Hypoxia Inducible Factor-1 plays a key transcriptional role in the adaptation of hypoxic solid
tumors to low
oxygen environment. Here, we aimed to investigate
galbanic acid (GBA) inhibitory effects on HIF-1 activation during
hypoxia and normoxia. MTT survival and
Annexin V assays were used to evaluate GBA cytotoxicity and apoptosis in treated cells. Quantitative real time PCR and western blotting were used to estimate
mRNA expression and translated
protein, respectively. Results showed that GBA dose- and time-dependently decreased the in vitro growth of OVCAR-3 human epithelial
carcinoma cells with an IC50 of approximately 37, 12.1 and 10μM GBA at 24, 48 and 72h, respectively. Following
phosphatidylserine of outer leaflet of the plasma membrane revealed occurrence of early/late apoptosis in GBA treated cells. In addition, we found that GBA down-regulates HIF-1α and HIF-1β
mRNA expression in both
hypoxia and normoxia. To determine the mechanism of action, we showed that GBA did not inhibit Akt and EGFR
mRNA expression, yet protein degradation investigation showed that GBA shortened the half-life of EGFR through decreasing its stability with a decrease of nearly 2 and 3h in A549 and OVCAR-3 cell lines, respectively. We also found that downstream genes contributed in glycolysis, including Eno 1 and GluT-1, are underexpressed in GBA treated cells in
hypoxia. Conclusively, GBA may inhibit HIF-1 activation through down-regulation of its subunit expression in
hypoxia, and increasing of EGFR degradation in normoxia.