Human Colon Tumor Cell Research Articles

Conjugated Eicosapentaenoic Acid (EPA) Inhibits Transplanted Tumor Growth via Membrane Lipid Peroxidation in Nude Mice

Both conjugated linoleic acid (CLA) and eicosapentaenoic acid (EPA) have an antitumor effect. Hence, we hypothesized that a combination of conjugated double bonds and an (n-3) highly unsaturated fatty acid would produce stronger bioactivity. To verify the antitumor effect of conjugated EPA (CEPA), we transplanted DLD-1 human colon tumor cells into nude mice, and compared the tumor growth between CEPA-fed mice and CLA- and EPA-fed mice. After tumor cell inoculation, mice were assigned to 1 of 4 groups (control, CLA, EPA, and CEPA) consisting of 10 mice each. The control group received only safflower oil fatty acids, whereas the remaining groups received a mixture of safflower oil fatty acids and 20 g/100 g of total fatty acids as CLA, EPA, or CEPA. Mice were fed once every 2 d for 4 wk at a dose of 50 mg/mouse at each feeding. After 4 wk, tumor growth in CEPA-fed mice was significantly suppressed, compared with that in CLA- (P < 0.005) and EPA-fed mice (P < 0.001). DNA fragmentation in the tumor tissues of the CEPA-fed mice occurred more frequently than in the CLA- (P < 0.001) and EPA-fed mice (P < 0.001), suggesting that CEPA induced apoptosis in the tumor tissues. To further investigate the mechanism, the level of oxidative stress in the tumor tissues was determined. The CEPA-fed mice showed significant lipid peroxidation, compared with the CLA- (P < 0.001) and EPA-fed mice (P < 0.001). Therefore, we verified that CEPA has a stronger in vivo antitumor effect than EPA and CLA, and that CEPA acts through induction of apoptosis via lipid peroxidation.

Highly Efficient Biomimetic Total Synthesis and Structural Verification of Bistratamides E and J from Lissoclinum bistratum.

The interesting biological activities of heterocycle-containing cyclic peptide-derived natural products, isolated from marine organisms over the past twenty years, have attracted the interest of many synthetic and natural products chemists. Bistratamides E–J, members of this class of natural products that were isolated very recently from Lissoclinum bistratum, exhibited cytotoxic activity against a human colon tumor (HCT-116) cell line. Here we report the first total syntheses of bistratamides E (1) and J (2) in overall yields of 19 and 34 %, respectively. The thiazole substructures have been synthesized by oxidation of their corresponding thiazoline substructures, which were obtained from cysteine containing peptides using a novel biomimetic approach wherein Val-Cys dipeptide units were converted to thiazolines by a bisphosphonium salt. The final macrocyclization was promoted efficiently using the combination of PyBOP and DMAP. This approach allows the use of readily available Fmoc-protected amino acids to make complex thiazole and oxazoline-containing natural products.

Identification of an N-terminal amino acid of the CLC-3 chloride channel critical in phosphorylation-dependent activation of a CaMKII-activated chloride current.

CLC-3, a member of the CLC family of chloride channels, mediates function in many cell types in the body. The multifunctional calcium-calmodulin-dependent protein kinase II (CaMKII) has been shown to activate recombinant CLC-3 stably expressed in tsA cells, a human embryonic kidney cell line derivative, and natively expressed channel protein in a human colonic tumour cell line T84. We examined the CaMKII-dependent regulation of CLC-3 in a smooth muscle cell model as well as in the human colonic tumour cell line, HT29, using whole-cell voltage clamp. In CLC-3-expressing cells, we observed the activation of a Cl(-) conductance following intracellular introduction of the isolated autonomous CaMKII into the voltage-clamped cell via the patch pipette. The CaMKII-dependent Cl(-) conductance was not observed following exposure of the cells to 1 microm autocamtide inhibitory peptide (AIP), a selective inhibitor of CaMKII. Arterial smooth muscle cells express a robust CaMKII-activated Cl(-) conductance; however, CLC-3(-/-) cells did not. The N-terminus of CLC-3, which contains a CaMKII consensus sequence, was phosphorylated by CaMKII in vitro, and mutation of the serine at position 109 (S109A) abolished the CaMKII-dependent Cl(-) conductance, indicating that this residue is important in the gating of CLC-3 at the plasma membrane.

