Malignant mesothelioma cells are rapidly sensitized to TRAIL-induced apoptosis by low-dose anisomycin via Bim.

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.

Gene expression profiles in human mesothelioma cell lines in response to interferon-gamma treatment.

Interferon-gamma (IFN-gamma) has been found to be antiproliferative and antitumoral in malignant mesothelioma (MM), but the MM cell response to IFN-gamma has not been fully characterized so far. We investigated gene expression profiles in human MM cell lines (HMCLs) exposed to IFN-gamma. Four HMCLs showing different sensitivities to the antiproliferative effect of IFN-gamma, two of them presenting a defect in the JAK/STAT signaling pathway, were treated with 500 IU/mL of IFN-gamma. Gene expression patterns were studied at 6 and 72 hours after exposure to the IFN-gamma, using a cDNA array technique. Six genes were studied with real-time reverse transcription-polymerase chain reaction. The gene expression profiles in response to IFN-gamma were shown to differ in different HMCLs. Numerous genes involved in cell proliferation and cell adhesion were regulated by IFN-gamma in JAK/STAT-deficient HMCLs. Genes possibly involved in regulation of cell proliferation included CDC2, PLK1, and IGFBP4, as well as several genes involved in cell-cell interactions and cell adhesion. The cDNA array technique revealed differences in expression pathways, especially those involved in cell growth, cell adhesion, and cell proliferation, between IFN-gamma-resistant and -sensitive MM cell lines. We found that the changes in gene expression profiles of HMCLs exposed to IFN-gamma were also related to features other than the antiproliferative response.

p21 (CDKN1A) is a negative regulator of p53 stability.

Cell cycle arrest in response to DNA damage involves protein stabilization and consequent upregulation of p53, which induces transcription of cyclin-dependent kinase inhibitor p21 (CDKN1A). We now show that p21 acts as a negative regulator of the cellular levels of p53. p21 knockdown by short hairpin RNA strongly increased p53 upregulation by a DNA-damaging drug doxorubicin in HT1080 fibrosarcoma cells. A protease inhibitor N-Ac-Leu-Leu-norleucinal (ALLN) drastically increased the amount of p53 in HCT116 colon carcinoma cells, but it had no effect on the already high p53 level in a p21(-/-) derivative of this cell line. Inhibition of transcription, which increases p53 levels in different cell lines due to the degradation of p53-destabilizing proteins such as Mdm2, failed to increase but instead decreased the amount of p53 in p21(-/-) cells, despite a drastic decrease in the level of Mdm2. These results indicate that p21 acts as a negative regulator of p53 stability in different cell types. p53 regulation by p21 may provide a negative regulatory loop that limits p53 induction.

c-Jun N-terminal kinase contributes to apoptotic synergy induced by tumor necrosis factor-related apoptosis-inducing ligand plus DNA damage in chemoresistant, p53 inactive mesothelioma cells.

Apoptotic resistance of cancer cells may be overcome by the combination of treatments that activate the two major apoptotic pathways: (i) the death receptor pathway activated by death ligands and (ii) the DNA damage pathway activated by chemotherapy. We have previously shown that mesothelioma cells, resistant to most treatments, are sensitive to the combination of the death ligand tumor necrosis factor-related apoptosis inducing ligand (TRAIL/Apo2L) plus chemotherapy. We investigated a possible role for c-Jun N-terminal kinase (JNK) in the synergistic effect, knowing that JNK can be activated separately by TRAIL and by DNA damage. We chose to study the M28 and REN human mesothelioma cell lines, which are p53-inactivated, to avoid an interaction between p53 and JNK. We showed that JNK was activated by TRAIL and by etoposide and that the activation was enhanced by the combination of the two treatments. We found this activation to be caspase-independent. To inhibit the JNK pathway, we used either dominant-negative constructs of JNK1 and JNK2 (compared with dominant-negative caspase 9) or a chemical inhibitor of the JNK pathway (SP600125). In cells treated with TRAIL plus etoposide, JNK inhibition increased cell survival and decreased apoptosis significantly. In transfected M28 cells, the effect of JNK inhibition was as great as that of the dominant-negative caspase 9 construct. We conclude that JNK contributes to the synergistic effect of TRAIL combined with DNA damage by mediating signals independent of p53 leading to apoptosis.