Inhibition of PI3K pathway using BKM120 intensified the chemo-sensitivity of breast cancer cells to arsenic trioxide (ATO).

Although conventional therapeutic approaches have brought remarkable advantages for the treatment of breast cancer (BC), drug-resistance still remains as a leading cause of tumor recurrence in this malignancy. In the present study, we designed experiments to evaluate the therapeutic value of PI3K inhibition in combination with Arsenic trioxide (ATO) in MCF-7 cells. The results of our study manifested that BKM120 sensitized MCF-7 cells to the lower concentrations of ATO. The significant anti-cancer effect of PI3K inhibition became even more evident when we found that BKM120, either as a single agent or in combination with ATO, reduced clonogenic ability of anoikis-resistant stem-like MCF-7 cells. Our findings also showed that BKM120 augmented ATO-induced anti-proliferative effects through inducing G1 arrest and reducing the incorporation of BrdU into the synthesized DNA of drugs-treated cells, which was coupled with c-Myc-mediated suppression of hTERT expression. Moreover, we found that in the presence of PI3K inhibitor, ATO is able to more profoundly induce apoptosis in MCF-7 cells, as revealed by the increment in the percentage of haplodiploid sub-G1 cells and the externalization of phosphatidylserine. Real-time PCR analysis also revealed that probably down-regulation of survivin coupled with up-regulation of forkhead family transcription factors is responsible for the enhancive effect of drugs in this cell line. Conclusively, this study shed lights on the effect of PI3K inhibition in chemo-sensitivity of MCF-7 cells, disclosing that combination of BKM120 and ATO could be a promising therapeutic approach in BC.

Glyphosate-based herbicides modulate oxidative stress response in the nematode Caenorhabditis elegans.

Glyphosate-based formulation is used as non-selective and post-emergent herbicides in urban and rural activities. In view of its recurring applications in agricultural producing countries, the increase of glyphosate concentration in the environment stresses the need to test the adverse effects on non-target organisms and assess the risk of its use. This paper analyzes the toxicological and oxidative stress and modulatory effects of a glyphosate commercial formulation (glyphosate F) on the nematode Caenorhabditis elegans. We detected ROS production and enhancement of oxidative stress response in glyphosate F-treated nematodes. Particularly, we found an increased ctl-1 catalase gene expression of a catalase specific activity. In addition, we showed that glyphosate F treatment activated the FOXO transcription factor DAF-16, a critical target of the insulin/IGF-1 signaling pathway, which modulates the transcription of a broad range of genes involved in stress resistance, reproductive development, dauer formation, and longevity. In summary, the exposure of glyphosate F induces an oxidative imbalance in C. elegans that leads to the DAF-16 activation and consequently to the expression of genes that boost the antioxidant defense system. In this regard, clt-1 gene and catalase activity proved to be excellent biomarkers to develop more sensitive protocols to assess the environmental risk of glyphosate use.

Targeting EZH2 reactivates a breast cancer subtype-specific anti-metastatic transcriptional program.

Emerging evidence has illustrated the importance of epigenomic reprogramming in cancer, with altered post-translational modifications of histones contributing to pathogenesis. However, the contributions of histone modifiers to breast cancer progression are unclear, and how these processes vary between molecular subtypes has yet to be adequately addressed. Here we report that genetic or pharmacological targeting of the epigenetic modifier Ezh2 dramatically hinders metastatic behaviour in both a mouse model of breast cancer and patient-derived xenografts reflective of the Luminal B subtype. We further define a subtype-specific molecular mechanism whereby EZH2 maintains H3K27me3-mediated repression of the FOXC1 gene, thereby inactivating a FOXC1-driven, anti-invasive transcriptional program. We demonstrate that higher FOXC1 is predictive of favourable outcome specifically in Luminal B breast cancer patients and establish the use of EZH2 methyltransferase inhibitors as a viable strategy to block metastasis in Luminal B breast cancer, where options for targeted therapy are limited.

Caenorhabditis elegans susceptibility to Daldinia cf. concentrica bioactive volatiles is coupled with expression activation of the stress-response transcription factor daf-16, a part of distinct nematicidal action.

