Human Colonic Epithelial Cell Line Research Articles

A serine protease extracted from Trichosanthes kirilowii induces apoptosis via the PI3K/AKT-mediated mitochondrial pathway in human colorectal adenocarcinoma cells.

Trichosanthes kirilowii exhibits various biological functions including anti-inflammatory, antidiabetic and anticancer activities. In this study, a novel protein with anti-cancer activity, named as TKP, was purified from Trichosanthes kirilowii fruit by cell-base screening. Mass spectrometry and protease assays revealed that TKP is a serine protease. TKP decreased cell viability in a concentration-and time-dependent manner in human colorectal adenocarcinoma cells. Notably, TKP presented very little effect on human colon epithelial cell line NCM460. Furthermore, TKP inhibited colorectal adenocarcinoma cells to aggregate into viable colony clusters and induced cell apoptosis. The loss of mitochondrial membrane potential, upregulation of cytochrome c and Bax, downregulation of Bcl-2, and activation of caspase-9 and -3 were observed, indicating mitochondria-dependent apoptosis induced by TKP. We found that TKP activated MAPK/ERK and suppressed PI3K/AKT signaling. Inhibition of MAPK/ERK signaling by employing inhibitor PD98059 did not antagonize the effects of TKP. Treatment with PI3K inhibitor LY294003 increased the impact of TKP on mitochondria-related apoptosis proteins (cytochrome c, Bcl-2, Bax, caspase-9 and caspase-3) and cell apoptosis. On the contrary, PI3K/AKT activator insulin-like growth factor-1 reversed the effects of TKP. Taken together, our results demonstrated that TKP has potential anti-colorectal cancer activity by inducing apoptosis, which was regulated by the PI3K/AKT-mediated mitochondria-dependent pathway.

MicroRNA-124 (MiR-124) Inhibits Cell Proliferation, Metastasis and Invasion in Colorectal Cancer by Downregulating Rho-Associated Protein Kinase 1(ROCK1)

Background/Aims: MiR-124 inhibits neoplastic transformation, cell proliferation, and metastasis and downregulates Rho-associated protein kinase (ROCK1) in Colorectal Cancer (CRC). The aim of this study was to further investigate the roles and interactions of ROCK1 and miR-124 and the effects of knockdown of ROCK1and MiR-124 in human Colorectal Cancer (CRC). Methods: Three Colorectal cancer cell lines (HCT116, HT29 and SW620) and one Human Colonic Mucosa Epithelial cell line (NCM460) were studied. The protein expression of ROCK1 was examined by Western-blot and qRT-PCR were performed to examine the expression levels of ROCK1 mRNA and miR-124. Furthermore, We performed transfection of cancer cell line (SW620) with pre-miR-124(mimics), anti-miR-124(inhibitor), ROCK1 siRNA and the control, then observed the affects of ROCK1 protein expression by westen-blot, cell proliferation by EDU (5-ethynyl-2'deoxyuridine assay) and expression levels of ROCK1mRNA by qRT-PCR . A soft agar formation assay, Migration and invasion assays were used to determine the effect of regulation of miR-124 and ROCK1, and survivin on the transformation and invasion capability of colorectal cancer cell. Results: MiR-124 expression was significantly downregulated in CRC cell lines compare to normal (P < 0.05). In contrast, ROCK1 protein expression was significantly increased in CRC cell lines compared to the normal (P < 0.05), whereas the gene (ROCK1mRNA) expression remained unaltered (P > 0.05). ROCK1 mRNA was unaltered in cells transfected with miR-124 mimic and miR-124 inhibitor, compared to normal controls. There was a significant reduction in ROCK1 protein in cells transfected with miR-124 mimic and a significant increase in cells transfected with miR-124 inhibitor (P < 0.05). Cell proliferation, transformation and invasion of cells transfected with miR-124 inhibitor were significantly increased compared to those in normal controls (P<0.05). However, cell proliferation, transformation and invasion of cells transfected with ROCK1 siRNA were significantly decreased compared to control (P < 0.05). Conclusions: In conclusion, our results demonstrated that miR-124 not only promoted cancer cell hyperplasia and significantly associated with CRC metastasis and progression, but also downregulated ROCK1 protein expression. More importantly, increased ROCK1 expression or inhibited miR-124 expression may constitute effective new therapeutic strategies for the treatment of renal cancer in the future.

