ICG-001 inhibited the growth of endometriotic lesions in the mouse model. CBP/β-catenin signaling plays an important role in the pathophysiology of endometriosis. Inhibiting the CBP/β-catenin signal can be a therapeutic target for endometriosis.Endometriosis exhibits unique characteristics, such as fibrosis, resistance to apoptosis, and promotion of cell proliferation; however, its pathophysiology is not fully understood. Recurrence rates after treatment are high, and the progression risk continues until menopause; hence, more effective therapy for endometriosis is needed. CREB-binding protein (CBP)/β-catenin signaling inhibitors have demonstrated antifibrogenetic effects in liver, lung, and skin diseases. The present study evaluated the effects of two CBP/β-catenin signaling inhibitors, ICG-001 and C-82, on the progression of endometriosis using endometriotic cyst stromal cells from the ovary and normal endometrial stromal cells from the uterus. ICG-001 was also evaluated in a mouse model. ICG-001 and C-82 inhibited cell proliferation, fibrogenesis, and cell migration, and promoted apoptosis  Pathologically, endometriosis is diagnosed by the presence of endometrial glands, endometrial stroma, and hemosiderin laden macrophages outside the uterus, mainly in the ovaries. Although the pathophysiology of endometriosis is not fully understood, it is characterized by fibrosis (scarring), resistance to apoptosis, and promotion of cell proliferation2. Pain from endometriosis can be treated by excising the peritoneal lesions and ovarian cysts, or using hormonal agents such as progestin, oral contraceptives, and gonadotropin-releasing hormone agonists. However, these treatment strategies are associated with high recurrence rates1; hence, more effective therapy is needed.Endometriosis is a benign estrogen-dependent disease in which endometrial tissue develops outside the uterus, such as in the ovaries, peritoneum, and rectovaginal space. Most patients with endometriosis are of reproductive age. The main symptoms of endometriosis are dysmenorrhea, chronic pelvic pain, subfertility, and dyspareunia, which often decrease quality of life significantly3. We hypothesized that targeting fibrosis can be a new treatment strategy for endometriosis. Studies on fibrosis in endometriosis have reported a possible correlation between aberrant activation of the Wnt/β-catenin signaling pathway and progression of endometriosis5. The Wnt/β-catenin signaling pathway activates fibrosis and is associated with fibrotic diseases in lung, skin, kidney, and liver8. While drugs with sufficient antifibrotic activity have not yet been identified, PRI-724 , a CBP/β-catenin-specific antagonist, is a promising antifibrotic drug against liver cirrhosis whose safety, tolerability, and antifibrotic effect were assessed in several patients with hepatitis C virus (HCV) cirrhosis10. An active metabolite of PRI-724 called C-82 was discovered from the early lead compound ICG-001, which binds to CBP and inhibits CBP/β-catenin binding11. ICG-001 was reported as an effective treatment for bleomycin-induced lung fibrosis in mice12.There are some reports about the relationship between fibrosis and the progression of endometriosis15. Therefore, in the present study, we evaluated ICG-001 and C-82 using ECSC and NESC in in vitro functional assays, and ICG-001 in in vivo experiments using an established mouse model of endometriosis.Our study group has already validated a cell culture experimental assay with endometriotic cyst stromal cells (ECSC) from ovarian endometriomas (chocolate cysts) and normal endometrial stromal cells (NESC) from the uterus for chemical screening studiesβ-catenin expression in endometriotic lesions from patients with ovarian endometriosis (n = 5) as well as uterine endometrium from patients without endometriosis (n = 5) were evaluated by immunohistochemistry Fig. . Figure We next examined cell proliferation and apoptosis in ECSC after treatment with CBP/β-catenin signaling inhibitors. ICG-001 significantly inhibited cell viability by 20.8% and 52.6% compared with the control at concentrations of 20 and 200 µM, respectively, in the methylthiazoletetrazolium (MTT) assay  mRNA was significantly higher in ECSC than in NESC , thereby disrupting the CBP/catenin interaction23. PRI-724 is a second generation specific CBP/catenin antagonist24.The Wnt/β-catenin signaling pathway is involved in normal reproduction and development, as well as maintenance of normal physiological functions. The Wnt/β-catenin signaling pathway is further involved in progression of diseases by regulating cell proliferation, migration, invasion, and fibrogenesis15. The present study revealed significant upregulation of β-catenin protein expression in ECSC, suggesting activation of Wnt/β-catenin signaling pathway in the pathophysiology of endometriosis. ICG-001 and C-82 inhibited in vitro cell growth, cell migration, and fibrosis, and promoted apoptosis. The mRNA expression of α-SMA with ICG-001 and C-82 was significantly downregulated in ECSC, but there was no difference in protein expression  appeared to be nontoxic to mice, but effectively modified disease progression in the murine model of endometriosis where ICG-001 dose-dependently inhibited intra-abdomen fibrosis as evaluated. However, the number of endometriotic lesions, intensity of the modified Masson’s staining, and the staining intensity of α-SMA were the weakest in the ICG-001 100 mg/kg group. Thus, further study is needed. Data generated from this study provide additional evidence to support the inhibition of the Wnt/β-catenin signaling pathway being an attractive therapeutic target to improve fibrosis and to reverse endometriotic lesions.et al. demonstrated that fibrosis, cell proliferation, and cell migration were inhibited in ECSC26 after blocking the Wnt/β-catenin pathway. The antifibrotic effect of CGP049040, a small molecule TCF/β-catenin antagonist, on fibrosis was shown in their mouse model of endometriosis. The staining scores by Sirius red and Masson trichrome stains were significantly lower in CGP049040 treated mice. Their data support our view that activation of the Wnt/β-catenin signaling pathway in endometriosis is associated with fibrosis. Although Matsuzaki et al. showed a reduction of collagen fiber by a small molecule antagonist for the TCF/β-catenin complex, they did not provide data on the reduction in size or the number of endometriotic lesions, whereas our study clearly demonstrated a reduction of endometriotic lesions in size and number with CBP/β-catenin inhibitors. CBP/β-catenin interaction is crucial in the pathological mechanism of endometriosis progression.Prior to the present study, the relationship between CBP/β-catenin binding and endometriosis had not been fully examined, although there are some reports illustrating that the Wnt/β-catenin pathway is activated in endometriosis. Matsuzaki 8. A high dose of ICG-001 was given intraperitoneally, and this dose may not be comparable to the clinical dose. In this study, the pharmacokinetics of ICG-001 were not evaluated; therefore, it is necessary to investigate the relationship between exposure and pharmacodynamics. The present endometriosis model was created by transplanting the uterine tissue of a normal mouse into the abdominal cavity of another mouse. It may not accurately reflect the condition of human endometriosis and its translationability is unknown. The menstrual cycle was different from what naturally occurs in the model mice because estradiol was continuously administered. Although this was translational research at the stage of confirming safety, there is a history of mice models being widely used for endometriosis studies in general, and it has been validated as a model that shows the dynamics of endometriosis.There are some limitations in the current study. Although no apparent drug-related toxicity was observed from the intraperitoneal findings or body weight, the effects on other organs have not been confirmed. There is no denying that this drug may affect the functions or morphology of other organs. PRI-724 has been administrated to humans, and a clinical trial of PRI-724 (C-82) revealed that most of the observed side effects to humans were mild, such as reaction at the injection site, nausea, and constipationIn conclusion, we have identified the CBP/β-catenin signaling inhibitors, ICG-001 and C-82, as leads to promising therapeutic agents for the treatment of endometriosis. The CBP/β-catenin inhibitors suppressed cell migration and cell proliferation and promoted apoptosis. Hence, our data suggest that CBP/β-catenin inhibitors have a possibility not only to prevent but reduce fibrosis in endometriosis. Future studies will need to focus on searching and synthesizing more effective CBP/β-catenin inhibitors. The promotion of research and practical application of new drugs for endometriosis is important, and a clinical trial of CBP/β-catenin inhibitors for endometriosis should be planned for the near future.29. In addition, we performed basic experiments using western blotting. CD10 and vimentin were positive, and we confirmed the cells were derived from endometrial stroma. Isolated cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 100 IU/mL penicillin , 50 mg/mL streptomycin , and 10% heat-inactivated fetal bovine serum  at 37 °C in air containing with 5% CO2. ECSC and NESC were confirmed to have >99% purity by immunocytochemical staining with vimentin , CD10 , cytokeratin , and factor VIII  after two to three passages. Cells from two to five passages were used. ICG-001 was synthesized in our laboratory and C-82 was provided by Prism Pharma Co., Ltd., Tokyo, Japan . Normal endometrium tissues were obtained from patients who had undergone hysterectomy because of myoma or other benign diseases without endometriosis . None of the patients had received hormonal treatments for at least 6 months before surgery. All specimens were confirmed to be in the proliferative phases based on pathological observation, the menstrual cycle, or both. ECSC and NESC were isolated from the ovarian endometriotic tissues and normal uterine tissues using enzymatic digestion with collagenase. They were then confirmed to be stromal cells from the endometrium as previously describedpan Fig. . We haveThis study was approved by the Institutional Review Board of the Faculty of Medicine, Oita University (no. P16–01), and written informed consent was obtained from all patients. All methods involving patients were performed in accordance with relevant guidelines and regulations.Immunohistochemical staining (using the streptavidin–biotin–peroxidase method) was performed with mouse monoclonal antibody against human β-catenin , and biotinylated goat anti-rabbit IgG . The staining intensities and sites of β-catenin in the endometriotic cysts of the ovaries compared with normal endometrial tissue were assessed.The protein levels of β-catenin and α-SMA in ECSC and NESC were evaluated by western blot analysis. Antibodies against β-catenin , α-SMA , and GAPDH  were used. Total protein from ECSC cells that were treated with ICG-001 (20 µM) or C-82 (2 µM) for 48 hours was extracted. The expression of each protein relative to the GAPDH protein in ECSC and NESC was analyzed using Image Lab software . The data were presented as the percentage of the values obtained for ECSC compared with NESC.et al.2 reported previously. Briefly, 5 × 103 ECSC were seeded into each well of a 96-well flat-bottom microplate . After 24 hours, the culture medium was replaced with medium containing ICG-001  or C-82 , and the cells were incubated for 24 hours. After that, 20 µL of the MTT reagent was added to each well, the cells were incubated for 4 hours, then 100 µL of detergent reagent was added to each well. Cell viability was determined by measuring the absorbance at 570 nm. The values obtained from the ICG-001 or C-82 treated ECSC are presented as a percentage of the values from untreated ECSC.The viability of ECSC after ICG-001 or C-82 treatment was determined by a modified MTT assay using Cell Proliferation Kit I  as Hirakawa et al.2 reported. ECSC (5 × 103) were seeded into each well of a 96-well flat-bottom microplate . After 24 hours, the culture medium was replaced with medium containing ICG-001  or C-82 , and the cells were incubated for 24 hours. Then, 10 µL BrdU (10 mM) was added to each