Min/+ mice intestinal organoids revealed that p38α acts as a β-catenin chromatin-associated kinase required for the regulation of a signaling platform involved in tumor proliferation, metastatic dissemination, and chemoresistance in these CRC model systems. In particular, the p38α kinase inhibitor ralimetinib, which has already entered clinical trials, promoted sensitization of patient-derived CRC-SCs to chemotherapeutic agents commonly used for CRC treatment and showed a synthetic lethality effect when used in combination with the MEK1 inhibitor trametinib. Taken together, these results suggest that p38α may be targeted in CSCs to devise new personalized CRC treatment strategies.The prognosis of locally advanced colorectal cancer (CRC) is currently unsatisfactory. This is mainly due to drug resistance, recurrence, and subsequent metastatic dissemination, which are sustained by the cancer stem cell (CSC) population. The main driver of the CSC gene expression program is Wnt signaling, and previous reports indicate that Wnt3a can activate p38 MAPK. Besides, p38 was shown to feed into the canonical Wnt/β-catenin pathway. Here we show that patient-derived locally advanced CRC stem cells (CRC-SCs) are characterized by increased expression of p38α and are “addicted” to its kinase activity. Of note, we found that stage III CRC patients with high p38α levels display reduced disease-free and progression-free survival. Extensive molecular analysis in patient-derived CRC-SC tumorspheres and APC The survival rate of affected patients largely depends on the stage at which the tumor is diagnosed. About one-third of CRC patients have stage III disease, which is characterized by spread to regional lymph nodes and absence of distant metastases2. Stage III CRC patients are at high risk for tumor recurrence, and their overall prognosis, for which the N stage has been found to be a reliable indicator, remains unsatisfactory even with curative surgery and adjuvant chemotherapy3. Indeed, it is reported that more than one-third of stage III CRC patients will develop recurrence or metastasis within 5 years of systemic therapy4.Colorectal cancer (CRC) is the third most frequent malignancy but the second leading cause of death for tumor worldwide5. Therefore, new treatment approaches targeting CRC-SCs are needed in order to achieve complete tumor eradication6.CRC stem cells (CRC-SCs) are involved in drug resistance, tumor recurrence, and metastasis after primary treatment. Indeed, conventional therapies wipe out bulk tumor populations, while CRC-SCs are resistant to chemotherapy and radiotherapy7. During carcinogenesis, increasing amounts of β-catenin resulting from APC inactivation translocate into the nucleus and modulate the transcriptional activity of TCF/LEF transcription factors8. High levels of nuclear β-catenin lead to constitutive activation of the Wnt pathway, loss of normal cellular architecture, and neoplastic conversion9.Several dysregulated signaling pathways confer to cancer stem cells (CSCs) a survival advantage over current therapies; among these pathways, the main driver controlling CSC fate is Wnt signaling10 and is the main p38 isoform in colorectal and ovarian cancer cells13. Importantly, Wnts can activate p38 MAPKs. Indeed, Wnt3a was recently shown to stimulate p38 activation in mouse F9 teratocarcinoma cells. Of note, Wnt-induced p38 activation appears to regulate canonical Wnt/β—catenin signaling8, and p38 was found to phosphorylate GSK3β at Thr390, which inactivates GSK3β kinase activity, leading to β-catenin accumulation14.Previous reports indicate that p38α, one of the four p38 isoforms , is highly expressed in colorectal neoplasms compared to normal mucosa17. Recently, we demonstrated the existence of a p38α-ERK synthetic lethality crosstalk that is crucial for CRC therapy response. Indeed, combined inhibition of p38α and MEK1 efficiently reduced the volume of xenografted tumors and colitis-associated orthotopic tumors in vivo12. Besides, resistance to cisplatin (CDDP), irinotecan (CPT-11), and 5-fluorouracil (5-FU) chemotherapy has been shown to involve MAPK signaling, and recent studies identified p38α MAPK as a mediator of resistance to various agents in CRC patients13. Our previous studies also revealed that p38α inhibition sensitizes chemoresistant CRC cells to CDDP, with the combined treatment inducing Bax-dependent apoptosis in both chemosensitive and chemoresistant cells18.Our previous results indicate that p38α is required for CRC cell proliferation and survival, and its inhibition induces growth arrest, autophagy, and cell death both in vitro and in vivo19. p38α also phosphorylates MSK1, which in turn phosphorylates Ser10 on histone H3, inducing a transcriptional activation-permissive chromatin modification20. Additionally, p38α can physically interact with RNA polymerase II and promote the transcription elongation step21.p38α is considered a prototypical chromatin-associated kinase. Indeed, it can associate with and phosphorylate several transcription factors and can recruit subunits of the SWI/SNF ATP-dependent remodeling complexes directly to the DNA, thereby modulating chromatin structure and transcription22 and mediates CSC drug resistance to oxaliplatin and anti-angiogenic agents23. Moreover, p38-inhibited cells showed decreased expression of CSC markers and reduced sphere-forming ability in head and neck squamous cell carcinoma24.In recent years, a role has emerged for the p38 pathway in CSC regulation. Indeed, p38 seems to promote survival in hypoxic and serum-starved CRC-SCsHere, we performed an extensive characterization of p38α in patient-derived stage III CRC-SCs, identifying it as a direct interactor of β-catenin, the key element of the Wnt pathway. p38α acts as a chromatin-associated kinase involved in the activation of β-catenin target gene transcription, and its pharmacological manipulation affects various cancer features. Importantly, we show that targeting p38α may overcome chemoresistance in a CRC-SC model, with p38α levels being a potential new marker of therapeutic efficacy in stage III CRC patients.Z-score and classified them as p38α high (80 patients) or p38α low (91 patients) to investigate the association between p38α expression and prognosis. We found that high expression of p38α was associated with worse disease-free survival  and progression-free survival   PanCancer Atlas. This dataset encompasses clinical data of 580 CRC patients with stage I–IV disease. We stratified the 171 stage III CRC patients based on p38α mRNA level 82) Fig. .Fig. 1p325. We thus characterized various CRC cell lines established from stage III CRC patients and grown as tumorspheres by analyzing their mutation status, chromosomal and microsatellite instability (MSI), and expression of a group of surface markers  or activator  confirmed p38α–β-catenin nuclear/cytoplasmic co-localization under all treatment conditions also in these cells Fig. S.27, and found that p38α directly interacts with β-catenin in vitro  experiments performed in HEK293 cells transiently transfected with HA-tagged p38α and FLAG-tagged β-catenin confirmed that this interaction also occurs ulo Fig. .Next, we evaluated whether endogenous p38α is a partner of β-catenin complexes in our colorectal model systems. Immunoprecipitation of whole-cell lysates with an antiserum against p38α or β-catenin, followed by immunoblotting, indicated that p38α is a molecular partner of β-catenin complexes in these cells Figs. ; S3A.Further immunoprecipitation experiments in HCEC-1CT, HT29 cells, and patient-derived CRC-SCs subjected to a cellular fractionation protocol revealed that p38α and β-catenin co-immunoprecipitate mainly in the cytoplasm in normal colonocytes but predominantly in the nucleus in CRC cells and patient-derived CRC-SCs Fig. ; S3B.in cellulo, we performed in vivo experiments in APCMin/+ mice, which are heterozygous for a missense mutation in the APC gene and model human familial adenomatous polyposis (FAP) as they develop multiple intestinal polyps that acquire carcinoma features after exposure to the carcinogen drug azoxymethane (AOM)28. We found increased levels of p-p38, p-ERK, β-catenin, and c-Myc in mouse colon adenoma and adenocarcinoma compared to normal colon mucosa . HT29 cells were serum-starved to inhibit β-catenin activity in the nucleus and then switched to a serum-containing medium. ChIP assays revealed that serum mitogens dramatically stimulated β-catenin and p38α recruitment to Wnt responsive elements (WREs) of several β-catenin target genes, including c-Myc, c-Met, Survivin, and CD44, which are all involved in CRC progression Fig. . SimilarSubsequently, we investigated the effect of p38α pharmacological inhibition  or genetic ablation (with two specific siRNAs) on the regulation of β-catenin target gene expression. Real-time PCR experiments showed that treatment of HT29 cells with ralimetinib or specific siRNAs leads to the downregulation of β-catenin target genes, including CD44 and Cyclin D1 (cell cycle markers), Survivin (apoptosis inhibition), c-MET (migration and invasion), and SOX9 and TCF7 (CSC proliferation markers) Fig. . These d30. We thus searched for novel β-catenin residues that could be directly phosphorylated by p38α. Since 85% of the p38α phosphorylation sites described so far are Ser-Pro or Thr-Pro motifs31, we performed an in silico phosphorylation prediction analysis with DISPHOS 1.3, NETPHOS 3.1, Phosida, iPTMnet, and Phosphosite Plus servers, focusing on serine and threonine residues. We identified eight putative phosphosites  that were recognized by at least four prediction servers  once a week for 5 weeks; one month later, they were subjected to daily intraperitoneal injections of SB202190 (0.05 μmol/kg body weight) or DMSO for 14 days and then sacrificed , such as 5-FU, CDDP, CPT-11, or trametinib, a MEK1 inhibitor that is already approved for clinical use Fig. .To this end, CRC-SCs pre-treated with ralimetinib for 48 h were subsequently treated with CHTs/trametinib for 24 h. Our results revealed that the combined therapeutic strategy  has nonlinear cumulative effects and is more effective than CHTs/trametinib alone. Indeed, pre-treatment with ralimetinib reduced CRC-SC proliferative index Figs. ; S7A andThen, we performed a soft agar assay to assess the ability of patient-derived CRC-SCs to form colonies of anchorage-independent tumor cells. Our results showed that combined treatment with ralimetinib and CHTs or trametinib almost completely abolishes CRC-SC clonogenic activity compared to each single treatment Figs. ; S7C.Since patient-derived CRC-SCs grow as spheres, we also performed a spheroid-based migration assay to assess their invasive capacity. We found that co-treatment with ralimetinib and CHTs or trametinib leads to a remarkable decrease in CRC-SC migratory ability Figs. ; S7D.34. Based on our results, pre-treatment with ralimetinib enhanced the growth-inhibitory activity of CHTs and trametinib .Here, we identified p38α as a new druggable member of β-catenin chromatin-associated kinase complexes in colorectal model systems . Moreover, we showed that CRC cells and patient-derived CRC-SCs have higher levels of activated p38α than normal colonocytes and are “addicted” to p38α activity.Interestingly, our meta-analysis on a cohort of colorectal tumors retrieved from TCGA PanCancer Atlas dataset correlated p38α mRNA levels to stage III disease prognosis, with high p38α expression being associated with worse DFS and PFS. Based on these findings, p38α may be used as a marker of resistance and a predictor of therapy response in CRC.We also demonstrated that p38α acts as a β-catenin chromatin-associated kinase involved in tumor proliferation, metastatic dissemination, and chemoresistance. Indeed, our data suggest that p38α serves as a regulator of gene expression by interacting with β-catenin-TCF/LEF transcriptional complexes that are recruited to WREs. The recruitment of protein kinases to promoter regulatory elements can have important functional consequences; in particular, these proteins may represent new therapeutic targets in cancer cell signaling.37, and the other at the chromatin level, by interacting with and phosphorylating β-catenin on WREs, as shown by our data.Our analysis of p38α recruitment on β-catenin target gene promoters using ChIP re-ChIP assays suggests that the Wnt signaling pathway likely converges on p38α and β-catenin, which co-regulate target gene expression. Indeed, p38α feeds into the Wnt pathway at least in two different ways: one in the cytoplasm, at the level of GSK3β, as demonstrated by Thornton and colleagues39. Interestingly, we found that