Evaluation of chemopreventive potential of Strobilanthes crispus against colon cancer formation in vitro and in vivo

BackgroundWith cancer being one of the major causes of death around the world, studies are ongoing to find new chemotherapeutic leads. There are common mechanisms for colorectal cancer (CRC) formation. Several are connected with oxidative stress-induced cell apoptosis and others are related to imbalanced homeostasis or intake of drugs/toxins. Plants that have been used for decades in folk and traditional medicine have been accepted as one of the commonest sources of discovered natural agents of cancer chemotherapy and chemoprevention. The aim was to study the antioxidant and chemopreventive effects of Strobilanthes crispus on colorectal cancer formation.MethodsFive groups of rats were injected subcutaneously with AOM, 15 mg/kg body weight, each once weekly for 2 weeks. The cancer group was continued on 10 % Tween-20 feeding for 8 weeks. The standard drug group was continued on 35 mg/kg 5-fluorouracil intraperitoneal injection twice a week for 8 weeks, and the experimental groups were continued on 250 and 500 mg/kg S. crispus extract oral feeding for 8 weeks, respectively. The normal group was injected subcutaneously with normal saline once a week for 2 weeks, followed by oral administration of 10 % Tween-20 for 8 weeks. All the rats were sacrificed after 10 weeks. The colons were evaluated grossly and histopathologically for aberrant crypt foci (ACF). Gene expression was performed for Bax, Bcl2, Defa24, Slc24a3, and APC genes by real-time PCR. S. crispus and its fractions were evaluated for their chemopreventive effects against human colorectal adenocarcinoma cell line HT29 and cytotoxicity for normal human colon epithelial cell line CCD 841, and the active fraction was assessed for its components.ResultsWe observed significant decrease in total colonic ACF formation, malonaldehyde (MDA) and lactate dehydrogenase (LDH), increase in superoxide dismutase (SOD), up-regulation of APC, Bax and Slc24a3, and down-regulation of Defa24 and Bcl-2 in rats treated with Strobilanthes crispus.ConclusionOur results support the in vivo protection of S. crispus against CRC formation (azoxymethane-induced aberrant crypt foci) and suggest that the mechanism is highly specific to protect from oxidative insults and the following apoptotic cascade.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0926-7) contains supplementary material, which is available to authorized users.

Human Colon Tumors Express a Dominant-Negative Form of SIGIRR That Promotes Inflammation and Colitis-Associated Colon Cancer in Mice

Single immunoglobulin and toll-interleukin 1 receptor (SIGIRR), a negative regulator of the Toll-like and interleukin-1 receptor (IL-1R) signaling pathways, controls intestinal inflammation and suppresses colon tumorigenesis in mice. However, the importance of SIGIRR in human colorectal cancer development has not been determined. We investigated the role of SIGIRR in development of human colorectal cancer. We performed RNA sequence analyses of pairs of colon tumor and nontumor tissues, each collected from 68 patients. Immunoblot and immunofluorescence analyses were used to determine levels of SIGIRR protein in primary human colonic epithelial cells, tumor tissues, and colon cancer cell lines. We expressed SIGIRR and mutant forms of the protein in Vaco cell lines. We created and analyzed mice that expressed full-length (control) or a mutant form of Sigirr (encoding SIGIRR(N86/102S), which is not glycosylated) specifically in the intestinal epithelium. Some mice were given azoxymethane (AOM) and dextran sulfate sodium to induce colitis-associated cancer. Intestinal tissues were collected and analyzed by immunohistochemical and gene expression profile analyses. RNA sequence analyses revealed increased expression of a SIGIRR mRNA isoform, SIGIRR(ΔE8), in colorectalcancer tissues compared to paired nontumor tissues. SIGIRR(ΔE8) is not modified by complex glycans and is therefore retained in the cytoplasm-it cannot localize to the cell membrane or reduce IL1R signaling. SIGIRR(ΔE8) interacts with and has a dominant-negative effect on SIGIRR, reducing its glycosylation, localization to the cell surface, and function. Most SIGIRR detected in human colon cancer tissues was cytoplasmic, whereas in nontumor tissues it was found at the cell membrane. Mice that expressed SIGIRR(N86/102S) developed more inflammation and formed larger tumors after administration of azoxymethane and dextran sulfate sodium than control mice; colon tissues from these mutant mice expressed higher levels of the inflammatory cytokines IL-17A and IL-6 had activation of the transcription factors STAT3 and NFκB. SIGIRR(N86/102S) expressed in colons of mice did not localize to the epithelial cell surface. Levels of SIGIRR are lower in human colorectal tumors, compared with nontumor tissues; tumors contain the dominant-negative isoform SIGIRR(ΔE8). This mutant protein blocks localization of full-lengthSIGIRR to the surface of colon epithelial cells and itsability to downregulate IL1R signaling. Expression of SIGIRR(N86/102S) in the colonic epithelium of mice increases expression of inflammatory cytokines and formation and size of colitis-associated tumors.

