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PubMed2000

The constituents of the root and stem of Aristolochia cucurbitifolia Hayata and their biological activity.

Four new compounds, three phenanthrene derivatives, aristolochic acid-III methyl ester (1), cepharanone C (2), and sodium 7-hydroxyl-8-methoxyaristolate (3), and the benzoate derivative, sodium 3,4-dimethoxybenzoate (4), together with 53 known compounds were isolated and characterized from the fresh root and stem of Aristolochia cucurbitifolia. Their structures were elucidated by spectral analyses and chemical transformations. The cytotoxicity and antiplatelet activity of the isolated compounds are also discussed.

Animals↗
PubMed2002

Use of adenoviruses to study isoform specificity of G-protein-receptor-coupled Ca2+ signaling in intact epithelial cells.

It is firmly established that the activation of many heptahelical receptors by extracellular agonists leads to the activation of effectors such as phospholipase Cbeta (PLCbeta), the subsequent production of inositol-1,4,5-trisphosphate (IP3), and a resultant increase in intracellular free Ca2+. Heterotrimeric G-proteins have a critical role in transducing the signal from the heptahelical receptor to PLCbeta and in determining the specificity and duration of the cellular responses. There remain, however, a number of areas of uncertainty regarding the exact mechanisms involved in regulating G-protein-mediated receptor-effector coupling in different cell types. For example, the molecular identity of the G-protein involved and the degree of isoform specificity among G-proteins of the same family and their receptors remains unclear. It is also not known in many cell types whether it is the alpha- or the betagamma-subunits of these G-proteins that activate PLCbeta. In order to address these issues, we have used replication-deficient adenoviruses as a tool to deliver, into intact epithelial cells, transgenes coding for proteins involved in G-protein-coupled receptor signaling pathways.

Adenoviridae↗
PubMed2000

Somatostatin through its specific receptor inhibits spontaneous and TNF-alpha- and bacteria-induced IL-8 and IL-1 beta secretion from intestinal epithelial cells.

Intestinal epithelial cells secrete proinflammatory cytokines and chemokines that are crucial in mucosal defense. However, this secretion must be tightly regulated, because uncontrolled secretion of proinflammatory mediators may lead to chronic inflammation and mucosal damage. The aim of this study was to determine whether somatostatin, secreted within the intestinal mucosa, regulates secretion of cytokines from intestinal epithelial cells. The spontaneous as well as TNF-alpha- and Salmonella-induced secretion of IL-8 and IL-1beta derived from intestinal cell lines Caco-2 and HT-29 was measured after treatment with somatostatin or its synthetic analogue, octreotide. Somatostatin, at physiological nanomolar concentrations, markedly inhibited the spontaneous and TNF-alpha-induced secretion of IL-8 and IL-1beta. This inhibition was dose dependent, reaching >90% blockage at 3 nM. Furthermore, somatostatin completely abrogated the increased secretion of IL-8 and IL-1beta after invasion by Salmonella. Octreotide, which mainly stimulates somatostatin receptor subtypes 2 and 5, affected the secretion of IL-8 and IL-1beta similarly, and the somatostatin antagonist cyclo-somatostatin completely blocked the somatostatin- and octreotide-induced inhibitory effects. This inhibition was correlated to a reduction of the mRNA concentrations of IL-8 and IL-1beta. No effect was noted regarding cell viability. These results indicate that somatostatin, by directly interacting with its specific receptors that are expressed on intestinal epithelial cells, down-regulates proinflammatory mediator secretion by a mechanism involving the regulation of transcription. These findings suggest that somatostatin plays an active role in regulating the mucosal inflammatory response of intestinal epithelial cells after physiological and pathophysiological stimulations such as bacterial invasion.

Caco-2 Cells↗
PubMed2004

IL-4 and IL-13 up-regulate intestinal trefoil factor expression: requirement for STAT6 and de novo protein synthesis.

