LS174T Human Colon Carcinoma Cells Research Articles

Site Specific Discrete PEGylation of 124I-Labeled mCC49 Fab′ Fragments Improves Tumor MicroPET/CT Imaging in Mice

The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2-4 kDa discrete, branched PEGylation reagents on mCC49 Fab' (MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab' (Fab'-NEM) was conjugated at a hinge region cysteine with maleimide-dPEG 12-(dPEG24COOH)3 acid (Mal-dPEG-A), maleimide-dPEG12-(dPEG12COOH)3 acid (Mal-dPEG-B), or maleimide-dPEG12-(m-dPEG24)3 (Mal-dPEG-C), and then radiolabeled with iodine-124 ((124)I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab' with Mal-dPEG-A (Fab'-A) reduced the binding affinity of the non PEGylated Fab' by 30%; however, in vivo, Fab'-A significantly lengthened the blood retention vs Fab'-NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p < 0.01), showed excellent tumor to background, better microPET/CT images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 ± 0.83 vs 3.83 ± 1.50%ID/g, p < 0.05). Despite the strong similarity of the three PEGylation reagents, PEGylation with Mal-dPEG-B or -C reduced the in vitro binding affinity of Fab'-NEM by 70%, blood retention, microPET/CT imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 ± 1.50 vs 1.97 ± 0.29%ID/g, p < 0.05) and 63% (3.83 ± 1.50 vs 1.42 ± 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A.

Treatment of Colorectal Cancer Using a Combination of Liposomal Irinotecan (Irinophore C™) and 5-Fluorouracil

PurposeTo investigate the use of liposomal irinotecan (Irinophore C™) plus or minus 5-fluorouracil (5-FU) for the treatment of colorectal cancer.Experimental DesignThe effect of irinotecan (IRI) and/or 5-FU exposure times on cytotoxicity was assessed in vitro against HT-29 or LS174T human colon carcinoma cells. The pharmacokinetics and biodistribution of Irinophore C™ (IrC™) and 5-FU, administered alone or in combination, were compared in vivo. A subcutaneous model of HT-29 human colorectal cancer in Rag2-M mice was utilized to assess the efficacy of IrC™ alone, and in combination with 5-FU.ResultsThe cytotoxicity of IRI and 5-FU were strongly dependent on exposure time. Synergistic interactions were observed following prolonged exposure to IRI/5-FU combinations. Pharmacokinetics/biodistribution studies demonstrated that the 5-FU elimination rate was decreased significantly when 5-FU was co-administered intravenously with IrC™, versus alone. Significant decreases in 5-FU elimination were also observed in plasma, with an associated increase of 5-FU in some tissues when 5-FU was given by intraperitoneal injection and IrC™ was given intravenously. The elimination of IrC™ was not significantly different when administered alone or in combination with 5-FU. Therapeutic studies demonstrated that single agent IrC™ was significantly more effective than the combination of IRI/5-FU; surprisingly, IrC™/5-FU combinations were no more effective than IrC™ alone. The administration of combinations of 5-FU (16 mg/kg) and IrC™ (60 mg IRI/kg) showed increased toxicity when compared to IrC™ alone. Treatment with IrC™ alone (60 mg IRI/kg) delayed the time required for a 5-fold increase in initial tumor volume to day 49, compared to day 23 for controls. When IrC™ (40 mg IRI/kg) was used in combination with 5-FU (16 mg/kg), the time to increase tumor volume 5-fold was 43 days, which was comparable to that achieved when using IrC™ alone (40 mg IRI/kg).ConclusionsSingle agent IrC™ was well tolerated and has significant therapeutic potential. IrC™ may be a suitable replacement for IRI treatment, but its use with free 5-FU is complicated by IrC™-engendered changes in 5-FU pharmacokinetics/biodistribution which are associated with increased toxicity when using the combination.

Microfluidic technique to measure intratumoral transport and calculate drug efficacy shows that binding is essential for doxorubicin and release hampers Doxil

