LS174T Cells Research Articles

Ascl2 knockdown results in tumor growth arrest by miRNA-302b-related inhibition of colon cancer progenitor cells.

BackgroundAchaete scute-like 2 (Ascl2), a basic helix-loop-helix (bHLH) transcription factor, controls the fate of intestinal stem cells. However, the role of Ascl2 in colon cancer progenitor cells remains unknown. The cell line HT-29 (47.5–95% of CD133+ population) and LS174T (0.45% of CD133+ population) were chosen for functional evaluation of Ascl2 in colon cancer progenitor cells after gene knockdown by RNA interference.Methodology/Principal FindingsImmunohistochemistry demonstrated that Ascl2 was significantly increased in colorectal adenocarcinomas. Downregulation of Ascl2 using RNA interference in cultured colonic adenocarcinoma HT-29 and LS174T cells reduced cellular proliferation, colony-forming ability, invasion and migration in vitro, and resulted in the growth arrest of tumor xenografts in vivo. The Ascl2 protein level in CD133+ HT-29 cells was significantly higher than in CD133− HT-29 cells. Ascl2 blockade via shRNA interference in HT-29 cells (shRNA-Ascl2/HT-29 cells) resulted in 26.2% of cells staining CD133+ compared with 54.7% in control shRNA-Ctr/HT-29 cells. The levels of ‘stemness’ associated genes, such as CD133, Sox2, Oct4, Lgr5, Bmi1, and C-myc, were significantly decreased in shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells in vitro as well as in the corresponding tumor xenograft (CD133 was not performed in shRNA-Ascl2/LS174T cells). The shRNA-Ascl2/HT-29 cells had inhibited abilities to form tumorspheres compared with control. The microRNA (miRNAs) microarrays, identified 26 up-regulated miRNAs and 58 down-regulated miRNAs in shRNA-Ascl2/HT-29 cells. Expression levels of let-7b, miRNA-124, miRNA-125b, miRNA-17, miRNA-20a and miRNA-302b, involved in the regulation of ‘stemness’, were quantified with qPCR, which confirmed their identities. Restoration of miRNA-302b, via its mimic, led to the restoration of shRNA-Ascl2/HT-29 ‘stemness’ characteristics, including tumorsphere formation and ‘stemness’ associated genes levels, and the recovery of cellular behaviors, including colony-forming ability, invasion and migration in vitro.Conclusions/SignificanceAscl2 may be a potential target for the inhibition of colon cancer progenitor cells, and functions through a miR-302b-related mechanism.

Indomethacin antagonizes EP2 prostanoid receptor activation in LS174T human colon cancer cells

Increases in the level of cyclooxygenase (COX)-2 and prostanoids such as prostaglandin E2 (PGE2) are considered biomarkers of colorectal cancer. Therefore, non-steroidal anti-inflammatory drugs (NSAID) have been used to reduce the risk of cancer development by reducing prostanoid biosynthesis as COX inhibitors. Along with their activity as COX inhibitors, NSAID have been reported to have other effects. One major NSAID, indomethacin, has been shown to have several effects independent of COX inhibition. To further examine the COX-inhibition-independent effects of indomethacin on colorectal cancer, we used human colon cancer LS174T cells, known to have express little COX-2 and have no detectable PGE2 production. Here we show that indomethacin has a potential antagonizing effect on human EP2 receptors. We believe this study raises the reasons to use indomethacin as a lead-compound for setting up another EP2 receptor-specific antagonist as a relatively cost-efficient strategy for anti-cancer medication in the future.

Chronic Anti-inflammatory Drug Therapy Inhibits Gel-Forming Mucin Production in a Murine Xenograft Model of Human Pseudomyxoma Peritonei

