thapsigargin and Lymphoma

3β,16β,17α-Trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranosyl)-(1→3)-2-O-acetyl-α-L-arabinopyranoside (OSW-1) is a natural product with potent antitumor activity against various types of cancer cells, but the exact mechanisms of action remain to be defined. In this study, we showed that OSW-1 effectively killed leukemia cells at subnanomolar concentrations through a unique mechanism by causing a time-dependent elevation of cytosolic Ca(2+) prior to induction of apoptosis. A mechanistic study revealed that this compound inhibited the sodium-calcium exchanger 1 on the plasma membrane, leading to an increase in cytosolic Ca(2+) and a decrease in cytosolic Na(+). The elevated cytosolic Ca(2+) caused mitochondrial calcium overload and resulted in a loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3. Furthermore, OSW-1 also caused a Ca(2+)-dependent cleavage of the survival factor GRP78. Inhibition of Ca(2+) entry into the mitochondria by the uniporter inhibitor RU360 or by cyclosporin A significantly prevented the OSW-1-induced cell death, indicating the important role of mitochondria in mediating the cytotoxic activity. The extremely potent activity of OSW-1 against leukemia cells and its unique mechanism of action suggest that this compound may be potentially useful in the treatment of leukemia.

Topics: Biological Products; Calcium; Calcium Channels; Calpain; Caspase 3; Cell Death; Cell Line, Tumor; Cholestenones; Cyclosporine; Cytochromes c; Cytosol; Drug Screening Assays, Antitumor; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Activation; Extracellular Space; Heat-Shock Proteins; Homeostasis; Humans; Leukemia; Lymphoma; Membrane Potential, Mitochondrial; Mitochondria; Saponins; Sodium-Calcium Exchanger; Thapsigargin; Time Factors

Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)-specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication. SAL showed ER-stress-dependent and -independent antiviral effects, preventing virus release in human LCLs and abrogating gp350 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated B95-8 cells. TG resulted in sustained BCL6 but not BLIMP1 or CD138 expression, which is consistent with maintenance of a germinal center B-cell, rather than plasma-cell, phenotype. Microarray analysis identified candidate genes governing lytic replication in LCLs undergoing ER stress.

Topics: Carcinogens; Cell Line; Cinnamates; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Epstein-Barr Virus Infections; Eukaryotic Initiation Factor-2; Gene Expression Profiling; Gene Expression Regulation, Viral; Genes, Immediate-Early; Germinal Center; Herpesvirus 4, Human; Humans; Immediate-Early Proteins; Lymphocytes; Lymphoma; Membrane Glycoproteins; Plasma Cells; Tetradecanoylphorbol Acetate; Thapsigargin; Thiourea; Trans-Activators; Viral Matrix Proteins; Virus Replication

ABCG2 is a transporter preferentially expressed in a primitive subpopulation of cells and recently reported as a surviving factor for trophoblasts. To date, manner of ABCG2 expression in lymphoid tissues is not known. Immunohistochemically, strong ABCG2 expression was found in a small proportion of plasma cells mainly located in the interfollicular space of lymphoid tissues. The number of ABCG2-high plasma cells increased in interleukin-6- (IL-6) rich lesions, such as Castleman's disease of plasma cell type. Plasma cells are subjected to endoplasmic reticulum (ER) stress when excess proteins are synthesized, and IL-6 stimulates protein synthesis. Therefore, the effect of IL-6 and ER stress on ABCG2 expression in plasma cells was examined. The expression level of ABCG2 increased by treatment with either IL-6 or ER stress inducers, and further increased with both. The promoter analysis revealed that the effect of IL-6 and ER stress inducers was mediated through the site overlapping XBP-1 and HIF-1 binding sequences. Knocked-down of ABCG2 by siRNA or ABCG2 inhibitor reduced plasma cell viability under ER stress. These suggest that ABCG2 is a surviving factor for plasma cells. To our knowledge, this is the first study reporting the effect of ER stress on ABCG2 expression.

