ADAM10 and Neprilysin level decreases in immune cells of mice bearing metastatic breast carcinoma: Possible role in cancer inflammatory response.

OBJECTIVE AND DESIGN: ADAM10 and Neprilysin, proteases, play critical role in inflammatory disease, however their role in cancer immune response is not clear. We here evaluated changes in immune response using an experimental model for breast cancer. MATERIAL AND METHOD: Highly metastatic breast cancer cells (4T1-derived) were injected orthotopically (mammary-pad of Balb-c mice) to induce tumors. Changes in enzyme level and activity as well as alterations in inflammatory cytokine release in the presence or absence of ADAM10 and NEP activity was determined using specific inhibitors and recombinant proteins. Cytokine response was evaluated using mix leucocyte cultures obtained from control and tumor-bearing mice. ANOVA with Dunnett's posttest was used for statistical analysis. RESULTS: ADAM10 and NEP expression was decreased markedly in lymph nodes and spleens of tumor-bearing mice. ADAM10 activity was reduced together with apparent alterations of ADAM10 processing. ADAM10 and NEP activity decreased TNF-α, IL-6 and IFN-É£ secretion. Suppression of these inflammatory cytokines were more prominent in cultures obtained from control mice demonstrating counteracting factors that are exist in tumor-bearing mice. CONCLUSION: Loss of ADAM10 and NEP activity in immune cells during breast cancer metastasis might be one of the main factors involved in induction of chronic inflammation by tumors.

Carcinogenesis and Metastasis: Focus on TRPV1-Positive Neurons and Immune Cells.

Both sensory neurons and immune cells, albeit at markedly different levels, express the vanilloid (capsaicin) receptor, Transient Receptor Potential, Vanilloid-1 (TRPV1). Activation of TRPV1 channels in sensory afferent nerve fibers induces local effector functions by releasing neuropeptides (most notably, substance P) which, in turn, trigger neurogenic inflammation. There is good evidence that chronic activation or inactivation of this inflammatory pathway can modify tumor growth and metastasis. TRPV1 expression was also demonstrated in a variety of mammalian immune cells, including lymphocytes, dendritic cells, macrophages and neutrophils. Therefore, the effects of TRPV1 agonists and antagonists may vary depending on the prominent cell type(s) activated and/or inhibited. Therefore, a comprehensive understanding of TRPV1 activity on immune cells and nerve endings in distinct locations is necessary to predict the outcome of therapies targeting TRPV1 channels. Here, we review the neuro-immune modulation of cancer growth and metastasis, with focus on the consequences of TRPV1 activation in nerve fibers and immune cells. Lastly, the potential use of TRPV1 modulators in cancer therapy is discussed.

Changes in TRPV1 Expression as Well as Substance P and Vasoactive Intestinal Peptide Levels Are Associated with Recurrence of Pterygium.

Pterygium, a disease of the ocular surface, is characterized by the proliferation and invasion of fibrovascular tissue. Chronic inflammation contributes to pterygium occurrence. Sensory neuropeptides of TRPV1-positive nerve fibers are involved in inflammation and corneal wound healing. The possible association between TRPV1 in nerve fibers and neuropeptides such as Substance P (SP) and Vasoactive Intestinal Peptide (VIP) in the recurrence of pterygium has not been examined before. The pterygia from 64 patients were used to determine changes in SP and VIP levels using 10 min acetic-acid extraction that yielded mainly neuronal peptides. There was a sufficient amount of pterygium tissues from the 35 patients for further immunohistochemical analysis of TRPV1 and S100, which is a glial marker to visualize nerve fibers. SP and VIP levels increased markedly in cases with primary and secondary recurrences, and there was a close correlation between SP and VIP levels. TRPV1 expression increased in the epithelium, while stromal expression decreased in recurrences. Nerve fibers were demonstrated mainly in the stroma, and serial sections confirmed the localization of TRPV1 with the nerve fibers. These results together with previous findings demonstrated that the increased epithelial expression of TRPV1 in recurrent pterygia might be involved in the pathogenesis, and the inhibition of epithelial TRPV1 activity may prevent recurrence.

Regulation of Carcinogenesis by Sensory Neurons and Neuromediators.

