Influence of oxygen partial pressure on the characteristics of human hepatocarcinoma cells

Abstract

Most of the in vitro studies using liver cell lines have been performed under atmospheric oxygen partial pressure (21% O2). However, the oxygen concentrations in the liver and cancer cells are far from this value. In the present study, we have evaluated the influence of oxygen on 1) the tumor cell lines features (growth, steady-state ROS levels, GSH content, activities of antioxidant enzymes, p66 Shc and SOD expressions, metalloproteinases secretion, migration, invasion, and adhesion) of human hepatocellular carcinoma cell lines, and b) the response of the cells to an oxidant stimulus (aqueous leaf extract of the V. baccifera plant species). For this purpose, three hepatocarcinoma cell lines with different p53 status, HepG2 (wild-type), Huh7 (mutated), and Hep3B (deleted), were cultured (6–30 days) under atmospheric (21%) and more physiological (8%) pO2. Results showed that after long-term culturing at 8% versus 21% O2, the cellular proliferation rate and the steady-state levels of mitochondrial O2- were unaffected. However, the intracellular basal ROS levels were higher independently of the characteristics of the cell line. Moreover, the lower pO2 was associated with lower glutathione content, the induction of p66 Shc and Mn-SOD proteins, and increased SOD activity only in HepG2. This cell line also showed a higher migration rate, secretion of active metalloproteinases, and a faster invasion. HepG2 cells were more resistant to the oxidative stress induced by V. baccifera. Results suggest that the long-term culturing of human hepatoma cells at a low, more physiological pO2 induces antioxidant adaptations that could be mediated by p53, and may alter the cellular response to a subsequent oxidant challenge. Data support the necessity of validating outcomes from studies performed with hepatoma cell cultures under ambient O2.

Defining Physiological Normoxia for Improved Translation of Cell Physiology to Animal Models and Humans

“…The liver is a highly vascularized and metabolically active tissue, and as such is relatively well oxygenated as illustrated in Figure 13. (587). Despite this, SOD activity and expression were significantly higher in these cells, and they exhibited greater resistance to oxidative challenge.…”

Unraveling the in vitro antitumor activity of Vismia baccifera against HepG2: role of hydrogen peroxide

“…Intracellular ROS levels were measured using the cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCF-DA) probe, as described in [11] . Inside the cells, the probe is deacetylated and oxidized forming the fluorescent compound, 2′,7′-dichlorofluorescein (DCF).…”

“…Glutathione (GSH) was evaluated with the glutathione reductase-DTNB recycling method, as reported previously [11] .…”

“…SOD activity was determined indirectly by the method of nitroblue tetrazolium, as is described in [11] .…”

“…The extract was rich in flavanols, particularly epicatechin (monomers, dimers and trimers), and flavonols. The plant infusion induced toxicity to human and rat hepatoma cell lines, increasing the intracellular levels of reactive oxygen species (ROS) at early times [ 10 , 11 ]. Results suggested that the cell death could be mediated by ROS.…”