More than the two decades, the question of whether vitamin D has a role in cancer frequency, development, and death has been premeditated in detail. Colorectal, breast, and prostate cancers have been a scrupulous spot of center, altogether, these three malignancies report for approximately 35% of cancer cases and 20% of cancer demises in the United States, and as such are a chief public health apprehension. The aim was to evaluate antitumor activity of Vitamin D-Nanoemulsion (NVD) in colorectal cancer cell lines and HCT116 xenograft model in a comprehensive approach.Two human colorectal cancer cell lines HCT116 and HT29 -2, 5-diphenyl tetrazoliumbromide protocol were performed to show the impact of NVD and β-catenin inhibitor (FH535) on the viability of HCT116 and HT29 cell lines. Apoptosis/cell cycle assay was performed. Analysis was done with a FACScan . About 10,000 cells per sample were harvested and Histograms of DNA were analyzed with ModiFitLT software . Western blotting and RT-PCR were performed for protein and gene expression respectively in in vitro and in vivo.catenin gene, AKT gene and Survivin gene and protein expression in in vitro and in vivo.We found that NVD induced cytotoxicity in colorectal cells in a dose-dependent manner and time dependent approach. Further, our data validated that NVD administration of human colorectal cancer HCT116 and HT29 cells resulted in cell growth arrest, alteration in molecules regulating cell cycle operative in the G2 phase of the cell cycle and apoptosis in a dose dependent approach. Further our results concluded that NVD administration decreases expression of β-β-catenin signaling pathway possibly takes part in the genesis and progression of colorectal cancer cells through regulating cell cycle and the expression of cell cycle regulators.Our findings suggest that targeting β-catenin gene may encourage the alterations of cell cycle and cell cycle regulators. Wnt/The online version of this article (10.1186/s13578-019-0277-z) contains supplementary material, which is available to authorized users. Various observations have illustrated that 1,25(OH)2D3 hampers the WNT/β -catenin pathway and the commencement of its candidate genes in colorectal cancer cells, which play a crucial part in inhibition of cell propagation and upholding of the distinguished phenotype [2D3 hampers the WNT/β-catenin pathway via numerous mechanisms. Primary, it swiftly raises the sum of Vitamin D receptor (VDR) attached to β-catenin, therefore tumbling the association involving β-catenin and the transcription factors of the TCF/LEF family. Thus resulting to the suppression of its candidate genes [2D3 [2D3 persuades β -catenin nuclear export allied to E-cadherin increase at the plasma membrane adherent junctions [2D3 hampers the creation of IL1β by THP-1 monocytic leukemia cells and, if extrapolated to tumor-allied macrophages, this would symbolize one more mechanism of prevention of β-catenin/TCF-dependent gene commencement in CRC cells, as IL1 β hampers β-catenin phosphorylation and tagging for deprivation by GSK-3 β [2D3 applies a composite set of dogmatic activities resulting in the prevention of the WNT/β-catenin pathway. Since this pathway is deviantly triggered in most adenomas and colorectal tumors and is known as the chief element in this neoplasia, its reticence is doubtless critical for the anti-tumoral activity of 1,25(OH)2D3 in CRC. Moreover, 1,25(OH)2D3 sensitizes CRC cells to the growth inhibitory activity of TGF-β by up-regulating the expression of its type I receptor (TGFBR1) [2D3 hampers the stimulation of cell propagation by Epidermal growth factor (EGF) through the attenuation of EGF receptor (EGFR) expression and the stimulation of its internalization after ligand binding [2D3 may also hamper EGFR action by enhancing the expression of E-cadherin [2D3 hinder the growth-stimulatory activity of insulin growth factor 2 (IGF2) via the inhibition of IGF2 discharge and the enunciation of the expression of various genes encoding IGF-binding proteins (IGFBP) [Colorectal cancer (CRC) is an all-inclusive problem with a yearly frequency of roughly 1 million reports and a yearly death of more than 100,000 . The utthenotype . 1, 25 . Further binding . 1,25 , 17.2D3 in countless cancer cell lines, the mechanism of action of the 1,25(OH)2D3 throughout tumor decline is predominantly veiled. Consequently, our study, investigated the anticancer properties of vitamin D-Nano-emulsion (NVD) in an efficient approach, using in vitro and in vivo model systems. It was seen that NVD encourages frequent fold advanced intensity of cytotoxicity in malignancy. Auxiliary, we showed growth obstruction and initiation of apoptosis in CRC cells encouraged by NVD, down regulation of Wnt/β-catenin signal transduction pathway, Anti-proliferative effect of administration of NVD, β-catenin Inhibitor (FH535) in HCT116 and HT29 cells, Flow cytometric analysis of colorectal cancer cells after NVD treatment for apoptosis and cell cycle, Inhibition of colony formation in HCT116 and HT29 cells after administration with NVD and amendment in CTNNB1 protein intensity after NVD administration. Therefore our data specify that NVD may possibly be developed further as a prospective anti-cancer agent, both in conventional and combination therapy.In spite of frequent studies suggesting the cytotoxicity of 1, 25(OH)® CCL-247 ™ and ATCC® HTB-38 ™ respectively) purchased in July 2017 from American Type Culture Collection , ethical approval was taken from ethics committee of preclinical studies, college of Pharmacy, King Saud University, KSA.Athymic nude mice studies were carried out according to the Institutional principles for the concern and use of animals. The experimental protocol was approved (BAS#0256) by the ethical board of Quaid-i-Azam University, Islamabad, Pakistan and College of Pharmacy , King Saud University, Riyadh, KSA. Before onset of the experiment on human colorectal cancer cell lines HCT116 and HT29 (ATCC2 atmosphere at 37 °C in medium containing Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC® 30–2002™), 10% fetal bovine serum  as well as 1% penicillin/streptomycin. NVD and β-catenin inhibitor (FH535) dissolved in DMSO was applied for cell treatment. Cells with 70% confluency were induced with NVD and β-catenin inhibitor at 10–100 µM for 48 h in cell culture medium and the dilution of DMSO applied for each treatment was 0.1% (V/V).Two human colorectal cancer cell lines HCT116 and HT29  were cultured in a 5% CO4 cells per well) in 1 ml of culture medium consisting of 10–200 µM dilution of in 24-well microtiter plates. Cells were kept in a humidified incubator for 48 h at 37 °C, 200 µl of 3-94,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide  was supplemented to each well and kept for 2 h, 200 µl of DMSO was added to each plate which was then spun (1800 × g for 5 min at 4 °C). The readings at 540 nm wavelength were noted on a microplate reader (Elx 800). Impact of NVD and FH535 β-catenin inhibitor on inhibition of growth was calculated as  % cell viability as DMSO-treated cells were kept as control. Absorbance numbers of media containing wells were subtracted from test sample values.3--2, 5-diphenyl tetrazoliumbromide protocol was carried out to show the effect of NVD and β-catenin inhibitor (FH535) on the viability of HCT116 and HT29 cell lines. The cells were plated  or 1000 cells (HT29) were plated in triplicate into 35 mm cell culture dishes. After 7 days in culture, colonies were stained with 0.5% crystal violet as described . Cell coAll HCT116 and HT29 were administrated with NVD  in inclusive medium were trypsinized. Then fixed in 1% paraformaldehyde:1xpbs. Then washed twice with cold PBS and spun. Chilled 70% ethanol was used to suspend cell pellet and incubated overnight. The cells were spun for 5 min at 1000 rpm and the pellet obtained was washed twice with Chilled PBS to remove ethanol then finally cells are labeled with FITC and propidium iodide (PI) using the Apo-Direct Kit . the analysis was done with a FACScan . About 10,000 cells per sample were harvested and Histograms of DNA were analyzed with ModiFitLT software .SDS-PAGE and western blot investigations were performed by previously developed protocol with slight modifications , 20. Bri−ΔΔCT. Primer sequences used were;Whole RNA was extracted (RNeasy Mini Kit (Cat No./ID: 74104) from the cells using the following method. RNA dilution was determined by using a spectrophotometer at 260 NM and cDNA was prepared by following the manufacturer protocol (BioLabs E6300) using the kit. The reaction mixture was prepared containing 10 µl FastStart Universal SYBR Green Master , 6 µM reverse primers, and 10 µg cDNA, with RNAase free water added to a total volume of 20 µl. The amplification and real time analysis were done for 40 cycles with following factors; 95 °C (10 min) in order to activate of FastStart Taq DNA polymerase; 60 °C (1 min) for amplification and real-time analysis. The gene expression levels were determined using 2CTNNB1 Sense 5ʹ-TGTGAATCCCAAGTACCAGTGT-3ʹCTNNB1 Antisense 5ʹ-CGTCAGACAAGGAGAAACATT-3ʹβ-Actin Sense 5ʹ-CCTCTTCCTCAATCTCGCTC-3ʹβ-Actin Antisense 5ʹ-GCTCAATGTCAAGGCAGGAG-3ʹHCT116 and HT29 cells were cultured in a two chamber tissue culture glass slides and were administrated with 40 µM of NVD at 75% confluence for 24 h. Once the chamber was removed, Phosphate buffer was used to rinse the slides, 2% paraformaldehyde was used to fix the cells and permeablized in methanol. Slides were rinsed with phosphate buffer and 2% serum was used as a blocking agent. Incubation with primary antibody overnight was followed by incubation with appropriate fluorophore-tagged secondary antibody. For mounting antifade 4, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) (invitrogen NY) was used to apply and hematoxylin for counter staining. The analysis was done by using Bio-Rad Radiance system (2100 MP Rainbow) for imaging at the college of pharmacy, King Saud University, KSA. To detect apoptotic and necrotic cells, the Annexin-V-fluos staining Kit  was used according to the kit’s procedure. Zeiss 410 confocal microscopy was used to measure fluorescence. Annexin V and propidium iodide were used to stain the cells. The unstained cells in a selected field were calculated to determine the level of necrosis as well as apoptosis.g, 15 min, 20 °C). Supernatants were collected for further analyses, including protein content, mean particle size (d32 and d43), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).Pea protein (3 g) with 90% pea protein (dry basis) was added to 100 ml deionized water. Stirred for 30 min at room temperature (23 °C). Pea protein dispersion was adjusted to pH 12 with 2 M NaOH. Ultrasound was applied using a laboratory scale mano-thermo-sonication (MTS) system. A VC-750 ultrasound generator at 20 kHz  was used to deliver acoustic energy to a probe (12.5 mm diameter) placed in a specially designed sonoreactor. Different ultrasound techniques will be applied after pH-shifting to pH 12 for 1–5 min, including manosonication (MS), thermosonication (TS), and mano-thermosonication (MTS). After ultrasound treatment, samples were held 1 h (room temp) prior to the adjustment to pH 7 with 2 M HCl. Protein dispersion without treatment was served as the control. Neutralized protein dispersions were centrifuged (120032 and d43) and stability of the nanoemulsions and nanocomplexes were calculated. Centrifugation was done to measure the Physical stability of nanoemulsions.Nano-emulsions were prepared with vitamin D (VD) and pea protein modified with above-mentioned methods as an emulsifier. VD concentrations are 0.5, 1.0, and 2.0% (v/v) in the nano-emulsions. A set dose of soluble pea protein  was utilized. The VD and soluble pea protein were assorted (5 min), followed by sonication (1–5 min) and/or homogenizing by IKA Labor Pilot colloidal mill to obtain the nanoemulsion. Pectin  was added to nanoemulsion as a stabilizer. Nanoemulsion without pectin was