The development of intestinal goblet cell hyperplasia/hypertrophy during nematode infection involves the Th2 cytokines IL-4 and IL-13 via STAT6 activation. This is thought to play an important role in host protective immunity against the infection. In this study we demonstrate that IL-4 and IL-13 up-regulate the specific goblet cell product trefoil factor-3 (TFF3) from the mucus-producing HT-29 CL.16E and HT-29 cells selected by adaptation to methotrexate. Up-regulation of TFF3 mRNA and protein levels occurred in a time- and dose-dependent fashion and was accompanied by up-regulation of the goblet cell product mucin 2 (MUC2). Addition of actinomycin D before IL-4/IL-13 stimulation led to decreases in TFF3 mRNA levels similar to those observed in controls without IL-4/IL-13. Furthermore, IL-4-mediated increased TFF3 transcription required de novo protein synthesis. Stable transfection of HT-29 CL.16E cells with a truncated dominant-negative form of STAT6 produced a cell line that was unresponsive to IL-4/IL-13. Although only one consensus STAT6 binding site is contained in the TFF3 gene, located in the intron 1, it did not operate as an enhancer in the context of an SV40 promoter/luciferase construct. Thus, STAT6 activation mediates a transcriptional enhancement of TFF3 by induction of de novo synthesized protein in goblet cells.

Goblet Cells↗
PubMed2005

V gamma 9V delta 2 T cell response to colon carcinoma cells.

During analysis of CD8 T cells derived from ascites of a colon cancer patient, we isolated a Vgamma9Vdelta2 T cell clone showing strong reactivity against autologous tumor cell lines. This clone killed a large fraction of allogeneic colon carcinoma and melanoma cell lines, but did not affect a normal colon cell line, colon fibroblasts, or melanocytes. Tumor cell recognition was TCR and NKG2D dependent and induced TNF-alpha and IFN-gamma secretion by the clone; accordingly, tumor targets expressed several NKG2D ligands, such as MHC class I chain-related gene A and UL16-binding protein molecules. Colon tumor recognition by Vgamma9Vdelta2 T cells was highly dependent on isopentenyl pyrophosphate production and ICAM-1 expression by target cells. Finally, similar reactivity patterns against colon carcinoma cell lines were observed using polyclonal Vgamma9Vdelta2 T cells of various origins, and Vgamma9Vdelta2 lymphocytes were present in the majority of colon tumor samples studied. Together, these results suggest that Vgamma9Vdelta2 T cells contribute to the natural immune surveillance against colon cancers. Therefore, this study provides a strong rationale for the use of Vgamma9Vdelta2 T cell agonists in immunotherapies targeting colon tumors.

Antigens↗
PubMed2005

Genotoxic and endoplasmic reticulum stresses differentially regulate TRB3 expression.

TRB3 has recently been identified as a potential pro-apoptotic protein that may modulate the Akt/PKB-dependent signaling pathway. Here we report that TRB3 expression is strongly upregulated by endoplasmic reticulum (ER) stress-inducing agents that (1) promote ER Ca2+ pool depletion or (2) disrupt protein trafficking. Genotoxic stress (DNA damage)-inducing agents, by contrast, downregulate TRB3 expression and appear to do so through both p53-dependent and -independent mechanisms. To the best of our knowledge, TRB3 is the first gene that is upregulated by ER stress and downregulated following genotoxic stress. Collectively, these findings highlight the importance of stress-specific signaling cascades as well as point out the seemingly divergent roles that TRB3 may play in the cellular stress response.

Cell Cycle Proteins↗
PubMed2006

Activating Ras mutations fail to ensure efficient replication of adenovirus mutants lacking VA-RNA.

Adenoviruses lacking their PKR-antagonizing VA RNAs replicate poorly in primary cells. It has been suggested that these virus recombinants still replicate efficiently in tumor cells with Ras mutations and might therefore be useful in tumor therapy. The ability of interferon-sensitive viruses to grow in Ras-mutant tumor cells is generally ascribed to a postulated inhibitory effect of mutant Ras on PKR. We have constructed a set of isogenic adenoviruses that lack either or both VA RNA species, and tested virus replication in a variety of cell species with different Ras status. In tendency, VA-less viruses replicated with higher efficiency in Ras-mutant cells, as compared to cell lines without Ras mutation. However, several exceptions to this rule were observed, arguing against a direct inhibition of PKR by mutant Ras. Phosphorylation of the PKR-substrate eIF2alpha was observed regardless of the Ras mutational status, upon infection with VA-less adenoviruses in the presence of interferon, but also upon addition of the PKR activator polyIC to cells. When comparing two isogenic cell lines that differ solely with regard to the presence or absence of mutant Ras, no difference was observed concerning the replication of VA-less adenoviruses or the phosphorylation of eIF2alpha. We conclude that mutant Ras does not directly affect eIF2alpha phosphorylation or the replication of interferon-sensitive adenoviruses. These results strongly suggest that the Ras mutational status is insufficient to predict the oncolytic effect of interferon-sensitive viruses. We propose that Ras mutations predispose tumor cells to undergo secondary changes that sometimes enable the replication of interferon-sensitive viruses.