well, followed by incubation for 2 hours. The incorporation of BrdU was determined by measuring the absorbance of the resultant solution at 450 nm. The values of the ICG-001 or C-82 treated ECSC are presented as a percentage of the values of untreated ECSC.The proliferation of ECSC following treatment with ICG-001 or C-82 was determined based on BrdU incorporation using an ELISA kit  as Hirakawa et al.2 described. ECSC (5 × 103 cells/well) were seeded in a 96-well flat-bottom microplate . After 24 hours, the culture medium was replaced with medium containing ICG-001  or C-82 , and the cells were incubated for 24 hours. Then, the cells were lysed and centrifuged at 200 × g for 10 minutes, and the mono- and oligo-nucleosomes in the supernatants were quantified using an antihistone–biotin antibody . The concentration of the nucleosome–antibody complex was determined by measuring the absorbance at 405 nm using 2,2′-azino-di(3-ethylbenzthiazolinesulfonate) as the substrate. The values of ECSC treated with ICG-001 or C-82 are presented as a percentage of the values from untreated ECSC.We determined the apoptosis levels of ECSC following ICG-001 or C-82 treatment by direct determination of nucleosomal DNA fragmentation using ELISA  as Hirakawa et al.2 reported. ECSC (5 × 103 cells/well) were seeded in a 96-well flat-bottom microplate. After 24 hours, the culture medium was replaced with medium containing ICG-001  or C-82 , and the cells were incubated for 24 hours. The supernatant of the medium and the Caspase-Glo 3/7 reagent were mixed in another 96-well flat-bottom microplate, the plate was shaken gently at 20–25 °C for 180 minutes, then the luminescence was measured using a plate-reading luminometer . The values of ECSC treated with ICG-001 or C-82 are presented as a percentage of the values of untreated ECSC.The caspase-3 and caspase-7 activity of ECSC following incubation with ICG-001 or C-82 was evaluated using the Caspase-Glo 3/7 assay  as Hirakawa The scratch assay was used to assess cell migration and proliferation. ECSC were incubated in 12-well culture plates. When cells were 80% confluent, each plate was scratched with the same straight line. The medium was replaced with medium containing ICG-001 (20 µM) or C-82 (2 µM). The concentrations were determined from the results of the cell proliferation assay and the assay of the anti-apoptotic effect. Photos were taken on a microscope and the gap distances were quantitatively evaluated using BZ9000  at 0 and 24 hours after the scratch. Narrow distance between scratch edges means great migration ability and strong cell growth. The ratio before and after migration was calculated. A migration ratio of 100% indicates the highest migration ability.29. In this system, ECSC are cultured in floating collagen lattices to induce the reorganization and compaction of collagen fibers, resulting in the contraction of collagen gels. This culture system provides models of mechanically relaxed tissue with low tensile strength and the early developmental stage of endometriotic lesions with high tensile strength. Collagen gel contraction assays were performed as previously described29. Forty-eight hours after ICG-001 (20 µM) or C-82 (2 µM) treatment, the ECSC were embedded in the collagen gel  and cultured three-dimensionally for a further 72 hours. ICG-001 (20 µM) or C-82 (2 µM) was also added to the culture medium. The collagen gels were then photographed and the area of the gel surface was measured with ChemiDoc XRS+ with Image Lab Software .We have established a three-dimensional collagen gel culture system with ECSC as a model of fibrosis formation in endometriosiset al.2 reported. The expression level of mRNA relative to GAPDH mRNA was calculated by the relative standard curve method. Standard curves for relative quantification were prepared. Quantity is expressed relative to an untreated control. The data from ECSC were presented as the percentage of the values compared with NESC.Total RNA of ECSC and NESC was extracted using a miRNeasy Mini kit  following the manufacturer’s instructions. In addition, total RNA from ECSC that were treated with ICG-001 (20 µM) or C-82 (2 µM) for 48 hours was extracted similarly to analyze the expression of α-SMA. Subsequently, cDNA was synthesized from 1 μg of total RNA using the Reverse Transcription System . Quantitative RT-PCR was carried out with a LightCycler 480  using TaqMan Universal PCR Master Mix II with the following specific primers : β-catenin (Assay ID: Hs00355045_m1); α-SMA (Assay ID: Hs04406862_m1); glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Assay ID: Hs02786624_g1) as Hirakawa 23.Before surgery, 40 8-week-old adult female mice  were habituated for 2 weeks at a controlled temperature (22–23 °C) on a 12-hour light, 12-hour dark cycle, and given food and water freely. The mice were divided into four groups: control group (ICG-001 0 mg/kg), ICG-001 10 mg/kg group, ICG-001 50 mg/kg group, and ICG-001 100 mg/kg group. The dosages were taken from previous studies31. Two weeks after the ovariectomy, donor mice were euthanized, the uterus was removed, minced, and injected peritoneally with Corning Matrigel into the recipient mice. The uterus from each donor mouse was divided into two parts and implanted into two recipient mice. One week after uterine implantation, the sites and numbers of endometriotic lesions in the abdomen were examined. After confirming implantation of endometriosis, we started drug administration. Intraperitoneal injections  to the right side of the abdomen were administered three times a week for 1 week after the implantation. Six weeks after ICG-001 administration, mice were euthanized and dissected to examine the abdomen. The sites of implantation and the numbers and weights of endometriotic lesions were recorded. An experimental assistant who was blinded to this experiment measured each resected specimen with an electronic analytical scale (SHIMADZU AG 204). Resected endometriotic lesions and uteruses were embedded in paraffin and stained by HE and anti-α-SMA antibody . The histological tissue of the endometriotic lesions was also observed with modified Masson’s staining  and Sirius red staining  to evaluate fibrosis. To assess the extent of fibrosis, we analyzed the stained areas of collagen fiber by modified Masson’s staining using BZ9000 . The analysis area was defined by the detection of both endometrial epithelial cells and endometrial stromal cells under high-power magnification.In addition, three mice were examined for endometriotic lesions 1 week after implantation, and an additional 44 mice were prepared as uterus donors. Mice were anesthetized with 2% isoflurane and, after bilateral dorsal incision, the bilateral ovaries were resected. The incision was sutured with 4–0 nylon. Estradiol  was injected intraperitoneally into the left side of the abdomen once a week after the ovariectomyt-test.The data were analyzed and presented as percentages relative to the corresponding control values as mean ± standard deviation. All statistical analyses were performed using the Excel statistical software package . Significance was set at p < 0.05 with the Bonferroni correction and Student’s In Vivo Experiments) guidelines.This study was approved by the Institutional Review Board of the Faculty of Medicine, Oita University (No. P16-01), and written informed consent was obtained from all patients. All methods involving humans were performed in accordance with relevant guidelines and regulations. The mouse model study was approved by Oita University Animal Ethics Committee (No. 172901). We followed the ARRIVE (Animal Research: Reporting of Supporting informationSupporting information 2Supporting information 3Supporting information 4Supporting information 5Supporting information 6


Toughening an epoxy resin by bio-based modifiers without trade-offs in its modulus, mechanical strength, and other properties is still a big challenge. This paper presents an approach to modify epoxy resin with tannic acid (TA) as a bio-based feedstock. Carboxylic acid-modified tannic acid (TA–COOH) was first prepared through a simple esterification between TA and methylhexahydrophthalic anhydride, and then used as a modifier for the epoxy/anhydride curing system. Owing to the chemical modification, TA–COOH could easily disperse in epoxy resin and showed adequate interface interaction between TA–COOH and epoxy matrix, in avoid of phase separation. The use of TA–COOH in different proportions as modifier of epoxy/anhydride thermosets was studied. The results showed that TA–COOH could significantly improve the toughness with a great increase in impact strength under a low loading amount. Moreover, the addition of TA–COOH also simultaneously improved the tensile strength, elongation at break and glass transition temperature. The toughening and reinforcing mechanism was studied by scanning electron microscopy (SEM), dynamic mechanical analysis (DMA) and thermal mechanical analysis (TMA), which should be owned to the synergistic effect of good interface interaction, aromatic structure, decreasing of cross linking density and increasing of free volume. This approach allows us to utilize the renewable tannic acid as an effective modifier for epoxy resin with good mechanical and thermal properties. Because of their excellent performance properties, good processability and low cost, epoxy resins are used as one of the most versatile thermosetting polymers with a wide range of applications including coatings, adhesives, structural composites and electronic materials ,2,3. HowTg) of the resin was reduced to a certain extent [In recent years, the fast depletion of petroleum reserve and increasing environmental problems have led to a growing interest in the use of bio-based sustainable feedstock in the synthesis of bio-based chemicals and products. In this regard, some researches had focused on the synthesis and utilization of renewable material as efficient epoxy modifier ,14,15,16n extent . SatheesTannic acid (TA) is water-soluble high molecular weight polyphenolic compounds, mostly extracted from plants and microorganisms. It has a macromolecular structure composed of gallic units and abundant terminal phenolic hydroxyl groups. Owing to such a structure, TA shows remarkable properties and is widely used in many application, such as coatings, adsorption and antibacterial materials, mucoadhesive compounds, separator for lithium-ion batteries, and nanomaterials ,19,20,21Tg and modulus of cured epoxy thermoset. In this context, the use of tannic acid as an epoxy modifier seems to be an interesting proposition because its architectural structure is similar to the hyperbranched aromatic polyester with abundant terminal phenolic hydroxyl groups. Furthermore, the utilization of bio-based tannic acid will contribute to the long-term sustainability including environmental and health safety issues [It has been proved that a highly branched structure can introduce more internal cavities or free volumes in cured thermosets, which is favor of improving toughness ,23,24. Iy issues .Therefore, the motivation for this work is to utilize TA as a bio-based modifier for epoxy resins with simultaneous improvement in toughness and other properties. We had tried to add TA into epoxy formula without any modification. However, because of intermolecular hydrogen bonds, Van der Waals interactions and π–π stacking of aromatic groups, TA is immiscible with epoxy resin and tends to precipitation during curing. Therefore, a certain extent of chemical modification of TA is necessary to improve the miscibility of TA in epoxy matrix and enhance the interface interaction between TA and epoxy matrix.Tg and strength. In addition, other thermal properties and fracture surfaces were also studied.In the study reported herein, a carboxylic acid-modified tannic acid (TA–COOH) was prepared through the simple esterification between TA and a commercial epoxy hardener methylhexahydrophthalic anhydride (MeHHPA). In addition, TA–COOH was added into the epoxy formula and used as an all-purpose epoxy modifier. It was found that TA–COOH could significantly improve the toughness of cured thermosets with an increase in impact strength under low loading amount, and simultaneously improve the elongation at break, TA  was added to the solution. The mixture was stirred at 80 °C for 36 h. After reaction, the mixture