Interactions between the immune system and the nervous system are crucial in maintaining homeostasis, and disturbances of these neuro-immune interactions may participate in carcinogenesis and metastasis. Nerve endings have been identified within solid tumors in humans and experimental animals. Although the involvement of the efferent sympathetic and parasympathetic innervation in carcinogenesis has been extensively investigated, the role of the afferent sensory neurons and the neuropeptides in tumor development, growth, and progression is recently appreciated. Similarly, current findings point to the significant role of Schwann cells as part of neuro-immune interactions. Hence, in this review, we mainly focus on local and systemic effects of sensory nerve activity as well as Schwann cells in carcinogenesis and metastasis. Specific denervation of vagal sensory nerve fibers, or vagotomy, in animal models, has been reported to markedly increase lung metastases of breast carcinoma as well as pancreatic and gastric tumor growth, with the formation of liver metastases demonstrating the protective role of vagal sensory fibers against cancer. Clinical studies have revealed that patients with gastric ulcers who have undergone a vagotomy have a greater risk of stomach, colorectal, biliary tract, and lung cancers. Protective effects of vagal activity have also been documented by epidemiological studies demonstrating that high vagal activity predicts longer survival rates in patients with colon, non-small cell lung, prostate, and breast cancers. However, several studies have reported that inhibition of sensory neuronal activity reduces the development of solid tumors, including prostate, gastric, pancreatic, head and neck, cervical, ovarian, and skin cancers. These contradictory findings are likely to be due to the post-nerve injury-induced activation of systemic sensory fibers, the level of aggressiveness of the tumor model used, and the local heterogeneity of sensory fibers. As the aggressiveness of the tumor model and the level of the inflammatory response increase, the protective role of sensory nerve fibers is apparent and might be mostly due to systemic alterations in the neuro-immune response. Hence, more insights into inductive and permissive mechanisms, such as systemic, cellular neuro-immunological mechanisms of carcinogenesis and metastasis formation, are needed to understand the role of sensory neurons in tumor growth and spread.

Olvanil activates sensory nerve fibers, increases T cell response and decreases metastasis of breast carcinoma.

BACKGROUND: Inactivation of sensory neurons expressing transient receptor potential vanilloid 1 (TRPV1) enhances breast cancer metastasis. Sensory neurons have profound effects on immune response to a wide range of diseases including cancer. Hence, activation of sensory nerves using feasible approaches such as specific TRPV1 agonists may inhibit breast cancer metastasis through neuroimmune pathways. TRPV1 agonists are considered for the treatment of pain and inflammatory diseases. METHODS: We here first determined the effects of four different TRPV1 agonists on proliferation of three different metastatic breast carcinoma cells since TRPV1 is also expressed in cancer cells. Based on the results obtained under in-vitro conditions, brain metastatic breast carcinoma cells (4TBM) implanted orthotopically into the mammary-pad of Balb-c mice followed by olvanil treatment (i.p.). Changes in tumor growth, metastasis and immune response to cancer cells were determined. RESULTS: Olvanil dose-dependently activated sensory nerve fibers and markedly suppressed lung and liver metastasis without altering the growth of primary tumors. Olvanil (5 mg/kg) systemically increased T cell count, enhanced intra-tumoral recruitment of CD8+ T cells and increased IFN-γ response to irradiated cancer cells and Con-A. Anti-inflammatory changes such as increased IL-10 and decrease IL-6 as well as S100A8+ cells were observed following olvanil treatment. CONCLUSIONS: Our results show that anti-metastatic effects of olvanil is mainly due to activation of neuro-immune pathways since olvanil dose used here is not high enough to directly activate immune cells. Furthermore, olvanil effectively depletes sensory neuropeptides; hence, olvanil is a good non-pungent alternative to capsaicin.

Distribution of transient receptor potential vanilloid-1 channels in gastrointestinal tract of patients with morbid obesity.