Adenoviruses, Human↗
PubMed1999

Cellular pharmacology of the combination of the DNA topoisomerase I inhibitor SN-38 and the diaminocyclohexane platinum derivative oxaliplatin.

CPT-11, a DNA topoisomerase I inhibitor, and oxaliplatin, a diaminocyclohexane platinum derivative, are cytotoxic agents that have demonstrated clinical antitumor activity in colorectal cancer. Given the therapeutic potential of their combination, we studied the cellular pharmacology of SN-38, the active metabolite of CPT-11, and oxaliplatin in the human colon cancer HT29 cell line. Growth inhibition was studied after a 1- or 24-h exposure to SN-38 or oxaliplatin, given alone or in combination. The cytotoxicity analysis by the isobolograms method elicited synergy. SN-38 delayed the reversion of oxaliplatin-induced DNA interstrand cross-links (ISCs), measured in cells by alkaline elution. The amount of detectable ISCs 15 h after a 1-h exposure to 10 microm oxaliplatin was 27% of the ISC peak levels and increased to 68% in the presence of 0.1 microM SN-38. The presence of oxaliplatin DNA adducts led to a 3.3-fold increase in the SN-38-induced DNA elongation inhibition, as measured by pulse-labeling alkaline elution. Inhibition of DNA and RNA synthesis was longer after exposure to the combination of oxaliplatin and SN-38 than after exposure to each agent alone. Consistently, flow cytometry analyses revealed that preexposure to oxaliplatin enhanced SN-38-induced S-phase arrest. Filter binding assays indicated that the cells arrested in S-phase at 48 h were undergoing apoptosis. Hence, supra-additive cytotoxicity appears related to major modifications in the cellular response to DNA damage rather than to changes in DNA damage formation. The combination of CPT-11 and oxaliplatin induced a 2-fold higher tumor growth reduction in vivo than did oxaliplatin alone at feasible nonlethal doses. This study provides a rationale for the optimal use of CPT-11 and oxaliplatin in combination.

Adenocarcinoma↗
PubMed1999

Organometallic 99mTc-aquaion labels peptide to an unprecedented high specific activity.

UNLABELLED: A new peptide labeling method that uses the organometallic aquaion [99mTc(H2O)3(CO)3]+ has been developed. METHODS: A selection of amino acids was labeled at different concentrations with the organometallic aquaion, and the labeling yield was determined by high-performance liquid chromatography. This investigation has shown histidine to be a very potent ligand, with specific activities of up to 6 TBq/micromol (160 Ci/micromol) ligand. Histidine derivatives have been coupled to neurotensin(8-13) (NT[8-13]) and have been labeled with the aquaion, resulting in high specific activities with (N(alpha)-histidinyl)acetic acid-NT(8-13) similar to those with histidine. RESULTS: Histidine derivatives of NT(8-13) labeled using this approach fully retained their receptor affinity, showing KD values of all investigated NT analogs below 1 nmol/L on colon carcinoma HT29 cells. Biodistrbution experiments in BALB/c mice showed complete clearance of (N(alpha)-histidinyl)acetic acid-NT(8-13) from the blood after 24 h and no unwanted accumulation in any tissue. CONCLUSION: The novel labeling method using the organometallic 99mTc-aquaion combines the advantage of highest specific activities with minimal functionalization of proteins and peptides under retention of biologic affinity.

Animals↗
PubMedDate not supplied

A novel use for the comet assay: detection of topoisomerase II inhibitors.

