Mechanisms of IKBKE Activation in Cancer

Cancer is the second leading cause of death in the USA and it is expected to surpass heart diseases making it important to understand the underlying mechanisms of cancer. The efforts to target single signaling molecule showed little success in increasing the patient survival and it can be due to increased compensation for cell survival by alternative pathway activations. Hence comprehensive understanding of the alternative signaling pathways may help us treat cancer better. Chronic inflammation is attributed to increased risk of cancer and emerging studies show the growing importance of both canonical and non-canonical IκB kinases such as IKKα, IKKβ, IKBKE and TBK1 in human cancer pathogenesis. Initially identified as activator of NFκB pathway, IKBKE was shown to play an important oncogenic role by regulating multiple pathways downstream. Although IKBKE is implicated in tumorigenesis for over a decade, therapeutic targeting of this pathway has been a challenge. Recently, amlexanox and CYT387, which are in clinical trials for Type II diabetes and myeloproliferative disorders respectively, were identified as potential IKBKE inhibitors. In this study, we uncovered specific novel mechanisms of activation of IKBKE in tumor cells and the outcomes of targeting IKBKE pathway. Oncogenic mutations are a cause of several human malignancies. Mutations in EGFR are observed in 15% of non-small cell lung cancer patients. While cells expressing these mutations respond better to the first generation TKIs, patients become resistant to these inhibitors due to secondary mutations in EGFR. These mutations were shown to make EGFR constitutively active even in the absence of ligands. Direct targeting of EGFR with secondary mutations has been challenging as EGFR acquires novel mutations upon inhibitor treatment, which confer resistance to the EGFR-TKIs. Hence, it is important to improve our knowledge of the downstream signaling pathways of EGFR. Although PI3K, MEK signaling are well established, mutant EGFR was shown to activate several novel signaling pathways such as miRNA processing and autophagy that are implicated in resistance to EGFR-TKIs. Here, we show that IKBKE acts downstream of mutant EGFR to activate the NFκB and AKT pathways. In addition, we show that mutant EGFR but not wildtype EGFR can directly phosphorylate IKBKE at Tyrosine 153 and Tyrosine 179 residues that are important for activation of IKBKE kinase. We also found that the IKBKE/TBK1 inhibitor Amlexanox exhibits increased efficacy in inhibiting cell viability in NSCLC cells with activating EGFR mutations. Furthermore, we also found that IKBKE inhibitors activate the MAPK pathway, and EGFR-TKI resistant NSCLCs exhibit enhanced response to co-treatment with IKBKE inhibitors and MEK inhibitors. Similar to lung cancer, pancreatic cancer is a challenging disease due to lack of direct inhibitors of the KRas mutations that are observed in more than 95% of pancreatic cancer patients. IKBKE/TBK1 pathway is important for KRas signaling, but the efficacy of IKBKE inhibitors in pancreatic cancers is not well studied. Here, we show that IKBKE is an important target in pancreatic cancers that regulates pancreatic cell viability, cell migration and cancer stem cells. Importantly, we provide mechanistic insights into the effects of IKBKE inhibitors on specific signaling pathways. We found that IKBKE inhibition results in significantly increased expression of RTKs, such as ErbB3 and IGF1-R, which increases ERK1/2 activation. Our findings provide support for novel combination strategies for pancreatic cancer. Metastasis is a poor prognostic factor for ovarian cancer. Although patients with early stage ovarian cancer with no distal metastasis exhibit a 70% 5-year survival rate, Stage IV patients with distal metastasis exhibit only 20% 5-year survival rate. Hence, ongoing efforts are aimed at targeting the pathways that regulate metastasis in ovarian cancers. IKBKE is upregulated in ovarian cancer patients, and IKBKE expression is known to regulate the expression of several genes important for cell motility in ovarian cancers. IKBKE is also implicated in chemo-resistance in ovarian cancer, and siRNA knockdown of IKBKE increases sensitivity towards chemotherapy. However, the mechanistic role of IKBKE in chemo-resistance in ovarian cancer is not known. EphA2 is another well studied oncogene in ovarian cancer as 70% of ovarian cancer patients exhibit elevated levels of EphA2. By activating Focal Adhesion Kinases (FAK), EphA2 can induce metastasis in ovarian cancers. In this study, we show that the clinical PARP inhibitor Olaparib (AZD2281) activates IKBKE by EphA2-mediated tyrosine phosphorylation. We also found that phosphorylation of EphA2 or IKBKE expression can be used as a biomarker for olaparib resistance. Together, these studies have shed light on novel mechanisms of regulation of IKBKE and their importance in therapy resistance. These observations form a strong pre-clinical proof-of-concept to study the inhibitors further in the clinic

