Tumor necrosis factor enhances SN38-mediated apoptosis in mesothelioma cells: The role of nuclear factor-κB pathway activation

BACKGROUND. Despite the best and most aggressive, often integrated, standard therapeutic approaches for mesothelioma, overall survival remains very poor. The actual failure points out clearly the need for the development of novel therapy. One of the promising paths of experimentation is artificial induction of apoptosis. A therapeutic strategy that relies on the down-regulation of BCL-XL inhibition nuclear factor κB (NF-κB) with a combination of SN38 and tumor necrosis factor (TNF) was studied in human mesothelioma cell lines (MSTO-221H, IST-MES1, IST-MES2, MPP89, H28, H513, H2052, and H290). METHODS AND RESULTS. Cell proliferation (clonogenic assay) was inhibited strongly by the combination of TNF and SN38. Examining the persistence of the NF-κB complexes using an electrophoretic mobility-shift assay, it appeared that they still were present at 24 hours in TNF-treated cells. In SN38-treated cells, NF-κB complexes persisted for 6 hours. In cells that were treated with combined SN38 and TNF, NF-κB complexes disappeared quickly and became undetectable at 6 hours. In flow cytometry analysis, only cells that were treated with combined SN38 and TNF demonstrated significant cellular accumulation in the sub-G₀-G₁ phase, suggesting a specific induction of apoptosis. Morphologic examination (4,6-diamidino-2- phenylindole staining and electron microscopy) and internucleosomal DNA fragmentation (gel ladder) confirmed rigorously the induction of apoptosis. CONCLUSIONS. Because of NF-κB inhibition with the combination of SN38 and TNF, the expression of BCL-XL (both the protein [Western blot analysis] and the mRNA [reverse transcriptase-polymerase chain reaction analysis]) was down-regulated, cytochrome c was released into the cytoplasm, caspase 3 was activated (Western blot analysis), and, consequently, apoptosis was triggered. The authors hope that the results of the current study may contribute to the design and implementation of a novel therapeutic approach that improves patients' responses to treatment for mesothelioma.