Intraperitoneal accumulation of mucinous ascites in pseudomyxoma peritonei (PMP) promotes an inflammatory/fibrotic reaction that progresses to bowel obstruction and eventual patient demise. Cytokines and inflammation-associated transcription factor binding sites, such as glucocorticoid response elements and COX-2, regulate secretory mucin, specifically MUC2, production. We hypothesized that anti-inflammatory drugs targeting inflammation-associated pathways may reduce mucin production and subsequent disease morbidity in PMP. The effects of dexamethasone and Celebrex were assessed in mucin-secreting human colon cancer LS174T cells in vitro and murine xenograft models of LS174T and human appendiceal PMP in vivo by serial parametric measurements, MUC2 transcripts via real-time RT-PCR, and MUC2 protein expression via immunofluorescence assays. Dexamethasone significantly inhibited basal MUC2 mRNA levels in LS174T cells, inhibited mucinous tumor accumulation in an intraperitoneal PMP xenograft model, and prolonged survival in a subcutaneous LS174T xenograft model. Celebrex significantly inhibited sodium butyrate-stimulated MUC2 mRNA levels in LS174T cells and demonstrated a statistically nonsignificant trend toward reduced mucinous tumor growth and prolonged survival in the xenograft models. MUC2 protein analysis by immunofluorescence demonstrated a dual effect of dexamethasone on mucin production and tumor cell count. Inflammatory mediators are known to regulate mucin production and may promote overexpression of MUC2 by neoplastic cells with goblet cell phenotype in PMP. Anti-inflammatory drugs, dexamethasone and Celebrex, could inhibit extracellular mucin production in PMP by targeting inflammatory cascades and, therefore, may decrease compressive symptoms, increase the disease-free interval, and reduce the extent or frequency of morbid cytoreductive surgeries.

Regulation of the Potential Marker for Intestinal Cells, Bmi1, by β-Catenin and the Zinc Finger Protein KLF4

B lymphoma Mo-MLV insertion region 1 (Bmi1) is a Polycomb Group (PcG) protein important in gene silencing. It is a component of Polycomb Repressive Complex 1 (PRC1), which is required to maintain the transcriptionally repressive state of many genes. Bmi1 was initially identified as an oncogene that regulates cell proliferation and transformation, and is important in hematopoiesis and the development of nervous systems. Recently, it was reported that Bmi1 is a potential marker for intestinal stem cells. Because Wnt signaling plays a key role in intestinal stem cells, we analyzed the effects of Wnt signaling on Bmi1 expression. We found that Wnt signaling indeed regulates the expression of Bmi1 in colon cancer cells. In addition, the expression of Bmi1 in human colon cancers is significantly associated with nuclear β-catenin, a hallmark for the activated Wnt signaling. Krüppel-like factor 4 (KLF4) is a zinc finger protein highly expressed in the gut and skin. We recently found that KLF4 cross-talks with Wnt/β-catenin in regulating intestinal homeostasis. We demonstrated that KLF4 directly inhibits the expression of Bmi1 in colon cancer cells. We also found that Bmi1 regulates histone ubiquitination and is required for colon cancer proliferation in vitro and in vivo. Our findings further suggest that Bmi1 is an attractive target for cancer therapeutics.

Plasma Half-life Extension of Small Recombinant Antibodies by Fusion to Immunoglobulin-binding Domains

Many therapeutic proteins possessing a small size are rapidly cleared from circulation. Half-life extension strategies have therefore become increasingly important to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Here, we performed a comparative analysis of the half-life extension properties of various bacterial immunoglobulin-binding domains (IgBDs) derived from Staphylococcus protein A (SpA), Streptococcus protein G (SpG), and Finegoldia (formerly Peptostreptococcus) protein L (PpL). These domains, composed of 50-60 amino acid residues, were fused to the C terminus of a single-chain Fv and a bispecific single-chain diabody, respectively. All fusion proteins were produced in mammalian cells and retained their antigen-binding properties. The half-lives of the antibody molecules were prolonged to varying extents for the different IgBDs. The strongest effects in mice were observed for domain C3 of SpG (SpG(C3)) followed by domains B and D of SpA, suggesting that SpG(C3) is particularly useful to extend the plasma half-life of small proteins.

MicroRNA-101 (miR-101) post-transcriptionally regulates the expression of EP4 receptor in colon cancers

