Chronic neuropathic pain, resulting from dysfunction of the nervous system, is a clinical concern in both humans and animal patients. Neuropathic pain is characterised by spontaneous pain, hypersensitivity, manifested as hyperalgesia and allodynia, and refractoriness to conventional analgesics such as non-steroidal anti-inflammatory drugs, thus representing an unmet therapeutic need. Equine laminitis is a disease that involves the disruption of the dermoepidermal junction within the hoof, leading to severe pain and lameness, with poor responsiveness to anti-inflammatory therapy. We developed a Quantitative Sensory Testing method, using a novel hydraulically-powered feedbackcontrolled hoof tester, in order to provide an objective tool for the assessment of mechanical hyperalgesia in laminitic horses. Hoof Compression Thresholds of laminitic horses were significantly lower than those of normal horses and variance component analysis of the data confirmed the reliability of the method. In order to investigate mechanisms underlying laminitis pain, we performed histological studies of peripheral nerves innervating the hoof. Electron micrographic analysis of the digital nerve of laminitic horses revealed a significant reduction in the number of unmyelinated and myelinated fibres together with abnormal morphology. Additionally, cell bodies of sensory neurons innervating the hoof in cervical C8 dorsal root ganglia showed an upregulated expression of the nerve injury marker activating transcription factor-3 (ATF3), neuropeptide Y (NPY), and the TRPM8 channel; each of which has been associated with laboratory models of neuropathic pain. Previous work has shown that, in a rodent model of neuropathic pain, the TRPM8 channel is upregulated in sensory neurons and its activation by cool temperature, menthol or icilin leads to reversal of the hypersensitive pain state. Further investigation of TRPM8-channel mediated analgesia was aimed at uncovering the molecular mechanisms involved in the activation of this system in sensitised states. It was hypothesised that serotonin, released following inflammation and nerve damage, can enhance TRPM8 channel activity through peripheral 5-HT1B receptors. Calcium fluorometry carried out in HEK293 cells transfected with the TRPM8 channel and the 5-HT1B receptor revealed that coadministration of a 5-HT1B receptor agonist facilitated the activation of the TRPM8 channel by icilin. Moreover, it appears that this effect is mediated through phospholipase D1 (PLD1), possibly leading to increased production of phosphatidylinositol (4,5-) bisphosphate (PIP2), a known positive modulator of TRPM8 channel activity. In vitro co-immunoprecipitation studies suggested that the TRPM8 channel, the 5-HT1B receptor and PLD1 physically interact with each other, further providing a molecular basis for their functional co-operation. Calcium imaging carried out in cultured rat DRG cells showed that the 5-HT1B receptor-mediated enhancement of icilin responses at the TRPM8 channel also occurs in sensory cells and is reversed by inhibition of PLD1. Moreover, TRPM8 and the 5-HT1B receptor appear to be physically associated in vivo as shown by their co-immunoprecipitation from spinal cord homogenates. Assessment of nociceptive behavioural reflexes following intrathecal injection of selective pharmacological agents provided further support for the idea of 5-HT1B receptor facilitation of TRPM8 channel responses in vivo. In addition to providing novel evidence of a neuropathic component to equine laminitis and validation of a novel QST method for pain assessment in horses, this study reveals for the first time a physical and functional interaction between the 5-HT1B receptor and the TRPM8 channel.

