Normal Colon Epithelial Cell Line Research Articles

Abstract 549: Gene expression changes of GAEC1 in a large cohort of human cancer tissues

Abstract Background: Gene amplified in esophageal cancer 1 (GAEC1) is a novel oncogene located at 7q22.1 and preliminary work on GAEC1 suggested that it has tumorigenic potential and over expression of GAEC1 is a critical step for carcinogenesis. However, because the initial work on this gene was undertaken in its oncogenic properties, there is currently a lack of information of the molecular roles of this novel gene in other cancers. This research is intended to investigate the expression pattern of GAEC1 mRNA in tissues and cell line models of cancer. Materials and methods: Approximately 856 human tissues and blood samples have been collected. This comprises 430 colorectal tissues 135 oesophageal tissues 254 thyroid tissues; 37 blood samples (25 oesophageal cancers and 12 healthy individuals). This study used nine cancer (oesophageal, colorectal and thyroid cancers) and two normal (oesophageal epithelial and colon epithelial cell line) cell lines. Histological samples (non-tumour, pre-cancerous and cancerous lesions) were collected and the pathology of these lesions were reviewed. The mRNAs of the selected samples were extracted by standard protocol. GAEC1 mRNA expression was studied by evaluating the quantitative amplification of GAEC1 RNA using quantitative real-time polymerase chain reaction. Results: GAEC1 mRNA expression levels were significantly altered in all selected cancer tissue models (colorectal, oesophageal and thyroid carcinomas). This level of GAEC1 was associated with tumour site, histological subtypes, and metastasis. At the mRNA level, 29% (n=61/210) of colorectal cancers, 22% (n=23/105)of oesophageal cancers and 7% (n=8/120) of thyroid cancers showed high GAEC1 expression (relative to control tissue). GAEC1 mRNA expression (mean) in oesophageal cancer blood samples showed 4 times higher expression compared to the control samples (p < 0.0001). Patients with high GAEC1 mRNA expression levels in their oesophageal cancer tissue had reduced survival period compared to patients with low GAEC1 mRNA levels (p=0.05). Conclusion: The current study noted multiple new findings, including relationships with clinical and pathological parameters with GAEC1 mRNA expression in three different types of cancers. The altered mRNA expression of GAEC1 and its correlation with different tissues samples and sites of tumour may also be important for the development of gene targeting therapies for cancers. Further research in unveiling GAEC1 protein structure and functional studies and post translational modifications will help in understanding the role of this oncogene in human carcinogenesis. Results of this study indicate that GAEC1 has an impact on human cancer pathogenesis. Citation Format: Vinod Gopalan, Robert A. Smith, Suja Pillai, Ali Salajegheh, Johnny Chuek-on Tang, Alfred KY Lam. Gene expression changes of GAEC1 in a large cohort of human cancer tissues. [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 549. doi:10.1158/1538-7445.AM2014-549

Implications of the colonic deposition of free hemoglobin-α chain: a previously unknown tissue by-product in inflammatory bowel disease.

We analyzed inflamed mucosal/submucosal layers of ulcerative colitis (UC = 63) and Crohn's colitis (CC = 50), and unexpectedly, we unveiled a pool of free hemoglobin alpha (Hb-α) chain. Patients with colitides have increased reactive oxidative stress (ROS), DNA oxidation products, free iron in mucosa, in preneoplastic, and in colitis-cancers and increased risks of developing colorectal cancer. All inflammatory bowel disease-related colorectal cancer lesions are found in segments with colitis. Linking this information, we investigated whether free Hb-α is key transformational stepping that increases colitis-related colorectal cancer vulnerability. UC/CC samples were profiled using matrix-assisted laser desorption/ionization mass spectrometry; protein identification was made by liquid chromatography. Diverticulitis was used as control (Ctrl). The presence of Hb(n) (n = α, β, or hemin)/Hb was validated by Western blotting and immunohistochemistry. We tested for DNA damage (DNAD) by exposing normal colonic epithelial cell line, NCM460, to 10 μM and 100 μM of Hb(n)/Hb, individually for 2, 6, and 12 hours. Quantification of Hb-α staining was done by Nikon Elements Advance Research Analysis software. ROS was measured by the production of 8-OHdG. DNAD was assessed by Comet assay. Colonic tissue homogenate antioxidants Nrf2-, CAT-, SOD-, and GPx-expressions were analyzed densitometrically/normalized by β-actin. Immunohistochemistry of CC/UC mucosal/submucosal compartments stained strongly positive for Hb-α and significantly higher versus Ctrl. NCM460 exposed to Hb(n)/Hb exhibited steadily increasing ROS and subsequent DNAD. DNAD was higher in 10 μM than 100 μM in Hb-β/hemin the first 2 hours then plateaued followed by DNAD repair. This may be likely due to apoptosis in the later concentration. Nrf2 enzyme activities among UC, CC, and ulcerative colitis-associated colon cancer (UCAC) were observed impaired in all inflammatory bowel disease subjects. Decreased levels of Nrf2 among patients with UC versus patients with CC with active disease were insignificant as well as versus Ctrls but significantly lower in UCAC versus Ctrl. SOD was decreased in UC and UCAC and GPx in CC but statistically not significant. Comparing CC versus UC, SOD was significantly lower in CC (P < 0.05). CAT was observed increased among patients with CC/UC/UCAC and GPx in UC and UCAC versus Ctrl, respectively, and significantly increased in CC versus Ctrl (P < 0.01). In the colitides, mucosal/submucosal tissue microenvironments demonstrated pool of free Hb-α chain. In vitro exposure of NCM460 cells to Hb(n)/Hb induced ROS and DNAD. Toxic effect of free Hb-α, in colonic epithelial cells, is therefore through production of ROS formation modulated by impairment of antioxidant effects. Targeting reduction-oxidation-sensitive pathways and transcription factors may offer options for inflammatory bowel disease-management and colitis-related cancer prevention.

