Muntane 2017 MiP2017

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The endoplasmic reticulum stress is the driving signaling promoting cell death in a context of reduced oxidative/nitrosative stress during treatment of liver cancer cells with Sorafenib.

Link: MiP2017

Muntane J, Gonzalez R, Ranguelova K, Cuadrado A, Padillo FJ, Rodriguez-Hernandez MA (2017)

Event: MiP2017

Hepatocarcinoma (HCC) represents more than 85% of primary tumors in liver, the sixth more frequent neoplasia and the second cause of death by cancer. 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 p53 genetic profile were used (HepG2, wild type; Hep3B, non-expression; and Huh7, mutated p53). We determined the effect of Sorafenib (0-100 μM) on different markers of ERS, cell survival, energetic sensor and redox signaling (Akt, AMPk, JNK and Nrf2), oxidative/nitrosative stress using fluorescent (2,7-diclorodihidrofluorescein diacetate or H2DCFDA, dihydroethidium or DHE, 4,5-diaminofluorescein diacetate or DAF-2) and spin (cyclic hydroxylamine 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine or CMH) probe and spin (5,5-dimethyl-1-pyrroline-N-oxide or DMPO) trap using bench-top EMXnano spectrometer (Bruker), mitochondrial polarization (5,5’,6,6-tetrachloro-1,1,3, 3-tetraethylbenzimidazolylcarbocyanine iodide or JC-1, and tetramethylrhodamine or TMR), 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, LC3I/LC3II 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 cell proliferation was also reduced from 12 h of Sorafenib treatment. The reduction of PERK and IRE1α signaling by siRNA downregulated autophagy and increased caspase-3 activity even at low Sorafenib concentration (10 nM) in HepG2. The inhibition of autophagy by 3-methyladenine and chloroquine potentiated Sorafenib-induced caspase-3 and cell proliferation inhibition in HepG2. The JNK inhibition by SP600125 reduced cell proliferation and increased caspase-3 induced by Sorafenib (10 µM). The reduction of Bim, but not Bak and Bax, reduced Sorafenib-induced caspase-3 in HepG2 cells.

Sorafenib reduced dose-dependently nitric oxide, O2.- and H2O2 generation, and the overall pattern of S-nitrosylated, carbonylated and tyrosine nitrated proteins in Sorafenib-treated HepG2 cells. The posttranslational modification of cell death receptors altered apoptosis. In this sense, Sorafenib (10 µM) 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. Nrf2 signaling was also altered by the treatment. Sorafenib reduced luciferase activity in control, as well as VEGF- and PDGF-stimulated ARE-Luc-transfected HepG2 cells. This effect correlated to mRNA reduced expression of 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.

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. The early Sorafenib-induced ER stress and JNK-AMPk-dependent survival autophagic process (3-12 h) derived upon unresolved ER stress up to PERK-CHOP-dependent increased of Bim-induced apoptosis in HepG2 cells. However, the kinetic of Bim expression profile and related apoptosis signaling were also the tightly balance between Akt- and AMPk-related signaling on Foxo3a-dependent Bim upregulation. The mitochondrial hyperpolarization is an early event of events 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 induced the shift from caspase-8 vs caspase-3 activity.

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)
  1. Inst Biomedicine Seville (IBIS), IBiS/“Virgen del Rocío” Univ Hospital/CSIC/Univ Seville, Spain
  2. Bruker BioSpin Corporation, Bruker, Billerica, MA, USA
  3. Inst Biomedical Research “Alberto Sols”, Madrid, Spain
  4. Dept General Surgery, “Virgen del Rocío” University Hospital/IBiS/CSIC/Univ Seville, Spain
  5. CENTRO INVESTIGACIÓN BIOMÉDICA EN RED Enfermedades Hepáticas y Digestivas (CIBERehd). - [email protected]