Human Colorectal Adenocarcinoma Cell Line Research Articles

P-0209 Ascorbic ACID and Its Therapeutic Potential Against Colorectal Adenocarcinoma

ABSTRACT Introduction Colorectal cancer (CRC) is a major health problem, with more than 800,000 new cases diagnosed worldwide every year. A better understanding of the processes involved in the transformation of normal cells into cancer cells has led to the development of new therapies which target cancer through different mechanisms. The use of vitamin C in the treatment of cancer has been the source of many controversies over the last 25 years. Vitamin C is available in its reduced form (ascorbic acid - AA) and in its oxidized form (dehydroascorbic acid). Although AA is most commonly known for its antioxidant properties, it is also known to function as a pro-oxidant at pharmacological concentrations, promoting the formation of reactive oxygen species (ROS). The increased production of hydrogen peroxide, coupled with the breakdown of the activity of antioxidant enzymes and the presence of transition metals in cancer cells, may result in the selective cytotoxicity of vitamin C and the subsequent revelation of its therapeutic potential. The aim of this study is to evaluate the therapeutic potential of AA in human colorectal adenocarcinoma cell lines in vitro and in vivo. Methods Two human colorectal adenocarcinoma cell lines, the cells WiDr and C2BBe1, were cultured in DMEM supplemented with 10% FBS. Cells were incubated in absence and with different concentrations of AA during different periods of time. The half maximal inhibitory concentration (IC50) was calculated after 24, 48, 72 and 96 hours by the sulforodamine B (SRB) assay. In order to evaluate cell survival, clonogenic assays were performed. At tenth day, the number of colonies was counted, being the plate efficiency and the survival factor determined. In order to verify in vivo the evolution of tumor growth after the daily injection of AA, WiDr cells were inoculated on the back of Balb/c nu/nu mice and, during several days, the body weight and tumor size of mice were monitored. AA was not administered to control animals. Results Our results obtained with SRB show that AA induces a decrease in cell proliferation in both cell lines, in a dose-dependent way. It was also verified that the IC50 of AA decreases in both cell lines in a time-dependent way. The clonogenic assays revealed that, as the concentration of AA increases, the survival factor decreases. This behaviour was observed in both cell lines, being more evident in WiDr cells (the reduction factor is 44.88% in cells treated with 0.5mM and above 80% when cells are treated with 2, 3 and 5mM). The in vivo studies suggest that AA administered daily at 150 mg/kg inhibits tumor growth since there are significant differences between the two murine groups (control vs treated mice). Conclusion Our study suggests that AA induces an anti-proliferative effect and a decrease in WiDr and C2BBe1 cells survival. Our results also indicate that AA stabilizes tumor growth in a mice model of CRC. The data obtained in the two different human cell lines of colorectal adenocarcinoma could contribute to the insights of the role of antioxidants as anti-cancer therapies.

Tumour Hypoxia and Technetium Tracers: In Vivo Studies

Hypoxia is a biochemical condition where reduced oxygen partial pressure at tissue level occurs. This metabolic situation can lead to resistance to radio and chemotherapy. In malignant solid tumours, hypoxia is a common characteristic, having a great impact at biological level, being of tremendous importance for complete understanding of tumour progression. We studied the behavior of 99mTc-HL91 in vivo, using an animal model based on Balb-c nu/nu mice with a xenotransplant of the human colorectal adenocarcinoma cell line, WiDr. In vivo studies using an animal model of xenograft on Balb/c nu/nu nude mice were carried out. This model, allowed us to evaluate the radiopharmaceutical biodistribution and to calculate tumour/muscle ratio, acquired after 99mTc-HL91 injection. We also performed ex vivo studies, using the excised tumours to access viability and to characterize the intracellular production of reactive oxygen species and the status of mitochondrial membrane potential through flow cytometry. The biodistribution after 99mTc-HL91 injection showed urinary and hepatobilliary excretion in similar proportions and tumour uptake around 4.4% of administered activity. This uptake was higher at the bigger tumours. Through flow cytometry we observed that larger tumours have a higher amount of reactive oxygen species and a decrease in mitochondrial membrane potential. 99mTc-HL91 allowed a non-invasive evaluation of the solid tumours oxidative state by nuclear medicine functional imaging. This information can be of high importance at the pre-treatment estimation of this type of tumours.