was precipitated into diethyl ether to remove pyridine and the unreacted MeHHPA. The precipitate was dissolved in THF and precipitated into diethyl ether twice, and then dried under vacuum at 30 °C to give a brown solid product.TA  was dissolved in 30 mL of pyridine in a round-bottomed flask with a magnetic stirrer and a gas inlet to fill the flask with NThe TA–COOH/epoxy thermosets were prepared by the following procedure. A required amount of TA–COOH was dispersed in epoxy resin (E-51) under mechanical stirring at 60 °C. Then, the curing agent (MeHHPA) and catalyst (ethyl triphenyl phosphonium bromide) were added to the above mixture . Finally, the mixture was degassed and poured into mold. The samples for thermal and mechanical characterization were cured using the following profile: 80 °C for 1.5 h, 100 °C for 1 h, 120 °C for 1 h, and 140 °C for 2 h. These thermosets with 0.5, 1.0 and 2.0 wt % of TA–COOH were coded as TA–COOH0.5, TA–COOH1.0 and TA–COOH2.0, respectively. Neat epoxy was prepared following the same procedure as mentioned above.13C-NMR spectra of TA–COOH were recorded on a Bruker AV400M nuclear magnetic resonance spectrometer . In order to calculate the degree of modification, an inverse gated decoupling technique with the time of 4 h was used. Fourier transform infrared (FTIR) spectra were obtained using a Thermo Fisher Scientific Nicolet iS50 spectrometer  at room temperature in the wavenumber range of 600–4000 cm−1. TGA experiment was carried out on a METTLER TOLEDO TGA/1  in N2 atmosphere with a heating rate of 10 °C min−1. DSC was recorded on a NETZSCH 204 F1 thermal analyzer . The samples of ~5 mg in weight were placed in aluminum pans under nitrogen atmosphere. Dynamic mechanical analysis (DMA) was conducted on a TA Instruments DMA Q800  at a heating rate of 3 °C min−1 and a frequency of 1 Hz under an air atmosphere. The linear coefficients of thermal expansion were measured using a METTLER TOLEDO TMA/SDTA841e thermal mechanical analyzer . The tensile strengths of the cured hybrids were characterized by an Instron 1185 test machine  according to ISO 527:1993. Un-notched impact strength tests were performed on a Ceast Resil impact tester  according to ISO 179:1982. For each composition, at least 5 samples were measured. The fracture surfaces from fracture toughness tests were investigated by a HITACHI S-4800 field-emission scanning electron microscope . Rheological measurements were performed on a discovery DHR-2 hybrid rheometer  equipped with cone and plate geometry . All the experiments were performed at 25 °C.The 76H52O46. Its chemical structure was given in −1 appears (as arrow has pointed out). The carbonylic region was also given in the inset. As can be seen, compared to the IR spectrum of TA, a new absorption peak at 1730 cm−1 occurrs, which could be assigned to the stretching vibration of C=O in the ester groups generated by the ring-opening of anhydride. Moreover, the bands at 2804 cm−1 and 2959 cm−1 are due to the aliphatic C–H vibrations of MeHHPA moiety in the TA–COOH. The commercial TA is given as C13C-NMR spectroscopy. 13C -NMR spectrum of TA–COOH with the corresponding assignments. It can be seen, after modification, TA–COOH shows a resonance at 176 ppm, which can be assigned to the –COOH groups generated by anhydride. The resonances at 23, 29, 33, 41, 119 and 133 ppm are assigned to the aliphatic C from MeHHPA moiety. The resonances at 109, 140, 146, 151 and 155 ppm are assigned to the C in benzene ring from TA. The structure of TA–COOH was further confirmed by Because of steric hindrance affect, not all the 25 hydroxyl groups of TA are equally active. Thus, the degree of modification of MeHHPA was calculated from the integration of the aliphatic and aromatic signals. After calculation, it is found that about 15 hydroxyl groups of TA reacted with MeHHPA. Although the modification degree is not as high as expected, TA–COOH still shows good solubility in epoxy resin by stirring at 60 °C for 2 h. In addition, the terminal carboxyl groups offer a good interface interaction between TA–COOH and epoxy matrix.Generally, when epoxy resin is toughened by a linear liquid rubber, its viscosity increases to a great extent. However, for hyperbranched toughener, it usually has a low melt viscosity because of its globular structure. Such a low melt viscosity is good for processing, which shows a very little increase in the viscosity than liquid rubber. The rheological studies of the neat epoxy and epoxy resin with different TA–COOH loading were performed. All of the samples have Newtonian behaviors and show similar rheological behavior in the range of frequencies studied . As expeAs an aim to maintain a certain interface interaction between the TA–COOH and the epoxy matrix after curing, we selected anhydride as curing agent. The use of anhydride as curing agents has been extensively reported ,30. HereEa) and pre-exponential factor (A) was calculated and the results were shown in The curing kinetics were studied by the non-isothermal integral isoconversional procedure. The activation energy (E’) and loss tangent (tan δ) as a function of temperature for the cured neat epoxy and the thermosets containing 0.5, 1.0 and 2.0 wt % TA–COOH are shown in Er). Following classical rubber elasticity, Er is proportional to the average crosslinking density. The crosslinking density (ρ) of a cured epoxy network can be calculated using the equation:The dynamic mechanical behaviors of neat epoxy and the TA–COOH modified epoxy resins were measured. The storage moduli (−3), Er is rubbery modulus (MPa), R is the gas constant, and T is the absolute temperature. Theoretically, adding of TA–COOH may enhance the crosslinking density due to its higher functionality. However, the thermosets with different TA–COOH loadings show lower crosslinking density than neat epoxy. This presumably can be explained as following. Although the carboxylic content of TA–COOH is relative low, which has been confirmed by 13C-NMR .Where ρ represents the crosslinking density per unit volume  and the rubber region (αr) as well as their difference (Δα = αr − αg) are listed in g in cured hybrids could lower the internal stress when processing and is very beneficial for the composite matrix, because it helps to maintain better interfacial strength during temperature cycles and shocks. On the other hand, as shown in g decreases, and αr increases. Thus, the Δα increases. Based on the free volume theory [T (fT) can be expressed as:The values of the coefficient of thermal expansion (CTE) measured in the glassy region  of the thermosets with different TA–COOH loadings are slightly lower than that of neat epoxy  had been synthesized through a simple esterification between TA and MeHHPA. Then TA–COOH was used as a modifier for anhydride cured epoxy system. Owing to the chemical modification, TA–COOH could easily disperse in epoxy resin and showed a good interface interaction between TA–COOH and epoxy matrix. When TA–COOH was used as modifier, it can simultaneously improve toughness, elongation at break,


Rhodococcus rhodochrous and Ochrobactrum anthropi. As epoxy was the only carbon source available for these bacteria, their measured growth rate reflected their ability to degrade epoxy resin. Bacterial growth took place only when the two bacteria were grown together, indicating a synergistic effect. The surface morphology of the epoxy droplets changed significantly due to the biodegradation process. The metabolic pathway of epoxy by these two microbes was investigated by liquid chromatography mass spectrometry. Bisphenol A, 3,3′-)bis(oxy))bis and some other constituents were identified as being consumed by the bacteria.Epoxy resins have a wide range of applications, including in corrosion protection of metals, electronics, structural adhesives, and composites. The consumption of epoxy resins is predicted to keep growing in the coming years. Unfortunately, thermoset resins cannot be recycled, and are typically not biodegradable. Hence, they pose environmental pollution risk. Here, we report degradation of epoxy resin by two bacteria that are capable of using epoxy resin as a sole carbon source. These bacteria were isolated from soil samples collected from areas around an epoxy and polyurethanes manufacturing plant. Using an array of molecular, biochemical, analytical, and microscopic techniques, they were identified as  Epoxy resins contain epoxide groups, and may be reacted (cross-linked) with a wide range of co-reactants , such as amines and phenols. This cross-linking reaction is commonly referred to as curing, and it forms thermosetting polymers. Epoxies are known for their excellent adhesion, chemical and heat resistance, good-to-excellent mechanical properties, and very good electrical insulating properties. They have a wide range of applications, including paints and coatings , electronics and electrical insulators, structural adhesives, and fiber-reinforced composites . The global demand for epoxy resins was estimated at ~21.5 billion USD in 2016, and is forecasted to increase to 37.3 billion USD by 2025 [Biodegradation is one of various polymer degradation routes. It is the process by which organic substances are broken down by microorganisms, such as bacteria, fungi, and algae. These microorganisms can degrade the polymers aerobically—producing carbon dioxide and water, or anaerobically—producing carbon dioxide, water, and methane [ methane . Degradathermoset resins cannot be recycled. Furthermore, they are typically not biodegradable, and can only be burned under strict precautions due to the release of toxic substances. Consequently, environmental pollution by their use and waste has been recognized as a severe problem, which must be globally addressed. A recently published paper describes a study of the fungal and bacterial microflora isolated from an epoxy art object, which can grow on the surface of the object [Pseudomonas putida, which was isolated from natural seawater with 2216E medium. The bacterium significantly decreased the corrosion resistance of the coating. The bacterium formed a mature biofilm on the coating and promoted extensive underfilm corrosion of the steel substrate. The authors attributed the degradation of the epoxy in the presence of the bacterium to oxidation of hydroxyl to the intermediate carbonyl .Unlike thermoplastics, e object . That ste object , we coule object  exploredHere, we describe our study aimed to isolate from the surroundings microorganisms that can degrade thermoset epoxy resins in an economically viable process under mild conditions, characterize these microorganisms, demonstrate the biodegradation process, and try understanding the metabolism of epoxy by these microorganisms. This study could open the door also to biodegradation of the cured end-products of epoxy resins, which might be a more challenging task. Such a microbial degradation process is a promising eco-friendly strategy, which represents a great opportunity to manage waste plastic materials with no adverse impacts. The discovery of such plastic-degrading microorganisms has great biotechnological potential, may become part of a larger screening program, and could aid natural bioremediation processes, favoring the natural cleaning of natural ecosystems.® LY 5052 . This low-viscosity resin is a blend of 1,4-butanediol diglycidyl ether (C10H18O4) and epoxy phenol novolac resin (C35H32O4), which contains bisphenol A. The content of these two constituents is 55–68 and 38–42 wt.%, respectively. The weight per epoxide (per ASTM D1652) of this resin is 148.33 g/eq. The number of epoxide groups in the phenol novolak resin is four, and its molecular weight is 345 g/mol. The functionality of 1,4-butanediol diglycidyl is two, and its molecular weight is 202.3 g/mol. The main applications of the Araldite® 5052 system are in aerospace and industrial composites, and in tooling for aircraft repair. (2) EPON™ resin 815C . This low-viscosity resin is a blend of 4,4-Isopropylidenediphenol-Epichlorohydrin copolymer (containing bisphenol A) and 2-(butoxymethyl)oxirane. The latter is also referred to as n-butyl glycidylether. The weight per epoxide of this resin is 180–195 g/eq, and its number average molecular weight is ≤700.Two types of epoxy resin were used: (1) Aralditehttp://www.polymer-g.com), including from the front door. This factory makes epoxy, polyurethanes, and silicones.Soil samples were collected from different regions outside the manufacturing factory of Polymer-G 2SO4, 0.5 g/L Na3-Cit·3H2O, 0.1 g/L MgSO4·7H2O, and its pH 7.0 ± 0.2 at 25 °C. Minimal media are those that contain essential minerals and a carbon source. We added 1 wt.