Proceedings of The Physiological Society

Mitochondria: Form and function (London, UK) (2017) Proc Physiol Soc 38, PC11

Poster Communications

Development of Sorafenib resistance in hepatocellular carcinoma is mediated by SIRT1

M. Penke2, A. Garten1,2, A. Barnikol-Oettler2, T. Gorski2, G. Lavery1, W. Kiess2

1. University of Birmingham, Birmingham, United Kingdom. 2. Hospital for Child and Adolescent Medicine, Leipzig University, Leipzig, Germany.


Sorafenib is a multi-kinase inhibitor as well as an inhibitor of oxidative phosphorylation (OXPHOS) (Fiume et al. 2011) and frequently the only treatment option for patients with non-resectable hepatocellular carcinomas (HCCs). Resistance to Sorafenib develops frequently and could be mediated by the NAD dependent deacetylase sirtuin (SIRT) 1 (Chen et al. 2012), a master regulator of cellular energy metabolism and stress responses. SIRT1 activity is regulated by cellular NAD+ concentrations and NAD salvage by nicotinamide phosphoribosyltransferase (NAMPT) (Revollo et al. 2004). We aimed to find out if Sorafenib treatment induces changes in cellular NAD levels and activity of Sirt1 and the cellular energy sensor adenosine monophosphate kinase (AMPK) and whether or not altering Sirt1 activity by overexpression or increasing cellular NAD levels could influence Sorafenib action on HCC in vitro. We could show that Sorafenib treatment of HCC cell lines (Huh7, Hep3B, HepG2) induced apoptosis 2-fold in Hep3B and HepG2 and 5-fold in Huh7 at 5 uM Sorafenib. Oxygen flow measured by high resolution respirometry in permeabilised cells was lower after Sorafenib treatment (1 and 5 uM for 24h) both in the leak (non-phosphorylating) and OXPHOS state. The ratio of H2O2/O as as measure for reactive oxygen species (ROS) production normalized to oxygen flow was increased after Sorafenib (1 uM) exposure for 24h, specifically in the leak state and after inhibiting complex III of the electron transport chain with Antimycin A. SIRT1 protein was downregulated and cellular NAD concentrations were significantly lower in Huh7 cells after exposure to Sorafenib for 24h. Concomitant to increasing phosphorylation of AMP kinase, activity of its downstream target mammalian target of rapamycin (mTOR) was decreased after Sorafenib treatment, which could indicate energy deprivation consistent with the decreased mitochondrial respiration. Both number of apoptotic cells and effects on AMPK/mTOR phosphorylation were reversed by supplementation of nicotinamide mononucleotide (NMN), the enzyme product of NAMPT. While pharmacologically inhibiting NAMPT by FK866 did not sensitise HCC to Sorafenib treatment, transient overexpression of Sirt1 decreased Sorafenib-induced apoptosis. We can therefore conclude that Sorafenib influences SIRT1 function and that overexpression of SIRT1 could be an underlying mechanism of resistance to Sorafenib treatment in HCC.

Where applicable, experiments conform with Society ethical requirements