Muntane 2017 MiP2017
Hepatocarcinoma (HCC) represents more than 85% of primary tumors in liver. Sorafenib is the unique accepted molecular targeted drug for the treatment of patients in advanced stage. The present study evaluated the molecular mechanism related to the induction of cell death by Sorafenib in HCC cell lines.
HCC cell lines with different cell differentiation stages and p53 genetic background were used. The effect of Sorafenib (0-100 μM) was assessed on endoplasmic reticulum stress (ERS), energetic sensor and redox signaling, oxidative/nitrosative stress using fluorescent and spin probe and spin traps, mitochondrial polarization, autophagy, cell proliferation and apoptosis. The role of ERS, JNK, autophagy and Bcl-2 family members was determined using siRNA strategies in Sorafenib-induced apoptosis. The therapeutic effect of Sorafenib was also assessed in vivo in tumors developed by subcutaneous implantation of HCC cells in athymic mice.
Sorafenib (10-100 μM) induced early (3-12 h) mitochondrial hyperpolarization, ERS characterized by an increase of Ser51eIFα/eIFα, CHOP, IRE1α and sXBP1, but a decrease of ATF6 expression, overall temporally associated with the increase of the Thr183JNK/JNK, Thr172AMPα, Thr308Akt/Akt and Thr32Foxo3a/Foxo3a ratios, as well as reduction of Ser2481mTOR/mTOR and protein translation. This pattern was related to an increase of autophagy markers (Beclin, LC3II/LC3I and p62), and reduction of Mcl-1 and Bcl-2 protein expression. The progressive increase of CHOP expression, and reduction of Thr308Akt/Akt and Ser473Akt/Akt ratios was related to the reduction of autophagic flux and increase of Bim expression and caspase-3 activity (24 h). The reduction of ERS downregulated autophagy and increased apoptosis. The reduction of Bim, but not Bak and Bax, reduced Sorafenib-induced caspase-3 in HepG2 cells.
Sorafenib (10 µM) reduced dose-dependently nitric oxide, O2.- and H2O2 generation, and reduced S-nitrosylation of cell death receptors (CD95, TNF-R1 and Trail-R1) that correlated to a shift from caspase-8- to caspase-3-related apoptosis. Sorafenib also downregulates Nrf2 signaling and Nrf2-related genes (thioredoxin-1, thioredoxin reductase, hemoxigenase-1, NQO1, glutathione cysteine ligase and glutathione peroxidase). The in vivo study confirmed the antitumoral properties (reduction tumor size, and pro-autophagic and apoptotic, anti-proliferative, anti-angiogenic and anti-fibrotic effects) of Sorafenib in xenograft mice model.
In conclusion, the study showed that the induction of ER stress by Sorafenib was the driving mechanism involved in the sequential induction of autophagy and apoptosis in HepG2 cells. In particular, the induction of cell death by Sorafenib was related to the PERK-CHOP-dependent increase of Bim-induced apoptosis in HepG2 cells. However, the kinetic of Bim expression profile and related apoptosis signaling were also the results of tightly balance between Akt- and AMPk-related signaling on Foxo3a-dependent Bim upregulation. The mitochondrial hyperpolarization is an early event associated with autophagy and cell death in Sorafenib-treated liver cancer cells. However, the treatment reduced oxidative and nitrosative intracellular stress, Nrf2-dependent signaling and S-nitrosylation of cell death receptors which promoted downstream apoptotic events.
• Bioblast editor: Kandolf G
Labels: MiParea: Pharmacology;toxicology Pathology: Cancer Stress:Cell death Organism: Human Tissue;cell: Liver
- Muntané J(1,4,5), González R(1), Ranguelova K(2), Cuadrado A(3), Padillo FJ(4,5), Rodríguez-Hernández MA(1)
- Inst Biomedicine Seville (IBIS), IBiS/“Virgen del Rocío” Univ Hospital/CSIC/Univ Seville, Spain
- Bruker BioSpin Corporation, Bruker, Billerica, MA, USA
- Inst Biomedical Research “Alberto Sols”, Madrid, Spain
- Dept General Surgery, “Virgen del Rocío” University Hospital/IBiS/CSIC/Univ Seville, Spain
- CENTRO INVESTIGACIÓN BIOMÉDICA EN RED Enfermedades Hepáticas y Digestivas (CIBERehd), Madrid, Spain. - [email protected]