Title:Combination of Hypoxia and RNA-Interference Targeting VEGF Induces Apoptosis in Hepatoma Cells Via Autocrine Mechanisms
Volume: 13
Issue: 11
Author(s): Esther Raskopf, Annabelle Vogt, Georges Decker, Sarah Hirt, Katjana Daskalow, Thorsten Cramer, Jens Standop, Maria-Angeles Gonzalez-Carmona, Tilman Sauerbruch and Volker Schmitz
Affiliation:
Keywords:
Apoptosis, autocrine mechanisms, hepatocellular carcinoma, hypoxia, RNA interference, VEGF signaling, HCC disease, antitumoral efficacy
Abstract: Control of VEGF signaling is an intense objective of pre-clinical and clinical studies in HCC disease with
steadily increasing clinical application. Despite its emerging role, several aspects of anti-VEGF based treatments are
poorly investigated, like the impact on tumor cells themselves, such as the effect on intracellular signaling and apoptosis
induction in hepatoma cells. Effects of siRNA-VEGF on VEGF, VEGF-receptor expression and VEGF-A signaling such
as AKT and JNK phosphorylation were determined under normoxic or hypoxic conditions in murine hepatoma cells.
Apoptosis induction was analyzed by SubG1-fraction, JC1-staining and caspase-8 activation. VEGF receptor expression
was analysed by semiquantitative real time PCR. Independent of oxygen status, siRNA-VEGF reduced VEGF levels resulting
in decreased AKT and increased JNK phosphorylation in Hepa129 cells. The VEGF-receptors neuropilin-1 (Nrp1)
and neuropilin-2 (Nrp2) were downregulated following siRNA-VEGF treatment or hypoxia induction respectively. Functionally,
hypoxia significantly increased the apoptosis rate (as analyzed by SubG1-fraction, JC1-staining and JNKphosphorylation)
which was further stimulated by siRNA-VEGF treatment. Our data indicate that antitumoral efficacy of
an anti-VEGF based treatment with siRNA is partly based on negative autocrine feedback mechanisms which are even
enhanced under hypoxic conditions. This observation helps to understand why antitumoral efficacy can be maintained despite
of counteracting stimulation of tumoral VEGF secretion due to hypoxia. The direct impact on tumor cells further underscores
the attractiveness of an anti-VEGF based siRNA treatment.
