Type 2 diabetes (T2D) is a health problem throughout the world. In 2010, there were nearly 230 million individuals with diabetes worldwide and it is estimated that in the economically advanced countries the cases will increase about 50% in the next twenty years. Insulin resistance is one of major features in T2D, which is also a risk factor for metabolic and cardiovascular complications. Epidemiological and animal studies have shown that the consumption of vegetables and fruits can delay or prevent the development of the disease, although the underlying mechanisms of these effects are still unclear. Brassica species such as broccoli (Brassica oleracea var. italica) and nasturtium (Tropaeolum majus) possess high content of bioactive phytochemicals, e.g. nitrogen sulfur compounds (glucosinolates and isothiocyanates) and polyphenols largely associated with the prevention of cancer. Isothiocyanates (ITCs) display their anti-carcinogenic potential by inducing detoxicating phase II enzymes and increasing glutathione (GSH) levels in tissues. In T2D diabetes an increase in gluconeogenesis and triglyceride synthesis, and a reduction in fatty acid oxidation accompanied by the presence of reactive oxygen species (ROS) are observed; altogether is the result of an inappropriate response to insulin. Forkhead box O (FOXO) transcription factors play a crucial role in the regulation of insulin effects on gene expression and metabolism, and alterations in FOXO function could contribute to metabolic disorders in diabetes. In this study using stably transfected human osteosarcoma cells (U-2 OS) with constitutive expression of FOXO1 protein labeled with GFP (green fluorescent protein) and human hepatoma cells HepG2 cell cultures, the ability of benzylisothiocyanate (BITC) deriving from benzylglucosinolate, extracted from nasturtium to modulate, i) the insulin-signaling pathway, ii) the intracellular localization of FOXO1 and iii) the expression of proteins involved in glucose metabolism, ROS detoxification, cell cycle arrest and DNA repair was evaluated. BITC promoted oxidative stress and in response to that induced FOXO1 translocation from cytoplasm into the nucleus antagonizing the insulin effect. BITC stimulus was able to down-regulate gluconeogenic enzymes, which can be considered as an anti-diabetic effect; to promote antioxidant resistance expressed by the up-regulation in manganese superoxide dismutase (MnSOD) and detoxification enzymes; to modulate autophagy by induction of BECLIN1 and down-regulation of the mammalian target of rapamycin complex 1 (mTORC1) pathway; and to promote cell cycle arrest and DNA damage repair by up-regulation of the cyclin-dependent kinase inhibitor (p21CIP) and Growth Arrest / DNA Damage Repair (GADD45). Except for the nuclear factor (erythroid derived)-like2 (NRF2) and its influence in the detoxification enzymes gene expression, all the observed effects were independent from FOXO1, protein kinase B (AKT/PKB) and NAD-dependent deacetylase sirtuin-1 (SIRT1). The current study provides evidence that besides of the anticarcinogenic potential, isothiocyanates might have a role in T2D prevention. BITC stimulus mimics the fasting state, in which insulin signaling is not triggered and FOXO proteins remain in the nucleus modulating gene expression of their target genes, with the advantage of a down-regulation of gluconeogenesis instead of its increase. These effects suggest that BITC might be considered as a promising substance in the prevention or treatment of T2D, therefore the factors behind of its modulatory effects need further investigation. / Diabetes mellitus Typ 2 stellt auf der ganzen Welt ein Gesundheitsproblem dar. Im Jahr 2010 waren annähernd 230 Millionen Personen weltweit an Diabetes erkrankt und innerhalb der nächsten 20 Jahre wird in industrialisierten Ländern