The bionematicidal effect of a synthetic volatile mixture (SVM) of four volatile organic compounds (VOCs) emitted by the endophytic fungus Daldinia cf. concentrica against the devastating plant-parasitic root-knot nematode Meloidogyne javanica has been recently demonstrated in both in vitro and greenhouse experiments. However, the mode of action governing the observed irreversible paralysis of J2 larvae upon exposure to SVM is unknown. To unravel the mechanism underlying the anthelmintic and nematicidal activities, we used the tractable model worm Caenorhabditis elegans. C. elegans was also susceptible to both the fungal VOCs and SVM. Among compounds comprising SVM, 3-methyl-1-butanol, (±)-2-methyl-1-butanol, and 4-heptanone showed significant nematicidal activity toward L1, L4 and young adult stages. Egg hatching was only negatively affected by 4-heptanone. To determine the mechanism underlying this activity, we examined the response of C. elegans mutants for glutamate-gated chloride channel and acetylcholine transporter, targets of the nematicidal drugs ivermectin and aldicarb, respectively, to 4-heptanone and SVM. These aldicarb- and ivermectin-resistant mutants retained susceptibility upon exposure to 4-heptanone and SVM. Next, we used C. elegans TJ356 strain zIs356 (daf-16::GFP+rol-6), LD1 ldIs7 [skn-1B/C::GFP + pRF4(rol-6(su1006))], LD1171 ldIs3 [gcs-1p::gfp; rol-6(su1006))], CL2166 dvIs19 (gst-4p::GFP) and CF1553 muIs84 (sod-3p::GFP+rol-6), which have mutations in genes regulating multiple stress responses. Following exposure of L4 larvae to 4-heptanone or SVM, there was clear nuclear translocation of DAF-16::GFP, and SKN-1::GFP indicating that their susceptibility involves DAF-16 and SKN1 regulation. Application of 4-heptanone, but not SVM, induced increased expression of, gcs-1::GFP and gst-4::GFP compared to controls. In contrast, application of 4-heptanone or SVM to the sod-3::GFP line elicited a significant decline in overall fluorescence intensity compared to controls, indicating SOD-3 downregulation and therefore overall reduction in cellular redox machinery. Our data indicate that the mode of action of SVM and 4-heptanone from D. cf. concentrica differs from that of currently available nematicides, potentially offering new solutions for nematode management.

The effect of 1, 25(OH)2 D3 (calcitriol) alone and in combination with all-trans retinoic acid on ROR-γt, IL-17, TGF-ß, and FOXP3 gene expression in experimental autoimmune encephalomyelitis.

OBJECTIVES: It has been shown that calcitriol and all-trans retinoic acid (ATRA) have modulatory effects on the immune system. The present study investigates the synergistic effects of combination treatment of calcitriol and ATRA in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). METHODS: The mice were allocated to four preventive groups, each consisting of eight animals, ATRA (250 µg/mouse), calcitriol (100 ng/mouse), combination of ATRA and calcitriol (125  µg/mouse and 50 ng/mouse) and vehicle groups. EAE was induced by MOG35-55 peptide in female C57BL/6 mice. Treatments were initiated at day 1 before immunization and continued every other day throughout the study until the day 21 post-immunization. Splenocytes were isolated from EAE-induced mice and the expression of retinoic acid receptor-related orphan receptor gamma t (ROR-γt), Interleukin-17 (IL-17), transforming growth factor beta (TGF-ß), and forkhead box P3 (FOXP3) genes was measured using real-time polymerase chain reaction. RESULTS: The expression of FOXP3 and TGF-ß genes in the splenocytes of combination-treated and calcitriol alone-treated mice was significantly increased compared to vehicle group (P < 0.05). The expression of ROR-γt and IL-17 genes in the splenocytes of ATRA, calcitriol and combination- treated mice was significantly reduced compared to those of vehicle- treated mice (P < 0.05). The relative expression level of ROR-γt was significantly (P < 0.05) lower in the combination group than in the mice treated by ATRA or calcitriol alone. DISCUSSION: This study demonstrated that treatment with combination of calcitriol and ATRA can be considered as a new strategy for MS prevention and treatment.

Migratory capacity and function of dendritic cells from mesenteric afferent lymph nodes after feeding a single dose of vitamin A.

Lamina propria dendritic cells (DCs) have a permanent turnover with constitutive migration to mesenteric lymph nodes and replenishment by progenitors. Luminal bacteria and dietary constituents provide key signals that endow DCs their unique properties in vivo. Taking into account that the intestinal immune system is greatly influenced by retinoids, we evaluated in B6 mice 3, 8, 16 and 24 h after feeding a single dose of vitamin A phenotype and function of cells present in mesenteric afferent lymph nodes as well as signals involved in migration. We studied the frequency of CD11c+MHC-II+CD103+CD86+ and RALDH+ DCs by flow cytometry, we determined CCL-21 and D6 levels in tissue homogenates by Western blot, and we co-cultured cells isolated from afferent lymphatics with sorted CD4+ lymphocytes to assess Foxp-3 induction and homing receptor expression. Sixteen hours after vitamin A administration, DCs isolated from afferent lymphatics were able to induce homing receptors and Foxp3 expression in CD4+ lymphocytes. Our results show that a single dose of vitamin A generated a stream of signals and amplified the tolerogenic activity of DCs migrating to lymphoid tissue.

FOXO1 inhibits osteoclastogenesis partially by antagnozing MYC.

FOXO transcription factors especially FOXO1 have profound roles in bone development and remodeling. The regulation of cells of the osteoblast lineage by FOXOs is suggested to be stage-specific or context dependent. Intriguingly, recent studies on the role played by FOXOs in osteoclastogenesis reached different conclusion. Bartell et al. showed that FOXOs restrained osteoclastogenesis and bone resorption partially by upregulation of the H2O2-inactivating enzyme catalase. Wang et al. demonstrated that FOXO1 activated osteoclast formation. In the present study, we confirmed the results of Bartell et al. that FOXO1 expression was reduced upon stimulation of RANKL; FOXO1 inhibition promoted and FOXO1 activation repressed, osteoclast differentiation and activity; the inhibitory effect of FOXO1 on osteoclastogenesis was partially mediated by ROS since treatment with ROS scavengers cancelled the effect of FOXO1 inhibition on osteoclastogenesis. We further investigated the mechanisms responsible for repressed osteoclastogenesis by FOXO1. We found that FOXO1 inhibition modulated MAPKs, NF-κB and AP-1. Finally, we proved that the inhibitory effect of FOXO1 on osteoclast formation was partially mediated by MYC suppression by showing that MYC repression almost totally abrogated the effect of FOXO1 inhibition on osteoclastogenesis. To conclude, our study confirmed FOXO1 as a cell-autonomous inhibitor of osteoclastogenesis.

Antiproliferative and pro-apoptotic effects of three fungal exocellular ß-glucans in MCF-7 breast cancer cells is mediated by oxidative stress, AMP-activated protein kinase (AMPK) and the Forkhead transcription factor, FOXO3a.

Fungal ß-d-glucans of the (1→3)-type are known to exhibit direct antitumor effects, and can also indirectly decrease tumor proliferation through immunomodulatory responses. The underlying molecular mechanisms involved in decreasing tumor formation, however, are not well understood. In this study, we examined the antiproliferative role and mechanism of action of three different fungal exocellular ß-glucans in MCF-7 breast cancer cells. The ß-glucans were obtained from Botryosphaeria rhodina MAMB-05 [two botryosphaerans; (1→3)(1→6)-ß-d-glucan; one produced on glucose, the other on fructose] and Lasiodiplodia theobromae MMPI [lasiodiplodan; (1→6)-ß-d-glucan, produced on glucose]. Using the cell proliferation-MTT assay, we showed that the ß-glucans exhibited a time- and concentration-dependent antiproliferative activity (IC50, 100µg/ml). Markers of cell cycle, apoptosis, necrosis and oxidative stress were analyzed using flow cytometry, RT-PCR and Western blotting. Exposure to ß-glucans increased apoptosis, necrosis, oxidative stress, mRNA expression of p53, p27 and Bax; the activity of AMP-activated protein-kinase, Forkhead transcription factor FOXO3a, Bax and caspase-3; and decreased the activity of p70S6K in MCF-7 cells. In the presence of hydrogen peroxide, the fungal ß-glucans increased oxidative stress, which was associated with reduced cell viability. We showed that these ß-glucans exhibited an antiproliferative effect that was associated with apoptosis, necrosis and oxidative stress. This study demonstrated for the first time that the apoptosis induced by ß-glucans was mediated by AMP-activated protein-kinase and Forkhead transcription factor, FOXO3a. Our findings provide novel mechanistic insights into their antiproliferative roles, and compelling evidence that these ß-glucans possess a broad range of biomodulatory properties that may prove useful in cancer treatment.

8­bromo­7­methoxychrysin induces apoptosis by regulating Akt/FOXO3a pathway in cisplatin­sensitive and resistant ovarian cancer cells.

8­bromo­7­methoxychrysin (BrMC), a novel chrysin analog, was reported to have anti­cancer activities. The aim of the present study was to investigate the molecular mechanism of 8­bromo­7­methoxychrysin (BrMC)­induced apoptosis via the Akt/forkhead box O3a (FOXO3a) pathway in cisplatin (DDP)­sensitive and ­resistant ovarian cancer cells. The human ovarian cancer cell lines A2780 and A2780/DDP were cultured in vitro. Various molecular techniques were used to assess the expression of FOXO3a and B cell lymphoma 2 (Bcl­2)­interacting mediator of cell death (Bim) in cisplatin­sensitive and ­resistant ovarian cancer cells. Different concentrations of BrMC induced apoptosis in cisplatin­sensitive and ­resistant ovarian cancer cells. BrMC­induced apoptotic cell death occurred mainly by the activation of Akt, which was accompanied by the overexpression of transcription factor FOXO3a, with a concomitant increase in the expression levels of Bim. Silencing Bim expression by using small interfering RNA, attenuated the induction of apoptosis by BrMC treatment. The results indicated that BrMC­induced apoptosis in cisplatin­sensitive and ­resistant ovarian cancer cells may occur via the regulation of Akt/FOXO3a, leading to Bim transcription.

The longevity effect of echinacoside in Caenorhabditis elegans mediated through daf-16.

Echinacoside (ECH), a natural polyphenolic compound, has been reported to possess important pharmacological activities. However, very little is known about whether or how ECH affects longevity in vivo. We have examined the effects of ECH on the life span and stress tolerance in Caenorhabditis elegans. Our studies demonstrate that the life span of wild-type worms could be extended in the presence of ECH. Furthermore, ECH was found to increase tolerance of worms to heat shock and oxidative stress, while not exerting any influence on pharyngeal pumping rate and progeny production. Our mechanistic studies indicate that supplementation of ECH increases the transcript level of daf-16. ECH treatment also modulates the nuclear localization and transcriptional activities of daf-16, thus fine tunes the expression of daf-16 target genes to promote longevity and increases stress response in C. elegans. Overall, this work reveals the longevity effect of ECH and elucidates the underpinning mechanisms.

Evaluation of the bioactive properties of avenanthramide analogs produced in recombinant yeast.

Saccharomyces cerevisiae has been proven to be a valuable tool for the expression of plant metabolic pathways. By engineering a S. cerevisiae strain with two plant genes (4cl-2 from tobacco and hct from globe artichoke) we previously set up a system for the production of two novel phenolic compounds, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid (Yeast avenanthramide I, Yav I) and N-(E)-caffeoyl-3-hydroxyanthranilic acid (Yeast avenanthramide II, Yav II). These compounds have a structural similarity with a class of bioactive oat compounds called avenanthramides. By developing a fermentation process for the engineered S. cerevisiae strain, we obtained a high-yield production of Yav I and Yav II. To examine the biological relevance of these compounds, we tested their potential antioxidant and antiproliferative properties upon treatment of widely used cell models, including immortalized mouse embryonic fibroblast cell lines and HeLa cancer cells. The outcomes of our experiments showed that both Yav I and Yav II enter the cell and trigger a significant up-regulation of master regulators of cell antioxidant responses, including the major antioxidant protein SOD2 and its transcriptional regulator FoxO1 as well as the down-regulation of Cyclin D1. Intriguingly, these effects were also demonstrated in cellular models of the human genetic disease Cerebral Cavernous Malformation, suggesting that the novel phenolic compounds Yav I and Yav II are endowed with bioactive properties relevant to biomedical applications. Taken together, our data demonstrate the feasibility of biotechnological production of yeast avenanthramides and underline a biologically relevant antioxidant activity of these molecules.

Platycodin D induces tumor growth arrest by activating FOXO3a expression in prostate cancer in vitro and in vivo.

Platycodin D (PD), a major saponin derived from Platycodin grandiflorum, exerted cytotoxicity against prostate cancer cell lines (PC3, DU145 and LNCaP cells) with IC50 values in the range of 11.17 to 26.13 µmol/L, whereas RWPE-1 cells (a non-malignant human prostate epithelial cell line) were not significantly affected. A further study in these cell lines showed that PD could potently affect cell proliferation (indicated by the bromodeoxyuridine assay), induce cell apoptosis (determined by Annexin V-FITC flow cytometry) and cause cell cycle arrest (indicated by PI staining). After being treated with PD for 48 hours, DU145 and LNCaP cells were arrested in the G0 /G1 phase, and PC3 cells were arrested in the G2/M phase. A Western blotting analysis indicated that PD increased the expression of the FOXO3a transcription factor, decreased the expression of p-FOXO3a and MDM2 and increased the expression of FOXO-responsive genes, p21 and p27. MDM2 silencing (transiently by siRNA-MDM2) increased the PD-induced FOXO3a protein expression, while MDM2 overexpression (in cells transiently transfected with a pcDNA3-MDM2 plasmid) decreased the PD-induced expression of the FOXO3a protein. Moreover, PD dose-dependently inhibited the growth of PC3 xenograft tumors in BALB/c nude mice. A Western blotting analysis of the excised xenograft tumors indicated that similar changes in protein expression also occurred in vivo. These results suggest that PD exhibits significant activity against prostate cancer in vitro and in vivo. The FOXO3a transcription factor appears to be involved in the activity of PD. Together, all of these findings provide a basis for the future development of this agent for human prostate cancer therapy.

Anti-neoplastic activity of low-dose endothelial-monocyte activating polypeptide-II results from defective autophagy and G2/M arrest mediated by PI3K/Akt/FoxO1 axis in human glioblastoma stem cells.

Glioblastoma multiforme (GBM) is a life-threatening brain tumor with fatal recurrence, for which glioblastoma stem cells (GSCs) are held responsible. Though endothelial-monocyte activating polypeptide-II (EMAP-II) has been confirmed as a possible antitumor agent that can induce apoptosis of endothelial cells and inhibit tumor angiogenesis, the direct cytotoxicity by EMAP-II on tumor cells and its underlying mechanism are largely unknown. In the present study, it was demonstrated that low-dose (0.05 nM) EMAP-II reduces cell viability and mitochondrial membrane potential in vitro. Likewise, EMAP-II suppressed tumor growth in GSC-xenografted mice. Though no apoptosis was detected, all these antitumor effects were attenuated when GSCs were pretreated with 3-methyladenine (3-MA). Analysis of EMAP-II-treated GSCs exhibited the morphological and biochemical changes typical of autophagy, which was further shown to be defective. Moreover, EMAP-II was found to suppress tumor growth by inducing G2/M arrest in GSCs. Our data further showed that EMAP-II inhibited PI3K/Akt activation with concomitant induction of FoxO1 activation. FoxO1 knockdown significantly attenuated the induction of autophagy and G2/M arrest. Excessive accumulation of lipid droplets was intriguingly detected by transmission electron microscope, which was accompanied by autophagosomes. Further investigation indicated that the transcriptional regulation of Atg2B by FoxO1 was responsible for the induction of autophagy and formation of lipid droplets. These results suggest that EMAP-II is an effective anticancer agent for glioblastoma therapy, which can induce direct growth suppression in GSCs through defective autophagy and G2/M arrest mediated by the PI3K/Akt/FoxO1 axis.

Mechanism of action for the cytotoxic effects of the nitric oxide prodrug JS-K in murine erythroleukemia cells.

