thapsigargin and Carcinoma--Hepatocellular

Drug resistance causes therapeutic failure in refractory cancer. Cancer drug resistance stems from various factors, such as patient heterogeneity and genetic alterations in somatic cancer cells, including those from identical tissues. Generally, resistance is intrinsic for cancers; however, cancer resistance becomes common owing to an increased drug treatment. Unfortunately, overcoming this issue is not yet possible. The present study aimed to evaluate a clinical approach using candidate compounds 19 and 23, which are sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) inhibitors, discovered using the evolutionary chemical binding similarity method. mRNA sequencing indicated SERCA as the dominant marker of patient-derived anti-cancer drug-resistant hepatocellular carcinoma (HCC), but not of patient-derived anti-cancer drug-sensitive HCC. Candidate compounds 19 and 23 led to significant tumor shrinkage in a tumor xenograft model of anti-cancer drug-resistant patient-derived HCC cells. Our results might be clinically significant for the development of novel combinatorial strategies that selectively and efficiently target highly malignant cells such as drug-resistant and cancer stem-like cells.

Topics: Calcium; Carcinoma, Hepatocellular; Drug Discovery; Endoplasmic Reticulum; Humans; Liver Neoplasms; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

Alpha-1 acid glycoprotein (AGP) is a major acute-phase protein that is involved in drug/ligand binding and regulation of immune response. In response to inflammation, AGP secretion from the liver increases, resulting in elevated concentration of plasma AGP. AGP exhibits multiple N-glycosylation sites, and thus, is highly glycosylated. Although AGP glycosylation is considered to affect its functions, the significance of AGP glycosylation for its secretion is unclear. In this study, we investigated the effects of AGP glycosylation using glycosylation-deficient mouse AGP mutants lacking one, four, or all five N-glycosylation sites. Furthermore, we examined the effects of endoplasmic reticulum (ER) stress-inducing reagents, including tunicamycin and thapsigargin, which induce ER stress in an N-glycosylation-dependent and -independent manner, respectively. Here, we found that glycosylation deficiency and ER stress induce a little or no effect on AGP secretion. Conversely, thapsigargin significantly suppressed AGP secretion in glycosylation-independent manner. These findings indicate that AGP secretion is regulated via thapsigargin-sensitive pathway that might be further controlled by the intracellular calcium concentrations.

Topics: Animals; Calcium; Carcinoma, Hepatocellular; Cell Line, Tumor; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Glycosylation; Mice, Inbred ICR; Mutation; Orosomucoid; Thapsigargin; Tunicamycin

Mitochondria-associated membranes (MAMs) are specific endoplasmic reticulum (ER) domains that enable it to interact directly with mitochondria and mediate metabolic flow and Ca. This work compares mitochondrial/ER contact during combined ER stress/mitochondrial dysfunction using a model of human hepatoma cells (Hep3B cell line) treated for 24 h with classic pharmacological inducers of ER stress (thapsigargin), mitochondrial dysfunction (carbonyl cyanide m-chlorophenyl hydrazone or rotenone) or both (the antiretroviral drug efavirenz used at clinically relevant concentrations).. Markers of mitochondrial dynamics (dynamin-related protein 1, optic atrophy 1 and mitofusin 2) were expressed differently with these stimuli, pointing to a specificity of combined ER/mitochondrial stress. Lon, a matrix protease involved in protein and mtDNA quality control, was up-regulated at mRNA and protein levels under all conditions. However, only efavirenz decreased the mitochondrial content of Lon while increasing its extramitochondrial presence and its localization to MAMs. This latter effect resulted in an enhanced mitochondria/ER interaction, as shown by co-immunoprecipitation experiments of MAMs protein partners and confocal microscopy imaging.. A specific dual drug-induced mitochondria-ER effect enhances the MAMs content of Lon and its extramitochondrial expression. This is the first report of this phenomenon and suggests a novel MAMs-linked function of Lon protease.

Topics: Alkynes; Benzoxazines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclopropanes; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Humans; Liver Neoplasms; Microscopy, Confocal; Mitochondria; Mitochondrial Membranes; Protease La; Rotenone; Thapsigargin

We have recently shown that the histidine-rich calcium binding protein (HRC) promotes the invasion and metastasis of hepatocellular carcinoma (HCC). In the current study, we evaluated whether HRC may also affect the growth of HCC. We found that ectopic expression of HRC obviously enhanced proliferation and colony formation, while suppression of HRC exhibited inhibitory effects. Furthermore, we demonstrated that HRC promoted tumor growth in nude mice. These effects may result from the ability of HRC to upregulate cyclinD1 and cyclin-dependent kinase 2 (CDK2) expressions and promote G1/S transition. Further study showed that MEK/ERK signaling pathway was involved in HRC-induced cell proliferation. Interestingly, overexpression or depletion of HRC revealed its regulation on endoplasmic reticulum stress (ERS) and apoptosis, which was partially dependent on PERK/ATF4/CHOP signaling pathway. In addition, blocking ERS using 4-phenylbutyric acid (4-PBA) not only downregulated the expression of PERK, ATF4 and CHOP, but also significantly decreased apoptosis induced by HRC silence, whereas ERS inducer thapsigargin (TG) exerted the opposite effects. Our study thus demonstrates a role of HRC in promoting HCC growth, besides its role in inducing HCC metastasis, and highlights HRC as a promising intervention target for HCC.

Topics: Activating Transcription Factor 4; Animals; Apoptosis; Calcium-Binding Proteins; Carcinoma, Hepatocellular; Caspase 3; Cell Proliferation; eIF-2 Kinase; Endoplasmic Reticulum Stress; G1 Phase Cell Cycle Checkpoints; Humans; Liver Neoplasms; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Phenylbutyrates; RNA Interference; RNA, Small Interfering; Thapsigargin; Transcription Factor CHOP; Transplantation, Heterologous

Endoplasmic reticulum stress (ERS) has been found in non-alcoholic fatty liver disease. The study was to further explore the mechanistic relationship between ERS and lipid accumulation. To induce ERS, the hepatoblastoma cell line HepG2 and the normal human L02 cell line were exposed to Tg for 48 h. RT-PCR and Western blot were performed to evaluate glucose-regulated protein (GRP-78) expression as a marker of ERS. ER ultrastructure was assessed by electron microscopy. Triglyceride content was examined by Oil Red O staining and quantitative intracellular triglyceride assay. The hepatic nuclear sterol regulatory element-binding protein (SREBP-1c), liver X receptor (LXRs), fatty acid synthase (FAS), and acetyl-coA carboxylase (ACC1) expressions were examined by real-time PCR and Western blot. 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) was used to inhibit S1P serine protease inhibitor, and SREBP-1c cleavage was evaluated under ERS. SREBP-1c was knockdown and its effect on lipid metabolism was observed. Tg treatment upregulated GRP-78 expression and severely damaged the ER structure in L02 and HepG2 cells. ERS increased triglyceride deposition and enhanced the expression of SREBP-1c, FAS, and ACC1, but have no influence on LXR. AEBSF pretreatment abolished Tg-induced SREBP-1c cleavage. Moreover, SREBP-1c silencing reduced triglycerides and downregulated FAS expression. Pharmacological ERS induced by Tg leads to lipid accumulation through upregulation of SREBP-1c in L02 and HepG2 cells.

