Mounting evidence has indicated microRNA (miR) dysregulation and the Wnt/β-catenin signaling pathway jointly drive carcinogenesis, cancer metastasis, and drug-resistance. The current review will focus on the role of the crosstalk between miRs and the Wnt/β-catenin signaling pathway in cancer development. MiRs were found to activate or inhibit the canonical Wnt pathway at various steps. On the other hand, Wnt activation increases expression of miR by directly binding to its promoter and activating transcription. Moreover, there are mutual feedback loops between some miRs and the Wnt/β-catenin signaling pathway. Clinical trials of miR-based therapeutic agents are investigated for solid and hematological tumors, however, challenges concerning low bioavailability and possible side effects must be overcome before the final clinical application. This review will describe current understanding of miR crosstalk with the Wnt/β-catenin signaling cascade. Better understanding of the regulatory network will provide insight into miR-based therapeutic development. MiR, as an evolutionarily conserved gene expression regulator, participates in many fundamental physiological processes. In cancer development, miR functions as a tumor suppressor or an oncogene by targeting specific genes in a 3′UTR dependent manner. This mechanism occurs in a variety of tumors. It is well known that Wnt/β-catenin drives the carcinogenesis, cancer progression, and metastasis in many tumors. Emerging evidence has indicated the importance of interaction between the Wnt/β-catenin signaling pathway (Wnt pathway for short) and miR mediated gene regulation in cancer development. In this paper, we would like to summarize current understanding of the crosstalk between miRs and the Wnt pathway with respect to oncogenesis, cancer metastasis, and drug-resistance.MiRs are short (18-25 nucleotides) non-coding RNAs. They repress gene expression through interaction with the 3′UTR of target genes, either inhibiting mRNA translation and/or promoting mRNA degradation. One miR family is potentially able to target 500 genes on average [Genome-wide profiling has shown that miRs are frequently aberrantly expressed in human neoplasms including gastric cancer, hepatocellular carcinoma (HCC), breast cancer, glioblastoma, prostate cancer and colorectal cancer (CRC) . SoftwarThe Wnt pathway is critical for embryo development and adult tissue homeostasis. Without Wnt protein stimulation, β-catenin is anchored by a destruction complex comprised of APC, GSK3β and Axin . β-catenAberrant activation of the Wnt pathway is highly associated with tumorigenesis and tumor metastasis. Wnt signaling components are commonly mutated in many human cancers. Exome-sequencing has revealed that most of CRC patients harbor loss of function mutations in APC . In someDysregulation of miR induces constitutively active Wnt signaling activity in cancer, while expression of miR, in turn, is intensely controlled by Wnt signaling. In order to identify miRs that regulate the Wnt pathway activity, 470 miRs were screened in a cell-based assay in human HEK293 cells and 38 candidate miRs were identified . MiRs acIt is well established that Wnt activation initiates from the Wnt proteins binding to the FZD and LRP5 or LRP6. If miRs target the Wnt ligand/ receptor such as WNT, FZD and LRP, the pathway is repressed. Both miR-122 and -148a were found to be down-regulated in HCC and to repress the Wnt pathway by targeting WNT1. MiR-122 was shown to suppress cell proliferation and promote cell apoptosis . MiR-148via targeting DKK, which resulted in either accelerating cancer cell proliferation/invasion or inducing resistance to chemotherapy. Additional examples of miR regulating Wnt ligands/receptors and their associated inhibitory proteins are listed in Table Conversely, Wnt signaling is activated when miRs target Wnt antagonists such as WIF, sFRP, DKK, and PR domain proteins (PRDM). MiR-374a , 31, -60The Wnt receptor is at the crux between extracellular ligands and intracellular responses such as survival or apoptosis, proliferation or growth arrest, drug susceptibility or drug resistance. Thus targeting the receptor and its associated proteins by miRs fundamentally influences signaling transduction at the initiation phase. As discussed, miR regulation of the Wnt/β-catenin cascade is multi-faceted with regards to receptor activation and warrants interest for possible therapeutic interventions.via targeting ZEB2 [β-catenin, the most important signaling factor in the Wnt pathway, transduces the signal by directly binding to TCF/LEF transcription factors and turning on downstream gene expressions which promote cell proliferation, migration, EMT, cancer cell metastasis and chemo-resistance . A numbeing ZEB2 . Moreoveing ZEB2  and downing ZEB2 .Many other miRs target β-catenin as well, besides the miR-200 family. MiR-214 , 49, -32Certain miRs not only target β-catenin alone, but also directly suppress a set of multiple components of the Wnt signaling cascade to regulate Wnt activity such as miR-34. MiR-34 trans-activated by p53 inhibits the Wnt pathway by targeting the 3′-UTRs of a set of conserved targets which are elements of the Wnt pathway. These genes include WNT1/3, β-catenin, LRP6, AXIN2 and LEF1 –59. The β-catenin is the central and most well studied signaling molecule in the Wnt pathway. By targeting β-catenin, miRs directly repress the Wnt signaling activity Figure . MoreoveDue to the significance of β-catenin in Wnt signaling, miRs targeting the β-catenin interacting proteins such as APC, GSKβ, AXIN and similar associates, consequently regulate Wnt signaling. APC is a scaffolding protein for the APC/GSK3β/CK1α/β-catenin/AXIN complex which mediates destruction of β-catenin. It is a common tumor suppressor, mutated in a variety of cancers. MiR-155 has been reported to negatively regulate APC in multiple carcinomas including HCC and papillary thyroid carcinoma , 62 to pIn regard to the kinase in the “destruction complex”, GSK-3β was targeted by miR-26a as revealed in target prediction and validated in biological experiments. MiR-26a targets GSK-3β to promote cholangiocarcinoma growth through concomitant activation of Wnt/β-catenin . GSK-3β Another scaffolding protein in the “destruction complex”, AXIN2 is targeted by miR-544a. MiR-544a was identified as an EMT-inducing miR in a cell-based reporter system with a 328-miRs library. In gastric cancer, it targets E-cadherin and AXIN2 to promote β-catenin translocation to the nucleus, resulting in active Wnt signaling . It suggIn summary, miRs regulate the Wnt pathway by targeting β-catenin directly and by inhibiting expression of β-catenin interacting proteins, revealing a regulatory network that is intertwined and complicated.via direct targeting of oncogenic transcription factor LEF1 [The established action mechanism of β-catenin is activation of downstream gene expression by binding the transcriptional factor TCF/LEF. MiRs that target the TCF/LEF transcription factors and other associated co-activators/co-repressors regulate transcription transduced by Wnt signaling Figure . For exator LEF1 . MiR-139tor LEF1 .Other transcriptional factors and co-activators/co-repressors besides the TCF family are modulated by miRs to affect Wnt signaling. P130 is a transcription factor associated with GSK3β which sequesters β-catenin in an inactive form as a P130/GSK3β/β-catenin complex in the cell nucleus . It was NLK (Nemo-like kinase), an evolutionarily conserved protein kinase, is an inhibitor for the Wnt pathway by binding to and phosphorylating TCF/LEF-1 family proteins . MiR-92bCollectively, research has documented the involvement of miRs in regulating the Wnt pathway at the last checkpoint-mRNA expression. Transcriptional regulation was observed by direct control of TCF/LEF expression and by inhibition of the formation of the active TCF/β-catenin complex. Thus, miR regulation of transcription adds another layer of fine tuning the signaling cascade.More recently, the crosstalk between Wnt signaling and other signaling pathways has been described in more detail. A great number of miRs have been found to regulate the Wnt signaling pathway indirectly by targeting elements in other pathways Table . The funTranscription factor E2F1 preferentially binds to retinoblastoma protein pRB to prevent cell-cycle progression. It mediates p53-dependent/independent apoptosis. E2F1 was shown to suppress the Wnt pathway by trans-activating Catenin Beta Interacting Protein 1 (CTNNBIP1)  or by upMyocyte enhancer factor 2D (MEF2D) is a transcription factor of the MEF2 family. The family members were found to be involved in carcinogenesis and cancer progression . MiR-19 Take another transcription factor for example, smad4 is an important transcription factor for TGFβ signaling which participates in multiple biological processes including cell survival, apoptosis, growth and differentiation. For the TGFβ/Smad pathway, smad4, smad7 and TGFβ are targeted by miR-182-5p , miR-93 In addition to transcription factors, miR regulates Wnt signaling indirectly by targeting enzymes in other signaling pathways. PTEN, a critical enzymatic protein in PI3K/AKT signaling pathway, was found to be targeted by miR-301a  and miR-via APC reduction. EZH2 was reported to be targeted by miR-506 [via Wnt pathway inhibition.Enhancer of zeste homolog 2 (EZH2) catalyzes the methylation of histone H3 in target gene promoters to repress gene expression. It regulates cell proliferation, migration, metastasis, and chemo-resistance by silencing tumor suppressor genes such as APC –91. EZH2 miR-506  and miR- miR-506  in colonIn another example, ZNRF3, an E3 ubiquitin ligase, inhibits Wnt pathway by increasing proteolysis of FZD and LRP6 . It was In addition to transcriptional factors and enzymes, miRs also regulate the Wnt pathway by targeting receptor associated proteins. For instance, ROBO receptors are members of the immunoglobulin superfamily of cell adhesion molecules (ICAMs). The ROBO pathway has been implicated in cancer development and progression . The molSLC34A2 is a plasma membrane protein that mediates sodium-dependent phosphate transport. SLC34A2 is involved in tumorigenesis. MiR-410 promotes the tumorigenesis and development of NSCLC by down-regulating SLC34A2 and activating the Wnt pathway. MiR-410 might be a new potential therapeutic target for NSCLC .Integrin beta-like 1 (ITGBL1), is a beta-integrin similar protein. The function of ITGBL1 is not clear to date. ITGBL1, targeted by miR-576-5p, inhibits NSCLC progression by suppressing the Wnt pathway .Additional examples of miRs that regulate Wnt by targeting elements in other signaling pathways are listed in Table Thus far, it has been shown that miRs regulate the Wnt pathway however, the reverse is true-Wnt pathway modulates the expression of miR positively and negatively in a variety of tumors to induce carcinogenesis, progression, and drug resistance Table . The abeIn colon cancer, Brian et al. have used quantitative PCR arrays as well as mathematical analysis to discover distinctively expressed miRs in tumors resulting from loss of function mutation of APC. MiR-215 and -137 are repressed and miR-708, -135b and -31 levels are increased in APC mutation induced tumors. Target prediction and pathway analysis suggest that these miRs control signaling pathways critical for transformation . ConversOther than CRC, 30% of gastric cancers carry nuclear accumulation of β-catenin, highlighting the importance of the Wnt pathway in gastric oncogenesis. To identify the miRs regulated by Wnt signaling, a miR microarray was conducted with β-catenin interference in gastric cancer cells to detect expression differences in the miR transcriptome . The expAdditionally, several studies explored the underlying mechanisms of HCC progression from the perspective of Wnt signaling, as β-catenin is a major oncogene in HCC and is activated in 30-40% of cases . Wnt/β-cvia trans-activating Lin28 in breast cancer stem cells [In breast cancer, miR-182 is highly expressed and is up-regulated by the Wnt pathway. The overexpression of miR-182 increases tumorigenicity and invasiveness by repressing RECK . In addiem cells . It was There are positive feedback circuits between miR and Wnt signaling. MiR-218 and Wnt pathway form a positive feedback loop to increase osteoblast differentiation and abnormal expression of osteoblastic genes in breast cancer cells . MiR-218Mutual inhibition between miR and Wnt pathway has also been observed. In glioma cells, miR-122 inhibits the Wnt pathway, which negatively regulates the expression of miR-122 . In CRC,In addition, a negative feedback loop was also found between miR and the Wnt signaling pathway. MiR-483-3p targets β-catenin which induces the expression of miR-483-3p . They foIn summary, miRs regulate the Wnt signaling pathway and Wnt signaling in turn modulates expression of miRs in human neoplasms. In addition, both form mutual feedback circuits, thereby increasing the connectivity and complexity of the regulatory network. Chartering this network will facilitate the development and advancement of miR-based clinical applications.ClinicalTrials.gov; NCT01829971). MRX34 is used for patients with advanced HCC, other selected solid cancers and hematologic malignancies. The scope of the trial is to establish the safety, pharmacokinetics and pharmacodynamics of MRX34. The company planned to finish the phase 1 trial by the end of 2016 and start the phase 2 clinical trial in 2017.Recently, miR-based therapeutics have been developed based on the regulatory network of miRs and Wnt/β-catenin. MiR-based anticancer therapeutic approaches have used several strategies such as miR sponges, anti-miR oligonucleotides, miR masks, and small molecule inhibitors (reviewed in ). MiR-34Pioneering work has certain challenges that must be overcome before miR-based therapies can be used in a clinical setting. Low bioavailability, specific tissue delivery, off target side effects, miR instability, immunogenicity and tumorigenicity are major problems to solve before the application in human clinical trials . DespiteCollectively, we have summarized the current understanding of crosstalk between miRs and the canonical Wnt signaling cascade in various types of tumors. Communication intersects in multiple planes to modulate cell proliferation, migration, cancer metastasis, and drug response. MiR-based therapeutics have entered clinical trials; a stage where opportunities and challenges co-exist for miR-based therapeutics for cancer. A better understanding of miRs and the Wnt signaling regulatory network will provide insights to further the development of miR-based remedies.


In the adult central nervous system (CNS), Wnt signaling regulates dendritic structure and synaptic plasticity. The Wnt signaling pathway can be divided into the canonical (β-catenin-dependent) and non-canonical pathways. In the canonical pathway, the binding of canonical ligands such as Wnt3a to the Frizzled receptor induces inactivation of glycogen synthase kinase-3β (GSK-3β), which stabilizes β-catenin and allows its translocation to the nucleus. However, to date, few studies have focused on β-catenin-independent Wnt signaling or explained the underlying mechanisms connecting Wnt signaling to cellular energy metabolism. A recent study demonstrated negative regulation of 5′ adenosine monophosphate-activated protein kinase (AMPK), a major target of GSK-3β that regulates cellular metabolism under diverse conditions. Mainly based on these observations, we evaluated whether Wnt3a ligand modulates autophagy by regulating the GSK-3β/AMPK axis.Cultured primary hippocampal neurons and slices of the CA1 region of rat hippocampus were used. GSK-3β inhibition, AMPK activation, PP2Ac expression, and LC3 processing were examined by western blotting. Autophagic compartments were studied using the CYTO-ID® fluorescent probe, and mature autophagosomes were observed via transmission electron microscopy (TEM).Wnt3a ligand, acting through the Frizzled receptor, promotes the rapid activation of AMPK by inactivating GSK-3β. Biochemical analysis of downstream targets indicated that Wnt3a ligand modulates autophagy in hippocampal neurons.Our results revealed new aspects of Wnt signaling in neuronal metabolism. First, AMPK is an additional target downstream of the Wnt cascade, suggesting a molecular mechanism for the metabolic effects previously observed for Wnt signaling. Second, this mechanism is independent of β-catenin, suggesting a relevant role for non-genomic activity of the Wnt pathway in cellular metabolism. Finally, these results have new implications regarding the role of Wnt signaling in the modulation of autophagy in neurons, with a possible role in the removal of accumulated intracellular proteins. Mechani GTPases . However GTPases . On the  GTPases –7. In th GTPases .S-succinyltransferase, and lysosomal cysteine proteinase [To date, data on the role of Wnt signaling at the cellular metabolic level include GSK-3β as a point of intersection with the insulin pathway , 10; traoteinase ; regulatoteinase ; mitochooteinase ; modulatoteinase , 15; andoteinase , 17. At oteinase . Therefo+ 2/CaM-dependent protein kinase β (CaMKKβ) [However, the precise mechanisms that connect Wnt signaling with cellular metabolic events remain unclear. In this sense, 5′ adenosine monophosphate-activated protein kinase (AMPK) is a good candidate. This heterotrimeric protein kinase is extensively expressed in all tissue types, including hippocampal neurons , 20, act(CaMKKβ) . AMPK is(CaMKKβ) , 24 and (CaMKKβ) , 26. In (CaMKKβ) –30.+ 2 release after Wnt activation [Interestingly, several reports have shown that autophagy can negatively modulate the Wnt signaling pathway through Dvl degradation –33. Moretivation . In the 2. After 3 and 7 days in vitro (DIV), cells were treated with AraC for 24 h to avoid glial cell growth. Neurons were ready for experimentation at 14 DIV as previously described [Rats were housed at the Animal Warehouse Facility of Pontificia Universidad Católica de Chile. The Bioethical and Biosafety Committee of the Faculty of Biological Sciences at our university approved the experimental procedures. Briefly, hippocampi were isolated and dissected from Sprague–Dawley rats at embryonic day 18. Primary rat hippocampal neurons were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% horse serum for 2 h at 37 °C. Then, the medium was substituted with Neurobasal medium supplemented with B27, 100 U/mL streptomycin, and 100 U/mL penicillin and cultured at 37 °C with 5% CORecombinant Wnt3a and Dkk1 were purchased from R&D Systems . Lithium chloride was from Sigma Chemicals . TCS-183 was obtained from Tocris Bioscience . Rapamycin was from Enzo Life Technologies , and metformin hydrochloride was purchased from Abcam Biochemicals® .Total protein extraction from cultured hippocampal neurons and immunoblot analysis were performed as previously described . The folSeveral previous reports have agreed that the Cyto-ID® Autophagy Detection probe  labels autophagosomes and have validated this probe as a good estimate of autophagic flux , 38. DyeWe prepared brain slices from rat brains by cutting transverse slices of 400 μm using a vibrating microtome; slices were maintained under cold artificial cerebrospinal fluid (ACSF), according to a previously described protocol and . The sliData are presented as the mean ± SEM. The number of experimental replicates is indicated by “n”. ANOVA followed by uncorrected Fisher’s LSD post hoc test was used to assess statistically significant differences. Statistical analysis was performed using GraphPad Prism 6.0v .We assessed whether stimulation with the canonical Wnt3a ligand modulates AMPK activity in hippocampal neurons. We treated neurons with 300 ng/ml of Wnt3a ligand and monitored AMPK activity by assessing Thr172 phosphorylation over 60 min. AMPK phosphorylation significantly increased (2–3-fold) from 15 to 60 min Fig. . In paraThe canonical Wnt pathway signals through the Fz receptor along with LRP5/6. This signaling can be antagonized using Dickkopf 1 (Dkk1), an LRP5/6 antagonist, which prevents GSK-3β inactivation and leads to β-catenin destruction. In our experiments, AMPK phosphorylation significantly increased following the addition of 300 ng/ml Wnt3a ligand at 30 and 60 min of treatment. Interestingly, decreased AMPK activity was observed in the presence of the canonical Wnt inhibitor Dkk1 Fig. . SimilarWe used lithium, a pharmacological inhibitor of GSK-3β, that produces the stabilization of β-catenin to verify GSK-3β-dependent activation of AMPK , 48. In Downstream of AMPK activation, autophagy is typically triggered upon mTORC1 inhibition , 51. Aut/neuron , which highlights autophagosomal compartments with green fluorescence . CompareFinally, we used TEM to determine the presence and number of autophagolysosomal structures in the rat hippocampal CA1 region, which is rich in synapses  where Wnt ligands have an effect on learning/memory processes. Wnt3a ligand has an especially marked influence on fear memory acquisition and consolidation . The numTo date, Wnt signaling has been shown to play an extensive role in neuroprotection that is particularly concentrated in the synaptic region –56. Wnt3Indeed, previous reports have described an alternative branch of Wnt/GSK-3β signaling, including the prevention of aberrant tau and microtubule-associated protein 1B (MAP1B) phosphorylation mediated by Wnt3a ligand and Wnt7a treatment, respectively , 62. MorIn the present work, for the first time, we demonstrate that Wnt3a ligand can modulate AMPK activation and autophagy in hippocampal neurons; these two pathways are directly connected Fig. . Accordi+ 2 from the extracellular space after Wnt3a ligand treatment potentially suggests an alternative pathway for AMPK activation through the Ca+ 2-Calmodulin-CaMKKβ axis [However, other regulatory mechanisms for the Wnt3a/AMPK axis have been described, e.g., a rapid influx of CaKKβ axis –66.Our findings also offer new insights regarding Wnt-autophagy crosstalk because Wnt3a ligand could modulate autophagy in cultured hippocampal neurons. According to assays using the Cyto-ID probe, Wnt3a ligand significantly increased autophagolysosomal staining, but Wnt3a ligand plus rapamycin behaved similarly to rapamycin alone and did not show any synergistic activity or inhibition of the Wnt pathway activated by a canonical Wnt ligand Fig. . Thus, oNevertheless, autophagy may be regulated by mTOR-dependent and mTOR-independent pathways that respond to different metabolic conditions . In the Although the Wnt pathway activates mTORC1 through GSK-3β following non-canonical Wnt/STOP signaling . By the Our studies allow us to suggest a hypothetical feedback loop between the Wnt signaling pathway and autophagy that positively modulates autophagy.Although further research is needed, we believe that our results suggest a novel GSK-3β/AMPK fast route in which Wnt signaling activated by canonical ligands has effects that do not involve the stabilization of β-catenin. Moreover, non-genomic activities, particularly the modulation of autophagy, may be a relevant player in the clearance of toxic intracellular protein accumulation, such as neurofibrillary tangles or intracellular Aβ species, which are highly relevant in different neuropathological conditions, particularly in Alzheimer’s disease.In summary, our results highlight at least three novel aspects of the influence of the Wnt pathway on hippocampal neuronal metabolism. First, our results reveal AMPK as an additional actor downstream of the Wnt signaling cascade, suggesting a molecular explanation for the underlying metabolic effects previously observed after Wnt ligand treatment. Second, this mechanism is probably independent of β-catenin, suggesting a role for the non-genomic activity of the Wnt pathway. Finally, our data suggest a novel role for the Wnt pathway in modulating the process of autophagy, which is a pivotal mechanism for preventing the accumulation of toxic intracellular proteins.


The objective of this study was to investigate the potential use of sand washing waste as filler for epoxy resin mortar. The mechanical properties of four series of mortars containing epoxy binder at 10, 15, 20, and 25 wt. % mixed with sand blended with sand washing waste filler in the range of 0–20 wt. % were examined. The compressive and flexural strength increased with the increase in epoxy and filler content; however, above epoxy 20 wt. %, slight change was seen in strength due to increase in epoxy and filler content. Modulus of elasticity also linearly increased with the increase in filler content, but the use of epoxy content beyond 20 wt. % decreased the modulus of elasticity of the mortar. For epoxy content at 10 wt. %, poor bond strength lower than 0.8 MPa was observed, and adding filler at 20 wt. % adversely affected the bond strength, in contrast to the mortars containing epoxy at 15, 20, 25 wt. %. The results indicate that the sand washing waste can be used as potential filler for epoxy resin mortar to obtain better mechanical properties by adding the optimum level of sand washing waste filler. Concrete structures were assumed to be durable and maintenance free; however, in the past few decades, sign of degradation are becoming apparent ,2 for vaVarious materials from conventional Portland cement to polymer binders have been used to perform the repair of concrete structures. Ordinary Portland cement (OPC) is by far the most widely consumed material for the repair and restoration of concrete structures. Despite their vast consumption, OPC-based composites present different shortcomings, such as longer curing time, weak flexural strength, poor bond strength, high shrinkage, low resistance to aggressive environment, and durability problems. These problems of OPC have instigated the use of polymer-based materials as a repair material, which resulted in the introduction of epoxy resin mortar in the repair industry .Epoxy resin mortar has been used as a repair material for the past couple of decades ,10,11,12The recycling of industrial wastes such as sand washing waste is important for sustainable development in the construction industry. Sand washing waste is a waste product of the sand production process, and creates a landfill problem. The potential reuse of sand washing waste as a supplementary material for sand is beneficial for reducing the depletion of natural sand ,25. HencIn this study, a mortar was prepared by using epoxy resin, sand, and filler; the mechanical properties of the mortars with different mix proportions were investigated.A two-part epoxy having a Grade 1 viscosity as per ASTM C881  was used3, water absorption of 0.41%, a specific gravity at oven dry condition of 2.6, and a void fraction of 41.28%. Since the epoxy resin mortar was also prepared to repair cracks with small widths, a fine-graded sand with high percentage of particle size less than 600 µm was used. In order to accurately determine the particle size of the sand, a consistent method to that of sand washing waste (laser diffraction technique) was utilized, and Sand used in this study was purchased from a local store. The physical properties of the sand were characterized according to ASTM C128  and ASTM3 and specific gravity at oven dry condition of 2.69 according to ASTM C29 and ASTM D854 [The filler used in this investigation was a sand washing waste that was collected from an aggregates producing plant located in Gyeonggi-do, Korea; the sludge material was a waste product of a sand washing process. The collected sludge was dried at 110 °C to remove any moisture, crushed to break off clusters, and sieved to remove any clusters. The sand washing waste used as filler  had a bu2/m3 and 140,625.65 m2/m3, and the mean size was 450.83 μm and 21.12 μm. The chemical composition of the filler and the sand examined using X-ray Fluorescence manufactured by Horiba, Japan is given in 1 target (λ = 1.54059 Å), U = 40 kV, and I = 200 mA. The XRD data is shown in Based on the particle size distribution of the sand and the filler, the specific surface area and the mean size were calculated. The specific surface area of sand and filler were found to be 1965.57 mFour mortar groups were prepared with epoxy content at 10%, 15%, 20%, and 25% by weight fraction of the mortar. The minimum epoxy content of 10 wt. % was selected from workability condition, and the maximum content was limited to 25 wt. % due to segregation of the epoxy from the mortar mixture. Filler was added to the mortar mix at 10 and 20 wt. % as sand replacement. The detailed mix proportion of the samples investigated in this study is presented in Specimens were prepared to study the effect of filler on the compressive strength, flexural strength and modulus of elasticity, and bond strength of epoxy resin mortar. The sample preparation was performed as follows. Except for control specimens, first dry mixing of sand and filler was performed for 2 min using an automatic mortar mixer to ensure a uniform distribution of the sand and filler. Epoxy resin and its hardener were manually mixed for 1 min in a separate bowl and were added to the mortar mixer. Mixing was continued for another 2 min, and the mortar mixture was casted as per KS F 4043 . Before The compressive test of the epoxy resin mortar was conducted according to KS F 4043/EN 12190 . Three cThe flexural strength and modulus of elasticity of three epoxy resin mortars of each mix proportion were evaluated in accordance to ASTM C580 . ContinuThe compressive strength is a traditionally examined property of repair material for concrete structures. Mostly it is expected that the ratio of the compressive strength of repair mortar to that of the substrate concrete should be equal to or higher than one. Furthermore, KS F 4043 suggests a minimum compressive strength of 40 MPa for epoxy resin mortars used for repair of concrete structures. All the samples except E10-F0 showed a compressive strength above 40 MPa.The flexural strength of mortar mixes prepared with varying filler content at given amount of epoxy is presented in The strength of repair materials to withstand a bending load is stated as flexural strength. Generally, the ratio of the flexural strength of the repair mortar and the substrate concrete should be higher than one, whereas KS F 4043 specifies 10 MPa as the minimum flexural strength value for epoxy resin mortars used for restoration of concrete structures. In our study, all mortar mixes prepared exhibited a flexural strength much higher than 10 MPa.2 value of 0.998, 0.999, 0.966, and 0.999 for epoxy content at 10, 15, 20, and 25 wt. %, respectively. The addition of filler resulted in an average increase in MoE of 51.5% for epoxy at 10 wt. %, 54.47% for epoxy at 15 wt. %, 11.10% for epoxy at 20 wt. %, and 7.29% for epoxy at 25 wt. % when compared to the control group. Besides the substantial improvement in the MoE of mortar containing epoxy at 10 wt. % and 15 wt. %, a higher rate of increase in MoE with the increase in filler content was observed when compared to mortars containing epoxy at 20 wt. % and 25 wt. %. The increase in MoE can be attributed to the increased stiffness of the mortar when the filler is added.The tangent modulus of elasticity of the epoxy resin mortars was determined from the load versus deflection relation obtained via three-point bending test. 2 value of 0.919 exists between the MoE and the density of the samples.The density of the epoxy resin mortar three-point bending test beams were determined by measuring the mass and volume in order to understand the relation between the MoE and the density of the hardened beam samples. The bond strength of epoxy resin mortar to substrate concrete was determined using a pull-off test. A high strength concrete with a 28-days compressive strength of 50 MPa was prepared as a substrate to execute this test. Depending on which section is the weakest, one of the three modes of failures (de-bonding)  could occur when the pull-off test on the epoxy resin mortar-bonded concrete is performed.Bond strength of repair material is an important property that holds the repair mortar and the substrate concrete bonded as a unit. Hence, in order to have a durable repair work of concrete structures, the mortars used should have adequate adhesion to avoid bond failure between the repair and the substrate because of the stresses developed due to internal and external loads. KS F 4043 suggests a minimum of 1.5 MPa bond strength of the epoxy resin mortar used for the maintenance of concrete structure. In this study, the epoxy resin mortar prepared with 10 wt. % epoxy showed poor bond strength lower than 0.8 MPa. Whereas all the samples with epoxy at 15, 20, and 25 wt. % showed a bond strength higher than 1.5 MPa.(1)The compressive strength, flexural strength and modulus of elasticity value of epoxy mortar containing filler up to 20 wt. % improved by an average of 1.08–1.66, 1.18–1.66, and 1.07–1.54 times, respectively, at different weight fraction of epoxy in the range of 10–25 wt. % when compared to the control specimens.(2)It was observed that when filler is used at optimum level, it can improve bond strength; all the mortars except those prepared with epoxy at 10 wt. % showed a good bond strength higher than 1.5 MPa.(3)It can be established that sand washing waste can be used as potential filler for epoxy resin mortar to obtain better compressive, flexural, stiffness, and bond strength.(4)This study does not cover all the parameters required to examine repair materials for concrete structures; further investigations on compatibility issues such as dimensional change stability of the epoxy resin mortar containing sand washing waste filler is required.This study investigated the effect of filler on the mechanical and adhesion properties of epoxy resin mortar. The following conclusions were drawn based on the experimental results obtained.


CTNNB1 gene that encodes β-catenin occur in a wide spectrum of cancers. These mutations alter the spatial characteristics of the β-catenin protein, leading to drastic reprogramming of the nuclear transcriptional network. Among the outcomes of this reprogramming are increased cell proliferation, enhanced immunosuppression, and disruption of metabolic regulation. Herein we review the current understanding of CTNNB1 mutations, their roles in tumorigenesis and discuss their possible therapeutic implications for cancer.The canonical Wnt/β-catenin signaling pathway, an important modulator of progenitor cell proliferation and differentiation, is highly regulated for the maintenance of critical biological homeostasis. Decades of studies in cancer genetics and genomics have demonstrated that multiple genes encoding key proteins in this signaling pathway serve as targets for recurrent mutational alterations. Among these proteins, β-catenin and adenomatosis polyposis coli (APC) are two key nodes. β-catenin contributes in transporting extracellular signals for nuclear programming. Mutations of the  APC, is one frequently mutated gene of Wnt signaling in human cancers [Cancer is a systems disease with a complicated pathogenesis. Tumorigenesis is characterized by abnormal regulation of cell growth and cell death. Mutations in proto-oncogenes and tumor suppressor genes underlie tumorigenesis by dysregulation of intracellular signaling pathways that are critical for the normal physiology of organisms . The can cancers . As one  cancers . β-caten cancers , 9. In tWnt gene was first discovered in 1982 by Nusse and Varmus while studying the transcription mechanisms for a tumor virus in a murine mammary tumor [Int1, the gene was determined to encode proteins that transfer growth and development signals between cells. Further studies demonstrated that this gene could make Drosophila flies wingless in normal embryonic development, and it was renamed Wnt [+ pathway, noncanonical planar cell polarity pathway, and canonical Wnt/β-catenin signaling pathway [The ry tumor . Initialamed Wnt , 12. Theamed Wnt . There aamed Wnt –16. Wnt  pathway , 17–19.  pathway , 20, 21.The complexity of the canonical Wnt/β-catenin pathway derives from the high number of ligands and receptors involved in signaling that can elicit a variety of intracellular responses , 23. As In the absence of Wnt ligands, cytoplasmic β-catenin is phosphorylated at N-terminal serine-threonine residues by the destruction complex and degraded by the proteasome via the ubiquitin-proteasome pathway , 27. In Alternatively, in the presence of Wnt, ligand binds to the cell-surface receptor Frizzled and acts on Dishevelled protein , 33. FriCTNNB1, which maps to 3p21 [CTNNB1 has 16 exons according to restricted mapping and partial sequence analysis [β-catenin is a multifunctional cytoplasmic protein composed of a polypeptide chain . It is e to 3p21  and withanalysis . The prianalysis –56. The analysis . The armanalysis , each ofanalysis . Generalanalysis , 60. Theanalysis –64.β-catenin is crucial for two important developmental processes: 1) establishment and maintenance of cell-type-specific cell-to-cell adhesion and 2) regulation of target gene expression via the Wnt signaling pathway , 65. It CTNNB1 is a key exon encoding serine-threonine phosphorylation sites for GSK-3β that activates degradation of β-catenin [CTNNB1 mutations are frequently missense mutations [Exon 3 of -catenin . The CTNutations . Nearly utations , 80–82. CTNNB1 and deregulation of the canonical Wnt pathway were in colorectal cancer cases [CTNNB1 mutation spots and rates in human cancers in detail [CTNNB1 mutations and abnormal nuclear β-catenin expression [Gene mutations leading to constitutive activation of the Wnt/β-catenin signaling pathway, especially the canonical one, are early events in the development of some cancer cases . Moreoveer cases , 86. Siner cases –92. Pauln detail . For exan detail . Such mun detail , 95. Then detail . Fbw1 isn detail , 96. In pression , 98.CTNNB1 mutates, once a threshold of β-catenin accumulates in the cytoplasm and nucleus, this status reflects abnormal expression of β-catenin [APC mutations can correlate with high expression levels of β-catenin whereas wild-type APC expression can reduce β-catenin levels in colorectal cancer cells [c-myc and cyclin D1. This process promotes cell proliferation [As described above, under normal physiological conditions, β-catenin is expressed mainly in the cell membrane. In contrast, the expression level of dissociative β-catenin in the cytoplasm is quite low owing to the combination with the destruction complex . Whether-catenin . When de-catenin . For exaer cells , 100. Ther cells –104. Theer cells . The resferation . Thus, aferation , 79.CTNNB1 mutations in EEC compared to nonendometrioid endometrial carcinoma cases (NEEC) [CTNNB1 mutations seemingly occurr in the early stages of endometrial carcinogenesis [CTNNB1 in endometrioid ovarian carcinoma [CTNNB1 mutations in EEC [CTNNB1 gene mutations. Eighty-seven percent of the tumors in this cluster carried CTNNB1 mutations. This cluster was associated with young, obese patients with grade 1 or 2 tumors that were early stage at diagnosis (stages I or II) and had decreased expression of estrogen and progesterone receptors. Importantly, this cluster was also associated with decreased overall survival. Table CTNNB1 mutations in these cases.For some cancers, such as endometrioid endometrial carcinoma (EEC), studies have demonstrated that 40-60% of cases exhibited nuclear accumulation of β-catenin in tumor cells , 108. Ots (NEEC) , 110. Dearcinoma –116. Froarcinoma . Liu ands in EEC . They peCTNNB1 mutations have been shown to induce the accumulation of nuclear β-catenin and activate the canonical Wnt pathway, some studies demonstrated that this accumulation can occur without CTNNB1 mutations [AXIN1 and AXIN2 can activate the Wnt pathway and have been associated with the development of a subset of colon, hepatic, and ovarian carcinomas and medulloblastomas [Although utations , 106. Thlastomas , 118–120lastomas . In addilastomas –124. In lastomas . Inhibitlastomas . Similarlastomas –130.cyclin D1 and c-myc, by binding with the 3’-untranslated region of β-catenin mRNA directly [CTNNB1 mutation can activate the canonical Wnt pathway.There are also critical upstream regulators of β-catenin, specifically p53 and microRNAs (miRNAs), that impact canonical Wnt signaling. As a tumor suppressor, loss of p53 function can activate canonical Wnt pathway by its transcriptional activity . miRNAs directly . Roman Adirectly . miRNA-1CTNNB1 mutations were associated with TGF-β2, which contributes to tumor progression. These mutations also influenced the numbers of cytotoxic cells that can kill tumor cells, such as lymphocytes and macrophages [Many types of tumors, including hematological malignancies and melanoma have been treated with immunotherapy , 137. Horophages . From thMetabolic regulation is essential to tumorigenesis, and metabolic reprogramming is one of the hallmarks of tumor cells . Metabollactate transporter MCT-1 (SLC16A1) and pyruvate dehydrogenase kinase, isozyme 1, are also downregulated due to this blockage [The Wnt/β-catenin signaling pathway can induce the Warburg effect and establish metabolic zonation Figure  153, 16–165. Howblockage . Pyruvatblockage . Therefoblockage . Authorsblockage .bone morphogenic protein and the activin membrane-bound inhibitor (Bambi), which can induce Wnt/β-catenin signaling pathway leading to cell proliferation and survival [Metformin has been used to treat hyperglycemia and type 2 diabetes mellitus . This drsurvival , 176. Alsurvival . Taken tin vitro and tumorigenesis in vivo were regulated by β-catenin in triple-negative breast cancer. In addition, they proved that the transcriptional activity of β-catenin was quite important for the chemosensitivity of triple-negative breast cancer cells. So at least in part, β-catenin can be considered as one of therapeutic targets for this cancer.Researchers revealed that β-catenin was required for the development of triple-negative breast cancer that is estrogen-dependent . In theiPast studies have elaborated that antagonists of β-catenin or other proteins in the Wnt pathway could block this pathway and inhibit cell proliferation in many different tumor types , 178–185Besides the specific antagonists, hormones can influence the expression of β-catenin. For example, progesterone can counteract the accumulation of β-catenin in the nucleus and inhibit the Wnt/β-catenin signaling pathway , 108. ThIn summary, the important canonical Wnt/β-catenin signaling pathway exerts potent biological functions. As evidence, induction of tumorigenesis occurs when any step in this pathway is deregulated. β-catenin is the key intracellular transducer of this signaling pathway. Abnormal expression and mutations of β-catenin have been regarded as important signs of malignancy , 79. ResAnother noteworthy problem about β-catenin is that some studies demonstrated that β-catenin activation alone could not induce spontaneous development of hepatocellular carcinoma in mouse models . β-caten


Oxygenic phototrophs have strategies to alleviate photo-oxidative damage to allow life in present atmospheric CO2 conditions. However, the mechanisms for CO2 limitation acclimation are diverse among the various oxygenic phototrophs, and many mechanisms remain to be discovered. In this study, we found that the gene encoding a CO2 limitation-inducible protein, ColA, is required for the cyanobacterium Synechococcus sp. PCC 7002 (S. 7002) to acclimate to limited CO2 conditions. An S. 7002 mutant deficient in ColA (ΔcolA) showed lower chlorophyll content, based on the amount of nitrogen, than that in S. 7002 wild-type (WT) under ambient air but not high CO2 conditions. Both thermoluminescence and protein carbonylation detected in the ambient air grown cells indicated that the lack of ColA promotes oxidative stress in S. 7002. Alterations in the photosynthetic O2 evolution rate and relative electron transport rate in the short-term response, within an hour, to CO2 limitation were the same between the WT and ΔcolA. Conversely, these photosynthetic parameters were mostly lower in the long-term response of a few days in ΔcolA than in the WT. These data suggest that ColA is required to sustain photosynthetic activity for living under ambient air in S. 7002. The unique phylogeny of ColA revealed diverse strategies to acclimate to CO2 limitation among cyanobacteria.A limitation in carbon dioxide (CO This process involves both light and dark reactions in the photosynthetic electron transport system on the thylakoid membrane and the Calvin-Benson cycle in the stroma in chloroplasts, respectively. In the photosynthetic electron transport system, photon energy excites the reaction center chlorophylls (Chl), P680 and P700 in photosystems (PS) II and I, respectively, to drive photosynthetic linear electron transport. In PSII, H2O is oxidized to oxygen (O2) by photo-oxidized P680, and electrons derived from P680 are transferred to photo-oxidized P700 in PSI through plastoquinone, cytochrome b6/f complex, and plastocyanin or cytochrome c6. These processes result in the formation of a proton gradient across the thylakoid membrane to produce ATP with chloroplast ATP synthase. Conversely, electrons originating in P700 are transferred to NADP+ via ferredoxin and ferredoxin-NADP+ reductase. Subsequently, NADPH and ATP are produced by photosynthetic linear electron transport. In the stroma, ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase, also known as Rubisco, catalyzes the carboxylation of RuBP with CO2 to drive the Calvin-Benson cycle, producing a variety of sugars with NADPH and ATP as reducing agents.Oxygenic photosynthesis is the most popular anabolic biological activity on earth. From photon energy, water  [2 to produce reactive oxygen species (ROS), causing photo-oxidative damage in PSII and PSI [Oxygenic phototrophs have developed diverse strategies to prevent and recover from photo-oxidative damage to allow acclimation to limited COow (AEF) ,8, non-pow (AEF) , and oxiow (AEF) . Without and PSI ,6,10. Th2 limitation in cyanobacteria and eukaryotic algae [2 limitation are diverse, even among cyanobacteria, which are known as the progenitors of oxygenic phototrophs. In the cyanobacterium Synechocystis sp. PCC 6803 (S. 6803), flavodiiron proteins (FLV) 2 and 4 are highly expressed in response to limited CO2 conditions [2 as the final electron acceptor [Synechococcus elongatus PCC 7942 (S. 7942) does not have FLV2/4 and shows little AEF in limited CO2 [Synechococcus sp. PCC 7002 (S. 7002) does not possess FLV2/4. Nevertheless, S. 7002 shows a substantial O2-dependent AEF under limited CO2 conditions with FLV1 and 3 alone, when the cells are grown under ambient air but not high CO2 [2 limitation in S. 7002 [2 limitation, even in the absence of FLVs, S. 7002 cannot oxidize P700 without FLV or it will suffer from photo-oxidative damage in PSI when the available CO2 is insufficient [2 limitation is substantially different from that in S. 6803 and S. 7942.We reported on the diverse responses of photosynthesis to COic algae ,12,13,14nditions ,15, and acceptor , which iacceptor . Conversited CO2 ,12. Simi S. 7002 . AlthougSYNPCC7002_A2492 that encodes a 20-kDa small membrane-associated protein as a candidate gene related to the unique ability of S. 7002 to acclimate to CO2-limited conditions. A global transcriptome analysis of S. 7002 by Bryant’s research group showed that expression of SYNPCC7002_A2492 is 690-fold higher in cells grown in ambient air than in those grown in high CO2, which is the greatest change in all genes, even though the p-value did not indicate significance [2 limitation-associated protein, ColA. To elucidate the physiological significance of ColA in S. 7002, we constructed a ColA knockout mutant (ΔcolA) and compared the growth, Chl and nitrogen contents, oxidative stress, and photosynthetic parameters with those of the wild type (WT) S. 7002.In the present study, we focused on the gene ificance . Here, wSYNPCC7002_A2492, ΔcolA  of the cultures, was not significantly different between the WT and ΔcolA in either ambient air or high CO2 conditions  content in S. 7002 WT and Δ content . Therefohigh CO2 A. The N nificant B. Furthenditions C, which high CO2 . The firnditions . This prcolA grown in ambient air. Typical high-temperature thermoluminescence of the cyanobacterial cells reflects the extent of oxidative stress originating from lipid peroxidation in the cells [colA than in the WT  and long-term (a few days) in S. 7002 WT and ΔcolA.Based on a lower conversion of N into Chl and the oxidative stress in Δ2 in the reaction medium and the relative Chl fluorescence of the cells, were simultaneously measured during the transition of photosynthesis from CO2-saturated to CO2-limited phases in S. 7002 WT and ΔcolA grown under high CO2  (approximately 20 μmol O2 mg−1 N h−1) were lower than those five minutes after turning on the actinic light (AL) (approximately 30 μmol O2 mg−1 N h−1), corresponding to the relative ETR , which is a marker of PSI activity [colA, which indicated that Pm in ΔcolA  was almost the same as that in the WT , three days after the transition to ambient air growth conditions. In this experiment, we adjusted the concentration of the cyanobacterial cells in the reaction medium to 80 μg N mL−1. These data suggest that the larger decrease in photosynthetic activity in ΔcolA may be due to the inactivation of PSII, rather than of PSI.Next, we investigated the photosynthesis response to CO0 to 3.0 B, and thn the WT A,B. The n the WT C−F. Althient air C,D, Δcolient air E,F. Unfoactivity , in the sll0218 as a ColA gene, whereas S. 7942 has no gene encoding ColA. The gene sll0218 in S. 6803 is highly expressed under CO2-limited conditions, like that observed for SYNPCC7002_A2492 in S. 7002 [flv4-2 operon, by Aro’ and colleagues, in cyanobacterial genomes [sll0218 is independent of that of FLV2/4, even though these genes exist in the same operon [Gene homologs encoding ColA are conserved in some, but not all, cyanobacteria other than S. 7002. For example, S. 6803 harbors  S. 7002 , and is  S. 7002 . We alig genomes ,28. In f genomes  possess 2-limited conditions. Thereafter, the genes for FLV2/4 were generated by the duplication of genes for FLV1/3 that merged into the location of the gene coding ColA to form the flv4-2 operon in the genomes of some cyanobacteria, which would have been advantageous for cyanobacteria requiring mechanisms to prevent photo-oxidative damage under CO2 limitation conditions. In this study, we constructed a mutant of S. 7942 (#HA-ColA) with the gene for ColA introduced into S. 7002 in the form of a hemagglutinin (HA)-fusion protein   . Unfortu S. 7942 , implyin2 conditions [flv4-2 operon  on A+ medium agar plates [+ liquid medium and were grown on a rotary shaker (100 rpm) in continuous light  at high CO2 (2% (v/v)) or ambient air. The optical density (OD) of the cultures at 750 nm was measured with a spectrophotometer .Cultures of S. 7002 were kept under continuous light conditions  methanol. After incubation at room temperature for 5 min, the suspensions were centrifuged at 10,000× g for 5 min. Total Chla in the supernatants were spectrophotometrically determined [For Chl measurement, cells from 0.1–1.0 mL of culture medium were centrifugally harvested and resuspended by vortexing in 1 mL 100% (termined .http://genome.microbedb.jp/CyanoBase) [All of the cyanobacterial gene sequence data used in this study were obtained from CyanoBase (anoBase) .To construct the phylogenetic tree of gene homologs for ColA, we searched for gene homologs among cyanobacteria using the BLAST tool in CyanoBase. Amino acid sequences of these gene products were aligned in ClustalW . The evoTo predict the transmembrane helices in ColA , we usedt-tests were applied to detect differences. All statistical analyses were performed using Microsoft Excel 2010  and JMP8 .Student’s colA, the genomic region encoding SYNPCC7002_A2492 was amplified from the genomic DNA of S. 7002 by PCR  and then cloned into the pGEM-T Easy vector . The recombinant plasmid was linearized and amplified by inverse polymerase chain reaction (PCR)  and then applied to the In-Fusion cloning system  with a chloramphenicol resistance gene cassette originating from the pACYC184 vector [+ medium agar plate containing chloramphenicol (15 µg·mL−1), and complete segregation was confirmed by PCR . The resulting plasmid was used to transform WT S. 7942 according to the method used for S. 7002. Heterologous expression of HA-tagged ColA was achieved in the light for 2 h in the presence of isopropyl β-d-1-thiogalactopyranoside, which was examined by immunoblotting as described below.To introduce the HA-tagged ColA into S. 7942, we used pNSHA based on pNSE1 harboring a spectinomycin resistance gene in the fragment derived from the neutral site . The cod2, and 1 mM phenylmethylsulfonyl fluoride; pH 7.5). The suspensions were homogenized with glass beads using Bug Crasher GM-01  at 4 °C for 20 min and thereafter were centrifuged at 8000× g for 15 min at 4 °C. The supernatants were incubated at room temperature for 1 h in Laemmli SDS sample buffer with 6 M urea and then centrifuged at 13,000× g for 10 min at 4 °C. The resulting supernatants (10 µL each) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were electrotransferred to a polyvinylidene fluoride membrane and detected by HA-specific antibody .For the immunodetection of HA-tagged ColA, cyanobacterial cell cultures (20 mL) were harvested by centrifugation, and the pellets were suspended in 500 µL extraction buffer . The suspensions were homogenized with glass beads using Bug Crasher GM-01  at 4 °C for 20 min, and thereafter were centrifuged at 8000× g for 15 min at 4 °C, resulting in supernatants and pellets. The supernatants were centrifuged twice more at 13,000× g for 30 min at 4 °C, and the resulting supernatants were treated as crude soluble fractions. Conversely, the pellets were mildly vortexed in the extraction buffer with 2% (w/v) octyl β-d-glucopyranoside for 30 min at 4 °C. After centrifugation at 13,000× g for 30 min at 4 °C, the resulting supernatants were treated as crude membrane fractions. Protein concentrations in both crude fractions were determined using a Pierce 660 nm Protein Assay  with bovine serum albumin as the standard. Both crude soluble and membrane fractions  were analyzed by SDS-PAGE. After electrophoresis and electrotransfer to a polyvinylidene fluoride membrane, the proteins were derivatized with 2,4-dinitrophenylhydrazine (DNPH) and then detected by polyclonal antibody specific to DNPH groups [To analyze protein carbonylation, 20 mL cyanobacterial cell cultures were harvested by centrifugation, and the pellets were suspended in a 500 µL extraction buffer . Total nitrogen (N) content was determined using Nessler’s reagent after adding sodium potassium tartrate and sodium hydroxide (NaOH) [Cyanobacterial cells were centrifugally harvested and dried overnight at 60 °C. Dried pellets were digested using the Kjeldahl method with sulfuric acid and hydrogen peroxide (He (NaOH) .−1 using a Thermoluminescence System TL 500  [Lipid peroxidation was monitored by measuring the in vivo thermoluminescence from 20 °C to 160 °C at a heating rate of 1 °C minepublic) .2 were simultaneously measured with Chl fluorescence. Cell samples in a freshly prepared medium  were stirred with a magnetic microstirrer and illuminated with red actinic light (AL) (620 < λ < 695 nm) at 25 °C. A halogen lamp  with an LS2 light  was used as the AL source. O2 was continuously monitored using an O2 electrode  [Net uptake and evolution of Oa was measured using a PAM-Chl fluorometer  [λ > 710 nm (Schott RG9 long-pass filter). The minimum Chl fluorescence (Fo) was determined based on illumination using a measuring light (ML). Steady-state fluorescence (Fs) was monitored under AL, and 1000-ms pulses of saturated light  were supplied to determine the maximum variable fluorescence (Fm′). The fluorescence terminology used in this study follows that used in a previous report [Fm′–Fs)/Fm′ [The relative Chl fluorescence originating from ChlGermany) . Pulse-ms report . The eff–Fs)/Fm′ . The relFor the S. 7942 mutants, the cells were measured similar to the method used for the S. 7002 cells, with the exception that the reaction medium was replaced with 50 mM HEPES-KOH at pH 7.5.2 and relative Chl fluorescence by the method previously reported [2 conditions, were applied to an O2 electrode chamber without an additional inorganic carbon source and were then illuminated with red AL. Illumination with AL stimulated photosynthesis, which was accompanied by an increase in O2 in the reaction medium  of the cyanobacterial cell samples was measured using a Dual-PAM-100  at room temperature (25 ± 2 °C). The reaction mixture consisted of fresh A+ medium and cells (10 μg Chl mL−1). The maximum P700 photo-oxidation level, obtained with saturated pulse light under far-red illumination, was measured and defined as Pm according to the standard method [−2·s−1) was supplied.Total oxidizable P700 (Pd method . For mea