Apoptotic-related gene expression in oxaliplatin-treated CCM-1 colorectal adenocarcinoma and MALT-nt-1 mucosa-associated lymphoid tissue lymphoma cell lines

The purpose of this study was to assess the apoptotic -related gene expression of single-agent oxaliplatin in vitro. Growth inhibition studies were performed using the human cerebral cavernous malformation-1 colorectal adenocarcinoma cell line and mucosa-associated lymphoid tissue-nt-1 lymphoma cell line. At 50% inhibitory concentrations, oxaliplatin displayed suppressive effects toward Bcl-2 and the surviving expression in human cancer cell lines, whereas the expression of Fas/FasL was completely abolished in peripheral blood cells. Our data showed that oxaliplatin exerts potent antiapoptotic effects in human normal peripheral blood mononuclear cells at the suprapharmacologic concentration, warranting further investigations in the role of autoimmune adverse events while being used in numerous cycles for the treatment of malignancies.

Paclitaxel Induced B7-H1 Expression in Cancer Cells via the MAPK Pathway

In this study, we investigated the mechanisms by which the chemotherapeutic agent paclitaxel (PTX) induced the expression of B7-H1 immunosuppressive molecules in the human colorectal adenocarcinoma cell line SW480 and the hepatocellular carcinoma cell line HepG2. We found PTX induced B7-H1 protein expression in SW480 and HepG2 cells as demonstrated by immunofluorescence and flow cytometry and mRNA expression by using real-time quantitative polymerase chain reaction (PCR). Moreover, PTX treatment induced Erk1/2 phosphorylation in both cell lines. PTX-increased B7-H1 MRNA expression was significantly blocked by meK inhibitor U0126. However, the protein expression caused by PTX was only partially blocked by U0126. Our results suggest that PTX upregulated B7-H1 expression in cultured SW480 and HepG2 cells via both transcriptional and post-transcriptional mechanisms. This may help us better understand PTX-related tumor immune evasion

56Co-labelled radioactive Fe3O4 nanoparticles for in vitro uptake studies on Balb/3T3 and Caco-2 cell lines

Magnetite nanoparticles (Fe3O4 NPs) are manufactured nanomaterials increasingly used in healthcare for different medical applications ranging from diagnosis to therapy. This study deals with the irradiation of Fe3O4 NPs with a proton beam in order to produce 56Co as radiolabel and also with the possible use of nuclear techniques for the quantification of Fe3O4 NPs in biological systems. Particular attention has been focused on the size distribution (in the range of 100 nm) and the surface charge of the NPs characterizing them before and after the irradiation process in order to verify if these essential properties would be preserved during irradiation. Moreover, X-ray diffraction studies have been performed on radioactive and non-radioactive NPs, to assess if major changes in NPs structure might occur due to thermal and/or radiation effects. The radiation emitted from the radiolabels has been used to quantify the cellular uptake of the NPs in in vitro studies. As for the biological applications two cell lines have been selected: immortalized mouse fibroblast cell line (Balb/3T3) and human epithelial colorectal adenocarcinoma cell line (Caco-2). The cell uptake has been quantified by radioactivity measurements of the 56Co radioisotope performed with high resolution γ-ray spectrometry equipment. This study has showed that, under well-established irradiation conditions, Fe3O4 NPs do not undergo significant structural modifications and thus the obtained results are in line with the uptake studies carried out with the same non-radioactive nanomaterials (NMs). Therefore, the radiolabelling method can be fruitfully applied to uptake studies because of the low-level exposure where higher sensitivity is required.