Topics: Acetyl-CoA Carboxylase; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Fatty Acid Synthases; Fatty Liver; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Heat-Shock Proteins; Humans; Lipid Metabolism; Liver; Liver Neoplasms; Liver X Receptors; Orphan Nuclear Receptors; Proteolysis; RNA, Messenger; Sterol Regulatory Element Binding Protein 1; Sulfones; Thapsigargin; Triglycerides; Up-Regulation

c-Met, the tyrosine-kinase receptor for hepatocyte growth factor, plays a critical role in the tumorigenesis of hepatocellular carcinoma (HCC). However, the underlying mechanism remains incompletely understood. The mature c-Met protein p190Met(αβ) (consists of a α subunit and a β subunit) is processed from pro-Met. Here we show that pro-Met is processed into p190Met(NC) by sarco/endoplasmic reticulum calcium-ATPase (SERCA) inhibitor thapsigargin. p190Met(NC) compensates for the degradation of p190Met(αβ) and protects human HCC cells from apoptosis mediated by endoplasmic reticulum (ER) stress. In comparison with p190Met(αβ), p190Met(NC) is not cleaved and is expressed as a single-chain polypeptide. Thapsigargin-initiated p190Met(NC) expression depends on the disturbance of ER calcium homeostasis. Once induced, p190Met(NC) is activated independent of hepatocyte growth factor engagement. p190Met(NC) contributes to sustained high basal activation of c-Met downstream pathways during ER calcium disturbance-mediated ER stress. Both p38 MAPK-promoted glucose-regulated protein 78 (GRP78) expression and sustained high basal activation of PI3K/Akt and MEK/ERK are involved in the cytoprotective function of p190Met(NC). Importantly, the expression of p190Met(NC) is detected in some HCC cases. Taken together, these data provide a potential mechanism to explain how c-Met promotes HCC cells survival in response to ER stress. We propose that context-specific processing of c-Met protein is implicated in HCC progression in stressful microenvironments.

Topics: c-Mer Tyrosine Kinase; Calcium; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Homeostasis; Humans; Liver Neoplasms; Male; Proteolysis; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction; Thapsigargin

Diabetes and obesity are associated with activation of endoplasmic reticulum (ER) stress; however a direct link between ER stress and increased hepatic gluconeogenesis remains unclear. Here we show that ER stress triggers a significant increase in expression of CCAAT/enhancer-binding protein (C/EBPβ) and phosphorylated CREB together with reduced phospho-AMP-activated protein kinase (pAMPK) in hepatoma cells. ER stress contributed to transcriptional activation of the gluconeogenic phosphoenolpyruvate carboxykinase (PEPCK) promoter in Huh7 and HepG2 cells via cAMP binding motif (CRE site). Chromatin immunoprecipitation assays demonstrate that C/EBPβ is recruited to the PEPCK promoter during ER stress and is reversed by pre-treatment with a JNK inhibitor that relieves ER stress. C/EBPβ but not pCREB was suppressed by the AMPK-activator AICAR or constitutively active AMPK, while dominant negative AMPK increased C/EBPβ expression. These data suggest that ER stress triggers suppression of AMPK while increasing C/EBPβ and pCREB expression which activates PEPCK gene transcription. Understanding how ER stress suppresses AMPK activation and increases C/EBPβ expression could lead to a potentially novel pathway for treatment of diabetes.

Topics: Adenylate Kinase; Animals; Carcinoma, Hepatocellular; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Endoplasmic Reticulum; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Models, Genetic; Phosphoenolpyruvate Carboxykinase (ATP); Protein Binding; Protein Kinase Inhibitors; Rats; Response Elements; Stress, Physiological; Thapsigargin; Up-Regulation

βig-h3 is a TGF-β (transforming growth factor β)-induced ECM (extracellular matrix) protein that induces the secretion of MMPs (matrix metalloproteinases). However, the mechanism of induction is yet to be established. In this study, siRNAs (small interfering RNAs) targeted against βig-h3 were transfected into SMMC-7721 cells [a HCC (human hepatocellular carcinoma) cell line] to knockdown the expression of βig-h3. We found that NiCl2, a potent blocker of extracellular Ca2+ entry, reduced βig-h3-induced secretion of MMP-2 and -9. Further investigation suggested that reduction in the levels of βig-h3 decreased the secretion of MMP-2 and -9 that was enhanced by an increase in the concentration of extracellular Ca2+. SNAP (S-nitroso-N-acetylpenicillamine), a NO (nitric oxide) donor, and 8-Br-cGMP (8-bromo-cGMP) inhibited thapsigargin-induced Ca2+ entry and MMP secretion in the invasive potential of human SMMC-7721 cells. Further, the inhibitory effects of 8-Br-cGMP and SNAP could be significantly enhanced by down-regulating βig-h3. βig-h3 attenuates the negative regulation of NO/cGMP-sensitive store-operated Ca2+ entry. Our findings suggest that the expression of βig-h3 might play an important role in the regulation of store-operated Ca2+ entry to increase the invasive potential of HCC cells.

Topics: Calcium; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclic GMP; Extracellular Matrix Proteins; Fluorescent Antibody Technique; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Nickel; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; S-Nitroso-N-Acetylpenicillamine; Thapsigargin; Transfection; Transforming Growth Factor beta

The effect of diindolylmethane, a natural compound derived from indole-3-carbinol in cruciferous vegetables, on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in HA59T human hepatoma cells is unclear. This study explored whether diindolylmethane changed [Ca(2+)](i) in HA59T cells. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Diindolylmethane at concentrations of 1-50 μM evoked a [Ca(2+)](i) rise in a concentration-dependent manner. The signal was reduced by removing Ca(2+). Diindolylmethane-induced Ca(2+) influx was not inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators but was inhibited by aristolochic acid. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise. Incubation with diindolylmethane inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca(2+)](i) rise. At concentrations of 10-75 μM, diindolylmethane killed cells in a concentration-dependent manner. The cytotoxic effect of diindolylmethane was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Propidium iodide staining data suggest that diindolylmethane (25-50 μM) induced apoptosis in a concentration-dependent manner. Collectively, in HA59T cells, diindolylmethane induced a [Ca(2+)](i) rise by causing phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx via phospholipase A(2)-sensitive channels. Diindolylmethane induced cell death that may involve apoptosis.