prepared as a control. Pea protein–VD-pectin nanocomplexes were prepared at different protein: VD ratios. VD was slowly added to soluble pea protein with strong stirring at the determined optimum protein to VD molar ratios. The pH values of the nanoemulsion and nanocomplex pH was accustomed from 3 to 7. Particle size (d6) subcutaneously mixed with Matrigel  in a ratio of 1:1.Athymic male mice were acquired from King Faisal Hospital and research center, Riyadh, KSA, were homed under contamination free environment (12 h day/12 h night schedule), and nourished with a sterilized food adlibitum. HCT116 cells were selected for evaluating the in vivo impact of NVD and β-catenin inhibitor (FH535) as they generate fast tumors in mice. Cells were harvested, suspended in complete DMEM. Tumor xenografts HCT116 cells in mice were established by injecting cells .Group2 Divided into two subgroups; Group 2a and 2b consisting of six animals each. Received NVD (15 and 25 mg/kg) intra-peritoneally (i.p) respectively, twice weekly.Group3 Divided into two subgroups; Group 3a and 3b consisting of six animals each. Received FH535 (15 and 25 mg/kg) intra-peritoneally (i.p) respectively, twice weekly.Thirty mice were categorized into three groups.Throughout the study, body weight of animals, food and water expenditure were documented twice a week. Tumor volume was measured by digital caliper and calculated using the formula: L1 × L2 × H × 0.5238, where (L1 = long diameter L2 = short diameter and Height = height of the tumor).3 animals were sacrificed by CO2 inhalation was used as anesthesia. Tumors were resected, weighed and frozen at − 80 °C for subsequent western blotting, RNA extraction and immunohistochemistry.Tumor sizes were recorded twice weekly. At the end of the experiment when tumor volume reached to ~ 1200 mmThe immunohistochemistry was done by following the protocol described in our previous work .50 values. Level of significance between different treatments groups relative to control was estimated by oneway analysis of variance followed by Tukey’s multiple comparison tests. Comparison between more than one parameter was accomplished using two way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test. p < 0.05 was considered statistically significant. Where required correlation analysis was done and R values were calculated.Densitometry of western blot images was performed using an image analysis program . Data of in vitro assays were analyzed GraphPad Prism 5 software to determine IC50 values of NVD and FH535 -treated HCT116 were 40.6 and 40.1 and 25.5 and 24.6 μM at 24 h and 48 h respectively while IC50 values of NVD and FH535 -treated HT29 cells were 50.1 and 43.5 at 24 h and 30.3 and 26.2 μM at 48 h -2, 5-diphenyl tetrazolium bromide (MTT) assay against HCT116 and HT29 colorectal cancer cells. It was observed that NVD and FH535 treatment (0–100 μM for 24 h and 48 h) to CRC cells results in inhibition of cell growth in a concentration and time-dependent manner. Time course scrutiny revealed CRC cells respond to NVD and FH535 treatment within 48 h. The ICTo conclude the consequence of NVD treatment on the clonogenicity of HCT116 and HT29 cells, clonogenicity assay was performed. HCT116 and HT29 cells were pre-treated with NVD at 40 and 60 µM doses substantiated a concentration dependent reticence of colonogenicity with deference to untreated control cells. Our results illustrate the clonogenicity of HCT116 and HT29 cells after treatment with NVD was patently condensed as compared to the control  inhibition of Bcl-xL protein after 24 h of administration. The inhibitory effect of NVD against Bcl-2 protein expression was also decreased as like as Bcl-xL , HCT116 and HT29 cells illustrated a decrease in β-catenin expression when compared with control (untreated) by immunofluorescence staining. Significant Alexa fluor staining of β-catenin (nucleus) of both cell lines (green fluorescence)  was experiential in control, while the expression of β-catenin as specified by staining was noticeably decreased in NVD treated cells as shown in Fig. FH535 β-catenin inhibitor prevents survivin and Akt phosphorylation resulting down regulation of β-catenin protein expression. Compared with NVD treatment 60 μM), pre-incubation of HCT116 and HT29 cells with FH535 β-catenin inhibitor (20 μM) for 2 h before NVD treatment resulted in noteworthy decline in the protein expression of β-catenin, p-Akt, and survivin as observed by Immunoblot analysis  is an evolutionary conserved, highly pleiotropic, a ubiquitous protein kinase with multi- substrates controlling a wide range of cellular processes. CK2 α expression plays a crucial role in cellular as well as in organismal endurance. HCT116 and HT29 cells administrated with NVD for 24 h drastically diminished expression of the CK2α protein in a dose-dependent approach Fig. a. NucleaIn HCT116 and HT29 Cells administrated with NVD inhibited the expression of both NFкB p65 P-Ser529 and total NFκB p65 protein in a dose and time dependent approach. Moreover, protein expression of phospho-IκBα Ser32/Ser36 was significantly declined in a concentration and time dependent approach following NVD treatment Fig. a, b.p < 0.001) suppression of protein expression was exhibited after administration for 48 h. Prominent STAT3 has been allied with the growth, progression, and maintenance of many human tumors. Results showed that treatment with NVD for 48 h significantly declined phospho-STAT3 Tyr705. Also, NVD led to noteworthy inhibition of total STAT3 protein expression  male nude mice were used for appraising antitumor effects of NVD. On the 8th day of injection of HCT116 cells, when an evident tumor was noticeable, nude mice were administered NVD (15 mg/kg and 25 mg/kg), intraperitoneally (i.p), twice weekly. The particular concentration of compound was chosen based on a pilot study. Result illustrated a notable diminution in tumor volume when administrated with NVD in contrast to untreated control animals bearing tumor after 15–17th day of administration. Though, the decrease in tumor size was more noteworthy in a case of 25 mg/kg NVD administrated mice.No death was witnessed in NVD and FH535 treated (15 and 25 mg/kg) tumor bearing mice during the experimental period. Whilst untreated control nude mice survived for a maximum of only ~ 50 days, the NVD and FH535 administrated groups led to ~ fourfold amplification in the lifespan for at least 43% of mice. The organs from NVD and FH535 revealed no obvious variations in histo-architecture , which manage cell cycle processes through phosphorylation. Dysregulation of cyclin D1 gene expression or function contributes to the loss of normal cell cycle control during tumorigenesis. Immunoblot analysis illustrated downregulation of cyclin D in NVD and FH535 treated group (25 and 15 mg/kg) as compared to a control group. A marked difference was observed among NVD, FH535 treated (25 and 15 mg/kg) and control in an expression of cyclin D in immunohistochemistry analysis Fig. a, b.Fig.β-catenin gene, alteration of β-catenin expression by NVD induction in HCT116 xenograft, a marked decrease in mRNA expression by employing RT-PCR, was observed to be in dose dependent manner. At 15 and 25 mg/kg of NVD, a significant decline in β-catenin expression was observed  on β-catenin and survivin expression in HCT116 xenograft tumors was corroborated by immunoblotting, immunohistochemical staining, and RT-PCR. An outstanding sink in expression was seen. The xenograft tumors (HCT116) revealed inclined levels of β-catenin protein. The dose-dependent effect of NVD and FH535 on HCT116 xenograft tumors confirmed an outstanding sink in β-catenin and survivin protein intensity at 2.5 and 1.25 mg doses Fig. a. A promved Fig. c.Fig. 12Akt and β-Catenin signaling pathways are a vital watchdog in cell proliferation, differentiation, and growth. AKT phosphorylation encourages β-Catenin transcriptional activity and hence AKT and β-Catenin association plays a vital role in tumor progression and invasion. The HCT116 xenografts administrated with NVD and FH535 in a dose-dependent approach buckets AKT phosphorylation. Immunoblotting showed down regulation of AKT protein expression by administration of NVD and FH535 (15 and 25 mg/kg), which in turn hampers proliferation. The control and treated groups illustrated prominent differences in AKT protein expression Fig. a.The chief herald lesions for CRC are adenomas, and a meta-analysis of the occurrence of adenomas among U.S. adults has anticipated a range from 22% to over 50%, with a pooled frequency of approximately 30% . Among iHCT116 and HT29 cells consequent from human colorectal adenocarcinoma at the metastatic stage are a generally used cell model for advanced metastatic colorectal cancer, which presently have no efficient alleviate. A perfect therapeutic loom is to develop drugs that are confined to aim tumors while scanting normal tissues.2D3 or vitamin D analogues, showed a prominent anticancer effect. This effect of 1α, 25(OH)2D3 and its derivatives, acts through the VDR to control proliferation and apoptosis. The study related to SCC cells revealed that treatment of 1α,25 (OH)2D3 induces G0/G1 cell-cycle arrest because of the transcriptional activation of CDKN1B subsequent pRb hypophosphorylation [Moreover, the studies working with breast cancer, lung cancer, prostate cancer, squamous cell carcinoma (SCC) illustrated that 1α, 25(OH)rylation .2D3 downregulates BCL2 expression in MCF-7 breast tumor and HL-60 leukemia cells and increase protein expression of BAX and BAK in prostate cancer, colorectal adenoma, and carcinoma cells. Also 1α,25(OH)2D3 has exposed to standardize protein expression of BCL2 family and illustrated that 1α,25(OH)2D3, might trigger caspase effector molecules [Reports revealed that treatment of 1α, 25(OH)olecules . KeepingCellular propagation consequential in tumor pattern might occur due to the alteration in cell cycle regulation , 32. A vP21 and p27, as members of the Cip/Kip family, have general structures and activities. Their mechanisms for cell cycle reticence may differ. In the current study, appraisal of apoptosis encouraged in HCT116 and HT29 cells demonstrated that NVD is extremely competent inducers of apoptosis in dose-dependent manner. Also our study revealed downregulation of Cyclin B1, D1, A and E, and Cdk-2, 4 and 6 expression and accompanied upregulation of cdk inhibitors WAF1/p21 and KIP1/p27 expression in NVD treated cells. In addition, NVD administrated CRC cells demonstrated arrest in the G2 phase of the cell cycle. These results are momentous since cell cycle regulation is a fundamental target for expectancy against CRC.PARP, a characteristic caspase substrate, is a central player of DNA repair against environmental stress and in the prolongation of cell viability. Cleavage of PARP is a feature of apoptosis . PARP clFrequent studies have demonstrated that PARP-1 is overexpressed in various human tumors  –36. BesiBcl-2 family connections are the principal overseer of apoptosis. Their overexpression stalwartly hampers apoptosis owing to cytotoxic injuries by the eradication of free radicals, preclusion of mitochondrial passage pattern, and the liberation of cytochrome c , 40. BclStudies illustrated that phosphorylation of β-catenin at Ser552 by protein kinase AKT results to its amplified transcriptional activity  and alsoThe results obtained in our study support a mechanism of CK2α-dependent regulation of β-catenin transcriptional activity that may bypass the negative complex formed by axin/APC/GSK3b, but it still deems numerous phosphorylation steps. One occurs at residue Thr393 of β-catenin by CK2 in a region of the ARM domain where APC interrelates to encourage β-catenin deprivation , 46. TheBecause of critical roles in crucial cellular processes, including the homeostasis, growth control, and regeneration of cells, the two signaling pathways, Wnt/β-catenin and RAS-ERK must be firmly synchronized –52. UnusThe communication