SERPINA1 gene, strongly lower CX3CR1 mRNA expression in human peripheral blood mononuclear cells (PBMCs). This parallels with increase of intracellular levels of CX3CR1 and Z-AAT proteins. Presented data indicate the involvement of the CX3CR1 pathway in the Z-AAT-related disorders and further support the role of misfolded proteins in CX3CR1 regulation.Expression levels of CX3CR1 (C-X3-C motif chemokine receptor 1) on immune cells have significant importance in maintaining tissue homeostasis under physiological and pathological conditions. The factors implicated in the regulation of CX3CR1 and its specific ligand CX3CL1  expression remain largely unknown. Recent studies provide evidence that host’s misfolded proteins occurring in the forms of polymers or amyloid fibrils can regulate CX3CR1 expression. Herein, a novel example demonstrates that polymers of human ZZ alpha-1 antitrypsin (Z-AAT) protein, resulting from its conformational misfolding due to the Z (Glu342Lys) mutation in  Proteins can lose their structure and form polymers because of mutations or changes in their immediate environment which can lead to cell damage and disease. Interestingly, polymers formed by a variety of proteins can reduce the levels of CX3C chemokine receptor 1 (CX3CR1 for short) that controls the behaviour of immune cells and is implicated in a range of illnesses.Inherited ZZ alpha-1 antitrypsin deficiency is a rare genetic condition that highly increases the risk of liver and lung diseases. This disorder is characterised by mutant alpha-1 antitrypsin proteins (AAT for short) reacting together to form polymers; yet it remains unclear how the polymers affect different cells or organs, and lead to diseases. To investigate this question, Tumpara et al. examined whether polymers of mutant AAT influence the level of the CX3CR1 protein in specific classes of immune cells.Experiments revealed that in people with AAT deficiency, certain blood immune cells express lower levels of CX3CR1. Regardless of age, clinical diagnosis, or treatment regimen, all individuals with ZZ alpha-1 antitrypsin deficiency had AAT polymers circulating in their blood: the higher the levels of polymers measured, the lower the expression of CX3CR1 recorded in the specific immune cells. When Tumpara et al. added polymers of mutant AAT to the immune cells of healthy donors, the expression of CX3CR1 dropped in a manner dependent on the polymer concentration. According to microscopy data, AAT polymers occurred inside cells alongside the CX3CR1 protein, suggesting that the two molecular actors interact. In the future, new drugs that remove these polymers, either from inside cells or as they circulate in the body, could help patients suffering from conditions associated with this abnormal protein aggregation. Interactions between the chemokine receptors and chemokines, but also other proteins, peptides, lipids, and microbial products, play a critical role in the recruitment of inflammatory cells into injured/diseased tissues . Many huSERPINA1 gene, lower CX3CR1 mRNA expression in human PBMCs, which parallels with increased intracellular CX3CR1 and Z-AAT protein levels.Current findings suggest that divergent proteins with a common propensity to form extracellular oligomers interact with chemokine receptors and affect their expression levels. For example, Alzheimer’s peptide, Aβ, interacts with CX3CR1 and significantly reduces its expression in cultured microglial cells and in Alzheimer’s brain . SimilarSERPINA1 gene mutations. Homozygous Z AATD mutation is the most clinically relevant among Caucasians  that is characterized by low plasma levels of AAT protein  and the presence of intracellular and circulating Z-AAT polymers  is a rare genetic condition caused by polymers . The livpolymers . The intpolymers . The accpolymers , (ii) papolymers , and  , or augmentation therapy, the p<0.001) . A previype mice . A diminype mice . Howeverype mice , and did insults  showing + NK cells . Next, plasma Z-AAT polymers were measured, as the biomarkers of all carriers of the Z allele   was included as a known reducer of CX3CR1 expression  . This lapression . Indeed,CR1 mRNA  whereas controls . AccordiNK cells .CX3CR1 expression. To achieve a definitive answer how Z-AAT or other types of protein polymers regulate CX3CR1 levels, detailed mechanistic studies are required. In general, along with transcriptional regulation, chemokine receptors trafficking is of great importance to understand  as compared to non-treated controls . The CX3derstand .Under the same experimental conditions, monomeric M-AAT had no effect on CX3CR1 mRNA expression relative to housekeeping gene HPRT1 , and protein levels . LikewisThe CX3CR1 helps to define the major subsets of human monocytes because classical monocytes express much lower levels of CX3CR1 than non-classical monocytes . After i++ monocytes have the lowest expression of CX3CR1  with ZZ AATD have significantly higher mRNA and surface expression of CD14 as compared to age matched MM subjects . Thus, higher levels of Z-AAT polymers and lower numbers of CX3CR1-positive cells may favor the development of lung injury and disease. A decrease in the expression of CX3CR1 on human monocytes has been shown in patients with atopic dermatitis  provided ethical approval for individuals recruited in Germany. For Z-AAT polymer determination, we added 12 ZZ AATD emphysema patients recruited at Leiden University Medical Center. In addition, ZZ AATD emphysema patients  were enrolled with mean (SD): age 51 (6.6) years, forced expiratory volume in 1 s percent predicted  using Lymphosep  discontinuous gradient centrifugation according to the manufacturer’s instructions as described previously . ThereafIsolation of total RNA, synthesis of cDNA and mRNA analysis using Taqman gene expression assays  equipped with Dynex Revelation 4.21 software. Measurements were carried out in triplicates.The AAT polymer ELISA using the monoclonal antibody LG96 (deposited under access number DSM ACC3092 at German Collection of Microorganisms and Cell Cultures) was developed by Candor Biosciences. Normal M-AAT was used for a negative control. Recovery ratio, signal-to-noise ratio, calibration curve, sample stability under different storage conditions were tested and all tests passed. A cross-reactivity with M-AAT was not reported in any of the tests. Nunc MaxiSorp flat-bottom 96-well plates  were coated overnight at 2–8°C with monoclonal antibody LG96, at 1 µg/ml in coating buffer pH 7.4 . After a 2 hr blocking step, the plasma samples were applied in the previously determined dilutions made in LowCross-Buffer (Candor Biosciences), which also served as a blank. Incubation was performed for 2 hr at room temperature (RT). For detection, the captured antigen was incubated with antibody (LG96)-horseradish peroxidase (HRP) conjugate (1:2000) for 2 hr. The conjugate was prepared in advance with the HRP Conjugation Kit Lightning-Link  according to the manufacturer’s instructions. For signal development SeramunBlau fast2 microwell peroxidase substrate  was used. The incubation was performed at RT for 12 min in the dark and the reaction was stopped with 2 M HPlasma M- and Z-AAT was isolated by affinity chromatography using the AAT-specific Alpha-1 Antitrypsin Select matrix  according to the manufacturer’s recommendations. For Z-AAT preparation plasma from volunteers not receiving AAT augmentation therapy was pooled. To change the buffer in the M- and Z-AAT protein pools to Hank’s balanced salt solution , Vivaspin centrifugal concentrators with 10,000 MWCO  were used. Plasma purified human AAT  was changed to HBSS by the same method. Protein concentrations were determined using Pierce BCA Protein Assay Kit . The quality of the M- and Z-AAT preparations was confirmed on Coomassie gels , and Vivaspin-20, 100 kDa centrifugal column units  were used to separate Z-AAT monomers from polymers. Protein concentrations were determined using the Pierce BCA Protein Assay Kit  according to manufacturer’s instructions. The Z-AAT protein monomers were confirmed by using 7.5% SDS-PAGE without sample heating and without β-mercaptoethanol .6 cells/ml) were incubated for 18 hr at 37°C, 5% CO2 either alone, or with Z- or M-AAT proteins, or LPS . In some experiments, a recombinant CX3CL1 protein  was used. Protein was reconstituted at a concentration of 25 µg/ml in sterile PBS containing 0.1% BSA (Sigma-Aldrich) and added to PBMCs at various concentrations up to 500 ng/ml either alone or together with Z-AAT (0.5 mg/ml) for 18 hr. Afterward, cells were used for RNA isolation, flow cytometry or Western blot analysis. For Western blot, PBMCs were lysed in RIPA buffer (Sigma-Aldrich), supplemented with protease inhibitor cocktail (Sigma-Aldrich). For some Western blot experiments, we extracted detergent resistant lipid raft associated proteins from insoluble cell fractions using UltraRIPA kit according to the supplier’s instructions .PBMCs  prior to transfer onto polyvinylidene difluoride membranes . Membranes were blocked for 1 hr with 5% low fat milk  followed by overnight incubation at 4°C with specific primary antibodies: polyclonal rabbit anti-human AAT (1:800) , mouse monoclonal anti-AAT polymer antibody , rabbit polyclonal anti-CX3CR1 , or HRP-conjugated monoclonal anti-β-actin antibody  for a loading control. The immune complexes were visualized with anti-rabbit or anti-mouse HPR-conjugated secondary antibodies (DAKO A/S) and enhanced by Clarity Western ECL Substrate . Images were acquired by using Chemidoc Touch imaging system (BioRad) under optimal exposure conditions and processed using Image Lab v5.2.1 software (BioRad). For quantification, the signal intensity of the CX3CR1 protein band in each lane was divided by the corresponding β-actin band intensity . Afterward, the normalized signal of each lane was divided by the normalized target signal observed in the control sample to get the abundance of the CX3CR1 protein as a fold change relative to the control.Plasma samples from 22 ZZ AATD and 21 non-AATD controls were analyzed for CX3CL1/Fractalkine using Duoset kit . Cell-free culture supernatants were analyzed directly or stored at −80°C. ELISA Duoset kits for TNF-α (assay detection range 15.6–1000 pg/ml), IL-1β/IL-1F2 (assay detection range 3.91–250 pg/ml), and IL-6 (assay detection range 9.38–600 pg/ml) were purchased from R&D Systems  and were used according to the manufacturer’s instructions. Plates were measured on Infinite M200 microplate reader . Measurements were carried out in duplicates.6 cells per condition) were incubated with LPS (1 µg/ml), M-AAT (1 mg/ml), or Z-AAT (0.5 mg/ml) for 18 hr. Staining was performed with phycoerythrin (PE)-conjugated mouse monoclonal anti-CX3CR1 antibody , fluorescein (FITC)-conjugated mouse monoclonal anti-CD14 antibody , allophycocyanin (APC)-conjugated mouse monoclonal anti-CD16 antibody , or BV-480-conjugated anti-CD56 mouse monoclonal antibody  alone or in combinations. Dead cells were excluded by a staining with 7-amino-actinomycin D. Samples were measured on a BD FACSAria Fusion machine and analyzed with FlowJo v10 . The gating strategy is shown in PBMCs (2 × 106) were plated onto glass coverslips and incubated alone or with Z-AAT polymers (0.5 mg/ml) in RPMI medium for 18 hr at 37°C and 5% CO2. Cells were then washed with PBS, fixed with 3% paraformaldehyde in PBS for 20 min, and continued with or without permeabilization with 0.5% Triton X-100 in PBS for 5 min at RT. For immunolabeling, cells were co-incubated with primary antibodies against human CX3CR1  and anti-AAT polymer antibody, LG96  for 1 hr, at RT. After washing, the cells were incubated with corresponding secondary antibodies (1:1000) conjugated to AlexaFluor-488 (goat anti-rabbit) or AlexaFlour-594 (goat anti-mouse) both from Thermo Fisher Scientific, Rockford, IL. After final wash, the cells were mounted on microscope slides using ProLong Gold Antifade Mountant with DAPI . Images were acquired using confocal laser microscope FluoView 1000  equipped with a 60× oil immersion objective and differential interference contrast in sequential mode. Confocal z-stacks were collected with a 0.25 µm increment.Human total PBMCs . If normality test failed, the nonparametric Kruskal-Wallis one-way analysis or Mann-Whitney rank sum test was performed, and data were presented as median (range). For correlation analysis, the Pearson’s linear correlation method was used to measure the correlation for a given pair. A p-value of less than 0.05 was considered significant. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by 2 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen Satyajit Rath as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Polymerization of misfolded protein lowers CX3CR1 expression in human PBMCs" for consideration by The reviewers have discussed the reviews with one another, and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife. Considering these concerns of the reviewers, we can only consider the manuscript further after a major revision if you sufficiently address all the concerns as outlined below.The reviewers agree that the implications of your work are broad and carry clinical significance, although there are major concerns over the lack of sufficient molecular level understanding of the regulation of CX3CR1's cell surface expression by Z-AAT polymers, for consideration for publication in eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:The manuscript by Tumpara S. et al. shows that polymers of mutated α-1 antitrypsin (AAT) affect expression and internalization of CX3CR1, presenting a novel example of CX3CR1 regulation by misfolded proteins and what could be a novel immunomodulatory function of protein α-1 antitrypsin. Using human PBMCs and plasma purified AAT in its wild type monomeric and mutated polymeric forms, authors show that Z AAT polymers reduce CX3CR1 expression and its surface expression. As CX3CR1 has been previously found to be regulated by Tau and amyloid-β, this finding poses a question if other conformationally altered extracellular proteins could have the same property. It also opens a question how this process is affected by inflammatory conditions and what are its implications for individuals with Z AAT. In addition to the novel mechanism of CX3CR1 regulation, these findings could be of relevance for better understanding of molecular pathophysiology associated with AAT deficiency (AATD) adding a potentially important step to the mechanism of AATD pathology. Furthermore, these findings could also have relevance in other conditions in which CX3CR1 axis and AAT have been implicated (e.g. cancer).Essential revisions:1. The protein level of CX3XR1 in total cell lysate is high although the gene expression is significantly low in AATD patients or Z-AAT treated cells which has been explained by showing Z-AAT mediated CX3XR1's association with lipid rafts. How specific is this effect? Would any oligomeric protein which can bind to CX3CR1, have similar effect? What is the effect with treatment with monomeric Z-AAT on CX3CR1 gene expression and protein level?2. There is no explanation provided for decreased expression CX3CR1 in AATD patients or after in vitro polymeric Z-AAT treatment except for indirect connection with increased CD14 expression (CD14++ cells are known to decrease CX3CR1 expression). What is the possible mechanism of such drastic decrease in expression level of CX3CR1?3. Are there any changes in CX3CR1 expression after fractaline challenge of the PBMCs treated with polymeric Z-AAT?4. Is there any change in CX3CR1 expression and cell surface expression in cells having intracellular Z-AAT polymers?eLife. Your revised article has been reviewed by two reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Satyajit Rath as the Senior Editor.Thank you for resubmitting your work entitled "Polymerization of misfolded Z α-1antitrypin protein lowers CX3CR1 expression in human PBMCs" for further consideration by Although both the reviewers have agreed that the manuscript has been improved by adding further experimental evidence for reduced cell surface expression of CX3CR1 by ZZ-AAT polymers and additional experiments, there are some issues, as outlined below, which need to be addressed before a final decision can be made.1. The explanation for significant decrease in mRNA expression levels of CX3CR1 in patient PBMCs by ZZ-AAT polymers is not properly supported experimentally. At the very least, an acknowledgement and a plausible explanation of such an important observation is essential.2. A discussion (supported by experiments if possible) on the fate of internalized CX3CR1 upon removal of ZZ-AAT polymers from surrounding media should be included to increase the impact of the study.3. The submission is being considered as a short report. It is critical to maintain the appropriate format 5, in the revision.The detailed recommendations from both reviewers are being provided below for ease of response.Reviewer #1:eLife-64881R1, Tumpara et al. has successfully addressed the observation of decreased cell surface expression of CX3CR1 on PBMCs in patients of α-1 antitrypsin deficiency (AATD) with circulating polymeric forms of AAT (Z-AAT) protein. They have shown the exclusive role of polymeric ZZ-AAT in CX3CR1 internalization in comparison to monomeric form of the protein or the CX3CL1. The authors mentioned that this finding is already known for other aggregated proteins like Tau, so this finding is not sufficiently novel. Importantly, the most intriguing finding of significant decrease in mRNA level expression of CX3CR1 is not explained except for an indirect correlation with increased CD14 expression by the ZZ-AATD treated cells. This finding needs further explanation. Authors have only explained about reduction of cell surface expression of CX3CR1 which is not satisfactory to explain the decreased mRNA level expression of CX3CR1 with polymeric ZZ-AATD exclusively and its implication in patients of AATD. To be acceptable for publication, this important observation needs to be explained and its implication in patients of AATD needs to be discussed in detail.In the revised version of manuscript 13-11-2020-RA-Reviewer #3:Manuscript provides data showing that Z AAT induces reduced CX3CR1 gene expression and protein surface expression while increasing intracellular levels of the protein and new data provided by the authors show that the effect on CX3CR1 expression is executed specifically by Z AAT polymer and not by its monomeric form or polymeric variant of WT protein. Experiments including CXCL1 were added and new colocalization experiment provides an explanation for increased amount of CX3CR1 in cell lysates treated with Z AAT and I believe the manuscript is suitable for publication. It would be interesting to know if upon removal of ZAAT from surrounding media CX3CR1 could be recycled back to the surface or is the interaction more permanent (ZAAT induces increase in intracellular CXCR1 compared to CX3CL1), however that would be beyond the scope of this manuscript. Given the limit of 5 figures for short report the authors can consider moving Figure 5 showing gene expression in presence of CX3CL1, as well as Key Resources Table to supplement or possibly merging Figure 4 with Figure 2. Essential revisions:1. The protein level of CX3XR1 in total cell lysate is high although the gene expression is significantly low in AATD patients or Z-AAT treated cells which has been explained by showing Z-AAT mediated CX3XR1's association with lipid rafts. How specific is this effect? Would any oligomeric protein which can bind to CX3CR1, have similar effect? What is the effect with treatment with monomeric Z-AAT on CX3CR1 gene expression and protein level?Thank you very much for this important point. Other pathogenic oligomeric proteins, which bind to CX3CR1, may have a similar effect as Z-AAT polymers. For example, it has been shown that CX3CR1 in microglia acts as surface receptor for extracellular Tau oligomers and promotes its internalization :59). However, it is difficult to predict if this phenomenon applies to all misfolded proteins.As suggested, we also tested Z-AAT monomers in our PBMCs model. However, monomer showed no effect on CX3CR1 mRNA and protein levels. We included these data into the manuscript. We also tested artificially generated M-AAT polymers by heating M-AAT for 3h at 60°C. This method is well-known and published many times as useful to induce polymers of normal M-AAT. However, M-AAT polymers showed no effect on CX3CR1 levels. This shows that only certain polymers, probably dependent on their size and/or structural properties can interact with CX3CR1 and regulate its surface expression and intracellular levels. However, the mechanisms behind this regulation requires detailed studies in vivo. We added new data regarding effects of Z-AAT monomer and M-AAT heat-induced polymer on CX3CR1 receptor levels in PBMCs, and included additional discussion and references into the manuscript.2. There is no explanation provided for decreased expression CX3CR1 in AATD patients or after in vitro polymeric Z-AAT treatment except for indirect connection with increased CD14 expression (CD14++ cells are known to decrease CX3CR1 expression). What is the possible mechanism of such drastic decrease in expression level of CX3CR1?Thank you for this point. We included additional data and putative explanations regarding reduced CX3CR1 expression in ZZ-AAT PBMCs. Sustained CX3CR1 down-regulation in ZZ-AATD individuals seems to result from the circulating Z-AAT polymers which interact with cells and get internalized. Similar like in case of Tau polymers, Z-AAT polymers seems to internalize together with CX3CR1 resulting in reduced cell surface expression of CX3CR1. We performed additional experiments using laser scanning confocal microscopy and demonstrate that in many cells Z-AAT polymers localize with CX3CR1. A new figure and additional data are included into the manuscript.We also included a statement into the manuscript that, although both Z-AAT polymers and LPS induce CD14 expression and similarly affect CX3CR1 expression and protein levels, our results imply that Z-AAT polymers and LPS do not share the same signalling mechanisms. LPS induces cell activation as measured by induced cytokine production whereas under the same experimental conditions Z-AAT polymers did not induce cytokines.3. Are there any changes in CX3CR1 expression after fractaline challenge of the PBMCs treated with polymeric Z-AAT?Thank you for this question. We performed additional experiments, in which total PBMCs were pre-incubated first with various concentrations of soluble CX3CL1 followed by incubation without or with constant amount of Z-AAT polymers for 18 hours. According to our results, soluble CX3CL1 alone was unable to affect the expression of its own receptor and had no effect on Z-AAT polymers. Independent of the presence of soluble CX3CL1 (even at very high concentration such as 500 ng/ml) Z-AAT polymers down-regulated CX3CR1 mRNA expression and induce intracellular levels of CX3CR1 protein. Our finding that soluble CX3CL1 has no effect on its receptor is in concordance with data published by Pachot et al. J Immunol, 2008, 180 (9) 6421-6429. However, there are reports showing that soluble CX3CL1 induces an internalization of CX3CR1 : 2554–2562). Data from our new experiments and additional discussion are included into the manuscript.4. Is there any change in CX3CR1 expression and cell surface expression in cells having intracellular Z-AAT polymers?Thank you for the question. Our findings are based on cells isolated from ZZ-AAT deficiency individuals who naturally contain Z-polymers in all cells producing AAT as well as extracellularly, in all biological fluids. Monocytes and macrophages express AAT, therefore our PBMCS from ZZ AAT deficiency individuals include cells having Z-AAT polymers and low CX3CR1 expression. We now show that circulating ZZ polymers, which are markers for Z- AAT deficiency, can be taken up by the cells. We performed confocal laser scanning microscopy and confirmed that Z-AAT polymer enters cells and in part localize together with CX3CR1 receptor. We included these data into the manuscript. Therefore, even if polymers are intracellularly not produced their can be taken up from the circulation. Our current experiments show that cells can take up Z-AAT and lower CX3CR1 expression. Hence, theoretically cells having intracellular Z-AAT polymers will have lower surface expression of CX3CR1.Unfortunately, due to limited access to rare ZZ patients, especially during the covid-19 times, we are not able to obtain new blood for monocyte or macrophage isolation and polymer analysis. We also do not know how cell isolation and sorting would affect intracellular polymers of AAT. A detailed comparison of cells with and without Z-AAT polymers regarding CX3CR1 expression would be of interest to perform in the further.


