NOX1 supports the metabolic remodeling of HepG2 cells

Abstract

NADPH oxidases are important sources of reactive oxygen species (ROS) which act as signaling molecules in the regulation of protein expression, cell proliferation, differentiation, migration and cell death. The NOX1 subunit is over-expressed in several cancers and NOX1 derived ROS have been repeatedly linked with tumorigenesis and tumor progression although underlying pathways are ill defined. We engineered NOX1-depleted HepG2 hepatoblastoma cells and employed differential display 2DE experiments in order to investigate changes in NOX1-dependent protein expression profiles. A total of 17 protein functions were identified to be dysregulated in NOX1-depleted cells. The proteomic results support a connection between NOX1 and the Warburg effect and a role for NOX in the regulation of glucose and glutamine metabolism as well as of lipid, protein and nucleotide synthesis in hepatic tumor cells. Metabolic remodeling is a common feature of tumor cells and understanding the underlying mechanisms is essential for the development of new cancer treatments. Our results reveal a manifold involvement of NOX1 in the metabolic remodeling of hepatoblastoma cells towards a sustained production of building blocks required to maintain a high proliferative rate, thus rendering NOX1 a potential target for cancer therapy.

Fig 1. NOX1 and ROS levels are decreased in HepG2 cells expressing shNOX1.

NOX1 levels were determined by Western blot analysis in HepG2 cells stably expressing shRNA against NOX1 or a control shRNA. The image exemplifies a representative NOX1 Western blot. Relative protein expression is expressed as ratio between the Western blot intensity and total protein content of the sample determined from the PonceauS staining (left). Differences between groups were assessed using Welsch t-test (N_shCtr = 8, N_shNOX1 = 7). The dependence of the abundance of the dysregulated proteins upon NOX1 protein levels was tested in a mixed effect model with spot ID as random factor. ROS production was measured using CM-H2DCFDA assay (right). Differences in ROS levels between NOX1 reduced HepG2 cells and control cells were tested in a mixed effect model with cell type nested in replicates (N = 3).

Fig 2. 2DE map displaying proteins differentially expressed in control vs. NOX1 reduced HepG2 cells.

Empty arrows denote up-regulated protein functions, filled arrows denote down-regulated protein functions. White arrows denote differentially regulated but un-identified protein spots.

Fig 3. Abundance of differentially expressed proteins depends upon NOX1 relative expression (plotted are 95% CI).

Fig 4. Western blot validation of NOX1 regulated proteins.

The major components of the 2DE spots which contained complex proteins mixtures were subjected to validation through Western blot analysis. Seven of the proteins were confirmed to be differentially expressed in control vs. NOX1 reduced HepG2 cells. Plotted are the mean and SEM of WB intensities normalized to the total protein content of the sample determined from the PonceauS staining. SET—protein SET, HMGC—HMG-CoA-synthase, AFP—Alpha-fetoprotein, Acsl1—Long chain fatty acid-CoA ligase, GDH1—Glutamate dehydrogenase 1, UDPGP—UTP-glucose-1-phosphate uridylyltransferase. Statistical results are presented in Table 1.

Fig 5. NOX1 regulates glycogen content in HepG2 cells.

Glycogen levels were assessed in HepG2 transiently transfected with two shRNAs against NOX1 or with control shRNA. NOX1 reduced HepG2 cells produce more glycogen than shCtr expressing cells (right). Differences in glycogen levels were tested in a linear mixed effect model with cell line as predictor and replicate (N = 4) as random factor (one-tailed post-hoc z test). NOX1 depletion was confirmed be Western blot analysis (left).

Fig 6. Overview of the metabolic pathways regulated by NOX1.

Proteins differentially expressed in control vs. NOX1 reduced HepG2 cells are involved in glucose, glutamine, nucleotide and lipid metabolism. α-KG—α-ketoglutarate, OAA—oxaloacetic acid, PEP—phosphoenolpyruvate, TAG—triacylglycerol, TCA cycle—tricarboxylic acid cycle.