between Wnt/β-catenin and RAS/ERK pathways has been confirmed. Studies illustrated that RAF-1-MEK-ERK pathway is instantaneously triggered by recombinant Wnt3a administration in NIH3T3 and L cells, specifies direct relations of the Wnt/β-catenin and RAF-1-MEK-ERK pathways . The P13Earlier study has proved that overexpressed β-catenin can actually interact with NF-κB indirectly and hamper its activity, signifying a narrative mechanism for β-catenin-mediated oncogenesis; namely, β-catenin hampers NF-κB activity, which may permit cancer cells to flee immune scrutiny and also the study stalwartly proposed that β-catenin is a chief mediator for the cross regulation of NF-κB by the GSK-3β pathway . The comAn observation illustrated that the significant interaction and proficient inhibition of NF-κB by β-catenin in colorectal cancer cells involves phosphatidylinositide 3-kinase (PI3 K) . ImpedimGenetic deviation in the JAK/STAT/SOCS-signaling pathway emerges to be allied with a colon as well as a rectal cancer risk. The JAK/STAT/SOCS-signaling pathway has a vital part in immune defense and control of inflammation specified its indispensable association with cytokine signaling. Furthermore, the machinery of the pathway, such as STAT3, is involved in promoting uninhibited cell growth and survival in the course of dysregulation of gene expression responsible for apoptosis, cell-cycle regulation, and angiogenesis . JAK1, JSurvivin (BIRC5), an allies of the inhibitor of apoptosis (IAP) protein family, is a allot anti-apoptotic protein which enhances tumor cell growth . SurviviWe confirmed related finding in in vivo system, Athymic mice implanted with HCT116 xenografts with NVD and FH535 β-catenin inhibitor, administration induced a dose-dependent downregulation of β-catenin, surviving, and Akt phosphorylation. Akt is a crucial element of signaling cascades for cell strength and propagation throughout growth and series of malignancy. Currently, protein expression of β-catenin, Survivin, and FH535 β-catenin inhibitor p-Akt was assessed in NVD and FH535 administrated against an untreated control group.β-Catenin, and down-regulation of Cyclin D1 and caspase 3 cleavage symbolize an encouraging validation of the NVD and FH535 potential in vivo in tumors inhibition and present adequate direct confirmation for the reticence of the canonical Wnt/β-Catenin pathway and of β-Catenin target genes by NVD and FH525 in human CRC. Results demonstrated stimulation of p-Akt protein and cyclin D expression in the control group as compared to both groups encouraged with NVD and FH535 administration. The decline in protein expression is in support of reticence of propagation in groups by NVD and FH525 administration. A Manifest discrepancy was present between expressions of p-Akt and cyclin D in control verses NVD and FH535 administrated group when checked by staining (Immunohistochemistry). The expression of Cyclin D and p-Akt was downregulated in NVD and FH535 administrated group as contrast to the control group. The decline in tumor growth and volume with a simultaneous decrease in β-catenin and survivin proteins expression levels were seen in the NVD and FH535 administrated groups by western blot analysis, may have various therapeutic significance. Further mRNA expression was checked by RT-PCR, revealed a decline in β-catenin gene expression in NVD and FH535 group as compared to control. The conclusion of the current observation can have a constructive suggestion and translational impact to colorectal cancer subjects as it demonstrates that NVD can affect tumor succession, which could amplify the continued existence and superiority lifespan of the subjects suffering from colorectal carcinoma.Our results displayed, for the foremost time, the in vivo potential of NVD and FH535 in reducing the HCT116 xenograft expansion. In tissue specimens from xenografts, the decreased expression of β-Catenin pathway, we suppose that to attain significant clinical results in the treatment of colorectal cancers harboring a stabilizing mutation of β-catenin, NVD could symbolize a superior prospect to amplify the efficacy of therapies.Furthermore, the conclusion of the current study is that the NVD hold ability for maturity as a possible therapeutic and chemopreventive agent against colorectal cancer. While auxiliary experiments are considered necessary to entirely scrutinize the association of NVD with Wnt/The present study proposed that NVD act as an inhibitor of the Wnt/β-catenin pathway has the prospective to restrain tumor invasion and metastasis in both in vitro (HCT116 and HT29 cells) and in vivo (Xenograft) CRC models: NVD demonstrated evident and constant reticence of colorectal cancer cell proliferation. This prospective to restrain CRC invasion and metastasis were evident by cell cycle regulators and apoptotic proteins (Pro apoptotic and ant apoptotic proteins). The anticancer effect of NVD has been recognized to the inhibition of β-catenin and AKT protein expression. Additionally to this, we observed that NVD diminish the expression of an anti-apoptotic protein survivin. Survivin encourages tumor proliferation by way of modulating multiple critical cell signaling pathways. Consequently, NVD is predictable to offer more powerful prospective to restrain tumor invasion and metastasis, representing as a promising strategy for future CRC treatment.Based on our finding, we recommend a mechanism by which NVD elicits its effect on CRC. We chiefly took under consideration different pathways like Wnt/β-catenin, Erk and PI3K/Akt. As reported NVD decrease protein expression of β-catenin, AKT and also reduces protein expression of survivin, the downstream target of Wnt/β-catenin signaling, consequently, we proposed that NVD may act as a β-catenin inhibitor.Additional file 1.Figure S1. A) Mice weight was taken twice weekly and values represent mean±SD of six mice. **, p < 0.02 (25 mg/kg),*, p < 0.01 (15mg/kg) vs control group;**, p < 0.001. (B) H&E staining of kidney brain, heart, liver and lung of NVD treated mice vs. control for toxicity studies. Table S1. Slide review: preliminary observations.


Depletion of endogenous Smad7 and β-catenin in muscle cells reduced ckm promoter activity indicating their role during myogenesis. Deletion of the β-catenin SID substantially reduced the effect of Smad7 on the ckm promoter and exogenous expression of SID abolished β-catenin function, indicating that SID functions as a trans dominant-negative regulator of β-catenin activity. β-catenin interaction with the Mediator kinase complex through its Med12 subunit led us to identify MED13 as an additional Smad7-binding partner. Collectively, these studies document a novel function of a Smad7-MED12/13-β-catenin complex at the ckm locus, indicating a key role of this complex in the program of myogenic gene expression underlying skeletal muscle development and regeneration.Recent reports indicate that Smad7 promotes skeletal muscle differentiation and growth. We previously documented a non-canonical role of nuclear Smad7 during myogenesis, independent of its role in TGF-β signaling. Here further characterization of the myogenic function of Smad7 revealed β-catenin as a Smad7 interacting protein. Biochemical analysis identified a Smad7 interaction domain (SID) between aa575 and aa683 of β-catenin. Reporter gene analysis and chromatin immunoprecipitation demonstrated that Smad7 and β-catenin are cooperatively recruited to the extensively characterized  During embryogenesis, pluripotent mesodermal stem cells commit to become myogenic progenitor cells6. Commitment to the myogenic lineage results in a binary state of either maintenance of proliferative potential and multipotency or, on appropriate cues, withdrawal from the cell cycle, activation of a battery of structural, contractile, and metabolic genes and ultimately formation of multi-nucleated, electrically excitable myofibers8. Also, in adult skeletal muscle, resident stem cells (satellite cells) located between the basal lamina and the plasma membrane of mature muscle fibers9 recapitulate this “myogenic program” of differentiation in response to injury or for routine maintenance of the muscle tissue10.Developmental myogenesis, the process of terminal differentiation of skeletal muscle progenitor cells, consists of a series of well-characterized highly regulated steps that has become a paradigm for lineage acquisition and cellular differentiation12. Extensive biochemical and genetic evidence has implicated a family of DNA-binding transcriptional regulatory proteins encoded by the myogenic regulatory factor (MRF) genes, myf5, myod1, myogenin (myog), and mrf4, in myogenesis13. In conjunction with the proteins encoded by the myocyte enhancer factor 2 (mef2a-d) gene family, the MRF’s activate an evolutionarily conserved program of gene expression, which leads to the generation of terminally differentiated skeletal muscle cells16. Understanding the trans-acting factors contributing to this process has been aided in tandem by extensive analysis of the cis-regulatory elements of muscle-restricted, differentiation-induced genes such as the muscle creatine kinase (ckm) gene19.How networks of transcriptional regulators exert molecular control over developmental and adult myogenesis has been a prevalent theme in understanding the molecular control of ontogeny, physiology, and pathology of the striated muscle20, other transcription factors have been implicated in the control of myogenic differentiation such as Six 1 and 423, AP-125, β-catenin28, and Smad731. Since our initial observations of the pro-myogenic role of Smad7 in cultured muscle cells29, we have documented a novel nuclear role for Smad7 in muscle that is essentially independent of its “canonical” role as a repressor of transforming growth factor (TGF)-β signaling31. In addition, other groups have documented an in vivo role for Smad7 in the skeletal muscle30. Understanding the nature of the ancillary role played by Smad7 and other transcriptional regulators at muscle-restricted genes is therefore of some importance for our overall understanding of the molecular programming of myogenic identity.In addition to the central role played by the MRF/MEF2 axis in myogenesis32 since both are known, independently, to be key regulators of muscle gene expression in a variety of contexts. We therefore assessed this putative PPI in cultured muscle cells. Our data support a robust interaction between Smad7 and β-catenin that contributes to the transcriptional control of a key myogenic promoter (ckm). We further extended these observations in characterizing the recruitment of Mediator components (Med12/13) by the β-catenin/Smad7 complex, thus connecting the muscle transcriptosome assembled on enhancers such as ckm to the basal transcription machinery. Integration of the Smad7-β-catenin complex into the network of proteins regulating the “myogenic program” expands our understanding of the unique molecular wiring encoding myogenic differentiation, growth and repair.In view of the “non-canonical” nuclear role of Smad7 alluded to above, the mechanistic basis by which it contributes to the myogenic differentiation program is so far incompletely understood. We were intrigued by a report identifying a protein–protein interaction (PPI) between Smad7 and β-catenin in human prostate cancer cells2, and 37 °C.C2C12 myoblasts were obtained from the American Type Culture Collection. Cells were cultured in growth medium (GM) consisting of high-glucose Dulbecco’s modified Eagle’s medium , 10% fetal bovine serum (FBS), and L-Glutamine (HyClone) supplemented with 1% penicillin–streptomycin . Myotube formation was induced by replacing GM with differentiation medium (DM), consisting of DMEM supplemented with 2% horse serum  and 1% penicillin–streptomycin. Cells were maintained in an incubator at 95% humidity, 5% COFor ectopic protein expression, cells were transfected using the calcium phosphate precipitation method for transcription reporter