thapsigargin and Lung-Neoplasms

Many cancer diseases, e.g., prostate cancer and lung cancer, develop very slowly. Common chemotherapeutics like vincristine, vinblastine and taxol target cancer cells in their proliferating states. In slowly developing cancer diseases only a minor part of the malignant cells will be in a proliferative state, and consequently these drugs will exert a concomitant damage on rapidly proliferating benign tissue as well. A number of toxins possess an ability to kill cells in all states independently of whether they are benign or malignant. Such toxins can only be used as chemotherapeutics if they can be targeted selectively against the tumors. Examples of such toxins are mertansine, calicheamicins and thapsigargins, which all kill cells at low micromolar or nanomolar concentrations. Advanced prodrug concepts enabling targeting of these toxins to cancer tissue comprise antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT), lectin-directed enzyme-activated prodrug therapy (LEAPT), and antibody-drug conjugated therapy (ADC), which will be discussed in the present review. The review also includes recent examples of protease-targeting chimera (PROTAC) for knockdown of receptors essential for development of tumors. In addition, targeting of toxins relying on tumor-overexpressed enzymes with unique substrate specificity will be mentioned.

Topics: Antineoplastic Agents; Apoptosis; Calicheamicins; Cell Proliferation; Delayed-Action Preparations; Drug Carriers; Drug Design; Drug Liberation; Enzyme Therapy; Gene Knockdown Techniques; Humans; Lung Neoplasms; Male; Maytansine; Molecular Targeted Therapy; Peptide Hydrolases; Prodrugs; Prostatic Neoplasms; Thapsigargin; Toxins, Biological

Mounting evidence indicates that activation of unfolded protein response (UPR) and metabolic reprogramming contribute to cancer cell migration and invasion, but the molecular mechanism of pro-EMT program through a coordinated action of UPR with metabolism has not been defined. In this study, we utilized ER stress-inducing reagent, thapsigargin (TG), to induced pharmacologic ER stress in lung cancer cells. Here. We report that the branch of UPR, IRE1α-XBP1 pathway plays a pivotal role in reprogramming lung cancer cell metabolism. At the molecular level, the expression of pyruvate dehydrogenase kinase-1 (PDK-1) is directly induced by XBP1 as a consequence of UPR activation, thus facilitating aerobic glycolysis and lactate production. We also demonstrated that PDK1 serves as a downstream element of UPR activation in induction of Snail and EMT program. In addition, PDK1-induced Snail was dependent on the lactate production derived from metabolic reprogramming. Our findings reveal a critical role of lactate in pro-invasion events and establishes a direct connection between ER-stress and metabolic reprogramming in facilitating cancer cell progression.

Topics: Carcinoma, Non-Small-Cell Lung; Endoplasmic Reticulum Stress; Endoribonucleases; Epithelial-Mesenchymal Transition; Humans; Lactates; Lung Neoplasms; Protein Serine-Threonine Kinases; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Thapsigargin; Unfolded Protein Response; X-Box Binding Protein 1

The endoplasmic reticulum (ER) is an organelle involved in various physiological processes such as lipid metabolism, protein synthesis and folding, and cellular calcium storage. In a physiological tumor microenvironment, hypoxia, nutrient deprivation, and calcium dysregulation cause accumulation of unfolded and misfolded proteins. Such accumulation induces ER stress and unfolded protein responses (UPRs). Increased UPR signaling pathways are associated with multiple types of cancer. The influence of ER stress on acyl‑CoA metabolic enzymes is not well understood. Evaluation of PRECOG and Kaplan‑Meier plotter databases in the present study suggested that high expression of acyl‑CoA thioesterase (ACOT)7, ACOT11, ACOT13, soluble carrier family 27 member A4 (SLC27A4) and SLC27A5 was associated with poor clinical outcomes. In addition, expression levels of ACOT7, ACOT11, SLC27A4 and SLC27A5 were not altered after induction of ER stress. By contrast, expression of some enzymes was decreased, such as those of long‑chain acyl‑CoA synthetase (ACSL)3, ACSL4 and SLC27A2. Fatty acid uptake capacity was suppressed in lung cancer cell lines A549 and CL1‑0 after thapsigargin treatment but intracellular reactive oxygen species levels were not suppressed. Gene enrichment and regulatory element analysis were performed; the results provided potential targets for further investigation. On the whole, our findings demonstrate the potential regulatory mechanism of high‑expression of acyl‑CoA metabolic enzymes, the biological effects of decreased enzyme expression levels, possible regulatory elements, and the interaction network involved in responses to ER stress in lung cancer.

