Oleanolic acid inhibits colon cancer cell stemness and reverses chemoresistance by suppressing JAK2/STAT3 signaling pathway

Background:Oleanolic acid(OA),a pentacyclic triterpenoid exhibiting specific anti-cancer properties and highly effective antioxidant activity,was isolated from traditional Chinese medicinal herbs.Conversely,the OA that impacts colon cancer(CC)cells and its underlying mechanisms remain poorly understood.Methods:The cytotoxic effect of OA alone or OA-5-Fluorouracil(5-FU)combination on normal and CC cells was analyzed by methyl thiazolyl diphenyl-tetrazolium bromide(MTT).Then,the impact of OA on CC cell lines(LoVo and HT-29)proliferation and stemness were measured using colon formation and tumorsphere formation assays.Octamer-binding transcription factor 4(Oct4),Prominin-1(CD133),Nanog,and transcription factor SOX-2(SOX2)are cell stemness-related indicators whose expression was assessed usingfluorescence qPCR assay,Western blotting,and immunohistochemistry.The effect of OA on the proliferative potency of CC cells was evaluated using an in vivo model.Results:The stem-like characteristics and clone production of colon cancer cells were markedly reduced by OA alone or in combination with OA-5-FU.Moreover,OA increases the susceptibility of CC cells to 5-FU by blocking the cell stemness-related markers(CD133,Nanog,SOX2,and Oct4)expression levels both in vitro and in vivo,as well as by inactivating the activator of transcription 3(STAT3 signaling)and Janus kinase 2/signal transducer(JAK2).Conclusion:Thesefindings imply that oleanolic acid,both in vitro and in vivo,suppresses the JAK2/STAT3 pathway,which in turn reverses chemoresistance and decreases colon cancer cell stemness.Therefore,by reducing the recommended amount of 5-FU,this strategy may improve chemotherapeutic effectiveness and minimize undesired side effects.

Mechanism of action of cordycepin in the treatment of hepatocellular carcinoma via regulation of the Hippo signaling pathway

Hepatocellular carcinoma(HCC)is one of the common most malignant tumors.This study aimed to determine the in vitro and in vivo anticancer activity of cordycepin and elucidate its mechanism of action.The results of in vitro and in vivo studies revealed that cordycepin inhibited proliferation and migration in HepG-2 cells and inhibited the growth of HepG-2 xenograft-bearing nude mice by inducing apoptosis.Transcriptome sequencing analysis revealed a total of 403 differential genes,which revealed that cordycepin may play an anti-HCC role by regulating Hippo signaling pathway.The regulatory effects of cordycepin on the Hippo signaling pathway was further investigated using a YAP1 inhibitor.The results demonstrated that cordycepin upregulated the expression of MST1 and LAST1,and subsequently inhibited YAP1,which activated the Hippo signaling pathway.This in turn downregulated the expression of GBP3 and ETV5,and subsequently inhibited cell proliferation and migration.Additionally,YAP1 regulated the expression of Bax and Bcl-2,regulated the mitochondrial apoptotic pathway,and induced apoptosis by upregulating the expression of the caspase-3 protein.In summary,this study reveals that cordycepin exerts its anti-hepatocarcinoma effect through regulating Hippo signaling pathway,and GBP3 and ETV5 may be potential therapeutic targets for hepatocarcinoma.

Down-regulation of mi R-30a-3p/5p promotes esophageal squamous cell carcinoma cell proliferation by activating the Wnt signaling pathway

AIM To investigate the potential role of micro RNA-30 a(mi R-30 a) in esophageal squamous cell carcinoma(ESCC).METHODS Expression of mi R-30 a-3 p/5 p was analyzed using microarray data and fresh ESCC tissue samples. Both in vitro and in vivo assays were used to investigate the effects of mi R-30 a-3 p/5 p on ESCC cell proliferation. Furthermore,Kyoto Encyclopedia of Genes and Genomes analysis was performed to explore underlying mechanisms involved in ESCC,and then,assays were carried out to verify the potential molecular mechanism of mi R-30 a in ESCC.RESULTS Low expression of mi R-30 a-3 p/5 p was closely associated with advanced ESCC progression and poor prognosis of patients with ESCC. Knock-down of mi R-30 a-3 p/5 p promoted ESCC cell proliferation. Increased mi R-30 a-3 p/5 p expression inhibited the Wnt signaling pathway by targeting Wnt2 and Fzd2.CONCLUSION Down-regulation of mi R-30 a-3 p/5 p promotes ESCC cell proliferation by activating the Wnt signaling pathway through inhibition of Wnt2 and Fzd2.