Neopetrosiquinones A and B, sesquiterpene benzoquinones isolated from the deep-water sponge Neopetrosia cf. proxima

Two new marine-derived sesquiterpene benzoquinones which we designate as neopetrosiquinones A (1) and B (2), have been isolated from a deep-water sponge of the family Petrosiidae. The structures were elucidated on the basis of their spectroscopic data. Compounds 1 and 2 inhibit the in vitro proliferation of the DLD-1 human colorectal adenocarcinoma cell line with IC50 values of 3.7 and 9.8μM, respectively, and the PANC-1 human pancreatic carcinoma cell line with IC50 values of 6.1 and 13.8μM, respectively. Neopetrosiquinone A (1) also inhibited the in vitro proliferation of the AsPC-1 human pancreatic carcinoma cell line with an IC50 value of 6.1μM. The compounds are structurally related to alisiaquinone A, cyclozonarone, and xestoquinone.

Reactive oxygen species mediate arsenic induced cell transformation and tumorigenesis through Wnt/β-catenin pathway in human colorectal adenocarcinoma DLD1 cells

Long term exposure to arsenic can increase incidence of human cancers, such as skin, lung, and colon rectum. The mechanism of arsenic induced carcinogenesis is still unclear. It is generally believed that reactive oxygen species (ROS) may play an important role in this process. In the present study, we investigate the possible linkage between ROS, β-catenin and arsenic induced transformation and tumorigenesis in human colorectal adenocarcinoma cell line, DLD1 cells. Our results show that arsenic was able to activate p47 phox and p67 phox, two key proteins for activation of NADPH oxidase. Arsenic was also able to generate ROS in DLD1 cells. Arsenic increased β-catenin expression level and its promoter activity. ROS played a major role in arsenic-induced β-catenin activation. Treatment of DLD1 cells by arsenic enhanced both transformation and tumorigenesis of these cells. The tumor volumes of arsenic treated group were much larger than those without arsenic treatment. Addition of either superoxide dismutase (SOD) or catalase reduced arsenic induced cell transformation and tumor formation. The results indicate that ROS are involved in arsenic induced cell transformation and tumor formation possible through Wnt/β-catenin pathway in human colorectal adenocarcinoma cell line DLD1 cells.

Antioxidant, antimicrobial, anti-inflammatory and anticancer activities of Carthamus tinctorius flowers

Carthamus tinctorius L. (Asteraceae) is an aromatic and folkloric medicinal plant thanks to its multiple virtues. However, few scientific studies investigated its biological activities. For that, this study aimed to investigate antioxidant, antibacterial, anti-inflammatory and anticancer activities of methanolic flower extracts of Carthamus tinctorius in order to validate some of its ethnopharmacological claims. Antioxidant activity was assessed via the ABTS radical scavenging and β-carotene inhibition tests, the antibacterial capacity were tested against human pathogen strains. Whereas, anti-inflammatory activity were estimated using inhibition NO release in LPS-stimulated Raw 264.7 macrophages, in comparison with N(G)-nitro-L-arginine methyl ester (L-NAME) which used as a positive control. In addition, anticancer activity was evaluated against Human lung carcinoma (A-549) and Human colorectal adenocarcinoma (DLD-1) cell lines. Main results showed that the flowers exhibit interesting biological activities. Indeed, flower extract displayed an inhibition percentage against ABTS equal to 30% and over 40% for the β-carotene inhibition assay. Antimicrobial activities were important especially against M. luteus strains (100% inhibition). Concerning anti-inflammatory activity, methanolic extract was able to inhibit NO release by 80% at 160µg/ml. Furthermore, C. tinctorius extract showed an anticancer activity against tumor cell lines DLD-1 with an IC50 value of 72±9µg/ml. These findings demonstrate the interesting potentiality of Carthamus tinctorius flowers as valuable source of antioxidant compounds which exhibit novel biological activities as antibacterial, anti-inflammatory and anticancer capacities.