Topics: Acyl Coenzyme A; Cell Line, Tumor; Coenzyme A Ligases; Computational Biology; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Fatty Acids; Humans; Lipid Metabolism; Lung Neoplasms; Protein Interaction Maps; Reactive Oxygen Species; Survival Analysis; Thapsigargin; Thiolester Hydrolases; Tumor Microenvironment; Unfolded Protein Response

Pterostilbene (PT), the natural dimethylated analog of resveratrol (RSV), is a potent anticarcinogen for non-small-cell lung cancer (NSCLC), but its anti-NSCLC mechanisms remain unclear. In this study, we show that PT treatment time- and dose-dependently enhanced the endoplasmic reticulum stress (ERS) signaling (i.e., p-PERK, IRE1, ATF4, CHOP), thus decreasing the cell viability and inducing apoptosis in human PC9 and A549 NSCLC cell lines. Moreover, the decreased migratory and adhesive abilities, downregulation of intracellular glutathione (GSH) level, enhanced reactive oxygen species (ROS) generation, Caspase 3 activity and mitochondrial membrane depolarization were observed in NSCLC cells treated with PT. These effects were reversed by CHOP siRNA which inhibited the ERS signaling pathway, but were promoted by thapsigargin (a classical ERS inducer) in vitro. Besides, in vivo studies also verify that PT exerted anticancer activity by mobilizing ERS signaling and apoptosis-related proteins, and these effects were enhanced by thapsigargin. Therefore, ERS activation may represent a new mechanism of anti-NSCLC action by PT, and a novel therapeutic intervention for lung cancer.

Topics: A549 Cells; Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; Cell Survival; Down-Regulation; Endoplasmic Reticulum Stress; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Stilbenes; Thapsigargin

Chemoresistance in cancer therapy is an unfavorable prognostic factor in non-small cell lung cancer (NSCLC). Elevation of intracellular calcium level in multidrug resistant (MDR) sublines leads to sensitization of MDR sublines to cell death. We demonstrated that a fungal protein from Ganoderma microsporum, GMI, elevates the intracellular calcium level and reduces the growth of MDR subline via autophagy and apoptosis, regardless of p-glycoprotein (P-gp) overexpression, in mice xenograft tumors. In addition, we examined the roles of autophagy in the death of MDR A549 lung cancer sublines by GMI, thapsigargin (TG) and tunicamycin (TM) in vitro. Cytotoxicity of TG was inhibited by overexpressed P-gp. However, TM-induced death of MDR sublines was independent of P-gp level. Combinations of TG and TM with either docetaxel or vincristine showed no additional cytotoxic effects on MDR sublines. TG- and TM-mediated apoptosis of MDR sublines was demonstrated on Annexin-V assay and Western blot and repressed by pan-caspase inhibitor (Z-VAD-FMK). Treatment of MDR sublines with TG and TM also augmented autophagy with accumulation of LC3-II proteins, breakdown of p62 and formation of acidic vesicular organelles (AVOs). Inhibition of ATG5 by shRNA silencing significantly reduced autophagy and cell death but not apoptosis following TG or TM treatment. GMI treatment inhibited the phosphorylation of Akt/S473 and p70S6K/T389. Interestingly, the phosphorylation of ERK was not associated with GMI-induced autophagy. We conclude that autophagy plays a pro-death role in acquired MDR and upregulation of autophagy by GMI via Akt/mTOR inhibition provides a potential strategy for overcoming MDR in the treatment of lung cancers.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Autophagy; Autophagy-Related Protein 5; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Docetaxel; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Fungal Proteins; Ganoderma; Humans; Lung Neoplasms; Male; Medicine, Chinese Traditional; Mice; Mice, Inbred NOD; Microtubule-Associated Proteins; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; RNA, Small Interfering; Taxoids; Thapsigargin; TOR Serine-Threonine Kinases; Tunicamycin; Vincristine; Xenograft Model Antitumor Assays