Stevia Genus: Phytochemistry and Biological Activities Update

The Stevia genus (Asteraceae) comprises around 230 species, distributed from the southern United States to the South American Andean region. Stevia rebaudiana, a Paraguayan herb that produces an intensely sweet diterpene glycoside called stevioside, is the most relevant member of this genus. Apart from S. rebaudiana, many other species belonging to the Stevia genus are considered medicinal and have been popularly used to treat different ailments. The members from this genus produce sesquiterpene lactones, diterpenes, longipinanes, and flavonoids as the main types of phytochemicals. Many pharmacological activities have been described for Stevia extracts and isolated compounds, antioxidant, antiparasitic, antiviral, anti-inflammatory, and antiproliferative activities being the most frequently mentioned. This review aims to present an update of the Stevia genus covering ethnobotanical aspects and traditional uses, phytochemistry, and biological activities of the extracts and isolated compounds.Fil: Borgo, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; ArgentinaFil: Laurella, Laura Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; ArgentinaFil: Martini, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; ArgentinaFil: Catalán, Cesar A. N.. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Orgánica; ArgentinaFil: Sülsen, Valeria Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; Argentin

The expression of Sirtuin1 and its role in ovarian cancer

SIRT1 has been a popular subject in the scientific research field since its detection in 1990. The function of this protein varies greatly depending on the tissue. Recent studies focused on SIRT1, especially in tumors. SIRT1 has been revealed to modulate cancer cell growth, metastasis, and invasion in numerous tumors. However, SIRT1 has been barely studied in gynecological tumors. The purpose of our study was to evaluate the effect of SIRT1 on ovarian cancer. First, we analyzed the relationship between the intensity of SIRT1 and RXR-alpha staining and clinical prognosis in ovarian cancer by immunohistochemistry. To further study the therapeutic effects of SIRT1 activator Resveratrol, we used MTT, M30, BrdU, and other methods to test the function of resveratrol on ovarian cancer cell lines A2780, UWB1, 289, and A2780cis, including proliferation and apoptosis. Finally, the relationship between RXR-alpha and SIRT1 proteins in ovarian cancer treated with Resveratrol was studied by western blot. Studies have shown that nuclear RXR-alpha and SIRT1-expression are significantly connected with a better outcome and overall survival rates in advanced ovarian cancer. Resveratrol had a direct effect on ovarian cancer: it suppressed the growth and proliferation of ovarian cancer cells and even increased their apoptosis. At the protein level, resveratrol (100 μM, 24h) up-regulated the expression of RXR-alpha in the anti-carboplatin cell line A2780cis and down-regulated the expression of SIRT1 in A2780. In conclusion, SIRT1 may have a suppressive role in ovarian cancer, especially in advanced ovarian cancer