Effect of Particle Size of Zinc Oxides on Cytotoxicity and Cell Permeability in Caco-2 Cells

The cell permeability and cytotoxic effects of different-sized zinc oxide (ZnO) particles were investigated using a human colorectal adenocarcinoma cell line called Caco-2. Morphological observation by scanning electron microscopy revealed that three zinc oxides with different mean particle sizes (ZnO-1, 20 ㎚; ZnO-2, 90~200 ㎚; ZnO-3, 1~5 ㎛) tended to aggregate, particularly in the case of ZnO-1. When cytotoxicities of all three sizes of zinc oxide particles were measured at concentration ranges of 1~1000 ㎍/㎖, significant decreases in cell viability were observed at concentrations of 50 ㎍/㎖ and higher. Among the three zinc oxides, ZnO-1 showed the lowest viability at 50 ㎍/㎖ in Caco-2 cells, followed by ZnO-2 and ZnO-3. The permeate concentration of ZnO-1 from the apical to the basolateral side in the Caco-2 model system after four hours was about three-fold higher than that of either ZnO-2 or ZnO-3. These results demonstrated that ZnO-1, with a 20 ㎚ mean particle size, had poorer viability and better permeability in Caco-2 cells than ZnO-2 and ZnO-3.

Abstract 2907: Protein Kinase D1 expression attenuates colon cancer progression

Abstract Objective: Protein Kinase D1 (PKD1) is an important modulator of several signal-transduction pathways in benign and malignant human diseases. Currently the role of PKD1 in colon cancer, which is the second-leading cause of cancer-related deaths, is not well established. We have found previously that advance stage prostate cancer has suppressed expression. This led us to analyze PKD1 expression patterns in various other cancer tissue samples including colon cancer. Materials and Methods: Tissue microarrays containing colon cancer samples with corresponding normal tissues were used to investigate the expression profile of PKD1 using immunohistochemistry. To determine PKD1 mediated effects on oncogenic β-catenin signaling in colon cancer, human colorectal adenocarcinoma cell line SW480. Stable transformants of SW480 were created with GFP and SW480-PKD1-GFP, PKD1 overexpression leads to attenuation of β-catenin/T cell factor (TCF) transcription activity measured by luciferase reporter assays. To determine the effect of PKD1 overexpression on other cancer associated genes, RT-PCR array (that had 84 cancer related genes) analysis was performed in SW480-GFP and SW480-PKD1-GFP cells. Results: Our expression analysis determined a significant decrease in PKD1 expression in advanced Dukes stage (II, III and IV) colon cancer samples as compared to non-neoplastic colon samples. We have also noticed downregulation and aberrant localization of β-catenin in colon cancer samples compared to non-neoplastic colon samples. Therefore, we also determined the function of PKD1 in oncogenic β-catenin signaling in colon cancer. Our confocal microscopy analyses demonstrates that PKD1 up-regulation caused translocation of β-catenin from the nucleus to the plasma membrane. PKD1 overexpression also reduced β-catenin/TCF interaction in the nucleus and suppressed the expression of its downstream signaling proteins. Phenotypic changes in the stable transformants were measured via proliferation assays, anchorage independent growth, anchorage dependent growth, cellular motility and invasion. The overexpression of PKD1 markedly suppressed cell proliferation in SW480 cells. The SW480-PKD1-GFP cells formed fewer colonies compared with SW480-GFP cells. The SW480-PKD-GFP cells showed very minimal or no motility and invasion compared to SW480-GFP cells. Our RT-PCR array data shows that PKD1 overexpression also modulates expression of tissue inhibitor of metalloproteinase 3 (TIMP3), interferon α, interferon β and tumor necrosis factor receptor (TNFR). Conclusion: In conclusion, PKD1 is down regulated in colon cancer and its downregulation is correlated with aberrant β-catenin subcellular localization. Our data suggest that the altered PKD1 expression may induce β-catenin dependent and independent signaling in colon cancer and suppression of PKD1 may play a critical role in colon carcinogenesis and colon cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2907. doi:10.1158/1538-7445.AM2011-2907