Topics: Apoptosis; Calcium; Calcium Channel Blockers; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Econazole; Endoplasmic Reticulum; Fura-2; Humans; Hydroquinones; Imidazoles; Indoles; Liver Neoplasms; Nifedipine; Phospholipases A2; Protein Kinase C; Tetrazolium Salts; Thapsigargin

The expression of genes encoding key hepatic gluconeogenic enzymes, including phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), is regulated at the transcriptional level by a network of transcription factors and cofactors, including cAMP response element-binding protein (CREB). It has been suggested that increased endoplasmic reticulum (ER) stress in the liver impairs hepatic glucose metabolism. However, the direct effect of ER stress on hepatic gluconeogenesis is still not clear. Here, we investigated whether ER stress influences hepatic gluconeogenesis and whether this process is mediated by activating transcription factor 6 (ATF6) through the inhibition of cAMP-mediated activation of CREB. A cAMP stimulant, forskolin, and 8-bromoadenosine-cAMP increased PEPCK and G6Pase mRNA expression in H4IIE rat hepatoma cells, and ER stress induced by tunicamycin or thapsigargin decreased the expression of these genes in forskolin or 8-bromoadenosine-cAMP-treated cells. In a transient transfection study, ATF6 inhibited the PEPCK and G6Pase promoters. Also, adenovirus-mediated overexpression of ATF6 in H4IIE cells decreased forskolin-stimulated PEPCK and G6Pase gene expression. Moreover, the inhibition of endogenous ATF6 expression by small interfering RNAs restored the ER stress-induced suppression of PEPCK and G6Pase gene expression. Transient transfection of ATF6 inhibited transactivation by CREB on the PEPCK and G6Pase promoters, and a gel shift assay showed that Ad-ATF6 inhibits forskolin-stimulated CREB DNA-binding activity. Finally, we found that expression of ATF6 decreased fasting-induced PEPCK, G6Pase mRNA expression, and blood glucose levels in mice. Taken together, these data extend our understanding of ER stress and the regulation of liver gluconeogenesis by ATF6.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Activating Transcription Factor 6; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Colforsin; CREB-Binding Protein; Cyclic AMP; Endoplasmic Reticulum; Gluconeogenesis; Hep G2 Cells; Humans; Liver; Liver Neoplasms; Mice; Rats; RNA Splicing; RNA, Messenger; RNA, Small Interfering; Thapsigargin; Tunicamycin

Adiponectin receptors play a key role in steatosis and inflammation; however, very little is known about regulation of adiponectin receptors in liver. Here, we examined the effects of palmitate loading, endoplasmic reticulum (ER) stress, and the hypolipidemic agent fenofibrate on adiponectin receptor R2 (AdipoR2) levels and AMP-activated protein kinase (AMPK) in human hepatoma Huh7 cells and in Huh.8 cells, a model of hepatitis C-induced steatosis. Palmitate treatment reduced AdipoR2 protein and basal AMPK phosphorylation in Huh7 cells. Fenofibrate treatment preserved AdipoR2 and phosphorylated AMPK (pAMPK) levels in palmitate-treated cells accompanied by reduced triglyceride (TG) accumulation and less activation of ER stress markers CCAAT/enhancer binding (C/EBPbeta) and eukaryotic translation initiation factor 2 alpha. ER stress agents thapsigargin and tunicamycin suppressed AdipoR2 and pAMPK levels in Huh7 cells, while fenofibrate and the chemical chaperone 4-phenylbutyrate (PBA) prevented these changes. AdipoR2 levels were lower in Huh.8 cells and fenofibrate treatment increased AdipoR2 while reducing activation of c-Jun N-terminal kinase and C/EBPbeta expression without changing TG levels. Taken together, these results suggest that fatty acids and ER stress reduce AdipoR2 protein and pAMPK levels, while fenofibrate and PBA might be important therapeutic agents to correct lipid- and ER stress-mediated loss of AdipoR2 and pAMPK associated with nonalcoholic steatohepatitis.

Topics: AMP-Activated Protein Kinases; Blotting, Western; Carcinoma, Hepatocellular; CCAAT-Enhancer-Binding Proteins; Cell Line, Tumor; Endoplasmic Reticulum; Enzyme Activation; Fatty Acids; Fatty Liver; Fenofibrate; Hepacivirus; Humans; Hypolipidemic Agents; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Phenylbutyrates; Phosphorylation; Receptors, Adiponectin; Thapsigargin; Triglycerides; Tunicamycin

Mannan-binding protein (MBP) is a C-type mammalian lectin specific for mannose and N-acetylglucosamine. MBP is mainly synthesized in the liver and occurs naturally in two forms, serum MBP (S-MBP) and intracellular MBP (I-MBP). S-MBP activates complement in association with MBP-associated serine proteases via the lectin pathway. Despite our previous study (Mori, K., Kawasaki, T., and Yamashina, I. (1984) Arch. Biochem. Biophys. 232, 223-233), the subcellular localization of I-MBP and its functional implication have not been clarified yet. Here, as an extension of our previous studies, we have demonstrated that the expression of human MBP cDNA reproduces native MBP differentiation of S-MBP and I-MBP in human hepatoma cells. I-MBP shows distinct accumulation in cytoplasmic granules, and is predominantly localized in the endoplasmic reticulum (ER) and involved in COPII vesicle-mediated ER-to-Golgi transport. However, the subcellular localization of either a mutant (C236S/C244S) I-MBP, which lacks carbohydrate-binding activity, or the wild-type I-MBP in tunicamycin-treated cells shows an equally diffuse cytoplasmic distribution, suggesting that the unique accumulation of I-MBP in the ER and COPII vesicles is mediated by an N-glycan-lectin interaction. Furthermore, the binding of I-MBP with glycoprotein intermediates occurs in the ER, which is carbohydrate- and pH-dependent, and is affected by glucose-trimmed high-mannose-type oligosaccharides. These results strongly indicate that I-MBP may function as a cargo transport lectin facilitating ER-to-Golgi traffic in glycoprotein quality control.

Topics: Amino Acid Sequence; Animals; Biological Transport; Carbohydrate Conformation; Carbohydrate Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; Endoplasmic Reticulum; Golgi Apparatus; HIV Envelope Protein gp120; Humans; Liver Neoplasms; Lysosomal Membrane Proteins; Mannose-Binding Lectin; Molecular Sequence Data; Oligosaccharides; Protein Isoforms; Recombinant Fusion Proteins; Sequence Alignment; Subcellular Fractions; Surface Plasmon Resonance; Thapsigargin

We present evidence that increases in intracellular calcium, induced by treatment with calcium ionophore A23187 or the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin, dephosphorylated histone H3 at serine10 (histone H3-Ser10) in a dose-dependent manner in human hepatoma HepG2 cells. Inhibition of p42/44MAPK, pp90RSK, or p38MAPK did not affect the ability of A23187 to dephosphorylate histone H3-Ser10. This response is significantly blocked by okadaic acid, indicating a requirement for protein phosphatase 2A (PP2A). A23187 increased the activity of PP2A towards phosphorylated histone H3-Ser10. Furthermore, pretreatment with calphostin C, a selective protein kinase C (PKC) inhibitor, blocked A23187-dependent dephosphorylation of histone H3-Ser10, and coimmunoprecipitation analysis showed PP2A association with the PKCbetaII isoform. Unlike untreated cells, coimmunoprecipitated complex from A23187-treated cells showed greater dephosphorylation of histone H3-Ser10 in a PP2A-dependent manner. Inhibition of PP2A increased phosphorylation at Ser660 that determines calcium sensitivity and activity of PKCbetaII isoform, thus supporting a role for intracomplex regulation. Finally, chromatin immunoprecipitation assays following exposure to A23187 and okadaic acid revealed regulatory role of histone H3-Ser10 phosphorylation in selective gene induction. Altogether, our findings suggest a novel role for calcium in modulating histone H3-Ser10 phosphorylation level and led us to propose a model emphasizing PP2A activation, occurring downstream following perturbations in calcium homeostasis, as key event in dephosphorylating histone H3-Ser10 in mammalian cells.