eine Steigerung der Fälle um 50% erwartet. Eines der Hauptmerkmale des Typ 2 Diabetes ist die Insulinresistenz, die auch als Risikofaktor für metabolische und kardio-vaskuläre Komplikationen gilt. Epidemiologische Studien und Tierversuche haben ergeben, dass durch Verzehr von Gemüse und Obst eine Prävention oder Verzögerung der Entwicklung dieser Krankheit erreicht werden kann, jedoch sind die zugrunde liegenden Mechanismen dieser Effekte noch nicht aufgeklärt. Brassica Spezies wie Broccoli (Brassica oleracea var. italica) und Nasturtium (Tropaeolum majus) enthalten einen hohen Anteil an bioaktiven Pflanzen-inhaltsstoffen, wie z. B. stickstoff- und schwefelhaltige Verbindungen (Glukosinolate und Isothiocyanate) und Polyphenole, die bisher hauptsächlich mit der Prävention von Krebs assoziiert wurden. Isothiocyanate (ITCs) erreichen ihr antikanzerogenes Potential durch die Induktion von entgiftenden Phase II Enzymen und eine Anhebung der Glutathion (GSH)-Spiegel im Gewebe. Diabetes Typ2 geht einher mit einem Anstieg der Glukoneogenese und Triglycerid-Synthese, sowie einer Reduktion der Fettsäure-Oxidation in Verbindung mit erhöhten Spiegeln an reaktiven Sauerstoffspezies (ROS) insgesamt als Resultat einer unangemessenen Insulinantwort. Forkhead box O (FOXO) Transkriptionsfaktoren spielen eine wesentliche Rolle in der Regulation der Insulineffekte in Bezug auf die vermittelte Genexpression und den Metabolismus, wobei Veränderungen in der Funktion von FOXO zu metabolischen Entgleisungen im Diabetes beitragen können. In dieser Studie wurde unter Verwendung von stabil transfizierten humanen Osteosarkoma-Zellen (U-2 OS) mit konstitutiver Expression von GFP (grün fluoreszierendes Protein)-markiertem FOXO1 und humanen Hepatoma-Zellen (HepG2) die Wirkung von Benzylisothiocyanat (BITC), dessen Vorstufe Benzylglukosinolat aus Nasturtium isoliert wurde, in Zellkulturen evaluiert wie Modulationen der i) Insulin-Signal-Kaskade, ii) intrazellulären Lokalisation von FOXO1 und iii) Expression beteiligter Proteine am Glucose Metabolismus, der ROS Detoxifikation, Zellzyklus-Fixierung und DNA-Reparatur. BITC erzeugte oxidativen Stress und induzierte als Antwort darauf eine Translokation von FOXO1 aus dem Zytoplasma in den Zellkern antagonisierend zum Insulin-Effekt. Eine Stimultion mit BITC war in der Lage, die Expression von Enzymen der Gluconeogenese herunter zu regulieren, was als antidiabetogener Effekt betrachtet werden kann, eine antioxidative Resistenz durch Induktion der Mangan-Superoxid-Dismutase (MnSOD) und entgiftender Enzyme zu erzeugen, Autophagie zu modulieren durch Induktion von BECLIN1 und Herunterregulation des „mammalian target of rapamycin complex1 (mTORC1)-Stoffwechselwegs, den Zellzyklus zu fixieren und DNA-Reparatur zu induzieren durch Hochregulation des Cyclin- abhängigen Kinase- Inhibitors p21CIP und GADD45 (growth arrest and DNA damage repair). Mit Ausnahme des nuklearen Faktors (erythroid derived)-like2 (NRF2) und dessen Einfluss auf die Genexpression von Entgiftungsenzymen waren alle beobachteten Effekte unabhängig von FOXO1, Proteinkinase B (PKB/AKT) und der NAD-abhängigen Deacetylase Sirtuin-1 (SIRT1). Die gegenwärtige Studie liefert Anhaltspunkte dafür, dass Isothiocayanate neben dem antikanzerogenen Potential eine Rolle bei der Prävention von Typ 2 Diabetes spielen könnten. BITC-Stimulationen ahmen einen Fastenzustand nach, in dem kein Insulin-Signal ausgelöst wird, FOXO Proteine im Zellkern verbleiben und die Expression von Target-Genen modulieren, mit dem Vorteil einer Herunterregulation der Glukoneogenese anstelle seiner Zunahme. Diese Effekte legen nahe, dass BITC als vielversprechende Substanz zur Prävention und Behandlung von Typ 2 Diabetes angesehen werden könnte. Deshalb benötigen die Faktoren, die dessen modulatorische Effekte hervorrufen, weitere Untersuchungen.