Downregulation of microRNA-498 in colorectal cancers and its cellular effects

miR-498 is a non-coding RNA located intergenically in 19q13.41. Due to its predicted targeting of several genes involved in control of cellular growth, we examined the expression of miR-498 in colon cancer cell lines and a large cohort of patients with colorectal adenocarcinoma. Two colon cancer cancer cell lines (SW480 and SW48) and one normal colonic epithelial cell line (FHC) were recruited. The expression of miR-498 was tested in these cell lines by using quantitative real-time polymerase chain reaction (qRT-PCR). Tissues from 80 patients with surgical resection of colorectum (60 adenocarcinomas and 20 non-neoplastic tissues) were tested for miR-498 expression by qRT-PCR. In addition, an exogenous miR-498 (mimic) was used to detect the miRNA׳s effects on cell proliferation and cell cycle events in SW480 using MTT calorimetric assay and flow cytometry respectively. The colon cancer cell lines showed reduced expression of miR-498 compared to a normal colonic epithelial cell line. Mimic driven over expression of miR-498 in the SW480 cell line resulted in reduced cell proliferation and increased proportions of G2-M phase cells. In tissues, miR-498 expression was too low to be detected in all colorectal adenocarcinoma compared to non-neoplastic tissues. This suggests that the down regulation of miR-498 in colorectal cancer tissues and the direct suppressive cellular effect noted in cancer cell lines implies that miR-498 has some direct or indirect role in the pathogenesis of colorectal adenocarcinomas.

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.

MACC1 promotes carcinogenesis of colorectal cancer via β-catenin signaling pathway.

Here we confirmed that metastasis-associated in colon cancer 1 (MACC1) and β-catenin expression were higher in colorectal cancer (CRC) cells and tissues than those in normal colonic epithelial cell line and adjacent non-tumour colorectal mucosa (ANM) tissues, respectively. MACC1 expression was significantly related to histological differentiation (p<0.001), UICC stage (p=0.029), T classification (p=0.017), and N classification (p=0.023). Cox regression analysis demonstrated that high MACC1/abnormal β-catenin expression was the strongest independent prognostic indicator for reduced overall survival in CRC patients. Significant positive correlation between MACC1 expression and abnormal β-catenin expression was found in CRC tissues. MACC1 knockdown dramatically inhibited cellular proliferation, migration, invasion, colony formation, and tumorigenesis, both in vitro and in vivo, but induced apoptosis in CRC cells. Further MACC1 over-expression increased Met, β-catenin, and its downstream genes including c-Myc, cyclin D1, and MMP9 expression, and its upstream gene phos-GSK3β (Ser9) expression. In addition, MACC1 increased vimentin and suppressed E-cadherin in HCT116 cells. Silencing of MACC1 reversed all these changes. Our results firstly suggest that MACC1 plays an important role in carcinogenesis and progression of CRC through β-catenin signaling pathway and mesenchymal-epithelial transition.