α-Linolenic acid (ALA) is an anti-inflammatory agent in inflammatory bowel disease

Studies suggest that consumption of omega-3 (n-3) polyunsaturated fatty acids (PUFA) plays a protective role in inflammatory bowel disease; however, the use of plant-derived oils rich in α-linolenic acid (ALA) has not been widely investigated. The aims of this study were to test the effects of two different sources of (n-3) PUFA, fish and plant-derived oils, in two animal models of experimental colitis and to determine whether the (n-3) PUFA-enriched diets could ameliorate the inflammatory status. Rats were fed diets rich in corn, fish or sage oil with or without vitamin A supplementation for 3weeks then colitis was induced by adding dextran sodium sulfate to the drinking water or by injecting 2,4,6-trinitrobenzene sulfonic acid. We show that colitic rats fed the sage oil diets had a lower inflammatory response, improved histological repair and had less necrotic damage in the mucosa when compared to the corn and fish oil groups. Colonic damage and myeloperoxidase activity were significantly lower. Colonic mRNA levels of pro-inflammatory genes including interleukin IL-6, cyclooxygenase 2 and tumor necrosis factor α were markedly down-regulated in rats fed fish and sage oils compared to control. These results were supported by experiments in the human colonic epithelial cell line Caco-2, where ALA supplementation was shown to be effective in inhibiting inflammation induced by IL-1β by down-regulating mRNA levels of pro-inflammatory genes including IL-8, COX2 and inducible nitric oxide synthase. Taken together, these results suggest that plant-derived oil rich in ALA could ameliorate the inflammatory damage in colitis.

Omega-3 fatty acids induce Ca(2+) mobilization responses in human colon epithelial cell lines endogenously expressing FFA4.

Aim:Free fatty acid receptor 4 (FFA4; formerly known as GPR120) is the G protein-coupled receptor (GPCR) for omega-3 polyunsaturated fatty acids. FFA4 has been found to express in the small intestines and colons of mice and humans. In this study we investigate the effects of omega-3 polyunsaturated fatty acids on FFA4 in human colon epithelial cells in vitro.Methods:HCT116 and HT-29 human colon epithelial cell lines endogenously expressing FFA4 were used. Intracellular Ca2+ concentration ([Ca2+]i) was measured in fura 2-AM-loaded cells with fluorescence spectrophotometry. RT-PCR and immunohistochemistry were used to detect FFA4.Results:Ten to 100 μmol/L of omega-3 polyunsaturated fatty acids α-linolenic acid (αLA) or eicosapentaenoic acid (EPA) induced dose-dependent [Ca2+]i increase in HCT116 and HT-29 cells, whereas docosahexaenoic acid (DHA) had no effect. In addition, the omega-6 fatty acids linoleic acid and γ-linoleic acid also dose-dependently increase [Ca2+]i, but the mono-unsaturated fatty acid oleic acid and saturated fatty acids such as stearic acid and palmitic acid had no effect. In HCT116 and HT-29 cells, the αLA-induced [Ca2+]i increase was partially inhibited by pretreatment with EGTA, phospholipase C inhibitor edelfosine, cADPR inhibitors 8-bro-cADPR or DAB, and abolished by pretreatment with Ca2+ATPase inhibitor thapsigargin, but was not affected by Gi/o protein inhibitor PTX or IP3R inhibitor 2-APB.Conclusion:Omega-3 and omega-6 long-chain polyunsaturated fatty acids (C18-20) induce Ca2+ mobilization responses in human colonic epithelial cells in vitro through activation of FFA4 and PTX-insensitive Gi/o protein, followed by Ca2+ release from thapsigargin-sensitive Ca2+ stores and Ca2+ influx across the plasma membrane.

Amyloid precursor protein mediated changes in intestinal epithelial phenotype in vitro.

BackgroundAlthough APP and its proteolytic metabolites have been well examined in the central nervous system, there remains limited information of their functions outside of the brain. For example, amyloid precursor protein (APP) and amyloid beta (Aβ) immunoreactivity have both been demonstrated in intestinal epithelial cells. Based upon the critical role of these cells in absorption and secretion, we sought to determine whether APP or its metabolite amyloid β (Aβ), had a definable function in these cells.Methodology/Principal FindingsThe human colonic epithelial cell line, Caco-2 cells, were cultured to examine APP expression and Aβ secretion, uptake, and stimulation. Similar to human colonic epithelium stains, Caco-2 cells expressed APP. They also secreted Aβ 1-40 and Aβ 1-42, with LPS stimulating higher concentrations of Aβ 1-40 secretion. The cells also responded to Aβ 1-40 stimulation by increasing IL-6 cytokine secretion and decreasing cholesterol uptake. Conversely, stimulation with a sAPP-derived peptide increased cholesterol uptake. APP was associated with CD36 but not FATP4 in co-IP pull down experiments from the Caco-2 cells. Moreover, stimulation of APP with an agonist antibody acutely decreased CD36-mediated cholesterol uptake.Conclusions/SignificanceAPP exists as part of a multi-protein complex with CD36 in human colonic epithelial cells where its proteolytic fragments have complex, reciprocal roles in regulating cholesterol uptake. A biologically active peptide fragment from the N-terminal derived, sAPP, potentiated cholesterol uptake while the β secretase generated product, Aβ1-40, attenuated it. These data suggest that APP is important in regulating intestinal cholesterol uptake in a fashion dependent upon specific proteolytic pathways. Moreover, this biology may be applicable to cells beyond the gastrointestinal tract.