Le rhabdomyosarcome de l'adulte est une tumeur rare au pronostic. Le présent travail propose d'étudier les caractéristiques cliniques et moléculaires et la prise en charge des adolescents et adultes atteints de rhabdomyosarcome ainsi que la possibilité de combinaison de thérapie ciblées sur lignées cellulaires in vitro. Nous avons anamysé rétrospectivement 239 patients âgés de 10 ans ou plus, atteints de rhabdomyosarcome au MD Anderson Cancer Center entre 1957 et 2003 et leur statut fusionnel pour PAX-FOXO1 par hybridation in situ en fluorescence. Trois lignées cellulaire de sarcome à petites cellules ont été soumises à des combinaisons de thérapies ciblées avec analyse de la viabilité. Les patients de plus de 50 ans avaient une survie globale à 5 ans de 13 % (médiane de survi à 1.7 ans) en dépit d'une maladie localisée. Approximativement 13 % des patients métastasiques de moins de 50 ans ont eu une survie prolongée de plus de 15 ans. L'utilisation d'une stratégie thérapeutique triple, intégrant chirurgie, chimiothérapie et radiothérapie était signifcativement associée à une survie prolongée. Auniveau molécualire, la présence du transcrit de fusio PAX3/7-FOXO1 était significativement liée à un risque accru de maladie métastatique. L'étude in vitro de thérapies ciblées a permis d'identifier la combinaison du vorinostat plus le 17DMAG associée à la doxorubicine comme ayant une meilleure efficacité. La prise en charge du rhabdomyosarcome de l'adolescent et de l'adulte semble souffrir d'une approche moins agressive comparée au rhabdomyosarcome pédiatrique. De plus, des combianaisons de thérapies ciblées peuvent être intégrées aux protocoles de chimiothérapies standards. / Rhabdomyosarcoma is a rare entity adult patient with unfavourable outcome. This work describes the clinical and molecular specificities of adolescent and adult type of rhabdomyosarcoma and investigates the optimal integration of targetd therapy combinations on small cell sarcoma cell lines in vitro. We retrospectively analyzed 239 patients, 10 years of age and greater, diagonsed withrhabdomyosarcoma at MD Anderson Cancer Center from 1957 trough 2003 and their PAX-FOXO1 fusion gene status by fluorescence in situ hybridization on tissues microarray. Three samll cell sarcoma cell lines were exposed to targetd agent combinations. PAtient with metastatic rhabdomyosarcoma were found to have a 18 % survival rate at 5 years from diagnosis with an 12 %survival past 15 years. This outcome was even poorer for patients over 50 of age, even with localized disease. Younger patients were more likely to receive multidisciplinary therapy than their older counterparts. The presence of PAX-FOXO1 tranlocation was significantly associated with a higher frequency of metastatic disease. The four agents with the exception of abacavir synergized two by two with each other in vitro but the triple combinations did not perform beter than the bitherapies. The dual therapies vorinostat 5HDAC inhibitor) plus 17-DMAG (Hsp90 inhibitor) added with doxorubicin achvied better results than dual or triple therapies. Adult patient with rhabdomyosarcoma present similar molecular and clinical characteristics compared pediatric patients but outcome decrease with age partly du to a less multimodal management. Moreover targeted combinations should be integrated to chemotherapy backbone.