Purpose: Expression of the PGE2 receptor, EP4, is up-regulated during colorectal carcinogenesis. However the mechanism leading to deregulation of the EP4 receptor is not known. The present study was conducted to investigate the regulation of EP4 receptor by miRNAs. Experimental Design: We analyzed 26 colon cancers (i.e. 15 adenocarcinomas and 9 adenomas) and 16 normal colon specimens for EP4 receptor expression by immunohistochemistry. A bioinformatics approached identified putative microRNA binding sites with the 3'-UTR of the EP4 receptor. Both colon cancer cell lines and tumor specimens were analyzed for miR-101 and EP4 expression by qRT-PCR and Western analysis respectively and simultaneously in situ hybridizations was used to confirm our results. In vitro and in vivo assays were used to confirm our clinical findings. Results: We observed an inverse correlation between the levels of miR-101 and EP4 receptor protein. Transfection of LS174T cells with miR-101 significantly suppressed a luciferase reporter containing the EP4 receptor-3'-UTR. In contrast, a mutant EP4 receptor-3'-UTR construct was unaffected. Ectopic expression of miR-101 markedly reduced cell proliferation and motility. Co-transfection of EP4 receptor could rescue colon cancer cells from the tumor suppressive effects of miR-101. Moreover, the pharmacologic inhibition of EP4 receptor signaling or silencing of EP4 receptor phenocopied the effect of miR-101. This is the first study to show that the EP4 receptor is negatively regulated by miR-101. Conclusions: These data provide new insights in the modulation of EP-4 receptor expression at the post-transcriptional level by miR-101 and suggests therapeutic strategies against miR-101 targets may be warranted.

Fucoxanthin Attenuates Rifampin-Induced Cytochrome P450 3A4 (CYP3A4) and Multiple Drug Resistance 1 (MDR1) Gene Expression Through Pregnane X Receptor (PXR)-Mediated Pathways in Human Hepatoma HepG2 and Colon Adenocarcinoma LS174T Cells

Pregnane X receptor (PXR) has been reported to regulate the expression of drug-metabolizing enzymes, such as the cytochrome P450 3A (CYP3A) family and transporters, such as multiple drug resistance 1 (MDR1). Fucoxanthin, the major carotenoid in brown sea algae, is a putative chemopreventive agent. In this study, we determined whether fucoxanthin could overcome drug resistance through attenuation of rifampin-induced CYP3A4 and MDR1 gene expression by PXR-mediated pathways in HepG2 hepatoma cells. We found that fucoxanthin (1–10 μM) significantly attenuated rifampin (20 μM)-induced CYP3A4, MDR1 mRNA and CYP3A4 protein expression at 24 h of incubation. Mechanistically, fucoxanthin strongly attenuated the PXR-mediated CYP3A4 promoter activity in HepG2 cells. In addition, fucoxanthin attenuated constitutive androstane receptor (CAR)- and rPXR-mediated CYP3A4 promoter activity in this cell line. Using the mammalian two-hybrid assay, we found that fucoxanthin significantly decreased the interaction between PXR and SRC-1, a PXR co-activator. Thus, fucoxanthin can decrease rifampin-induced CYP3A4 and MDR1 expression through attenuation of PXR-mediated CYP3A4 promoter activation and interaction between PXR and co-activator. These findings could lead to potentially important new therapeutic and dietary approaches to reduce the frequency of adverse drug reactions.

The histone deacetylase inhibitor trichostatin A induces cell cycle arrest and rapid upregulation of gadd45β in LS174T human colon cancer cells

Histone deacetylase (HDAC) inhibitors are considered as promising therapeutic agents against several malignant diseases because they inhibit cancer cell proliferation. The stress sensor genes of the growth arrest and DNA damage-inducible protein (gadd45) family exhibit disordered expression in several types of malignant diseases and are thus a novel target for cancer therapy. However, there have been only few investigations of whether HDAC inhibitors affect the expression of gadd45 genes. We examined the effects of a HDAC inhibitor, trichostatin A (TSA), on the time-dependent expression of gadd45 genes in the human colon cancer cell line LS174T. Addition of TSA to LS174T cells induced inhibition of cell proliferation by arresting the cell cycle. We found that TSA treatment of LS174T cells induced rapid upregulation of gadd45β mRNA expression within 15 min, reaching a peak level at 3 h. Although the time-dependent expression pattern of gadd45β mRNA was similar to that of gadd45β mRNA, the peak level of gadd45β was lower than that of gadd45β. TSA treatment also upregulated the mRNA level of p21Waf1/Cip1, a prolif- eration inhibitor, after 3 h, but downregulated the mRNA levels of cyclin D1, a proliferation inducer, after 3 h, and of c-Myc after 1 h. TSA treatment induced a certain level of apoptosis, but the mRNA level of p53, a potent apoptosis inducer, was down-regulated after 3 h. These results suggest that the up-regulation of p21Waf1/Cip1 and apoptosis was independent of p53 and that the early upregulation of gadd45β gene, which precedes the upregulation of p21Waf1/Cip1 and the downregulation of cyclin D1, are important in TSA-treated LS174T cells.