Purpose: In this study, we independently synthesised and labelled a novel bidentate bifunctional chelating agent, 177Lu-3,4-HOPO-Cetuximab, that achieved tight binding between targeting and radioactivity, and evaluated its targeted killing ability of cells in vitro and in vivo.Method: 3,4-HOPO was successfully synthesised through a series of chemical steps using malt phenol as the raw material, which was then coupled with Cetuximab labelled with 177Lu. 177Lu-3,4-HOPO-Cetuximab and 177Lu-DOTA-Cetuximab was tested for its cell viability and cell-binding rate after different times and at different doses by CCK-8 and cell-binding experiments. 177Lu-3,4-HOPO-Cetuximab (~500 μCi) and 177Lu-DOTA-Cetuximab (~500 μCi) were injected into the tail vein of a subcutaneous metastasis mouse model of triple-negative breast cancer with a single injection, and tumour volume growth and body weight changes were regularly monitored for 20 days. The radioactivity distribution in nude mice was analysed after sacrifice, and the treated and untreated tumour tissues were analysed by HE staining.Result: The cell viability of 177Lu-3,4-HOPO-Cetuximab declined exponentially after treatment for 48 h at 50 Bq/mL to 500 kBq/mL, respectively; the cell activity was slowed down from 8 to 96 h at a dose of 500 kBq; while the binding rate of 4T1 cells in 177Lu-3,4-HOPO-Cetuximab from 1 to 24 h, respectively, increased logarithmically, which was similar with 177Lu-DOTA-Cetuximab. After 20 days of treatment, the body weight of nude mice with 177Lu-3,4-HOPO-Cetuximab and 177Lu-DOTA-Cetuximab were hardly changed, while the body weight with physiological saline decreased significantly. The tumour inhibition rate of the 177Lu-3,4-HOPO-Cetuximab and 177Lu-DOTA-Cetuximab were (37.03 ± 11.16)% and (38.7 ± 5.1)%; HE staining showed that tumour cells were affected by the action of 177Lu causing necrosis.Conclusion: The experiments showed that 177Lu-3,4-HOPO-Cetuximab has a certain targeted therapeutic ability for triple-negative breast cancer, and it is expected to become a potential targeted nuclear medicine treatment for triple-negative breast cancer. Several peptides and monoclonal antibodies (mAbs) have been conjugated to bifunctional chelators and the labelled radionuclide 177Lu. Ramli et al. ; FJ-391A4 radioactivity metre (Beijing Nuclear Instrument Factory); LB 2111 Multi Crystal Gamma Counter ; KDC-20 low-speed centrifuge .The nude mouse triple-negative breast cancer cell line 4T1 cells were provided by Soochow university laboratory. Female BALB/c nude mice were purchased from Nanjing Skrui Biotechnology Co., Ltd., aged 4–6 weeks, and were raised at the Experimental Animal Centre of Suzhou University according to SPF requirements and approved by the animal ethics committee of Soochow University.177LuCl3 was purchased from ITG Germany.3-hydroxy-2-methyl-4-pyrone ; DOTA-NHS purchased from MACROCYCLICS, Europe; Cetuximab Purchased from Shanghai Saima Biotech Co., Ltd.; The synthesis process of bidentate 3,4-HOPO is shown in Accurately weigh 43 g of maltol (1) dissolved in 285 mL of methanol and 35 mL of 10.25 mol/L NaOH solution, add 45 mL of benzyl chloride, and reflux at 100°C for 48 h to obtain orange oily liquid benzyl maltol (2).Take benzyl maltol (2) 73.23 g dissolved in 210 mL mixed solution of methanol and water (volume ratio 1:1), add sodium glycinate powder, reflux reaction at 100°C for 36 h, then extract with 40–100 mL dichloromethane, water phase The concentrated HCl (12 mol/L) was adjusted to a pH 2, and the precipitated yellow crystals were filtered and dried in a vacuum oven for 30 h to obtain the product Bn-3,4-HOPO (3).After the Bn-3,4-HOPO was dissolved in methanol, a certain amount of palladium carbon (about 5% by mass of the reactant) was weighed and added slowly to the reaction solution, and hydrogen was introduced and stirred for 4 h. After completion of the reaction, the mixture was filtered to obtain palladium carbon and a product mixture; after adding DMF , the mixture was heated and stirred at 80°C until the solution became a black suspension, and the filtered filtrate was steamed to obtain rice. The white solid was dried in vacuo to give the product as 3,4-HOPO (4). The results of the nuclear magnetic resonance test were as follows: 1H NMR : δ 7.53 , 6.11 , 4.82 , 2.15 ; LC-MS [M + H+] m/z: 183.87.3COONH4 solution for 5 min, add 3,4- HOPO or DOTA ratio 1.2 equivalent EDC continued to activate for 30 min; molar ratio 3,4-HOPO or DOTA: Ab = 50:1 coupling; addition of 0.25 mol/L CH3COONH4 solution to 0.5 mL; constant temperature mixing at room temperature for 24 h to complete coupling Union. The coupled 3,4-HOPO-Ab solution or DOTA solution was centrifuged at 4,500 rpm for 45 min at room temperature, followed by ultrafiltration at 6,000 rpm for 15 min; the conjugate was collected, 0.5 mL of 0.25 mol/L CH3COONH4 solution was added, and placed. Store this at 4°C.Take 200 μL of Ab solution from the −80°C refrigerator and thaw it in a water bath. The ratio of 3,4-HOPO:NHS = 1:1.2 in 0.25 mol/L CH177Lu-DOTA-Ab solution from a 4°C refrigerator; add 20 μL of 177LuCl3 dilution solution to the 3,4-HOPO-Ab solution or 177Lu-DOTA-Ab solution (activity is about 1 mCi); add CH3COONH4 buffer with pH 5. The solution was 500 μL; the mixture was shaken for 30 min at 37°C under a vortex constant temperature mixer to complete the labelling. The reaction solution after the completion of the labelling was centrifuged at 4,000 rpm for 15 min at room temperature; the buffer was discarded, the label was rinsed from the inner liner with a 0.25 mol/L CH3COONH4 solution, and the collected liquid was stored in an EP tube. The radiochemical purity was measured by a TLC method in a test tube in which the developing solvent was acetic acid: ammonium acetate (pH 3). The labelling rates of the 177Lu-3,4-HOPO-Ab and 177Lu-DOTA-Ab solutions were (89.7 ± 0.9)% and (92.6 ± 1.0)%. After purification, the radiochemical purity were both higher than 95%.Remove 350 μL of purified 3,4-HOPO-Ab solution or 5/mL, and 1 mL is added to each well in the 12-well plate and cultured overnight in the cell incubator at 37°C. When the cells adhered to the logarithmic growth phase on the second day after administration, 0.37 kBq markers were added to each hole. After 1, 2, 4, 8, and 24 h, the supernatant was added to the supernatant tube and washed twice by PBS, the cleaning fluid was also added to the supernatant tube; the cells were digested with trypsin, and the cell suspension was inhaled into the cell tube, washed with PBS for 2 times, and the radioactivity count (B) of the cell tube and the radioactivity count (F) of the supernatant tube were measured by γ counter. The cell-binding rate of the marker is B/(B+F) × 100%.When the cells grow to more than 90%, the cell concentration is adjusted to about 1.0 × 102 saturated humidity incubator; the cells were fully attached for about 24 h. Six sets of parallel wells were randomly selected from each group, the medium was discarded; the medium containing 177LuCl3, 177Lu-3,4-HOPO-Ab, and 177Lu-DOTA-Ab was, respectively, added to a 96-well plate by 50 Bq/mL~ 500 kBq/mL, 100 μL per well, the medium containing 2 μg/mL of Ab was a positive control group and the blank control group was drug-free. After 4 h, replace the drug with a drug-free solution, and re-dispose it in a 37°C, 5% CO2 saturated humidity incubator. After 48 h, discard the medium, and add 100 μL CCK-8 per well. About 0.5 h until the culture medium showed an orange colour, and the absorbance was measured by a microplate reader.4T1 cells were uniformly inoculated into 96-well plates and the number of cells was 8,000/well. The cells were placed in a 37°C, 5% CO177LuCl3, 177Lu-3,4-HOPO-Ab, and 177Lu-DOTA-Ab with a radioactive dose of 500 kBq/mL were added to a 96-well plate and then placed at 37°C, 5% CO2 saturated humidity. After the box was cultured for 4 h, the culture solution was discarded, the drug-free culture solution was replaced, and it was then discarded in the incubator for 8, 24, 48, 72, and 96 h, respectively. In total, 100 μL of medium containing 10% CCK-8 was added to each well. About 0.5 h until the culture medium showed an orange colour and was placed on a microplate reader. Cell viability (%) = [A (dosing) − A (blank)]/[A (control) − A (blank)]; A (dosing) is the absorbance of wells with cells, CCK-8 solution and drug solution; A (blank) is the absorbance of wells with medium and CCK-8 solution without cells; A (control) is the absorbance of wells with cells and CCK-8 solution without drug.Afterwards, 100 μL of 5 cells/mL, and 1 mL was added to each well of a 12-well plate, and cultured in a 37°C cell culture incubator overnight. When the cells were attached to the next day and were in the logarithmic growth phase, 1 mL of the culture medium containing the radioactive label was replaced (about 0.37 kBq per well), and three replicate wells were set in each group. After the markers were treated with 4T1 cells for 1, 2, 4, 8, and 24 h, the supernatant was aspirated and transferred to the supernatant EP tube. After washing twice with PBS, the washing solution was also incorporated into the supernatant EP tube. The cells were digested with a trypsin digestion solution. After the cells were completely digested, the cell suspension was inhaled into the EP tube of the cells, washed twice with PBS, and the washing solution was also added to the EP tube, and the cells were separately measured in a γ counter. The radioactivity count of the tube (B) and the radioactivity count of the supernatant tube (F). The cell-binding rate of the marker was B/(B+F) × 100%.After the cells were grown to more than 90% of the digested cells, the adjusted cell concentration was about 1.0 × 10In total, 30 nude mice bearing tumours with a diameter of ~0.4–0.6 cm were randomly divided into two groups (treatment group and control group). There was no significant difference in body weight between the two groups.177Lu-3,4-HOPO-Ab was injected into the tail vein at 100 μL/mouse (~500 μCi), and the control group was injected with an equal volume of normal saline via the tail vein. Treatment was administered once. After the completion of the tail vein injection, the state and activity of the nude mice were observed every day. The nude mice were weighed on day 0, day 3, day 5, day 7, day 10, and day 20 after dosing. Additionally, the long diameter and short diameter of each group of tumours were measured with a Vernier calliper, and the volume of the tumour was calculated for a total of 20 days.In the treatment group, The calculation method of the tumour volume was as follows:where X is the long diameter and Y is the short diameter.The tumour growth inhibition rate  is the tumour inhibition rate = [(A − B)/A] × 100%, where A is the control group and B is the treatment group.6 cpm = 1 μCi.On the 20th day after administration, blood was collected from the retro-orbital sinus, and the nude mice were sacrificed. The nude mice were dissected, and organs such as the heart, liver, spleen, lung, kidneys, stomach, intestine, tumour, bone, muscle, and skin were taken, weighed, and detected by radioactive gamma count. The result was converted to %ID/g [%ID/g = radioactive count of the organ/(radioactive count rate of the injected drug × organ mass)], and the gamma counter was calibrated to be 1.2 × 104T1 tumour tissues were extracted without treatment or with treatment, fixed by 4% paraformaldehyde for 24 h. HE staining and immunohistochemical analysis were entrusted to Wuhan Sevier Biotechnology Co., Ltd. The rate of EGFR positive cells is the average number of positive cells in 3 visual fields (200×) of a certain type of cells: 0–5% is grade 0, 6–25% is grade 1, 26–50% is grade 2, 51–75% is grade 3, >75% is grade 4.SPSS 19.0 software. One-way analysis of variance (ANOVA) was performed in a completely randomised design. P < 0.05 was considered statistically significant.The experimental results were expressed as mean ± standard deviation (x̄ ± s). The data were analysed by +] m/z: 184.00. Since it is the test result in H+ mode, it is inferred that the actual molecular weight is 183.00 Infrared spectrum  indicates that 3,4-HOPO will be protonated at low pH and reach a deprotonation equilibrium at pH = 3.7.While the dissociation constants pKa1, pKa2, pKa3, pKa4, and pKa5 of DOTA are 2.5 ± 0.2, 2.5 ± 0.2, 4.6 ± 0.2, 8.7 ± 0.2, and 10.1 ± 0.2, respectively. The results showed that DOTA reached the first-order and second-order dissociation equilibrium at pH = 2.5, the third-order dissociation equilibrium at pH = 4.6, the fourth-order dissociation equilibrium at pH = 8.7, and the fifth-order dissociation equilibrium at pH = 10.1.As shown in 3+ ions were studied by potentiometric titration. The cumulative formation constants of logβ0lh and pM were obtained by Hyperquad 2008 as shown in Hyss 2009. The maximum enrichment zone of chelators with Lu3+ (enriched density above 95%) was pH < 8.2 in the form of Lu2LH for 3,4-HOPO and pH < 6.0 in the form of Lu2L for DOTA, respectively.The complexation behaviours of 3,4-HOPO and Lu177Lu-DOTA-Ab and 177Lu-3,4-HOPO-Ab on the proliferation of 4T1 cells is shown in 177Lu has a certain inhibitory effect on cell proliferation. With the increase of dose % and (93.75 ± 0.64)% of 50 Bq/mL to (17.82 ± 1.03)% and (14.70 ± 0.99)% of 500 kBq/mL. Compared with 177Lu, the 177Lu-3,4-HOPO-Ab had a statistically significant difference in cell viability at a dose of 500 Bq/mL (P < 0.05), while the dose was increased to 5 kBq/mL, the difference of cell activity was statistically significant (P < 0.05) between 177Lu and 177Lu-3,4-HOPO-Ab or 177Lu-DOTA-Ab.The effect of  of dose , the cel177Lu at 500 kBq and cell culture for 8 h had a slight inhibitory effect on cell proliferation. With the increase in time, the cell activity gradually increased after the initial plateau, it was from (82.10 ± 1.75)% after 8 h of drug-free medium replacement to (90.40 ± 1.12)% at 96 h. However, 177Lu-DOTA-Ab and 177Lu-3,4-HOPO-Ab were cultured for 8 h had a good inhibitory effect on cell proliferation. With the increase of time, the cell activity decreased continuously (49.20 ± 1.40)% for 177Lu-DOTA-Ab and (50.47 ± 1.83)% for 177Lu-3,4-HOPO-Ab at 8 h after replacing the drug-free medium were changed to (7.50 ± 0.28)% and (9.24 ± 0.68)% at 96 h. And the differences at each time point were statistically significant (P < 0.05) compared with the cell activity at 8 h, we also found that 177Lu-DOTA-Ab had a better therapeutic effect than 177Lu-3,4-HOPO-Ab from 48 to 72 h, but the difference was not statistically significant (P > 0.05) at 96 h between 177Lu-DOTA-Ab and 177Lu-3,4-HOPO-Ab.As shown in 177Lu-3,4-HOPO and 177Lu in 177Lu-3,4-HOPO-Ab and 177Lu-DOTA-Ab.The results showed that 4T1 cell was no binding effect with 177Lu-3,4-HOPO-Ab and 177Lu-DOTA-Ab to 4T1 cells with time is shown in 177Lu-3, 4-HOPO-Ab, and 177Lu-DOTA-Ab were (1.45 ± 0.17)% and (1.67 ± 0.08)% at 1 h, increased to (10.33 ± 0.82)% and (10.17 ± 0.46)% at 24 h. There were statistically significant differences (P < 0.05) in the cell-binding rate between 177Lu-3,4-HOPO and 177Lu-3,4-HOPO-Ab as well as 177Lu-DOTA-Ab all the time.The change of the binding rate of 177Lu-DOTA-Ab, and 177Lu-3,4-HOPO-Ab over time after administration are shown in 177Lu-DOTA-Ab, and 177Lu-3,4-HOPO-Ab were (19.74 ± 0.35) g, (18.9 ± 0.21) g, and (19.11 ± 0.28) g, respectively, with the difference between each group were not statistically significant (P > 0.05). After a period of feeding, the weight change trend of the 177Lu-labelled monoclonal antibody treatment groups was increased, while it was decreased in the saline group. After 20 days, the weights of nude mice with 177Lu-3,4-HOPO-Ab and 177Lu-DOTA-Ab were (18.02 ± 1.20) g and (18.7 ± 1.38) g, (15.21 ± 0.78) g in saline, and there were statistically significant differences (P < 0.05) between the treatment groups and the saline group from the beginning at 7th day.The body weight changes of the 4T1 tumour-bearing nude mice in physiological saline, 177Lu-DOTA-Ab, and 177Lu-3,4-HOPO-Ab were (58.60 ± 7.53) mm3, (51.2 ± 6.3) mm3, and (59.64 ± 9.60) mm3, respectively, and there were no differences (P > 0.05) between each of two groups. After 20 days of treatment, the volume of tumours in 177Lu-3,4-HOPO-Ab and 177Lu-DOTA-Ab were (299.20 ± 105.70) mm3 and (272.2 ± 17.8) mm3, which in saline was (465.04 ± 87.02) mm3. There were statistically significant differences (P < 0.01) in the volume of transplanted tumours within the treatment groups and the saline group from the beginning of the 7th day. The tumour inhibition rates of 177Lu-3,4-HOPO-Ab and 177Lu-DOTA-Ab were (37.03 ± 11.16)% and (38.7 ± 5.1)%, respectively. Tumor treatment changes in nude mice is shown in As shown in 177Lu-3,4-HOPO-Ab and 177Lu-DOTA-Ab were (3.20 ± 0.30)%ID/g and (3.69 ± 0.42)%ID/g in liver and (1.83 ± 0.30)%ID/g and (2.7 ± 0.6)%ID/g in bone. However, the 177Lu-3,4-HOPO-Ab group and 177Lu-DOTA-Ab group had radioactivity were, respectively (1.90 ± 0.32)%ID/g and (2.2 ± 0.2)%ID/g in tumours.After 20 days of injection in the tail vein, the radioactivity profile of the 4T1 tumour-bearing nude mice  showed t177Lu with and without treatment of 4T1 tumour, as shown in HE staining of EGFR is mainly expressed on the cell membrane, and particles ranging from pale yellow to tan are representative of EGFR expression. For the untreated group (left), there was more EGFR expression in the tumour tissues; after treatment (mid and right), the expression of EGFR in the tumour decreased, and the proportion of positive cells was less than that in the control group.177Lu-3,4-HOPO-Ab and 177Lu-3,4-HOPO-Ab groups were both grade 2.According to the semi-quantitative scoring method, the EGFR positive rate of the untreated group was grade 10. The EGFR positive rate of the Hydroxypyridone (HOPO) is an outstanding building block for the development of various reagents in the field of metal chelation. At present, in-depth analysis of the synthesis methods of HOPO-based ligands and many methods for achieving optimal biological activity have been reported  and Lu2L (99.8). In this state, the complexation constant of 3,4-HOPO is 5 orders of magnitude higher than that of DOTA.The pH of human body fluid is close to 7.4, so we analysed the species distribution relationship and complexation constant of 3,4-HOPO interacting with different metal ions at pH 7.4. 3,4-HOPO and DOTA in LuIn 1907, the French chemist G. Urbain recrystallised several times with a solution of niobium nitrate, from which a new element “Lutecium” was isolated and named “Paris” in ancient Latin-Lutetia named it Lutetium (Lu) (Yokel et al., 177Lu had no killing effect for 4T1 cells, while 177Lu-3,4-HOPO or 177Lu-DOTA conjugated with antibody displayed little damage to cells at a low dose and cell lethality with increasing dose. It also increased exponentially because Cetuximab binds to the EGFR receptor on the surface of 4T1 cells, allowing 177Lu to act on cells for a long time while free 177Lu is washed after 4 h.CCK-8 experiments showed that free 177Lu-3,4-HOPO-Cetuximab can specifically bind to EGFR-positive receptors in 4T1 cells, and the corresponding cell-binding rate increases as the duration of action increases. Since 177Lu cannot penetrate the cell membrane, the β-particles emitted during cell culture damage the cells, resulting in a decrease in cell proliferation ability. However, after washing with the medium, there is almost no 177Lu in the 4T1 cell culture system, and this is illustrated by the cell-binding assay. Since the cells only interacted with 500 kBq 177LuCl3 for 4 h, the damaged cells were gradually repaired, and the cell activity gradually increased with time, reaching 90.40% at 96 h, which was significantly increased compared with 82.10% at 8 h. The cells were treated with 500 kBq 177Lu-3,4-HOPO-Cetuximab for 8 h, 177Lu-3,4-HOPO-Cetuximab was retained in the cells by binding of the antibody Cetuximab to the EGFR receptor positively expressed on the surface of 4T1 cells, and sustained beta-ray damage Cells, causing cell activity to decrease over time.Due to the presence of Cetuximab, 177Lu-3,4-HOPO-Cetuximab in the treatment of 4T1 nude mice xenografts showed that the weight of nude mice in the treatment group will decrease in a short period of time because of the strong growth of tumour cells at this stage and rapid consumption of nutrition in nude mice. Substance, after 7 days, showed the weight of nude mice in the treatment group began to rise, indicating that the tumour cell activity in nude mice decreased, and the metabolic capacity decreased. From the growth of transplanted tumour volume, the growth rate of xenografts in the treated group compared with the control group. As it slows down significantly, the growth curve slope prediction is expected to reach a negative value after a certain time, and it reaches a significant anti-tumour effect after 20 days. This series of data confirms the second part of 177Lu-3,4-HOPO-Cetuximab for 4T1 The killing effect of 177Lu in vitro and the targeting ability of Cetuximab. After 20 days of administration, the data of the radioactive distribution of the nude mice in the treatment group showed that the liver, tumour, and bone were the three organs with the highest radioactivity accumulation, and the 177Lu accumulation of 177Lu-3,4-HOPO-Cetuximab in the bone site was relatively low. The clinical case shows that 177Lu-labelled DOTA-coupled targeting material can cause severe bone metastasis after treatment for a period of time, in vivo radioactivity distribution data show that the dose deposition of HOPO in the liver and bone were lower than that of DOTA, and this indicates that HOPO has a better ability to promote the excretion of metal ions than DOTA. 3,4-HOPO is expected to become a new targeted therapy drug for clinical nuclear medicine to solve the problem of bone metastasis.Intravenous injection of 227Th marker was visible in the in situ 4T1 model of invasive nude mice. The anti-tumour activity (Tinianow et al., 89Zr labelling both result in the in vivo stability being stronger than DTPA; in addition, there exists a report (Deri et al., 177Lu-3,4-HOPO-Cetuximab has obvious killing and lethal effect on 4T1 tumours.3,4-HOPO is a bidentate chelating agent synthesised by malt phenol as a template through a series of chemical synthesis steps. At present, the medical community has little research and application on HOPO chelate. Hagemann et al.  showed tWe have confirmed that bidentate 3,4-HOPO has shown strong chelation application value, and it is necessary to synthesise four or more teeth 3,4-HOPO must have a more powerful clinical medicine role.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Soochow University.KL was responsible for collecting data for cell and animal experiments and writing article. YZ and XW were responsible for the production and purification of molecular materials. RZ was responsible for data collation. CM and RH were responsible for proposing the experimental ideas and revising the article. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The Wnt/β-catenin signaling pathway plays a pivotal role during embryogenesis and its deregulation is a key mechanism in the origin and progression of several tumors. Wnt antagonists have been described as key modulators of Wnt/β-catenin signaling in cancer, with Dickkopf-1 (DKK-1) being the most studied member of the DKK family. Although the therapeutic potential of DKK-1 inhibition has been evaluated in several diseases and malignancies, little is known in pediatric tumors. Only a few works have studied the genetic inhibition and function of DKK-1 in rhabdomyosarcoma. Here, for the first time, we report the analysis of the therapeutic potential of DKK-1 pharmaceutical inhibition in rhabdomyosarcoma, the most common soft tissue sarcoma in children. We performed DKK-1 inhibition via shRNA technology and via the chemical inhibitor WAY-2626211. Its inhibition led to β-catenin activation and the modulation of focal adhesion kinase (FAK), with positive effects on in vitro expression of myogenic markers and a reduction in proliferation and invasion. In addition, WAY-262611 was able to impair survival of tumor cells in vivo. Therefore, DKK-1 could constitute a molecular target, which could lead to novel therapeutic strategies in RMS, especially in those patients with high DKK-1 expression. DKK-1, DKK-2, DKK-3 and DKK-4, with DKK-1 being the most studied and characterized [The Wnt/β-catenin signaling pathway plays a pivotal role during embryogenesis and its deregulation is a key mechanism in many tumors . Among tIn recent years, the therapeutic potential of targeting DKK-1 has been evaluated for several diseases, such as rheumatoid arthritis . AlthougDespite the fact that DKK-1 has been studied in several cancers, little is known about its function in pediatric tumors. Thus, only a few works have explored DKK-1 implication in medulloblastoma , neuroblHerein, for the first time, we analyzed the therapeutic potential of DKK-1 pharmacological inhibition in RMS. Thus, after genetic or pharmacological inhibition in RMS cell lines, our results indicated that DKK-1 inhibition affects differentiation with after effects on proliferation and invasiveness. Furthermore, the compound WAY-262611 was able to impair the survival of tumor cells after tail-vein injection of RMS cells in an in vivo murine experimental model. Taken together, our results highlight DKK-1 as an interesting target that could lead to novel therapeutic strategies for RMS.DKK-1, DKK-3 and DKK-4 were significantly increased in RMS patients compared with muscle tissue from healthy donors. No differences between the ERMS and ARMS subtypes were observed (data not shown). However, the most prominent overexpression was observed for DKK-1 and DKK-3. This last observation was also confirmed when DKK expression was analyzed in a set of RMS cell lines  and CW9019 (ARMS subtype). Two shRNAs (sh#1 and sh#2) induced a clear DKK-1 downregulation in both cell lines A. ConcomIn order to investigate the Wnt signaling status after DKK-1 knockdown, β-catenin levels in total cell lysate and in the nuclear fraction were analyzed by Western blot. Levels of β-catenin displayed an increase in both fractions and in both cell lines in response to DKK-1 silencing. The increase observed was particularly significant for nuclear β-catenin and with a particularly intense upregulation with sh#2 C. In addThe results of our analysis revealed that the RD and CW9019 cell lines were both sensitive to WAY-262611 treatment, with IC50 values as low as 0.30 μM and 0.25 μM, respectively A. DespitRegarding β-catenin, both protein levels and promoter activity (measured by luciferase activity) were significantly increased by WAY-262611 in both cell lines E,F. HoweIn order to analyze the effects of WAY-262611 treatment on tumor growth in vivo, we established an orthotopic RMS mouse model and tested the antitumor activity of both genetic and pharmacological inhibition (with WAY-262611 at two concentrations). Neither the tumor volume nor tumor weight were affected by the genetic inhibition nor treatment A,B. OwinThe Wnt/β-catenin signaling pathway has been described as an important player in tumorigenesis in several malignancies . Its regIn our gene expression analyses, we observed an upregulation of different members of the DKK family in RMS, both in patients and in cultured cell lines. Presumably, the overall high levels of DKK family components in RMS would entail an inactivation of the Wnt/β-catenin signaling pathway. These findings are in accordance with previous studies that reported a low expression of β-catenin in RMS, evidencing an inactive state of Wnt/β-catenin signaling ,16. We hIn our work on RMS, the observed increase in β-catenin expression and the promotion of its nuclear location, both observed after genetic or pharmacological DKK-1 inhibition, did not produce significant variations in the canonical target c-Myc. This result is in agreement with previous observations that point out a relevance of the non-canonical Wnt signaling pathway in RMS ,24. HoweIn addition to the induction of myogenic markers, the consistent FAK downregulation observed after genetic DKK-1 inhibition or WAY-262611 treatment may also contribute to the observed anti-oncogenic effects. In support of this observation, a previous work showed that treatment with WAY-262611 was able to inhibit the expression of FAK in a rheumatoid arthritis model . FAK is Despite the effects on proliferation and myogenic markers observed in vitro, genetic inhibition or treatment with WAY-262611 did not produce significant effects on tumor growth in the primary mouse models. These results coincide with a previous work where they show that reactivation of the β-catenin pathway did not produce significant effects on proliferation in a RMS in vivo model . Indeed,Despite the lack of activity of the compound WAY-262611 in mouse primary RMS tumors, its notable effects on FAK expression and in cell invasiveness observed in vitro led us to hypothesize a possible role of DKK-1 in RMS metastasis process. With the aim of testing this hypothesis, we analyzed the effect of DKK-1 inhibition produced by the compound WAY-262611 in an experimental mouse model of metastasis, by which it is possible to quantify the cells that reach the lung. Our results suggest that DKK-1 inhibition reduce the number of tumor cells in lungs, thereby evidencing an essential role for DKK-1 in this early metastasis step, which would not be completely compensated—in contrast to the results in the primary tumor model—by other DKK proteins and/or other Wnt antagonist proteins. In fact, the relationship between DKK-1 and metastasis has been previously described ,37. In thttp://r2.amc.nl, accessed on 15 December 2020). Muscle tissue was considered as the “normal” counterpart. For this latter group, data from two datasets were used: “Normal Muscle—Gordon—22—MAS5.0—u133p2” and “Normal Muscle—Hofman—121—MAS5.0—u133a”. Only data from control donors were taken into account for the analysis. For the Gordon database, only the young subset of patients was included to avoid age-related bias. The “Tumor Rhabdomyosarcoma—Davicioni—147—MAS5.0—u133a” set was used for RMS patients.Analysis of gene expression data from patients was performed on publicly available data from the R2: Genomic Analysis and Visualization Platform   in order to obtain the relative expression values for each DKK gene in each cell line.Raw expression data from RMS cell lines were obtained from the public database GEO2R ( GSE8840 ). Raw da2 with humidified atmosphere. Cell lines were purchased from the American Type Culture Collection, except CW9019, which was kindly provided by Dr. Jacklyn Biegel’s laboratory.All RMS cell lines were grown in MEM media . Culture media were supplemented with 10% fetal bovine serum , 2 mM L-glutamine, 1 mM sodium pyruvate, 1× non-essential amino acids, 100 U/mL penicillin and 0.1 mg/mL streptomycin . Cell lines were cultured at 37 °C in a 5% CO5 cells (for RD and CW9019) were seeded in 60 mm dishes and incubated overnight. Then, 48 h after infection, selection with puromycin  was performed to select positive-transduced cells. The knockdown efficiency was tested by Western blot (WB). The selected DKK-1 shRNAs were clones V2LHS_19944 (for sh#1) and V3LHS_412074 (for sh#2) .Genetic knockdown of DKK-1 was carried out by shRNA technology using the lentiviral vector pGIPZ . Lentiviral particles were generated in HEK293T as previously described . Briefly4 cells/well for RD, and at 2.5 × 104 cells/well for CW9019. At different time points , cells were trypsinized and resuspended in coulter