Topics: Apoptosis; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Caspase 3; Cell Line; DNA Methylation; Endoplasmic Reticulum; Gene Expression; Humans; Immunohistochemistry; Interleukin-6; Lymph Nodes; Lymphoma; Neoplasm Proteins; Palatine Tonsil; Plasma Cells; Promoter Regions, Genetic; Stress, Physiological; Thapsigargin; Tunicamycin

Disablement of cell death programs in cancer cells contributes to drug resistance and in some cases has been associated with altered translational control. As eukaryotic translation initiation factor 4E (eIF4E) cooperates with c-Myc during lymphomagenesis, induces drug resistance, and is a genetic modifier of the rapamycin response, we have investigated the effect of dysregulation of the ribosome recruitment phase of translation initiation on tumor progression and chemosensitivity. eIF4E is a subunit of eIF4F, a complex that stimulates ribosome recruitment during translation initiation by delivering the DEAD-box RNA helicase eIF4A to the 5' end of mRNAs. eIF4A is thought to prepare a ribosome landing pad on mRNA templates for incoming 40S ribosomes (and associated factors). Using small molecule screening, we found that cyclopenta[b]benzofuran flavaglines, a class of natural products, modulate eIF4A activity and inhibit translation initiation. One member of this class of compounds, silvestrol, was able to enhance chemosensitivity in a mouse lymphoma model in which carcinogenesis is driven by phosphatase and tensin homolog (PTEN) inactivation or elevated eIF4E levels. These results establish that targeting translation initiation can restore drug sensitivity in vivo and provide an approach to modulating chemosensitivity.

Topics: Animals; Apoptosis; Benzofurans; Cell Line; Cell Line, Tumor; Disease Models, Animal; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Eukaryotic Initiation Factor-4A; Eukaryotic Initiation Factor-4E; Female; HeLa Cells; Humans; Lymphoma; Mice; Mice, Inbred C57BL; Peptide Chain Initiation, Translational; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Polyribosomes; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Sirolimus; Thapsigargin; Triterpenes

The purpose of this study was to determine the roles of calcium-dependent phospholipase A2 (cPLA2) and calcium-independent phospholipase A2 (iPLA2) in thapsigargin-induced membrane susceptibility to secretory phospholipase A2 (sPLA2) and programmed cell death. 3H-arachidonic acid release was observed in the presence of thapsigargin. This release was inhibited partially by an inhibitor of iPLA2 (BEL) and completely by an inhibitor of both cPLA2 and iPLA2 (MAFP) suggesting that these enzymes were active during apoptosis. The process of cell death did not require the activity of either enzyme since neither inhibitor impeded the progression of apoptosis. However, both inhibitors increased the susceptibility of the membrane to sPLA2 in the presence of thapsigargin. In the case of BEL, this effect appeared to involve direct induction of apoptosis in a sub-population of the cells independent of the action of iPLA2. In conclusion, the results suggested that cPLA2 and iPLA2 are active during thapsigargin-induced apoptosis in S49 cells and that cPLA2 tempers the tendency of the cells to become susceptible to sPLA2 during apoptosis.

Topics: Animals; Apoptosis; Arachidonic Acid; Arachidonic Acids; Cell Membrane; DNA Fragmentation; Enzyme Inhibitors; Lymphoma; Mice; Organophosphonates; Phospholipases A; Phospholipases A2; Thapsigargin; Tritium; Tumor Cells, Cultured