BACKGROUND: Transient receptor potential vanilloid-1 (TRPV1), a nonselective cation channel, is activated by capsaicin, a pungent ingredient of hot pepper. Previous studies have suggested a link between obesity and capsaicin-associated pathways, and activation of TRPV1 may provide an alternative approach for obesity treatment. However, data on the TRPV1 distribution in human gastric mucosa are limited, and the degree of TRPV1 distribution in the gastric and duodenal mucosal cells of obese people in comparison with normal-weight individuals is unknown. AIM: To clarify gastric and duodenal mucosal expression of TRPV1 in humans and compare TRPV1 expression in obese and healthy individuals. METHODS: Forty-six patients with a body mass index (BMI) of > 40 kg/m2 and 20 patients with a BMI between 18-25 kg/m2 were included. Simultaneous biopsies from the fundus, antrum, and duodenum tissues were obtained from subjects between the ages of 18 and 65 who underwent esophagogastroduodenoscopy. Age, sex, history of alcohol and cigarette consumption, and past medical history regarding chronic diseases and medications were accessed from patient charts and were analyzed accordingly. Evaluation with anti-TRPV1 antibody was performed separately according to cell types in the fundus, antrum, and duodenum tissues using an immunoreactivity score. Data were analyzed using SPSS 17.0. RESULTS: TRPV1 expression was higher in the stomach than in the duodenum and was predominantly found in parietal and chief cells of the fundus and mucous and foveolar cells of the antrum. Unlike foveolar cells in the antrum, TRPV1 was relatively low in foveolar cells in the fundus (4.92 ± 0.49 vs 0.48 ± 0.16, P < 0.01, Mann-Whitney U test). Additionally, the mucous cells in the duodenum also had low levels of TRPV1 compared to mucous cells in the antrum (1.33 ± 0.31 vs 2.95 ± 0.46, P < 0.01, Mann-Whitney U test). TRPV1 expression levels of different cell types in the fundus, antrum, and duodenum tissues of the morbidly obese group were similar to those of the control group. Staining with TRPV1 in fundus chief cells and antrum and duodenum mucous cells was higher in patients aged ≥ 45 years than in patients < 45 years (3.03 ± 0.42, 4.37 ± 0.76, 2.28 ± 0.55 vs 1.9 ± 0.46, 1.58 ± 0.44, 0.37 ± 0.18, P = 0.03, P < 0.01, P < 0.01, respectively, Mann-Whitney U test). The mean staining levels of TRPV1 in duodenal mucous cells in patients with diabetes and hypertension were higher than those in patients without diabetes and hypertension (diabetes: 2.11 ± 0.67 vs 1.02 ± 0.34, P = 0.04; hypertension: 2.42 ± 0.75 vs 1.02 ± 0.33, P < 0.01 Mann-Whitney U test). CONCLUSION: The expression of TRPV1 is unchanged in the gastroduodenal mucosa of morbidly obese patients demonstrating that drugs targeting TRPV1 may be effective in these patients.

Effects of in-vitro modulation of TRPV1 activity on immune response of mice bearing metastatic breast carcinoma: Enhanced inflammatory response may hinder therapeutic potentials of TRPV1 agonists.

AIMS: Activation of transient receptor potential vanilloid 1 (TRPV1) ion channels inhibits inflammation, enhance cytotoxic immune response, and may have therapeutic potential in treatment of cancer characterized by increased systemic inflammation. We here determined how activation of TRPV1 alters immune response of tumor-bearing mice. MAIN METHODS: Three different metastatic subset of 4 T1 breast carcinoma cells were used to induce tumors in Balb-c mice. Mix leukocyte cultures (MLCs) using spleens and draining lymph nodes were prepared and stimulated with various challenges. Effects TRPV1 agonists including capsaicin, antagonist (AMG9810) and Gambogic Amide (GA), a TrkA agonist that sensitizes TRPV1, on secreted levels of cytokines were determined. KEY FINDINGS: MLCs of tumor-bearing mice secreted markedly higher levels of IL-6 and lower levels of IFN-γ compared to control mice. We observed differential effects of TRPV1 agonists in control and mice bearing different subset of metastatic cells. TRPV1 increased IFN-γ and IL-17 secretion in control mice while they markedly increased IL-6 secretion and suppressed IFN--γ secretion in tumor-bearing mice. Unexpectedly, AMG9810 acted as an inverse agonist and did not antagonize the effects of TRPV1 agonists. SIGNIFICANCE: Our results demonstrate constitutive activity of TRPV1 in immune cells, suggesting cross activation. To prevent excessive chronic activation of TRPV1 in immune cells in the presence of metastatic breast carcinoma, lower doses of TRPV1 agonist should be considered. Unexpected findings further document that a drug can have multiple intrinsic activities depending on surrounding factors can act on the same receptor as an agonist, antagonist or inverse agonist.

Apoptosis Deregulation and the Development of Cancer Multi-Drug Resistance.

The ability of tumor cells to evade apoptosis is established as one of the hallmarks of cancer. The deregulation of apoptotic pathways conveys a survival advantage enabling cancer cells to develop multi-drug resistance (MDR), a complex tumor phenotype referring to concurrent resistance toward agents with different function and/or structure. Proteins implicated in the intrinsic pathway of apoptosis, including the Bcl-2 superfamily and Inhibitors of Apoptosis (IAP) family members, as well as their regulator, tumor suppressor p53, have been implicated in the development of MDR in many cancer types. The PI3K/AKT pathway is pivotal in promoting survival and proliferation and is often overactive in MDR tumors. In addition, the tumor microenvironment, particularly factors secreted by cancer-associated fibroblasts, can inhibit apoptosis in cancer cells and reduce the effectiveness of different anti-cancer drugs. In this review, we describe the main alterations that occur in apoptosis-and related pathways to promote MDR. We also summarize the main therapeutic approaches against resistant tumors, including agents targeting Bcl-2 family members, small molecule inhibitors against IAPs or AKT and agents of natural origin that may be used as monotherapy or in combination with conventional therapeutics. Finally, we highlight the potential of therapeutic exploitation of epigenetic modifications to reverse the MDR phenotype.

