SAT-124 Optimization of Experimental Conditions for Mimicking Hypoxia in Cultured Breast Cancer Cells by Using Cobalt(II) Chloride (CoCl(2))

Hypoxia is a common phenomenon in solid tumor development caused by a decrease in either oxygen concentration or oxygen pressure as a result of rapid tumor cell growth. Hypoxia is characterized by stabilization of the alpha subunit of the hypoxia-inducible factor (HIF-1α) and its nuclear translocation and heterodimerization with HIF-1β. Activation of this signaling pathway involves multiple downstream effectors including carbonic anhydrase 9 (CA9, s. CAIX). A reliable method to mimic hypoxia utilizes cobalt(II) chloride (CoCl(2)), which directly induces the expression of HIF-1α. The aim of this study was to optimize the experimental conditions for CoCl(2) treatment of breast cancer cells in vitro using three human breast cancer cell lines (MDA-MB-231, T-47D, and MCF-7 cells). We performed time- and concentration-response experiments, using various concentrations of CoCl(2) (50, 100, 200, and 300 μM) for 24 and 48 hours, and measured the expression of HIF-1α and CA9 by qRT-PCR and Western blot analyses. Results demonstrated that CoCl(2) downregulated HIF-1α mRNA levels but upregulated CA9 mRNA levels in a concentration- and time-dependent manner. Concomitantly, CoCl(2) treatment resulted in a significant induction of HIF-1α protein levels. We further investigated the effect of the CoCl(2) concentrations listed above on cell apoptosis using an in situ apoptosis detection kit. The results demonstrated that concentrations of CoCl(2) up to 100 μM had no significant effect on cell apoptosis.