Although many behavioral studies have reported associative memory was different from item memory, evidence coming from ERP researches has been in debate. In addition, directed forgetting effect for items has been fully discussed, but whether association between items can be directed-forgotten was unclear. The directed forgetting effect was important for dissociating the item retrieval and associative retrieval because of the one-to-one mapping relationship both between item retrieval and familiarity and between associative retrieval and recollection. Thus, the aim of this study was to investigate the dissociation between item retrieval and associative retrieval and test directed forgetting effect for associative information. Associative recognition paradigm combined with directed forgetting paradigm by ERP recording was employed. Old/rearranged effect in to-be-remembered condition, which was associated with associative memory, was significant at 500–800 ms (LPC) but not at 300–500 ms interval (FN400), indicating that item information was retrieved prior to associative information. The ERP wave calculated by subtracting the to-be-forgotten old pairs with “old” response from those with “rearranged” response, which reflected associative retrieval in the to-be-forgotten condition, was negative from 500 to 800 ms (reversed old/new effect), indicating that association between items can be directed-forgotten. Similar evidence was obtained by contrasting “rearranged” responses aimed to the to-be-forgotten old pairs with those aimed to the to-be-remembered rearranged pairs, which actually represented the complete failure of associative retrieval. Therefore, item retrieval and associative retrieval were indexed by FN400 and LPC respectively, with associative retrieval more inhibited than item retrieval. Episode memory consists of item memory, which refers to memory for item information, and associative memory, which refers to memory for associative information between single item and other item . Taking word pairs for example, item information comes from individual words of a word pair, and associative information concerns the relationship or link between the two words. Increasing evidence coming from cognitive psychology . At test, participants were asked to make three kinds of judgments for all studied names regardless of the instruction . On the basis of previous studies  who are all native Chinese speakers participated in the experiment. One dataset was excluded due to excessive muscle artifacts and electrode drift, leaving 16 for analyses. All the participants have normal or corrected visual acuity, and reported no current or past neurological or psychiatric disease. Each subject signed an informed consent form before experiment and received monetary compensation after experiment. This research was approved by the Human Research Ethics Committee at Capital Normal University, methods were carried out in accordance with its relevant guidelines.F = 1.29, p > 0.05).The stimuli consisted of a set of 388 two-character Chinese full names, such as “王筠” (Wang Jun) and “刘蔷” (Liu Qiang) Figure . It shouThe experiment included a study phase and a test phase Figure . In the old”; if both of them were studied, but not encoded together at study, they would be judged as “rearranged”; if both of them were not studied at all, they would be judged as “new.” Participants were reminded that they could not make a judgment for old and rearranged pairs according to instruction (“记住” or ”忘记”). The order of trials was also pseudo-random with the constraint of no more than three consecutive trials coming from the same type. Both accuracy and speed of response were emphasized. Response buttons for “old” and “new” were counterbalanced across participants.During the test phase, participants also firstly received practice in order to adapt to the task demanding. Formal test consisted of 5 blocks, each with 108 names . Each test trial began with a fixation cross lasting for 500 ms, then a name  was presented for 2000 ms followed by a 1000 ms blank screen  data were recorded in test phase of the experiment and measured from 62 Ag/AgCl electrodes embedded in an elastic cap with NeuroScan SynAmps system . The electrode locations adhered to the extended international 10–20 system. All channels were referenced to left mastoid on-line and re-referenced off-line to averaged mastoids. EEG and EOG were amplified using a 0.01–100 Hz band pass and sampled at 500 Hz. Impedance was kept below 5 kΩ. Data were band pass filtered from 0.05 to 40 Hz off-line. EOG blink artifacts were corrected using a linear regression estimate. The averaged epoch was 1200 ms, including 200 ms prior to stimulus onset. Baseline corrections were performed using mean amplitudes of pre-stimulus onset. Trials exceeding ±90 μV were rejected.We analyzed all data with SPSS 21.0. Repeated-measures ANOVA adjusted by Greenhouse-Geisser corrections when assumptions of sphericity in the repeated measures analyses were violated. The alpha level was 0.05. The analysis of behavioral data focused on accuracy and reaction times (RTs). The ERP amplitudes were averaged at two latency intervals in the test phase (300–500 ms and 500–800 ms) and over sets of midline electrode clusters along the anterior-posterior axis , which were selected on the basis of previous studies  × 3 (Stimulus Type: old vs. rearranged vs. new) × 3 (Response Type: old vs. rearranged vs. new) incomplete within-subjects design. This design was necessarily incomplete because the instruction manipulation was not varied for new stimulus. Thus, we sorted the EEG data into 15 experimental conditions to examine the event-related potentials elicited by the names. For old names in TBR condition, if they were correctly remembered and judged by participants as “old”—TBR_R; if they were forgotten and were judged as “new”—TBR_F; finally, they could be judged as “rearranged”—TBR_r. For old names in TBF condition, if they were remembered and judged by participants as “old”—TBF_R, if they were forgotten and were judged as “new”—TBF_F; finally, they could be judged as “rearranged”—TBF_r. For rearranged names which consisted of items from names following by the same Remember or Forget instruction, they were denoted as TBRr and TBFr respectively. They could be judged as “old” , “new”  or “rearranged” . For new names, they could be judged as “old” (N_R), “new”  or “rearranged” (N_r). All abbreviations for each condition were shown in Table According to our purpose, we selectively focused on 6 conditions: TBR_R, TBF_R, TBF_r, TBRr_r, TBFr_r, and N_CR condition. The EEG of the selected 6 conditions were separately overlapped and averaged. The mean numbers (standard deviation) of artifact-free trials for them were as follows: TBR_R 61 (10), TBF_R 26 (9), TBF_r 33 (10), TBRr_r 39 (7), TBFr_r 29 (6), and N_CR 130 (25). The mean trial numbers (standard deviation) of the rest 9 conditions were as fol1ows: TBR_F 7 (5), TBR_r 19 (6), TBF_F 29 (12), TBRr_R 13 (5), TBRr_F 7 (5), TBFr_R 10 (5), TBFr_F 19 (6), N_R 9 (7), N_r 35 (20). There were two main reasons why we abandoned these conditions: firstly, the correlation of most conditions with our experiment purpose was low; secondly, trial numbers of these conditions were not sufficient to support further analysis (less than 16).The analysis of ERP results was conducted including the following three steps. First, in order to investigate the dissociation between item retrieval and associative retrieval, we examined overall differences among TBR_R, TBRr_r, and N_CR condition. Both the stimuli and responses in these conditions differed from each other, but participants made correct responses in each condition. In Lyu et al.  and TiboSecond, in order to investigate the DF effect, we examined overall differences among TBR_R, TBF_R, and N_CR condition. In Nowicka et al.  and Van Third, we examined the DF effect for associative information. In Nowicka et al.  and Van In addition, to exclude the possibility that the between-condition difference in the 500–800 ms time window may be correlated with the difference in the 300–500 ms time window, we planned to confirm the independency between the two time windows by dissociating the two ERP components . According to previous studies  = 10.406, p < 0.001]. Mean accuracy in TBF_R condition was lower than TBR_R condition . Mean accuracy in TBF_r condition was greater than TBF_R condition . Mean accuracy in TBF_r condition was greater than TBFr_r condition . Mean accuracy in TBF_r condition was lower than TBRr_r condition .For mean accuracy, paired t-test showed that RTs in TBR_R condition was shorter than TBRr_r condition . RTs in TBF_R condition was shorter than TBR_R condition . RTs in TBF_r condition was longer than TBF_R condition . RTs in TBF_r condition was not shorter than TBFr_r condition . RTs in TBF_r condition was longer than TBRr_r condition .For RTs, paired Participants in TBR_R condition made response more accurately and quickly than in TBRr_r condition, which indicated identifying rearranged stimuli was a difficult task. Overall, performance in TBF_R condition was inferior to that in TBR_R condition, indicating that DF effect was present. Associative information may be directed-forgotten by comparing TBF_r condition with TBF_R, TBFr_r, and TBRr_r condition.The neural activity elicited by TBR_R condition, TBRr_r condition, and N_CR condition are shown in Figure F = 17.095, p < 0.001]. Post-hoc comparison showed that N_CR condition was more negative than TBR_R condition and TBRr_r condition (both p < 0.001), whereas no difference was observed between TBR_R condition and TBRr_r condition (p = 0.487). The reliability of the null hypothesis was confirmed with Bayesian factor analysis  = 1.512, p = 0.196], the condition × location and condition × laterality interaction were significant . In order to dissolve the interaction involving the factors “location” and “laterality,” separate ANOVA with the factor “condition” was performed for each electrode site. Significant effects of condition were obtained for electrodes F3, Fz, F4, C3, Cz, and C4 . Subsequent post-hoc comparison obtained similar modulation for each electrode except for the magnitude of p value. Specifically, N_CR condition was more negative than TBR_R condition for electrodes F3, Fz, F4, C3, Cz, and C4 . N_CR condition was more negative than TBRr_r condition for electrodes F3 (p = 0.008), Fz (p = 0.007), F4 (p = 0.004), C3 (p = 0.001), Cz (p < 