Changes in ghrelin, substance P and vasoactive intestinal peptide levels in the gastroduodenal mucosa of patients with morbid obesity.

AIMS: The aim of the study was to assess changes in levels of substance P (SP), vasoactive intestinal peptide (VIP) and ghrelin in the gastroduodenal mucosa of obese individuals, which has not been studied before. METHODS: Forty-six patients with a body mass index (BMI) of >40 kg/m2 and 20 patients with a BMI of 18-25 kg/m2 were included in the study. VIP and SP levels in the fundus, antrum and duodenal mucosa were measured in freshly frozen tissues using enzyme-linked immunosorbent assay (ELISA). Fasting levels of ghrelin in blood were also measured with ELISA. Tissue levels of ghrelin were assessed by immunohistochemical staining, and immunoreactivity scores were used for ghrelin evaluation in tissues. RESULTS: Antrum SP levels were higher in the obese group than in the control group. A significant number of obese patients had low VIP levels in the fundus and antrum. Intense ghrelin staining was observed in a limited number of cells in the mucosal area of the gastric fundus that was similar in the control and patient groups. In the antrum and duodenum, ghrelin staining was low in all the samples examined. CONCLUSION: Here, we found that SP levels are increased, while VIP levels are decreased in the antrum of morbidly obese individuals. Previous studies show that SP increases gastroduodenal motility, that VIP slows it down, and that the gastric emptying rate is higher in obese individuals, preventing negative feedback mechanisms upon food intake. Therefore, increases in SP and decreases in VIP levels in the antrum may contribute to obesity by accelerating gastric emptying.

Rebound increases in chemokines by CXCR2 antagonist in breast cancer can be prevented by PKCδ and PKCε activators.

Activation of CXCR2 by chemokines such as CXCL1 and CXCL2 increases aggressiveness of breast cancer, inducing chemoresistance, hence CXCR2 antagonists are in clinical trials. We previously reported that inhibition of CXCR2 increases MIP-2 (CXCL2), which may inhibit anti-tumoral effects of CXCR2 antagonists. This seems to be due to inhibition of protein kinase C (PKC) by CXCR2 antagonist since specific inhibitor of PKC also enhances MIP-2 secretion. We here examined whether CXCR2 inhibitor also increases KC (CXCL1) secretion, ligand for CXCR2 involved in metastasis and PKC activators can prevent increases in chemokine secretion. We used SB 225002, which is a specific CXCR2 antagonist. The effects of PKC activators that have documented anti-tumoral effects and activates multiple isozymes of PKC such as Ingenol-3-angelate (I3A) and bryostatin-1 were examined here. In addition, FR236924, PKCε selective and 7α-acetoxy-6ß-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz), PKCδ selective activators were also tested. The effects of activators were determined using brain metastatic (4TBM) and heart metastatic (4THM) subset of 4T1 breast carcinoma cells because these aggressive carcinoma cells with cancer stem cell features secrete high levels of KC and MIP-2. Inhibition of CXCR-2 activity increased KC (CXCL1) secretion. PKC activators prevented SB225002-induced increases in KC and MIP-2 secretion. Different activators/modulators induce differential changes in basal and SB225002-induced chemokine secretion as well as cell proliferation and the activators that act on PKCδ and/or PKCε such as bryostatin 1, FR236924 and Roy-Bz are the most effective. These activators alone also decrease cell proliferation or chemokine secretion or both. Given the role of KC and MIP-2 in drug resistance including chemotherapeutics, activators of PKCε and PKCδ may prevent emerging of resistance to CXCR2 inhibitors as well as other chemotherapeutics.

Changes of HMGB-1 and sTLR4 levels in cerebrospinal fluid of patients with febrile seizures.

PURPOSE: Fever-induced inflammatory processes and pro-inflammatory cytokines have gained importance in recent years in the pathogenesis of febrile convulsion. Increased levels of HMGB1 (high mobility group box 1), one of the most important pro-inflammatory cytokines, are associated with prolongation of seizure duration, recurrence of seizures and the development of epilepsy. Changes in the sTLR4 level (soluble toll-like receptor 4) in the cerebrospinal fluid (CSF) are thought to be associated with memory and learning functions. In our study, we aimed to evaluate changes in HMGB1 and sTLR4 levels in patients who had febrile seizures between 6 months and 6 years. METHODS: Forty patients who were admitted to Akdeniz University Medical Faculty Hospital between April 2016 and April 2018 with a complaint of febrile seizure and 45 patients whose CSF samples were taken for complaints other than febrile convulsion (control group) were included in our study. RESULTS: Comparison of the CSF HMGB1 levels of the febrile convulsion group and control group revealed a statistically significant increase in patients with febrile convulsions (p: 0.001). Comparison of the subgroups revealed that the mean value of CSF HMGB1 level was highest in the complex FS group with a mean value of 3363.9 ± 835,47 pg/mL. Comparison of the patient and control groups revealed that the changes in CSF sTLR4 levels were not statistically significant. CONCLUSION: HMGB1 level, a key inflammatory molecule, was significantly higher in the CSF of children with febrile seizures. Our data suggest that the HMGB1 network may contribute to the generation of febrile seizures in children.