In this study, we investigated the anticancer activity of icariin (ICA) against human lung adenocarcinoma cells in vitro and in vivo and explored the role of endoplasmic reticulum (ER) stress (ERS) signaling in this process. ICA treatment resulted in a dose- and time-dependent decrease in the viability of human lung adenocarcinoma A549 cells. Additionally, ICA exhibited potent anticancer activity, as evidenced by reductions in A549 cell adhesion, migration and intracellular glutathione (GSH) levels and increases in the apoptotic index, Caspase 3 activity, and reactive oxygen species. Furthermore, ICA treatment increased the expression of ERS-related molecules (p-PERK, ATF6, GRP78, p-eIF2α, and CHOP), up-regulated the apoptosis-related protein PUMA and down-regulated the anti-apoptosis-related protein Bcl2. The down-regulation of ERS signaling using PERK siRNA desensitized lung adenocarcinoma cells to ICA treatment, whereas the up-regulation of ERS signaling using thapsigargin (THA) sensitized lung adenocarcinoma cells to ICA treatment. Additionally, ICA inhibited the growth of human lung adenocarcinoma A549 cell xenografts by increasing the expression of ERS-related molecules (p-PERK and CHOP), up-regulating PUMA, and down-regulating Bcl2. These data indicate that ICA is a potential inhibitor of lung adenocarcinoma cell growth by targeting ERS signaling and suggest that the activation of ERS signaling may represent a novel therapeutic intervention for lung adenocarcinoma.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Flavonoids; Glutathione; Heterografts; Humans; Lung Neoplasms; Male; Mice, Nude; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Thapsigargin

The objective of this study was performed to investigate the effects of thapsigargin on apoptosis, actin cytoskeletal dynamics, and actin cytoskeletal proteins in human lung adenocarcinoma cell. Thapsigargin is a specific irreversible inhibitor of ER calcium-ATPase, which may promote ER stress by depletion of lumenal calcium stores and show potential to induce cell death. The effects of thapsigargin on the apoptosis in A549 cells were assayed by Hoechst staining. Moreover, the F-actin staining by Rhodamine-phalloidin and RhoA antibody for cytoskeleton organizations were applied to A549 cells. To confirm the impairment of cytoskeletal dynamics treated with thapsigargin, western blots were applied to analyze the protein levels of p-Cofilin-1 (Ser3), Cofilin-1, and pPaxillin (Tyr118), as well as RhoA and pS6 (S240/244). Results suggest that thapsigargin may induce cell death in A549 cells with a time- and dose-dependent manner. The F-actin fibers and RhoA signals are also reduced with a time- and dose-dependent manner by thapsigargin treatment. The phosphorylation forms of Cofilin-1 and paxillin are attenuated by 1 μM thapsigargin treatment for 24 h. These alternations may be caused by the inhibition of of mTORC1 activities (indicated by pS6 (Ser240/244)) and RhoA pathways after thapsigargin treatment. The present findings highlight important roles of calcium entry in cytoskeleton organization and apoptosis in human lung adenocarcinoma cells and will help to set a stage to the clinical treatment of cancer cell metastasis.

Topics: Adenocarcinoma; Apoptosis; Blotting, Western; Calcium; Calcium-Transporting ATPases; Cell Line, Tumor; Cofilin 1; Cytoskeletal Proteins; Cytoskeleton; Endoplasmic Reticulum Stress; Humans; Lung Neoplasms; Paxillin; Thapsigargin

Endoplasmic reticulum (ER) stress is increasingly recognized as an important mechanism in a wide range of diseases including cystic fibrosis, alpha-1 antitrypsin deficiency, Parkinson's and Alzheimer's disease. Therefore, there is an increased need for reliable and quantitative markers for detection of ER stress in human tissues and cells. Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum can cause ER stress, which leads to the activation of the unfolded protein response (UPR). UPR signaling involves splicing of X-box binding protein-1 (XBP1) mRNA, which is frequently used as a marker for ER stress. In most studies, the splicing of the XBP1 mRNA is visualized by gel electrophoresis which is laborious and difficult to quantify. In the present study, we have developed and validated a quantitative real-time RT-PCR method to detect the spliced form of XBP1 mRNA.