p38α pharmacological inhibition does not prevent the formation of the p38α–β-catenin complex but downregulates several β-catenin target genes, including c-Myc, in a manner comparable to the Wnt/β-catenin inhibitor PRI-724, which is currently being evaluated in clinical trials . Moreover, our results revealed that both the CSC and the differentiated cell compartments of CRC-SC cultures are efficiently targeted by treatment with the p38α kinase inhibitor ralimetinib.Activation of p38α and nuclear β-catenin is observed in many tumors, and several genes targeted by these signaling pathways are crucial for cancer development and progression. For instance, it has been demonstrated that c-Myc, the main Wnt/β-catenin target, is consistently overexpressed in CRC-SCs and its downregulation suppresses CRC-SC self-renewal and xenograft growthIn our model systems, the combined use of CHTs and ralimetinib proved more effective than CHTs alone.Min/+ mice organoids.In particular, ralimetinib promoted sensitization of patient-derived CRC-SCs to CHTs commonly used in CRC therapy, such as 5-FU, CDDP, and CPT-11, as shown by a reduction in Ki67-positive cells and induction of apoptosis. These combined treatments also affected key CRC-SC features that contribute to tumor aggressiveness and metastatization, including their colony-forming and migratory ability. Moreover, ralimetinib showed a synthetic lethality effect when used in combination with the MEK1 inhibitor trametinib in patient-derived CRC-SC tumorspheres, in a human 3D culture system, and in APC45. In particular, ralimetinib displayed a tolerable safety profile, with preliminary evidence of antineoplastic activity in a recent phase I trial  in patients with advanced or metastatic cancer. Moreover, it is currently being tested in combination with other agents in an ongoing phase I study in CRC patients .Overall, our results confirmed the crucial role of the p38α–β-catenin axis in the regulation of intestinal tumorigenesis, suggesting that p38α manipulation could be an effective therapeutic approach in stage III CRC patients. Indeed, it may be used to target CRC-SCs in addition to bulk tumor populations to counter uncontrolled proliferation, metastatic dissemination, and chemoresistance. Several p38α inhibitors passed phase I clinical trials and are currently in phase II or III for inflammatory diseases and cancerAltogether, our preclinical data support further clinical development of ralimetinib as a sensitizing agent to commonly used CHTs and suggest the potential of a synthetic lethality approach based on p38α and MEK1 inhibition.CRC tissues were obtained from four patients at the time of resection, in accordance with the ethical standards of the Institutional Committee on Human Experimentation  after informed consent.38. All cell lines were tested to be mycoplasma-free . Human intestinal 3D cultures were generated from CRC-SCs as previously described46. Mouse intestinal organoids were generated from APCMin/+ male mice as previously described48. All cell cultures were performed in a 37 °C and 5% CO2 incubator.HCT116 and HT29 cells were cultured in DMEM  with 10% FBS  and 100 IU/ml penicillin–streptomycin . HEK293 cells were supplemented with 1% pyruvate  and 1% NEAA . HCEC-1CT cells were cultured in COLO-UP (Evercyte) medium supplemented with 100 IU/ml penicillin–streptomycin . #8, #9, #21, and #40 cells were isolated and propagated from CRC patients as previously described5-FU (F6627), Wnt3a (H17001), LiCl (L9650), and Trypan blue (T8154) were purchased from Sigma-Aldrich. Cisplatin (S1166), Ralimetinib (S1494), Trametinib (S2673), Irinotecan (S2217), PRI-724 (S8262), and SB202190 (S1077) were purchased from SelleckChem.E. coli (C2527I) were purchased from New England Biolabs and were used for all cloning experiments. Cells were grown in standard LB media. Plasmids were generated as previously described49. The pcDNA-human-β-catenin (#16828) plasmid was purchased from Addgene. Primer sequences are listed in Supplementary Table Efficiency DH5α Competent Cells (C2987H) and BL21 (DE3) Competent HEK293 cells were transfected with mammalian expression plasmids using Lipofectamine 3000  according to the manufacturer’s instruction. For RNA interference, HT29 cells were transfected with 50 nM validated siRNAs (Ambion) directed against MAPK14 using the HiPerfect reagent  according to the manufacturer’s instructions. siCTRL (Eurofins) was used as a non-silencing control. siRNA sequences are listed in Supplementary Table BL21 competent cells, transformed with different constructs, were grown in LB medium with antibiotics and induced with IPTG. Cells were lysed with B-PER lysis buffer . GST-fusion proteins were purified with Pierce Glutathione Magnetic Agarose Beads  according to the manufacturer’s instructions. His-fusion proteins were purified with Dynabeads His-Tag Isolation and Pulldown  according to the manufacturer’s instructions.10. Primary antibodies: anti-β-actin (#3700), anti-c-Myc (#9402), anti-p44/42 MAPK (Erk1/2) (#9102), anti-phospho p44/42 MAPK (Erk1/2) (Thr202/tyr204) (#9106S), anti-p38 MAPK (#9212), anti-p38α MAPK (#9228), anti-phospho-p38 MAPK (Thr180/Tyr182) (#9211), anti-Lamin B1 (#12586), anti-Keratin20 (BK13063S), anti-PDI (#2446S), anti-β-catenin (#9562), anti-GST (#2625), anti-CD133 (#5860), anti-CD44 (#3570), anti-Musashi (#2154) all from Cell Signaling Technologies, anti-HIS-tag (H1029), anti-FLAG (F1804), anti-HA-tag (H3663) all from Sigma-Aldrich, anti-lgr5 (GPCR) (75732) from Abcam, HSP90 α/β (sc13119) from Santa Cruz Biotechnology, p-p38 MAPK α  (MA5-15177) from Invitrogen and anti-phospho-p38α (MAB8691) from R&D Systems. Rabbit IgG HRP and Mouse IgG HRP  were used as secondary antibodies and revealed using the ECL-plus chemiluminescence reagent . Densitometric evaluation was performed by ImageJ software.Nuclear and cytoplasmic fractions were obtained by using the Nuclear Extraction Kit  according to the manufacturer’s instructions. Immunoblots were carried out as previously described10. Cells were lysed with the Nuclear Extraction Kit  according to the manufacturer’s instructions. Primary antibodies: p38α , β-catenin , and p-p38 MAPK α  . IgG was used as a negative control.Co-IP was carried out as previously described4 cells/plate were stained with Muse Annexin V and Dead Cell Reagent (Luminex MCH100105) according to the manufacturer’s instructions.2 × 10Cells were seeded on glass coverslips, fixed in 4% paraformaldehyde, and permeabilized using 0.01–0.1% Triton X-100. Coverslips were incubated with the indicated primary antibodies and then with Alexa Fluor 488  and 647 (#A-32728) secondary antibodies; nuclei were counterstained using DAPI . Slides were sealed using Vectashield Mounting Medium . Images were acquired using a Zeiss fluorescence microscope. Primary antibodies: p38α  and β-catenin .10. Primer sequences are listed in Supplementary Table RNA extraction and real-time PCR were performed as previously described2O. Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 10 min. Antigen retrieval was conducted in 10 mM sodium citrate buffer (pH 6.0) for 30 min. Sections were incubated overnight with the primary antibodies: p-p38α , β-catenin , c-Myc , cyclin D1 , and CD44v6 . Then, they were incubated with secondary biotinylated antibodies and subsequently with streptavidin–biotin–peroxidase . Samples were developed with DAB and mounted with permanent mounting media. Negative controls were used in each experiment. p-p38α, β-catenin, c-Myc, Cyclin D1, and CD44v6 immunoreactivity was evaluated by a semiquantitative approach by two independent pathologists, in a blinded manner, who scored the percentage of positive-stained cells and the intensity of the staining .Tissue specimens were formalin-fixed in 4% buffered formalin, embedded in paraffin, sectioned at 4 mm thickness, and stained with hematoxylin and eosin. Additional sequential sections (3–5 μm) were cut and used for immunohistochemical analysis. Sections were dewaxed and rehydrated in dHImmunocytochemical analysis was performed on cytospins using p-p38 MAPK α  (MA5-15177) (1:100) from Invitrogen. Single staining was revealed using a biotin–streptavidin system (Dako) and detected with 3-amino-9-ethylcarbazole (Dako). Nuclei were counterstained with aqueous hematoxylin (Sigma).Chromatin isolated from HT29 cells and CRC-SCs was subjected to chromatin immunoprecipitation using the MAGnify Chromatin Immunoprecipitation System  according to the manufacturer’s instructions. Chromatin was sonicated to a fragment length of about 200–500 bp and immunoprecipitated with 1 µg of rabbit IgG: p38α  and β-catenin . For re-ChIP, immune complexes were eluted with elution buffer  for 30 min at 37 °C, diluted with the dilution buffer provided in the kit, and subjected to immunoprecipitation with a second antibody of interest. Primer sequences are listed in Supplementary Table His-β-catenin recombinant human protein was incubated with GST-p38α fusion protein. p300 (302-530)-GST fusion protein was used as a positive control. Fusion proteins were precipitated with Dynabeads His-Tag Isolation and Pulldown  according to the manufacturer’s instructions. Primary antibodies: polyHistidine  and GST . Rabbit IgG HRP and Mouse IgG HRP  were used as secondary antibodies and revealed using the ECL-plus chemiluminescence reagent .6 cells, BD Biosciences). Cells were washed with 2% BSA and 2 mM EDTA in PBS and filtered with a 70 µm mesh to prevent cell clogging. Isolation of CD44v6low and CD44v6high cells was performed by using the FACSMelody cell sorter. Post-sorting analysis was performed to verify the purity of sorted populations.For CRC-SC sorting, cells were collected, washed in PBS, and stained for 1 h at 4 °C with conjugated antibodies specific for CD44v6  or a corresponding isotype-matched control (IMC). Dead cell exclusion was performed by using 7-AAD (0.25 µg/1 × 106 cells/ml). After 24 h, colcemid-treated CRC-SCs were incubated in a hypotonic solution, fixed in a chilled fixative solution, and then washed extensively. Chromosomes were counted using an Olympus microscope.CRC-SCs were seeded at high density  using the MSI Analysis System kit . Amplified fragments were detected by loading the PCR products for capillary electrophoresis using an ABI Prism 3500 Genetic Analyser and the POP-4 polymer  according to the manufacturer’s instructions. MSI status was determined upon analysis with GeneMapper software, Version_4.1 (Applied-Biosystems).For targeted deep DNA re-sequencing, the sequencing library was prepared as previously described® AQueous One Solution Cell Proliferation Assay  (MTS) was performed according to the manufacturer’s instructions and analyzed by using the GDV MPT reader (DV 990 BV6).For cell viability assays, viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay Kit  according to the manufacturer’s instructions. The luminescent signal was read using a SPECTROstar Omega microplate reader (BMG Labtech). The CellTiter 9617. Human intestinal 3D cultures and APCMin/+ mouse intestinal organoids were stained using the LIVE/DEAD® Cell Imaging Kit  according to the manufacturer’s instructions.For cell death assays, cell death was assessed by cell counting as previously describedFor clonogenic assays, dissociated CRC-SCs were plated in triplicate at 500 cells/well suspended in 0.3% agarose over a layer of 0.5% agarose and treated as indicated.4 control or treated CRC-SCs were suspended in 200 μl of non-supplemented stem cell medium and plated into the upper wells of Matrigel-coated Boyden chambers containing 8 μm diameter polycarbonate membranes . Lower wells contained 600 μl of stem cell medium supplemented with 20 ng/ml EGF and 10 ng/ml basic FGF and the relevant drugs.For motility assays, 1 × 10For proliferation assays, CRC-SCs treated as indicated were analyzed to determine the percentage of proliferating cells based on Ki67 expression using the Muse Ki67 Proliferation Kit  according to the manufacturer’s instructions.For the luciferase assay, HEK293 cells were lysed with 100 µl Passive Lysis  and the assay was performed according to the manufacturer’s instructions.Analysis of p38α kinase activity was performed using the ADP-Glo Kinase Assay  according to the manufacturer’s instructions. p38α active protein  was assayed in a kinase reaction buffer with 50 µM ATP and varying concentrations of human recombinant β-catenin . 