Burkholderia pseudomallei and B. mallei are Gram-negative, facultative intracellular bacteria that cause melioidosis and glanders, respectively. Currently, there are no vaccines for these two diseases. Animal models have been developed to evaluate vaccines and therapeutics. Tissues from infected animals, however, must be fixed in formalin and embedded in paraffin (FFPE) before analysis. A brownish staining material in infected tissues that represents the exopolysaccharide of the pathogen was seen by bright field microscopy but not the actual microorganism. Because of these results, FFPE tissue was examined by laser scanning confocal microscopy (LSCM) in an attempt to see the microorganism. Archival FFPE tissues were examined from ten mice, and five nonhuman primates after exposure to B. pseudomallei or B.mallei by LSCM. Additionally, a historical spleen biopsy from a human suspected of exposure to B. mallei was examined. B. pseudomallei was seen in many of the infected tissues from mice. Four out of five nonhuman primates were positive for the pathogen. In the human sample, B. mallei was seen in pyogranulomas in the spleen biopsy. Thus, the presence of the pathogen was validated by LSCM in murine, nonhuman primate, and human FFPE tissues. Burkholderia pseudomallei. It is endemic in Southeast Asia and northern Australia and appears to be much more widely distributed than originally reported [B. pseudomallei can be through cutaneous inoculation, ingestion of contaminated water or food, or by inhalation. Infection by B. pseudomallei is the third leading cause of disease in Southeast Asia behind tuberculosis and AIDs, and pneumonia is the most common clinical presentation of melioidosis. Host risk factors, such as diabetes, excess alcohol consumption, and renal and lung disease, significantly influence the susceptibility to infection [B. pseudomallei and its closely related species B. mallei, which causes glanders, as Tier 1 biological agents.Melioidosis is caused by the Gram-negative, facultative intracellular pathogen reported . It can reported . Exposurnfection ,4. Treatnfection . At presB. pseudomallei or B. mallei infections. This positive signal comes from the recognition of the exopolysaccharide that is common between these two pathogens, but the actual microorganism was not visualized [B. mallei whole-cell antigen was used as the primary antibody to analyze FFPE tissue. Furthermore, different antibody preparations were evaluated with LSCM to visualize the pathogen in FFPE infected tissue. In the following report, examples of the presence of B. pseudomallei or B. mallei in archival tissues from our animal model studies using LSCM were presented. In addition, the possible presence of B. mallei in a historical biopsy of a spleen from a human suspected of exposure to B. mallei was presented. Finally, different antibody preparations were shown to be used with LSCM to visualize the pathogen in FFPE tissues.Animal models of melioidosis and glanders were developed for the evaluation of therapeutics or vaccines ,7,8,9. Fsualized ,11,12. TB. mallei GB18-3 was obtained from the Bacteriology Division culture collection at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, Maryland, and it had been passed through hamsters 3 times [B. pseudomallei K96243 were obtained from the Unified Culture Collection (UCC) at USAMRIID. A rabbit antibody preparation made against an extract of irradiated, whole-B. pseudomallei K96243 (IRBpK) cells was a kind gift from Robert Ulrich (USAMRIID). Human tissue from a patient suspected of exposure to B. mallei was obtained from the Joint Pathology Center . 3 times . Single B. mallei [B. pseudomallei. All procedures were performed under biosafety level 3 (BSL3) conditions and culture/cell manipulations were carried out in a biosafety hood. Two hundred milliliters of 4% glycerol tryptone broth (GTB)  in a 1L flask was inoculated with 5 µl of a stock culture of the organism, and the culture incubated with shaking at 200 rpm overnight at 37 °C (16–18 h). After overnight growth, the culture was placed into 50 mL conical tubes , and the tubes were centrifuged for 25 min at 3700 rpm in a swinging bucket rotor at 4 °C. The culture supernatants were discarded and cell pellets were suspended in 1.0 mL of Hanks Balanced Salt Solution . The cell suspensions were combined and formaldehyde (ThermoFisher Scientific) was added to a final concentration of 4%. The cells were left in the formalin solution for 24 h at 4 °C, and then the cells were washed twice with cold HBSS. Ten percent of the total volume was used to test for sterility on sheep blood agar plates that were incubated at 37 °C for 3 days. After validation of the sterility of the formalin-fixed cells, the cells were dialyzed for 3 days against 1 L of water with daily changes  at 4 °C. The dialyzed cells were centrifuged and cell pellets were suspended in sterile water. The absorbance of the cells suspension was compared to a standard curve to calculate the protein concentration, and the cells were stored in aliquots at −70 °C. The following procedure describes the general growth conditions and preparation of a whole-cell, bacterial antigen of . mallei  or B. psB. mallei (fBm) GB18-3 cells were formulated with Ribi TriMix as the adjuvant  [B. pseudomallei K96243 (fBpK) cells were formulated with Freund’s complete adjuvant (FCA) or Freund’s incomplete adjuvant (FIA) . The general procedure to generate polyclonal antibodies in 2 female NZW rabbits (~2.5 kg) were as follows : prebleed, 21 days before the primary vaccination; primary vaccination, 250 µg of fBpK in FCA; 3 boost (21 days apart) vaccinations starting 21 days after the primary vaccination, 125 µg of fBpK in FIA; terminal bleed, 14 days after the last boost. Antibody (IgG) titers against IRBpK, fBpK, and fBm cells were determined at least twice by ELISA as previously described [Two methods were used to produce rabbit polyclonal antibodies against formalin-treated whole-cells. In the first method, formalin-treated MT, USA) . In the B. mallei or B. pseudomallei antibody  at a dilution of 1:6000 and incubated at room temperature for forty five minutes. They were rinsed, and the peroxidase-labeled polymer (secondary antibody) was applied for thirty minutes. Slides were rinsed and a brown chromogenic substrate 3,3′ Diaminobenzidine (DAB) solution (Dako Agilent Pathology Solutions) was applied for eight minutes. The substrate–chromogen solution was rinsed off the slides, and the slides were counterstained with hematoxylin and rinsed. The sections were dehydrated, cleared with Xyless, and then coverslipped. Stained sections were digitized and examined with Aperio Image Scope software . The specimens were examined with an Olympus BX53 microscope . Immunohistochemistry (IHC) was performed using the Dako Envision system . Briefly, after deparaffinization, peroxidase blocking, and antigen retrieval, sections were covered with a rabbit polyclonal anti-B. pseudomallei or anti-B. mallei polyclonal antibody (1:1000–1500) for 2 h at room temperature. After rinsing with PBS, the sections were incubated with a secondary Alexa Fluor 488 conjugated goat anti-rabbit antibody for 1 h at room temperature. Sections were cover slipped using the Vectashield mounting medium with or without 4′,6-diamidino-2-phenylindole (DAPI)  to stain nuclei. Additionally, in some cases, to visualize the presence of macrophages in the nonhuman primate samples, a mouse anti-human/NHP CD68 antibody was used (Dako Agilent Pathology Solutions) or CD45 antibody for lymphocytes (Dako). To visualize B. pseudomallei/B. mallei, z-stacks (multiple slices) were used. Images were captured on a Zeiss LSM 880 confocal system  and processed using ImageJ software .Formalin-fixed paraffin embedded (FFPE) tissue sections were deparaffinized using xylene and a series of ethanol washes before staining single sections with H&E. 0.1% Sudan black B (Sigma-Alrich) treatment was used to eliminate the autofluorescence background, and sections were heated in a citrate buffer (pH 6.0) for 15 min to reverse formaldehyde crosslinks. After rinsing with PBS (pH 7.4), the sections were blocked with PBS containing 5% normal goat serum overnight at 4 °C. The sections were incubated with rabbit anti-B. pseudomallei K96243 polyclonal antibody was used as the primary antibody to detect the presence of B. pseudomallei in the FFPE tissue. An example is shown of a spleen from a C57BL/6 mouse 47 days post-infection (PI) that was exposed to B. pseudomallei 22 by aerosol  were positive for B. pseudomallei. Tissues that were positive were also those with local pyogranulomatous inflammation, and areas without pyogranulomatous inflammation were negative for the pathogen  for each NHP  were examined by LSCM that were exposed to the lung A–C and sthe lung D–F of onthe lung G–I. For the lung J–L, whilhe lungs M–O or inB. mallei. A historical spleen biopsy (FFPE) from a human suspected of having been exposed to B. mallei was obtained. B. mallei in FFPE tissue was demonstrated from a human suspected of being exposed to B. mallei by LSCM. Other FFPE tissues were examined to determine if LSCM could identify the presence of B. mallei GB18 (fBm) whole cell. B. mallei FMH that was examined with two other antibody preparations  that was used in the previous study. In B. pseudomallei K96243 (extBpK). Similarly, the presence of B. mallei was seen in the lung of the AGM with an antibody raised against formalin-treated B. pseudomallei K96243 whole-cells (fBpK). Therefore, it appears that antibodies raised against three different Burkholderia antigen preparations will work with LSCM of infected FFPE tissue.A question arose if other types of antibodies would work with LSCM when examining FFPE tissue. In the previous study above with LSCM, an antibody was used that was developed against a formalin-treated B. pseudomallei or B. mallei bacterial cells in FFPE tissue by LSCM was demonstrated that it was not by bright field microscopy. Burkholderia cells in FFPE tissues were seen from mice and nonhuman primates exposed to B. pseudomallei or B. mallei, and in a historical spleen biopsy from a human suspected of being exposed to B. mallei. The difference in the technology of image formation between bright field microscopy and LSCM made it possible to clearly visualize the pathogen in FFPE tissue. Briefly, in LSCM the excitation (laser) and emission light sources are limited (either both or only emission) by pinhole apertures with the focus on a point(s) within a single plane of the sample. Excitation and emission light derived from above, below, and away from the point or plane of focus are generally excluded by the pinhole apertures that result in a higher resolution image. As the image is scanned, the point of focus stays in the same plane  [The presence of tioning) . In contB. pseudomallei or B. mallei in FFPE tissue samples, it was reported that the presence of the pathogen was seen after immunohistochemical (IHC) staining of paraffin sections [B. pseudomallei or B. mallei present in FFPE tissue in infected murine [B. pseudomallei specific primers to determine if the pathogen was present in the samples [B. pseudomallei in experimental mouse studies [In earlier reports on the visualization of sections ,16, whicsections , they red murine  or humand murine . Further samples ,20. In t studies  or clini studies  without B. pseudomallei or B. mallei in infected tissue. LSCM was used to visualize B. mallei, B. pseudomallei, or B. thailandensis in murine macrophage-like RAW 264.7 cells [B. pseudomallei infection of A549 human lung epithelial cells [B. mallei in an infected mouse FFPE spleen sample that was examined by LSCM was reported [Mycobacterium tuberculosis in lung tissue from human patients [There are few reports of the use of LSCM to visualize .7 cells ,24, and al cells ,25. A repatients . Burkholderia appear to accumulate in the spleen and cause the formation of pyogranulomas that are in most cases easy to observe upon autopsy. A mouse spleen with a pyogranuloma may contain from 104 to 109 CFU [One caution to this report is that the figures presented in this report may show more microorganisms than present than seen in human cases of melioidosis or glanders. Animal tissues are easier to recover and manipulate than human tissue, for example the mouse spleen.  109 CFU ,8.B. pseudomallei or B. mallei in FFPE tissue from different animal or human sources. This technology may be diagnostic for melioidosis (or glanders) or complement the diagnosis of the disease with the isolation of the pathogen. In addition, it would be useful to have B. pseudomallei and B. mallei capsule-specific monoclonal antibodies, if there is a question between diagnosis of melioidosis or glanders [This report established the feasibility of using LSCM to validate the presence of glanders ,29,30. O


