Overexpression of 2-oxoglutaratecarrier in hepatocellular carcinoma overcomes cholesterol-mediated mitochondrial GSH depletion to evade cell death and promote in vivo tumor growth

Abstract

Mitochondrial cholesterol is emerging as a critical player in liver pathobiology. While mitochondrial cholesterol contributes to the transition from steatosis to steatohepatitis through sensitization to TNF/Fas via mitochondrial GSH (mGSH) depletion, hepatocellular carcinoma (HCC) cells exhibit increased mitochondrial cholesterol loading which is paradoxically associated with unimpaired GSH transport in mitochondria and hence undepleted mGSH levels. Since the 2-oxoglutarate carrier (SLC25A11, OG) has been shown to contribute to the transport of GSH into hepatic mitochondria, our aim was to analyze the expression of OG in HCC and its impact in hypoxia susceptibility. [Methods]: OG expression was determined in HCC cell lines and in samples from patients with HCC. HepG2 and H35 cells were transfected for 48 h with the OG siRNA or the control siRNA, examining GSH levels by recycling method. HepG2 and H35 cells with or without OG mRNA silencing were cultured under hypoxic (2% O2) or normoxic (21% O2) conditions for 48 h, analyzing ROS generation, cardiolipin peroxidation and cell viability. Moreover, we generated a Hep3B clone with stable downregulation of OG (Hep3B-OG) carrier to determine in vivo tumor growth. [Results]: While cholesterol loading in rat or mice hepatic mitochondria impairs GSH transport leading to mGSH depletion, mitochondria from HepG2, Hep3B or H35 cells exhibit unrestricted GSH transport despite increased cholesterol loading and decreased membrane fluidity. Interestingly, both HepG2 and H35 cells as well as samples from patients with HCC display enhanced OG mRNA and protein expression compared to rat liver or human hepatic extracts. Moreover, OG silencing by siRNA led to mGSH depletion (50–60%), sensitizing HCC cells to hypoxia-induced ROS generation, cardiolipin peroxidation, cytochrome c release and apoptosis. These findings were reproduced by pharmacological depletion of mGSH by BSO, which potentiated hypoxia-induced cardiolipin peroxidation and cell death. Finally, the rate of tumor growth in vivo of Hep3B-OG clones was lower compared to Hep3B cells, which was accompanied by increased TUNEL staining of corresponding tumors. [Conclusions]: OG overexpression is a novel strategy of HCC to resist and adapt to hypoxia, and its silencing sensitizes them to hypoxia by depleting mGSH levels.