calcimycin and Neoplasm-Metastasis

We have isolated a novel soluble factor(s), neutrophil activator of matrix metalloproteinases (NAM), secreted by unstimulated normal human peripheral blood neutrophils that causes the activation of cell secreted promatrix metalloproteinase-2 (proMMP-2). Partially purified preparations of NAM have been isolated from the conditioned media of neutrophils employing gelatin-Sepharose chromatography and differential membrane filter centrifugation. NAM activity, as assessed by exposing primary human umbilical vein endothelial cells (HUVEC) or HT1080 cells to NAM followed by gelatin zymography, was seen within one hour. Tissue inhibitor of metalloproteinase-2 (TIMP-2) and hydroxamic acid derived inhibitors of MMPs (CT1746 and BB94) abrogated the activation of proMMP-2 by NAM, while inhibitors of serine and cysteine proteases showed no effect. NAM also produced an increase in TIMP-2 binding to HUVEC and HT1080 cell surfaces that was inhibited by TIMP-2, CT1746, and BB94. Time-dependent increases in MT1-MMP protein and mRNA were seen following the addition of NAM to cells. These data support a role for NAM in cancer dissemination.

Topics: Adult; Amides; Calcimycin; Cathepsin G; Cathepsins; Cells, Cultured; Culture Media, Conditioned; Endothelium, Vascular; Enzyme Activation; Enzyme Precursors; Humans; Inflammation; Ionomycin; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Neoplasm Metastasis; Neutrophils; Oligopeptides; Pancreatic Elastase; Phenylalanine; Protease Inhibitors; Proteins; RNA, Messenger; Serine Endopeptidases; Substrate Specificity; Tetradecanoylphorbol Acetate; Thiophenes; Tissue Inhibitor of Metalloproteinase-2; Umbilical Veins

We investigated whether alterations in the mechanisms involved in intracellular pH (pHin) and intracellular calcium ([Ca2+]in) homeostasis are associated with the metastatic potential of poorly (A375P) and highly (C8161) metastatic human melanoma cells. We monitored pHin and [Ca2+]in simultaneously, using the fluorescence of SNARF-1 and Fura-2, respectively. Our results indicated that steady-state pHin and [Ca2+]in between these cell types were not significantly different. Treatment of cells with NH4Cl resulted in larger pHin increases in highly than in poorly metastatic cells, suggesting that C8161 cells have a lower H+ buffering capacity than A375P. NH4Cl treatment also increased [Ca2+]in only in C8161 cells. To determine if the changes in [Ca2+]in triggered by NH4Cl treatment were due to alterations in either H+- or Ca2+-buffering capacity, cells were treated with the Ca2+-ionophore 4Br-A23187, to alter [Ca2+]in. The magnitude of the ionophore-induced [Ca2+]in increase was slightly greater in C8161 cells than in A375P. Moreover, A375P cells recover from the ionophore-induced [Ca2+]in load, whereas C8161 cells did not, suggesting that A375P may exhibit distinct [Ca2+]in regulatory mechanisms than C8161 cells, to recover from Ca2+ loads. Removal of extracellular Ca2+ ([Ca2+]ex) decreased [Ca2+]in in both cell types at the same extent. Ionophore treatment in the absence of [Ca2+]ex transiently increased [Ca2+]in in C8161, but not in A375P cells. Endoplasmic reticulum (ER) Ca2+-ATPase inhibitors such as cyclopiazonic acid (CPA) and thapsigargin (TG) increased steady-state [Ca2+]in only in C8161 cells. Together, these data suggest that the contribution of intracellular Ca2+ stores for [Ca2+]in homeostasis is greater in highly than in poorly metastatic cells. Bafilomycin treatment, to inhibit V-type H+-ATPases, corroborated our previous results that V-H+-ATPases are functionally expressed at the plasma membranes of highly metastatic, but not in poorly metastatic cells (Martínez-Zaguilán et al., 1993). Collectively, these data suggest that distinct pHin and [Ca2+]in regulatory mechanisms are present in poorly and highly metastatic human melanoma cells.