Topics: Anti-Bacterial Agents; Base Sequence; Biomarkers; Cells, Cultured; DNA-Binding Proteins; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Enzyme Inhibitors; Epithelial Cells; Humans; Lung Neoplasms; Molecular Sequence Data; Oxidative Stress; Polymerase Chain Reaction; Regulatory Factor X Transcription Factors; RNA Splicing; RNA, Messenger; Stress, Physiological; Thapsigargin; Transcription Factors; Tunicamycin; X-Box Binding Protein 1

The molecular carcinogenesis of lung cancer has yet to be clearly elucidated. We investigated the possible oncogenic function of SEC62 in lung cancer, which was predicted based on our previous findings that lung and thyroid cancer tissue samples exhibited increased Sec62 protein levels. The SEC62 gene locus is at 3q26.2, and 3q amplification is reportedly the most common genomic alteration in non-small cell lung cancer. We analyzed SEC62 mRNA and protein levels in tissue samples from lung cancer patients by real-time quantitative PCR, Western blot, and IHC and found significantly increased SEC62 mRNA and protein levels in tumors compared with tumor-free tissue samples from the same patients. Correlation analyses revealed significantly higher Sec62 levels in tumors with lymph node metastases compared with nonmetastatic tumors, as well as in poorly compared with moderately differentiated tumors. On the basis of these promising results, we examined the role of Sec62 in cancer cell biology in vitro. Cell migration assays with lung and thyroid cancer cells showed distinct stimulation of migration in SEC62-overexpressing cells and inhibition of migration in Sec62-depleted cells. Moreover, we found that SEC62 silencing sensitized the cells to thapsigargin-induced endoplasmic reticulum stress. Thus, our results indicate that SEC62 represents a potential candidate oncogene in the amplified 3q region in cases of non-small cell lung cancer and harbors various functions in cancer cell biology.

Topics: Adaptor Proteins, Signal Transducing; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Chromosomes, Human, Pair 3; DNA Topoisomerases, Type II; DNA-Binding Proteins; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Gene Amplification; Gene Silencing; Humans; Immunohistochemistry; Lung Neoplasms; Membrane Transport Proteins; Real-Time Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Thapsigargin; Thyroid Neoplasms

Endoplasmic reticulum (ER) stress is activated under severe cellular conditions. GADD153, a member of the C/EBP family, is an unfolded protein response (UPR) responsive transcription factor. Increased levels of lipocalin 2, an acute phase protein, have been found in several epithelial cancers. The aim of this study is to investigate the function of lipocalin 2 in lung cancer cells under ER stress. Treatment with thapsigargin, an ER stress activator, led to increases in cytotoxicity, ER stress, apoptosis, and lipocalin 2 expression in A549 cells. GADD153 silencing decreased lipocalin 2 expression in A549 cells. On chromatin immunoprecipitation assay, ER stress increased GADD153 DNA binding to lipocalin 2 promoter. Furthermore, silencing of lipocalin 2 mitigated ER stress-mediated apoptosis in A549 cells. Our findings demonstrated that lipocalin 2 is a new GADD153 target gene that mediates ER stress-induced apoptosis.

Topics: Acute-Phase Proteins; Apoptosis; Cell Line, Tumor; Chromatin Immunoprecipitation; Endoplasmic Reticulum Stress; Gene Silencing; Humans; Lipocalin-2; Lipocalins; Lung Neoplasms; Proto-Oncogene Proteins; Thapsigargin; Transcription Factor CHOP

