Inhibition of nitric oxide synthase (NOS) reduces the effect of stress hormone signalling in breast cancer

Expression of nitric oxide synthase (NOS) has been found to correlate with tumour progression in breast cancer, indicating that NO activity may drive malignant growth. Previously we have shown that the stress hormone cortisol acts through a nitric oxide synthase (NOS) mediated pathway to induce production of nitric oxide (NO), and can induce DNA damage in breast cancer. Breast cancer cell lines MCF-7 and MDA-MB-231 as well as the mouse mammary tumour cell line 66CL4 were exposed to cortisol and levels of intracellular NO were measured using composite electrochemical sensors. DNA damage was quantified using immunofluorescence and expression of iNOS and metastatic markers VEGF and TWIST were examined using qPCR. An in vivo syngeneic breast cancer model was also used to examine the effect of L-NAME, a NOS inhibitor, on tumour aggressiveness and metastasis in conjunction with daily restraint stress (2hrs) (n=4/group repeated in duplicate). Cortisol significantly increased the expression of iNOS, the generation of NO and DNA damage in breast cancer cells and this was blocked by the NOS inhibitor L-NAME. A significant increase in VEGF and TWIST expression was also observed in response to cortisol. Furthermore, L-NAME also significantly reduced primary tumour growth in stressed mice and reduced the number of metastatic sites/mouse. Tumour microvasculature (as evidenced by CD31 expression) was significantly increased in stressed mice and this was reduced with L-NAME treatment. We demonstrated that L-NAME through inhibition of NO signalling is effective in reducing primary tumour formation and metastatic potential in stressed mice. This data may have impact for patients with breast cancer experiencing extreme stress and further genomic analysis are ongoing

The role of psychological stress in cancer initiation:clinical relevance and potential molecular mechanisms

The hypothesis that the physiological response to psychological stress influences the initiation of cancer is highly controversial. The link between initiating stressors, the psychological stress response, and disease is plausible considering that the stress response is associated with defined physiological outcomes and molecular mechanisms. In light of this, we review the clinical relevance of psychological stress on the risk of cancer, and we propose potential molecular pathways that may link the stress response to early stages of malignant cell transformation

Thermoregulation is not impaired in breast cancer survivors during moderate-intensity exercise performed in warm and hot environments

This study aimed to assess how female breast cancer survivors (BCS) respond physiologically, hematologically, and perceptually to exercise under heat stress compared to females with no history of breast cancer (CON). Twenty‐one females (9 BCS and 12 CON [age; 54 ± 7 years, stature; 167 ± 6 cm, body mass; 68.1 ± 7.62 kg, and body fat; 30.9 ± 3.8%]) completed a warm (25℃, 50% relative humidity, RH) and hot (35℃, 50%RH) trial in a repeated‐measures crossover design. Trials consisted of 30 min of rest, 30 min of walking at 4 metabolic equivalents, and a 6‐minute walk test (6MWT). Physiological measurements (core temperature (T (re)), skin temperature (T (skin)), heart rate (HR), and sweat analysis) and perceptual rating scales (ratings of perceived exertion, thermal sensation [whole body and localized], and thermal comfort) were taken at 5‐ and 10‐min intervals throughout, respectively. Venous blood samples were taken before and after to assess; IL‐6, IL‐10, CRP, IFN‐γ, and TGF‐β(1). All physiological markers were higher during the 35 versus 25℃ trial; T (re) (~0.25℃, p = 0.002), T (skin) (~3.8℃, p  0.05). Both groups covered a greater 6MWT distance in 25 versus 35℃ (by ~200 m; p = 0.003). Nevertheless, the control group covered more distance than BCS, regardless of environmental temperature (by ~400 m, p = 0.03). Thermoregulation was not disadvantaged in BCS compared to controls during moderate‐intensity exercise under heat stress. However, self‐paced exercise performance was reduced for BCS regardless of environmental temperature

An individual-based model to explore the impact of psychological stress on immune infiltration into tumour spheroids

In recent in vitro experiments on co-culture between breast tumour spheroids and activated immune cells, it was observed that the introduction of the stress hormone cortisol resulted in a decreased immune cell infiltration into the spheroids. Moreover, the presence of cortisol deregulated the normal levels of the pro- and anti-inflammatory cytokines IFN-{\gamma} and IL-10. We present an individual-based model to explore the interaction dynamics between tumour and immune cells under psychological stress conditions. With our model, we explore the processes underlying the emergence of different levels of immune infiltration, with particular focus on the biological mechanisms regulated by IFN-{\gamma} and IL-10. The set-up of numerical simulations is defined to mimic the scenarios considered in the experimental study. Similarly to the experimental quantitative analysis, we compute a score that quantifies the level of immune cell infiltration into the tumour. The results of numerical simulations indicate that the motility of immune cells, their capability to infiltrate through tumour cells, their growth rate and the interplay between these cell parameters can affect the level of immune cell infiltration in different ways. Ultimately, numerical simulations of this model support a deeper understanding of the impact of biological stress-induced mechanisms on immune infiltration