Regulation of TWIK-related potassium channel-1 (Trek1) restitutes intestinal epithelial barrier function.

The disruption of epithelial barrier integrity is an important factor in the pathogenesis of various immune disorders. However, the restitution of the compromised barrier functions is difficult. This study investigates the regulation of TWIK-related potassium channel-1 (Trek1) in the restitution of intestinal epithelial barrier functions. The human colon epithelial cell line T84 was cultured in monolayers and used to observe epithelial barrier functions in vitro. An intestinal allergy mouse model was created. Cytokine levels were determined by enzyme-linked immunosorbent assay and western blotting. The results showed that Trek1 deficiency induced T84 monolayer barrier disruption. Allergic responses markedly suppressed the expression of Trek1 in the intestinal epithelia via activating the mitogen-activated protein kinase pathways and increasing the expression of histone deacetylase-1. The inhibition of histone deacetylase-1 by sodium butyrate or the administration of a butyrate-producing probiotic (Clostridium butyricum) restored the intestinal epithelial barrier functions and markedly enhanced the effect of antigen-specific immunotherapy. The data suggest that Trek1 is required for the maintenance of intestinal epithelial barrier integrity. Allergic responses induce an insufficiency of Trek1 expression in the intestinal epithelia. Trek1 expression facilitates the restoration of intestinal epithelial barrier functions in an allergic environment.

Exome Sequencing of Normal and Isogenic Transformed Human Colonic Epithelial Cells (HCECs) Reveals Novel Genes Potentially Involved in the Early Stages of Colorectal Tumorigenesis.

BackgroundWe have generated a series of isogenically derived immortalized human colonic epithelial cell (HCEC 1CT and HCEC 2CT) lines, including parental un-immortalized normal cell strains. The CDK4 and hTERT immortalized colonic epithelial cell line (HCEC 1CT) is initially karyotypically normal diploid and expresses a series of epithelial cell markers including stem cell markers. Under stressful tissue culture conditions, a spontaneous aneuploidy event occurred in the HCEC 1CT line, resulting in a single chromosomal change leading to a stable trisomy 7 cell line (1CT7). Trisomy 7 occurs in about 40% of all benign human adenomas (polyps) and thus this specific chromosomal change in diploid HCEC 1CT cells appears to be non random. In addition, we have partially transformed the HCEC 1CT line by introducing stable knockdown of wild type APC and TP53, and ectopically introducing a mutant Krasv12 and a mutant version of APC (A1309), all commonly found mutations in colorectal cancer (CRC).MethodsWhole exome sequencing and bioinformatic analyses were performed to comprehensively examine the genetic background of these isogenic cell lines.ResultsExome sequencing of these experimentally progressed cell lines recapitulates a list of genes previously reported to be involved in CRC tumorigenesis. In addition, sequencing revealed a collection of novel genes specifically detected in 1CT7 and A1309 cells but not normal diploid 1CT cells.ConclusionThis study demonstrates the utility of using isogenic experimentally derived HCEC lines as a model to recapitulate CRC initiation and progression. Exome sequencing reveals a collection of novel genes that may play important roles in CRC tumorigenesis.

P-183 Kindlin 1and Kindlin 2 Regulate Adhesion and Migration of Colonic Epithelial Cells

The Intestinal epithelial barrier plays an important role in the pathophysiology of inflammatory bowel disease (IBD). Integrity and barrier properties of the intestinal epithelium are determined by specialized adhesive structures such as intercellular junctions and focal adhesions, which respectively mediate cell-cell and cell-matrix interactions. Disassembly of epithelial junctions and matrix adhesion mediates disruption of the gut barrier during acute inflammation, whereas restoration of these adhesion structures is critical for epithelial restitution. Kindlins are unique cytosolic adaptor proteins that are enriched in both epithelial junctions and matrix adhesions. They have multiple binding partners including major classes of integrins and different cytoskeletal regulators. Importantly, mutations in the kindlin-1 gene result in Kindler syndrome, a genetic disorder associated with severe colitis, and deletion of kindlin-2 gene in mice is embryonically lethal. Despite these dramatic phenotypes caused by kindlin dysfunctions, little is known regarding the role of these adaptor proteins in regulation of the intestinal mucosal barrier disruption and restitution. This study was performed using a well-differentiated clone of HT29 human colonic epithelial cell line (HT29cF8). Transient downregulation of kindlin isoforms was achieved using RNA interference. Stable overexpression of kindlins 1 and 2 was achieved using a lentiviral expression system. Cell lines were cultured either on collagen-coated, permeable polycarbonate filters (0.4 mm pore size), or on 6-well plastic plates, for permeability, adhesion and motility assays. Barrier function of epithelial cell monolayers was determined via transepithelial electrical resistance (TEER) measurements Focal adhesions and cytoskeleton were visualized by immunofluorescence labeling and confocal microscopy. Overexpression of either kindlin 1 and/or kindlin 2 resulted in a significant increase in TEER, while siRNA-mediated knockdown of either isoform caused a substantial increase in paracellular permeability. Interestingly, both overexpression and depletion of kindlin 1 attenuated collective cell migration as determined by wound closure assay. By contrast, downregulation of kindlin 2 expression did not affect cell motility. Analysis of cell adhesion and spreading on a collagen matrix revealed significant attenuation of matrix adhesion and spreading of kindlin 1 and kindlin 2 overexpressing cells. These effects correlate well with a marked reduction of the phosphorylated form of focal adhesion protein, paxillin. Additionally, kindlin 1 overexpression regulated an increased level of non-muscle myosin IIB, which is indicative of increased contractile forces. Our data highlights kindlins as important regulators of barrier properties and restitution of human intestinal epithelium.