Changes in substance P levels of inferior turbinate in patients with mucosal contact headache / Alterações nos níveis da substância P da concha inferior de pacientes com cefaleiapor ponto de contato da mucosa

Abstract Introduction Mucosal contact headache is a referred pain that arises from contact between the nasal septum and the lateral nasal wall. Evidence supports the role of substance P in a contact headache such that release of substance P from sensory nerve endings causes inflammation and allergy. Objectives This study aimed to determine possible differences in substance P levels in inferior turbinate hypertrophy creating a contact headache. Methods 28 patients who had contact headaches (study group) and 16 volunteers with no complaints were included in the study. Substance P levels in the inferior turbinate tissue samples were quantified using a commercially available substance P EIA kit. Results In the study group average substance P levels were 2.65 ± 0.27 pg/mg tissue (range: 0.61-5.44) and in the control group it was 1.77 ± 0.27 pg/mg tissue (range: 0.11-4.35). The difference was statistically significant between the two groups (p = 0.0215). Average preoperative headache group visual analog scale scores was 5.93 ± 0.38 (2-9) and the turbinate volume was 6.56 ± 0.35 cm3 (3.50-10.30). The control group turbinate volume was 4.71 ± 0.39 cm3 (2.50-7.70). We found a correlation between the visual analog scale scores and substance P levels such that substance P levels were higher in visual analog scale scores above 5 (p = 0.001). Conclusion This study demonstrates the relationship between intranasal contact headaches and increased mucosal substance P levels. We also found that there is no correlation with substance P levels and volume of the inferior turbinate.

Resumo Introdução A cefaleia por ponto de contato da mucosa é uma dor direcionada que surge do contato entre o septo nasal e a parede nasal lateral. Evidências corroboram o papel da substância P na cefaleia de contato, de tal forma que a liberação da mesma a partir de terminações nervosas sensoriais possa causar inflamação e alergia. Objetivo Determinar possíveis diferenças nos níveis da substância P na hipertrofia de conchas inferiores em relação à cefaleia de contato. Método Foram incluídos no estudo 28 pacientes que apresentaram cefaleia por ponto de contato (Grupo Estudo) e 16 voluntários sem queixas. Os níveis de substância P nas amostras de tecido da concha inferior foram quantificados com um kit substância P EIA, comercialmente disponível. Resultados No grupo do estudo, os níveis médios de substância P foram 2,65 ± 0,27 pg/mg de tecido (variação: 0,61-5,44) e no grupo controle foram de 1,77 ± 0,27 pg/mg de tecido (variação: 0,11-4,35) e a diferença foi estatisticamente significante entre os dois grupos (p = 0,0215). O escore médio da escala visual analógica do grupo de cefaleia pré-operatória foi de 5,93 ± 0,38 (2-9) e o volume das conchas foi de 6,56 ± 0,35 cm3 (3,50-10,30). O volume da concha do grupo controle foi de 4,71 ± 0,39 cm3 (2,50 ± 7,70). Encontramos uma correlação entre o escore da escala visual analógica e os níveis de substância P, de modo que os níveis de substância P foram maiores nos escores da escala visual analógica acima de 5 (p = 0,001). Conclusão Este estudo demonstra a relação entre cefaleias por contato intranasais e níveis aumentados de substância P nas mucosas. Também observamos que não há correlação com os níveis de substância P e o volume da concha inferior.

Tumor microenvironment and epithelial mesenchymal transition as targets to overcome tumor multidrug resistance.