0.001), and C4 (p = 0.03). No difference was observed between TBR_R condition and TBRr_r condition for electrodes F3 (p = 0.103), Fz (p = 0.067), F4 (p = 0.276), C3 (p = 0.57), Cz (p = 0.388), and C4 (p = 0.726).For 300–500 ms interval, the main effect of condition was significant . Post-hoc comparison revealed that more positive amplitude was found for TBR_R condition relative to N_CR condition (p < 0.001), but TBRr_r condition was not more positive than N_CR condition (p = 0.141). Critically, TBR_R condition was more positive than TBRr_r condition (p = 0.032). The condition × location, condition × laterality and condition × location × laterality interaction were insignificant .For 500–800 ms interval, the main effect of condition was significant . Post-hoc comparison showed that N_CR condition was more negative than TBR_R condition and TBF_R condition (both p < 0.001), whereas no difference was observed between TBR_R condition and TBF_R condition (p = 0.082). Although the condition × location interaction was insignificant , the condition × laterality and condition × location × laterality interaction were significant . Dissolving the interactions involving the factors of location and laterality, separate condition ANOVA was calculated for each electrode site yielding significant effects of condition for all electrodes . Subsequent post-hoc comparison showed that ERP for TBF_R condition did not differ from TBR_R condition for all electrodes , except for electrode F3 (p = 0.033). ERP for N_CR were more negative-going than ERP for TBF_R condition for electrodes Fz (p = 0.003), F4 (p = 0.001), C3 (p = 0.045), Cz (p = 0.001), C4 (p = 0.001), P3 (p = 0.011), Pz (p = 0.003), and P4 (p = 0.011).For 300–500 ms interval, the main effect of condition was significant . Post-hoc comparison showed that TBR_R condition and TBF_R condition were more positive than N_CR condition , whereas no difference was observed between TBR_R condition and TBF_R condition (p = 0.167). Although the condition × location and condition × laterality interaction were insignificant , the condition × location × laterality interaction was significant . To dissolve the three-way interaction, separate ANOVA with the factor “condition” was conducted for each electrode site yielding significant effects of condition for all electrodes . Subsequent post-hoc comparison revealed that TBF_R condition elicited more negative-going waves than TBR_R condition for electrodes P3 (p = 0.027) and Pz (p = 0.041). However, ERP for TBF_R were more positive-going than ERP for N_CR condition for electrodes Fz (p = 0.016), F4 (p = 0.013), Cz (p = 0.043), and P3 (p = 0.037).For 500–800 ms interval, the main effect of condition was significant . Both the two-way condition × location and three-way interaction were insignificant . However, the condition × laterality interaction was significant . To dissolve the two-way interaction, separate ANOVA with the factor “condition” was conducted for each electrode site yielding significant effects of condition for electrodes Fz (p = 0.029), F4 (p = 0.018), Cz (p = 0.01), and C4 (p = 0.039). For 500–800 ms interval, the main effect of condition was significant, ERP for TBF_r condition were more negative than ERP for TBF_R condition . Although the condition × laterality and condition × location × laterality interaction were insignificant , the condition × location interaction was significant . To dissolve the two-way interaction, separate ANOVA with the factor “condition” was conducted for each electrode site yielding significant effects of condition for electrodes Fz (p = 0.006), F4 (p = 0.002), and Cz (p = 0.039).For 300–500 ms interval, the main effect of condition was significant, ERP for TBF_r condition were more negative than ERP for TBF_R condition . The null hypothesis was also confirmed with Bayesian factor analysis (Bayesian factor = 5.35). All interactions concerning “condition” were insignificant: the condition × location interaction , the condition × laterality interaction , and the condition × location × laterality interaction . For 500–800 ms interval, the main effect of condition was insignificant, that is, ERP for TBF_r condition did not differ from ERP for TBFr_r condition . The null hypothesis was also confirmed with Bayesian factor analysis (Bayesian factor = 7.76). Although the condition × laterality and condition × location × laterality interaction were insignificant , the condition × location interaction was significant . To dissolve the two-way interaction, separate ANOVA with the factor “condition” was conducted for each electrode site yielding significant effects of condition only for electrode Fz (p = 0.017).For 300–500 ms interval, the main effect of condition was insignificant, that is, ERP for TBF_r condition did not differ from ERP for TBFr_r condition . All interactions with “condition” did not reach significance: the condition × location interaction , the condition × laterality interaction , and the condition × location × laterality interaction . For 500–800 ms interval, the main effect of condition was significant, that is, ERP for TBF_r condition were more negative-going than ERP for TBRr_r condition . The condition × location, condition × laterality, and condition × location × laterality interaction were insignificant .For 300–500 ms interval, the main effect of condition was significant, that is, ERP for TBF_r condition were more negative-going than ERP for TBRr_r condition , demonstrating the observation that the old/new effects tended to be more anterior for 300–500 ms and more posterior for 500–800 ms.To dissociate the two ERP components , topographical analyses for old/new effects were performed on the old and new differences using rescaled data with the vector normalization approach  was uncertain. The ERP difference between rearranged and new pairs is thought to reflect item information as both associations in the two type of pairs were not studied in the study phase Hockley, . LikewisThe idea that associative retrieval was indexed by the ERP difference between old and rearranged pairs was supported by indirect evidence. Donaldson and Rugg  showed tItem retrieval was demonstrated to occur earlier than associative retrieval. Meanwhile, we provided evidence for the viewpoint that item retrieval relied only on familiarity and associative retrieval relied only on recollection. A lot of researches have revealed that associative retrieval depended only on recollection. Yonelinas  reportedThe amplitudes of ERP for TBF_r condition were lower than amplitudes of TBF_R, TBFr_r, TBRr_r condition at 500–800 ms interval (the reversed effect), which was similar to the findings of Nowicka et al.  and Van According to selective rehearsal account, the rote rehearsal for associative information is stopped at the time of presentation of Forget instruction (Bailey and Chapman, We found that ERP of TBF_R condition was more negative than that of TBR_R condition at the two selected time window. It can be inferred that the whole name can be directed-forgotten. Unfortunately, we cannot determine which kind of information was directed-forgotten. As directed forgetting for associative information was indexed by three reversed effect at 500–800 ms interval in our experiment, the ERP difference between TBF_R and TBR_R condition at 300–500 ms interval can be taken as an indication of directed forgetting for item information. In addition, the ERP difference between TBF_r condition and TBRr_r condition in 300–500 ms interval reflected the success of item retrieval under Forget instruction. Meanwhile, there was no ERP difference between TBF_r condition and TBFr_r condition in the time window of 300–500 ms. These results in concert reflected that item information was retrieved in the early time window.In ERP results, the above evidence strongly suggested that remembered item information in TBF context evoked more negative-going potentials relative to that in TBR context. This was in line with two previous studies, which reflected that remembered TBR items are more likely to give rise to a sense of conscious recollection than remembered TBF items (Nowicka et al., The amplitudes of ERP for TBF_R condition were higher than amplitudes of N_CR condition at 300–500 ms interval, that is, remembered item information in TBF context evoked more positive potentials than new item information. This is inconsistent with Nowicka et al.  and Van The discrepancy between the studies of Nowicka et al.  and Van The amplitudes of ERP for TBF_r condition were lower than amplitudes of TBFr_r condition at 500–800 ms interval, that is, forgotten associative information in TBF context evoked more negative potentials than new associative information. This seemed to indicate that the neural pattern of directed forgetting for associative information differed from that for item information. As already discussed, in our experiment, item retrieval only based on familiarity which is lack of details, but associative retrieval only engendered recollection which contains numerous details. Thus, associative retrieval retrieves more details than item retrieval. In addition, there is a possibility that the degree of inhibition for item retrieval is equal to that for associative retrieval at the very beginning. Because it was acknowledged by Lyu et al.  and our In summary, the present study focused on resolving the time window wherein dissociation appeared. On the one hand, item retrieval and associative retrieval were dissociated by different ERP components referred to FN400 and LPC respectively. On the other hand, however, item retrieval and associative retrieval were dissociated by different ERP effect referred to DF effect and pronounced reversed old/new effect. Thus, we can conclude that different ERP correlates are associated with item retrieval and associative retrieval. Unfortunately, in the context of current study, it was impossible to determine in which stage inhibition occurred. In order to resolve this problem, future research should aim at encoding phase. Furthermore, it is interesting to investigate whether our results are interacted with the type of association, as between-domain inter-item association is different from within-domain inter-item association used here (Mayes et al., YW designed the experiment, collected data, drawed the figures, and wrote the manuscript. XM, BL, and WW revised the manuscript. CG instructed the whole research.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.