Plusieurs études ces dernières décennies suggèrent l’importance des canaux TRPs dont TRPM8 dans le développement et la dissémination du cancer de la prostate. Néanmoins, les différentes études menées sur ce canal sont contradictoires. L’objectif de ma thèse a été d’étudier le rôle précis de TRPM8 dans le cancer prostatique par des études in vivo afin d’évaluer l’impact de TRPM8 sur la croissance tumorale, la dissémination de ces cellules et la formation de métastases. De plus, nous avons approfondi les mécanismes moléculaires sous-jacents régulant l’effet anti-migratoire de TRPM8.Grâce à l’utilisation de nanocapsules lipidiques contenant un agoniste du canal TRPM8, nous avons confirmé par des études in vitro et in vivo un rôle inhibiteur de TRPM8 sur les capacités de migration et d’invasion des cellules cancéreuses prostatiques. De plus, nous avons également déterminer un rôle de TRPM8 sur la croissance tumorale grâce à l’utilisation de greffes orthotopiques dans des prostates murines. Nous avons également déterminé les mécanismes de régulation de la migration cellulaire par le canal TRPM8 et des protéines partenaires à ce canal. Dans ce cadre, nous avons défini et déterminé une régulation du canal TRPM8 par les androgènes modulant la migration cellulaire mais également les mécanismes sous-jacents de l’inhibition de la migration cellulaire induite par TRPM8 et impliquant la petite GTPase Rap1.L’ensemble de nos résultats démontrent un rôle antiprolifératif et anti-migratoire du canal TRPM8 sur les cellule cancéreuses prostatiques, suggérant ainsi une action protectrice de ce canal dans la dissémination des métastases prostatiques. / Several studies in recent decades suggest the importance of TRP channel including TRPM8 in the development and metastatic dissemination of prostate cancer. Nevertheless, the different studies conducted on this channel are contradictory. The aim of my thesis was to study the precise role of TRPM8 in prostate cancer by in vivo studies to study the impact of TRPM8 on tumor growth and metastatic dissemination. In addition, we determined the underlying molecular mechanisms regulating the anti-migratory effect of TRPM8.Due to the use of lipid nanocapsules containing a TRPM8 channel agonist, we have confirmed by in vitro and in vivo studies an inhibitory role of TRPM8 on the migration and invasion capacities of prostatic cancer cells. In addition, we also determined an inhibitory role of TRPM8 on tumor growth through the use of orthotopic grafts in murine prostates. We also determined the regulatory mechanisms of cell migration by TRPM8 channel and its partner proteins. In this context, we defined and determined a regulation of the TRPM8 channel by androgens modulating cell migration. Moreover, we determined also the mechanisms underlying the inhibition of TRPM8-induced cell migration involving the small Rap1 GTPase.All of my results demonstrate an anti-proliferative and anti-migratory role of the TRPM8 channel on prostatic cancer cells, suggesting a protective action of this channel in the dissemination of prostatic metastases.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A Riparina IV, uma alcamida sintetizada de Aniba riparia, foi testada em modelos animais padronizados de dor, bem como os possÃveis mecanismos de aÃÃo envolvidos. Foram utilizados camundongos Swiss (20-30g), e a Riparina IV foi administrada de forma aguda em todos os testes, nas doses de 25 e 50 mg/kg, por via oral. Foram utilizados os testes de contorÃÃes abdominais induzidas por Ãcido acÃtico; placa quente; teste da formalina; hipernocicepÃÃo mecÃnica induzida pela carragenina; teste da nocicepÃÃo induzida por capsaicina, cinamaldeÃdo, mentol; teste da nocicepÃÃo induzida por glutamato, bem como em modelos comportamentais que permitam excluir a possibilidade de uma atividade relaxante muscular ou induzir resultados falso-positivos nos modelos anteriores, tais como testes do campo aberto e rota Rod. Os resultados demonstraram que a Riparina IV possui uma atividade antinociceptiva no modelo de nocicepÃÃo visceral induzida por Ãcido acÃtico. A Riparina IV nÃo demonstrou atividade no modelo de nocicepÃÃo tÃrmica da placa quente. O prÃ-tratamento com a Riparina IV reduziu significativamente a nocicepÃÃo inflamatÃria induzida pela segunda fase da formalina, porÃm nÃo alterou a nocicepÃÃo neurogÃnica induzida pela primeira fase do teste da formalina. Os animais prÃ-tratados com a Riparina IV tambÃm exibiram uma reduÃÃo significativa na hipernocicepÃÃo mecÃnica induzida pela carragenina. Em relaÃÃo à participaÃÃo dos receptores de potencial transitÃrio (TRP), a Riparina IV demonstrou atividade nos modelos de nocicepÃÃo induzida pela administraÃÃo de capsaicina e mentol, porÃm nÃo apresentou atividade na nocicepÃÃo induzida por cinamaldeÃdo. TambÃm reduziu a nocicepÃÃo induzida pela administraÃÃo intraplantar de glutamato. Para o estudo dos mecanismos de aÃÃo da Riparina IV foi utilizada somente a dose de 50 mg/kg da substÃncia. Na avaliaÃÃo da participaÃÃo dos canais de potÃssio