BACKGROUND: The simple and quick comet assay can quantitatively detect DNA cleavage in cells. This study aimed to determine whether the comet assay could be used to detect topoisomerase (topo) II inhibitors. MATERIALS AND METHODS: HT-29 colon cancer cells were pre-incubated with aclarubicin, a topo II antagonist, then treated with topo II poisons: etoposide (VP-16), teniposide (VM-26), 4'-(acridinylamino) methansulfon-m-anisidide (m-AMSA) and adriamycin (doxorubicin). We also tested a topo I poison (camptothecin) and a microtubule depolymerization inhibitor (taxol). RESULTS: Aclarubicin significantly reduced DNA cleavage induced by topo II poisons, but not that induced by camptothecin. In HL-60/MX2 cells (containing no topo II beta and reduced topo II alpha), DNA breakage induced by topo II poisons was lower. Also, aclarubicin antagonized topo I-mediated camptothecin-induced DNA cleavage in these resistant cells. CONCLUSIONS: The comet assay can be used to detect topo II poisons in cultured cells. Also, aclarubicin has a dual topo I and topo II antagonism, with "preferential antagonism" of topo II when topo II beta catalytic activity is normally expressed.

Aclarubicin↗
PubMed2001

The Sp family of transcription factors in the regulation of the human and mouse MUC2 gene promoters.

Modulation of mucin gene expression is an important component both in the early steps of colon cancer development and in later tumor progression. Previous work from our laboratory and others has suggested that the Sp family of transcription factors may play an important role in the regulation of the human MUC2 gene. To determine whether this was an essential element, we extended our work to the cloning and analysis of 3.5 kb of the 5'-flanking region of the mouse Muc2 (mMuc2) gene. Comparative analysis between the mouse and human MUC2 promoter regions has identified a strong sequence homology between the mouse and human genes, including the presence of GC-rich boxes, the location and composition of which are maintained in the mouse and human genes. We show that these GC boxes are binding sites for Sp-family transcription factors and are functionally important since mithramycin, an inhibitor of Sp1/Sp3 binding, blocks MUC2 gene expression in HT29 cells. Furthermore, by a combination of gel shift analysis and site-directed mutagenesis, we have identified the relative contribution of individual GC boxes, and of the factors they bind, to the regulation of the mouse Muc2 promoter, which appears to be different in the mouse and human genes. Finally, we demonstrate by overexpressing Sp1 and Sp3 that the functional difference between the proximal promoter region of the MUC2 gene in the two species is not attributable to differential ability of this region to bind members of the Sp family of transcription factors, but rather to the different anatomy of the individual GC boxes in the mouse and human proximal promoters.

Animals↗
PubMed2001

Quantitative reverse transcription-polymerase chain reaction detection of cytokeratin 20 in noncolorectal lymph nodes.

PURPOSE: Unexpected reverse transcription-PCR detection of cytokeratin 20 (CK20) in samples from healthy individuals and cancer types not expected to express CK20 has cast uncertainty on the role of CK20 as a specific marker of disseminated colorectal cells. We aimed to clarify the specificity of CK20 by examining its expression profile by real-time reverse transcription-PCR. EXPERIMENTAL DESIGN: A quantitative real-time PCR assay on the LightCycler instrument was developed and used to examine CK20 expression in tumors and lymph nodes from subjects with colorectal and breast carcinoma, head and neck and vulval squamous cell carcinoma, and melanoma. To select a method for reproducible quantification, four approaches were evaluated. RESULTS: The developed assay allowed rapid, convenient-to-use, specific, sensitive, and reproducible CK20 quantification amenable to large-scale analysis. For quantity calculation, an efficiency-adjusted relative ratio method was selected that controls for RNA loading and integrity as well as inefficient PCR reactions and provides a platform for standardization across laboratories. Using this assay, we detected CK20 in 41 of 89 (46%) lymph nodes from noncolorectal cancer types. There was a strong association between CK20 detection and lymph node metastasis determined by histology (P < 0.0001). Quantitatively, CK20 expression levels in colorectal cancer lymph nodes significantly exceeded the levels obtained in lymph nodes of extracolonic carcinomas (P < 0.05). Mean CK20 levels in lymph nodes and tumors from subjects with colorectal and breast cancers were similar in a tumor-type specific fashion. CONCLUSIONS: These results characterize low-level, epithelial cell-specific CK20 expression in infiltrated lymph nodes from subjects with noncolorectal cancer types and demonstrate the potential advantages of detecting circulating epithelial cells by quantitative PCR.

