment with caprine milk induces MUC1, MUC3, MUC4 and MUC5B genes expression. Optimal concentrations for caprine milk was determined to be 25% and 50% from cell viability assay. IL-22 induced helminth infection model was confirmed. However, the indirect anthelmintic effect of caprine milk was only relative as treatment of caprine milk in LS174T cells and IL-22 in vitro did not significantly induce MUC1, MUC3, MUC4 and MUC5B genes expression when compared to treatment with IL-22 alone. In conclusion, caprine milk was not significantly associated with the mechanism of increased mucus production through upregulation of mucin genes by intestinal cells. Caprine milk may possess direct anthelmintic effect rather than indirect.

Lectin drug conjugate therapy for colorectal cancer.

Drug resistance represents an obstacle in colorectal cancer (CRC) treatment because of its association with poor prognosis. rBC2LCN is a lectin isolated from Burkholderia that binds cell surface glycans that have fucose moieties. Because fucosylation is enhanced in many types of cancers, this lectin could be an efficient drug carrier if CRC cells specifically present such glycans. Therefore, we examined the therapeutic efficacy and toxicity of lectin drug conjugate therapy in CRC mouse xenograft models. The affinity of rBC2LCN for human CRC cell lines HT‐29, LoVo, LS174T, and DLD‐1 was assessed in vitro. The cytocidal efficacy of a lectin drug conjugate, rBC2LCN‐38 kDa domain of pseudomonas exotoxin A (PE38) was evaluated by MTT assay. The therapeutic effects and toxicity for each CRC cell line‐derived mouse xenograft model were compared between the intervention and control groups. LS174T and DLD‐1 cell lines showed a strong affinity for rBC2LCN. In the xenograft model, the tumor volume in the rBC2LCN‐PE38 group was significantly reduced compared with that using control treatment alone. However, the HT‐29 cell line showed weak affinity and poor therapeutic efficacy. No significant toxicities or adverse responses were observed. In conclusion, we demonstrated that rBC2LCN lectin binds CRC cells and that rBC2LCN‐PE38 significantly suppresses tumor growth in vivo. In addition, the efficacy of the drug conjugate correlated with its binding affinity for each CRC cell line. These results suggest that lectin drug conjugate therapy has potential as a novel targeted therapy for CRC cell surface glycans.

Role of wzxE in Salmonella Typhimurium lipopolysaccharide biosynthesis and interleukin-8 secretion regulation in human intestinal epithelial cells

In Salmonella Typhimurium (S. Typhimurium), lipopolysaccharide (LPS) anchored on the bacterial outer membrane is a major immune stimulus that can broadly activate immune cells and induce innate immune responses. wzxE is involved in bacterial LPS biosynthesis but has rarely been reported in Salmonella; wzxE encodes a flipase that can flip the precursor of LPS across the membrane into the periplasm space. Our preliminary data showed that the wzxE transposon mutant of S. Typhimurium could not significantly adhere to and invade into HEp-2 cells, but the mechanism remains unknown. In this study, we infected human LS174T, Caco-2, HeLa, and THP-1 cells with the wild-type S. Typhimurium strain SL1344, its wzxE mutant, and its complemented strain. wzxE depletion significantly attenuated bacterial adhesion and internalization in the four cell types. In addition, the postinfectious production of interleukin-8 (IL-8) was significantly decreased in the Caco-2 cells infected with the wzxE mutant. Bacterial LPS stained with polymyxin B probe also exhibited a reduced signal in the wzxE mutant. The silver staining of purified LPS demonstrated a significant reduction of the O-antigen (OAg) chain in the wzxE mutant. To confirm the role of OAg in the wzxE mutant during infection, we treated the HT-29 cells with the S. Typhimurium strain SL1344, its wzxE mutant, and their purified LPS, which revealed significantly decreased IL-8 secretion in the HT-29 cells treated with purified LPS from the wzxE mutant and with the wzxE mutant. In conclusion, wzxE mediates LPS biosynthesis and plays a major role in bacterial pathogenesis by regulating OAg flipping.