Substitution of lysine 133 to alanine in β-catenin almost completely abolishes its nuclear localization. We also demonstrate the K133 methylation is critical for the interaction of β-catenin with FOXM1. Furthermore, after treatment with a SMYD2 inhibitor, significant reduction of nuclear β-catenin and subsequent induction of cancer cell death are observed. Accordingly, our results imply that β-catenin methylation by SMYD2 promotes its nuclear translocation and activation of Wnt signaling.Accumulation of β-catenin in the nucleus is a hallmark of activation of the Wnt/β-catenin signaling pathway, which drives development of a large proportion of human cancers. However, the mechanism of β-catenin nuclear translocation has not been well investigated. Here we report biological significance of SMYD2-mediated lysine 133 (K133) methylation of β-catenin on its nuclear translocation. Knockdown of SMYD2 attenuates the nuclear localization of β-catenin protein in human cancer cells. Consequently, transcriptional levels of well-known Wnt-signaling molecules,  Wnt/β-catenin pathway is a canonical Wnt signaling pathway that is critical for stem cell regulation and is also known to be activated in a large proportion of human cancers such as colorectal cancer and hepatocellular carcinoma (HCC). β-catenin functions as the transcriptional co-activator in the Wnt signaling pathway and transmits the signal by regulating the downstream genes including key developmental genes or those involved in development/progression of human cancer –5. NucleSMYD2 was first identified as one of the SMYD family members and functions as an oncogene, which is highly expressed in various types of human cancer. Our group has reported critical roles of SMYD2-mediated methylation in tumorigenesis as a methyltransferase –9.Since SMYD2 protein is mainly located in the cytoplasm and overexpressed frequently in colorectal and liver cancers where the Wnt/β-catenin pathway is also frequently activated, we aimed to investigate a possible relationship between SMYD2 and β-catenin.Here we report biological significance of SMYD2-mediated lysine 133 (K133) methylation of β-catenin on its nuclear translocation. We show the K133 methylation is critical for the interaction of β-catenin with FOXM1. After treatment with a SMYD2 inhibitor, significant reduction of nuclear β-catenin and subsequent induction of cancer cell death are observed. Our results imply that β-catenin methylation by SMYD2 promotes its nuclear translocation and activation of Wnt signaling.in vitro methyltransferase assay using recombinant His-SMYD2 protein and recombinant GST-WT (wild type)-β-catenin to examine a possibility whether β-catenin can be a potential substrate of SMYD2, and found that SMYD2 could methylate β-catenin in a dose-dependent manner - or K133A-substituted-β-catenin, with HA-SMYD2 vector into 293T cells. Then we immunoprecipitated cell lysates with the anti-FLAG antibody and performed western blot analysis using the anti-K133-methylated β-catenin antibody Figure . The antβ-catenin mutation and APC mutation, respectively, as well as two HCC cell lines, SNU449 and SNU475, with β-catenin mutation and Axin1 mutation, respectively.Since it is known that the majority of SMYD2 protein is located in the cytoplasm, we hypothesized that methylation of a lysine residue in β-catenin might affect its subcellular localization in cancer cells as we reported examples in our review , 11 and SMYD2-specific siRNAs , and conducted western blot analysis after fractioning cytoplasmic and nuclear components. We found that nuclear β-catenin as well as endogenously methylated β-catenin (both in the cytoplasm and nucleus) in these two cell lines was significantly decreased by SMYD2 knockdown  and cMYC, which are well known for their involvement in cell cycle progression and proliferation of cancer cells. When we treated two HCC cells and two colon cancer cells with siRNA for SMYD2 (siSMYD2), significant downregulation of both CCND1 and cMYC was observed in concordance with the decrease of nuclear β-catenin in all of these four cell lines  that SMYD2 methylates Lys133 of β-catenin, (2) that methylated β-catenin can bind to FOXM1, and then (3) that β-catenin can be translocated into the nucleus with FOXM1. Concordantly, Lys133Ala-substituted β-catenin could not be methylated  and could not enter into the nucleus. Knockdown of SMYD2 abolished β-catenin methylation and nuclear localization of β-catenin through the loss of the interaction with FOXM1, resulted in the downregulation of the Wnt/β-catenin/TCF downstream genes such as It has been known for many years that dysfunction of β-catenin degradation machinery or β-catenin activating-mutations cause the stabilization and nuclear accumulation of β-catenin in many types of human cancer including colon cancer and hepatocellular carcinoma. The presence of nuclear β-catenin accumulation is considered as a hallmark of the activation of the Wnt/β-catenin/TCF pathway. Somehow, we thought that the stabilization of β-catenin in the cytoplasm could spontaneously cause the translocation of β-catenin into the nucleus and the accumulation of a large amount of β-catenin in the nucleus, and activate the Wnt/β-catenin/TCF pathway. However, our results clearly imply that methylation of β-catenin by SMYD2 is critically important for its nuclear translocation and the activation of the Wnt/β-catenin/TCF pathway. The fact that K133A-substituted β-catenin, which lacked a methylation site, could interact with SMYD2, but lost the interaction with FOXM1, indicated the significance of K133 methylation for the interaction with FOXM1, and the subsequent nuclear translocation of β-catenin with FOXM1. In addition, a region including the methylation site of β-catenin is suggested to be important for the interaction with α-catenin Figure . Hence, Although we need to further elucidate the detailed mechanism using an animal model, our data strongly support the significance of SMYD2-medicated β-catenin methylation for the Wnt/β-catenin/TCF pathway activation. Hence, our results should shed light on a novel and critical process for the activation of the Wnt/β-catenin/TCF pathway. Since SMYD2 is overexpressed in a large proportion of human cancers including colon cancer, breast cancer, and hepatocellular carcinoma, development of novel anti-cancer drugs targeting SMYD2 should be a very effective way to treat various types of human cancer in which the Wnt/β-catenin/TCF pathway is activated.2 condition except for SW480 cells (without CO2).293T, SNU449, SNU475, HCT116 and SW480 cell lines were obtained from American Type Culture Collection ATCC, tested and authenticated by DNA profiling for polymorphic short tandem repeat (STR) markers  in a mixture of methylase activity buffer  for 2 h at 30°C. After denaturing, samples were separated by SDS-PAGE, blotted to PVDF membrane and visualized by MemCode Reversible Stain  and fluorography.eviously , 20. BriThe bands corresponding to β-catenin were exacted from the gel, and digested with sequencing grade TPCK-trypsin  at 37°C for 12 h. The digestion mixture was analyzed by nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS) using Q-Exactive mass spectrometer . The peptides were separated using nano ESI spray column on an NTCC analytical column  at a flow rate 300 nL/min. The mass spectrometer was operated in the positive-ion mode, and the spectra were acquired in a data-dependent TOP 5 MS/MS mode. The MS/MS spectra were searched against the in-house database using local MASCOT server .Full-length β-catenin cDNA was cloned into the pCAGGS-n3FC (FLAG tag) vector. Full-length SMYD2 cDNA was cloned into the pCAGGS-nHC (HA tag) vector. Substitution of β-catenin at lysine 133 to alanine was introduced using Q5 site direct mutagenesis kit (New England Biolabs) according to the manufacture's protocol. Expression vectors were transfected into cells using FuGENE HD  according to manufacturer's protocols.TM M cell lysis reagent  supplemented with a protease inhibitor . Whole cell lysates, fractionated cell lysates or immunoprecipitated samples were separated by SDS-PAGE and blotted to nitrocellulose membrane. Nitrocellulose membrane was firstly incubated with each primary antibody as described in the above Antibodies section, and then protein bands were detected by incubating secondary antibody: horseradish peroxidase (HRP)-conjugated antibodies  and visualizing with enhanced chemiluminescence (Thermo Fisher Scientific) and Prime enhanced chemiluminescence . Nuclear and cytoplasmic fractions were prepared from SNU449 and SNU475 cells 72 h after siRNA transfection and were prepared from 293T cells 48 h after transfection of an indicated expression vector(s). All the samples were fractionated by using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) according to the manufacture's protocol.Samples were prepared from the cells lysed with CelLyticTM M cell lysis reagent  and a subsequent immunoprecipitation reaction was conducted. Before lysing the cells, a protease inhibitor  was added to cell lysis reagent under instructions of the manufacture. For fractionated cytoplasmic and nuclear components, immunoprecipitation reaction was conducted directly after cell fractionation. In an immunoprecipitation reaction, 300 μg of cell extract were incubated with 30 μL of anti-FLAG M2 affinity gel , which is a purified monoclonal anti-FLAG antibody covalently attached to agarose beads. After the beads were washed 3 times in 1 ml of TBS buffer (pH 7.6), proteins that bound to the beads were eluted by boiling in Lane Marker Reducing Sample Buffer (Thermo Fisher Scientific).For whole-cell extract, transfected 293T cells were lysed by CelLyticsiNegative control (siNC) from Sigma-Aldrich, a mixture of three different oligonucleotide duplexes was used as a control siRNA. Two siRNA oligonucleotide duplexes were purchased from Sigma-Aldrich for targeting SMYD2 transcripts, and named as siSMYD2#1 and -#2. The siRNA sequences are described in https://imagej.nih.gov/ij).Cells were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer at 4°C for 1 h, permeabilized in 0.1% Triton X-100 (Sigma-Aldrich) for 3 min at room temperature and blocked with 3% BSA for 1 h at room temperature. Fixed cells were incubated with anti-α-tubulin, anti-β-catenin, anti-FLAG and anti-HA antibodies overnight at 4°C followed by incubation with Alexa Fluor-conjugated secondary antibody , and observed using Leica confocal microscopy . Signal intensity  of exogenously expressed β-catenin was quantified using ImageJ software (GAPDH (housekeeping gene), SMYD2, CCND1, cMYC, c-JUN, VEGF, Axin2 and claudin-1 were designed  or 7 days  of transfection with siRNAs. The Cell counting kit-8  was used for methyl thiazolyl tetrazolium reaction. After reaction for 2 h, the numbers of cells were counted in a microplate reader at 450 nm.Cancer cells were seeded into 96-well flat-bottom plates  at 4000 cells per well, and treated with a SMYD2-specific inhibitor, LLY-507 , at concentration of 0, 3, 5, or 7 μM, and cultured at 37°C under 5% COt-test (two groups). Significant difference between groups was noted when P-value was < 0.05.Values were presented as the means plus or minus one standard deviation. Statistical analyses were performed using unpaired Student's


