Metastatic Human Colon Research Articles

Comparative Analysis of Structural Analogs of Dipyridothiazines with m-Xylene and a Lutidine Moiety—In Silico, In Vitro, and Docking Studies

Dimers of dipyridothiazines with an m-xylene moiety are presented in terms of a comparative analysis with anticancer active structural analogs containing a lutidine system. The synthesis of new isomeric dimers was described, the structure of which was confirmed by 1H, 13C and 2D NMR, and HR MS spectroscopic methods. The preliminary prediction of the pharmacological profile using the Way2Drug server indicated the anticancer potential of the tested derivatives. In vitro biological activity tests were performed on a normal skin cell line (HaCaT) and five cancer cell lines, including human primary colon cancer (SW480), human metastatic colon cancer (SW620), human breast adenocarcinoma (MDA-MB-231), human lung carcinoma (A-549), and human glioblastoma (LN-229), which indicated low cytotoxic activity. In order to explain the surprisingly low activity, a comparative structural analysis of the tested analogs compared to the dimers with the lutidine system was performed using quantum mechanics and molecular docking in relation to histone deacetylase. Molecular docking indicated the different binding sites of the analyzed dimers, which explained the differences in the activity.

The Role of Neuropilin-2 in the Epithelial to Mesenchymal Transition of Colorectal Cancer: A Systematic Review.

Neuropilin-2 (NRP-2) expression has been found in various investigations on the expression and function of NRP-2 in colorectal cancer. The link between NRP-2 and colorectal cancer, as well as the mechanism that regulates it, is still mostly unclear. This systematic review was carried out according to the Cochrane guidelines for systematic reviews. We searched PubMed, Embase®, MEDLINE, Allied & Complementary MedicineTM, Medical Toxicology & Environmental Health, DH-DATA: Health Administration for articles published before 1 October 2021. The following search terms were used: “neuropilin-2” “neuropilin 2”, “NRP2” and “NRP-2”, “colorectal cancer”, “colon cancer”. Ten articles researching either tumor tissue samples, cell lines, or mice models were included in this review. The majority of human primary and metastatic colon cancer cell lines expressed NRP-2 compared to the normal colonic mucosa. NRPs have been discovered in human cancers as well as neovasculature. The presence of NRP-2 appears to be connected to the epithelial–mesenchymal transition’s function in cancer dissemination and metastatic evolution. The studies were heterogeneous, but the data assessed indicates NRP-2 might have an impact on the metastatic potential of colorectal cancer cells. Nevertheless, further research is needed.

Influence of oxygen availability on expression of glutaminolysis genes in human colon cancer cells

Abstract Introduction Glutaminolysis, beside glycolysis, is a key metabolic pathway of a cancer cell that provides energy and substrates for the synthesis of nucleic acids, proteins, and lipids. The pathway is mediated by both mitochondrial and cytosolic enzymes. Neither expression of glutaminolysis enzymes in colon cancer cells nor the influence of various oxygen concentrations on their expression has been studied so far. Objectives The aim of the study was to determine and compare the mRNA expression of enzymes involved in glutaminolysis at various oxygen levels in human primary (SW480) and metastatic (SW620) colon cancer cells cultured in 1% O2 (hypoxia), 10% O2 (tissue normoxia), 21% O2 (atmospheric normoxia). Methods Cell viability was determined by Trypan Blue exclusion (TB) and Thiazolyl Blue Tetrazolium Bromide (MTT). The expression of HIF1α, GLUT1, GLS1, AST1, AST2, ACL, PC and GC1, GC2 at mRNA levelwas determined by RT-qPCR. Results. Correlation between increasing oxygen concentration and cell count was not observed. In both cell lines the number of viable cells was the lowest at 10% oxygen. The enzyme profile and expression of proteins involved in glutaminolysis varied depending on oxygen pressure and type of cell lines. In summary, our findings suggest differences in metabolic adaptation to oxygen availability in vivo between primary and metastatic colon cancer cells.

Lymph Node Subcapsular Sinus Microenvironment-On-A-ChipModeling Shear Flow Relevant to Lymphatic Metastasis and Immune Cell Homing.