Diallyl disulfide (DADS) increases histone acetylation and p21(waf1/cip1) expression in human colon tumor cell lines.

Diallyl disulfide (DADS) is a naturally occurring organosulfur compound, from garlic, which exerts pleiotropic biological effects. In rodents, DADS inhibits colon chemically induced carcinogenesis. DADS anti-promoting effect may partly result from its ability to inhibit tumoral cell proliferation in vivo and in vitro. As far as DADS may modulate the expression of a subset of genes, we investigated DADS effect on histone acetylation, in two human colon tumor cell lines. Our study demonstrates that in Caco-2 and HT-29 cells treated for 6 h, 200 microM DADS increases histone H3 acetylation (x2 and x1.4, respectively). In Caco-2 cells, we also observed histone H4 hyperacetylation, preferentially at the lysine residues 12 and 16. We explored the effects of DADS and one of its metabolites, allyl mercaptan (AM), on histone deacetylase (HDAC) activity: using nuclear extracts of Caco-2 cells, 200 microM DADS decreased HDAC activity by 29% and AM at the same concentration was more efficient (92% inhibition). We also observed that DADS induced an increase in p21(waf1/cip1) expression, at mRNA and protein levels, in both cell lines. This effect was associated with an accumulation of cells in the G2 phase of the cell cycle. Our results suggest that in Caco-2 and HT-29 cells, DADS could inhibit cell proliferation through the inhibition of HDAC activity, histone hyperacetylation and increase in p21(waf1/cip1) expression. The present study provides evidence for cellular and molecular responses triggered by DADS that could be linked to its effect on histone acetylation and play a role in its protective properties on colon carcinogenesis.

Radiosensitive Variants Derived from a Human Colon Tumor Cell Line

Radiosensitive Variants Derived from a Human Colon Tumor Cell Line

Highly efficient biomimetic total synthesis and structural verification of bistratamides E and J from Lissoclinum bistratum.

The interesting biological activities of heterocycle-containing cyclic peptide-derived natural products, isolated from marine organisms over the past twenty years, have attracted the interest of many synthetic and natural products chemists. Bistratamides E-J, members of this class of natural products that were isolated very recently from Lissoclinum bistratum, exhibited cytotoxic activity against a human colon tumor (HCT-116) cell line. Here we report the first total syntheses of bistratamides E (1) and J (2) in overall yields of 19 and 34 %, respectively. The thiazole substructures have been synthesized by oxidation of their corresponding thiazoline substructures, which were obtained from cysteine containing peptides using a novel biomimetic approach wherein Val-Cys dipeptide units were converted to thiazolines by a bisphosphonium salt. The final macrocyclization was promoted efficiently using the combination of PyBOP and DMAP. This approach allows the use of readily available Fmoc-protected amino acids to make complex thiazole and oxazoline-containing natural products.

A profile of the in vitro antitumor activity of lissoclinolide

Lissoclinolide is a small non-nitrogenous lactone isolated from the marine ascidian Lissoclinum patella. Previous studies of lissoclinolide (isolated from a fungus and an actinomycete) have identified varying activity against both Gram-negative and Gram-positive bacteria. In this study, lissoclinolide was able to inhibit cell growth in various mammalian tumor lines at an average IC 50 of 395 nM (determined by MTT conversion after 48-h treatment). Treatment of HCT 116 human colon tumor cells with 2.4 μM lissoclinolide resulted in a strong arrest in the G 2/M phase of the cell cycle after 24-h exposure. A daughter cell line lacking p53 showed an identical response while there was a slight increase in cytotoxicity towards a p21 null cell line. Although treatment with 2.4 μM lissoclinolide did not result in apoptosis after 48 h, this arrest was not reversible when drug wash out was attempted. The mechanism of action does not appear to involve tubulin, ubiquitin-specific isopeptidases, p53 or p21. COMPARE analysis in the NCI 60 cell line tumor panel revealed a moderate selectivity towards colon tumor cell lines.

