Tumor necrosis factor enhances SN38-mediated apoptosis in mesothelioma cells: the role of nuclear factor-kappaB 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 kappaB (NF-kappaB) 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).


RESULTS
Cell proliferation (clonogenic assay) was inhibited strongly by the combination of TNF and SN38. Examining the persistence of the NF-kappaB 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-kappaB complexes persisted for 6 hours. In cells that were treated with combined SN38 and TNF, NF-kappaB 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-kappaB 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.
Russo
Patrizia
P
Laboratory of Translational Research B(Lung Cancer), Department of Integrated Medical Oncology, National Cancer Institute, Genoa, Italy. patrizia.russo@istge.it
Catassi
Alessia
A
Malacarne
Davide
D
Margaritora
Stefano
S
Cesario
Alfredo
A
Festi
Luigi
L
Mulé
Antonino
A
Ferri
Luigi
L
Granone
Pierluigi
P
D017209:Apoptosis / Q000187:drug effects*; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D004249:DNA Damage; D053938:DNA Fragmentation; D004357:Drug Synergism; D006801:Humans; D000077146:Irinotecan; D008654:Mesothelioma / Q000188:drug therapy*; D016328:NF-kappa B / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D014407:Tumor Cells, Cultured; D048069:Tumor Necrosis Factors / Q000494:pharmacology*; D051020:bcl-X Protein
C494811:BCL2L1 protein, human; D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D048069:Tumor Necrosis Factors; D051020:bcl-X Protein; D000077146:Irinotecan; D045304:Cytochromes c; D020169:Caspases; D002166:Camptothecin