Topics: Calcimycin; Calcium; Carcinoma, Hepatocellular; Cell Line, Tumor; Histones; Humans; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Phosphoserine; Promoter Regions, Genetic; Protein Binding; Protein Kinase C; Protein Kinase C beta; Protein Phosphatase 2; Receptors, LDL; Signal Transduction; Thapsigargin; Transcription, Genetic

The process by which store-operated Ca2+ channels (SOCs) deliver Ca2+ to the endoplasmic reticulum (ER) and the role of (Ca2++Mg2+)ATP-ases of the ER in the activation of SOCs in H4-IIE liver cells were investigated using cell lines stably transfected with apo-aequorin targeted to the cytoplasmic space or the ER. In order to measure the concentration of Ca2+ in the ER ([Ca2+]er), cells were pre-treated with 2,5-di-tert-butylhydroquinone (DBHQ) to deplete Ca2+ in the ER before reconstitution of holo-aequorin. The addition of extracellular Ca2+ (Cao2+) to Ca2+-depleted cells induced refilling of the ER, which was complete within 5 min. This was associated with a sharp transient increase in the cytoplasmic Ca2+ concentration ([Ca2+]cyt) of about 15 s duration (a Cao2+-induced [Ca2+]cyt spike) after which [Ca2+]cyt remained elevated slightly above the basal value for a period of about 2 min (low [Ca2+]cyt plateau). The Cao2+-induced [Ca2+]cyt spike was inhibited by Gd3+, not affected by tetrakis-(2-pyridymethyl) ethylenediamine (TPEN), and broadened by ionomycin and the intracellular Ca2+ chelators BAPTA and EGTA. Refilling of the ER was inhibited by caffeine. Neither thapsigargin nor DBHQ caused a detectable inhibition or change in shape of the Cao2+-induced [Ca2+]cyt spike or the low [Ca2+]cyt plateau whereas each inhibited the inflow of Ca2+ to the ER by about 80%. Experiments conducted with carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) indicated that thapsigargin did not alter the amount of Ca2+ accumulated in mitochondria. The changes in [Ca2+]cyt reported by aequorin were compared with those reported by fura-2. It is concluded that (i) there are significant quantitative differences between the manner in which aequorin and fura-2 sense changes in [Ca2+]cyt and (ii) thapsigargin and DBHQ inhibit the uptake of Ca2+ to the bulk of the ER but this is not associated with inhibition of the activation of SOCs. The possible involvement of a small sub-region of the ER (or another intracellular Ca2+ store), which contains thapsigargin-insensitive (Ca2++Mg2+)ATP-ases, in the activation of SOCs is briefly discussed.

Topics: Aequorin; Animals; Ca(2+) Mg(2+)-ATPase; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Channels; Carcinoma, Hepatocellular; Cytoplasm; Drug Delivery Systems; Endoplasmic Reticulum; Ethylenediamines; Fura-2; Hydroquinones; Liver; Mitochondria; Rats; Thapsigargin

SelS is a newly identified selenoprotein and its gene expression is up-regulated in the liver of Psammomys obesus after fasting. We have examined whether SelS is regulated by glucose deprivation and endoplasmic reticulum (ER) stress in HepG2 cells. Glucose deprivation and the ER stress inducers tunicamycin and thapsigargin increased SelS gene expression and protein content several-fold in parallel with glucose-regulated protein 78. The overexpression of SelS increased Min6 cell resistance to oxidative stress-induced toxicity. These results indicate that SelS is a novel member of the glucose-regulated protein family and its function is related to the regulation of cellular redox balance.

Topics: Amino Acid Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, Reporter; Glucose; HSP70 Heat-Shock Proteins; Humans; Hydrogen Peroxide; Luciferases; Membrane Proteins; Molecular Sequence Data; Mutagenesis, Insertional; Oxidants; Promoter Regions, Genetic; Proteins; RNA, Messenger; Selenoproteins; Sequence Homology, Amino Acid; Stress, Physiological; Thapsigargin; Time Factors; Tunicamycin

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

To investigate the effect of Ca(2+) mobilization on release and activation of matrix metalloproteinases (MMPs) in human hepatocellular carcinoma cells.. Ca(2+) and chemicals which can induce or inhibit Ca(2+) mobilization were added into human SMMC-7721 hepatoma cells in vitro. SDS-PAGE protein electrophoresis and gelatin zymography analysis were carried out to detect the changes of release and activation of MMPs in the cell culture supernatant.. Addition of CaCl(2) into culture system resulted in an enhanced secretion and activation of MMP-2 and MMP-9 in a dose-dependent manner. At a dose of 0.8 mmol/L CaCl(2), it maintained a stable high level of MMPs, especially of MMP-2 with (109.71 +/- 27.93)% elevation as compared to the cells without CaCl(2) addition (P < 0.001). SDS-PAGE analysis showed that most secreted proteins were MMPs (MMP-2 and MMP-9) when the cells cultured in media without serum. Thapsigargin (Tg, 4 micromol/L), an inducer of intracellular Ca(2+) stores depletion, significantly enhanced the release and activation of MMP-2 and MMP-9, compared to the control with (58.63 +/- 31.04)% elevation (P < 0.05), while the inducing effect of Tg on MMPs release and activation was significantly inhibited by S-nitro-N-acetylpenicillamine (SNAP, 200 micromol/L), an NO donor.. Intracellular Ca(2+) regulation pathways may play an important role in the process of release and activation of MMPs.

Topics: Calcium; Carcinoma, Hepatocellular; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Nitric Oxide Donors; Penicillamine; Thapsigargin

Endoplasmic reticulum (ER) was recently suggested as a third subcellular compartment in apoptotic execution. Survivin is a member of inhibitors of apoptosis and ursodeoxycholic acid (UDCA) prevents apoptosis from various apoptotic stimuli. To assess the activity of survivin and the effect of UDCA on the survivin in ER stress-mediated apoptosis, we treated hepatoma cell lines with thapsigargin (TG). TG-induced apoptosis was assessed by morphological changes, DNA fragmentation, cleavages of poly(ADP-ribose)polymerase (PARP), and activation of calpain and caspase-12. The level of survivin was decreased after TG treatment in hepatoma cell lines indicating that survivin play an important role in ER stress-mediated apoptosis. UDCA prevented decrease in survivin levels and inhibited TG-induced apoptosis and caspase-12 activation suggesting an anti-apoptotic effect of UDCA.