Topics: Ammonium Chloride; Anti-Bacterial Agents; Benzopyrans; Calcimycin; Calcium; Calcium-Transporting ATPases; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Homeostasis; Humans; Hydrogen-Ion Concentration; Indoles; Ionophores; Macrolides; Melanoma; Naphthols; Neoplasm Metastasis; Proton-Translocating ATPases; Rhodamines; Thapsigargin; Tumor Cells, Cultured

Adhesion of tumor cells (TC) to endothelial cells (EC) is necessary for movement of TC out of the interstitium to form metastatic deposits. This interaction may be influenced by proadhesive molecules such as lipoxygenase products of arachidonic acid metabolism. We studied the effect of inflammatory stimuli, A23187 calcium ionophore, n-formyl-methionyl-leucine-phenylalanine (FMLP) and phorbol myristate acetate (PMA) on TC-EC interaction. Adherence of metastatic breast tumor cell line (MCF-7), choriocarcinoma cell line (JEG-3), and non metastatic pituitary cell (GH-3) were assayed as the number of radiolabeled TC attached to EC (cpm/well). TC and EC were incubated with A23187, FMLP, and PMA for varying time periods. Lipoxygenase products (LTB4, 5-HETE) were measured under basal and stimulated conditions using RP-HPLC and RIA. There were no differences in basal adherence of TC lines to EC. When EC were incubated with stimuli, there were significant increases in the numbers of MCF-7 and JEG-3 cells adherent to EC compared to GH-3. Light and phase contrast microscopy confirmed that TC were attached to EC. Upon stimulation, GH-3 preferentially produced prostaglandins (PGI1(2)) while MCF-7 and JEG-3 produced lipoxygenase products (LTB4 and 5-HETE). Pre-incubation of MCF-7 and JEG-3 with the lipoxygenase inhibitor nordihydroguiaretic acid resulted in partial inhibition of adhesion to EC. Our data strongly indicate a role for lipoxygenase products of arachidonic acid in adherence of TC to EC.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonic Acid; Calcimycin; Cattle; Cell Adhesion; Endothelium, Vascular; Humans; Lipoxygenase; Lipoxygenase Inhibitors; Masoprocol; N-Formylmethionine Leucyl-Phenylalanine; Neoplasm Metastasis; Neoplasms; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

In the present study, we described the effects of three different drugs, A23187 calcium ionophore (A23187), indomethacin (IND) and phorbol myristate acetate (PMA), on the secretion pattern and the metastatic potential in the low metastatic 3LL tumor cell line. The results evidence that a low molecular weight protein fraction is always secreted in an higher amount in drug treated cells than in untreated cells independently of the drug used. In addition to their effects on secretion pattern, the three drugs assayed enhance noticeably the metastatic rate in vivo of the 3LL cells. The data confirm our earlier observations showing that our high performance gel permeation chromatography (HPGPC) method is suitable for discriminating even between cells with a different degree (low and high) of metastatic potential by its chromatographic secretion pattern and that alterations in the secretion pattern from treated cells are related with changes in the degree of metastatic potential of these cells.

Topics: Animals; Calcimycin; Female; Indomethacin; Lung Neoplasms; Mice; Mice, Inbred BALB C; Molecular Weight; Neoplasm Metastasis; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Cultured Lewis Lung carcinoma cells (3LL) treated with A23187 calcium ionophore were studied by transmission electron microscopy and HPLC analysis. Results showed that A23187 calcium ionophore induces on osmiophilic inclusion bodies of 3LL cells similar process of lamellar bodies formation to those reported in the development of the osmiophilic inclusions bodies within the granular pneumocyte of normal lungs. HPLC analysis shows that calcium ionophore generates quantitative changes in the 3LL cytoplasmic protein content expressed by an increase of 18-22 kDa and 4-9 kDa proteins.

Topics: Animals; Calcimycin; Cytoplasm; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Proteins; Tumor Cells, Cultured

Evidence is provided to show that two secondary cell-signaling pathways, Ca2+ mobilization and the activation of protein kinase C (PKC), are involved in the induction of spontaneous metastasis in mouse adenocarcinoma cell line SP1. Unlike the parental cells, which were found to be tumorigenic but unable to metastasize from a s.c. site, SP1 cells treated with ionophore A23187 (to mobilize Ca2+) or phorbol 12-myristate 13-acetate (to activate PKC) were able to metastasize spontaneously. Analysis of SP1 cells treated with either agent separately or with both agents simultaneously revealed that both pathways contributed to the final response in a separate and nonsynergistic way. The induced metastatic phenotype in most cases appeared to be heritable. Examination of Ca2+ sources during cell activation by ionophore A23187 suggested that internal Ca2+ was sufficient for the process of induction. Examination of PKC activity and its intracellular distribution during and after treatment of SP1 cells with ionophore A23187 and phorbol 12-myristate 13-acetate were also evaluated. The results suggested that the basal levels of PKC and the activation of the enzyme appear to be involved in the induction of spontaneous metastasis. Taken together, these observations are consistent with the hypothesis that cell-signaling pathways exist which can induce the metastatic phenotype and that this may be related to phosphatidylinositol turnover.