ATP-dependentes, o prÃ-tratamento com glibenclamida nÃo foi capaz de reverter a aÃÃo antinociceptiva da Riparina IV, descartando-se o seu envolvimento; da mesma forma, o prÃ-tratamento com ioimbina, um antagonista α2-adrenÃrgico, e pCPA, um depletor das reservas de serotonina, tambÃm nÃo foram capazes de reverter tal aÃÃo, nÃo havendo envolvimento com o mecanismo de aÃÃo da Riparina IV. O prÃ-tratamento com L-arginina, um precursor do Ãxido nÃtrico, reverteu a aÃÃo antinociceptiva da Riparina IV, sugerindo, em parte, a participaÃÃo da via do Ãxido nÃtrico no seu mecanismo de aÃÃo. Os resultados mostraram que essa substÃncia nÃo alterou a atividade locomotora no teste do campo aberto, nem diminuiu o nÃmero de quedas no teste do rota Rod, descartando a possibilidade de haver sedaÃÃo ou incoordenaÃÃo motora por parte da Riparina IV. Em sÃntese, os resultados demonstraram que a Riparina IV possui uma atividade em modelos animais de nocicepÃÃo, possivelmente envolvendo os receptores TRPV1, TRPM8, glutamatÃrgicos e a via do Ãxido nÃtrico. / Riparin IV, an alkamide synthesized from Aniba riparia, was tested in standard animal models of pain, as well as the possible mechanisms of action involved. It was used Swiss mice (20-30g), and Riparin IV was administred acutely in all tests, at the doses of 25 and 50 mg/kg, by gavage. It was used the tests of abdominal writhing induced by acetic acid, hot plate test, formalin test, mechanical hypernociception induced by carrageenan, nociception test induced by capsaicin, cinnamaldehyde and menthol, nociception test induced by glutamate, as well as models of behavior that ruled out the possibility of a muscle relaxing activity or induce false-positive results in previous models, such as the open field test and the rota Rod test. The results showed that Riparin IV has an antinociceptive activity at the model of visceral nociception induced by acetic acid. Riparin IV did not show any activity at the hot plate thermal nociception model. Pretreatment with Riparin IV reduced significantly the inflammatory nociception induced at the second phase of formalin test, but did not alter the neurogenic nociception induced at the first phase of formalin test. The animals pretreated with Riparin IV also exhibited a significant reduction at the mechanical hypernociception induced by carrageenan. Related to the participation of the Transient Potential Receptors (TRP), Riparin IV showed an activity at the models of nociception induced by capsaicin and menthol, but did not show any activity at the nociception induced by cinnamaldehyde. Also reduced the nociception induced by administration of glutamate at the rind paw. To study the mechanisms of action of Riparin IV, it was used only the dose of 50 mg/kg of the substance. At the evaluation of participation of the ATP-dependent potassium channels, pretreatment with glibenclamide was not able to reverse the antinociceptive action of Riparin IV, discharging its involvment; at the same way, pretreatment with yohimbine, an a2-adrenergic antagonist, and pCPA, a depletor of the serotonin reservations, were not able of reverse such action, not having any involvement with the mechanism of action of Riparin IV. Pretreatment with L-arginine, a precursor of Nitric Oxide, reversed the antinociceptive action of Riparin IV, suggesting, in part, the participation of nitric oxide pathway at the mechanism of action. The results showed that this substance did not alter the locomotor activity at the open field test, neither diminished the number of falls at the rota Rod test, discharging the possibility of sedation or incoordination by Riparin IV. In summary, the results showed that Riparin IV has an action in animal models of nociception, possibly involving the receptors TRPV1, TRPM8, glutamatergic receptors and the nitric oxide pathway.

No description available.

La détection de la température externe par la peau est le point de départ de nombreuses adaptations cellulaires et comportementales permettant de maintenir notre température interne constante. Selon ce concept, les fibres sensorielles cutanées sont les seuls récepteurs sensoriels de la peau pour la détection de la température. Plusieurs canaux ioniques activés directement par des températures chaudes ou froides ont été identifiés, ce sont les canaux TRPs. Le froid peut-il modifier le fonctionnement des organes du toucher?Nous montrons chez l’homme et la souris que les cellules de Merkel (CMs), qui sont les cellules tactiles des complexes de Merkel, peuvent être activées par le froid. Chez les souris dépourvues du canal TRPM8 (KO M8) la réponse au froid des CMs diminue. Le BCTC et le M8B, 2 bloqueurs du canal TRPM8, diminuent également la réponse au froid des CMs. Pour déterminer l’impact de cette sensibilité au froid sur la performance tactile, nous avons enregistré les variations de l’activité nerveuse des récepteurs de Merkel chez les souris WT et KO M8. Un froid modéré (20°C) appliqué sur la peau diminue le train de potentiels d’action issu d’un récepteur de Merkel stimulé