% of epoxy resin as the carbon source. The samples were kept under aerobic conditions at 30 °C while shaking at 150 rpm for one week. Some cultures were grown on Difco™ LB Broth, Lennox. Exponentially growing cultures were diluted to an optical density at 600 nm (OD600nm) of 0.03. In order to enrich the bacteria growing on the epoxy media, 100 μL of a one-week old culture was added to a fresh 10 mL epoxy resin growth medium. The soil was allowed to settle for one hour before a sample was taken from the top of the suspension for a second enrichment in Davis medium. Overall, five enrichment cycles were run at one-week intervals, and the turbidity was measured at OD600nm after each cycle.1 g of soil sample was added to 10 ml of Davis minimal medium (broth) . The com® Eon™ microplate monochromator-based spectrophotometer . The underlying principle is that most of the light scattered by the bacteria no longer reaches the photoelectric cell, so that the electric signal is weaker than for a bacteria-free cuvette. The choice of 600 nm wavelength is due to the fact that many of the components that comprise the cell culture media remain transparent at this wavelength, so that optical density is proportional to the density of cells in the light path. OD600nm is preferable to ultraviolet (UV) spectroscopy when measuring the growth over time of a cell population because, at this wavelength, the cells will not be killed as they would under too much UV light. UV light has also been shown to cause small to medium sized mutations in bacteria, potentially altering or destroying genes of interest.Turbidity measurements were made at 10 min steps, using a BioTek® Genomic DNA Purification Kit . DNA fragments were separated via 1% agarose gel electrophoresis. The gels were prepared by dissolving the agarose in a Tris-acetate-EDTA (TAE) buffer. Polymerase chain reaction (PCR) was used to amplify the DNA segment that can provide bacterial identification. The primers used for amplifying the conserved regions within 16S rRNA genes were: forward primer AGAGTTTGATCCTGGCTCAG, reverse primer GGTTACCTTGTTACGACTT. For sequencing of an amplified material, a colony was mixed with 1 μL of each primer, 5 μL Taq polymerase buffer, 5 μL deoxynucleotide triphosphates (dNTPs), 0.5 μL TaKaRa Ex Taq™ DNA polymerase enzyme, and water to 50 μL. The amplification conditions were as follows: 30 cycles at 95 °C for 5 min, 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min, a final extension at 72 °C for 5 min, and cooling to 10 °C for 1 min [® NIH genetic sequence database. Partial 16S rRNA sequences were compared with sequences deposited in the database, using the standard nucleotide-nucleotide Basic Local Alignment Search Tool (BLAST®) code (http://www.ncbi.nlm.nih.gov/blast/). To avoid errors due to the alignment of DNA sequences of varying lengths, only the first 1500 nucleotides in each sequence were used for the alignment.Genomic identification of specific bacteria was based on sequencing of the gene coding for 16S RNA ,8,9,10. or 1 min . The DNA® 2  [® 2 system with anaerobic and corynebacteria identification card (ANC) was then used for the automated identification of the bacteria according to the manufacturer’s procedure.VITEKNC, USA) ,12 is an® MS system , following the manufacturer’s procedure. Briefly, a portion of a fresh colony was smeared on a FlexiMass™ disposable target plate, and was then immediately covered with 1 µL of ready-to-use α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution. After drying at room temperature, the target plate was loaded into the Axima Assurance mass spectrometer. Spectra were generated and compared to a database that contains 193 clinically relevant microorganisms.In matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) ,14,15, aThe shape and size of the bacteria were characterized using a Nikon Eclipse TE2000-U  inverted light microscope equipped with bright field, phase contrast, and epi-fluorescence cubes. Colonies were transferred from LB growing plates to a glass slide with double-distilled water (DDW). A coverslip was placed over the root or entire seedling, to ensure minimal trapped air bubbles, and the excess liquid was drained from the slide.The surface morphology of epoxy droplets, in the absence as well as in the presence of bacteria, was characterized by FEI Quanta 200 field-emission gun (FEG) environmental scanning electron microscope (ESEM™), operated in the wet mode with a gaseous secondary electrons detector (GSED). The ESEM vacuum system was pumped down to 5.3 Torr, which provided relative humidity of 100% for a sample temperature of 2 °C, as described elsewhere . The cle® G1 high-resolution Q-TOF LC-MS mass spectrometer equipped with an ACQUITY™ UPLC™ system and a tunable UV (TUV) detector, which is capable of UV-vis detection and electrospray ionization in the same run . The stationary phase consisted of a C18  column , and the mobile phase compositions were: (A) 95% H2O + 5% acetonitrile + 0.1% formic acid; (B) acetonitrile + 0.1% (v/v) formic acid. The elution gradient consisted of a linear increase to 100% B in 12 min and hold for 2 min, then return to the starting conditions for additional 1 min. 10 µL samples of analyzed materials were injected, and the flow rate was 0.3 mL/min. The temperature in the sample chamber was preset to 10 °C, and remained stable (±1 °C) throughout the measurement. The UV detector was set to 254 nm. The mass spectrometer was operated both in the negative and in the positive ion modes. Data interpretation was done for both ion modes, using MassLynx™ software .The metabolic pathways of the epoxy resins by bacteria were studied by the liquid chromatography–mass spectrometry (LC-MS) technique. LC-MS combines the resolving power of LC with the detection specificity of MS. LC separates a sample to its components, and then introduces them to the mass spectrometer for further analysis. The LC-MS data can be used to provide information on the molecular weight, structure, identity and quantity of specific sample components. In this study, we used a SYNAPT® LY 5052 and EPON™ resin 815C—with Davis minimal medium and soil samples. After a week of incubation at 30 °C, the cultures were diluted into fresh medium, and incubations were continued. At one-week intervals, the turbidity (OD600nm) of suspensions was measured, as an indicator to the growth of microorganisms. It should be noted that the oily-epoxy resin appeared as large droplets, without forming a dispersion. Since the droplets were large, they did not affect the OD value. Pictures of the flasks before and after the first week of incubation are shown in 600nm values surprisingly decreased, just opposite to the expected trend. There are two possible explanations to this phenomenon. First, it is possible that some nutrients derived from the soil ran out. The second explanation is the effect of the diversity of microorganisms in the soil. Initially, there was a large variety of microorganisms that were either specific or nonspecific to epoxy as a carbon source. As the process continued, the diversity decreased, and only microorganisms that are specific to epoxy remained.For the first screening stage, we incubated two different epoxy resins—AralditeA culture sample after the third enrichment cycle was used to determine the bacterial growth rate. When the epoxy resin is the only carbon source available for the bacteria, their growth rate can be related to the extent or rate of biodegradation of the epoxy resin. The results presented in In order to determine the effect of the concentration of epoxy resin LY 5052 on bacterial growth, cultures were grown for 8 days, using media with progressively increased epoxy resin concentrations of 0.4 wt.%, 0.8 wt.%, 1 wt.%, and 1.2 wt.%. The maximal growth rate was evident at 1 wt.% ; lower cIn order to isolate and characterize the bacteria capable of degrading the epoxy, a sample of the bacteria (after the second enrichment cycle) was grown on rich medium agar plates (LB). The plates were examined after 24 h of incubation, and it was possible to observe a clear growth of three different types of colonies, which could be distinguished based on their color: orange, yellow, or white. Because we found that the yellow-colored bacteria could be grown on different growth media only in the presence of the bacteria producing orange or white color, it was decided not to use further yellow-colored bacteria in this study.The typical colonies of the other two bacteria are shown in Ochrobactrum anthropi (chromosomes 1 and 2), ATCC® 49188™, accession NC 009668.1. Sequencing data for the bacteria giving orange colonies showed 96% homology with Bacillus sp. 1NLA3E bacterium (accession NC 021171.1). The sequence analyses were made using the standard nucleotide-nucleotide BLAST® search tool.Identification of the bacteria was done by 16S ribosomal RNA (rRNA) gene sequencing ,8,9. SeqAs our isolations started from a soil sample, and because culturable bacteria represent only a very small fraction of the bacterial population in soil, the question arises whether the two isolated bacteria are the ones who are degrading the epoxy. Our results indicate that the bacteria we cultured are indeed the ones involved in the degradation, as we could not detect additional bacteria by 16S RNA sequencing. O. anthropic was supported by additional tests: it is Gram negative [® 2 [l-lactate alkalinisation, citrate, d-tagatose, urease, tyrosine arylamidase, l-proline arylamidase (ProA), l-pyrrolydonyl-arylamidase (PyrA), and adonitol. These results are in line with the report of Laffineur et al. [l-pyrrolydonyl-arylamidase (PyrA) and alkaline phosphatase (PHO) can be useful in identification of Gram-negative bacteria; 100% of the Ochrobactrum anthropi strains were found to be positive for PyrA and negative for PHO assays. The identification of negative  , using it as a carbon source [acterium ,23,24. Age tubes ,26,27. Tn source .Rhodococcus is a bacterial genus that can be isolated from a range of sources, including soil, marine sediments, rocks, and groundwater [Rhodococcus exhibits a large metabolic diversity, and as a result can degrade a variety of pollutants, which may afford both environmental and industrial purposes, in transformation and degradation chemistries. Rhodococci can catabolize short- and long-chain alkanes, aromatic compounds , as well as heterocyclic and polycyclic aromatic compounds. Surprising is the extent to which these bacteria extend their biodegradative abilities to compounds that could be described as more difficult with respect to recalcitrance and potential microbial toxicity [Rhodococcus stems from the diverse range of reactions, sometimes novel, which their enzymes can catalyze. Their hydrophobic, mycolate-containing cell envelope structure also seems to be relevant to many potential applications  [Rhodococcus has been attributed both to its genomics and morphology [Rhodococci DNA undergoing recombination easily and at high frequency with heterologous sequences. On the other hand, the morphology of the genus also makes classification complicated. Rhodococcus that are in exponential phase of growth exhibit filament or short-rod shape, while those in the stationary phase tend to be in cocci [Rhodococcus also has recalcitrant cell walls, which makes nucleic acids extraction difficult [undwater ,30,31. Iundwater . Rhodocotoxicity . Much ofactants) . Difficurphology ,35. It sifficult .Once the two bacteria were successfully isolated and identified unambiguously, the growth rate of each individual bacterium was measured on various growth media. Each of the two bacteria could easily be grown on a variety of media, including a minimal medium with sodium acetate as a carbon source. In contrast, none of these bacteria could grow on a minimal medium containing epoxy resin 815C or LY 5052 as a sole carbon source. Growth on epoxy resin required the presence of the two bacteria together .Ochrobactrum anthropi and Rhodococcus rhodochrous. This would worth further research.The nature of this obligatory synergy is not yet understood. The