1 μg of p38 peptide substrate was used as a control. The generated luminescence was measured using a luminometer .low- and CD44v6high-enriched cells were plated at 1, 2, 4, 8, 16, 32, 64, and 128 cells per well in 96-well plates. Clonal frequency was calculated using the extreme limiting dilution analysis (ELDA) tool (http://bioinf.wehi.edu.au/software/elda/index.html).CD44v6n = 12), APCMin/+ mice (n = 12), and APCMin/+ mice (n = 24) treated with 14 mg/kg of AOM , respectively. Four-month-old APCMin/+ male mice were administered with AOM (14 mg/kg body weight) once a week for 5 weeks; one month later, they were subjected to daily intraperitoneal injections of the p38α inhibitor SB202190 (0.05 μmol/kg body weight) or DMSO for 14 days and then sacrificed. Body weight was recorded daily. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that comply with national and international laws and policies.For in vivo studies, normal, adenoma, and adenocarcinoma colon mucosa tissues were obtained from C57BL/6 mice (Z-scores) of 592 CRC patients and TNM stage clinical data of 580/592 patients were obtained from TCGA PanCancerAtlas through the cBioPortal website50. Patients were stratified based on p38α mRNA Z-score into two groups with high  or low  p38α mRNA expression. Statistical analysis was performed using R (version 3.6.2), an open-source freely available software environment for statistical computing and graphics. Survival curves of stage III CRC patients (n = 171) were assessed according to the Kaplan–Meier method, and DSF and PFS were used as the endpoint. Differences between stage III CRC patients with high p38α mRNA (n = 80/171) and low p38α mRNA (n = 91/171) were assessed using the log-rank test and R packages “survival” and “survminer”53.To study the association between p38α mRNA expression levels and CRC aggressiveness, RNA-seq gene expression data (In silico prediction analysis was performed using DISPHOS 1.3, NETPHOS 3.1, Phosida, iPTMne, and Phosphosite Plus servers.t-tail test, and P < 0.05 was considered statistically significant. Results are representative of at least three independent experiments.The statistical significance of the results was analyzed using the Student’s supplementary tablesupplementary figure legendsSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplementary Figure 7


S2 splitting interval was significantly correlated with IRD in each experiment (p < .001). In conclusion, the START algorithm can accurately assess S2 splitting and may serve as a useful tool to assess interventricular dyssynchrony.Second heart sound (S2) splitting results from nonsimultaneous closures between aortic (A2) and pulmonic valves (P2) and may be used to detect timing differences (dyssynchrony) in relaxation between right (RV) and left ventricle (LV). However, overlap of A2 and P2 and the change in heart sound morphologies have complicated detection of the S2 splitting interval. This study introduces a novel S‐transform amplitude ridge tracking (START) algorithm for estimating S2 splitting interval and investigates the relationship between S2 splitting and interventricular relaxation dyssynchrony (IRD). First, the START algorithm was validated in a simulated model of heart sound. It showed small errors (<5 ms) in estimating splitting intervals from 10 to 70 ms, with A2/P2 amplitude ratios from 0.2 to 5, and signal‐to‐noise ratios from 10 to 30 dB. Subsequently, the START algorithm was evaluated in a porcine model employing a wide range of paced RV‐LV delays. IRD was quantified by the time difference between invasively measured LV and RV pressure downslopes. Between LV pre‐excitation to RV pre‐excitation, mean S2 splitting interval decreased from 47 ms to 23 ms ( We introduce an S‐transform amplitude ridge tracking (START) algorithm for estimation of 2nd heart sound (S2) splitting and investigated the relationship between S2 splitting and time difference in contraction between right and left ventricle in a porcine model. START was accurate in estimating S2 splitting interval in chirp‐model‐generated heart sounds. S2 splitting correlated well in pacing‐induced time differences in contraction of the ventricles in an animal model. PhysiolEstimating heart sound splitting interval has long been a challenge because of the overlap of heart sound components. Auscultatory detection of S2 splitting is possible at intervals of at least 40 ms, whereas detection of intervals of 20–40 ms is only feasible with low ambient noise and extensive listener's experience . It firThe duration of each component, t, is defined as 0 ≤ t ≤ 60 ms. A and φ represent the amplitude and phase function of A2 and P2, respectively:ampA and ampP represent the normalized amplitude of A2 and P2, respectively. In this study, we used a constant normalized amplitude of P2, so ampP = 1. Phase functions (5) and (6) control the frequency range of A2 and P2, respectively. To validate the START algorithm, we varied the splitting interval from 10 ms to 70 ms; ampA from 0.2 to 5.0; and signal‐to‐noise ratios (SNRs)from 10 to 30 decibels (dB).2.4Open‐chest sacrifice pig experiments were performed in accordance with the Dutch Law on Animal Experimentation and the European Directive for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes. The protocol was approved by the Central Committee for Animal experiments (CCD) in The Netherlands and the Animal Experimental Committee of Maastricht University.Five male adult pigs (weight: 64 ± 1 kg) were premedicated with prophylactic antibiotics (ampicillin 1000 mg I.V.) and thiopental  for induction of general anesthesia  to RV (A‐RV) pacing delay (150 ms) and varying A to LV (A‐LV) pacing delays (50 ms to 250 ms), using 25 ms in the 1st experiment or 50 ms per step in the remaining 4 experiments.During each pacing setting, ECG and hemodynamic signals were collected for 20–30 seconds using the IDEEQ data acquisition system (IDEE Maastricht University / Maastricht Instruments BV). Accelerometer signals were collected using a custom‐made data acquisition system. Hemodynamic signals and accelerometer signals were aligned using a synchronous pulse signal. Hemodynamic analysis was performed using the IDEEQ software, developed at Maastricht University.2.5IRD was defined as the time difference between the downslopes of LV and RV pressure curves Figure . Pressur2.6From data acquired during the animal experiments, a combined accelerometer signal was calculated from the raw signals of X, Y, and Z directions, and double integrated to obtain a displacement signal. A second‐order Butterworth bandpass filter of 50–250 Hz was applied. Locations of S1 and S2 were identified with reference to lead II electrocardiogram (ECG). To reduce the effect of any sudden vibrations and background noises on manual identification, we overlapped all heartbeats and calculated a median heart sound signal as a reference. Premature ventricular contraction beats and their two subsequent heartbeats as well as heartbeats with too much noise were discarded. Finally, the resulting S2 signal was processed with the START algorithm to obtain splitting interval.2.7To clarify the role of our START algorithm in comparison with other existing heart sound splitting detection algorithms, we searched the PubMed database using terms of “heart sound” and “split*” on October 27, 2020. Date of publication was from the year 1970. Each publication was first judged by title and abstract. Publications including reviews, letters, and case reports were excluded. Potential articles were further screened for full text. The search approach was complemented by consulting experts in the field for potential related studies. We included original studies which described heart sound splitting detection algorithms. The following information was extracted from each eligible article: first author name, year of publication, brief description of the algorithm, whether the algorithm works on a single heartbeat, and whether a validation study was performed. Validation study could be any of the following: 1) validation of the algorithm in various splitting intervals; 2) validation of the algorithm in various A2/P2 amplitude ratios; or 3) validation of the algorithm in various SNRs.2.8Hemodynamic data and summary of correlation data were expressed as mean and standard deviation. S2 splitting interval and IRD were expressed as median  to reduce the potential effect of respiration on our analysis. Pearson's correlation was calculated between START‐estimated splitting interval and simulated splitting interval as well as between estimated splitting interval and IRD during animal experiments. Spearman's rank correlation was calculated between splitting interval and A‐LV delay as well as between IRD and A‐LV delay. A P value less than 0.05 was assumed to indicate a statistically significant difference. All statistical analyses were performed using MATLAB R2018b and Stata/MP 14.0.33.12 = 1, p < .001)  Figure . Mean es) Figure . From SN) Figure .3.2Table p < .001).The left panels of Figure p < .001). It is noteworthy that like S2 splitting interval in Figure p < .001). Table Figure 3.3Searching the PubMed database using “heart sound” and “split*” resulted in 120 publications. After excluding six reviews, three letters and 35 case reports, the remaining 76 publications were checked for description of heart sound splitting detection algorithm. Two publications were recommended by experts in the field  the START algorithm for estimating S2 splitting interval is accurate in a wide range of splitting intervals, A2/P2 amplitude ratios, and SNRs; and 2) START‐estimated S2 splitting interval is significantly correlated with paced interventricular delays and IRD, though no correlations are observed for long A‐LV delays in porcine models probably because of the fact that the LV is activated even before the moment of pacing. To our knowledge, our study is the first to investigate the possibility of using a heart sound indicator to monitor interventricular dyssynchrony in CRT‐like settings. As a simple measurement, heart sound is likely to serve as a promising real‐time monitoring approach for patients with interventricular dyssynchrony.4.1Distinguishing the time difference between the two components of S2, that is, A2 and P2, has long been a challenge because of their overlap within a short period of time. Our proposed algorithm works for a small splitting interval down to 10 ms, for various A2/P2 amplitude ratios and for low SNRs. Previous studies using modeling approaches to extract splitting interval assumed a standard template of heart sound which remains debatable  of patients with RV pacing did not show any reversed splitting of S2 , left ventricular systolic time (time difference between S1 and S2), and S3 strength (Taha et al., While these parameters mainly reflect contractility, the START‐based S2 splitting is the only indicator of interventricular dyssynchrony from heart sound so far and may therefore be of additional value on top of the aforementioned indicators. It can be imagined that S2 splitting can be used to guide pacing lead location during CRT implantation as well as in CRT optimization during follow‐up, either manually during outpatient visits or, when accelerometers are incorporated in pacing leads and or pacemakers, in ambulatory fashion.5Translating results from animal experiments to clinical applications should be done with caution. First of all, the conduction system in the pigs differs to some extent from that of humans, resulting in narrower QRS complexes during ventricular pacing. This likely also results in smaller interventricular dyssynchrony. The fact that START algorithm can detect the small S2 splitting intervals in pigs suggests therefore, that this certainly will be possible in humans. Second, the pigs had normal cardiac function, whereas humans eligible for pacing treatments may have depressed cardiac function. Third, the number of experiments was small and measurements were performed during anesthesia and with an open chest.6The proposed START algorithm is accurate in estimating S2 splitting under a wide range of conditions as shown by heart sound simulation. Our pilot animal experiment in the AV block porcine model demonstrates that the START‐based estimated S2 splitting interval can be used as an indicator of interventricular dyssynchrony. The estimated S2 splitting is well correlated with paced interventricular delays and with invasively measured IRD.We declare no competing interests. R.C. is an employee of Medtronic.H.L., P.W., L.H., M.K., R.C., and F.P. conceived and designed research; H.L., P.W., L.B., and M.K. performed experiments; H.L. analyzed data, prepared figures, and drafted manuscript; H.L., P.W., M.S., R.C., T.D., and F.P. interpreted results of experiments; H.L., R.C., T.D., L.H., and F.P. edited and revised manuscript; H.L., P.W., M.S., L.H., M.K., R.C., T.D., and F.P. approved final version of the manuscript.