mécaniquement. A 20°C ni le seuil de déclenchement des potentiels d’action, ni le train de potentiels d’action en réponse à une stimulation électrique ne sont modifiés. En revanche chez les souris KO M8 cette réponse mécanique tactile n’est plus diminuée. Ce résultat montre pour la première fois qu’une cellule non nerveuse de la peau, la cellule de Merkel, contient un récepteur au froid, le canal TRPM8, qui ajuste l’activité des récepteurs de Merkel lors d’une stimulation tactile. / In the skin, Merkel cells (Mcs) are connected to keratinocytes and A sensory nerve fibers and the complexes works as a slow adaptive mechanoreceptor (SA1 receptor). We observe that cooling human and mouse Merkel cells to 15°C increases intracellular Ca2+ ions concentration. The TRPM8 agonist’s provoke intracellular Ca2+ increases. The responses to cooling and TRPM8 agonist’s are reduced in absence of extracellular Ca2+ ions, by the TRPM8 antagonist’s and in KO M8 mouse. These results show that MCs sense cooling through TRPM8 channels. We hypothesize that cooling sensitivity modulate mechano-transduction and we investigate the modulation of SA1 response using the skin nerve and microneurography techniques in mouse and human, respectively. In mouse, cooling the skin at 22°C reduces the frequency of the SA1 discharge, without modifying the nerve conduction. This reduction disappeared in KO M8 mouse. These results suggest that MCs activity reduced the discharge of SA1 receptor at mild fresh temperature, anticipating effect of lower temperature on A nerve fiber excitability.This study is the first report about the sensitivity of MCs to cold temperature and its consequences on the SA1 receptor activity in mouse and human. We conclude that cold sensitivity of Merkel cells mediated by TRPM8 regulates the SA1 mechanical response, particularly at mild fresh temperature, when the nerve conduction is not significantly modified by cold. This is the first description of an active inhibitory process, driven by a TRP channel, during sensory transduction in the skin.

Das Prostatakarzinom ist die häufigste Krebserkrankung des Mannes. Bei den zu Tode führenden Tumoren wird es im Jahre 2003 nach dem Bronchialkarzinom an 2. Stelle stehen. Diese Inzidenz zeigt, dass dringend neue diagnostische Marker und therapeutische Zielgene zur Behandlung von Prostatakrebs benötigt werden. Ziel dieser Dissertation war es, mit Hilfe der DNA-Chiptechnologie neue tumorrelevante Gene für eine Small-Molecule- und Antikörper-Basierte Therapie des Prostatakarzinoms zu identifizieren. Auf einen proprietären Tumor-Chip der Firma metaGen Pharmaceuticals GmbH wurde mikrodissektiertes Normal- und korrespondierendes Tumorgewebe von 52 Prostatatumorpatienten hybridisiert. Mit Hilfe bioinformatischer Analysen der Chipergebnisse konnte das Gen TRPM8 identifiziert werden, das in Prostatatumoren in mehr als 56% der Patienten überexprimiert ist. Northern-Blot, Dot-Blot und Chipexperimente zeigten, dass TRPM8 ungewöhnlich gewebespezifisch exprimiert wird. In mehr als 400 getesteten Tumorpatienten und in 23 Normalgeweben wurde TRPM8 ausschließlich in der Prostata und neuroendokrinen Tumoren nachgewiesen. TRPM8 gehört zur Familie der Transient Receptor Potential Channel Proteins. Es konnte hier erstmals in Fluoreszenz-Resonanz-Energie-Transfer-Experimenten (FRET) gezeigt werden, dass TRPM8 Multi-Homomere bildet. Dies wurde bisher nur für Kanäle anderer TRP-Subfamilien (TRPV und TRPC) gezeigt. Weiterhin konnten erstmals mehrere Spleißvarianten von TRPM8 identifiziert werden. Quantitative RT-PCR Experimente zeigten, dass diese noch stärker in Prostatatumoren überexprimiert sind als TRPM8 selbst. Des Weiteren wurde ein neues Gen auf dem DNA-Gegenstrang von TRPM8 entdeckt, das mit Exon 11 von TRPM8 100% komplementär ist und an der Regulation von TRPM8 beteiligt sein könnte. Der Promotor von TRPM8 wurde durch eine in silico Analyse identifiziert und in vitro bestätigt. Obwohl eine starke androgenabhängige Expression von TRPM8 in LNCaP Zellen gezeigt werden konnte, wurden keine Bindungsstellen für androgenabhänginge Elemente gefunden. Allerdings ließen sich drei Bindungsstellen des androgenregulierten Homeoboxgens NKX3.1 identifizieren. Die Ergebnisse dieser Arbeit zeigen, dass TRPM8 und seine Isoformen aufgrund ihrer Gewebspezifität ausgezeichnete Angriffspunkte für eine zielgerichtete Prostatakrebstherapie sind. / Prostate cancer is the most commonly diagnosed malignancy in men in the Western World. In 2003 malignancies of the prostate will be the second most common fatal cancer in men after lung cancer as estimated by the American Cancer Society. Despite the tremendous efforts made in the past to improve the treatment of prostate cancer patients, there is still an urgent need for new markers and therapeutic targets for medication. The aim of this thesis was the identification of new genes relevant in prostate cancer, which could be used in a small-molecule or antibody based therapy of prostate cancers. Microdissected matched prostate cancer and normal tissues of 52 