assays. Cells were re-fed 16 h post-transfection and harvested. For small interfering RNA (siRNA) experiments, cells were transfected with Lipofectamine 2000 (Life Technologies) using instructions provided by the manufacturer and harvested 48 h later, unless otherwise indicated.ctnnb1 siβ-catenin#1 (SASI_Rn01_00099925), siβ-catenin#2 (SASI_Rn01_00099923), siβ-catenin#3 (SASI_Rn01_00099924), siSmad7#1 (SASI_Mm02_00290887), siSmad7#2 (SASI_Mm02_00290886), siSmad7#3 (SASI_Mm02_00290885) and universal scrambled siRNA (SIC001) were used at 75 nM concentrations.MISSION siRNA (Sigma-Aldrich) for rat and mouse pckm-luc have been described previously33. β-catenin mutant expression plasmids were constructed by the ligation of PCR-amplified nucleotides corresponding to the indicated amino acid (aa) regions  at Hind III and Xho I sites of pcDNA3-EYFP or pcDNA3-3Xflag-8XHis, respectively. Constructs for expression of glutathione-S-transferase (GST)-fused β-catenin fragments were described previously34.Expression plasmids for Myc-His-tagged full-length Smad7, β-catenin-myc, transcription reporter assay constructs Renilla plasmid  as an internal control. Cells were washed with 1× phosphate-buffered saline and harvested in Luciferase Lysis Buffer (20 mM Tris pH 7.4 and 0.1% Triton X-100). Enzymatic activity was measured in each sample on a luminometer  using Luciferase assay substrate  or Renilla assay substrate . Luciferase activity values obtained were normalized to Renilla activity in the same cell extracts and expressed as fold activation to the control.Transcriptional reporter assays were performed using luciferase reporter plasmids along with expression constructs (indicated in the figure legends) and a Nuclear and cytoplasmic extraction was obtained using the NE-PER Kit , as per the instructions provided by the manufacturer. Immunoblotting of extracellular signal-regulated kinase and c-Jun was used as the positive control for cytoplasmic and nuclear fractions, respectively.Total cellular protein extracts were prepared in NP-40 lysis buffer (0.5% (vol/vol)), 50 mM Tris-HCl (pH 8),150 mM NaCl, 10 mM sodium pyrophosphate, 1 mM EDTA (pH 8), and 0.1 M NaF supplemented with 1× protease inhibitor cocktail  and 0.5 mM sodium orthovanadate. Protein concentrations were determined by a standard Bradford assay. Equivalent amounts of protein were denatured in sodium dodecyl sulfate (SDS) loading buffer at 100 °C for 5 min and then run in SDS-polyacrylamide gels, followed by electrophoretic transfer to an Immobilon-FL polyvinylidene difluoride membrane (Millipore) as directed by the manufacturer. Blots were incubated with blocking buffer that consisted of 5% milk in Tris-buffered saline (TBS)-T  prior to the incubation with primary antibody at 4 °C overnight with gentle agitation. After three washes with TBS-T, appropriate horseradish peroxidase-conjugated secondary antibody  were added for 2 h at room temperature. Protein/antibody immune-complexes were detected with Enhanced Chemiluminescence western blotting substrate .Rabbit monoclonal for αSmad7 (ab124890) and polyclonal for αMED13 (ab76923) and αMED12 (ab70842) were purchased from Abcam. A rabbit polyclonal antibody was raised against GST-Smad7 according to the protocol approved by York University of Animal Care Committee. This was used for endogenous Smad7 immunoprecipitation (IP) and detection in cellular and nuclear extract. αβ-Catenin (pAb9562) and ChIP-grade αFlag antibody (mAb 14793S) were purchased from Cell signaling. Monoclonal αFlag antibody (F1804) was from Sigma. αMyc (9E10), αMyHC (MF20), and αMyogenin (F5D) were purchased from DSHB. αActin (Sc1616), αMyoD (sc304), and αCKM (sc-69878) were purchased from Santa Cruz.For Co-IP, cells were harvested, and proteins were extracted as described above. IP was performed using an ImmunoCruz Optima Kit (Santa Cruz Biotechnology) according to the manufacturer’s instructions. Eluates were analyzed by western blotting as described above.2). The raw images were processed by ZEN (Carl Zeiss) to obtain pseudo-colored micrographs.C2C12 cells were seeded onto the glass-bottom dishes (MatTek Corp). The cells were transfected for the expression of fluorescent-tagged proteins. Before imaging the cells, the media was replaced for FluoroBrite DMEM (Thermo Fisher Scientific) supplemented with 10% FBS. To mark nuclei, Hoechst 33342 (Sigma-Aldrich) was added to 2.5 µM into the media. After 30 min, the stained cells were visualized by a Carl Zeiss Spinning disc system (Zeiss Observer Z1 with Yokogawa CSU-X1 and AxioCam MRm camera) in the environment chamber  using primers specific for the For GST pull down assays, GST-Smad7 and 6XHis-β-catenin fusion proteins were produced in bacteria using standard protocol. Briefly, GST-Smad7- and 6XHis-β-catenin-expressing cells were sonicated and the proteins were purified with glutathione-agarose beads (Sigma-Aldrich G-4510). Protein concentrations were estimated by SDS-polyacrylamide gel electrophoresis and following Coomassie blue staining, using BSA for comparative estimation. GST-β-catenin fragments corresponding to aa (full length (FL), 1–100, 120–683, 422–683, 575–696, 575–683, 1–574) were utilized for mapping study. Five micrograms of GST FL Smad7, β-catenin , and 25 µl glutathione-agarose beads (50% slurry) were incubated in 600 µl NETN buffer  0.5 mM EDTA, 0.5% (vol/vol) NP-40) overnight at 4 °C. In experiments where GST-β-catenin was used to co-precipitate Smad7, GST-Smad7 was thrombin  digested to yield the immune-complex without the GST tag (10 µl thrombin per mg fusion protein was incubated at room temperature for 17 h), and eluates were analyzed by western blotting.32, we assessed whether Smad7 and β-catenin form a complex under these cellular conditions in cultured muscle cells and muscle tissue ). Total protein lysates from mouse TA and C2C12 muscle cells were subjected to Co-IP with β-catenin and Smad7 antibodies. Interestingly, Smad7 and β-catenin were precipitated together in muscle tissue (TA) and in muscle cells , muscle creatine kinase (CKM), and myogenin levels during differentiation Fig. . Endogenlls Fig. . The intlls Fig. .Fig. 1Sm35 , CBP (CREB-binding protein/p300), and proteins associated with chromatin regulation35.As the Co-IP analysis indicated Smad7 and β-catenin were in the same complex, we next clarified whether this was a direct interaction by utilizing GST pull down assays with bacterially expressed purified proteins. GST-Smad7 and 6xHistidine (6xHis)-β-catenin fusion protein were produced to conduct the assay. A Coomassie stained blot confirmed the purification of the fusion proteins Fig. . β-caten35 Fig . GST-β-c35 Fig  is requi35 Fig . This reckm gene (ckm) has been extensively characterized and has served as a paradigm for tissue-restricted transcriptional control during myogenesis36. Our laboratory previously documented that Smad7 associates with the promoter proximal elements of ckm29. We therefore used this as a test promoter to assess the function of the β-catenin:Smad7 interaction and to interrogate the role of this complex during myogenic differentiation. First, ChIP coupled with qPCR for ckm and gapdh (control) was utilized to assess whether Smad7 and β-catenin are enriched on the ckm promoter proximal region. In the absence of a Smad7 antibody efficacious for ChIP, Smad7 enrichment was confirmed by Flag-Smad7 recruitment to the ckm promoter  promoter fragment driving a firefly luciferase reporter gene. These data indicate that Smad7 and β-catenin transactivate this promoter region both alone and in combination , β-catenin  along with a ckm luciferase construct were ectopically expressed in C2C12 cells alone or in combination. These data indicate that, while Smad7 and the β-catenin-FL enhanced the ckm promoter, this activity was substantially reduced in cells expressing Smad7 in combination with β-catenin aa1–574 or β-catenin ΔSID  of endogenous β-catenin activity. To test this idea, we constructed a mammalian expression plasmid for tagged β-catenin aa575–683. Smad7 and β-catenin were ectopically expressed in C2C12 cells along with increasing concentrations of either EYFP-β-catenin aa575–683 (EYFP-SID) or Flag β-catenin aa575–683 (Flag-SID). Reporter gene analysis revealed that ckm promoter activity was significantly reduced in Smad7- and β-catenin-expressing cells in the presence of either EYFP-SID or Flag-SID  was unaffected by SID expression, suggesting that the effect of SID on ckm is specific  assembly and stabilization renders many Mediator subunits essential for life, since gene targeting in mice has revealed that many subunits prove embryonic lethal when deleted and in yeast all Pol II-regulated genes are dependent on Mediator57. The experimental dissection of Mediator has been aided by the characterization of four relatively stable sub-complexes that have been designated as the head, middle, tail, and kinase modules. In our study, we have documented that the Smad7:β-catenin complex specifically associates with the MED12 and 13 subunits of the Mediator kinase module. The kinase domain is speculated to fulfill a transient regulatory function in Mediator by promoting recruitment of all four Mediator domains to enhancers which then transitions to a core promoter-bound Mediator complex in which the kinase module is absent. We therefore propose that Mediator recruitment to the ckm enhancer is mediated by the Smad7:β-catenin complex in muscle cells  activity is a feature of the embryonal form of RMS  and embryonal (ERMS)). The result of this GSK3 activity is perpetual proteosomal degradation of β-catenin since this is the primary function of the APC-GSK3 complex in canonical Wnt signaling59. Upon Wnt signaling stimulation, GSK3 activity is substantially reduced and proteosomal degradation of β-catenin ceases, resulting in its cytoplasmic accumulation and translocation to the nucleus where it activates transcription in combination with other transcriptional regulators, such as TCF/LEF. Based on these previous observations and the current study, it is worth speculating that the absence of nuclear β-catenin function due to constitutive GSK3 activity contributes to the differentiation defect in ERMS. Promoting terminal differentiation of the myo-like cells in RMS is seen as a potentially effective therapeutic target, since differentiation, by nature, results in cell cycle withdrawal and cessation of proliferation which would cause tumor regression. There are several lines of indirect evidence supporting this idea; we recently reported that Myogenin is expressed at high levels in RMS but, despite the role of this MRF as a key terminal effector of the myogenic differentiation program, it is functionally inactive in these cells. It is therefore possible that the lack of nuclear β-catenin underlies this functional impairment due to the failure to recruit Mediator to connect Myogenin function to PIC assembly in these ERMS cells. In support of this possibility, we observed that Smad7:β-catenin is tethered to muscle promoters by its interaction with MRFs (such as MyoD and Myogenin) bound to E boxes on muscle promoter/enhancer regions. Another line of circumstantial evidence supporting this notion is that pharmacological GSK3 inhibitors have been reported to force RMS cells into a more differentiated cellular phenotype, suggesting that re-constitution of β-catenin function in RMS cells promotes their differentiation and could potentially be antitumorigenic. Further clarification of the role of β-catenin:Smad7 as a therapeutic target in RMS is therefore warranted.In view of the role played by β-catenin: Smad7, it is perhaps worth consideration of the implications of this in the context of the pathology of RMS, a soft tissue pediatric cancer with features of muscle33. Specifically, we recently reported that a p38 MAPK-dependent interaction with the MEF2 transcription factor enhances β-catenin nuclear retention and activity33. During differentiation, MyoD and MEF2 bind muscle-specific promoters and enhancers, leading to the recruitment of co-activators (including p300) and the basal transcriptional machinery to establish a transcriptionally poised promoter. The previously implicated mechanism is that p38 MAPK activates ckm expression by phosphorylation-dependent recruitment of the histone methyltransferase Ash2L by MEF2D60. In view of the current observations, it is also possible that p38 MAPK may promote β-catenin recruitment to ckm through its interaction with MEF2.Phospho-dependent protein interactions are a signature of β-catenin function. There is ample evidence of kinase-mediated phosphorylation modulating the affinity of PPIs in the canonical Wnt pathway, resulting in important outcomes for Wnt-dependent target gene activationIn summary, we have documented a PPI between Smad7 and β-catenin that may serve a fundamental role in the control of myogenic gene transcription. These observations may have implications for our understanding of the molecular control of myogenic differentiation during embryonic development and adult muscle regeneration.Supplementary dataSupplementary figures