Detection of β-defensins and Other Antibacterial Peptides in P-glycoprotein Expressing Human Gastrointestinal Cell Lines and Their Secretions

Our laboratory has shown previously [1] that activity of surface P-glycoprotein (P-gp) was inversely associated with bacterial adhesion to the human gastrointestinal cell lines, Caco-2 and LS174T. To attempt a detailed analysis of the rationale for our observations, filtered conditioned medium, that contained only low molecular weight proteins, was taken from these human cell lines with or without treatment with rifampicin, a P-gp inducer. The conditioned medium samples were tested against pathogenic microflora and screened for antimicrobial factors with and without exposure to lipopolysaccharides of E. coli 0111:B4. Human β-defensin-1 mRNA expression increased with the pregnane X receptor (PXR) inducing rifampicin suggesting a positive association with PXR stimulation. In conclusion, antimicrobial activity was detected in the conditioned medium of the gastrointestinal cells, however, a single antimicrobial peptide responsive to PXR-mediated regulation and acting as a substrate for P-gp, was not conclusive.

Alterations to Mucus Production and Secretion during Giardia Infection Involve Giardia Protease Activity and PAR2 Activation

BackgroundThe protozoan parasite Giardia duodenalis has been shown to disrupt the intestinal mucus barrier via unknown mechanisms, potentially disrupting host mucosal immunity and contributing to development of post‐infectious disorders. Protease‐activated receptor 2 (PAR2) has been identified as a potential target for secreted Giardia cysteine proteases, and may play a role in disruption of mucus production and secretion in the intestines.MethodsThe human colonic epithelial cell line LS174T was infected with Giardia trophozoites (isolates NF, WB, S2, and GSM). Prior to infection, trophozoites were treated with E64, a broad‐spectrum cysteine protease inhibitor, and LS174T were treated with a pepducin PAR2 antagonist, BAPTA, a calcium chelator, or U0126, an ERK1/2 inhibitor. MUC2 mucin gene expression was assessed using quantitative PCR (qPCR), and intracellular mucin content was quantified by staining with fluorescein‐coupled wheat germ agglutinin (WGA) and imaging using confocal microscopy.Chinese hamster ovary cells transfected with nano‐luciferase tagged PAR2 were incubated with Giardia trophozoites. Release of enzymes due to cleavage at the receptor N‐terminus by Giardia resulted in a luminescent signal proportional to the number of cleaved receptors.Wild type (WT) and PAR2 deficient (PAR2 −/−) mice were infected with Giardia trophozoites. The colonic mucus layer was stained using WGA and qPCR was performed for Muc2 and Muc5ac in the small and large intestines.ResultsGiardia NF, S2, and WB, but not GSM, upregulate MUC2 expression in LS174T cells. This increase was attenuated by inhibition of Giardia cysteine proteases and by antagonism of PAR2 or inhibition of calcium release or ERK1/2 signaling in LS174T cells. Preliminary results suggest that WGA staining is decreased upon exposure of LS174T to Giardia NF trophozoites.Giardia trophozoites cleaved PAR2 at the N‐terminus, suggesting they are capable of activating the receptor. The amount of cleavage was isolate‐dependent, reflecting differences in protease activity, and cleavage was significantly reduced by E64 treatment for all isolates.Giardia infected WT mice show increased Muc2 and Muc5ac expression in the colon, and increased Muc5ac but decreased Muc2 expression in the jejunum. These changes were not seen in PAR2−/− mice. Both WT infected and PAR2−/− non‐infected mice showed thinning of the colonic mucus layer compared to WT controls. There was some recovery in thickness in PAR2−/− infected mice.ConclusionsGiardia‐induced alterations to mucin gene expression in vitro and in vivo are dependent on PAR2 signaling. Giardia cysteine proteases cleave and activate PAR2, leading to both calcium release and MAPK pathway activation, which in turn contributes to altered gene transcription. Further, Giardia induces altered mucus secretion in vitro, which may contribute to thinning of the colonic mucus gel in vivo. Further study is required to determine the role of PAR2 in mucus secretion during Giardia infection.Support or Funding InformationCrohn’s Colitis Canada

LKB1 Represses ATOH1 via PDK4 and Energy Metabolism and Regulates Intestinal Stem Cell Fate