Wnt signalling is involved in the formation, metastasis and relapse of a wide array of cancers. However, there is ongoing debate as to whether activation or inhibition of the pathway holds the most promise as a therapeutic treatment for cancer, with conflicting evidence from a variety of tumour types. We show that Wnt/β-catenin signalling is a bi-directional vulnerability of neuroblastoma, malignant melanoma and colorectal cancer, with hyper-activation or repression of the pathway both representing a promising therapeutic strategy, even within the same cancer type. Hyper-activation directs cancer cells to undergo apoptosis, even in cells oncogenically driven by β-catenin. Wnt inhibition blocks proliferation of cancer cells and promotes neuroblastoma differentiation. Wnt and retinoic acid co-treatments synergise, representing a promising combination treatment for MYCN-amplified neuroblastoma. Additionally, we report novel cross-talks between MYCN and β-catenin signalling, which repress normal β-catenin mediated transcriptional regulation. A β-catenin target gene signature could predict patient outcome, as could the expression level of its DNA binding partners, the TCF/LEFs. This β-catenin signature provides a tool to identify neuroblastoma patients likely to benefit from Wnt-directed therapy. Taken together, we show that Wnt/β-catenin signalling is a bi-directional vulnerability of a number of cancer entities, and potentially a more broadly conserved feature of malignant cells. Wnt/β-catenin signalling is a key developmental pathway which is dysregulated in a wide variety of cancers –7. ActivA recent study of neuroblastoma survival rates in Europe between 1999 and 2007 showed no improvement , despiteMalignant melanoma is the most deadly form of skin cancer and its global incidence continues to increase , 34. WhiColorectal cancer (CRC) has one of the highest incidences of all cancers , 44. DesThere is an unmet clinical need for novel therapies in each of these three cancer entities, with the targeting of the Wnt pathway representing a potentially useful strategy. However, a greater understanding of the effect of Wnt signalling modulation is required. In this study we revealed that both Wnt inhibition and hyper-activation are therapeutically valid approaches to neuroblastoma, once high levels of modulation are achieved, with each resulting in distinct beneficial outcomes. We also report numerous novel functional interactions between the MYCN and Wnt pathways. In line with the rapid advance of precision medicine in clinical oncology , to helpTo address the conflicting literature reports and assess whether both Wnt activation and inhibition can drive similar outcomes in the same neuroblastoma cell lines we treated IMR32 cells with the small molecules ICG-001 and Wnt agonist 1. ICG-001 inhibits β-catenin functioning by inhibiting β-catenin from binding to CBP , therefore acting as an inhibitor of the canonical Wnt signalling pathway . ConversGiven the similar outcome of Wnt activation and inhibition on neuroblastoma cell viability we tested these small molecules in a zebrafish developmental screen to confirm that they can produce opposing effects. GSK3 inhibitors (BIO and azakenpaullone) were also used as a positive control for Wnt activation phenotypes, as GSK3 negatively regulates Wnt signalling by promoting β-catenin protein degradation . Wnt actProviding further evidence of the specificity of Wnt agonist 1, Wnt activation also disrupted anterior development, inducing a headless phenotype Figure . This isWe next confirmed that modulation of Wnt signalling in either direction can reduce neuroblastoma cell viability using non-pharmaceutical approaches. When KCN cells were treated with Wnt3a protein they showed a dose dependent loss of cell viability Figure . The Wnthttp://r2.amc.nl) in two large neuroblastoma tumour datasets , with responses to the knockdown of these genes varying between the un-induced and induced MYCN overexpression conditions. Further supporting its therapeutic potential, β-catenin was also an inferred transcriptional regulator  of the 674 genes that strongly reduced SY5Y-MYCN cell viability when knocked down in the high-throughput RNAi screen . In summR = −0.9, p-value = 0.03739).Having established that the observed reductions in viability were indeed Wnt specific, we next examined the cellular response to Wnt inhibition and activation in more detail. Wnt inhibition increased MYCN mRNA levels, whereas Wnt activation decreased them Figure . NeuroblHaving shown that both inhibition and activation of Wnt/β-catenin signalling preferentially reduced cell viability of high MYCN expressing neuroblastoma cells, we next explored how oncogenic MYCN and Wnt are functionally linked. To achieve this we mined our omic datasets, consisting of RNA-seq, 4sU-seq (labelled) ChIP-seq and interaction proteomics data which we had generated to globally profile overexpressed and amplified MYCN's signalling networks , 71. 4sUhttp://www.ingenuity.com/), which allows the interrogation of high-throughput data at the pathway, network, function and regulator levels. Wnt/β-catenin signalling pathway components were significantly enriched in the differentially expressed genes of the MYCN overexpression time-course (24 h and 48 h) compared with un-induced cells  analysis, using IPA's ITRs algorithm . Using pInterestingly, ITR analysis showed that Wnt/β-catenin signalling was repressed in the MNA cells lines Figure , S3. Thiwww.string-db.org). This analysis revealed a small network, where β-catenin (CTNNB1) and HDAC2 were the best connected hubs  of overexpressed MYCN protein using mass spectrometry-based interaction proteomics, and assessed which putative interaction partners were also ITRs of the MYCN overexpression time-course RNA-seq. We thereby identified proteins which bound MYCN and transcriptionally regulated their target genes upon MYCN overexpression. A protein-protein interaction map of these regulators was then generated using the String database  bound to a number of genes encoding Wnt/β-catenin pathway components Figures . ImportaCell fractionation analysis revealed that both MYCN and β-catenin are present in the nuclei of IMR32 cells, which further supports our hypothesis that β-catenin protein and elevated MYCN protein interact in the nucleus. While nuclear β-catenin is generally considered a readout for Wnt signalling activation, our findings suggest that MYCN-β-catenin protein interaction reverses the normal transcriptional regulation of β-catenin target genes. Neither ICG-001 nor Wnt Agonist 1 treatment strongly altered nuclear β-catenin levels, which is in line with the drugs' mode of action: blocking β-catenin from binding to its nuclear co-factors rather than regulating β-catenin sub-cellular localisation. However, interestingly, ICG-001 treatment did reduce both cytosolic and nuclear MYCN levels Figure . This ICThe above data were obtained with neuroblastoma cell lines and hence may be cell line specific or may represent adaptations to cell culture conditions. Therefore, we examined whether these results were more generally applicable to neuroblastoma tumour biology by testing the salient findings in a well-characterised neuroblastoma tumour microarray dataset comprising 478 patients .2FC) between the patient-matched lines, KCN and KCNR. In addition, LEF1 was identified as a top transcription factor associated with overexpressed MYCN's genomic targets using the DiRE  strains) were maintained on a 14 h light/10 h dark lighting cycle at 28.5°C. Drug treatments were conducted for 48 h (4hpf - 52hpf) or for 3 h (4hpf - 7hpf). For Wnt activators, treatments for 3 h (4hpf - 7hpf) were also performed and produced axial patterning results comparable with 48 h (4hpf - 52hpf) treatments. Embryos studies were approved by the UCD Animal Research Ethics Committee.Zebrafish (www.ebi.ac.uk/arrayexpress) under accession numbers, E-MTAB-2690, E-MTAB-2691, E-MTAB-1684, E-MTAB-2787, E-MTAB-4100 and E-MTAB-2689. The sequence of the mβ-catenin/Tcf3 fusion construct was deposited in Genbank under accession number KM189193.Sequencing data were deposited in ArrayExpress (Our findings explain the previously contradictory reports on the role of Wnt signalling in cancer, revealing that Wnt can have both oncogenic and tumour suppressor functions within the same cancer entities. Furthermore, we demonstrate that inhibition and hyper-activation of Wnt signalling are viable therapeutic approaches in their own right, even within the same tumour type. The relevance of the MYCN-β-catenin omics data to neuroblastoma patient outcome was verified both in terms of predicting correlations in expression between genes and by segregating patient outcome. This provides further confidence that our results are applicable to the clinical setting. We revealed not only multiple levels of cross-talk between MYCN and the canonical Wnt signalling pathway but simultaneously provided a predictive gene signature to inform personalised treatment strategies that harness Wnt modulating therapeutics.


The bioflavonoid apigenin has been shown to possess cancer-preventive and anti-cancer activities. In a drug screening, we found that apigenin can inhibit Wnt/β-catenin signaling, a pathway that participates in pivotal biological functions, which dis-regulation results in various human diseases including cancers. However, the underlying mechanism of apigenin in this pathway and its link to anti-cancer activities remain largely unknown. Here we showed that apigenin reduced the amount of total, cytoplasmic, and nuclear β-catenin, leading to the suppression in the β-catenin/TCF-mediated transcriptional activity, the expression of Wnt target genes, and cell proliferation of Wnt-stimulated P19 cells and Wnt-driven colorectal cancer cells. Western blotting and immunofluorescent staining analyses further revealed that apigenin could induce autophagy-mediated down-regulation of β-catenin in treated cells. Treatment with autophagy inhibitors wortmannin and chloroquine compromised this effect, substantiating the involvement of autophagy-lysosomal system on the degradation of β-catenin during Wnt signaling through inhibition of the AKT/mTOR signaling pathway. Our data not only pointed out a route for the inhibition of canonical Wnt signaling through the induction of autophagy-lysosomal degradation of key player β-catenin, but also suggested that apigenin or other treatments which can initiate this degradation event are potentially used for the therapy of Wnt-related diseases including cancers. Plant preparation that contains apigenin as traditional medicines for centuries in Europe is routinely used for the therapy of asthma, insomnia, neuralgia, shingles, Parkinson’s disease, and degeneration-related diseases2. Epidemiological investigation has shown that food stuff rich in flavones is related to a reduced risk of several cancers, especially cancers of skin, breast, prostate, digestive tract, and certain hematological malignancies1. Apigenin has been shown to interfere with the process of carcinogenesis and is regarded as a cancer-chemo-preventive agent. Besides, apigenin can inhibit tumor growth, invasion, and metastasis3.The natural flavone apigenin  is abundant in fruits and vegetables. It is shown to be a bioactive flavonoid that possesses anti-inflammatory, antioxidant and anti-cancer activities5. Dis-regulation in Wnt signaling causes a wide variety of human diseases such as leukemia, tetra-amelia, schizophrenia, kidney damage, bone morbidity, pulmonary fibrosis, and different kinds of cancers6. In