Abstract 848: Prevention of colitis-associated colorectal cancer with 8-hydroxydeoxyguanosine

Abstract Colitis-associated colorectal cancer (CAC) is one of clear examples of inflammation-carcinogenesis sequence, based on which strict control of colitis provides the right rationale of the intervention of anti-inflammatory drug on the chemoprevention. We have shown that 8-OHdG, which traditionally has been acknowledged as a marker of oxidative stress-related mutagenesis, could paradoxically exerted potent anti-inflammatory and anti-oxidant action in various inflammation models (Ock CY et al, Biochem Pharmacol, 2010). Based on the fact that Rac1 binds and activates STAT3 which is significantly up-regulated in inflammatory bowel disease (IBD) as well as CAC, we hypothesized that attenuation of Rac1-STAT pathway by exogenous 8-OHdG treatment may block inflammatory bouts in dextran sodium sulfate (DSS) induced colitis and can reduce carcinogenesis promoted by azoxymethane (AOM) followed by DSS ingestion. First we have checked whether exogenous 8-OHdG can ameliorate IBD, for which IL10 knock-out mice, IBD prone mice, are designed to ingest 5% DSS in tap water ad libitum for a week, and 8-OHdG (50, 100, or 150 mg/kg) is given intraperitoneal route daily. Treatment with 8-OHdG significantly reduced complication rates, pathologic grade of inflammation and edema compared with DSS only group. On pathogenic basis, the expressions of various inflammatory mediators such as TNF-α, IL-6, COX-2 and iNOS were decreased, as well as Rac1 activity and phosphorylation of STAT3 in 8-OHdG treated group in a dose-dependent manner. Next, in order to document the chemoprevention effects of 8-OHdG, mice were injected AOM (10 mg/kg) intraperitoneally followed by drinking 2.5% DSS for a week, after which AIN76 diet that contains 0.03 % and 0.06 % 8-OHdG was given for 20 weeks. As results, mice which consumed 8-OHdG-contained diet were significantly reduced tumor incidence and tumor multiplicity. Increased activity of Rac1 and phosphorylation of STAT3 with AOM plus DSS administration were significantly attenuated in 8-OHdG treatment group. These results were further validated with human colorectal adenocarcinoma cell line Caco-2 in vitro model. Conclusively, for the very first time, we clearly showed chemopreventive effect of exogenous 8-OHdG against CAC. Since 8-OHdG, one of well-known by-products generated by oxidative stress, paradoxically blocks inflammation-carcinogenesis sequence in colitis-associated cancer model, 8-OHdG could be a promising chemopreventive drug for the patient suffering from longstanding and extensive IBD, high risk group for CAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 848. doi:10.1158/1538-7445.AM2011-848

Efflux Pumps Modulation in Colorectal Adenocarcinoma Cell Lines: The Role of Nuclear Medicine

Introduction: Multidrug resistance (MDR) is one of the major problems of chemotherapy. Overexpression of efflux pumps, such as P-glycoprotein (Pgp), multiple resistance-related protein 1 (MRP-1) and lung resistance protein (LRP) can lead to MDR. Verapamil and L-buthionine-sulfoximine (BSO) are two modulators of these proteins. This study aims to compare 99mTc-Sestamibi transport kinetics in human colorectal adenocarcinoma cell lines, in the presence and absence of the MDR modulators verapamil and BSO. Material and Methods: MDR proteins expression was evaluated in sensitive (WiDr) and resistant (LS1034) human colorectal adenocarcinoma cell lines. Intracellular and plasma membrane Pgp and MRP1, and LRP expression was analyzed by flow-cytometry and western blot. Cellular transport kinetics was assessed using 99mTc-Sestamibi. MDR modulation was evaluated though retention studies in resistant cells after incubation with the modulators. Results: Pgp expression was significantly higher (p≤0.001) in resistant cells. These results were confirmed by western blot analysis. 99mTc-Sestamibi uptake and retention percentage were significantly higher (p99mTc-Sestamibi, there were differences among the MDR modulators used (p99mTc-Sestamibi could indicate MDR phenotype in colorectal adenocarcinoma cells. As the modulators used showed a reversion of the retention profile only for the first minutes, their administration should occur immediately before the administration of cytotoxic drugs.