In addition to the Notch and Wnt signaling pathways, energy metabolism also regulates intestinal stem cell (ISC) function. Tumor suppressor and kinase STK11 (also called LKB1) regulates stem cells and cell metabolism. We investigated whether loss of LKB1 alters ISC homeostasis in mice. We deleted LKB1 from ISCs in mice using Lgr5-regulated CRE-ERT2 (Lkb1Lgr5-KO mice) and the traced lineages by using a CRE-dependent TdTomato reporter. Intestinal tissues were collected and analyzed by immunohistochemical and immunofluorescence analyses. We purified ISCs and intestinal progenitors using flow cytometry and performed RNA-sequencing analysis. We measured organoid-forming capacity and ISC percentages using intestinal tissues from Lkb1Lgr5-KO mice. We analyzed human Ls174t cells with knockdown of LKB1 or other proteins by immunoblotting, real-time quantitative polymerase chain reaction, and the Seahorse live-cell metabolic assay. Some intestinal crypts from Lkb1Lgr5-KO mice lost ISCs compared with crypts from control mice. However, most crypts from Lkb1Lgr5-KO mice contained functional ISCs that expressed increased levels of Atoh1 messenger RNA (mRNA), acquired a gene expression signature associated with secretory cells, and generated more cells in the secretory lineage compared with control mice. Knockdown of LKB1 in Ls174t cells induced expression of Atoh1 mRNA and a phenotype of increased mucin production; knockdown of ATOH1 prevented induction of this phenotype. The increased expression of Atoh1 mRNA after LKB1 loss from ISCs or Ls174t cells did not involve Notch or Wnt signaling. Knockdown of pyruvate dehydrogenase kinase 4 (PDK4) or inhibition with dichloroacetate reduced the up-regulation of Atoh1 mRNA after LKB1 knockdown in Ls174t cells. Cells with LKB1 knockdown had a reduced rate of oxygen consumption, which was partially restored by PDK4 inhibition with dichloroacetate. ISCs with knockout of LKB1 increased the expression of PDK4 and had an altered metabolic profile. LKB1 represses transcription of ATOH1, via PDK4, in ISCs, restricting their differentiation into secretory lineages. These findings provide a connection between metabolism and the fate determination of ISCs.

Humanized Fluorescent Tumor-associated Glycoprotein-72 Antibody Selectively Labels Colon-cancer Liver Metastases in Orthotopic Mouse Models.

Fluorescence imaging has been shown to improve intra-operative detection of liver metastasis. The present study aimed to determine whether humanized anti-TAG-72 antibody (huCC49) conjugated to a near-infrared dye provides selective labeling of colorectal-cancer liver metastasis in orthotopic mouse models. Humanized anti-TAG-72 (huCC49) was conjugated to IRDye800CW (huCC49-IR800). Orthotopic liver-metastasis nude-mouse models (n=5) were established with the human colon-cancer LS174T cell-line. Three weeks later, mice were administered huCC49-IR800 and intra-vital imaging was performed 48 h later. The mean tumor-to-liver ratio (TLR) was calculated. Intra-vital imaging demonstrated clear tumor margins with minimal liver fluorescence 48 h after administration of 50 μg huCC49-IR800 with mean TLR=7.53 (SD±2.76). Anti-TAG-72 monoclonal antibody conjugated to IRDye800 provides distinct and bright labeling of colorectal tumors in orthotopic nude-mouse models of liver metastasis. TAG-72 may be a useful target for intra-operative imaging of colorectal cancer liver metastasis in the clinic.

Citrus auraptene induces drug efflux transporter P-glycoprotein expression in human intestinal cells.

P-glycoprotein (encoded by MDR1) is a membrane transport protein expressed in the intestine, liver, kidney, placenta, and blood-brain barrier. It excludes various clinically important drugs from cells, such as verapamil, digoxin, tacrolimus, and vinblastine. Therefore, human P-glycoprotein plays important roles in drug absorption, distribution, and excretion. We reported previously that auraptene, a natural compound occurring widely in citrus fruit (e.g., grapefruit), inhibited P-glycoprotein-mediated drug transport. In this study, we investigated the effects of auraptene and other phenylpropanoids on P-glycoprotein expression using human intestinal epithelial LS174T cells and a reporter plasmid expressing 10.2 kbp of the upstream regulatory region of MDR1. Auraptene (7-geranyloxycoumarin), a prenylated coumarin, and several phenylpropanoids, such as 3-(4'-geranyloxy-3'-methoxyphenyl)-2-trans propenoic acid, derricidin [2'-hydroxy-4'-(prenyloxy)chalcone], and 3-(4'-geranyloxyphenyl)-propanoic acid, induced MDR1 promoter activity in LS174T cells. Overexpression of the nuclear receptor human pregnane X receptor gene (NR1I2) enhanced auraptene-induced MDR1 activation. Nuclear factor-kappaB inhibitors, Bay11-7082 and JSH-23, repressed MDR1 activation by auraptene. Western blot analyses showed the induction of P-glycoprotein expression in the auraptene-treated LS174T cells. The citrus phytochemical auraptene can induce the drug efflux transporter P-glycoprotein in human intestinal cells, and thus has the potential to cause food-drug interactions.