New chemical inhibitors of protein–protein interactions are needed to propel advances in molecular pharmacology. Peptoids are peptidomimetic oligomers with the capability to inhibit protein-protein interactions by mimicking protein secondary structure motifs. Here we report the in silico design of a macrocycle primarily composed of peptoid subunits that targets the β-catenin:TCF interaction. The β-catenin:TCF interaction plays a critical role in the Wnt signaling pathway which is over-activated in multiple cancers, including prostate cancer. Using the Rosetta suite of protein design algorithms, we evaluate how different macrocycle structures can bind a pocket on β-catenin that associates with TCF. The in silico designed macrocycles are screened in vitro using luciferase reporters to identify promising compounds. The most active macrocycle inhibits both Wnt and AR-signaling in prostate cancer cell lines, and markedly diminishes their proliferation. In vivo potential is demonstrated through a zebrafish model, in which Wnt signaling is potently inhibited. Small molecules and peptide inhibitors have their benefits and faults when it comes to inhibiting protein-protein interactions. Here, the authors designed a peptoid-peptide hybrid that inhibited β-catenin/TCF interactions, leading to inhibition of Wnt signalling in models of prostate cancer. Despite these important advances, additional design strategies are urgently needed. Small molecules may lack sufficient surface area to abrogate protein–protein binding effectively, as these interfaces are typically broad and flat2. In addition, peptides often lack desirable pharmacological characteristics and are readily susceptible to degradation in vivo. These shortcomings can potentially be addressed by development of peptidomimetic oligomers that exhibit steric and chemical complementarity with protein surfaces6. Such molecules may populate an attractive middle ground between small molecules and biomacromolecular therapeutics. Crafting peptidomimetics suitable for targeting PPIs is a formidable and intriguing challenge. However, specific binding will require conformationally ordered oligomers that present diverse chemical groups in a predictable orientation.Protein–protein interactions (PPIs) provide the structural and functional basis for many critical biological processes. Extensive efforts have been made to identify small molecules or peptides capable of inhibiting particular PPIs that play a role in the pathogenesis of disease7. One particularly promising class of foldamers is peptoids, which are composed of sequences of N-substituted glycine monomer units. Although peptoids are oligo-amides, like native peptides, the side chains are presented from the backbone amide nitrogens instead of the α-carbons. This backbone modification confers peptoids with a broad resistance to proteolytic cleavage9. Peptoid synthesis efficiently employs the use of myriad primary amines as submonomer reagents, leading to rapid solid-phase generation of oligomers that exhibit extraordinary chemical diversity and tunable physicochemical properties11. These synthetic and structural features endow peptoids with the capability to satisfy the physicochemical requirements for a variety of biomedical applications.Foldamers are sequence-specific oligomers that can mimic a range of biomolecular secondary structure motifs13. Strategies have now been identified to direct the organization of linear peptoid secondary structures, such as inclusion of bulky branched residue types16. Head-to-tail macro-cyclization can further rigidify peptoids in a β-hairpin conformation and can enhance biological activity19. Additional structural motifs can be attained in hybrid oligomer systems upon incorporation of α-amino acid residues20. This study evaluates the potential for peptoid-containing macrocycles to address outstanding challenges in molecular pharmacology, including targets that are currently considered undruggable.Initially, attempts to establish conformational order for peptoids proved challenging, due to the lack of backbone hydrogen bonding, along with facile isomerization about the peptide backbone amide ω dihedral angle23. Recent advances have elaborated protein design tools, such as the Rosetta molecular design suite to allow the modeling, docking, and sequence optimization of both peptide and peptidomimetic macrocyclic systems23.The ability to employ computational design algorithms that can reliably identify particular foldamer sequences capable of binding a specific protein surface would provide a significant advance in targeting PPIs relevant to a wide array of disease states. Computational and experimental studies have demonstrated that there are discrete low energy conformations for cyclic peptides which are highly amenable to computational prediction24. While patients with localized disease have a positive prognosis, those with metastatic disease have a 5 year survival rate of 30%. The main therapeutic target in prostate cancer is the androgen receptor (AR). Unfortunately, most patients treated with anti-androgens will develop resistance and continued disease progression, a state known as metastatic castration-resistant prostate cancer (mCRPC). Second generation anti-androgens, including Enzalutamide and Abiraterone, have been developed but extend life expectancy on average less than 6 months, as resistance can continue to develop26. There are multiple mechanisms of resistance, such as gene amplification or alternative splicing of the AR27. Additionally, the activation of auxiliary pathways can be oncogenic and/or upregulate AR signaling28. For instance, the AR gene is under transcriptional control of the Wnt signaling pathway30. The central Wnt regulator, β-catenin, can also interact with the AR and act as a co-activator31.Among men, prostate cancer is the most prevalent form of cancer and the third most common cause of cancer-related death33. The most common Wnt mutations found were in APC or the phosphorylation domain of β-catenin, both of which cause the constitutive stabilization of β-catenin35. Following its stabilization, β-catenin translocates to the nucleus where it binds the T-cell factor (TCF) family of transcription factors. This causes a conformational change in TCF that activates transcription of a set of genes involved in cell proliferation and differentiation. β-catenin interacts with most of its binding partners including TCF, APC, Axin, E-cadherin, and the AR through its alpha-helical armadillo domain36. While the protein contacts partially overlap, there are distinct differences that can potentially enable specific inhibition of one PPI without affecting others. For example, a co-crystal structure of β-catenin and TCF shows there is a unique β-hairpin loop motif in TCF that binds to a cleft in the β-catenin armadillo domain37.Recent genome-wide sequencing studies found the Wnt signaling pathway to be mutated in upwards of 20% of tumors from patients with mCRPC, making it one of the most mutated pathways after AR, P53, and PTEN39. High-throughput screening has identified multiple classes of small-molecule β-catenin:TCF inhibitors43. These screens use either cell based luciferase assays or assays that monitor β-catenin:TCF interactions. Small-molecule virtual screening has also been used to identify inhibitors45. Some rational design techniques have been implemented that focused on modifying large peptide fragments associated with TCF’s β-catenin binding domain in order to enhance their stability48. Nevertheless, there is still a need to establish a robust rational design strategy for discovery of β-catenin:TCF interaction inhibitors with promising pharmacological properties that can be advanced beyond pre-clinical testing.Previously efforts have targeted the β-catenin:TCF interaction50 values in both 2D and 3D cell culture models. In addition, we demonstrate that the peptoid macrocycle inhibits Wnt signaling in vivo through a zebrafish model.In this study we apply computational protein design protocols to design macrocyclic peptoid–peptide hybrids to target the N-terminal TCF β-hairpin binding pocket of β-catenin and demonstrate its potential for use as a therapeutic in prostate cancer models. Using the Rosetta suite of computational tools, we first generate a small library of peptoid–peptide macrocycles designed in silico that are predicted to bind β-catenin. We use cell culture based luciferase assays to test the oligomers and select a promising compound. We show that the compound potently inhibits binding between β-catenin and TCF proteins. It also inhibits the proliferation of prostate cancer cells with nano-molar ICXenopus laevis TCF3, which forms a β-hairpin structure at the site of interaction in the X-ray structure (PDBid: 1G3J)37. The β-catenin binding domain of xTCF3 aligns very closely with the sequence of human TCF4  and automated docking protocols utilizing Rosetta’s InterfaceAnalyzer53.We applied rational design techniques to target the xTCF3 β-hairpin binding pocket of β-catenin. In order to identify suitable oligomer scaffolds, we evaluated high-resolution structures experimentally determined for a set of peptoid and peptoid-α-amino acid hybrid macrocyclic structures1 suggested that the D-Alanine and “N-Smpe” residues played a critical role in directing the overall fold of the oligomer. We hypothesize these bulky α-chiral peptoid side chains and a D-amino acid confer a unique structure to the oligomer backbone  was used as a control compound. It incorporates the identical chemical moieties as its cyclic counterpart, but is likely not constrained in an optimal binding conformation and was therefore anticipated to demonstrate weaker affinity. Indeed, as observed in luciferase assays  for 13 compared to 3.27 µM ± 0.99 for the linear oligomer. Compound 13 also showed better inhibition of the AR luciferase reporter (IC50 of 1.02 µM ± 0.20) compared to linear 13 (IC50 of 7.63 µM ± 1.22). While these values may suggest that the compounds inhibit Wnt signaling with increased potency relative to the AR pathway, the experimental conditions used with each reporter, including siRNA knockdown of APC to activate the Wnt reporter and DHT treatment to activate AR signaling, confound direct comparison of the values. To confirm that 13 does inhibit the β-catenin:TCF protein interaction, we developed a sandwich ELISA that detected recombinant β-catenin binding to a GST fusion protein containing the TCF4 N-terminal β-catenin binding domain  and 782 nM ± 34 (LNCaP-95). We also tested compound 13 in non-prostate cell lines that have been shown to be Wnt independent  and A549 (lung adenocarcinoma) cells.To test the effect of nes Fig. . The mRNent Fig. . Compounent Fig. 46,47. Co13, or linear 13 administered at the time of seeding at low cell density in a 384-well plate. In a separate experiment, to test the