The impact of stress on tumor growth: peripheral CRF mediates tumor-promoting effects of stress

<p>Abstract</p> <p>Introduction</p> <p>Stress has been shown to be a tumor promoting factor. Both clinical and laboratory studies have shown that chronic stress is associated with tumor growth in several types of cancer. Corticotropin Releasing Factor (CRF) is the major hypothalamic mediator of stress, but is also expressed in peripheral tissues. Earlier studies have shown that peripheral CRF affects breast cancer cell proliferation and motility. The aim of the present study was to assess the significance of peripheral CRF on tumor growth as a mediator of the response to stress in vivo.</p> <p>Methods</p> <p>For this purpose we used the 4T1 breast cancer cell line in cell culture and in vivo. Cells were treated with CRF in culture and gene specific arrays were performed to identify genes directly affected by CRF and involved in breast cancer cell growth. To assess the impact of peripheral CRF as a stress mediator in tumor growth, Balb/c mice were orthotopically injected with 4T1 cells in the mammary fat pad to induce breast tumors. Mice were subjected to repetitive immobilization stress as a model of chronic stress. To inhibit the action of CRF, the CRF antagonist antalarmin was injected intraperitoneally. Breast tissue samples were histologically analyzed and assessed for neoangiogenesis.</p> <p>Results</p> <p>Array analysis revealed among other genes that CRF induced the expression of SMAD2 and β-catenin, genes involved in breast cancer cell proliferation and cytoskeletal changes associated with metastasis. Cell transfection and luciferase assays confirmed the role of CRF in WNT- β-catenin signaling. CRF induced 4T1 cell proliferation and augmented the TGF-β action on proliferation confirming its impact on TGFβ/SMAD2 signaling. In addition, CRF promoted actin reorganization and cell migration, suggesting a direct tumor-promoting action. Chronic stress augmented tumor growth in 4T1 breast tumor bearing mice and peripheral administration of the CRF antagonist antalarmin suppressed this effect. Moreover, antalarmin suppressed neoangiogenesis in 4T1 tumors in vivo.</p> <p>Conclusion</p> <p>This is the first report demonstrating that peripheral CRF, at least in part, mediates the tumor-promoting effects of stress and implicates CRF in SMAD2 and β-catenin expression.</p

A Novel Role of Peripheral Corticotropin-Releasing Hormone (CRH) on Dermal Fibroblasts

Corticotropin-releasing hormone, or factor, (CRH or CRF) exerts important biological effects in multiple peripheral tissues via paracrine/autocrine actions. The aim of our study was to assess the effects of endogenous CRH in the biology of mouse and human skin fibroblasts, the primary cell type involved in wound healing. We show expression of CRH and its receptors in primary fibroblasts, and we demonstrate the functionality of fibroblast CRH receptors by induction of cAMP. Fibroblasts genetically deficient in Crh (Crh−/−) had higher proliferation and migration rates and compromised production of IL-6 and TGF-β1 compared to the wildtype (Crh+/+) cells. Human primary cultures of foreskin fibroblasts exposed to the CRF1 antagonist antalarmin recapitulated the findings in the Crh−/− cells, exhibiting altered proliferative and migratory behavior and suppressed production of IL-6. In conclusion, our findings show an important role of fibroblast-expressed CRH in the proliferation, migration, and cytokine production of these cells, processes associated with the skin response to injury. Our data suggest that the immunomodulatory effects of CRH may include an important, albeit not explored yet, role in epidermal tissue remodeling and regeneration and maintenance of tissue homeostasis

Role of neuropeptide corticotropin releasing hormone (CRH) and glucocorticoids in wound healing