L’efficacité de la réponse immunitaire adaptative repose tout particulièrement sur la motilité des lymphocytes T naïfs entre la circulation sanguine et les organes lymphoïdes secondaires, leur permettant ainsi de rencontrer un antigène spécifique. De nombreuses voies de signalisation sont impliquées dans ce phénomène. En particulier, les Rho GTPases y jouent un rôle central, par leur capacité à moduler le cytosquelette d’actine. Nous avons identifié la protéine Fam65b comme nouveau régulateur de la circulation des lymphocytes T. En effet, nous avons montré que la diminution de l’expression de Fam65b dans des LT primaires humains induit une augmentation de leur polarisation, leur adhésion et leur migration in vitro. Afin d’étudier son rôle dans un contexte plus physiologique, nous avons développé au laboratoire une souris Fam65b-/-, dans laquelle l’expression de Fam65b est supprimée dans le lignage T. Les lymphocytes T issus de ces souris présentent un contenu global en F-actine réduit, une plus grande quantité de L-sélectine et d’intégrines actives à leur surface, et une migration moins rapide et moins rectiligne que leurs équivalents WT. Nous n’avons pu observer, avec nos méthodes, aucune différence significative de polarisation, de migration in vitro ou d’entrée dans les organes lymphoïdes secondaires pour les LT Fam65b-/-. Nous avons identifié les Rho GTPases comme médiateurs de ces effets de Fam65b. Nous avons observé, en cytométrie de flux, que les niveaux de RhoA-GTP et de Rac-GTP sont plus élevés dans les LT murins Fam65b-/-, et que cela est aussi vrai pour RhoA-GTP dans les LT humains exprimant de faibles niveaux de Fam65b. Nous avons identifié, dans des expériences in vitro, le mécanisme par lequel Fam65b inhibe l’activité de RhoA, puisqu’il ralentit sa charge en GTP par les protéines GEF. Nous avons montré, par des techniques de biochimie, que l’activation de RhoA en aval d’une stimulation chimiokine est permise par la dissociation de RhoA et de Fam65b, probable conséquence de la phosphorylation de Fam65b. Cette dissociation a aussi été observée pour Fam65b et Rac1, mais les mécanismes mis en jeu restent à déterminer. D’autre part, l’expression de Fam65b est sous le contrôle du facteur de transcription FOXO1, connu pour son rôle dans le contrôle de l’écotaxie (homing) via la régulation de l’expression de molécules permettant l’entrée dans les ganglions lymphatiques. Fam65b, régulateur atypique de l’activité des Rho GTPases, représente donc un lien inédit entre la voie PI3K/FOXO1 et les Rho GTPases. / The motility of naive T lymphocytes between the blood and secondary lymphoid organs is essential to the efficiency of the adaptative immune response, and allows those cells to meet their cognate antigen. Numerous signaling pathways are involved in this phenomenon, such as Rho GTPases, modulators of the actin cytoskeleton. We have identified Fam65b as a new regulator of T lymphocytes recirculation. We have shown that a decrease of Fam65b expression in human primary T cells increases the morphological polarization, the adhesion and the in vitro migration of those cells. Looking for a more physiological model, we developed, in the lab, a Fam65b KO (Knock-Out) mouse, specific to the T lineage. In those animals, T cells showed decreased levels of F-actin, an increase in the display of L-selectin and integrins, and a slower and less straight migration, compared to WT (Wild-Type) T cells. On the other hand, we weren't able to see any significant differences in the morphological polarisation, the in vitro migration or the homing capacity of the Fam65b KO T cells. We have identified Rho GTPases as mediators of the effects of Fam65b. We showed, in flow cytometry, that the amount of RhoA-GTP and Rac-GTP are increased in the Fam65b KO cells. The RhoA-GTP levels are also increased in human primary T cells expressing low levels of Fam65b. We have identified, in in vitro experiments, that Fam65b slows down RhoA loading with GTP by its GEF proteins, thus inhibiting RhoA activity. Moreover, we showed that Fam65b dissociates from RhoA after chemokine stimulation of T cells, thus allowing RhoA activation. The phosphorylation of Fam65b is a probable cause to this phenomenon. Fam65b also dissociates from Rac1 in these conditions, although no mechanism is yet known. Furthermore, the transcription factor FOXO1 controls the expression of Fam65b. FOXO1 is also known to control the homing capacity of T cells, since it controls the expression of molecules involved in the entry of lymphocytes in the lymph nodes. Fam65b, an atypical regulator of Rho GTPases activity, thus represents a new connection between the PI3K/FOXO1 and the Rho GTPases pathways.