Long-Chain Acetylenic Ketones from the Micronesian Sponge Haliclona sp. Importance of the 1-yn-3-ol Group for Antitumor Activity

Two new long-chain C33 polyacetylenic compounds, halicynones A and B were isolated from the marine sponge Haliclona sp. along with known analogs. The known compound pellynol A possessing a 1-yn-3-ol terminus, exhibited strong antitumor activity against the human colon tumor cell line HCT-116 (IC50 0.026 μg/mL), however, the corresponding 1-yn-3-one, halicynone A, was inactive, which suggests an important role for the terminal 1-yn-3-ol functional group in mediating cytotoxic activity.

Increased activity of catalase in tumor cells overexpressing IGFBP-2.

Elevated levels of IGFBP-2 are found in serum and tissues under various stressful conditions and in many malignancies. In previous studies, we have shown that overexpression of IGFBP-2 results in increased tumorigenic potential in Y-1 mouse adrenocortical tumor cells, and that these effects are presumably mediated through IGF-independent mechanisms. Here, we show that highly proliferative IGFBP-2-overexpressing Y-1 cells, but not control Y-1 cells, grow to very high cell densities. In order to evaluate whether the increased cell densities in IGFBP-2-transfected Y-1 cells were accompanied by alterations in the oxidative stress system, we analyzed the effect of IGFBP-2 overexpression on the activity of various antioxidative enzymes in two malignant cell lines. Among the tested antioxidative enzymes (catalase, superoxide-dismutase, glutathione peroxidase, glutathione S-transferase), only catalase enzyme activity was significantly higher in IGFBP-2-transfected Y-1 mouse adrenocortical tumor cells and in IGFBP-2-transfected human colon tumor cells (Caco-2) compared to control-transfected Y-1 and Caco-2 cells and non-tumor 293 human epithelial cells. However, overexpression of catalase in malignant cells did not result in increased resistance to oxidative stress as measured by cell viability and protein oxidation after treatment of the cells with hydrogen peroxide. This might be due to an upregulation of the GST enzyme activity after treatment with H (2)O (2) that we observed selectively in the control-transfected Y-1 cells and which might compensate for the higher catalase activity in the IGFBP-2 overexpressing cells. In summary, we found a strong and selective upregulation of the catalase activity in IGFBP-2 overexpressing malignant Y-1 and Caco-2 cell lines that might contribute to the highly malignant phenotype of IGFBP-2 overexpressing tumors through as yet unknown mechanisms.

Rapid in vitro chemosensitivity analysis of human colon tumor cell lines

In oncology, diagnostic assays have the potential to individualize treatment. Due to a large number of chemotherapeutic agents available, a chemosensitivity assay would be of great value for patients receiving chemotherapy. However, no broadly accepted test exists to date. Our work aims at establishing a chemosensitivity test using a microphysiometer, a device that measures the extracellular acidification rate of cells. The test assesses chemosensitivity by comparing the acidification rate of cells treated with cytostatic agents with that of nontreated cells. We performed experiments with two human colon tumor cell lines, HCT 116 and SW620, and cytostatic agents commonly used in therapy of colon carcinoma. We showed that a chemosensitivity test can be reproducibly performed using a microphysiometer. Suitable media and conditions for the assay were found. Test results were further correlated with the crystal violet assay and a tetrazolium salt assay. In comparison, cytostatic effects were rapidly and most clearly displayed with the microphysiometer. Our results suggest that microphysiometry is a promising platform for chemosensitivity testing.

