Acute lymphoblastic leukemia (ALL) is an aggressive hematologic neoplastic disorder that arises from the clonal expansion of transformed T-cell or B-cell precursors. Thanks to progress in chemotherapy protocols, ALL outcome has significantly improved. However, drug-resistance remains an unresolved issue in the treatment of ALL and toxic effects limit dose escalation of current chemotherapeutics. Therefore, the identification of novel targeted therapies to support conventional chemotherapy is required. The Wnt/β-catenin pathway is a conserved signaling axis involved in several physiological processes such as development, differentiation, and adult tissue homeostasis. As a result, deregulation of this cascade is closely related to initiation and progression of various types of cancers, including hematological malignancies. In particular, deregulation of this signaling network is involved in the transformation of healthy HSCs in leukemic stem cells (LSCs), as well as cancer cell multi-drug-resistance. This review highlights the recent findings on the role of Wnt/β-catenin in hematopoietic malignancies and provides information on the current status of Wnt/β-catenin inhibitors with respect to their therapeutic potential in the treatment of ALL. Acute lymphoblastic leukemia (ALL) is an aggressive hematological disorder that can originate from either B-lineage (B-ALL) or T-lineage (T-ALL) lymphoid precursors and is characterized by a marked heterogeneity at both molecular and clinical levels.B-ALL is the most frequent neoplasia in childhood, which represents 80% of pediatric ALL . Thanks T-ALL is caused by clonal transformation of T-lineage lymphoid precursors and comprises 10–15% of pediatric ALL cases and around 25% of adults . T-ALL hRecently, it has been demonstrated that several key drivers of cell growth, proliferation, survival, and differentiation networks contribute to the pathogenesis of ALL. This opens new opportunities for future innovative therapies that could ameliorate the prognosis of ALL patients, especially for the high-risk or relapsed ones .The Wnt/β-catenin network is an evolutionarily conserved signaling pathway that plays a fundamental role in many physiological processes such as differentiation, proliferation, and cell fate determination. Hence, aberrant activation of this cascade may underlie the initiation and progression of various types of cancers, including hematologic malignancies .In this review, the current insights into the relevance of Wnt/β-catenin signaling in hematopoiesis and ALL development will be highlighted. The pharmacological application of Wnt/β-catenin inhibitors in view of therapeutic strategies will also be described.The Wnt/β-catenin pathway regulates several physiological processes such as embryogenic development, adult tissue homeostasis, wound healing, and stem cell maintenance by regulating the cell fate, differentiation, apoptosis, polarity, and migration .The Wnt family is a group of secreted cysteine-rich glycoproteins (19 in humans) that carry out their functions through three different signaling pathways named β-catenin-independent, “non-canonical” β-catenin-dependent, and “canonical” β-catenin-dependent (hereinafter Wnt/β-catenin) which is the most studied [In mammals, some consecutive steps characterize the Wnt/β-catenin axis: Wnt/β-catenin activation at the membrane level, its stabilization in the cytoplasm, where β-catenin can accumulate, and translocation of β-catenin to the nucleus where it activates Wnt/β-catenin-dependent target genes  Figure .The activation of the pathway occurs at the cellular membrane, where Wnt ligands bind to the seven transmembrane-domain protein receptors Frizzled (Fzd) and/or to the low-density lipoprotein receptor-related protein (LRP) 5/6. This interaction leads to the inhibition of the axin degradasome destruction complex, which is a multiprotein complex that controls the cytoplasmic amount of β-catenin via phosphorylation, and, thereby, triggers β-catenin degradation by the proteasome in the absence of Wnt . The desWhen Wnt binds to Fzd and/or LRP5/6 receptors, the Wnt/β-catenin pathway is activated and the axin degradasome is inhibited . As a coC-MYC, CCND1, BIRC5, and CDKN1a [Moreover, inactivation of GSK3β through Akt-dependent Ser9 phosphorylation prevents the phosphorylation of β-catenin, which allows its stabilization and accumulation in the cytoplasm. Stabilized β-catenin translocates to the nucleus where it binds to transcription factors, notably T-cell factor (TCF) and lymphoid enhancing factor (LEF), TCF/LEF. This interaction displaces the co-TCF/LEF repressor Groucho, whose function under basal conditions is to compact chromatin . Grouchod CDKN1a ,18.Under physiological conditions, Wnt/β-catenin signaling pathway is strictly and efficiently regulated at many levels through multiple positive and negative feedback mechanisms .R-spondins represent the main activators of Wnt/β-catenin axis. They are a family of secreted proteins that prevent LRP5/6 internalization and increase the activation of the Wnt/β-catenin cascade through a synergism with Wnt ligands. It has been shown that R-spondin 1 improves Wnt/β-catenin pathway activity by enhancing β-catenin stabilization and phosphorylation of LRP6 . ImportaZNRF3 and RNF43 are single-pass transmembrane E3 ligases carrying intracellular RING domains. They act as powerful negative regulators of the Wnt/β-catenin pathway through their ability to promote the ubiquitination and lysosomal degradation of Fzd and LRP5/6. Of note, RNF43 and ZNRF3 are encoded by Wnt target genes, which leads to a negative feedback loop ,23. LossNorrin is an extracellular growth factor that represents another key activator of the Wnt/β-catenin pathway, which interacts with Fzd4 and requires LRP5/6 for its activation ,29,30.Several negative regulators finely tune the Wnt/β-catenin network via their binding to Wnt ligands. For instance, secreted Frizzled-related proteins (SFRPs), in concert with Wnt inhibitory factor (WIF) and adenomatosis polyposis down-regulated 1 (APCDD1), inhibit Wnt/β-catenin signaling by preventing Fzd and Wnt binding ,32.Sclerostin (SOST) and sclerostin domain containing 1 (SOSTDC1), alias WISE, counteract Wnt/β-catenin signaling by binding to LRP5/6 ,34,35. CMoreover, it has been demonstrated that the positive Wnt/β-catenin regulator Dvl also acts by recruiting ZNRF3 and RNF43 to Fzd receptors and, thus, inhibits the pathway . The ZNRHematopoiesis is a lifelong and tightly regulated process that gives rise to all blood cell types. Hematopoiesis depends on hematopoietic stem cells (HSCs), which is a pool of rare stem cells characterized by pluripotency and self-renewal through asymmetric division, quiescence, and multi-lineage differentiating potential. Even though dormancy is the preferred status for HSCs, upon specific stimuli, these cells can self-renew, and yield hematopoietic progenitors that, in turn, give rise to the mature hematopoietic cell lineages . HSCs reWnt/β-catenin network controls the delicate balance between self-renewal and lineage commitment of HSCs as well as the HSCs maintenance . This tiThe specific roles played by Wnt/β-catenin axis on healthy HSCs are still a matter of debate as several studies, based on loss-of-function and gain-of-function approaches, resulted in conflicting results.It has been shown that aberrant overexpression of β-catenin, in both in vitro and in vivo studies, activated the long-term growth of HSCs . On the Luis and colleagues  gave an Likewise, loss-of-function studies produced conflicting data. β-catenin-deficient mice displayed a decreased HSCs self-renewal capacity, long-term growth, and maintenance , while a−/− mice [In Dkk1 transgenic mice  or Wnt3a−/− mice , Wnt/β-c−/− mice  demonstrSeveral studies showed that, during hematopoiesis, the expression and modulation of different Wnt proteins, including Wnt3, Wnt5, and Wnt9, seem to have different effects during differentiation stages, which suggests that they are non-redundant ,59. For In general, all these findings suggest that a complex balance of the Wnt/β-catenin pathway is necessary to maintain HSC integrity. Moreover, it has been indicated that the lack of Wnt/β-catenin signaling is detrimental to HSCs function. However, a weak activity of the Wnt/β-catenin pathway is sufficient to restore the HSCs capacity .Aberrant proliferation and differentiation of HSCs is one of the key features of leukemic transformation . Due to Even though the precise cellular mechanisms underlying leukemic transformation are still unclear, mutations of Wnt ligands or β-catenin-dependent genes have a fundamental role in hematological malignancies pathogenesis, which suggests that these diseases may be “Wnt addicted.”The aberrant activation of Wnt/β-catenin signaling is related to different hematological malignancies, either by mutations or ectopic activation, including acute myeloid leukemia (AML) , chronicThe hyperactivation of the Wnt/β-catenin pathway in ALL may be due to a variety of dysregulated mechanisms, including aberrant expression of Wnt proteins , epigeneC-MYC, BIRC5, TCF1, and LEF [C-MYC represents a target gene of Wnt/β-catenin and Notch signaling cascades and it is a promising target for eradicating LSCs in T-ALL [BIRC5 gene, is downstream of c-myc and its aberrant expression has been observed in ALL primary cells [Wnt/β-catenin pathway deregulation is a frequent event in T-ALL pathogenesis. About 80% of pediatric T-ALL patients showed higher levels of β-catenin compared to healthy controls, which leads to aberrant activation of β-catenin-dependent genes, including  and LEF ,80. It i and LEF ,82. Of nin T-ALL . Surviviry cells .