Topics: Apoptosis; Calpain; Carcinoma, Hepatocellular; Caspase 12; Caspases; DNA Fragmentation; Endoplasmic Reticulum; Enzyme Activation; Genes, bcl-2; Humans; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Microtubule-Associated Proteins; Neoplasm Proteins; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Stress, Physiological; Survivin; Thapsigargin; Tumor Cells, Cultured; Ursodeoxycholic Acid

The effect of NPC-14686, a potential anti-inflammatory drug, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in HA22/VGH human hepatoma cells was explored by using fura-2 as a fluorescent Ca(2+) indicator. NPC-14686 at concentrations above 10 microM increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 100 microM. The Ca(2+) signal was reduced by removing extracellular Ca(2+) or by 10 microM nifedipine and was not changed by verapamil or diltiazem. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) abolished 200 microM NPC-14686-induced Ca(2+) release; and conversely pretreatment with NPC-14686 abolished thapsigargin-induced Ca(2+) release. The Ca(2+) release induced by 200 microM NPC-14686 was not changed by inhibiting phospholipase C with 2 microM U73122. Together, the results suggest that in human hepatoma cells, NPC-14686 induced a [Ca(2+)](i) increase by causing store Ca(2+) release from the endoplasmic reticulum in an phospholipase C-independent manner, and by inducing nifedipine-sensitive Ca(2+) influx.

Topics: Aminobutyrates; Calcium; Calcium Channel Blockers; Calcium Signaling; Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Drug Combinations; Estrenes; Fluorescent Dyes; Fura-2; Hepatocytes; Humans; Pyrrolidinones; Thapsigargin; Tumor Cells, Cultured; Type C Phospholipases

The TRPC1 (transient receptor potential canonical 1) protein, which is thought to encode a non-selective cation channel activated by store depletion and/or an intracellular messenger, is expressed in a number of non-excitable cells. However, the physiological functions of TRPC1 are not well understood. The aim of these studies was to investigate the function of TRPC1 in liver cells using small interfering RNA (siRNA) to ablate the TRPC1 protein. Treatment of H4-IIE liver cells with siRNA targeted against TRPC1 caused an approx. 50% decrease in expression of the human TRPC1 protein in cells transfected with cDNA encoding human TRPC1, and a 50% decrease in expression of the endogenous TRPC1 protein (assessed by Western blot and immunofluorescence). The decrease in endogenous TRPC1 protein in cells transfected with TRPC1 siRNA was associated with a greater increase in cell volume (compared with the increase observed in control cells) immediately after cells were placed in a hypotonic medium, and an enhanced regulatory cell volume decrease after exposure to hypotonic medium. Treatment with siRNA targeted against TRPC1 also led to a 25% inhibition of thapsigargin-stimulated Ca(2+) inflow, a 40% inhibition of ATP and maitotoxin-stimulated Ca(2+) inflow, and a 50% inhibition of maitotoxin-stimulated Mn(2+) inflow. The idea that, in liver cells, TRPC1 encodes a non-selective cation channel involved directly or indirectly in the regulation of cell volume is consistent with the results obtained.

Topics: Animals; Biological Transport; Blotting, Western; Calcium; Calcium Channels; Carcinoma, Hepatocellular; Cell Size; DNA, Complementary; Enzyme Inhibitors; Fluorescent Antibody Technique; Humans; Ion Channels; Liver; Manganese; Polymerase Chain Reaction; Precipitin Tests; Rats; RNA, Messenger; RNA, Small Interfering; Thapsigargin; Transfection; TRPC Cation Channels; Tumor Cells, Cultured

We have previously demonstrated that insulin and G(q)-coupled receptor agonists individually activate mitogen-activated protein kinase (MAPK) in liver cells and both effects involve an influx of extracellular Ca(2+). Yet, these agonists have opposing physiological actions on hepatocyte glucose metabolism. We thus investigated the interaction between insulin and the P2Y(2) purinergic agonist adenosine triphosphate (ATP) on MAPK in HTC cells, a model hepatocyte cell line, and determined the involvement of cytosolic Ca(2+). Insulin and ATP each induced a dose-dependent phosphorylation of p44/42 MAPK that was partially inhibited by EGTA. However, pretreatment with insulin markedly increased the MAPK phosphorylation response to ATP. This potentiation was canceled by chelation of extracellular Ca(2+) with EGTA. We used patch clamp electrophysiology and fluorescence microscopy to understand the role of intracellular Ca(2+) in this effect. Insulin and ATP, respectively, induced monophasic and multiphasic changes in membrane potential and intracellular Ca(2+) as expected. Pretreatment with 10 nmol/L insulin significantly decreased the initial rapid depolarization (inward nonselective cation current [NSCC]), as well as the compounded Ca(2+) response induced by 100 micro mol/L ATP. However, in Ca(2+)-free conditions, insulin did not modify the Ca(2+) mobilized from internal pools after stimulation with ATP. Upon Ca(2+) readmission, internal store depletion by ATP or thapsigargin doubled the rate of capacitative Ca(2+) influx, whereas insulin increased this influx 1.32-fold. On the other hand, insulin pretreatment counteracted the increased rate of Ca(2+) influx induced by ATP but not by thapsigargin. In summary, insulin counteracts the membrane potential and Ca(2+) responses to ATP in HTC cells. However, insulin and ATP effects on MAPK activation are synergistic and Ca(2+) influx plays a permissive role. Therefore, the opposing metabolic actions of insulin and G(q)-coupled receptor agonists involve an interaction in signaling pathways that resides downstream of Ca(2+) influx.

Topics: Adenosine Triphosphate; Animals; Calcium; Carcinoma, Hepatocellular; Cells, Cultured; Chelating Agents; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Homeostasis; Hypoglycemic Agents; Insulin; Ion Channels; Liver; Liver Neoplasms; Mitogen-Activated Protein Kinases; Patch-Clamp Techniques; Phosphorylation; Rats; Thapsigargin; Tumor Cells, Cultured

Gossypol is a natural toxicant present in cottonseeds, and is hepatotoxic to animals and human. The effect of gossypol on cytosolic free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatocytes was explored using fura-2 as a fluorescent Ca2+ indicator. Gossypol increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 2 microM. The Ca2+ signal was reduced by removing extracellular Ca2+ or by 10 microM La3+, but was not affected by nifedipine, verapamil or diltiazem. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ partly reduced 10 microM gossypol-induced Ca2+ release; and conversely pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. The Ca2+ release induced by 10 microM gossypol was not changed by inhibiting phospholipase C with 2 microM U73122 or by depleting ryanodine-sensitive Ca2+ stores with 50 microM ryanodine. Together, the results suggest that in human hepatocytes, gossypol induced a [Ca2+]i increase by causing store Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and by inducing Ca2+ influx.