The role of psychological stress and nitric oxide synthase inhibition in breast cancer

Psychological stress has been implicated as a risk factor in the progression of breast cancer, however the biological mechanisms are not well understood. In animal models of psychological stress, anincrease in mammary tumour burden and metastatic spread has been observed.1 Furthermore, in breast cancer cells, the stress hormones cortisol and noradrenaline, released as part of the stress response, have been shown to promote DNA damage through the generation of reactive oxygen/nitrogen species.2 The research presented here aimed to first explore the in vitro effects of cortisol by measuring the induction of DNA damage and the generation of nitric oxide (NO). An in vivo mouse model of psychological stress was also employed to study the impact of stress on the progression of breast cancer, alongside an inhibitor of nitric oxide synthase (NOS) (L-NAME). Through pharmacological inhibition of the glucocorticoid receptor (GR), as well as selective and non-selective inhibition of NOS, this research aimed to elucidate a mechanism through which stress may impact breast cancer progression. In mammary tumour cells treated with cortisol, an increase in both NO and DNA damage was observed. This was abrogated with inhibition of the GR, as well as pan-inhibition of NOS and, specifically, the isoform inducible NOS (iNOS). Cortisol upregulated the expression of iNOS, a marker previously suggested as an indicator of poor survival in breast cancer.3 Induction of chronic stress in a syngeneic mouse model of breast cancer had no effect on the growth of the primary tumour;however, inhibition of NOS in stressed mice significantly decreased tumour volume compared to stress alone. A significant increase in tumour microvasculature was observed in the primary tumours, as there was an increase in metastatic sites per mouse in stressed mice, compared to the control. This was also reversed through the inhibition of NOS. This study indicated that stress may impact tumourigenic progression through the induction of DNA damage, mediated by the release of NO. Inhibition of NOS was able to negate the effects of stress on tumour growth and metastasis, providing an insight into the potential benefits of NOS inhibitors in breast cancer treatment. However, presentation of this research at the ESMO congress raised some potential implications of inhibition of NO signalling. Primarily, these included the effects of NOS inhibition on haemodynamics, because L-NAME has been used previously in a clinical setting to increase blood pressure. In future studies, this limitation could be resolved through cotreatment with antihypertensives, as was thecase in a previous study that demonstrated the growth inhibitory effects of iNOS inhibition in breast cancer.4 Discussion also focussed on the effects of glucocorticoid signalling in the immune system, since previous research has demonstrated that stress can adversely affect the immune response in breast cancer models, and in patients.5,6Furthermore, an increase in the expression of the GR has been observed to correlate with poor prognosis in oestrogen receptor-negative breast cancer patients, indicating that glucocorticoid-mediated signalling can influence cancer progression.7 As such, current trials using novel GR inhibitors in breast cancer patients are underway. Future perspectives for this work would seek to use pharmacological therapeutics, including using selective GR inhibitors in combination with NOS inhibitors, in highly stressed patients with breast cancer

An integrated framework for quantifying immune-tumour interactions in a 3D co-culture model

Investigational in vitro models that reflect the complexity of the interaction between the immune system and tumours are limited and difficult to establish. Herein, we present a platform to study the tumour-immune interaction using a co-culture between cancer spheroids and activated immune cells. An algorithm was developed for analysis of confocal images of the co-culture to evaluate the following quantitatively; immune cell infiltration, spheroid roundness and spheroid growth. As a proof of concept, the effect of the glucocorticoid stress hormone, cortisol was tested on 66CL4 co-culture model. Results were comparable to 66CL4 syngeneic in vivo mouse model undergoing psychological stress. Furthermore, administration of glucocorticoid receptor antagonists demonstrated the use of this model to determine the effect of treatments on the immune-tumour interplay. In conclusion, we provide a method of quantifying the interaction between the immune system and cancer, which can become a screening tool in immunotherapy design

Sexual Size Dimorphism and Body Condition in the Australasian Gannet

Funding: The research was financially supported by the Holsworth Wildlife Research Endowment. Acknowledgments We thank the Victorian Marine Science Consortium, Sea All Dolphin Swim, Parks Victoria, and the Point Danger Management Committee for logistical support. We are grateful for the assistance of the many field volunteers involved in the study.Peer reviewedPublisher PD