Compared to a normal LWA with an aggregate impact value (AIV) of 38.7%, the AIV of the proposed epoxy–TiO2-embedded high-strength functional LWA was reduced by approximately half to 21.1%. In addition, the reduction rates of NOx and SOx gases resulting from the photocatalytic properties of TiO2 nanoparticles coated with epoxy were approximately 90.9% and 92.8%, respectively. Epoxy–TiO2, embedded in LWAs through a mixture, exhibited stability, high strength, and a reduction in air pollutant characteristics, despite repeated water washing. The LWA proposed herein offers excellent structural and functional properties and is expected to be used in functional lightweight concrete that can be practically applied in high-rise and large-scale architectural structures.With the increasing trend of high-rise, large-scale, and functional modern architectural structures, lightweight aggregate (LWA) concrete that exhibits excellent strength and high functionality has garnered active research attention. In particular, as the properties of concrete vary considerably with the raw materials and the proportions of aggregates in the mix, in-depth research on weight reduction, strength improvement, and functional enhancements of aggregates is crucial. This study used the negative pressure coating of a mixed solution comprising epoxy (mixture of epoxy resin and crosslinker), hyper-crosslinked polymer, and titanium oxide (TiO Recently, lightweight aggregate (LWA) concrete, which is lightweight yet strong, has received increasing attention owing to the accelerating trend of high-rise, large-scale, non-standardized, and specialized building structures. When LWAs are mixed with cement, the aggregates act as reinforcements and fillers for concrete, improving its strength and reducing its weight. Therefore, the self-weight of the structure and the cross section of the structural members can be reduced along with improvements in the exclusive space of the structure and earthquake resistance.In general, LWAs are divided into natural LWAs using natural minerals and artificial LWAs made of fly ash, cinder ash, and bottom ash. Recently, artificial LWAs have received attention from precast concrete industries, such as panels with higher insulation and structural elements including walls and slabs, because the lower density of LWA concrete can lead to lower-cost transportation and easier handling in the construction site. However, compared to natural LWAs, artificial LWAs generally have a low strength due to high moisture absorption . The spe2, a representative photocatalytic material, was applied to LWAs to impart NOx and SOx reduction functionality [2 is superior to other photocatalytic materials, such as ZnO, SnO2, Fe2O3, CuO, NiO, and CeO2, in its ability to remove NOx and SOx by the photo-decomposition principle under UV light irradiation [2 in the cement-based composite [It is advantageous that LWAs with various functionalities can be produced simply by introducing a few additives to the raw material mixture. For instance, TiOionality ,13,14,15adiation ,17. In aomposite , the impomposite , the impomposite , the hydomposite , and theomposite ,22 have x and SOx was also carried out by adding TiO2 nanoparticles (NPs) with photocatalytic properties, along with epoxy. The strength and NOx/SOx reduction ability of the aggregates were confirmed based on the maintenance of the epoxy–TiO2 coating of the pores that were present in the inner and outer surfaces of the LWA which remained unaffected despite harsh external environment conditions such as rain. This study aims to introduce a new method to fabricate LWA that is structurally and functionally superior by addressing the brittle fracture problem, a fundamental problem of artificial LWAs, and by enhancing the pollutant-reduction functionality by targeting additional air pollutants.The aim of this study is to maintain the light weight of the aggregates while simultaneously enhancing the strength of the pores by bonding a hyper-crosslinked polymer (HCP) to the inner pores of LWAs. Accordingly, a solution containing epoxy and HCP was embedded in the inner pores of the LWA under negative pressure conditions, and crosslinking and polymerization were employed as coating methods for the post-treatment of the LWA. In addition, the reduction of NO3 and 14.1%, respectively. To remove pollutants including dust on the surface of the artificial LWA, the pristine LWA was washed with running water and dried in air for 15 h. An epoxy solution was prepared by mixing epoxy in isopropyl alcohol  in six different ratios from 0 to 15 wt.%, and the crosslinker was fixed at 25 wt.% of epoxy resin. The ATR–FTIR spectrum of the mixture of epoxy resin and crosslinker showed the typical epoxy-related peaks, a stretching O–H peak of 3370.53 cm−1, a C–H stretching vibration peak of 2928.98 cm−1 of CH3, aromatic peaks of 1609.54 and 1506.62 cm−1, and C–O–C stretching peaks of 1229.58 and 1031.47 cm−1. Thereafter, the epoxy solution and TiO2 NPs , rutile structure, ReagentPlus®, Sigma–Aldrich, St. Louis, MI, USA) were mixed at a mass ratio of 10:1 and were sonicated to prepare an epoxy–TiO2 composite solution. The prepared epoxy–TiO2 composite solution was embedded in the LWA under normal or negative pressure coating conditions. In the case of the normal pressure coating, LWAs were dipped in an epoxy–TiO2 composite solution for 60 min at 20 °C under normal pressure conditions (1 atm). Subsequently, the LWAs were removed from the solution and dried in air for 8 h. For the negative pressure coating, the epoxy–TiO2 composite solution containing LWAs was placed in a vacuum oven  and held at a negative pressure of 600 Torr for 30 min. Then, the LWAs were removed from the solution and dried for 8 h under room temperature (20 °C) and normal pressure conditions (1 atm).High-strength functional LWA was fabricated by simultaneously embedding epoxy  into the internal pores (~0.3–300 μm) of pristine artificial LWA  and by coating the external surface. The density and water absorption of LWA were 1.4 g/cm2 coated on the pristine LWA, the LWA was weighed and its aggregate impact value (AIV) was measured via an aggregate impact testing machine  before and after the epoxy–TiO2 coating. According to BS 812–112 (Testing aggregates-Part 112: Methods for determination of AIV), the method of the determination of AIV is obtained by vertically dropping a weight of 14 kg from 381 mm above the surface of the aggregates 15 times to crush them. Thereafter, the crushed aggregates are sieved using a sieve with a 2.36 mm-sized aperture, and the weight difference of the aggregates before and after the crushing is calculated; the value is converted into a percentage. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy  was employed to determine if the epoxy was evenly embedded into the LWA pores. Field emission scanning electron microscopy  and energy-dispersive X-ray spectrometry  were employed to determine the shape of the inner side of the LWA and the presence of coated TiO2.To determine the amount of epoxy–TiOx/SOx gas analysis system  was used to determine the effect of reducing NOx and SOx gases on the epoxy–TiO2-embedded LWA. A total of 500 g of epoxy–TiO2-embedded LWA was placed in an open box with the dimensions 705 mm  × 210 mm  × 105 mm ; thereafter, it was placed inside the main chamber of the NOx/SOx gas analysis system. Subsequently, UV light (UV intensity ~1.2 mW/cm2) was irradiated at a distance of 220 mm from the LWA. The amount of NO and SO2 gas injected into the chamber was controlled using a mass flow controller  and readouts . For the measurement of NOx reduction, 303.0 μmol/mol of NO and N2 gases was injected into the chamber at a rate of 3.8 sccm and 3 slm, respectively. For the measurement of SOx reduction, 300.0 μmol/mol of SO2 and N2 gases was injected into the chamber at a rate of 19.5 sccm and 0.5 slm, respectively. After 2 h of injection, the NO or SO2 inside the chamber attained a concentration of 1 ppm, which was maintained for another 3 h. Then, the NOx or SOx concentration inside the chamber was measured under UV light irradiation. A NOx analyzer  and a SOx analyzer  were used for the measurement of concentrations.A NO2-embedded LWA, the following process was conducted to confirm the change in NOx removal efficiency. First, 500 g of epoxy–TiO2-coated LWA that was tested for NOx removal was transferred to a sieve with an aperture of 2 mm (diameter ~175 mm). After placing a spray gun (AS ONE) at a height of 300 mm above the epoxy–TiO2-embedded LWA, water was sprayed on the entire anterior surface of the epoxy–TiO2-embedded LWA at a rate of 1250 mL/min for 10 min. The wet epoxy–TiO2-embedded LWA was thoroughly air-dried at room temperature for 12 h. Finally, the NOx removal effect of the dried epoxy–TiO2-coated LWA was measured.After washing the epoxy–TiOx and NOx by uniformly mixing HCP with TiO2.HCP is highly applicable in energy and environment sectors such as for gas storage, carbon capture/removal, pollutant isolation/removal, and catalysts due to the advantages of a variety of synthesis methods, easy functionalization, high surface area, and mild reaction conditions ,24,25,262 NPs as a photocatalyst were mixed with an epoxy solution to remove NOx and SOx, which are typical air pollutants. During the preparation of the epoxy–TiO2 composite solution, IPA was added to the high-viscosity epoxy resin to lower the viscosity and reduce the reaction rate with the crosslinker; thereafter, the TiO2 NPs were mixed and ultrasonic homogenization was performed. When the prepared epoxy–TiO2 composite solution is embedded in the LWA, the composite solution cannot penetrate into the pores owing to the presence of air, and therefore it only coats the outer surface of the aggregates. In order to tackle this problem, a vacuum state (600 Torr) is maintained while dipping the LWA in the composite solution; this causes the air inside the LWA to escape and the composite solution can therefore easily penetrate into the pores of the LWA through a capillary phenomenon.2, Al2O3, Fe2O3, CaO, MaO, and Na2O) in the LWA, and due to bonding with the porous surface of the aggregates, the strength of the LWA is enhanced. Despite the addition of TiO2 NPs with photocatalytic properties into the epoxy, the inflow of SOx and NOx gases remains unaffected due to the microporous network structure, an inherent property of HCP, and therefore the functionality of SOx and NOx gas decomposition is imparted [imparted ,28,29,302 solution while fabricating a high-strength LWA for the adsorption of air pollutants. An aggregate impact testing machine which compares the difference between impact resistance and resistance due to static compressive load was used to measure the LWA strength. The hardness of the LWA can be determined by measuring the impact resistance and the amount of crushed LWA. In addition, the degree of penetration of epoxy–TiO2 into the LWA was obtained by crushing the coated LWA and examining the central part of the LWA through ATR–FTIR spectroscopy.2 under normal pressure, and the LWA coated with epoxy–TiO2 under negative pressure (600 Torr). The air present in the pores of the LWA hinders the penetration of the epoxy–TiO2 composite solution into the pores under normal pressure. Therefore, the coating of epoxy–TiO2 on the LWA was conducted under a negative pressure condition of 600 Torr in order to remove the air present in the LWA pores. As the brown-colored pristine LWA was coated with epoxy–TiO2, the overall appearance of white TiO2 NPs was displayed on a gray background. When examining the change in strength according to the coating conditions of the LWA, the AIV value of 38.7% ± 1.5% for the pristine LWA decreased to 31.6% ± 1.3%  and 21.1% ± 0.7% (negative pressure coating) according to the epoxy–TiO2 coating  and the coating method. The concentration of the epoxy–TiO2 composite solution was changed by increasing the concentration of epoxy (epoxy resin and crosslinker) to 0, 3, 6, 9, 12, and 15 wt.% while keeping the amount of TiO2 NPs constant. Note that the ratio of epoxy resin to crosslinker was equal to 4:1. 2 composite solution coated on the LWA under negative pressure. TiO2 was mixed with an epoxy solution containing IPA and epoxy at a mass ratio of 10:1 to ensure a constant TiO2 ratio in the total epoxy–TiO2 composite solution. 2-embedded high-strength functional LWA coated with a solution prepared using varying concentrations of epoxy at 0, 3, 6, 9, 12, and 15 wt.% with respect to the total epoxy–TiO2 composite solution. Epoxy not only acts as a binder in the inner pore surface of the aggregates, but also positions TiO2 ensuring it is stably embedded along the inner and outer surfaces of the aggregates, as shown by the uniformly distributed white TiO2 NPs occurring as the pristine LWA is coated with epoxy–TiO2. Furthermore, when the concentration of epoxy in the epoxy–TiO2 solution exceeds 12 wt.%, the viscosity increases as the amount of epoxy resin increases; the rate of crosslinking accelerates with the increase in the concentration, resulting in a drastic increase in the molecular weight of the crosslinked epoxy polymer. In this case, the volume of the epoxy polymer increases, making it difficult for the solution to penetrate the pores of the LWA; as the solubility of the epoxy polymer decreases, phase separation occurs and the layers are separated [2 in the epoxy–TiO2 solution decreased, epoxy–TiO2 was not uniformly coated on the inside and on the surface of the LWA but rather showed an agglomerated form.When the epoxy–TiOeparated . In addi2 was uniformly distributed inside the LWA. 2 solution containing 9 wt.% epoxy. FE–SEM images showed that the epoxy–TiO2 penetrated into the pores inside the LWA. Elemental C and Ti measured by EDS mapping showed that the epoxy–TiO2 was uniformly distributed inside the LWA.An EDS measurement was conducted to investigate whether the epoxy–TiO2-embedded high-strength functional LWA according to the epoxy concentration. AIV is an index to examine the strength of LWAs. To produce concrete with higher compressive strength and a longer lifespan, the use of LWAs with higher fracture resistance is essential. As the epoxy concentration increased, the AIV of the epoxy–TiO2-embedded high-strength functional LWA decreased from 35.8% ± 0.7, 27.2% ± 1.8%, 24.7% ± 1.7%, 21.1% ± 0.7%, and 21.8% ± 0.7% to 18.4% ± 1.3% . . With an epoxy concentration of 9 wt.%, an optimal coating condition that does not cause agglomeration of epoxy–TiO2, the AIV decreased by approximately 50%, while only 0.2 times the weight was gained.tively). c As the 2 NPs contained in the epoxy–TiO2-embedded high-strength functional LWA are typical photocatalytic materials and have the ability to decompose and reduce NOx and SOx gases when irradiated with UV light. TiO2, with a band gap of 3.2 eV, absorbs UV light at wavelengths below 386 nm, and excites electrons from the valence band (VB) to the conduction band (CB). Thus, holes (h+) are formed in the VB, whereas electrons (e−) are generated in the CB. The VB hole reacts with water molecules adsorbed on the aggregates to produce hydroxyl radicals (•OH). Meanwhile, electrons in CB reduce oxygen in the atmosphere to generate superoxide species (•O2−). Hydroxyl radicals and superoxide species chemically react to transform NOx gas to nitrates and SOx gas to sulfite and sulfate [TiO sulfate ,34,35.x/SOx gas analysis system. The epoxy–TiO2-embedded high-strength functional LWA was placed in the main chamber, and the NOx or SOx gas removal rate was measured under a UV intensity condition of 1.2 mW/cm2. The epoxy–TiO2-embedded high-strength functional LWA was fabricated with an epoxy concentration of 9 wt.% and showed excellent strength characteristics without causing the agglomeration of epoxy–TiO2. x and SOx reduction performance of the epoxy–TiO2–embedded high-strength functional LWA. After injecting NOx or SOx gas (1 ppm) into the NOx/SOx gas analysis system, the concentration inside the chamber was kept constant for 5 h, and thereafter UV light was irradiated. Immediately, the concentration of NOx or SOx gas in the chamber began to decrease, and the maximum removal rates were 90.3% (NOx) and 92.8% (SOx). The concentration of NOx gas in the chamber rapidly decreased until approximately 100 min after the UV irradiation was initiated, and then the concentration was kept constant at approximately 0.1 ppm for 12 h. The concentration of SOx gas in the chamber continuously decreased until approximately 6 h after the UV irradiation was initiated, and then a concentration of approximately 0.1 ppm was maintained for the next 12 h.2-embedded high-strength functional LWA exhibits stable durability under wet environments owing to the presence of epoxy in the composition of TiO2 NPs. x gas reduction capacity of the epoxy–TiO2-embedded high-strength functional LWA was measured through five repeated washing cycles, and the NOx gas removal rates observed were 87.7, 86.5, 85.7, 84.0, and 86.1%. Notably, the ~3% variation in this value is likely to be an error attributed to the degree of surface exposure of aggregates placed on the measuring equipment. Therefore, stable reduction characteristics were confirmed.As LWAs are applied to buildings exposed to the external environment, they are constantly exposed to harsh weather conditions such as precipitation. The fabricated epoxy–TiO2 NPs to epoxy, a reduction in NOx and SOx (air pollutants) was noted. Although epoxy is coated on the surface of TiO2 NPs, NOx/SOx and the gas decomposed by TiO2 NPs can be easily eliminated owing to the ability of HCP to form micropores during polymerization and crosslinking. Through the negative pressure coating method, epoxy–TiO2 completely penetrated the pores of the LWA, and by controlling the epoxy concentration from zero to 15 wt.%, an optimal condition was discovered at which the light weight of the LWA was maintained and its strength was enhanced. The AIV of the epoxy–TiO2-embedded high-strength functional LWA coated with 9 wt.% concentration, an optimal condition for no agglomeration of epoxy, is reduced by half to 21.1% when compared with the 38.7% of the pristine LWA. In addition, due to the photocatalytic properties of TiO2 NPs, it was confirmed that NOx and SOx gases were reduced by ~90.9% and ~92.8%, respectively, under UV light. Since TiO2 NPs are mixed with epoxy, stable NOx gas reduction properties were maintained despite continuous and repeated washing. In the case of the negative pressure coating method used in this study, the surfaces of LWAs should be exposed to air. Accordingly, if the exposure of the surface of the LWAs is limited due to overlapping LWAs during the large-capacity coating process, it is difficult to remove air completely in the LWAs with a negative pressure of 600 Torr and some pores of the LWAs may not be coated. Therefore, in order to uniformly coat a large amount of LWAs, further research is needed on the development of equipment that can evenly expose a large amount of LWAs to the air in a negative pressure ambient. Epoxy–TiO2-embedded high-strength functional LWAs proposed in this study are expected to be widely applied in multifunctional LWA concrete as they offer excellent strength and durability as well as the functionality of reducing air pollution.In summary, epoxy, an HCP, was used as a reactive binder for LWA surface bonding and as an external shock absorber in order to address the challenging drawback of the weak strength of LWAs. In addition, by adding TiO


This study investigated the role of Forkhead box Q1 (FOXQ1) in the osteogenic differentiation of bone mesenchymal stem cells.Foxq1-overexpressing mBMSCs, Foxq1-suppressed mBMSCs, and mBMSC controls. The activity of osteogenic differentiation was evaluated with alizarin red staining, alkaline phosphatase activity assay, and RT-qPCR. Wnt/β-catenin signaling activities were compared among groups by TOPFlash/FOPFlash assay, immunofluorescence staining, and western blot assay of beta-catenin (CTNNB1). Coimmunoprecipitation mass spectrometry was also carried out to identify proteins binding with FOXQ1.Mouse bone mesenchymal stem cells (mBMSCs) were transfected with lentivirus to generate Our data showed that FOXQ1 expression was positively correlated with the osteogenic differentiation of the mBMSCs. FOXQ1 also promoted the nuclear translocation of CTNNB1 in the mBMSCs, enhancing Wnt/β-catenin signaling, which was also shown to be essential for the osteogenic differentiation-promoting effect of FOXQ1 in the mBMSCs. Annexin A2 (ANXA2) was bound with FOXQ1, and its depletion reversed the promoting effect of FOXQ1 on Wnt/β-catenin signaling.These results showed that FOXQ1 binds with ANXA2, promoting Wnt/β-catenin signaling in bone mesenchymal stem cells, which subsequently promotes osteogenic differentiation. Mesenchymal stem cells constitute a group of multipotent cells capable of differentiating into various types of cells, including osteoblasts, adipocytes, and various other types of cells. This process is modulated by various signaling pathways, including BMP , TGF-β , and theForkhead box Q1  is a member of the forkhead box (FOX) family of proteins. It was first identified as a regulator of hair follicle development . Later iIn the current study, we aimed to investigate the influence of FOXQ1 on the osteogenic differentiation of mesenchymal stem cells and to elucidate the underlying molecular mechanisms. Our findings suggested that FOXQ1 promotes osteogenic differentiation of mouse bone mesenchymal stem cells via the Wnt/β-catenin signaling pathway.To obtain mouse embryo and alveolar bone tissue for histological evaluation, pregnant Chinese Kunming (KM) mice (4 weeks old) and Chinese Kunming mice (7 days old) were purchased from Sun Yat-sen University. Chinese Kunming mice (7 days old) came from the same pregnant mouse to minimize the genetic difference between groups. The study protocol was approved by the Ethics Committee of the Hospital of Stomatology, Sun Yat-sen University .To observe FOXQ1 expression in alveolar bone tissue, a pregnant KM mouse was sacrificed to obtain 3 mouse embryos at embryonic day 15.5 (E15.5), and 3 KM mice were sacrificed at each time point  and postnatal day 11 (P11)), whose mandibles were isolated surgically. The whole embryos of E15.5 mouse and the mandibles from P7 and P11 mice were fixed with 4% paraformaldehyde at room temperature for 72 h. Then, the samples were dehydrated with graded solutions of alcohol and embedded. Anti-FOXQ1 polyclonal antibody  was used as the primary antibody.Immunofluorescence analyses were carried out to evaluate the transnucleation of CTNNB1. Cells were incubated overnight with anti-CTNNB1 polyclonal antibody  at 4 °C. Then, the sections were incubated with secondary antibody  for 1 h in a dark chamber. Finally, the sections were counterstained with 4′6-diamidino-2-phenylindole  for 15 min for nuclear labeling.Commercially available mouse bone mesenchymal stem cells (mBMSCs) derived from the bone marrow of Balb/c mice  were purchased and cultured with alpha-modified Eagle medium  supplemented with 10% fetal bovine serum , 100 U/mL penicillin , and 100 mg/mL streptomycin . After the 3rd passage, the cells were used for experiments.Foxq1 cDNA was amplified with Flag-tagged primers from total RNA and then cloned into a pCDNA3.1 vector  to produce the bait, Flag-tagged FOXQ1 protein, for the coimmunoprecipitation study. Full-length Foxq1 cDNA was also cloned from total RNA and inserted into a pGLV5 vector . PGLV3 lentivirus containing the Foxq1 shRNA sequence and lentivirus particles with an empty pGLV3 vector and with a pGLV5 plasmid were purchased from GenePharma . MBMSCs were then transfected with lentiviral particles containing Foxq1-sh pGLV3, Foxq1-overexpressing pGLV5, an empty pGLV3, or an empty pGLV5 vector, creating 4 groups of cells denoted as Foxq1-sh mBMSCs, Foxq1-over mBMSCs, lv3 mBMSCs, and lv5 mBMSCs, respectively. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blot assay were carried out to assess Foxq1 expression in each group of mBMSCs.Full-length 6 cells per well. An osteogenic induction medium was prepared according to previous studies [Mouse bone mesenchymal stem cells were seeded into 6-well plates at a density of 1.0 × 10 studies . The celAlizarin red staining (ARS) was conducted to visualize the mineral deposition in each group after osteogenic induction. The cells were first fixed with cold methanol at room temperature for 10 min, rinsed twice with deionized water, and stained with alizarin red  at room temperature for 30 min. Then, the excess dye was removed with deionized water. For quantification of ARS, stain was desorbed with 10% cetylpyridinium chloride (CPC) in PBS, pH 7.0, for 15 min at room temperature. Then, ARS concentration was determined by absorbance at 560 nm with a spectrometer.Alkaline phosphatase activity assay was also carried out to evaluate the osteogenic differentiation of mBMSCs from 4 groups. Cultured in the osteogenic induction medium for 7 days, 4 groups of cells were then lysed with 200 μL 1% Triton X-100. Cell lysates were then measured for alkaline phosphatase (ALP) activity with ALP measuring kit . The ALP activity was measured at 405-nm wavelength with a spectrometer.Total RNA was extracted from 4 groups of mBMSCs using TRIzol  according to the manufacturer’s protocol. Complimentary DNA synthesis was performed with random 6-mer primers using a PrimeScript 1st Strand cDNA synthesis kit . Messenger RNA expression was measured by RT-qPCR using SYBR® Green.Foxq1-overexpressing mBMSCs, while 6-bromoindirubin-3′-oxime  was used to activate Wnt signaling in the Foxq1-sh mBMSCs. The mRNA levels of Wnt1, Wnt3, and Wnt10a were then evaluated with RT-qPCR. The relative fold change for the expression of the target gene was normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (Gapdh). The primers employed are listed in Table Various markers indicating activities of mesenchymal stem cell osteogenic differentiation were evaluated. Also, to investigate the effect of counterregulating Wnt signaling on FOXQ1’s osteogenic differentiation-promoting action in mBMSCs, Dickkopf WNT signaling pathway inhibitor 1  was used to suppress Wnt signaling in the Foxq1-sh mBMSCs, Foxq1-over mBMSCs, lv3 mBMSCs, and lv5 mBMSCs were seeded at a density of 1 × 106 cells per well. Total proteins were extracted using RIPA buffer following the manufacturer’s protocol. Nuclear protein was isolated with a nuclear protein extraction kit according to the manufacturer’s instructions . The primary antibody was anti-CTNNB1  and anti-FOXQ1 . Anti-THOC1 , anti-EEF1A1 , and anti-GAPDH  were used as controls for the nuclear proteins, cytoplasmic proteins, and total proteins, respectively. All assays were performed in triplicate. Quantitative analysis of western blot assays was carried out with ImageJ software [software .Foxq1-sh mBMSC, Foxq1-over mBMSC, lv3 mBMSC, and lv5 mBMSC groups and Foxq1-over+siAnxa2 mBMSCs were seeded on 96-well plates at a density of 4 × 103 cells per well. Then, they were transiently transfected with TOPFlash or FOPFlash luciferase reporter plasmid  according to the manufacturer’s protocol. The firefly luciferase activity level was normalized against the Renilla luciferase activity level. The fold increase indicating the TOPFlash activity compared to the FOPFlash is reported.To assess the influence of FOXQ1 levels on Wnt/β-catenin signaling activities, a TOPFlash/FOPFlash reporter assay was performed. Cells from the Foxq1-overexpressing mBMSCs and lv5 mBMSCs were transfected with the Flag-tagged Foxq1 pCDNA3.1 vector. Total protein was obtained from cell lysates from each group of cells. Coimmunoprecipitation (co-Ip) was carried out with a Pierce co-IP kit  following the manufacturer’s protocol. In brief, 10 μg of anti-FLAG antibody  was first incubated with coupling resin. The antibody-conjugated resin was then incubated overnight with 200 μL mBMSC total protein lysates at 4 °C. Then, the resin was washed, and protein complexes bound to the anti-FLAG antibody were eluted. A western blot analysis was subsequently performed as described above to confirm that the anti-FLAG antibody pulled down FOXQ1 proteins.To investigate the potential mechanism of the interaction between FOXQ1 and Wnt/β-catenin and subsequent signaling, coimmunoprecipitation was performed and the resultant data analyzed. Liquid chromatography–mass spectrometry (LC-MS) analysis was conducted with a NanoLC 400 system, and a TripleTOF 5600-Plus  system was used for the mass spectrometry (MS) analysis. ProteinPilot software  was used to analyze data from the TripleTOF 5600-Plus and identified proteins bound to FOXQ1.The potential interactions among identified proteins were evaluated with the STRING pathway database . Specifit tests (comparison between two groups) or one-way ANOVA, and Dunnett’s test was used as a post hoc test (comparison between 3 or more groups). P < 0.05 was considered significant. Statistical analyses were carried out using R 3.4.2 .Upon confirmation of a normal distribution of data, all the quantitative data were subjected to Student’s Foxq1 expression increase in the mBMSCs after osteogenic induction treatment (Fig. Immunohistochemical staining demonstrated a high level of FOXQ1 in alveolar bone tissue from the E15.5 mouse embryos (Fig. Foxq1 mRNA level in the Foxq1-over mBMSC group was 28-fold greater than that in the lv5 mBMSC group (Fig. Foxq1-sh mBMSC group was one-half that of the lv3 mBMSC groups (Fig. To further study the role of FOXQ1 in the osteogenic differentiation of bone mesenchymal stem cells, FOXQ1 expression was manipulated with a lentivirus plasmid. The RT-qPCR results showed that the Foxq1-overexpressing mBMSCs was 116%, and it was 71% in the Foxq1-sh mBMSCs (Fig. Seven days after osteogenic induction, ALP activity assay showed that FOXQ1 expression level was associated with ALP level in mBMSCs (Fig. Alp (Fig. Ocn (Fig. Opg (Fig. Runx2 (Fig. Foxq1-overexpressing mBMSC group and downregulated in the Foxq1-sh mBMSC group, compared to the levels of the control groups. These results demonstrated a dose-dependent relationship between FOXQ1 levels and osteogenic marker expression in response to the osteogenic induction.We then investigated whether FOXQ1 is able to influence the expression of osteogenesis-related molecules with RT-qPCR. On the 7th day and 14th day after osteogenic induction, various osteogenic markers, namely, Foxq1-over mBMSCs (Fig. Foxq1-sh mBMSCs, the transcriptional activity was one-half that of the lv3 mBMSCs (Fig. Foxq1-overexpressing mBMSCs, demonstrating an increase in the nuclear translocation of CTNNB1 (Fig. Wnt/β-catenin is an important pathway in the regulation of osteogenic differentiation. The TOPFlash/FOPFlash assay of β-catenin/TCF/LEF transcriptional activity demonstrated a 5.7-fold increase in the Wnt1, Wnt3, and Wnt10a are indicators of Wnt/β-catenin activity level, and their expression levels were evaluated with RT-qPCR. The overexpression of FOXQ1 upregulated Wnt1 (Fig. Wnt3 (Fig. Wnt10 (Fig. The treatment with DKK-1 attenuated the FOXQ1-induced increase in osteogenic differentiation (Fig. Foxq1-over mBMSC group, and 199 proteins were identified in both types of samples to be binding with FOXQ1(Fig. Anxa2 mRNA depletion with siAnxa2 reversed the increase in the Wnt/β-catenin signaling in the Foxq1-overexpressing mBMSCs, suggesting that ANXA2 is an integral part of the mechanism by which FOXQ1 promotes Wnt/β-catenin signaling.The coimmunoprecipitation and LC-MS analysis identified various proteins that were pulled down with FOXQ1. A total of 484 proteins were identified in the samples from the lv5 mBMSC group, while 240 proteins were identified in the samples from the FOXQ1 is a member of the forkhead box family, and a recent study showed that various members of this family were involved in the osteogenesis process. FOXO1 was found to be an early molecular regulator during mesenchymal stem cell differentiation into osteoblasts . FOXC1 wFoxq1-over mBMSC group or the Wnt/β-catenin signaling activator 6BIO in the Foxq1-sh mBMSC group. From these results, it can be inferred that FOXQ1 promotes the osteogenic differentiation of mesenchymal stem cells via the Wnt/β-catenin signaling pathway.Our results showed that FOXQ1 overexpression or suppression led to enhanced or attenuated Wnt signaling activity in mBMSCs, respectively. Wnt/β-catenin signaling is an important regulator of osteogenic differentiation , 23. BetAnxa2 mRNA depletion reversed the promoting effect of FOXQ1 overexpression, demonstrating that binding with ANXA2 is important for FOXQ1 function in Wnt signaling regulation to promote osteogenic differentiation of mesenchymal stem cells.Previous studies have shown that FOXQ1 expression is mediated by Wnt/β-catenin . InteresIn summary, our study highlights the importance of FOXQ1 as a mediator of mesenchymal stem cell osteogenic differentiation. To the best of our knowledge, this is the first study to demonstrate that FOXQ1 regulates the activities of Wnt/beta-catenin signaling by binding with ANXA2. These results provide novel insights into the mechanism underlying the osteogenic differentiation of mesenchymal stem cells.