Diallyl Disulfide Induces ERK Phosphorylation and Alters Gene Expression Profiles in Human Colon Tumor Cells

Diallyl disulfide (DADS), a compound found in processed garlic, has been shown to arrest unsynchronized human colon tumor cells (HCT-15) in the G(2)/M phase of the cell cycle. The present studies were designed to examine whether this cell cycle block related to alterations in protein kinase C (PKC), Ca(2+)/calmodulin-dependent protein kinase II (CAMK II) or extracellular signal-regulated kinase (ERK) activity. Exposing double thymidine synchronized HCT-15 cells to DADS (25, 50 and 100 micromol/L) for 4 h increased the G(2)/M population by 30, 31 and 63%, respectively, compared with controls (P < 0.05). PKC and CAM KII activities were not influenced by increasing DADS exposure and thus did not correlate with the block of cells in the G(2)/M phase. Although ERK activity increased by 44 and 60% after treatment with 100 and 500 micromol/L DADS (P < 0.05), it was not influenced by exposure to 25 or 50 micromol/L DADS. Western blot analysis revealed that although DADS (25, 50, 100 and 500 micromol/L) did not influence the quantity of ERK protein expressed, it did increase its phosphorylation by 39, 52, 73 and 61%, respectively, compared with controls (P < 0.05). These studies provide evidence that early alterations in ERK pathway signaling may contribute to the G(2)/M arrest observed after DADS exposure. Preliminary data generated using the Clonetech Atlas Human Cancer cDNA Expression Array suggest that alterations in cell cycle, DNA repair and cellular adhesion factors accompany DADS exposure and may also be involved in mediating the block in G(2)/M progression.

Nitric oxide prevents UV-induced phosphorylation of the p53 tumor-suppressor protein at serine 46: a possible role in inhibition of apoptosis

Ser-46 of p53 is phosphorylated in response to DNA-damage in vivo, and it plays a pivotal role for apoptotic signaling by p53 through regulating the transcriptional activation of genes involved in apoptosis. We found that the presence of the nitric oxide (NO) donor S-nitroso- N-acetyl- d, l-penicillamine (200–800 μM) during UV-irradiation of MCF-7 cells resulted in a significant reduction in the Ser-46 phosphorylation, compared to the UV-irradiated cells without NO. This reduction occurred independently of cyclic GMP generation and without affecting activities of p53 kinases such as the PI3K family, p38 MAPK, and HIPK2. The presence of NO was found to protect HCT116 human colon tumor cells containing wild-type p53 from UV-induced apoptosis, whereas no apparent inhibitory effect of NO on UV-induced apoptosis was observed in those deficient in p53. Our results suggest that NO-mediated protection of apoptosis is p53-dependent, occurring at least partly through NO-inhibition of phosphorylation of p53 on Ser-46.

Overexpression of the beta-catenin binding domain of cadherin selectively kills colorectal cancer cells.

The beta-catenin pathway is involved in growth, differentiation and tumor formation. Suppression of pathway activity by expressed inhibitors can cause growth arrest or apoptosis in certain colon carcinoma lines. We compare the effects of 2 pathway inhibitors, a VE-cadherin cytoplasmic domain fragment (Cad5CD) and a truncated, dominant-negative Tcf4 (TcfDN), using a microplate assay for cell death and microarray gene expression analysis. The cell-lethal assay shows that Cad5CD, when expressed in HT29 human colon tumor cells and 3 non-colon lines, selectively kills the HT29 cells. Cad5CD overexpression inhibits beta-catenin/Tcf4 transcriptional activity, as determined by results from microarray experiments. Our results support the view that beta-catenin is an attractive anti-cancer target, especially if the Cad5CD binding site or Cad5CD itself can be exploited for drug development. In addition, therapeutically relevant phenotypes such as drug selectivity may be difficult to predict from gene expression analysis alone. Other more specialized phenotypic tests such as cell-lethal assays may be required.