Vitamins as regulators of immune cells and immune functions

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2022As vitaminas são micronutrientes essenciais que o ser humano não consegue sintetizar ou que não sintetiza em quantidades suficientes. Têm de ser fornecidas através da dieta, ou através da sua produção pelas bactérias comensais. As vitaminas têm um papel em diversos eventos biológicos, nomeadamente na regulação de respostas imunes, pelo que, quando se encontram em concentrações deficientes aumentam o risco de desregulação imunológica. Nos últimos anos, nas sociedades ocidentais, temos assistido a um aumento de incidência de doenças autoimunes/inflamatórias e cancro, apontando para uma maior influência de fatores ambientais em oposição aos fatores genéticos. O cancro é a segunda causa de morte no mundo. Desta forma, é importante perceber as interações entre o microambiente tumoral e o sistema imune; como é que as células tumorais conseguem escapar à vigilância imune e proliferar. Um dos tratamentos mais inovadores contra o cancro é a imunoterapia, capaz de estimular as células imunes contra as células tumorais. Quando funciona, pode ser revolucionária, no entanto, apenas uma minoria de pacientes beneficia dela. As vitaminas influenciam as respostas das células imunes e as células imunes são essenciais no desenvolvimento do cancro. Será que componentes da dieta, como as vitaminas, podem influenciar células tumorais através da sua ação nas células imunes? Nesta revisão, pretendemos abordar os principais conhecimentos existentes acerca das funções das vitaminas A, C e D nas respostas imunes, com particular foco na ação destas sobre as principais células imunes na homeostase, bem como o seu potencial modulador das respostas linfocitárias com impacto no crescimento e tratamento neoplásico. Utilizou-se o PubMed para pesquisa bibliográfica e selecionou-se artigos originais e algumas revisões de 2011 até 2021.Vitamins are essential micronutrients that human organism cannot synthesize or does not synthesize in sufficient quantities. Consequently, vitamins need to be supplied by the diet or commensal bacteria. They have diverse functions in many biological events, including regulation of immune responses, leading to increased susceptibility to immune dysregulation and immune-related diseases, when lacking. Clinical evidence points to a continuous rise in autoimmune/inflammatory diseases and cancer throughout westernized societies over the past decade, suggesting to a stronger influence of environmental factors as opposed to genetic factors. Cancer is the second most common cause of death worldwide. Hence, it is urgent to understand the tumour microenvironment, the interactions between tumour and immune cells, and how cancers manage to escape the immune surveillance and progress. Immunotherapies are one of the most innovative treatments in cancer, enhancing immune system to fight against tumour cells. When immunotherapy works, the result can be life-changing, unfortunately, only a minority of patients benefit from it. Vitamins influence immune cells responses and immune cells are key players in the initiation and development of cancer. Hence, can dietary components, such as vitamins, impact the cancer growth by their action on immune cells? In this review, we intend to discuss the progress regarding the immune function of vitamins A, C and D with a particular focus on how they influence key immune players in steady state, as well as their potential role as immune modulators of antitumoral lymphocytes responses and as co-adjuvant of immunotherapies, such as immune checkpoint inhibitors. The literature research was conducted using the electronic database PubMed and were selected original articles and few reviews from 2011 up to 2021

Mechanisms of T-Cell Exhaustion in Pancreatic Cancer.

T-cell exhaustion is a phenomenon that represents the dysfunctional state of T cells in chronic infections and cancer and is closely associated with poor prognosis in many cancers. The endogenous T-cell immunity and genetically edited cell therapies (CAR-T) failed to prevent tumor immune evasion. The effector T-cell activity is perturbed by an imbalance between inhibitory and stimulatory signals causing a reprogramming in metabolism and the high levels of multiple inhibitory receptors like programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and Lymphocyte-activation gene 3 (Lag-3). Despite the efforts to neutralize inhibitory receptors by a single agent or combinatorial immune checkpoint inhibitors to boost effector function, PDAC remains unresponsive to these therapies, suggesting that multiple molecular mechanisms play a role in stimulating the exhaustion state of tumor-infiltrating T cells. Recent studies utilizing transcriptomics, mass cytometry, and epigenomics revealed a critical role of Thymocyte selection-associated high mobility group box protein (TOX) genes and TOX-associated pathways, driving T-cell exhaustion in chronic infection and cancer. Here, we will review recently defined molecular, genetic, and cellular factors that drive T-cell exhaustion in PDAC. We will also discuss the effects of available immune checkpoint inhibitors and the latest clinical trials targeting various molecular factors mediating T-cell exhaustion in PDAC

