thapsigargin and Cell-Transformation--Neoplastic

Alveolar rhabdomyosarcoma comprises a rare highly malignant tumor presumed to be associated with skeletal muscle lineage in children. The hallmark of the majority of alveolar rhabdomyosarcoma is a chromosomal translocation that generates the PAX3-FOXO1 fusion protein, which is an oncogenic transcription factor responsible for the development of the malignant phenotype of this tumor. Alveolar rhabdomyosarcoma cells are dependent on the oncogenic activity of PAX3-FOXO1, and its expression status in alveolar rhabdomyosarcoma tumors correlates with worst patient outcome, suggesting that blocking this activity of PAX3-FOXO1 may be an attractive therapeutic strategy against this fusion-positive disease. In this study, we screened small molecule chemical libraries for inhibitors of PAX3-FOXO1 transcriptional activity using a cell-based readout system. We identified the Sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCA) inhibitor thapsigargin as an effective inhibitor of PAX3-FOXO1. Subsequent experiments in alveolar rhabdomyosarcoma cells showed that activation of AKT by thapsigargin inhibited PAX3-FOXO1 activity via phosphorylation. Moreover, this AKT activation appears to be associated with the effects of thapsigargin on intracellular calcium levels. Furthermore, thapsigargin inhibited the binding of PAX3-FOXO1 to target genes and subsequently promoted its proteasomal degradation. In addition, thapsigargin treatment decreases the growth and invasive capacity of alveolar rhabdomyosarcoma cells while inducing apoptosis in vitro. Finally, thapsigargin can suppress the growth of an alveolar rhabdomyosarcoma xenograft tumor in vivo. These data reveal that thapsigargin-induced activation of AKT is an effective mechanism to inhibit PAX3-FOXO1 and a potential agent for targeted therapy against alveolar rhabdomyosarcoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Calcium; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Drug Screening Assays, Antitumor; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Mice; Oncogene Proteins, Fusion; Paired Box Transcription Factors; Phenotype; Phosphorylation; Protein Binding; Proteolysis; Proto-Oncogene Proteins c-akt; Rhabdomyosarcoma, Alveolar; Small Molecule Libraries; Thapsigargin; Transcription, Genetic; Tumor Burden; Xenograft Model Antitumor Assays

Rotavirus infection modifies the metabolism and ionic homeostasis of the host cell. First, there is an induction of viral synthesis with a parallel shutoff of cell protein production, followed by an increase of plasma membrane Ca2+ permeability, thereby inducing an increase of free cytoplasmic and sequestered Ca2+ concentrations. Cell death follows at a later stage. We studied the role of the increase in Ca2+ concentration in cell death. An elevation of extracellular Ca2+ concentration during infection induced an increase in [Ca2+]i and potentiated cell death. Buffering the increases in [Ca2+]i with BAPTA added at 6 h p.i. reduced the cytopathic effect without inhibiting viral protein synthesis and infectious particle production. Metoxyverapamil (D600), a Ca2+ channel inhibitor, added at 1 h p.i. reduced Ca2+ permeability, the increases in [Ca2+]i, and cell death produced by infection without modifying viral protein synthesis and infectious titer. Thapsigargin, the inhibitor of Ca(2+)-ATPase of endoplasmic reticulum, potentiated the increase of [Ca2+]i and accelerated the time course of cell death. Double staining with fluorescein diacetate and ethidium bromide or acridine orange and ethidium bromide showed that infected MA104 cells had lost plasma membrane integrity without DNA fragmentation or formation of apoptotic bodies. These results support the hypothesis that the increase in [Ca2+]i due to a product of viral protein synthesis triggers the chain of events that leads to cell death by oncosis.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium-Transporting ATPases; Cell Death; Cell Line; Cell Membrane Permeability; Cell Transformation, Neoplastic; Chelating Agents; Cytopathogenic Effect, Viral; Egtazic Acid; Enzyme Inhibitors; Gallopamil; Haplorhini; Homeostasis; Microscopy, Fluorescence; Rotavirus Infections; Thapsigargin; Viral Proteins