The nitric oxide (NO) prodrug JS-K, a promising anti-cancer agent, consists of a diazeniumdiolate group necessary for the release of NO as well as an arylating ring. In this study, we research the mechanism by which JS-K kills a murine erythroleukemia cell line and determine the roles of NO and arylation in the process. Our studies indicate that JS-K inhibits the PI 3-kinase/Akt and MAP kinase pathways. This correlates with the activation of the tumor suppressor FoxO3a and increased expression of various caspases, leading to apoptosis. The arylating capability of JS-K appears to be sufficient for inducing these biological effects. Overall, these data suggest that JS-K kills tumor cells by arylating and inactivating signaling molecules that block the activation of a tumor suppressor.

FoxO proteins: regulation and molecular targets in liver cancer.

Human forkhead box class O (FoxO) transcription factors, activated in response to a wide range of external stimuli, like growth factors, insulin, nutrient levels and oxidative stress, are able to control several specific geneexpression programs. Besides their clear implication in metabolic processes, they appear to play a relevant role in tumour suppression by upregulation of genes involved in cell cycle arrest or apoptosis. Recent research efforts provide new insights into the molecular modulation of FoxO in liver cancer and disclose potential opportunities for developing new antitumor drugs. Through an intricate regulatory model, achieved via several post-translational modifications, including phosphorylation, acetylation, and ubiquitination, which control their subcellular localization and DNA binding activity, FoxO factors act as tumour suppressors. Low levels of FoxOs are associated with poor prognosis in cancer patients, and seem to confer chemotherapy resistance. Within FoxO members, FoxO3a appears to present anti-tumour properties in hepatocellular carcinoma, inducing the expression of pro-apoptotic genes, or interfering with signaling cascades commonly altered in this disease such as Wnt/ß-catenin, PI3K/AKT/mTOR or MAPKs pathways. Here, we describe the main mechanisms of FoxO proteins regulation, and their cross-link with altered pathways in liver cancer. Moreover, based on the current knowledge of FoxO modulation, emphasis is placed on the development of novel agents which specifically activate FoxO family members and could be useful in the treatment of hepatocarcinoma.

Integrative toxicoproteomics implicates impaired mitochondrial glutathione import as an off-target effect of troglitazone.

Troglitazone, a first-generation thiazolidinedione of antihyperglycaemic properties, was withdrawn from the market due to unacceptable idiosyncratic hepatotoxicity. Despite intensive research, the underlying mechanism of troglitazone-induced liver toxicity remains unknown. Here we report the use of the Sod2(+/-) mouse model of silent mitochondrial oxidative-stress-based and quantitative mass spectrometry-based proteomics to track the mitochondrial proteome changes induced by physiologically relevant troglitazone doses. By quantitative untargeted proteomics, we first globally profiled the Sod2(+/-) hepatic mitochondria proteome and found perturbations including GSH metabolism that enhanced the toxicity of the normally nontoxic troglitazone. Short- and long-term troglitazone administration in Sod2(+/-) mouse led to a mitochondrial proteome shift from an early compensatory response to an eventual phase of intolerable oxidative stress, due to decreased mitochondrial glutathione (mGSH) import protein, decreased dicarboxylate ion carrier (DIC), and the specific activation of ASK1-JNK and FOXO3a with prolonged troglitazone exposure. Furthermore, mapping of the detected proteins onto mouse specific protein-centered networks revealed lipid-associated proteins as contributors to overt mitochondrial and liver injury when under prolonged exposure to the lipid-normalizing troglitazone. By integrative toxicoproteomics, we demonstrated a powerful systems approach in identifying the collapse of specific fragile nodes and activation of crucial proteome reconfiguration regulators when targeted by an exogenous toxicant.

Indoles mitigate the development of experimental autoimmune encephalomyelitis by induction of reciprocal differentiation of regulatory T cells and Th17 cells.

