anisomycin and Mesothelioma

Pleural fluid accumulation is a frequent clinical observation in diffuse malignant pleural mesothelioma (MPM). The cytological analysis of pleural fluid often reveals the presence of free spheroid aggregates of malignant cells, giving rise to the question of the ability of non-adherent tumor cells to resist the loss of anchorage-induced apoptosis (termed as anoikis), and to develop new tumor foci in the pleural cavity. Here, we show that MPM cells cultured under non-adherent conditions form well-organized aggregates composed of viable cells, which progressively enter in G(0). Although the PI3K/Akt, ERK and SAPK/JNK signaling pathways are activated in adherent MPM cells, loss of anchorage results in the inactivation of these pathways. By comparison, we show that the non-tumoral mesothelial cells MeT-5A enter anoikis in an SAPK/JNK-, Bim- and caspase-9-dependent pathway. The survival of MPM cells can be reversed by activating SAPK/JNK with anisomycin, according to a Bim-dependent mitochondrial pathway. Finally, our findings show that impairment of cell aggregation activates SAPK/JNK and Bim and induces anoikis. Our results underline the importance of intercellular contacts in the anoikis resistance of MPM cells.

Topics: Anisomycin; Anoikis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Cell Line; Extracellular Signal-Regulated MAP Kinases; Humans; JNK Mitogen-Activated Protein Kinases; Membrane Proteins; Mesothelioma; Pleural Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Resting Phase, Cell Cycle; RNA, Small Interfering; Signal Transduction

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) holds promise for the treatment of tumors; however, many tumors are resistant to TRAIL alone. We previously showed that resistant malignant mesothelioma cells are sensitized to TRAIL-induced apoptosis by diverse toxic insults including chemotherapy, irradiation, or protein translation inhibitors such as cycloheximide. In seeking nontoxic sensitizers for TRAIL, we tested the protein translation inhibitor anisomycin at subtoxic concentrations 10- to 100-fold below those reported to inhibit protein translation. At these low concentrations (25 ng/mL), anisomycin potently and rapidly sensitized mesothelioma cells to TRAIL-induced apoptosis. Moreover, such sensitization occurred in malignant but not in nonmalignant mesothelial cells. Sensitization by anisomycin was dependent on Bid, indicating a role for mitochondrial amplification in the apoptotic synergy with TRAIL signaling. Consistent with this, we found that anisomycin induces rapid accumulation of the BH3-only protein Bim; moreover, small interfering RNA knockdown of Bim inhibits anisomycin-induced sensitization. Bim accumulation seems not to be transcriptional; instead, it is associated with Bim phosphorylation and increased stability, both consistent with the activation of c-jun NH2-terminal kinase signals by anisomycin. Overall, our data indicate that the rapid and selective sensitization by anisomycin in mesothelioma cells is mediated by posttranslational potentiation of Bim, which primes the cells for apoptosis via the death receptor pathway. Such subtoxic approaches to sensitization may enhance the value of TRAIL in cancer therapy.

Topics: Anisomycin; Annexin A5; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Combined Modality Therapy; Cycloheximide; Drug Synergism; Electrophoresis, Gel, Two-Dimensional; Etoposide; Humans; Immunoblotting; JNK Mitogen-Activated Protein Kinases; Ligands; Membrane Proteins; Mesothelioma; Phosphorylation; Protein Synthesis Inhibitors; Proto-Oncogene Proteins; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured

The urokinase-type plasminogen activator (uPA) interacts with its receptor (uPAR) to promote proteolysis as well as cell proliferation and migration. These functions contribute to the pathogenesis of neoplastic growth and invasiveness. Expression of uPAR in tumor extracts also inversely correlates with prognosis in many forms of cancer. In this study, we sought to determine if differences in uPAR expression were distinguishable between cultured human lung carcinoma and malignant mesothelioma subtypes. We also sought to determine if, as in malignant mesothelioma cells, uPAR expression is regulated at the posttranscriptional level in cultured malignant lung carcinoma cells. Using 125I-uPA binding and ligand blotting techniques, uPAR was expressed by phenotypically diverse lung carcinoma cell lines, including the H460, H157 and H1395 non-small cell lines and the H146 small cell lung carcinoma line. Increased uPAR expression was also detected in spindle-shaped (M33K) and epithelioid (M9K and MS-1) malignant mesothelioma cells. Selected mediators, including TGF-beta, TNF-alpha, LPS and PMA, uniformly enhanced uPAR expression in each of the tumor cell lines. Steady state uPAR mRNA expression was determined by RNase protection assay and correlated directly with the changes in cell surface uPAR expression. By gel mobility shift and UV-cross linking assays, a uPAR mRNA binding protein (uPAR mRNABp) implicated in the posttranscriptional control of message stability, was identified in each of the cell lines. Expression of uPAR and its message in cultured lung carcinoma and malignant mesothelioma cells is similarly influenced by effectors present in the tumor microenvironment. Regulation of the uPAR message occurs at the posttranscriptional level in cultured small and non-small cell lung carcinoma cells as well as spindle-shaped and fibrous malignant mesothelioma cell lines. Posttranscriptional regulation of uPAR in all these cells involves the interaction of the uPAR mRNABp with uPAR mRNA, which promotes uPAR mRNA destabilization.

Topics: Anisomycin; Carcinoma; Cell Movement; Cycloheximide; DNA; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mesothelioma; Pleura; Protein Biosynthesis; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA Processing, Post-Transcriptional; RNA Stability; RNA-Binding Proteins; RNA, Messenger; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator