Finding a source from which high-energy-density biofuels can be derived at an industrial scale has become an urgent challenge for renewable energy production. Some microorganisms can produce free fatty acids (FFA) as precursors towards such high-energy-density biofuels. In particular, photosynthetic cyanobacteria are capable of directly converting carbon dioxide into FFA. However, current engineered strains need several rounds of engineering to reach the level of production of FFA to be commercially viable; thus new chassis strains that require less engineering are needed. Although more than 120 cyanobacterial genomes are sequenced, the natural potential of these strains for FFA production and excretion has not been systematically estimated.FFA SC (FFASC), an in silico screening method that evaluates the potential for FFA production and excretion of cyanobacterial strains based on their proteomes. A literature search allowed for the compilation of 64 proteins, most of which influence FFA production and a few of which affect FFA excretion. The proteins are classified into 49 orthologous groups (OGs) that helped create rules used in the scoring/ranking of algorithms developed to estimate the potential for FFA production and excretion of an organism. Among 125 cyanobacterial strains, FFASC identified 20 candidate chassis strains that rank in their FFA producing and excreting potential above the specifically engineered reference strain, Synechococcus sp. PCC 7002. We further show that the top ranked cyanobacterial strains are unicellular and primarily include Prochlorococcus  and marine Synechococcus  that cluster phylogenetically. Moreover, two principal categories of enzymes were shown to influence FFA production the most: those ensuring precursor availability for the biosynthesis of lipids, and those involved in handling the oxidative stress associated to FFA synthesis.Here we present the in silico method to screen cyanobacteria proteomes for their potential to produce and excrete FFA, as well as the first attempt to parameterize the criteria derived from genetic characteristics that are favorable/non-favorable for this purpose. Thus, FFASC helps focus experimental evaluation only on the most promising cyanobacteria.To our knowledge FFASC is the first The online version of this article (doi:10.1186/s12864-016-3389-4) contains supplementary material, which is available to authorized users. As potential feedstock for renewable energy, the use of microbes that produce free fatty acid (FFA) has been strongly suggested [Escherichia coli (E. coli) for FFA production [E. coli produces FFA, it requires fixed carbon sources that are too costly to be exploited as feedstock. As an alternative, lignocellulosic biomass was also considered as a feedstock, however this process demands huge amounts of fresh water and farmland [The grand challenges of the 21uggested . Substanoduction –9. Howevfarmland , 11. Thufarmland .Synechococcus sp. PCC 7002 [Synechocystis PCC 6803 [Synechococcus elongatus PCC 7942 [Arthrospira (Spirullina) platensis NISE-39 [Synechocystis PCC 6803 has received the most research attention because of its ability to grow photoautrophically and heterotrophically. Moreover, it was the first cyanobacterial genome to be completely sequenced [E. coli) [Saccharomyces cerevisiae) [S. cerevisiae) [E. coli) [Synechocystis sp. PCC 6803) [Synechocystis sp. PCC 6803) [There are several aspects to consider when evaluating the potential of a cyanobacterial strain as a candidate chassis strain for FFA production in the context of biofuel production. Some of these aspects include: 1/native biosynthetic capability for FFA production and excretion, 2/environmental robustness, 3/strain turnover rate, 4/the necessary gene expression levels, 5/metabolic fluxes, and 6/established genetic engineering tools. The primary aspect to consider is the strain’s natural potential to produce and excrete FFA, as when this potential is weak the strain would be considered as less useful. For simplicity in what follows we will refer to ‘FFA production and excretion’ as ‘FFA production’. In cyanobacteria, fatty acids are synthesized via the type II fatty acid synthases (FAS). Focal to fatty acids synthesis are acyl carrier protein (ACP) that covalently binds all fatty acyl intermediates during the synthesis process. Fatty acid synthesis represents a central, conserved process by which acyl chains are produced and core enzymes required for fatty acids initiation and elongation are well characterized , 13. FFAPCC 7002 , SynechoPCC 6803 , 15, 16,PCC 7942  and Arth NISE-39 , 19. Of equenced , 21. Curequenced . HoweverE. coli) , 2,3-butevisiae) , succinaevisiae) , malonylE. coli) , acetyl-CC 6803) , ethanolCC 6803) , constraCC 6803) , OptKnocCC 6803) , OptGeneCC 6803)  and CiEDCC 6803)  have beeExperimental evaluations , 13, 17 in silico screening method FFASC. FFASC estimates and ranks the potential of cyanobacterial strains for FFA production, and hence indirectly biofuel production, based on their predicted proteomes. FFASC has been established based on: 1/a compilation of protein orthologous groups  that impact FFA production; 2/a compilation of relevant assessment criteria; 3/the development of an algorithm that uses the criteria derived from OGs to rank candidate chassis strains based on their estimated potential to produce and excrete FFA. We used FFASC to screen and rank cyanobacterial proteomes for this purpose and indirectly screen their potential as candidates for cyanobacterial biofuel cell factories. The FFASC ranking for the top candidates is supported by their phylogenetic relationship, and by additional indirect in silico evidence. Thus, our study suggests that FFASC allows selecting the most promising candidates for experimental validation, whereas the established selection criteria might provide useful insight for efficient metabolic engineering. Moreover, although the methodology developed in our study is focused on FFA production, it can be applied in a similar way to other processes  as well as to other bacteria, fungi or plants.In this study we provide such an E. coli and diatoms [The common procedures used to enhance the biotechnological production of FFA include the introduction of heterologous pathways, as well as the modification of the candidate cell factory metabolism via deletion of genes or enhancing gene expression. However, genetic engineering was not based on the consideration of the collective effects of different criteria that characterize a good cyanobacterial cell factory for FFA production, even though experimental outcomes have shown that not all cyanobacteria are suitable producers , 14, 32. diatoms –44. ThroAcyl-ACP synthetase/long-chain-fatty-acid CoA ligase (AAS/FadD) is an example of one of the cyanobacterial proteins from this group. Kaczmarzyk and Fulda [aas knockout mutants for Synechocystis sp. PCC 6803 and S. elongatus PCC 7942 (strain SE01) exhibited increased secretion of FFA into the culture medium compared to the wild-type strains [aas gene negatively impacts the efficiency of the candidate cell factory.In total, we identified 13 OGs (based on reported knockout or knockdown experiments) whose presence in the organisms negatively impacts FFA production. These proteins we collectively named nOG  is an example from this group. It was previously demonstrated that TesA cleaves the acyl-carrier-protein from the FA moiety, and in this manner increases FA biosynthesis in E. coli by reducing feedback inhibition [E. coli-derived truncated thioesterase (‘tesA) and inserted it into the S. elongatus PCC 7942 genome along with the aas knockout, thereby generating a mutant strain SE02. SE02 produced a higher percentage of saturated FFA and a lower percentage of unsaturated FFA compared to the wild type [tesA positively impacted the efficiency of the biofuel production. The remaining 12 OGs identified are required for FA production, but are not included in pOG, and we named them rOG (‘required OGs’). The difference between these two groups is that rOGs are essential for FFA production, while pOGs can be considered as ‘enhancers’.Based on reported gene insertion and overexpression experiments, we also identified 24 OGs that contain proteins whose presence in the organisms positively impacts FFA production capability  platensis NISE-39. Thus, our estimated weights are skewed and not optimal. However, they still provide better qualitative ranking of species for FFA production potential than in the case when all weights are assumed to be equal . In order to estimate an organism’s potential for FFA production, we used all of these derived criteria to generate an overall score that reflects FFA potential. For this purpose we developed FFASC. Our optimization process through which we estimated the optimized weights of the criteria used, is based on two species, To evaluate the FFA production potential of the 120 cyanobacterial strains that have not been considered for FFA/biofuel production and the five cyanobacterial strains included in the reference dataset, the proteomes of all 125 cyanobacterial strains were screened using FFASC. The number of protein hits obtained from the sequence homology and domain search were used as an input to generate the OG hit numbers associated with each OG, and then applied to the derived set of criteria  to predict suitability of cyanobacterial strains for FFA production. The strains were ranked based on the sum of scores generated by all criteria. The higher the score, the better the rank  are placed in one major clade along with cyanobacterial positive reference strains, while the diatoms, used as an outgroup needed for hierarchical clustering, are placed in a clade of their own. Also, the negative reference strains do not mix with the clade that contain the top ranked strains, that is, the heatmap shows a clear separation between these clades. Moreover, the major clade that contains the top ranked strains generally has a higher number of pOGs (represented by the reddish shaded area) and lower numbers of nOGs (represented by the greenish shaded area), which contrasts with the clade in which negative reference strains are placed. Taken together, the clade with top ranked strains displays more favorable traits for FFA production based on the 49 OGs assessed.The subsequent list of ranked cyanobacterial strains demonstrates that the positive reference strains rank above the negative reference strains. However, they are not the top ranked strains. The positive reference strains A more in depth assessment of the weights assigned to the 49 OGs  association required for incorporation into SEED models. For the four enzymes missing from FFASC, another modification made by Liu et al. include the knockout of the slr1710 (PBP2) gene responsible for peptidoglycan layer assembly [slr1710 is known to be present in Synechocystis sp. PCC 6803, and is correctly found by both Model SEED and FFASC. However, we found that Model SEED identified slr1710 in 22 additional cyanobacterial strains, whereas FFASC only identified slr1710 in 11 additional cyanobacteria screened. We found FFASC filtered out the other slr1710 hits as a consequence of the stringent protein-domain condition applied to increase the accuracy underlying FFASC predictions, that is, only homologous protein sequences that have all domains of the associated protein from the group of 64 proteins were recorded as OG hits. Moreover, all the core enzymes of the general fatty acid biosynthesis pathway were identified using FFASC, whereas Model SEED did not identify FabZ due to the lack of GPR association required for incorporation into SEED models. Here, the differences between Model SEED and FFASC are a consequence of: 1/Model SEED is a generic method in which all pathways are treated equally, whereas FFASC is specialized and focuses on FFA production and is built based on proteins known to either positively or negatively affect FFA production; 2/Model SEED provides the presence or absence of the enzymes, whereas FFASC takes the copy number into account when assessing potential for FFA production; and 3/FFASC include all proteins (not just enzymes) that directly or indirectly affect FFA production. Taken together, FFASC is more refined in assessing the “natural” cyanobacterial strains potential for FFA production, whereas Model SEED was developed for a more generic purpose.To analyze this data, we tabulated the engineered genes in model organisms e et al.  made sixthetases . This shhat RAST  correctlassembly . This shk clusters, where distance of species within a single cluster is minimized and distance between clusters or cluster centers is maximized, a value for k has to be set in away that reflects the natural groupings. That is, if k is too small, the clustering algorithms will reduce the total number of groups to the specified value of k, which forces some natural clusters to combine, thereby producing artificial fusions [k is too large, natural clusters will start dividing in an artificial way, to match the specified k value.To provide additional support that the predictions obtained by FFASC are reasonable, we used K-means clustering  based on fusions . Likewisk = 6 and k = 7, the point at which diatoms start to separate is when the number of clusters is k = 8. Additionally, using an average silhouette width as the measure of ‘natural’ clustering [k = 6, 7 or 8, the highest average silhouette width of 0.41  index for k = 6 (67.43), k = 7 (56.91) and k = 8 (61.89) . CH index results verify that k = 6 is the appropriate cluster number. A visual illustration of the case k = 6 , k = 7 6.91 and Bacillariales, Thalassiosirales and Naviculales), while the 125 cyanobacterial strains are classified under only seven orders, namely Chroococcales, Gloeobacterales, Nostocales, Oscillatoriales, Pleurocapsales, Prochlorales and Stigonematales   and marine Synechococcus , which are reported to have diverged from common ancestry [Prochlorococcus genome is further thought to have ‘streamlined’ [Synechococcus and other cyanobacteria is larger than Prochlorococcus genome sizes [Prochlorococcus from Synechococcus is their divergent light-harvesting strategies [Synechococcus uses the phycobilisome as their light-harvesting antenna that are not found in Prochlorococcus. These phycobilisome antenna systems are used by Synechococcus to adjust to changes in temperature, likely contributing to its greater geographical occupancy range [Prochlorococcus main light-harvesting antenna complex is made up of divinyl chlorophyll a and b, prochlorophyte chlorophyll-binding protein (Pcb), as well as accessory pigment [Prochlorococcus to warmer, oligotrophic oceans [Prochlorococcus is reported to be a leading example of a naturally 'streamlined' genome [Prochlorococcus can be inexpensively cultivated using seawater [This result is supported by literature, since the top ranked cyanobacterial strains primarily include ancestry . Followiamlined’ , thus, tme sizes . Anotherrategies : Synechocy range , 63. Ins pigment , 64. Colc oceans . Since P' genome , 66, thiseawater .Chroococcales, including Synechococcus PCC 7002, Synechocystis PCC 6803 and S. elongatus PCC 7942, were engineered, and demonstrate the production and secretion of FFA, which provides proof-of-concept. However, none of the predicted top ranked strains of the order Chroococcales has been shown to produce FFA. Nonetheless, Synechococcus UTEX 2973 , has recently been reported to be a fast growing chassis strain for biosynthesis using light and carbon dioxide, growing two times faster than S. elongatus PCC 7942 [Prochlorococcus strain MIT9313 produces lipid-containing vesicles that are released into the surrounding seawater. These released lipid-containing vesicles maybe collected without disturbing the growth of the Prochlorococcus, as opposed to other cyanobacteria or algae that require destroying one batch of cells and starting with a new batch, to retrieve lipids. FFASC ranked the Prochlorococcus strain MIT9313 at position 41, suggesting that if the MIT9313 mechanism is a Prochlorococcal trait, there are several other possible vesicle-releasing Prochlorococcus strains that may be a better chassis for FFA production. Moreover, the fact that the candidate chassis strains are clustered primarily in orders Synechococcus and Prochlorococcus, is a welcomed surprise that could constitute an additional criterion for positive prediction.Reference strains of the order PCC 7942  have repProchlorococcus marinus, Candidatus Atelocyanobacterium thalassa (isolate ALOHA), Synechococcus sp. CB0101, Synechococcus sp. RS9917, Thermosynechococcus elongates BP-1, Synechococcus sp. WH 8109, Synechococcus sp. WH 5701, Thermosynechococcus sp. NK55, Synechococcus sp. JA-3-3Ab and Synechococcus sp. CB0205, as potentially favorable chassis FFA producers. It would also be reasonable to consider other strains with a phylogenetic closeness to the above strains as potential FFA producers as well. Moreover, the methodology developed can be adopted for other metabolic production, and for other species.In this study we developed FFASC, a first screening method that ranks the potential of candidate cyanobacteria for FFA production and excretion based on favorable/non-favorable genetic characteristics. Ranking the candidate species enables narrowing the experimental focus on more likely candidates for good FFA producers. Thus FFASC might prove a useful tool in highlighting candidate strains for industrial-scale biofuel production . The outcome of this analysis suggests unicellular cyanobacterial species such as We plan to follow-up this research by: 1/expanding the orthologous group to other cyanobacterial genes that are closely related to FFA production such as CO2-fixation, photosynthesis, cell division, environment tolerance genes and 2/develop the FFASC database to classify and evaluate the FFA production potential of cyanobacterial strains based on their proteomes.Escherichia coli, cyanobacteria, algae, diatoms, plants, and yeast : these OGs contain proteins whose encoding genes have been knocked out or knocked down during genetic engineering experiments to increase the organisms’ potential for FFA production.OGsthat positively impact FFA production (pOG): these OGs contain proteins whose encoding genes have been inserted or forced to overexpress to increase the organisms’ potential for FFA production.Required OGs (rOG): these are a set of proteins required for FA production, not included in pOG.The OGs were categorized as follows .Lyngbya sp. PCC 8106 in the control set. This strain is not engineered, but it produces biodiesel, although less than S. elongatus PCC 7942 [Synechococcus sp. PCC 7002 [, Synechocystis PCC 6803 [S. elongatus PCC 7942 [Lyngbya sp. PCC 8106 [A. platensis NISE-39 [Phaeodactylum tricornutum [Thalassiosira psedonana [Fragilariopsis cylindrus [Our control dataset includes cyanobacteria that have been genetically engineered for FFA/biofuel production. The connection to FA production is that the biodiesel is produced from triacylglycerols that are synthesized from three FAs joined together by one glycerol molecule. However, since there are not many cases of engineered cyanobacteria for FA production, we have also included cyanobacteria PCC 7942 . CyanobaPCC 7002 , SynechoPCC 6803 , 15, 16,PCC 7942 . On the PCC 8106  and A. p NISE-39 , 19  of C represents hitN of j-th OG in i-th species.The number of BLASTp hits of all proteins from an OG to the proteome of a species represents an OG hit number (hitN). hitNs are used to define criterion for the OGs. In determining hitNs, only proteins matched by BLASTp that have all domains of the source protein were used. One can conveniently describe species and OGs in terms of hitNs as follows. Suppose that The quantification rules are defined as follows. Proteins from nOGs receive the values equal to “–hitN” that correspond to the considered species and the OG. Proteins from pOGs receive the values of “hitN” that correspond to the considered species and the OG. If, however, a pOG has “hitN = 0”, then we assign to it a value of “-1” as a penalty. Proteins from rOGs receive the values of “hitN” that correspond to the species and the OG Fig. .Fig. 8Fli to produce biofuel based on this approach will be described as:Consequently, the score that would quantify the potential of species c is an element of C. While this is not the only possible way to calculate this score we find it simple and suitable. Note that in (where  that in  we assumRanking Algorithm (Algorithm 1)C matrix and use it as the input to the algorithm. This algorithm evaluates each of the considered species and generates scores according to  of the global optimization toolbox in MATLAB. For the PSS, a generalized pattern search algorithm was used with default values. The optimized solutions for the weights found by the optimizer were between 0.001 and 1, where p (ranking effect coefficient) is equal to 0.010241 at 1744 iterations, with the objective function value at the solution equal to 0.0232 (convergence level). The proposed objective function to achieve our goal is based on maximizing the difference in scores for the two species used; Synechocystis sp. PCC 6803 and A. platensis NIES.39 as defined below:In Algorithm 1 we assume that all criteria considered have the same level of influence to the potential of an organism for FFA production as expressed through Equation  added to the set we considered. The pseudocode of Algorithm 2 that describes ranking based on score determined by )*10^-10)by adding a small level of noise to avoid numerical problems with singular matrices.The EC numbers corresponding to the 49 OGs were used for comparison with Model SEED. In addition, we submitted 25 cyanobacteria  to the Model SEED resource  and obtained the SEED metabolic models and corresponding genome annotations. Similarly, we had binary (presence/absence) output from our FFASC method. We compared the identified EC numbers of 41 OGs in both models and generated the comparison data for Model SEED and FFASC with binary values (0/1) . The 16S rRNA sequences for the 124 strains and outgroup were aligned using MAFFT (Multiple Alignment using Fast Fourier Transform) [RAxML (Randomized Axelerated Maximum Likelihood), with default parameters and 1000 bootstrap runs for the GTRCAT substitution model [In order to see if the ranking obtained as described above reflects any phylogenetic similarities, we performed phylogenetic analyses of cyanobacteria. We used 16S rRNA sequences for the 124 cyanobacterial strains retrieved from INDIGO . Synechoansform)  with defansform) . A maximansform)  was thenon model . The maxon model  and editon model .k clusters, we established 1/the point at which artificial fusions are omitted, that is, when diatoms fall into a separate cluster (determined to be where k = 6) and 2/the point at which the natural clusters are divided in an artificial way, that is, when diatoms start to separate into individual clusters (determined as k = 8). Thus, based on the properties of the dataset, natural clustering was found to range from cluster 6 to 8   was used


In Caenorhabditis elegans, mild developmental mitochondrial stress activates UPRmt reporters and extends lifespan. We show that similar developmental stress is necessary and sufficient to extend Drosophila lifespan, and identify Phosphoglycerate Mutase 5 (PGAM5) as a mediator of this response. Developmental mitochondrial stress leads to activation of FoxO, via Apoptosis Signal-regulating Kinase 1 (ASK1) and Jun-N-terminal Kinase (JNK). This activation persists into adulthood and induces a select set of chaperones, many of which have been implicated in lifespan extension in flies. Persistent FoxO activation can be reversed by a high-protein diet in adulthood, through mTORC1 and GCN-2 activity. Accordingly, the observed lifespan extension is prevented on a high-protein diet and in FoxO-null flies. The diet-sensitivity of this pathway has important implications for interventions that seek to engage the UPRmt to improve metabolic health and longevity.The mitochondrial unfolded protein response (UPR A wide range of studies in genetically accessible model systems have led to the realization that aging is a malleable process, responsive to both genetic and pharmacological interventions. An integrated view of the aging process has emerged from these efforts, spurred by the identification of a select group of biological processes and pathways that drive, influence, and regulate the physical decline characteristic of the aging process . Many ofmt) has emerged as a unifying mechanism for several of these pathways  and endoplasmic reticulum unfolded protein responses  was overexpressed, resulting in upregulation of mitochondrial chaperones and induction of mitophagy  . In Droitophagy . Furtherhagy (mt . HoweverO (FoxO) . FoxO acO (FoxO) .mt has also been implicated in the lifespan-extending effects of various drugs, including resveratrol  is required to activate a pathway that includes ASK1, JNK, Relish and FoxO and promotes protective gene expression in response to mitochondrial stress. Activation of this pathway during development leads to persistent FoxO activation and increased expression of chaperones in adult flies, and is required for longevity. We further find that lasting FoxO activation is sensitive to dietary conditions, as it can be abolished by elevated protein intake and elevated mTORC1 and GCN-2 activity. Our findings identify a new diet-sensitive pathway of lifespan regulation by mitochondrial stress. Since the identified pathway components are evolutionarily conserved, we anticipate that these results inform our understanding of similar interactions in vertebrate systems, including humans.Here, we have explored the signaling pathway regulating the transcriptional response to mitochondrial proteostatic stress in mt in Drosophila, we sequenced mRNA extracted from adult female thoracic tissue after 24 hr of mitochondrial stress. We induced mitochondrial stress ubiquitously, using GeneSwitch for temporal control, either by overexpressing a misfolding variant of the human mitochondrial enzyme ornithine transcarbamylase (ΔOTC) (mt in Drosophila (Toll and Imd pathways.To gain an overview of the gene expression changes induced by the UPRe (ΔOTC)  or by kne (ΔOTC) . 23% of osophila . The maiosophila . Activatmt, under conditions used in earlier studies , and further demonstrated that transcriptional induction of these genes was specifically triggered by mitochondrial stress and not by conditions that trigger the cytoplasmic- (heat shock) or endoplasmic reticulum  unfolde studies . As furt studies . Initialmt has previously been noted in worms, where both immune and proteostatic gene induction is dependent on the ATFS-1 transcription factor  as a reporter of mitochondrial stress. Knocking down the transcription factors foxo and Relish/NF-κB, but not jun or kayak/Fos, strongly suppressed Mtk induction (Drosophila JNK basket (bsk) would prevent Mtk induction. Indeed, Mtk induction by mitochondrial stress was significantly impaired in bsk loss-of-function conditions  experiencing mitochondrial stress confirmed this finding , had been reported to interact with and be activated by the mitochondrial Serine/Threonine phosphatase Phosphoglycerate Mutase 5 (PGAM5) . Knockin factors . The lac finding . To detend hsp10 .PGAM5 or ASK1 also play a role in canonical pathways for antimicrobial peptide (AMP) induction, and measured induction of Mtk following exposure to the pathogenic Pseudomonas entomophila. Ubiquitously knocking down either PGAM5 or ASK1 had no effect on this response  driver to express ΔOTC throughout larval development only, and measured adult lifespan. As shown in C. elegans, ΔOTC expression limited to adulthood did not lead to extended lifespan  to eliminate microbes, then transferred to a sterile hood and reared on autoclaved food throughout development and adult life. In the second condition, we added a cocktail of antibiotics shown to eliminate all culturable strains of the microbiome  was lethal, but expressing bskDN simultaneously with ΔOTC in adult flies limited FoxO nuclear translocation  during developmental ΔOTC expression, however, did not affect FoxO nuclear localization, and neither Sir2 overexpression nor supplementation with the NAD precursor nicotinamide riboside were sufficient to induce nuclear translocation of FoxO  precursors activate the UPR sir-2.1 . Knockin of FoxO . Drosophby HDAC4 , but knoby HDAC4 .Drosophila Insulin-like Peptide (dilp) family  for 5 days did not abolish the persistent nuclear localization of FoxO caused by developmental ΔOTC expression  family . During pression .mt in C. elegans , ASK1 RNAi (#32646), 21foxo (Δ94foxo (bskDN (#6409).The following lines were obtained from the Bloomington , foxo21 , foxoΔ94, PGAM5 RNAi (#51657), ASK1 RNAi (#110228), TSC1 RNAi (#110811), bsk RNAi (#34138), foxo RNAi (#30556).The following lines were obtained from the Vienna 1PGAM5  at 1:500. Secondary antibodies were from Jackson Immunoresearch. TUNEL staining was performed using In Situ Cell Death Detection Kit (Roche). DNA was stained using Hoechst, and tissue mounted on slides using Mowiol mounting medium (Sigma). Imaging was done on a Zeiss LSM700 confocal microscope at 40x magnification.Fat bodies were dissected from adult females or from third instar larvae pre-wandering. Tissue was fixed at room temperature for 45 min in 100 mM glutamic acid, 25 mM KCl, 20 mM MgSO4, 4 mM Sodium Phosphate, 1 mM MgCl2, 4% formaldehyde. Washes were done in PBS with 0.5% BSA, 0.1% Triton X-100 at 4°C. Primary incubation was done overnight 4°C using a rabbit N-terminal FoxO antibody (RRID:All assays were done on samples of five whole flies/larvae. NAD and NADH levels were quantified from whole flies using the NAD/NADH Quantitation Colorimetric kit from BioVision. Glucose and glycogen were quantified using the Glucose (HK) Assay kit from Sigma. Lipids were quantified with the Triglyceride LiquiColor kit from Stanbio. Protein levels were quantified with the BCA assay.P. entomophila and P. aeruginosa were cultured in LB medium for 16 hr at 37°C and 10 hr at 30°C, respectively. For oral infections, flies were starved for 2 hr before treatment. 6 mL culture was pelleted at 5000 g for 10 min, then resuspended in 500 uL 5% sucrose and added to a vial containing Whatman filter paper. 20 flies per vial were maintained at 29°C, and deaths tracked daily or twice daily. 100 uL 5% sucrose was added to the vials each day. For humeral infections, a tungsten needle was dipped in 30x concentrated culture and flies were pricked in the periphery of the abdomen. They were then transferred to normal food vials. Negative controls were fed/pricked with 5% sucrose alone.RP49.Muscle tissue was isolated by removal of head, abdomen, wings, legs and intestine from fly thoraces. Fat bodies were manually isolated from fixed abdomens/larvae. 5–8 flies were used per sample, and total RNA was extracted from using Trizol (Life Sciences) according to manufacturer’s protocol. cDNA was synthesized using Superscript III (Life Sciences). Results represent four biological samples, each with triplicate technical repeats. Real-time PCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) on a Bio-Rad Real-Time CFX96 system. Expression levels were calculated as ΔΔCt normalized to SCR_013194) and reads were mapped to Drosophila genome release 5.2. Expression was recorded as FPKM: fragments per kilo-base per million reads.Muscle or fat body tissue was isolated and RNA extracted as described above, using 15 flies per sample. TruSeq RNA Library Prep Kit (Illumina) was used to prepare libraries, and sequencing was performed on an Illumina MiSeq system. Raw data were analyzed with the Tuxedo suite (RRID:SCR_005825) to trim redundant terms (Allowed similarity: Medium).Significance in two-condition experiments was evaluated by student’s t-test. Multiple condition experiments were evaluated by one-way ANOVA, with Dunnett’s post-hoc comparing to induced sample or Tukey’s post-hoc when multiple conditions are compared. Gene Ontology analysis of RNAseq experiments was done at flymine.org, using REVIGO .Flies were washed in 70% EtOH for 60 s to kill external bacteria, and intestines dissected out in sterile PBS. Ten guts were collected per sample. Commensal genomic DNA was extracted using UltraClean Microbial DNA Isolation Kit (MO BIO). This DNA was used as template for limited cycle PCR with primers targeting V3/V4 regions (Forward 5’-CCTACGGGNGGCWGCAG-3’ and Reverse 5’-GACTACHVGGGTATCTAATCC −3’) to generate a 16S metagenomic sequencing library. The following reaction conditions were used: 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 48°C for 2 min, and 72°C for 2 min, and a final extension at 72°C for 5 min. Illumina Miseq paired-end (2 × 300 bp) sequencing was performed and Miseq Reporter Software was used for primary analysis and classification based on Greengenes database  and three reviewers, one of whom, Utpal Banerjee, is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Edward Owusu-Ansah (Reviewer #2).Thank you for submitting your article "PGAM5 promotes lasting FoxO activation after developmental mitochondrial stress and extends lifespan in The reviewers have discussed the reviews with one another and appreciate the importance of the findings and the overall quality of the data. The summary of these reviews and discussions are summarised below by the Reviewing Editor.A set of specific issues raised by the reviewers that we believe can be addressed by new experiments and/or rewriting the manuscript are summarized below as "other concerns". However, there is one major issue that needs special attention. During the post-review discussions, the reviewers unanimously agree that the data lack direct evidence for a sustained FOXO activation .Summary:Drosophila development leads to a stress response that has lasting effects on the flies, most notably on extension of lifespan. The authors characterize the steps that signal mitochondrial dysfunction to modulate nuclear gene expression, using an immune response gene as an output. In adult flies that were subjected to mitochondrial stress during development, a subset of stress response genes are expressed ; it is proposed that these genes  mediate lifespan extension. The lasting effects of developmental mitochondrial dysfunction can be reversed through exposure to a high protein diet during adulthood.In this paper Jensen et al. investigate how mitochondrial dysfunction during Essential revisions:(major concerns):The key weakness regards the study's (implied) inference on the role of lasting FoxO activation as the longevity mechanism. Epistasis analysis is needed to test this idea.The important required study is to conduct mtUPR (in any way) in FoxO-null epistasis. Alternatively, the authors could test the effect of co-expression of the OTC construct and RNAi to FoxO, using the gene-switch system. This approach allows the genotype without RU to serve as its own co-isogenic control. One needs to measure the longevity of the OTC expression alone, using geneswitch again, to confirm there is a longevity benefit in that period. And, one should do the same by driving the FoxO RNAi with the same geneswitch .Essential revisions:(other concerns):mt stress response per se? How much is due to a more general response caused by mitochondrial dysfunction. This is unclear because of the nature of mitochondrial stressors. ND75 knockdown will cause mitochondrial dysfunction . The ΔOTC mutant in flies also severely impacts mitochondrial function . Therefore, it is not clear that a UPRmt or some other type of mitochondrial stress response is being studied. Presumably studies in worms, where chaperone up-regulation has clear linkage to UPRmt provides the inspiration, but this is not yet in flies. For example, ΔOTC causes ETC dysfunction in flies . If this is not true for this system, then some functional data are required, or the description of the model should be modified.1) Are phenotypes due to a UPRmt pathway ? If a chaperone important in the UPRmt response affecting proteostasis directly was chosen as a marker rather than a gene involved in the immune response, then the relation of this new signaling pathway to UPRmt could be better assessed. Either a more specific UPRmt output marker should be used, or discussion of UPRmt should be reserved for the Discussion.2) Is the PGAM5/ASK/JNK signaling pathway a bona fide UPRmt, such as Hsp60, Hsp10, mortalin (mt-Hsp70) and the ClpP protease are induced under the conditions used to activate the UPRmt in this study. The authors should comment on this, and provide a possible explanation for this apparent disparity.3) In the same vein, it is unclear whether the canonical markers of the UPR4) The exact dosages of RU456 used in the gene-switch experiments should be listed in the figure legends.mt and longevity is not yet settled, and while this work provides important advancements, the authors will do well to tone down the enthusiasm with which they link expression of the OTC construct with UPRmt induction. OTC induces other genes as well . Therefore several bold statements such as the subheading "UPRmt-mediated longevity is not caused by improved adult immune function, or by changes to the microbiome" and "Developmental UPRmt affects the metabolic state of adult flies and leads to persistent FoxO" or (based on point 1 above), "Of the two methods initially used to induce the UPRmt, we opted to use ΔOTC for further experiments to avoid potential secondary effects of inducing ETC dysfunction" should be modified to reflect the fact that the results observed are due to OTC expression and not necessarily all due to the UPRmt.5) The link between UPR6) ADaGSxOTC itself yields less than 50% eclosion. Survivors of this cohort may live longer because of frailty selection: weak larvae that would produce shorter-lived adults do not eclose. Some rescue-type data  argues amt induced in adults activates FoxO, but such cohorts are not long-lived when RU is constantly applied. That FoxO is only transiently activated when RU is given for just one week is not a satisfactory explanation.7) UPRmt impact on adult longevity.8) The data with HPD is unclear. Presumably, HPD resets both this TF and the lifespan, and this is used to infer causality between the induced longevity and FoxO activation. But in the sole survival experiment to this point , the sha Essential revisions:(major concerns):The key weakness regards the study's (implied) inference on the role of lasting FoxO activation as the longevity mechanism. Epistasis analysis is needed to test this idea.The important required study is to conduct mtUPR (in any way) in FoxO-null epistasis. Alternatively, the authors could test the effect of co-expression of the OTC construct and RNAi to FoxO, using the gene-switch system. This approach allows the genotype without RU to serve as its own co-isogenic control. One needs to measure the longevity of the OTC expression alone, using geneswitch again, to confirm there is a longevity benefit in that period. And, one should do the same by driving the FoxO RNAi with the same geneswitch .Δ94/21 transheterozygotes already described in the manuscript). In parallel, we replicated the DaGS x ΔOTC lifespan experiment already included in the paper. In support of our model, developmental mitochondrial stress leads to a significant shortening of lifespan in FoxO null flies, in sharp contrast to the lifespan extension observed in normal flies. The FoxO null data is presented in the revised We agree that this is an important experiment. Per the reviewer’s suggestions, we have now carried out duplicate lifespan experiments where DaGS was used to drive ΔOTC expression in a FoxO null background :mt stress response per se? How much is due to a more general response caused by mitochondrial dysfunction. This is unclear because of the nature of mitochondrial stressors. ND75 knockdown will cause mitochondrial dysfunction . The ΔOTC mutant in flies also severely impacts mitochondrial function . Therefore, it is not clear that a UPRmt or some other type of mitochondrial stress response is being studied. Presumably studies in worms, where chaperone up-regulation has clear linkage to UPRmt provides the inspiration, but this is not yet in flies. For example, ΔOTC causes ETC dysfunction in flies . If this is not true for this system, then some functional data are required, or the description of the model should be modified.1) Are phenotypes due to a UPRmt is difficult in any organism. In our original manuscript, we decided to follow the nomenclature of previous studies . However, to our knowledge (and as suggested by the reviewers), all methods of inducing the UPRmt in any organism leads to mitochondrial dysfunction of some sort. It does not yet seem clear whether and which such dysfunctions should be classified as causative or part of the UPRmt. For example, some parts of the UPRmt have been characterized as ROS-dependent. We originally chose to define the UPRmtas any response occurring in two distinct types of mitochondrial proteostatic stress, but we agree with the reviewers’ point that the Drosophila UPRmt remains imperfectly described. We have thus rewritten several parts of the manuscript to refer to ‘mitochondrial stress’ rather than UPRmt.We agree that distinguishing general mitochondrial stress from a more specific UPRmt pathway ? If a chaperone important in the UPRmt response affecting proteostasis directly was chosen as a marker rather than a gene involved in the immune response, then the relation of this new signaling pathway to UPRmt could be better assessed. Either a more specific UPRmt output marker should be used, or discussion of UPRmt should be reserved for the Discussion.2) Is the PGAM5/ASK/JNK signaling pathway a bona fide UPRmt, such as Hsp60, Hsp10, mortalin (mt-Hsp70) and the ClpP protease are induced under the conditions used to activate the UPRmt in this study. The authors should comment on this, and provide a possible explanation for this apparent disparity.3) In the same vein, it is unclear whether the canonical markers of the UPRhsp-6 and hsp-60 in C. elegans respond strongly to induction of the UPRmt, Drosophila studies  have shown only moderate (~1.5-fold) induction of these genes. For this reason, the markers mentioned by reviewers were not included in the set of 3-fold upregulated genes in metchnikowin as our marker for subsequent screens due to its higher dynamic range of induction.Although the fluorescent reporters available for To address the pertinent comments by the reviewers, we have added a supplement to 4) The exact dosages of RU456 used in the gene-switch experiments should be listed in the figure legends.200 μm RU486 was used throughout, and this has now been added to figure legends.mt and longevity is not yet settled, and while this work provides important advancements, the authors will do well to tone down the enthusiasm with which they link expression of the OTC construct with UPRmt induction. OTC induces other genes as well . Therefore several bold statements such as the subheading "UPRmt-mediated longevity is not caused by improved adult immune function, or by changes to the microbiome" and "Developmental UPRmt affects the metabolic state of adult flies and leads to persistent FoxO" or (based on point 1 above), "Of the two methods initially used to induce the UPRmt, we opted to use ΔOTC for further experiments to avoid potential secondary effects of inducing ETC dysfunction" should be modified to reflect the fact that the results observed are due to OTC expression and not necessarily all due to the UPRmt.5) The link between UPRmt. In response to the comment that “OTC induces other genes as well .”, we note that Pellegrino et al. 2014 showed that AMPs are induced by ATFS-1 activation even absent mitochondrial stress, and were thus described as part of the core UPRmt in C. elegans.As noted in our response to comment #1, we have rewritten parts of the manuscript to refer to mitochondrial stress rather than UPR6) ADaGSxOTC itself yields less than 50% eclosion. Survivors of this cohort may live longer because of frailty selection: weak larvae that would produce shorter-lived adults do not eclose. Some rescue-type data  argues amt by constitutively active ATFS-1 in C. elegans also leads to reduced survival through development . However, our new FoxO null lifespan data shows that FoxO activity not only correlates with, but is required for lifespan extension. It is pertinent to this specific concern that while FoxO null mutants show strongly reduced survival through developmental ΔOTC expression  UPRWe apologize for not making this clear in the original manuscript, as indeed the transience of FoxO activation by mitochondrial stress does not explain why continuous mitochondrial stress in adults does not extend lifespan. Our interpretation is that the negative effects of chronically impairing mitochondrial function through expression of ΔOTC has detrimental effects that overshadow the improved proteostasis conferred by activating the FoxO pathway. This is consistent with observations by Pimenta-Castro et al. 2012, who show impaired mitochondrial function and survival with chronic adult ΔOTC expression. In the case of developmentally expressed ΔOTC, on the other hand, the mitochondrial stress is eliminated in adults while FoxO activation is retained chronically.mt impact on adult longevity.8) The data with HPD is unclear. Presumably, HPD resets both this TF and the lifespan, and this is used to infer causality between the induced longevity and FoxO activation. But in the sole survival experiment to this point , the shaAs mentioned above, we have now obtained additional evidence that FoxO activity is required for lifespan extension by developmental ΔOTC expression (new lifespan data for null mutants). We have edited the manuscript to merely note that the lack of extension on HPD is consistent with our demonstration that HPD blocks FoxO activation and with the fact that FoxO is required for lifespan extension by developmental mitochondrial stress.


Androgen receptor (AR) signaling remains the major oncogenic pathway in prostate cancer (PCa). Androgen-deprivation therapy (ADT) is the principle treatment for locally advanced and metastatic disease. However, a significant number of patients acquire treatment resistance leading to castration resistant prostate cancer (CRPC). Epigenetics, the study of heritable and reversible changes in gene expression without alterations in DNA sequences, is a crucial regulatory step in AR signaling. We and others, recently described the technological advance Chem-seq, a method to identify the interaction between a drug and the genome. This has permitted better understanding of the underlying regulatory mechanisms of AR during carcinogenesis and revealed the importance of epigenetic modifiers. In screening for new epigenomic modifiying drugs, we identified SD-70, and found that this demethylase inhibitor is effective in CRPC cells in combination with current therapies. The aim of this review is to explore the role of epigenetic modifications as biomarkers for detection, prognosis, and risk evaluation of PCa. Furthermore, we also provide an update of the recent findings on the epigenetic key processes  involved in AR expression and their possible role as therapeutic targets. Prostate cancer (PCa) is the most prevalent cancer in men and the third cause of cancer-specific mortality in Western countries . To undeHistone methylation is an important and complex method of transcriptional control mediated by histone methyltransferase (HMT) and histone demethylase (HDM) enzymes. Methylation changes to the local chromatin encourage or repress transcription according to the site of modification . For exaOne of the most extensively studied HMT enzymes in PCa is SET9, which seems to improve gene expression by inducing histone H3K4me1 and obstructing histone H3K9 methylation and the nucleosome remodeling deacetylase (NURD) complex ,20,21. DIL-6, IL-8, VEGFA and survivin in CRPC cells.The nuclear receptor-binding SET domain-protein 2 (NSD2) is a histone methyltransferase that cooperates with the DBD of the AR . High leHistorically, EZH2 has been considered an AR transcriptional repressor. This peculiarity has been related to the ability of EZH2 to catalyze two repressive histone markers, H3K27me3 and H3K4me3, via AR recruitment . Other wAnother methyltransferase involved in PCa growth is PRMT6 . PRMT6 hA relevant enzyme for the AR regulation is the lysine specific demethylase 1 (LSD1). It has been targeted for its dual ability to suppress or stimulate AR expression . The expAKR1C3 and HSD17B6, two genes responsible for the androgen synthesis [Historically, despite its aforementioned role as coactivator, LSD1 has been considered a corepressor. LSD1 acts as a demethylase for H3K4me1,2  enhancinynthesis ,44. The ynthesis ,46 and rynthesis ,48.Furthermore, it has been shown that other HMT enzymes such as the lysine demethylase 4B (KDM4B) , KDM4C , and KDMTMPRSS2-ERG [TMPRSS2 and ERG loci near genomic breakpoints, leading to TOP2B-mediated DNA double strand break formation [It is well known that one of the tumorigenic mechanisms in PCa cells is the fusion gene RSS2-ERG  and seveRSS2-ERG . Androgeormation .TMPRSS2-ERG fusion, Metzeger et al. [TMPRSS2-ERG oncogenic fusion [Yu et al. , throughr et al.  showed tc fusion . The mecThe histone acetyltransferases (HAT) and histone deacetylases (HDAC) are two groups of enzymes that regulate acetylation and deacetylation . In geneAs described in other reviews ,60,61,62N-acetyltransferase arrest-defect 1 protein (ARD1) [Many works have described several co-regulators of the AR transcription machinery with a HAT activity such as p300/CAF , p160/SRn (ARD1) .CBP and p300 are proteins with HAT activity, and they are able to regulate transcription ,69. It wPSA and TMPRSS2 [The Steroid Receptor Coactivator-1 (SRC1) is responsible for the activation of AR due to its HAT domain . Moreove TMPRSS2 .PSA) [As previously suggested in another review , in addiPSA) . At the PSA) . IL-6 isPSA) , and it PSA) .TIP60, an AR factor acetyl transferase (FAT), has a specificity for the LBD of the AR . More reArrest defective-1 protein (ARD1) is another acetyltransferase  which haInterestingly, another study suggests that the levels of AR potentiate the recruitment of AR and the components of the transcription machinery to chromatin in order to enhance the acetylation on H3K9 and on H3K14 in CRPC cells even in an androgen deprivation environment . These fAs mentioned above, acetylation of particular residues determines the enhancement of the AR activity, and it is normal to expect that the opposite process can lead to inhibition. Within the HDAC family, we encounter several proteins with a similar enzymatic activity. For example, HDAC1 interacts with the PSA promoter and suppresses AR signaling  while HDSirtuin 1 (SIRT1), a NAD-dependent deacetylase, has been described as a repressor of AR activity . Fu et aIn the last decade, several articles corroborated by the use of new technologies to reveal that a major portion of the non-coding genome is transcribed with many regulatory functions. This brought about a change in thinking that non-coding RNA can have a role in cancer . Non-codMicroRNAs (miRNAs) are a class of small non-coding RNAs with an important role in cell development, differentiation and signal transduction. Generally, miRNAs cause mRNA translational repression or mRNA degradation by binding to the 3′ untranslated region (3′-UTR) . FurtherIn 2011, Waltering et al. presented one of the first miR microarray studies to examine androgen regulation of miRNAs , and theGenome-wide screenings of androgen target genes have identified miR-125b as androgen-inducible miRNA  and in pROCK1) and ROCK2 (implicated in cytoskeleton regulation) at mRNA and protein levels [RBAK) and matrix metalloproteinase 11 (MMP11), two genes involved in migration pathway, are controlled by miR-135a. They showed that PCa progression is associated with low levels of miR-135a and high levels of RBAK and MMP11.MiR-135a has been found to be upregulated in androgen sensitive PCa cells and AR, as previously reported for miR-125, directly activates transcription by using a functional ARE in the miR-135a promoter region . To expln levels . Coarfa n levels  also foun levels  describeBTG2), a member of the antiproliferative (APRO) gene family [MiR-32 is also reportedly an androgen-regulated miRNA. The transfection of pre-miR-32 into LNCaP cells confers significant cell growth and reduces apoptosis. In CRPC, miR-32 is regulated by androgen through targeting the B-cell translocation gene 2  and member RAS oncogene family (RAB1A). In this study, downregulation of HECTD2 affected androgen related transcription, and downregulation of HECTD2 and RAB1A altered the expression of many cell cycle genes and pathways, promoting tumor metastasis and leading to the development or maintenance of the CRPC phenotype.MiR-221 and miR-222 are encoded on the X chromosome , but curMany investigations have been conducted for documenting the role of miRs in controlling the AR pathway. By using a miR library in 2011, Ostling et al. demonstrated the ability of 71 unique miRs (52 decreasing and 19 increasing) to influence the AR . Since tIL-8 and EDN1) are responsible for improving the expression of the AR, and others are involved in the MAPK/ERK, mTOR, and IL-6 signaling pathways [MiR-205 is deregulated in PCa compared to benign prostate tissues, it is inversely associated to advanced disease and short life expectancy, and miR-205 levels exhibit a negative correlation to AR . Moreovepathways .MiR-34 family includes three miRNAs that have been previously reported to suppress tumorigenesis by different mechanisms, including modulation of cell cycle, epithelial to mesenchymal transition, or metastasis . In PCa,RAS and MYC [c-MYC, a molecule required for the correct transcription of AR [LET7 levels are frequently decreased in human cancers ,135. The and MYC ,137. A won of AR . In detaon of AR . These ron of AR  reportedERBB-2 mRNA 3′-UTR contains two specific miR-331-3p target sites and that miR-331-3p suppresses ERBB-2 expression at both the transcript and protein levels. MiR-331-3p expression was found to be lower in ERBB-2 overexpressing PCa tissue compared to normal adjacent tissue. The same group also explained that miR-331-3p is involved in the downstream PI3K/AKT signaling in multiple PCa cell lines. Interestingly, it has been shown that miR-331-3p acts specifically to decrease PSA promoter activity and PSA levels without reducing AR expression [Not only do miRNAs have direct effects, but they can also use other pathways to control androgen signaling. Two of these pathways are mediated by the ERBB-2 and PI3K/AKT. The tyrosine receptor ERBB-2 is often elevated in PCa, whereas the activation of PI3K/AKT signaling is linked to proliferation, metastasis, apoptosis resistance and angiogenesis in PCa . A work pression .MiR-488* directly targets AR by targeting the AR in 3′-UTR. MiR-488* down-regulates AR protein expression in both androgen-sensitive and insensitive PCa cells, inhibiting cellular growth and increasing apoptosis as observed after the transfection of miR-488* .MiR-17-5p has been shown to target PCAF, a coactivator of AR, and to support PCa development . The autMiR-124 has been described as a tumor suppressor miRNA in several cancer types including PCa ,145,146.HMGA) gene which, as previously reported, can inactivate p53 [The same authors propose an explanation of this phenomenon. The upregulation of p53 may in part be due to the capacity of miR-124 to inhibit the AR/miR-125b signaling pathway or by targeting the 3′-UTR of the high mobility group A  in controlling various biological processes or having a central role in various cancers ,153,154,In 2000, Srikantan et al.  characteAn elegant study performed by Yang et al.  discoverNevertheless, the efficacy of these findings has been questioned. In fact, Prensner et al.  disagreeRecently, Ho et al.  describeThe lncRNA PCA3, one of the most important prostate-specific genes, has been extensively studied as a tumor biomarker  due to iAnother lncRNA named CTBP1 antisense (CTBP1-AS) has been identified as a promoter of the AR transcriptional activity . To explCui et al.  firstly The lncRNA HOTAIR is a 2.2-kb-long transcript localized to the boundaries of the HOXC gene cluster . Tsai etProstate cancer transcript-associated 18 (PCAT18) is a lncRNA reported to be prostate specific and up-regulated in PCa compared to other tumors . An RNA Another novel lncRNA, PCAT29, was recently discovered, and its relationship to the AR explained. Malik et al.  describeProstate specific antigen (PSA) is the most important screening technique used for PCa diagnosis and tumor monitoring. Despite being organ specific, PSA is not cancer specific, and its level changes in the presence of several conditions such as prostatitis, hyperplasia, prostate biopsies and surgeries . All of The epigenetic alterations in PCa, as in part described above, can provide effective biomarkers for early detection and cancer relapse, for prognosis and, finally, to predict then response to specific therapies . In thisSeveral reviews report the role of the DNA methylation asset as a biomarker for PCa detection/diagnosis or prognosis and response to therapy ,182.GSTP1) gene. In fact, GSTP1 has been considered one of the most promising candidates for a DNA methylation biomarker because it appears in more than 90% of cases [One of the most frequent epigenetic alterations in PCa is the aberrant promoter methylation of the glutathione S-transferase pi 1  .GSTP1, APC, and RARB2) has been evaluated in a prospective study named ProCaM [Moreover, the combination of the methylation pattern of three genes  in men with a low risk for disease after a negative biopsy. ConfirmMDx, after a validation in a European and a US population, achieved a negative predictive value of 88%–90% [A methylation marker genetic test, ConfirmMDx , is a tissue-based assay that studies the epigenetic alteration surrounding the tumor lesions. This test identifies the methylation pattern of three genes  is a prostate cancer-specific antigen mapped to chromosome 9q21-22 . It is aThe PCA3-test involves collection of a urine sample after DRE to mobilize prostatic cells. The PCA3 score is a mathematical operation, and it can be acquired by dividing PCA3 RNA by PSA RNA levels in order to normalize PCA3 signals.Despite these suggestive findings, the best cut-off to use is still controversial. A urine PCA3 score more of than 35 has been linked with a sensitivity comprised between 47%–57% and a specificity around 70% ,197. A rIn 2013, GenomeDx Biosciences  and Mayo Clinic  co-developed and validated a tissue-based genomic classifier that is able to evaluate the risk of developing clinical metastases at 5 years postoperatively named Decipher . The autSeveral studies suggest the use of miRNAs in the clinical setting and many reviews have been published about the argument ,212,213.Larne et al.  focused As mentioned above, several miRNAs, regulated by or involved in AR transcriptional activity, are able to predict biochemical failure, clinical relapse, and castration resistant status. Interestingly, the overexpression of miR-21 has been reported in patients with castration-resistant disease , and it Androgen deprivation therapy (ADT) is the principle treatment for advanced PCa and induces remission in 80%–90% of patients . DespiteUncovering the locations of proteins throughout the genome helped physicians to understand the biology of both healthy and tumoral prostates. Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) is considered a novel technique to discover transcription factor binding sites, chromatin regulators, and the identification of genomic histone marks. Moreover, mapping the interactions of small molecules with chromatin, a technique named Chem-seq, has not only helped build the understanding of novel mechanisms underlying the biology of diseases but in discovering new specific treatments .Chem-seq, ChIP-seq, and RNA-seq methods were used to evaluate the role of a small molecule, termed SD70, originally recognized as an inhibitor of DHT and chromosomal translocations events in PCa . The 8-hRNA-seq analysis in KDM4C knockdown cells confirmed the central role of KDM4C on AR target-gene regulation. Moreover, a Chip-seq in androgen dependent cells revealed that SD70 represses the methylation activity of KDM4C at the AR-regulated enhancers . In consHistone deacetylase inhibitors (HDACi) are a group of molecules with anticancer activity against hematologic and solid tumors . DiffereA recent review  extensivRomidepsin is a cyclic depsipeptide, enhancing the acetylation of HSP90, that reportedly interferes with the correct folding of AR determining its degradation . DespitePanobinostat is a cinnamic hydroxamic acid class molecule with an HDACi activity. In vivo studies in AR-positive PCa cell lines showed a significant degradation of the AR mediated by the acetylation and subsequent inhibition of the HSP90 chaperone function . In 2010In the last decade, several findings have documented the role of miRNAs as new oncogenes or tumor suppressor genes, thus supporting their use as therapeutic tools. Artificial miRNA mimics and inhibitors are considered a good way in which to “block or boost” the production of several proteins . MiRNA mThe androgen receptor is the central regulator of nominal and tumor prostate biology. Prostate carcinogenesis is a complex event due to genetic mutations and epigenetic alterations. In the past decade, the role of epigenetic regulation has become evident, and considerable progress has been made defining its role in the onset and progression of prostate cancer. In this review we focused our attention mainly on the AR epigenetic alterations. A better understanding of AR transcriptional pathway is indispensable to develop diagnostic and therapeutic procedures exploiting these epigenetic changes. Although PSA remains the prevalent test for prostate cancer screening and prognosis, the new generation of biomarkers can help physicians in their clinical decisions. The PCA3 test is widely used in clinical practice but chromatin remodeling marks and miRNA panels, as well as genomic tests, are becoming new promising predictive tools. New technologies for global epigenomic analyses and integration with genomic and transcriptomic data are extending our knowledge on prostate tumorigenesis. A new approach named “Chem-seq” permitted us to uncover the site and the mechanism of action of a small molecule named SD70. The demethylase SD70, targeting a key regulator of AR function, is effective in CRPC cells in combination with current therapies. Furthermore, the optimization of the stability of miRNAs and the improvement of the efficacy of HDAC inhibitors are also challenges for the future treatment of prostate cancer. Knowing the specific molecular mechanisms underlying tumors will be desiderable for the identification of more effective approaches allowing to personalize therapy.


Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is associated with an increased risk of colorectal cancer in 8-10 years after disease onset. Current colitis treatment strategies do not offer a cure for the disease, but only treat the symptoms with limited success and dangerous side-effects. Also, there is no preventive treatment for either UC or colorectal cancer. Quinacrine is an anti-malarial drug with versatile use in the treatment of diseases involving inflammatory response such as rheumatoid arthritis and lupus erythematosus. It also has putative anti-cancer effect. Quinacrine's anti-inflammatory, anti-oxidant properties, and anti-tumorigenic properties make it a potential small molecule preventive agent for both UC and associated colorectal cancer.There were obvious changes in the CDI, histology, and inflammatory load in quinacrine-treated groups in a dose and time dependent manner in both models of UC, induced by chemical or haptenating agent.We tested quinacrine at two different doses as a colitis treatment agent in two mouse models of UC - the dextran sulfate sodium and oxazolone. The clinical disease index (CDI), histological changes of the colon, levels of inflammatory markers  and overall health vitals were evaluated.We demonstrate that quinacrine successfully suppresses colitis without any indication of toxicity or side-effects in two mouse models of UC. Ulcerative colitis (UC) is a chronic disease that causes inflammation and ulcers in the colon and the rectum. Colorectal cancer is the most serious complication of ulcerative colitis . The risQuinacrine (IUPAC name 4-N-(6-chloro-2-methoxyacridin-9-yl)-1-N,1-N-diethylpentane-1,4-diamine), approved by the FDA, is a heterocyclic three-ring compound that was widely used during World War II as an anti-malarial agent. Over the last half century, it has been used for the treatment of giardiasis, tapeworm infestations, and connective tissue diseases, such as lupus erythematosus and rheumatoid arthritis . MechaniRecognizing these anti-cancer and anti-inflammatory properties of quinacrine, we hypothesized that quinacrine can suppress colitis in mice. In this article we test this hypothesis in two different mouse models: a DSS model, and Oxazolone model of colitis, showing a significant decrease in the overall colonic inflammatory load in the quinacrine treated animals.in vitro, using the ANA-1 murine macrophages cell line. Western blot analysis shows that pre-incubation of ANA-1 cells with quinacrine effectively attenuates the induction of iNOS for up to 24 hours  and associated reactive nitrogen species (RNS) are driving factors of inflammation and cancers associated with chronic inflammation . Since qs Figure  and 1B.in vitro; are highly likely to suppress colitis in vivo. We have demonstrated this with Resveratrol, American Ginseng, a protein arginine deiminase inhibitor, Chlor-Amidine, and with a Hexane Fraction of American Ginseng [in vivo, we first asked whether quinacrine can induce apoptosis in inflammatory cells. Consistent with this understanding, Figure We have previously shown that compounds that both: (a) suppress iNOS induction in inflammatory cells; and (b) induce apoptosis in inflammatory cells  Ginseng -26. Therin vitro studies where quinacrine suppresses iNOS induction and drives apoptosis of inflammatory cells, we have tested it on mice to check if it suppressed DSS induced colitis. Quinacrine was given to mice ad libitum at the two doses of 50 mg/kg and 10 mg/kg equivalent in the water after 1 week exposure to 2% DSS. The mice that received no quinacrine treatment showed moderate to severe inflammation and mild ulceration with a histology score rising gradually over the exposure time from 7.8 ± 1.04 at 7 days to 26.2 ± 1.7 at 14 days and to 27.11 ± 1.6 at 17 days time points. In contrast, although colitis did not return to that of the water-only treated group, the quinacrine-treated mice demonstrate mild inflammation and ulceration at both doses, as reflected by histology score of 16.5 ± 3.0 for the quinacrine dose of 10 mg/kg and 13.1 ± 2.6 for the quinacrine dose of 50 mg/kg at the 14 day time point , which progressed to a more severe state by experimental day 15 as indicated by average inflammation score of 28.5 ± 5.6 (group Oxazolone 5/10)  Figure . In contOverall, treatment of mice with quinacrine in oxazolone model of colitis leads to suppression of colitis in both time-and dose dependent manner, similarly to DSS model.in vivo, we examined Cox-2, iNOS, and p53 expression in the colon of mice. Immunohistochemical staining was accomplished by rocking slides using the Antibody AmplifierTM  to ensure even, consistent, sensitive and reproducible staining. Figure To further assess the impact of quinacrine on inflammatory markers UC is a painful disease and the complications of increased risk of colorectal cancer only add to the distress. Other than surgery, treatment available now can only alleviate the symptoms, giving temporary relief, but cannot cure the disease. Our search for a novel treatment for this disease led us to quinacrine; widely used as anti-malarial and anti-protozoal drug. Since its discovery for use as an anti-parasitic, multiple studies have identified a putative anti-inflammatory mechanism for quinacrine , 13, 28.in vitro  was purchased from MP Biomedicals.2 atmosphere. Experiments with quinacrine were carried out by pre-incubating cells with indicated concentrations of quinacrine for specified times. Quinacrine was dissolved in DMEM medium (0.1% NBCS) containing 1% DMSO. Following a wash step, cells were activated by exposure to 100 U/ml interferon (IFN)-γ .ANA-1 murine macrophage cells and TK6 human lymphoblastoid cells were maintained in Dulbecco's modified Eagle's media  supplemented with 10% New Born Calf serum (NBCS) , 2 mM glutamine (Biofluids), penicillin (10 U/ml) and streptomycin  in growing suspension culture at 37°C in a humidified 5% COWestern blots were carried out as described previously , 57. AntTUNEL labeling was performed as we have done previously . BrieflyMale C57BL/6 mice, 8 weeks of age, weighing 20 to 25g were obtained from The Jackson Laboratories. All mice were kept in dedicated and clean animal quarters and provided food and water. Care and use of animals was overseen by the Animal Resource Facility (ARF) of the University of South Carolina under the direction of a veterinarian. The ARF is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, is registered with the U.S. Department of Agriculture (56-R-003) and has an active letter of Assurance of Compliance on file at the NIH. Animal Care and Use Committee (IACUC) of the University of South Carolina approved this study.The dextran sulfate sodium (DSS) mouse model used here is similar to the one used previously by our lab . Animalset al. [For our oxazolone model of UC we used a modified protocol generally following the methods described by Wirtz et al. . BrieflyThe harvested colon was washed in PBS, Swiss-rolled, fixed in formalin and embedded in paraffin. The sections were then stained with hematoxylin and eosin. The slides were examined by two individuals in a blind fashion and the histopathologic changes were recorded using the previously described scoring system . InflammFor immunohistochemical staining, serial sections of mouse colon tissues (processed as described above) were incubated with antibodies against p53 , cyclooxygenase-2 (Cox-2)  or inducible nitric oxide synthase (iNOS) . To ensure even staining and reproducible results, sections were incubated by slow rocking overnight in primary antibodies (4°C) using the Antibody Amplifier™ . Following incubation with primary antibody, sections were processed with EnVision+ System-HRP kits  according to the kit protocol. The chromogen was diaminobenzidene and sections were counter stained with 1% methyl green. The negative control was carried out without primary antibody incubation.Immunohistochemistry was quantified as we described previously , with a CDI was assessed as described previously . BrieflyWith inflammation as an end point, a χ2 contingency table analysis was done on the DSS and DSS + Quinacrine groups to determine if there was a statistically significant difference in their inflammation scores.


Niclosamide, an FDA-approved anti-helminthic drug, has activity in preclinical models of castration-resistant prostate cancer (CRPC). Potential mechanisms of action include degrading constitutively active androgen receptor splice variants (AR-Vs) or inhibiting other drug-resistance pathways . Published pharmacokinetics data suggests that niclosamide has poor oral bioavailability, potentially limiting its use as a cancer drug. Therefore, we launched a Phase I study testing oral niclosamide in combination with enzalutamide, for longer and at higher doses than those used to treat helminthic infections.We conducted a Phase I dose-escalation study testing oral niclosamide plus standard-dose enzalutamide in men with metastatic CRPC previously treated with abiraterone. Niclosamide was given three-times-daily (TID) at the following dose-levels: 500, 1000 or 1500mg. The primary objective was to assess safety. Secondary objectives, included measuring AR-V expression from circulating tumor cells (CTCs) using the AdnaTest assay, evaluating PSA changes and determining niclosamide’s pharmacokinetic profile.20 patients screened and 5 enrolled after passing all screening procedures. 13(65%) patients had detectable CTCs, but only one was AR-V+. There were no dose-limiting toxicities (DLTs) in 3 patients on the 500mg TID cohort; however, both (N = 2) subjects on the 1000mg TID cohort experienced DLTs . The maximum plasma concentration ranged from 35.7–82 ng/mL and was not consistently above the minimum effective concentration in preclinical studies. There were no PSA declines in any enrolled subject. Because plasma concentrations at the maximum tolerated dose (500mg TID) were not consistently above the expected therapeutic threshold, the Data Safety Monitoring Board closed the study for futility.Oral niclosamide could not be escalated above 500mg TID, and plasma concentrations were not consistently above the threshold shown to inhibit growth in CRPC models. Oral niclosamide is not a viable compound for repurposing as a CRPC treatment.NCT02532114Clinicaltrials.gov:  Nearly 30,000 American men die as a result of their prostate cancer each year. Since tAR amplification, AR overexpression and/or maintenance of intratumoral androgens; ii) AR-signaling activation via feedback pathways ; and iii) activation of the AR program via mutations (e.g. AR ligand binding domain mutation) or AR substitutions . This study was approved by the University of Washington/Fred Hutchinson Cancer Research Center Institutional Review Board and written informed consent was obtained from all enrolled subjects. Study participants were recruited through medical oncology clinics at the University of Washington and Seattle Cancer Care Alliance , and the study was open for recruitment from October 2015 to November 2017, with all patient follow up completing in December 2017. Niclosamide was given three-times-daily (TID) by mouth (PO) for four weeks at one of the following dosing cohorts: 500, 1000, or 1500 mg. All patients were required to have mCRPC  and had received prior abiraterone. Patients were originally required to have detectable AR-V transcripts in circulating tumor cells (CTC) as determined using the AdnaTest assay. However13C6 in methanol) and methanol (200 μL) in a 0.5 mL snap-cap micro-centrifuge tube. After vortexing, the samples were centrifuged at 20000 x G for 10 minutes at 4°C. The supernatant was transferred to a 96-well plate and 2 μL were injected on the liquid chromatography mass spectrometry (LCMS) system. The LCMS system was an ultrapressure liquid chromatography  1290 series) coupled to an Agilent G6410B triple-quadrupole mass spectrometer. The column was an Agilent Zorbax SB-C18 2.1 mm x 150 mm x 5μ maintained at 45°C. Mobile phase A was 10 mM ammonium formate (pH = 3) and mobile phase B was acetonitrile in the ratio of 35% A: 65% B at 0.4 mL/min.The pharmacokinetic samples were quantitated for plasma niclosamide concentrations using a modification of previously published methods. Blood fm/z (niclosamide) and 330.9→176.9 m/z (niclosamide 13C6).The mass spectrometer was operated in the ESI-negative mode with the following transitions: 324.8→170.9 13C6) and were fitted using a polynomial fit with 1/x weighting. The correlation coefficient was used to evaluate the linearity of the calibration curves and was >0.99 in all experiments. The limit of quantitation was 40.8 ng/mL  and the limit of detection was 16.3 ng/mL, with a signal to noise ratio of 53.75.A ten-point calibration curve was created by spiking blank plasma with niclosamide to make standards in the range of 40.8 to 8160 ng/mL and processing the standards identically to samples. Calibration curves were based upon the height ratio of the niclosamide to the internal standard  mRNA. The AdnaTest ProstateCancerSelect/ProstateCancerDetect assay  and methods similar to those described by Antonarakis and colleagues were used. AR-V PCmax, Cmin, Css and t1/2) were extracted.We utilized the continual reassessment method (CRM) to evaluate dose-related toxicities and to determine the recommended Phase II dose. We targFrom December 2015 to October 2017, 20 patients were screened and 5 passed all screening procedures and enrolled onto the study . The 15 Niclosamide was well tolerated in combination with enzalutamide in the first dose cohort , and only one out of three patients at this dose level experienced any toxicities deemed at least possibly related to niclosamide . Both patients treated at the second dose level  experienced dose-limiting toxicities. Patient #19 had Grade 3 nausea, vomiting, and diarrhea lasting >72 hours. Symptoms began on Day 26 of treatment, and he discontinued the study drugs that day. He was ultimately hospitalized between Days 32 to 40 as a result of this AE. This patient also experienced a Grade 2 lipase elevation without other signs of pancreatitis, which was felt to be possibly related to niclosamide. At the time of discharge he had increased oral intake without nausea or vomiting. Patient #20 had Grade 3 colitis, abdominal pain and diarrhea, with the colitis constituting a DLT. Symptoms began on Day 8 of treatment and abdominal CT on Day 9 revealed evidence of colitis. He was subsequently admitted to the hospital between Days 9 to 12 where he received aggressive supportive care (i.e. IV hydration and antibiotics). This AE had returned to Grade 1 at the time of discharge. It should be noted that this patient had received an immune checkpoint inhibitor as part of a clinical trial immediately prior to enrolling onto this study, and a colonoscopy with biopsy was performed to evaluate for delayed onset immune-mediated colitis. Pathologic findings from this biopsy were not consistent with an immune-mediated adverse event, however, and it was felt that niclosamide was likely the causative factor. A summary of all AEs deemed at least possibly related to niclosamide are provided in th day of treatment in four patients (0-∞) after the first dose, which should be evaluated with caution in those participants whose AUC0-∞ was over 20% extrapolated. The apparent oral clearance of niclosamide, which is clearance divided by the oral bioavailability , ranged from 9.09 to 37.8 L/hr per kg of ideal body weight. Because niclosamide plasma concentrations in the maximal tolerated dosing cohort  were below those expected to exert an anti-tumor effect, the study was closed for futility.Considering the enthusiasm for developing niclosamide for AR-V+ mCRPC, we chose to include all pharmacokinetic data. Pharmacokinetic data were obtained after the first dose in all patients and after the morning dose on the 15patients . It shoumax data agreeing with previous reports[Our main findings are: 1) that niclosamide doses could not be escalated above 500 mg PO TID because of toxicity; 2) niclosamide is not a viable oral compound for repurposing as a mCRPC treatment because the dosing cohort with acceptable toxicity  does not consistently yield concentrations above those shown to inhibit tumor growth in mCRPC models; and 3) niclosamide pharmacokinetics had moderate variability with our Cs reports, 28–32. s reports, 29–32. s reports. Ultimatmax ≥163.5 ng/mL. There was no evidence of clinical activity in any patient enrolled and toxicity prevented dose escalation above the 500 mg PO TID cohort. Therefore, the Data Safety Monitoring Board for the trial recommended that the trial terminate prematurely for futility.Prior preclinical studies testing niclosamide reported decreased DU145 proliferation at a half-maximal inhibitory concentration (IC50) of 330 ng/mL, and niclosamide’s anti-tumor effects are apparent across a range of prostate cancer models, with decreased cellular viability documented at concentrations from 81.8 to 327 ng/mL. Liu and colleagues have published a series of papers examining the effects of niclosamide in abiraterone and/or enzalutamide-resistant models, including several with AR-V7 expression, 38, 39.This study was originally designed to include AR-V positivity as a novel integral biomarker given that: 1) AR-V7 has been hypothesized to promote resistance to drugs that interfere with the AR-ligand/AR interaction ; 2) niclosamide has been shown to be effective in preclinical models of AR-V+ CRPC; and 3) prior studies documented AR-V positivity in >45% of mCRPC patients post-abiraterone and/or enzalutamide, 40, 41.14C-labeled niclosamide[The small sample size of this trial represents its primary limitation, and while relatively few patients were treated per protocol, our data still represent the largest cohort of niclosamide-treated patients with contemporary safety and pharmacokinetic data. Prior conclusions regarding the pharmacokinetic profile of niclosamide all trace back to one internal Bayer document from 1971 in which an undisclosed number of healthy volunteers received a single dose of closamide. UltimatOverall, we conclude that the development of the current oral formulation of niclosamide as a cancer therapy should not be pursued. Attention should be turned to developing niclosamide analogs with improved oral bioavailability and enhanced antitumor effects.S1 TREND Checklist(PDF)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.