Tg very similar to that of NC514 and thus could be useful in formulating epoxy with renewable cardanol content.Cardanol is a renewable resource based on cashew nut shell liquid (CNSL), which consists of a phenol ring with a C15 long aliphatic side chain in the meta position with varying degrees of unsaturation. Cardanol glycidyl ether was chemically modified to form side-chain epoxidized cardanol glycidyl ether (SCECGE) with an average epoxy functionality of 2.45 per molecule and was cured with petroleum-based epoxy hardeners, 4-4′-methylenebis(cyclohexanamine) and diethylenetriamine, and a cardanol-based amine hardener. For comparison, cardanol-based diphenol diepoxy resin, NC514 (Cardolite), and a petroleum-based epoxy resin, diglycidyl ether of bisphenol-A (DGEBA) were also evaluated. Chemical and thermomechanical analyses showed that for SCECGE resins, incomplete cure of the secondary epoxides led to reduced cross-link density, reduced thermal stability, and reduced elongation at break when compared with difunctional resins containing only primary epoxides. However, because of functionality greater than two, amine-cured SCECGE produced a  Thermosetting polymers are being used extensively in coatings, adhesives, and as matrices in polymer composites because of their good mechanical properties, remarkable chemical and thermal resistance, as well as good processability ,2. RenewModification of cardanol for thermoset synthesis has been achieved by the functionalization of the phenolic ring as well as the alkyl side chain. In previous work, cardanol has been functionalized at the phenol moiety to synthesize monofunctional epoxy using epichlorohydrin. This molecule can be used as a chain-capping agent in thermoset formulations ,7,8. FunPrevious studies of the reactivity of epoxidized soy-bean oil (ESO) and its derivatives showed that secondary epoxies are not as reactive as primary glycidyl moieties and cannot be effectively cured to a high extent with amine curing agents ,17,18. I1H NMR) and epoxy equivalent weight (EEW) titrations. Earlier studies concerning the epoxidation of primary unsaturation sites show that formic acid/hydrogen peroxide methods are not effective for epoxidizing terminal double bonds [Herein, functionalization of the unsaturation sites of the cardanol glycidyl ether (CGE) side chain was achieved via hydrogen peroxide and formic acid to obtain side chain epoxidized CGE (SCECGE) with secondary epoxies on the side chain. Structural verification of the SCECGE monomer was obtained using proton nuclear magnetic resonance , diethylenetriamine , sodium chloride , sodium bicarbonate (99.9%), magnesium sulfate (99.9%), and monocyclohexylamine  were obtained from Sigma-Aldrich, USA; 4-4′-methylenebis(cyclohexanamine)  was obtained from Air Products, Allentown, PA, USA. Cardanol-based di-phenyl di-epoxy (NC514), mono-epoxy, cardanol glycidyl ether  and cardanol-based epoxy hardener NX2003  were kindly supplied by Cardolite, Newark, NJ, USA. All chemicals in this work were used as-received. Structures of the primary materials used in this work are presented in Formic acid , toluene (99.8%), hydrogen peroxide   was added to the flask drop-wise over 1 h at 20 °C. After the addition was completed, the temperature of the system was slowly increased to 40 °C over 1 h, and the contents of the reaction mixture were allowed to react at that temperature for 24 h. At the end of the reaction time, the contents were washed with 5 wt.% sodium bicarbonate solution followed by deionized water to remove the excess acid and 5 wt.% NaCl solution three times. The remaining residue was dried with anhydrous magnesium sulfate and rotary evaporated to yield a brownish-yellow liquid product (90% yield).The double bonds of the monoepoxy CGE were epoxidized using peroxy acids, which are produced from FA and H1H NMR spectroscopy was used for characterization of reactants and products. A 1H-NMR  unit was used with a spectral window of ±2000 Hz, 90° pulse width, and 32 scans at 25 °C. The epoxide functionality of NC514 and DGEBA were obtained using the 1H NMR method of Jaillet et al. [−1 using parallel plate geometry.EEW titrations were performed in accordance with the ASTDM D1652-97 standard to determine the epoxy content of the CGE, SCECGE, as well as DGEBA and NC514 epoxies. t et al.  These we−1 range with an 8 cm−1 resolution and 32 scans per spectra at ambient conditions.Polymer samples were prepared by thermally cross-linking SCECGE, NC514, and DGEBA epoxy monomers with different petroleum-based (PACM and DETA) and cardanol-based (NX2003) epoxy hardeners at stoichiometry. The epoxy-amine combinations were mixed and degassed via a high shear mixer (Thinky ARE-310), cast into rectangular and dog-bone shaped rubber molds, and then thermally cured at 90 °C for 12 h and at 180 °C for 12 h. The conversion of epoxy and amine groups were measured using transmission near-IR in the 4000–8000 cm1H NMR spectra and EEW of the mixture throughout the curing process. In addition, using SCEC instead of SCECGE monomer yielded clearer FT-IR and 1H-NMR spectra since the secondary epoxy peak of SCECGE overlaps with the primary epoxy peak, making sample analysis difficult. For mid-IR studies, the samples were sandwiched between inert NaCl and scanned with a resolution 8 cm−1 and 32 scans per spectrum. 1H-NMR and EEW studies were also used to determine the overall secondary epoxy conversion before and after the curing and postcuring steps.To determine the conversion of the secondary epoxies, cardanol-derived secondary epoxy containing model compound (SCEC) was utilized. The side chain epoxidation of the cardanol model compound was carried out using the same chemistry applied to functionalize the side chain of CGE molecule. The representative structure of this model compound is shown in ABS(t) and ABS(t = 0) represent the reduced absorbance for the FTIR studies before and after the curing reaction, respectively. In Equation (2), I(t) and I(t = 0) also represent the reduced area of the 1H NMR peak after a certain time t, and the reduced peak area at the start of curing process. For the mid-IR studies, the secondary epoxy peak observed around 840 cm−1 was followed and referenced to the invariant aromatic peak at 1610 cm−1. For the 1H-NMR studies, the secondary epoxy peaks observed between 2.8–3.1 ppm were tracked and referenced to the invariant terminal methyl proton peak found at 0.88 ppm.In Equation (1), v), glass transition temperature (Tg), as well as the room-temperature storage modulus (E′) of the cured polymers. Rectangular samples with approximate dimensions of 35.5 × 12 × 1.0 mm3 were tested isothermally at 25 °C and 1 Hz with a deflection of 15 µm by using a single cantilever arrangement to obtain E′ accurately. To determine the Tg and calculate cross-link density of the cured samples, rectangular DMA bars with approximate dimensions of 35.5 × 12.0 × 3.5 mm3 were tested from −100 °C to well above their glass transition temperature by ramping at 2 °C/min at 1 Hz with a deflection of 15 µm. The temperature value of the maximum of the loss modulus peak was taken as the Tg of the cured polymer. The v values were calculated from the rubbery plateau of the storage modulus curves at 50 °C above Tg using Equation (3) where R is the universal gas constant [Dynamic mechanical analysis  was used to evaluate the cross-link density . Tensile tests were performed on type IV samples. Samples were tested at ambient conditions using an extensometer to measure strain with a constant crosshead speed of 1 mm/min and a gauge length of 45 mm. For each formulation, at least 6 tensile specimens were tested and analyzed.1H-NMR spectra of CGE and its epoxidation product SCECGE with the corresponding peak assignments for the different types of protons of CGE and SCECGE, respectively. The epoxidation of the side chain was confirmed by the shifting of the peak at 5.3 ppm, corresponding to the methylene protons (–CH=CH–) in the spectrum of CGE to 3.2–3.0 and 2.95 ppm (c and c′), representing the secondary epoxy protons on the side chain. In addition, the peak at 2.8 8 ppm (E) assigned to the protons between two double bonds (–CH=CH–CH2–CH=CH–) in the spectrum of CGE shifted to 1.73–1.91 91 ppm (e peak), representing the protons between the two epoxies, and G peak at 2.00 ppm (–CH2 protons next to double bonds) present in the spectrum of CGE shifted to 1.5 ppm (g) in the spectrum of SCECGE, which was attributed to the protons next to the epoxy ring. More detailed 1H NMR and 12C NMR analysis of the CGE and SCECGE polyepoxy monomer can be found elsewhere [lsewhere ,15.1H NMR peaks. The four aromatic protons of (A) were used as the internal invariant. The internal and terminal double bond functionality of the CGE was determined as 1.45 and 0.38 double bond/molecule, respectively, as shown in The double bond and epoxy functionalities of the CGE were determined via the integration of the related 1H NMR spectra of the cardanol-based di-phenyl di-epoxy NC514. The epoxy functionality of the NC514 epoxy resin was calculated as 1.35 epoxy/cardanol unit via its 1H NMR spectra. A minor amount of unreacted cardanol (~4%) was also observed in NC514. In addition, size exclusion chromatography analysis of this resin showed that NC514 is effectively a mixture of phenolated, epoxidized cardanol and higher molecular weight species, as determined previously [Detailed structure analysis previously performed on cardanol-based NC514 epoxy resin showed that this resin contains a significant amount of impurities in the form of higher molecular weight oligomers formed during the phenolation and epoxidation of cardanol ,12. Strueviously . The higeviously . The EEWeviously ,12, sugg−1 shear rate, given in Viscosity measurements were a performed on NC514, DGEBA, CGE, and SCECGE resins at 25 °C. The viscosity of all the epoxy resins did not show significant dependence of shear rate, suggesting Newtonian behavior. Viscosity values—measured at 20 s−1, the primary and secondary amine peak at 6570 cm−1, or the epoxy peak at 4540 cm−1 were observed in the cured samples of DGEBA and NC514. The absence of these peaks indicates a complete epoxy/amine reaction in the cured samples within the limits of IR spectroscopy measurements. For SCECGE, no primary amine peak was observed after cure either. However, even after the prolonged curing times at elevated temperatures, a slight peak at 6570 cm−1 associated with primary and secondary amine was still observed, suggesting that SCECGE-based samples were not cured fully as was the case for DGEBA and NC514. The incomplete curing of SCECGE-based samples may be explained by the lower reactive nature of the secondary epoxies presented on the molecule, which do not react easily with the secondary amines to form a highly cross-linked network, as demonstrated by previous studies. Although this extent of conversion would indicate that the conversion of the secondary epoxies is around 60–70% for different curing agents—assuming complete conversion of the glycidyl primary epoxides—there are no distinct peaks visible for the secondary epoxies in near-IR, so this method does not allow for direct measurement. Thus, the investigation with side chain epoxidized model compound (SCEC) described in the next section was designed to evaluate the conversion of the secondary epoxies with amines at temperatures typical of the cure and postcure of epoxy-amines.1H-NMR, and EEW titrations as described in the experimental section.Although near-IR can provide valuable information about the curing reaction between epoxies and amine systems, it is not possible to specifically monitor the secondary epoxies with this technique. To determine the conversion of the secondary epoxies and the extent of the cure reaction between secondary epoxy and amines, SCEC (EEW: 275 g/eq) model epoxy was cured with the monoamine MHA in 100% molar excess. The secondary epoxy and amine reaction was followed via near-IR, −1 shows that the secondary epoxy reduced in intensity during the curing process. However, even after prolonged curing times at elevated temperatures, this peak did not completely disappear, suggesting the secondary epoxies were not fully converted. The conversion of the secondary epoxies was determined to be 60% after 12 h of curing at 90 °C and increased to 72% after postcuring at 180 °C for 24 h. Postcuring the mixture for longer times did not increase the conversion any further. In addition, representative 1H-NMR spectra of the SCEC-MHA mixture are presented in Tg compared to SCECGE and NC514 because of their higher cross-link density and rigid, aromatic backbone [Tg of the NC514- and SCECGE-based formulations had significantly lower glass transition temperatures than the DGEBA-based systems, most likely because of the presence of the more flexible alkyl chain found between cross-links [Tg of this monomeric system. Formulations prepared with NC514 had only slightly higher glass transition temperatures than formulations prepared with SCECGE. A 5 °C difference in Tg was observed between the NC514 and SCECGE resins when cured with PACM. The Tg difference was 3 °C between the two monomers when cured with DETA. This is a surprisingly small difference considering the added higher aromatic content between cross-links found in networks formed with NC514 and the incomplete cure of the secondary epoxide groups in SCECGE. The reason for this could be related to an overall higher molecular weight between cross-links formed with NC514 and the nonreactive aliphatic side chain that is found these systems. Thus, the topological differences of the SCECGE networks relative to NC514 appear to narrow the presumed Tg gap associated with the greater aromatic content in NC514. Future studies aimed at epoxidizing the terminal double bonds of SCECGE will help to test this assertion.The glass transition temperature of the fully cured epoxy amine combinations presented in backbone . The valss-links . FurtherTg . In addition, these room temperature storage modulus values obtained via DMA are in a good agreement with the Young’s modulus values obtained via tensile tests, also shown in Room-temperature storage modulus values were also determined via DMA by testing the rectangular DMA bars isothermally at 25 °C, and these results are also given in E), tensile strength, and tensile strain of the fully cured samples were obtained from tensile tests, and the results are shown in The tensile modulus (The type of amine curing agent used has a significant effect on the modulus of the cured samples. PACM gave the highest tensile modulus and strength values despite their lower cross-link densities relative to DETA because of the stiffer nature of the cycloaliphatic amine PACM. Epoxy resins cured with the cardanol-based amine hardener NX2003 have shown the lowest tensile modulus and tensile strength values among the curing agents due to the plasticization effect of the C15 alkyl chain of the hardener and the lower cross-link densities for the NX2003 networks. This effect is more pronounced when the cardanol-based epoxies were cured with cardanol-based curing agents because of the large plasticization effect caused by the combination of the C15 alkyl chains, low extent of cure, and low cross-link density of the network. The elongation at break of the epoxy amine formulations presented in Tg very similar to the cardanol-derived NC514 diepoxy, which contains only phenolic glycidyl ether reactive groups. Moreover, the viscosity of SCECGE is significantly lower than that of the other systems studied. Thus, SCECGE is a promising candidate for making useful epoxy resins with renewable content based on CNSL.The cardanol-based epoxy monomer SCECGE was prepared, and contained epoxy groups on the aliphatic side chain as well as the glycidyl ether on the phenolic ring. The structural characterization of SCECGE showed that epoxidation of the inner secondary double bonds was achieved almost completely, while the terminal ones were minimally epoxidized. Mid-IR analysis of the different epoxy-amine systems showed that the primary phenolic glycidyl ethers were almost fully reacting with the amine curing agents, but the secondary epoxies found on the SCECGE-based samples required a higher temperature for curing and were not observed to fully cure with the amines investigated. Model compound studies were used to evaluate secondary epoxy conversion by a number of methods, and it was found that for the reaction conditions investigated, it was possible to react between 60–70% of the secondary epoxy groups. Though it is well known that the secondary epoxide groups on aliphatic chains are significantly less reactive than glycidyl ethers, in this investigation the reactivity of these groups has been quantified, adding to our understanding of the cure of resin systems that incorporate these reactive groups. Results of the investigation showed that incomplete cure of the secondary epoxides led to a reduced cross-link density for SCECGE resins and reduced elongation at break. Otherwise, amine-cured SCECGE resin produced a


Magnesium alloys are highly strain rate sensitive and exhibit good workability in a narrow forging temperature range. Consequently, parts made of these materials are usually forged with low-speed hydraulic presses, using specially designed tool heating systems in order to ensure near-isothermal conditions. This study investigates whether popular magnesium alloys such as Mg-Al-Zn can be forged in forging machines equipped with high-speed forming tools. Experimental upset forging tests on AZ31B, AZ61A and AZ80A specimens were conducted, using a screw press with a ram speed of 0.5 m/s and a die forging hammer with a ram speed at stroke of about 5 m/s. Test specimens were preheated to 350 °C, 410 °C and 450 °C. After the upset forging process, they were air- or water-cooled and then examined for their workability, hardness and grain size. To validate the results, a forging process for a producing handle was designed and modelled by the finite element method. Distributions of strain, temperature and fracture criterion were analysed, and energy and force parameters of the forging process were calculated. After that, experimental tests were performed on AZ31B and AZ61A specimens in order to determine mechanical properties of forged parts and examine their micro- and macrostructure. Results have demonstrated that AZ80A is not suitable for forging with either the screw press or the die forging hammer, that AZ61A can be press- and hammer-forged but to a limited extent, and that AZ31B can be subjected to forging in both forging machines analysed in the study. Due to their low specific weight and high relative strength, magnesium alloys are used as cast or wrought alloys in many industries such as aviation and aerospace, automotive, sports and recreation, electronics, and medical ,2,3. NewStrain rate and temperature of the billet and tools are the most important parameters of the forging process. Owing to their high sensitivity to strain rate, magnesium alloys should be forged predominantly using hydraulic presses or mechanical presses with low operating speeds, under isothermal conditions. Put simply, it can be stated that the higher the strength properties of an alloy, the lower the strain rate should be applied ,9,10,11.−1. After the forging process, forged parts were aged at a temperature of 177 ± 5 °C for 24 h and then cooled in air. Parts obtained with this forging method had correct shape and dimensions. Compared to the billet, the forged part showed a slight increase in the strength properties, but its workability would remain unchanged. The rim is one of the parts that are made of magnesium alloys. This part is widely used in cars, airplanes and helicopters. An example of a forming process for a rim made of AZ80 alloy is given in [A large number of existing studies in the specialist literature have examined forging processes for magnesium alloys, conducted with hydraulic presses. A great practical value of these studies is that they specify parameters that should be used for a given alloy to obtain defect-free products. Płonka et al.  investiggiven in ,16. In tgiven in  investiggiven in ,20 descrgiven in ,22 presegiven in ,32,33,34In the light of reported results, it is justified to investigate forging processes for selected magnesium alloys conducted using standard high-speed forging machines, such as forging hammers and screw presses. The scientific aim of this study is to determine the relationships between forging temperature and deformation speed related to forging machines as well as between cooling method and properties  of selected magnesium alloys subjected to forging with industrial forging machines such as screw presses and forging hammers. The main practical goal of the study is to develop the technological assumptions of a hot forging process for selected magnesium alloys, conducted with screw presses and forging hammers that are available in many standard forging plants.The study was conducted on magnesium alloys containing aluminium and zinc, i.e., AZ31B, AZ61A and AZ80A. Chemical compositions of these alloys are given in AZ31B (MgAl3Zn) is the most widely used magnesium alloy and is suitable for hot working. It has the following properties: ultimate tensile strength TS ≈ 240 MPa, yield strength YS ≈ 150 MPa, elongation E ≈ 9% and hardness 50–55 HB. It is used in structures that do not carry loads, e.g., various types of hinges, flaps and handles used in the design of helicopters and airplanes.AZ61A (MgAl6Zn) is a moderately hot-workable alloy. Its properties are similar to those of low-strength structural aluminium alloys and are as follows: TS ≈ 275 MPa, YS ≈ 165 MPa and E ≈ 7% and hardness of about 60 HB. This material is used for structures that carry low static and dynamic loads, e.g., pylons and levers in the aircraft design.AZ80A (MgAl8Zn) is hard to deform and difficult to hot-work. Its properties are similar to those of structural 6000 series aluminium alloys and are as follows: TS ≈ 330 MPa, YS ≈ 250 MPa and E ≈ 4% and hardness approx. 72 HB.The study was conducted using an F1736A screw press with a ram speed at stroke of about 0.5 m/s and an MPM 3150 forging hammer with an average ram speed at stroke of about 5 m/s. The research was divided into three stages.Stage 1 of the study involved performing experimental upset forging tests for the selected magnesium alloys. The objective of this stage of the study was to determine the effect of temperature, tool speed of a given forging machine and cooling method on the workability, microstructure and hardness of forged parts. Results were used as a basis for estimating whether the selected alloys could be subjected to forging with the analysed forging machines. Cylindrical specimens with a diameter of 20 mm and a height of 30 mm were preheated to temperatures of 350 °C, 410 °C and 450 °C, and then upset forged with the hammer and screw press to the height of 10 mm. The preheating temperatures of specimens were selected based on data reported in the specialist literature ,20,21,22Stage 2 of the study consisted of first designing screw press and hammer forging processes and then modelling these processes to verify whether the design assumptions were correct. The study investigated a forging process for a handle shown in For the selected forging it has been assumed that it must meet the technical requirements given in Cutting the billet (bar) into the dimensions of ø22 × 170 mm;Preheating the billet to 410 °C for 22 min (1 min per 1 mm of the billet diameter);Forging operation in a bending cavity;Preliminary forging operation in a finish forging cavity, leaving an underforged portion of about 2 mm;Preheating the forged part;Forging in a finish forging cavity;Cooling the forged part in air or water.The forging process consisted of the following:It was assumed that the dies would be heated to 300 °C.Analyse the distribution of temperature and effective strain in the forged part;Identify the regions of the forging that are most prone to crack formation;Examine the flow of material in individual operations, predominantly in terms of potential underfill, overlap and other shape defects;Determine stroke energy (hammer forging) and forming force (press forging) in order to select the most suitable forging machine.The designed forging process was verified via numerical simulations in DEFORM 3D ver. 11.1 software based on the finite element method. The objectives of the numerical verification were the following:σ—flow stress, MPa;ε—strain;−1;T—temperature, °C.Parameters applied in the numerical simulation were compliant with the designed process. The calculations were made for specimens of alloys AZ31 and AZ61, under the three-dimensional strain assumption. The workpiece and dies are respectively defined as rigid-plastic and rigid bodies. The workpiece was meshed using 100,000 tetrahedral elements. The material model of AZ31 was described by equation:2·K, while that between the material and environment was 20 W/m2·K [C is, described by the equation [1σ—maximum principal stress;eσ—equivalent stress;ε—strain;C—integral value.Function (1) was determined based on combined results reported in ,36,37,380 W/m2·K . The Coc0 W/m2·K  was usedequation :(3)C=∫0εlimitC, which can be expressed with the following equation:fε is the fracture strain.Fracture occurs when the integral reaches the critical value limitC in an unambiguous way because this value depends on factors such as the state of stress or strain paths. Consequently, the C–L criterion was only employed to determine the most fracture-prone regions of the sample; we did not, however, undertake to determine whether fracture would occur, and if so—at what moment.Nevertheless, it is problematic to determine the value of In the employed simulation program, the boundary conditions relating to strains are defined by the geometry of forming tools, whereas the boundary conditions relating to loads are defined by the models of forging machines used. The screw press model was defined by a ram speed of 0.5 m/s, while the die forging hammer model was described with a drop part weight of 1200 kg (1000 kg ram weight + 200 kg die weight), blow energy of 36 kJ and efficiency of 80%.Stage 3 of the study included the verification of the forging process under industrial conditions. A series of experimental die forging tests for producing a handle were carried out. The main goal of the tests was experimental verification of the designed die forging process based on the most important technological parameters identified in the first stage of this study (upset forging tests) and determined via FEM numerical modelling. The press and hammer forging tests were carried out using the same process parameters as in the second stage of the study. A lubricant based on grease and graphite was used in the preliminary and final forging operations.The magnesium alloys tested in the third stage (AZ31B and AZ61A) belong to a group of materials which—due to a low content of alloy components—are not subjected to precipitation hardening (supersaturation and aging), because such heat treatment would not significantly improve their strength properties. Consequently, some forgings were only subjected to stress relief annealing and others were left as manufactured, i.e., without heat treatment, in order to determine the effect of annealing on the properties of the tested alloys. The forged parts were subjected to annealing at 350 °C for 1 h. The main aim of this treatment was to relieve stresses induced by the forging process.Determination of strength properties via static tensile testing in room temperature;Measurement of Brinell hardness;Examination of the macrostructure in the longitudinal and cross sections relative to the fibre direction;Metallographic examination of the microstructure in the etched condition.The forged parts were then examined for quality. The examination involved the following:The experimental results were used to investigate the possibility of forging the tested magnesium alloys with the use of high-speed forging machines, i.e., the screw press and die forging hammer.The results of the AZ61A specimens show that this alloy retains satisfactory workability when subjected to screw press forging, with the specimens preheated to 410 °C . The hamThe results of the AZ80A specimens  demonstrMacrostructure of the upset forged parts was examined for internal and subsurface defects that were impossible to identify via visual inspection. The results of the upset forging tests demonstrate that AZ31B shows relatively good workability at an appropriate preheating temperature, the workability of AZ61A is limited, while AZ80A is not suitable for press and hammer forging at all. Therefore, further part of the study focused on the first two magnesium alloys.The grain size in the AZ31B specimens corresponds to the grain size numbers 7–12;The grain size in the AZ61A specimens corresponds to the grain size numbers 9–12.It must be stressed that the lower the grain size number specified in the standard, the bigger the size of the grain is see .The designed forging process for producing a handle was modelled numerically. Separate simulations were carried out for the screw press forging process conducted with a pressure of 6.3 MN and for the hammer forging process conducted with a blow energy of 36 kJ. Individual operations of the forging process  are shown in Distributions of effective strain during the final forging operations of the hammer forging process are plotted in A characteristic of the die hammer forging process is that high values of blow energy are converted into plastic deformation work which, in turn, is predominantly converted into heat. The temperature of the forging increases, especially in the area of the highest strains. Example of temperature variations toward the end of the final forging operation is plotted in The FEM analysis involved determination of the energy required to form a part in the preliminary and final forging operations of the hammer forging process and calculation of the maximum forming force for these operations in the press forging process. The calculations were made for AZ61A alloy which requires the use of forging machines with higher energy and load parameters than in the case of AZ31B. The results demonstrate that the preliminary forging operation of the hammer forging process was conducted with a stroke energy of approx. 20.7 kJ, while the forging operation in the finish forging cavity was conducted with a stroke energy of approx. 30.3 kJ. This means that in the hammer forging process conducted with a stroke energy of 36 kJ and an efficiency of 80%, the final forging operation must be performed at least with two strokes. The maximum force in the preliminary forging operation of the press forging process for AZ61A is approx. 1.76 MN, and it increases to approx. 2.56 MN in the final forging operation. The results of the force in the press forging process show that the screw press is suitable for conducting the designed forging process.The third stage of the study consisted of performing experimental tests of the screw press and hammer forging processes, according to the designed technique. Individual stages of the die forging process are given in The screw press forging process is very stable. After visual inspection, the forged parts made of AZ31B and AZ61A are classified as correct—they have no defects such as cracks and delamination either in the forging or the flash .The quality of the hammer-forged parts is lower, particularly in the case of the AZ61A specimens. Numerous cracks are present in the flash area. The simulation results show that the strain and, consequently, material temperature in this area are the highest, with the temperature exceeding 450 °C . Under sThe results demonstrate that the stress relief annealing operation has no significant effect on the mechanical properties of the alloys under study. The only observed difference is that the workability of AZ61A has decreased from approx. 12% (press) and 14.4% (hammer) without heat treatment to 10% after annealing.2 each. These defects are related to the purity of the billet and thus have no effect on the examination results whatsoever. Another surface defect to be observed is a graphite residue from the lubricant used in the forging process. Due to the presence of these surface contaminants, it is difficult to cleanse the surface of the forged parts in the etching operation. As a result, the surface of the forged parts should be first cleansed by sandblasting and then etched.The forged parts were also subjected to macroscopic examination for internal defects. The examination was performed on unetched metallographic sections that were cut either perpendicular or longitudinal to the fibre direction, depending on the requirement. The location of the sections subjected to macrostructural examination is shown in Forging tests were also made for other types of forgings. Detailed results of these studies are presented in ,44,45.The experimental results demonstrate that the forged parts are of good quality and have no defects such as cracks, overlap and underfill. Their mechanical properties are within the ranges specified by the standards. The tested alloys should be preheated to 410 °C, which is a temperature at which they exhibit good workability in both die hammer and press forging. It is worth mentioning that the temperature in the real forging process is slightly lower due to the heat transfer occurring when the workpiece is removed from the furnace and exposed to contact with the colder dies. The parts forged with the use of both forging machines have recrystallized microstructure.It should be noted, however, that with increasing speed of deformation, the workability of the magnesium alloys decreases. Consequently, in terms of fracture, the hammer forging process is more difficult to conduct than the press forging process.It is technically feasible to obtain correctly shaped forged parts with required quality from the selected AZ magnesium alloys , using high-speed tool forging machines such as screw presses and die hammers. AZ31B alloy exhibits good workability when preheated to 410 °C and forged with the use of both machines. For the same temperature, AZ61A alloy exhibits satisfactory workability when the die forging process is conducted with a screw press; when subjected to hammer forging, the workability of this alloy is acceptable only in forging processes for simple-shape parts. What is more, to obtain correctly shaped forged parts, the tools must be preheated to 300 °C.Due to its low workability at high deformation speed, AZ80A alloy cannot be forged on screw presses and forging hammers, as this would lead to crack formation. AZ80A alloy parts should be forged in compliance with the guidelines presented in the specialist literature, i.e., with the use of slow hydraulic presses and tool preheating systems for ensuring near-isothermal conditions.The mechanical properties of the forged parts obtained with the forging hammer and screw press are similar and meet the requirements for the tested alloys. The application of stress relief annealing does not affect their mechanical properties to any significant extent. It only slightly increases the grain size.The results of microstructural examination have shown that the magnesium alloy specimens subjected to hammer forging are more fine-grained than those forged with the screw press. The grain in the water-cooled forged parts is slightly finer than in the air-cooled forgings.The fact that some magnesium alloys containing zinc and aluminium can be forged using screw presses and forging hammers is of great practical importance. The results demonstrate that die forging processes for AZ31B and AZ61A can be performed in forging plants equipped with standard forging machines, and that the use of expensive tool preheating systems is not necessary. Therefore, the proposed forging technique is more cost-effective than isothermal forging with hydraulic presses.The results of this study lead to the following conclusions:


Despite their differences, pleural and peritoneal mesothelioma are frequently lumped together to describe epidemic curves and to forecast future mesothelioma trends. This study aims to describe the malignant peritoneal mesothelioma (MPeM) epidemic in Italy (1996–2016) and to forecast future trends up to 2040 in order to contribute to the assessment of MPeM future burden. All MPeM deaths in Italy from 1996–2016 were collected ) in order to estimate MPeM mortality rates for each 3-year period from 1996 to 2016. Poisson age-period-cohort (APC) models were then used to forecast MPeM future trends. Between 2017 and 2040, 1333 MPeM deaths are expected. The number of MPeM deaths, as well as mortality rates, are expected to constantly decrease throughout the considered period. Based on considering the information from this study, it can be concluded that the MPeM epidemic has probably already reached its peak in Italy. Peritoneum is the second most frequent site of malignant mesothelioma following the pleura. Malignant peritoneal mesothelioma (MPeM) has some peculiar features differentiating it from the pleural form , such asAccording to the Italian National Mesothelioma Register (ReNaM), the incidence of MPeM in Italy in 2014 was 1.7 per 1,000,000 in men and 1.0 in women [Despite the clinical and epidemiological differences, pleural and peritoneal mesothelioma are often either lumped together to describe epidemic curves and to forecast overall mesothelioma trends, or MPeM cases are dropped and estimations are carried out based only on pleural mesothelioma data. Both approaches translate into a lack of information on MPeM epidemiology.The aim of this paper is to describe MPeM mortality in Italy within the period 1996–2016 and to forecast future trends of this disease up to 2040 in order to contribute to the estimation of MPeM future burden.Registrations of all MPeM deaths in Italy in 1996–2016 were collected from the Italian National Statistical Institute (ISTAT). A specific death code for MPeM C45.1) was not available in Italy until 2003, when the tenth revision of the International Classification of Diseases was implemented. Thus, identification of cases for the period 1996–2003 was based on the individual assessment of death certificates, taking advantage of a study on MPeM previously conducted in the Italian National Multiple Causes of Death database of ISTAT [.1 was nohttp://demo.istat.it/index_e.html).Actual (1996–2016) and predicted (2017–2040) population data, stratified by year, gender, and age, were downloaded from the website of the National Institute of Statistics  were observed in Italy, both in men and women) and diagnosis is less certain at older ages.We estimated the MPeM mortality rates of men and women for each three-year period from 1996 to 2016, and we used Poisson age-period-cohort (APC) models to forecast MPeM future trends. To reduce collinearity issues, we used the parametrization and the sequential approach proposed in Carstensen [We ran different models with a number of spline parameters varying from 2 to 5, and we selected the model with the minimum Akaike information criterion (AIC) . The conapc and Epi R package . In all the three-year periods considered, the number of MPeM deaths was always higher in males, with M:F ratios between 1.45 and 1.88 .Observed mortality rates among men were broadly constant . Observed mortality rates among women never attained as high of values as those found in males, with the highest value of 1.01 per 1,000,000 p-y in 1999–2001 and rates reducing thereafter .Mortality rates increased by age, reaching over 5 and 2 per 1,000,000 p-y after 60 years of age, respectively, in men and women. The highest mortality rates were observed within the age class of 75–77 years for men born between 1931 and 1935  and within the age class of 72–74 years for women born between 1925 and 1929  . According to predictions of the APC model, in the period 2017–2040, 1333 MPeM deaths are expected, with 885 (66.4%) deaths in men and 448 (33.6%) deaths in women . In bothOur study evaluated the evolution of the MPeM epidemic in Italy, providing both observed (1996–2016) and predicted mortality (2017–2040) figures.2A total of 1417 MPeM deaths were observed in Italy from 1996 to 2016, 992 of which occurred in the period 1996–2010. Considering that in the same period, 12,337 pleural mesotheliomas were observed [Over the considered period, the observed mortality rates ranged from 1.29 to 1.59 per 1,000,000 p-y and from 0.76 to 1.01 per 1,000,000 p-y for men and women, respectively. Considering that in Italy there is usually good agreement between incidence and mortality data for MPeM .Our results suggest that the number of MPeM cases is decreasing, with a peak of deaths already reached in 2014–2016 for men and in 1999–2001 for women. This outcome is different from predictions for pleural mesothelioma, whose peak has been predicted to occur sometime around 2020–2024 in Italy . A potenBased on the results of this study, it can be concluded that the MPeM epidemic in Italy has probably already reached its peak, both in terms of the number of deaths and mortality rates. Nonetheless, 1333 MPeM deaths are expected in the period up until 2040. These figures are likely related to the high occupational asbestos exposure levels during the 1960s and the 1970s.


Our results showed that Tan IIA inhibited lung metastases of CRC cells in vivo and extended the survival time of mice with CRC. In vitro, Tan IIA increased the expression of E-cadherin, decreased the expression of Snail, N-cadherin and Vimentin, thus suppressed EMT and the migratory ability of CRC cells. Further study found that the mechanism of action of Tan IIA in regulating EMT and metastasis is associated with the suppression of β-arrestin1 expression, resulting in the increase of GSK-3β expression, reduction of β-catenin nuclear localization, thereby decreased the activity of β-catenin signaling pathway. Our data revealed a new mechanism of Tan IIA on the suppression of EMT and metastasis in CRC via β-arrestin1-mediated β-catenin signaling pathway and provided support for using Tan IIA as anti-metastatic agents in CRC.Tanshinone IIA (Tan IIA) is a major active ingredient extracted from Salvia miltiorrhiza, which has been proved to be able to inhibit metastasis of various cancers including colorectal cancer (CRC). However, the mechanisms of anti-metastatic effect of Tan IIA on CRC are not well explored. A number of studies indicate that epithelial-to-mesenchymal transition (EMT) plays an important role in CRC metastasis, and our previous studies demonstrate that β-arrestin1could regulate EMT in CRC partly through β-catenin signaling pathway. In this work, we investigate whether Tan IIA could regulate EMT in CRC through β-arrestin1-mediated β-catenin signaling pathway both  Colorectal cancer (CRC) is the third most common cancer and the second cause of cancer-associated death in the world, consisting of over 1.7 million new cases and 861,663 deaths per year . CRC hasEpithelial-to-mesenchymal transition (EMT) plays a key role in CRC metastasis . Generalin vivo and in vitro. This study will provide a rational for application of Tan IIA for the treatment of CRC.Tanshinone is an ether or ethanol extract from the root of Salvia miltiorrhiza, and Tan IIA is its major active constituent. Several studies have showed the pharmacological effects of Tan IIA on cell apoptosis, metastasis, drug resistance, and angiogenesis in different cancers . Zhou et2 incubator. Rabbit monoclonal antibodies against human E-cadherin, N-cadherin, Vimentin, Snail, β-arrestin1, GSK-3β, β-catenin, c-Myc, and CyclinD1 were purchased from Cell Signaling Technology (United States). Rabbit monoclonal antibodies against human MMP2 and MMP9 were purchased from Santa Cruz Biotechnology (United States). Mouse monoclonal antibody against human GAPDH was purchased from Proteintech (China). Tanshinone IIA (S2365) with 99% purity (HPLC) was purchased from Selleck .Human colorectal cancer cell line HCT-116  was cultured in 1640 medium and LoVo  was cultured in F-12K medium, containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in a 5% CO4 cells/well were seeded into 96-well plates. When the cell density reached 60%, different concentrations of Tan IIA were added into the wells and incubated for 24, 48, and 72 h. Then the medium containing CCK-8 reagent was added into the cells and incubated for 4 h. The light absorption was measured at 450 nm with a microplate reader . All experiments were carried out in six wells, and each experiment was repeated at least three times.Cell Counting Kit-8 (CCK-8) was used to detect cell proliferation. Cells at 1 × 10In brief, all cells were lyzed with RIPA Lysis buffer, and the extracted protein was quantified by BCA protein assay . Approximately 50 μg of proteins were added into the 10% SDS-PAGE gels for electrophoresis and then transferred onto a polyvinylidene difluoride (PVDF) membrane. After that, PVDF was blocked with 5% BSA, and incubated with the primary antibodies followed by the HRP-conjugated secondary antibodies. The results were examined with enhanced chemiluminescence , and the target bands were analyzed using the Scion Imaging application (Scion Corporation).HCT-116 and LoVo cells were fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with Triton X-100 (0.5%) for 15 min, then blocked with 5% BSA solution for 1 h. The cells were incubated with primary antibodies for 1 h, followed by secondary antibodies for 1 h at room temperature. After incubation, nucleus was labeled with DAPI for 5 min. Finally, cells were examined with a DMI3000B inverted microscope .HCT-116 and