Effects of two monoclonal antibodies, MLS128 against Tn-antigen and 1H7 against insulin-like growth factor-I receptor, on the growth of colon cancer cells

MLS128 is an anti-carbohydrate monoclonal antibody (mAb) that binds three or two consecutive Tn-antigens. MLS128 bound 110-210 kDa glycoproteins (GPs) and inhibited the growth of LS180 and HT29 colon and MCF-7 breast cancer cells. One possible mechanism of MLS128's inhibition of growth may be via insulin-like growth factor-I receptor (IGF-IR) down-regulation (Morita et al. BioScience Trends. 2009; 3:32-37). The current study examined the role of IGF-IR signaling in the growth of colon cancer cells and its possible interaction with MLS128-induced inhibition of cell growth in LS180, LS174T, and HT29 human colon cancer cells treated with MLS128 or anti-IGF-IR 1H7. Both MLS128 and 1H7 treatment significantly inhibited the growth of colon cancer cells. All three colon cancer cell lines expressed IGF-IR. Their growth was in part IGF-I dependent, but inhibition by MLS128 was independent of IGF-IR signaling. All of the colon cancer cell lines expressed an 110kDa GP for MLS128 binding, but MCF-7 cells expressed MLS128-detectable bands with higher molecular masses. 1H7 treatments caused down-regulation of IGF-IR but did not affect 110kDa GP levels. MLS128 treatments resulted in partial disappearance of the 110kDa band but did not affect IGF-IR levels. Western blotting analyses of colon and breast cancer cell lysates revealed that colon and breast cancer cells differed significantly in patterns of expression of growth-related molecules while colon cancer cells were similar but distinctive. In conclusion, MLS128 inhibited the growth of colon cancer cells by binding to the 110kDa GP receptor. Inhibition of growth by MLS128 did not appear to affect IGF-IR signaling and instead only affected other growth signaling pathways.

Anti-CEA loaded maghemite nanoparticles as a theragnostic device for colorectal cancer

Nanosized maghemite particles were synthesized, precoated (with dimercaptosuccinic acid) and surface-functionalized with anticarcinoembryonic antigen (anti-CEA) and successfully used to target cell lines expressing the CEA, characteristic of colorectal cancer (CRC) cells. The as-developed nanosized material device, consisting of surface decorated maghemite nanoparticles suspended as a biocompatible magnetic fluid (MF) sample, labeled MF-anti-CEA, was characterized and tested against two cell lines: a high-CEA expressing cell line (LS174T) and a low-CEA expressing cell line (HCT116). Whereas X-ray diffraction was used to assess the average core size of the as-synthesized maghemite particles (average 8.3 nm in diameter), dynamic light scattering and electrophoretic mobility measurements were used to obtain the average hydrodynamic diameter (550 nm) and the zeta-potential (−38 mV) of the as-prepared and maghemite-based nanosized device, respectively. Additionally, surface-enhanced Raman spectroscopy (SERS) was used to track the surface decoration of the nanosized maghemite particles from the very first precoating up to the attachment of the anti-CEA moiety. The Raman peak at 1655 cm−1, absent in the free anti-CEA spectrum, is the signature of the anti-CEA binding onto the precoated magnetic nanoparticles. Whereas MTT assay was used to confirm the low cell toxicity of the MF-anti-CEA device, ELISA and Prussian blue iron staining tests performed with both cell lines (LS174T and HCT116) confirm that the as-prepared MF-anti- CEA is highly specific for CEA-expressing cells. Finally, transmission electron microscopy analyses show that the association with anti-CEA seems to increase the number of LS174T cells with internalized maghemite nanoparticles, whereas no such increase seems to occur in the HCT116 cell line. In conclusion, the MF-anti-CEA sample is a biocompatible device that can specifically target CEA, suggesting its potential use as a theragnostic tool for CEA-expressing tumors, micrometastasis, and cancer-circulating cells.

Delta-like ligand 4-notch blockade and tumor radiation response.