Sorbitol Dehydrogenase Overexpression and Other Aspects of Dysregulated Protein Expression in Human Precancerous Colorectal Neoplasms: A Quantitative Proteomics Study

Colorectal adenomas are cancer precursor lesions of the large bowel. A multitude of genomic and epigenomic changes have been documented in these preinvasive lesions, but their impact on the protein effectors of biological function has not been comprehensively explored. Using shotgun quantitative MS, we exhaustively investigated the proteome of 30 colorectal adenomas and paired samples of normal mucosa. Total protein extracts were prepared from these tissues (prospectively collected during colonoscopy) and from normal (HCEC) and cancerous (SW480, SW620, Caco2, HT29, CX1) colon epithelial cell lines. Peptides were labeled with isobaric tags (iTRAQ 8-plex), separated via OFFGEL electrophoresis, and analyzed by means of LC-MS/MS. Nonredundant protein families (4325 in tissues, 2017 in cell lines) were identified and quantified. Principal component analysis of the results clearly distinguished adenomas from normal mucosal samples and cancer cell lines from HCEC cells. Two hundred and twelve proteins displayed significant adenoma-related expression changes (q-value < 0.02, mean fold change versus normal mucosa ±1.4), which correlated (r = 0.74) with similar changes previously identified by our group at the transcriptome level. Fifty-one (∼25%) proteins displayed directionally similar expression changes in colorectal cancer cells (versus HCEC cells) and were therefore attributed to the epithelial component of adenomas. Although benign, adenomas already exhibited cancer-associated proteomic changes: 69 (91%) of the 76 protein up-regulations identified in these lesions have already been reported in cancers. One of the most striking changes involved sorbitol dehydrogenase, a key enzyme in the polyol pathway. Validation studies revealed dramatically increased sorbitol dehydrogenase concentrations and activity in adenomas and cancer cell lines, along with important changes in the expression of other enzymes in the same (AKR1B1) and related (KHK) pathways. Dysregulated polyol metabolism might represent a novel facet of metabolome remodeling associated with tumorigenesis.

Two Natural ent-kauranoids as Novel Wnt Signaling Inhibitors.

Constitutively active Wnt signaling frequently occurs in most colon cancers. Therefore, inhibitors of Wnt signaling pathway could provide rational therapeutic effects for colorectal malignancy. Within this paper, we identified two inhibitors of Wnt signaling pathway, rabdoternin B and maoecrystal I from a natural ent-kauranoid library by a dual-luciferase reporter gene assay. The two compounds inhibited Wnt signaling pathway in a concentration-dependent manner and exhibited selective cytotoxicity toward a number of colon carcinoma cell lines SW480, HCT116, and HT29, with only weak cytotoxicity towards the normal colonic epithelial cell line CCD-841-CoN. Rabdoternin B and maoecrystal I treatment induced G2/M phase arrest efficiently in SW480 cells as revealed by flow cytometry analysis. A further study found that maoecrystal I decreased the expression of Wnt signaling target genes, including c-myc, cyclin D1, survivin and Axin2 in colon cancer cells. Collectively our data suggests that rabdoternin B and maoecrystal I are novel inhibitors of canonical Wnt signaling pathway and may possess potentials for colon cancer therapy.

Quantitative shotgun proteomics reveals early markers of colorectal carcinogenesis (591.1)

Colorectal adenomas are cancer precursor lesions of the large bowel. Using iTRAQ 8‐plex labelling, OFFGEL electrophoresis, and LC‐coupled tandem MS, we investigated the proteome of 30 colorectal adenomas and paired normal mucosal samples (prospectively collected during colonoscopy), and that of normal (HCEC) and cancerous (SW480, SW620, CACO2, HT29, CX1) colon epithelial cell lines. A total of 4325 and 2017 non‐redundant protein families were identified and quantified in tissue and cell lines, respectively. Principal component analysis clearly distinguished adenomas from normal mucosa, and cancer cell lines from HCEC cells. Two hundred twelve proteins displayed significant adenoma‐related expression changes (q‐value&lt;0.02, mean fold change +/‐1.4), which correlated (r=0.74) with similar changes previously identified by our group at the transcriptome level. Fifty one proteins displayed directionally similar expression changes in colorectal cancer cells (vs HCEC cells) and were therefore attributed to the epithelial component of adenomas. Although benign, adenomas already exhibited protein expression changes previously documented only in advanced colorectal cancers. They also disclosed several novel changes with potentially important roles in early‐stage large bowel tumorigenesis, including the marked upregulation of a key enzyme in the polyol pathway.Grant Funding Source: Supported by the Swiss National Science Foundation

Regulation of microRNA‐1288 in colorectal cancer: Altered expression and its clinicopathological significance