SummaryA lymph node sinus-on-a-chip adhesion microfluidic platform that recapitulates the hydrodynamic microenvironment of the lymph node subcapsular sinus was engineered. This device was used to interrogate the effects of lymph node remodeling on cellular adhesion in fluid flow relevant to lymphatic metastasis. Wall shear stress levels analytically estimated and modeled after quiescent and diseased/inflamed lymph nodes were experimentally recapitulated using a flow-based microfluidic perfusion system to assess the effects of physiological flow fields on human metastatic cancer cell adhesion. Results suggest that both altered fluid flow profiles and presentation of adhesive ligands, which are predicted to manifest within the lymph node subcapsular sinus as a result of inflammation-induced remodeling, and the presence of lymph-borne monocytic cells may synergistically contribute to the dynamic extent of cell adhesion in flow relevant to lymph node invasion by cancer and monocytic immune cells during lymphatic metastasis.

Engineering the Extracellular Matrix to Model the Evolving Tumor Microenvironment.

SummaryClinical evidence supports a role for the extracellular matrix (ECM) in cancer risk and prognosis across multiple tumor types, and numerous studies have demonstrated that individual ECM components impact key hallmarks of tumor progression (e.g., proliferation, migration, angiogenesis). However, the ECM is a complex network of fibrillar proteins, glycoproteins, and proteoglycans that undergoes dramatic changes in composition and organization during tumor development. In this review, we will highlight how engineering approaches can be used to examine the impact of changes in tissue architecture, ECM composition (i.e., identity and levels of individual ECM components), and cellular- and tissue-level mechanics on tumor progression. In addition, we will discuss recently developed methods to model the ECM that have not yet been applied to the study of cancer.

Proteomic Analysis of Protein Ubiquitination Events in Human Primary and Metastatic Colon Adenocarcinoma Tissues

Protein ubiquitination is essential for multiple physiological processes through regulating the stability or function of target proteins and has been found to play critical roles in human cancers. However, the protein ubiquitination profile of human metastatic colon adenocarcinoma tissue has not been elucidated yet. In this study, a proprietary ubiquitin branch (K-ε-GG) antibody-based label-free quantitative proteomics and bioinformatics were performed to identify the global protein ubiquitination profile between human primary (Colon) and metastatic colon adenocarcinoma (Meta) tissues. A total of 375 ubiquitination sites from 341 proteins were identified as differentially modificated (| Fold change| > 1.5, p < 0.05) in Meta group compared with the Colon group. Among them, 132 ubiquitination sites from 127 proteins were upregulated and 243 ubiquitination sites from 214 proteins were downregulated in Meta group. Fifteen ubiquitination motifs were found. Furthermore, GO and KEGG pathway analysis indicated that proteins with altered ubiquitination in Meta group were enriched in pathways highly related to cancer metastasis, such as RNA transport and cell cycle. We speculate that the altered ubiquitination of CDK1 may be a pro-metastatic factor in colon adenocarcinoma. This study provides novel scientific evidences to elucidate the biological functions of protein ubiquitination in human colon adenocarcinoma and insights into its potential mechanisms of colon cancer metastasis, which would be helpful to discover novel biomarkers and therapeutic targets for effective treatment of colon cancer.

Anticancer and antimicrobial effects of novel ciprofloxacin fatty acids conjugates