Here, we show that CX3CR1 plays a role in double-strand DNA break response and repair by regulating expression of RAD50 by a MYC-dependent mechanism. We demonstrate that downregulation of CX3CR1 alone and in a combination with irradiation affects peritoneal metastasis in an organ-specific manner; we show that CX3CR1 regulates lipid uptake which could control omental metastasis. This study identifies CX3CR1 as a novel potential target for sensitization of ovarian carcinoma to DNA damage therapies and reduction of peritoneal carcinomatosis.Failure of currently used cytotoxic chemotherapy is one of the main reasons behind high mortality from metastatic high grade serous ovarian carcinoma. We found that high expression of a receptor for fractalkine (CX Among all cancers affecting women ovarian carcinoma is the deadliest gynecologic malignancy1. Among all of its subtypes, high grade serous ovarian carcinoma (HGSOC) is the most predominant and lethal.Cancer metastasis remains a largely unsolved health problem worldwide2. Novel approaches are urgently needed to restore and maintain efficacy of DNA damage therapy to treat this deadly malignancy.There are no effective therapeutic options to treat recurrent chemotherapy-resistant metastatic HGSOC. Patients with metastatic HGSOC undergo debulking surgery and first-line cytotoxic chemotherapy with a microtubule stabilizing taxane and a DNA damage-inducing platinum agent. However, intrinsic and acquired resistance to DNA damaging agents remains a barrier for successful application of these powerful drugs3. Chemokine receptors regulate the metastasis, and, therefore they have been used as anti-cancer drug targets in both pre-clinical and clinical studies6.Chemokine receptors are seven transmembrane G protein-coupled receptors that are activated by their ligands (chemokines) resulting in stimulation of the downstream signaling pathways and changes in cell behavior and function. Chemokine receptors serve as key regulators of many disease states, thus, providing a strong rationale for their development as drug targets for various illnesses3CR1 is activated by only chemokine ligand fractalkine (CX3CL1), which could be present in both secreted and membrane-tethered forms8. Our previous studies demonstrated that interaction of CX3CR1 expressed in ovarian carcinoma cells with membrane-tethered CX3CL1 expressed in peritoneal mesothelial cells mediates peritoneal adhesion of disseminating cells9. We and others have shown that secreted CX3CL1 facilitates ovarian cancer cell proliferation and migration in vitro10. We have demonstrated that ovarian cancer cell proliferation at metastatic sites is dependent on CX3CL1 expressed by parenchyma of intraperitoneal organs and tissues11.A receptor for fractalkine, CX3CR1 can regulate double-strand DNA break repair in HGSOC cells subjected to therapeutic agents inducing DNA damage, such as x-ray radiation and platinum-based agents. Our studies also suggest that CX3CR1 facilitates fatty acid (FA) uptake. Together, these mechanisms converge in promoting omental metastasis in a xenograft mouse model of the disease.Here, we demonstrate that CX13 to analyze survival of patients treated with the standard of care and second-line chemotherapies, including platinum, gemcitabine, and topotecan, as a function of CX3CR1 expression. We have found that both overall (OS) and progression-free (PFS) survivals were significantly shorter when CX3CR1 expression was high , which, if not repaired, lead to cell death17. Hence, these data suggest that CX3CR1 may be involved in double-strand DNA repair (DDR).We used Kaplan−Meier Plotter database for serous ovarian cancer3CR1 in DDR, we used x-ray radiation as a tool to induce DSBs in HGSOC cell lines. Expression of CX3CR1 in cell lines OVCAR-4, Caov-3, and SKOV-3 was transiently downregulated using a pool of CX3CR1-specific siRNAs. The choice of these cell lines as models of HGSOC was motivated by studies demonstrating that the genomic and proteomic profiles of ovarian carcinoma cell lines OVCAR-4 and Caov-3 bear close resemblance to those of HGSOCs, while proteomic profiling of SKOV-3 suggested that this cell line mimics a more deadly mesenchymal subtype of HGSOC19. Transient downregulation was given preference to the stable downregulation to avoid reduction of proliferation due to loss of the functional CX3CL1/CX3CR1 axis, as reported before10, which would greatly impact the outcomes of the clonogenic assay; instead, our goal was to discern the role of CX3CR1 in cellular ability to tolerate DNA damage. Downregulation of CX3CR1 in all tested cell lines synergized with x-ray radiation in significantly reducing their clonogenic ability  was required to achieve this level of reduction of clonogenic ability in the parental cells. These data further suggested a potential role for CX3CR1 in DNA damage response (DDR).To determine the role of CX3CR1 in DDR, we determined levels of a phosphorylated form of H2A histone family, member X (γ-H2AX), a surrogate biomarker for DSBs20, shortly after (20 min) and 24 h following exposure to x-ray using immunofluorescence staining to define a role of CX3CR1 in double-strand DNA damage following irradiation. Although immunoreactivity of nuclear γ-H2AX in cells transfected with control siRNAs was high 20 min post x-ray exposure, significantly fewer γ-H2AX-immunoreactive foci were detected in irradiated cells transfected with CX3CR1 siRNAs at the same time point, indicating that detection of the DSBs in irradiated cells with downregulated CX3CR1 may have been delayed. In fact, 24 h following radiation exposure, γ-H2AX signal in the nuclei of control cells was very low, indicating that DSBs were successfully repaired. On the contrary, γ-H2AX immunoreactivity was very strong in the nuclei of cells with downregulated CX3CR1  kinase activation and decreased intensity of DNA damage response foci in irradiated lung fibroblastsion Fig. . Further25, we tested activation of DNA damage response kinases, including ATM kinase, checkpoint kinases 1 and 2 (CHEK1 and CHEK2), and DNA-dependent protein kinase, catalytic subunit (DNA-PKcs or PRKDC), using western blot. All of the examined kinases were activated in response to x-ray radiation in the control cells (NT and Ctrl si); however, downregulation of CX3CR1 resulted in significantly reduced activation of ATM, CHEK1, CHEK2, and PRKDC in irradiated cells  and homologous recombination (HR)lls Fig. , suggest27. Our data demonstrate that expression of RAD50 was significantly reduced in cells with downregulated CX3CR1 expression , RAD50 double-strand break repair protein (RAD50), and nibrin (NBN), or the MRN complex, is one of the complexes responsible for sensing the double-strand DNA damage, activation of ATM, and, ultimately, DNA repair by HR and NHEJion Fig. , suggest30. We hypothesized that these mechanisms could play a role in CX3CR1-dependent downregulation of RAD50 in ovarian cancer cells. Our studies suggest that MYCN, and MEF2C are not involved in CX3CR1-dependent downregulation of RAD50 or other proteins of MRN complex  values were <1, indicating that downregulation of CX3CR1 synergized with platinum-based drugs in reducing clone formation of treated cells. Analysis of post-progression survival (PPS) of platinum-, gemcitabine-, and topotecan-treated patients with serous ovarian carcinoma expressing high CX3CR1 using KM Plotter database13 showed it was significantly lower compared to that for patients with low CX3CR1 , irradiated cells transfected with control siRNAs (designated “3 gray CTRLsi”), and unirradiated cells transfected with either CX3CR1-specific or control siRNAs   Fig. . Consistly) Fig. .Fig. 6Tr3CR1, either alone or in combination with each other, affected tumor formation at intraperitoneal organs covered by visceral peritoneum as well as at retroperitoneal organs. Both irradiation and CX3CR1 downregulation resulted in significantly reduced tumor burden at omentum; moreover, a combined action of irradiation and downregulation of CX3CR1 led to significant reduction of tumor burden as well as the number of animals bearing omental metastasis compared to irradiation alone  QBT FA uptake assay, (2) incubation with linoleic acid, counterstaining with Oil Red O dye, and fluorescence imaging. Transient downregulation of CX3CR1 led to a significant reduction in FA uptake , ATPase phospholipid transporting 8B4 (ATP8B4), bridging integrator 2 (BIN2), free fatty acid receptor 4 (FFAR4), G protein-coupled receptor 65 (GPR65), macrophage scavenger receptor 1 (MSR1), and oxidized low density lipoprotein receptor 1 (OLR1)  Fig. , which rNAs Fig. . High exNAs Fig. . Collect3CR1 either alone or together with x-ray radiation results in a measurable long-term effect, as it affects organ-specific metastatic colonization of the abdomen. Transient downregulation of CX3CR1 significantly reduces formation of lesions at the liver and omentum. At the same time, formation of lesions at the peritoneal wall, pancreas, and spleen is not affected by transient reduction of CX3CR1 alone, suggesting that neither attachment to mesothelial monolayer, nor cell growth, nor cell migration and invasion are affected, implying the existence of other yet unknown organ-specific mechanisms supporting metastatic dissemination to the liver and omentum that are abrogated upon transient downregulation of CX3CR1. Our studies can at least partially explain the mechanisms involved in CX3CR1-dependent reduction of omental metastasis. Our studies indicate that expression of CX3CR1 correlates with that of several proteins responsible for FA uptake, and, thus, might contribute to reduced tumor growth. However, it remains to be investigated whether CX3CR1 can also participate in direct lipid uptake or regulate other mechanisms leading to reduced omental metastasis.Our in vivo data indicate that transient downregulation of CX3CR1 allows using significantly lower doses of x-ray to achieve the same effects, which may carry a promise of reducing radiation dose and associated toxicities while maintaining the same efficacy in killing tumor cells.Interestingly, irradiation alone induced opposite effects on organ-specific colonization. Irradiated cells were not capable of forming tumors at the liver, and colonization of the pancreas, spleen, and omentum was partially reduced, suggesting that radiation treatment can affect formation of recurrent tumors in an organ-specific manner. Together, our data demonstrate that DNA damage induced by irradiation is significant enough to either completely prevent or reduce tumor formation at visceral and retroperitoneal organs. Irradiation is currently used in palliative care of terminally ill ovarian carcinoma patients; hence, patients whose metastases are localized to visceral and retroperitoneal sites may potentially draw benefits from irradiation. Moreover, our studies suggest that downregulation of CX3CR1 together with irradiation brought about the most deleterious effect. Our studies show that dissemination to sites affected by radiation, such as the pancreas, spleen, and omentum, was further negatively affected by CX3CR1 downregulation, suggesting that increased DNA damage in cells subjected to both stimuli is the underlying reason for reduced metastatic burden at these sites. Thus, the approach to downregulate CX3CR1 together with administration of a DNA damaging agent carries a promise to more effectively kill metastatic cells. However, this approach has to be extensively evaluated using multiple in vivo models and different CX3CR1-specific targeting