Effects of 2-methoxyestradiol on apoptosis and HIF-1 alpha and HIF-2 alpha expression in lung cancer cells under normoxia and hypoxia

摘要

Hypoxic tumor cells are known to be more resistant to conventional chemotherapy and radiation than normoxic cells. However, the effects of 2-methoxyestradiol (2-ME), an anti-angiogenic, antiproliferative and pro-apoptotic drug, on hypoxic lung cancer cells are unknown. The aim of the present study was to compare the effects of 2-ME on cell growth, apoptosis, hypoxia-inducible factor 1 alpha (HIF-1 alpha) and HIF-2 alpha gene and protein expression in A549 cells under normoxic and hypoxic conditions. To establish the optimal 2-ME concentration with which to carry out the apoptosis assay and to examine mRNA and protein expression of HIFs, cell growth analysis was carried out through N-hexa-methylpararosaniline staining assays in A549 cell cultures treated with one of five different 2-ME concentrations at different times under normoxic or hypoxic growth conditions. The 2-ME concentration of 10 mM at 72 h was selected to perform all further experiments. Apoptotic cells were analyzed by flow cytometry. Western blotting was used to determine HIF-1 alpha and HIF-2 alpha protein expression in total cell extracts. Cellular localization of HIF-1a and HIF-2 alpha was assessed by immunocytochemistry. HIF-1 alpha and HIF-2 alpha gene expression was determined by real-time PCR. A significant increase in the percentage of apoptosis was observed when cells were treated with 2-ME under a normoxic but not under hypoxic conditions (p=0.006). HIF-1 alpha and HIF-2 alpha protein expression levels were significantly decreased in cells cultured under hypoxic conditions and treated with 2-ME (p<0.001). Furthermore, 2-ME decreased the HIF-1 alpha and HIF-2 alpha nuclear staining in cells cultured under hypoxia. The HIF-1 alpha and HIF-2 alpha mRNA levels were significantly lower when cells were exposed to 2-ME under normoxia and hypoxia. Our results suggest that 2-ME could have beneficial results when used with conventional chemotherapy in an attempt to lower the invasive and metastatic processes during cancer development due to its effects on the gene expression and protein synthesis of HIFs.