LoVo cells were inoculated into the upper chamber of transwell plates and cultured in 600 μl 1640 or F12K medium with 10 μg/ml fibronectin. Medium containing 15% FBS was added in the lower chamber of the transwell plate. Then different concentrations of Tan IIA were added into the upper chamber and incubated for 48 h. Migrated cells were detected by crystal violet staining and observed using the DMI3000B inverted microscope . Five random views were selected to count the migrated cells.HCT-116 or LoVo cells were seeded into 6-well plates for 24 h, then an artificial scratch wound was created using a 20 μl pipette tip and detached cells were removed by washing with PBS three times. After 48 h incubation, cell migration was photographed using the inverted microscope and evaluated by measuring the difference in wound width.6 Luc-labeled HCT-116 cells through the tail vein. Two weeks later, the mice were randomly divided into five groups, eight in each group. The mice were treated with different concentrations of Tan IIA  for 4 weeks by tail vein injection. For bioluminescence imaging, mice were anesthetized with 1% pentobarbital sodium (200 μl/per), and D-luciferin (15 mg/ml) was injected intraperitoneally. The data were captured by using an IVIS Lumina system . After that, the lungs were excised, fixed in 4% paraformaldehyde, and paraffin-embedded. The paraffin-embedded lung tissues were cut into 5 μm sections. All the lung sections were stained with hematoxylin-eosin (HE). E-cadherin, Vimentin, Snail, and β-arrestin1 were detected with immunohistochemistry (IHC). The animal experiments were performed under the approval of the animal ethics committee of Shuguang Hospital, Shanghai University of Traditional Chinese Medicine.Male BALB/c nude mice (5 weeks old) were purchased from the Department of Experimental Animals of Shanghai University of Traditional Chinese Medicine  and maintained in the specific pathogen-free condition for 1 week. The mice were injected with 2 × 10p < 0.05 was considered as statistically significant.All the data were presented as means ± SD of at least three independent experiments and analyzed using SPSS22 Software. The mean values of two groups were compared by Student’s t test. in vivo, we established a CRC lung metastasis mouse model by tail vein injection of CRC cells. The mice were treated with different concentrations of Tan IIA. In vivo imaging results indicated that HCT-116/luc cells migrated to lung tissues after tail injection, and Tan IIA at concentrations of 0.5, 1, and 2 mg/kg  of low, medium and high dose of Tan IIA in tumor-bearing mice increased 8.73%, 19.09% and 34.63%, respectively compared to control group . Furthermore, the numbers of lung metastases in Tan IIA treated mice were significantly fewer than control group. The number of lung metastases was the least in the high-dose Tan IIA treated group . HE staining was used to observe lung metastases in nude mice, which was consistent with in vivo imaging results .Tan IIA is a major active ingredient extracted from Salvia miltiorrhiza, which has been proved to be able to inhibit metastasis of various cancers. In the present study, to assess the effect of Tan IIA on CRC metastasis  2 mg/kg  inhibite mg/kg . E-cadherin, N-cadherin, Vimentin, and Snail are the protein molecules responsible for EMT  is the physical barrier in the process of tumor metastasis. The change of composition or structure of ECM is closely associated with invasion and metastasis of tumor cells. It was found that, the expression of MMP-2 and MMP-9 decreased after cells were treated with Tan IIA . This result was in consistent with a previous analysis, which reported that abnormal expression of matrix metalloproteinases (MMPs) especially MMP-2 and MMP-9 accounts for degradation of ECM (The above results confirmed the effect of Tan IIA on EMT. We then investigated the role of Tan IIA on CRC metastasis l group . Similaor 48 h . ExtracTan IIA . This rn of ECM ..Inactivation of GSK-3β phosphorylation resulted in nuclear localization of β-catenin, which leads to the activation of GSK/β-catenin signaling pathway and promotes tumor metastasis . To stud.. Consistent with the previous results, lung metastatic tumor treated with Tan IIA had a higher expression of E-cadherin and lower expression of N-cadherin, Vimentin, and Snail compared with those from the tumor without Tan IIA treatment . Further detection of metastasis-related proteins found that the levels of MMP-2 and MMP-9 were reduced after Tan IIA treatment . The Wnt/β-catenin signaling activity was partly blocked by Tan IIA. The result showed that Tan IIA down-regulated the expression of β-arrestin1, β-catenin and the downstream gene of β-catenin, such as c-Myc and CyclinD1, while up-regulated the protein expression of GSK-3β in a concentration-dependent manner .The metastasis-related proteins in each group of lung metastases were investigated by immunohistochemistry and Western blot. Immunohistochemical staining showed that Tan IIA decreased the level of β-arrestin1, Vimentin, and Snail protein, while increased expression of E-cadherin protein in a concentration-dependent manner . Consiseatment . Furtheeatment . The Wn manner .in vitro. EMT-related factors Slug and ZEB1 have been found to play important roles as facilitators of brain metastasis (Tan IIA, which is from Salvia miltiorrhiza, has been proved to have coronary artery dilatation, antioxidant and anti-inflammatory effects . Recent tastasis . Therefotastasis . In addiin vivo and in vitro. The mechanism of β-arrestin1 regulating EMT and metastasis is partly through decreasing the expression of GSK-3β, leading to the accumulation of β-catenin in the cytoplasm and promoting β-catenin to get into the nucleus. In the present study, in vitro experimental results demonstrated that Tan IIA inhibited the expression of β-arrestin1 while promoted the expression of GSK-3β, thus blocking β-catenin entry into the nucleus. Then, the expression of c-Myc and CyclinD1 were reduced as a result of the inhibition of β-catenin. Moreover, in vivo experiments obtained a similar result of Tan IIA, demonstrated the effects of Tan IIA on EMT, β-catenin signaling pathway and its related proteins.A number of studies have found that Wnt/β-catenin is involved in EMT and tumor invasion and metastasis . When caIn summary, this study found that Tan IIA inhibits EMT and metastasis of CRC through repressing β-arrestin1-mediated β-catenin signaling pathway. The potential anti-metastatic mechanism is summarized in


Farnesoid X receptor , a critical regulator of bile acid homeostasis, is widely implicated in human tumorigenesis. However, the functional role of FXR in colorectal cancer (CRC) and the precise molecular mechanism remain unclear. In this study, we demonstrated that FXR expression was downregulated in colon cancer tissues and decreased expression of FXR predicted a poor prognosis. Knockdown of FXR promoted colon cancer cell growth and invasion in vitro, and facilitated xenograft tumor formation and distant metastasis in vivo, whereas ectopic expression of FXR had the reserved change. Mechanistic studies indicated that FXR exerted its tumor suppressor functions by antagonizing Wnt/β-catenin signaling. Furthermore, we identified an FXR/β-catenin interaction in colon cancer cells. The FXR/β-catenin interaction impaired β-catenin/TCF4 complex formation. In addition, our study suggested a reciprocal relationship between FXR and β-catenin, since loss of β-catenin increased the transcriptional activation of SHP by FXR. Altogether, these data indicated that FXR functions a tumor-suppressor role in CRC by antagonizing Wnt/β-catenin signaling. Globally, ~1,800,000 new cases are diagnosed as CRC every year. With the change in lifestyle, including high-fat diets (HFDs), tobacco use and less or lack of exercise, the incidence of CRC has increased rapidly in developing countries3. Although great progress has been achieved in multimodality therapy of CRC, the prognosis of late-stage CRC is still unsatisfactory due to distant metastasis and relapse5. The molecular pathogenesis of CRC is considered a multistep and consecutive process with the accumulation of various aberrant genetic and epigenetic variations7. Dissecting the precise mechanisms of colorectal tumorigenesis is crucial for developing better prognostic and therapeutic strategies.Colorectal cancer (CRC) ranks the second leading cause of cancer-related death8. Persistent activation of Wnt signaling featured by nuclear accumulation of β-catenin is an early event of colorectal tumorigenesis9. Wnt-related targets, including c-Myc, cyclin D1, MMP-7, and VEGF, are critical contributors to tumor cell proliferation, invasion, and migratory potential12. Epithelial-mesenchymal transition (EMT) is a biological process that involves the malignant transformation of epithelial cells with a loss of an epithelial and gain of a mesenchyme-like phenotype13. EMT plays a significant role in tumor progression via endowing tumor cells with the potential for invasive and metastatic growth. In invasive regions, tumor cells undergoing EMT exhibit a strong accumulation of nuclear β-catenin14. Recent study further demonstrates a direct link between β-catenin and EMT by identifying slug, a strong inducer of EMT, as the target of β-catenin15. These data strongly indicate that Wnt signaling participated in EMT process.Canonical Wnt/β-catenin signaling plays essential roles in embryonic development and maintaining gut homeostasis17, gastric cancer18, esophageal cancer20, and pancreatic cancer22. Exposure to elevated fecal bile acids is associated with the occurrence of colon cancer24. The farnesoid X receptor , a nuclear receptor of bile acids, is widely present in the gastrointestinal tract and liver26. In addition to its essential role in regulating bile acid homeostasis27, accumulating evidence supports a critical role of FXR in human tumorigenesis29. Reduced FXR at the mRNA level is found in colon polyps and is even more remarkable in CRC30. Restoration of FXR has been shown to suppress abnormal intestinal cell growth and curtail CRC progression31. However, the functional role of FXR in CRC and the precise molecular mechanism remain to be further elucidated.Bile acids are widely involved in the pathogenesis of human malignancies, including hepatocellular carcinoma (HCC)In this study, we aim to investigate the correlation between FXR and Wnt/β-catenin signaling during colorectal tumorigenesisOne hundred and twenty-three human colon cancer tissues were obtained from patient diagnosed with colon cancer and received surgery at the First Affiliated Hospital of Xi’an Jiaotong University from January 2011 to May 2013. No patient had received preoperative chemotherapy or radiotherapy. The procedure of this study was approved by the institutional review board of the First Affiliated Hospital of Xi’an Jiaotong University. Informed consent was obtained by all participants.2 at 37 °C.Human colon cancer cells HT-29, Caco-2, HCT116, RKO, SW480, and Lovo were maintained in DMEM medium  supplemented with 10% FBS  at 5% COThe phU6-EGFP-shRNA-FXR, β-catenin, and SHP lentiviral vectors and their control vectors were used to inhibit FXR, β-catenin, and SHP expression, while the pUbi-EGFP- FXR, β-catenin, and SHP lentiviral vectors and their control vectors were used to increase FXR, β-catenin, and SHP expression. All transfections were conducted in accordance with the manufacturer’s protocol. All the lentiviral vectors were constructed and prepared by GeneChem Co., Ltd. 32. For colony formation assay, three hundred cells were seeded and cultured for 14 days. Colonies (≥50 cells/colony) were counted. Cell cycle distributions were evaluated by flow