Polymers with moderate molecular weights and relatively broad dispersity (Ð) were obtained. Kinetic studies revealed that the polymerization followed first-order kinetics.The preparation, structural characterization, luminescence, and catalytic activity of three palladium(II) complexes bearing imino phenoxide ligands are reported. The X-ray studies revealed that the complexes are mononuclear with palladium centres coordinated in a square-planar coordination environment. Two of the complexes are emissive in solution at room temperature. The catalytic activities towards the ring-opening polymerization of The online version of this article (10.1007/s00706-017-2119-1) contains supplementary material, which is available to authorized users. Tetradentate salen-type ligands are omnipresent in coordination chemistry. They are easy to synthesize and they exhibit a high structural variability due to a large number of commercial available building blocks which can be used as starting materials. In many cases, they form highly stable metal complexes throughout the whole periodic table and a plethora of metal complexes have been reported in the past , 2. Rela8 configuration and hence prefers a square-planar coordination. It is well known that such complexes form polymeric columnar structures, often with infinite and close Pd–Pd bonds. Contrary to their platinum congeners + and [L2PdNa]+ are detected. The IR spectra of the free ligands show intense signals in the region from 1623 to 1632 cm−1 . In the complexes, the spectra are shifted towards lower frequencies (1607–1624 cm−1), which eventually revealed the coordination of imine nitrogen atoms to the metal . In the 1H NMR spectra, the formation of the metal complexes is shown by the disappearance of the phenol –OH signals of the ligands. In addition, the signals corresponding to the protons of the CH=N groups appear low field shifted (7.52–7.59 ppm) compared to those of the ligands (8.4–8.6 ppm) which indicates the coordination of nitrogen and oxygen atoms of the ligands to the metal.Complexes 1 crystallizes in the monoclinic space group P21/n, complex 2 in the triclinic space group P2 is isostructural to the homologues copper(II) complex . This might be due to steric reasons and packing effects. The palladium atoms in all complexes are not engaged in any further Pd–Pd interactions.Single crystals suitable for X-ray diffraction of all three complexes were grown by slow evaporation of the solvent from their solutions in dichloromethane over a period of 1 week. All complexes were obtained as red crystalline solids. The crystallographic data are summarized in Table mentclasspt{minimaractions , 47. Sim2 carries the moderately electron-withdrawing bromine substituent, whereas the ligand in the non-emissive complex 1 is substituted by a strongly electron-donating methoxy group. For 3, the ligand is substituted with a very bulky t-butyl group which leads not only to a strongly distorted coordination geometry as elucidated by the solid state structure, but also to a more rigid structure with respect to a distortion in the excited state. Minimizing the excited state structural distortion has been found to be effective in increasing the luminescence quantum yield. It should be noted that analogous Pt-complexes often feature intensive luminescence [The electronic spectra of all the complexes were recorded in solution at room temperature Fig. . The datnescence .Fig. 2UV1–3 were investigated as catalysts for ROP of rac-LA (Scheme 1H NMR spectroscopy. The polymerization was performed in a 200:1 ratio ([rac-LA]0/[Cat]0 = 200:1) catalysed by complexes 1–3. The plot of % conversion of rac-LA against time gives a sigmoid curve meaning that initially the conversion rate is high but decreases significantly at later stage  vs. time indicative for a first-order dependency on the monomer concentrations with the absence of any induction period  were extracted from the linear plot of ln([rac-LA]0/[rac-LA]t) vs. time and were found to be 3.47 × 10−2 min−1, 2.14 × 10−2 min−1, and 2.84 × 10−2 min−1 for 1–3, respectively  and relatively broad dispersities (Mw/Mn = 1.91–1.98). The large difference between the experimental and theoretic values and broad Ð compared to literature values +) 637.13, found 637.33.Yield: 0.07 g (79%); 1H NMR : δ = 1.17–1.20 2, 12H), 1.29–1.32 2, 12H), 3.52–3.66 2, 4H), 7.15–7.16 , 7.18-7.19 , 7.21 , 7.28–7.29 , 7.44 , 7.52–7.53  ppm; 13C{1H} NMR : δ = 23.48 (CH(CH3)2), 24.42 (CH(CH3)2), 28.81 (CH(CH3)2), 105.27 (Ar–C), 114.32 (Ar–C), 120.93 (Ar–C), 124.70 (Ar–C), 127.54 (Ar–C), 135.44 (Ar–C), 140.31 (Ar–C), 142.02 (Ar–C), 145.07 (Ar–C), 159.98 (Ar–O), 163.25 (CH=N) ppm; IR (ATR): −1; MS (ESI): m/z calculated for C38H41Br4N2O2Pd ([M + H]+) 983.780, found 983.497.Yield: 0.07 g (73%); 1H NMR : δ = 1.37 3, 18H), 2.18 , 3.84 , 4.98 , 6.79–6.80 , 6.86–6.89 , 7.06–7.07 , 7.48 , 7.53–7.56  ppm; 13C{1H} NMR : δ = 20.40 (CH3), 29.52 (C(CH3)3), 35.22 (C(CH3)3), 55.38 (Ar–O–CH3), 60.87 (Ar–CH2), 114.31 (Ar–C), 122.32 (Ar–C), 123.14 (Ar–C), 129.35 (Ar–C), 130.23 (Ar–C), 131.16 (Ar–C), 131.83 (Ar–C), 133.35 (Ar–C), 139.33 (Ar-C), 159.12 (Ar–O), 165.95 (CH=N) ppm; IR (ATR): −1; MS (ESI): m/z calculated for C40H49N2O4Pd ([M + H]+) 727.27, found 727.60.Yield: 0.06 g (75%); λ = 0.71073 Å). The structures were solved by direct methods  [F2 (SHELXL-97) [http://www.ccdc.cam.ac.uk. The crystal data are given in Table Suitable single crystals for X-ray diffraction were grown from concentrated dichloromethane solution of the respective compounds over a period of 7 days. Single crystal analysis were carried out on a Bruker SMART APEX and a Bruker Smart X2S diffractometer operating with Mo Kα radiation ( SIR-97) , 54 and ELXL-97) . The H aSupplementary material 1 (PDF 122 kb)Supplementary material 2 (PDF 150 kb)Supplementary material 3 (PDF 208 kb)Supplementary material 4 (CIF 14 kb)Supplementary material 5 (CIF 16 kb)Supplementary material 6 (CIF 48 kb)Supplementary material 7 (DOCX 1576 kb)Below is the link to the electronic supplementary material.


Cisplatin (CDDP)-based combination chemotherapy remains the mainstream treatment for advanced bladder cancer. However, its efficacy is often limited due to the development of resistance for which underlying mechanisms are poorly understood. Meanwhile, emerging evidence has indicated the involvement of androgen-mediated androgen receptor (AR) signals in bladder cancer progression. In this study, we aimed to investigate whether AR signals have an impact on sensitivity to CDDP in bladder cancer cells. UMUC3-control-short hairpin RNA (shRNA) cells with endogenous AR and AR-negative 647V/5637 cells stably expressing AR were significantly more resistant to CDDP treatment at its pharmacological concentrations, compared with UMUC3-AR-shRNA and 647V-vector/5637-vector control cells, respectively. A synthetic androgen R1881 significantly reduced CDDP sensitivity in UMUC3, 647V-AR, or 5637-AR cells, and the addition of an anti-androgen hydroxyflutamide inhibited the effect of R1881. In these AR-positive cells, R1881 treatment also induced the expression levels of NF-κB, which is known to involve CDDP resistance, and its phosphorylated form, as well as nuclear translocation of NF-κB. In CDDP-resistant bladder cancer sublines established following long-term culture with CDDP, the expression levels of AR as well as NF-κB and phospho-NF-κB were considerably elevated, compared with respective control sublines. In bladder cancer specimens, there was a strong trend to correlate between AR positivity and chemoresistance. These results suggest that AR activation correlates with CDDP resistance presumably via modulating NF-κB activity in bladder cancer cells. Targeting AR during chemotherapy may thus be a useful strategy to overcome CDDP resistance in patients with AR-positive bladder cancer. A previous study also demonstrated that addition of dihydrotestosterone in androgen-depleted culture medium resulted in reduction of the cytotoxic effect of doxorubicin on AR-positive bladder cancer cell proliferation . SimilarIt has been shown that the levels of AR expression are significantly lower in bladder tumors than in non-neoplastic urothelial tissues as well as in high-grade/muscle-invasive bladder carcinomas than in low-grade/superficial tumors , 22. NonNF-κB that normally resides in the cytoplasm as an inactivated form translocates to the nucleus and thereby activates its target genes involving cell proliferation/apoptosis, angiogenesis, and cell invasion. In prostate cancer , 35 and NF-κB, as a transcription factor that induces anti-apoptotic proteins, has been considered as a key molecule for CDDP resistance –28. CorrIn contrast to our current findings, in a study where the effects of androgen treatment and AR silencing on cytotoxicity of doxorubicin, 5-fluorouracil, and CDDP in bladder cancer cells were examined, androgen failed to modulate sensitivity to CDDP . DiffereIn conclusion, we demonstrate preclinical evidence indicating that AR activation contributes to CDDP resistance in bladder cancer cells presumably via NF-κB activation. Therefore, patients with AR-negative and p-NF-κB-negative tumor are more likely to respond to CDDP-based chemotherapy. In contrast, those with AR-positive or p-NF-κB-positive tumor may be resistant to CDDP therapy and are anticipated to benefit from combined anti-AR therapy. Thus, depending on the gender and hormone milieu of the patients as well as AR expression profile in the tumors, comedication with ADT may be useful for overcoming chemoresistance. Further investigations are required to determine the effects of anti-AR therapy on chemosensitivity, especially in animal models for bladder cancer, and to elucidate the precise mechanisms of how AR signals involve CDDP resistance in bladder cancer cells.Anti-AR (N-20), anti-NF-κB (sc-109), and anti-GAPDH (6c5) antibodies and an anti-p-NF-κB antibody (Ser536) were purchased from Santa Cruz Biotechnology and Cell Signaling Technology, respectively. We obtained methyltrienolone (R1881) from PerkinElmer, and HF and CDDP from Sigma-Aldrich.Human urothelial carcinoma cell lines, UMUC3 and 5637, were originally obtained from the American Type Culture Collection . Another human urothelial carcinoma cell line, 647V, was used in our previous studies . All these lines were recently authenticated, using GenePrint 10 System (Promega), by the institutional core facility.Cell lines stably expressing a full-length wild-type human AR (5637-AR and 647V-AR) or vector only (5637-V and 647V-V) were established, using a lentivirus vector (pWPI-AR or pWPI-control) with psPAX2 envelope and pMD2.G packaging plasmids, as we described previously , 15. Sime.g. 0.2–2.0 μM) for at least 12-weeks.CDDP-resistant cell lines, 647V-CR, 647V-AR-CR, 5637-AR-CR, and UMUC3-CR, were then established from 647V, 647V-AR, 5637-AR, and UMUC3 cells, respectively, by stepwise, continuous treatment with CDDP (All these cell lines were maintained in Dulbecco's modified Eagle's medium (Mediatech) supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO2. Cells were then cultured in phenol red-free medium supplemented with either 10% FBS or 10% CS-FBS at least 24 hours before experimental treatment.Equal amounts of proteins obtained from cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes electronically, blocked, and incubated with a specific primary antibody. The membrane was then incubated with a secondary antibody, and specific signals were detected, using a LI-COR imaging system.3) seeded in 96-well plates were cultured for 72 h in the presence or absence of CDDP and then incubated with 0.5 mg/mL of MTT (Sigma) in 100 μL of medium for 3 h at 37°C. MTT was solved by DMSO, and the absorbance was measured at a wavelength of 570 nm with background subtraction at 630 nm.We used the MTT (thiazolyl blue) assay to assess cell viability, as described previously , with miTotal RNA isolated from cultured cells by TRIzol  was reverse transcribed, using 1 μmol/l oligo(dT) primers and four units of Ominiscript reverse transcriptase (Qiagen). Real-time PCR was then performed, using RT2 SYBR Green FAST Mastermix (Qiagen), as described previously . The priImmunofluorescent staining was performed, as described previously , 16, 44,n = 24) and non-responders (n = 31) to the neoadjuvant therapy were pathologically defined as the absence (≤ pT1N0M0) and presence , respectively, of muscle-invasive, extravesical, or metastatic disease at the time of cystectomy [We retrieved bladder tissue specimens obtained by transurethral resection for TMA construction. Appropriate approval from the institutional review board was obtained before construction and use of the TMAs. These bladder TMAs constructed previously , 24, 45 stectomy .Immunohistochemistry was performed on the sections (5 μm thick) from the bladder TMAs, using a primary antibody to AR (dilution 1:200), NF-κB (dilution 1: 200), or p-NF-κB (dilution 1: 50) and a broad spectrum secondary antibody (Invitrogen), as described previously , 21. Allt-test was used to analyze differences in variables with a continuous distribution. The Fisher exact test or chi-square test was used to evaluate the associations between categorized variables. P values less than 0.05 was considered statistically significant.Student's