The online version of this article (doi:10.1186/s13058-017-0823-8) contains supplementary material, which is available to authorized users.
Psychological stress increases the circulating levels of the stress hormones cortisol and norepinephrine (NE). Chronic exposure to elevated stress hormones has been linked to a reduced response to chemotherapy through induction of DNA damage. We hypothesize that stress hormone signalling may induce DNA damage through the production of reactive oxygen species (ROS)/reactive nitrogen species (RNS) and interference in DNA repair processes, promoting tumourigenesis.
Breast cancer cell lines were incubated with physiological levels of cortisol and NE in the presence and absence of receptor antagonists and inducible nitric oxide synthase (iNOS) inhibitors and DNA damage measured using phosphorylated γ-H2AX. The rate of DNA repair was measured using comet assays and electrochemical sensors were used to detect ROS/RNS in the cell lysates from cells exposed to stress hormones. A syngeneic mouse model was used to assess the presence of iNOS in mammary tumours in stressed versus control animals and expression of iNOS was examined using western blotting and qRT-PCR.
Acute exposure to cortisol and NE significantly increased levels of ROS/RNS and DNA damage and this effect was diminished in the presence of receptor antagonists. Cortisol induced DNA damage and the production of RNS was further attenuated in the presence of an iNOS inhibitor. An increase in the expression of iNOS in response to psychological stress was observed in vivo and in cortisol-treated cells. Inhibition of glucocorticoid receptor-associated Src kinase also produced a decrease in cortisol-induced RNS.
These results demonstrate that glucocorticoids may interact with iNOS in a non-genomic manner to produce damaging levels of RNS, thus allowing an insight into the potential mechanisms by which psychological stress may impact breast cancer.
Additional file 1: Figure S1. ROS/RNS detection controls. Untreated MCF-7 (a) and MDA-MB-231 (b) were incubated alongside treatment wells and lysed at 0 and 60 minutes. Cell lysates were collected and electrochemical sensors used to measure levels of hydrogen peroxide (H 2 O 2 ) and nitrogen dioxide (NO 2 ). (PPTX 111 kb)13058_2017_823_MOESM1_ESM.pptx
Additional file 2: Figure S2. Stress hormones do not induce DNA damage or iNOS expression in a non-tumourigenic mammary epithelial cell line. a MCF10A cells were exposed to cortisol (1 μM) and NE (1 μM) for 30 minutes and assessed for DNA damage using the Comet assay. Comet tails indicating DNA strand breaks were visually scored according to intensity (0–4). Representative images shown. b MCF10A cells were exposed to cortisol (1 μM) for 30 minutes and 24 h and mRNA extracted. cDNA was synthesised and amplified in the presence of gene specific primers for NOS2 and β-actin using qRT-PCR. Ct values for NOS2 were normalised against β-actin and fold change calculated using the delta-Ct method. Mean ± SEM is expressed and significance was determined using one-way ANOVA (post hoc Tukey multiple comparisons); *significant increase, *p < 0.05, **p < 0.01, ***p < 0.001. Technical replicate (n = 3). (PPTX 125 kb)13058_2017_823_MOESM2_ESM.pptx
Additional file 3: Figure S3. Expression of iNOS protein is unchanged in response to cortisol. MCF-7 cells were exposed to cortisol (1 μM) for 30 minutes or 24 h. iNOS protein expression was visualised using western blotting. Optical density values were normalised against β-actin. Mean ± SEM is shown. (PPTX 186 kb)13058_2017_823_MOESM3_ESM.pptx
Additional file 4: Figure S4. Glucocorticoid receptor localisation in mice mammary tumours. The 4T1 mouse mammary gland cells were transplanted into the fourth mammary fat pad of female BALB/C mice and the animals randomised into groups either exposed to acute restraint stress or no stress. Tumours were harvested, fixed in paraffin and sectioned subsequent to immunofluorescent detection of glucocorticoid receptor (GR). Representative panels are shown. (PPTX 414 kb)13058_2017_823_MOESM4_ESM.pptx
Additional file 5: Figure S5. Cortisol induces the dissociation of Src from HSP90. MCF-7 and MDA-MB-231 cells were exposed to cortisol (1 μM) for 30 minutes alongside PP2 (10 μM). Cell lysates were immunoprecipitated for HSP90 and protein levels of Src were visualised using western blotting. (PPTX 52 kb)13058_2017_823_MOESM5_ESM.pptx
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- Glucocorticoids induce production of reactive oxygen species/reactive nitrogen species and DNA damage through an iNOS mediated pathway in breast cancer
Renée L. Flaherty
Marcus C. Allen
Bhavik A. Patel
Melanie S. Flint
- BioMed Central
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