Differential Impact of Intermittent vs. Sustained Hypoxia on HIF-1, VEGF and Proliferation of HepG2 Cells

Abstract

Obstructive sleep apnea (OSA) is an emerging risk factor for cancer occurrence and progression, mainly mediated by intermittent hypoxia (IH). Systemic IH, a main landmark of OSA, and local sustained hypoxia (SH), a classical feature at the core of tumors, may act separately or synergistically on tumor cells. Our aim was to compare the respective consequences of intermittent and sustained hypoxia on HIF-1, endothelin-1 and VEGF expression and on cell proliferation and migration in HepG2 liver tumor cells. Wound healing, spheroid expansion, proliferation and migration were evaluated in HepG2 cells following IH or SH exposure. The HIF-1α, endothelin-1 and VEGF protein levels and/or mRNA expression were assessed, as were the effects of HIF-1 (acriflavine), endothelin-1 (macitentan) and VEGF (pazopanib) inhibition. Both SH and IH stimulated wound healing, spheroid expansion and proliferation of HepG2 cells. HIF-1 and VEGF, but not endothelin-1, expression increased with IH exposure but not with SH exposure. Acriflavine prevented the effects of both IH and SH, and pazopanib blocked those of IH but not those of SH. Macitentan had no impact. Thus, IH and SH stimulate hepatic cancer cell proliferation via distinct signaling pathways that may act synergistically in OSA patients with cancer, leading to enhanced tumor progression.

Keywords: HepG2; hypoxia inducible factor 1 (HIF-1); intermittent hypoxia (IH); liver cancer; obstructive sleep apnea syndrome (OSA); sustained hypoxia (SH); vascular endothelial growth factor (VEGF).

Figure 1

In vitro sustained hypoxia and intermittent hypoxia increase hepatic tumor cell expansion. (a) Representative illustrations of HepG2 cell invasiveness in 2D, assessed by wound healing, before and after 7 days of normoxia, intermittent hypoxia or sustained hypoxia exposure (white scale bar = 600 µm). (b) Wound healing, expressed as a % of repaired area compared with normoxia, of HepG2 cells exposed to 7 days of normoxia, intermittent hypoxia or sustained hypoxia; n = 5 experiments per group with at least 3 wells/experiment. Intermittent hypoxia global effect p < 0.00001, ** p < 0.01 on D4 and D7 for IH vs. N and on D5 and D7 for SH vs. N and *** p < 0.001 for IH vs. N on D5, one-way repeated measures ANOVA. Sustained hypoxia global effect p = 0.006, one-way repeated measures ANOVA and ** p < 0.01 on D5 and on day 7. (c) Representative illustrations of HepG2 spheroid expansion before and after 5, 7, 14 and 21 days of normoxia, intermittent hypoxia or sustained hypoxia exposure (white scale bar = 1000 µm). (d) HepG2 spheroid expansion in response to 21 days of intermittent hypoxia; n = 5 experiments per group (6 to 12 wells/experiment). * p < 0.05, repeated measures ANOVA). Post hoc analysis showed significant differences * p < 0.05 on days 7 and 21 of exposure. HepG2 spheroid expansion in response to 21 days of sustained hypoxia; n = 5 experiments per group (6 to 12 wells/experiment). * p < 0.01, repeated measures ANOVA. Post hoc analysis showed significant differences * p < 0.05 on days 7, 14 and 21 of exposure.

Figure 2

Sustained hypoxia and intermittent hypoxia increase cell proliferation in vitro. (a) Proliferation, expressed as a % of normoxia values, of viable HepG2 cells quantified by MTT staining after 5 days of normoxia, intermittent hypoxia or sustained hypoxia exposure; n = 3 independent experiments/group (at least 18 wells/group). * p < 0.05 and ** p < 0.01 on D5 for IH cells compared with normoxia cells, and p < 0.05 for SH cells compared with N cells, Mann–Whitney U test. (b) Migration, expressed as a % of normoxia values, of HepG2 cells after 2 days of normoxia, intermittent hypoxia or sustained hypoxia exposure; n = 3 independent experiment/group (6 wells/group: 2 transwells/experiments). Mann–Whitney U test.

Figure 3

In vitro effects of IH and SH on HIF-1α gene expression, and VEGF gene and protein expression. (a) Intermittent hypoxia is associated with an increase in HIF-1α and VEGF gene expressions in HepG2 cells after 5 days of exposure. The levels of HIF-1α and VEGF gene expression were measured by RT-QPCR; n = 3 independent experiments/group. * p < 0.05, Mann–Whitney U test. (b) VEGF expression increases in HepG2 cells after 5 days of IH but not SH exposure. The levels of VEGF protein in the cell’s supernatant were measured by ELISA; n = 3 independent experiments/group. * p < 0.05) Mann–Whitney U test.

Figure 4

Effects of acriflavine and pazopanib on wound healing and proliferation under sustained hypoxia or intermittent hypoxia. (a) Wound healing, expressed as % of repaired area, of HepG2 cells exposed to 5 days of normoxia (N), intermittent hypoxia (IH) or sustained hypoxia (SH) and treated or not by acriflavine (Acri); n = 3 experiments per group with at least 3 wells/experiment. ** p < 0.01 on D5 for IH acriflavine-treated cells compared with IH untreated and * p < 0.05 on D5 for SH acriflavine-treated cells compared with SH untreated cells, Mann–Whitney U test. (b) Wound healing, expressed as a % of repaired area, of HepG2 cells exposed to 5 days of normoxia, intermittent hypoxia or sustained hypoxia and treated or not by pazopanib (Pazo); n = 3 experiments per group with at least 3 wells/experiment. ** p < 0.01 on D5 for IH pazopanib-treated cells compared with IH untreated, Mann–Whitney U test. (c) Proliferation, expressed as a % of Normoxia values, of viable HepG2 cells quantified by MTT staining after 5 days of normoxia, intermittent hypoxia or sustained hypoxia and treated or not by acriflavine (Acri); n = 3 independent experiments/group, 6 to 12 wells/group: at least 2 wells/experiment. * p < 0.05 on D5 for IH acriflavine-treated cells compared with IH untreated, p < 0.05 for CH acriflavine-treated cells compared with CH untreated cells, Mann–Whitney U test. (d) Proliferation, expressed as a % of control values (respectively N, IH or CH), of viable HepG2 cells quantified by MTT staining after 5 days of normoxia, intermittent hypoxia or sustained hypoxia and treated or not by pazopanib (Pazo); n = at least 3 independent experiments/group (9 wells/group: at least 2 wells/experiment). * p < 0.05 on D5 for IH pazopanib-treated cells compared with IH untreated, Mann–Whitney U test.