thapsigargin and Neoplasm-Metastasis

Topics: Apoptosis; Gene Expression; Humans; Male; Neoplasm Metastasis; Prodrugs; Prostatic Neoplasms; Sesquiterpenes; Thapsigargin

HAb18G/CD147 is a heavily glycosylated protein containing two immunoglobulin superfamily domains. Our previous studies have indicated that overexpression of HAb18G/CD147 enhances metastatic potentials in human hepatoma cells by disrupting the regulation of store-operated Ca2+ entry by nitric oxide (NO)/cGMP. In the present study, we investigated the structure-function of HAb18G/CD147 by transfecting truncated HAb18G/CD147 fragments into human 7721 hepatoma cells. The inhibitory effect of HAb18G/CD147 on 8-bromo-cGMP-regulated thapsigargin-induced Ca2+ entry was reversed by the expression of either C or N terminus truncated HAb18G/CD147 in T7721deltaC and T7721deltaN cells, respectively. The potential effect of HAb18G/CD147 on metastatic potentials, both adhesion and invasion capacities, of hepatoma cells was abolished in T7721deltaC cells, but not affected in T7721deltaN cells. Release and activation of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were found to be enhanced by the expression of HAb18G/CD147, and this effect was abolished by both truncations. Thapsigargin significantly enhanced release and activation of MMPs (MMP-2 and MMP-9) in non-transfected 7721 cells, and this effect was negatively regulated by SNAP. However, no effects of thapsigargin or SNAP were observed in T7721 cells, and expression of HAb18G/CD147 enhanced secretion and activation of MMPs at a stable and high level. Taken together, these results suggest that both ectodomain and intracellular domains of HAb18G/CD147 are required to mediate the effect of HAb18G/CD147 on the secretion and activation of MMPs and metastasis-related processes in human hepatoma cells by disrupting the regulation of NO/cGMP-sensitive intracellular Ca2+ mobilization although each domain may play different roles.

Topics: Antigens, CD; Antigens, Neoplasm; Basigin; Calcium; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Neoplasm Metastasis; Protein Structure, Tertiary; Thapsigargin

The present study examined the effect of hepatoma-associated antigen HAb18G (homologous to CD147) expression on the NO/cGMP-regulated Ca(2+) mobilization and metastatic process of human hepatoma cells. HAb18G/CD147 cDNA was transfected into human 7721 hepatoma cells to obtain a cell line stably expressing HAb18G/CD147, T7721, as demonstrated by Northern blot and immunocytochemical studies. 8-Bromo-cGMP (cGMP) inhibited the thapsigargin-induced Ca(2+) entry in a concentration-dependent manner in 7721 cells. The cGMP-induced inhibition was abolished by an inhibitor of protein kinase G, KT5823 (1 microm). However, expression of HAb18G/CD147 in T7721 cells decreased the inhibitory response to cGMP. A similar concentration-dependent inhibitory effect on the Ca(2+) entry was observed in 7721 cells in response to a NO donor, (+/-)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca(2+) entry was significantly reduced in HAb18G/CD147-expressing T7721 cells, indicating a role for HAb18G/CD147 in NO/cGMP-regulated Ca(2+) entry. Experiments investigating metastatic potentials demonstrated that HAb18G/CD147-expressing T7721 cells attached to the Matrigel-coated culture plates and invaded through Matrigel-coated permeable filters at the rate significantly greater than that observed in 7721 cells. Both the attachment and invasion rates could be suppressed by SNAP, and the inhibitory effect of SNAP could be reversed by NO inhibitor, N(G)-nitro-l-arginine methyl ester. The sensitivity of the attachment and invasion rates to cGMP was significantly reduced in T7721 cells as compared with 7721 cells when cells were pretreated with thapsigargin. The difference in the sensitivity between the two cells could be abolished by a Ca(2+) channel blocker, Ni(2+) (3 mm). These results suggest that HAb18G/CD147 enhances metastatic potentials in human hepatoma cells by disrupting the regulation of store-operated Ca(2+) entry by NO/cGMP.

Topics: Alkaloids; Antigens, CD; Antigens, Neoplasm; Antigens, Surface; Avian Proteins; Basigin; Blood Proteins; Blotting, Northern; Calcium; Carbazoles; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line; Cell Movement; Collagen; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; DNA, Complementary; Dose-Response Relationship, Drug; Drug Combinations; Enzyme Inhibitors; Humans; Immunohistochemistry; Indoles; Laminin; Liver Neoplasms; Membrane Glycoproteins; Neoplasm Metastasis; NG-Nitroarginine Methyl Ester; Nickel; Nitric Oxide; Penicillamine; Proteoglycans; Signal Transduction; Thapsigargin; Time Factors; Transfection; Tumor Cells, Cultured

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

The aim of this study was to determine whether stable differences in apoptosis sensitivity were selected for in nonmetastatic and metastatic variants of the LNCaP human prostate carcinoma line that had been isolated from tumors grown orthotopically in the prostate glands and regional lymph nodes of nude mice. The nonmetastatic LNCaP-Pro5 cells were significantly more sensitive to thapsigargin-induced apoptosis than were the metastatic LNCaP-LN3 cells, as measured by viability, DNA fragmentation, and interleukin 1beta-converting enzyme family-mediated cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase. Apoptosis resistance in the metastatic cells was associated with higher levels of expression of the cell death suppressor BCL-2 and lower levels of the death promoters BAX and BAK than were detected in the nonmetastatic LNCaP-Pro5 cells, whereas levels of two other BCL-2 family members (BCL-X(L) and BAD) were indistinguishable. Our data support the hypothesis that apoptosis resistance contributes to prostate cancer metastasis and that elevated expression of BCL-2 is involved.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Carcinogens; Doxorubicin; Drug Resistance; Humans; Male; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Proteins; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proteins; Proto-Oncogene Proteins c-bcl-2; Thapsigargin; Tumor Cells, Cultured

MTS1 is a metastasis-associated gene highly expressed in highly metastatic tumours. NM23 has been described as a putative metastasis suppressor gene. Here we show that thapsigargin (which raises intracellular calcium [Ca2+]i from intracellular stores) and verapamil (which blocks Ca2+ influx) both down-regulate MTS1 and NM23 gene expression in the poorly metastatic F1 and highly metastatic ML8 variants of the B16 murine melanoma without altering their metastatic behaviour. The data presented here suggest that Ca2+ released from intracellular stores could be functionally differentiated from influxed Ca2+ and could be activating different components of the Ca2+ signalling system. Many of the cellular responses to calcium are mediated through calmodulin. We have therefore further investigated the role of Ca2+ in the regulation of the MTS1 and NM23 genes using the calmodulin inhibitor W-7. Both these genes were down-regulated after treatment of the F1 and ML8 cell variants. We have shown previously that retinoic acid reduces lung colonization by the highly metastatic variant ML8 and that melanocyte stimulating hormone (MSH) enhances lung colonization by the poorly metastatic variant F1, with corresponding changes in the relative expression of NM23 and MTS1. Here we have found that verapamil and thapsigargin have no effect on lung colonization, possibly due to both genes being down-regulated. These data support the concept that NM23 and MTS1 gene expression is linked and that metastatic potential may be determined by their relative expression.

Topics: Animals; Blotting, Northern; Calcium; Calmodulin; Female; Gene Expression; Genetic Variation; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Proteins; RNA, Neoplasm; Sulfonamides; Terpenes; Thapsigargin; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured; Verapamil