Ciprofloxacin (CP) has a confirmed cytotoxic action on some cancerous cells, but in high, non-pharmacological concentrations. Considering features of natural fatty acids, such as biocompatibility, biodegradability and their increased cellular uptake by cancer cells, it seems that combining them with a drug could improve its bioavailability, and thus cytotoxicity. Therefore, the aim of this study was coupling of CP with saturated and unsaturated fatty acids, and evaluation of their cytotoxicity, apoptosis-inducing effects and inhibition of IL-6 release in human primary (SW480) and metastatic (SW620) colon cancer, metastatic prostate cancer (PC3) and normal (HaCaT) cell lines. The PC3 cell line was the most sensitive to the presence of the obtained conjugates. The value of IC50 for oleic acid conjugate (4) was 7.7 μM, and it was 12 times lower than for CP alone (101.4 μM). The studied derivatives induced late apoptosis in all cancer cell lines, but not in normal cells. The most potent apoptosis inducer was conjugate 4, that resulted in the highest percentage of PC3 cells in late apoptosis (81.5% ± 3.9), followed by elaidic acid amide 5 (75% ± 4.8). The strongest pro-apoptic effects on SW480 cells were demonstrated by conjugates of DHA (8) and sorbic (2) acids, whereas in SW620 cell lines, compounds 2 and 5 appeared to be the most effective. To establish the mechanism of cytotoxic action of derivatives 2, 4, 5, the level of interleukin-6 (IL-6) was measured. The compounds with the highest cytotoxic potential significantly decreased the release of IL-6 by cancer cells. Additionally, all conjugates were evaluated for their in vitro antimicrobial activity. Short chain amides - crotonic (1) and sorbic (2) - were the most active against Staphyloccoci. The second-mentioned amide has shown both strong antistaphylococcal and antitumor properties.

Telmisartan Influences the Antiproliferative Activity of Linoleic Acid in Human Colon Cancer Cells

Aim: Linoleic acid (LA) and telmisartan as PPARgamma agonists exhibit anticancer activity. The LA effect is observed for high non-achievable in vivo concentrations and in short treatment period, therefore we evaluate the effect of supplemental LA and pharmacological telmisartan plasma concentrations on human primary (SW480) and metastatic (SW620) colon cancer cells and immortal keratinocytes (HaCaT) cells in long-term treatment. Methods: Cell viability and proliferation were determined by TB and MTT and pro-apoptotic effect was measured by Annexin V binding assays, respectively.Results: LA decreased cancer cell viability and proliferation in a concentration-dependent manner, whereas no significant effect was found for HaCaT cells. Telmisartan (0.2 µM) suppresses antiproliferative effect of 60 µM LA on cancer cells in short-term treatment. Long-term administration of 60 µM LA reduced cancer cells viability after one week, while telmisartan delayed this effect by two weeks. Growth of all cell lines with 20 µM LA was unchanged during all treatment time. Telmisartan decreased late apoptosis of cancer and normal cells with 60 and 120 µM LA. Conclusion: The cytotoxic LA action depends not only on its concentration but also duration of treatment. Telmisartan exhibits biphasic but not synergistic effect on LA cytotoxicity in cancer cells.

An N-terminal truncated carboxypeptidase E splice isoform induces tumor growth and is a biomarker for predicting future metastasis in human cancers.

Metastasis is a major cause of mortality in cancer patients. However, the mechanisms governing the metastatic process remain elusive, and few accurate biomarkers exist for predicting whether metastasis will occur, something that would be invaluable for guiding therapy. We report here that the carboxypeptidase E gene (CPE) is alternatively spliced in human tumors to yield an N-terminal truncated protein (CPE-ΔN) that drives metastasis. mRNA encoding CPE-ΔN was found to be elevated in human metastatic colon, breast, and hepatocellular carcinoma (HCC) cell lines. In HCC cells, cytosolic CPE-ΔN was translocated to the nucleus and interacted with histone deacetylase 1/2 to upregulate expression of the gene encoding neural precursor cell expressed, developmentally downregulated gene 9 (Nedd9)--which has been shown to promote melanoma metastasis. Nedd9 upregulation resulted in enhanced in vitro proliferation and invasion. Quantification of mRNA encoding CPE-ΔN in HCC patient samples predicted intrahepatic metastasis with high sensitivity and specificity, independent of cancer stage. Similarly, high CPE-ΔN mRNA copy numbers in resected pheochromocytomas/paragangliomas (PHEOs/PGLs), rare neuroendocrine tumors, accurately predicted future metastasis or recurrence. Thus, CPE-ΔN induces tumor metastasis and should be investigated as a potentially powerful biomarker for predicting future metastasis and recurrence in HCC and PHEO/PGL patients.

Protein corona-resistant SERS tags for live cell detection of integrin receptors.