Hormonemate, a new cytotoxic and apoptosis-inducing compound from the endophytic fungus Hormonema dematioides. I. Identification of the producing strain, and isolation and biological properties of hormonemate.

In a search for new fungal compounds inducing apoptosis of the colon cancer derived cell line COLO-320, hormonemate (1) was purified from fermentations of an endophytic fungus isolated from living needles of a Pinus species. The producing strain was identified as Hormonema dematioides by microscopy and ITS rDNA sequence analysis. The structure of hormonemate was determined by spectroscopic methods. The compound exhibited cytotoxic effects against the human colon tumor cell lines COLO-320, DLD-1 and HT-29 as well as five other human cell lines (HL-60, JURKAT, HEP-G2, MCF-7, HeLa S3). It also induced apoptosis in COLO-320 cells as detected by a caspase-activity assay and morphological changes, and it triggered morphological and physiological differentiation of HL-60 cells into granulocytes, which subsequently died by apoptosis.

Detection of differentially expressed HES-6 gene in metastatic colon carcinoma by combination of suppression subtractive hybridization and cDNA library array.

The molecular mechanisms involved in the progression of colon carcinomas from a primary to a metastatic tumor have been only partially elucidated and poorly understood. This study combines suppression subtractive hybridization and cDNA array hybridization to identify genes with expression differences between a primary human colon tumor cell line (HT29) and three isogenic lung tumor metastases. The positive clones isolated in this screen were further validated and quantitated with real-time reverse transcription polymerase chain reactions. HES-6 was identified as up-regulated in each of the individual tumor metastases, as well as in a panel of primary human tumors derived from the lung, breast and kidney. These findings demonstrate that it is possible to utilize longitudinal samples from an in vivo model of colon carcinoma to identify genes up-regulated in metastases and that HES-6 may be an important marker of a range of primary cancers as well as metastatic colon carcinoma.

Total synthesis of phorboxazole A.

Total synthesis of phorboxazole A.

In Vitro and In Vivo Pharmacology and Pharmacokinetics of a Human Engineered™ Monoclonal Antibody to Epithelial Cell Adhesion Molecule

ING-1(heMAb), a Human Engineered™ monoclonal antibody to epithelial cell adhesion molecule (Ep-CAM ), was evaluated for its in vitro and in vivo activity. The dissociation constant of ING-1(heMAb) for binding to Ep-CAM on HT-29 human colon tumor cells was 2 to 5 nM, similar to chimeric ING-1. In antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays, ING-1(heMAb) caused a concentration-dependent lysis of BT-20 breast, MCF-7 breast, HT-29 colon, CACO-2 colon tumor cells, with maximum cytolysis at approximately 1 μg/ml. After an intravenous injection in rats, plasma ING-1(heMAb) levels declined with an alpha half-life of 8 to 11 hours, a beta half-life of 20 days, typical of an IgG in a species without the target for ING-1. In nude mice with human HT-29 colon tumors, plasma ING-1(heMAb) levels declined more rapidly than in non-tumor-bearing mice, suggesting an enhanced clearance via the tumor-associated human Ep-CAM. In nude mice, intravenous treatments with ING-1(heMAb) twice a week for 3 weeks significantly suppressed the growth of human HT-29 colon and PC-3 prostate tumors in a dose-dependent manner, with 1.0 mg/kg providing the greatest benefit. These results indicate that Human Engineered™ ING-1(heMAb) is a high-affinity antibody with potent in vitro activity that targets and suppresses the growth of human tumors in vivo.