Topics: Calcium; Calcium Channel Blockers; Calcium Signaling; Carcinoma, Hepatocellular; Cell Line, Tumor; Contraceptive Agents, Male; Enzyme Inhibitors; Gossypol; Hepatocytes; Humans; Liver Neoplasms; Thapsigargin

We have developed an assay system suitable for assessment of compound action on the Edg4 subtype of the widely expressed lysophosphatidic acid (LPA)-responsive Edg receptor family. Edg4 was stably overexpressed in the rat hepatoma cell line Rh 7777, and a Ca(2+)-based FLIPR assay developed for measurement of functional responses. In order to investigate the mechanisms linking Edg4 activation to cytosolic Ca(2+) elevation, we have also studied LPA signalling in a human neuroblastoma cell line that endogenously expresses Edg4. LPA responses displayed similar kinetics and potency in the two cell lines. The Ca(2+) signal generated by activation of LPA-sensitive receptors in these cells is mediated primarily by endoplasmic reticulum. However, there is a substantial inhibition of the LPA response by FCCP, indicating that mitochondria also play a key role in the LPA response. Partial inhibition of the response by cyclosporin A could indicate an active Ca(2+) release role for mitochondria in the LPA response. The inositol 1,4,5-triphosphate receptor antagonist 2-aminoethyl diphenyl borate markedly inhibits, but does not abolish, the Ca(2+) response to LPA, suggesting further complexity to the signalling pathways activated by Edg receptors. In comparing Edg signalling in recombinant and native cells, there is a striking overall similarity in receptor expression pattern, agonist potency, and the effect of modulators on the Ca(2+) response. This indicates that the Edg4-overexpressing Rh7777 cell line is a very useful model system for studying receptor pharmacology and signalling mechanisms, and for investigating the Edg4 receptor's downstream effects.

Topics: Calcium; Calcium Signaling; Carcinoma, Hepatocellular; Cell Line, Tumor; Coloring Agents; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Immunohistochemistry; Inositol 1,4,5-Trisphosphate; Liver Neoplasms; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; Recombinant Proteins; Signal Transduction; Thapsigargin

A novel approach to reducing organ toxicity of anticancer agents is the application of nontoxic glucuronide prodrugs from which the active drug is released by human beta-glucuronidase, an enzyme present at high levels in many tumors. In view of high interindividual variability in beta-glucuronidase expression, regulation of this enzyme is an essential factor modulating bioactivation of glucuronide prodrugs. However, data on regulation of human beta-glucuronidase expression are not available. Preliminary evidence from animal experiments points to a role of intracellular calcium in regulation of beta-glucuronidase activity. Therefore, we investigated regulation of beta-glucuronidase by the calcium ionophore A23187 and the calcium ATPase inhibitor thapsigargin in the human hepatoma cell line HepG2. The enzyme was characterized on activity, protein, and mRNA levels by cleavage of 4-methylumbelliferyl-beta-D-glucuronide, Western blotting, Northern blotting, and nuclear run-on transcription. Incubation of HepG2 cells with A23187 and thapsigargin, respectively, revealed a time and concentration dependent down-regulation of beta-glucuronidase activity to about 50% of the control level. This effect could also be demonstrated in several other cell lines (e.g., HL-60, ECV 304, 32M1, Caco-2/TC7). Effects on protein and mRNA levels paralleled those obtained on enzymatic activity. In line with these data, A23187 and thapsigargin decreased beta-glucuronidase transcriptional rate. Our data demonstrate regulation of human beta-glucuronidase by xenobiotics. Down-regulation of beta-glucuronidase by A23187 and thapsigargin is at least partly mediated by a transcriptional mechanism. Based on our findings, we speculate that beta-glucuronidase activity and hence bioactivation of glucuronide prodrugs in humans can be modulated by exogenous factors.

Topics: Blotting, Northern; Blotting, Western; Calcimycin; Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Glucuronidase; Humans; Ionophores; Lung Neoplasms; RNA, Messenger; Thapsigargin; Time Factors; Transcription, Genetic; Tumor Cells, Cultured

Tumor necrosis factor-alpha (TNF-alpha) is an important component of the early signaling pathways leading to liver regeneration and proliferation, but it is also responsible for several hepatotoxic effects. We have investigated the effect of TNF-alpha on thapsigargin (TG)-induced store-mediated Ca2+ entry (SMCE) in the human hepatocellular carcinoma cell line HepG2. In these cells, short-term (10 min) exposure to TNF-alpha slightly increased SMCE. In contrast, long-term (12 h) exposure to TNF-alpha significantly reduced SMCE. This effect was reversed by coincubation with atrial natriuretic peptide (ANP), which itself had no effect on SMCE. Cytochalasin D and latrunculin A, inhibitors of actin polymerization, abolished SMCE. Long-term exposure of HepG2 cells to TNF-alpha abolished TG-induced actin polymerization and membrane association of Ras proteins. When TNF-alpha was added in combination with ANP, these effects were reduced. These findings suggest that in HepG2 cells, TNF-alpha inhibits SMCE by affecting reorganization of the actin cytoskeleton, probably by interfering with the activation of Ras proteins, and that ANP protects against these inhibitory effects of TNF-alpha.

Topics: Actins; Atrial Natriuretic Factor; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Carcinogens; Carcinoma, Hepatocellular; Cell Compartmentation; Cytochalasin D; Cytoskeleton; Humans; Liver Neoplasms; Nucleic Acid Synthesis Inhibitors; Polymers; ras Proteins; Thapsigargin; Thiazoles; Thiazolidines; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

This study aimed to investigate cGMP-regulated store-operated Ca(2+)entry in human 7721 hepatoma cells. [Ca(2+)](i)was measured using Fura2/AM. After incubation of the cells with 4 microm thapsigargin, Ca(2+)entry was evoked by application of 1 mMm Ca(2+)to extracellular solution and was blocked by 3 m m Ni(2+), indicating the presence of store-operated Ca(2+)entry in human 7721 hepatoma cell line. Application of 8-Br-cGMP reduced the [Ca(2+)](i)in hepatoma 7721 cells by 80%. These data demonstrated for the first time that store-operated Ca(2+)entry pathway is present in human hepatoma cells, which is regulated by cGMP.