It is well established that multifactorial drug resistance hinders successful cancer treatment. Tumor cell interactions with the tumor microenvironment (TME) are crucial in epithelial-mesenchymal transition (EMT) and multidrug resistance (MDR). TME-induced factors secreted by cancer cells and cancer-associated fibroblasts (CAFs) create an inflammatory microenvironment by recruiting immune cells. CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSCs) and inflammatory tumor associated macrophages (TAMs) are main immune cell types which further enhance chronic inflammation. Chronic inflammation nurtures tumor-initiating/cancer stem-like cells (CSCs), induces both EMT and MDR leading to tumor relapses. Pro-thrombotic microenvironment created by inflammatory cytokines and chemokines from TAMs, MDSCs and CAFs is also involved in EMT and MDR. MDSCs are the most common mediators of immunosuppression and are also involved in resistance to targeted therapies, e.g. BRAF inhibitors and oncolytic viruses-based therapies. Expansion of both cancer and stroma cells causes hypoxia by hypoxia-inducible transcription factors (e.g. HIF-1α) resulting in drug resistance. TME factors induce the expression of transcriptional EMT factors, MDR and metabolic adaptation of cancer cells. Promoters of several ATP-binding cassette (ABC) transporter genes contain binding sites for canonical EMT transcription factors, e.g. ZEB, TWIST and SNAIL. Changes in glycolysis, oxidative phosphorylation and autophagy during EMT also promote MDR. Conclusively, EMT signaling simultaneously increases MDR. Owing to the multifactorial nature of MDR, targeting one mechanism seems to be non-sufficient to overcome resistance. Targeting inflammatory processes by immune modulatory compounds such as mTOR inhibitors, demethylating agents, low-dosed histone deacetylase inhibitors may decrease MDR. Targeting EMT and metabolic adaptation by small molecular inhibitors might also reverse MDR. In this review, we summarize evidence for TME components as causative factors of EMT and anticancer drug resistance.

NK1R antagonist decreases inflammation and metastasis of breast carcinoma cells metastasized to liver but not to brain; phenotype-dependent therapeutic and toxic consequences.

Substance P a neuro-immune mediator acts on Neurokinin-1 and -2 receptors (NK1R and NK2R). Inhibitors of NK1R are considered to be safe and effective approaches for cancer treatment since Aprepitant, a non-peptide antagonist of NK1R is widely used for chemotherapy-induced emesis and has cytotoxic and antitumor effects in various models for cancer. On the other hand, our previous findings demonstrated that systemic inhibition of NK1R may decrease cytotoxic anti-tumoral immune response. Hence, actual consequences of inhibition of neurokinin receptors under in vivo conditions in a syngeneic model of carcinoma should be determined. The effects of highly potent and selective non-peptide mouse NK1R and NK2R antagonists RP 67580 and GR 159897, respectively, on metastatic breast carcinoma were evaluated. Specifically, 4T1 breast cancer cells metastasized to brain (denoted as 4TBM) and liver (denoted as 4TLM) were used to induce tumors in Balb-c mice. Changes in tumor growth, metastasis and immune response to cancer cells were determined. We here observed differential effects of NK1R antagonist depended on the subset of metastatic cells. Specifically, inhibition of NK1R markedly increased liver metastasis of tumors formed by 4TBM but not 4TLM cells. On the contrary, NK1R antagonist decreased inflammatory response and liver metastasis in 4TLM-injected mice. 4TLM tumors act more aggressively inducing more inflammatory response compared to 4TBM tumors. Hence, differential effects of NK1R antagonist are at least partly due to extend and type of the inflammatory response evoked by specific subset metastatic cells. These findings demonstrate the necessity for understanding the immunological consequences of tumor-microenvironment interactions.

Role of sensory neurons, neuroimmune pathways, and transient receptor potential vanilloid 1 (TRPV1) channels in a murine model of breast cancer metastasis.

Sensory nerves sensitive to capsaicin are afferent nerve fibers which contain TRPV1 channels. Activation of these channels induces release of neuropeptides which regulate local blood flow and immune response. Inactivation of sensory neurons either with high-dose capsaicin treatment or local ablation of vagal sensory nerve activity markedly increases metastasis of breast carcinoma formed by 4T1 derivative cells. These cancer cells also induce an extensive systemic inflammatory response. Further findings have documented that lack of local sensory neuromediators alters phenotype of cancer cells within primary tumor leading to overgrowth of metastatic subsets. This might be due to decreases in local and systemic immune response to growing tumor. Specifically, Substance P, one of the most abundant sensory neuropeptides, enhances anti-tumoral immune response evoked by radiotherapy under in vivo conditions. These findings further suggest that activation of TRPV1 channels on sensory neurons may induce an anti-tumoral immune response. We are testing this hypothesis. Our initial results as reported here demonstrate anti-inflammatory consequences of low-dose systemic capsaicin treatment. In conclusion, sensory nerve fibers sensitive to capsaicin have important roles in defense against metastatic breast carcinoma; hence, controlled activation of these neural pathways might be effective in cancer therapy. Specifically, activation of sensory fibers of left vagus nerve using a perineuronal stimulation may inhibit metastasis of breast carcinoma. Likewise, pharmacological modulators of TRPV1 channels may induce anti-tumoral immune response. Exact players of this newly explored defense system are, however, only partly validated, and further studies are required.