approaches to find the most effective and safest combinations for various patients cohorts. The major side effect of irradiation in treatment of ovarian cancer is irradiation of normal cells, which could be potentially lethal to the patient. Recent studies demonstrated the efficacy of novel methods of radiation delivery that spare normal tissues from radiation damage. Downregulation of CX3CR1 expression in this setting could allow reducing the radiation dose and associated toxicities while keeping the efficacy of treatment at the same high level.Finally, transient downregulation of CX3CR1 is instrumental in double-strand DNA damage response and repair by regulating expression of RAD50. Building on these mechanistic studies, one of the most important messages taken from these findings is a potential to use CX3CR1 as a target for sensitization to DNA damage chemotherapies frequently used to treat ovarian and other solid cancers. In support, our in vitro studies indicated that cisplatin synergized with downregulation of CX3CR1 to reduce clone formation. Importantly, our data indicate that even transient downregulation of CX3CR1 in combination with other tested therapies brings about measurable DNA damage resulting in long-term deleterious effects on tumor growth. As CX3CR1 is a surface receptor, manipulating MRN expression by targeting CX3CR1 could be a promising approach in the future to increase sensitivity to DNA damaging therapies. Hence, it would be important to evaluate combinations of CX3CR1-targeting agents with cisplatin and other platinum agents in their efficacy against peritoneal metastasis in future studies.Our in vitro studies provide mechanistic explanation for some of the effects seen in the in vivo studies. We show that CX3CR1 in ovarian carcinoma could be beneficial from multiple standpoints: (1) it could disrupt FA uptake, further depriving growth of rapidly proliferating metastatic ovarian cancer cells, and, most importantly, (2) it could increase efficacy of DNA damage agents, including x-ray radiation and cisplatin.Collectively, our data suggest that impairment of CXMycoplasma fermentans, as was routinely determined using The LookOut Mycoplasma PCR Detection kit (Sigma).Human-derived ovarian carcinoma cell lines OVCAR-4 and SKOV-3 were obtained from National Cancer Institute Tumor Cell Repository . Human-derived ovarian carcinoma cell line Caov-3 was obtained from Dr. M.S. Stack . The cell lines were maintained in minimal essential medium or Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum for no longer than 20 consecutive passages and were routinely assessed by cell morphology and the average doubling time; identity of the cell lines was confirmed using a short tandem repeat analysis that indicated 100% match of cell line-specific DNA loci reported in the ATCC database to the tested samples. The cell lines were free from contamination by Athymic nude FOXN1nu mice were obtained from Harlan Laboratories . All experimental procedures were performed according to the Institutional Animal Care and Use Committee protocols approved by the Animal Care Committee of University of Illinois at Chicago. Animals were fed ad libitum and maintained in Association for Assessment and Accreditation of Laboratory Animal Care International-approved facilities on a 12 h light−12 h dark cycle.3CR1, anti-phospho-ATM, anti-phospho-PRKDC, anti-PRKDC, anti-ATM, and anti-phospho-H2AX antibodies were obtained from Abcam . Anti-mouse IgG HRP-linked antibody, anti-phospho-ATM, anti-CHEK2, anti-MYC, and anti-phospho-H2AX were obtained from Santa Cruz Biotechnology . Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG were from ThermoFisher Scientific . Anti-GPR65 and anti-FFAR4 antibodies were obtained from One World Lab . Anti-RAD50 antibodies were purchased from NeoBiolab .Anti-TUBB and anti-ACTB were purchased from Developmental Studies Hybridoma Bank . Anti-rabbit IgG HRP-linked antibody, anti-phospho-CHEK2, anti-phospho-CHEK1, and anti-CHEK1, antibodies were obtained from Cell Signaling Technology . Anti- CX3CR1-specific siRNAs, and ATM kinase inhibitor were purchased from Santa Cruz Biotechnology . Cisplatin was obtained from Sigma-Aldrich . Dharmafect reagent 1 was obtained from GE Dharmacon . QBTTM Fatty Acid Uptake Assay Kit was purchased from Molecular Devices .ECL reagent was purchased from Thermo Scientific . Control siRNA, CX6 cells were resuspended in minimal essential media supplemented with 2% fetal bovine serum, transferred on top of the agarose, and incubated at 37 °C at 5% CO2 for 48 h.Plates were coated with 0.5% agarose and allowed to solidify at room temperature for 30 min. 1×10Cells were grown to 70−80% confluency and transfected with siRNA as suggested by the manufacturer using Dharmafect reagent I as a transfection reagent.Transfected cells as well as controls plated in tissue culture treated 6-well plates were subjected to a single dose x-ray radiation at 0.8 gray/min using a linear accelerator (UIC Radiation Oncology) 3 days following transfection.2 diameter tissue culture plates and allowed to grow for up to 2 weeks until visible colonies of more than 50 cells formed. Cells were fixed with 4% para-formaldehyde (PFA) and stained with 0.05% crystal violet solution. Colonies were quantified and used to create survival curves. Surviving fraction of cells was found by detecting OD450 values and plotting using SigmaPlot 12.5 software . Both quantification methods produced similar results.After radiation 1–5% of the cells were replated on 100 mm48. Radiation enhancement factor (REF) and dose modifying factor (DMF) were used to compare radiosensitivity between the control and the treatment groups. REF is defined by the surviving fraction of the control group compared to that of the treatment groups at 2 gray (SF2control/SF2treatment). DMF is defined as the dose that is required to kill 90% of the population in the control group compared to that in the treatment group (DMF10control/DMF10treatment). When both REF2 and DMF10 were larger than 1.1, the treatment group was considered to be radiosensitized.The parameters of the linear quadratic model were used to compare radiosensitivity provided that the data were statistically significant by one-way ANOVA test50. Primary antibodies at 1:50 dilution in 2% goat serum were incubated with cells for 2 h at room temperature. All images across the same experiment were obtained using the same exposure time to enable quantitative comparison of immunofluorescence signals using ImageJ software (NIH).The procedures were performed as we described before50. Anti-CX3CR1 and anti-ATM antibodies were used at 1:2000, anti-phospho-ATM was used at 1:5000, anti-FFAR4, anti-RAD50, anti-phospho-CHEK1, anti-phospho-CHEK2, and anti-PRKDC were used at 1:1000, anti-phospho-PRKDC was used at 1:500, anti-CHEK1, anti-CHEK2, anti-TUBB and anti-ACTB were used at 1:200 dilutions.The procedures were performed as we described before4 cells were mixed with 1% solution of molten low melting agarose at 1:10 ratio. Cell-agarose complexes were transferred onto agarose-coated glass slides, allowed to solidify, kept at 4 °C for 15 min, and submerged into pre-chilled neutral lysis solution for DSB detection containing 2% sarkosyl, 0.5 M disodium ethylenediaminetetraacetate dihydrate (Na2EDTA-2H2O), 0.5 mg/ml proteinase K (pH 8.0) for 18 h at 37 °C. Agarose slides were submerged into running buffer containing 90 mM Tris, pH 8.5, 90 mM boric acid, 2 mM Na2EDTA-2H2O for 30 min with two additional washes followed by DNA electrophoresis at 1 V per cm, wash in deionized H2O and ice-cold 70% ethanol, air dried and stained with 1:10,000 dilution of Vista Green DNA Dye  for 15 min. Slides were imaged using Zeiss Axiovert fluorescence microscope. At least ten random fields were imaged per each slide. Fifty comets per slide were analyzed using CaspLab.com software.Transfected cells and their controls subjected to radiation, or not, were placed on ice immediately after x-ray exposure. Cells were collected, counted, and 2×106 cells/mouse were injected intraperitoneally (i.p.) into 6−8 week-old female athymic nude mice (n = 6). Animals were monitored three times weekly for tumor formation, ascites development, and survival up to 40 days. At the end of the experiments animals were sacrificed and dissected. Two co-authors (H.G.M. and J.X.) conducted the animal experiments, but did not analyze the outcomes. The co-author (M.V.B.) who did not directly conduct animal experiments and did not have information on the conditions pertaining to each experimental group has analyzed the outcomes. Tumors formed in the abdominal region were collected, paraffin-embedded, and analyzed with hematoxylin & eosin staining as we described before50.For generation of intraperitoneal tumors 3×10Lipid uptake was assessed using two methods. QBT FA uptake assay was used as suggested by the manufacturer; fluorescence signal was detected using GloMax Multi Detection System (Promega) and plotted. FA uptake was also monitored by incubating cells in the presence of 500 μM linoleic acid for 24 h, fixing with 4% PFA, and staining with Oil red O solution for 30 min followed by red fluorescence imaging.t test and Microsoft Excel software. A series of non-parametric tests, including Tukey HSD, Scheffe, Bonferroni and Holm, and Mann−Whitney U tests were used to compare two datasets with abnormal distribution. Data belonging to three or more independent groups were analyzed using one-way ANOVA and GraphPad Prism software. The findings were considered statistically significant at p < 0.05.For analysis of survival a non-parametric test, the log-rank (Mantel−Cox) test, was employed using cBioportal and KM Plotter software. Comet assay parameters, including percent of tail DNA, tail length, tail moment, and olive tail moment, were analyzed using one-way ANOVA and MANOVA and GraphPad Prism software. Comparisons between two datasets with normal distribution were conducted using Student’s supplementary figure legendssupplementary resultssupplementary figure 1supplementary figure 2supplementary figure 3supplementary figure 4supplementary figure 5supplementary figure 6supplementary figure 7supplementary figure 8supplementary figure 9supplementary figure 10