Intratumoral transport and binding are important mechanisms that determine the efficacy of cancer drugs. Current drug screening methods rely heavily on monolayers of cancer cells, which overlook the contribution of tissue-level transport and binding. To quantify these factors, we developed a method that couples an in vitro, drug-delivery device containing a three-dimensional cell mass and a mathematical model of drug diffusion, binding to DNA, release from carriers, and clearance. Spheroids derived from LS174T human colon carcinoma cells were inserted into rectangular chambers to form rectangular cell masses (tissue) and subjected to continuous medium perfusion. To simulate drug delivery and clearance, the tissues were treated with doxorubicin followed by drug-free medium. To evaluate the effect of liposome encapsulation, tissues were treated with liposome-encapsulated doxorubicin (Doxil). Spatiotemporal dynamics of drug distribution and apoptosis was measured by fluorescence microscopy. The diffusivity and DNA binding constant of doxorubicin were determined by fitting experimental data to the mathematical model. Results show that an ideal combination of diffusivity, binding constant, clearance rate, and cytotoxicity contribute to the high therapeutic efficacy of doxorubicin. There was no detectable release of doxorubicin from Doxil in the tissues. The rate of doxorubicin release, evaluated by fitting experimental data to the mathematical model, was below therapeutically effective levels. These results show that despite enhanced systemic circulation obtained by liposome encapsulation, the therapeutic effect of Doxil is limited by slow intratumoral drug release. The experimental and computational methods developed here to calculate drug efficacy provide mechanisms to explain poor performance of drug candidates, and enable design of more successful cancer drugs.

Anti‐invasion effect of rosmarinic acid via the extracellular signal‐regulated kinase and oxidation–reduction pathway in Ls174‐T cells

Rosmarinic acid is a major phenylpropanoid isolated from Prunella vulgaris L., which is a composition of herbal tea for centuries in China. However, the anti-invasion activity on Ls174-T human colon carcinoma cells has not been studied. In this study, we investigated the anti-metastasis functions according to wound healing assay, adhesion assay, and Transwell assay and found that rosmarinic acid could inhibit migration, adhesion, and invasion dose-dependently. Rosmarinic acid also could decrease the level of reactive oxygen species by enhancing the level of reduced glutathione hormone. In addition, rosmarinic acid repressed the activity and expression of matrix metalloproteinase-2,9. According to Western blot and quantitative real-time PCR assay, rosmarinic acid may inhibit metastasis from colorectal carcinoma mainly via the pathway of extracellular signal-regulated kinase. In animal experiment, intraperitoneal administration of 2 mg of rosmarinic acid reduced weight of tumors and the number of lung nodules significantly compared with those of control group. Therefore, these results demonstrated that rosmarinic acid can effectively inhibit tumor metastasis in vitro and in vivo.

VEGF165b the anti‐angiogenic isoform of VEGF inhibits tumor growth in vivo.

To determine the potential of the anti‐angiogenic isoform,VEGF165b for cancer therapy, we compared the inhibitory effects of recombinant human VEGF165b on mouse model of colon cancer with bevacizumab. 2x106 LS174T human colon carcinoma cells were injected subcutaneously (SC) into the intrascapular dorsal region of nude mice. Tumor‐bearing mice were injected intraperitoneally (IP) with saline or bevacizumab (50 ?g), or SC in the lumbar region with 100 ?g VEGF165b bi‐weekly for 2 weeks. The inhibitory concentration of the IC50 was calculated. Growth of tumors was significantly suppressed by SC VEGF165b injection compared to those treated by saline (p&lt;0.001) or bevacizumab (p&lt;0.001), which also significantly reduced tumor growth (p&lt;0001, 2 way ANOVA). SC injection of VEGF165b resulted in a significant reduction of tumor volume from 254±51mm3 to 35.4±17mm3 after injection (p&lt;0.01 compared with before treatment, rmANOVA, Dunnets), whereas bevacizumab stabilised tumor growth (231±22 before compared with 342.± 117mm3, NS). Tumors in saline treated mice grew significantly from 149±46 to 853±362mm3, (p&lt;0.001). The IC50 of VEGF165b was calculated to be 48µg. Administration of VEGF165b inhibits the growth of colon cancer in vivo and indicates that systemic therapy of VEGF165b may be an effective anti‐VEGF tool for the treatment of colon cancer. This work supported by AICR and PhiloGene Inc.

Docosahexaenoic acid suppresses arachidonic acid-induced proliferation of LS-174T human colon carcinoma cells

To investigate the impact of arachidonic acid (AA) and docosahexaenoic acid (DHA) and their combination on colon cancer cell growth. The LS-174T colon cancer cell line was used to study the role of the prostaglandin precursor AA and the omega-3 polyunsaturated fatty acid DHA on cell growth. Cell viability was assessed in XTT assays. For analysis of cell cycle and cell death, flow cytometry and DAPI staining were applied. Expression of cyclooxygenase-2 (COX-2), p21 and bcl-2 in cells incubated with AA or DHA was examined by real-time RT-PCR. Prostaglandin E(2) (PGE(2)) generation in the presence of AA and DHA was measured using a PGE(2)-ELISA. AA increased cell growth, whereas DHA reduced viability of LS 174T cells in a time- and dose-dependent manner. Furthermore, DHA down- regulated mRNA of bcl-2 and up-regulated p21. Interestingly, DHA was able to suppress AA-induced cell proliferation and significantly lowered AA-derived PGE(2) formation. DHA also down-regulated COX-2 expression. In addition to the effect on PGE(2) formation, DHA directly reduced PGE(2)-induced cell proliferation in a dose-dependent manner. These results suggest that DHA can inhibit the pro-proliferative effect of abundant AA or PGE(2).