Alkaline stress transforms Madin-Darby canine kidney (MDCK) cells as indicated by loss of epithelial structure, multilayer cell growth and formation of foci. In the present study we report that transformed MDCK cells (MDCK-F cells) exhibit spontaneous and lasting oscillations of intracellular Ca2+ concentration ([Ca2+]i), which are absent in non-transformed cells. Oscillations, as revealed by Fura-2 video imaging, were due to the activity of an inositol 1,4,5-trisphosphate-(InsP3)-sensitive Ca2+ store since their frequency was dependent on bradykinin concentration and they were abolished by the phosphoinositidase C inhibitor U73122. Moreover, blockers of the cytoplasmic Ca(2+)-ATPase, thapsigargin and 2,5-di-(tetr-butyl)-1,4-benzohydroquinone inhibited oscillatory activity. In contrast, neither injection of ruthenium red, ryanodine nor caffeine had any effect on oscillations. Analysis of the spatial distribution of [Ca2+]i showed that Ca2+ transients originated from an initiation site constant for a given cell and spread through the cell as an advancing Ca2+ wave. Oscillations started in a random manner from single cells and spread over neighbouring cells, suggesting a kind of intercellular communication. We conclude that MDCK-F cells have acquired the ability for endogenous Ca2+ release through transformation. Oscillations are primarily due to the activity of an InsP3-sensitive cytosolic Ca2+ oscillator.

Topics: Animals; Biological Clocks; Bradykinin; Calcium; Calcium-Transporting ATPases; Cell Communication; Cell Line; Cell Transformation, Neoplastic; Cytoplasm; Dogs; Estrenes; Hydrogen-Ion Concentration; Inositol 1,4,5-Trisphosphate; Ionomycin; Pyrrolidinones; Terpenes; Thapsigargin; Type C Phospholipases

Transformed Mardin-Darby canine kidney-focus (MDCK-F) cells exhibit spontaneous Ca2+ oscillations from an inositol 1,4,5-triphosphate-sensitive cytoplasmic Ca2+ store. In this study, Ca2+ entry from the extracellular space and its role in generation of oscillations were investigated by means of Ca2+ video imaging and the Fura-2/Mn2+ quenching technique. Oscillations were dependent on extracellular Ca2+ concentration and were inhibited by extracellularly applied La3+, Co2+ and Ni2+. Depolarization of the cell membrane with high K+ concentrations and the L-type Ca2+ channel blocker nifedipine had no effect on oscillations, indicating the lack of involvement of voltage-gated Ca2+ channels. Mn2+ quenching experiments disclosed significant Ca2+ influx into MDCK-F cells. The rate of this influx was constant between Ca2+ spikes, but markedly increased during the spontaneous Ca2+ spikes. Similar transient increases in Ca2+ entry could be mimicked by agents triggering intracellular Ca2+ release such as bradykinin and thapsigargin. We conclude that the plasma membrane of MDCK-F cells exhibits a marked voltage-independent Ca2+ permeability permitting Ca2+ entry into the cytoplasm. The rate of Ca2+ entry which determines the frequency of oscillations is most likely to be regulated by the cytoplasmic Ca2+ concentration.

Topics: Animals; Biological Clocks; Bradykinin; Calcium; Calcium-Transporting ATPases; Cell Line; Cell Membrane Permeability; Cell Transformation, Neoplastic; Cytoplasm; Dogs; Extracellular Space; Inositol 1,4,5-Trisphosphate; Ion Transport; Manganese; Terpenes; Thapsigargin

Here we describe differences in the formation of the epinephrine-induced Ca2+ transients between parent and Ki-ras-transformed NIH3T3 fibroblasts. These transients proved to be the results of an efflux from both the thapsigargin-sensitive and -insensitive intracellular pools. While the epinephrine-induced detachment of the ras-transformed cells might be due to cytoskeletal and/or cell-matrix alterations.

Topics: 3T3 Cells; Animals; Calcium; Cell Transformation, Neoplastic; Cytoskeleton; Epinephrine; Extracellular Matrix; Genes, ras; Mice; Signal Transduction; Terpenes; Thapsigargin; Transfection