Extracellular ATP is a potent surfactant secretagogue but its origin in the alveolus, its mechanism(s) of release and its regulatory pathways remain unknown. Previously, we showed that hypotonic swelling of alveolar A549 cells induces Ca(2+)-dependent secretion of several adenosine and uridine nucleotides, implicating regulated exocytosis. In this study, we examined sources of Ca(2+) for the elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) evoked by acute 50% hypotonic stress and the role of autocrine purinergic signalling in Ca(2+)-dependent ATP release. We found that ATP release does not directly involve Ca(2+) influx from extracellular spaces, but depends entirely on Ca(2+) mobilization from intracellular stores. The [Ca(2+)](i) response consisted of slowly rising elevation, representing mobilization from thapsigargin (TG)-insensitive stores and a superimposed rapid spike due to Ca(2+) release from TG-sensitive endoplasmic reticulum (ER) Ca(2+) stores. The latter could be abolished by hydrolysis of extracellular triphospho- and diphosphonucleotides with apyrase; blocking P2Y(2)/P2Y(6) receptors of A549 cells with suramin; blocking UDP receptors (P2Y(6)) with pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS); emptying TG-sensitive stores downstream with TG or caffeine in Ca(2+)-free extracellular solution; or blocking the Ca(2+)-release inositol 1,4,5-triphosphate receptor channel of the ER with 2-aminoethyldiphenylborinate. These data demonstrate that the rapid [Ca(2+)](i) spike results from the autocrine stimulation of IP(3)/Ca(2+)-coupled P2Y, predominantly P2Y(6), receptors, accounting for approximately 70% of total Ca(2+)-dependent ATP release evoked by hypotonic shock. Our study reveals a novel paradigm in which stress-induced ATP release from alveolar cells is amplified by the synergistic autocrine/paracrine action of coreleased uridine and adenosine nucleotides. We suggest that a similar mechanism of purinergic signal propagation operates in other cell types.

Topics: Adenosine Triphosphate; Apyrase; Autocrine Communication; Caffeine; Calcium; Calcium Signaling; Calcium-Transporting ATPases; Cell Line, Tumor; Endoplasmic Reticulum; Enzyme Inhibitors; Epithelial Cells; Humans; Hydrolysis; Hypotonic Solutions; Inositol 1,4,5-Trisphosphate Receptors; Lung Neoplasms; Osmotic Pressure; Paracrine Communication; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; Suramin; Thapsigargin; Time Factors; Uracil Nucleotides

NF-kappaB is critical for determining cellular sensitivity to apoptotic stimuli by regulating both mitochondrial and death receptor apoptotic pathways. The endoplasmic reticulum (ER) emerges as a new apoptotic signaling initiator. However, the mechanism by which ER stress activates NF-kappaB and its role in regulation of ER stress-induced cell death are largely unclear. Here, we report that, in response to ER stress, IKK forms a complex with IRE1alpha through the adapter protein TRAF2. ER stress-induced NF-kappaB activation is impaired in IRE1alpha knockdown cells and IRE1alpha(-/-) MEFs. We found, however, that inhibiting NF-kappaB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner. Gene expression analysis revealed that ER stress-induced expression of tumor necrosis factor alpha (TNF-alpha) was IRE1alpha and NF-kappaB dependent. Blocking TNF receptor 1 signaling significantly inhibited ER stress-induced cell death. Further studies suggest that ER stress induces down-regulation of TRAF2 expression, which impairs TNF-alpha-induced activation of NF-kappaB and c-Jun N-terminal kinase and turns TNF-alpha from a weak to a powerful apoptosis inducer. Thus, ER stress induces two signals, namely TNF-alpha induction and TRAF2 down-regulation. They work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.

Topics: Adenoviridae; Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Caspase 8; Caspases; Cell Line, Tumor; Cell Survival; Down-Regulation; Electrophoretic Mobility Shift Assay; Endoplasmic Reticulum; Endoribonucleases; Enzyme Activation; Female; Fibroblasts; Gene Expression Regulation; Genes, Reporter; Humans; I-kappa B Proteins; JNK Mitogen-Activated Protein Kinases; Kinetics; L Cells; Luciferases; Lung Neoplasms; Male; Membrane Proteins; Mice; Mice, Knockout; NF-kappa B; Precipitin Tests; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Thapsigargin; TNF Receptor-Associated Factor 2; Tumor Necrosis Factor-alpha; Tunicamycin