the condition of without Wnt, members of the Wnt signaling pathway such as Axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β), casein kinase 1α (CK1α), microtubule actin crosslinking factor 1 (MACF1)7 and beta-catenin (CTNNB1) form a protein complex termed the “β-catenin destruction complex” or “Axin complex” in the cytoplasm. In this complex, β-catenin will be phosphorylated by GSK3β and CK1α on serines 33, 37, 45 and threonine 41 and subsequently be tagged with polyubiquitin before its destruction by the 26S proteasome degradation system. In the presence of Wnt, Wnt binds to its membranous receptor frizzled and co-receptor low-density lipoprotein receptor-related protein 5/6 (LRP5/6). Dishevelled (DVL), another member of Wnt signaling, will be phosphorylated and recruited to the cell membrane by binding to the receptor Frizzled. Thereafter the Axin complex will be translocated from the cytoplasm to the cell membrane with the help of MACF17 and bind to phosphorylated LRP5/6 through Axin, and finally Axin will be degraded. β-catenin will be released, accumulated in the cytoplasm, move into the nucleus, bind to T-cell factor/lymphoid enhancer factor (TCF/LEF), and then activate the expression of Wnt target genes, such as c-Myc, cyclin D1 and Axin28.Wnts are a group of secreted lipoglycoproteins that function as signaling molecules to regulate embryonic development at different stages and participate in adult tissue homeostasis10. Major causes for committing colorectal cancers include eating processed meat and red meat, smoking and drinking, obesity, a history of inflammatory Bowel diseases, and genetic variations that contribute to the inherited CRC, familial adenomatous polyposis (FAP), and hereditary nonpolyposis colorectal cancer (HNPCC)13. Traditional treatments for CRC are surgery, chemotherapy, radiotherapy, and targeting therapies14. Recently, a flurry in the progress of screening and prevention such as the developments in genomic analysis and biomarker, and the advancement in other non-traditional therapies such as immunotherapy and nutritional supplement therapy, has greatly reduced the mortality rates14. However, patients with an advanced and metastatic CRC are still hard to tackle with, suggesting that there is an urgent need to look for novel ways for the therapy of the disease.With more than 1.3 million of people diagnosed each year, colorectal cancer (CRC) was among the most frequent cancers and was also one of the top cause of cancer-related death16, and thus autophagy can maintain cellular homoeostasis. Autophagy can also be induced by certain environmental stresses such as nutrient deficiency, oxygen deprivation, and cytotoxic agents17. There are at least two ways of autophagy-mediated degradation: one is the degradation of specific cellular components and invading micro-organisms, the other is the non-specific bulk degradation of cytoplasm. The detailed mechanisms underlying these specificities largely remain to be determined17. The process is initiated by the sequestration of intracellular candidate components into the small membrane structures called phagophores which then develop into the double–membrane vesicular structures termed autophagosomes, finally fusing with lysosomes to become autolysosomes and initiate the degradation. During the formation of autophagosomes, the mammalian homologues of yeast autophagy related protein Atg8, the microtubule-associated protein LC3, is processed by another autophagy related protein Atg4 to produce the active cytosolic LC3-I, which in turn activated by Atg3 to become the membrane-bound form, LC3-II. LC3-II then binds tightly to preautophagosomal, autophagosomal and autolysosomal membranes with its bound lipid moiety18. On the route to autophagy, LC3 binds to the autophagy adaptor sequestosome 1 (SQSTM1/p62) to facilitate the autophagic flux19. At the end of autophagy, cargos, LC3 and SQSTM1/p62 will eventually be degraded in the autolysosome.Autophagy is a self-eating mechanism to recycle damaged proteins and organelles through the lysosomal degradation systemIn this paper, we found that the bioflavonoid apigenin can down-regulate β-catenin through activation of the autophagy-lysosomal degradation system by inhibiting the AKT/mTOR signaling pathway in Wnt-stimulated cells and Wnt-driven colorectal cancer cells, contributing to the suppression in Wnt signaling and thus the inhibition of cancer cell proliferation. These results suggest the potential application of harmless apigenin for the therapy of colorectal cancer as well as Wnt-related diseases, and also provide a novel therapeutic target for rational drug design.20 and drugs that can block aberrant Wnt signaling would be promising for the therapy of these diseases. In order to look for potential drugs that inhibit Wnt/β-catenin signaling, we have set up a drug screening platform based on the β-catenin/TCF-mediated transcriptional activity assay21 and have successfully picked up several candidates that can inhibit Wnt signaling from natural compounds, modified compounds, and Chinese herbs, and one of them is apigenin. Apigenin is a flavonoid rich in parsley, celery, and chamomile and has been shown to possess cancer-preventive and anti-cancer activities22. To confirm the effects of apigenin on the Wnt/β-catenin signal transduction pathway, we treated Wnt-reporter-transfected P19 cells in control-conditioned medium, Wnt-3a-conditioned medium, or with apigenin in Wnt-3a-conditioned medium, and analyzed the Wnt-induced β-catenin/TCF-mediated luciferase activity. P19 cells are pluripotent embryonic carcinoma cells with stem cell properties that can be differentiated into three germ layers upon stimulation23. Our previous data have shown that P19 cells have good Wnt response and are suitable for the study of Wnt signaling21. As shown in Fig. 24, we explored the effects of apigenin on this activity. As shown in Fig. Wnt signaling is dis-regulated in many human diseasescyclin D1, c-Myc, T (brachyury), and Axin2 (conductin). RT-PCR analyses showed that Wnt-3a treatment induced the mRNA expression of Wnt target genes including T, Axin2, and cyclin D1 in Wnt-stimulated P19 cells. Treatment with apigenin decreased the expression of these genes  or β-catenin genesade Figs , we want27. Ectopic expression of LRP5ΔN indeed induced the β-catenin/TCF-mediated luciferase activity and treatment with apigenin decreased this activity . To address this, P19 cells were transfected with dual reporters, the 3X-Flag-tagged DVL228 and treated with different concentrations (0~60 μM) of apigenin. Cell lysates were then obtained for dual luciferase activity assay. As shown in Fig. Since apigenin inhibited Wnt/β-catenin signaling and cell proliferation, we began to look for the cellular targets of apigenin in the Wnt/β-catenin signaling pathway. We first checked whether apigenin acts upstream or downstream of LRP5/6. To this end, P19 cells were transfected with a Wnt reporter, a normalization vector, the constitutive active mutant of LRP5, pLRP5ΔN, and treated with 50 μM of apigenin for dual reporter activity assay. Over-expression of LRP5ΔN will cause the constitutive activation of Wnt signaling downstream of LRP5ity Fig. , suggest29. As compared to cells without any treatment, treatment with LiCl induced a strong β-catenin/TCF-mediated luciferase activity. Treatment with apigenin substantially suppressed the LiCl-triggered β-catenin/TCF-mediated transcriptional activity, with more than 75% of inhibition at concentrations higher than 10 μM , p70 S6 kinase, phosphorylated p70 S6 kinase (Thr389), 4E-BP-1, and phosphorylated 4E-BP-1 (Ser65). As shown in Fig. Previous data have shown that autophagy-mediated lysosomal degradation system is involved in the apigenin-elicited down-regulation of β-catenin during Wnt signaling. It is known that the AKT/mammalian target of rapamycin (mTOR) signaling pathway regulates autophagyase Thr38, 4E-BP-1ase Thr38, 4E-BP-120. For example, mutation in APC gene that defects in β-catenin degradation can ultimately result in the formation of colorectal cancers38. Thus, any strategies that can down-regulate the deregulated β-catenin will contribute to the therapy of the diseases. The natural flavonoid apigenin has been shown to disturb the process of carcinogenesis, inhibit tumor cell growth, make cancer cells sensitive to apoptosis, and hinder the development of metastasis22. Moreover its low toxicity to normal cells and strong activities to cancerous cells39 make apigenin appear to be an ideal agent for chemoprevention and cancer therapy. We are therefore interested to know whether the effect of apigenin on tumorigenesis, especially in colorectum, is through the inhibition of canonical Wnt signaling. In this paper, we discovered that the phytochemical apigenin is able to suppress Wnt/β-catenin signaling  the mobilization of β-catenin into the nucleus; (2) the association of β-catenin with the transcription factor TCF/LEF, (3) the association of β-catenin with co-activators such as CBP; (4) the binding of β-catenin transcription activation complex onto the cognate promoter region, or (5) the events after complex binding onto the target DNA. Drugs that target on these processes have been identified. For example, a small molecule drug ICG-001 acts on the binding of β-catenin to co-activator CBP (cyclic AMP response element-binding protein)52. Resveratrol can disrupt the association of β-catenin with TCF21. Here we have shown that the harmless apigenin is able to drive the accumulated wild type  and dissolved in dimethyl sulfoxide (DMSO) at the concentration of 50 mM. Chloroquine and wortmannin was obtained from Sigma-Aldrich and dissolved in sterile water and DMSO respectively. 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Sigma-Aldrich and prepared in phosphate-buffered saline. Paraformaldehyde was purchased from Electron Microscopy Sciences . 