Isoton II diluent (Beckman Coulter). To evaluate the number of cells on each time point, cell suspensions were analyzed in a Z series Coulter counter  following the manufacturer’s instructions. Each condition was analyzed in triplicate in three independent assays.For the proliferation assay, cells were seeded in 6-well plates at a density of 3 × 103 cells/well for RD and 6 × 103 for CW9019). The following day, WAY-262611 was added at different concentrations. After 72 h, cells were dyed with 0.5% crystal violet . After overnight drying, crystals were dissolved with 15% acetic acid solution and absorbance was quantified at 590 nm with an Epoch Microplate Spectrophotometer . Absorbance data were used to calculate the percentage survival under each condition. Three independent assays with six replicates per condition were performed.For IC50 determination, cells were seeded in 96-well plates (2 × 105 RD or CW9019 cells were seeded in an 8 μm pore size transwell (Corning), previously coated with 25 µL of BD Matrigel™ (Corning). After 24 h of incubation, migrated cells were washed, fixed and stained with Hoechst-33342 (5 ng/mL). Cells were counted under the fluorescent microscope. For each condition, three wells from three independent assays were analyzed.Invasion assay was performed as previously described in detail . BrieflyProteins were extracted using RIPA buffer , and the protein content was measured using the DC Protein Assay Kit . Then, 20–40 μg of total protein were resolved by SDS-PAGE electrophoresis and transferred onto a PVDF membrane. After blocking, membranes were incubated with the following primary antibodies: Anti-DKK-1 , Anti-c-Myc , Anti-MYOD1 , Anti-Myogenin , Anti-FAK , Anti-β-catenin , Anti-Lamin A  and Anti-Actin . Anti-goat , anti-mouse  and anti-rabbit  were used as secondary antibodies. Uncropped membranes and values of intensity ratio for each band are shown in To analyze the activation of Wnt/β-catenin signaling, growing RD and CW9019 cells were co-transfected with M50 Super 8x TOPFlash plasmid  or M51 Super 8x FOPFlash plasmid  together with Renilla reporter plasmid pRL-TK , using X-tremeGENE 9 DNA Transfection Reagent , following the manufacturer’s instructions. After 24 h, cells were harvested and a luciferase assay was performed using the Dual-Glo Luciferase Assay System  following the manufacturer’s instructions. Luminescence was measured using the Appliskan reader . The luciferase activity of each condition was normalized for transfection efficiency to its Renilla luciferase activity.6 RD cells were injected into the right gastrocnemius muscle of SCID mice . All mice were maintained under pathogen-free conditions. To analyze the effects of DKK-1 genetic inhibition on tumor growth in vivo, mice were injected with RD cells carrying the control plasmid or infected with sh#2. For the pharmacological inhibition, mice injected with RD wt cell line were randomized into 3 groups and treated daily with subcutaneous vehicle solution, 2.5 mg/kg or 10 mg/kg of the WAY-262611 compound. Limb volume was measured with a caliper to calculate the tumor volume using the formulaFor the primary orthotopic tumor model, 1 × 106 RD cells previously transduced with a GFP reporter vector pGIPZ (control plasmid) were intravenously injected through the tail vein of SCID-beige mice. Prior to injection, RD cells were pre-treated in vitro with DMSO  or with WAY-262611 (0.2 µM) (treated group) for 48 h. After cell injection, mice were treated daily with subcutaneous injection (for 7 days) with the vehicle  (control group) or with 10 mg/kg of WAY-262611 in the same solvent (treated group). At the end of the experiment, the lungs were enzymatically dissociated with collagenase A  and analyzed by FACS . Analyses were carried out as previously described [For the lung metastasis model, 3 × 10escribed . All prot-test or ANOVA, considering * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Bonferroni correction was applied for multiple comparisons when more than two datasets were analyzed.Statistical analyses were performed using GraphPad Prism Software (version 6.01). At least three independent replicates were performed for each experiment. The mean ± SD was graphed, and statistical significance was determined by Student’s In this work, we provide an initial characterization of the DKK family in RMS with a special emphasis on DKK-1. However, the possible role of other DKK members in RMS needs to be further investigated. Our results indicated that DKK-1 inhibition produces anti-oncogenic effects in RMS, both in vitro and in vivo. Furthermore, our data suggest a possible regulatory link between reactivation of Wnt/β-catenin signaling—promoted by DKK-1 inhibition—and the differentiation of RMS cells via FAK downregulation and a concomitant promotion of MYOD1 and myogenin. Therefore, we identify DKK-1 as a pro-oncogenic protein in RMS and as a putative molecular target, which may have potential in the treatment of this soft tissue sarcoma. The most remarkable effect observed by DKK-1 pharmacological inhibition was the impairment of the survival of tumor cells in lungs, while primary tumor growth was not affected by genetic inhibition or WAY-262611 treatment. Our work suggests that DKK-1′s specific role is essential for early metastasis steps; however, the fact that tumor growth is not affected by DKK-1 inhibition makes us think that the most plausible hypothesis is that other DKKs, or even Wnt antagonists belonging to other families, may compensate the lack of DKK-1 function. Therefore, to completely unravel the molecular mechanisms involved, more studies are needed. These studies should take into account the intricate regulation of the pathway to permit the implementation of a therapy based on the reactivation of Wnt/β-catenin signaling.


Gambogic acid (GA) has been reported to induce apoptosis in cholangiocarcinoma (CCA) cell lines. However, the molecular mechanisms underlying its anti-cancer activity remain poorly understood. This study was aimed to investigate GA’s effect on human CCA cell lines, KKU-M213 and HuCCA-1, and its associated mechanisms on Wnt/β-catenin signaling pathway. Cell viability, apoptosis, and cell cycle analysis were conducted by MTT and flow cytometry. The effect of GA mediated Wnt/β-catenin and ER stress were determined by luciferase-reporter assay, qRT-PCR, and western blot analysis. c-Myc, a downstream target gene of Wnt/β-catenin signaling. GA activated genes involved in ER stress mechanism in KKU-M213 and enhanced CCA’s sensitivity to gemcitabine.GA exhibited potent cytotoxicity in CCA cells which was associated with significantly inhibited cell proliferation, promoted G1 arrest, and activated caspase 3 mediated-apoptosis. GA attenuated β-catenin transcriptional levels, decreased β-catenin protein, and suppressed the expression of Our findings reveal that the molecular mechanism underpinning anti-cancer effect of GA is partially mediated through the inhibition of Wnt/β-catenin signaling pathway and induction of ER stress induced-apoptosis. GA may serve as a promising therapeutic modality for amelioration of gemcitabine-induced toxicity in CCA.  Cholangiocarcinoma (CCA) is the second most common primary liver tumor with a morphological feature of the aggressive malignant proliferation of biliary duct epithelial cells. While CCA incidence rates are increasing globally, the highest incidence and mortality rates are in the North-eastern Thailand specifically in the endemic area of liver fluke infestations . Indeed, surgery is the principal treatment that provided the best survival prognosis of CCA patients , however, 40-85% of all patients have recurrent disease even after tumor excision  and surgical resection has become an incurable option for most CCA patients in advanced stages of the disease . Due to rapid progression of CCA, therapeutic effort has been directed to identifying and modulating key molecular pathways relevant to cancer growth. Chemotherapy for advanced CCA is largely ineffective due to the emerging of resistance in CCA . Therefore, targeting essential signaling pathways mediated CCA progression is critical for understanding and developing treatments. c-Myc and CCND1. Hyper-activation of Wnt/β-catenin signaling observe in several types of human cancers which drives tumor initiation and progression . Thus, the discovery of compounds targeting Wnt/β-catenin signaling has potential as a therapeutic strategy for CCA. Wnt/β-catenin signaling pathway plays important roles in normal tissue development, including cell proliferation and differentiation . Cytosolic β-catenin protein is a principal mediator of Wnt/β-catenin signaling and its phosphorylation, ubiquitination, and degradation are governed by a destruction complex comprising of Axin, adenomatous polyposis coli (APC), and glycogen synthase kinase-3β (GSK-3β). Aberrant activation of this complex gives rise to the accumulation of β-catenin in nucleus which functions as a transcriptional coactivator of transcription factors included in the T-cell factor and lymphoid enhancer factor (TCF/LEF) family and promotes the transcription of Wnt responsive genes including Garcinia hanburyi . Among the naturally occurring caged xanthones, Gambogic acid (GA) has been identified as an effective anti-cancer agent in a variety of tumors . GA has a unique structure in which the planar structure of xanthone   was isolCell culture 2 incubator. KKU-M213 cells were purchased from JCRB . HEK 293T cells were obtained from American Type Culture Collection . HuCCA-1 cell line was kindly provided by Dr. Stitaya Sirisinha, Department of Microbiology, Faculty of Science, Mahidol University. KKU-M213 and HuCCA-1 cells were cultured in Ham’s F-12 medium . HEK 293T cells were maintained in Minimum Essential Medium (MEM). All culture mediums were supplemented with 10% Fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin and cells were cultured at 37°C in a humidified atmosphere containing 5% COCell proliferation and cytotoxic assay Cell proliferation was determined by a Cell Proliferation ELISA and BrdU colorimetric assay according to the manufacturer’s instructions. GA treated-cells were incubated in BrdU labelling solution for 2 h at 37°C and BrdU-POD antibody was added and incubated for 90 min at room temperature (RT). Cell proliferation was determined by measuring the absorbance at 450 nm using a Microplate Spectrophotometer. Cell viability was determined by MTT assay. Briefly, cells were incubated with 0.5% MTT reagent for 4 h at 37°C to allow the formation of a violet formazan