Recent studies have identified novel actions for 2-aminoethoxydiphenyl borate (2-APB) in triggering calcium release and enhancing calcium influx induced by the depletion of intracellular calcium stores. In this study, we have examined the effects of 2-APB on the human lung adenocarcinoma A549 cell line, which we have previously shown displays a unique calcium influx response, when ER calcium stores are depleted by thapsigargin (TG) treatment. Here, we show that low concentrations of 2-APB failed to induce the rapid augmentation of TG-activated calcium influx previously reported for other cell types. We observed that store-operated calcium (SOC) channels in the A549 cell line exhibited short-term sensitivity to low doses of 2-APB, perhaps reflecting a delayed augmentation of SOC channel activity or the recruitment of 2-APB-insensitive SOC channels. In both intact and permeabilized cells, 2-APB effectively discharged a subset of A549 calcium pools corresponding to the hormone-sensitive intracellular calcium stores. The 2-APB-induced calcium release produced a long-lasting perturbation of the adenosine triphosphate (ATP)-releasable calcium pools, effectively uncoupling ATP-activated calcium release even, when stores are replenished with calcium. In contrast to previous reports, we found that disruption of either the actin or microtubule-based cytoskeleton failed to block the 2-APB-induced effects on calcium signaling in A549 cells. Our study describes novel cytoskeletal-independent effects of 2-APB on Ca2+-signaling pathways, revealing differentially sensitive Ca2+-influx pathways and long-term perturbation of hormone-sensitive Ca2+ stores.

Topics: Actins; Adenocarcinoma; Adenosine Triphosphate; Antineoplastic Agents, Phytogenic; Boron Compounds; Calcium; Calcium Channels; Calcium Signaling; Cell Line, Tumor; Cytochalasin D; Cytoskeleton; Digitonin; Dose-Response Relationship, Drug; Enzyme Inhibitors; Green Fluorescent Proteins; Humans; Indicators and Reagents; Inositol 1,4,5-Trisphosphate; Ionomycin; Lung Neoplasms; Microscopy, Confocal; Nucleic Acid Synthesis Inhibitors; Paclitaxel; Thapsigargin; Time Factors

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

Heterologous expression of the transient receptor potential-1 gene product (Trp1) encodes for a Ca2+ entry pathway, though it is unclear whether endogenous Trp1 contributes to a selective store-operated Ca2+ entry current. We examined the role of Trp1 in regulating both store-operated Ca2+ entry and a store-operated Ca2+ entry current, I(SOC), in A549 and endothelial cells. Twenty different 'chimeric' 2'-O-(2-methoxy)ethylphosphothioate antisense oligonucleotides were transfected separately using cationic lipids and screened for their ability to inhibit Trp1 mRNA. Two hypersensitive regions were identified, one at the 5' end of the coding region and the second in the 3' untranslated region beginning six nucleotides downstream of the stop codon. Antisense oligonucleotides stably decreased Trp1 at concentrations ranging from 10 to 300 nM, for up to 72 h. Thapsigargin increased global cytosolic Ca2+ and activated a I(SOC), which was small (-35 pA @ -80 mV), reversed near +40 mV, inhibited by 50 microM La3+, and exhibited anomalous mole fraction dependence. Inhibition of Trp1 reduced the global cytosolic Ca(2+) response to thapsigargin by 25% and similarly reduced I(SOC) by 50%. These data collectively support a role for endogenously expressed Trp1 in regulating a Ca2+-selective current activated upon Ca2+ store depletion.