4′,6-diamidino-2-phenylindole (DAPI) was from Invitrogen . Dual-Luciferase Reporter Assay System was from Promega . Complete™ Protease Inhibitor Cocktail Tablet, EDTA-free, and PhosSTOP Phosphatase Inhibitor Cocktail Tablets were from Roche . Triton X-100 was from Sigma-Aldrich. SuperScript III First-Strand Synthesis SuperMix and Platinum PCR SuperMix were from Invitrogen. Primary antibodies for β-tubulin , GAPDH, brachyury (T), Axin2 (Conductin), c-Myc (9E10), lamin A, SQSTM1/p62 , β-catenin , LC3B, cyclin D1, 4E-BP1 (53H11), phospho-4E-BP1 (Ser65), p70 S6 kinase, phospho-p70 S6 kinase (Thr389), pan Akt (C67E7), and phospho-Akt (Ser473) (D9E)  were obtained from the indicated vendors. The human colorectal cancer cell lines HCT-116 (BCRC 60349), SW480 (BCRC 60249), WiDr (BCRC 60157) cells, the mouse embryonal carcinoma P19 cells (BCRC 60052) and the African green monkey fibroblast COS-7 cells (BCRC 60094) were obtained from Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan. 3X-Flag-tagged human DVL2 cDNA was from Dr. Jeff Wrana (Addgene plasmid number: 24802). All other chemicals were from either Merck or Sigma unless specified. pLRP5ΔN was constructed by inserting the PCR-amplified cDNA fragment containing LRP5 transmembrane and C-terminal region into the pcDNA3-HA(3) plasmid, a pcDNA-based vector with an engineered HA tag, and the insert was completely sequenced to confirm its identity.2. The culture of L cells and L-Wnt-3a cells and preparation of control and Wnt-3a conditioned media were as described previously21. Cells were transfected with Lipofectamine 2000 (Life Technologies) according to the company’s brochure.HCT-116, SW480, WiDr, and COS-7 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% of fetal bovine serum , 1% of penicillin and streptomycin (Gibco), and sodium pyruvate. P19 cells were cultured in Minimum Essential Medium (MEM) alpha with 2.5% of fetal bovine serum (Hyclone), 7.5% of bovine calf serum (Hyclone), 1% of penicillin and streptomycin, and sodium pyruvate. All cells were incubated in a 37 °C humidified chamber with 5% of CO21. Cells were seeded at the density of 2 × 103 cells per well in a 96-well dish and kept in the culture chamber for overnight to allow the cells to attach. Cells were treated with different concentrations of apigenin in the culture medium or Wnt-conditioned medium (for P19 cells) and cultured for additional four days. Cultured medium was then removed and 50 μL of MTT solution (2 mg/mL) was added into the cells carefully and the dish was placed in the culture chamber for additional 4 h. At the end of incubation, the dish was spun at 2,500 rpm for 20 min at 4 °C. MTT solution was carefully removed and 100 μL of DMSO was pipetted into each well to dissolve the developed formazan crystals. After 20 min of incubation, the dish was read at 570 nm in an ELISA reader  and the data was further processed by Excel to calculate the relative cell viability.We used MTT assay to measure the cell viabilityCells were treated as described in the text and fractionation of cells into nuclear and cytosolic fractions was performed by using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to the procedures provided by the company.Cells were seeded on coverslips, treated with drugs, and fixed with 4% of paraformaldehyde (PFA) for 15 min at room temperature. After three washes in PBS for 10 min, cells were permeabolized with 0.1% of Triton X-100 in PBS and washed with PBS for three times. For the detection of autophagosomes, cells were further permeabolized with cold methanol at −20 °C for 10 min after PFA fixation but omit the Triton X-100 step. Cells were then blocked in 10% of normal goat serum  in PBS for 30 min at ambient temperature before the incubation with primary antibodies in 1.0% of NGS in PBS at 4 °C for overnight. On the next day, cells were warmed up for 10 min at room temperature and washed three times with PBS for 10 min each time. Cells were then incubated with secondary antibody Alexa Flor 488 goat anti-mouse IgG, Alexa Flor 488 goat anti-rabbit IgG, or Alexa Flor 546 goat anti-mouse IgG  for 1 h at room temperature. To prevent the quench from light, cells were always kept in the dark after the step with secondary antibodies. After incubation with DAPI  for 10 min, cells were washed three more times with PBS, mounted in 50% of glycerol in PBS, and sealed upside-down onto the slide with nail polish covered around the coverslip. Images were recorded by using a Zeiss AXIO Observer. D1 Fluorescence microscope equipped with an imaging system, AxioCam MRm .5/well) were seeded onto a 24-well dish and incubated for overnight. For HCT-116 and SW480 cells, cells were transfected with the Wnt reporter pGL3-OT and the normalization vector pTK-Renilla  and treated with different concentrations of apigenin before the dual luciferase activity assay. To test the effects of apigenin on upstream or downstream of DVL and LRP5, P19 cells were transfected with dual reporters, the effector plasmid (DVL2 and pLRP5ΔN) or empty vector, and treated with different concentrations of apigenin for the indicated time. To test the effects of apigenin on Wnt signaling, P19 cells or COS-7 cells were transfected with dual reporters, and either treated with different concentrations of apigenin in Wnt-3a-conditioned medium or incubated in control-conditioned medium. For LiCl treatment, Wnt-reporter-transfected P19 cells were incubated in culture medium, treated with LiCl in culture medium, or treated with LiCl and different concentrations of apigenin in culture medium before the dual luciferase activity assay. Each treatment was in three replicates. Cells were then washed once with PBS and lysed in 1X passive lysis buffer. Cell lysates were collected and spun at 12,000 rpm for 1 min. Supernatant was transferred into a new eppendorf tube and kept at −80 °C or proceeded to the measurement of dual luciferase activities using the Dual Luciferase Reporter Assay System on a GLOMAX 20/20 luminometer . Firefly luciferase activity was normalized by Renilla luciferase activity7.P19, COS-7, HCT-116 or SW480 cells (1 × 10GAPDH gene by using the PCR SuperMix (Invitrogen). Gene-specific primers used were: mouse Axin2 , forward, 5′-GTTAGTGACTCTCCTTCCAGATCC-3′, reverse, 5′-GAGTGTAAAGACTTGGTCCACCTG-3′, human Axin2 , forward, 5′-GTTGGTGACTTGCCTCCCGGACCC-3′, reverse, 5′-GAGTGTAAGGACTTGGTCCACCGG-3′, human and mouse GAPDH , forward, 5′-CGTATTGGGCGCCTGGTCACC-3′, reverse, 5′-GAGGGGCCATCCACAGTCTTC-3′, human c-Myc , forward, 5′-CCAGGACTGTATGTGGAGCGG-3′, reverse, 5′-CTTGAGGACCAGTGGGCTGTG-3′, mouse T , forward, 5′-GTGACCAAGAACGGCAGGAGGATG-3′, reverse, 5′-AAGCAGTGGCTGGTGATCATGCG-3′, and human and mouse cyclin D1 , forward, 5′-ATGGAACACCAGCTCCTGTGCTGCG-3′, reverse, 5′-TCCAGGTAGTTCATGGCCAGCGGG-3′.P19 cells or HCT-116 cells were treated with different concentration of apigenin or carrier reagent (DMSO) in culture medium (HCT-116 cells) or in Wnt-3a-conditioned media (P19 cells) for the indicated time. Total RNAs were isolated from treated cells by using the Trizol reagent (Invitrogen) according to the company’s protocol. 5 μg of RNAs were used for the synthesis of cDNAs by using a cDNA synthesis kit (Invitrogen) and the resulting cDNAs were used as the template for the amplification of Wnt target genes or Treated cells were collected, resuspended in protein lysis buffer  supplemented with the protease inhibitor and phosphatase inhibitor (Roche), and incubated for 10 min at 4 °C with gentle shaking. After centrifugation at 12,000 rpm for 15 min at 4 °C, clear cell lysate was transferred to a new tube. Protein concentration was determined by the Bradford method using Protein Assay Dye Reagent . Equal amount of cell lysate was resolved on a SDS-PAGE gel and transferred onto PVDF membrane . The transblot was incubated in the blocking solution  for 1 h at room temperature. After a 10-minute incubation in TBS containing 1% skim milk, the blot was then incubated in primary antibody for 2 h at room temperature or overnight at 4 °C on a rotatory shaker. The blot was washed three times in TBST (0.1% [v/v] Tween-20 in TBS) for 10 min each time, and soaked in HRP-coupled goat anti-mouse or rabbit secondary antibodies for 1 h at room temperature. After three additional washes in TBST and one wash in TBS, the blot was layered with ECL reagent  and exposed to X-ray film  to reveal the signals.Supplementary Information


The WNT-beta-catenin pathway is known to regulate cellular homeostasis during development and tissue regeneration. Activation of WNT signaling increases the stability of cytoplasmic beta-catenin and enhances its nuclear translocation. Nuclear beta-catenin function is regulated by transcriptional co-factors such as CREB binding protein (CBP) and p300. Hyper-activated WNT-beta-catenin signaling is associated with many cancers. However, its role in inducing stemness to liver cancer cells, its autoregulation and how it regulates tumor suppressor pathways are not well understood. Here we have investigated the role of CBP-beta-catenin signaling on the expression of CD133, a known stem cell antigen and PP2A-PTEN pathway in tumor initiating liver cancer cells.. Western blotting and real time PCR (qPCR) were used to quantify protein expression/phosphorylation and mRNA levels, respectively. CBP and CD133 gene silencing was performed by siRNA transfection. Fluorescence Activated Cell Sorting (FACS) was performed to quantify CD133 positive cells. Protein Phosphatase (PP2A) activity was measured after PP2AC immunoprecipitation.Human hepatoblastoma cell line HepG2 and clonally expanded CD133 expressing tumor initiating liver cells (TICs) from premalignant murine liver were used in this study. CBP-beta-catenin inhibitor ICG001 was used to target CBP-beta catenin signaling in liver cancer cells in vitroCBP silencing significantly reduced CD133 expression and anchorage independent growth in HepG2 and murine TICs. CD133 silencing in TICs decreased cell proliferation and expression levels of cell cycle regulatory genes, CyclinD1 and CyclinA2. ICG001 treatment and CBP silencing reduced the levels of phosphoSer380/Tyr382/383PTEN, phosphoSer473-AKT, Phospho-Ser552beta-catenin in TICs. ICG001 mediated de-phosphorylation of PTEN in TICs was PP2A dependent and partly prevented by co-treatment with PP2A inhibitor okadaic acid.CBP inhibitor ICG001 and CBP-beta-catenin signaling promotes stemness via CD133 induction and cell proliferation in TICs. We found a novel functional link between CBP-beta-catenin and PP2A-PTEN-AKT pathway in liver TICs. Therefore, CBP-beta-catenin-PP2A-PTEN-AKT signaling axis could be a novel therapeutic target to prevent liver tumor initiation and cancer recurrence.The online version of this article (10.1186/s12964-018-0222-5) contains supplementary material, which is available to authorized users. WNT-beta-catenin signaling is one of the most conserved signaling pathways that is critical for the development and regeneration of various organs. It is highly deregulated in many cancers. Beta-catenin is the central component of the canonical WNT pathway. Secreted WNT ligands interact with Frizzled/LRP (low density lipoprotein receptor-related protein) receptors to activate intracellular WNT signaling , 2. CytoRecent studies have shown that cancer recurrence is associated with the presence of tumor initiating cells (TICs) , 5. TICsICG001 is a small molecule inhibitor that disrupts CBP-beta-catenin signaling . ICG001 All general buffers and chemicals were purchased from Sigma-Aldrich and were of molecular biology grade.