Topics: Calcium; Calcium Channels; Calcium-Transporting ATPases; Carcinoma, Hepatocellular; Cyclic GMP; Enzyme Inhibitors; Humans; Liver Neoplasms; Nickel; Thapsigargin; Tumor Cells, Cultured

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

1. The human hepatoma cell line Hep3B is a widely used model for studies of hypoxia-related gene expression. Cytosolic free calcium concentration ([Ca2+]i) has been implicated in cellular oxygen-sensing processes. We investigated whether calcium ions have a significant impact on the production of erythropoietin (EPO) and vascular endothelial growth factor (VEGF). 2. We found that the calcium ionophore ionomycin induced a rapid and sustained increase of [Ca2+]i while thapsigargin, an inhibitor of endoplasmic reticulum calcium ATPase, only caused a 20 % elevation of [Ca2+]i within 10 min after application. However, the calcium content of intracellular stores was considerably reduced by thapsigargin after an incubation period of 24 h. 3. Variations in [Ca2+]o did not result in altered EPO or VEGF secretion rates. Ionomycin decreased EPO production while the lowering of VEGF production was not statistically significant. In the presence of extracellular Ca2+ the membrane permeant calcium chelator BAPTA-AM stimulated the production of EPO (P < 0.05) but not of VEGF while EGTA-AM, a closely related agent, affected neither EPO nor VEGF formation under these conditions. Incubation with thapsigargin resulted in decreased EPO synthesis (P < 0.05) but stimulated VEGF secretion (P < 0.05). 4. In the absence of extracellular calcium, EGTA-AM led to an accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha). This treatment significantly stimulated VEGF synthesis but also decreased EPO secretion (P < 0.05). 5. Our data suggest that the calcium transient and the cytosolic Ca2+ concentration do not play a key role in hypoxia-induced EPO and VEGF production in Hep3B cells.

Topics: Blotting, Northern; Calcium; Calcium-Transporting ATPases; Carcinoma, Hepatocellular; Cell Hypoxia; Chelating Agents; Egtazic Acid; Endothelial Growth Factors; Enzyme Inhibitors; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Ionomycin; Kinetics; Liver Neoplasms; Lymphokines; Thapsigargin; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

In normal subjects and in patients with cardiovascular disease, plasma triglycerides are positively correlated with plasminogen activator inhibitor type 1 (PAI-1) levels. Moreover, in vitro studies indicate that VLDLs induce PAI-1 synthesis in cultured cells, ie, endothelial and HepG2 cells. However, the signaling pathways involved in the effect of VLDL on PAI-1 synthesis have not yet been investigated. We report that VLDLs induce a signaling cascade that leads to an enhanced secretion of PAI-1 by HepG2 cells. In myo-[(3)H]inositol-labeled HepG2 cells, VLDL (100 microg/mL) caused a time-dependent increase in [(3)H]inositol phosphates, the temporal sequence being tris>bis>monophosphate. VLDL brought about a time-dependent stimulation of membrane-associated protein kinase C (PKC) activity and arachidonate release. Finally, VLDL stimulated mitogen-activated protein (MAP) kinase, and this effect was reduced by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), which suggests that PKC plays a pivotal role in MAP kinase phosphorylation. VLDL-induced PAI-1 secretion was completely prevented by U73122, a specific inhibitor of phosphatidylinositol-specific phospholipase C, by H7 or by PKC downregulation, and by mepacrine (all P<0.01 versus VLDL-treated cells). 3,4,5-Trimethoxybenzoic acid 8-(diethylamino)-octyl ester, which prevents Ca2+ release from intracellular stores, inhibited VLDL-induced PAI-1 secretion by 60% (P<0.05), and the MAP kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 completely suppressed both basal and VLDL-induced PAI-1 secretion. These data demonstrate that VLDL-induced PAI-1 biosynthesis results from a principal signaling pathway involving PKC-mediated MAP kinase activation.

Topics: Antioxidants; Arachidonic Acid; Calcium Channel Blockers; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Hepatocellular; Cholesterol, VLDL; Endothelium, Vascular; Enzyme Inhibitors; Estrenes; Fibrinolysis; Flavonoids; Gallic Acid; Phosphatidylinositol 3-Kinases; Phosphatidylinositols; Phosphodiesterase Inhibitors; Phospholipase D; Phosphorylation; Plasminogen Activator Inhibitor 1; Protein Kinase C; Pyrrolidinones; Signal Transduction; Thapsigargin; Tritium; Tumor Cells, Cultured; Tyrosine

Copper overload and deficiency are known to cause morphological and functional mitochondrial abnormalities. The reverse transcriptase-polymerase chain reaction (RT-PCR)-based method of differential display of mRNA was used to identify genes with altered expression in cultured human hepatoma cells (Hep G2) exposed to increasing concentrations of copper (0-100 microM, 24 h). Copper regulation of a cloned PCR product, identified as the gene for the mitochondrially encoded cytochrome b, was confirmed by Northern analysis and in situ hybridization. Copper toxicity increased cytochrome b mRNA abundance up to 3.6-fold, and copper chelation reduced it by 50%. Hepatic cytochrome b mRNA was also increased in rats fed a high-copper diet. Thapsigargin treatment resulted in a significant increase in cytochrome b mRNA, suggesting that an increase in intracellular calcium may be involved in the mechanism of copper action. Furthermore, although cyclohexamide (CHX) alone did not increase cytochrome b mRNA, the addition of CHX and copper resulted in a sixfold increase. These data suggest a role for cytochrome b in the response to increases or decreases in hepatic copper.

Topics: Animals; Carcinoma, Hepatocellular; Chelating Agents; Copper; Cycloheximide; Cytochrome b Group; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation; Humans; Hydrogen Peroxide; Liver; Male; Mitochondria, Liver; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sequence Analysis, DNA; Thapsigargin; Tumor Cells, Cultured; Zinc

Some investigators have reported previously that phorbol esters inhibit in vitro erythropoietin production stimulated by hypoxia; whereas others have reported that phorbol esters enhanced Epo production during exposure to hypoxia. We have demonstrated in the present experiments that hypoxia significantly increased diacylglycerol levels in cultured human hepatocellular carcinoma (Hep3B) cells. 1-oleoyl-2-acetyl-ras-glycerol (OAG) and N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9), two well-known protein kinase C activators, significantly increased medium levels of erythropoietin as well as erythropoietin messenger RNA levels in normoxic Hep3B cells. A potent protein kinase C inhibitor, chelerythrine chloride, significantly decreased hypoxia-induced increases in medium levels of erythropoietin as well as erythropoietin messenger RNA levels in Hep3B cells. A cis-unsaturated free fatty acid, oleic acid, significantly enhanced OAG-induced medium levels of erythropoietin in normoxic Hep3B cells, whereas a phospholipase A2 inhibitor, mepacrine, significantly decreased hypoxia-induced erythropoietin production in Hep3B cells. These results provide strong support for a positive role for protein kinase C in the hypoxic regulation of erythropoietin production.