We aim to examine the miR-1288 expression in cancer cell lines and a large cohort of patients with colorectal cancer. Two colon cancer cell lines (SW480 and SW48) and one normal colonic epithelial cell line (FHC) were recruited. The miRNA expressions of miR-1288 were tested on these cell lines by using quantitative real-time polymerase chain reaction (qRT-PCR). An exogenous miR-1288 (mimic) was used to detect cell proliferation and cell cycle changes in SW480 using MTT calorimetric assay and flow cytometry, respectively. In addition, tissues from 122 patients with surgical resection of colorectum (82 adenocarcinomas, 20 adenomas, and 20 non-neoplastic tissues) were tested for miR-1288 expression by qRT-PCR. The colon cancer cell lines showed reduced expression of miR-1288 compared to normal colonic epithelial cell line. Over expression of miR-1288 in SW480 cell line showed increased cell proliferation and increased G2-M phase cells. In tissues, reduced miR-1288 expression was noted in majority of colorectal adenocarcinoma compared to colorectal adenoma and non-neoplastic tissues. Reduced or absent expression of miR-1288 was noted in 76% (n = 62/82) of the cancers. The expression levels of miR-1288 were higher in distal colorectal adenocarcinomas (P = 0.013) and in cancers of lower T staging (P = 0.033). To conclude, alternation of miR-1288 expression is important in the progression of colorectal cancer. The differential regulation of miR-1288 was found to be related to cancer location and pathological staging in colorectal cancers.

Clinical impacts of mammalian target of rapamycin expression in human colorectal cancers

This study investigated the clinicopathologic roles of mammalian target of rapamycin (mTOR) expression and its relationship to carcinogenesis and tumor progression in a colorectal adenoma-adenocarcinoma model. Two colon cancer cell lines with different pathologic stages (SW480 and SW48) and 1 normal colonic epithelial cell line (FHC) were used, in addition to 119 colorectal adenocarcinomas and 32 adenomas. mTOR expression profiles at messenger RNA (mRNA) and protein levels were investigated in the cells and tissues using real-time quantification polymerase chain reaction and immunohistochemistry. The findings were correlated with the clinicopathologic features of the tumors. The colon cell line from stage III cancer (SW48) showed higher expression of mTOR mRNA than that from stage II cancer (SW480). At the tissue level, mTOR showed higher mRNA and protein expression in colorectal carcinoma than in adenoma. The mRNA and protein expression was correlated with each other in approximately one-third of the carcinomas and adenomas. High levels of mTOR mRNA expression were noted more in carcinoma or adenoma arising from the distal portion of the large intestine (P = .025 and .019, respectively). Within the colorectal cancer population, a high level of expression of mTOR mRNA was related to the presence of lymph node metastases (P = .031), advanced pathologic stage (P = .05), and presence of persistent disease or tumor recurrence (P = .035). To conclude, the study has indicated that mTOR is likely to be involved in the development and progression of colorectal cancer and is linked to cancer initiation, invasiveness, and progression.

CDC20 overexpression predicts a poor prognosis for patients with colorectal cancer

BackgroundThe cell division cycle 20 homolog (CDC20) is an essential cofactor of the anaphase-promoting complex (APC/C). CDC20 overexpression has been detected in many types of human cancers; however, its clinical role in colorectal cancer remains unknown.MethodsWestern blotting and immunohistochemistry were used to compare CDC20 expression in adjacent non-cancerous, cancerous and liver metastatic tissues as well as in colon cancer cell lines and normal colon epithelial cell lines. Additionally, the correlation of CDC20 expression with patient clinical parameters and its diagnostic value were statistically analyzed.ResultsCDC20 was overexpressed in colon cancer cell lines/primary cancer tissues compared with normal colon epithelial cell lines/adjacent noncancerous tissue samples. Interestingly, CDC20 expression was further increased in metastatic liver tissues. CDC20 protein expression was significantly correlated with clinical stage (P = 0.008), N classification (P = 0.020), M classification (P = 0.013) and pathologic differentiation (P = 0.008). Patients with higher CDC20 expression had a shorter overall survival than those with lower CDC20 expression. Univariate and multivariate analyses indicated that CDC20 expression was an independent prognostic factor (P < 0.001).ConclusionCDC20 may serve as a potential prognostic biomarker of human colorectal cancer.