impact on pre-formed spheroids, cells were treated 5 days after initial seeding. Spheroids from both experiments were grown for 22 days, with compound refreshed on days 5, 9, 13, 16, and 20. Figure 13, or 2 µM linear 13 at 20 days. PrestoBlue was used to measure cell viability on day 22 . It also reduced spheroid proliferation when added on day 5, with somewhat lower potency and efficacy and an IC50 of 1.34 µM ± 0.11. Figure 13  prevented spheroid formation when added on day 0 with seeding and 0.5 µM 13 also slowed growth , concentrations of 13 at or above 2 µM stalled the growth of pre-formed spheroids but caused less of a decrease in spheroid area  model. The relevance of the model for our studies is that zebrafish embryos with Wnt activating mutations in Axin1, a component of the β-catenin destruction complex, fail to develop eyes and a forebrain56. The phenotype can be recapitulated by treatment with an inhibitor (termed BIO) for another β-catenin destruction complex member, GSK3, in embryos at 6 h post-fertilization (hpf)57. It would then be anticipated that 13 could inhibit the chemically overactivated Wnt signal, and potentially rescue eye and forebrain development. To test this, BIO was added to zebrafish embryos at 6 hpf with and without 13 as described in Fig. 13 inhibited Wnt signaling as anticipated, as only one of the eight fish treated with GSK3 inhibitor BIO alone developed eyes, while all the fish treated with both BIO and 13 developed eyes  is established in a zebrafish model of Wnt signaling. The hypothesis that the conformational constraint imposed on the macrocyclic oligomer enforces steric and chemical complementarity for the β-catenin surface is strongly supported by comparisons to a linear analog, which exhibits markedly diminished activities in vitro and in vivo.In this study, we demonstrate how computational tools can facilitate the design of oligomers that target β-catenin and disrupt its interaction with TCF. We evaluate peptoid–peptide hybrid macrocycles designed to fold into structures complementary to a region on the β-catenin surface that interacts with TCF. We demonstrate that an oligomeric macrocycle exhibits potent antiproliferative effects on prostate cancer cell lines in both 2D and 3D models. In vivo efficacy of this compound , it may have a different therapeutic profile than a drug targeting β-catenin:TCF and there may be opportunities for combination therapy with compound 13.The β-catenin:TCF PPI has been targeted previously13 in mouse xenograft models of prostate cancer will be important to determine its therapeutic potential, as demonstrated for other bioactive peptoid oligomers71. The ability to introduce a broad range of alternative oligomer side chains should facilitate further improvements of activity along with enhanced pharmacokinetic and pharmacodynamic properties towards the identification of an optimized lead compound. In addition to promising activity versus prostate cancer, macrocycles targeting β-catenin:TCF could also be of therapeutic benefit in other forms of cancer for which Wnt signaling is similarly pro-tumorigenic, such as colon and breast cancers. More generally, this study portends further computer-assisted discovery of increasingly complex folded oligomers to address the vast number of different PPI relevant to human disease.Future testing with 13 used the following amines: S-(-)-4-methoxy-α-methylbenzylamine (Sigma), 3-isopropoxypropylamine (Sigma), 3,5-dimethoxyaniline , and 2-phenoxyphenethylamine (Sigma). The resin was cleaved with 8:1:1 DCM:Acetic acid:HFIP for 1 h. The DCM was removed via rotary evaporation and the remaining solid was dissolved in 50:50 water:ACN and lyophilized overnight. Reverse phase HPLC was used to purify the linear product. Cyclization was carried out using 25 mg of linear compound with 5X PyBOP and 10X DIEA in dry DCM with less than 1 mg/ml compound overnight under argon. The DCM was removed via rotary evaporation and the remaining solid was dissolved in 50:50 water:ACN and lyophilized overnight. The final product was purified to > 95% by reverse phase HPLC and verified by liquid chromatography-mass spectrometry (Agilent LCMSD Trap XCT). Mass spectrometry peak identified for compound 13 (m/z): [M + Na]+ calcd. for C59H72N6O12Na 1079.5; found, 1078.346. A key for chemical abbreviations is supplied in Supplementary Table 2-chlorotrityl resin (Anaspec) was incubated with Fmoc-D-Alanine (Sigma) for 1.5 h followed by washing 5× with DMF. Deprotection was carried out with 20% piperidine in DMF. Peptoid residues were then added with iterative steps of 1.2 M bromoacetic acid + DIC for 20 min and 1 M amine for 1 h. Compound -ΔΔCt) in triplicate of biological replicates. RT-qPCR primers are given in Supplementary Table LNCaP-abl and LNCaP-95 cells were cultured in indicator free RPMI 1640 media (Life) supplemented with 10% charcoal stripped FBS, 1% pennicillin/streptomycin, and 1% L-Glutamine. HEK-293 Cells were cultured in DMEM media (Life) supplemented with 10%FBS and 1% pennicillin/streptomycin. A549 and U2OS cells were cultured in RPMI 1640 media (Life) and McCoy’s 5 A modified media (Life), respectively, both supplemented with 10%FBS and 1% pennicillin/streptomycin. LNCaP-abl, and LNCaP-95 cell lines were generous gifts from Z. Culig  and J. Isaacs , respectively. Cell lines were authenticated by STR profiling . A549 and U2OS were purchased from ATCC (product numbers CCL-185 and HTB-96). Cell lines were routinely screened for mycoplasma. Luciferase assays were conducted using stably transfected TOP-flash and 3xARE LNCaP-abl cell lines. For the Wnt pathway, TOP-Flash LNCaP-abl cells were treated with 1 nM siAPC  for 24 h and seeded into 384-well plates at 5000 cells/well, followed by 24 h of treatment with compound. For the AR-pathway, 3xARE LNCaP-abl cells were seeded into 384-well plates at 5000 cells per well, treated for 48 h with compound, with the addition of R1881 for the final 8 h. Both were measured using the ONE-Glo detection kit (Promega) and data is standardized to CellTiter-Fluor (Promega) viability readings to account for potential variations in well to well cell number. Control Wnt inhibitors were purchased from Sigma with the exception of PRI-724 which was purchased from Selleckchem. Co-immunoprecipitation was carried out using HEK293 cells transfected with either myc-TCF (addgene #32738) or myc-TCF dominant negative (addgene #32739) followed by 48 h of treatment with compound. Cells were lysed using Triton lysis buffer and 1 mg total protein was incubated overnight at 4 C with 40 µl anti-cMyc magnetic beads (Thermo). Total protein concentration in cell lysates were determined using Pierce Rapid Gold BCA Protein Assay Kit (Thermo). The beads were washed 3× with lysis buffer then boiled in 2X sample loading dye. SDS-PAGE and western blotting were performed using anti-Myc (CST 2276) and anti-β-catenin (CST 8480). Both antibodies were diluted in blocking buffer 1:1000 and incubated overnight at 4 C with the membrane. Full blots from IP Fig.  are depi600 0.6 and induced with 0.5 mM IPTG (Sigma) for β-catenin and 0.1 mM IPTG for TCF. The cells were then shaken for 8 h at 30 C. The bacteria were then pelleted, resuspended in PBS, and lysed with a bacterial FRENCH pressure cell press (Thermo). The lysates were incubated overnight with Ni-NTA or glutathione affixed resin (Thermo), washed with TBS, and eluted using 250 mM imidazole or 20 mM glutathione in 20 mM Tris-HCl pH 8.0 and 200 mM NaCl. The imidazole or glutathione were removed by dialysis into a buffer containing 20 mM Tris-HCL pH 8.0, 200 mM NaCl, and 10% glycerol. Lysates were then aliquoted and frozen at −80 °C. The ELISA was initiated with adsorption of 5 µg/ml GST-TCF CBD into a 96-well Nunc Maxisorp plate (Fisher) overnight at 4 °C. BSA was adsorbed to control wells. The wells were washed, blocked with 5% milk for 1 h, washed, incubated with 5 nM His6-β-catenin with varying concentrations of compound in washing buffer for 1 h, washed, incubated with anti-His6 (Thermo MA1-135) diluted 1:10000 in blocking buffer for 1 h, washed, incubated with anti-mouse-HRP (Santa Cruz) diluted 1:15000 in blocked buffer for 1 h, washed, and then developed using 1-Step Ultra TMB-ELISA (Thermo). His6-β-catenin was pre-incubated with compound for 30 min prior to its addition to the plate.The ELISA was conducted using recombinant GST-tagged catenin binding domain (CBD) of hTCF4, AA8-54, and His6-β-catenin armadillo domain, AA134-668. Both proteins were expressed in BL21 DL3 (NEB) bacteria grown to ODDynamic light scattering was measured with a Wyatt DynaPro NanoStar. Aliquots of recombinant frozen His6-β-catenin were thawed fresh for each experiment and centrifuged at high speed for 10 min. Compound 13 or α-cyclodextrin (Sigma) were added to a concentration of 10 μM and compared with the addition of 1% DMSO (vehicle). Ten readings were measured per experiment, each with an integration time of 5 s. Three experiments were performed per condition. Data was collected and analyzed using DYNAMICS software (Wyatt).LNCaP-abl cells were seeded into 384-well ultra-low attachment plates (Corning), 1000 cells/well. Varying concentrations of compound were added either on the same day as seeding or on day 5. Media was changed every 3–4 days using a Biotek automated media exchanger. Images were taken using an ArraySca VTI (Cellomics) on days 5, 9, 13, 16, 20, and 22. Images were analyzed for spheroid area with the HCS Studio Cellomics Scan Version 6.6.0 (Thermo) software. A final viability measurement was taken on day 22 using PrestoBlue reagent (Thermo).13 or Linear 13 was added. About 30 min later at shield stage, 50 µl of 6 nM (3×) BIO was added. The embryos were then visualized for the presence of eyes and imaged at 30 hpf. Zebrafish embryos were randomly assigned to non-blinded treatment groups, none were excluded. Sample size was determined with a power calculation to achieve statistical significance  if only 60% of the fish had developed eyes. Zebrafish experiments were approved by the NYULMC Animal Care & Use Program under IACUC Protocol Number: 170105-02. All ethical regulations were complied with.The zebrafish phenotypic assay was conducted using wild-type zebrafish embryos seeded into a 96-well round bottom plate at 4 hpf in 50 µl of Fish Water  supplemented with 1 mg per L Methylene Blue (Sigma-Aldrich). The embryos were not dechorionated. At 50% epiboly, roughly 5.5 hpf, 50 µl of 30 µM Supplementary InformationPeer Review File