The existence of a robust, mature CD4 T cell population is essential in orchestration of an immune response. CD4 T cell activation is a result of antigenic stimulation of a unique cell pool that is normally resting. Termed “naïve”, these CD4 T cells lack effector function and are maintained long term in the periphery. Expression of key cell surface receptors and transcription factors dictates their ability to survive, home and differentiate into effector subsets. However, transcriptional regulation of these processes in naïve CD4 T cells is only partly characterized. Ikaros has been identified as a transcriptional activator and repressor of T cell lineage fate decisions and polarization into T helper cell subsets. In this dissertation, a role for Ikaros in regulation of naïve CD4 T cells is revealed as in its absence, cells exhibit decreased survivability, impaired migration to lymph nodes and failure to develop into induced regulatory T cells (iTreg). Defects are linked to decreased expression of IL- 7Rα, CD62L and Foxp3, respectively, all identified as targets of a transcription factor important in naïve CD4 T cell homeostasis, Foxo1. Analogous consequences on T cell survival, homing and differentiation have been reported for Foxo1- deficient T cells. Furthermore, results from Western blot and qRT-PCR analyses of protein and mRNA from Ikaros null (IK-/-) CD4 T cells demonstrated decreased Foxo1 levels, prompting investigation into mechanisms for regulation of Foxo1 expression by Ikaros. Retroviral transductions were performed, beginning with delivery of Ik-7 and Foxo1-shRNA, interfering with Ikaros and Foxo1 activity in wild type cells, respectively. Similar decreases in CD62L and IL-7Rα levels indicated the need for both Ikaros and Foxo1 for expression. However, re-introduction of either Foxo1 or Ikaros into IK-/- CD4 T cells highlighted differential modes of Ikaros and Foxo1 regulation for IL-7Rα and CD62L expression. qRT-PCR analyses revealed increased levels of Foxo1 mRNA with Ikaros transduction into IK-/- CD4 T cells. My studies have thereby identified Ikaros to be the first transcriptional regulator of Foxo1 gene expression in ensuring survival, homing and iTreg differentiation of the naïve CD4 T cell compartment.

Type 2 diabetes mellitus (T2DM) is a disease of multi-complications affecting more than 20 million US adults. Hyperglycemia is the classic clinical feature of diabetes, and uncontrolled hyperglycemia leads to deadly health complications. Thus, control of blood glucose represents a major goal for diabetics. Human and rodent studies revealed another clinical feature; diabetics have low tissue and plasma levels of polyunsaturated fatty acids (PUFAs), an effect often attributed by impaired endogenous PUFA synthesis. In this context, rodent studies have revealed a possible link between PUFA synthesis and high fat diet induced obesity and diabetes. These studies have shown that obese and diabetic mice have low hepatic expression and activity of fatty acid elongase-5 (Elovl5), a key enzyme involved in the PUFA synthesis pathway. Over-expression of Elovl5 in livers of chow fed C57BL/6J mice decreased fasting blood glucose and increased hepatic glycogen contents. Therefore, my hypothesis for the current work is that elevated hepatic Elovl5 activity or improved hepatic PUFA synthesis will improve systemic and hepatic carbohydrate metabolism in a mouse model of diet induced obesity and diabetes. Using a recombinant adenovirus approach, we over-expressed Elovl5 in livers of high fat diets (60% calories derived from fat as lard, Research Diets) induced obese-diabetic mice. Elevated hepatic Elovl5 activity increased hepatic and plasma C��������������� PUFA contents, reduced homeostatic model assessment for insulin resistance (HOMA-IR), improved glucose tolerance and lowered fasting blood glucose to euglycemic levels in obese-diabetic mice. The mechanism for insulin mimetic effect of Elovl5 on hepatic glucose metabolism was correlated with increased phosphorylation of Akt-S��������, FoxO1-S�������� and PP2Acat-Y��������, decreased nuclear content of FoxO1, and decreased expression of Pck1 and G6Pase; important enzymes involved in gluconeogenesis (GNG) and glucose production. Phospho-FoxO1 is excluded from nuclei, ubiquitinated and degraded by the proteasome. Loss of nuclear FoxO1, due to its increased phosphorylation, leads to the reduction in the expression of key genes involved in gluconeogenesis, i.e., Pck1 and G6Pase. Using obese-diabetic mice liver extracts and HepG2 cells, I established that Elovl5 uses two mechanisms to control hepatic GNG. The first mechanism involves Elovl5 mediated increased Akt2-S�������� and FoxO1-S�������� phosphorylation via mTORC2-rictor pathway. The second mechanism involves Elovl5 mediated attenuation of de-phosphorylation of FoxO1 via PP2A inhibition. Together, these mechanisms increase FoxO1 phosphorylation status in livers of fasted obese-diabetic mice, lower hepatic FoxO1 nuclear abundance and FoxO1 capacity to sustain transcription of GNG genes and inhibit GNG and restore blood glucose levels in fasted obese-diabetic mice. Results of these studies showed Elovl5 corrected high fat diet induced hyperglycemia in C57BL/6J mice, identified the molecular mechanism of Elovl5 control of GNG and explained how Elovl5 or PUFA synthesis controls GNG. Therefore, these findings will be eventually helpful in developing a therapeutic target to combat hyperglycemia. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from July 27, 2012 - July 27, 2013

No description available.