prostate cancer patients were hybridized to a proprietary high density Cancer-Chip based on Affymetrix GeneChip technology. Using a bioinformatic analysis, it was possible to identify TRPM8, which was highly overexpressed in 56% of prostate cancer patients. Northern blot, dot blot and gene chip experiments revealed that TRPM8 expression is extremely tissue specific. Of 400 patients and 23 tissues tested, TRPM8 expression could only be detected in the prostate and neuroendocrine tumors. Functionally, the protein belongs to the transient receptor potential channel family of non-voltage gated proteins. It could be shown for the fist time that TRPM8 subunits form homomers using FRET technology. Molecular characterization of TRPM8 transcription revealed multiple splice forms of TRPM8. Further, it was possible to identify a new mRNA present on the opposite strand of TRPM8, which was 100% complementary to exon 11 of TRPM8, thus it could possibly function as a regulatory RNA of TRP channel. All of these isoforms were found to be even higher overexpressed in prostate tumors than TRPM8 itself. The promoter region of TRPM8 was identified using in silico methods and confirmed in promoter reporter assays. Although a high androgen dependent transcriptional activation of TRPM8 could be found by RT-PCR in LNCaP cells, no androgen responsive elements was identifiable within the promoter region. On the other hand three binding sites for the androgen dependent homeobox gene NKX3.1 and several other homeobox genes were discovered. The results of the thesis show that TRPM8 and its isoforms are, due to their tissue specificity, ideal targets for the development of new therapeutic drugs for the treatment of prostate cancer.

Le développement des nanotechnologies dans le domaine médical a suscité un engouement considérable ces dernières années, notamment l’utilisation de nanoparticules pour la vectorisation de principes actifs. Les nanoparticules offrent des perspectives uniques pour la vectorisation et la délivrance de principes actifs qu’ils soient des gènes (thérapie génique),des anti-cancéreux (chimiothérapie) ou encore des agents photosensibilisateurs (photothérapie dynamique, TPD). Le défi majeur reste cependant l’acheminement des molécules thérapeutiques jusqu’à leur site d’action, tout en gardant leur intégrité ainsi que leur effet thérapeutique.L’axe de recherche de cette thèse est l’utilisation des nanocapsules lipidiques comme plateforme multifonctionnelle pour la délivrance de principes actifs. Un des objectifs étant le développement de nanocapsules lipidiques, stables de point de vue physico-chimique, et fonctionnalisées avec du polyéthylèneimine capables de délivrer efficacement un plasmide ADN et un anti-cancéreux (paclitaxel) dans le cadre d’une thérapie combinée. Les applications de ces nanovecteurs pour la transfectionde gènes et la vectorisation de chimiothérapeutique in vitro ont été réalisées.Par ailleurs, l’aptitude de nanocapsules lipidiques à vectoriser des agents photosensibilisants pour la thérapie photodynamique a été aussi étudiée in vitro, et les résultats ont montré que l’encapsulation de deux molécules de PS dans les nanocapsules permet une synergie de l’effet photodynamique tout en gardant les propriétés physico-chimiques de chaque PS. Enfin, l’encapsulation d’un agoniste au canal ionique TRPM8, le menthol, fait l’objet du dernier chapitre. L’étude par imagerie calcique du relargage de cette molécule lipophile in vitro a permis de confirmer le potentiel des NCL comme nanovecteurs de principes actifs. / The development of nanotechnology in the medical field has attracted considerable interest in recent years, including the use of nanoparticles for drug delivery. Nanoparticles offer unique opportunities for delivery of active drugs such as genes (gene therapy), anti-cancer (chemotherapy) or photosensitizers (photodynamic therapy, PDT). The major challenge, however, remains the delivery of therapeutic molecules to their site of action while keeping their integrity and their therapeutic effect.The research focus of this thesis is the use of lipid nanocapsules as a multifunctional platform for the delivery of drugs. One goal is the development of stable lipid nanocapsules, functionalized with polyethyleneimine and capable of effectively delivering a plasmid DNA and an anti-cancer (paclitaxel) as part of a combination therapy. The applications of these nanocarriers for transfection and delivery of chemotherapeutic were performed in vitro.Moreover, the ability of lipid nanocapsules to encapsulate photosensitizers for photodynamic therapy has been studied in vitro, and the results showed that the encapsulation of two molecules of PS in the nanocapsules allows a synergy photodynamic effect while protecting the PS from photo degradation.Finally, encapsulating an ion channel TRPM8 agonist (menthol) is the subject of the last chapter. The study by calcium imaging of the release of this lipophilic molecule in vitro confirmed the potential of lipid nanocapsules as nanocarriers of drugs.