MiR-19a-3p regulates the Forkhead box F2-mediated Wnt/β-catenin signaling pathway and affects the biological functions of colorectal cancer cells

BACKGROUND Colorectal cancer(CRC)is one of the most common malignancies worldwide.AIM To explore the expression of microRNA miR-19a-3p and Forkhead box F2(FOXF2)in patients with CRC and the relevant mechanisms.METHODS Sixty-two CRC patients admitted to the hospital were enrolled into the study group,and sixty healthy people from the same period were assigned to the control group.Elbow venous blood was sampled from the patients and healthy individuals,and blood serum was saved for later analysis.MiR-19a-3p mimics,miR-19a-3p inhibitor,miR-negative control,small interfering-FOXF2,and short hairpin-FOXF2 were transfected into HT29 and HCT116 cells.Then quantitative polymerase chain reaction was performed to quantify the expression of miR-19a-3p and FOXF2 in HT29 and HCT116 cells,and western blot(WB)analysis was conducted to evaluate the levels of FOXF2,glycogen synthase kinase 3 beta(GSK-3β),phosphorylated GSK-3β(p-GSK-3β),β-catenin,p-β-catenin,α-catenin,Ncadherin,E-cadherin,and vimentin.The MTT,Transwell,and wound healing assays were applied to analyze cell proliferation,invasion,and migration,respectively,and the dual luciferase reporter assay was used to determine the correlation of miR-19a-3p with FOXF2.RESULTS The patients showed high serum levels of miR-19a-3p and low levels of FOXF2,and the area under the curves of miR-19a-3p and FOXF2 were larger than 0.8.MiR-19a-3p and FOXF2 were related to sex,tumor size,age,tumor-nodemetastasis staging,lymph node metastasis,and differentiation of CRC patients.Silencing of miR-19a-3p and overexpression of FOXF2 suppressed the epithelialmesenchymal transition,invasion,migration,and proliferation of cells.WB analysis revealed that silencing of miR-19a-3p and FOXF2 overexpression significantly suppressed the expression of p-GSK-3β,β-catenin,N-cadherin,and vimentin;and increased the levels of GSK-3β,p-β-catenin,α-catenin,and Ecadherin.The dual luciferase reporter assay confirmed that there was a targeted correlation of miR-19a-3p with FOXF2.In addition,a rescue experiment revealed that there were no differences in cell proliferation,invasion,and migration in HT29 and HCT116 cells co-transfected with miR-19a-3p-mimics+sh-FOXF2 and miR-19a-3p-inhibitor+si-FOXF2 compared to the miR-negative control group.CONCLUSION Inhibiting miR-19a-3p expression can upregulate the FOXF2-mediated Wnt/β-catenin signaling pathway,thereby affecting the epithelial-mesenchymal transition,proliferation,invasion,and migration of cells.Thus,miR-19a-3p is likely to be a therapeutic target in CRC.

Chaihu Longgu Muli Decoction relieving temporal lobe epilepsy in rats by inhibiting TLR4 signaling pathway through miR-146a-3p and miR-146a-5p

Objective To explore the effect and mechanism of Chaihu Longgu Muli Decoction(柴胡龙骨牡蛎汤,CHLGMLD)in rats with temporal lobe epilepsy(TLE).Methods A total of 80 Sprague-Dawley(SD)male rats were randomized into control(CON),model(MOD),carbamazepine(CBZ,0.1 g/kg),CHLGMLD low dose(CHLGMLD-L,12.5 g/kg),and high dose(CHLGMLD-H,25 g/kg)groups,with 16 rats in each group.TLE rat models were established in the four groups with the use of lithium-pilocarpine except for the CON group.After the successful establishment of TLE models,all drugs were administered through gavage,and distilled water was given to rats in the CON and MOD groups for four weeks.The frequency and duration of seizures before and after treatment were recorded for the evaluation of the alleviation degree.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression levels of miR-146a-3p and miR-146a-5p.The expression levels of toll-like receptor 4(TLR4),interleukin-1 receptor-associated kinase 1(IRAK1),tumor necrosis factor(TNF)receptor-associated factor 6(TRAF6),TAK1-binding protein(TAB),nuclear factor-kappa B(NF-κB),and interleukin-1 beta(IL-1β)in hippocampus were tested by immunofluorescence assay.Correlation analysis between the above factors and expressions of miR-146a-3p and miR-146a-5p were performed separately.Results CHLGMLD decreased the frequency(P<0.05)and duration(P<0.01)of seizures in rats.CHLGMLD down-regulated the expression levels of miR-146a-5p and miR-146a-3p(P<0.05),and inhibited the expression levels of TLR4,IRAK1,TRAF6,TAB,NF-κB,and IL-1β(P<0.01).The correlation analysis revealed that the expression levels of TLR4,IRAK1,TRAF6,TAB,NF-κB,and IL-1β were positively correlated with the expression levels of miR-146a-3p and miR-146a-5p detected by qRT-PCR,respectively(P<0.01).Conclusion CHLGMLD can inhibite the TLR4 signaling pathway by lowering the expression levels of miR-146a-3p and miR-146a-5p to alleviate hippocampal dentate gyrus inflammation in TLE rats,thus relieving seizures.