The microenvironment plays an important role in regulating tumor response to radiotherapy. Ionizing radiation can disrupt tumor vasculature, and Notch pathway inhibition can interfere with functional angiogenesis. We explored the potential cooperativity between Notch inhibition and ionizing radiation in delaying tumor growth. Human colorectal carcinoma LS174T cells, which express the Notch ligand delta-like ligand 4 (DLL4), and human head and neck cancer FaDu cells, which do not, were grown as subcutaneous xenografts in nude mice. The mice were treated with dibenzazepine (DBZ), a γ-secretase inhibitor that blocks all Notch signaling, or a DLL4-specific blocking monoclonal antibody, alone or in combination with ionizing radiation (n = 5-10 mice per group), and response was assessed by tumor growth delay. Microbubble contrast Doppler ultrasound was used to measure tumor blood flow. Tumor Notch activity was monitored by in vivo bioluminescence from a Notch luciferase reporter. Vessel density was assessed using Chalkley vessel counting. All statistical tests were two-sided. In LS174T xenografts, the average time for tumor volumes to reach four times the starting volume was longer for mice treated with the DLL4 monoclonal antibody than for mice treated with DBZ (16.4 vs 9.5 days, difference = 6.9 days, 95% confidence interval [CI] = 3.7 to 10.1 days, P < .001). Both Notch inhibitors suppressed tumor Notch activity within 24 hours of administration compared with vehicle (change in luciferase activity, vehicle vs DBZ: 103% vs 28%, difference = 75%, 95% CI = 39% to 109%, P = .002; vehicle vs DLL4 antibody: 172% vs 26%, difference = 146%, 95% CI = 86% to 205%, P < .001). Administration of the DLL4 antibody or DBZ after ionizing radiation resulted in a supra-additive growth delay compared with vehicle (vehicle vs DLL4 antibody + ionizing radiation: 6.8 vs 44.3 days, difference = 37.5 days, 95% CI = 32 to 43 days, P < .001; vehicle vs DBZ + ionizing radiation: 7.1 vs 24.4 days, difference = 17.3 days, 95% CI = 15.9 to 18.6 days, P < .001). Treatment of mice with the DLL4 antibody alone or in combination with ionizing radiation increased tumor vessel density but reduced tumor blood flow. Combination therapy with DLL4 antibody and ionizing radiation resulted in extensive tumor necrosis in LS174T xenografts and enhanced tumor growth delay in FaDu xenografts. The combination of specific DLL4-Notch blockade and ionizing radiation impairs tumor growth by promoting nonfunctional tumor angiogenesis and extensive tumor necrosis, independent of tumor DLL4 expression.

Inflammatory cues modulate the expression of secretory product genes, Golgi sulfotransferases and sulfomucin production in LS174T cells

The signals that mediate goblet cell expression of specific mucin chemotypes are poorly defined. Animal and in vitro studies show that acidomucin chemotypes may be altered by inflammation and changes in intestinal microbiota. To examine factors that may elicit this response, human adenocarcinoma-derived LS174T cells, which have a goblet cell-like phenotype and produce both sulfo- and sialomucins, were used to examine the effects of selected microbial and host factors on expression of goblet cell secretory product genes, sulfotransferases and sulfomucin production. Expression of genes encoding mucin 2 (MUC2), resistin-like molecule β (RETNLB), and trefoil factor 3 (TFF3) and Golgi sulfotransferases, carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 5 (CHST5) and galactose-3-O-sulfotransferase 2 (GAL3ST2), was measured by quantitative reverse transcriptase-polymerase chain reaction following treatment with bacterial flagellin, tumor necrosis factor α (TNF-α) or the mucogenic cytokine interleukin-13 (IL-13). Expression of the toll-like receptor 5 (TLR5) gene was also analysed. Sulfomucin expression was examined via high-iron diamide/alcian blue (HID/AB) histochemistry and immunofluorescent staining for the Sulfo Le(a) antigen, which is synthesized in part by GAL3ST2. Flagellin, IL-13 and TNF-α all significantly increased GAL3ST2, MUC2, TFF3 and TLR5 expression, while only IL-13 increased RETNLB and CHST5 expression. Based on HID/AB histochemistry, mucin sulfation was significantly increased in response to both flagellin and IL-13 but not TNF-α. Only treatment with flagellin increased the expression of the Sulfo Le(a) antigen. Collectively, these results indicate that bacterial flagellin, IL-13 and TNF-α differentially modulate the expression of goblet cell secretory product genes, sulfotransferases and sulfomucin production.

