Role for the ATPase inhibitory factor 1 in the environmental carcinogen-induced Warburg phenotype

Most tumors undergo metabolic reprogramming towards glycolysis, the so-called Warburg effect, to support growth and survival. Overexpression of IF1, the physiological inhibitor of the F0F1ATPase, has been related to this phenomenon and appears to be a relevant marker in cancer. Environmental contributions to cancer development are now widely accepted but little is known about the underlying intracellular mechanisms. Among the environmental pollutants humans are commonly exposed to, benzo[a]pyrene (B[a]P), the prototype molecule of polycyclic aromatic hydrocarbons (PAHs), is a well-known human carcinogen. Besides apoptotic signals, B[a]P can also induce survival signals in liver cells, both likely involved in cancer promotion. Our previous works showed that B[a]P elicited a Warburg-like effect, thus favoring cell survival. The present study aimed at further elucidating the molecular mechanisms involved in the B[a]P-induced metabolic reprogramming, by testing the possible involvement of IF1. We presently demonstrate, both in vitro and in vivo, that PAHs, especially B[a]P, strongly increase IF1 expression. Such an increase, which might rely on β2-adrenergic receptor activation, notably participates to the B[a]P-induced glycolytic shift and cell survival in liver cells. By identifying IF1 as a target of PAHs, this study provides new insights about how environmental factors may contribute to related carcinogenesis.


Western Blot immunoassays
For whole-cell lysates, cells were harvested and lysed for 20 min on ice in RIPA buffer supplemented with 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 1 mM orthovanadate, and a cocktail of protein inhibitors (Roche). Cells were then centrifuged at 13,000g for 15 min at 4 °C. The resulting supernatants were collected and frozen at −80 °C or used immediately. Ten to 30 µg of whole-cell lysates were heated for 5 min at 100 °C, loaded in a 4% stacking gel, and then separated by 10% sodium dodecyl sulfate-polymerase gel electrophoresis (SDS-PAGE). Gels were electroblotted overnight onto nitrocellulose membranes (Millipore).
For mitochondrial lysates, 30 µg were heated at 100°C for 5 min and then loaded on Any kD mini format precast gels (Biorad). After migration process, gels were then electroblotted on PVDF membrane (Biorad) using the Trans-Blot Transfer System (Biorad). After membrane blocking with a Tris-buffered saline (TBS) solution supplemented with 5% bovine serum albumin, membranes were then hybridized with primary antibodies overnight at 4 °C and next incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 1 hour. Immunolabeled proteins were visualized by chemiluminescence using the LAS-3000 analyzer (Fujifilm). Image processing was performed using Multi Gauge software (Fujifilm).

Cell culture
The mouse hepatoma cell line Hepa1c1c7 (purchased from the European Collection of Cell Culture) was maintained in MEMα medium with l-glutamine without ribonucleosides and deoxyribonucleosides (Gibco, Cergy Pontoise, France), and supplemented with 10% fetal calf serum and 0.1 mg/ml gentamycin, as previously described (Holme et al., 2007;Podechard et al., 2011). Cells were seeded near confluence (90 × 103 cells/cm 2 ) a day before treatment and the medium changed before exposures. When using inhibitors, these were added for 1 h before B[a]P treatment, for the indicated time points.

Mitochondria network visualization using fluorescence microscopy
F258 cells, grown on coverslips, were stained with 10 nM Mitotracker Red (a mitochondria selective dye; Molecular Probes, Invitrogen Life Technologies) during the last 30 min of B[a]P exposure. Cells were then washed with PBS and fixed in 4% paraformaldehyde (Sigma-Aldrich) for 20 min and then washed with PBS. Samples were digitized with 63x or 40x fluorescence objectives (Zeiss) on the IX83 inverted microscope (Olympus; Rungis, France) equipped with an ultra-high-speed wavelenght switching system Lambda DG4 (Sutter Instrument; Novato, USA) and an ORCA Flash 4.0 CMOS camera (Hamamatsu; Massy, France) using cellSense Dimension software (Olympus).

Analysis of the mitochondria network ultrastructure using Transmission electron microscopy
After drug exposure, cells were rinsed with 0.15 M Na cacodylate buffer and fixed by dropwise addition of glutaraldehyde (2.5%) for 1 h. After fixation, the specimens were rinsed several times with 0.15 M Na cacodylate buffer and postfixed with 1.5% osmium tetroxide for 1 h. After further rinsing with cacodylate buffer, the samples were dehydrated through a series of graded ethanol from 70 to 100%. The specimens were infiltrated in a mixture of acetone-Eponate (50/50) for 3 h and then in pure Eponate for 16 h. Finally, the specimens were embedded in DMP30-Eponate for 24 h at 60 °C. Sections (0.5 μm) were cut on a Leica UC7 microtome and stained with toluidine blue. Ultrathin sections (90 nm) were obtained, collected onto copper grids, and counterstained with 4% uranyl acetate and then with lead citrate. Examination was performed with a JEOL 1400 transmission electron microscope operated at 120 kV.