Topics: Animals; Calcimycin; Calcium; DNA; Enzyme Activation; Mammary Neoplasms, Experimental; Mice; Mice, Inbred CBA; Neoplasm Metastasis; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The peptide N-formyl-Met-Leu-Phe stimulates chemotaxis and metastasis in rat Walker carcinosarcoma cells by a receptor-mediated pathway. Since oxygen radical generation follows chemotactic stimulation in leukocytes, we looked for similar responses in the Walker tumor. Upon incubation with 10(-6) M N-formyl-Met-Leu-Phe, Walker cells elicited chemiluminescence in the presence of 5 X 10(-5) M luminol. The response peaked within 2 min and was maintained for greater than 20 min; it was dose dependent with a 50% maximal effective dose (ED50) value of 4.5 X 10(-8) comparable to the 50% maximal effective dose value for chemotaxis. The responses were significantly reduced but not abolished in the absence of calcium in the external medium and were elicited by the ionophore A23187. The lipoxygenase inhibitor nordihydroguaiaretic acid had almost no effect in decreasing the response, while flurbiprofen, a cyclooxygenase inhibitor was very effective at 10(-6) M. Evidence for the generation of oxygen radicals included: (a) marked inhibition of light emission in the absence of oxygen; (b) inhibition in the presence of superoxide dismutase, catalase, and mannitol; and (c) dose-dependent reduction of acetylated cytochrome c. We postulate that activation of circulating tumor cells may facilitate metastasis by the release of toxic oxygen species.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Carcinoma 256, Walker; Cell Line; Colchicine; Dose-Response Relationship, Drug; Free Radicals; Lipid Peroxides; Luminescent Measurements; Luminol; Male; N-Formylmethionine Leucyl-Phenylalanine; Neoplasm Metastasis; Oxygen; Rats; Rats, Inbred Strains; Superoxides

The purpose of this investigation was to determine whether cells transformed by herpes simplex virus type 2 (HSV-2) can be stimulated to synthesize prostaglandins (PG). Stimulation was determined by measuring the release of PG into overlay fluids from cell monolayers prelabeled with [3H]arachidonic acid. Results showed that Ca2+ ionophore A-23187 markedly stimulated arachidonic acid release starting 30 min after treatment of HSV-2-transformed and nontransformed rat embryo fibroblast cells. However, only HSV-2-transformed cells were stimulated in production of PG. HSV-2-transformed, nontumorigenic, rat embryo fibroblast, line G, clone 2.0 cells synthesize nearly equal amounts of prostaglandin E2 (PGE2) and prostaglandin F2 alpha, while tumor (rat fibrosarcoma) cells synthesize primarily PGE2. Stimulation of PGE2 synthesis by Ca2+ ionophore A-23187 or 12-O-tetradecanoyl-phorbol-13-acetate decreased as rat fibrosarcoma cells were serially passaged in tissue culture. At low passage of parental rat fibrosarcoma cells, four distinct morphological clonal cell lines were isolated, which varied markedly in their capacity to be stimulated in PG synthesis by 12-O-tetradecanoyl-phorbol-13-acetate. There was correlation between the capacity of clone 1 cells to be stimulated in PGE2 synthesis by serum alone and capacity of the tumors produced by the clone 1 cells to metastasize to the lungs of syngeneic tumor-bearing rats. In summary, cell transformation by HSV-2 appears to be essential for stimulation of PG synthesis in cells. The capacity to be stimulated in arachidonic acid metabolism and PG synthesis may be important in the process of carcinogenesis by a putative human cancer virus.

Topics: Animals; Calcimycin; Cell Division; Cell Transformation, Neoplastic; Clone Cells; Dinoprost; Dinoprostone; Embryo, Mammalian; Fibrosarcoma; Kinetics; Neoplasm Metastasis; Phorbols; Prostaglandins; Prostaglandins E; Prostaglandins F; Rats; Simplexvirus; Tetradecanoylphorbol Acetate