calcimycin and Stomach-Neoplasms

Previous studies have revealed the critical role of transglutaminase 2 (TGM2) as a potential therapeutic target in cancers, but the oncogenic roles and underlying mechanisms of TGM2 in gastric cancer (GC) are not fully understood. In this study, we examined the role and potential mechanism of TGM2 in GC.. Western blotting, immunohistochemistry, CCK8, colony formation and transwell assays were used to measure TGM2 expression in the GC cells and tissues and to examine the in vitro role of TGM2 in GC. Xenograft and in vivo metastasis experiments were performed to examine the in vivo role of TGM2 in GC. Gene set enrichment analysis, quantitative PCR and western blotting were conducted to screen for potential TGM2 targets involved in GC. Gain/loss-of-function and rescue experiments were conducted to detect the biological roles of STAT1 in GC cells in the context of TGM2. Co-immunoprecipitation, mass spectrometry, quantitative PCR and western blotting were conducted to identify STAT1-interacting proteins and elucidate their regulatory mechanisms. Mutations in TGM2 and two molecules (ZM39923 and A23187) were used to identify the enzymatic activity of TGM2 involved in the malignant progression of GC and elucidate the underlying mechanism.. In this study, we demonstrated elevated TGM2 expression in the GC tissues, which closely related to pathological grade, and predicted poor survival in patients with GC. TGM2 overexpression or knockdown promoted (and inhibited) cell proliferation, migration, and invasion, which were reversed by STAT1 knockdown or overexpression. Further studies showed that TGM2 promoted GC progression by inhibiting STAT1 ubiquitination/degradation. Then, tripartite motif-containing protein 21 (TRIM21) was identified as a ubiquitin E3 ligase of STAT1 in GC. TGM2 maintained STAT1 stability by facilitating the dissociation of TRIM21 and STAT1 with GTP-binding enzymatic activity. A23187 abolished the role of TGM2 in STAT1 and reversed the pro-tumor role of TGM2 in vitro and in vivo.. This study revealed a critical role and regulatory mechanism of TGM2 on STAT1 in GC and highlighted the potential of TGM2 as a therapeutic target, which elucidates the development of medicine or strategies by regulating the GTP-binding activity of TGM2 in GC.

Topics: Calcimycin; Cell Line, Tumor; Guanosine Triphosphate; Humans; Protein Glutamine gamma Glutamyltransferase 2; STAT1 Transcription Factor; Stomach Neoplasms; Ubiquitination

After incubation with 2-butylamino-2-demethoxy-hypocrellin A (2-BA-2-DMHA), photodynamically induced change in the cytoplasmic free calcium concentration ([Ca(2+)](i)) and its effect on cell damage were investigated in human gastric cancer (MGC-803). Fluorescence spectrophotometry measurement indicated that the photosensitization of MGC-803 by 2-BA-2-DMHA caused an increase in intracellular calcium [Ca(2+)](i), and this increase in [Ca(2+)](i) showed a dependence on the concentration of 2-BA-2-DMHA, light dose and extracellular [Ca(2+)](e). This phenomenon of intracellular calcium accumulation was further confirmed by using laser scanning confocal microscopy (LSCM). Furthermore, the results from MTT assay and flow cytometry analysis suggested that chelation of extracellular calcium by EGTA or intracellular calcium by BAPTA could inhibit photodynamically induced cell killing, while increase of [Ca(2+)](i) by thapsigargin (TG), a highly specific inhibitor of the Ca(2+)-ATPase, or by A23187, a calcium ionophore could enhance this action. Meanwhile, the nucleus morphology was also investigated by fluorescence microscopy. The results indicated that the increase in intracellular Ca(2+) concentration was responsible for 2-BA-2-DMHA photodynamically induced damage to MGC-803.

Topics: Calcimycin; Calcium; Cell Survival; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Humans; Ionophores; Light; Microscopy, Confocal; Microscopy, Fluorescence; Perylene; Photochemotherapy; Photosensitizing Agents; Quinones; Stomach Neoplasms; Thapsigargin; Tumor Cells, Cultured

HGT cells are a human gastric tumor cell line. Preliminary data have shown that HGT cells incorporate exogenous arachidonic acid (AA) in their membrane lipids. However, we found that HGT cells are unable to produce significant amounts of AA metabolites after stimulation with calcium ionophore A23187. Furthermore, no lipoxygenase activity was detected in crude HGT cell extracts by employing an assay monitoring the in vitro utilization of linoleic acid. The meaning of these results is discussed in respect of the role of eicosanoids during cell proliferation.

Topics: Calcimycin; Eicosanoids; Humans; Linoleic Acid; Linoleic Acids; Stomach Neoplasms; Tumor Cells, Cultured

The receptor for hepatocyte growth factor/scatter factor (HGF/SF) is an alpha beta tyrosine kinase of 190 kDa which mediates growth and motility in several cell types. We have previously shown that tyrosine autophosphorylation enhances the receptor kinase activity, while serine phosphorylation by protein kinase C or other Ca(2+)-dependent kinase(s) is inhibitory. We now identify Ser985 as the major phosphorylation site for the protein kinases responsible for such inhibition. Both phorbol esters or Ca2+ ionophore treatment induces phosphorylation of the same tryptic phosphopeptide corresponding to the sequence Leu983-Arg987 located in the juxta-membrane domain of the receptor beta chain. Purified protein kinase C phosphorylates in vitro a synthetic peptide (V14S) including Ser985. Trypsin digestion of the phosphorylated V14S generates a single phosphopeptide comigrating in reverse-phase high performance liquid chromatography with the tryptic peptide phosphorylated in vivo. Phorbol ester treatment of cultured cells inhibits the ligand-induced tyrosine autophosphorylation of the receptor. In vitro, Ser985 phosphorylation inhibits the receptor tyrosine kinase activity on exogenous substrates. Substitution of Ser985 by site-directed mutagenesis results in increased tyrosine phosphorylation of the receptor and abolishes down-modulation by protein kinase C. These data show that phosphorylation of Ser985 is a key mechanism for the negative regulation of HGF/SF receptor.

Topics: Amino Acid Sequence; Calcimycin; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Consensus Sequence; Hepatocyte Growth Factor; Humans; Kinetics; Molecular Sequence Data; Peptide Fragments; Phosphates; Phosphopeptides; Phosphorylation; Protein Kinase C; Proto-Oncogene Proteins c-met; Proto-Oncogenes; Receptor Protein-Tyrosine Kinases; Sequence Homology, Amino Acid; Serine; Stomach Neoplasms; Substrate Specificity; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The influence of recombinant human tumor necrosis factor-alpha (TNF-alpha) and calcium ionophore A23187 on luminol- and lucigenin-dependent chemiluminescence capacity (CL) of human polymorphonuclear leukocytes (PMN) has been studied. The CL response of TNF-alpha treated PMN is amplified by lucigenin, but not luminol. TNF-alpha and A23287 synergistically induced both the luminol- and lucigenin-dependent early CL response. The combination of A23187 and activator of protein kinase C--phorbol (myristoyl-13-acetyl)--also provoked early CL response. While the combination of TNF-alpha and A23187 decreased late CL response compared to A23187 alone. The obtained results suggests that synergistic CL response of PMN induced by TNF-alpha and A23187 is connected with activation of protein kinase by TNF-alpha.

Topics: Acridines; Calcimycin; Cells, Cultured; Humans; Luminescent Measurements; Luminol; Neutrophils; Oxygen; Precancerous Conditions; Recombinant Proteins; Stomach Neoplasms; Tetradecanoylphorbol Acetate; Time Factors; Tumor Necrosis Factor-alpha