Putative colon cancer risk factors damage global DNA and TP53 in primary human colon cells isolated from surgical samples

This study describes a novel in vitro method in genetic toxicology that is based on detection of chemical-induced DNA damage connected with altered migration of TP53 in primary human colonocytes. Techniques were developed to isolate high numbers of human epithelial colon cells from surgical tissues. High quantities of viable cells were obtained per donor. The primary cells were treated with the endogenous risk factors trans-2-hexenal, and hydrogen peroxide. Global DNA damage and repair were measured by single-cell gel electrophoresis (Comet assay). We compared responses of primary colon cells to HT29clone19A, a differentiated human colon tumour cell line, for which the karyotype was analysed with 24-colour FISH. Both compounds were genotoxic in both cell types and most of the induced DNA damage was repaired after 30 min. Specific migration of TP53 was determined by fluorescence in situ hybridization (Comet FISH). Using primary colon cells, we quantified the migration of TP53 signals into the comet tails. In these cells TP53 was more sensitive than global DNA for genotoxicity induced by trans-2-hexenal and H 2O 2. HT29clone19A cells cannot be used for Comet FISH because of their aberrant karyotype. The approach described allows us to obtain more knowledge of putative risk factors in colon carcinogenesis.

Growth-inhibitory effects of the chemopreventive agent indole-3-carbinol are increased in combination with the polyamine putrescine in the SW480 colon tumour cell line.

BackgroundMany tumours undergo disregulation of polyamine homeostasis and upregulation of ornithine decarboxylase (ODC) activity, which can promote carcinogenesis. In animal models of colon carcinogenesis, inhibition of ODC activity by difluoromethylornithine (DFMO) has been shown to reduce the number and size of colon adenomas and carcinomas. Indole-3-carbinol (I3C) has shown promising chemopreventive activity against a range of human tumour cell types, but little is known about the effect of this agent on colon cell lines. Here, we investigated whether inhibition of ODC by I3C could contribute to a chemopreventive effect in colon cell lines.MethodsCell cycle progression and induction of apoptosis were assessed by flow cytometry. Ornithine decarboxylase activity was determined by liberation of CO2 from 14C-labelled substrate, and polyamine levels were measured by HPLC.ResultsI3C inhibited proliferation of the human colon tumour cell lines HT29 and SW480, and of the normal tissue-derived HCEC line, and at higher concentrations induced apoptosis in SW480 cells. The agent also caused a decrease in ODC activity in a dose-dependent manner. While administration of exogenous putrescine reversed the growth-inhibitory effect of DFMO, it did not reverse the growth-inhibition following an I3C treatment, and in the case of the SW480 cell line, the effect was actually enhanced. In this cell line, combination treatment caused a slight increase in the proportion of cells in the G2/M phase of the cell cycle, and increased the proportion of cells undergoing necrosis, but did not predispose cells to apoptosis. Indole-3-carbinol also caused an increase in intracellular spermine levels, which was not modulated by putrescine co-administration.ConclusionWhile indole-3-carbinol decreased ornithine decarboxylase activity in the colon cell lines, it appears unlikely that this constitutes a major mechanism by which the agent exerts its antiproliferative effect, although accumulation of spermine may cause cytotoxicity and contribute to cell death. The precise mechanism by which putrescine enhances the growth inhibitory effect of the agent remains to be elucidated, but does result in cells undergoing necrosis, possibly following accumulation in the G2/M phase of the cell cycle.

5-(2'-oxoheptadecyl)-resorcinol and 5-(2'-oxononadecyl)-resorcinol, cytotoxic metabolites from a wood-inhabiting basidiomycete.

5-(2'-oxoheptadecyl)-resorcinol [structure: see text] and 5-(2'-oxononadecyl)-resorcinol [structure: see text] were isolated from fermentations of an imperfect basidiomycete. The structures of the compounds were determined by spectroscopic techniques. Both compounds exhibit cytotoxic effects against the human colon tumor cell lines COLO-320, DLD-1 and HT-29 and the human promyeloid leukemia cell line HL-60, the human leukemia T cell JURKAT, the human hepatocellular carcinoma cell line HEP-G2 as well as the J774 mouse macrophage cell line. The compounds induce morphological and physiological differentiation of HL-60 cells into granulocytes, which subsequently die by apoptosis. Both compounds show no antibacterial and antifungal activity.