Interferon-γ induces a tryptophan-selective amino acid transporter in human colonic epithelial cells and mouse dendritic cells

IDO1, which encodes the immunosuppressive and tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase-1 (IDO1), is a target for interferon-γ (IFN-γ). IDO1-mediated tryptophan catabolism in dendritic cells and macrophages arrests T cell proliferation, thereby providing a molecular basis for the immunosuppressive function of IDO1. Whether the entry of tryptophan into IDO1-expressing cells is also regulated by IFN-γ is not known. Here we used a human colonic epithelial cell line (CCD841) and a mouse dendritic cell line (DC2.4) to test the hypothesis that IFN-γ, which induces IDO1, also induces a tryptophan transporter to promote substrate availability to IDO1. Upon treatment with IFN-γ, there was a marked increase in IDO1 mRNA and a concomitant increase in tryptophan uptake in both cell lines. The induced uptake system was selective for tryptophan and saturable with a Michaelis constant of 36±3μM in CCD841 cells and 0.5±0.1μM in DC2.4 cells. The induction by IFN-γ and the tryptophan-selectivity of the induced transport system were demonstrable even in the presence of physiologic concentrations of all other amino acids. Since kynurenine, the catabolic end product of IDO1, is a signaling molecule as an agonist for the aryl hydrocarbon receptor (AhR), we examined if AhR signaling induces the tryptophan-selective transporter. Treatment of the cells with kynurenine and other AhR agonists increased tryptophan uptake. The present studies demonstrate that IFN-γ coordinately induces IDO1 and a tryptophan-selective transporter to maximize tryptophan depletion in IDO1-expressing cells and that the process involves a positive feedback mechanism via kynurenine-AhR signaling.

Abstract 2322: β-catenin-dependent TCF/LEF transcriptional regulation of phosphodiesterase expression in colon cancer cells

Abstract Cyclic nucleotide phosphodiesterases (PDE) regulate cyclic nucleotides, cAMP and cGMP, blocking signaling by hydrolyzing the 3′,5′-phosphodiester bond resulting in their degradation to inactive forms. We have previously reported that certain PDE isozymes (e.g. PDE5) are elevated in several types of cancer, which can disrupt cyclic nucleotide signaling to suppress apoptosis. The inhibition of PDE5 by agents such as sulindac sulfide (SS) results in increased cGMP levels and reduced β-catenin/TCF/LEF mediated transcription leading to the selective inhibition of colon cancer cell proliferation and induction of apoptosis. Currently, little is known regarding the transcriptional regulation of PDE5 in cancer cells. We therefore analyzed the promoter regions of PDE5, as well as PDE10, for transcription factor binding sites using the Matinspector Genomatix Software. Matinspector analysis predicted that both PDE5 and PDE10 promoters contain binding sites for TCF/LEF-1, an important transcriptional factor that is regulated by Wnt/β-catenin signaling, which plays an important role in colorectal cancer. To determine if Wnt/β-catenin signaling can regulate PDE5 and PDE10 expression, human HCT116 colon tumor cells and normal colonic epithelial cell line NCM-460 were treated with the GSK-3β inhibitor, lithium chloride (LiCl) to inhibit β-catenin degradation and stimulate TCF/LEF transcriptional activity. TCF/LEF transcriptional activity was measured by mRNA levels of genes (e.g. survivin, cyclin D1 and c-myc) that are known to be regulated by Wnt/β-catenin signaling. Survivin, cyclin D1 and c-myc mRNA were confirmed to be significantly increased after LiCl treatment in comparison to control cells treated with NaCl. Translocation of β-catenin to the nucleus was also evident after treatment with LiCl. Levels of PDE5 and 10 mRNA were found to be significantly elevated after LiCl treatment in comparison to NaCl treated cells. Inhibitors such as SS, the novel derivative SS-D1 and a tadalafil (a selective PDE5 inhibitor) derivative TA-D1 demonstrated selective inhibition of colon cancer cell growth in comparison to normal colonocytes. The potential mechanism of these selective inhibitors may be through the inhibition of Wnt/β-catenin signaling and thus the inhibition of β-catenin/TCF/LEF mediated transcription. This study demonstrates a novel regulatory mechanism of PDE5 and PDE10 expression by the β-catenin-dependent TCF/LEF transcription factor in colon cancer cells and identifies a potential mechanism for novel chemopreventive agents. Defining the role of this regulatory mechanism in cancer progression may provide valuable insight into targeting PDE5 and/or PDE10 by small molecules for cancer chemoprevention. Citation Format: Alexandra M. Fajardo, Bernard D. Gary, Kevin J. Lee, Adam B. Keeton, Bing Zhu, Xi Chen, Ashraf H. Abadi, Gary A. Piazza. β-catenin-dependent TCF/LEF transcriptional regulation of phosphodiesterase expression in colon cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2322. doi:10.1158/1538-7445.AM2014-2322