Olfactomedin-4 is a glycoprotein secreted into mucus in active IBD

BackgroundOlfactomedin-4 (OLFM4) is a glycoprotein characteristic of intestinal stem cells and apparently involved in mucosal defense of the stomach and colon. Here we studied its expression, regulation and function in IBD. MethodsThe expression of OLFM4, mucins Muc1 and Muc2, the goblet cell differentiation factor Hath1 and the proinflammatory cytokine IL-8 was measured in inflamed or noninflamed colon in IBD patients and controls. OLFM4 protein was located by immunohistochemistry, quantified by Dot Blot and its binding capacity to defensins HBD1-3 was investigated. The influence of bacteria with or without the Notch blocker dibenzazepine (DBZ) and of several cytokines on OLFM4 expression was determined in LS174T cells. ResultsOLFM4 mRNA and protein were significantly upregulated in inflamed CD (4.3 and 1.7-fold) and even more pronounced in UC (24.8 and 3.7-fold). OLFM4 expression was correlated to IL-8 but not to Hath1. In controls immunostaining was restricted to the lower crypts but in inflamed IBD it expanded up to the epithelial surface including the mucus. OLFM4 bound to HBD1-3 without profoundly inactivating these defensins. In LS174T-cells OLFM4 mRNA was significantly augmented after incubation with Escherichia coli K12, Escherichia coli Nissle and Bacteroides vulgatus. DBZ downregulated OLFM4 expression and blocked bacterial induction whereas IL-22 but not TNF-α was stimulatory. ConclusionsOLFM4 is overexpressed in active IBD and secreted into mucus. The induction is triggered by bacteria through the Notch pathway and also by the cytokine IL-22. OLFM4 seems to be of functional relevance in IBD as a mucus component, possibly by binding defensins.

Hypoxia inducible factor 1α expression in colorectal cancer and its correlation with Survivin

Objective To investigate the correlation between the expression levels of hypoxia inducible factor-1α (HIF-1α) and Survivin in human the colorectal carcinomas (CRCs).Methods Plasmid expressing small interfering RNA (siRNA) targeting HIF-1 α mRNA (pSilence-2.1-U6-siRNA ) was constructed and transfected into LS174T cells by Lipofectamine methods.The effect of HIF-1α on the expression of survivin by siRNA targeting HIF-1α mRNA was studied.The expression of survivin and HIF-1α was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.The expression of HIF-1 α and Survivin was investigated by immunohistochemistry in 69 patients with CRCs.Results The successful construction of pSilence-2.1-U6-siRNA plasmid was confirmed by sequencing analysis.HIF-1 α and Survivin expression levels were markedly down-regulated after the siRNA expression vector targeting HIF-1α was transfected into the LS174T cells.The positive rate of HIF-1α protein in CRCs was significantly higher than that in colorectal adenoma lesions ( P ＜ 0.05 ).There was significant difference in the expression of HIF-1α protein between stage Ⅲ and stage Ⅰ-Ⅱ CRCs (P ＜0.01 ).The level of HIF-1αwas positively correlated with that of Survivin protein ( P ＜ 0.05 ) during colorectal tumor progression.Conclusion There is a positive correlation between the expression levels of HIF-1αt and Survivin in tumor progression of human CRCs. Key words: Hypoxia inducible factor-1 α; Survivin; Small interfering RNA; Colorectal carcinoma

Nuclear Factor of Activated T Cells (NFAT) Proteins Repress Canonical Wnt Signaling via Its Interaction with Dishevelled (Dvl) Protein and Participate in Regulating Neural Progenitor Cell Proliferation and Differentiation

The Ca(2+) signaling pathway appears to regulate the processes of the early development through its antagonism of canonical Wnt/β-catenin signaling pathway. However, the underlying mechanism is still poorly understood. Here, we show that nuclear factor of activated T cells (NFAT), a component of Ca(2+) signaling, interacts directly with Dishevelled (Dvl) in a Ca(2+)-dependent manner. A dominant negative form of NFAT rescued the inhibition of the Wnt/β-catenin pathway triggered by the Ca(2+) signal. NFAT functioned downstream of β-catenin without interfering with its stability, but influencing the interaction of β-catenin with Dvl by its competitively binding to Dvl. Furthermore, we demonstrate that NFAT is a regulator in the proliferation and differentiation of neural progenitor cells by modulating canonical Wnt/β-catenin signaling pathway in the neural tube of chick embryo. Our findings suggest that NFAT negatively regulates canonical Wnt/β-catenin signaling by binding to Dvl, thereby participating in vertebrate neurogenesis.