Biomarkers, Tumor↗
PubMed2001

[Synthesis of phosphoramide mustard analogues belonging to the L-sugar series].

During the past decades numerous cyclophoshamide (mustard) derivatives of nucleosides and aminodeoxy sugars have been prepared for investigating their antitumor activities. The cyclophosphamide analogues of aminotrideoxy hexoses belonging to the D-series of sugars have been prepared by Monneret et al. The present paper reports the synthesis of the new phosphoramide mustards 16-17 from 12 and 15 (belonging to the L-sugar series). First compound 10 was synthesized from the L-rhamnose (9). Methyl 3-azido-2,3,6,-trideoxy-alpha-L-ribo-hexopyranoside (11) was obtained by the replacement of the 3-O-p-toluene-sulfonyl group of 10 with sodium azide. Methyl 3-azido-2,3,6,-trideoxy-alpha-L-arabino-hexopyranoside (14) was synthesized by rign opening of 13 with sodium azide. The corresponding amino sugars (12, 15) were obtained by catalytic hydrogenation (over palladium on carbon) of 11 and 14. Our compounds 12 and 15 were transformed into the cyclophosphamide derivatives 16a,b-17a,b upon treatment with bis(2-chloroethyl)phoshoramidic dichloride in the presence of triethylamine (36 h, r.t.). The approximately 1:1 mixtures of isomers (due to the different steric position of the P=O group) could be readily separated by chromatography. The 1H NMR assignments of compounds 16a, 16b, 17a and 17b, were based on one-dimensional selective decoupling experiments or two-dimensional chemical shift-correlated spectroscopy (COSY-60). The assignment of configuration to the isomeric phosphoramidates was based on the magnetic anisotropy of the P=O bond. The distinctly different chemical shift patterns of sugar protons observed for the two isomers allowed the unambiguous assignment of the P=O stereochemistry. The compounds 16a,b-17a,b (mixture of isomers) were tested for inhibitory activity using L1210 and HT29 cell lines.

Antineoplastic Agents, Alkylating↗
PubMed2002

[Antitumor effect of anuoning].

BACKGROUND & OBJECTIVE: The authors' previous studies have demonstrated that anuoning bullatacin and squamocin have anticancer activity in vitro. Squamocin could induce apoptosis of HL-60 cells. The purpose of this paper was to investigate cytotoxicity and antitumor effect of anuoning. METHODS: MTT assay was used to examine the growth inhibition of anuoning on human colon carcinoma cell line (HT-29), human nasopharyngeal carcinoma cell line (SUNE1, CNE2), human liver carcinoma (bel-7402), human breast adenocarcinoma cell line (MCF-7) and human lung adenocarcinoma cell line (GLC-82). The models of mice S-180 sarcoma and HepS were used for in vivo antitumor test. RESULTS: The IC50 of anuoning on CNE2, bel-7402, HT-29, SUNE1 cell were 0.044, 0.068, 0.446, and 1.617 micrograms/ml, respectively. The IC50 of anuoning on MCF-7 cell and GLC-82 cell were 1.857 and 3.481 micrograms/ml, respectively. Under the doses of 15, 30, and 60 micrograms/kg, i.p., qd x 10 d, the average tumor inhibitory rates of anuoning to mice tumor HepS were 36.9%, 51.8%, and 57.9%, respectively (P < 0.05). Under the same concentration, the average tumor inhibitory rates of anuoning on mice S-180 sarcoma were 43.0%, 52.1%, and 61.0%, respectively (P < 0.05). CONCLUSIONS: The results indicate that the anuoning possess cell growth inhibition activity to various human tumor cells in vitro and antitumor effect in vivo.

Animals↗
PubMed2003

New glycosylated porphyrins for PDT applications.