Cutaneous wound healing is a complex process tightly related to inflammation, which requires the cooperation of several different cell populations and factors. Corticotropin releasing hormone (CRH) is the main regulator of the hypothalamic‐pituitaryadrenal (HPA) axis, which mediates the body's response to stress, including inflammation. The stimulation of CRH by stressful stimuli leads to the release of adrenal glucocorticoids, a potent endogenous anti‐inflammatory agent. CRH exerts its effects through two main types of receptor, receptor 1, (CRF1) and receptor 2 (CRF2). The mRNA of CRH and the immunoreactive peptide and its receptors have been identified in a plethora of peripheral tissues including the skin. Although it has been shown that peripheral CRH possesses direct pro‐inflammatory action, its role in the process of cutaneous wound healing remains unclear. To study the role of CRH in cutaneous wound healing we used mice with genetic deletion of CRH (Crh‐/‐). CRH mRNA was detected in both normal and wounded skin of Crh+/+ mice. Crh‐/ ‐ mice characterized by accelerated wound healing which was accompanied by rapid progression of inflammation and re‐epithelialization from the second to the fifth day following the induction of wound compared to wild type mice. Moreover, Crh‐/‐ mice had higher levels of TGF‐β1 on day 3 when compared with Crh+/+ mice. The replacement of glucocorticoids on Crh‐/‐ mice accelerated the wound healing even more during the first day following the induction of wound compared with vehicle alone‐treated Crh‐/‐ mice and simultaneously accelerated the resolution of the inflammatory response and increased the number and organization speed of fibroblasts. Finally, administration of corticosterone restored the tissue levels of TGF‐β1 in Crh‐/‐ mice to those observed in Crh+/+ mice. Our results suggest that genetic deficiency of CRH is associated with accelerated wound healing characterized by increased epithelialization and presence of fibroblasts in the area of the wound in a manner independent of glucocorticoids insufficiency. To clarify the role of endogenous CRH in the function of fibroblasts during wound healing we used primary cultures of fibroblasts derived from neonatal skin of Crh +/+ and Crh‐/‐ mice. Our studies showed that Crh is expressed in wild type fibroblasts. Moreover, fibroblasts of both genotypes have functional receptors, as they induce the production of cAMP. Fibroblasts with genetic deficiency of CRH have higher proliferation and migration rate and decreased production of IL‐6, TGF‐β1 and collagen in comparison with the corresponding wild type fibroblasts. The pharmacological blockade of CRF1 using antalarmin in cultured human fibroblasts led to similar changes on the rate of proliferation and migration and secretion of IL‐6, with those observed in Crh ‐/‐ fibroblasts. These results reinforce the importance of CRH in the biology of skin fibroblasts during wound healing. In summary, our findings from in vivo and in vitro experiments demonstrate for the first time the important role of locally produced CRH in cutaneous wound healing. The exact mechanism of action has not been clarified. However it appears that fibroblasts are an important component in this process.Η δερματική τραυματική επούλωση είναι μία σύνθετη διαδικασία, άρρηκτα συνδεδεμένη με τη φλεγμονή, που απαιτεί τη συνεργασία πολλών διαφορετικών κυτταρικών πληθυσμών και παραγόντων. Η εκλυτική ορμόνη της κορτικοτροπίνης (CRH) αποτελεί τον κύριο ρυθμιστή του υποθαλαμικού‐υποφυσιακού‐επινεφριδιακού (HPA) άξονα, ο οποίος μεσολαβεί την απόκριση του οργανισμού στο στρες, συμπεριλαμβανομένης και της φλεγμονής. Η διέγερση της CRH από στρεσογόνα ερεθίσματα οδηγεί στην απελευθέρωση των επινεφριδιακών γλυκοκορτικοειδών, ενός ισχυρού ενδογενούς αντιφλεγμονώδους μέσου. Η CRH ασκεί τις δράσεις της μέσω δύο κύριων τύπων υποδοχέων, τον υποδοχέα 1 της CRH (CRF1) και τον υποδοχέα 2 της CRH (CRF2). Το mRNA της CRH καθώς και το ανοσοδραστικό πεπτίδιο και οι υποδοχείς της έχουν εντοπιστεί σε πληθώρα περιφερικών ιστών συμπεριλαμβανομένου του δέρματος. Αν και έχει δειχθεί ότι η περιφερική CRH ασκεί άμεση προ‐φλεγμονώδη δράση, εντούτοις ο ρόλος της στη διαδικασία της δερματικής τραυματικής επούλωσης παραμένει αδιευκρίνιστος. Προκειμένου να μελετηθεί ο ρόλος της CRH στην επούλωση του δερματικού τραύματος χρησιμοποιήθηκαν μύες στους οποίους είχε γίνει απαλοιφή του γονιδίου της CRH (Crh/ ). Η CRH ανιχνεύθηκε τόσο σε φυσιολογικό όσο και τραυματισμένο δέρμα Crh+/+ μυών. Οι Crh/ μύες χαρακτηρίζονταν από επιταχυμένη τραυματική επούλωση η οποία συνοδευόταν από ταχύτερη εξέλιξη της φλεγμονής και της επαναεπιθηλιοποίησης από τη δεύτερη έως την πέμπτη ημέρα μετά την επαγωγή του τραύματος συγκριτικά με τους μύες αγρίου τύπου. Επιπλέον οι Crh/ μύες είχαν υψηλότερα επίπεδα TGF‐β1 την 3η ημέρα όταν συγκρίνονταν με τους Crh+/+ μύες. Η αποκατάσταση των γλυκοκορτικοειδών στους Crh/ μύες επιτάχυνε την τραυματική επούλωση ακόμα περισσότερο την πρώτη ημέρα μετά την επαγωγή του τραύματος συγκριτικά με τους Crh/ μύες στους οποίους δε χορηγήθηκε κορτικοστερόνη και ταυτόχρονα επιτάχυνε τη λύση της φλεγμονώδους αντίδρασης και αύξησε τον αριθμό και την ταχύτητα οργάνωσης των ινοβλαστών. Τέλος, η χορήγηση κορτικοστερόνης επανέφερε τα ιστικά επίπεδα του TGF‐β1 στους Crh‐/‐ μυς σε αυτά που παρατηρούνταν Crh/ στους μύες. Τα παραπάνω αποτελέσματα δείχνουν ότι η γενετική έλλειψη CRH συνδέεται με επιταχυμένη τραυματική επούλωση που χαρακτηρίζεται από αυξημένη επιθηλιοποίηση και παρουσία ινοβλαστών στην τραυματική περιοχή κατά τρόπο ανεξάρτητο των γλυκοκορτικοειδών. Για να αποσαφηνιστεί ο ρόλος της ενδογενούς CRH στη λειτουργία των ινοβλαστών κατά τη διάρκεια της επούλωσης χρησιμοποιήθηκαν πρωτογενείς καλλιέργειες ινοβλαστών προερχόμενες από δέρμα νεογνών Crh+/+ και Crh/ μυών. Οι μελέτες μας έδειξαν ότι η Crh εκφράζεται στους ινοβλάστες μυών αγρίου τύπου. Επιπλέον, οι ινοβλάστες και των δύο γονοτύπων διαθέτουν λειτουργικούς υποδοχείς, καθώς επάγουν την παραγωγή cAMP. Οι ινοβλάστες με γενετική έλλειψη CRH παρουσίαζαν υψηλότερο ρυθμό πολλαπλασιασμού και μετανάστευσης και μειωμένη παραγωγή IL‐6, TGF‐β1 και κολλαγόνου σε σύγκριση με τους ινοβλάστες αγρίου τύπου. Ο φαρμακολογικός αποκλεισμός του CRF1 με τη χρήση ανταλαρμίνης σε καλλιέργειες ανθρώπινων ινοβλαστών επέφερε παρόμοιες αλλαγές αναφορικά με το ρυθμό πολλαπλασιασμού και μετανάστευσης και την έκκριση IL‐6, με αυτές που παρατηρούνταν στους Crh / ινοβλάστες. Τα αποτελέσματα αυτά ενισχύουν το σημαντικό ρόλο που διαδραματίζει η CRH στη βιολογία των δερματικών ινοβλαστών κατά τη διάρκεια της αποκατάστασης του τραύματος. Συνοψίζοντας, τα ευρήματά μας, από in vivo και in vitro πειράματα, καταδεικνύουν για πρώτη φορά το σημαντικό ρόλο της τοπικά παραγόμενης CRH στη δερματική τραυματική επούλωση. Ο ακριβής μηχανισμός της δράσης της αυτής δεν έχει διασαφηνισθεί. Παρόλα αυτά φαίνεται ότι οι ινοβλάστες αποτελούν σημαντική συνιστώσα στη διαδικασία αυτή