Coming Full Circle: Epithelial Plasticity And The Natural History Of Metastasis

The primary cause of cancer-related deaths is metastasis— the spread of cancer cells to distant organs— and yet the mechanisms underlying this process remain elusive due to the difficulty in detecting early metastatic events, which are rare, stochastic and microscopic. To investigate the cellular and molecular mechanisms of metastasis, I utilized an autochthonous mouse model of pancreatic cancer (KPCY) in which all tumor cells are genetically labeled with yellow fluorescent protein (YFP). The YFP lineage label allows for the detection and isolation of disseminated tumor cells as they delaminate from epithelial structures within the primary tumor, invade into the stroma and circulation, and colonize distal organs. Using this system, I characterized the development of metastatic lesions from single disseminated cells to grossly macroscopic lesions in the murine liver. I found that gross metastases closely resembled primary tumors in terms of differentiation and microenvironment— these large lesions are well differentiated, containing primarily epithelial tumor cells, and accumulate stroma consisting of myofibroblasts, leukocytes and extracellular matrix (ECM). In contrast, single disseminated cells tend to be poorly differentiated and lack any association with stromal cells, and must build up a microenvironment around them as they grow. Despite the presumably protective stroma surrounding large lesions, gross metastasis was significantly reduced with chemotherapy, while single cells were unaffected. Interestingly, residual lesions were enriched for epithelial features, suggesting that EMT confers chemosensitivity in this context. I also used the KPCY model to investigate the molecular mechanisms of epithelial-mesenchymal transition (EMT), which is widely considered to be the first step in the metastatic cascade. The YFP lineage label made it possible to identify and isolate tumor cells that have undergone EMT for transcriptional profiling. Surprisingly, I found that in a majority of pancreatic tumors, conventional transcriptional repressors were not involved in EMT. Although a mesenchymal transcriptional program was significantly enriched in cells that had undergone this “non-canonical” mechanism of EMT, the epithelial program was downregulated at the protein level by a mechanism involving protein internalization. Because cells retain both epithelial and mesenchymal properties during non-canonical EMT, this phenomenon represents an attractive explanation for the ability of tumor cells to cycle between epithelial and mesenchymal states and adapt to the changing microenvironment on their way to metastatic sites. The journey from primary tumor to metastatic site requires cancer cells to overcome many obstacles and a better understanding of how they navigate the numerous steps of the metastatic cascade could open the door to desperately needed anti-metastatic therapies

Transcriptional Governance of Hair Follicle Stem Cell Quiescence and Niche Maintenance in Long-Term Tissue Regeneration

Adult stem cells are endowed with the remarkable ability to maintain, regenerate and repair tissues throughout the lifetime of the organism. Whether parsimonious utilization of adult stem cells is necessary to preserve their long-term potential has not been fully explored. I investigated this issue using the adult murine hair follicle stem cell (HFSC) as my paradigm. HFSCs reside in their niche called the bulge, and mostly remain in a quiescent state, becoming mobilized only transiently to fuel cyclical bouts of hair follicle regeneration. By ablating a key HFSC transcription factor, Forkhead Box C1 (FOXC1), I discovered that hair follicles underwent more rounds of regeneration and yet were unable to result in a thickening of the animal’s hair coat. Mechanistically, unlike WT HFSCs, FOXC1-deficient HFSCs failed to remain in prolonged durations of quiescence. Instead, they were primed to re-enter the cell cycle and launch new rounds of hair regeneration prematurely. After activation, they failed to re-establish quiescence promptly, and remained in a primed state to proliferate. In turn, their expression of cell adhesion proteins remained low. As new hairs grew, wild-type (WT) HFSCs that had returned to quiescence and restored their repertoire of adhesion-associated proteins were able to anchor their bulge niche and the older hairs in place. However, FOXC1- deficient HFSCs were unable to do so, resulting in the gradual loss of their bulge and old hair coats. As the bulge is also a cellular source of HFSC-inhibitory factors, its loss exacerbated the inability of FOXC1-deficient HFSCs to maintain quiescence. Consequently, as these mutant mice aged, their hair coat appeared sparse. Indeed, their HFSC numbers and ability to regenerate new hairs upon stimulation had declined. Therefore, through FOXC1, HFSCs couple their quiescence to an adhesion-mediated niche maintenance to achieve long-term tissue homeostasis