Sa1693 The Role of C9orf140 in Colorectal Cancer Invasion

Background: c9orf140 is a novel gene that has been recently isolated and indentified by the mRNA differential display (mRNAD), which is associated with cell proliferation and tumorigenesis in Gastric caner. Moreover, it was reported that the expression of c9orf140 is significantly elevated in colorectal caner (CRC) tissues when compared with normal tissues. However, the role of c9orf140 in the invasion of CRC and the mechanism of c9orf140 overexpression are still unclear. Method: c9orf140, EZH2, beta-catanin, STAT5 and pSTAT5 expressions were measured by Western Blot and immunohistochemical staining. To establish stable cells, CRC cells were infected with c9orf140 shRNA, c9orf140 overexpression or control lentivirus. Stable cell lines with different protein or shRNA expressions were introduced into nude mice respectively via tail veins injection. The photos were taken by the NightOWL II LB 983 in vivo Imaging system. Chromatin Immunoprecipitation (ChIP) and Luciferase reporter gene assay were carried out according to the protocol of manufactures. Result: there is almost no expression of C9orf140 in a normal human colon epithelial cell line, CRL1790, but the expression of C9orf140 is significantly increased in all CRC cell line especially in those highly invasive CRC cells. Immunofluorence analysis showed that C9orf140 is mainly detected in the cytoplasm of CRC HCT116 and HT29 cells. Immunohistochemical analysis of 150 patients' tissue sections showed that the staining of C9orf140 was found at higher expression levels in CRC samples than normal samples. Furthermore, the expression of C9orf140 was gradually increased from well differentiated to poor differentiated in CRC tissues. Knockdown of C9orf140 dramatically increased E-cadherin expression and decreased N-cadherin expression. Knockdown of C9orf140 significantly reduced the CRC cell invasion. More lung metastasis and shorter overall survival were detected after overexpression of C9orf140 compared with control in vivo. Knockdown of C9orf140 dramatically increased overall survival and decreased metastasis of lung in vivo. Western Blot data showed that C9orf140 expressions may be regulated by EZH2, STAT5 and β-catanin. Moreover, Immunoprecipitation, ChIP and Luciferase reporter gene assay showed that EZH2, STAT5 and βcatanin may colocalized together and directly bind to the promoter of C9orf140 gene to upregulate this gene expression in CRC cells. Conclusion: we firstly revealed the role of C9orf140 in the metastatic progression of CRC in vitro and in vivo. C9orf140-induced CRC cell migration and invasion may depend on promote the progression of EMT. Furthermore, we firstly identified that c9orf140 expression may be directly regulated by EZH2, STAT5 and β-catanin. Our data may provide potential targets to prevent and/or treat aggressive CRC.

Sa1694 Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) Is Upregulated in Primary Colorectal Cancer and Affects Cancer Signaling Pathways

Background: c9orf140 is a novel gene that has been recently isolated and indentified by the mRNA differential display (mRNAD), which is associated with cell proliferation and tumorigenesis in Gastric caner. Moreover, it was reported that the expression of c9orf140 is significantly elevated in colorectal caner (CRC) tissues when compared with normal tissues. However, the role of c9orf140 in the invasion of CRC and the mechanism of c9orf140 overexpression are still unclear. Method: c9orf140, EZH2, beta-catanin, STAT5 and pSTAT5 expressions were measured by Western Blot and immunohistochemical staining. To establish stable cells, CRC cells were infected with c9orf140 shRNA, c9orf140 overexpression or control lentivirus. Stable cell lines with different protein or shRNA expressions were introduced into nude mice respectively via tail veins injection. The photos were taken by the NightOWL II LB 983 in vivo Imaging system. Chromatin Immunoprecipitation (ChIP) and Luciferase reporter gene assay were carried out according to the protocol of manufactures. Result: there is almost no expression of C9orf140 in a normal human colon epithelial cell line, CRL1790, but the expression of C9orf140 is significantly increased in all CRC cell line especially in those highly invasive CRC cells. Immunofluorence analysis showed that C9orf140 is mainly detected in the cytoplasm of CRC HCT116 and HT29 cells. Immunohistochemical analysis of 150 patients' tissue sections showed that the staining of C9orf140 was found at higher expression levels in CRC samples than normal samples. Furthermore, the expression of C9orf140 was gradually increased from well differentiated to poor differentiated in CRC tissues. Knockdown of C9orf140 dramatically increased E-cadherin expression and decreased N-cadherin expression. Knockdown of C9orf140 significantly reduced the CRC cell invasion. More lung metastasis and shorter overall survival were detected after overexpression of C9orf140 compared with control in vivo. Knockdown of C9orf140 dramatically increased overall survival and decreased metastasis of lung in vivo. Western Blot data showed that C9orf140 expressions may be regulated by EZH2, STAT5 and β-catanin. Moreover, Immunoprecipitation, ChIP and Luciferase reporter gene assay showed that EZH2, STAT5 and βcatanin may colocalized together and directly bind to the promoter of C9orf140 gene to upregulate this gene expression in CRC cells. Conclusion: we firstly revealed the role of C9orf140 in the metastatic progression of CRC in vitro and in vivo. C9orf140-induced CRC cell migration and invasion may depend on promote the progression of EMT. Furthermore, we firstly identified that c9orf140 expression may be directly regulated by EZH2, STAT5 and β-catanin. Our data may provide potential targets to prevent and/or treat aggressive CRC.

