Galbanic acid inhibits HIF-1α expression via EGFR/HIF-1α pathway in cancer cells.

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.