+ CD8+ double positive thymocytes that predispose it to malignant transformation, which leads to a c-myc aberrant activation and develops a Notch-independent leukemia form [It has been shown that β-catenin overexpression targeted the highly proliferative CD4mia form ,85. It hmia form . In partmia form . Notablymia form . This fumia form .PTEN deletions cooperated with β-catenin in leukemia development, which demonstrated that activation of the Wnt/β-catenin pathway is related to a subtype of Notch-independent T-ALL, characterized by C-MYC rearrangements and PTEN mutations [Furthermore, utations .LEF1, which is a member of the LEF/TCF complex, may act as either a tumor suppressor or an oncogene in different cellular contexts. On one side, a significant increase of LEF1 has been observed in more than 25% of adult T-ALL samples  and, in The role played by the Wnt/β-catenin network in T-ALL pathogenesis is very complex and the precise mechanisms underlying leukemia development remain to be elucidated.Dysregulation of the Wnt/β-catenin pathway is involved in B-ALL development as demonstrated in a pre-B ALL subtype characterized by  translocation, where Wnt16b was hyper-activated by the expression of the aberrant E2A-Pbx1 fusion protein. This contributes to leukemia development . SubsequIt has also been shown that Wnt proteins increased proliferation of B-ALL cell lines  and thatTherefore, activation of the Wnt/β-catenin network may be a general phenomenon that characterizes B-ALL, acting as a mean for the microenvironment to sustain the survival of the LSCs.Nygren et al.  suggesteIt has been reported that Wnt5a, which promotes GSK3β-independent β-catenin degradation , has an It has been demonstrated that mice overexpressing a constitutive active LEF1 mutant developed B-ALL . This ob+) B-ALL, which is a leukemia subset characterized by an extremely poor outcome [+ B-ALL, the over-activation of Wnt/β-catenin pathway in B-ALL may be caused by epigenetic alterations, including hypermethylation of promoters of the Wnt/β-catenin antagonists SFRP, WIF1, and Dkk3 [− and HCO3− [Last, but not least, the Wnt/β-catenin network is dysregulated in Philadelphia positive  antagonist was developed for pediatric patients with relapses of B-ALL (NCT01186328) [It has been demonstrated that, in ALL, Wnt/β-catenin antagonism has a role in depleting the self-renewal of drug-resistant LSCs ,105. Inh1186328) .Different ways to inhibit Wnt/β-catenin signaling are currently under investigation for acute leukemias see .Several Wnt/β-catenin modulators are now undergoing clinical trials to treat different tumors, including ovarian cancer (NCT02092363), pancreatic cancer (NCT01764477), and advanced solid tumors (NCT02521844). These studies have shown some promising outcomes .In B-ALL cells, it has been observed that the interaction between Wnt/β-catenin pathway and BM stromal cells contributed to cytarabine resistance . TreatmeBIRC5, and abrogated drug-resistance in primary leukemia cells [When added to classical chemotherapeutic drugs, ICG-001, which is a CBP/β-catenin transcription inhibitor, led to decreased self-renewal capacity of ALL cells, downregulated ia cells . Authorsia cells . TherefoThese findings have been confirmed through the inhibition of Wnt by iCRT14 (an inhibitor of β-catenin-dependent transcription) that caused marked cytotoxic effects in ALL cell lines and relapsed ALL samples and restored chemo-sensitivity .Moreover, PKF115-584  prevented and partially reverted leukemogenesis, by inducing apoptosis and reducing proliferation in human T-ALL cells .In the last few years, crosstalk and correlation between up-regulation of Wnt/β-catenin and multiple signaling pathways have emerged in ALL.Inhibition of Wnt/β-catenin and phosphatidylinositol 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathways is considered a possible innovative therapeutic strategy for cancer treatment ,110.Independent studies have identified that both PI3K/Akt/mTOR and Wnt/β-catenin signaling networks contribute to leukemia by sustaining neoplastic cell proliferation and drug-resistance ,112,113.Our group recently showed that co-targeting Wnt/β-catenin and PI3K/Akt/mTOR pathways could be a potentially promising treatment in T-ALL settings . InhibitWnt/β-catenin and Notch pathways are linked each other both in development and oncogenesis . Notch1-PTEN loss is frequently associated with Wnt/β-catenin dysregulation in leukemia settings [PTEN deletion clustered together in a mouse model with Notch-independent T-ALL [Another paper demonstrated that settings . β-catennt T-ALL .In addition, the aberrant activation of the mitogen-activated protein kinase (MAPK) pathway, which is a common event in cancer, has been shown to be associated with the activation of the Wnt/β-catenin network in relapsed ALL .The bromodomains (BRD) extra terminal proteins (BET) inhibitors directly target BRD proteins, while downregulating c-myc transcription. Given the pivotal role of c-myc in cancer, the effects of BET inhibitors in leukemia were described in several studies and these drugs represent a new therapeutic option. Several BET inhibitors are currently being evaluated in clinical trials in a range of diseases, including hematological malignancies (NCT02158858), even if molecular and cellular mechanisms that govern sensitivity and chemo-resistance remain unknown.Recently, it has been shown that c-myc inhibition reduced LSCs in mice by demonstrating the involvement of c-myc in LSCs maintenance in ALL ,119. TreCombined treatment with a β-catenin inhibitor and a BET inhibitor had strong cytotoxic effects in in vivo and in vitro post-myeloproliferative neoplasms (MPN) AML blast progenitor cells (BPCs) ,122. MorInhibition of Wnt/β-catenin cascade resulted in restoration of sensitivity to BET inhibitors both in vitro and in vivo , which fAll these data together provide new insights into the combination of BET and Wnt/β-catenin inhibitors, which represents a potential new therapeutic strategy to overcome chemo-resistance in leukemia.Although a large number of compounds inhibiting Wnt/β-catenin signaling have been explored as anti-cancer therapeutics, only two have been tested in clinical studies for hematological disorders. CWP232291 is a β-catenin inhibitor that interferes with the β-catenin-dependent gene expression and is currently being tested for AML (NCT01398462). PRI-724, an ICG-001-derived compound, has entered early-phase clinical trials for hematological malignancies, where it displayed a tolerable toxicity profile .ALL is an aggressive blood disorder that needs more effective and less toxic targeted therapies, especially for primary resistant and relapsed patients. Chemo-resistance is due to persistence of residual LSCs, which are characterized by quiescence that allow them to escape chemotherapy-induced cell death. Increasing attention has been focused to eradicate the bulk of LSCs by developing novel strategies targeting aberrant signaling networks that lead to LSCs survival and to drug-resistance.Wnt/β-catenin axis has a central but controversial role in hematopoiesis, which suggests the possible existence of unidentified mechanisms through which the Wnt/β-catenin pathway regulates blood cell production. Moreover, overexpression of Wnt/β-catenin signaling axis has been reported in several cancers, including hematological malignancies and contributes to chemo-resistance. In this context, inhibition of the Wnt/β-catenin pathway offers alternative and interesting possibilities for blood disorders therapeutic intervention, including ALL.There is now considerable proof that targeting the Wnt/β-catenin network at the level of the receptor/ligand is an attractive therapeutic strategy for inhibiting tumor growth, chemo-resistance, and potentially invasiveness. In particular, the development of Wnt/β-catenin inhibitors at the level of the ligand/receptor, including Wnt secretion inhibitors or antibodies blocking Fzd receptors, are now under investigation and have entered clinical trials.However, further investigations on the mechanisms underlying the controversial functions of the Wnt/β-catenin pathway will provide new insights in better understanding the processes controlled by Wnt/β-catenin, by identifying those that may safely benefit from administration of selective drugs. Moreover, these studies could provide insight toward other molecules, controlled by the Wnt/β-catenin signaling axis, which would serve as targets for therapeutic intervention.Another critical step will be to shed light on the reciprocal interactions between Wnt/β-catenin and the BM microenvironment that support the LSCs’ survival. Genetic and pharmacological approaches showed that inhibition of Wnt secretion had no effects on maintenance, self-renewal, and differentiation of adult HSCs . These dImportantly, in line with this finding, a recent study showed that LSCs express high levels of the long isoform of LEF1 compared with immature and more quiescent HSCs. This differential expression of the LEF isoform in LSC versus normal HSCs offers the opportunity to preferentially target LSCs by impairing LEF1-β-catenin interaction, and targeting Wnt/β-catenin axis. This differential dependency of LSC versus HSCs on LEF1-mediated Wnt/β-catenin signaling could potentially be exploited for targeting LSCs while sparing the healthy HSCs .Considering the clinical implications of all these findings, the involvement of Wnt/β-catenin signaling in normal and malignant hematopoiesis should be better addressed.