HSP90 inhibitor PU-H71 increases radiosensitivity of breast cancer cells metastasized to visceral organs and alters the levels of inflammatory mediators.

Heat shock protein 90 (HSP90) inhibitors are considered as new radiosensitizing agents. PU-H71, a novel HSP90 inhibitor, is under evaluation for the treatment of advanced cancer. It is however not known whether PU-H71 alters radiosensitivity of metastatic breast cancer. Hence, we here evaluated mechanisms of possible anti-tumoral and radiosensitizing effects of PU-H71 on breast carcinoma cells metastasized to vital organs such as the liver and brain. The effect of PU-H71 on proliferation of breast carcinoma cells was determined using 4T1 cells and its brain (4TBM), liver (4TLM), and heart (4THM) metastatic subsets as well as non-metastatic 67NR cells. Changes in radiation sensitivity were determined by clonogenic assays. Changes in client proteins and levels of angiogenic and inflammatory mediators from these cancer cell cultures and ex vivo cultures were detected. PU-H71 alone inhibited ERK1/2, p38, and Akt activation and reduced N-cadherin and HER2 which further documented the anti-tumoral effects of PU-H71. The combination of PU-H71 and radiotherapy induced cytotoxic effect than PU-H71 alone, and PU-H71 showed a radiosensitizing effect in vitro. On the other hand, PU-H71 and radiation co-treatment increased p38 phosphorylation which is one of the hallmarks of inflammatory response. Accordingly, IL-6 secretion was increased following PU-H71 and radiotherapy co-treatment ex vivo. Levels of angiogenic and inflammatory factors such as MIP-2, SDF-1, and VEGF were increased under in vitro conditions but not under ex vivo conditions. These results demonstrated for the first time that PU-H71 enhances therapeutic effects of radiotherapy especially in highly metastatic breast carcinoma but a possible increase in inflammatory response should also be considered.

Changes in substance P levels of inferior turbinate in patients with mucosal contact headache.

INTRODUCTION: Mucosal contact headache is a referred pain that arises from contact between the nasal septum and the lateral nasal wall. Evidence supports the role of substance P in a contact headache such that release of substance P from sensory nerve endings causes inflammation and allergy. OBJECTIVES: This study aimed to determine possible differences in substance P levels in inferior turbinate hypertrophy creating a contact headache. METHODS: 28 patients who had contact headaches (study group) and 16 volunteers with no complaints were included in the study. Substance P levels in the inferior turbinate tissue samples were quantified using a commercially available substance P EIA kit. RESULTS: In the study group average substance P levels were 2.65±0.27pg/mg tissue (range: 0.61-5.44) and in the control group it was 1.77±0.27pg/mg tissue (range: 0.11-4.35). The difference was statistically significant between the two groups (p=0.0215). Average preoperative headache group visual analog scale scores was 5.93±0.38 (2-9) and the turbinate volume was 6.56±0.35cm3 (3.50-10.30). The control group turbinate volume was 4.71±0.39cm3 (2.50-7.70). We found a correlation between the visual analog scale scores and substance P levels such that substance P levels were higher in visual analog scale scores above 5 (p=0.001). CONCLUSION: This study demonstrates the relationship between intranasal contact headaches and increased mucosal substance P levels. We also found that there is no correlation with substance P levels and volume of the inferior turbinate.

CD200 mimetic aptamer PEG-M49 markedly increases the therapeutic effects of pegylated liposomal doxorubicin in a mouse model of metastatic breast carcinoma: an effect independent of CD200 receptor 1.

We previously reported that CD200 overexpression in the host decreases progression and metastasis of the highly aggressive metastatic 4THM breast carcinoma. We have explored a possible synergistic interaction between the CD200 mimetic PEG-M49 and pegylated liposomal doxorubicin (Peg-Dox) in wild-type CD200 knockout (CD200-/-) and CD200 Receptor 1 knockout (CD200R1-/-) mice for the first time. A 4THM breast carcinoma model and three groups of BALB/c mice (wild type, CD200-/- and CD200R1-/-) were used. Five days after injection of tumor cells, mice were injected with Peg-Dox (ip, once a week) and PEG-M49 or a control aptamer (iv, every 3 days). Necropsies were performed either 12 (mid-point) or 24 (endpoint) days after injection and the extent of tumor growth, visceral metastasis and changes in the tumor-directed immune response were evaluated. PEG-M49 and Peg-Dox co-treatment induced complete tumor regression and loss of macroscopic lung metastasis in four out of seven WT mice. This synergistic anti-tumoral effect is thought to be due to Peg-M49-induced inhibition of Gr1 + CD11b + cells and Peg-Dox-induced increases in tumor-infiltrating CD8 + and CD8CD4 double-positive cells. Similar changes were observed in CD200R1-/- mice indicating that the primary effects of Peg-M49 are mediated by non-CD200R1 receptors. We also demonstrated for the first time that tumor growth, metastasis, and tumor infiltrating GR1 + CD11b + cells were markedly increased in CD200R1-/- mice, indicating an anti-inflammatory and protective role of CD200. CD200 mimetics might be a safe and effective immunomodulatory treatment in conjunction with classical chemotherapeutics for therapy of aggressive metastatic breast carcinoma.