Analysis of glycoproteins in human colon cancers, normal tissues and in human colon carcinoma cells reactive with monoclonal antibody NCL-19-9

The expression of sialyl Lewis(a) antigen, also known as Ca 19-9, in colon cancer, normal tissues and LS174T human colon carcinoma cells were studied. In colon adenocarcinoma and cell plasma membranes this antigen is expressed on various glycoproteins with different molecular weights ranging in size from over 200 kDa to about 100 kDa. In addition, there is very low expression in peritumoral tissues. In cytosol and culture medium this epitope is carried by a single complex-glycoprotein with a very high molecular weight resembling a mucin. In cells the rise in cAMP levels elevate the synthesis and release of the carbohydrate antigen 19-9; whereas the treatment with 1,9-dideoxyforskolin, a diterpene, which does not activate adenylate cyclase, has no effect on content of the antigen. These results suggest that cAMP is involved on the expression of glycoprotein-associated sialyl Lewis(a) antigen in LS174T cells.

Potential new antitumor agents from an innovative combination of camphorato, a ramification of traditional Chinese medicine, with a platinum moiety

Eight new camphorato platinum complexes have been synthesized and evaluated for their in vitro cytotoxicity against HL-60 human leukemia, 3AO human ovarian carcinoma, BEL-7402 human hepatocarcinoma, and A549 human lung carcinoma cell lines. Most complexes showed good cytotoxic activity against the above-selected cell lines. Among the complexes, two compounds were assayed for their in vivo antitumor activity against LS-174T human colon carcinoma cells implanted in mice. One complex exhibited not only higher in vivo antitumor activity, but also less toxicity than oxaliplatin when it was administered intravenously at a dose of 6 mg/kg three times.

Cyclic AMP-dependent secretion of Ca 19-9 by LS174T human colon carcinoma cells

Prolonged increase of cyclic adenosine-monophosphate (cAMP) level in culture medium of a human colon cancer cell (LS174T) inhibits cellular growth and stimulates Ca 19-9 expression. The raise in cAMP level was produced by dibutyryl cyclic AMP (DBcAMP) or by forskolin an agent acting at the level of cAMP generation. Both these agents in a range of concentration between 10(-3)-10(-5) M have an inhibitory effect on the growth which is dose and time dependent. The inhibition was reversible as demonstrated by complete restoration of cell growth soon after the withdrawal of the substances from the culture medium. When cAMP levels in culture medium was raised, an increase in Ca 19-9 expression was observed and it appears that cyclic nucleotides have at least two effects: the first to cause rapid release of already synthesized Ca 19-9 and second to stimulate new antigen synthesis. The findings of the present study demonstrated that LS174T cells are unable to proliferate upon sustained accumulation of intracellular cyclic AMP suggesting the use of strategies able to increase cAMP levels for therapy of colon cancer. Furthermore, the finding that cAMP may also be a regulator of Ca 19-9 synthesis and release indicates the utility of cell line LS174T as a model for studies on the mechanism of synthesis and secretion of specific tumoral markers in colon cancer.

Extracellular protein kinase A as a cancer biomarker: its expression by tumor cells and reversal by a myristate-lacking Calpha and RIIbeta subunit overexpression.

Overexpression of cAMP-dependent protein kinase (PKA) type I isozyme is associated with cell proliferation and neoplastic transformation. The presence of PKA on the external surface of LS-174T human colon carcinoma cells has been shown. Here, we show that cancer cells of various cell types excrete PKA into the conditioned medium. This extracellular PKA (ECPKA) is present in active, free catalytic subunit (C subunit) form, and its activity is specifically inhibited by PKA inhibitory protein, PKI. Overexpression of the Calpha or RIalpha subunit gene of PKA in an expression vector, which up-regulates intracellular PKA type I, markedly up-regulates ECPKA expression. In contrast, overexpression of the RIIbeta subunit, which eliminates PKA type I, up-regulates PKA type II, and reverts the transformed phenotype, down-regulates ECPKA. A mutation in the Calpha gene that prevents myristylation allows the intracellular PKA up-regulation but blocks the ECPKA increase, suggesting that the NH(2)-terminal myristyl group of Calpha is required for the ECPKA expression. In serum of cancer patients, the ECPKA expression is up-regulated 10-fold as compared with normal serum. These results indicate that the ECPKA expression is an ordered cellular response of a living cell to actively exclude excess intracellular PKA molecules from the cell. This phenomenon is up-regulated in tumor cells and has an inverse relationship with the hormone dependency of breast cancer. Thus, the extracellular PKA may serve as a potential diagnostic and prognostic marker for cancer.