Analgesic and Anti-Inflammatory Effects of the Synthetic Neurosteroid Analogue BNN27 during CFA-Induced Hyperalgesia

Dehydroepiandrosterone (DHEA), an adrenal and neurosteroid hormone with strong neuroprotective and immunomodulatory properties, and ligand for all high-affinity neurotrophin tyrosine kinase receptors (Trk), also exerts important effects on hyperalgesia. Its synthetic, 17-spiro-epoxy analogue, BNN27, cannot be converted to estrogen or androgen as DHEA; it is a specific agonist of TrkA, the receptor of pain regulator Nerve Growth Factor (NGF), and it conserves the immunomodulatory properties of DHEA. Our study aimed to evaluate the anti-nociceptive and anti-inflammatory properties of BNN27 during Complete Freund’s Adjuvant (CFA)-induced inflammatory hyperalgesia in mice. Hyperalgesia was evaluated using the Hargreaves test. Inflammatory markers such as cytokines, NGF and opioids were measured, additionally to corticosterone and the protein kinase B (AKT) signaling pathway. We showed for the first time that treatment with BNN27 reversed hyperalgesia produced by CFA. The effect of BNN27 involved the inhibition of NGF in the dorsal root ganglia (DRG) and the increased synthesis of opioid peptides and their receptors in the inflamed paw. We also found alterations in the cytokine levels as well as in the phosphorylation of AKT2. Our findings strongly support that BNN27 represents a lead molecule for the development of analgesic and anti-inflammatory compounds with potential therapeutic applications in inflammatory hyperalgesia

