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.

Mitochondrial NDUFA4L2 protein promotes the vitality of lung cancer cells by repressing oxidative stress

“…To confirm the relationship between HIF‐1α and NDUFA4L2 in lung cancer cells cultured in hypoxia, we used an HIF‐1α inhibitor (2‐ME, 20 μM) and an agonist (DMOG, 1 mM) to pretreat A549 and H1299 cells. DMOG repressed and 2‐ME activated the activation of prolyl hydroxylases and were used to investigate the role of HIF‐1α . First, we confirmed that NDUFA4L2 was located in mitochondria (Fig a).…”

“…DMOG repressed and 2-ME activated the activation of prolyl hydroxylases and were used to investigate the role of HIF-1α. [25][26][27] First, we confirmed that NDUFA4L2 was located in mitochondria (Fig 2a). Cytochrome C was marked with green, and NDU-FA4L2 was marked with red.…”

Inhibition of proliferation and invasion in 2D and 3D models by 2-methoxyestradiol in human melanoma cells

“…2ME is a metabolite of estrogen and has antitumoral activity against many cancer cells, such as lung cancer (9), ovarian cancer (10), hepatocellular carcinoma (11), colon cancer (12) and melanoma (13). Herein, 2ME affected cell lines with different genetic backgrounds, confirming that its effects are independent of BRAF and NRAS mutational status.…”

“…Moreover, Fotsis and collaborators revealed the high cytotoxicity of 2ME in vascular endothelial cells leading to inhibition of tumor angiogenesis and growth in vivo (8). Multiple in vitro and in vivo studies have shown the potential of 2ME to induce cell death and inhibit angiogenesis across many different tumor types (9–13). In melanoma, the antitumor effect of 2ME has been little explored.…”

Mitochondrial Dysfunction and Redox Imbalance as a Diagnostic Marker of “Free Radical Diseases”

“…2-ME is a natural endogenous metabolite of 17β-estradiol that exerts anti-proliferative, anti-angiogenic, pro-apoptotic and transcriptional activity in various cells, including induction of cell-cycle arrest (72)(73)(74)(75)(76). Experiments have demonstrated that 2-ME is involved in the inhibi-tion of the polymerization of tubulin in vitro, thus disrupting normal microtubule function (77).…”