Aberrant mechanical factor is one of the etiologies of the intervertebral disc (IVD) degeneration (IVDD). However, the exact molecular mechanism of spinal mechanical loading stress-induced IVDD has yet to be elucidated due to a lack of an ideal and stable IVDD animal model. The present study aimed to establish a stable IVDD mouse model and evaluated the effect of aberrant spinal mechanical loading on the pathogenesis of IVDD. Eight-week-old male mice were treated with lumbar spine instability (LSI) surgery to induce IVDD. The progression of IVDD was evaluated by μCT and Safranin O/Fast green staining analysis. The metabolism of extracellular matrix, ingrowth of sensory nerves, pyroptosis in IVDs tissues were determined by immunohistological or real-time PCR analysis. The apoptosis of IVD cells was tested by TUNEL assay. IVDD modeling was successfully produced by LSI surgery, with substantial reductions in IVD height, BS/TV, Tb.N. and lower IVD score. LSI administration led to the histologic change of disc degeneration, disruption of the matrix metabolism, promotion of apoptosis of IVD cells and invasion of sensory nerves into annulus fibrosus, as well as induction of pyroptosis. Moreover, LSI surgery activated Wnt signaling in IVD tissues. Mechanical instability caused by LSI surgery accelerates the disc matrix degradation, nerve invasion, pyroptosis, and eventually lead to IVDD, which provided an alternative mouse IVDD model. Intervertebral disc (IVD) degeneration (IVDD) is clinically associated with LBP and recognized as the main pathogenic factor for LBP3. Previous studies on tissue mechanics showed that persistent aberrant mechanical stress generated by body weight and muscle force is one of the common causes of disc degeneration and associated LBP, and there is a positive relationship between changes in mechanical properties and abnormal changes of IVD structure and composition5. However, due to a lack of an ideal animal model of persistent instability of IVDD, the exact molecular biological mechanisms by which aberrant spinal mechanical loading initiates and promotes IVDD has not been fully elucidated.Low back pain (LBP) is a highly prevalent disabling musculoskeletal disease, affecting as much as 84% of the population worldwide at some point in their lifetime6. Each IVD consists of the central nucleus pulposus (NP) surrounded by lamellated collagenous ring, the annulus fibrosus (AF), and cartilage endplates (EP) comprises of the superior and inferior boundaries of the IVD separating the disc from the vertebrae. On the inner border of the AF, the fibers form a tissue mainly composed of type II collagen (Col2). Constrained by the tight AF, NP is critical water-retaining tissue, producing Aggrecan and other proteoglycans. The complex structural features of IVDs enable to absorb and disperse loads from physical activities and other body parts, as well as maintenance of disc height7. Aberrant changes in extracellular matrix (ECM) composition are key to the development of IVDD. Mechanical test on cadaveric lumbar motion segments suggested that complex mechanical loading simulating typical activities in vivo could damage IVDs, including gross structural disruption as well as cell-mediated matrix composition changes, and eventually result in IVDD8. However, the mechanical factors driving IVD cell transition are still unclear, particularly how the mechanical load influences cell signaling.IVDs are the major cartilaginous joints of the vertebral column, contributing about 1/3 of spinal length11. Pyroptosis, also known as cell inflammatory necrosis, is a newly discovered programmed cell death that manifests as cells continue to swell until the cell membrane ruptures, resulting in the release of cell contents and activation of a strong inflammatory response12. Pyroptosis is mediated by the formation of inflammasome complexes, which are cytosolic heptameric oligomers composed of the nucleotide-binding domain and leucine-rich repeat (NLR) pattern recognition receptors. The best-characterized NLR, NLRP3, is a redox-sensitive cytosolic sensor, which triggers the docking and activation of pro-Caspase-1, thus cleaving pro-IL-1β into 17-kDa mature active forms, IL-1β14. Nevertheless, during the development of IVDD, the role of pyroptosis is remains obscure. Moreover, a previous clinical study found that nerve ingrowth is abundant and maximal in degenerate IVDs of patients expressing pain15. Similarly, a puncture-induced disc herniation in the rodent also showed that puncture-induced damage to IVDs resulted in pinched nerves, which is potential pathogenesis of pain in the lower back and legs16. However, the underlying mechanism has not yet entirely understood.Recent studies have postulated that both chronic inflammation and innervation are both key contributors to LBP19. Both canines with an age-related propensity for degeneration and IVDD patients have up-regulated levels of β-catenin20. The analysis of Wnt signaling using mouse models has been indispensable in further elucidating the development of IVD and the occurrence of disc diseases21. Mice haploinsufficient for β-catenin in osteocytes and/or late-stage osteoblasts showed no measurable anabolic response to mechanical stimulation in vivo22. The activation of Wnt signaling allows nuclear-translocated β-catenin to combine with the transcription factors TCF proteins and activates expression of target genes, such as Dkk1 and Lef124. Specifically blocking Wnt/β-catenin signaling pathway by Dkk-1 could reverse TNF-α induced disc degeneration and protect the normal metabolism of intervertebral disc tissue25.Although the biological mechanisms controlling mechanical transduction has not been completely defined, considerable research have revealed that Wnt/β-catenin signaling plays an essential role in mechanically induced signal transduction, thus participating in the regulation of normal disc metabolism and diseased states26. Therefore, the purpose of this study was to construct and characterize an aberrant spinal mechanical loading induced IVDD model which can help to gain a better understanding of the pathophysiologic events during mechanical instability-induced pathogenesis of IVDD.To the best of our knowledge, the most common IVDD model was created by needle puncturing in IVDs, which is difficult to simulate the non-injured degeneration of the human IVDD27. We firstly evaluated the disc height of the L4-L5 IVD at 1-, 2-, 4- and 8-week after LSI surgery by micro-computed tomography (μCT) analysis. We found that the disc height of LSI mice was significantly decreased 8-week post-surgery  model by removing spinous processes, posterior supraspinous and interspinous ligaments, so as to construct a mouse IVDD model Fig. a,b. Sinc28. The score results suggested that NP and EP had significant decreases since 2-week after surgery, followed by decreases of AF scores 4- and 8-weeks after surgery  by immunofluorescent (IF) analysis. The IF results showed that LSI surgery significantly increased the number of Cgrp-positive cells in AF region 42 could induce aberrant mechanical loading of lumbar spine, and finally lead to IVDD. Moreover, decompensated changes in lumbar spine structure result from abnormal mechanical environment, such as proliferation of facet joints43, formation of osteophytes44, hypertrophy of ligamentum flavum45 or calcification of longitudinal ligament47, will fail to resist aberrant mechanical loading and eventually accelerate the process of IVDD in patients. Since there is no desirable model to mimic the clinical development process of aberrant mechanical loading induced-IVDD more accurately, the underlying molecular events of IVDD pathogenesis remains largely elusive. In this study, we constructed a reproducible mouse IVDD modeling with direct in vivo evidence of medical iconographic and morphological changes in the IVD after loading axial anomalous stress. LSI surgery could promote the progression of IVD degeneration, including notable decreases in IVD height and histological score, ectopic new bone formation in EP, folds and tears in AF, reduction of the vacuole volume in NP, acceleration of ECM degradation, innervation into AF, and induction of pyroptosis after IVDD modeling. In addition, LSI treatment resulted in the activation of Wnt/β-catenin signaling in IVDD mice, which might be responsible for the above changes 50, which was also significantly elevated in IVD samples obtained from patients with disc degeneration52. To develop a mouse model to mimic human IVDD, our previous study has generated an inducible β-catenin conditional activation (cAct) mice by breeding β-cateninfx(Ex3)/fx(Ex3) mice with Col2a1-CreERT2 transgenic mice, according to the up-regulation of β-catenin in disc tissue of IVDD patients. Although these mice have multiple features that resemble some of the features of human IVDD, NP cells were not targeted by Col2a1-CreERT2 transgenic mice and the severely disorganized defect seen in NP tissues may result from the disruption of nutrient and solute supplies to this region and structural changes in AF tissues after the loss of the growth plate cartilage in β-catenin cAct mice20. Therefore, the phenotype of these mice may only partially resemble the feature of human IVDD. In the present study, we found that LSI surgery significantly increased β-catenin and its target proteins both in NP and AF tissues, which provided an alternative method for simulation of human IVDD.The β-catenin adhesion complex is one of the central components of the cell–cell adhesion junctions, transmitting mechanical stress from cell to cell. Mechanical stress could upregulate β-catenin protein53. The latest research indicated that pyroptosis activation in IVD was likely responsible for the inflammatory pathology of IVDD54. And mechanical stress could drive inflammatory responses associated with IVDD and LBP in vitro55. The important finding in our study is that the pyroptosis was significantly facilitated in outer AF and ectopic bone formation zone inside EP after LSI surgery treatment. The increase of IL-1β induced by pyroptosis, a main inflammatory factor, will further inhibit ECM anabolism and promotes its catabolism in IVDs, as well as the boost of apoptosis in IVD cells.Clinical evidence has reported that IVD inflammation and axonal growth of afferent fibers innervating the disc are main factors of discogenic LBP, and IL-1β is a pain-related molecule, which is significantly elevated in painful human IVD10. Whether mechanical stress could directly promote nerve ingrowth in AF is an under-investigated aspect. Navone et al.found that mechanical stress could affect nerve proliferation by changing the disc microenvironment56. Impaired AF could lead to the loss of focal proteoglycan, and leave a matrix favorable for nerve ingrowth57. In this study, we found that IVDD mice exhibited a significant increase in the number of Cgrp-positive sensory nerves both in outer and inner AF. This finding is very similar to the phenomenon in clinical patients, that is, IVDD patients had more nociceptive sensory innervation in AF, which is an important origin of LBP in IVDD patients. In addition, a large number of cytokines released by pyroptosis cells also stimulate sensory nerves, which further leading to more severe LBP. Therefore, anti-pyroptosis and anti-innervation may serve as potential therapeutic strategies for