Evaluation of HSP70 deficiency in the inflammatory response in vivo and in vitro: The impact of HSP90 expression, glucocorticoid release, and glutamine administration

The present study aimed to explore the pathway through which Heat Shock Protein 70 (HSP70) is involved in inflammation and sepsis and the possible interaction with glutamine, an essential nutrient during sepsis. We also evaluated the possible interaction of HSP70 with HSP90, another HSP involved in sepsis. Therefore, we utilized mice with specific deletion of the Hsp70.1 and Hsp70.3 genes (Hsp70-/-) and their wild type littermates (Hsp70+/+) for the in vivo studies and primary macrophages collected from Hsp70+/+ and Hsp70-/- mice for the in vitro studies. We found that HSP70 deficiency was associated with lower inflammatory cytokine levels in plasma and lung tissue and increased levels of glucocorticoid secretion during inflammation in vivo. On the contrary, Hsp70-/- murine peritoneal macrophages showed increased inflammatory response compared to Hsp70+/+ macrophages, following LPS stimulation. Glutamine administration repressed the in vivo secretion of plasma cytokines from Hsp70-/- mice while enhanced the in vitro secretion of cytokines from macrophages of the same genotype (Hsp70-/-), but it did not affect the secretion of cytokines from wild type mice either in vivo or in vitro, following LPS treatment. Our data also demonstrated that HSP70 deficiency was associated with increased glucocorticoid release, in vivo. In vitro, pre-treatment of macrophages from both genotypes with HSP90 inhibitor significantly attenuated IL-6 secretion only from Hsp70-/- cells, following LPS treatment. In summary, in vivo, the lack of HSP70 leads to an anti-inflammatory phenotype, while in vitro, the lack of HSP70 in macrophages gives rise to a proinflammatory phenotype, which may be mediated by the HSP90. Both in vivo and in vitro, glutamine treatment responses in Hsp70-/- mice suggest alternative pathways bypassing HSP70

Structures of Spiritual Needs Questionnaire Versions in Countries with Different Cultural and Religious Backgrounds

Both the interpretation and extent of personal spiritual concerns might be different as spiritual needs are multidimensional, and dependent on religious and cultural background. Importantly, for rigorous spiritual needs assessment well-prepared, valid and reliable measurement tools are needed. Initially developed in Germany and published in English, the SpNQ has been translated and validated so far in different languages and used in various countries. In each of these translations, the cross-cultural validation and adaptation process has brought differences in the proposed domains, reflecting the cultural aspects of each of the societies in which the instrument was applied. In this chapter we will discuss findings from Lithuania, Poland, Portugal, Brazil, Iran and China. The intention of this comparative chapter is to underline that, depending on sample size, in a group of persons with different health conditions, cultures and religious background, the factorial structure of spiritual needs measurement may vary

Corticotropin Releasing Factor promotes breast cancer cell motility and invasiveness

Abstract Introduction Cancer cells secrete bioactive peptides that act in an autocrine or paracrine fashion affecting tumor growth and metastasis. Corticotropin-releasing factor (CRF), a hypothalamic neuropeptide that controls the response to stress, has been detected in breast cancer tissues and cell lines. CRF can affect breast cancer cells in an autocrine or paracrine manner via its production from innervating sympathetic neurons or immune cells. Methods In the present study we report our findings regarding the impact of CRF on breast cancer cell motility and invasiveness. For this purpose we used the MCF7 breast cancer cell line and evaluated the effect of CRF on motility and invasiveness using the wound-healing and boyden-chamber assays. In addition, we measured the effect of CRF on molecules that mediate motility by western blot, immunofluorescence, ELISA and RT-PCR. Results Our findings show that: 1. CRF transiently inhibited the apoptosis of MCF7 cells. 2. CRF enhanced MCF7 cell motility in a wound healing assay and their invasiveness through extracellular matrix. 3. CRF increased actin polymerization, phosphorylation of Focal Adhesion Kinase (FAK), providing a potential mechanism for the observed induction of MCF7 motility. 4. CRF induced the expression of Cox-1 but not Cox-2 in MCF7 cells as well as the production of prostaglandins, factors known to promote invasiveness and metastasis. Conclusion Overall, our data suggest that CRF stimulates cell motility and invasiveness of MCF7 cells most probably via induction of FAK phosphorylation and actin filament reorganization and production of prostaglandins via Cox1. Based on these findings we postulate that the stress neuropeptide CRF present in the vicinity of tumors (either produced locally by the tumor cells themselves or by nearby normal cells or secreted from the innervations of surrounding tissues) may play an important role on breast tumor growth and metastatic capacity, providing a potential link between stress and tumor progression.</p

Corticotropin Releasing Factor promotes breast cancer cell motility and invasiveness

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