Abstract 505: Jagged 2 knockdown inhibits invasion in colorectal cancer cell lines

Abstract Background: The notch pathway is involved in control of cell differentiation in various tissues including pancreas, muscle and bone marrow. Jagged 2 (JAG2) is a ligand for the notch pathway and was found to be overexpressed in malignant plasma cells from patients with multiple myeloma, causing the secretion and release of IL-6, VEGF, and IGF-1 in the microenvironment supporting myeloma cell growth. In breast cancers, it was found to be overexpressed at the hypoxic invasive front and was significantly correlated with metastasis free survival. Furthermore, up-regulation of JAG2 expression in mouse lung adenocarcinoma cells promoted its metastasis in mice. However, the expression pattern and function of JAG2 in colorectal cancer (CRC) is not clear. Therefore in this study, we examined the expression of JAG2 in CRC cell lines and determined the effect(s) of JAG2 knockdown by RNA interference in CRC cell lines. Materials and Methods: The expression of JAG2 protein was examined in four CRC cell lines (SW480, SW620, HCT116, DLD-1) and also in 1 normal colon epithelial cell line (CCD18co) using the western blot. To investigate the effects JAG2 knockdown in CRC cell lines, a pool of 4 siRNAs against JAG2 was transfected into the 4 CRC cell lines listed above. The effects on cell growth was then determined by the CellTiter 96® AQueous One Solution Cell Proliferation Assay (the MTS assay) and the Matrigel invasion assay was performed to assess its effects on invasive capability. Results: Western blot results showed that JAG2 protein was expressed in all 4 colorectal cancer cell lines including SW480, SW620, HCT116 and DLD-1 but not in the CCD18co normal colon epithelial cell line. The MTS assay showed that cell growth was not affected by JAG2 knockdown in the CRC cell lines. Interestingly, the invasive capability of HCT116 and DLD-1cells was reduced after JAG2 knockdown. Conclusions: Results from this study suggest that JAG2 may play a role in the invasiveness of CRC cell lines but not in cell proliferation. JAG2 may be a promising therapeutic target for inhibition of metastasis in colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 505. doi:1538-7445.AM2012-505

Abstract 4134: miR1288 in colorectal cancers: Altered expression and its clinicopathological significance

Abstract Introduction: MicroRNA-1288 (miR-1288) is a non-coding RNA located in 17p11.2 and not been studied solely in cancer. We aim to examine the miR-1288 expression in a large cohort of patients with colorectal carcinoma, adenoma and non-neoplastic colorectal tissues. The implications of miR-1288 expression and clinicopathological features were also studied. Methods: Tissues from 102 patients with surgical resection of colorectum (82 adenocarcinomas, 10 adenomas and 10 non-neoplastic tissues), two colon cancer cell lines (SW480 and SW48) and one normal colonic epithelial cell line (FHC) were recruited. miRNA was extracted from all these samples and were converted in to cDNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for the detection of miR-1288 expression. The results were correlated with the clinical and pathological data. Results: miR-1288 expression was decreased in majority of colorectal adenocarcinoma when compared to colorectal adenoma and non-neoplastic tissues. Reduced or absence expression of miR-1288 was noted in 78% (n=64) of the cancers. The colon cancer cell lines showed reduced expression of miR-1288 compared to normal colonic epithelial cell line. Distal colorectal cancers showed higher expression levels of miR-1288 than proximal cancers (p= 0.007). The expression levels of miR-1288 were significantly altered in different levels of cancer invasion (T staging) (p= 0.023). Conclusions: Altered expression pattern of miR-1288 in colorectal cancers compare to non-neoplastic tissues was identified. Also, differential regulation of miR-1288 was found to be related to location and pathological staging of colorectal cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4134. doi:1538-7445.AM2012-4134

Abstract 3847: Lupeol, a novel inhibitor of Wnt/β-catenin signaling: Implications in colon cancer therapy