obligatory synergy could be due to a situation where each of the bacteria produces only part of the required enzymes for breaking down the polymer. A more complex situation may be that one of them is producing enzymes that can assist the other one via a regulatory process, such as quorum sensing (QS). QS allows bacteria to communicate via exchange of extracellular signaling molecules, called autoinducers (AIs), and act as multicellular organism. At certain concentration levels in bacterial surroundings, these AIs bind to specific receptors and initiate the expression of target genes required for biofilm formation. Acyl homoserine lactones (AHLs) are among the most extensively studied AIs produced by Gram-negative bacteria, whereas autoinducing peptides (AIPs) are the signals found in Gram-positive bacteria ,37,38. MFinally, the biodegradation of epoxy resin by bacteria was demonstrated by ESEM  and LC-MIn order to better understand the metabolic pathway of epoxy resin by the two bacteria, LC-MS analyses were performed. Three sample types were analyzed: (1) Davis epoxy resin LY 5052 with bacteria, (2) Davis epoxy resin LY 5052 without bacteria, and (3) Davis and bacteria without epoxy resin. LC-MS analyses were conducted after one week of incubation. Mass spectrometry analysis of each chromatogram was done both in the positive and in the negative modes )bis(oxy))bis, which is promoted by bacteria, was suggested. This study could pave the way also to biodegradation of the cured end-products of epoxy resins, which might be a more challenging task. Such a microbial degradation process is a promising eco-friendly strategy, which represents a great opportunity to manage waste plastic materials with no adverse impacts.This article describes our study aimed to isolate from the surroundings microorganisms that can degrade thermoset epoxy resins, which have been considered to be typically non-recyclable and non-biodegradable, in an economically viable process under mild conditions, characterize these microorganisms, demonstrate the biodegradation process, and try understanding the metabolism of epoxy by these microorganisms. Two different epoxy resins were studied: Araldite


Resistance to molecular targeted therapies is a significant challenge for advanced colorectal cancer (CRC). Understanding the underlying mechanisms and developing effective strategies against regorafenib resistance are highly desired in the clinic. Here, we screened the expression of chemokine receptors and identified CC chemokine receptor 2 (CCR2) as a top upregulated gene in regorafenib-resistant cells. CCR2 silencing alleviated drug tolerance in regorafenib-resistant cells, while overexpression of CCR2 enhanced CRC cells resistance to regorafenib. Moreover, CCR2-mediated regorafenib tolerance was demonstrated to be associated with AKT/GSK3β-regulated β-catenin stabilization. In turn, β-catenin modulation is sufficient to trigger the transcriptional activation of CCR2 expression. Clinically, high-CCR2 expression was correlated to shorter overall survival and disease-free survival of patients. A positive correlation between CCR2 and nuclear β-catenin expression was observed in a cohort of CRC tissues. Altogether, these findings suggest β-catenin and CCR2 are part of a positive-feedback loop, which sustains a high CCR2 expression level, conferring CRC cells resistance to regorafenib. Thus, targeting CCR2 may be a useful therapeutic strategy to alleviate regorafenib tolerance to increase the efficacy of CRC treatments. In the past 20 years, the treatment for CRC has evolved to the combination of cytotoxic therapy and target-specific vehicles2. Current chemotherapeutic regimens utilized in stage IV CRC include fluoropyrimidines, oxaliplatin, irinotecan, and molecular targeted agents . Despite these impressive advances, recurrence remains common due to the development of drug resistance3.Colorectal cancer (CRC) is the third leading causes of cancer-related deaths worldwide4. Regorafenib inhibits c-Raf, b-Raf, vascular endothelial growth factor receptors (VEGFR), platelet-derived growth factor receptor (PDGFR), and other oncogenic kinases5. The antitumor activity of regorafenib has been demonstrated to be correlated with induction of apoptosis, suppression of tumor angiogenesis and proliferation5. Although many progressions have been made, the activity of regorafenib is limited by primary and acquired drug resistance. To date, several studies have investigated the mechanisms underlying regorafenib tolerance in human malignancies. It is shown that isomerase Pin1 inhibition reverses the resistance of hepatocellular carcinoma cells to regorafenib6. Moreover, antiapoptotic BCL-2 proteins play a key role for regorafenib tolerance in hepatocellular carcinoma7. In human CRC, it is reported that FBW7 mutational status mediates cells resistance to regorafenib by blocking Mcl-1 degradation8. However, the specific mechanisms in cancer resistance to regorafenib remain unknown.Regorafenib, a multikinase inhibitor targeting the RAS/RAF/MEK/ERK pathway, has been approved to treat metastatic colorectal cancer9. Recently, it is also reported that Wnt/β-catenin signaling plays a role in cancer resistance to targeted therapies. For instance, the destabilization of Ras overcomes erlotinib tolerance in non-small cell lung cancer through inhibition of Wnt/β-catenin pathway10. The study by et al. suggests sorafenib-resistant cells can be eliminated via attenuation of β-catenin signaling11. Although Wnt/β-catenin pathway is associated with the effect of regorafenib on tumorigenesis12, its function in cancer resistance to regorafenib has not been revealed.The Wnt/β-catenin pathway modulates a variety of processes in tumor progression, including cell proliferation, invasion, and metastasis13. Growing evidences have elucidated the critical functions of chemokines and their receptors in tumor biology14. Our previous work suggests that CCR4 promotes CRC metastasis via ERK/NF-κB/MMP13 pathway15. Moreover, CCR6 facilitates tumor angiogenesis through the AKT/NF-κB/VEGF signaling in colorectal cancer16. Recently, the role of ectopic expression of chemokine receptors on cancer cells has been reported to be involved in drug resistance. It is shown that activation of mitogen-activated protein kinase (MAPK) signaling by CXCR7 contributes to enzalutamide resistance in prostate cancer17. In esophageal squamous cell carcinoma, cancer-associated fibroblasts derived IL-6 promotes chemoresistance by upregulating CXCR7 expression of tumor cells18. Furthermore, CCL2/CCR2 axis is demonstrated to be a contributor to cabazitaxel resistance in prostate cancer cells19.Chemokines are a superfamily of small molecules that are regulated by their interaction with chemokine receptorsIn this study, we hypothesized that chemokine receptors might play important roles in cancer resistance to targeted therapies. Interestingly, the results identified CC chemokine receptor 2 (CCR2) as a top upregulated gene in regorafenib-resistant (regR) cancer cells. Thus, we focused on the function and the underlying mechanism of CCR2 in drug tolerance. We found that CCR2 promoted cells resistance to regorafenib via β-catenin stabilization, and that β-catenin modulation was sufficient to positively regulate CCR2 mRNA and protein expression, by a direct recruitment onto TCF/LEF consensus-binding sites located in CCR2 promoter. Overall, these data suggest targeting CCR2 may be an effective method to alleviate regorafenib resistance, thus increasing the therapeutic efficacy of regorafenib in CRC patients.Primary CRC cell lines  were cultured with regorafenib to generate regR cells Fig. . To confTo explore the function of CCR2 in regorafenib resistance of CRC cells, stable clones were generated with either shRNA-mediated CCR2 silencing in regorafenib-resistant cells or a protein ectopic expression in nonresistant cells. The effect of stable transfection was confirmed by western blot Fig. . As show2 Profiler PCR Array to profile differentially expressed genes between each group . The heatmap produced by this array shows gene-expression levels relative to β-actin on a log2 scale  to drive the transcription of target geneser9 Fig. . In contnin Fig. . Furtherlls Fig. . AltogetTo explore whether β-catenin signaling underlies the function of CCR2 in cells resistance to regorafenib, regR/sh-CCR2 and regR/sh-NC cells were cultured with CT99021, a GSK-3 inhibitor that stabilizes β-catenin expression by protecting it from proteasomal degradation. Furthermore, si-RNA was used to silence β-catenin expression in HCT116/CCR2, SW480/CCR2, and their controls. In fact, both CT99021 and si-RNA caused a significant change in total β-catenin expression relative to the control Fig. . TOP/FOPWe further assessed the effect of β-catenin alterations on the expression of downstream target genes  in regR/sh-CCR2, CCR2-overexpressing cells and their controls. The results demonstrated that CT99021 increased the expression levels of downstream genes in both regR/sh-CCR2 and regR/sh-NC cells Fig. . MoreoveNext, we investigated the mechanism responsible for the upregulation of CCR2 in regorafenib-resistant cells. As revealed by above-mentioned PCR array, the β-catenin signaling was significantly activated in HCT116/regR cells versus non-resistant controls Fig. . MoreoveWe first tested the effect of β-catenin knockdown in regorafenib-resistant cells, where CCR2 is highly expressed. We found that an efficient silencing of β-catenin led to a significant decrease of CCR2 mRNA expression Fig. . As β-caWe then explored whether β-catenin regulates CCR2 expression directly, through the two putative TCF/LEF consensus-binding sites found in CCR2 promoter. These sites are located in upstream  relative to the transcription start site of CCR2. A luciferase reporter assay showed that β-catenin transactivated CCR2 promoter activity Fig.  in HCT11The preceding data raised a question concerning whether upregulation of CCR2 could sufficiently promote regorafenib resistance in vivo. To this end, the tumor cells were subcutaneously injected into nude mice to establish a xenograft model. Following tumor generation, we treated these mice with regorafenib or vehicle. The results showed the xenografts derived from CCR2-overexpressing cells exhibited increased volume and weight than that from controls in response to regorafenib Fig. . Moreove25. Representative images of CCR2 and nuclear β-catenin staining are shown in Fig. positive cases, 62 (83.8%) displayed prominent nuclear β-catenin expression. Meanwhile, of the 42 CCR2negative cases, 18 (42.9%) were nuclear β-catenin negative  in this cohort. Patients in the CCR2positive group had a significantly poorer overall survival than those in the CCR2negative group , the expression of p-GSK3βSer9 and β-catenin was significantly blocked in tumor cells. Thus, β-catenin could be stabilized by CCR2 through the AKT/GSK3β pathway in CRC cells.To investigate the molecular mechanisms responsible for regorafenib tolerance in CRC cells, we used a PCR array to profile differentially expressed signal transduction-related genes in two groups of cells . Our results suggested β-catenin/TCF signaling might play a crucial role in CCR2-mediated drug resistance. To date, β-catenin signaling has been demonstrated to be critical for maintaining chemoresistance in tumor cellsWe then investigated whether β-catenin stabilization regulated by CCR2 could contribute to regorafenib resistance. CT99021 was used to restore β-catenin expression in CCR2-silencing regR cells, while si-RNA was used to knockdown β-catenin in CCR2-overexpressiong CRC cells. Interestingly, CT99021 treatment in regR cells with CCR2 silencing led to the activation of β-catenin transcriptional activity, upregulation of downstream target genes and promotion of cells tolerance to regorafenib. Conversely, the inhibition of β-catenin in CRC cells with CCR2 overexpression resulted in the inactivation of β-catenin transcriptional activity, downregulation of downstream target genes and alleviation of regorafenib resistance.The preceding findings promoted us to explore the mechanism for the upregulation of CCR2 in regorafenib-resistant cells. First, we found that silencing of β-catenin or Lef-1 led to a significant decrease of CCR2 mRNA and protein levels. On the contrary, ectopic expression of β-catenin was sufficient