IVDD disease and LBP.Besides, the onset of discogenic pain is characterized by ingrowth of nerve fibers into an otherwise aneural tissues58. And activation of Wnt/β-catenin signaling led to apoptosis of IVD cells and increase of ECM degradation enzymes, thereby accelerating matrix degradation and promoting IVDD progression59. However, the relationship between LSI surgery and Wnt/β-catenin signaling in IVDD mice is largely elusive. Our results found that LSI administration up-regulated Wnt/β-catenin signaling, accompanied by increased Adamts5 and Mmp3 in IVDs. Recent evidence also indicated that down-regulating Wnt/β-catenin signaling could suppress Nlrp3 inflammasome activation60. Consistent with the above findings, we found that the activation of Wnt/β-catenin signaling preceded the increase of Nlrp3, Caspase-1 and IL-1β in IVD tissues of IVDD mice. These findings indicated that LSI treatment might promote pyroptosis via activation of Wnt signaling.Previous research has reported that sustained mechanical load could activate Wnt/β-catenin signaling in the development of osteoarthritisCol2a1-CreERT2 transgenic mice cannot effectively target all disc cells, especially NP cells. We have constructed Aggrecan-CreERT2 transgenic mice to specifically target disc cells61, which enable us to further explore the relationships between Wnt/β-catenin signaling and mechanical stress, pyroptosis and nerve ingrowth during the pathological process of IVDD.As stated above, one limitation of this study is that not all aspects of pathological factors of IVDD can be taken into consideration in experimental animal models, and the experimental animal model can only partially resemble complex human conditions. However, this simplified model is extremely useful in investigating the reciprocal interactions between mechanical loading and IVDD progression. In this study, there was a significant induction effect on pyroptosis and innervation of IVDs in IVDD mice, which suggested that the therapy of anti-pyroptosis and anti-innervation may have therapeutic potential for clinical IVDD. However, whether the therapy was able to directly target the intervertebral disc, as well as serious side effects on the human body is still masked. In addition, To sum up, we found a mouse IVDD modeling by operating the LSI surgery. Aberrant mechanical stress-induced lumbar IVDD model may expand our knowledge regarding the occurrence and development of IVDD, and also may help explain why osteophytes and spinal stenosis occur. Simultaneously, the study reminded us that restoring the normal mechanical environment of IVD, mitigating IVD pyroptosis, or inhibiting Wnt/β-catenin signaling pathway could have therapeutic potential for clinical IVDD.62. All the animal procedures were approved by the Ethical Committee of Zhejiang Chinese Medical University (IACUC-20190930–03).A total of 48 C57BL/6 J male mice  were purchased from Shanghai Laboratory Animal Center and housed at the Animal Care Facility of Chinese Medical University according to the institutional guidelines for laboratory animals. The ARRIVE  guidelines for reporting animal research were carried outAll mice were randomly divided into the LSI model group and the Sham group (n = 24 per group). After being anaesthetized with sodium pentobarbital, mice were placed on the surgical table in a prone position. The L5 vertebra was located on the anterior superior iliac spine. The lower dorsal skin was shaved and disinfected using iodine solution, then a longitudinal incision was created along the midline of the back to expose the lower lumbar spine. The posterior paravertebral muscles from the L3–L5 vertebrae were separated to expose the lumbar 3rd–5th spinous processes, which were further resected along with the supraspinous and interspinous ligaments using a fine scissor. Mice in the Sham group only received a detachment of the posterior paravertebral muscles from the L3–L5 vertebrae Fig. b. After 63. By using a high-resolution μCT  with 90 kVp source and 300 μA current, we scanned vertebrae at a resolution of 9 μm. Three-dimension model visualization software, CTVol v2.0 , was employed to analyze parameters of the L4-L5 IVD with half-height of the L4 and L5 vertebrae. To quantify disc space, we measured the distance between L4 and L5 from μCT images by Image Pro Plus 4.5 . L5 vertebral body TV was described to figure out the medial compartment excluding cortical bone, transverse and spinous processes. Three-dimensional structural parameters analyzed included: BS/TV, Tb.N, Tb.Sp.Before histological processing, we firstly evaluated paraformaldehyde-fixed vertebrae by μCT analysis as we previously described63. Histological score was graded in a blind fashion using the definition established by Norcross et al.28, with some modifications  for 3 weeks at room temperature, dehydrated and then embedded in paraffin. The lumbar tissues were processed into 5-mm-thick coronal-oriented sections for Safranin O/Fast green staining. Briefly, sections were incubated with primary antibodies to Aggrecan , Col2 , Mmp3 , Adamts5 , Nlrp3 , Caspase-1 , IL-1β , Cgrp , β-catenin , Tcf4 , Dkk1  and Ccn2  at 4 °C overnight. For IHC staining, a horseradish peroxidase streptavidin detection system  was subsequently used to detect the immunoactivity, followed by counterstaining with hematoxylin . For IF analysis, the slides were incubated with secondary antibodies conjugated with fluorescence at room temperature for 1 h. The morphometric study was performed using the Image Analysis System . Triplicates of each sample were used for staining. Quantitative histomorphometric analysis was conducted in a blinded manner with Image-Pro Plus Software version 6.0  as we previously described63. Negative controls were incubated in a TdT free-enzyme solution. The number of positive cells was quantified in three randomly selected fields of view using three sections from each sample. DAPI staining was used to estimate the total cell number.The TUNEL assay for detecting DNA breaks was performed with TUNEL Bright Green Apoptosis Detection Kit according to the manufacturer’s instruction  as we previously described-ΔΔCT method64.Total RNA was extracted from L4-L5 IVD using TRIzol reagent  following the manufacturer’s protocol. The yield and purity of RNA were quantified using a Nanodrop 2000C spectrophotometer  and 2 μg of RNA were reverse-transcribed into cDNA using the Prime Script RT Reagent Kit protocol . Real-time PCR amplification was performed using murine gene-specific primers and TB Green Premix Ex Taq II . The PCR program was: initial 2 min at 95 °C denaturing; 10 s at 95 °C denaturing; and 30 s annealing/elongation for a total of 40 cycles. The primers used for the PCR are listed in Table t-test. All data analysis was performed using SPSS 15.0 analysis software  and the level of significance was defined as P < 0.05.All the data were expressed as mean ± standard error of mean (sem), as indicated in the figure legends. Statistical significance was determined using Student’s


This research investigated the application of epoxy resin polymer as a self-healing strategy for improving the mechanical and durability properties of cement-based mortar. The epoxy resin was added to the concrete mix at various levels , and the effectiveness of healing was evaluated by microstructural analysis, compressive strength, and non-destructive (ultrasonic pulse velocity) tests. Dry and wet-dry conditions were considered for curing, and for generating artificial cracks, specimens at different curing ages (1 and 6 months) were subjected to compressive testing (50 and 80% of specimen’s ultimate compressive strength). The results indicated that the mechanical properties in the specimen prepared by 10% epoxy resin and cured under wet-dry conditions was higher compared to other specimens. The degree of damage and healing efficiency index of this particular mix design were significantly affected by the healing duration and cracking age. An optimized artificial neural network (ANN) combined with a firefly algorithm was developed to estimate these indexes over the self-healing process. Overall, it was concluded that the epoxy resin polymer has high potential as a mechanical properties self-healing agent in cement-based mortar.  Fissures and cracks are the main concerns for concrete due to its relatively low tensile strength, and this is one of the most severe problems affecting the durability and service life of concrete structures in the natural climate. At the early age of cementitious materials such as concrete, mortar, and paste, the loss of moisture from fresh mixture results in a reduction in volume. The restraint of mortar to volume changes caused initial shrinkage cracks. Initial shrinkage cracks in concrete normally occur in all building materials or components that are cement/lime-based such as concrete, mortar, masonry units, masonry and plaster, etc., and are one of the leading causes of cracking in structure. Initial shrinkage in concrete and mortar occurs during the construction of structural members due to drying out of moisture. The initial shrinkage of concrete is partly reversible if the moisture is maintained in concrete, but it becomes irreversible when concrete becomes dry ,2. DurinFrom a crack treatment perspective, conventional systems concentrate on the application of regular maintenance and scheming inspection. Nevertheless, in large-scale concrete structures, such conventional systems required a considerable amount of funds and labor. Furthermore, the treatment process somehow may be difficult or even impossible to implement where the affected structures are in service, i.e., large dams and tunnels. In these circumstances, self-healing concrete was proposed and has become increasingly attractive .In concrete structures, self-healing of micro-cracks has already been introduced as a phenomenon that cracks networks could be automatically repaired by rehydration of insufficiently hydrated or un-hydrated cement particles in cracked areas. However, the previous research indicated that the crack width that can be healed in such technique is limited to a maximum of 0.1 mm, which is not enough for practical application . In addiSelf-healing properties can improve in concrete using different techniques including encapsulation of polymers, inclusion of fibers, and secondary hydration of un-hydrated cement . The exi2 without any hardener [The epoxy resin’s unique properties such as strong adhesion, high corrosion-resistance, and chemical durability made it attractive in the construction sector to produce modified concretes. Additionally, epoxy resin is advantageous as the admixture in the concretes because it can impart many emergent properties absent in the pure concretes. However, to produce the epoxy resin-modified mortar hardeners are essential ,18,19. Ahardener .2) to produce the hardened epoxy motivated the researchers to apply epoxy resin as self-healing agent in concrete industries. It is established that the inclusion of the epoxy resin in the hydration process of the OPC matrix is responsible for enhancing the mechanical properties of the product. In the OPC hydration process, the produced hydroxyl ions are essential for the self-healing mechanism and C-(A)-S-H gels formation. In the bacteria-based self-healing concretes, the bacterial strain reacts with the oxygen to produce the calcite, thus