Comparing two TAG-72 binding peptides previously identified by phage display as potential imaging agents

To evaluate the targeting property in vitro and in vivo of two tumor-associated glycoprotein 72 (TAG-72) binding peptides, previously identified in this laboratory by phage selection using different elution conditions. The peptides GGVSCMQTSPVCENNL (A2-6) and NPGTCKDKWEICLLNGG (A3-10) were radiolabeled with technetium-99m ((99m)Tc) using N-hydroxysuccinimidyl-S-acetyl-mercaptoacetyltriglycine (NHS-MAG(3)) as a chelator or were biotinylated. The specificity of the two peptides for the TAG-72 positive LS-174T cancer cells was demonstrated in vitro both by flow cytometry analysis using the biotinylated peptides and by competitive binding using the (99m)Tc-labeled peptides. The in-vivo biodistributions of the peptides were evaluated in TAG-72 positive LS-174T tumor-bearing mice by small-animal single photon emission computed tomography/computed tomography imaging. As evidence of specific binding, both peptides showed a significant increase in percentage binding with increasing peptide concentration by flow cytometry analysis to LS-174T cells, but not to TAG-72 negative HT-29 cells. The (99m)Tc-labeled A2-6 peptide bound LS-174T cells with an inhibition constant at 50% of 46.5 nmol/l compared with 420 nmol/l for the A3-10 peptide. In mice, accumulation of both peptides was highest in kidneys and gallbladder. Tumors were clearly visible by single photon emission computed tomography imaging for both (99m)Tc-labeled peptides through 60 min, although the tumor accumulation was higher for the A3-10 peptide. The A3-10 peptide, with lower, yet reasonable binding affinity compared with the A2-6 peptide, showed sufficiently favorable specific binding and tumor accumulation to be considered further as a potential imaging agent for TAG-72 positive cancers.

Suppression of mucin 2 enhances the proliferation and invasion of LS174T human colorectal cancer cells

Altered expression of MUC2 (mucin 2) is related to tumour development in colorectal cancer. Colorectal mucinous carcinomas are positive for MUC2 expression, whereas MUC2 is down-regulated in non-mucinous adenocarcinomas. In the present study, we down-regulated MUC2 expression by RNAi (RNA interference) and investigated the in vitro and in vivo effects on the proliferation and invasion/migration potential of the LS174T human colorectal cancer cells. The LS174T cell line is a goblet-cell-like colorectal cancer cell line that continuously produces high levels of MUC2. Inhibition of MUC2 expression in vitro by transfection of LS174T cells with the recombinant plasmid pcDNA6.2-GW/EmGFP-miR-MUC2 led to the production of a stably transfected MUC2-RNAi LS174T cell line. The proliferation and invasion/migration of MUC2-RNAi cells in vitro were significantly higher than those in control cells, as assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], colony formation and transwell assays. Subcutaneous injection of MUC2-RNAi LS174T cells into nude mice resulted in the development of subcutaneous tumours visible to the naked eye after 1 week. The growth rate of tumours derived from MUC2-RNAi LS174T cells was greater than that of tumours derived from control cells. Ki67 and matrix metalloproteinase-9 proteins were detected by immunohistochemistry in the xenografts. The expression levels of these proteins were higher in the MUC2-RNAi-derived xenografts than in xenografts derived from control cells. Although the role of MUC2 in colorectal tumorigenesis is not fully understood, these results strongly suggest a relationship between the proliferation and invasion of LS174T cells and the expression of MUC2.

