title: Effects of 2-methoxyestradiol on apoptosis and HIF-1α and HIF-2α expression in lung cancer cells under normoxia and hypoxia.
abstract: 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 1α (HIF-1α) and HIF-2α 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-1α and HIF-2α protein expression in total cell extracts. Cellular localization of HIF-1α and HIF-2α was assessed by immunocytochemistry. HIF-1α and HIF-2α 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-1α and HIF-2α 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-1α and HIF-2α nuclear staining in cells cultured under hypoxia. The HIF-1α and HIF-2α 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.
authors.affiliation: Instituto Nacional de Enfermedades Respiratorias 'Ismael Cosío Villegas', Mexico City, DF, Mexico.
authors.forename: Arnoldo
authors.initials: A
authors.lastname: Aquino-Gálvez
authors.affiliation: Instituto Nacional de Enfermedades Respiratorias 'Ismael Cosío Villegas', Mexico City, DF, Mexico.
authors.forename: Georgina
authors.initials: G
authors.lastname: González-Ávila
authors.affiliation: Instituto Nacional de Enfermedades Respiratorias 'Ismael Cosío Villegas', Mexico City, DF, Mexico.
authors.forename: Javier
authors.initials: J
authors.lastname: Delgado-Tello
authors.affiliation: Instituto Nacional de Enfermedades Respiratorias 'Ismael Cosío Villegas', Mexico City, DF, Mexico.
authors.forename: Manuel
authors.initials: M
authors.lastname: Castillejos-López
authors.affiliation: Instituto Nacional de Enfermedades Respiratorias 'Ismael Cosío Villegas', Mexico City, DF, Mexico.
authors.forename: Criselda
authors.initials: C
authors.lastname: Mendoza-Milla
authors.affiliation: Instituto Nacional de Enfermedades Respiratorias 'Ismael Cosío Villegas', Mexico City, DF, Mexico.
authors.forename: Joaquín
authors.initials: J
authors.lastname: Zúñiga
authors.affiliation: Instituto Nacional de Enfermedades Respiratorias 'Ismael Cosío Villegas', Mexico City, DF, Mexico.
authors.forename: Marco
authors.initials: M
authors.lastname: Checa
authors.affiliation: Instituto Nacional de Cancerología, Mexico City, DF, Mexico.
authors.forename: Héctor Aquiles
authors.initials: HA
authors.lastname: Maldonado-Martínez
authors.affiliation: Instituto Nacional de Enfermedades Respiratorias 'Ismael Cosío Villegas', Mexico City, DF, Mexico.
authors.forename: Axel
authors.initials: A
authors.lastname: Trinidad-López
authors.affiliation: Instituto Nacional de Enfermedades Respiratorias 'Ismael Cosío Villegas', Mexico City, DF, Mexico.
authors.forename: José
authors.initials: J
authors.lastname: Cisneros
authors.affiliation: Instituto Nacional de Pediatría, Mexico City, DF, Mexico.
authors.forename: Luz María
authors.initials: LM
authors.lastname: Torres-Espíndola
authors.affiliation: Instituto Nacional de Enfermedades Respiratorias 'Ismael Cosío Villegas', Mexico City, DF, Mexico.
authors.forename: Claudia
authors.initials: C
authors.lastname: Hernández-Jiménez
authors.affiliation: Instituto Nacional de Enfermedades Respiratorias 'Ismael Cosío Villegas', Mexico City, DF, Mexico.
authors.forename: Bettina
authors.initials: B
authors.lastname: Sommer
authors.affiliation: Instituto Nacional de Enfermedades Respiratorias 'Ismael Cosío Villegas', Mexico City, DF, Mexico.
authors.forename: Carlos
authors.initials: C
authors.lastname: Cabello-Gutiérrez
authors.affiliation: Instituto Nacional de Enfermedades Respiratorias 'Ismael Cosío Villegas', Mexico City, DF, Mexico.
authors.forename: Luis H
authors.initials: LH
authors.lastname: Gutiérrez-González
mesh_terms: D000077584:2-Methoxyestradiol; D017209:Apoptosis / Q000187:drug effects; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism; D015687:Cell Hypoxia / Q000187:drug effects; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D000077584:2-Methoxyestradiol; D051792:Basic Helix-Loop-Helix Proteins; D004958:Estradiol; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D000099162:Endothelial PAS Domain-Containing Protein 1; C497442:HIF1A protein, human