A good sensitizer for photodynamic therapy (PDT) should be a very selective reagent, which should fastly eliminate from healthy tissues. Furthermore it should have a strong absorption in the red light and good energy transfer properties to molecular oxygen to produce singlet oxygen. All these specifications imply that many second generation photosensitizers (porphyrins, chlorins, bacteriochlorins...) have been modified in order to improve their properties, but however, few have received the approval by regulatory authorities. One way to increase the selectivity of a photosensitizer is coupling it to a vector. Carbohydrate-bound porphyrins were found to be of great interest because of the specific affinity of particular carbohydrates for some tumour cells, the increasing of plasmatic life time and solubility. In this study, we report the synthesis and the photophysical properties (absorption, fluorescence, singlet oxygen formation) of new glycosylated porphyrins. Then, in vitro photocytotoxic properties were evaluated on the human colon adenocarcinoma cell line HT29.

Computer Graphics↗
PubMed2003

Human epithelial cancers secrete immunoglobulin g with unidentified specificity to promote growth and survival of tumor cells.

Immunoglobulins (Igs) are found thus far only to be produced by differentiated B lymphocytes. By immunohistochemistry analysis, in situ hybridization, and laser capture microdissection-assisted single-cell PCR, we demonstrate that human cancers of epithelial origin, including carcinomas of breast, colon, liver, lung, established epithelial cancer lines, as well as some normal lung tissues, also produce IgG in both cytoplasmic and secreted forms. Furthermore, blockade of tumor-derived IgG by either antisense DNA or antihuman IgG antibody increased programmed cell death and inhibited growth of cancer cells in vitro. More importantly, administration of antihuman IgG antibody also suppressed the growth of an IgG-secreting carcinoma line in immunodeficient nude mice. Our results support a role of tumor-derived IgG as growth factor for epithelial cancers. Prevalent expression of IgG in human carcinomas and its growth-promoting functions may have important implications in growth regulation and targeted therapy of human cancers.

Amino Acid Sequence↗
PubMed2003

[Construction and expression analysis of recombinant vector pcDNA3.1+-HIF-1alpha].

OBJECTIVE: To construct the eukaryotic expression vector for human hypoxia-inducible factor-1alpha (HIF-1alpha) gene and examine its expression. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on the total RNA extracted from HT29 cells to obtain the cDNA of HIF-1alpha, which was inserted into T vector pUC18. DNA sequencing was performed before the amplified products were cloned into the eukaryotic expression vector pcDNA3.1+ identified by endonuclease digestion. This recombinant vector was transfected into HEK293 cells by means of liposome and its expression examined. RESULTS: The amplified products were confirmed as the cDNA of HIF-1alpha by DNA sequencing, and pcDNA3.1+ -HIF-1alpha obtained was verified by endonuclease digestion, being capable of expression in HEK293 cells. CONCLUSION: We have successfully constructed the eukaryotic expression vector for HIF-1alpha, which can be expressed in HEK293 cells.

Cloning, Molecular↗
PubMedDate not supplied

The antitumor activity of the somatostatin structural derivative (TT-232) on different human tumor xenografts.

A somatostatin structural derivative (TT-232) has been developed in our laboratory with strong antiproliferative effect but no GH- release inhibitory activity. TT-232 inhibited tyrosine kinase activity of tumor cells lines and this inhibition correlated well with the inhibition of cell proliferation. The antineoplastic activity of TT-232 has been found to be associated with induction of programmed cell death (apoptosis) in tumor cell, resulting in highly selective elimination of neoplastic tissue. The aim of this study was the therapeutic efficacy of TT-232 on different human models: PC-3 prostate carcinoma, MDA-MB-231 (ER-) and MCF-7 (ER+) breast carcinoma, HT-29 colon carcinoma, HT-18 melanoma, HL-60 promyelocytic leukemia. We studied the therapeutic efficacy of the novel somatostatin analog, it for 30 days with intermittent injection once daily and for 14 days with s.c. infusion using the Alzet osmotic minipump (model 2002). The antitumor activity of TT-232 was evaluated on the basis of survival time and tumor growth inhibition. The tumor growth inhibitory effect of TT-232 on human tumor xenografts proved to be significant, resulting in 30%-80% decrease in tumor volume and in 20-60% tumor free animals. This antitumor efficacy of the novel somatostatin analog was observable in almost all tumors investigated. These data suggest that the novel somatostatin analog (TT-232) is an effective and promising antitumor agent.

Antineoplastic Agents↗