Chemical signals are conveyed to cells through ligand-receptor binding, triggering cascades of biochemical reactions and resulting in pivotal cellular functions. These binding events are important in understanding membrane signaling and drug interactions. To probe ligand-receptor binding, surface enhanced Raman scattering (SERS) tags are a promising tool. SERS tags are plasmonic nanostructures functionalized with a protective coating, a Raman reporter molecule, and a biorecognition element. In biological fluids, native proteins have affinity for bare nanoparticles and form a protein corona. SERS tags have a protective shell which eliminates this complication. It is important to analyze ligand-receptor binding with SERS tags in live cells since cell fixatives alter protein structure, leading to spectral changes and data misinterpretation. In this study, we synthesized a novel SERS tag by creating a mixed monolayer of the small cyclic arginine-glycine-aspartic acid-phenylalanine-cysteine (RGDFC) peptide and 4-mercaptobenzonitrile (MBN) on the surface of spherical gold nanoparticles (Au NP). Au-RGDFC-MBN NP showed resistance to PC formation and were successfully detected in both fixed and living human metastatic colon cancer cells.

Label-free plasmonic nanostar probes to illuminate in vitro membrane receptor recognition.

Protein-ligand recognition is a key activity where chemical signals are communicated to cells to activate various biochemical pathways, which are important for understanding membrane signaling and drug interactions. Gold nanostars are highly attractive for biological applications due to their readily modified surface chemistry, facile synthesis and optical properties. The increase in electromagnetic field at their branches increases the surface enhanced Raman scattering (SERS) making them ideal candidates as label free in vitro probes that can be used to detect a variety of cellular activities. However, the use of particles in vitro is complicated by the adsorption of proteins, which forms the protein corona. In this paper we demonstrate gold nanostars as label free in vitro probes to study the interaction between αvβ3 integrin and RGD. Nanostars functionalized with cyclic-RDGFC reduced the formation of the protein corona, due to its zwitterionic nature, indicating a small peptide approach to minimizing protein absorption. Additionally, the functionalized nanostars evince a SERS response from their interaction with αvβ3 integrin representative of the amino acids present at the binding site which is also retained in a complex biological matrix. The nanostars were used in vitro to selectively detect αvβ3 integrin on the membrane of human metastatic colon cancer cells. By exploiting the intense SERS and tunable plasmon resonance properties of gold nanostars functionalized with cyclic RGDFC, we have demonstrated a label free approach to investigate the chemical interactions associated with protein-ligand binding from both purified proteins and membrane bound receptors in cells.

Effects of meta-tetrahydroxyphenylchlorin photodynamic therapy on isogenic colorectal cancer SW480 and SW620 cells with different metastatic potentials

The aim of this study is to investigate the antitumor effects and possible mechanisms of meta-tetrahydroxyphenylchlorin-mediated photodynamic therapy (m-THPC-PDT) on human primary (SW480) and metastatic (SW620) colon cancer cell lines. SW480 and SW620 cells were incubated with various concentrations of m-THPC, followed by photodynamic irradiation. Subcellular localization of m-THPC in cells was observed with confocal laser scanning microscopy (CLSM). Photocytotoxicity of m-THPC in the two cells was investigated by using MTT assay. The flow cytometry was employed to detect the cell apoptosis. The migration and long-term recovery ability were determined by scratch test and colony formation assay respectively. CLSM showed that m-THPC was mainly distributed within the endoplasmic reticulum (ER) and lysosome of SW480 cells and within the lysosome and mitochondria of SW620 cells. m-THPC-PDT induced a dose-dependent and light energy-dependent cytotoxicity in SW480 and SW620 cells. Apoptosis rate was approximately 65 and 25% in SW480 and SW620 respectively when the concentration of m-THPC increased to 11.76 μM. However, the rate of necrotic cells had no significant changes in two cell lines. The colony formation and migration ability of the two cell lines were decreased with m-THPC-PDT treatment in a dose-dependent manner. PDT with m-THPC not only could effectively inhibit cell proliferation and decrease migration ability and colony formation ability, but also could effectively kill SW480 and SW620 cells in a dose-dependent manner in vitro. These results suggest that m-THPC is a promising sensitizer that warrants further development and extensive studies towards clinical use of colorectal cancer.