to turn on CCR2 gene expression levels. Subsequent luciferase reporter assay suggested β-catenin transactivated CCR2 promoter activity in HCT116 and SW480 cells. Moreover, both binding sites A and B were demonstrated to be critical for β-catenin-induced CCR2 activation. A ChIP assay further confirmed the enrichment of β-catenin on CCR2 promoter in CRC cells and tissues. Thus, β-catenin is directly recruited onto the TCF/LEF consensus-binding sites located in CCR2 promoter, leading to CCR2 expression activation. Clinically, IHC analysis revealed a positive correlation between CCR2 and nuclear β-catenin expression in a cohort of tissues. High expression of CCR2 in CRC tissues was significantly associated with shorter overall survival and disease-free survival. Furthermore, combination of CCR2 and nuclear β-catenin was a more powerful prognostic marker for patients with CRC.In conclusion, this study identifies CCR2 as a top upregulated chemokine receptor in regorafenib-resistant cancer cells. Moreover, β-catenin and CCR2 are part of a positive feedback loop, which sustains a high CCR2 expression level, conferring CRC cells resistance to regorafenib. Our findings thus identify new roles for CCR2 in the development of regorafenib tolerance and suggest that CCR2 may be a potential target for overcoming CRC drug resistance.8. Briefly, cancer cells were cultured at a concentration of 40 μM regorafenib for 3 days, followed by recovery for 5 days. Then the period of treatment and recovery was repeated for a total of 4 cycles. Regorafenib, LY294002 (PI3K/AKT pathway inhibitor) and CT99021 (GSK-3 inhibitor that stabilizes β-catenin expression) were purchased from Selleck Chemicals (USA) and used according to manufacturer’s instructions.CRC cells  were purchased from the ATCC  and authenticated by STR/DNA profiling by BIOWING BIOTECHNOLOGY . Cells were cultured in RPMI 1640 media containing 10% FBS at 37 °C and confirmed as mycoplasma free using a Mycoplasma Detection Kit (Roche). Stable resistant cells were generated as previously described2 Profiler PCR Array (QIAGEN) was conducted according to the manufacturer’s instructions. Target gene expressions were compared to β-actin as a control. The sequences of primers were depicted in Supplementary Table Total RNA was extracted using TRIzol reagent. We then used a reverse transcription kit (Invitrogen) to perform reverse transcription. Finally, q-PCR was performed in an Applied Biosystems 7500 System with primers, mixture of cDNA and SYBR Green PCR Master Mix (Applied Biosystems). In addition, the Human Signal Transduction PathwayFinder RTNuclear proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). Protein lysates were collected from cells in RIPA buffer with 1% protease inhibitor, and protein lysate concentration was measured by bicinchonininc acid. Proteins were detected following an overnight incubation at 4 °C with primary antibodies, followed by 2 h incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature. A LI-COR Odyssey Infrared fluorescence scanner was used to capture the images. Antibodies against CCR2 (12199), β-catenin (8480), β-actin (4970), Histone H3 (4499), AKT (40D4), p-Ser473-AKT (4060), and p-Ser9-GSK3β (9322) were purchased from Cell Signaling Technology. Antibodies against GAPDH (ab181602) and GSK3β (ab32391) were purchased from Abcam.5 cells per well. When the cell density reached about 70%, lentivirus particles were transfected into cells in the presence of polybrene. Stably transfected cells were selected with 5 μg/ml puromycin and tested regularly by immunoblotting to ensure downregulation or upregulation of CCR2. In some experiments, cells were transiently transfected with β-catenin or Lef-1 siRNA  using Lipo3000 (Invitrogen) according to the manufacturer’s instructions.The short hairpin RNA (shRNA) targeting CCR2 and pGL3-CCR2 lentivirus vector were established by Genepharma (China). Tumor cells were seeded onto six-well plates at 3 × 1025. All protocols were authorized by the Ethics Committee of Shanghai Ruijin Hospital. Tissue staining was performed as previously described25. Ten randomly chosen fields were captured for each slide. The staining score was independently evaluated by two pathologists according to the percentage of positive cells and staining intensity. Notably, the evaluation of nuclear β-catenin was based on percentage of positive nucleus and intensity of nucleus. Scoring for percentage was: 0% (0), 1–5% (1), 6–29% (2), 30–59% (3), and more than 60% (4). Scoring for staining intensity was: no staining (0), slight staining (1), moderate staining (2), and intense staining (3). These values were multiplied together to provide a score for each case. Cases were grouped as either negative (score 0–4) or positive (score >4) for analysis.The tissue microarray used in this study has been previously reported3 cells per well and treated with the indicated doses of regorafenib  for 48 h. Then, the cells were incubated with 10 µL CCK-8 for 60 min at 37 °C, 5% CO2. The absorption value was detected at 450 nm with a spectrophotometer. Each assay was conducted in triplicate and repeated three times. In addition, cell proliferation rates were analyzed by measuring DNA synthesis using the Click-iT EdU Assay .Cells were seeded in a 96-well plate overnight at a density of 5 × 10n = 10 per group). When tumor volumes reached approximately 0.2 cm3, mice were intravenously treated vehicle or 45 mg/kg regorafenib  twice a week for three weeks. Body weights and tumor volumes were measured every other day. Tumor volume was calculated using the formula: length × width2 × 0.5. All experiments were performed according to the official recommendations of the Chinese animal community.Four-week-old male nude BALB/C mice were subcutaneously injected with CCR2-overexpressing or control cells (Tumor cells were seeded in 24-well plates and transfected with 2 ng of pRL-TK Renilla luciferase vector and either TOP flash plasmid or FOP flash plasmid (200 ng) using Lipofectamine 3000. Forty-eight hour later, luciferase activity was measured using the Dual Luciferase Reporter Assay  according to the manufacturer’s protocol. The results were presented as the relative TOP/FOP value.CCR2 promoter fragments were amplified from human genomic DNA, and were inserted into pGL3-basic vector. The β-catenin-overexpressing or control cells were cotransfected with pGL3-CCR2-promotor constructs. Luciferase assays were performed 24 h after transfection using Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions. The ratios of luminescence intensities were measured relative to that of the pGL3 empty vector. The primers are presented in Supplementary Table 15. Briefly, after crosslinking with 1% formaldehyde, protein and DNA were extracted by SDS lysis buffer and sheared by sonication. The supernatants were incubated overnight at 4 °C with 2 µg anti-β-catenin  or control rabbit immunoglobulins. After purification of precipitated DNA fragments, q-PCR was performed using the listed primers . All experiments were conducted at least three times. ANOVA and Student’s t test were performed to compare differences among experimental groups. Supplementary Table.Figure S1Figure S2Figure S3Supplementary figure legends.


Journal of Analytical Methods in Chemistry has retracted the article titled “Antitumor Molecular Mechanism of Chlorogenic Acid on Inducting Genes GSK-3β and APC and Inhibiting Gene β-Catenin” [Catenin” . ConfideCatenin” .


Triple-negative breast cancer (TNBC) is a severe and heterogeneous disease that lacks an approved targeted therapy and has a poor clinical outcome to chemotherapy. Although the RAS-ERK signaling axis is rarely mutated in TNBC, ~50% of TNBCs show an increased copy number and overexpression of epidermal growth factor receptor (EGFR). However, EGFR-targeted therapies have offered no improvement in patient survival, underscoring the need to explore downstream targets, including RAS. We found that both β-catenin and RAS, as well as epidermal growth factor receptor (EGFR), are overexpressed and correlated with one another in tumor tissues of TNBC patients. KYA1797K, an Axin-binding small molecule reducing β-catenin and RAS expression via degradation and suppressing EGFR expression via transcriptional repression, inhibited the proliferation and the metastatic capability of stable cell lines as well as patient-derived cells (PDCs) established from TNBC patient tissues. KYA1797K also suppressed the stemness of 3D-cultured PDCs and xenografted tumors established by using residual tumors from TNBC patients and those established by the TNBC cell line. Targeting both the Wnt/β-catenin and RAS-ERK pathways via small molecules simultaneously reducing the levels of β-catenin, RAS, and EGFR could be a potential therapeutic approach for TNBC. The triple-negative breast cancer (TNBC), a highly aggressive form of breast cancer may be susceptible to drug therapy that targets two critical signaling pathways, one that governs cell fate and the other that controls the cell cycle. Researchers from Yonsei University in Seoul, South Korea, had previously synthesized a small-molecule called KYA1797K and found that could lower levels of the proteins, β-catenin (a master regulator of cell fate decisions) and RAS (which controls cell division), in colorectal cancer cells. Now, Kang-Yell Choi, Soonmyung Paik, and colleagues have shown that KYA1797K has the same effects on β-catenin and RAS levels in cells taken from patients with TNBC. In cell lines and mouse models, KY1797K suppressed the proliferation and metastatic capability of TNBC, highlighting the therapeutic potential of this strategy degrading both β-catenin and RAS. Without these receptors, neoadjuvant chemotherapy is the standard treatment for TNBC. Although ~30% of TNBC patients undergo pathological complete response (pCR) after chemotherapy with excellent survival, those without pCR suffer a grave clinical outcome5. Despite the recent success of postneoadjuvant capecitabine or immune checkpoint inhibitors in improving the clinical outcome of patients without pCR, there is still a critical need for further reduction in the recurrence rate of these high-risk tumors.Triple-negative breast cancer (TNBC) is a breast cancer subtype that lacks expression of estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2 (HER2), with a diagnosis rate of 15–20% in breast cancer patients8. Approximately 50% of TNBCs show an increased EGFR copy number, which is associated with poor clinical outcome9. However, many clinical trials of drugs targeting EGFR, such as cetuximab, a recombinant EGFR monoclonal antibody, did not yield successful outcomes11.One of the potential treatment targets in TNBC is the EGFR signaling axis13. EGFR overexpression can occur through increases in gene copy number9 or transcriptional induction via the Wnt/β-catenin pathway14. The Wnt/β-catenin pathway is active in TNBC patient tissues, and is correlated with tumorigenesis, metastasis, cancer stemness, and poor prognosis in TNBC patients16. Residual tumors after neoadjuvant chemotherapy were found to have an increased proportion of cancer stem cells (CSCs), suggesting the involvement of CSCs in chemotherapy resistance18. Therefore, targeting both β-catenin and RAS, as well as upstream EGFR, could be an ideal approach for the treatment of TNBC.The RAS-ERK  pathway is activated in TNBC patient tissues, which is not attributed to mutations of the genes in this pathway, but to EGFR overexpression19, in TNBC both in vitro and in vivo. Due to the major problem of EGFR overexpression in TNBC, the use of KYA1797K, which represses EGFR transcription by β-catenin degradation, provides further advantages.In this study, we identified that the expression levels of β-catenin and RAS were highly increased alongside accumulated EGFR in patient tumor tissues, and their expression levels were correlated in these samples. These results led us to test the efficacy of KYA1797K, a small molecule that simultaneously degrades both β-catenin and RAS proteins via Axin–RGS domain binding21.KYA1797K dose-dependently inhibited the growth and transforming capabilities of various TNBC cell lines and primary patient-derived cells (PDCs) with reductions in β-catenin, pan-RAS, and EGFR levels. In addition, KYA1797K further suppressed the invasive characteristics of migratory TNBC cells, which provided support for