Hypoxia-induced expression of HIF-1a and GPR30, in relation to survival, proliferation and apoptosis in MCF-7 and SkBr3 breast cancer cells.

Abstract: Introduction: Hypoxia that accompanies tumor growth induces the expression of hypoxiainducible factor a (HIF-1a), in turn mediating the expression of other genes, including G protein-coupled receptor 30 (GPR30), to promote adaption to the hypoxic microenvironment and enable cell proliferation and survival. Nonetheless, this correlation has not been causally explained. Objective: To further investigate how breast cancer cells with expression of GPR30 adapt to hypoxia, by studying survival, proliferation, and ability to induce apoptosis. Method: Human breast cancer cells, MCF-7 (ER-positive) and SkBr3 (ER-negative), were seeded and transfected with either GPR30 or an empty pcDNA plasmid. Transfection efficiency was assessed by transfecting the cells with enhanced green fluorescent protein (EGFP). Cells were incubated in either a hypoxic or normoxic condition, and molecular biological methods as Western blot and FACS were applied to investigate the cell´s survival, proliferation, and apoptosis induction. Mann-Whitneys U test was performed to statistically analyze intra- and inter group variations. Results: Recombinant expression of GPR30 was reduced in hypoxic cells, and β-actin staining of those cells, as well as their PARP-cleavage, suggested cell death. Significant increased cleaving of PARP in hypoxic GPR30+ SkBr3 cells (15.5% vs 62.95%, p = 0.0286) and a significant difference between hypoxic GPR30+ SkBr3 and MCF-7 cells (p = 0.0286). GPR30+ cells also had a lower cleavage of PARP. SkBr3 cellularity was affected negatively, whilst MCF-7 cellularity was generally increased, plus more MCF-7 cells in the S-phase, in hypoxia. GPR30+ cells had a lower survival rate in hypoxia, but an increased proliferation in normoxia. Conclusion: Our results propose a trend showing an interaction between hypoxia and GPR30, a correlation between GPR30 and a promoted proliferation in normoxia, as well as a significant difference between SkBr3 and MCF-7 in those aspects. However, our results are not enough evidence of these correlations and requires additional research.