Pretargeted radioimmunotherapy of colorectal cancer metastases: models and pharmacokinetics predict influence of the physical and radiochemical properties of the radionuclide

We investigated influences of pretargeting variables, tumor location, and radionuclides in pretargeted radioimmunotherapy (PRIT) as well as estimated tumor absorbed doses. LS-174T human colonic carcinoma cells expressing carcinoembryonic antigen (CEA) were inoculated in nude mice. Biodistribution of a bispecific anti-CEA x anti-hapten antibody, TF2, and of a TF2-pretargeted peptide was assessed and a multi-compartment pharmacokinetic model was devised. Tissue absorbed doses were calculated for (131)I, (177)Lu, (90)Y, (211)At, and (213)Bi using realistic specific activities. Under conditions optimized for tumor imaging (10:1 TF2 to peptide molar ratio, interval time 15-24 h), tumor uptake reached ∼9 ID/g in subcutaneous tumors at 2 h with very low accretion in normal tissues (tumor to blood ratio >20:1 after 2 h). For a low dose of peptide (0.04 nmol), (211)At is predicted to deliver a high absorbed dose to tumors [41.5 Gy considering a relative biologic effect (RBE) of 5], kidneys being dose-limiting. (90)Y and (213)Bi would also deliver high absorbed doses to tumor (18.6 for (90)Y and 26.5 Gy for (213)Bi, taking RBE into account, for 0.1 nmol) and acceptable absorbed doses to kidneys. With hepatic metastases, a twofold higher tumor absorbed dose is expected. Owing to the low activities measured in blood, the bone marrow absorbed dose is expected to be without significant toxicity. Pretargeting achieves high tumor uptake and higher tumor to background ratios compared to direct RIT. Short-lived radionuclides are predicted to deliver high tumor absorbed doses especially (211)At, with kidneys being the dose-limiting organ. (177)Lu and (131)I should be considered for repeated injections.

The targeted gene (KDRP-CD/TK) therapy of breast cancer mediated by SonoVue and ultrasound irradiation in vitro

Suicide gene therapy has become an effective therapy for breast cancer, and ultrasound targeted microbubble destruction (UTMD) has become a popular topic in the gene therapy field. In this study, MCF-7 cells with the KDR promoter and LSl74T cells without the KDR promoter were transfected with the recombinant plasmid pEGFP-KDRP-CD/TK using UTMD. The recombinant plasmid pEGFP-KDRP-CD/TK was transfected into MCF-7 and LS174T cells successfully with no significant difference in transfection efficiency ( p > 0.05). By RT-PCR, the CD/TK fusion gene was shown to be expressed in MCF-7 cells but not expressed in LS174T cells. In a cytotoxicity experiment, transgenic MCF-7 cells were sensitive to the prodrugs 5-FC and GCV. When both 5-FC and GCV were administered, the rate of cellular inhibition was significantly greater than that achieved when only one of the prodrugs was administered ( p < 0.001). Moreover, the inhibition rates achieved administering 5-FC, GCV and both 5-FC and GCV were all significantly greater than the gene transfection rate of 21.92 ± 3.64% ( p < 0.001). However, transgenic LS174T cells were not sensitive to any prodrug. These results demonstrated that UTMD is a safe, effective and targeted gene delivery system. Also, the KDR promoter can drive expression of the CD/TK double suicide gene target in MCF-7 cells, and the targeted killing effect of the KDRP-CD/TK gene on MCF-7 cells in vitro has good synergy with expression of the CD/TK fusion gene.

The proto-oncogene Pim-1 is a target of miR-33a

The constitutively active serine/threonine kinase Pim-1 is upregulated in different cancer types, mainly based on the action of several interleukines and growth factors at the transcriptional level. So far, a regulation of oncogenic Pim-1 by microRNAs (miRNAs) has not been reported. Here, we newly establish miR-33a as a miRNA with potential tumor suppressor activity, acting through inhibition of Pim-1. A screen for miRNA expression in K562 lymphoma, LS174T colon carcinoma and several other cell lines revealed generally low endogenous miR-33a levels relative to other miRNAs. Transfection of K562 and LS174T cells with a miR-33a mimic reduced Pim-1 levels substantially. In contrast, the cell-cycle regulator cyclin-dependent kinase 6 predicted to be a conserved miR-33a target, was not downregulated by the miR-33a mimic. Seed mutagenesis of the Pim-1 3'-untranslated region in a luciferase reporter construct and in a Pim-1 cDNA expressed in Pim-1-deficient Skov-3 cells demonstrated specific and direct downregulation of Pim-1 by the miR-33a mimic. The persistence of this effect was comparable to that of a small interfering RNA-mediated knockdown of Pim-1, resulting in decelerated cell proliferation. In conclusion, we demonstrate the potential of miR-33a to act as a tumor suppressor miRNA, which suggests miR-33a replacement therapy through delivery of miR mimics as a novel therapeutic strategy.