In HCC, autophagic cell death was found to be a major contributor to drug-induced antiprolioferation (12�C13) of tumor cells. A previous study also proved that selleck chemicals Bortezomib the celecoxib derivative, OSU-03012, could induce reactive oxygen species-related autophagy to inhibit HCC tumor growth (14�C15). Nilotinib is a second generation tyrosine kinase inhibitor (TKI) that functions via ATP-competitive inhibition. In the treatment of drug-resistant chronic myeloid leukemia (CML), nilotinib possesses an in vitro Bcr-Abl binding potency 30 times higher than imatinib in imatinib-resistant cells and a 5�C7 times higher potency in imatinib-sensitive leukemic cells (8). In addition to Bcr-Abl inactivation, nilotinib also inhibits kinases including KIT, DDR, MAPK, ZAK, and PDGFR with less potency (6).
Its broad spectrum of kinase-suppression activity makes nilotinib further applicable to the treatment of other types of cancers such as gastrointestinal stromal tumors (GIST), breast cancer, and melanoma. Nilotinib was demonstrated to have significant clinical activity in imatinib- and sunitinib-resistant GIST (10�C11). Nilotinib also exerted antiproliferative effects in an estrogen-deprived breast cancer cell line MCF-7 (15). Metastatic melanoma cells expressing c-Abl/Arg kinase activity are also susceptible to nilotinib-mediated cell growth inhibition (13). In this study, we explored whether nilotinib exerts any antitumor activity against HCC. Our data show that nilotinib is an impressive killer of HCC cells. Surprisingly, this cell death was mediated by activation of autophagy, rather than apoptosis.
We validated nilotinib induction of autophagic cell death through deactivating phosphatase PP2A and subsequently increasing AMPK phosphorylation. The antitumor activity exerted by nilotinib-mediated autophagy was further confirmed in an in vivo nude mouse model. In light of the identification of the PP2A-AMPK axis as a novel target of nilotinib-induced autophagy, further studies are warranted to assess nilotinib as an anti-HCC treatment. EXPERIMENTAL PROCEDURES Reagents and Antibodies Nilotinib (Tasigna) was kindly provided by Novartis Pharmaceuticals (Basel, Switzerland). For in vitro studies, nilotinib at various concentrations was dissolved in DMSO and then added to cells in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% fetal bovine serum (FBS).
The final DMSO concentration was 0.1% after addition to medium. Okadaic GSK-3 acid (OA), forskolin, and 3-methyladenine (3-MA) were purchased from Cayman Chemical (Ann Arbor, MI). Hydroxychloroquine (HCQ) was from Sigma-Aldrich (Seelze, Germany). Antibodies for immunoblotting as anti-LC3, -P-Akt (Ser473), -4EBP1, -P-4EBP1, -P-mTOR, -mTOR, -P-S6K, -S6K, anti-S6, -P-S6, -ATG3, -ATG5, -ATG7, and -Beclin 1 were from Cell Signaling (Danvers, MA). Cell Culture and Western Blot Analysis The PLC5 and Hep3B cell lines were obtained from American Type Culture Collection (Manassas, VA).
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