Establishment of human metastatic colorectal cancer model in rabbit liver: A pilot study

Rationale and objectivesTo develop a human metastatic colorectal cancer (mCRC) model in a rabbit liver.Materials and methodsImmunosuppression in 4 adult New Zealand White rabbits weighing 3.5 to 4.5 kg was induced with daily subcutaneous injection of 15 mg/kg Cyclosporine A (CsA). On day 3 open mini-laparotomy was performed and 0.2 ml (1.8x105 cells) suspension of HCT-116 and HT-29 human CRC cells were injected into the left and right medial lobe respectively. On day 10 the CsA dose was reduced to 10 mg/kg daily maintenance dose. Rabbits were weighed weekly, closely monitored for CsA side effects (weight loss, gingival hyperplasia and gut modification). Rabbits were sacrificed 5, 6, 7, and 8 weeks after cells injection. Liver tumors were collected for histopathology and immunohistochemical analysis.ResultsHT-29 Tumor growth was observed in 3 rabbits (75%). Tumors measured 3, 4 and 6 mm after 5, 6 and 8 weeks respectively. Microscopically, tumors contained hyperchromatic, pleomorphic cells that stained for monoclonal carcinoembryonic antigen (CEA), polyclonal CEA, cytokeratin 20, vascular markers (CD31, CD34), and vascular endothelial growth factor (VEGF) by immunohistochemistry, supporting involvement by the poorly differentiated HT-29 colorectal cancer cell line. No gross tumor growth or microscopic viability was observed from HCT-116 cell injection. CsA extra-hepatic manifestations included minimal gum hyperplasia and decrease in gut motility in 3 rabbits (75%), which was treated with Azithromycin 15 mg/kg and Cisapride 0.5 mg/kg every 12 hours, respectively.ConclusionWe successfully developed a human metastatic colon cancer model in immunosuppressed rabbit liver using HT-29 cells.

Lactate Formation in Primary and Metastatic Colon Cancer Cells at Hypoxia and Normoxia.

High glucose consumption and lactate synthesis in aerobic glycolysis are a hallmark of cancer cells. They can form lactate also in glutaminolysis, but it is not clear how oxygen availability affects this process. We studied lactate synthesis at various oxygen levels in human primary (SW480) and metastatic (SW620) colon cancer cells cultured with L-Ser and/or L-Asp. Glucose and lactate levels were determined colorimetrically, amino acids by HPLC, expression of AST1-mRNA and AST2-mRNA by RT-PCR. In both lines glucose consumption and lactate synthesis were higher at 10% than at 1% oxygen, and lactate/glucose ratio was increased above 2.0 by L-Asp. AST1-mRNA expression was independent on oxygen and cell line, but AST2-mRNA was lower at hypoxia in SW480. We conclude that, in both cell lines at 1% hypoxia, lactate is formed mainly from glucose but at 10% normoxia also from L-Asp. At 10% normoxia, lactate synthesis is more pronounced in primary than metastatic colon cancer cells.

Abstract 3839: Vitamin E delta-tocotrienol targets human colon cancer stem cells and inhibits colon cancer metastasis and induces apoptosis

Abstract Background: Colon cancer is the most commonly diagnosed cancer both in men and women and cause of cancer related deaths worldwide. Treatment for the recurrent and metastatic colon cancer remains a center of clinical attention. Cancer stem cells (CSCs) are suggested to be the only cells within tumor with tumorigenic capacity and implicated in metastasis. We have shown that vitamin E delta-tocotrienol (VEDT) is the most bioactive tocotrienols against cancers. This study aimed to evaluate the effect of VEDT on colon cancer growth, metastatic epithelial to mesenchymal transition (EMT), migration/ invasion/angiogenesis and colon CSCs growth and apoptosis. Methods: Human colon cancer cells HCT-116 and metastatic colon cancer cells SW-620 and colon CSCs were treated with VEDT (10-100 μM) for 72 h and cell viability recorded. VEDT (50 μM) was used for malignant transformation; migration/invasion, CSCs spheroid formation, and pluripotency were determined. Azoxymethane treated Fisher 344 rats were used as colon carcinogenesis model. They were treated with 1) vehicle control and 2) VEDT (200 mg/kg, orally twice a day) for 20 weeks. After 20 weeks the polyps and tumor formation were recorded. Results: VEDT significantly inhibited the anchorage-dependent and independent cancer cell growth, migration/invasion and induced apoptosis compared to control. VEDT inhibited the CSC spheroid formation EMT (E-cadherin to vimentin), metastasis (MMP9), angiogenesis (VEGF), CSCs transcription factors (Nanog, Oct4, Sox2 and KLF4) compared to control. Conclusion: Vitamin E delta-tocotrienol inhibited colon cancer growth and metastasis through inhibition of cancer stem cell pluripotency, EMT, migration, invasion, angiogenesis and induction of apoptosis. Citation Format: Kazim Husain, Domenico Coppola, Said M. Sebti, Mokenge P. Malafa. Vitamin E delta-tocotrienol targets human colon cancer stem cells and inhibits colon cancer metastasis and induces apoptosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3839.