precipitating and healing the concretes’ microcracks. In the epoxy resin-based self-healing concretes, similar mechanisms are involved, and also the epoxy resin can react with the hydroxyl groups, leading to the healing of the concrete cracks.The reaction ability between the epoxy resin and hydroxyl ions from the OPC hydration (Ca(OH)In this research, the epoxy resin without any hardener called diglycidyl ether of bisphenol A was adopted as the self-healing agent to produce a sustainable mortar for several construction applications with long life span and high durability. The mixes were designed with various ratios of the epoxy resin to OPC to assess the efficacy of the epoxy as the self-healing agent. The modified mortars were subjected to the dry and wet-dry conditions of curing to determine their improved properties such as flowability, CS development, porosity, degree of damage, and healing efficiency. The CS, ultrasonic pulse velocity (UPV), and SEM morphologies were recorded to determine the feasibility of implementing the epoxy resin as the self-healing agent in mortar manufacturing. In addition, an optimized artificial neural network (ANN) combined with a firefly algorithm was developed to estimate the degree of damage and healing efficiency output parameters over the self-healing process.2 (20.1%), and Al2O3 (5.4%), where the total of loss on ignition (LOI) was 2.2 of the weight%. The mineral compounds of OPC, provided by the X-ray diffraction  pattern analyses, consist of Dicalcium Silicate (C2S), Tricalcium Silicate (C3S), tricalcium aluminate (C3A), and calcium aluminoferrite (C4AF) with the proportion of 9.3, 74.9, 6.7, and 9.1%, respectively, as shown in 2S and C3S. These kind of elements will contribute to formulate the portlandite (Ca(OH)2) during the hydration process. Silicate minerals in non-crystalline or an amorphous state are highly reactive with Ca(OH)2 produced from the hydration of cement to form additional calcium silicate hydrate (C-S-H) gels. In this study, the OPC was collected from the Holcim Cement Manufacturing Company  following ASTM C150 standard  to prepaTo develop self-healing properties in the mix design, epoxy resin was used without any hardener for the easy reaction with the hydroxyl ions and hardening. Therefore, the epoxy resin with high viscosity was selected where it was obtained using a Digital Brookfield Viscometer  while the epoxy equivalent, molecular weight, and its flash point were provided by the manufacturing company . −1). For the OPC, the bands at 462.9, 824, and 1033 cm−1 corresponded to the Si-O, Si-O-Al, and Si-O-Si bending. The previous literature reported that the broad band’s existence around 500–650 cm−1 was related to the glassy phase (short-ranged ordering of the network structures) of the silicates [−1 were due to the C-H bending and H-O-H (water bands) stretching, respectively. The epoxy resin’s FTIR bands indicated the presence of different crosslinking mechanisms due to the chemical reactions between one or two kinds of monomers with different functional groups. The reactivity of the epoxies became completely different because of the alteration of the molecular structure. González et al. [−1 were assigned to the C-O-C, C-O, and C-O-C stretching. The C-H and O-H stretching were observed in the range of 2871 to 3600 cm−1. In addition, the FTIR spectra of the epoxy resin without hardener appeared to be a valuable tool for both qualitative and quantitative analysis of these processes. It provided the specific information and reaction mechanisms to identify the epoxy resin components and the existence of different vibration bands. Moreover, the O-H band analysis rendered some information about the intermolecular interactions within the components of the epoxy resin.The Fourier transform infrared  of the epoxy resin, and OPC were considered to identify and quantify their bonding structures, compositions, functional groups, and hydration properties. ilicates . Meanwhiz et al.  showed tThe sieve analysis result of the river sand is shown in (1)Following to ASTM C1329 and ASTM C109 standards, the ratio of the cement to river sand and water to cement was fixed to 0.33 and 0.48, respectively for all mix designs;(2)Municipal tap water was added to the concretes during the mixing and curing;(3)During the preparation stage, a saturated surface dry condition of the river sand was adopted.To evaluate epoxy resin’s adequacy as a self-healing agent, five batches of mix designs containing various epoxy resin levels were prepared in the laboratory, During the preparation stage, following ASTM C150 , first tThe workability of the mortars was measured using the flow table test. After the blend was mixed properly, the fresh mortar was poured onto the molds with two layers, where each layer was exposed to 15-s vibration to liberate the air and reduce the pores. The cubical samples with the dimension of 70 mm × 70 mm × 70 mm was used for the compressive strength (CS) test, cylindrical samples of dimension 75 mm × 150 mm was taken for the splitting tensile strength and the prism specimen of dimension 40 mm × 40 mm × 160 mm was prepared for the flexural strength test. Two types of curing conditions were applied on the obtained specimens including the dry and wet-dry. In the wet-dry state, the mortar was immersed in water for five days at (20 ± 3 °C). After five days, the specimens were taken out and left in the ambient condition (24 ± 3 °C) until the testing. In the dry curing, the mortar was left in the ambient state till testing. The epoxy-modified mortars’ fresh characteristics were evaluated in terms of the workability using the flow table test in compliance with ASTM C230 . The staThe CS of specimens were measured at the age of 28 days using the universal testing machine  with a 0.8 kN/s loading rate in compliance with ASTM C109 . Similarts) and after that, oven-dried for 24 h at 105 °C (Wtd). Each sample’s weight was measured and percentage of the water absorption (WA %) was calculated using the following equation in compliance with ASTM D 6532 [For the water absorption test, first three cubical epoxy-modified specimens were submerged inside water for 72 h (WM D 6532 .(1)WA =ts and Wtd (in kg) denote the weight of the treated mortar unit after the immersion in water and oven-dried. WTo evaluate the self-healing performance of the studied mortars, the artificial cracks were generated using a compression test machine  (loading speed 0.8 kN/s) at the ages of 1 and 6 months. After generating artificial cracks, the pre-loaded and non-loaded (control samples) were left for curing at ambient temperature (27 ± 1.5 °C) under the relative humidity of 75%. The main reason to have a similar environment condition for all specimens was to ensure no external factor can affect the self-healing process. a and Vb are the velocities after and before the peak loading (m/s) in the mortars.Suaris et al.  recommen1 is the CS before the loading; CS2 is the CS after the loading; CSn and CSm are the CS of pre-loaded and non-loaded specimens at same curing age.The self-healing efficiency can also be determined using the following equation:The effects of the epoxy resin on the workability of the prepared mortars were measured using setting time and the flow table test. − ions from the hydration of Ca(OH)2. Meanwhile, the results confirm that the mechanical properties tend to decrease by increasing epoxy resin content to from 10 to 15 and 20% significantly. For instance, the CS declined by 54% by increasing the epoxy resin from 10 to 20%. The previous literature indicated that by increasing the epoxy content by more than 10% in the mix design, the residual unhardened epoxy within the mortar matrix might have interrupted the hydration and polymerization processes [rocesses . It was − content that improve the hydration process and increasing the C-S-H gel product [ product . Using multi-linear regression analysis, The proposed empirical equations by multi-linear regression analysis are in good agreement with ACI equations.2 in the control specimen that are primary products of the cement hydration. 2 was generated, and it was consumed partially over C-(A)-S-H production. In the 10% epoxy resin modified specimen, the remaining Ca(OH)2 was reacted with the unhardened epoxy resin, creating strong bonds between the hydroxyl ions and epoxy to increase the CS. The continuous hydration reaction in the epoxy-modified mortar initiated the polymerization reactions. These reactions further strengthened the bonds and filled the pores within the mortar. Consequently, the improved mechanical behavior was achieved in the epoxy-modified mortars, particularly the one containing 10% of the epoxy resin.2 absorption due to residual hydroxyl ions used in the epoxy resin reaction. The FTIR spectrum indicated the Si-O, Al-O, and C-H reaction regimes in the mortar metric. The Si-O and Al-O bond vibration energy levels were increased from 698.8 and 787.8 cm−1 to 706.3 and 789.1 cm−1 with a corresponding rise in the epoxy resin level from 0 to 10%. Increasing demand of the OH− ion and its consumption in the production of the hardened epoxy (hydroxyl-ions-epoxy resin) affected the total amount of Ca (OH)2, which further reduced the consumption of the silica-aluminum in the hydration process. The increase in the bond vibration energy 875 to 1045 cm−1 was attributed to the generation of the high quantity of Al(OH)4, C-S-H, and C-A-S-H gels apart from the hydroxyl-ions-epoxy resin, improving the mechanical performance. The distortion of the bonds in the range of 1100 and 1800 cm−1 might be relevant to the H-O-H’s bending vibrations in the H2O molecules, whereas the C-C ring of the phenol group showed stretching in the epoxy-modified specimen. The addition of 10% of the epoxy resin activated some interactions with the hydroxyl ions, and the reaction of the Ca(OH)2 with the epoxy resin caused the polymerization processes. The amount of Ca(OH)2 was found to decrease as the polymer became reactive.− with the unhardened epoxy resin in the modified mortars. The self-healing efficiency of epoxy resin on CS and pulse velocity under 50 and 80% pre-loading is presented in For the specimen adopted as a control sample, the strength was dropped from 30.8 MPa to 15.4 MPa after pre-loading 50% of the maximum load a. HoweveThe results indicate that the healing efficiency on CS and UPV is much higher for specimens containing 10% epoxy resin than conventional mortar. The CS in epoxy-modified specimen significantly improved, from 18.1 MPa after artificial crack generation using 50% pre-load to around 35 MPa over 11 months of healing duration. Almost the same trend was observed for pulse velocity, where the velocity in epoxy-modified specimens was increased by an average of 23.7% after spending 11 months of healing duration. This is an indication that the structure of the specimen is dense with fewer pores and cracks. The results also acknowledged that the CS and pulse velocity in epoxy resin modified specimen substantially recovered after generation artificial cracks by 80% pre-load and reached initial condition after spending 11 months of healing duration. On the other hand, a low rate of enhancement was observed by conventional specimen after 11 months of pre-loading age (12 months of curing age), which significantly affects its durability performance. 