Aplysia kurodai as an antagonist of Wnt/β-catenin signaling. Aplykurodin A inhibited β-catenin responsive transcription, which was stimulated by a Wnt3a-conditioned medium or a glycogen synthase kinase 3β inhibitor by accelerating intracellular β-catenin degradation. Aplykurodin A downregulated the level of oncogenic β-catenin and decreased the expression of β-catenin-dependent gene, leading to inhibition of human hepatoma Hep3B and SNU475 cell proliferation. Moreover, apoptosis and autophagy were elicited by aplykurodin A, as indicated by an increase the number of Annexin V-FITC-stained cells and the formation of microtubule-associated protein 1 light chain 3 puncta, respectively, in Hep3B and SNU475 cells. Our findings suggest that aplykurodin A provides a novel therapeutic strategy for human hepatocellular carcinoma via stimulation of oncogenic β-catenin degradation.Dysregulation of the Wnt/β-catenin signaling pathway is involved in the development of human hepatocellular carcinoma and has thus emerged as a therapeutic target for this malignant tumor. In this study, we employed sensitive cell-based assays to identify aplykurodin A isolated from  Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the third leading cause of cancer-mediated mortality worldwide . Recent β-Catenin is an essential constituent of the Wnt/β-catenin pathway, which regulates cell growth, differentiation and development ,6,7 and AXIN gene or infection of hepatitis B virus, is frequently observed in hepatocellular carcinoma [The aberrant β-catenin accumulation, which is caused by mutation in arcinoma ,13. Thenarcinoma ,15,16,17Aplysia kurodai (family Aplysiidae), is distributed in the coasts of Northeast Asia. Because of its unique texture and flavor, A. kurodai has been consumed as seafood in South Korea. The sea hare is also used as a traditional medicine to treat inflammation and wounds. Previous study revealed that Aplysia species protect themselves by releasing toxic compounds stored in the digestive glands [A. kurodai were reported to have cytotoxic activity against human cervical cancer cells [A. kurodai in 1986 [A. kurodai-derived compounds, there are few reports on its biological activity on aplykurodin A. Based on the fact that a degraded sterol, 3-epi-aplykurodinone B, displayed cytotoxicity against human tumor carcinoma cells, our study was focused on the evaluation of cytotoxic activity against HCC in association with Wnt/β-catenin signaling pathway. In the present study, we demonstrated that aplykurodin A antagonized Wnt/β-catenin signaling and inhibited proliferation of hepatocellular carcinoma cells by destabilizing intracellular β-catenin.The sea hare, e glands , and suce glands . The macer cells . Moreoveer cells . Aplykur in 1986 , was obtTo examine whether aplykurodin A inhibits the Wnt/β-catenin pathway, we used HEK293-firefly luciferase (FL) reporter cells previously established . IncubatIn Wnt/β-catenin signaling, CRT primarily relies on the amount of intracellular β-catenin  that is GSK-3β catalyzes β-catenin phosphorylation at Ser33, Ser37, and Thr41 residues, which is prerequisite event for acceleration of β-catenin turnover. We, thus, investigated whether GSK-3β is involved in aplykurodin A-induced β-catenin degradation. As previously reported , incubatAXIN1, a critical component of destruction complex [AXIN1 gene, were incubated with aplykurodin A. Western blot analysis revealed that aplykurodin A downregulaed the expression of β-catenin-dependent genes, cyclin D1, c-myc, and axin-2 , we evaluated whether aplykurodin A was able to downregulate CRT in SNU475 and Hep3B hepatoma cells that displayed elevated CRT because of an inactivation mutation in  complex ,28. Whend axin-2 C. Given t manner D. Under t manner . AXIN1-mutated hepatoma cells. SNU475 and Hep3B cells were exposed to aplykurodin A and then the number of apoptotic cells was counted using Annexin V/PI staining. The proportion of Annexin V positive or Annexin V/PI double positive cells was significantly increased in a concentration-dependent manner. Moreover, treatment of these AXIN1-mutated hepatoma cells with aplykurodin A activated caspase-3/7, which is consistent with an increase in apoptosis  and cyclin-dependent kinase 2 (CDK2)/cyclin A phosphorylate β-catenin at N-terminal residues Ser33/37/Thr41) and N-terminal phosphorylation motif is essential for β-catenin degradation ,32,33. L/37/Thr41Several studies have reported that tumor suppressor p53 involved in regulation of the Wnt/β-catenin pathway. Activation of p53 induces expression of Siah-1 E3 ubiquitin ligase, which promotes β-catenin degradation in a complex with APC. We reported previously that hexachlorophene accelerated β-catenin decomposition by activating the expression of Siah-1 in HCT116 and LS174T colon cancer cells . In contThe Wnt/β-catenin pathway regulates both autophagy and apoptosis ,37. AutoAXIN1-mutated HCC cells. Although aplykurodin A exhibited cytotoxic effect against HCC cells at high concentration, it is likely to be development into chemopreventive or therapeutic agent against CRT-positive cancers caused by AXIN1 mutation.In summary, we identified a novel Wnt/β-catenin signaling inhibitor aplykurodin A and demonstrated its anti-proliferative activity aganist HCC cells. Aplykurodin A promoted proteasomal β-catenin decomposition through a mechanism independent of GSK-3β and Axin1, major components of β-catenin destruction complex. In addition, aplykurodin A induced apoptosis and autophagy, thereby suppressing the growth of Aplysia kurodai  were extracted by CH2Cl2/EtOH (1:1) three times under reflux. The extract (800 g) was suspended in water and partitioned successively with CH2Cl2, yielding CH2Cl2 (140 g) and water-soluble (620 g) fractions. The CH2Cl2 fraction (140 g) was subjected to silica gel vacuum liquid column chromatography (VLC) (1400 g) and eluted with stepwise gradient of n-hexane-EtOAc , n-hexane-EtOAc-MeOH (1:1:0.2), CHCl3-MeOH (3:1), CHCl3-MeOH-H2O (1:1:0.1), and MeOH (100%) to give eight fractions (Fr. A-H). Fr. D (2.7 g) was combined with Fr. E (5 g) based on their similar TLC patterns and continue to separate into 10 fractions (Fr. DE-1—DE-10) by MPLC  with a gradient of acetone-MeOH-H2O (17:17:66 → 47:47:6). Fraction DE-5 (1.02 g) was separated using silica gel column (1.5 cm × 80 cm) chromatography with a gradient of n-hexane-EtOAc-acetone (6.5:1:0.1 → 4:1:0.1) to give six subfractions (Fr. DE-5-1—DE-5-6). Fraction DE-5-6 (337.7 mg) was purified by a silica gel column (1.0 × 70 cm) and eluted with n-hexane-EtOAc-acetone (5:1:0.2) to get aplykurodin A (210 mg). Aplykurodin A was obtained as white amorphous powder. The protonated-molecular ion was detected at m/z 322.25 in ESI-MS, corresponding to the molecular formula C20H34O3. The 1H NMR spectroscopic data showed typical signals for two oxygenated methine protons at δH 5.06 , and 3.86 , one tertiary methyl group at δH 1.02 , and three secondary methyl groups at δH 1.02 , 0.94 . The 13C NMR spectroscopic data displayed 20 carbon signals, which were assigned to one carboxyl group at δC 175.2 (C-1), two oxygenated methines at δC 82.5 (C-9) and 67.4 (C-4), one tertiary methyl at δC 23.4 (C-12), three secondary methyls at δC 23.2 (C-20), 22.9 (C-19), and 19.2 (C-14), in addition to one quaternary carbon, five methines, and seven methylenes. Based on the NMR spectroscopic data analysis  were cultured in the Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The HEK293-FL, HEK293-SEAP reporter cells, and Wnt3a-CM were prepared as previously described ,24. The Western blot analyses were performed as previously described . Anti-β-w/v) agarose, stained with Dyne LoadingSTAR and detected using Gel document system .RNA was isolated from HEK293-FL reporter cells using TRIzol Reagent , according to the manufacturer’s instructions. Complementary DNA (cDNA) synthesis, amplification, and polymerase chain reaction (PCR) were performed as previously described . AmplifiCells were innoculated into 96-well plates and incubated with aplykurodin A (20 and 40 μM) for 48 h. The cell viability of each treated sample was measured in triplicate using the CellTiter-Glo assay kit (Promega) according to the manufacturer’s instructions. The value of time 0 was subtracted to calculate growth inhibition of hepatoma cells.After treatment with aplykurodin A for 48 h, the cells were stained with ApoScanTM annexin V-FITC apoptosis detection kit (Promega), according to the instructions and then analyzed by Cellometer Vision image cytometer (Nexcelom Bioscience). The activity of caspase was determined using the Caspase-Glo 3/7 Assay (Promega) according to the manufacturer’s instructions.The GFP-LC3-transfected cells were incubated with aplykurodin A for 48 h and fixed with 4% paraformaldehyde in PBS at room temperature. Fluorescence signals were visualized and captured using the Leica 4000 confocal microscope.p < 0.05 or p < 0.01. Results are presented as mean ± standard deviation (SD).The Student’s t-test was used to compare the means between control and experimental groups. All experiments were performed three times. Statistical significance was set at