Cigarette smoke extracts induced the colon cancer migration via regulating epithelial mesenchymal transition and metastatic genes in human colon cancer cells.

There was considerable evidence that exposure to cigarette smoke is associated with an increased risk for colon cancer. Nevertheless, the mechanism underlying the relationship between cigarette smoking and colon cancer remains unclear. Moreover, there were only a few studies on effects of complexing substance contained in cigarette smoke on colon cancer. Thus, we further investigated whether cigarette smoke extract (CSE) affects the cell cycle, apoptosis and migration of human metastatic colon cancer cells, SW-620. MTT assay revealed that SW-620 cell proliferation was significantly inhibited following treatments with all CSEs, 3R4F, and two-domestic cigarettes, for 9 days in a concentration-dependent manner. Moreover, CSE treatments decreased cyclin D1 and E1, and increased p21 and p27 proteins by Western blot analysis in SW-620 cells. Additionally, the treatment of the cells with CSE contributed to these effects expressing by apoptosis-related proteins. An increased migration or invasion ability of SW-620 cells following CSE treatment was also confirmed by a scratch or fibronectin invasion assay in vitro. In addition, the protein levels of E-cadherin as an epithelial maker were down-regulated, while the mesenchymal markers, N-cadherin, snail, and slug, were up-regulated in a time-dependent manner. A metastatic marker, cathepsin D, was also down-regulated by CSE treatment. Taken together, these results indicate that CSE exposure in colon cancer cells may deregulate the cell growth by altering the expression of cell cycle-related proteins and pro-apoptotic protein, and stimulate cell metastatic ability by altering epithelial-mesenchymal transition (EMT) markers and cathepsin D expression. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 690-704, 2017.

Human Milk Oligosaccharides and Synthetic Galactosyloligosaccharides Contain 3′-, 4-, and 6′-Galactosyllactose and Attenuate Inflammation in Human T84, NCM-460, and H4 Cells and Intestinal Tissue Ex Vivo

Background: The immature intestinal mucosa responds excessively to inflammatory insult, but human milk protects infants from intestinal inflammation. The ability of galactosyllactoses [galactosyloligosaccharides (GOS)], newly found in human milk oligosaccharides (HMOS), to suppress inflammation was not known.Objective: The objective was to test whether GOS can directly attenuate inflammation and to explore the components of immune signaling modulated by GOS.Methods: Galactosyllactose composition was measured in sequential human milk samples from days 1 through 21 of lactation and in random colostrum samples from 38 mothers. Immature [human normal fetal intestinal epithelial cell (H4)] and mature [human metastatic colonic epithelial cell (T84) and human normal colon mucosal epithelial cell (NCM-460)] enterocyte cell lines were treated with the pro-inflammatory molecules tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) or infected with Salmonella or Listeria. The inflammatory response was measured as induction of IL-8, monocyte chemoattractant protein 1 (MCP-1), or macrophage inflammatory protein-3α (MIP-3α) protein by ELISA and mRNA by quantitative reverse transcriptase-polymerase chain reaction. The ability of HMOS or synthetic GOS to attenuate this inflammation was tested in vitro and in immature human intestinal tissue ex vivo.Results: The 3 galactosyllactoses (3′-GL, 4-GL, and 6′-GL) expressed in colostrum rapidly declined over early lactation (P < 0.05). In H4 cells, HMOS attenuated TNF-α– and IL-1β–induced expression of IL-8, MIP-3α, and MCP-1 to 48–51% and pathogen-induced IL-8 and MCP-1 to 26–30% of positive controls (P < 0.001). GOS reduced TNF-α– and IL-1β–induced inflammatory responses to 25–26% and pathogen-induced IL-8 and MCP-1 to 36–39% of positive controls (P < 0.001). GOS and HMOS mitigated nuclear translocation of nuclear transcription factor κB (NF-κB) p65. HMOS quenched the inflammatory response to Salmonella infection by immature human intestinal tissue ex vivo to 26% and by GOS to 50% of infected controls (P < 0.01).Conclusion: Galactosyllactose attenuated NF-κB inflammatory signaling in human intestinal epithelial cells and in human immature intestine. Thus, galactosyllactoses are strong physiologic anti-inflammatory agents in human colostrum and early milk, contributing to innate immune modulation. The potential clinical utility of galactosyllactose warrants investigation.