Topics: Alkaloids; Benzophenanthridines; Calcimycin; Carcinoma, Hepatocellular; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Erythropoietin; Humans; Ionophores; Liver Neoplasms; Models, Chemical; Oleic Acid; Phenanthridines; Phospholipases A; Phospholipases A2; Protein Kinase C; Quinacrine; Thapsigargin; Tumor Cells, Cultured

Using reverse transcriptase-PCR and Northern analysis, we have shown that the H4IIE cell line, derived from the Reuber H35 rat hepatoma, contains significant amounts of transcripts for the CaCh3 (neuroendocrine) and CaCh1 (skeletal muscle) L-type voltage-operated calcium channel alpha 1-subunits. Two of the CaCh3 transcripts have a 45 bp deletion in the IVS4 membrane-spanning region which is the result of a mutation in genomic DNA. The deduced amino acid sequences of the PCR-derived clones of CaCh3 indicate that the mutation causes the loss of 15 amino acids from the IVS4 region, including three of the six positively charged residues, which are thought to be part of the voltage-sensing mechanism of voltage-operated Ca2+ channels. Quantitative-PCR and Northern analysis indicate that one of the novel CaCh3 transcripts is present in sufficient amounts to imply it could play a functional role in Ca2+ inflow. RT-PCR analysis of hepatocytes isolated from rat liver detected transcripts of CaCh3 (without the IVS4 mutation) and CaCh2, but at considerably lower levels than observed for the isoforms in the H4IIE cell line. Transcripts of CaCh1 and CaCh2 were also detected at low levels in Jurkat T lymphocytes. Fluorimetric studies with the Ca(2+)-sensitive probe, Fluo-3, have shown that H4IIE cells exhibit receptor-activated and store-activated (thapsigarin-induced), but not depolarisation (extracellular KCl)-induced Ca2+ inflow. The mutant transcripts are unlikely to produce Ca2+ channels that are opened by membrane depolarisation. The idea that they may be opened by other mechanisms is briefly discussed.

Topics: Amino Acid Sequence; Animals; Blotting, Northern; Calcium Channels; Calcium Channels, L-Type; Carcinoma, Hepatocellular; Cell Line; Cell Membrane; DNA Primers; Enzyme Inhibitors; Humans; Liver; Mice; Molecular Sequence Data; Mutation; Polymerase Chain Reaction; Rats; RNA Splicing; Sequence Deletion; Sequence Homology, Amino Acid; Thapsigargin; Transcription, Genetic

Anticancer drugs etoposide and mitomycin C increased nuclear p53 protein and decreased proliferating cell nuclear antigen (PCNA) of PLC/PRF/5 human hepatoma cells. These changes were followed by DNA fragmentation and apoptosis. Teleocidin antagonized both apoptosis and alterations of nuclear p53 protein and PCNA induced by these anticancer drugs. In contrast, thapsigargin antagonized only drug-induced nuclear accumulation of p53 protein. Therefore, the inhibition of apoptosis appears not to be the common mechanism of tumor promotion. Both tumor promoters suppressed the increase in nuclear p53 protein, suggesting that an inadequate DNA repair due to the reduced nuclear accumulation of p53 protein might be playing important role in enhancing carcinogenesis.

Topics: Apoptosis; Carcinogens; Carcinoma, Hepatocellular; DNA Damage; Etoposide; Flow Cytometry; Humans; Lyngbya Toxins; Mitomycin; Proliferating Cell Nuclear Antigen; Terpenes; Thapsigargin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We found that thapsigargin (Tg), a non-phorbol ester type tumor promoter that specifically inhibits endoplasmic reticulum Ca(2+)-ATPase, transiently increased the level of cytosolic free calcium ([Ca2+]i) and subsequently induced chromatin condensation, nuclear fragmentation, and internucleosomal DNA cleavage in cultured PLC/PRF/5 human hepatoma cells. These alterations were followed by the loss of plasma membrane integrity and by cell death. Epidermal growth factor (EGF) and vasopressin similarly elevated [Ca2+]i without causing DNA fragmentation which is characteristic of apoptosis. Consequently, the elevation of [Ca2+]i itself was not sufficient for causing Tg-induced cell death. On the other hand, preculturing the cells with Tg completely suppressed Ca2+ mobilization induced by EGF and vasopressin; a result that strongly suggests that Tg depleted the endoplasmic reticulum Ca2+ pool. Such depletion is hypothesized to induce apoptotic cell death in this hepatoma cell line by changing the nuclear Ca2+ levels which probably produce a structural change in chromatin.

Topics: Apoptosis; Calcium; Carcinoma, Hepatocellular; Cell Division; DNA Topoisomerases, Type II; DNA, Neoplasm; Epidermal Growth Factor; Humans; Liver Neoplasms; Terpenes; Thapsigargin; Tumor Cells, Cultured

Topics: Apoptosis; Calcium; Calcium-Transporting ATPases; Carcinoma, Hepatocellular; Epidermal Growth Factor; Humans; Liver Neoplasms; Terpenes; Thapsigargin; Tumor Cells, Cultured

Mobilization of Ca2+ from the endoplasmic reticulum (ER) suppresses translational initiation and inhibits post-translational processing and secretion of glycoproteins. This study explores the mechanism whereby ionomycin, a Ca2+ ionophore, and thapsigargin, an ER Ca(2+)-ATPase inhibitor, promote retention of alpha 1-antitrypsin (alpha 1-AT) bearing high mannose, endoglycosidase H (Endo H)-sensitive oligosaccharide side chains within the ER of HepG2 cells. Arrest occurred at the removal of mannose residues such that intermediates with Man7-9GlcNAc2 side chains accumulated with the Man8-9GlcNAc2 structures predominating. Maturation of alpha 1-AT bearing Man5-6GlcNAc2 side chains was unaffected. Inhibition of alpha 1-AT processing by ionomycin occurred independently of translational suppression. Forms of alpha 1-AT identical to those retained with ionomycin or thapsigargin were observed upon treatment with the alpha-1,2-mannosidase inhibitor 1-deoxymannojirimycin whereas castanospermine, an inhibitor of ER alpha-glucosidase I, produced different forms of the glycoprotein. Neither inhibitor impaired transport or secretion of alpha 1-AT. With brefeldin A, which causes redistribution of Golgi enzymes to the ER, alpha 1-AT was retained intracellularly but acquired resistance to Endo H. With ionomycin, thapsigargin, or 1-deoxymannojirimycin-treated cells, however, brefeldin A failed to promote further processing of the glycoprotein. Possible mechanisms for the suppression of alpha 1-AT processing at the alpha-1,2-mannosidase step by Ca(2+)-mobilizing agents are discussed. Excepting tunicamycin, traditional inhibitors of protein processing did not affect amino acid incorporation.

Topics: 1-Deoxynojirimycin; alpha 1-Antitrypsin; alpha-Mannosidase; Brefeldin A; Calcium; Calcium-Transporting ATPases; Carbohydrate Conformation; Carcinoma, Hepatocellular; Cyclopentanes; Endoplasmic Reticulum; Glycoside Hydrolase Inhibitors; Hexosaminidases; Humans; Indolizines; Ionomycin; Liver Neoplasms; Mannose; Mannosidases; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Oligosaccharides; Terpenes; Thapsigargin; Tumor Cells, Cultured