The requirement for caspases (ICE-like proteases) were investigated in mediating apoptosis of WEHI7.2 mouse lymphoma cells in response to two death inducers with different mechanisms of action, the glucocorticoid hormone dexamethasone (DX) and the calcium-ATPase inhibitor thapsigargin (TG). Apoptosis induction by these agents followed different kinetics, and was closely correlated with in vivo activation of caspase-3 (CPP32/Yama/Apopain) and cleavage of the caspase target protein poly(ADP-ribose) polymerase (PARP). Caspase activation and PARP cleavage were inhibited by Bcl-2 overexpression. Cell extracts from DX- and TG-treated cells cleaved the in vitro synthesized baculovirus p35 ICE-like protease target, producing 25 and 10 kDa fragments. p35 cleavage was inhibited by mutating the active site aspartic acid to alanine, and by a panel of protease inhibitors that inhibit caspase-3-like proteases, including iodoacetamide, N-ethylmaleimide, and Ac-DEVD-cho. Treatment of cells in vivo with two cell permeant peptide fluoromethylketone inhibitors of caspase activity, Z-VAD-fmk and Z-DEVD-fmk, inhibited DX- and TG-induced apoptotic nuclear changes and maintained plasma membrane integrity, whereas the cathepsin inhibitor, Z-FA-fmk, and two calpain inhibitors failed to inhibit apoptosis. An unexpected observation was that due to the delayed time course of DX-induced apoptosis, optimal preservation of plasma membrane integrity was achieved by adding caspase inhibitors beginning 8 h after DX addition. In summary, the findings indicate that two diverse apoptosis-inducing signals converge into a common Bcl-2-regulated pathway that leads to caspase activation and apoptosis.

Topics: Animals; Apoptosis; Calcium-Transporting ATPases; Caspase 3; Caspases; Cell Death; Cell Membrane; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dexamethasone; Enzyme Activation; Enzyme Inhibitors; Glucocorticoids; Inhibitor of Apoptosis Proteins; Lymphoma; Mice; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Thapsigargin; Time Factors; Tumor Cells, Cultured; Viral Proteins

grp78/grp94 induction is critical for maintaining the viability of epithelial cells and fibroblasts following treatment with thapsigargin (TG), an inhibitor of Ca2+ uptake into the endoplasmic reticulum. In contrast to these cell types, WEHI7.2 mouse lymphoma cells undergo apoptosis when treated with TG, prompting us to examine the grp78/grp94 stress response in WEHI7.2 cells. TG treatment failed to induce grp78/grp94 transcription in WEHI7.2 cells, measured by Northern hybridization and nuclear run-on assays, even if the cells were protected from apoptosis by overexpressing bcl-2. However, grp78/grp94 transcription was induced by the glycosylation inhibitor tunicamycin, suggesting that there are at least two grp78/grp94 signaling pathways, one in response to TG-induced endoplasmic reticulum Ca2+ pool depletion, which is inoperable in WEHI7.2 cells, and one in response to glycosylation inhibition, which is operable in WEHI7.2 cells. Studies of additional lymphoid lines, as well as several nonlymphoid lines, suggested a correlation between grp78/grp94 induction and resistance to apoptosis following TG treatment. In conclusion, the vulnerability of TG-treated WEHI7.2 cells to apoptosis may be due to failure to signal a grp78/grp94 stress response.

Topics: Animals; Apoptosis; Calcium; Carrier Proteins; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Lymphoma; Membrane Proteins; Mice; Molecular Chaperones; Nerve Tissue Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Thapsigargin; Tunicamycin

The oncogene bcl-2 encodes a 26-kD protein localized to intracellular membranes, including the ER, mitochondria, and perinuclear membrane, but its mechanism of action is unknown. We have been investigating the hypothesis that Bcl-2 regulates the movement of calcium ions (Ca2+) through the ER membrane. Earlier findings in this laboratory indicated that Bcl-2 reduces Ca2+ efflux from the ER lumen in WEHI7.2 lymphoma cells treated with the Ca2+-ATPase inhibitor thapsigargin (TG) but does not prevent capacitative entry of extracellular calcium. In this report, we show that sustained elevation of cytosolic Ca2+ due to capacitative entry is not required for induction of apoptosis by TG, suggesting that ER calcium pool depletion may trigger apoptosis. Bcl-2 overexpression maintains Ca2+ uptake in the ER of TG-treated cells and prevents a TG-imposed delay in intralumenal processing of the endogenous glycoprotein cathepsin D. Also, Bcl-2 overexpression preserves the ER Ca2+ pool in untreated cells when extracellular Ca2+ is low. However, low extracellular Ca2+ reduces the antiapoptotic action of Bcl-2, suggesting that cytosolic Ca2+ elevation due to capacitative entry may be required for optimal ER pool filling and apoptosis inhibition by Bcl-2. In summary, the findings suggest that Bcl-2 maintains Ca2+ homeostasis within the ER, thereby inhibiting apoptosis induction by TG.