precipitate. Then, supernatant was removed and formazan crystals were dissolved in 100% DMSO prior to measuring absorbance at 540 nm by a Multiskan GO Microplate Spectrophotometer . Annexin V and Propidium iodide (PI) staining Cells were treated with GA for 24 h. They were harvested and incubated with Annexin V-FITC and PI for 15 min at RT in the dark. The frequency of apoptotic cells was determined by BD FACSCanto™ flow cytometer and analyzed with BD FACSDiva software .Cell cycle analysis CCA cells were treated with GA for 24 h, fixed with cold 70% ethanol overnight at 20°C and stained in PI/RNase Staining Buffer in the dark for 30 min at 37°C. Cell cycle analysis was performed by BD FACSCanto™ flow cytometer and analysed with a BD FACSDiva software.Luciferase reporter assay HEK 293T cells were transiently transfected with FLAG-β-catenin, TOPflash/TCF reporter, FOPflash, and Renilla luciferase reporter plasmids using lipofectamine 3000 according to the manufacturer’s instructions . Twenty-four hours after transfection, cells were treated with GA for 24 h and subjected to reporter assay analysis using Dual-luciferase reporter kit.Western blot analysis Total protein lysates were harvested using a modified lysis buffer as previously described . Samples were resolved by 10% of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The following antibodies were used: β-catenin (H-102) , β-actin (Sigma-Aldrich), Cleaved-caspase 3, p-GSK3 (Ser 9), and gH2AX . Membranes were then probed with HRP-conjugated secondary antibodies for 1 h. The signal was detected using SuperSignal West Pico Chemiluminescent ECL system .RNA extraction and real time PCR GAPDH and sequences of primers are GRP78 , IRE-1 , XBP-1 , CHOP , and GAPDH .Total RNAs were isolated using Trizol reagent according to the manufacturer’s instructions. The quantity of total RNA was determined by NanoDrop 2000 . First-strand complementary DNA (cDNA) synthesis was performed using iScriptTM cDNA synthesis kit . For real-time quantitative PCR, cDNA samples were amplified using SYBR Green I dye and conducted with ABI PRISM7500 Sequence Detection System and analysis software . The mRNA expression was normalized to the levels of endogenous Statistical analysis Data are represented as mean and standard deviation (mean ± SD) or mean ± standard error of mean (SEM) for IC50 (Supplementary Table 1). Data were analysed using the statistical software package, GraphPad Prism version 8.2 . The statistical differences among groups were analysed by a one-way ANOVA followed by Tukey-Kramer post-hoc test and paired t-test was used when applicable. Each value is represented as means ± SD of three independent experiments. Statistical significance was considered when values of *, p < 0.05, **, p < 0.01, ***, p < 0.001 compared with vehicle control. GA exhibits cytotoxic and anti-proliferative effects in CCA cells To investigate the effects of GA in CCA, KKU-M213 and HuCCA-1 cells were treated with various concentrations of GA for 24-72 h and cell viability was determined by MTT assay. Exposure of GA markedly reduced cell viability in time and dose-dependent manners in both CCA cell lines. The IC50 values of GA in KKU-M213 and HuCCA-1 cells are shown in Supplementary Table 1. These results aligned with a significantly decreased cell proliferation assessed by a BrdU assay . GA signGA induces cell cycle arrest and apoptosis in CCA cells To demonstrate the effect of GA on cell cycle distribution, GA-treated CCA cells were subjected to flow cytometry analysis. As shown in GA inhibits Wnt/β-catenin signaling Wnt/β-catenin signaling pathway is activated in multiple cancers  and caspase 3-dependent apoptosis has been reported to be partially mediated through Wnt/β-catenin signaling pathway . To address the probable anti-cancer mechanism of GA on Wnt/β-catenin signaling pathway, we examined the effect of GA mediated-Wnt/β-catenin transcriptional activity using a luciferase reporter assay in HEK 293T cells which is lack of endogenous levels of β-catenin. Cells were transiently transfected with TOPflash, FLAG-β-catenin, and Renilla plasmids, and then treated with GA. We observed that TCF/β-catenin transcriptional activity was significantly inhibited by GA in a dose-dependent manner . The inhAXIN2 and c-Myc. GA induced a dose-dependent activation of AXIN2, a negative feedback regulator of Wnt/β-catenin signaling , whereas markedly suppressed c-Myc mRNA expression . These results indicate that GA potentially inhibited the TCF/LEF-dependent activation of the canonical Wnt/β-catenin signaling and its downstream target gene c-Myc. β-catenin phosphorylation is mediated by GSK-3β and it results in targeting for degradation by the proteasome pathway . Mechanistically, to address whether GA attenuated-Wnt/β-catenin signaling is mediated through GSK-3β, we evaluated the effect of GA on Wnt/β-catenin signaling using a constitutively active β-catenin mutant, S33Y in HEK 293T cells. S33Y is insensitive to GSK-3β-mediated phosphorylation, thereby blocking proteasome-induced protein degradation. We observed that the sublethal concentration of GA at 1 µM significantly decreased Wnt/β-catenin activity in HEK 293T cells overexpressing wild-type β-catenin  but did not affected in S33Y-β-catenin mutant. These results were confirmed by co-treatment with LiCl, a known GSK-3β inhibitor and we observed that LiCl treatment resulted in greater activation of TOPflash luciferase activity. However, GA failed to inhibit Wnt/β-catenin signaling in the presence of LiCl in HEK 293T cells  suggesting that GA inhibited Wnt/β-catenin signaling is a GSK-3β dependence activation in HEK 293T cells. We further determined the effect of GA on phosphorylation GSK-3β (Ser 9) protein in KKU-M213. In the contrary to HEK 293T, exposure to GA did not alter the levels of p-GSK-3β in KKU-M213 . This result implies that inhibition of Wnt/β-catenin signaling by GA in KKU-M213 cells might be independent from GSK-3β’s action. We speculated that the modulation of GA on the GSK-3β is a cell line specific. However, the pathophysiological consequences of Wnt/β-catenin signaling mainly depend on the expression of downstream target genes . To address this significance, we therefore investigated the effect of GA on the expression of downstream Wnt target genes including GA induces endoplasmic reticulum (ER) stress in CCA cells GRP78/BiP, IRE1α, XBP1, and CHOP were determined. GRP78/BiP, IRE1α, and CHOP mRNAs were significantly up-regulated in KKU-M213 cells exposed to GA for 24 h, with a maximal functional response at 2.5 μM . ER stress induced-apoptotic cell death h as been reported in CCA . To determine whether GA induces ER stress promoted-apoptosis cell death in KKU-M 213cells, the mRNA levels of critical genes involved in ER stress mechanism including t 2.5 μM  in accorreatment . These rGA enhances the sensitivity of CCA cells to GemcitabineGemcitabine (GEM) has been used as an established regimen for advanced biliary cancer . GEM decreased cell viability of KKU-M213 in a dose-dependent manner . To evalIn this present study, we demonstrated that the anti-cancer effects of GA against KKU-M213 partially mediates through the inhibition of Wnt/β-catenin signaling pathway. GA potently inhibited CCA cell proliferation, arrested cell cycle, and substantially induced ER stress and apoptosis which significantly impacted on CCA cell survival. To our knowledge, this is the first report showing that the inhibitory effect of GA on Wnt/β-catenin signaling pathway and its activation to induce ER stress mediated-apoptosis may cooperate to suppress the progression of CCA cells. We observed potent cytotoxic effect of GA in both KKU-M213 and HuCCA-1 cell lines. Despite GA exhibited cytotoxicity and anti-proliferative effect in HuCCA-1 cells, but GA (at 0.5 - 2.5 µM) failed to induce apoptosis and cell cycle arrest in HuCCA-1 cells  which raises an intriguing question of whether acquired resistant mechanism mediated by GA is a cell line dependence. However, further investigation of the molecular mechanisms govern HuCCA-1 resistance should be addressed.AXIN2 which is one of an important Wnt/β-catenin target gene and acts as a negative regulator of Wnt/β-catenin signaling through a negative feedback mechanism . The expression of AXIN2 was up-regulated during activation of Wnt/β-catenin signaling to promote the differentiation of mouse pre-osteoblastic cells , while in our current study, AXIN2 was significantly increased, despite a decrease in β-catenin protein expression by GA in KKU-M213 cells. The results corroborate well with the previous study showing that AXIN2 has been demonstrated to promote degradation of β-catenin . At present, the underlying mechanism is still unclear. However, elucidating the effect of GA on upstream components of Wnt signaling pathway which directly regulate the degradation of β-catenin should be considered in the future for a better understanding of the molecular mechanisms of GA.Aberrant activation of Wnt/β-catenin signaling cascade is implicated in a variety of tumors, including hepatocellular tumor  and CCA . Unlike in HEK293 cells, we observed that the inhibition of β-catenin was not directly regulated by the phosphorylation of GSK-3β in CCA . We thus anticipated the possibility that GA-inhibited β-catenin expression in this cell line may be regulated by other components of Wnt signaling pathway such as CHOP is one of the key downstream molecules involved in ER stress-mediated apoptosis . It should be noted that the XBP1 mRNA was significantly decreased by GA . Thus, we anticipated that the reduction of XBP1 by GA may cooperate the result in the induction of apoptosis and cell cycle arrest through activating CHOP. In addition, GEM is a first-line therapy for pancreatic ductal adenocarcinoma and it has been explored as a single agent or in combination treatments . However, gemcitabine resistance appears to be a major obstacle of chemotherapy in CCA . Here we showed that GA sensitized CCA cells to GEM (ed by GA  but therpression . These ds to GEM  and the In summary, our data demonstrates the significant biological activities of GA against CCA cell growth through the inhibition of Wnt/β-catenin signaling and modulation of ER stress induced-apoptosis. GA holds promise as an anticancer and it may represent as a novel therapeutic option for CCA which could be exploited to minimize the use of gemcitabine in the setting of the combination treatment with the current chemotherapy.K.B., K.S., K.J., and A.C. conceived and planned the experiments. K.S, K.J., S.R., and K.B. performed the experiments. K.B., K.S, K.J., N.A., V.R., N.T., and A.C contributed to analysis and interpretation of the results. K.B., K.S, K.J., N.T., and A.C wrote the original manuscript. All authors reviewed and approved the final manuscript


