AKT as a molecular target of fenretinide activity in glioblastoma in vitro

Glioblastoma multiforme is the most aggressive of the primary central nervous system tumours, with a median patient’s survival of less then 1 year. In this tumour, a constitutive activation of the serine/threonine protein kinase Akt/PKB pathway is associated with increase in tumour grade, decreased level of apoptosis and adverse clinical outcome. Fenretinide, a synthetic derivative of retinoic acid, is a modulator of cell proliferation and inductor of apoptosis in vitro; it is a very promising agent because of easy administration, long-term tolerability and low incidence of long-term side effects. It is currently under evaluation in clinical trials as a chemopreventive agent against a variety of cancers. Fenretinide has been shown to inhibit glioblastoma cells in vitro, but the mechanism of its antiproliferative action remains elusive. The present study was designed to investigate the role of Akt/PKB in the molecular mechanism of action of fenretinide in human glioblastoma in vitro, and for this purpose CRS-A2 and A-172 cell lines were chosen, which do highly express Akt/PKB. The dose- and time-dependent significance of cell survival inhibition was determined by Trypan Blue exclusion test. Apoptosis was checked by DNA fragmentation and caspase induction. Protein expression was evaluated by Western blotting analysis. Results show that the antiproliferative activity of Fenretinide in human glioblastoma in vitro, at pharmacologically achievable doses, is correlated with a downregulation of Akt protein expression as well as an inhibition of constitutively active Akt phosphorylation. In addition, the drug induced a down-regulation of cyclin D1/Cdk4 and a decrease of p21CIP1 protein expression.. These events preceded activation of caspases, proteolysis of the nuclear enzyme poly(ADP-ribose)polymerase (PARP) and DNA fragmentation in CRS-A2 glioma cells. No induction of apoptosis was evident in A-172 glioblastoma cells. Our data identified in the Akt/PKB pathway a new molecular target of fenretinide activity and provides a molecular rationale for therapeutic strategies in human glioblastomas.