Abstract 2003: CPE-delta-N promotes metastasis by regulating Nedd9 and Yap1 expression

Abstract Cancer mortality is often from metastatic disease rather than the direct effect of the primary tumor. Identification of metastasis inducing genes may offer valuable mechanistic insight for guiding specific therapeutic strategies. We report here that the carboxypeptidase E gene (CPE) is alternatively spliced in human tumors to yield an N-terminal truncated protein (CPE-DELTA-N) that drives metastasis. CPE-DELTA-N mRNA was elevated in human metastatic colon, breast and HCC cell lines. Suppression of CPE-DELTA-N expression in these cell lines by si-RNA significantly inhibited their growth migration and invasion. To confirm these observations in vivo, an orthotopic nude mouse model was established. The mice implanted with a tumor derived from HCC cells transfected with si- CPE-DELTA-N RNA in the liver did not show tumor growth or metastasis, compared to scrambled controls. In HCC cytosolic CPE-DELTA-N protein was translocated to the nucleus and upregulated the expression of neural precursor cell expressed, developmentally downregulated gene 9 (Nedd9), through interaction with histone deacetylase (HDAC) 1/2. Inhibition of HDAC activity by the HDAC inhibitors suppressed expression of NEDD9, without effecting CPE-DELTA-N expression. The enhanced invasive phenotype of HCC cells stably transfected with CPE-DELTA-N was suppressed when Nedd9 was silenced by si-RNA. Furthermore CPE-DELTA-N protein up-regulation also increased Yes Associated Protein 1 (YAP1), Carcinoembryonic Antigen-related Cell Adhesion Molecule 5 (ceacam5) and Carbonyl Reductase 1(cbr1) and silencing CPE-DELTA-N with siRNA suppressed YAP1, CEACAM5 and CBR1 protein expression. cDNA Microarray studies of HCC cells overexpressing CPE-DELTA-N showed elevated expression of 27 genes associated with metastasis, while 30 genes associated with tumor suppressor function, which included insulin-like growth factor binding protein 5 and 3 (igfbp5 and igfbp3) and h19 were down-regulated. These data were further confirmed by qRTPCR.. In clinical studies, CPE-DELTA-N mRNA quantification in 99 patients with primary tumor (T) and normal surrounding tissue (N) established a T/N cut off value which predicted future intra-hepatic metastasis with high sensitivity and specificity, independent of cancer stage. Additionally, prospective study on 46 resected PHEO/PGLs having very high CPE-DELTA-N mRNA copy numbers accurately predicted future metastasis or recurrence in patients diagnosed with benign tumors at time of surgery, versus low copy numbers in post-surgery, disease-free patients. Additionally, CPE-DELTA-N is a diagnostic biomarker for metastasis in papillary thyroid carcinoma and colorectal cancer. Thus CPE-DELTA-N induces tumor metastasis and is a powerful biomarker for predicting future metastasis and recurrence in HCC and PHEO/PGL patients, superior to histopathological diagnosis. Citation Format: Saravana R.K Murthy, Terence K. Lee, Niamh X. Cawley, Stephen M. Hewitt, Peng Y. Loh. CPE-delta-N promotes metastasis by regulating Nedd9 and Yap1 expression. [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 2003. doi:10.1158/1538-7445.AM2014-2003

Extracellular matrix signatures of human primary metastatic colon cancers and their metastases to liver.