Genetic evidence has indicated that β-catenin plays a vital role in glucose and lipid metabolism. Here, we investigated whether pyrvinium, an anthelmintic agent previously reported as a down-regulator of cellular β-catenin levels, conferred any metabolic advantages in treatment of metabolic disorders. Glucose production and lipid accumulation were analyzed to assess metabolic response to pyrvinium in hepatocytes. The expression of key proteins and genes were assessed by immunoblotting and RT-PCR. The in vivo efficacy of pyrvinium against metabolic disorders was evaluated in the mice fed with a high fat diet (HFD). We found that pyrvinium inhibited glucose production and reduced lipogenesis by decreasing the expression of key genes in hepatocytes, which were partially elicited by the downregulation of β-catenin through AXIN stabilization. Interestingly, the AMPK pathway also played a role in the action of pyrvinium, dependent on AXIN stabilization but independent of β-catenin downregulation. In HFD-fed mice, pyrvinium treatment led to improvement in glucose tolerance, fatty liver disorder, and serum cholesterol levels along with a reduced body weight gain. Our results show that small molecule stabilization of AXIN using pyrvinium may lead to improved glucose and lipid metabolism, via β-catenin downregulation and AMPK activation. The liver has large metabolic plasticity and plays an important role in maintaining body homeostasis of glucose and lipid metabolism. During starvation, the liver is the primary source of energy and maintains blood glucose levels through gluconeogenesis. In contrast, after a large energy intake, the liver suppresses the production of glucose, promotes the absorption of glucose, and enhances lipogenesis, thus maintaining normoglycemia . The hepTCF7L2, which encodes a crucial nuclear binding protein of β-catenin, has been well shown to be an important risk factor for T2DM [The multifunctional armadillo repeats containing protein β-catenin is a critical effector protein for multiple pathways, such as the Wnt/β-catenin and FOXO1 pathways, to cross talk in various physiological processes ,5,6. Metfor T2DM ,11,12. Tfor T2DM . Upon β-for T2DM . DespiteOne approach to elucidate the role of β-catenin in glucose, lipid metabolism, and its therapeutic implication in metabolic complications is through the use of its small molecule modulators ,16. OnlyPyrvinium is an anthelmintic agent previously approved for the treatment of pinworm infections . As a poThe cell lines studied in the present study were obtained from the American Type Culture Collection (ATCC) and cultured according to the ATCC’s guidelines. For the growth of HEK293, HepG2, and Huh7 cells, the Dulbecco’s Modified Eagle’s Medium was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P/S) . The cells were seeded 24 h prior to transfection and treatment. Transfection was carried out with specified transfection reagents in Opti-MEM Reduced Serum Media .Cells were transfected with TCF/LEF-based reporter plasmid, TOPFlash, along with the plasmid for renilla luciferase as the internal control using Lipofectamine 2000 . The M50 Super 8x TOPFlash was a gift from Dr. Randall Moon (Addgene plasmid # 12456). Treatment with the compounds was carried out 16 h after transfection for additional 24 h. The Dual-Luciferase Reporter Assay System  was used to assess the luciferase activity on POLARstar Omega plate reader .g for 15 min at 4 °C. Bicinchoninic acid (BCA) assay was used to normalize protein concentrations across the samples, and the proteins were resolved on a 7.5–12% SDS-PAGE gels. Western blot analysis was carried out with primary antibodies specific to the proteins of interest as followed: β-catenin , AMPKα , phospho-AMPKα , β-actin , AXIN1 . Chemiluminescence detection of proteins was carried out and images were captured using an Odyssey-Fc Imaging System .Cell lysates were prepared using NP-40 lysis buffer as previously described . The lysTotal RNA was extracted from the cells using TRIzol reagent  following the manufacturer’s instructions. The total RNA was reverse transcribed and real-time PCR were carried out with SYBR green master mix  using specific primers as listed in RNAi transfection was performed with Lipofectamine RNAimax  for siRNAs. The pcDNA3 plasmid containing human beta-catenin cDNA was a gift from Dr. Eric Fearon , which was overexpressed using Lipofectamine 2000. Following 48 h of transfections, cells were treated with pyrvinium for additional 24–48 h unless otherwise stated. siRNA against AXIN1, CK1α, AMPKα1 were purchased from Sigma .Primary hepatocytes were isolated from the livers of C57BL/6J mice of 8–12 weeks old using a published method . After 2The staining method has been described previously ,23. BrieAll animal experiments were approved by the University of Maryland at Baltimore Institutional Animal Care and Use Committee (IACUC #0617011). Six-week-old male C57BL/6J mice were purchased from the Jackson Laboratories and allowed to acclimate to our animal facility for a week prior to experiments. The mice were fed 45% high fat diet or normal chow diet for 5 weeks. Pyrvinium or vehicle  was intraperitoneally administered every two days. It was weekly dose escalated from 0.2 mg/kg to 0.5 mg/kg in both normal chow group and high fat diet group in a span of one month. Glucose tolerance tests were performed on the mice with 2 g/kg glucose following 6 h of fasting as described . Liver tt-test for two groups or analysis of variance (ANOVA) for more than two groups, with statistical significance presented with p-value < 0.05.Statistical analysis was carried out using GraphPad Prism. Data are presented as mean ± standard deviations and are representative of three independent experiments. Analysis was performed with Student 50 of viability by pyrvinium in mouse primary hepatocytes was 904 nM). In a concentration-dependent manner, pyrvinium decreased glucose production in primary mouse hepatocytes . Four groups of mice were used, two groups fed normal chow diet (NCD) and the other two fed HFD. Due to the extremely low bioavailability of pyrvinium , the micThe function of β-catenin has been explored quite extensively by using genetic animal models ,32. To dβ-catenin plays an important role in regulation of hepatic glucose and lipid metabolism. It has been reported that fatty acid treatment will lead to β-catenin accumulation in hepatocytes, which enhances lipogenesis, eventually causing steatohepatitis . ConsistCTNNB1 gene. Lee’s group has reported that pyrvinium could bind allosterically and activate CK1α, thereby increasing phosphorylation and subsequent degradation of β-catenin [Nevertheless, the detail mechanism accounting for β-catenin downregulation upon pryvinium treatment remains unclear. In our study, pyrvninium treatment did not affect the transcript level of -catenin . However-catenin . Instead-catenin . Interes-catenin ,19. Like-catenin ,39. The G6P and PEPCK, and subsequently an increase in glucose production [IGFBP1. However, future studies are needed to demonstrate if FOXO1 mediates the effects of pyrvinium on gluconeogenesis and lipogenesis via β-catenin downregulation.The signaling pathway downstream to β-catenin also remains to be illustrated. β-catenin is an important protein involved in multiple signaling pathways. For example, it can collaborate with the transcriptional factor TCF7L2 to enhance the expression of canonical Wnt target genes. It can also bind with the transcriptional factor FOXO1 to promote certain gene expression ,27,40. TCTNNB1 gene lacking of exons 3–4 [The effects of pyrvinium treatment on the expression of gluconeogenic and lipogenic genes were moderate in HepG2 cells. Consistently, the effects by pyrvinium on the expression of Wnt and FOXO1 target genes were lost in HepG2 cells, which carry a somatic mutation in the xons 3–4 . Both GSxons 3–4 , all of AXIN stabilization may be critical to the pharmacological mechanism of pyrvinium in regulation of hepatic metabolism. Altering cellular AXIN protein levels has metabolic consequences. For instance, following its deletion in vitro and in vivo, there are defects in glucose and lipid metabolism resulting from deactivation of AMPK, a master protein regulator of energy metabolism ,51. The AXIN1 gene was not affected by pyrvinium treatment in hepatocytes. We have synthesized a variety of pyrvinium analogs [The signaling pathway of AMPK activation by pyrvinium also remains to be detailed. Among AMPK activation-induced metabolic benefits are decreased de novo lipogenesis, increased fatty acid oxidation, and reduced reactive oxygen species (ROS) generation, via phosphorylation of key downstream enzymes such as acetyl-CoA carboxylase (ACC), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), glycerol-3-phosphate acyltransferases (GPAT), the lipogenic transcription factor SREBP-1c, and the antioxidant transcription factor NRF2 ,57. It w analogs ,59. Curr50 in viability assays in hepatocytes. Using the chemical structure of pyrvinium as a template, we have synthesized and tested novel classes of analogs of which a lead compound has showed metabolic benefits against HFD-induced metabolic disorders [Given the observed metabolic effects of pyrvinium in vitro resulting from β-catenin downregulation and AMPK activation, we further evaluate its potential as a pharmacological strategy to treat metabolic disorders. Our studies in HFD-fed mice indicated that pyrvinium treatment led to improvement in glucose tolerance and a corresponding decrease in HFD-induced body weight gain. The data are consistent with various genetic studies showing that deactivation of the β-catenin leads to decrease in body weight gain of mice ,27. The isorders ,59. WithIn conclusion, our findings that small molecule downregulation of β-catenin may confer metabolic benefits is a significant step to resolving certain inconsistencies in previously reported genetic studies. Pyrvinium may increase AXIN levels, subsequently decrease β-catenin level and simultaneously activate AMPK to affect glucose and lipid metabolism as well as perhaps other nutrient metabolism in the liver. Further studies are needed to characterize detail molecular mechanisms underlying the metabolic effects of pyrvinium and to develop its desirable analogs that are useful in treatment of metabolic disorders.