Anti‐Cancer Effects of Protein Extracts from Calvatia lilacina, Pleurotus ostreatus and Volvariella volvacea

Calvatia lilacina (CL), Pleurotus ostreatus (PO) and Volvariella volvacea (VV) are widely distributed worldwide and commonly eaten as mushrooms. In this study, cell viabilities were evaluated for a human colorectal adenocarcinoma cell line (SW480 cells) and a human monocytic leukemia cell line (THP-1 cells). Apoptotic mechanisms induced by the protein extracts of PO and VV were evaluated for SW480 cells. The viabilities of THP-1 and SW480 cells decreased in a concentration-dependent manner after 24 h of treatment with the protein extracts of CL, PO or VV. Apoptosis analysis revealed that the percentage of SW480 cells in the SubG1 phase (a marker of apoptosis) was increased upon PO and VV protein-extract treatments, indicating that oligonucleosomal DNA fragmentation existed concomitantly with cellular death. The PO and VV protein extracts induced reactive oxygen species (ROS) production, glutathione (GSH) depletion and mitochondrial transmembrane potential (ΔΨm) loss in SW480 cells. Pretreatment with N-acetylcysteine, GSH or cyclosporine A partially prevented the apoptosis induced by PO protein extracts, but not that induced by VV extracts, in SW480 cells. The protein extracts of CL, PO and VV exhibited therapeutic efficacy against human colorectal adenocarcinoma cells and human monocytic leukemia cells. The PO protein extracts induced apoptosis in SW480 cells partially through ROS production, GSH depletion and mitochondrial dysfunction. Therefore, the protein extracts of these mushrooms could be considered an important source of new anti-cancer drugs.

MMP13 is a potential prognostic marker for colorectal cancer

Matrix metalloproteinase 13 (MMP13), a member of the matrix metalloproteinase family, is considered to play a role in the tumor cell proliferation and invasion. The purpose of this study was to verify the expression of MMP13 in colorectal cancer (CRC) in vitro and in vivo, and subsequently analyze whether the MMP13 expression levels correlate with the clinicopathological features and prognosis of CRC patients. We assessed MMP13 mRNA expression profile in human colorectal adenocarcinoma cell lines by quantitative RT-PCR, and further verified if it was a secreted protein or not by Western blot analysis of cell culture medium. By immunohistochemical staining the immunoreactivity of MMP13 showed that MMP13 was localized in the cytoplasm of CRC cells. MMP13 mRNA expression of 80 cancerous tissues collected from UICC stage I to III CRC patients were examined by membrane array. The correlations between MMP13 mRNA expression and patients' clinicopathological features were analyzed. MMP13 was confirmed to be a secreted protein by Western blot analysis. The larger tumor size (P<0.0001), advanced clinical stage (P=0.002), tumor invasive depth (P=0.039), lymph node metastasis (P=0.001) and post-operative relapse (P<0.0001) were significantly correlated with the MMP13 mRNA overexpression. Patients with MMP13 mRNA overexpression have a higher risk of postoperative relapse (P<0.0001; OR=7.989; 95% CI, 2.607-24.481). The results of the present study highly suggest that MMP13 is a secreted protein with a significant correlation to development of postoperative relapse; hence it could be a potential prognostic marker for CRC patients.