Clinical translation, especially the transplantation of MN precursors in spinal lesions, has thus far been elusive because of stem cell heterogeneity and protocol variability, as well as a hostile microenvironment such as inflammation and scarring, which yield inconsistent pre-clinical results without a consensus best-practice therapeutic strategy. Induced pluripotent stem cells (iPSCs) in particular have lower ethical and immunogenic concerns than other stem cells, which could make them more clinically applicable. In this review, we focus on the differentiation of iPSCs into neural precursors, MN progenitors, mature MNs, and MN subtype fates. Previous reviews have summarized MN development and differentiation, but an up-to-date summary of technological and experimental advances holding promise for bench-to-bedside translation, especially those targeting individual MN subtypes in SCI, is currently lacking. We discuss biological mechanisms of MN lineage, recent experimental protocols and techniques for MN differentiation from iPSCs, and transplantation of neural precursors and MN lineage cells in spinal cord lesions to restore motor function. We emphasize efficient, clinically safe, and personalized strategies for the application of MN and their subtypes as therapy in spinal lesions.Spinal cord injury (SCI) is a devastating neurological disorder that damages motor, sensory, and autonomic pathways. Recent advances in stem cell therapy have allowed for the  Spinal cord injury (SCI) results in a debilitating loss of motor, sensory, and autonomic function. SCI comprises two phases of injury: the initial mechanical damage to sensorimotor tracts and vasculature and the secondary inflammatory-ischemic cascade, leading to infarction and scarring . Many trin vivo survival and integration, neurotrophic capabilities, and well-established capability to differentiate into MNs and glia  and stage of differentiation . ESC transplants have been the most studied because of their plasticity, self-renewal, and glia . Howeverand glia , umbilicand glia , and adiand glia . Howeverand glia  and matuand glia . Finallyand glia . Becauseand glia , iPSC-deand glia .Challenges remain, however, in translating these findings to the clinic. These include concerns over methodological differences, variable stages of maturity, differentiation efficiency, scalability, purity, and teratoma formation . Some ofIn this review, we aim to address this need by summarizing the biological mechanisms, current techniques and derivation methods for generating MNs and MN subtypes, and SCI transplantation applications of iPSC-MN lineage cells. In an effort to maximize protocol standardization and clarity, we review each of these in relation to three levels of MN differentiation: neural precursors, cells committed to a MN-lineage, and mature MN subtypes. We emphasize recent techniques which are efficient, scalable, MN subtype-specific, and clinically-relevant. Of particular significance, we discuss the challenges of targeting specific MN subtypes in SCI, as well as strategies for directly or indirectly replacing damaged MN subpopulations through personalized SCI transplantations.in vivo and in vitro. PSCs are able to differentiate into endoderm, mesoderm, and ectoderm fates. Ectoderm consists of the neural tube and neural crest and will differentiate into a neural fate. Mesoderm differentiates into muscle and bone, while endoderm becomes luminal epithelia. Both ESCs and iPSCs have been found to have equivalent differentiation potential into MNs, likely driven by highly similar mRNA profiles . Graded levels of Shh are responsible for regulating several transcription factors, namely bHLH and HD factors, to induce a more ventral cell identity . Shh conThe most important markers of MN progenitors specific only to the pMN domain are OLIG2 and NGN1/2 . The marFollowing MN progenitor formation, NGN2 is highly expressed, and post-mitotic lower MNs are generated. NGN2 induces the expression of HB9, which works to consolidate MN identity . In addiAfter MNs exit the cell-cycle, additional specification occurs. HOX-family genes are differentially expressed along the rostral-caudal axis to form distinct lower MN columns in the cervical, thoracic, lumbar, and sacral spinal cord. The order of HOX chromosomal loci determines HOX gene rostral-caudal expression. Thus, HOX4-8 are expressed in the brachial MN column. Similarly, HOX8/9 are expressed in the thoracic region and HOX10/11 genes are highly expressed in lumbar regions . This paPost-mitotic MNs have distinct gene expression signatures. General MNs are most often defined by HB9 expression, but they also express ISL1, ISL2, and LHX3 . MMC MNsin vitro differentiation of upper MNs yields generic, immature, progenitor-like cortical neurons. These limitations have recently undergone review by The RA/Wnt/FGF pathways reviewed thus far form lower MNs, which are cholinergic, releasing acetylcholine into the synaptic cleft. Upper MNs and upper MN tracts, which include corticospinal, reticulospinal, tectospinal, rubrospinal, and vestibulospinal tracts among others, are glutamatergic, and release glutamate onto lower MNs or interneurons . As we reviewed, the generation of lower MNs occurs in three stages of differentiation: neural precursor, MN-progenitor, and post-mitotic MN. Less is known about the more complex embryogenesis of glutamatergic upper MN tracts and the complex, and often uncharacterized, mechanisms guiding their development and subtyping. Since multiple facets of the differentiation of iPSCs to upper MNs have not been characterized, and since upper MN precursors have not yet been transplanted in models of SCI, we do not focus on upper MNs in this article. The current standard for It is well known that certain MN pools have increased susceptibility to MN diseases. As a result, iPSC-derived MN subtypes may be a promising personalized therapy to target certain susceptible MNs. This is because iPSCs have lower ethical and immunogenic concerns than ESCs. In addition, like ESCs, iPSCs are able to differentiate into a wide variety of lower MN subtypes. For example, some MNs have increased susceptibility to ALS – spinal MNs in particular exhibit graded vulnerability to ALS, with fast-twitch MNs affected first . SusceptIn contrast to MN diseases like ALS, significantly less attention is given to differential MN subtype vulnerability in SCI. This is especially relevant considering that a hallmark of SCI is inter-individual heterogeneity. The fine-tuned packaging and organization of neuronal tracts, lower MNs, interneurons, decussations, nerve roots, and microvasculature in the spinal cord allows for inter-subject variability regarding structural damage, dislocation, compression, transection, or concussion. Today, SCI is most often incomplete, meaning that some level of function below the primary injury is retained . CompletWhile the degree of SCI severity plays the primary role in the level of damage that MNs undergo, studies have largely supported the theory that intrinsic differential MN susceptibility may also contribute to SCI severity and recovery of function. For example, the level at which SCI occurs can influence which MN pools are affected. In pediatric populations, lower MNs have been found to have increased vulnerability in caudal lesions . FurtherThese observations suggest that personalized iPSC-MN transplants, designed to exploit inter-subject heterogeneity by targeting certain vulnerable or selectively injured MN populations, could be effective therapies in SCI. Recent studies have demonstrated how strategically targeting propriospinal neurons, for example, can lead to robust axonal outgrowth that passes through scarring to establish new connections across lesions . Stem cein vitro from a variety of PSCs and cell resources. These include PSCs (ESCs and iPSCs) as well as MSCs. ESC-MNs and iPSC-MNs and their MN subtypes are increasingly well characterized and confirmed through downstream electrophysiology and grafting. MSC-MNs, however, have only been poorly characterized, and concerns exist over whether MSCs can be differentiated into mature MNs. Some studies seem to have succeeded in generating MNs from MSCs, but these are not always confirmed with electrophysiology, purity analysis, synapse-formation, or spinal grafting  formation in feeder culture, monolayer differentiation, and viral or synthetic mRNA delivery of transcription factors – each of which can be combined with small-molecule neural induction. These methods vary in efficiency, clinical applicability, and duration. These methods have undergone significant revision over the past decade with the goal of clinical translation because they need to be scalable, efficient, safe, non-tumorigenic, and non-integrating. Successful differentiation into clinically-useful MN fates requires good manufacturing practice during the initial steps of iPSC-generation, neuralization, and neural precursor induction.Once iPSCs have been generated, the next step in MN differentiation is the induction of a neural fate. To date, a variety of methods have been used to generate neural precursors for MN differentiation. Neural precursor formation is a critical first step to generating MNs in vitro morphogen coverage. They combined the steps of neuralization and ventralization by culturing iPSC-derived EBs for 1 week before treating them with RA and Purmorphamine (Shh agonist) for one additional week, thus generating spinal precursors expressing PAX6, POU class 3 homeobox 2 (POU3F2), and SOX3 in 2 weeks. EBs are clusters of iPSCs or ESCs representing all three germ layers, and an ectodermal  fate is the default specification without extrinsic morphogen treatment. Early MN differentiation methods used EB generation as a crucial first step in neural induction. These methods used a variety of neurotrophic factors to initiate neuralization and to culture neural precursors. Neuralization can be initiated by detaching PSCs from pluripotent media (which contain the factors leukemia inhibitory factor and bFGF), thus causing a termination of pluripotency and generating aggregate embryoid bodies (EB) . ProtocoThe formation of EB remains a concern to generating clinically-relevant iPSC-NPCs or iPSC-MNs. Although effective, EBs pose certain challenges, including contamination with non-neural cells, variation between cell lines, scalability, and cost . MultiplMore recent protocols have improved upon previous advances by generating NPCs in a chemically-defined fashion. After neuralization is induced and NPCs are formed, additional specification takes place to commit cells to a lower MN lineage. Caudalization and ventralization take place to generate a spinal and anterior, ventral MN character, respectively. These MN-committed progenitors most commonly express OLIG2, NGN1/2, NKX6.1, NKX6.2, and PAX6. Once maturation takes place, general MNs express the canonical markers HB9, ISL1/2, and LHX3. The most commonly employed methods to generate MN-committed progenitors and mature MNs include treating NPCs with RA, Shh pathway agonists, and neurotrophic factors.Early MN differentiation protocols used a wide variety of small molecules, temporal adjustments, and neurotrophic factors to generate cells committed to a MN fate . After gSignificant advances regarding the optimization of morphogen combinations and temporal treatment have taken place to improve the efficiency and reduce the duration of MN differentiation . HoweverRecent methods further increased efficiency and clinical-applicability, using synthetic mRNA, xeno-free, feeder-free, and EB-free methods of MN generation. After 7 days of synthetic mRNA induction, in vitro. While early protocols relied on expensive, inefficient, and lengthy methodology, recent advances have allowed for the large-scale generation of functionally mature MNs – using integration-free, feeder-free, chemically-controlled, highly efficient (>98%), and rapid (∼7 days from iPSC to MN) experimental strategies , which express canonical markers of MN-lineage – HB9, ISL1, ISL2, and LHX3. However, the differentiation of PSCs into MN subtypes, each expressing unique gene signatures along the spinal rostra-caudal axis, has received significantly less attention. This being said, several studies have succeeded in manipulating MN subtypes during their differentiation.In their study, in vitro. in vivo. These studies demonstrate that MN subtypes can be patterned from PSCs and associate with their appropriate subtype in vivo.Some MN subtype derivation methods have primarily focused on ESCs rather than iPSC to generate LMC, MMC, and PMC MN subtypes. As many of the protocols described thus far have shown, RA is one of the most widely used caudalizing factors in neural induction. Though past and future experimental advances, PSCs have been successfully differentiated into several MN subtypes, namely MMC, LMC, and PMC columns. In addition, recent progress in RA-mediated HOX patterning revealed the role of RA in inducing a neuromesodermal to neuroectodermal transition, thus committing neural precursors to their subtype fate . Future in vivo grafting.Multiple factors should be considered when selecting an iPSC-MN differentiation protocol. The protocols reviewed here each have advantages and disadvantages based on the goals of a proposed study. In particular, research groups should weigh factors that include but not limited to clinical relevance, viral integration, duration of iPSC-MN