Glucocorticoids are widely used in clinical therapy. However, they cause adverse effects including insulin resistance and Type 2 diabetes, which are characterised by decreased glucose transport into the muscles and fat. How glucocorticoids inhibit glucose transport remains unclear. Insulin stimulates glucose uptake via the insulin receptor substrate (IRS)-1 / phosphoinositide-3-kinase (PI3K) / protein kinase B (AKT) pathway and promotes the redistribution of GLUT4 from intracellular storage compartments to the plasma membrane (PM). Insulin-stimulated phosphorylation of AKT substrate of 160 kDa (AS160), a Rab-GTPase activating protein is downstream of AKT and appears to be essential for exposure of GLUT4 at the PM and glucose uptake. This is mediated through the association of phosphorylated AS160 (at the key residue T642) with 14-3-3 in the cytosol. The mildly insulin-responsive GLUT1 mediates basal glucose uptake in adipocytes. It is also subject to regulated trafficking like GLUT4. This study aimed to determine the level at which glucocorticoids inhibit glucose uptake in adipocytes. Effects of the synthetic glucocorticoid dexamethasone (Dex) and the natural glucocorticoid cortisol, on GLUT1 and GLUT4 function were examined. Candidates for the glucocorticoid-mediated inhibition of GLUT1- and GLUT4-mediated glucose uptake were investigated. These were glycogen synthase kinase (GSK) 3β (an AKT substrate) for GLUT1-mediated glucose transport; and adaptor protein containing PH domain, PTB domain, and leucine zipper motif (APPL)-1 (an AKT-interacting protein) and AS160 for GLUT4-mediated glucose transport. Dex and cortisol significantly decreased basal glucose uptake by 50% (p<0.05) in SGBS and 3T3-L1 adipocytes. Similarly, insulin-stimulated glucose uptake was decreased by 50% (p<0.001 for SGBS; p<0.05 for 3T3-L1) and 30% (p<0.05 for both) at 1 nM and 100 nM insulin respectively. Similar results were observed with differentiated primary human preadipocytes and human adipose explants. Dex-mediated inhibition of basal glucose uptake was limited to insulin-sensitive cell types implying that glucocorticoids may regulate GLUTs at steps common to GLUT1 and GLUT4 trafficking. Dex-mediated reduction in glucose uptake correlated with the reduction in basal and insulin-stimulated expression of GLUT1 and GLUT4 to the PM without changes in total GLUT1/4 expression. Dex did not alter total expression or phosphorylation of proximal insulin-signalling molecules up to and including AKT but increased FOXO1 expression, and modified GSK3β-S9 phosphorylation. Dex did not alter total APPL1 expression or subcellular distribution. Dex significantly decreased 1nM-insulin stimulated AS160-T642 phosphorylation by 50% (p<0.05) in SGBS and 3T3-L1 adipocytes via the glucocorticoid repector (GR). This correlated with reduced AS160:14-3-3 interaction. Similar results were obtained for AS160-T642 basal phosphorylation. At 1nM insulin, AS160-T642 phosphorylation is maximal at sub-maximal glucose uptake, i.e. AS160 phosphorylation significantly contributes to glucose uptake. RU486 significantly prevented but did not fully abrogate the Dex-mediated reduction in glucose uptake suggesting additional Dex-induced defects. In conclusion, glucocorticoids inhibit glucose uptake at a level distal to AKT by GR-dependent mechanisms. A role for GSK3β or APPL1 in glucocorticoid-mediated inhibition of glucose uptake requires further investigations. FOXO1 represents a suitable candidate for mediating the Dex-induced defects. Of significance, perturbation in AS160-T642 phosphorylation contributes to Dex-mediated inhibition of glucose uptake. Thus, AS160 presents a novel therapeutic target in the improvement of glucocorticoid-mediated inhibition of glucose uptake.