Abstract 5244: MicorRNA-27a functions as a tumor suppressor in colorectal cancer through targeting SPGG1 and Smad2

Abstract Colorectal cancer is one of the most common malignant diseases worldwide, but the causes of colorectal carcinogenesis are largely unknown. Using a miRNA array we have identified differential expression of some microRNAs in colorectal cancer and adjacent normal mucosa. Most of these miRNAs have been validated in colorectal tissues by quantitative RT-PCR. One of the most changed miRNAs is miR-27a. The level of miR-27 was significantly reduced in colon cancers in compared with matched normal mucosa, the reduced expression of miR-27a was also associated with colorectal cancer pathological stages - miR-27a was more reduced at stages III/IV, compared to stage II. The levels of miR-27a in human colon cancer cell lines were also less than that in human normal colon epithelial cell lines. Using bioinformatic and systemic analysis, two targets of miR-27a were predicted, they were SPGG1 and Smad2. The expression levels of SPGG1 and Smad2 were negatively correlated with miR-27a. Dual luciferase assay showed that overexpression of miR-27 significantly repressed SPGG1 and Smad2 transcriptional activities. Increasing miR-27 expression also inhibited SPGG1 and Smad2 protein levels in colon cancer cells. Functional studies revealed that overexpression of miR-27 inhibited colon cancer cell proliferation, promoted apoptosis and attenuated cell migration, which were linked to downregulation of p-STAT3 and upregulation of phosphorylated JNK1. Taken together, our results demonstrated that the expression levels of miR-27a were lower in colorectal cancer tissues in comparison to the non-tumor tissues and was related to pathological stages, suggesting that down-regulation of this miRNA could contribute to colorectal tumorigenesis and the acquisition of a more aggressive phenotype in colorectal cancer, which could be through targeting SPGG1 and Smad2. Note: This abstract was not presented at the meeting. Citation Format: Yonghua Bao, Zhiguo Chen, Yongchen Guo, Tiesuo Zhao, Yansheng Feng, Yunjuan Jiao, Zexin Li, Jianguo Wang, Weixing Zhao, Kai Li, Qian Wang, Jiaqi Wang, Huijuan Zhang, Wancai Yang, Wancai Yang. MicorRNA-27a functions as a tumor suppressor in colorectal cancer through targeting SPGG1 and Smad2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5244. doi:10.1158/1538-7445.AM2014-5244

Differential Effects of Escherichia coli Subtilase Cytotoxin and Shiga Toxin 2 on Chemokine and Proinflammatory Cytokine Expression in Human Macrophage, Colonic Epithelial, and Brain Microvascular Endothelial Cell Lines

Subtilase cytotoxin (SubAB) is the prototype of a recently emerged family of AB5 cytotoxins produced by Shiga-toxigenic Escherichia coli (STEC). Its mechanism of action involves highly specific A-subunit-mediated proteolytic cleavage of the essential endoplasmic reticulum (ER) chaperone BiP. Our previous in vivo studies showed that intraperitoneal injection of purified SubAB causes a major redistribution of leukocytes and elevated leukocyte apoptosis in mice, as well as profound splenic atrophy. In the current study, we investigated selected chemokine and proinflammatory cytokine responses to treatment with SubAB, a nontoxic derivative (SubAA272B), or Shiga toxin 2 (Stx2) in human macrophage (U937), brain microvascular endothelial (HBMEC), and colonic epithelial (HCT-8) cell lines, at the levels of secreted protein, cell-associated protein, and gene expression. Stx2 treatment upregulated expression of chemokines and cytokines at both the protein and mRNA levels. In contrast, SubAB induced significant decreases in secreted interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in all three tested cell lines and a significant decrease in secreted IL-6 in HBMECs. The downregulation of secreted chemokines or cytokines was not observed in SubAA272B-treated cells, indicating a requirement for BiP cleavage. The downregulation of secreted chemokines and cytokines by SubAB was not reflected at the mRNA and cell-associated protein levels, suggesting a SubAB-induced export defect.