Topics: Animals; Apoptosis; Calcium; Calcium-Transporting ATPases; Cell Division; Cell Survival; Cytoplasm; Endoplasmic Reticulum; Enzyme Inhibitors; Homeostasis; Lymphoma; Mice; Proto-Oncogene Proteins c-bcl-2; Thapsigargin; Time Factors; Tumor Cells, Cultured

Transient elevation of cytosolic Ca2+ induces the expression of a variety of genes involved in cell growth and transformation, including the early response gene c-fos. Previously, we reported that Bcl-2 inhibits the transient elevation of cytosolic Ca2+ induced by thapsigargin (TG), a selective inhibitor of the endoplasmic reticulum-associated Ca2+-ATPase. Therefore, to determine if the effect of Bcl-2 on cytosolic Ca2+ elevation modulates Ca2+ signaling, we investigated the induction of c-fos by TG in WEHI7.2 mouse lymphoma cells, control transfectants (WEHI7.2-neo), and transfectants that stably express a high level of Bcl-2 (W.Hb12 and W.Hb15). TG induced 20-fold elevation of c-fos mRNA in WEHI7.2 and WEHI7.2-neo cells, but c-fos mRNA induction by TG was only fivefold in W.Hb12 and W.Hb15 cells. In contrast, phorbol 12-myristate acetate induced marked c-fos mRNA elevation in both WEHI7.2 and W.Hb12 cells, indicating that the inhibitory effect of Bcl-2 is selective for induction of c-fos by Ca2+. To measure c-fos promoter activity, WEHI7.2 and W.Hb12 cells were transiently transfected with a c-fos promoter-luciferase reporter plasmid. TG induced c-fos promoter activity in WEHI7.2 cells, but not in W.Hb12 cells. In WEHI7.2 cells, the signal for c-fos induction by TG was cytosolic Ca2+ elevation, as the increase in both c-fos mRNA level and promoter activity were prevented by lowering extracellular Ca2+ concentration, a condition that inhibits cytosolic Ca2+ elevation by reducing the TG-mobilizable Ca2+ pool. In summary, the findings indicate that Bcl-2 regulates Ca2+ signaling.

Topics: Animals; Calcium; Cytosol; Extracellular Space; Gene Expression Regulation, Neoplastic; Genes, fos; Lymphoma; Mice; Mice, Inbred BALB C; Promoter Regions, Genetic; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tetradecanoylphorbol Acetate; Thapsigargin; Transfection; Tumor Cells, Cultured

The mechanism by which Bcl-2 inhibits apoptosis is unknown. The Bcl-2 protein is localized to intracellular membranes, including the endoplasmic reticulum (ER). The ER is the major intracellular reservoir of Ca2+ in non-muscle cells, sequestering Ca2+ for use in intracellular signaling, and is a prime target of oxidative damage. Because of the recent suggestion that Bcl-2 acts in an antioxidant pathway, we wondered whether Bcl-2 might protect the ER Ca2+ pool in cells exposed to reactive oxygen species. To test this hypothesis, we assessed the effect of hydrogen peroxide (H2O2) treatment on the ER Ca2+ pool in WEH17.2 cells, which do not express Bcl-2, and two stable transfectants, W.Hb13 and W.Hb12. The Bcl-2 level by Western blotting is 4-fold higher in W.Hb12 cells compared to W.Hb13 cells. The ER Ca2+ pool in H2O2-treated and untreated cells was measured according to the amount of Ca2+ mobilized from the ER lumen into the cytoplasm by thapsigargin (TG), a selective inhibitor of the ER (Ca2+)-ATPase. H2O2 treatment produced a significant reduction in the TG-mobilizable Ca2+ pool in WEH17.2 and W.Hb13 cells, but not in W.Hb12 cells, indicating that overexpression of Bcl-2 preserves the integrity of the ER Ca2+ pool in cells exposed to reactive oxygen species.