− 7 M dexamethasone (Sigma-Aldrich), and 10 mM nicotinamide (Sigma-Aldrich) in a cell culture incubator maintained at 37 °C and 5% CO2. Human hepatocellular carcinoma cell line HepG2 and cholangiocarcinoma cell line MzCha-1  were grown as described previously [Clonally expanded murine CD133 positive (TICs) cells (kindly provided by Dr. Bart Rountree) originally described by Rountree et al.  was culteviously . Cells weviously , 21, 23.FACS analysis was performed as described previously . HepG2 cCBP, CD133 and control scrambled siRNA were purchased from Thermo Scientific . The siRNA was reverse transfected into 1 × 105 cells at a dose of 20 nM in 6-well plates using the Lipofectamine RNAiMAX™ transfection reagent  for 48 h as described previously [Pre-validated Silencer® select siRNAs against eviously .3 per well) were grown in 0.7% top soft agar prepared on a 0.5% base soft agar layer in a 6-well plate for two weeks in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum with and without ICG001. Colonies formed at the end of two weeks were stained with 0.005% crystal violet for 30 min and washed thoroughly with water, and images were acquired using an Evos Advanced transmitted light microscope coupled with Evos xl software . Number of colonies was counted manually from five different images captured from six independent experiments.Anchorage-independent growth assay was performed as described . BrieflyImmunocytochemistry was performed as described previously . BrieflyTotal protein lysates were prepared from cells using Radio Immuno Precipitation Assay (RIPA) buffer , Phosphatase inhibitor cocktail (Thermo Scientific) and protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were measured by Bradford’s protein assay kit (Bio-Rad Laboratories) using bovine serum albumin as standard. Equal amounts of protein samples were separated on a 10% SDS-PAGE at 100 V and transferred onto nitrocellulose membrane (Bio-Rad). After blocking with 5% BSA or BLOTTO (Santa Cruz Biotechnology) prepared in Tris-buffered saline, Tween, 0.1%, (TBST), membranes were incubated with respective primary antibody diluted in blocking buffer for 16 h at 4 °C. Membranes were then washed in TBST and incubated with horseradish peroxidase-conjugated secondary antibody. Primary antibodies used are listed in Additional file t. Gapdh was used to normalize the gene expression.DNA-free RNA was isolated using a column-based purification method according to the manufacturer’s protocol . One microgram of total RNA was reverse-transcribed using 100 units of NxGen® M-MuLV Reverse Transcriptase according to the manufacturer’s protocol . Quantitative real-time PCR (qPCR) was performed using gene specific Taq-Man probes . Samples were spun at 10,000×g and clear supernatant was used for immunoprecipitation. Each sample  was subjected to immunoprecipitation with PP2Ac antibody for 16 h. Immuno-beads with PP2A were added to a phosphatase reaction mix containing threonine phospho-peptide in a shaking incubator for 15 min. Samples were then spun down and supernatant was transferred into a 96-well plate, into which malachite green detection solution was added. Plates were incubated for 15 min at room temperature and then read the absorbance at 650 nm in an automated plate reader .PP2A activity was measured using PP2A Immunoprecipitation-Phosphatase Assay Kit from EMD Millipore  according to manufacturer’s protocol. Briefly, 1 × 10t tests. Statistical significance was defined by P < 0.05.All data shown represent the mean ± SEM. The statistical significance of differences between two groups was analyzed with two-sided unpaired Student’s CD133 mRNA levels compared to Mat1a−/− murine liver tumor cells  as determined by BrdU incorporation assay   Fig. . CBP sil SC Fig. . Westernels Fig. . CBP silnin Fig. . In summProtein phosphorylation in vivo is a dynamic and highly regulated process. This is mainly achieved by the balanced activities of protein phosphatases and kinases. We hypothesized that PTEN de-phosphorylation in response to ICG001 treatment might be mediated via PP2A. PP2A is one of the serine/threonine phosphatases involved in the regulation of many cellular processes and is an important regulator of canonical WNT signaling. Its activity is inhibited in many cancers and hence is believed to be a tumor suppressor , 32. To Better understanding of the signaling pathways that induce stemness and promote the survival of tumor initiating cells is critical for preventing cancer recurrence and drug resistance. The presence of cancer stem cell or tumor initiating cell phenotype has been identified across multiple human cancers including lung, gastric, breast, pancreas and liver –39. TumoCD133 expression is regulated by CBP-beta-catenin signaling in murine TICs and HCC cells. In TICs, CD133 expression was positively correlated with cell proliferation and expression of multiple cell cycle regulatory genes. A recent study showed that CD133 interacts with PI3K subunit p85 and activates AKT pathway in glioma cancer stem cells and enhance cell proliferation [Using the specific CBP-beta catenin inhibitor ICG001 and in vitro gene silencing methods, we show that feration . Upregulferation . CD133 eferation , 49. TheCBP silenced samples. The difference in the fold activation level of PTEN in the presence of ICG001 and CBP silencing could  be due to decreased total PTEN protein levels and potentially a time dependent effect.Our previous study has demonstrated a cross talk between AKT and CBP-beta-catenin signaling in TICs that favors cell proliferation and survival . Here wePP2A is an important regulator of canonical WNT signaling . Its expTargeting a specific signaling pathway that induces stemness to tumor initiating cells is critical for developing novel therapies to prevent cancer initiation and recurrence. Our study demonstrates a regulatory role for CBP-beta catenin signaling in inducing stem cell phenotype and highlights a novel mechanistic link between CBP-beta-catenin and the tumor suppressor proteins PP2A-PTEN in liver TICs. A deeper understanding of cancer stem cell biology will facilitate in elucidating the mechanisms of tumor cell origin and resistance to targeted therapy.Additional file 1:Table S1. List of primary antibodies. (ZIP 125 kb)Additional file 2:Table S2. List of qPCR probes. (ZIP 50 kb)Additional file 3:Figure S1. Effect of ICG001 on activation of ERK1/2. Western blot analysis for phospho-ERK and total ERK in DMSO and ICG001 treated samples. Protein levels were normalized to beta-actin and ratio of pERK1/2 to total ERK1/2 was calculated to determine the level of activation. Results represent four independent experiments. Densitometry quantification of protein intensity was performed by Image J Software, NIH. (ZIP 319 kb)Additional file 4:Figure S2. PP2A is induced by ICG001 in TICs. Western blot analysis shows the effect of ICG001 and OA alone or in combination on PTEN phosphorylation. Densitometry quantification of protein intensity was performed by Image J Software, NIH. N = 3, *p < 0.05. (ZIP 366 kb)Additional file 5:Figure S3. ICG001 mediated PP2A activation is independent of posttranslational modification of PP2Ac. Western blot analysis for phospho-PP2Ac and methyl PP2Ac in DMSO and ICG001 treated samples. Results represent four independent experiments. (ZIP 524 kb)


Here we examined mRNA and protein expression of components of the Wnt signaling throughout the differentiation of islet β-cells from adipose-derived stem cells (ADSCs). After induction, ADSCs expressed markers of β-cells, including the insulin, PDX1, and glucagon genes, and the PDX1, CK19, nestin, insulin, and C-peptide proteins, indicating their successful differentiation. Compared with pancreatic adult stem cells (PASCs), the quantities of insulin, GLUT2, and Irs2 mRNA decreased, whereas Gcg, Gck, and Irs1 mRNA increased. Over time, during differentiation, insulin mRNA and protein expression increased, Gcg and Gck mRNA expression increased, Irs1 mRNA expression decreased and then increased, and Irs2 mRNA increased and then decreased . The expression of Dvl-2, LRP5, and GSK3β mRNA as well as the Dvl-2, GSK3β, and p-GSK3β proteins also increased (P < 0.05). Expression of TCF7L2 (6–10 d) and β-catenin mRNA as well as the β-catenin protein increased but not significantly (P > 0.05). Our results indicate that the Wnt signaling is activated during ADSC differentiation into islet β-cells, but there was no obvious enrichment of nonphosphorylated β-catenin protein. The Wnt signaling is critical for pancreatic development and islet function; however, its precise effects on the development and function of the  Furthermore, after the Wnt pathway, activator (Wnt-3a) or β-catenin protein was added to β-cells or pancreatic islets in culture, expression of PITX2  and Cyclin D2 (an important regulator of the β-cell cycle) was promoted, β-cells were proliferated, and serum insulin increased in vitro [β-cell clusters decreased, and glucose tolerance was damaged [β-catenin signaling pathway) gene increased the apoptosis of pancreatic β-cells, reduced β-cell proliferation, and, thereby, reduced insulin secretion [ TCF7L2 allele further indicate that the susceptibility and pathogenesis of diabetes mellitus may be caused by disturbance of the Wnt signaling pathway [β-catenin/TCF7L2 in pancreatic development, islet function, and generation and secretion of insulin have been determined, the effects of the Wnt/β-catenin signaling pathway on the development and function of the endocrine cells of the pancreatic islet (including the β-cells generating insulin) remain controversial [The Wnt/of cells  and is cof cells . β-Catenet genes , 12) haset genes . For exain vitro . However damaged . Moreoveecretion . Indeed, pathway . While toversial .β-catenin signaling pathway components and related factors at a transcriptional and translational level over time. The aim of our study was to confirm the role of the Wnt/β-catenin signaling pathway in the development and function of pancreatic β-cells generating insulin.Therefore, in this study, we induced the differentiation of ADSCs into insulin-producing cells and monitored the changes in the Wnt/β (ab93926) antibodies were from Abcam . Phospho-GSK3β (Ser9) (D3A4) (9322S), non-phospho- (active) β-catenin (Ser33/37/Thr41) (D13A1) (8814S), and Dvl-2 (30D2) (3224P) antibodies were from Cell Signaling Technology . CD13 (BA0718), CD44 (BA0321), CD106 (BA0406), and CD49d (BA1640) antibodies were from Boster Biological . Pancreatic and duodenal homeobox-1  antibody was from Bioss . Cytokeratin 19  and nestin (BA1289) antibodies were from Boster Biological .Low glucose Dulbecco's modified Eagle's medium (DMEM) (10567-014) and DMEM containing 4.5 g/L glucose (11965-092) were purchased from Gibco-BRL . Dithizone (D5130) was from Sigma . DMSO (042-21765) was from Wako . Recombinant human DKK-1 (5439-DK-010) and recombinant human Wnt-3a (5036-WN-010) was from R&D Inc. . Insulin antibody (sc-9168) was from Santa Cruz Biotechnology . C-peptide (ab14181) and GSK3Wistar rats (aged 5-6 weeks) were purchased from the National Research Center for Animal Transgenic Biotechnology, Inner Mongolia University. All studies adhered to procedures consistent with the National Research Council Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Inner Mongolia University. All rat treatments were performed with efforts to minimize suffering and rats were killed by cervical dislocation.The epididymal fat from male Wistar rats aged 5-6 weeks was obtained under aseptic conditions. ADSCs were then isolated and cultured according to our previous method . Surfaceβ-cells, and PDX1 is essential for maintaining the normal function of pancreatic islets.Pancreatic tissue was harvested from Wistar rats aged 5-6 weeks under aseptic conditions. PASCs were then isolated and cultured as described previously . Cell suβ-cell differentiation was then performed using 4.5 g/L D-glucose DMEM culture medium containing 10% FBS for 7 days. The process of induction was conducted at 37°C in 5% CO2 and saturated humidity. After induction, cells were dyed for 30 min at 37°C with the addition of 10 mg/mL dithizone stock solution to the culture medium at a dilution of 1 : 100. After discarding the culture medium, cells were washed three times with PBS and observed under the microscope. Then, cell surface markers of islet β-cells were detected immunohistochemically using the following four antibodies: PDX1, CK19, nestin, and insulin. Reverse-transcription (RT) PCR identification of islet β-cells was conducted using a commercial kit  and all PCR primers . Specific primer pairs (cross-intron) were designed based on the mRNA sequences of Rattus norvegicus (provided by NCBI) for insulin, glucagon (Gcg), glucokinase (Gck), glucose transporter type 2 (GLUT2), insulin receptor substrate 1 (Irs1), and insulin receptor substrate 2 (Irs2) and synthesized by TaKaRa  