Progesterone enhances cytokine-stimulated nitric oxide synthase II expression and cell death in human breast cancer cells

Abstract

The presence of hormone receptors is related to survival outcome in breast cancer. Previous results from our laboratory established a correlation between the presence of nitric oxide synthase II (NOSII) and nitric oxide (NO) production with progesterone receptors in a series of human breast tumours. Furthermore, this was directly related to a lower tumour grade and a lower proliferation rate of the tumour cells. To examine these results in further detail, the effect of progesterone (Pg) and 17b-oestradiol (E2) on NOSII expression was analysed in the human breast cancer cell line MCF-7. By Northern blot and promoter activity, we show that a cytokine mix (TNF-a, IL-b, and IFN-c) induces NOSII transcription after 6 h stimulation. In the absence of cytokines, neither hormone affects NOSII expression. However, Pg but not E2, enhances cytokine-induced NOSII transcription as well as NO synthesis, mainly by cooperation with gamma-interferon. The increase in NO accumulation in the media induced by addition of Pg to the cytokine treatment significantly increases cell death, mainly accounted for by apoptosis, as compared to the effect of cytokines alone. Our findings help clarify the role of steroid hormones in NOSII expression as well as the effect on cell viability and may suggest novel approaches towards hormonotherapy and the treatment of cancer. Keywords: NOSII; progesterone; transcription; breast cancer; apoptosis; nitric oxide Endogenous nitric oxide (NO) is produced in cells by the nitric oxide synthases (NOS), which exist as three isoforms. The calcium-dependent endothelial (eNOS or NOSIII) and neuronal (nNOS or NOSI) enzymes are constitutively present in various cell types and produce low levels of short acting NO. 1 The third isoform (iNOS or NOSII) is induced in most cell types only in response to external stimuli such as bacterial lipopolysaccharides (LPS) or diverse cytokines, and generates high sustained levels of NO. 2 NO is a transcellular messenger that plays an important role in diverse physiological processes such as vascular homeostasis, immunity and neurotransmission. The effect of endogenous NO on cellular homeostasis is highly dependent on the level of its synthesis. Thus, at low levels it can promote cell proliferation and angiogenesis whereas at high concentrations, such as those produced by NOSII, it can lead to cell damage and death. 3 Therefore, it is important to understand the regulation of the NOS enzymes in order to unravel the role of NO in physiological and pathological conditions. So far it is known that expression of NOSII is regulated mainly at the transcriptional level, although additional post-transcriptional and posttranslational controls have been documented. 4 The most important transcription factors required for NOSII expression are nuclear factor kappa B (NFkB) 5 and activator protein 1 (AP-1). 6 These transcription factors are induced or activated by cytokines such as tumour necrosis alpha (TNF-a), interleukin-1beta (IL-1b), gamma interferon (IFN-g) or LPS. 7 However, differenc...

Candidate pathways linking inducible nitric oxide synthase to a basal-like transcription pattern and tumor progression in human breast cancer

“…NOS2 is an inflammation responsive enzyme and bacterial lipopolysaccharide and cytokines have been shown to induce NOS2 in a variety of cell types through mechanisms that involve NF-kB33–35. Hormones may interact with cytokines in NOS2 induction and progesterone has been shown to enhance cytokine-stimulated NOS2 expression in MCF7 human breast cancer cells36. However, the level of NOS2 induction can be different between murine and human cell lines, with human cell lines commonly showing a lower expression of NOS2 following cytokine exposure.…”

Cloning and identification of EDD gene from ultraviolet‐irradiated HaCaT cells

“…Aberrant promoter hypermethylation of several known or putative tumour suppressor genes occurs frequently during the pathogenesis of lung cancer (26), prostate cancer (27), gastric carcoma (28), hepatoma (29) and so on. The most investigated change of DNA methylation in neoplasms is the silencing of tumour suppressor genes by CpG island promoter hypermethylation, which targets genes such as p16, BRCA1 and HMLH1 (30). It has been inferred that a combination of methylation of four genes (p16, ARF, MGMT and GSTP1) would theoretically allow detection of at least 87% of cancer with 100% specificity (27).…”

Oct-2 forms a complex with Oct-1 on the iNOS promoter and represses transcription by interfering with recruitment of RNA PolII by Oct-1

“…Cells were plated on 6-well plates at 1 × 106 cells per cm 2 to achieve 80% confluence. Fugene6 (Qiagen, UK) was used for transfection and reporter gene transcription was determined using the dual luciferase assay from Promega (Madison, USA) as described previously ( 13 ).…”

“…Cells were plated in 6-well plates in a proportion of 5 × 10E3/well in complete medium. They were depleted of hormones for 24–48 h and stimulated for 24 and 48 h. The supernatants were recovered and the concentration of NO 2 − was determined by the Griess reaction (as described in 13 ). The NO3- were reduced to NO 2 − by the nitrate reductase and these were measured using the same system.…”