2 available in the specimen’s matrix. After the generation of artificial cracks, the unhardened epoxy started reacting with the OH− from calcium hydroxide that becomes hardened and filling the cracks. −. Additionally, the high quantity of the C-S-H gel and Ca (OH)2 were observed from SEM results, where they are the main components associated with strength enhancement and healing the cracks.2, and hardened epoxy resin were produced, negatively affected the healing efficiency. In the self-healing mechanism, the OH− generated in the hydration process was significant for producing the C-(A)-S-H gels and self-healing system. The hydroxyl ion was needed for the occurrences of the self-healing action. In the bacteria-activated self-healing concretes, the bacterial strain reacts with the oxygen to generate the calcite, further precipitates to heal the cracks. Using a similar mechanism, the epoxy-modified mortars were prepared where the epoxy resin reacted with the OH− that enabled the hardening and healing of the cracks. Half of the epoxy resin was hardened, and the remaining one reacted as the healing agent at a later age. The reaction pathway followed the following mechanism:On the other hand, the SEM morphology shows once the artificial cracks were generated at the later age, This section develops an ANN combined with a metaheuristic algorithm to estimate the degree of damage and healing efficiency of studied epoxy-modified specimens. The multilayer feed-forward network provides a reliable feature for the ANN structure and, therefore, was used in this research. The multilayer feed-forward network comprises three individual layers: the input layer, where the data are defined to the model; the hidden layer/s, where the input data are processed; and finally, the output layer, where the results of the feed-forward ANN are produced. Each layer contains a group of nodes referred to as neurons that are connected to the proceeding layer. The neurons in hidden and output layers consist of three components; weights, biases, and an activation function that can be continuous, linear, or nonlinear. Standard activation functions include nonlinear sigmoid functions  and linear functions  . Once th0 represents the intensity of the source light and γ is the absorption of light by approximating the constant coefficient. The attractiveness β define using the following equation.0 is the attractiveness at the Euclidean distance γ is define using the following equation between two fireflies si and sj, and is equal to 0:The fireflies, also known as lightning bugs, are nocturnal, luminous beetles. Several researchers have studied the behavior of this creature in nature . The nat0), and if γ→∞ the firefly movement follows at a random walk. The firefly algorithm is being attractive in civil engineering and material sciences. Bui et al. [According to Equations (7) and (8), the firefly algorithm has two asymptotic behaviors. If γ→0, the attractiveness tend to be constant , Average Absolute Error (AAE), Model Efficiency (EF), and Variance Account Factor (VAF) that are defined as follows.2 to estimate the two output parameters as shown in After examining different topologies, it was found that the network with 6-6-4-2 topology has the lowest value of error in RMSE, AAE, EF, VAF and the highest value of RTo optimize the ANN’s weights and biases, the FOA has been used to provide the least prediction error for trained structure. The properties of the FOA parameters are shown in 2, for both input parameters, indicating the proposed model’s high potential and accuracy.The results of the FOA-ANN models are shown in This research investigated the application of epoxy resin polymer as a self-healing strategy for improving the mechanical and durability properties of cement-based mortar. Several mixes were designed with various ratios of the epoxy resin to OPC to assess the efficiency of the epoxy as the self-healing agent. The mechanical properties, ultrasonic pulse velocity (UPV), and SEM morphologies were recorded to determine the feasibility of implementing the epoxy resin as the self-healing agent in mortar manufacturing. In addition, by using the available experimental test database, an optimized ANN model combined with the firefly optimization algorithm (FOA-ANN) was developed to estimate the degree of damage and healing efficiency of mix designs. The following provides the main findings of this research.(1)The final setting time and water absorption were significantly decreased by increasing the epoxy resin content in the modified mortar compared to a conventional specimen.(2)− ions from the hydration of Ca(OH)2. However, the mechanical properties significantly decreased by increasing epoxy resin content from 10 to 15 and 20%. Such phenomenon can explain by the residual unhardened epoxy within the mortar matrix that may interrupt the hydration and polymerization processes.The best mechanical properties were achieved by a specimen containing 10% epoxy resin, attributed to the presence of OH(3)2 was reacted with the unhardened epoxy resin, creating strong bonds between the hydroxyl ions and epoxy to increase the CS.The SEM images indicated that the specimen prepared with 10% epoxy resin contain hydroxyl–ion–epoxy resin that led to the microstructure’s improvement, reduced the porosity, and providing high strength performance compared to the control specimen. In addition, in this specimen, the Ca(OH)(4)The healing efficiency on CS and UPV is much higher for the specimen containing 10% epoxy resin than the conventional mortar. Furthermore, it was concluded that there is a direct relationship between artificial crack age and self-healing efficiency, where a higher healing efficiency was achieved at a younger crack age. Similarly, the degree of damage and healing efficiency were recorded 2.3 and 71.8%, respectively for the epoxy-modified specimen once the artificial cracks were generated at an early age.(5)The ANN combined with the metaheuristic firefly algorithm provided satisfactorily results to estimate the degree of damage and healing efficiency in epoxy-modified specimens. Furthermore, the firefly algorithm optimization can also be used as a powerful tool in optimizing ANN weights. By using the optimized weight and bias of FOA-ANN, it is possible to design mixes with targeted degree of damage and healing efficiency depending on the particular environment.


The Wnt pathway is an integral cell-to-cell signaling hub which regulates crucial development processes and maintenance of tissue homeostasis by coordinating cell proliferation, differentiation, cell polarity, cell movement, and stem cell renewal. When dysregulated, it is associated with various developmental diseases, fibrosis, and tumorigenesis. We now better appreciate the complexity and crosstalk of the Wnt pathway with other signaling cascades. Emerging roles of the Wnt signaling in the cancer stem cell niche and drug resistance have led to development of therapeutics specifically targeting various Wnt components, with some agents currently in clinical trials. This review highlights historical and recent findings on key mediators of Wnt signaling and how they impact antitumor immunity and maintenance of cancer stem cells. This review also examines current therapeutics being developed that modulate Wnt signaling in cancer and discusses potential shortcomings associated with available therapeutics. Int1, discovered in 1982 [Int1, some of the most impactful discoveries were made which linked the Wnt pathway with developmental biology. Many of the genes regulated via the Wnt pathway, which were initially discovered to be important for development, later turned out to be oncogenes and tumor suppressors when studied in human cancer. Subsequently, some of the early genetic screens involving mutations in armadillo (β-catenin in vertebrates) and dishevelled (dsh in Drosophila) were similar to the Wingless mutants and thus shown to play an important role in segment polarity and embryonic development. Soon developmental assays, such as axis duplication assays in Xenopus, were shown to be excellent experimental model systems to characterize different components of the Wnt pathway. This led to classification of positive regulators of the Wnt pathway, for instance, wherein injection of murine Wnt1 mRNA into the embryo could induce axis duplication. Similarly, axis duplication was also induced by other components such as β-catenin, TCF, LEF, and GSK3β. In contrast, injection of Axin and APC mRNA resulted in complete loss of duplication, thereby identifying the negative regulators of the Wnt pathway. There was no major connection between the Wnt pathway and human cancer, until 1993 when a few research groups [Wnt signaling and its components have been implicated in a wide spectrum of important biological phenomena, where either a deficiency or overactivation of key effectors can lead to developmental disorders or impact cancer risk. The emergence of Wnt signaling, an integral mode of cell-to-cell communication, can be traced back to the early discovery of the first mammalian Wnt gene, h groups  reportedSince the early discoveries, several genetic and biochemical studies have identified novel signaling components and provided deeper insights into the Wnt pathway. The known components of Wnt signaling include Wnt ligands, receptors and co-receptors (Frizzled and LRP), components of β-catenin destruction complex, and other transcriptional regulators within the nucleus. With recent advancements in structural biology and comprehensive genomic studies, it is clearly evident that the Wnt pathway is integral for developmental biology and plays a critical role in human cancer. In this review, we provide an overview of the key components of the Wnt signaling pathway and the milestones in both developmental and cancer biology. We also describe some recently discovered components and functions and emerging links of Wnt signaling with cancer stemness and antitumor immunity. Finally, we touch upon some strategies in development being tested in clinical trials to target the Wnt signaling pathway.The Wnt pathway is one of the major signaling cascades that contributes to both normal development and pathophysiology. Herein, we will discuss the key components of the Wnt pathway and refer to some excellent reports which have laid the foundation and some recent findings that provide deeper insights into the complexities of this integral pathway.The Wnt pathway is divided into two branches: β-catenin dependent  and independent , as outlined in 2+ pathway. In the PCP pathway, Wnt ligands  bind to panel of receptors including FZD and tyrosine kinase co-receptors such as ROR1/2 and Ryk. DVL relays noncanonical Wnt signals and interacts with Rac1 and DVL-associated activator of morphogenesis 1 (DAAM1). Rac1 activates downstream c-Jun kinases, while DAAM1 activates Rho to further activate Rho-associated kinase (ROCK), eventually regulating actin polymerization and cellular cytoskeletal arrangements. In the Wnt/Ca2+ pathway, the Wnt ligands bind to FZD which interacts with heterotrimeric G-proteins and DVL, resulting in activation of phospholipase C (PLC) and an intracellular increase in Ca2+ levels. This cascade results in downstream activation of downstream signaling proteins such as protein kinase C (PKC), calcineurin, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) that regulate cell