MDR modulation in colorectal adenocarcinoma cell lines: kinetic studies using 99mTc-sestamibi

Multidrug resistance (MDR) represents one of the major setbacks to chemotherapy and can occur due to the overexpression of efflux pumps as P-glycoprotein (Pgp), multiple resistance-related protein 1 (MRP1) and lung resistance-related protein (LRP). Verapamil, a substrate for Pgp, modulates its activity. L-buthionine-sulfoximine (BSO) can be used as blocker for MRP1. In this study we aim to compare transport kinetics for sensitive and resistant human colorectal adenocarcinoma cell lines, in the presence and absence of verapamil and BSO, through 99mTc-Sestamibi. Pgp, MRP1 and LRP expression was evaluated in resistant (LS1034) and sensitive (WiDr) human colorectal adenocarcinoma cell lines using flow-cytometry. Western blot was also used to analyze Pgp expression. Cellular transport kinetics was assessed in the presence and absence of verapamil and BSO. Retention studies were performed after cell incubation with those drugs, for different time intervals (10 and 60 minutes) and concentrations (10, 25, 50 and 100 µM), using 99mTc-Sestamibi. Pgp expression was significantly higher (p < 0.05) in resistant cells, although LRP was also expressed. Western blotting analysis confirmed flow-cytometry results. 99mTc-Sestamibi retention percentage was significantly higher (p < 0.05) in the resistant cell line for all time-points considered. In resistant cells incubated with MDR modulators there were statistically significant differences (p < 0.05) among the modulators used but only for the first minutes after incubation with the radiotracer. The data obtained suggest that these modulators should be used immediately before the cytotoxic drugs are administrated.

Lactoferrin inhibits E. coli O157:H7 growth and attachment to intestinal epithelial cells

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 strains are associated with haemorraghic colitis and haemolytic uremic syndrome (HUS) in humans. Cattle are a reservoir of E. coli O157:H7. We studied the ability of bovine and human lactoferrin, two natural antimicrobial proteins present in milk, to inhibit E. coli O157:H7 growth and attachment to a human epithelial colorectal adenocarcinoma cell line (Caco-2). The direct antibacterial effect of bLF on E. coli O157:H7 was stronger than that of hLF. Nevertheless, both lactoferrins had bacteriostatic effects even at high concentrations (10 mg/ml), suggesting blocking of LF activity by a yet undefined bacterial defence mechanism. Additionally, both lactoferrins significantly inhibited E. coli O157:H7 attachment to Caco-2 cells. However, hLF was more effective than bLF, probably due to more efficient binding of bLF to intelectin present on human enterocytes leading to uptake and thus removal of bLF from the extracellular environment. Inhibition of bacterial attachment to Caco-2 cells was at least partly due to the catalytic effect of lactoferrins on the type III secreted proteins EspA and EspB

Potential of Xanthones from Tropical Fruit Mangosteen as Anti-cancer Agents: Caspase-Dependent Apoptosis Induction In Vitro and in Mice

The pericarp of mangosteen (Garcinia mangostana L.) is rich in various xanthones that are known to possess unique biological activities. In this work, we characterized the anti-proliferative and cytotoxic activities of mangosteen xanthones both in vitro and in mice. In vitro analysis with a human colorectal adenocarcinoma cell line, COLO 205, showed that mangosteen xanthones not only inhibit the proliferation of target cells but also induce their death by apoptosis that involves the activation of the caspase cascade. In vivo analysis using a mouse subcutaneous tumor model with COLO 205 cells showed that, at relatively low doses, the growth of tumors was repressed upon intratumoral administration of mangosteen xanthones. When a higher dose of mangosteen xanthones was administered, the size of tumors was reduced gradually, and, in some mice, the disappearance of tumors was seen. Histopathological evaluation and biochemical analysis of tumors that received mangosteen xanthones indicate the induction of apoptosis in tumors, which resulted in the repression of their growth and the reduction of their sizes. These results demonstrate the potential of mangosteen xanthones to serve as anti-cancer agents for the chemotherapy of cancer.