Abstract Introduction: Wnt/β-catenin signaling pathway which regulates a variety of cellular processes has been reported to play a critical role in the pathogenesis of human colon cancer and found in almost 90% of the colon cancer patients. Recent studies from our laboratory and others showed that Lupeol a natural triterpene acts as a potent and selective inhibitor of Wnt signaling in prostate and melanoma tumor cells (Saleem et al, Carcinogenesis, 30: 801-17, 2009). Keeping in view these reports, we hypothesized that Lupeol will inhibit the tumorigenic growth of colon cancer cells (exhibiting aberrant Wnt/α signaling) under in vitro and in vivo conditions. Objectives: We investigated the effect of Lupeol on growth of human normal colon and carcinoma cells (exhibiting varied Wnt profile) and determined if Lupeol could enhances the efficacy of Sulindac, (Wnt signaling inhibitor and chemotherapeutic agent) that is used in clinics to treat human colon cancer. Methods: To test our hypothesis we employed in vitro and in vivo strategies. Firstly, normal human colon epithelial cells (FHC) and colon carcinoma cell lines (SW-480, HCT-116, HT-29) treated with either Lupeol (10-50 μM), or Sulindac (100-200 μM), or combination (Lupeol + Sulindac) were tested for growth, proliferation, clonogenicity and surrogate biomarkers of Wnt signaling by employing 3[H]thymidine uptake, MTT, Soft-agar colony, FACS, immunoblot and luciferase-reporter assays. Secondly, we investigated whether Lupeol is bio-available to mice after oral and intraperitoneal (i.p.) administration by using mass spectrometry. Next, we treated athymic mice bearing colon tumors-derived from HT-29 cells (highly chemoresistant among all colon cells) with either Lupeol (40 mg/kg) or Sulindac (20 mg/kg) or combination and measured tumor growth. In vivo proliferation and biochemical analysis of harvested tumors for surrogate biomarkers of apoptosis, proliferation and Wnt signaling were measured by employing BrdU uptake, immunoblot and immunhistochemical analysis. Results: Lupeol treatment was observed to (i) inhibit the proliferation of colon cancer cells (exhibiting activated Wnt) while sparing normal cells, (ii) induce G0/G1 arrest and apoptosis, (iii) decrease cytoslic as well as nuclear β-catenin levels (iv) increase degradation of cytosolic β-catenin, (iv) decrease TCF-transcriptional activity and (v) decrease the expression of Wnt target genes. Lupeol was observed to bioavailable in mice after oral and i.p. administration. Lupeol significantly inhibited the tumorigenicity of colon tumor cells in a xenograft mouse model. Most importantly, Lupeol treatment significantly sensitized highly chemoresistant colon cancer cells to Sulindac therapy, in vitro and in vivo. Conclusion: Lupeol alone or as an adjuvant to current therapies could be developed as an agent to treat subtype of human colon cancer exhibiting constitutive activation of Wnt signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3847. doi:1538-7445.AM2012-3847

Decreased Diamine Oxidase Release Into the Intestinal Lymph in Rats With Experimental Colitis

Background: Intestinal epithelial cells (IECs) are increasingly recognized as an essential player in the regulation of intestinal immune homeostasis. During inflammatory bowel diseases (IBDs) several chemokines have a dysregulated pattern of expression on IEC contributing to the sustained mucosal inflammation in IBD. Avoiding inappropriate activation of TLR4 via NF-kB inhibition, during IBD, might have a translational readout because epithelial cells are in constant contact with a dense and complex milieu of commensal microorganisms. In addition to the antimicrobial activity, rifaximin a poorly absorbed antibiotic, acts as a gut-specific human PXR ligand Aims. To investigate whether rifaximin regulates epithelial intestinal immune homeostasis through a PXR-dependent mechanism. Methods. CRL-1831 cells, a normal colonic human epithelial cell line (ATCC), were exposed to LPS (1 μg/ml for 20 h) alone and in combination with rifaximin (50 μM). Cytokines and chemokines generation wasmeasured by RT-PCR and ELISA. NF-kB binding activity by EMSA. Additional experiments were carried out in CRL-1831 cells in after PXR silencing (siPXR). Biopsy specimens obtained from CD patients, were cultured with LPS (100 mg/ml) alone or in combination with rifaximin (100 μM) for 18h. Results. CRL-1831 cells express PXR. In comparison to wild type cells, PXR silencing decreased the TGF-β and IP-10 production (P< 0.05) while generation of TNF-α, IL-8, RANTES, PGE2 and MIP-3α and IL-6 mRNA levels were increased (P< 0.05). LPS stimulation further enhanced the production of TNFα, IL-8, Rantes, PGE2, and MIP-3α mRNA levels in siPXR cells (P<0.05). LPS stimulation increased TGF-β production in cell line and siPXR cells. Rifaximin causes a robust attenuation of inflammatory response caused by LPS (P< 0.05), while increased TGF-β (P< 0.05) and IL-6 mRNA (P < 0.05). si PXR abrogated completely the ability of rifaximin to change the IEC's immune profile. Rifaximin cotreatment LPS induced NF-κB DNA binding activityin a PXR-dependent manner. Exposure of colon biopsies to rifaximin reduces the generation of IL-8, Rantes and TNFα caused by LPS and iboosted TGF-β production. Conclusions. Rifaximin regulates IEC immune functions by a PXR mediated mechanism. Attenuation of endotoxin-induced immune activation of epithelial cells might contribute to the immunomodulatory activities of rifaximin in IBDs.