adhesion and cell migration. Wnt5a-mediated activation of the Wnt/Ca2+ branch antagonizes canonical Wnt/β-catenin signaling by phosphorylating TCF4 via Nemo-like kinase, thus preventing the binding of the β-catenin-TCF4 complex to DNA [The noncanonical branch is independent of β-catenin and, even though less characterized, regulates more diverse cellular function such as cell organization and polarity, allowing it to be further classified into the planar cell polarity (PCP) pathway and Wnt/Cax to DNA ,13, suggTogether, Wnt signals incorporated into the canonical and noncanonical pathways regulate complex normal cellular processes such as cell differentiation, development, tissue homeostasis, and wound healing; however, when the Wnt pathway is aberrantly regulated, it can be associated with developmental disorders, tumorigenesis, and other diseases.Wnt1 results in synaptic rearrangement in patients with schizophrenia. Tetra-amelia, a rare human genetic disease characterized by absence of four limbs, has been proposed to be caused by a nonsense mutation in the Wnt3 gene. Similarly, mutation in the Wnt4 gene is linked to intersex phenotype, Mullerian-duct regression, and kidney developmental defects. Other development disorders such as Fuhrmann syndrome, Al-Awadi/Raas-Rothschild/Schinzel phocomelia syndrome, and Santos Syndrome are linked with loss-of-function mutation in Wnt7A gene. Mutations in Wnt5B and Wnt10B may be linked to type II diabetes and obesity, respectively. Aberrant Wnt signaling may play a role in precancerous conditions such as hypohidrotic ectodermal dysplasia and odonto-onycho-dermal dysplasia as a result of Wnt10A mutations. Mutations in either Fzd4 or LRP5 genes are associated with familial exudative vitreoretinopathy (FEVR). Moreover, heterozygous mutations in Fzd2 are implicated to be involved with cardiovascular diseases and a rare skeletal disorder (known as omodysplasia) characterized by severe limb shortening and facial dysmorphism. The Wnt pathway is also involved in controlling bone mass, a discovery that stemmed from analysis of LRP5 mutation in patients with osteoporosis-pseudoglioma syndrome (OPPG), a disorder characterized by low bone-mass density. Conversely, patients who harbor LRP5 gain-of-function mutations experience high bone mass disease. Moreover, LRP6 mutations are also linked with neuronal and metabolic disorders such as Alzheimer’s and coronary artery disease. The central mediators of the Wnt pathway, Dishevelled genes, are also implicated in human disease. Early discoveries suggested that DVL genes were imperative for segment polarity in wing hair of Drosophila and Xenopus embryos [DVL knockout mice were shown to exhibit abnormal social interaction in nest building, home cage huddling, neural tube closure, and cardiovascular malformations [DVL are associated with severe disorders such as Schwartz-Jampel syndrome, Charcot-Marie-Tooth disease type 2A, DiGeorge syndrome, and Hirschsprung’s disease [Since Wnt signaling coordinates cell development processes and adult tissue homeostasis, it is certain that its deregulation can be linked with developmental disorders, cancer, and other diseases.  embryos . Moreovermations ,16,17. I disease ,19. Rece disease ,22,23,24 disease .APC, Axin1, and Axin, have been well documented in the cancer types mentioned above. Conversely, gain-of-function mutation of β-catenin has been established in colorectal cancer with wild-type APC. In contrast, attenuated β-catenin signaling may be implicated in the development of Alzheimer’s disease, a neurodegenerative disease characterized by deposits of the amyloid β-peptide and selective death of neurons. Recently, increasing evidence has shown that GSK-3β may be a key link between diabetes mellitus (DM) and Alzheimer’s disease (AD), where GSK-3β controls glycogen synthesis, thereby regulating blood glucose [TCF4 in these seemingly diverse disorders. TCF4 gene has been strongly implicated in type II diabetes; however, recent data suggest that TCF4 is also an important regulator of neurodevelopment disorders such as schizophrenia, Fuchs’ endothelial corneal dystrophy, and primary sclerosing cholangitis. However, rare TCF4 mutations causing Pitt–Hopkins syndrome, a disorder characterized by intellectual disability and developmental delay, have also been described in patients with other neurodevelopmental disorders [Deregulation of Wnt/β-catenin signaling also predisposes patients to multiple cancer types such as colorectal, hepatocellular carcinoma, ovarian, and lung cancer. In particular, loss-of-function mutations in  glucose . Genome-isorders .RAC2, have been found to cause a human disease, a severe phagocytic immunodeficiency characterized by life-threatening infections in infancy. Interestingly, the phenotype was predicted by a mouse knockout of Rac2 and resembles leukocyte adhesion deficiency (LAD) [TIAM1 has also shown to play a pivotal role in cardiac hypertrophy associated with heart failure [Interestingly, since components of noncanonical branch also play a central role in the cell processes and stress response, mutations in JNK, Rho/Rac, TIAM1, and NFAT have been associated with various disorders, including metabolic, neurodegenerative, and cardiovascular diseases. JNK is one of the most investigated signal transducers, and emerging evidence suggests that different isoforms of JNK (JNK1 and JNK2) may promote the development of obesity to insulin resistance in a cell-specific manner, NAFLD, and type II diabetes. This has led to development of isoform-specific JNK inhibitors with specific tissue distribution as possible drug targets for the treatment of type II diabetes . Moreovecy (LAD) . The actcy (LAD) ,33. Addi failure . Mathema failure . More geBlood or hematological malignancies such as leukemia, lymphoma, and myeloma originate from uncontrolled expansion of hematopoietic stem cells (HSCs) which exhibit deregulation of the Wnt signaling pathway ,87. In mWnt4, Wnt5B, Wnt6, Wnt7B, Wnt9A, Wnt10A, Wnt14, and Wnt16 are robustly expressed in CLL B-cells and acute lymphoblastic leukemia (ALL) cells, whereas these can be scarcely detected in normal pre-B cells [Dickkopf (Dkk) and secreted Frizzled receptor protein (SRFP) families, are reported to be silenced by promoter hypermethylation, thereby demonstrating a novel mechanism of Wnt activation in t-leukemia cells [BCR-ABL chronic myeloid leukemia (CML) cells rely on both β-catenin-mediated signaling and Wnt-mediated calcium signaling for self-renewal, proliferation, and survival [Leukemia is frequently associated with mutations of receptor tyrosine kinases (RTKs) and chromosomal translocations, where both mutations have been associated with aberrant Wnt signaling. Expression of WNT3 and LEF1 is amplified in chronic lymphocytic leukemia (CLL) B-cells, a subset of leukemias, compared to normal B-cells ,89. Othe-B cells ,91,92. Sia cells ,95,96,97survival ,99. Recesurvival .cyclinD1, TCF7, FZD7, LRP5, AXIN1, APC, and DVL3 were upregulated in MCL cells. Moreover, MCL cells show inactivation of GSK3β resulting in increased active β-catenin levels in the nucleus [Blood cancers arising from lymph nodes are collectively termed lymphoma, including Hodgkin’s lymphoma, mantle cell lymphoma (MCL), and Burkitt’s lymphoma. Several Wnt components such as  nucleus ,102. Pat nucleus ,104,105. nucleus ,107. Fur nucleus ,109. HowMyeloma is cancer of the plasma cells, and similar to leukemia and lymphoma, Wnt activation is observed in myeloma cells. It has been established that several Wnt ligands are overexpressed in multiple myeloma (MM) cell lines . In addiWnt1, although Wnt 3 and Wnt 10B were also found to be activated in similar manner [APC, Axin, and CTNNB1 (encoding β-catenin) are very rare. However, dysregulated canonical and noncanonical Wnt signaling branches have been implicated in various breast cancer subtypes. It is considered that alteration of Wnt signaling in breast tumorigenesis occurs via epigenetic changes, wherein the positive components of the Wnt pathway are increased and the antagonists (negative regulators) are decreased at the levels of ligands, receptors, and transducers [FZD7 promoters in breast cancer models [Wnt 3A genes are highly enriched in breast cancer stem cell population in comparison to normal-breast stem cell population, resulting in high metastatic potential and recurrence rate in breast cancer patients [One of the early links between Wnt signaling and breast cancer was made using genetically modified mouse models, where mouse mammary tumor virus (MMTV) was integrated into loci of Wnt genes . Some ofr manner ,114. Unlnsducers ,115. Fewnsducers . Some ofnsducers ,117,118.nsducers . Moreovensducers . Additionsducers . Furtherr models . LEF1, ar models . Moreover models . Mechanipatients . A clinipatients .APC gene along with β-catenin mutations that result in hyperactivation of the Wnt signaling pathway [APC (most being frameshift and nonsense mutations) result in a protein truncation which is believed to hyperactivate Wnt signaling by disrupting the destruction complex mediating β-catenin degradation. Other negative regulators (or tumor suppressors) such as Axin1 and Axin2 often undergo mutations and lead to genetic disposition to CRC development. Negative regulators, such as RNF43 and ZNRF3, promote FZD receptor turnover resulting in proliferation of Paneth cells by activating the Wnt cascade [The Wnt pathway is one of the key signaling cascades responsible for carcinogenesis in colorectal tissues. It is widely accepted that almost 80% of the colorectal cancer (CRC) patients harbor inactivating mutations in the  pathway . The pro cascade ,128. Act cascade ,130. Fur cascade . Other m cascade .APC loss and is associated with poor clinical outcomes in human CRC cells. For more information about miRNA and its association with Wnt signaling in the pathogenesis of CRC, we refer the readers to a review by Rahmani et al. [Furthermore, recent studies have elucidated the importance of oncogenic Wnt signals and their effect on metastasis and chemoresistance in CRC. Recently, a study reported that Wnt2B co-operates with FZD7 receptor to initiate mesenchymal-to-epithelial transition (MET), a reversible transition at a secondary site, using an LIM 1863-Mph three-dimensional in vitro model . The roli et al. .Wnt5a and its receptor Ror2 reports attenuation of inflammatory cytokine production and a decrease in interferon-γ (IFNγ)-producing CD4+ Th1 cells in the colon. Likewise, in vitro experiments demonstrated that the Wnt5a and Ror2 signaling axis promotes IFNγ production, augmenting IL-12 expression in dendritic cells and thereby inducing Th1 differentiation in colitis [Dishevelled 1 knockout mice displayed reduction and mislocalization of Paneth cells and an increase in CD8+ T cells in the lamina propria, resulting in abnormal gut microbiota [Wnt signaling is an important regulator of proliferation and differentiation of T-cell development and peripheral immune responses. Since this section will briefly overview the role of the Wnt pathway in cancer immunomodulation, we point the readers to previous reviews that have highlighted the role of Wnt signaling in the regulation of immune cells ,139. Dys colitis . Moreovecrobiota . Moreovecrobiota . Immunohcrobiota . In anotcrobiota ,146. Morcrobiota . CollectCD44, a Wnt target gene, can be detected in primary and metastatic