Topics: Animals; Antioxidants; Calcium; Calcium-Transporting ATPases; DNA; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Hydrogen Peroxide; Lymphoma; Mice; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Terpenes; Thapsigargin; Transfection; Tumor Cells, Cultured

The mechanism by which Bcl-2 inhibits apoptosis is unknown. One proposal is that Bcl-2 regulates intracellular Ca2+ fluxes thought to mediate apoptosis. In the present study, we investigated Bcl-2's mechanism of action by determining the effect of Bcl-2 on intracellular Ca2+ fluxes in the WEHI7.2 mouse lymphoma cell line, which does not express Bcl-2, and its stable transfectant, W.Hb12, which expresses a high level of Bcl-2. Treatment with the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin produced marked alterations in intracellular Ca2+ homeostasis in both WEHI7.2 and W.Hb12 cells, including elevation of free cytosolic Ca2+, endoplasmic reticulum Ca2+ pool depletion, capacitative entry of extracellular Ca2+, and increased loading of Ca2+ into mitochondria. Similar changes in intracellular Ca2+ occurred spontaneously in both cell lines following exponential growth. In both situations, W.Hb12 cells maintained optimal viability despite marked alterations in intracellular Ca2+, whereas WEHI7.2 cells underwent apoptosis. Treatment with the glucocorticoid hormone, dexamethasone, induced apoptosis in WEHI7.2 cells, but not in W.HB12 cells, even though dexamethasone treatment did not alter intracellular Ca2+ homeostasis in either cell line. These findings indicate that Bcl-2 acts downstream from intracellular Ca2+ fluxes in a pathway where Ca(2+)-dependent and Ca(2+)-independent death signals converge.

Topics: Animals; Apoptosis; Blotting, Western; Calcium; Cell Survival; Cytosol; Dexamethasone; Enzyme Inhibitors; Genes, bcl-2; Glucocorticoids; Homeostasis; Humans; Intracellular Fluid; Lymphoma; Mice; Temperature; Thapsigargin; Time Factors; Transfection; Tumor Cells, Cultured

There is growing evidence for the involvement of Ca2+ in the programmed cell death (apoptosis) of lymphocytes, but the nature of glucocorticoid-induced Ca2+ fluxes and their role in the cell death pathway are poorly understood. In the study reported here, we assessed the effect of glucocorticoid treatment on intracellular Ca2+ homeostasis in W7MG1 mouse lymphoma cells. Levels of cytosolic Ca2+ were measured using the intracellular Ca2+ indicator fura2 AM, and total cellular Ca2+ was measured by atomic absorbance spectroscopy. The level of Ca2+ within internal stores, including the endoplasmic reticulum (ER), was estimated by measuring the increase in cytosolic Ca2+ induced by either ionomycin, an ionophore that mobilizes Ca2+ from a variety of internal stores, and by thapsigargin, a specific inhibitor of the ER-associated Ca(2+)-ATPase that mobilizes Ca2+ from the ER. Glucocorticoid treatment induced a significant decrease in ionomycin- and thapsigargin-mobilizable Ca2+ stores that was accompanied by an initial decrease in total cellular Ca2+, followed by a modest increase in both total cellular Ca2+ and cytosolic Ca2+. The glucocorticoid-induced depletion of internal Ca2+ stores was receptor mediated and occurred after a delay corresponding to the time required for glucocorticoid receptor complexes to regulate gene transcription. Mobilization of ER-associated Ca2+ stores by thapsigargin treatment induced DNA fragmentation and cell death similar to that observed after glucocorticoid treatment. These findings suggest that a mobilization of Ca2+ from internal stores may be a critical step in the apoptotic pathway of mouse lymphoma cells.

Topics: Animals; Apoptosis; Calcium; Cytosol; Dexamethasone; DNA; Fluorescent Dyes; Fura-2; Homeostasis; Ionomycin; Kinetics; Lymphoma; Mice; Terpenes; Thapsigargin; Tumor Cells, Cultured