the potential effectiveness of KYA1797K in preventing metastasis. The inhibition of stem cell characteristics by KYA1797K was also indicated by growth suppression of tumor organoids, with reductions in the cancer stem cell (CSC) markers CD44 and aldehyde dehydrogenase 1 (ALDH1) A3Additional support for KYA1797K in TNBC suppression was indicated by the growth reduction of tumors generated by MDA-MB-468 TNBC cells or the residual tumor tissues of TNBC patients treated with neoadjuvant chemotherapy. We also confirmed the inhibitory effects of KYA1797K on TNBC patient-derived xenograft (PDX) tumors; these effects occurred through the modulation of β-catenin, RAS, and EGFR expression. Collectively, the small molecule induced destabilization of β-catenin and RAS, which leads to inhibition of their respective pathways and to inhibition of EGFR expression; this could provide insight into a potential therapy for TNBC patients.22.TMAs for normal-adjustment breast and TNBC tissues (BC081120b) were purchased from US Biomax (Rockville). Immunohistochemistry was performed with β-catenin, pan-RAS, or EGFR antibodies. Bright-field microscopy  was used to obtain images of each specimen. For quantification of the expression levels of nuclear or cytoplasmic proteins, the TMA images were quantified using the IHC profiler plugin for NIH Image software23. All studies were approved by the Institutional Review Board of Severance Hospital, Seoul, South Korea (4-2012-0705). Patient tumor samples were collected from patients in accordance with the relevant IRB guidelines. Briefly, 5-week-old female Balb/c nude mice  or female NOG mice  were purchased and acclimatized for 1 week, and used for the generation of xenograft mice with MDA-MB-468 cell lines or patient tumor tissues.PDX mice were established from the residual tumor tissues of two TNBC patients after neoadjuvant chemotherapy, as described previously6 MDA-MB-468 cells in 200 µl 2:1 PBS:Matrigel . Patient tumors were sliced into 3 × 3 × 3 mm3 fragments, and then subcutaneously implanted into the flanks of NOG mice. Drug treatment was initiated when the mean tumor volume reached between 150 and 200 mm3. Mice were randomly assigned to specific treatment groups. KYA1797K was injected intraperitoneally at a dose of 25 mg/ml daily. The sizes of the implanted tumors were measured 2–3 times a week using Vernier calipers, and the tumor volume was calculated as follows: (length × width2)/2. Mice were sacrificed, and the tumors were isolated, weighed, sliced, and fixed in formalin or liquid nitrogen for further analyses.The Balb/c nude mice were injected subcutaneously in the dorsal flank with 5 × 1024. Epithelial cells were cocultivated with irradiated (3000 rad) Swiss 3T3 fibroblasts (J2 strain) in F medium  with the addition of 5 to 10 µM/L Y-27632 .PDCs were established from residual tumor tissue from primary TNBC after neoadjuvant chemotherapy; the protocol for PDC establishment was as described by Liu et al.2 at 37 °C. All chemicals were dissolved in dimethyl sulfoxide  for the in vitro studies. Unless otherwise indicated, KYA1797K was used at a concentration of 25 µM for 24 h.Human TNBC stable cell lines  were obtained from the American Type Culture Collection . BT549 cells were provided by S.-J. Lee . Normal-like breast cells, MCF10A, were provided by D.S. Min . Cells were cultured in DMEM or RPMI (Gibco) containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco), and 5% CO25. Briefly, primary TNBC patient cells were suspended in modified M87 medium, mixed with Matrigel (BD Biosciences), and plated in 48-well plates. After Matrigel polymerization, modified M87 medium  was overlain. On the first day after seeding, the medium was also supplemented with 10 mM ROCK inhibitor Y-27632 (Sigma-Aldrich) to avoid anoikis. Fresh media with growth factors was changed every 2 days for maintenance. For treatment of tumor organoids with KYA1797K, media with KYA1797K or DMSO was changed every 2 days from the second day after seeding.The detailed protocol and reagents for primary tumor organoids were as described by DeRose et al.TNBC cells were seeded in six-well plates coated with collagen (500 μg/ml). After reaching confluence, cells were scratched with a 200 µl tip, and culture media was exchanged with 10% RPMI containing KYA1797K or DMSO. Migrated cells were quantified using NIS-Elements AR 3.1 software (Nikon). Screenshots were captured from video files and were represented as images at several time points. Mean ± SD are reported based on three or five biological replicates.5 onto noncoated or Matrigel-coated chambers (BD Bioscience) with KYA1797K or DMSO. Cells were allowed to invade for 24 h. After clearing cells on the inner surface of the chamber, the cells on the outer surface were fixed in 4% paraformaldehyde (PFA) for 15 min and stained with crystal violet for 20 min. The chambers were dipped in distilled water to remove excess staining and allowed to dry. Representative images were captured by microscopy . Mean ± SD are reported based on three biological replicates.PDCs or TNBC cell lines were seeded at a density of 3 × 104 cells/well. CSC media was supplemented with DMEM/F12 (Invitrogen) containing human recombinant EGF  and human recombinant basic fibroblast growth factor . After 24 h, spheroids were treated with KYA1797K or DMSO for 7 days, with whole media changed every 2 days. Dead cells were removed by centrifugation, and the remaining spheroids were washed with cold PBS and fixed with acetone for 24–48 h.BT549 or 4T1 cells were seeded in 90-mm Petri dishes at 1 × 10Cells were washed in ice-cold PBS and lysed using radioimmunoprecipitation assay (RIPA) buffer containing 20 mM NaF, 1 mM sodium vanadate, 10 mM Tris-HCl at pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 1 mM PMSF, and protease inhibitor cocktail . Tissues were homogenized and dissolved in RIPA buffer. Proteins were separated by a 10–12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to a nitrocellulose membrane . Immunoblotting was performed with the following primary antibodies: anti-pan-RAS monoclonal , anti-β-catenin , anti-p-ERK , anti-ERK , anti-p-Akt , anti-EGFR , anti-N-cadherin , anti-α-SMA , anti-Vimentin  and anti-β-actin . Horseradish peroxidase-conjugated anti-mouse  or anti-rabbit  secondary antibodies were used. Bands were detected by enhanced chemiluminescence  using a luminescent image analyzer .2O2  was applied to sections to block endogenous peroxidase activity for 15 min. Before incubating sections with mouse primary antibody, mouse IgG was blocked using the mouse-on-mouse (M.O.M.) IgG blocking kit (Vector Laboratories). Sections were incubated with anti-β-catenin, anti-pan-RAS, or anti-EGFR primary antibodies overnight at 4 °C, followed by incubation with biotinylated anti-mouse  or biotinylated anti-rabbit  secondary antibodies for 1 h at room temperature. The samples were then incubated in avidin biotin complex solutions (Vector Laboratories), stained with 3,3-diaminobenzidine  for a maximum of 5 min, and counterstained with Mayer’s hematoxylin . All incubations were performed in humid chambers. Signals were analyzed using a bright-field microscope .For immunohistochemistry (IHC) staining, 4-µm paraffin-embedded tissue sections were treated with citrate buffer (pH 6.0) and autoclaved for 15 min. The sections were then blocked with 5% bovine serum albumin (BSA) and 1% normal goat serum  in PBS for 1 h for human and mouse tumor samples. For fluorescent IHC, sections were incubated with primary antibody  overnight at 4 °C, followed by incubation with anti-mouse Alexa Fluor 488  or anti-rabbit Alex Fluor 555  secondary antibodies for 1 h at room temperature. Sections were counterstained with 4,6-diamidino-2-phenylindole  and mounted in gel/mount media . All processes were conducted in the dark, humid chambers. A confocal microscope  was used to visualize the fluorescence signal at excitation wavelengths of 488 nm (Alexa Fluor 488), 543 nm (Alexa Fluor 555), or 405 nm (DAPI). At least three fields per section were analyzed. For peroxidase IHC analysis, 3.45% HCells grown on gelatin-coated cover glasses were fixed in 4% PFA for 10 min, followed by permeabilization with 0.1% Triton X-100 for 15 min, blocking in 5% BSA for 1 h, and primary antibody incubation overnight at 4 °C. Primary antibodies were removed, and cells were washed with PBS and incubated for 1 h at room temperature with either Alexa Fluor 488- or Alexa Fluor 555-conjugated IgG secondary antibodies (Invitrogen). Cell nuclei were counterstained by incubating the cells in DAPI. Immunofluorescence images were captured using a confocal microscope .3 cells/well in a 24-well plate. Cells were then treated with 5 or 25 µM KYA1797K or with DMSO for 72 or 120 h. Next, 3--2-5-diphenyltetrazolium bromide  reagent was added to each well at a concentration of 0.5 mg/ml. After incubation for 1 h at 37 °C, insoluble purple formazan was obtained by incubating in 500 μl (24-well plate) of DMSO for 20 min. The absorbance of the formazan product was determined at 590 nm every 24 h. For colony-formation assays, cells were seeded in 12-well plates (100–500 cells/well for TNBC cells). DMSO control or KYA1797K was added to the cells with medium changes every 3 days until visible colonies formed. At the end of the experiment, cells were fixed in 4% PFA for 30 min and stained with 0.5% crystal violet in 20% ethanol for 30 min.To assay cell proliferation, TNBC PDCs and stable cell lines  were plated at a density of 8–10 × 104 cells on gelatin-coated chambers. After 24 h, single cells were imaged using a time-lapse video microscope. The videos and the migratory paths of single cells were constructed using NIS-Elements AR 3.1 (Nikon).PDCs were seeded at a density of 1 × 105 cells/well in six-well plates and then treated with KYA1797K (25 µM) for 24 h. The cells were washed with PBS, and the total RNA was isolated using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. The total RNA (2 μg) was reverse-transcribed using 200 units of reverse transcriptase (Invitrogen) in a 40 μl reaction performed at 42 °C for 1 h. The resulting cDNA (2 μl) was amplified in a 40 μl reaction mixture containing 10 mM dNTP , 10 pmol of the primer set (Bioneer), and 1 unit of Taq DNA polymerase (Invitrogen). The following primer sets were used: CTNNB1 (which encodes β-catenin), forward 5′-ACA AGC CAC AAG ATT ACA AGA A-3′ and reverse 5′-GCA CCA ATA TCA AGT CCA AGA-3′; H-RAS, forward 5′-GGA AGC AGG TGG TCA TTG-3′ and reverse 5′-AGA CTT GGT GTT GTT GAT GG-3′; N-RAS, forward 5′-AAG AGT TAC GGG ATT CCA TTC-3′ and reverse 5′-CCA TCA TCA CTG CTG TTG A-3′; K-RAS, forward 5′-AAA CAG GCT CAG GAC TTA G-3′ and reverse 5′-GTA TAG AAG GCA TCA TCA AC-3′; EGFR, forward 5′-ATG CCC GCA TTA GCT CTT AG-3′ and reverse 5′-GCA ACT TCC CAA AAT GTG CC-3′; and β-actin, forward 5′-AGA GCT ACG AGC TGC CTG AC-3′ and reverse 5′-AGC ACT GTG TTG GCG TAC AG-3′.CRCs and BT549 cells were seeded at a density of 3 × 10t tests, and statistically significant P-values are presented as follows: *P < 0.05; **P < 0.005; and ***P < 0.0005.Statistical analyses were performed using Microsoft Excel or GraphPad Prism 5 software (GraphPad Software). All data are represented as the mean ± SD. For the TMA analysis of normal-adjacent tissue (NAT) and TNBC tumor tissue, statistical analysis was also performed using GraphPad Prism 5 software. Statistically significant differences were determined using Student’s To validate the involvement of β-catenin and RAS in the tumorigenesis of TNBC, their expression levels and relationship with EGFR expression in breast cancer patient tissues were investigated by IHC analyses of TMA. Histoscores (H-scores) for nuclear β-catenin, which reflects Wnt/β-catenin pathway activity, alongside pan-RAS and EGFR expression were obtained using analysis with the IHC-Profiler plugin for NIH Image software.n = 34) compared with adjacent normal tissues    and pan-RAS , respectively  Fig.  and sign69) Fig. . To furtMoreover, we also observed a correlated change in Wnt/β-catenin and RAS-ERK/RAS-Akt pathway status in MCF10A cell lines by modification of Wnt/β-catenin signaling. The levels of β-catenin, RAS, and EGFR, as well as the RAS downstream effectors ERK and Akt, were significantly increased after Wnt3a treatment in MCF10A cells  were not affected   Fig. , and the01) Fig. . KYA179701) Fig. . In addi01) Fig. . TherefoThere