Interaction of Human Macrophage C-type Lectin withO-Linked N-Acetylgalactosamine Residues on Mucin Glycopeptides

A fluorescein-labeled synthetic peptide, PTTTPITTTTK, was converted into O-glycosylated glycopeptides with various numbers of attached N-acetyl-D-galactosamines (GalNAcs) by in vitro glycosylation with UDP-GalNAc and a microsomal fraction of LS174T human colon carcinoma cells. Glycopeptides with 1, 3, 5, and 6 GalNAc residues (G1, G3, G5, and G6) were obtained, and their sizes were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Their sequences were determined by a peptide sequencer to be PTTTGalNAcPITTTTK for G1, PTGalNAcTTPITGalNAcTGalNAcTTK for G3, PTTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK for G5, and PTGalNAcTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK for G6. A calcium-type human macrophage lectin (HML) was prepared in a recombinant form, and its interaction with these glycopeptides was investigated by surface plasmon resonance (SPR) spectroscopy and fluorescence polarization. The affinity of recombinant HML (rHML) for immobilized glycopeptides increased, as revealed by SPR, in parallel with the number of GalNAc. The highest affinity was obtained when the G6-peptide was immobilized at high density. Fluorescence polarization equilibrium-binding assays also revealed that the affinity of rHML for soluble gly-copeptides increased, depending on the number of attached GalNAcs. Carbohydrate recognition domain (CRD) fragments of HML were prepared, and their affinity for these four glycopeptides was also determined, this affinity was apparently lower than that of rHML. Affinity constants of rHML for the G3- and G5-peptides were 11- and 38-fold higher, respectively, than for the G1-peptide, whereas those of CRD fragments were only 2- and 6-fold higher, respectively. A chemical cross-linking study revealed that rHML but not recombinant CRD forms trimers in an aqueous solution. Thus, preferential binding of densely glycosylated O-linked glycopeptides should be due to the trimer formation of rHML.

Malignant and other properties of human colon carcinoma cells after suppression of sulfomucin production in vitro.

Although the loss of sulfomucins was known as an indicator of carcinogenesis and malignant progression of colonic epithelia, it was not known whether the loss was directly related to the malignant behavior of colon carcinoma cells. We have studied the biological properties of LS174T human colon carcinoma cells before and after suppression of sulfomucin production. Incorporation of [35S]-sulfate into high molecular weight mucins decreased after carcinoma cell treatment with 1.5% dimethylsulfoxide (DMSO) for 8 days. The amounts of sulfomucin determined using a sulfomucin-specific monoclonal antibody (mAb 91.9H), in Western blot and flowcytometric analyses, also decreased. In addition, the levels of MUC2 and MUC5B mucin gene expression measured by RT-PCR were reduced after DMSO-treatment, whereas the levels of MUC1, MUC5AC, and MUC6 mucin gene expression were not. The DMSO-treated cells were tested in vitro and in vivo for their properties. Differences were not detected in their anchorage-independent growth, anchorage-dependent growth, E-selectin-dependent cell adhesion or sensitivity to interleukin (IL)-2-activated lymphocyte cytolysis. When untreated or DMSO-treated LS174T cells were injected intrasplenically into nude mice, the treated cells lacking certain cell surface sulfomucins formed fewer metastatic colonies in the liver. These results suggest that the loss of sulfomucins by colonic epithelial cells during progression is not directly related to the enhanced malignant behavior.

Cyclic Adenosine 3‘:5‘-Monophosphate-Dependent Protein Kinase on the External Surface of LS-174T Human Colon Carcinoma Cells

The analysis of purified plasma membranes and the surface of intact cells revealed the presence of cyclic adenosine 3':5'-monophosphate-(cAMP) dependent protein kinase (PKA) on the external surface of LS-174T human colon carcinoma cells. Photoaffinity labeling of intact cells at confluence with 8-azido-[32P]cAMP identified the cAMP-binding proteins on the surface. Immunoprecipitation identified the photoaffinity-labeled cAMP-binding proteins as the RIIalpha regulatory subunit of PKA. During the logarithmic stage of growth, both the RIalpha and RIIalpha subunits of PKA were localized on the cell surface. Intact LS-174T cells catalyzed the phosphorylation of Kemptide in a cAMP-dependent manner; upon substitution of cAMP in the medium with 8-chloroadenosine, which did not compete with cAMP for the binding on intact cells, the ecto-PKA was no longer activated. The specific inhibitory protein for PKA, PKI, abolished the stimulation of phosphorylation by cAMP. Forskolin, which elevates intracellular levels of cAMP, activated ecto-PKA. Moreover, probenecid, which blocks the export of cAMP, inhibited the forskolin-mediated activation of ecto-PKA. These results demonstrate that LS-174T colon carcinoma cells possess an ecto-PKA on the external surface. This ecto-PKA is similar, if not identical, to the soluble intracellular PKA.