abstract: All organisms need to be able to sense and respond to their environment. Much of this process takes place via proteins embedded in the cell membrane, the border between a living thing and the external world. Transient receptor potential (TRP) ion channels are a superfamily of membrane proteins that play diverse roles in physiology. Among the 27 TRP channels found in humans and other animals, TRP melastatin 8 (TRPM8) and TRP vanilloid 1 (TRPV1) are the primary sensors of cold and hot temperatures, respectively. They underlie the molecular basis of somatic temperature sensation, but beyond this are also known to be involved in body temperature and weight regulation, inflammation, migraine, nociception, and some types of cancer. Because of their broad physiological roles, these channels are an attractive target for potential therapeutic interventions. This dissertation presents experimental studies to elucidate the mechanisms underlying TRPM8 and TRPV1 function and regulation. Electrophysiology experiments show that modulation of TRPM8 activity by phosphoinositide interacting regulator of TRP (PIRT), a small membrane protein, is species dependent; human PIRT attenuates TRPM8 activity, whereas mouse PIRT potentiates the channel. Direct binding experiments and chimeric mouse-human TRPM8 channels reveal that this regulation takes place via the transmembrane domain of the channel. Ligand activation of TRPM8 is also investigated. A mutation in the linker between the S4 and S5 helices is found to generally decrease TRPM8 currents, and to specifically abrogate functional response to the potent agonist icilin without affecting icilin binding. The heat activation thermodynamics of TRPV1 are also probed using temperature-controlled electrophysiology. The magnitude of the gating enthalpy of human TRPV1 is found to be similar to other species reported in the literature. Human TRPV1 also features an apparent heat inactivation process that results in reduced heat sensitivity after exposure to elevated temperatures. The work presented in this dissertation sheds light on the varied mechanisms of thermosensitive TRP channel function and regulation. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019

Breast cancer is the most prevalent cancer type among Canadian women. Breast cancers originate from the malignant transformation of mammary epithelial cells, which causes them to adopt an uncontrolled cell proliferation phenotype. My research suggests that the activity of specific ion channels (KV10.1, KCa3.1 and TRPM8) contribute to the proliferation of MCF-7 cells, a cell line commonly used to study breast cancer in vitro. Pharmacologically inhibiting the activities of KV10.1 or KCa3.1 channels decreased basal, but not estrogen-stimulated [3H]-thymidine incorporation, demonstrating that these channels contribute to MCF-7 cell proliferation. One way K+ channel activity is hypothesized to control cell proliferation is via regulation of membrane potential-dependent Ca2+ influx. Inhibition of KCa3.1 but not KV10.1 channel activity resulted in a membrane potential-dependent decrease in basal Ca2+ influx, suggesting that the way in which KCa3.1 channels contribute to cell proliferation is via regulating Ca2+ influx. In addition, my research also demonstrated that TRAM-34 increased or decreased cell proliferation depending on the concentration used and mitogenesis by TRAM-34 was blocked by estrogen receptor antagonists. TRAM-34 increased progesterone receptor mRNA expression, decreased estrogen receptor-alpha mRNA expression and reduced the binding of radiolabelled estrogen to estrogen receptor protein, in each case mimicking the effects of estrogen. Our finding that TRAM-34 is able to activate the estrogen receptor suggests a novel action of this supposedly specific K+ channel inhibitor and raises concerns of interpretation in its use. TRPM8 channels were also identified in MCF-7 cells, where they appeared to be important Ca2+ entry pathways. Inhibiting the activity of TRPM8 pharmacologically, as well as knocking down TRPM8 mRNA expression decreased cell proliferation, indicating that TRPM8 also contributed to MCF-7 cell proliferation. In conclusion, my research demonstrates that the activities of KV10.1, KCa3.1 and TRPM8 channels contribute to basal breast cancer cell proliferation. These findings suggest that the activity of specific ion channels may be potential targets for future therapeutic agents to treat breast cancer.