Docosahexaenoic and Eicosapentaenoic Acids Prevent Altered-Muc2 Secretion Induced by Palmitic Acid by Alleviating Endoplasmic Reticulum Stress in LS174T Goblet Cells.

Diets high in saturated fatty acids (FA) represent a risk factor for the development of obesity and associated metabolic disorders, partly through their impact on the epithelial cell barrier integrity. We hypothesized that unsaturated FA could alleviate saturated FA-induced endoplasmic reticulum (ER) stress occurring in intestinal secretory goblet cells, and consequently the reduced synthesis and secretion of mucins that form the protective mucus barrier. To investigate this hypothesis, we treated well-differentiated human colonic LS174T goblet cells with palmitic acid (PAL)—the most commonly used inducer of lipotoxicity in in vitro systems—or n-9, n-6, or n-3 unsaturated fatty acids alone or in co-treatment with PAL, and measured the impact of such treatments on ER stress and Muc2 production. Our results showed that only eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids protect goblet cells against ER stress-mediated altered Muc2 secretion induced by PAL, whereas neither linolenic acid nor n-9 and n-6 FA are able to provide such protection. We conclude that EPA and DHA could represent potential therapeutic nutrients against the detrimental lipotoxicity of saturated fatty acids, associated with type 2 diabetes and obesity or inflammatory bowel disease. These in vitro data remain to be explored in vivo in a context of dietary obesity.

Giardia‐Induced Alterations to Intestinal Mucus Production Involve Protease‐Activated Receptor‐2‐Mediated Activation of MAPK and Calcium Release

IntroductionDisruption of the intestinal mucus layers is associated with increased rates of infection and inflammation and has been implicated in a wide variety of intestinal disorders. Giardia duodenalis has been shown to disrupt mucus production and secretion [1] and may therefore be used as a model system to study regulation of these processes in health and disease.AimsDetermine the mechanisms involved in Giardia mediated disruptions to mucus production by intestinal goblet cells. The roles of protease‐activated receptor‐2 (PAR2) and Giardia cysteine proteases were investigated.MethodsThe human colonic epithelial cell line LS174T was infected with Giardia trophozoites (isolates NF, WB, S2, and GSM). Prior to infection, trophozoites were treated with E64, a broad‐spectrum cysteine protease inhibitor, and LS174T were treated with a pepducin PAR2 antagonist, BAPTA, a calcium chelator, or U0126, an ERK1/2 inhibitor. MUC2 mucin gene expression was assessed using quantitative PCR (qPCR).Chinese hamster ovary cells transfected with nano‐luciferase tagged PAR2 were incubated with Giardia trophozoites. Release of enzymes due to cleavage at the receptor N‐terminus by Giardia resulted in a luminescent signal proportional to the number of cleaved receptors.Wild type (WT) and PAR2 deficient (PAR2 −/−) mice were infected with Giardia trophozoites. The colonic mucus layer was stained using fluorescein‐coupled wheat germ agglutinin, and qPCR was performed for Muc2 and Muc5ac.ResultsGiardia isolates NF, S2, and WB, but not GSM, increased MUC2 expression in LS174T cells. This increase was attenuated by inhibition of Giardia cysteine proteases and by antagonism of PAR2 or inhibition of calcium release or MAPK signaling in LS174T cells.Giardia trophozoites cleaved PAR2 at the N‐terminus, suggesting they are capable of activating the receptor. The amount of cleavage was isolate‐dependent, reflecting differences in protease activity, and cleavage was significantly reduced by E64 treatment for all isolates.Giardia infected WT mice show increased Muc2 and Muc5ac expression in the colon, and increased Muc5ac but decreased Muc2 expression in the jejunum. These changes were not seen in PAR2−/− mice. Both WT infected and PAR2−/− non‐infected mice showed thinning of the colonic mucus layer compared to WT controls. There was some recovery in thickness in PAR2−/− infected mice.ConclusionsResults demonstrate that PAR2 plays a significant role in the regulation of mucin gene expression in mice and a human colonic cell line, and indicate a potential pathway in which Giardia cysteine proteases cleave PAR2 at the N‐terminus, activating canonical PAR2 signaling which involves both calcium release and MAPK activation. These pathways in turn contribute to altered mucin gene expression.Support or Funding InformationCrohn's Colitis Canada (CCC)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bovine milk-derived exosomes enhance goblet cell activity and prevent the development of experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is characterized by intestinal injury and impaired mucin synthesis. We recently showed that breast milk exosomes from rodents promote intestinal cell viability, epithelial proliferation, and stem cell activity, but whether they also affect mucus production is unknown. Therefore, the aim of this study was to investigate the effects of bovine milk-derived exosomes on goblet cell expression in experimental NEC and delineate potential underlying mechanisms of action. Exosomes were isolated from bovine milk by ultracentrifugation and confirmed by Nanoparticle Tracking Analysis and through the detection of exosome membrane markers. To study the effect on mucin production, human colonic LS174T cells were cultured and exposed to exosomes. Compared to control, exosomes promoted goblet cell expression, as demonstrated by increased mucin production and relative expression levels of goblet cell expression markers trefoil factor 3 (TFF3) and mucin 2 (MUC2). In addition, exosome treatment enhanced the expression of glucose-regulated protein 94 (GRP94), the most abundant intraluminal endoplasmic reticulum (ER) chaperone protein that aids in protein synthesis. Furthermore, experimental NEC was induced in mouse pups by hyperosmolar formula feeding, lipopolysaccharide administration and hypoxia exposure on postnatal days 5–9. Milk exosomes were given with each gavage feed. NEC was associated with ileal morphological injury and reduction in MUC2+ goblet cells and GRP94+ cells per villus. Exosome administration to NEC pups prevented these changes. This research highlights the potential novel application of milk-derived exosomes in preventing the development of NEC in high-risk infants when breast milk is not available.