using the following conditions: 95°C for 30 s and 50 cycles of 95°C for 5 s and 60°C for 34 s.The mRNA levels of genes relating to insulin secretion, as well as islet y TaKaRa . The relμg total) were added to each well of a SDS-polyacrylamide gel and separated by electrophoresis. Insulin and C-peptide antibodies were diluted at 1 : 500 and 1 : 100, respectively. Antibody binding was visualized using the ECL chemiluminescence reaction and the Tanon 5200 instrument. The difference in insulin and C-peptide protein expression was determined using ImageJ software.Protein concentrations were measured using the BCA method after extracting the total protein from the insulin-producing cells induced from ADSCs. The total protein from PASCs was used as a reference. Proteins  or DKK-1  to the culture medium  and cultivated the cells for 3, 6, and 10 days to monitor the changes in the Wnt signaling pathway over time. The mRNA levels of genes relating to Wnt signaling were analyzed by qPCR. Specific primer pairs (cross-intron) were designed based on the mRNA sequences of R. norvegicus (provided by NCBI) for dishevelled 2 (Dvl-2), low-density lipoprotein receptor-related protein 5 (LRP5), glycogen synthase kinase 3 beta (GSK3β), β-catenin, and TCF7L2 and synthesized by TaKaRa , non-phospho- (active) β-catenin (Ser33/37/Thr41) (non-p-β-cat) (1 : 1000), GSK3β (1 : 1000), phospho-GSK3β (Ser9)  (1 : 1000), and insulin (1 : 500). Insulin levels in insulin-producing cells induced from ADSCs at different time points were also detected by immunostaining (insulin antibody diluted at 1 : 300). Stained cells were observed by confocal microscopy .Total protein was isolated from the insulin-producing cells induced from ADSCs over time. The samples were labeled L3, H3, and H7 in the islet P < 0.05 indicating statistical significance.All data are the result of at least three independent experiments and are indicated by the mean ± standard deviation. Data were analyzed using SPSS 17.0 software . The least significant difference (LSD) method was applied for comparison among groups, with Parts of the cells started to adhere to the wall when the ADSCs were subjected to inoculation for 4–6 h. They had a small circle or short stick-like shape and were unequal in size. ADSCs gradually expanded into spindles or irregular polygons after 48 h. They presented as fibroblast-like after 3 days and reached 80%–90% confluence after 6-7 days. After subculturing, ADSCs presented as gyrate or parallel spindle-shaped adherent cells, which grew densely Figures .After digestion with collagenase V, some cell aggregates and single scattered cells were present in the cell suspension. After being cultivated for 60 h, the single cells adhered to the wall and were short and spindle-shaped in appearance. After replacing the DMEM no glucose culture medium containing 0.5% of FBS 2 weeks later for screening and purification, the majority of the cells were dead. Residual cells became proliferative after 4 days in the culture medium containing basic FGF (bFGF), and subculturing was required every 5-6 days. Subcultured PASCs were dense and fusiform or cobblestone-like in appearance Figures .No obvious changes were observed when ADSCs were subjected to induction for 3 days in DMEM low glucose culture medium containing 1% DMSO and still presented as spindles or polygons, with clear cell nuclei. However, the cells started to aggregate when ADSCs were subjected to induction for 3 days in 4.5 g/L D-glucose DMEM culture medium containing 10% FBS. The cells grew densely after being induced for 5 days, with circular clusters and significantly increased refractivity. After 7 days, the majority of ADSCs formed clusters Figures .The surface markers of CD13, CD44, and CD49d in the ADSCs were positive. CD13 showed weak expression, CD49d showed strong expression, and CD106 showed little expression Figures . These rAfter induction of rat ADSCs into insulin-producing cells, the majority of the cell clusters were brown/red after dyeing with dithizone, indicating that zinc ions were rich in the cytoplasm, proving the presence of insulin-producing cells (IPCs) . The maj GAPDH, insulin, PDX1, and glucagon genes were detected in the insulin-producing cells induced from ADSCs , 2.12 times (P < 0.01), 1.15 times (P > 0.05), 0.58 times (P < 0.01), 1.38 times (P < 0.01), and 0.83 times (P < 0.01) those in PASCs, respectively  during the induction of insulin-producing cells from ADSCs. In addition, Gcg and Gck mRNA expression increased (P < 0.05), while Irs1 mRNA expression decreased and then increased (P < 0.01), and Irs2 mRNA expression increased and then decreased (P < 0.01). Dvl-2, LRP5, and GSK3β mRNA expression also increased (P < 0.05) during the induction of insulin-producing cells from ADSCs over time. While the expression of β-catenin mRNA increased, the change was not statistically significant (P > 0.05). Similarly, the expression of TCF7L2 mRNA increased from day 6 to day 10 but not significantly (P > 0.05). Correspondingly, Dvl-2, GSK3β, and p-GSK3β protein expressions significantly increased (P < 0.01), while non-p-β-catenin protein expression did not (P > 0.05). Therefore, although the Wnt signaling pathway was activated, enrichment of the nonphosphorylated β-catenin protein was not obvious during the differentiation of ADSCs into the insulin-producing cells by DMSO and a high content of glucose.Insulin mRNA and protein expression increased , and TCF7L2 mRNA expression increased on day 3. Correspondingly, insulin mRNA and protein expression increased gradually after adding Wnt-3a; Gcg and Gck mRNA expression increased during this period; and GLUT2 mRNA expression increased obviously on day 10. When the inhibitor of the Wnt signaling pathway (DKK-1) was added, Dvl-2 mRNA and protein expression decreased, LRP5 mRNA expression decreased on day 6, GSK3β mRNA and protein expression decreased on day 10, p-GSK3β protein expression decreased on day 10, non-p-β-catenin protein expression decreased on day 10, and TCF7L2 mRNA expression decreased on day 6. Correspondingly, insulin protein expression decreased gradually after adding DKK-1  was added, Dvl-2 mRNA and protein expression increased transitorily on day 3, Figures .Consistent with the Western blot analysis, insulin protein expression increased over time when examined immunohistochemically Figures . Insulinβ-cells. We confirmed that ADSCs could be induced to differentiate into insulin-producing cells using DMSO and a high glucose condition [β-cells. Moreover, our results with the Wnt activator and inhibitor indicate that the Wnt signaling pathway may be important for this differentiation process. Therefore, our study provides evidence for the role of the Wnt/β-catenin signaling pathway in the differentiation of pancreatic β-cells generating insulin from ADSCs.In this study, we examined changes in the mRNA and protein expression of components of the Wnt signaling pathway when ADSCs were inducted to differentiate into islet ondition . The expβ-cells. PDX1 has previously been shown to be the crucial transcriptional factor involved in the differentiation and development of pancreatic islet cells [ PDX1 gene show increased secretion of glucose-stimulated insulin, indicating that hADSCs were induced to differentiate into IPCs by the expression of exogenous PDX1 [β-cell replacement therapy in T1DM.The expression of PDX1 protein observed in this study provides further evidence for its role in the differentiation of pancreatic et cells . Indeed,ous PDX1 . Moreoveous PDX1 . Therefoβ-cells , and found a dialogue between the FOXA2 and TCF7L2 transcription factors [The induction method in the current study was relatively economical, simple, and quick; however, different in vitro induction methods have been used previously for the differentiation of mesenchymal stem cells (MSCs) into islet cells , 29. A p factors  and thenβ-catenin is known to play a role in the development and function of insulin producing β-cells, its precise function remains controversial [β-catenin from being degraded by complexes composed of Axin, GSK3, APC, and CK1, among others. Without Wnt ligand stimulation, β-catenin is phosphorylated and degraded by the proteasome. In the current study, we found that the expression of Dvl-2, GSK3β, and p-GSK3β proteins increased during differentiation of ADSCs into insulin-producing cells over time, indicating that the Wnt signaling pathway was activated. However, we found no obvious enrichment of the nonphosphorylated β-catenin protein. Indeed, Baumgartner et al. [β-catenin on the development of β-cells is controversial. The number of β-cell clusters decreased as a result of specific deletion of β-catenin in the pancreas, which correlated with an early and specific loss of multipotent pancreatic progenitor cells [β-catenin gene was deleted, the pancreas became hypoplastic, but the function of the islet endocrine cell mass was not significantly perturbed. Furthermore, Keefe et al. [β-catenin is continuously required for the establishment and maintenance of the pancreatic acinar cell mass, but this requirement is not shared with islet cells, which can proliferate and function normally in the absence of β-catenin. Therefore, loss of Wnt/β-catenin activity is unlikely to drive islet dysfunction. In this study, we found that β-catenin mRNA expression increased over time when the Wnt-3a activator of the Wnt signaling pathway was added; however, the β-catenin protein expression did not change obviously. Despite this, the expression quantities of insulin mRNA and protein increased gradually when Wnt-3a was added. Therefore, our study provides further evidence that the Wnt signaling pathway is activated during differentiation of ADSCs into insulin-producing cells and promotes the expression of insulin over time, as summarized in While the classical Wnt/oversial . In thisr et al.  recentlyor cells . Murtaugor cells  also shoe et al.  proved tβ-catenin signaling pathway have focused on osteoblast differentiation of stem cells, and the results remain disputed. D'Alimonte et al. [β-catenin signaling pathway might trigger osteoblast differentiation of human amniotic fluid MSCs, and the expressions of Dvl-2, nonphosphorylated β-catenin, and p-GSK3βSerine9 increased in the early stage of osteoblast differentiation. However, Cho et al. [β-catenin and promote osteoblast differentiation of ADSCs. Furthermore, treatment with Wnt-3a conditioned media increased cellular β-catenin levels and the rate of cellular proliferation but inhibited osteogenic differentiation [β-catenin signaling pathway should be further studied with respect to stem cell differentiation, as different differentiation effects may occur in different types of stem cells.To date, studies of the Wnt/e et al.  showed to et al.  reportedntiation . Therefoβ-cell autoantigen IA-2/phogrin increased in the differentiation stage. In addition, developmentally important pathways including notch/jagged, Wnt/frizzled, TGFβ superfamily  and retinoic acid  were differentially regulated during the expansion/differentiation stage [β-cells in vitro for the use in treatment of diseases such as T1DM and T2DM.In conclusion, this study confirmed the positive effect of the Wnt signaling pathway on the differentiation of insulin-producing cells from ADSCs. After analyzing the gene expression profiles at three different stages  during in vitro islet generation, Dodge et al.  showed ton stage . Therefo