Effect of Photobiomodulation on Secretion of IL-6 and IL-8 by Human Gingival Fibroblasts In Vitro.

Objective: The aim of this study was to evaluate the effects of 940-nm diode laser irradiation on proinflammatory cytokine secretions [interleukin (IL)-6 and IL-8] by human gingival fibroblasts in vitro. Background: Photobiomodulation has been routinely used in many dental procedures; however, the exact biological action mechanism of photobiomodulation and its therapeutic benefits have not been established. Methods: Cells derived from systemically healthy individuals were treated with three different laser parameters-6 J for 20 sec [0.84 J/cm2 (0.04 W/cm2)], 10 J for 20 sec [1.4 J/cm2 (0.07 W/cm2)], and 14 J for 20 sec [1.97 J/cm2 (0.09 W/cm2)]-in the presence and absence of 1 µg/mL lipopolysaccharide (LPS) stimulation. Laser irradiations were carried out by a 940-nm diode laser device in continuous pain therapy mode with a deep tissue handpiece. Changes in cell viability, cytokine secretions, and mitogen-activated protein kinase pathway expressions were investigated, and results were compared with negative (medium) and positive control (1 µg/mL LPS) groups. The data obtained were statistically analyzed by the Mann-Whitney U test for pairwise comparisons among groups at the 0.05 level of significance. Results: Laser therapy with 0.84-1.4 J/cm2 amplified IL-6 and IL-8 secretions, whereas 1.97 J/cm2 suppressed IL-6 and IL-8 release in LPS-stimulated cells. Cell viability did not show a variation with photobiomodulation. Conclusions: These results demonstrate that photobiomodulation can alter IL-6 and IL-8 release, with cytokine suppression potency at a relatively high dose, as demonstrated previously. However, in contrast, we found that a low level of stimulation (6 J) in the presence of inflammation (LPS stimulation) may further enhance IL-6 and IL-8 release. We also found that p38 and ERK1/2 pathways are activated by LPS as well as by photobiomodulation.

Differential consequences of neurokinin receptor 1 and 2 antagonists in metastatic breast carcinoma cells; Effects independent of Substance P.

INTRODUCTION: The biological action of Substance P (SP) is mediated mainly by NK-1 receptors (NK1R) followed by NK2 receptors (NK2R). Aberrant expression of NK1R and NK2R has been identified in various carcinomas. The role of Substance P and its receptors, especially NK2R in cancer progression is not entirely known and there are conflicting results in the literature demonstrating the need for further investigation. In the current study, we examined the effects of SP and antagonists selective for the NK1R and NK2R in breast carcinoma cells metastasize to vital organs. METHODS: The effects of highly potent and selective non-peptide mouse NK1R and NK2R antagonists RP 67,580 and GR 159897, respectively, as well as SP and SP methyl ester, on both metastatic (4THM, 4TBM, 4TLM, 4T1) and non-metastatic (67NR) breast cancer cells were determined. RESULTS: NK1R and NK2R were over expressed in metastatic breast cells compared to non-metastatic cells. The NK1R antagonist at a 30 µM dose inhibited cell growth and induced cell death in metastatic cells while enhancing phosphorylation of Akt, the latter response not observed in the non-metastatic 67NR cells. Blocking the action of SP at the NK2R (30 µM antagonist) suppressed cellular proliferation in all the cell lines examined, with a response less prominent than that of the NK1R antagonist. Differently, the NK2R antagonist increased phosphorylation of p38 and enhanced MIP-2 secretion. SP and the SP methyl ester neither altered cell proliferation nor the effects of NK1R and NK2R antagonists in the metastatic cell lines. CONCLUSIONS: Increased sensitivity of metastatic breast carcinoma cells to NK1R and NK2R antagonists suggest potential therapeutic value of antagonists in metastatic disease. NK1R and NK2R in metastatic breast carcinoma cells react differently to agonists and antagonists. These findings together with previously published data demonstrate that differential consequences of receptor antagonists and SP may inhibit breast cancer growth and metastasis.