Colorectal cancer (CRC) is the fourth leading cause of cancer mortality worldwide. Aberrant activation of WNT/β-catenin signaling present in the vast majority of CRC cases is indispensable for CRC initiation and progression, and thus is a promising target for CRC therapeutics. Hispolon is a fungal-derived polyphenol with a pronounced anticancer effect. Several hispolon derivatives, including dehydroxyhispolon methyl ether (DHME), have been chemically synthesized for developing lead molecules with stronger anticancer activity. Herein, a DHME-elicited anti-CRC effect with the underlying mechanism is reported for the first time. Specifically, DHME was found to be more cytotoxic than hispolon against a panel of human CRC cell lines, while exerting limited toxicity to normal human colon cell line CCD 841 CoN. Additionally, the cytotoxic effect of DHME appeared to rely on inducing apoptosis. This notion was evidenced by DHME-elicited upregulation of poly (ADP-ribose) polymerase (PARP) cleavage and a cell population positively stained by annexin V, alongside the downregulation of antiapoptotic B-cell lymphoma 2 (BCL-2), whereas the blockade of apoptosis by the pan-caspase inhibitor z-VAD-fmk attenuated DHME-induced cytotoxicity. Further mechanistic inquiry revealed the inhibitory action of DHME on β-catenin-mediated, T-cell factor (TCF)-dependent transcription activity, suggesting that DHME thwarted the aberrantly active WNT/β-catenin signaling in CRC cells. Notably, ectopic expression of a dominant–active β-catenin mutant (∆N90-β-catenin) abolished DHME-induced apoptosis while also restoring BCL-2 expression. Collectively, we identified DHME as a selective proapoptotic agent against CRC cells, exerting more potent cytotoxicity than hispolon, and provoking CRC cell apoptosis via suppression of the WNT/β-catenin signaling axis. Colorectal cancer (CRC) is the third most frequently diagnosed and the fourth most lethal human cancer globally . Aging, APC gene or activating mutations in the β-catenin-encoding gene CTNNB1 [Accumulating evidence has delineated a number of signaling pathways whose deregulation contributes to CRC pathogenesis, including epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK), Hedgehog, Notch, phosphoinositide 3-kinase/protein Kinase B (PI3K/AKT), transforming growth factor-β (TGF-β), and WNT/β-catenin . Notablye CTNNB1 . Conseque CTNNB1 ,6,7,8. Ie CTNNB1 .Phellinus linteus [Hispolon is a polyphenolic compound structurally analogous to curcumin, and is a bioactive constituent present in the fruiting body and mycelium of medicinal mushroom  linteus ,11. Prev linteus ,13,14. I linteus ,16,17. G linteus .In the present study, we investigated the anti-CRC effect and the underlying mechanisms of dehydroxyhispolon methyl ether (DHME), a hispolon derivative designated as V5 in Balaji et al. . Our fin50 values of 12.25 ± 1.20 μM, 7.73 ± 0.25 μM, and 7.13 ± 0.35 μM for HCT 116, HCT-15, and LoVo cells, respectively. However, the viability of the CCD 841 CoN cells failed to drop to 50%, even when treated with 50 μM of DHME (p < 0.001), respectively  for 48 h, followed by cell viability determination using an MTS assay. It was noticed that DHME curtailed the survival of all tested CRC cell lines in a dose-dependent way, with IC of DHME A. Appare of DHME B. Specifectively C. Overal50 of hispolon for LoVo cells was 34.18 ± 1.93 μM, almost a six-fold increase compared to that of DHME (6.41 ± 0.80 μM). The difference between hispolon and DHME in HCT 116 cytotoxicity was even more obvious: while hispolon at 50 μM failed to lower HCT 116 cell viability to 50%, the IC50 of DHME was 10.56 ± 0.75 μM  (p < 0.001) and LoVo  cells, respectively (p < 0.001)  polymerase (PARP) cleavage, a canonical hallmark of caspase activation and hence apoptosis, in DHME-treated CRC cells was evaluated by immunoblotting. We noted an evident, dose-dependent increase in cleaved PARP levels in all DHME-treated CRC cell lines, illustrating the induction of CRC cell apoptosis by DHME A. To fur< 0.001) D–F. TakeGiven the fundamental contribution of aberrant WNT/β-catenin signaling to CRC pathogenesis and progression, we were eager to define the effect of DHME on this particular CRC oncogenic pathway using the TOPFlash assay system . To thisp < 0.001). Altogether, our findings identified suppression of WNT/β-catenin signaling as an integral mechanism of action of DHME-induced CRC cytotoxicity.We next addressed the functional significance of WNT/β-catenin signaling blockage in DHME- elicited anti-CRC action. In this regard, CRC cell lines stably expressing a constitutively active β-catenin mutant  ,24) wereIn this study, the DHME-induced, pro-apoptotic effect and the mechanism of DHME’s pro-apoptotic action in the context of human CRC cell lines was delineated for the first time. Specifically, we began by showing the cytotoxic effect of DHME on CRC cells while sparing normal colon epithelial cells , and fur3 in Balaji et al. vs. 8 × 103 in ours), as well as different length of drug incubation time  were applied. Alternatively, there might be certain disparities in the HCT 116 cell line between the one used by Balaji et al. and ours, such as growth status and passage numbers, which likely led to different sensitivity of cells to drug treatment.The data presented here indicate that DHME was cytotoxic against a panel of human CRC cell lines and exhibited stronger CRC cytotoxicity than hispolon . It is nIn this study, we demonstrated that DHME is pro-apoptotic to CRC cells, and further confirmed the induction of CRC cell apoptosis as a central mechanism underlying DHME-mediated cytotoxic action . Still, APC gene, as seen in HCT-15 and LoVo cells [CTNNB1 gene, as found in HCT 116 cells [Mounting evidence has underscored the fundamental role of aberrant WNT/β-catenin signaling in CRC genesis and malignant progression. It is well-established that WNT/β-catenin signaling controls pleiotropic events of malignancy, including sustaining stemness and promoting cell proliferation, survival, and invasion . Along tVo cells ,27 or ga16 cells . The resBCL-2 gene in human CRC cell lines HT-29 and SW620 [As a central regulator of cell survival, WNT/β-catenin signaling is known to inhibit apoptosis by activating β-catenin/TCF-mediated transcription of antiapoptotic genes, such as BCL-xL and survivin ,30, or bnd SW620 ,33. In vIn conclusion, we herein present the first report that documents DHME’s selective cytotoxicity to CRC cells while sparing normal colon epithelial cells, reveals a stronger CRC cytotoxicity of DHME than that of its parental structure hispolon, and establishes the mechanism of DHME-elicited cytotoxic action involving the induction of CRC cell apoptosis via suppression of the WNT/β-catenin signaling axis . CollectDehydroxyhispolon methyl ether (DHME) and hispolon were chemically synthesized and prepared as reported in Balaji et al. . Pan-casThe luciferase reporter vector for β-catenin-mediated, TCF/LEF-mediated, transcriptional activity M50 Super 8x TOPFlash (Addgene plasmid #12456) and the TOPFlash mutant vector M51 Super 8x FOPFlash (Addgene plasmid #12457) were gifts from Professor Randall Moon. The pBabe-HA–∆N90-β-catenin plasmid, a pBabe-based vector for the ectopic expression of an N-terminal, hemagglutinin (HA) epitope-tagged, dominant–active β-catenin mutant, has been described in detail in our previous report . Product2.Human colorectal carcinoma (CRC) cell lines HCT 116 (ATCC CCL-247), HCT-15 (ATCC CCL-225), and LoVo (ATCC CCL-229) were obtained from the Bioresource Collection and Research Center (BCRC) , and were cultured in McCoy’s 5a, RPMI-1640, and F-12K media , respectively. Normal human colon epithelial cell line CCD 841 CoN (ATCC CCL-1790) was purchased from the American Type Culture Collection (ATCC) , and was grown in Eagle’s Minimum Essential Medium (MEM) medium. All of the culture media were replenished with 10% fetal bovine serum, 1% penicillin–streptomycin, and 1% sodium pyruvate . Cells were grown at 37 °C in a humidified environment with 5% COThe in vitro cytotoxicity of DHME and hispolon was determined by the levels of cell viability after drug treatment for 48 h by the use of a CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS assay) , in addition to evaluating the capacity of drug-treated cells to form colonies (clonogenicity assay). Assays for cell viability and clonogenicity were conducted following our established protocol ,36.Immunoblotting was carried out as previously reported . PrimaryDHME-induced apoptosis was quantitatively evaluated by flow cytometry analysis to identify the levels of annexin V-stained (apoptotic) cells on the Muse Cell Analyzer, using Muse annexin V and a Dead Cell Assay Kit , in accordance with the procedures reported previously . To elucRenilla luciferase  was transfected into cells along with TOPFlash or FOPFlash plasmids for normalization of transfection efficiency. Dual luciferase activity assay was executed in accordance to our established protocol [The effect of DHME on the activity of WNT/β-catenein signaling was determined by the luciferase-based TOPFlash reporter system, where the amount of β-catenein-mediated TCF/LEF-dependent transcription activity was revealed by the activity of the luciferase reporter. A plasmid expressing protocol .t-tests were employed to determine the difference between two independent experiments, and the difference was considered statistically significant when p < 0.05.All data were obtained from at least three independent experiments, and are expressed as means ± SD. Unpaired, two-tailed