Abstract 5645: Growth inhibitory activity of ethanol extracts of Chinese and Brazilian propolis in human colon carcinoma cell lines

Abstract Propolis is a natural product derived from plant resins collected by honeybees, and has been used globally as a folk medicine for centuries. More than 300 bio-active compounds have been identified from bee propolis. In the current study, we examined whether the ethanol extracts of Chinese and Brazilian propolis exert anticancer activities in four human colon carcinoma cell lines, CaCo2, HCT116, HT29 and SW480. Propolis samples were extracted with ethanol, and the crude extracts were dissolved in dimethylsulfoxide and used for the following experiments. We also examined the effects of the ethanol extract of Chinese propolis on cell proliferation, cell cycle progression, apoptosis, and the levels of expression of cell cycle control molecules in the HCT116 and HT29 cell lines. In the HCT116, HT29 and SW480 cell lines, the extracts of both Chinese and Brazilian propolis caused a marked dose-dependent growth inhibition, with IC50 values in the range of 4-41 μg/ml. In the FHC normal human colonic epithelial cell line, the extract of Chinese propolis failed to cause a marked growth inhibition, the growth inhibition rate was about 20%, whereas it was about 90% in the HCT116 and HT29 cell lines. In the HCT116 cell line, at concentrations of 10 and 20 μg/ml the extract of Chinese propolis caused a marked increase in sub-G1 fraction of cells in a dose-dependent fashion. The propolis extract (20 μg/ml) caused a significant increase of cells in sub-G1 and this increase was associated with a concomitant relative decrease of cells in the G1, S and G2-M phases of the cell cycle, indicating that Chinese propolis extract induced apoptosis in the HCT116 cell line. In addition, Chinese propolis extract caused a dose-dependent increase in the cellular mRNA levels of p21CIP1 and p53 in the HCT116 cell line. These findings indicate that the ethanol extracts of propolis contain components that may have anticancer activity. Thus, propolis and related products may provide a novel approach to the chemoprevention and/or chemotherapy of human colon carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5645.

Abstract 4838: Alpha-methyltryptophan, a blocker of the amino acid transporter SLC6A14, induces autophagy and apoptosis in SLC6A14-positive breast cancer and colon cancer cell lines

Abstract SLC6A14 (also known as ATB0,+) is a Na+/Cl− -coupled amino acid transporter that transports 18 of the 20 proteinogenic amino acids, including all essential amino acids. Normal tissues express this transporter at low levels, whereas tumor tissues (colon, breast, and cervix) upregulate this transporter several-fold. The unique functional features of SLC6A14 (broad substrate specificity and multiple driving forces) make it an ideal transporter for tumor cells to maintain amino acid nutrition necessary for enhanced cell proliferation. We speculate that SLC6A14 could not only be an ideal tumor-specific delivery system for amino acid-based anti-cancer drugs but also be a direct drug target for cancer chemotherapy. Recently we identified alpha-methyltryptophan (ALT) as a blocker of SLC6A14. Here we examine the potential of this blocker as an anti-cancer agent in SLC6A14-positive breast and colon cancer cells. Treatment of MCF7 (a breast cancer cell line), HCT116 and LS174T (colon cancer cell lines) with ALT causes amino acid starvation as evidenced by the upregulation of asparagine synthetase and CHOP. These three cell lines are SLC6A14-positive. In contrast, ALT treatment of breast cancer cell line MDA-MB231, which is SLC6A14-negative, does not show any evidence of amino acid starvation. Similarly, ALT treatment has no effect on normal breast and colon epithelial cell lines (MCF10A and CCD841, respectively). These cell lines also do not express SLC6A14. These data show that ALT-induced amino acid starvation is mediated specifically through the blockade of SLC6A14. Since nutrient signaling is also associated with changes in mTOR pathway, we examined the influence of ALT treatment on phosphorylation of S6 and S6kinase. Blockade of SLC6A14 with ALT in SLC6A14-positive cancer cell lines leads to decreased mTOR signaling as evidenced by decreased phosphorylation of S6 and S6kinase. Again, such changes are not seen in SLC6A14-negative cancer or normal cell lines. The initial response of the SLC6A14-positive cancer cell lines to ALT treatment is induction of autophagy. ALT-induced autophagy is documented in these cells by light and electron microscopic examination as well as by biochemical means. Inhibition of autophagy with 3-methyladenine under these conditions leads to apoptosis. We conclude that SLC6A14 is a potential drug target for cancer chemotherapy and that ALT could serve as a lead compound for the future design of high-affinity blockers of SLC6A14. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4838.