cytometry as previously described32. Each experiment was performed in triplicate.CCK8 assays were performed as described previouslyCells were cultured in six-well plates until confluent. Then, three artificial vertical lines were created with pipette tips (10 µL) in each well. The wells were washed with phosphate-buffered saline (PBS) to remove cell debris. The cells were then cultured for an additional 48 h. The scratch lines were imaged under a microscope, and the scratch distances were measured. Each experiment was performed in triplicate.4 cells per upper chamber. The cells were incubated at 37 °C for 48 h. After incubation, non-migratory cells on the upper surface of the Transwell inserts were removed by washing with fresh PBS. The invading cells on the underside of the membrane were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The number of cells was counted in three randomly selected fields of fixed cells under an inverted microscope. Each experiment was performed in triplicate.Cell migration and invasion were measured by using Transwell plates  with or without Matrigel . In both assays, the lower chamber was filled with 600 μl of DMEM medium containing 20% FBS. The upper chamber filters were pre-coated with 50 µL of Matrigel and plated at 10 × 106 cells into the right flanks to establish xenograft tumor model. Tumor size was were monitored using callipers every 3 days, and the tumor volume was calculated according to the formula (length × width2 × 0.5). At the end of the experiment, the mice were killed and the xenograft tumors were isolated and weighted. Lung metastasis models were established via tail vein injections into each nude mouse. The weight of the nude mice was monitored every 3 days. At 60 days post-injection, the mice were killed by cervical dislocation, the lungs were excised, and the number of tumor nodules in the lung was recorded.All animal experiments were performed in accordance with the institutional guidelines, and were approved by the Laboratory Animal Center of Xi’an Jiaotong University. The 5-week-old female BALB/c-nude mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. . The mice were injected with 5 × 1032. The sequences of primers were summarized in Supplementary Table RNA isolation, complementary DNA (cDNA) synthesized, and real-time PCR were performed as described previously32. The extent of Ki67- and active caspase-3-stained cells was divided into 4 score ranks: 0–5% (0), 6–25% (1), 26–50% (2), 51–75% (3), and 76–100% (4). The staining intensity was divided into 4 score ranks: negative (0), light brown (1), brown (2), and dark brown (3). The immunoreactivity scores (IRSs) = extent score × intensity score. An IRS of ≤3 was defined as negative, and a score of >3 was defined as positive.The protocol was performed as previously describedNuclear extract was prepared with the protocol in the the Nuclear Extraction Kit . The nuclear protein was quantified and used for downstream applications.32. The antibody information was presented in Supplementary Table The detailed protocol was performed as described previouslyThe cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 10 min. After blocking with 5% bovine serum albumin (BSA) for 30 min at room temperature, the cells were incubated at 4 °C overnight with primary antibodies against E-cadherin and vimentin (1:100 dilution). The dishes were washed three times with PBS for 10 min each and then incubated with Alexa Fluor 594-conjugated secondary antibodies  for 1 h at room temperature. The nuclei were stained with DAPI (10 mg/ml) for 10 min. The samples were examined via microscopy  to analyze the expression of E-cadherin and vimentin.32. Each experiment was performed in triplicate.Fragments of the SHP 5′-flanking sequence were amplified by PCR using special primers Table  and clon33. Each experiment was performed in triplicate.The cells were washed three times with ice-cold PBS and harvested at 4 °C in immunoprecipitation lysis buffer. Co-immunoprecipitation (co-IP) assay was then performed as described previouslyCells were subjected to ChIP using the EZ-ChIP Kit 。32. Real-time PCR was conducted to amplify the regions of DNA fragments by using special primers . An Affymetrix PrimeView Human Gene Expression Array was used to investigate the changes in transcriptional profiles. The experiment was performed based on the manufacturer’s standard protocols. Genes with ≥1.5-fold change between two groups were identified as differentially expressed genes.t test, one-way ANOVA or χ2 test was conducted to compare the differences among the groups. Correlations were analyzed by using Pearson linear-regression analysis. OS and RFS rates were plotted using the Kaplan–Meier method and compared with log-rank test. Multivariate statistical analysis was performed using a Cox regression model. Statistical analyses were performed with SPSS 18.0 software . P < 0.05 was considered statistically significant.Each experiment was repeated three times. Data are presented as the mean ± SD. The Student’s To determine whether FXR correlates with CRC development and progression, we first employed immunohistochemistry (IHC) assay to detect the expression of FXR in 123 colon cancer tissues and paired normal tissues. FXR immunostaining was seen in the nuclei of colonic cells Fig. . The ratNext, western blotting analysis was conducted to evaluate the expression of FXR in eight colon cancer tissues and paired normal tissues Fig. . The resTo further gain insight into the impact of FXR in colorectal tumorigenesis, a series of in vitro experiments were performed in colon cancer cells with gain-of-function and loss-of-function of FXR. Knockdown of FXR in HT-29 and Caco-2 cells, and ectopic expression of FXR in SW480 and HCT116 cells were validated by western blotting analysis Fig. .Fig. 2FXCCK8 assays were employed to assess the effect of modulating FXR expression on the viability of colon cancer cells. Our data indicated that knockdown of FXR in HT-29 and Caco-2 cells resulted in an enhanced cell viability Fig. , whereasWe next conducted xenograft mouse model to assess in vivo tumor-suppressor role of FXR. The xenograft tumors in the FXR-knockdown group showed a decline in growth rate in relative to the control group . Hence, Wnt/β–catenin signaling was chosen for further research.We further explore the mechanism of FXR-mediated tumorigenicity and EMT inhibition by using microarray analyses of HT-29-shFXR cells and the control cells. Hierarchical clustering analysis showed that knockdown of FXR led to a difference in gene expression profile with the upregulation of 310 genes and the downregulation of 206 genes Fig. . PathwayTo further validate the impact of FXR on the activity of Wnt/β-catenin signaling in colon cancer cells, we first performed a TOP/FOP-Flash luciferase reporter assay. Knockdown of FXR in HT-29 and SW480 cells elevated the luciferase intensities compared with the control Fig. . MoreoveFurthermore, XAV-939, a selective inhibitor of Wnt signaling, was adopted to block activated Wnt signaling in FXR-knockdown cells. As expected, XAV-939 treatment attenuated the proliferative and invasive abilities inhibited by FXR knockdown . Since FXR is a mainly located in the nucleus, we hypothesized that the FXR might affect the β-catenin/TCF complex38. First, the co-IP assay showed that FXR did not bind with TCF4 in the nucleus of HEK293 cells , the well-known target gene of FXRsis Fig. . To furtsis Fig. . Westernsis Fig. . Converssis Fig.  as well sis Fig. . These f41. We then questioned whether modulation of β-catenin affected the FXR/RXRα complex. The co-IP assay showed that knockdown of β-catenin in SW480-FXR and HCT116-FXR cells resulted in a reduction in the FXR/β-catenin complex but a promotion in the FXR/RXRα complex  assay was employed to determine whether modulation of β-catenin affected the occupancy of FXR on the SHP promoter in vivo. Our data revealed that knockdown of β-catenin led to an enhancement of FXR binding to the SHP promoter in SW480-FXR and HCT116-FXR cells Fig. . ConversCorrelations among FXR and cell cycle- and EMT-related proteins were further explored in 30 colon cancer tissues by IHC assay  and NR inhibition of the Wnt/β-catenin/TCF cascade (theme II). Glucocorticoid receptor (GR) could inhibit tumor proliferation by repressing cyclin D1 expression via targeting of the β-catenin/TCF complex56 (theme II); and β-catenin acts as a coactivator of androgen receptor (AR) transcription and promotes cell proliferation and prostate pathogenesis57 (theme I). Our study revealed that a nontranscriptional mechanism of FXR that FXR forms a complex with β-catenin and subsequently disturbs the transcriptional activity of the β-catenin/TCF complex in colon cancer lines (theme II). FXR appears to regulate β-catenin activity without affecting β-catenin localization and protein stability, since modulation of FXR expression did not affect the translocation of β-catenin (data not shown). On the other hand, our study found that modulation of β-catenin affected the transcriptional activation of SHP by FXR, indicating that β-catenin negatively regulates FXR activity through direct binding (theme I). Based on these observations, we propose a model in which an event initiated in tumor cells activates Wnt signaling at the early phase of colorectal tumorigenesis, thus elevating the levels of nuclear β-catenin, forming β-catenin/FXR complex and subsequently impairing the tumor-suppressor effect of FXR. Furthermore, due to DNA hypermethylation30, miRNA regulation58, or transcription factor regulation59, loss of FXR enhances the β-catenin/FXR complex and leads to persistent activation of Wnt signaling to further promote tumorigenesis  has been highlighted in the field of cancer researchIn summary, we found that FXR is downregulated in colon cancer and is negatively associated with poor prognosis. Functional studies indicated that FXR exerts a tumor-suppressive function in CRC. Mechanistically, FXR suppresses the activity of Wnt/β-catenin signaling via interaction with β-catenin. Furthermore, the FXR/β-catenin interaction retards FXR-mediated transcriptional activation of its target gene SHP. These novel findings have identified a heretofore unrecognized relationship between FXR and β-catenin in tumorigenesis, thus providing a novel interventional opportunity.Supplementary Figures legendSupplementary TablesSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplementary Figure 7Supplementary Figure 8Supplementary Figure 9Supplementary Figure 10