DYRK1A in cancer: good or evil? : Defining properties of DYRK1A kinase as a novel tumor driver

DYRK (dual-specificity tyrosine-regulated kinases) is an evolutionary conserved family of protein kinases involved in the regulation of cellular processes, such as proliferation and survival, which play pivotal role in tumor development. In this Thesis work, the potential role of DYRK genes as tumor drivers has been explored through an extensive analysis of The Cancer Genome Atlas data. DYRKs were found altered in tumor samples at different levels. In particular, the dosage sensitive member DYRK1A emerged as a potential tumor driver. Functional screens on DYRK1A cancer somatic mutations showed that most of the mutants analyzed have a strong impact on the catalytic activity and/or stability of the protein, suggesting that DYRK1A loss-of-function is positively selected in cancer. Reversion, by a CRISPR/Cas9 strategy, of a DYRK1A mutant allele to wt in an endometrial cancer cell line strongly impaired tumor cell growth. The DYRK1A tumor suppressive role was confirmed in vivo using mouse tumor xenografts. Finally, an integrated transcriptomic, proteomic and phospho-proteomic analysis has uncovered potential molecular mechanisms underlying the DYRK1A-mediated tumor driver function.Las proteínas quinasas DYRK (dual-specificity tyrosine-regulated kinases) son una familia evolutivamente conservada que participan en la regulación de procesos celulares con funciones fundamentales en la transformación maligna. En este trabajo de tesis, el papel de los genes DYRK como conductores (drivers) de tumores se ha explorado mediante un extenso análisis de los datos de The Cancer Genome Atlas. El análisis encontró alterados los genes DYRK en muestras de tumores a diferentes niveles, e identificó al miembro de la familia DYRK1A como un potencial gen conductor en un grupo de tumores. Mediante ensayos funcionales se ha demostrado que las mutaciones somáticas de DYRK1A en cáncer investigadas tienen un fuerte impacto en la actividad catalítica y/o la estabilidad de la proteína, lo que sugiere que la perdida de función de DYRK1A se selecciona positivamente en la célula tumoral. La corrección, mediante la técnica CRISPR/Cas9, de una línea celular de cáncer de endometrio mutada en DYRK1A tiene un fuerte impacto en el crecimiento de estas células tumorales, sugiriendo un papel para DYRK1A como supresor de tumores en cáncer de endometrio, que se ha confirmado in vivo en modelos tumorales de xenoinjerto en ratón. Finalmente, un análisis transcriptómico, proteómico y fosfo-proteómico integrado ha desvelado posibles mecanismos moleculares que participan en la función de DYRK1A como supresor tumoral

Characterization of signalling cross-talk between the EP2 and FP receptors in endometrial epithelial cells

