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Polyamine depletion inhibits etoposide-induced NF-kappaB activation in transformed mouse fibroblasts
B Tantini1, C Pignatti, M Fattori
1Department of Biochemistry G. Moruzzi, University of Bologna, Bologna, Italy. benedetta.tantini@unibo.it
Abstract:
In a previous research, we have shown that adequate levels of polyamines are required in transformed mouse fibroblasts for the correlated activations of MAPK subtypes (ERK and JNK) and caspases induced by etoposide and leading to apoptosis. We report now that the treatment of fibroblasts with etoposide also elicited a progressive and sustained increase of NF-kappaB activation. The DNA binding activity of p65 NF-kappaB subunit was increased up to approximately 4-fold and was accompanied by enhancement of p65 phosphorylation. A two days pre-treatment of fibroblasts with alpha-difluoromethylornithine (DFMO), which caused polyamine depletion, provoked a slight activating effect when given alone, but markedly inhibited the etoposide-induced increases in p65 DNA binding and phosphorylation. The NF-kappaB inhibiting effect of DFMO was prevented by the addition of exogenous putrescine, which restored the intracellular content of polyamines. Selective inhibitors of the etoposide-stimulated MAPK subtypes also reduced NF-kappaB activation. Moreover, pharmacological NF-kappaB inhibition reduced the increase in caspase activity and cell death elicited by etoposide, suggesting that NF-kappaB is involved in signaling to apoptosis. The results of the present study, together with our previous findings, suggest that polyamines play a permissive role in the pathways triggered by etoposide and leading to cell death of fibroblasts, by supporting the activation of MAPKs, NF-kappaB and caspases.
Insights
Polyamines support cell death signaling by enabling the activation of key proteins like MAPKs, NF-kappaB, and caspases. This research clarifies polyamines
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Previous research established polyamines' necessity for MAPK and caspase activation in etoposide-induced apoptosis.
- Etoposide treatment in fibroblasts was observed to induce a progressive increase in NF-kappaB activation.
Purpose of the Study:
- To investigate the role of polyamines in etoposide-induced NF-kappaB activation and its subsequent signaling to apoptosis.
- To elucidate the interplay between polyamines, MAPKs, NF-kappaB, and caspases in the context of etoposide-induced cell death.
Main Methods:
- Fibroblasts were treated with etoposide, and NF-kappaB activation (DNA binding and phosphorylation) was assessed.
- Polyamine depletion was induced using alpha-difluoromethylornithine (DFMO), and its effects on NF-kappaB were evaluated.
- The impact of exogenous putrescine, MAPK inhibitors, and NF-kappaB inhibitors on etoposide-induced responses was examined.
Main Results:
- Etoposide treatment increased p65 NF-kappaB DNA binding activity and phosphorylation.
- DFMO-induced polyamine depletion inhibited etoposide-stimulated NF-kappaB activation, an effect reversed by putrescine.
- MAPK inhibitors reduced NF-kappaB activation, and NF-kappaB inhibition decreased etoposide-induced caspase activity and cell death.
Conclusions:
- Polyamines play a permissive role in etoposide-induced fibroblast apoptosis by supporting the activation of MAPKs, NF-kappaB, and caspases.
- NF-kappaB acts as a crucial signaling component in the pathway leading to etoposide-induced cell death.