Subcellular distribution of the R-subunits of cAMP-dependent protein kinase in LS-174T human colon carcinoma cells.

The analysis of the particulate preparations of LS-174T human colon carcinoma cells in confluent stage of growth revealed different distribution for regulatory subunits (R) of cAMP-dependent protein kinase (PKA) in the subcellular compartments. The LS-174T cell lysates were subjected to differential and discontinuous sucrose density gradient centrifugation. The obtained fractions were assayed for marker enzymes and photoaffinity labeled with 8-N3[32P]cAMP. The whole lysates and cytoplasmic fraction exhibited the presence of both RI alpha and RII alpha--subunits of PKA. The fractions exhibiting high activity of the marker enzymes for plasma membranes, Golgi apparatus and mitochondria contained mainly RII alpha. In the fractions of lysosomes and microsomes RI alpha and RII alpha were found in nearly equal amounts.

Overexpresson of RIIβ, Regulatory Subunit of Protein Kinase A in Human Colon Carcinoma Cell Induces Growth Arrest and Phenotypic Changes that are Abolished by Site‐Directed Mutation of RIIβ

LS-174T human colon carcinoma cells that contain approximately equal amounts of cAMP-dependent protein kinase (PKA) isozymes, PKA-I and PKA-II, were infected with retroviral vectors coding for regulatory (R) and catalytic (C) subunits of human PKA. In cells overexpressing RII alpha, RII beta and RII beta-P (a RII beta mutant at the autophosphorylation site), PKA-II levels increased while PK-A levels decreased. PKA-I was almost completely eliminated in cells overexpressing RII beta or RII beta-P. In contrast, overexpression of either RI alpha or C alpha had little or no effect on PKA isozyme levels. Although all infectants expressed high levels of PKA subunit mRNAs in accordance with gene introduction, the R subunit protein expression was reflected in PKA isozyme levels rather than in subunit mRNA levels. Only RII beta infectants demonstrated marked growth inhibition in monolayer culture, reduced thymidine incorporation into DNA, and inability to grow in semisolid medium or in serum-free medium. Conversely, all other infectants displayed growth properties similar to uninfected parental cells. The growth-retardation properties of RII beta infectants were reflected in their altered phenotypic appearances. Our findings that the mutant RII beta-P could not mimic the growth-inhibitory effect of RII beta suggest the functional importance of the authophosphorylation site in RII beta. Our results suggest a role for RII beta in the suppression of neoplastic cell growth, and thus abnormal expression of R subunit isoforms of PKA may be involved in neoplastic transformation.

Multifactorial resistance in LS174T human colon carcinoma cells selected with doxorubicin

A series of doxorubicin-resistant variants of the human LS174T colon carcinoma cell line was generated by stepwise selection. These variants also exhibited increased resistance to vinblastine, etoposide, cis-platinum, and melphalan, suggesting that resistance was multifactorial. The parental LS174T cell line and 3 resistant variants were examined for over-expression of P-glycoprotein, changes in total cellular glutathione content, and the level of topoisomerase-II expression. Changes in all of these parameters were observed in the doxorubicin-selectants, along with a marked shift in the intracellular distribution of doxorubicin. P-glycoprotein RNA and protein levels were increased 2- to 3-fold in the resistant variants, while total glutathione levels increased 1.4- to 2.1-fold. Treatment with DL-buthionine-[S,R]-sulfoximine, an inhibitor of glutathione biosynthesis, was able to reverse resistance to cis-platinum and melphalan in these variants, but had little effect on doxorubicin resistance. Immunoblot analysis of cell extracts indicated that the level of DNA topoisomerase II (EC 5.99.1.3) in the doxorubicin-resistant LS174T cells was decreased by approximately 50% compared with the parental cell line. Doxorubicin was mainly localized to the cytoplasm in resistant cells, while in the parent line it was mostly found in the nucleus. This constellation of changes suggests that selection with doxorubicin activated several mechanisms of resistance involving drug transport, metabolism, and ability to reach nuclear target sites.