Made available in DSpace on 2015-05-14T12:59:31Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3977582 bytes, checksum: 6a5b8750e2be83e492617d4f9954fa26 (MD5) Previous issue date: 2011-02-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Introduction: The Transient Receptor Potential (TRP) superfamily of cation channels is remarkable since it displays greater diversity in activation mechanisms, and are targets for plant-derived compounds. Aim: To investigate the role of TRP channels in the vasorelaxant response of rotundifolone in the superior mesenteric artery from Lyon Normotensive (LN) rats. Methods and Results: Endothelium-denuded artery rings were suspended by platinum hooks for isometric tension recordings. In nominally free-Ca2+ medium, the rings were submitted to successive phenylephrine (Phe) contractions to deplete Ca2+- stores and contracted to CaCl2 (10-2 M). The maximum response (MR) of CaCl2-contractions in presence of nifedipine (10-6 M) (MR = 31.66 ± 2.27 %) were significantly attenuated in the presence of nifedipine plus rotundifolone (3 x 10-4 and 3 x 10-3 M) (MR = 9.30 ± 2.38 and 1.12 ± 0.31 %) or nifedipine plus menthol (10-4 and 10-3 M) (MR = 10.96 ± 1.34 and 1.52 ± 0.82 %). Rotundifolone caused relaxation of vessels pre-contracted with Phe (MR = 100.32 ± 3.88 %; pD2 = 3.59 ± 0.04, n = 6). The vasorelaxant effect induced by rotundifolone was significantly atenuated in the presence of Gd3+ (10-4 M) (MR = 83.74 ± 5.71 %; pD2 = 3.15 ± 0.06); Gd3+ (2.25 x 10-5 or 2 x 10- 6 M) (pD2 = 3.18 ± 0.06 and 3.32 ± 0.03 %) or BCTC (MR = 76.30 ± 2.15 %; pD2 = 3.46 ± 0.04), but no in the presence of ruthenium red, La3+ or Mg2+, nor after TRPV1 desensitization with capsaicin. Menthol caused relaxation of vessels pre-contracted with Phe (MR = 105.07 ± 3.07 %; pD2 = 3.72 ± 0.02). The vasorelaxant effect induced by menthol was significantly potentiated in the presence of ruthenium red (10-5 M), a non-selective TRP channels blocker (pD2 = 4.12 ± 0,04, n = 6). Also, the vasorelaxant response of menthol was significantly attenuated in the presence of La3+ (8 x 10-5 M), non-selective TRP channels blocker (MR = 89.05 ± 1.61 %); Mg2+ (2.25 x 10-3 M), TRPM3, 6 and 7 selective blocker (MR = 90.76 ± 2.94 %); Gd3+ (10-4 M), TRPV4, TRPC1, 3 and 6, TRPM3 and 4 channels blocker (MR = 73.82 ± 5.44 %); Gd3+ (2.25 x 10-5 M), TRPC3 and 6, TRPV4 channels blocker (MR = 88.04 ± 2.33 %); Gd3+ (2 x 10- 6 M), TRPC6 selective blocker (MR = 89,30 ± 3,61 %) or BCTC (2 x 10-6 M), TRPM8 and TRPV1 channels blocker (MR = 66.77 ± 6.05 %), and after TRPV1 desensitization with capsaicin (10-5 M) (RM = 88.96 ± 4.50). The basal tension was reduced by change in the thermostat temperature from 37 ºC to 25ºC and 18ºC (MR = 21.15 ± 0.78 and 28.84 ± 1.03 %). This response was significantly potentiated by rotundifolone (3 x 10-3 M) (MR = 28.01 ± 1.81 and 38.45 ± 1.98 %) or menthol (10-3 M) (MR = 29.87 ± 1.25 and 43.03 ± 2.22 %). In the way similar to menthol, the effects induced by rotundifolone were attenuated in free-Ca2+ medium plus EGTA (MR = 20.42 ± 1.97 and 30.90 ± 2.58 %) or in the presence of BCTC (MR = 17.05 ± 1.94 and 26.48 ± 3.39 %), but not when the vessels were pre-treated with ruthenium red or capsaicin. The RNAm and the protein of the TRPM8 channel are expressed in the superior mesenteric artery from LN rats. Conclusions: These data suggest that rotundifolone induces concentration-dependent