differentiation, cost-effectiveness, efficiency, scalability, MN subtype generated, and if MNs have previously been confirmed with electrophysiology or in vivo grafting (mouse striatum). However, iPSCs were generated by using a pMX integrating viral transfection. As a result, combining this protocol with iPSCs generated by non-integrating means would further increase clinical applicability. For research groups interested in clinical applications with a shorter protocol duration, the method developed by First of all, for clinical applications, the recent protocol developed by Secondly, for applications prioritizing scalability, protocols developed by Finally, for applications prioritizing specific MN subtypes, research groups may consider the in vivo differentiation capacity into MNs and glia, thus replacing damaged axonal tracts and exerting a variety of neurotrophic, remyelinating, and relay-forming effects  have been transplanted in models of SCI . Neural  effects . For theWhile iPSC-NPCs have beneficial effects, the hostile microenvironment of SCI remains a challenge to MN growth. Immune and ischemic processes combine forces to recruit cytokines/chemokines, generate free-radicals, and stunt nascent cell development. Neither acute nor chronic stem cell transplants have yet been largely successful due mainly to the early inflammatory response, which is particularly damaging to cell grafts, and formation of the glial scar, which inhibits axonal regrowth. Thus, transplants occurring during the subacute phase, considered the optimal timeframe, have been shown to be more effective . In the 5), intraspinal routes of administration, and neural precursor transplants . As these new methods evolve, the use of integration-free, feeder-free, and virus-free reagents is crucial at every step for streamlined clinical applications.Future advances in mechanistically understanding CNS development, stem cell induction, and SCI pathophysiology are critical. Targeting specific MN subtypes, especially those most vulnerable following SCI, is a previously underappreciated therapeutic strategy. Future stratification of individuals and injury types according to the individual MN subpopulations affected will allow for personalized stem cell transplantation. In addition, advances in differentiating additional MN subtypes, particularly the differentiation of upper MNs, are needed to effectively target upper MN tracts. This is because upper MN tracts  are differentially vulnerable during SCI. Significant advances need to be made regarding iPSC-derived neural-committed cells are powerful therapeutic tools in stem cell transplantation for SCI. While previous stem cell sources (ESCs and MSCs) continue to be dogged by ethical, immunogenic, and clinical concerns, iPSCs are an alternative with significant advantages. iPSCs may be differentiated into MNs and different MN subtypes by a wide variety of methods. These protocols have undergone significant revision and improvement, and the most recent methods strive to be as translatable as possible. Significant technological and methodological advances have taken place that may now allow for easier clinical translation. Further, the recent differentiation strategies that generate individual MN subtypes should continue to become more relevant as personalized stem cell transplantation becomes reality. Future advances are needed to take advantage of these methods and develop novel treatment strategies able to target individual MN subtypes – performed with the ultimate goal of restoring motor function and quality of life for individuals with SCI.MT developed the idea in detail, reviewed the literature, and wrote the manuscript. GL designed the original project idea and guided the development of the review throughout the writing process. BD provided valuable feedback and helped to revise the manuscript. RF provided the leadership for the project and furnished the valuable feedback for revising the manuscript.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.


Tg parameter. The incorporation of IL-silica simultaneously enhanced the tensile strength, toughness, and thermal stability of the epoxy composites. The considerable improvements in mechanical and thermal properties are ascribed to the improved dispersion of IL-silica and the enhanced interfacial interactions between epoxy matrix and IL-silica by strong covalent bonding, which results in an effective load transfer.The present study aims to improve the mechanical properties of epoxy composite by incorporating supported ionic liquid silica (IL-silica). The IL-silica not only showed improved interfacial interaction and reinforcement, but also served as cure agent of epoxy composites. The differential scanning calorimetry analysis revealed that epoxy composites could be successfully cured with IL-silica without any routine curing agents. IL-silica/epoxy composites presented higher mechanical and thermal properties compared with epoxy composite containing un-functionalized silica (u-silica). The dynamic mechanical analysis showed that the storage modulus of composites significantly increased with the addition of IL-silica in comparison to that with added u-silica, as well as the variation of  At present, silica particles are widely employed as fillers to reinforce polymer composites. Silica particles can enhance the mechanical properties and overall performance of polymer composites ,2. HowevEpoxy resins have evolved as one of the most important thermosetting resins because of their good creep resistance, high strength, good thermal stability, and structural stiffness ,11. HoweThis work focuses on supported ionic liquid silica (IL-silica), in an attempt to achieve uniform dispersion and strengthened interfacial bonding of epoxy composites. The surface of silica was modified with 3-chloropropyltrimethoxysilane and 1-methylimidazole see . The eff2) was obtained from Wanzai County Huiming Chemical Industry Co., Ltd., Yichun, China. 3-Chloropropyltrimethoxysilane (CPTMS), 1-methylimidazole (1-MI), and xylene were supplied by Aladdin Industrial Corporation . Diglycidyl ether of bisphenol A (DGEBA)  was purchased from Yueyang Resin Factory, Yueyang, China. Alkyl (C12-C14) glycidyl ether (AGE)  was supplied by Hexion Inc., Shanghai, China.Silica  was used to mix fillers with epoxy resin. The IL-silica was mechanically mixed with DGEBA and AGE on a hotplate and then transferred on the three-roll mill. The gap width between the rolls was set to 2 μm and the velocities of the three rolls were set to 32/89/250 rpm. Three processing cycles were performed. After each cycle, the mixture was refilled into the feed opening and the rollers were cleaned. The mixture was finally transferred to a silicone rubber mold and cured at 80 °C for 2 h and 120 °C for 2 h, followed by a post-curing at 150 °C for 2 h.The detailed components of epoxy composites are shown  in 2 atmosphere. Tensile tests were carried out on an AI-700M universal test machine  according to ASTM D638 (2014) at a crosshead speed of 1 mm/min. Flexural tests were performed on an AI-700M universal test machine in accordance with ASTM D790 (2017) with a speed of 1 mm·min−1. The un-notched Izod impact strength was measured on a GT-7045-MDL pendulum impact test machine  according to ASTM D4812 (2011). Dynamic mechanical analysis (DMA) was analyzed using a DMA Q800  in single cantilever mode with a heating rate of 5 °C/min and a frequency of 1 Hz over a temperature range from room temperature to 200 °C. Scanning electronic microscope (SEM) was carried out with a SU8010 .Fourier transform infrared (FT-IR) spectroscopy was measured on a Nicolet NEXUS470 spectrometer . Themogravimetric analysis was carried out on Netzsch TG209 F3 instrument  with a heating rate of 10 °C/min from 30 to 600 °C. X-ray photoelectron spectroscopy (XPS) was performed on an X-ray photoelectron spectrometer VG Multilab 2000  with an aluminum Kα source (1486.6 eV). Evaluation of cure kinetic parameters was performed by differential scanning calorimetric analysis . About 5–10 mg of sample was heated from room temperature to 250 °C in N−1 correspond to the absorption of silanol groups and hydroxyl groups of the adsorbed water. The stretching vibration of Si–OH group is located at 961 cm−1. The peaks at 1110 and 804 cm−1 are assigned to the stretching vibrations of Si-O-Si group. In the FT-IR spectra of IL-silica, the bands at 2940 and 2839 cm−1 are due to the symmetric stretching band νs(C–H) and asymmetric stretching band νas(C–H) of alkyl groups. The imidazole ring vibrations are observed at 1575 and 1510 cm−1. The peak at 1446 cm−1 is assigned to the combined band of imidazole ring vibration and deformation band δ(C–H).The thermogravimetric analysis (TGA) curves of u-silica and IL-silica are shown in The XP Survey and N1s high resolution XPS spectra of u-silica and IL-silica are presented in N-heterocyclic carbon structure. In the initiation step of curing, the pyridine-type nitrogen atom of imidazole ionic liquids reacts with epoxy groups to form 1:1 adducts. In the following process, the pyrrole-type nitrogen of 1:1 adducts transforms to pyrridine-type nitrogen with H+ transfer. The formation of the above pyridine-type nitrogen can further react with epoxy polymer chains by combination of 1:1 adducts via anionic polymerization. Furthermore, two possible options would form in the cross-linked networks, both of which lead to the regeneration of 1-alkylimidazoles: (i) the cyclic epoxy polymer chains are formed through N-dealkylation reaction; (ii) the unsaturated structures are formed from the epoxy cross-link structure with carbonyl groups through tautomerization.−2 to 46.1 MPa, 70.2 MPa, 2.13 GPa, and 16.4 kJ·m−2. The improvement is due to the uniform dispersion of fillers and chemical bonds at the matrix/filler interface via polymerization of epoxy resin with the active imidazole groups of IL-silica. The reinforcement effect of polymer composites largely depends on the efficiency of load transfer at the interface [−2. A further increase in IL-silica content (up to 40 phr) generated a slight decrease of mechanical properties. This can be explained by the agglomeration of IL-silica in high content.nterface ,30. In tnterface ,32. By uTg value of the IL-silica20 sample shifted toward higher temperature by 9.2 °C compared to that of the silica20 sample. The presence of IL-silica led to a higher cross-linking degree of the resultant network than u-silica/epoxy composites. The Tg value of IL-silica/epoxy composites continuously shifted to higher temperature with increasing IL-silica content. This can be attributed to the increase of crosslinks density of epoxy resin with increasing IL-silica content, which would restrict the mobility of the epoxy chains for the enhanced interfacial interactions.Here the thermomechanical properties of epoxy composites are investigated based on the DMA results. The storage modulus and tan δ of various epoxy composites are shown in Tonset) and T10% shifted toward higher temperatures than that of the composite containing u-silica. For the composites containing various loadings of IL-silica, the Tonset and T10% increased with increasing IL-silica content. This can be explained in terms of the improved interfacial interaction and increased cross-linking density of epoxy composites [The TGA curves of epoxy composites are presented in mposites ,36,37.This study demonstrated supported ionic liquid silica (IL-silica) as curing agent for epoxy composite with improved dispersion and interfacial interaction. Epoxy composites were successfully cured with IL-silica without the addition of any routine curing agent. The IL-silica were observed to be highly dispersed and well integrated in epoxy resin compared to un-functionalized silica (u-silica). The uniform dispersion and improved interfacial adhesion provide for an efficient load transfer from the epoxy matrix to silica and thus enhance the overall mechanical and thermal properties of epoxy composites.