Topics: Base Sequence; Calcium; Calcium Channels; Cytosol; Electric Conductivity; Endothelium, Vascular; Gene Expression; Humans; Lung Neoplasms; Molecular Sequence Data; Oligonucleotides, Antisense; Protein Biosynthesis; Pulmonary Artery; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thapsigargin; Transfection; TRPC Cation Channels; Tumor Cells, Cultured

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Bucladesine; Calcium Channels; Carcinoma, Small Cell; Cell Division; Cyclic AMP; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Humans; Kinetics; Lung Neoplasms; Naloxone; Neurons; Receptors, Opioid, delta; Serotonin; Thapsigargin; Time Factors; Tumor Cells, Cultured

Human small-cell lung carcinoma (SCLC) cells express neuronal-like voltage-operated calcium channels (VOCCs) and release mitogenic hormones such as serotonin (5-HT). Opioid peptides, on the other hand, have been shown to reduce SCLC cell proliferation by an effective autocrine pathway. Here we show that in GLC8 SCLC cells, only delta-opioid receptor subtype mRNA is expressed. Consistently, the selective delta-opioid agonist [D-Pen2-Pen5]-enkephalin (DPDPE), but not mu and kappa agonists, potently and dose-dependently inhibits high-threshold (HVA) VOCCs in these cells. As in peripheral neurons, this modulation is largely voltage-dependent, mediated by pertussis toxin (PTX)-sensitive G-proteins, cAMP-independent, and mainly affecting N-type VOCCs. With the same potency and selectivity, DPDPE also antagonizes the Ca(2+)-dependent release of [3H]serotonin ([3H]5-HT) from GLC8 cells. However, DPDPE inhibits not only the depolarization-induced release, but also the Ca(2+)-dependent secretion induced by thapsigargin or ionomycin. This suggests that besides inhibiting HVA VOCCs, opioids also exert a direct depressive action on the secretory apparatus in GLC8 cells. This latter effect also is mediated by a PTX-sensitive G-protein but, contrary to VOCC inhibition, it can be reversed by elevations of cAMP levels. These results show for the first time that opioids effectively depress both Ca2+ influx and Ca(2+)-dependent hormone release in SCLC cells by using multiple modulatory pathways. It can be speculated that the two mechanisms may contribute to the opioid antimitogenic action on lung neuroendocrine carcinoma cells.

Topics: Analgesics; Base Sequence; Calcium; Calcium Channel Blockers; Calcium Channels; Carcinoma, Small Cell; Cyclic AMP; Electrophysiology; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Enzyme Inhibitors; GTP-Binding Proteins; Humans; Ion Channel Gating; Ionomycin; Ionophores; Lung Neoplasms; Membrane Potentials; Molecular Sequence Data; Opioid Peptides; Pertussis Toxin; Potassium Chloride; Receptors, Opioid, delta; Serotonin; Terpenes; Thapsigargin; Tritium; Tumor Cells, Cultured; Virulence Factors, Bordetella

The human lung small cell adenocarcinoma cell line, A549, demonstrates a concentration-dependent rise in [Ca2+]i in response to extracellular nucleotides. The cells show Ca2+ mobilization on addition of various nucleotides, with an order of agonist potency: UTP > or = ATP > ADP > ADP beta S > AMP; adenosine is ineffective. The EC50 values for UTP and ATP are 12.5 +/- 0.4 microM and 18.9 +/- 0.5 microM, respectively. Together, these results are strongly indicative of the P2U subclass being the major nucleotide receptor expressed in these cells. The Ca2+ response was typically biphasic consisting of an initial spike, representing release of Ca2+ from internal stores, and a subsequent plateau representing Ca2+ influx. The majority of cells showed an agonist-induced Ca2+ increase that was unaffected by pretreatment with the Ca(2+)-ATPase inhibitors 2,5-di(tert-butyl)1,4-benzohydroquinone or thapsigargin. Caffeine did not raise [Ca2+]i above basal levels and applied in conjunction with nucleotide did not attenuate the agonist-mediated response. The Ca2+ influx was sensitive to protein kinase C, and agonist addition in the presence of a protein kinase C inhibitor, D-erythrosphingosine, produced a significantly potentiated Ca2+ influx. Furthermore, agonist-mediated Ca2+ influx was abolished in the presence of a protein kinase C activator, phorbol 12,13-dibutyrate. It is concluded that these cells posses a functional P2U receptor that, upon activation, causes Ca2+ mobilization from TBQ and thapsigargin insensitive stores followed by protein kinase C regulated Ca2+ influx.