is a critical need to develop new therapeutic approaches for TNBC with residual tumors after neoadjuvant chemotherapy. In this study, we demonstrate that the inhibition of both Wnt/β-catenin and RAS/ERK pathways, especially via degradation of β-catenin and RAS, could be an effective therapeutic strategy for treating TNBC patients. This approach could be especially beneficial for TNBC treatment, since EGFR, which is overexpressed in approximately 50% of TNBCs, can be transcriptionally suppressed through Wnt/β-catenin pathway inhibition. The pathological significance of our small-molecule approach, which simultaneously reduced RAS, β-catenin, and EGFR levels, was supported by their correlated overexpression in TNBC patient tumor tissues compared with the adjacent normal mammary glands in our survey using tissue microarray.30 is also conserved in TNBC, as confirmed by in vitro experiments that showed the effects of Wnt3a treatment in MCF10A.We demonstrated that the mechanism for RAS/ERK pathway regulation by Wnt/β-catenin signaling, and the co-regulation of β-catenin and RAS stability in CRCEGFR were shown in primary and established TNBC cells. This transcriptional inhibition of EGFR by KYA1797K may be caused via suppression of Wnt/β-catenin signaling, as indicated by the finding that EGFR is a transcription target of the pathway14.The suppressive effects of KYA1797K on proliferation and transformation via β-catenin and RAS degradation and the transcriptional inhibition of 13.In addition, the inhibition of mobility and invasion of TNBC cells by KYA1797K and the reduction in the corresponding biomarkers indicated that inhibition of both pathways provides a potential approach to overcome metastasis, which frequently occurs in TNBC patients31. The inhibition of 3D-cultured organoids or spheroids derived from TNBC patient cells or established TNBC cell lines after KYA1797K treatment indicates its role in CSC suppression. Finally, we also confirmed the effectiveness of KYA1797K by the growth reduction of transplanted tumors in mice that were established from MDA-MB-468 or TNBC patient tissues. The clinical significance of β-catenin and RAS destabilization, and ultimately EGFR transcriptional suppression, was confirmed by the PDX experiment using residual tumors from TNBC patients after neoadjuvant chemotherapy. Although the kinetics and degree of growth reduction after KYA1797K treatment varied between PDX-1 and PDX-2 (Supplementary Fig. Regarding the causes of recurrence and metastasis after chemotherapy in TNBC, CSCs have been reported as a major triggering factorOverall, we found that β-catenin, RAS, and EGFR proteins are correlatively overexpressed in human TNBC. We further demonstrated that KYA1797K lowered the expression of these three proteins, which suppressed the growth of TNBC cells in both in vitro and in vivo systems, as using PDX and patient primary cells. Our small-molecule approach, which suppressed both Wnt/β-catenin and RAS-ERK pathways with a subsequent reduction in EGFR expression, could be a potential therapeutic strategy for TNBC.Supplementary information


Rhynchophorus ferrugineus, is a polyphagous insect that caused economic damage in various palm species, particularly coconut plantation in Malaysia. Therefore, entomopathogenic fungus Metarhizium anisopliae was being introduced in attempts to control biologically the RPW. The entomopathogenicity of an indigenous (Met-Gra4) and foreign (Met-TH) strains of M. anisopliae isolated from the soil of Malaysia and Thailand, were tested against RPW adults in laboratory bioassays at 50, 70, 90% relative humidity (RH). Bioassays indicate no significance differences in efficacy between both the conidia of M. anisopliae strains against RPW adults. Met-Gra4 showed the highest efficacy at 90% RH (LT50 = 6.17 days). However, LT50 only slightly differed from Met-TH . Scanning el ectron microscopy for the treated RPWs showed that Met-Gra4 (90% RH) was densely sporulated within the abdomen, while Met-TH can be found mainly across cuticular surface of RPW.Red palm weevil (RPW),  Specifications table•These data provide information regarding geographical regions and fungal habitat condition influencing entomopathogenic fungal efficacy (pathogenesis and epizootiology), in this case, relative humidity that affects fungal germination and host infectivity.•Metarhizium anisopliae MetGra-4 against red palm weevil in Malaysia, as influenced by mycelial growth corresponding to the relative humidity, as the first step to select effective fungal propagule.These data show the potential effect of •Hypervirulent fungal strains will be applicable in further developing suitable mycoinsecticide formulation to improve their shelf life and enhance its viability in fluctuated environmental conditions for insect pest biocontrol.1M. anisopliae which isolated from the soil of Felda Tenang, Terengganu (Met-Gra4) and soil of Muang Chum, Kanchanaburi (Met-TH) were tested against the adults of RPW. The subsequent susceptibility test achieved zero control mortality, which confirmed the entomopathogenic effect of M. anisopliae isolates on RPW at three different relative humidity. Overall, at 50 - 90% of relative humidity, data showed that Met-Gra4 required the shortest time period to reach 50% mortality of RPW as compared to Met-TH, of which the shortest time period was achieved at 90% RH (LT50 = 6.17 days) .Two selected virulent strains of 17 days) . HoweverM. anisopliae. The observations on the cadavers of RPW illustrated that Met-Gra4 was able to germinate and cause white  and green sporulation (late stage) with slightly higher proportion as compared to Met-TH, recorded 93.33% and 86.67%, at 50% and 90% RH, respectively  clearly revealed adhesion and penetration structures in the infected adult. Growth relative humidity was at 90%; temperature 25 - 28 °C. Generally, adhesion of the ungerminated conidia for both the Met-Gra4 and Met-TH can be found on the wing scales and the appendage segments of adult RPW  of adult RPW treated with the fungus dult RPW  and 3. Tdult RPW b. In thedult RPW a.Fig. 2SOn the other hand, at day five after the death of RPW, Met-TH as declared by SEM showed a less dense network of hyphal growth within the abdominal region with least decomposed fat and muscle tissues a. While Although fungal-induced mortality of RPW occurred at all humidity level tested, the formation of the characteristic white mycelial growths and conidia only occurred within the 70 to 90% RH ranges (figures not shown). Mycelial growth first appeared at seven days post-treatment. No external fungal growth of any kind was found on cadavers within the 50% RH.22.1Hypervirulent fungal strain from Malaysia, named as Met-Gra4 was kindly provided by Grace Lee Ern Lin, Universiti Malaysia Terengganu −8 dilution using standard techniques. Afterwards, 10 μm diluted suspensions of 10−7 and 10−8 dilutions were pipetted and spread on PDA medium plates. Two plates for each soil samples were incubated for 5 – 7 days at room temperature, and based on the morphological appearance, the potential fungal colonies of readily sporulating M. anisopliae which characterized by green conidia, were then subcultured by aseptically transferring the inoculum for streaking onto the freshly prepared PDA, plating in Petri dishes, and incubated at room temperature until mycelial growth has appeared. The 5th day after incubation, the fungal culture plates were observed and examined for any sorts of undesirable contaminants, in order to obtain a pure fungal culture. Contemporarily, successive monoxenic subculturing on artificial media recurrently leads to attenuation of fungal virulence. In an attempt to restore fungal virulence after prolonged culture on PDA, each isolate was passed through RPWs prior to culture on plates. Each of the dead RPWs was removed and taken to Petri dishes with moist filter paper-lined bottom, and sealed with Parafilm “M”  to enhance sporulation, as for the use in the consecutive bioassays.Isolation of EPF was performed by using 10X serial dilution method followed by spread plating of soil sample on artificial media. Soil samples were prepared by first grinding to smaller particles or in a powdery form using sterilised mortar and pestle. About 1 g of each soil sample was diluted in a master tube containing 10 mL of distilled water, and these suspensions were diluted up to 102.22.2.1RPWs were caught by using pheremone traps placed at coconut plantation in/ near Kampung Kubang Badak, UMT campus and Pantai Tok Jembal. In addition, adult weevils that were field-collected using pheromone traps, were baited with high release formulated lures, including ferrugineol (4-methyl-5-nonanol), Ferrolure+ (90% 4-methyl-5-nonanol + 10% 4-methyl-5-nonanone) and kairomone-releasing food bait (pineapple). Captured adult weevils were reared under 20 °C in air-conditioned culture room, placing in plastic rearing containers. Sugarcanes were baited and supplanted twice a week. In the subsequent tests, the field-collected RPWs without any defect or damage, with its body length within the range of 3.0 – 3.5 cm were selected. Mixed-sex adult RPWs were precleaned with running tap water before surface sterilized with 70% alcohol for 10 s, continued by immersing in sterile distilled water 3 times consecutively.2.3M. anisopliae on PDA plates were incubated at 28 °C for 2 weeks. In preparing and making initial conidia suspension, the conidia was gently scraped off from the PDA surface by adding sterilised 0.02% aqueous Tween 80Ⓡ solution into the culture plates. The premixed conidia suspensions were then centrifuged at 5000 rpm for 15 min to separate the conidia from the mycelium and other waste 8 conidia mL−1 based on the formula below:C1 = Concentration of the stock solution, conidia per mLV1 = Calculated volume required to be taken out from the stock solution, mLC2 = Desired concentration to be prepared, conidia per mLV2 = Volume a of desired concentration, mLFor conidia production, subcultures of 2.48 conidia/ insect, thereafter inoculated RPWs were maintained in containers at different relative humidity and room temperature. Each container was perforated with only two small holes to minimize gaseous exchange. The deliquescence relative humidity was thermodynamically manipulated using supersaturated salt solutions with pure hygroscopic salt minerals: potassium carbonate (50%), sodium chloride (70%), potassium chloride (90%), in accordance to the experimental value provided 2 and distilled water, provided 0% and 100% relative humidity, respectively. Approximately 100 mL of saturated salt solution was placed within the container according to the treatment. Each RH treatment was conducted using: three replicates with five inoculated RPWs per container, and three replicates with five uninoculated RPWs as control treatment. Entomopathogen-induced behavior alterations among the inoculated RPWs for each fungal isolates were observed and assessed daily for 2 weeks post-treatment. Dead cadavers were surface-sterilised for 3 min in 2.5% NaClO solution to avoid secondary fungal contaminants prior to incubation under darkness with corresponding RH condition at 25 ± 3 °C. Observation and photographic record of sporulating cadavers were conducted for 10 days.Disinfected RPWs were dipped in the prepared conidial suspension for 120 s, providing a dose of 102.5M. anisopliae. Micromorphology of the R. ferrugineus cuticular cross-section was observed and photographed using SEM. High quality reference images of fungal germ tube penetration and development were documented.The surface morphological investigation was in accordance to ,5 with s2.6The mortality mean value of the five replicates for each treatment was used throughout the statistical analysis. The fungal infectivity percentages towards RPW in four different treatments was corrected by eliminating the natural mortality in control treatment (0 − 5%) conforming to the Schneider Orelli's formula:50 at different treatment of relative humidity. Two-way ANOVA with Tukey's post hoc analysis was conducted to determine whether there were significant differences in the total mortality of RPW between fungal isolates at the different treatments. All tests were carried out by using IBM SPSS Statistics 24.Subsequently, the corrected mortality data was subjected to probit analysis to determine the significance for LT