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13377号 / 論医博第2211号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 妹尾 浩, 教授 稲垣 暢也 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Irrespective of plentiful efforts to enhance primary prevention and early detection, the number of melanoma cases in the United States has increased steadily over the past 30 years, thus greatly affecting public health and the economy. We have investigated the effects of five novel aPKC inhibitors; 2-acetyl-1,3-cyclopentanedione (ACPD), 3,4-Diaminonaphthalene-2,7-disulfonic acid (DNDA), [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1T) along with its nucleoside analog 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocyte cell lines. Molecular docking data suggested that both ACPD and DNDA specifically bind to protein kinase C-zeta (PKC-ζ) and PKC-iota (PKC-ι) while both ICA-1 compounds specifically bind to PKC-ι, and ζ-Stat showed a high affinity towards PKC-ζ. Kinase activity assays were carried out to confirm these observations. Results suggest that PKC-ι is involved in melanoma malignancy than PKC-ζ. Both isoforms promote the activation of nuclear factor (NF)-κB and protein kinase B (AKT) thereby supporting survival and progression. In addition, we demonstrated that PKC-ι induced the metastasis of melanoma cells by activating Vimentin, and PKC-ι inhibition downregulated epithilial-mesencymal transition (EMT), while inducing apoptosis. Of note, PKC-ἱ specific inhibitors downregulated the expression of both PKC-ι and phosphorylated PKC-ι, suggesting that PKC-ι plays a role in regulating its own expression in melanoma. We also report the underlaying mechanisms of the transcriptional regulation of PKC-ι (PRKCI gene) expression in melanoma. c-Jun, interferon-stimulated gene factor 3 (ISGF3), paired box gene 3 (PAX3), early growth response protein 1 (EGR1) and forkhead box protein O1 (FOXO1), which bind on or near the promoter sequence of the PRKCI gene, were analyzed for their role in PKC-ι regulation in SK-MEL-2 and MeWo cell lines. We silenced selected transcription factors using siRNA, and the results revealed that the silencing of c-Jun and FOXO1 significantly altered the expression of PRKCI. The levels of both phosphorylated and total PKC-ι increased upon FOXO1 silencing and decreased upon c-Jun silencing, suggesting that c-Jun acts as an upregulator, while FOXO1 acts as a downregulator of PRKCI expression. We also used a multiplex ELISA to analyze multiple pathways other than NF-κB that were affected by treatment with PKC-ι inhibitor. The silencing of NF-κB p65 and PKC-ι by siRNA suggested that the regulation of PKC-ι expression was strongly associated with FOXO1. In addition, we observed a significant decrease in the mRNA levels of both interleukin (IL)-6 and IL-8, with a significant increase in the levels of IL-17E and intercellular adhesion molecule 1 (ICAM-1) upon the knockdown of expression of PKC-ι in both cell lines. This suggested that PKC-ι expression was affected by these cytokines in an autocrine manner. Overall, the findings of this study suggest that PKC-ι inhibition suppresses its own expression, diminishing oncogenic signaling, while upregulating anti-tumor signaling, thus rendering it an effective novel biomarker for use in the design of novel targeted therapeutics for melanoma.

No description available.