Topics: Adenocarcinoma; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Benzoquinones; Caffeine; Calcium; Down-Regulation; Enzyme Inhibitors; Humans; Lung Neoplasms; Phosphodiesterase Inhibitors; Protein Kinase C; Purinergic P2 Receptor Agonists; Receptors, Purinergic P2; Thapsigargin; Thionucleotides; Tumor Cells, Cultured; Uridine Triphosphate

To the breast-fed infant, human milk is more than a source of nutrients; it furnishes a wide array of molecules that restrict microbes, such as antibodies, bactericidins, and inhibitors of bacterial adherence. However, it has rarely been considered that human milk may also contain substances bioactive toward host cells. While investigating the effect of human milk on bacterial adherence to a human lung cancer cell line, we were surprised to discover that the milk killed the cells. Analysis of this effect revealed that a component of milk in a particular physical state--multimeric alpha-lact-albumin--is a potent Ca(2+)-elevating and apoptosis-inducing agent with broad, yet selective, cytotoxic activity. Multimeric alpha-lactalbumin killed all transformed, embryonic, and lymphoid cells tested but spared mature epithelial elements. These findings raise the possibility that milk contributes to mucosal immunity not only by furnishing antimicrobial molecules but also by policing the function of lymphocytes and epithelium. Finally, analysis of the mechanism by which multimeric alpha-lactalbumin induces apoptosis in transformed epithelial cells could lead to the design of antitumor agents.

Topics: Animals; Apoptosis; Bacterial Adhesion; Breast Feeding; Calcium; Calcium-Transporting ATPases; Cattle; Cell Line; Cell Survival; Chromatin; Chromatography, Ion Exchange; Dogs; Electrophoresis, Polyacrylamide Gel; Embryo, Mammalian; Female; Humans; Infant; Lactalbumin; Lung Neoplasms; Lymphocytes; Milk Proteins; Milk, Human; Terpenes; Thapsigargin; Tumor Cells, Cultured

Small cell lung carcinoma is an aggressive neuroendocrine tumor that secretes several hormones, some of which act as autocrine growth factors. In order to obtain more information on the process of hormone secretion from this tumor, we have studied the role of intracellular free Ca2+ concentrations and voltage-operated calcium channels in the control of [3H]serotonin release from in vitro growing cell lines. We found that the Ca2+ ionophore ionomycin and the Ca(2+)-ATPase antagonist thapsigargin induced a dose-dependent increase of intracellular Ca2+ and a parallel enhancement of [3H]serotonin release. KCl-induced depolarization also stimulated a dose- and Ca(2+)-dependent [3H]serotonin release that in the GLC8 cell line was effectively inhibited by Ca2+ channel antagonists (Cd2+, nitrendipine, verapamil, omega-conotoxin GVIA, and omega-agatoxin IVA) and potentiated by the Ca2+ channel agonist BayK8644. Autoantibodies against Ca2+ channels present in the sera of Lambert-Eaton myasthenic patients antagonized KCl- but not ionomycin-induced [3H]serotonin release. Polymerase chain reaction analysis indicated that GLC8 cells express L-, N-, and P-type neuronal Ca2+ channel alpha 1 subunits, together with two types of Ca2+ channel beta subunits. The presence of three functionally distinct high threshold Ca2+ channels was also revealed by patch clamp experiments; high threshold Ca2+ channels were identified as dihydropyridine-sensitive (L-type), omega-conotoxin GVIA-sensitive (N-type), and omega-agatoxin IVA-sensitive (P-type). Our data demonstrate that [3H]serotonin is released by small cell lung carcinoma cells in a Ca(2+)-dependent manner and that depolarization-induced [3H]serotonin release is mediated by Ca2+ influx through distinct, neuron-like, Ca2+ channel subtypes.

Topics: Autoantibodies; Base Sequence; Calcium Channel Blockers; Calcium Channels; Calcium-Transporting ATPases; Carcinoma, Small Cell; Dihydropyridines; DNA Primers; Fura-2; Humans; Ionomycin; Lambert-Eaton Myasthenic Syndrome; Lung Neoplasms; Molecular Sequence Data; omega-Conotoxin GVIA; Peptides; Potassium Chloride; Serotonin; Terpenes; Thapsigargin; Tritium; 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