Stability and biological activity of wild blueberry (Vaccinium angustifolium) polyphenols during simulated in vitro gastrointestinal digestion

Wild blueberries are rich in polyphenols and have several potential health benefits. Understanding the factors that affect the bioaccessibility and bioavailability of polyphenols is important for evaluating their biological significance and efficacy as functional food ingredients. Since the bioavailability of polyphenols such as anthocyanins is generally low, it has been proposed that metabolites resulting during colonic fermentation may be the components that exert health benefits. In this study, an in vitro gastrointestinal model comprising sequential chemostat fermentation steps that simulate digestive conditions in the stomach, small intestine and colon was used to investigate the breakdown of blueberry polyphenols. The catabolic products were isolated and biological effects tested using a normal human colonic epithelial cell line (CRL 1790) and a human colorectal cancer cell line (HT 29). The results showed a high stability of total polyphenols and anthocyanins during simulated gastric digestion step with approximately 93% and 99% of recovery, respectively. Intestinal digestion decreased polyphenol- and anthocyanin- contents by 49% and 15%, respectively, by comparison to the non-digested samples. During chemostat fermentation that simulates colonic digestion, the complex polyphenol mixture was degraded to a limited number of phenolic compounds such as syringic, cinnamic, caffeic, and protocatechuic acids. Only acetylated anthocyanins were detected in low amounts after chemostat fermentation. The catabolites showed lowered antioxidant activity and cell growth inhibition potential. Results suggest that colonic fermentation may alter the biological activity of blueberry polyphenols.

Carboxymethylcellulose–tetrahydrocurcumin conjugates for colon-specific delivery of a novel anti-cancer agent, 4-amino tetrahydrocurcumin

Several curcumin derivatives are now becoming increasingly of interest because of their bioactive attributes, especially their action as antioxidants and anti-carcinogenic activities. Tetrahydrocurcumin (THC), an active metabolite of curcumin, was selected to be a proper starting material for the work presented here as it is stable in physiological pH and has the typical pharmacological properties of curcumin. We have now reported that novel synthesized water-soluble polymeric macromolecule prodrugs can specifically deliver the drug to the colon. To study the drug loading and drug release, THC was conjugated with a hydrophilic polymer, carboxymethylcellulose (CMC) with the degree of substitution (DS) values of 0.7 and 1.2. THC was also attached to two different spacers including p-aminobenzoic acid (PABA) and p-aminohippuric acid (PAH) via an azo bond that was cleaved by the azoreductase activities of colonic bacteria. The novel active molecule, 4-amino-THC, was readily released from the conjugates in the colon (>62% within 24h) with only very small amounts released in the upper GI tract (<12% over 12h). The polymer conjugates showed chemical stability at various pH values along the gastrointestinal tract and increased water solubility of up to 5mg/mL. 4-Amino-THC demonstrated cytotoxic ability against the human colon adenocarcinoma cell lines (HT-29) with an IC50 of 28.67±1.01μg/mL, and even greater selectivity (∼4 folds) to inhibit HT-29 cells than to normal human colon epithelial cell lines while curcumin was a non-selective agent against both cell lines. Our study has demonstrated that the use of THC–CMC conjugates may be a promising colon-specific drug delivery system with its sustained release in the colon to be an effective treatment for colonic cancer.

Reciprocal actions of Wnt9A and BMP4 on chondroitin‐4‐sulfotransferase (CHST11) expression in relation to arylsulfatase B activity (1005.2)

Initial experiments in human colonic epithelial cell lines and in the arylsulfatase B (N‐acetylgalactosamine‐4‐sulfatase; ARSB)‐deficient mouse showed that reduction in ARSB and the resultant increase in chondroitin‐4‐sulfate (C4S) were associated with reduction in chondroitin‐4‐sulfotransferase expression and ~50% reduction in sulfotransferase activity with overall increase in total sulfated glycosaminoglycans and C4S. Our experiments have also shown that decline in ARSB leads to increase in Wnt9A expression in human colonic epithelial cells and mouse intestine. Since modeling of the extracellular matrix by Wnt and BMP4 involves their interactions with sulfated glycosaminoglycans, including chondroitin sulfate, we evaluated the interactions among ARSB, Wnt9A, BMP4, and chondroitin‐4‐sulfotransferase (CHST11). Following silencing of Wnt9A by siRNA, the CHST11 expression increased to ~3 times baseline and the sulfotransferase activity increased to ~1.6 times the baseline. In contrast, when BMP4 was neutralized by blocking antibody, the CHST11 expression declined to ~50% of the baseline level. Exogenous administration of BMP4 increased the CHST11 expression to 2‐3 times the baseline level. In ARSB‐deficient mice, the BMP4 expression was ~50% of the value in the wild‐type control, and consistent with a decline in CHST11 expression. Both BMP4 and Wnt exert crucial effects in modeling of the extracellular environment, and these observed opposing effects on CHST11 expression and in relation to ARSB activity may be fundamental to their interaction and to their structural and functional effects.

