Molecular mechanism of Mitomycin C-dependent caspase-8 regulation: implications for apoptosis and synergism with interferon-α signalling

Abstract

Caspase-8 is frequently mutated or silenced in several tumors including hepatocellular carcinomas (HCC) thereby potentially contributing to chemoresistance. The aim of our present study was to evaluate if chemotherapeutic drugs may mediate their effects through up-regulation of caspase-8 gene transcription. Huh7 hepatoma cells were transfected with a caspase-8 promoter construct fused to a luciferase reporter gene followed by stimulation with a subset of different chemotherapeutic drugs. Several drugs slightly induced caspase-8 promoter activity. However, strong caspase-8 promoter induction was found after Mitomycin C (MMC) treatment and this correlated with an increase in endogenous caspase-8 mRNA expression. Further molecular analysis demonstrated that MMC controls caspase-8 transcription via a c-jun/AP1 site located in the promoter in close proximity to the transcription start site. Inactivation of this c-jun/AP1 site using a dominant-negative c-jun adenovirus or site-directed mutagenesis inhibited MMC-dependent promoter induction. MMC treatment resulted in higher caspase-8 enzymatic activity and apoptosis and could be synergistically enhanced by co-stimulation with interferon-alpha (IFNalpha) via independent transcriptional mechanisms. In summary MMC controls caspase-8 expression via a c-jun/AP1 element in its promoter region. MMC-induced up-regulation of caspase-8 triggers apoptosis in target cells which can be further enhanced by IFNalpha. Therefore these findings also provide a potential new therapeutic approach to treat cancer cells.

miR‐181b modulates multidrug resistance by targeting BCL2 in human cancer cell lines

“…Our study was in concordance with the studies by Fanini et al . and Shi et al ,29, 30 and we also found that exogenous overexpression of miR‐181b sensitizes SGC7901/VCR and A549/CDDP cells to VCR‐ and CDDP‐induced apoptosis, respectively, and except for VCR and CDDP, exogenous overexpression of miR‐181b also altered sensitivity of both SGC7901/VCR and A549/CDDP cells to ADR, VP‐16, 5‐Fu, but not to MMC, a possible explanation for this phenomenon could be that MMC triggers apoptosis of cancer cells via a pathway where BCL2 is not necessarily involved 32–34…”

10-Formyltetrahydrofolate Dehydrogenase–Induced c-Jun-NH2-Kinase Pathways Diverge at the c-Jun-NH2-Kinase Substrate Level in Cells with Different p53 Status

“…Of note, such a phenomenon was observed in PC-3 cells, in which JNK activation appears to be upstream of caspase 8 (38). In support of this conclusion, it has been recently shown that caspase 8 promoter has a c-Jun/AP1 regulatory site, which controls transcription in response to mitomycin C in Huh7 hepatoma cells (41). …”

Pro-apoptotic Sorafenib signaling in murine hepatocytes depends on malignancy and is associated with PUMA expression in vitro and in vivo

“…Alternatively, apoptosis was determined by quantification of specific caspase-3 enzyme activity (fluorescence units/ μ g protein) in protein lysates from cultured cells as described recently. 33 In brief, protein lysates were incubated with the artificial substrate AFC-DEVD (Enzo Life Sciences, Farmingdale, NY, USA) and the caspase-3 mediated release of AFC from DEVD was quantified by UV spectrometry. For determination of cleaved caspase-3 activity in whole-cell extracts of isolated primary hepatocytes and livers, murine Hepa1-6 hepatoma cells were treated with 50  μ m Sorafenib for 24 h and were used as a positive control (pos.…”