BACKGROUND AND PURPOSE: Dietary indole derivatives, indole-3-carbinol (I3C) and diindolylmethane (DIM), possess anti-cancer properties and exhibit the characteristics of aryl hydrocarbon receptor (AhR) ligands. Because AhR activation has recently been shown to regulate T cell differentiation, we tested the hypothesis that I3C and DIM may mediate anti-inflammatory properties by promoting regulatory T cell (T-regs) differentiation while inhibiting Th17 cells. EXPERIMENTAL APPROACH: We investigated the therapeutic efficacy of I3C and DIM against experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). The efficacy was evaluated based on clinical scores of paralysis, histopathology, serum cytokines and infiltration of T cells in the CNS. We next studied the mechanism of induction of T cells against myelin oligodendrocyte glycoprotein (MOG35₋55 ) peptide, both in vivo and in vitro, specifically investigating the differentiation of T-regs and Th17 cells, and determined if indoles were acting through AhR. KEY RESULTS: Pretreatment of EAE mice with I3C or DIM completely prevented the clinical symptoms and cellular infiltration into the CNS. Also, post-treatment of EAE with I3C or DIM proved highly effective in curtailing the overall severity of the disease. In addition, I3C or DIM promoted the generation of T-regs, while down-regulating the induction of MOG-specific Th17 cells. The regulation of FoxP3 induction and suppression of Th17 cells by indoles in vivo and in vitro were found to be AhR-dependent. CONCLUSIONS AND IMPLICATIONS: Together, our studies demonstrate for the first time that I3C and DIM may serve as novel therapeutics to suppress neuroinflammation seen during MS through activation of AhR.

The transcriptional targets of mutant FOXL2 in granulosa cell tumours.

BACKGROUND: Despite their distinct biology, granulosa cell tumours (GCTs) are treated the same as other ovarian tumours. Intriguingly, a recurring somatic mutation in the transcription factor Forkhead Box L2 (FOXL2) 402C>G has been found in nearly all GCTs examined. This investigation aims to identify the pathogenicity of mutant FOXL2 by studying its altered transcriptional targets. METHODS: The expression of mutant FOXL2 was reduced in the GCT cell line KGN, and wildtype and mutant FOXL2 were overexpressed in the GCT cell line COV434. Total RNA was hybridised to Affymetrix U133 Plus 2 microarrays. Comparisons were made between the transcriptomes of control cells and cells altered by FOXL2 knockdown and overexpression, to detect potential transcriptional targets of mutant FOXL2. RESULTS: The overexpression of wildtype and mutant FOXL2 in COV434, and the silencing of mutant FOXL2 expression in KGN, has shown that mutant FOXL2 is able to differentially regulate the expression of many genes, including two well known FOXL2 targets, StAR and CYP19A. We have shown that many of the genes regulated by mutant FOXL2 are clustered into functional annotations of cell death, proliferation, and tumourigenesis. Furthermore, TGF-ß signalling was found to be enriched when using the gene annotation tools GATHER and GeneSetDB. This enrichment was still significant after performing a robust permutation analysis. CONCLUSION: Given that many of the transcriptional targets of mutant FOXL2 are known TGF-ß signalling genes, we suggest that deregulation of this key antiproliferative pathway is one way mutant FOXL2 contributes to the pathogenesis of adult-type GCTs. We believe this pathway should be a target for future therapeutic interventions, if outcomes for women with GCTs are to improve.

FOXP3+ regulatory T cells: Current controversies and future perspectives.

Regulatory T cells (Treg) provide protection from autoimmune disease, graft-versus-host disease, transplant rejection and overwhelming tissue destruction during infections. Conversely, high Treg numbers enable cancer cells to evade the host immune response. Thus, Treg are seen as an important tool to manipulate the immune response. However, as the immunological community is trying to move this knowledge from mice to humans, contradictory results regarding the number and function of Treg in various diseases are appearing. This problem arises because we cannot clearly define Treg populations on the basis of expression of CD25 and other cell surface markers in humans. This review addresses the utility of the FOXP3 forkhead transcription factor for the identification of Treg populations and summarizes recent data on the expression of FOXP3 in lymphomas. It is crucial to really understand Treg biology before attempting therapies, including (i) the injection of expanded Treg to cure autoimmune disease or prevent graft-versus-host disease or (ii) the depletion or inhibition of Treg in cancer therapy. For instance, new data arising from the study of haematological malignancies highlight the additional complexity of systems where malignant cell populations may also be direct Treg targets.