BackgroundColorectal cancer is the third most frequently diagnosed cancer and the third cause of cancer deaths in the United States. Despite the fact that tumor cell-intrinsic mechanisms controlling colorectal carcinogenesis have been identified, novel prognostic and diagnostic tools as well as novel therapeutic strategies are still needed to monitor and target colon cancer progression. We and others have previously shown, using mouse models, that the extracellular matrix (ECM), a major component of the tumor microenvironment, is an important contributor to tumor progression. In order to identify candidate biomarkers, we sought to define ECM signatures of metastatic colorectal cancers and their metastases to the liver.MethodsWe have used enrichment of extracellular matrix (ECM) from human patient samples and proteomics to define the ECM composition of primary colon carcinomas and their metastases to liver in comparison with normal colon and liver samples.ResultsWe show that robust signatures of ECM proteins characteristic of each tissue, normal and malignant, can be defined using relatively small samples from small numbers of patients. Comparisons with gene expression data from larger cohorts of patients confirm the association of subsets of the proteins identified by proteomic analysis with tumor progression and metastasis.ConclusionsThe ECM protein signatures of metastatic primary colon carcinomas and metastases to liver defined in this study, offer promise for development of diagnostic and prognostic signatures of metastatic potential of colon tumors. The ECM proteins defined here represent candidate serological or tissue biomarkers and potential targets for imaging of occult metastases and residual or recurrent tumors and conceivably for therapies. Furthermore, the methods described here can be applied to other tumor types and can be used to investigate other questions such as the role of ECM in resistance to therapy.

Increased diacylglycerol kinase ζ expression in human metastatic colon cancer cells augments Rho GTPase activity and contributes to enhanced invasion

BackgroundUnraveling the signaling pathways responsible for the establishment of a metastatic phenotype in carcinoma cells is critically important for understanding the pathology of cancer. The acquisition of cell motility is a key property of metastatic tumor cells and is a prerequisite for invasion. Rho GTPases regulate actin cytoskeleton reorganization and the cellular responses required for cell motility and invasion. Diacylglycerol kinase ζ (DGKζ), an enzyme that phosphorylates diacylglycerol to yield phosphatidic acid, regulates the activity of the Rho GTPases Rac1 and RhoA. DGKζ mRNA is highly expressed in several different colon cancer cell lines, as well as in colon cancer tissue relative to normal colonic epithelium, and thus may contribute to the metastatic process.MethodsTo investigate potential roles of DGKζ in cancer metastasis, a cellular, isogenic model of human colorectal cancer metastatic transition was used. DGKζ protein levels, Rac1 and RhoA activity, and PAK phosphorylation were measured in the non-metastatic SW480 adenocarcinoma cell line and its highly metastatic variant, the SW620 line. The effect of DGKζ silencing on Rho GTPase activity and invasion through Matrigel-coated Transwell inserts was studied in SW620 cells. Invasiveness was also measured in PC-3 prostate cancer and MDA-MB-231 breast cancer cells depleted of DGKζ.ResultsDGKζ protein levels were elevated approximately 3-fold in SW620 cells compared to SW480 cells. There was a concomitant increase in active Rac1 in SW620 cells, as well as substantial increases in the expression and phosphorylation of the Rac1 effector PAK1. Similarly, RhoA activity and expression were increased in SW620 cells. Knockdown of DGKζ expression in SW620 cells by shRNA-mediated silencing significantly reduced Rac1 and RhoA activity and attenuated the invasiveness of SW620 cells in vitro. DGKζ silencing in highly metastatic MDA-MB-231 breast cancer cells and PC-3 prostate cancer cells also significantly attenuated their invasiveness.ConclusionElevated DGKζ expression contributes to increased Rho GTPase activation and the enhanced motility of metastatic cancer cells. These findings warrant further investigation of the clinical relevance of DGKζ upregulation in colon and other cancers. Interfering with DGKζ function could provide a means of inhibiting invasion and metastasis.