calpain 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

Calpains are cysteine proteases that have been implicated as both effectors and suppressors of apoptosis. Previously, we showed that c-myc transformation regulated calpain activity and sensitized cells to apoptosis induced by calpain inhibition. The objective of this study was to investigate the role of calpain in the Eμ-myc transgenic model of B-cell lymphoma. Calpain activity assays, apoptosis, cell cycle assays, and expression measurements were used to determine the activity and role of calpain in vitro and in vivo. We found that Eμ-myc transgenic cells have highly elevated calpain activity. Calpastatin, the negative calpain regulator, was expressed at much lower levels in Eμ-myc lymphoma cells compared to normal splenic B cells. The primary isoform in Eμ-myc lymphoma is calpain 1. Treatment of Eμ-myc lymphoma cells with the calpain inhibitors PD150606 or calpain inhibitor III induced caspase-3-dependent apoptosis in vitro. General caspase inhibitors or caspase-3/7 inhibitor protected cells from death induced by calpain inhibitor, whereas caspase-9 inhibitors failed to rescue cells. Human Burkitt's lymphoma (BL2) cells display a pattern of sensitivity and caspase-3 dependence similar to calpain inhibition. Treatment of Eμ-myc lymphoma-bearing mice with PD150606 inhibited calpain activity in vivo and induced cell death in these cells as determined by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining. Multiple daily treatments resulted in reduced tumor load, particularly in combination with etoposide. In conclusion, calpain is highly elevated in the Eμ-myc lymphoma and calpain inhibition has therapeutic potential.

Topics: Acrylates; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; B-Lymphocytes; Calcium-Binding Proteins; Calpain; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Etoposide; Humans; Lymphoma; Mice; Mice, Transgenic

Precise control of the level of c-Myc protein is important to normal cellular homeostasis, and this is accomplished in part by degradation through the ubiquitin-proteasome pathway. The calpains are a family of calcium-dependent proteases that play important roles in proteolysis of some proteins, and their possible participation in degradation of intracellular c-Myc was therefore investigated. Activation of calpain with the cell-permeable calcium ionophore A23187 in Rat1a-myc or ts85 cells in culture induced rapid cleavage of c-Myc. This degradation was both calpain- and calcium-dependent since it was inhibited by preincubation with either the calpain-inhibitory peptide calpeptin or the calcium-chelating agent EGTA. A23187-induced c-Myc cleavage occurred in a time-dependent manner comparable to that of FAK, a known calpain substrate, and while calpeptin was able to significantly protect c-Myc from degradation, inhibitors of the proteasome or caspase proteases could not. Exposure of Rat1a-myc or ts85 cells in culture to calpeptin, or to the thiol-protease inhibitor E64d, resulted in the accumulation of c-Myc protein without an impact on ubiquitin-protein conjugates. Using an in vitro assay, calpain-mediated degradation occurred rapidly with wild-type c-Myc as the substrate, but was significantly prolonged in some c-Myc mutants with increased transforming activity derived from lymphoma patients. Those mutants with a prolonged half-life in vitro were also more resistant to A23187-induced cleavage in intact cells. These studies support a role for calpain in the control of c-Myc levels in vivo, and suggest that mutations impacting on sensitivity to calpain may contribute to c-Myc-mediated tumorigenesis.

Topics: Animals; Blotting, Western; Calcimycin; Calcium; Calpain; Cell Line; Cell Transformation, Neoplastic; Chelating Agents; Cysteine Endopeptidases; Dipeptides; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Ionophores; Lymphoma; Mammary Neoplasms, Experimental; Mice; Multienzyme Complexes; Mutation; Peptide Hydrolases; Proteasome Endopeptidase Complex; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-myc; Rats

Apoptosis induced by cadmium has been shown in many tissues in vivo and in cultured cells in vitro. However, its molecular mechanism is not fully understood. When the human histiocytic lymphoma cell line U937 was treated with cadmium for 12 h, evidence of apoptotic features, including change in nuclear morphology, DNA fragmentation, formation of DNA ladder in agarose gel electrophoresis, and phosphatidylserine externalization, were obtained. Moreover, loss of the mitochondrial membrane potential (Deltapsi(m)) was observed in the cadmium-treated cells and was inhibited by a broad caspase inhibitor (Z-VAD-FMK). Caspase inhibitors suppressed the DNA fragmentation in the order of Z-VAD-FMK > caspase-8 inhibitor > caspase-3 inhibitor. Expression of Bcl-x(L) and Bid decreased significantly in the cadmium-treated cells, although no apparent change in Bcl-2 and Bax expression was found. Tetrakis-(2-pyridylmethyl) ethylendiamine, a cell-permeable heavy metal chelator, partially reversed the increase of fluorescence of Fura-2 in the cadmium-treated cells. In addition, verapamil (70 microm), a voltage-dependent Ca(2+) channel blocker, inhibited the DNA fragmentation induced by cadmium less than 100 microm and decreased the fluorescence of Fura-2. Cadmium up-regulated the expression of type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R) but not type 2 or type 3 IP(3)R. Calpain inhibitors I and II partially prevented DNA fragmentation. No effects of Z-VAD-FMK on the expression of type 1 IP(3)R or of calpain inhibitors on the loss of Deltapsi(m) were observed. These results suggest that cadmium possibly induced apoptosis in U937 cells through two independent pathways, the Ca(2+)-calpain-dependent pathway and the caspase-mitochondria-dependent pathway.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Cadmium; Calcium Channel Blockers; Calcium Channels; Calpain; Carrier Proteins; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Nucleus; Chelating Agents; DNA Fragmentation; Dose-Response Relationship, Drug; Ethylenediamines; Humans; Inositol 1,4,5-Trisphosphate Receptors; Lymphoma; Membrane Potentials; Mitochondria; Models, Biological; Phosphatidylserines; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; Time Factors; U937 Cells; Up-Regulation; Verapamil