Uterine fibroids are benign tumors that arise from the smooth-muscle uterine cells (myometrium) and are the most common uterine disorder occurring in as many as 30% of women over 35 years of age. Despite their frequent occurrence, the etiology of uterine fibroids is not well elucidated. Several studies have shown that numerous tumors can be regulated by cyclooxygenase (COX) enzyme products but their role in uterine fibroids is not well established. The initial aim of the study was to determine the expression level of COX enzymes and prostaglandin receptors in fibroids and autologous myometrium samples from women with fibroids. Real-Time reverse-transcriptase polymerase chain reaction (RT-PCR) revealed that the expression of COX enzymes, EP1, EP2 and EP4 prostanoid receptors and prolactin were not significantly altered while the EP3 subtype receptor was significantly down-regulated in fibroids compared to adjacent myometrium samples. The EP3 receptor has a protective role in tumor development suggesting the role for down-regulation of the receptor in uterine fibroids pathology. In addition, the expression of COX enzymes, prostaglandin receptors and prostaglandin-mediated genes were assessed in endometrium samples from women with and without uterine fibroids in different stages of the menstrual cycle. COX-2 and interleukin-8 (IL-8) mRNA expressions were significantly higher in both proliferative stage and early-mid secretory, EP2 receptor and IL-11 were elevated in the proliferative stage, vascular endothelial growth factor (VEGF) was highly expressed in the early-mid secretory phase while FP receptor was up-regulated in all stages of the menstrual cycle in endometrium samples from women with fibroids. These data suggest that up-regulation of COX-2 and prostaglandin receptors (EP2 and FP) in endometrium can induce expression of angiogenic and mitogenic factors such as VEGF, IL-8 and IL-11 which might act in a paracrine manner on neighboring myometrial/fibroid tissue to promote angiogenesis and facilitate tumor growth. XVII Furthermore, since EP2 and FP receptors were up-regulated in the proliferative phase of endometrium from uterine fibroid patients and the receptors are co-expressed in endometrial adenocarcinoma (Ishikawa) cells, this study investigated a possible cross-talk that influences intracellular signalling by using Ishikawa cells stably expressing the EP2 and FP receptors (FPEP2 cells) as a model cell line. Real-Time RT-PCR, Western blot analysis and immunofluorescence microscopy confirmed stable expression of the EP2 and FP receptors in FPEP2 cells localized to the perinuclear and plasma membrane. Using FPEP2 cells, the integrated effect of Butaprost (EP2 receptor ligand) and PGF (FP receptor ligand) co-administration on inositol phosphate (IP3) and adenosine 3-,5-cyclic monophosphate (cAMP) release was assessed to study a possible heterologous-interaction or cross-talk between the EP2 and FP receptors. The study showed that in FPEP2 cells, PGF alone does not alter cAMP production, but in combination with Butaprost augments EP2 receptor-mediated cAMP release. PGF-mediated potentiation of cAMP release was abolished by antagonism of the FP receptor, inhibition of phospholipase C (PLC) and IP3-receptor whereas inhibition of protein kinase C (PKC) had no effect suggesting the cross-talk is mediated by FP receptor activation of IP3 release. Moreover, inhibition of calcium effectors using calmodulin antagonist (W7) or Ca2+/calmodulin-dependent kinase II (CaMK-II) inhibitor (KN-93) abolished PGF potentiation of Butaprost-mediated cAMP release. Using short interfering RNA (siRNA) molecules targeted against the adenylyl cyclase 3 (AC3) isoform, the study showed the isoform to be responsible for the cross-talk between the FP and EP2 receptors. In order to determine the integrative effects of the EP2 and FP receptors co-activation on gene expression, a whole genome array profiling in FPEP2 cells in response to Butaprost and/or PGF was performed. The gene array revealed 228 genes that are regulated by co-activation of the EP2 and FP receptors that are involved in cell morphology, proliferation and differentiation. XVIII In addition, co-activation of EP2 and FP receptors with their respective ligands enhanced or repressed a set of EP2 receptor-regulated genes. One of the genes identified, SAT1 (Spermidine/ N1-acetyltransferase), was regulated by the EP2 and FP receptors cross-talk via the calcium sensitive AC3 isoform. SAT1, with known role in regulation of tumorigenesis was also up-regulated in the proliferative stage of endometrium samples from women with uterine fibroids suggesting the EP2 and FP receptor cross-talk characterized in vitro can also happen in vivo. In conclusion, this study reports that COX-2, EP2 and FP receptors, VEGF, IL-8, IL-11 and SAT1 are up-regulated in endometrium from women with uterine fibroids. These genes play a major role in development of fibroids by facilitating angiogenesis and cell growth and by inhibiting apoptosis via autocrine/paracrine mechanisms. In addition, this study demonstrates that co-activation of the EP2 and FP receptors results in enhanced release of cAMP via the FP receptor-G +-q-Ca2+-calmodulin pathway by activating the calcium-sensitive AC3 isoform and modulates a molecular switch which alters the trans-activation of a subset single-receptor induced genes that have important functions in the pathogenesis of reproductive pathologies