Therapeutic targeting truncated adenomatous polyposis coli (APC) proteins for the selective killing of colorectal cancer cells

The adenomatous polyposis coli (APC) gene is a multifunctional tumor suppressor mutated in over 80% of colorectal cancers (CRC). Mutations in APC are believed to be one of the earliest events that contribute to CRC initiation. APC has been implicated in the negative regulation of the canonical Wnt signaling pathway, cell cycle control, cell migration, differentiation, and apoptosis. However, there remain many unanswered questions about the precise roles of APC in CRC progression. For example, over 90% of APC mutations result in truncated proteins, among which termination mutations at codons 1309 and 1450 occur the most frequently. It is completely unclear why the vast majority of human colon cancers would retain truncated APC proteins unless there was some survival/growth advantage to the emerging tumor. We hypothesized that these truncated APC proteins may actually play a dominant (gain of function) role in the initiation and/or progression of CRC and have conducted experiments to test this idea. Our rationale for these experiments is there are no disease-specific targeted treatments for CRC and therapeutic agents targeting APC truncations could be beneficial. We developed a unique series of telomerase (TERT) and CDK4 immortalized human colonic epithelial cell (HCEC) lines and experimentally made isogenic derivatives by ectopic expression of the most commonly observed APC truncations found in patients. In addition, we made TERT and CDK4 expressing HCECs with TP53 and APC knockdowns as well as ectopic expression of oncogenic KRASv12. We have completed a cell-based high-throughput screen within UT Southwestern's 200,000 compound library utilizing the most cancer progressed isogenic cell line and tested the initial hits against the TERT immortalized normal diploid HCECs. A small molecule lead compound, TASIN-1 (truncated APC selective inhibitor), was identified from this screen that is selectively toxic towards a panel of colon cancer cell lines with APC truncations while sparing normal diploid HCECs and cancer cells with wild type APC. TASIN-1 and its analogs (with IC50s from 0.01nM to 5µM) represent a novel strategy for the treatment of colon cancer. In vitro and in vivo preclinical results will be presented.

Intracellular Signaling Mechanisms Pharmacological Action of Jasminum amplexicaule Buch.-Ham. (Oleaceae) on Gastrointestinal Secretion.

Jasminum amplexicaule Buch-Ham. (Oleaceae) has been commonly used in the traditional medicine in dysentery, diarrhoea and bellyache in China. In the present work, the methanol extract of Jasminum amplexicaule (JME) was examined for pharmacology on human colonic epithelial cell line T84 by the short-circuit current technique. The results showed that pretreatment of T84 cells with JME produced a concentration-dependent (0-1000 μg/mL. EC50 = 0.055 mg/ mL) inhibition effect on adrenalin (Adr.)-induced Cl- secretion. The maximal response was observed at 200 μg/mL. It has been demonstrated that JME has a direct effect on the enterocyte. Our results also demonstrated that the JME exerted inhibitory effect on gastrointestinal Cl(-)secretion that effected by acting on basolateral β-adrenoreceptors. These results suggested that the Chinese traditional medicine of JME can be used for the treatment of acute diarrhea and bellyache.

Molecular identification of potential Th1/Th2 responses-modulating bacterial genes using suppression subtractive DNA hybridization

Background and objectivesWe characterized the immunomodulating potential of a number of lactobacilli isolated from an African fermented food by co-incubation with peripheral blood mononuclear cells (PBMCs). Two strains with different immune modulating properties were genetically compared by suppression subtractive hybridization (SSH). MethodsFrom 48 Lactobacillus strains isolated from Kimere, African fermented pearl millet dough, 10 were selected based on their bile salt tolerance. Their effects on secretion by PBMCs of the T-helper cells Th1- and Th2-cytokines IFN-γ and IL-4, respectively, in the presence or absence of staphylococcal enterotoxin A (SEA) were assessed. To study the genetic basis of different immune-modulating properties, a subtracted cDNA library for L. fermentum strains K1-Lb1 (Th1 inducer) and K8-Lb1 (Th1 and Th2 suppressor) was constructed using SSH. Finally, adhesion of these strains to hydrocarbons (relative hydrophobicity) and to human HT-29 colonic epithelial cell line was assessed. ResultsTwo strains, K1-Lb1 and K4-Lb6, induced basal IFN-γ secretion. Four strains, K1-Lb6, K6-Lb2, K7-Lb1, and K8-Lb1 diminished INF-γ secretion by SEA-stimulated PBMCs. All strains, except K1-Lb1, K2-Lb4, and K9-Lb3, inhibited SEA-stimulated IL-4 secretion. Comparing the genomes of K1-Lb1 and K8-Lb1 by SSH indicated that K1-Lb1 is able to synthetize polysaccharides, for the synthesis of which K1-Lb8 appears to lack enzymes. A difference in the hydrophobicity properties of the surfaces of both strains indicated that this has impact on their surface. ConclusionThe K1-Lb1-specific sequences encoding putative glycosyltransferases and enzymes for polysaccharides synthesis may account for the observed differences in immunomodulation and surface properties between the two strains and for mediating potential probiotic effects.