relaxation in the mesenteric artery due to inhibition of ROC and SOC channels (probably TRPC1 and TRPC6) and activation of TRPM8 channels. / Introdução: A superfamília Potencial Receptor Transiente (TRP) de canais catiônicos se destaca por exibir uma grande diversidade de mecanismos de ativação, e são alvos de compostos derivados de plantas. Objetivo: Investigar o papel de canais TRP na resposta vasorrelaxante de rotundifolona em artéria mesentérica superior de ratos Normotenso de Lyon (LN). Métodos e Resultados: Anéis de artéria sem endotélio foram suspensos em hastes metálicas para registro de tensão isométrica. Em meio nominalmente sem Ca2+, os anéis foram submetidos a contrações sucessivas com FEN para depleção dos estoques de Ca2+ e contraídos com CaCl2 (10-2 M). O efeito máximo (Emáx) das contrações com CaCl2 na presença de nifedipino (10-6 M) (Emáx = 31,66 ± 2,27 %) foi significativamente atenuado na presença de nifedipino mais rotundifolona (3 x 10-4 e 3 x 10-3 M) (Emáx = 9,30 ± 2,38 e 1,12 ± 0,31 %) e nifedipino mais mentol (10-4 e 10-3 M) (Emáx = 10,96 ± 1,34 and 1,52 ± 0,82 %). Rotundifolona causou relaxamento de vasos pré-contraídos com FEN (Emáx = 100,32 ± 3,88 %; pD2 = 3,59 ± 0,04, n = 6). O efeito vasorrelaxante induzido por rotundifolona foi signigficativamente atenuado na presença de Gd3+ (10-4M) (Emáx = 83,74 ± 5,71 %; pD2 = 3,15 ± 0,06); Gd3+ (2,25 x 10-5 ou 2 x 10-6 M) (pD2 = 3,18 ± 0,06 e 3,32 ± 0,03 %) ou BCTC (Emáx = 76,30 ± 2,15 %; pD2 = 3,46 ± 0,04), mas não na presença de vermeho de rutênio, La3+ or Mg2+, nem após dessensibilização do TRPV1 com capsaicina. Mentol também causou o relaxamento de vasos pré-contraídos com FEN (Emáx = 105,07 ± 3,07 %; pD2 = 3,72 ± 0,02). O efeito vasorrelaxante induzido por mentol foi significativamente potencializado na presença de vermelho de rutênio (10-5 M), um bloqueador não seletivo de canais TRP (pD2 = 4,12 ± 0,04, n = 6) e significativamente atenuada na presença de La3+ (8 x 10-5 M), bloqueador não seletivo de canais TRP (Emáx = 89,05 ± 1,61 %); Mg2+ (2,25 x 10-3 M), bloqueador seletivo dos canais TRPM3, 6 e 7 (Emáx = 90,76 ± 2,94 %); Gd3+ (10-4 M), bloqueador de canais TRPV4, TRPC1, 3 and 6, TRPM3 and 4 (Emáx = 73,82 ± 5,44 %); Gd3+ (2,25 x 10-5 M), bloqueador de canais TRPC3 and 6, TRPV4 (Emáx = 88,04 ± 2,33 %); Gd3+ (2 x 10-6 M), bloqueador seletivo do TRPC6 (Emáx = 89,30 ± 3,61 %) ou BCTC (2 x 10-6 M), bloqueador dos TRPM8 e TRPV1 (Emáx = 66,77 ± 6,05 %), e após a dessensibilização do TRPV1 com capsaicina (10-5 M) (Emáx = 88,96 ± 4,50). A tensão basal foi reduzida por mudança na temperature do banho de 37 ºC para 25ºC e 18ºC (Emáx = 21,15 ± 0,78 e 28,84 ± 1,03 %). Essa resposta foi significativamente potencializada por rotundifolona (3 x 10-3 M) (Emáx = 28,01 ± 1,81 e 38,45 ± 1,98 %) ou mentol (10-3 M) (Emáx = 29,87 ± 1,25 e 43,03 ± 2,22 %). Semelhante ao mentol, os efeitos induzidos por rotundifolona foram atenuados em meio sem Ca2+ mais EGTA (Emáx = 20,42 ± 1,97 e 30,90 ± 2,58 %) ou na presença de BCTC (Emáx = 17,05 ± 1,94 e 26,48 ± 3,39 %), mas não quando os vasos foram pré-tratados com vermelho de rutênio ou capsaicina. O RNAm e a proteína do canal TRPM8 são expressos em artéria mesentérica de ratos LN. Conclusões: Esses dados sugerem que rotundifolona induz relaxamento dependente de concentração em artéria mesentérica devido à inibição de canais ROC e SOC (provavelmente TRPC1 e TRPC6) e ativação de canais TRPM8.