RNAi-mediated silencing of insulin receptor substrate-4 enhances actinomycin D- and tumor necrosis factor-alpha-induced cell death in hepatocarcinoma cancer cell lines.
Insulin receptor substrate-4 (IRS-4) transmits signals from the insulin-like growth factor receptor (IGF-IR) and the insulin receptor (IR) to the PI3K/AKT and the ERK1/2 pathways. IRS-4 expression increases dramatically after partial hepatectomy and plays an important role in HepG2 hepatoblastoma cell line proliferation/differentiation. In human hepatocarcinoma, IRS-4 overexpression has been associated with tumor development. Herein, we describe the mechanism whereby IRS-4 depletion induced by RNA interference (siRNA) sensitizes HepG2 cells to treatment with actinomycin D (Act D) and combined treatment with Act D plus tumor necrosis factor-alpha (TNF-alpha). Similar results have been obtained in HuH 7 and Chang cell lines. Act D therapy drove the cells to a mitochondrial-dependent apoptotic program involving cytochrome c release, caspase 3 activation, PARP fragmentation and DNA laddering. TNF-alpha amplifies the effect of Act D on HepG2 cell apoptosis increasing c-jun N-terminal kinase (JNK) activity, IkappaB-alpha proteolysis and glutathione depletion. IRS-4 depleted cells that were treated with Act D showed an increase in cytochrome c release and procaspase 3 and PARP proteolysis with respect to control cells. The mechanism involved in IRS-4 action is independent of Akt, IkappaB kinase and JNK. IRS-4 down regulation, however, decreased gamma-glutamylcysteine synthetase content and cell glutathione level in the presence of Act D plus TNF-alpha. These results suggest that IRS-4 protects HepG2 cells from oxidative stress induced by drug treatment.
Unidad de Toxicología Molecular Hepática, Departamento de Bioquímica y Biología Molecular, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD). Universidad de Alcalá, E-28871 Alcalá de Henares, Spain.
Eva P
EP
Cuevas
Oscar
O
Escribano
Jorge
J
Monserrat
Javier
J
Martínez-Botas
María G
MG
Sánchez
Antonio
A
Chiloeches
Borja
B
Hernández-Breijo
Verónica
V
Sánchez-Alonso
Irene D
ID
Román
Maria Dolores
MD
Fernández-Moreno
Luis G
LG
Guijarro
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D055504:Insulin Receptor Substrate Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism*; D034622:RNA Interference; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D055504:Insulin Receptor Substrate Proteins; D014409:Tumor Necrosis Factor-alpha; D003609:Dactinomycin