Rottlerin inhibits the nuclear factor kappaB/cyclin-D1 cascade in MCF-7 breast cancer cells.
In the course of a project aimed to clarify the molecular mechanisms by which phorbol 12-myristate 13-acetate (PMA)-activated forms of protein kinase C (PKC) promote growth arrest in an MCF-7 cell line, we found that the PKCdelta inhibitor Rottlerin was able by itself to block cell proliferation. In the current study, we investigated further the antiproliferative response to Rottlerin. Western blotting analysis of cytoplasmic/nuclear extracts showed that the drug did not prevent either extracellular signal-regulated kinase (ERK) activation by PMA or Akt phosphorylation, but did interfere with the NFkappaB activation process (both basal and PMA-stimulated), by lowering the levels of phospho-IkappaBalpha and preventing p65 nuclear migration. The growth arrest evoked by Rottlerin was not mediated by cell-cycle inhibitors p21 and p27 but was accompanied by a dramatic fall in the cyclin-D1 protein, the levels of which were not altered by the pan-PKC inhibitor GF 109203X, thus excluding a PKC-mediated mechanism in the Rottlerin effect. The parallel drop in cyclin-D1 mRNA suggested a down-regulation of the gene caused by the inhibition of nuclear factor-kappa B (NFkappaB), which occurs via a PKC-, Akt-, ERK- and mitochondrial uncoupling-independent mechanism. We provide preliminary evidence that the interference on the NFkappaB activation process likely occurs at the level of calcium/calmodulin-dependent protein kinase II (CaMKII), a known Rottlerin target. Indeed the drug prevented calcium-induced CaMKII autophosphorylation which, in turn, led to decreased NFkappaB activation.
Torricelli
C
C
Department of Physiology, University of Siena, via Aldo Moro, 7-53100 Siena, Italy. torricellic@unisi.it
Fortino
V
V
Capurro
E
E
Valacchi
G
G
Pacini
A
A
Muscettola
M
M
Soucek
K
K
Maioli
E
E
D000098:Acetophenones / Q000378:metabolism* / Q000494:pharmacology; D001578:Benzopyrans / Q000378:metabolism* / Q000494:pharmacology; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D045744:Cell Line, Tumor / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D006801:Humans; D007211:Indoles / Q000378:metabolism / Q000494:pharmacology; D008301:Maleimides / Q000378:metabolism / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
D000098:Acetophenones; D001578:Benzopyrans; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D016328:NF-kappa B; D019938:Cyclin D1; C085746:rottlerin; D051057:Proto-Oncogene Proteins c-akt; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D048049:Extracellular Signal-Regulated MAP Kinases; C070515:bisindolylmaleimide I