Belinostat, at Its Clinically Relevant Concentrations, Inhibits Rifampicin-Induced CYP3A4 and MDR1 Gene Expression.

Activation of human pregnane X receptor (hPXR) has been associated with induction of chemoresistance. It has been proposed that such chemoresistance via cytochrome P450/drug transporters can be reversed with the use of antagonists that specifically abrogate agonist-mediated hPXR activation. Unfortunately, proposed antagonists lack the specificity and appropriate pharmacological characteristics that allow these features to be active in the clinic. We propose that, ideally, an hPXR antagonist would be a cancer drug itself that is part of a "cancer drug cocktail" and effective as an hPXR antagonist at therapeutic concentrations. Belinostat (BEL), a histone deacetylase inhibitor approved for the treatment of relapsed/refractory peripheral T-cell lymphoma, and often used in combination with chemotherapy, is an attractive candidate based on its hPXR ligand-like features. We sought to determine whether these features of BEL might allow it to behave as an antagonist in combination chemotherapy regimens that include hPXR activators. BEL represses agonist-activated hPXR target gene expression at its therapeutic concentrations in human primary hepatocytes and LS174T human colon cancer cells. BEL repressed rifampicin-induced gene expression of CYP3A4 and multidrug resistance protein 1, as well as their respective protein activities. BEL decreased rifampicin-induced resistance to SN-38, the active metabolite of irinotecan, in LS174T cells. This finding indicates that BEL could suppress hPXR agonist-induced chemoresistance. BEL attenuated the agonist-induced steroid receptor coactivator-1 interaction with hPXR, and, together with molecular docking studies, the study suggests that BEL directly interacts with multiple sites on hPXR. Taken together, our results suggest that BEL, at its clinically relevant therapeutic concentration, can antagonize hPXR agonist-induced gene expression and chemoresistance.

Inhibition of cell proliferation, migration and colony formation of LS174T Cells by carbonic anhydrase inhibitor.

Metastasis is the leading cause of cancer deaths. Migration of tumor cells is an important stage in metastasis. Therefore, recent studies have focused on clarifying migration and migration-dependent cell functions such as angiogenesis, wound healing, and invasion. In the present study, we aimed to investigate the effect of acetazolamide, which is a classical carbonic anhydrase inhibitor, on the cell viability, migration, and colony forming capacity of human LS174T colorectal cancer cells. Three different cell culture techniques (MTT test, wound healing and clonogenic assay) were performed in this in vitro study on colorectal cancer cells. Acetazolamide reduced the cell viability, migration and colony formation ability of cells depending on dose. There was no significant difference between the cells treated with acetazolamide with 1 µM dose and the control. However, it can be concluded that acetazolamide exerts its effect on human colorectal cancer cells at 10-1000 µM concentrations. Acetazolamide was observed to significantly inhibit the cell viability, colony forming capacity, and migration ability in the culture medium of LS174T cells. This inhibitor effect of acetazolamide was observed to be dependent on the concentration in medium.