sq-23377 and Stomach-Neoplasms

Hypoxia-inducible factor 1alpha (HIF-1alpha) has been reported to be expressed aberrantly in gastric cancer cells. Stability and transactivation of HIF-1 were associated with the change of intracellular calcium. We hypothesized that KCl depolarization may modulate HIF-1 activity in gastric cancer cells through calcium involvement.. HIF-1alpha expression and its transcriptional activity were determined in SGC7901 gastric cancer cells treated with KCl and/or CoCl2 under normoxia. KCl induced change in the intracellular free calcium concentration and its effect on HIF-1 activity was investigated subsequently.. Exposure of SGC7901 cells to KCl (50 mM) could induce HIF-1alpha expression and its nucleus accumulation under normoxic conditions, reaching the peak at 8 and 2 h, respectively. KCl could also induce transactivation of the HIF-1 reporter gene and its target gene VEGF secretion at 8 h. Further experiments confirmed that depolarization of SGC7901 cells with KCl caused an increase in intracellular free calcium concentration. Chelation of intracellular calcium by BAPTA [1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid] induced HIF-1alpha accumulation and HIF-1 activity. However, elevation of cytosolic calcium level by ionomycin, a calcium ionophore, failed to induce HIF-1 transcriptional activity.. KCl depolarization would act through the calcium-independent pathway leading to enhanced HIF-1 transcriptional activity in gastric cancer cells.

Topics: Cell Line, Tumor; Egtazic Acid; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Ionomycin; Luciferases; Potassium Chloride; Reverse Transcriptase Polymerase Chain Reaction; Stomach Neoplasms; Transcription, Genetic; Transfection

Dihydropyrimidine dehydrogenase is the most extensively investigated predictive marker for individual response to 5-fluorouracil. Clinical responses to the anticancer agent, along with various reports, have clearly shown that dihydropyrimidine dehydrogenase activity is closely correlated to its mRNA levels, but the regulatory mechanisms of its expression have remained unclear. We attempted to clarify the mechanisms and found that activator protein (AP-1) is probably one of the key factors in the transcriptional regulation of DPYD in cancer cells, and that phorbol 12-myristate 13-acetate (PMA) plus ionomycin treatment enhances transcription of DPYD via AP-1 activation. In this study, we characterized our previously subcloned 5' region of human DPYD, an approximately 3.0-kb fragment (accession no. AB162145). Luciferase reporter assay showed that the clone showed strong promoter activities in 293T and HSC42 cells, and comparative analysis using 5' deletion mutants suggested the existence of several positive and negative regulatory regions, including putative binding sites for AP-1, SP-1, and nuclear factor-kappaB. PMA/ionomycin treatment increased the mRNA level of DPYD in HSC42 cells, and electrophoretic gel mobility shift assay showed that the complex on the putative AP-1 binding site was drastically induced by PMA/ionomycin treatment. The complexes formed were competed out by preincubation with the cold-consensus AP-1 binding site, and the DNA binding complex formed on the site contained c-Jun and c-Fos, which are components of AP-1 transcription factor. We further identified the functional AP-1 binding site (nucleotide positions from -290 to -280), whose nucleotide mutations abolished PMA/ionomycin-induced DPYD promoter activation.

Topics: Binding Sites; Biomarkers, Tumor; Cell Line, Transformed; Cell Line, Tumor; Dihydrouracil Dehydrogenase (NADP); Humans; Ionomycin; Kidney; Promoter Regions, Genetic; RNA, Messenger; Stomach Neoplasms; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription, Genetic; Transcriptional Activation; Transfection

Resveratrol is a dietary phytochemical that has been shown to inhibit proliferation of a number of cell lines, and it behaves as a chemopreventive agent in assays that measure the three stages of carcinogenesis. We tested for its chemopreventive potential against gastric cancer by determining its interaction with signaling mechanisms that contribute to the proliferation of transformed cells. Low levels of exogenous reactive oxygen (H(2)O(2)) stimulated [(3)H]thymidine uptake in human gastric adenocarcinoma SNU-1 cells, whereas resveratrol suppressed both synthesis of DNA and generation of endogenous O(2)(-) but stimulated nitric oxide (NO) synthase (NOS) activity. To address the role of NO in the antioxidant action of resveratrol, we measured the effect of sodium nitroprusside (SNP), an NO donor, on O(2)(-) generation and on [(3)H]thymidine incorporation. SNP inhibited DNA synthesis and suppressed ionomycin-stimulated O(2)(-) generation in a concentration-dependent manner. Our results revealed that the antioxidant action of resveratrol toward gastric adenocarcinoma SNU-1 cells may reside in its ability to stimulate NOS to produce low levels of NO, which, in turn, exert antioxidant action. Resveratrol-induced inhibition of SNU-1 proliferation may be partly dependent on NO formation, and we hypothesize that resveratrol exerts its antiproliferative action by interfering with the action of endogenously produced reactive oxygen. These data are supportive of the action of NO against reactive oxygen and suggest that a resveratrol-rich diet may be chemopreventive against gastric cancer.

Topics: Adenocarcinoma; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Division; DNA; Humans; Hydrogen Peroxide; Ionomycin; Ionophores; NADP; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Oxidants; Protein Kinase C; Reactive Oxygen Species; Resveratrol; Stilbenes; Stomach Neoplasms; Superoxides; Tumor Cells, Cultured

Analysis of serum cytokine levels has shown that cancer-bearing hosts have lower levels of IL-2 and IFN-gamma, suggesting that Th1-type immunity is impaired by cancer. However, the mechanisms of the Th1 dysfunction are not clearly understood.. The frequencies of Th1 cells in CD4(+) helper T cells were evaluated with an intracytoplasmic cytokine staining method in peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL) of patients with gastrointestinal cancer.. Activation of lymphocytes with PMA + Ionomycin induced the expression of IL-2 and IFN-gamma in each lymphocyte population. Compared with PBL of non-malignant donors, PBL in cancer patients contained significantly lower frequencies of CD4(+) T cells that produced IL-2 and IFN-gamma. LNL in cancer patients also contained lower levels of IL-2- and IFN-gamma-producing CD4(+) T cells, although the percentages did not show significant differences from those of PBL in the same patients.. Our data suggest that suppression of Th1 cytokine in cancer patients is, at least in part, due to the decreased frequency of Th1 cells with CD4(+) phenotype.

Topics: CD4-Positive T-Lymphocytes; Colorectal Neoplasms; Cytokines; Humans; Interferon-gamma; Interleukin-2; Ionomycin; Lymph Nodes; Lymphocyte Activation; Phenotype; Phytohemagglutinins; Stomach Neoplasms; T-Lymphocytes, Helper-Inducer; Th1 Cells