malignancies, where tumor cells with elevated levels of CD44 have been shown to behave as CSCs. Canonical Wnt3a-mediated signaling has been shown to support stem-cell-like properties and sphere-forming ability of breast tumor cells [Lgr5 is another example of a Wnt target gene that is prominently discussed in the CSC context. Lgr5 is a cell surface G-protein-coupled receptor which binds R-spondin to augment Wnt-ligand-mediated Wnt signaling. Evidence indicates the R-spondin and Lgr5 complex inhibits the E3 ubiquitin ligases, namely Rnf43 and Znrf3, and thus enhances Wnt signaling, thereby likely promoting stem cell properties. Interestingly, overexpression or inhibition of Lgr5 results in increased or decreased stem cell functionality in breast cancer, respectively [AXIN2, and DKK1 have shown to enhance the CSC pool, potentially by activating Wnt activity [Cancer stem cells (CSCs) are endowed with self-renewal capacity that results in tumor initiation, drug resistance, metastasis, and cancer relapse, eventually leading to cancer-related deaths. Some of the early reports linking cancer stem cells were in breast and colorectal cancer models , a main or cells . Similarectively . In coloectively . Togetheactivity ,153,154.activity ,158,159.activity . CollectAberrant regulation of the Wnt pathway has been linked with worse cancer outcomes, and therefore, current strategies aim at targeting this signaling cascade in multiple tumor subtypes . PharmacThe Wnt-pathway-associated transmembrane proteins and ligands are targeted by monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), decoy receptors, and small-molecule inhibitors. Currently, only a few of these agents have reached the clinical stages of development. Monoclonal antibodies developed against Wnt1 ligand have been shown to induce apoptosis and reduction in downstream protein components, leading to tumor suppression in human cancer cells ,168.Vantictumab (OMP-18R5), a first-in-class antibody, targets the extracellular domain of five Fzd receptors , thereby suppressing the induction of canonical Wnt signaling. This agent exhibited reduction in cancer stem cell population and decreased tumor growth in PDX models of various tumor types, both alone and in combination with standard-of-care chemotherapeutic agents . VantictIpafricept (OMP-54F28) is a first-in-class recombinant fusion protein which comprises of extracellular domains of human Fzd8 fused to a human IgG1 Fc fragment, designed to block Wnt signaling through binding of Wnt ligands . XenograFoxy-5 is a Wnt5A-mimicking peptide that activates the Wnt5A receptors Fzd2 and Fzd5 to modulate downstream Wnt5A signaling. In thyroid, prostate, colon, and breast cancer, Wnt5A is suggested to have a tumor-suppressor role wherein reconstitution of Wnt5A impairs tumor metastasis by targeting cell motility and, therefore, is associated with a higher disease-free survival . By contRNF43/ZNRF3 mutations [Porcupine inhibitors are selective inhibitors such as WNT974 and inhibitors of Wnt production (IWPs) that have been developed to disrupt Wnt secretion and tumor growth. Wnt proteins are post-translationally palmitoylated by a membrane-bound O-acyltransferase (MBOAT) called Porcupine, which is critical for Wnt ligand secretion. Porcupine inhibitors block tumor growth by retention of immature, nonpalmitoylated Wnt ligands in the endoplasmic reticulum. Preclinical studies have shown promising results with Porcupine inhibitors such as WNT974  that antagonize Wnt signaling and enhance targeting of chronic myeloid leukemia (CML) stem cell population. The efficacy of WNT974 was further enhanced in combination with tyrosine kinase inhibitor (nilotinib) and significantly inhibited the proliferation and growth of CML stem and progenitor cells in vivo, while sparing the normal counterparts . WNT974 utations . ETC-159utations .DKK and Dishevelled proteins.This class of inhibitors includes small-molecule antagonists that target components of the β-catenin destruction complex, including Inhibitors of Wnt response (IWRs), such as IWR-1, and XAV939 induce protein stability of Axin by inhibiting the poly(ADP)-ribosylating enzymes tankyrase 1 and tankyrase 2 ,182. MouDishevelled inhibitors target the PDZ domain of DVL proteins which interacts with the carboxyl terminal end of the FZD receptors. Some compounds, such as NSC668036, FJ9, and 3289-8625, identified through in silico screening and nuclear magnetic resonance (NMR) have shown the ability to block Wnt signaling in the micromolar range in vitro ,189,190.PRI-724 is an antagonist that interrupts interaction between β-catenin and transcriptional co-activator CREB-binding protein (CBP). Preclinical studies of PRI-724 in patients with advanced pancreatic cancer suggest this agent can decrease metastatic potential but has only modest clinical outcomes . E7386 iD. melanogaster identified three potential compounds, namely iCRT3, iCRT5, and iCRT14, as being able to disrupt β-catenin-TCF interaction. These Wnt inhibitors downregulate the expression of Wnt target genes but have been specifically shown to be cytotoxic to human colorectal cancer cells [PFK115-584, CGP049090, and PKF222-815 are compounds identified by high-throughput ELISA that can perturb β-catenin interaction with both TCF and APC proteins . Anotherer cells .Significant advances have been made, enabling a better understanding of canonical and noncanonical signaling networks of the Wnt pathway. We now understand the dynamic range of consequences of aberrant Wnt signaling that impact oncogenesis and developmental biology. From the literature cited and discussed at length here, it is clear that small-molecule or antibody-based drugs that regulate various branches of the Wnt pathway will likely prove beneficial in the treatment of a wide range of diseases. Combination therapies involving Wnt inhibitors and immunotherapy will likely represent an area ripe for further future investigation which may provide yet another means for treating a wide range of disorders, including autoimmune disease, chronic inflammation, and cancer. Moreover, targeting the Wnt pathway might prove effective in a preventive setting for individuals who have not yet acquired alterations that lead to advanced stages of disease. Another area for further investigation involves understanding the functional consequences of DVL post-translational regulation, which remains poorly studied; however, there is enough evidence to conclude that this area of investigation will likely lead to major insights into understanding how the various branches of Wnt signaling are engaged. Finally, it will be important to further investigate the role of Wnt signaling in innate and adaptive immunity associated with a wide range of pathophysiology. Several exciting emerging links of Wnt biology have been discussed with regard to cancer stemness, metastasis, and antitumor immunity, and future investigation of how Wnt signaling impacts these areas will be very important.


In the present review, we comprehensively discuss the natural product β-catenin inhibitors, their in vitro and in vivo anticancer activities, and underlying molecular mechanisms. We also discuss the current β-catenin-targeting strategies and other potential strategies that may be examined for identifying new β-catenin inhibitors as cancer preventive and therapeutic drugs.The mutations and deregulation of Wnt signaling pathway occur commonly in human cancer and cause the aberrant activation of β-catenin and β-catenin-dependent transcription, thus contributing to cancer development and progression. Therefore, β-catenin has been demonstrated as a promising target for cancer prevention and therapy. Many natural products have been characterized as inhibitors of the β-catenin signaling through down-regulating β-catenin expression, modulating its phosphorylation, promoting its ubiquitination and proteasomal degradation, inhibiting its nuclear translocation, or other molecular mechanisms. These natural product inhibitors have shown preventive and therapeutic efficacy in various cancer models  The WntAs a well-recognized cancer driver gene, β-catenin has been demonstrated as a molecular target for developing anticancer drugs. Small-molecule inhibitors have been designed and synthesized for inhibiting the β-catenin/TCF binding or promoting β-catenin phosphorylation and ubiquitination . NaturalCortex periplocae has been reported to down-regulate the expression of β-catenin and its downstream target gene Survivin and Cyclin D1 at both protein and mRNA levels -related proteins, including β-catenin -dependent manner -25-OCH3-PPD inhibits the viability and induces apoptosis in colon and lung cancer cells by downregulating the protein expression of β-catenin and its targets CDK4, cyclin D1, c-Myc, and TCF4 (S)-ginsenoside Rh2 inhibits cell viability and induces cell cycle arrest at the G0/G1 phase and apoptosis in leukemia KG−1a cells by down-regulating the expression of β-catenin at both protein and mRNA levels  Benth. (Rutaceae) has been reported to inhibit the proliferation, invasion, and migration of hepatocellular carcinoma (HCC) HepG2 and SMMC-7721 cells in vitro and suppress tumor growth and angiogenesis in mice bearing H22 and SMMC-7721 xenograft tumors in vivo , a natural withanolide from Physalis peruviana increases the level of phosphorylated β-catenin and decreases the levels of active nonphosphorylated form and total β-catenin in colon cancer HCT116 cells  inhibits the viability and proliferation of colon cancer HT-29 cells in vitro and suppresses the growth of HT-29 xenograft tumors in vivo  as an inducer of β-catenin protein degradation , inhibits proliferation, and induces apoptosis in colon cancer cells -resistant K562 or primary chronic myeloid leukemia (CML) xenograft tumors in vivo  enhances the formation of E-cadherin/β-catenin complex, inhibits β-catenin phosphorylation, and reduces invasion and motility of prostate cancer PC3 and DU145 cells  down-regulating β-catenin expression, 2) modulating β-catenin phosphorylation and inducing its inactivation, 3) promoting β-catenin protein ubiquitination and proteasomal degradation, 4) inhibiting β-catenin nuclear translocation, and 5) other.Thanks to the substantial advances in understanding the molecular basis of cancer initiation, progression, metastasis, and drug resistance, several promising molecular targets have been characterized for cancer drug discovery, including β-catenin . Considein vitro and in vivo. However, the detailed molecular mechanisms for the down-regulation of β-catenin expression by these natural products are not clear yet, which largely hinders the further development of these natural products as anticancer agents. Of note, simply reducing the expression of an oncogene may not only induce cancer cell death but also cause other adverse effects  by using these compounds for cancer prevention and therapy . MoreoveJ-JQ conceptualized the manuscript. W-KY, Z-YX, LY, SM, BX, and X-DC collected the literature, wrote the manuscript and made the figures. J-JQ edited and made significant revisions to the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by National Natural Science Foundation of China (81903842), Program of Zhejiang Provincial TCM Sci-tech Plan (2020ZZ005), and Zhejiang Chinese Medical University Startup Funding (111100E014).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.