The Effect of Phytoestrogens on Bone and T Cells Differentiation and Activity

Full version unavailable due to 3rd party copyright restrictions.The fall in circulating oestrogen (E2) after the menopause leads to an increased rate of bone remodelling, excessive osteoclast activity and a greater fracture risk. Until recently hormone replacement therapy (HRT) was prescribed to post-menopausal women to prevent bone loss, however HRT is associated with an elevated incidence of cardiovascular disease, stroke and cancer. These side-effects led to an interest in naturally occurring compounds with oestrogenic action such as phytoestrogens (PEs), which are non-steroidal-plant derived compounds. Human trials and animal studies suggest a beneficial effect of PEs on bone mass, although their ability to modify osteoclast formation in response to key inflammatory cytokines has not been examined. The aim of the following studies was to determine the effect of physiologically relevant concentrations of genistein, coumestrol and daidzein on TNF-α-induced osteoclast formation, osteoblasts differentiation and T cell activity. Genistein (10-7 M), daidzein (10-5 M), and coumestrol (10-7 M) significantly reduced TNF-α-induced TRAP positive osteoclast formation and bone resorption, which was prevented by the E2 antagonist ICI 182,780. The suppressive action on osteoclast formation was associated with a significant reduction in TNF-α-induced c-fos and NFATc1 mRNA expression and NFATc1 nuclear translocation. Constitutive c-fos expression prevented the inhibitory action of PEs on osteoclast differentiation, resorption and NFATc1 expression. The effect of PEs, in the presence or absence of the anabolic nutritional factor zinc, on osteoblasts differentiation and bone nodule formation was examined in-vitro. Coumestrol (10-5 to 10-7 M), daidzein (10-5 to 10-6 M) and genistein (10-5 M) enhanced bone nodule formation and ALP activity in human osteoblasts, and this effect was significantly augmented in the presence of zinc (10-5 M). Furthermore, PEs and zinc increased Runx2 mRNA expression and Zn2+ augmented the inhibitory effect of PEs on RANKL/OPG ratio. This suggests that in addition to the direct inhibitory effect on osteoclast formation PEs also in-directly reduce the osteoblastsic stimulus for osteoclast formation and promote bone formation. E2 deficiency is thought to promote osteoclastogenesis by modifying Thelper1 (Th1) cell proliferation and inflammatory cytokine production in particular TNF-α. I therefore examined the effect of PEs on T cell proliferation and inflammatory cytokine production. All PEs prevented the augmentative effect of con A stimulated T cells on osteoclast formation in co-culture. However the mechanism of action varied, genistein reduced con A stimulated TNF-α, IL-1β and RANKL expression with little effect on viability, coumestrol decreased cell viability and TNF-α expression whereas the inhibitory effect of daidzein was mediated via suppression of viable T cell number. This study provides novel evidence that PEs have multiple effects on bone cell activity, directly inhibiting TNF-α-induced osteoclast formation, reducing the osteoblasts and T cell derived stimulus for osteoclast formation and augmenting osteoblasts differentiation and bone formation. Thus, PEs have a potential role in the treatment of post-menopausal osteoporosis and inflammatory skeletal disorders and that the beneficial effect noted in previous studies is mediated through multiple mechanisms.Ministry of Higher education and scientific research/Republic of Ira