Background and aims: Inhibition of Wnt/β-catenin signaling by specific inhibitors is currently being investigated as an antitumoral strategy for various cancers. The role of Wnt/β-catenin signaling in neuroendocrine tumors still needs to be further investigated. Methods: This study investigated the antitumor activity of the porcupine (PORCN) inhibitor WNT974 and the β-catenin inhibitor PRI-724 in human neuroendocrine tumor (NET) cell lines BON1, QGP-1, and NCI-H727 in vitro. NET cells were treated with WNT974, PRI-724, or small interfering ribonucleic acids against β-catenin, and subsequent analyses included cell viability assays, flow cytometric cell cycle analysis, caspase3/7 assays and Western blot analysis. Results: Treatment of NET cells with WNT974 significantly reduced NET cell viability in a dose- and time-dependent manner by inducing NET cell cycle arrest at the G1 and G2/M phases without inducing apoptosis. WNT974 primarily blocked Wnt/β-catenin signaling by the dose- and time-dependent downregulation of low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation and non-phosphorylated β-catenin and total β-catenin, as well as the genes targeting the latter (c-Myc and cyclinD1). Furthermore, the WNT974-induced reduction of NET cell viability occurred through the inhibition of GSK-3-dependent or independent signaling . Similarly, treatment of NET cells with the β-catenin inhibitor PRI-724 caused significant growth inhibition, while the knockdown of β-catenin expression by siRNA reduced NET tumor cell viability of BON1 cells but not of NCI-H727 cells. Conclusions: The PORCN inhibitor WNT974 possesses antitumor properties in NET cell lines by inhibiting Wnt and related signaling. In addition, the β-catenin inhibitor PRI-724 possesses antitumor properties in NET cell lines. Future studies are needed to determine the role of Wnt/β-catenin signaling in NET as a potential therapeutic target. Neuroendocrine tumors (NET) frequently occur in the gastroenteropancreatic system and lung ,2. The t+ T, CD4+ T and regulatory T cells  . All expNET cells were grown and treated with WNT974 (1–16 µM) for 72 h and then harvested through trypsinization, centrifugation and two washing steps in phosphate-buffered saline (PBS). After that, the cells were re-suspended in Nicoletti solution containing propidium iodide  and then analyzed with the BD Accuri C6 flow cytometer and quantified using BD Accuri C6 Analysis software  for cell cycle distribution. All experiments, consisting of technical triplicates, were repeated at least three times.To assess changes in tumor cell apoptosis, we utilized the Apo-ONE Homogeneous Caspase-3/7 Assay . In brief, NET cells were seeded into 96 well plates at 10,000 cells per well and grown overnight, treated with 1 µM and 16 µM of WNT974 for 72 h, and then subjected to the Apo-ONE Homogeneous Caspase-3/7 Assay  according to the manufacturer’s instructions. The experiments, consisting of technical duplicates, were repeated at least three times.NET cells were seeded into six-well plates containing cell culture inserts  at a density of 120,000–140,000 cells per chamber and grown for 24 h. Afterwards the cell culture inserts were removed, fresh medium with 1% FBS was added, and the cells were treated with WNT974 for 24 h. The wound gap (created with the cell culture inserts) was observed and photographed using a Zeiss Axiovert 135 TV microscope fitted with a Zeiss AxioCam MRm camera . The NET cell migration activity was calculated by measuring the relative gap at each time point with Image–J software . The experiments, consisting of technical duplicates, were repeated at least three times.NET cells were grown and then lysed in lysis buffer M-PER  containing the HALT protease and phosphatase inhibitor cocktail from Thermo Scientific . Cell protein was quantified, and for all Western blot experiments, samples were adjusted to the same protein concentration . For Western blotting, the protein samples were first separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene fluoride membranes . Equal protein loading was verified in all Western blots by total protein staining and normalization, and using the housekeeping protein β-actin. After blocking in Clear Milk Blocking Buffer  for 30 min, the membranes were incubated at 4 °C overnight with different primary antibodies. The used antibodies were against Axin-1, Dvl-2, pLRP6S1490, LRP6, SFRP, Wnt5a/b, cyclinD1, cyclinD3, cyclinB1, cdk1, cdk4, cdk6, Chk1, c-Myc, PCNA, vimentin, ZO-1 (zona occludens proteins), pEGFR Y1068, EGFR, non-pho-β-catenin S45, pho-β-catenin Ser33/37/Thr41, pho-β-catenin S552, total β-catenin, pIGFR Y1135, IGFR, pAkt S473, Akt, pmTOR S2448, pp70S6K T389, p70S6K, pERK1/2 T202/Y204, ERK1/2, pJNK T138/Y185, JNK, pGSK3 S21/9, and GSK3 ; Bcl-2, and p21 Waf1/Cip1, ; Menin and Neurotensin ; Wnt3a ; and β-actin, and p53 . The following day, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody  at a dilution of 1:25,000 for 2 h. The protein bands were visualized using a chemiluminescence Western blotting detection system . The chemiluminescence was detected by an imaging system  and quantification was performed using the gel macros of ImageJ .We obtained small interfering RNA (siRNA) pools containing four different siRNAs, each. The siRNA pools were as follows: β-catenin siRNA , GSK3β siRNA , or non-targeting control siRNA . To knockdown the expression of these genes, we grew NET cells overnight and then transfected them with 50 nM of each siRNA using DharmaFECT 2 or 3 transfection reagent  for 72 h. Thereafter, the cells were subjected to Western blot and other assays.t-tests using SPSS 16.0 software for Windows . A p value < 0.05 indicated statistical significance.Our data were summarized as the mean ± SD and statistically analyzed by one-way analysis of variance (ANOVA) or two-sample In pre-experiments, the population doubling time (PDT) was calculated as 0.895 ± 0.066 d for BON1, 1.536 ± 0.051 d for QGP-1, 1.781 ± 0.295 d for NCI-H727 cells and 15.48± 1.757 d for GOT1 cells respectively. Our results were in accordance with the short PDTs in BON1 and QGP-1 cells previously reported by Hofving et al , while tFollowing the pre-experiments, we first assessed the effect of WNT974 (1–32 μM) on the regulation of cell viability. As shown in Based on these observations, BON1 exhibited the most pronounced response to WNT974. Due to the long PDT of GOT1 cells, and, thus, their limited availability, all further experiments were performed using only BON1, QGP-1, and NCI-H727 cells.We next used FACS and Western blot to assess the effect of WNT974 treatment on the regulation of cell cycle distribution and apoptosis in order to better understand the WNT974-induced reduction of NET cell viability . Treatmep < 0.001).No induction of apoptosis was observed following incubation of BON1, QGP-1, and NCI-H727 cells with WNT974 at concentrations of 1–16 µM . FollowiIn summary, WNT974 induced NET cell cycle arrest at the G1/G2 phase of the cell cycle but did not induce apoptosis.Treatment of BON1 and QGP-1 cells with WNT974 at concentrations of 0.25–16 µM significantly downregulated the expression of LRP/pLRP6, DVL2, and Wnt5a/b and reduced the level of non-phosphorylated β-catenin, total β-catenin, and β-catenin phosphorylation at Ser33/Ser37/Thr41  and its Because the inhibitory effects of WNT974 on canonical Wnt/β-catenin signaling in NET cells were not unique in all three NET cell lines, we further assessed whether inhibition of non-canonical Wnt/receptor tyrosine kinase signaling, i.e., PI3K/-Akt/-mTOR, could mediate the effect of WNT974.Treatment of NET cells with WNT974 at concentrations of 0.25–16 µM for 72 h caused a significant reduction in the levels of pAKT and downstream p4EBP1, and p70S6K in BON1 and QGP-1 cells, respectively . FurtherAs the PORCN inhibitor WNT974 demonstrated inhibition of Wnt-signaling and antitumor activity in NET cells in vitro, we explored the effects of the β-catenin inhibitor PRI-724 as well as β-catenin siRNA on NET cells.PRI-724, a selective inhibitor of the CBP/β-catenin interaction ,40,41,42We transfected β-catenin siRNA to knock down β-catenin expression. The transfection of β-catenin siRNA decreased the protein expression of β-catenin in BON1 and NCI-H727 cells to 42 ± 16% and 49 ± 8%, compared to control transfection of non-targeting control (β-actin) siRNA A,B. β-CaWe studied the effects of GSK3β siRNA on the regulation of NET cell viability and protein expression. In the canonical Wnt signaling cascade, GSK3 phosphorylates β-catenin at S33/S37/T41 and subsequently causes proteasomal degradation of β-catenin. Inhibition of GSK3 activity can activate canonical Wnt/β-catenin signaling ,46,47.We transfected GSK3β siRNA to knock down GSK3β expression. The transfection of GSK3β siRNA significantly decreased the protein expression of GSK3β in BON1 and NCI-H727 cells to 10% ± 5% and 19% ± 4%, compared to control transfection of non-targeting control (β-actin) siRNA A,B. As eOn the other hand, phosphorylation of GSK3 is well known for inhibiting GSK3 enzyme activity ,49,50,51Treatment with WNT974 at concentrations of 0.25–16 µM significantly decreased protein expression of p53 and p21 in BON1 cells, and of p21 in QGP1 cells .Neurotensin (NT) is able to induce NET cell proliferation ,19,52,53Therefore, we investigated whether WNT974 can regulate the expression of either neurotensin (NT) or Menin. Western blot data showed expression of both NT and Menin to be modestly downregulated by WNT974 .To further understand WNT974 antitumor activity, we next determined the effect of WNT974 (8 and 16 µM) on regulation of the NET cell migration capacity, and found that WNT974 only modestly reduced the migration of BON1 and QGP-1 cells and had no effect on migration in NCI-H727 cells .At the molecular level, we analyzed the expression of the mesenchymal marker vimentin and the epithelial marker E-cadherin, as well as the tight junction protein ZO-1, and found that WNT974 treatment decreased the expression level of vimentin and of ZO-1, whereas E-cadherin expression underwent no significant change in these cell lines .The current study demonstrates that the PORCN inhibitor WNT974 reduced NET cell viability in a dose- and time-dependent manner  and induThe potential role of Wnt/β-catenin signaling in neuroendocrine tumors (NETs) has recently been reviewed ,16. MeniAnalysis of human NET tumor samples demonstrated mutations of the MEN1 gene in up to 35% of lung carcinoids ,22,23 anWe revealed that the expression of pLRP6, DVL2, and Wnt5a/b was downregulated by WNT974 in NET cells , and corCell cycle progression is tightly controlled by a complex of cell cycle regulatory molecules, such as cyclin-dependent kinases (CDKs), CDK inhibitors, and cyclins. Indeed, Wnt/β-catenin signaling controls cell proliferation and migration, and an increase in β-catenin expression or activity will initiate transcriptional activation of cyclinD1/D3, CDK1/2/4, and c-Myc proteins, which control the cell cycle transition from G1 to S phase ,57. In tInterestingly, incubation with WNT974 caused a decrease in the expression of p53 and p21 proteins . At firsInhibitory effects of WNT974 on the non-canonical Wnt/receptor tyrosine kinase signaling pathway  and on GWNT974 decreased the expression of pEGFR, pIGFR, pAKT, pERK, and pJNK in a GSK3β-independent and cell-line-dependent manner .GSK3 has been implicated in the pathogenesis of various diseases, including cancer . GSK3 caOn the other hand, GSK3 is known to play a pivotal role in regulating the canonical Wnt pathway . In the The β-catenin inhibitor PRI-724 inhibited NET cell viability in vitro  and causOur in vitro study has several limitations. A major limitation is the limited number of human neuroendocrine cell lines investigated, and the fact that the established human neuroendocrine tumor cell lines differ from neuroendocrine tumors in vivo with respect to tumor genetics, tumor biology, proliferation rate, and population doubling time ,45,55,68In conclusion, the PORCN inhibitor WNT974 and the β-catenin inhibitor PRI-724 exert antiproliferative activities in human NET tumor cell lines in vitro. WNT974 inhibits canonical Wnt/β-catenin signaling and exerts an inhibitory effect on the non-canonical Wnt/receptor tyrosine kinase signaling pathways, PI3K/AKT/mTOR, EGFR, and IGFR, as well as on GSK3 activity. All these mechanisms might be involved in the inhibition of NET tumor cell growth and, in future, the molecular mechanisms of WNT974 on NET tumor cells need further in-depth investigation. Targeting Wnt/β-catenin signaling by various approaches, such as through the use of PORCN inhibitors or direct β-catenin inhibitors, might be a promising molecular targeted therapeutic strategy in NETs. Potential subgroups of patients with neuroendocrine tumors who may actually be responsive to personalized molecular targeted therapy based on inhibitors of Wnt/β-catenin signaling  still ne