Detection of early colorectal cancer imaged with peanut agglutinin-immobilized fluorescent nanospheres having surface poly( N-vinylacetamide) chains

Peanut agglutinin (PNA)-immobilized fluorescent nanospheres were designed as a novel imaging agent for colonoscopy. PNA is a targeting moiety that binds to β- d-galactosyl-(1–3)- N-acetyl- d-galactosamine, which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. The in vivo performance of the imaging agent was evaluated using a human colorectal cancer orthotopic animal model. Human colorectal adenocarcinoma cell lines, HT-29, HCT-116, and LS174T, were implanted on the cecal serosa of immune-deficient mice. A loop of the tumor-bearing cecum was made, and the luminal side was treated with the imaging agent. Strong fluorescence was observed at several sites of the cecal mucosa, irrespective of cancer cell type. Microscopic histological evaluation of the cecal mucosa revealed that bright areas with fluorescence derived from the imaging agent and dark areas without the fluorescence well denoted the presence and absence, respectively, of the invasion of implanted cancer cells on the mucosal side. This good correlation showed that PNA-immobilized fluorescent nanospheres recognized millimeter-sized tumors on the cecal mucosa with high affinity and specificity.

Ribozyme to TGF-β1 mRNA abrogates immunosuppressive effects of human colorectal adenocarcinoma HRT-18 cells in vitro and in vivo

Transforming growth factor-beta1 (TGF-beta1) is overexpressed in a variety of malignant epithelial tumors and was suggested to be a marker of colorectal cancer. Moreover, there is growing evidence that TGF-beta1 contributes to tumor progression by regulating tumor cell proliferation and differentiation, inducing a favorable tumor microenvironment, promoting migration and invasion, and suppressing macrophage cytotoxicity. Therefore, we stably transfected an anti-TGF-beta1 hammerhead ribozyme into the human colorectal adenocarcinoma cell line HRT-18. Expression of this ribozyme resulted in significant inhibition of TGF-beta1 expression on mRNA and protein level. This was associated with an enhanced tumor cell differentiation and a reduced tumor growth in vivo. The capability of tumor cells to suppress ROI production of co-cultivated human macrophages was abrogated in transfectants. Taken together, inhibition of TGF-beta1 in colorectal carcinoma cells might be an interesting therapeutic tool leading to reduced tumor cell growth and increased macrophage cytotoxicity. Thus, a gene-therapeutic approach using anti-TGF-beta1 ribozyme in combination with established anti-tumor agents is of great promise.

Semaphorin3F Down-Regulates the Expression of Integrin αvβ3 and Sensitizes Multicellular Tumor Spheroids to Chemotherapy via the Neuropilin-2 Receptor in vitro

Background: Multicellular resistance (MCR), i.e. decreased sensitivity to anticancer drugs compared with common monolayer cell (MC) cultures, depends partly on tumor cell-cell adhesion. Previous studies have shown that anti-adhesive therapies, including integrin α_v, β₁ and α_vβ₃ targeting, induced apoptosis and reversed the sensitivity of MCR. Methods: A model of three-dimensional cell culture was used to establish HT29 multicellular spheroid cells (MCS) and explore the effect of semaphorin3F (Sema3F) on integrin-mediated cell-cell interactions in MCS of a human colorectal adenocarcinoma cell line (HT29) and sensitization of HT29 MCS to 5-fluorouracil and oxaliplatin via a decrease in integrin α_vβ₃. Results: Elevated expression of Sema3F led to the up-regulation neuropilin-2 (Nrp2) receptor expression and the down-regulation of integrin α_vβ₃ expression. Furthermore, short interfering RNA of Nrp2 could reverse MCR. Conclusion: Our study demonstrates that Sema3F can sensitize MCR by decreasing integrin α_vβ₃expression via the Nrp2 receptor.