Together, HF has a dual role in autophagic modulation depending on nutritional conditions for anti-CRC.Autophagy has a key role in metabolism and impacts on tumorigenesis. Our previous study found that halofuginone (HF) exerts anticancer activity in colorectal cancer (CRC) by downregulating Akt/mTORC1 (mechanistic target of rapamycin complex 1) signaling pathway. But whether and how HF regulates autophagy and metabolism to inhibit cancer growth remains an open question. Here, we unveil that HF activates ULK1 by downregulation of its phosphorylation site at Ser757 through Akt/mTORC1 signaling pathway, resulting in induction of autophagic flux under nutrient-rich condition. On the other hand, HF inactivates ULK1 by downregulation of its phosphorylation sites at Ser317 and Ser777 through LKB1/AMPK signaling pathway, resulting in autophagic inhibition under nutrient-poor condition. Furthermore, Atg7-dependent autophagosome formation is also induced under nutrient-rich condition or blocked in nutrient-poor environment, respectively, upon HF treatment. More interestingly, we also found that HF inhibits glycolysis under nutrient-rich condition, whereas inhibits gluconeogenesis under nutrient-poor condition in an Atg7-dependent manner, suggesting that autophagy has a pivotal role of glucose metabolism upon HF treatment. Subsequent studies showed that HF treatment retarded tumor growth in xenograft mice fed with either standard chow diet or caloric restriction through dual regulation of autophagy  However, this method could not estimate overall autophagic flux or rate of flow, as LC3-II is both induced and degraded during autophagy. Therefore, it would be necessary to conduct several approaches to analyze autophagy in mammalian cells, including immunoblotting analysis of LC3 and SQSTM1 (p62), and monitoring autophagosome maturation by tandem mRFP-GFP fluorescence microscopy for autophagic vacuoles.4Macroautophagy (referred to hereafter as autophagy) is a catabolic cellular recycling process, which is a routine ‘garbage disposal’ service to cells, eliminating damaged components that could otherwise become toxic. It involves the sequestration of cytoplasmic material within double-membraned organelles, so-called autophagosomes, followed by lysosomal digestion.6 The metabolic states also impact the autophagic process. In normal (nutrient-replete) conditions, mechanistic target of rapamycin complex 1 (mTORC1) possesses kinase activity and interacts with a complex that contains ULK1, Atg13, FIP200, and Atg101, which inhibits the initiation of autophagy.7 Under metabolic stress, AMP-activated protein kinase (AMPK) acts as a central node that could impinge directly on core components including ULK1 of the autophagy machinery to initiate autophagy.8 Notably, AMPK is sensing to nutrient deprivation condition, particularly glucose limitation. On the other hand, the autophagic pathway could also provide metabolic substrate in solid cancers.10 In this context, the interplay between autophagy and metabolism is still largely unknown. Nevertheless, many tumors become addicted to autophagy for survival, suggesting inhibition of autophagy as a potential broadly applicable cancer therapy.11 The compound SBI-0206965, a highly selective ULK1 kinase inhibitor, regulates autophagy for potentiating cancer treatment.12 Another more advanced approach to inhibit autophagy for cancer therapy is the use of hydroxychloroquine to block autophagic flux and cargo degradation and suppress tumor growth,13 suggesting that autophagy is a key target for cancer treatment.14The role of autophagy in cancer is complex and is likely dependent on tumor type, stage, and genetic context. The current consensus is that autophagy acts as a double-edged sword in cancer. On one hand, autophagy impedes tumor initiation in some models; on the other hand, it can also promote tumorigenesis by facilitating the survival of cancer cells under duress.15 Notably, inhibition of mTORC1 may induce autophagy by phosphorylating ULK1. Thus, we suspected that HF could regulate autophagy in colorectal cancer (CRC) cells. To this end, autophagic flux was investigated in CRC cells upon HF treatment.Because autophagy serves a dichotomous role in cancer, it would be intriguing to develop an approach to dually regulate autophagy for cancer treatment. In our previous work, the compound halofuginone (HF) was shown to potentiate its bioactivity to inhibit Akt/mTORC1 and retard tumor growth under nutrient-rich condition.16 whereas HF inhibits AMPK-mediated phosphorylation of ULK1 at Ser317 and Ser777 to inhibit autophagy under nutrient-poor condition.17 Furthermore, we also interrogated the interplay between autophagy and glucose metabolism including glycolysis in nutrient-rich condition as well as gluconeogenesis in nutrient-poor condition, and found that HF modulates glucose metabolism in an ATG7-dependent manner. Moreover, our result also showed that HF dually modulates autophagy to retard tumor growth in vivo.Here, we report that HF regulates mTORC1-mediated phosphorylation of ULK1 at Ser757 to initiate autophagy under nutrient-replete condition;18 prompting us to ask if and how HF regulates autophagy in CRC cells. Thus, GFP-LC3-II, a fluorescent autophagosomal marker, was conducted to monitor autophagy in HCT116 cells. As shown in 19 We found that HF significantly reduced SQSTM1, whereas markedly elevated LC3-II protein levels under nutrient-rich condition  medium (α (ACCα), a downstream target of AMPK, was also validated  medium . Changes) medium . As AMPK) medium . The phoalidated . To furt with HF . These rAs mentioned above, HF can dually regulate the initiation step of autophagic flux. We further investigated whether HF could exert a marked impact on later steps including elongation of the double-membrane structure to form the autophagosome under both nutrient-rich and -poor conditions. In this regard, the lysosome inhibitor chloroquine (CQ) that allows the accumulation of the endogenous LC3-II was used to analyze the autophagic flux in CRC cells upon HF treatment. Either HF or CQ caused accumulation of LC3-II puncta and protein expression under nutrient-rich condition . In EBSS3 Because GFP loses its fluorescence from deprotonation in acidic lysosomes, autophagosomes display both green and red fluorescence, whereas autolysosomes are red only. Hence, a marked increase in the number of red-only indicates maturation of autophagosomes. As shown in To further provide evidence of autophagosome formation in autophagic flux, we performed tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) puncta formation assay.Because monodansylcadaverine (MDC) is proposed to be an autophagosome indicator, we utilized MDC assay for further investigation of HCT116 cells. As expected, HF induced more green fluorescence accumulation under nutrient-rich condition. By comparing with the CRC cells cultured in EBSS medium without HF treatment, we also observed that EBSS medium induced more green fluorescence, whereas HF reduced green fluorescence . Taken tAtg5, Atg7, Atg10, and Atg12. In this regard, we performed quantitative PCR to measure mRNA levels of these autophagy-related genes for a better understanding of molecular events at the stage of autophagosomal formation. Of particular interest, HF treatment pronouncedly enhanced the expression level of Atg7 under nutrient-rich condition, whereas decreasing the mRNA level of Atg7 in CRC cells under nutrient-poor condition, but Atg5, Atg10, and Atg12 were not significantly altered upon HF treatment  and mitochondrial phosphoenolpyruvate carboxykinase (PCK2), both of which are the key enzymes in gluconeogenesis  was performed in mice fed with 70% of normal food intake. As a result, HF treatment retarded tumor growth in xenograft-bearing nude mice fed with standard chow diet , ACCα, ATG7, phosphor-LKB1, LKB1, phosphor-CaMKKβ (Ser511), and β-actin were purchased from Cell Signaling Technology, Inc. . Antibodies against phosphor-ULK1 (Ser777), CaMKKβ, and goat anti-mouse IgG-HRP secondary antibody were purchased from Santa Cruz Biotechnology . HRP-goat anti-rabbit secondary antibody was purchased from Invitrogen .HF hydrobromide, MDC, EBSS, CQ, Pierce (R) BCA Protein Assay Kit and SBI-0206965 were obtained from Sigma-Aldrich . Antibodies against SQSTM1/p62, LC3-II, phosphor-ULK1 (Ser317), phosphor-ULK1 (Ser757), phosphor-mTOR (Ser2448), Glut1, hexokinase II, PCK2, PCB, phosphor-AMPK-/- MEFs were kindly provided by Professor Kevin Ryan in Beatson Institute for Cancer Research. All cells were cultured in DMEM  supplemented with 10% FBS , 10 U/ml penicillin–streptomycin  in a humidified atmosphere containing 10% CO2 and 90% air at 37 °C. The medium was changed every 3 days, and cells were passaged using 0.05% trypsin/EDTA. For nutrient starvation, cells cultured in DMEM were washed three times with PBS, and then cultured in EBSS medium for 2 h.HCT116 and SW480 were purchased from American Type Culture Collection . WT MEFs and Atg7HCT116 cells were transfected with pEGFP-LC3-II and mRFP-GFP-LC3 plasmids using lipofectamine 2000, respectively . One day after transfection, cells were treated with 20 nM HF for 12 h in high-glucose medium and for 2 h in EBSS medium prior to fixation, respectively. Then cells were imaged for GFP and RFP by using a Leica TCS SP8 (Leica) confocal microscope in different channels .g for 15 min at 4 °C, total protein concentration was measured using a Pierce(R) BCA Protein Assay Kit. 10–25 μg of protein was separated on 10% sodium dodecylsulphate-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes. After blocking (5% skim milk powder in TBS-Tween 20) for 1 h at room temperature, the membranes were then incubated with primary antibody overnight at 4 °C. The membranes were incubated with secondary antibody for 1 h at room temperature. All antibodies were diluted in TBS-Tween 20 containing 5% dry milk. The immune-reactive proteins were detected by enhanced chemiluminescence (ECL) using X-ray film and ECL reagent.After treated with 20 nM HF in high-glucose medium for 12 h and in EBSS medium for 2 h, respectively, cells were suspended in lysis buffer to obtain whole cell lysates. Following centrifugation at 13 500 × ad libitum. All of the experimental protocols were carried out with the approval of the Committee on Use of Human and Animal Subjects in Teaching and Research of Hong Kong Baptist University and according to the Regulations of the Department of Health, Hong Kong SAR, China. HCT116 cells (8 × 106 cells per mouse) were suspended in PBS and inoculated subcutaneously into the right flank of each mouse, and tumor growth was monitored regularly. Once tumors were palpable (~100 mm3), mice were divided at random into four groups with five mice in each group. The groups were as follows: (1) vehicle group in nutrient-rich condition, normally fed, receiving daily i.p. saline; (2) HF group in nutrient-rich condition, normally fed, receiving daily i.p. 0.1 mg/kg of HF; (3) vehicle group in nutrient-poor condition, fed with 70% of their normal food intake, receiving daily i.p. saline; (4) HF group in nutrient-poor condition, fed with 70% of their normal food intake, receiving daily i.p. 0.1 mg/kg of HF. The tumors were measured with calipers every day, and the tumor volumes were calculated by the following formula: a2 × b × 0.4, where ‘a’ is the smallest diameter and ‘b’ is the diameter perpendicular to ‘a’. Other indicators of general health, such as body weight, feeding behavior, and motor activity of each animal, were also monitored. After administration of HF or saline for 2 weeks, the mice were euthanized, and the tumor xenografts were immediately dissected, weighed, stored, and fixed.Male BALB/c nude mice, 6-weeks old, were obtained from the Laboratory Animal Services Centre, The Chinese University of Hong Kong. Mice were kept at room temperature 23± 2 °C with an alternating 12-h light–dark cycle, and were allowed access to food and water g for 15 min at 4 °C, the supernatants were collected and regarded as the total soluble proteins subsequently used for western blot analysis.To measure the SQSTM1/p62 and LC3-II activity in tumor tissue, three tumors in every group were collected and dispersed in lysis buffer by sonication for protein extraction. After centrifuging at 13 500 × Xenograft tumors were resected immediately and fixed in 10% neutral buffered paraformaldehyde at 4 °C for 24 h. Selected samples were embedded in paraffin, sectioned and stained with SQSTM1/p62 and LC3-II. All primary antibodies were used for dilution at 1:100. After overnight incubation at 4 °C, the sections were incubated with flurochrome-conjugated secondary antibody for 1 h and stained with DAPI for 10 min. The sections were then mounted with DPX mountant  for analysis.P<0.05 were considered as statistically significant.Each experiment was performed at least three times. GraphPad Prism 5.0 software was used for statistical analysis. The values of