骨碎补总黄酮上调骨质疏松症模型大鼠Wnt/LRP-5/β-catenin通路表达的研究

目的 验证骨质疏松症模型大鼠与假手术大鼠Wnt/LRP-5/β-catenin信号通路相关基因表达是否存在差异,观察骨碎补总黄酮给药干预对骨质疏松症模型大鼠该通路基因表达的影响。方法 取40只3月龄的健康雌性SD大鼠,随机分为假手术组(SHAM组)、骨质疏松症模型组(OVX组)、骨质疏松模型+骨碎补总黄酮灌胃组(OVX+OTF组)、骨质疏松症模型+雌激素灌胃组(OVX+USP组),每组各10只。除SHAM组外,各组均通过卵巢切除术进行骨质疏松症造模,SHAM组进行假手术。常规饲养10周后采用苏木精-伊红染色法(hematoxylin-eosin staining,HE)验证造模,成功后开始药物干预。OVX+OTF组给予108 mg/(kg·d)剂量的骨碎补总黄酮进行灌胃;OVX+USP组给予0.1 mg/(kg·d)剂量的结合雌激素溶液灌胃;OVX组及SHAM组给予适量生理盐水灌胃,每日一次,给药2个月后以脱颈法处死各组大鼠。采用RT-PCR法检测骨质疏松症模型大鼠与SHAM组大鼠之间Wnt/LRP5/β-catenin信号通路相关基因表达差异;采用免疫组化法观察骨碎补总黄酮灌胃干预对骨质疏松症模型大鼠Wnt/LRP5/β-catenin信号通路表达的影响。结果 与SHAM组相比,骨质疏松症模型大鼠的Wnt3α、LRP-5及β-catenin、Runx2 mRNA表达均显著降低(P<0.01);与OVX组相比,OVX+OTF组及OVX+USP组大鼠的Wnt1、LRP-5及β-catenin、Runx2的蛋白表达均呈显著上升趋势(P<0.01)。结论 骨质疏松症模型大鼠的Wnt3α、LRP-5、β-catenin基因表达均显著下调,骨碎补总黄酮可能通过提高OVX大鼠胫骨中Wnt/LRP-5/β-catenin通路中关键蛋白表达发挥抗骨质疏松作用。

Neuroprotective effect of rapamycin on spinal cord injury via activation of the Wnt/β-catenin signaling pathway

The Wnt/β-catenin signaling pathway plays a crucial role in neural development, axonal guid- ance, neuropathic pain remission and neuronal survival. In this study, we initially examined the effect of rapamycin on the Wnt/β-catenin signaling pathway after spinal cord iniury, by intraperitoneally injecting spinal cord injured rats with rapamycin over 2 days. Western blot analysis and immunofluorescence staining were used to detect the expression levels of β-catenin protein, caspase-3 protein and brain-derived neurotrophic factor protein, components of the Wnt/β-catenin signaling pathway. Rapamycin increased the levels of β-catenin and brain-derived neurotrophic factor in the injured spinal cord, improved the pathological morphology at the injury site, reduced the loss of motor neurons, and promoted motor functional recovery in rats after spinal cord injury. Our experimental fndings suggest that the neuroprotective effect of rapamycin intervention is mediated through activation of the Wnt/β-catenin signaling pathway after spinal cord injury.

Involvement of the Wnt signaling pathway and cell apoptosis in the rat hippocampus following cerebral ischemia/reperfusion injury

We investigated the role of the Wnt signaling pathway in cerebral ischemia/reperfusion injury by examining β-catenin and glycogen synthase kinase-3β protein expression in the rat hippocampal CA1 region following acute cerebral ischemia/reperfusion. Our results demonstrate that cell apoptosis increases in the CA1 region following ischemia/reperfusion. In addition, β-catenin and glycogen synthase kinase-3β protein expression gradually increases, peaking at 48 hours following reperfusion. Dickkopf-1 administration, after cerebral ischemia/reperfusion injury, results in decreased cell apoptosis, and β-catenin and glycogen synthase kinase-3β expression, in the CA1 region. This suggests that β-catenin and glycogen synthase kinase-3β, both components of the Wnt signaling pathway, participate in cell apoptosis following cerebral ischemia/reperfusion injury.

Role of Wnt/β-catenin Signaling Pathway in the Mechanism of Calcification of Aortic Valve

Aortic valve calcification is a common disease in the elderly, but its cellular and molecular mechanisms are not clear. In order to verify the hypothesis that Wnt/β-catenin signaling pathway is involved in the process of calcification of aortic valve, porcine aortic valve interstitial cells（VICs） were isolated, cultured and stimulated with oxidized low density lipoprotein（ox-LDL） for 48 h to induce the differentiation of VICs into osteoblast-like cells. The key proteins and genes of Wnt/β-catenin signaling pathway, such as glycogen synthase kinase 3β（GSK-3β） and β-catenin, were detected by using Western blotting and real-time polymerase chain reaction（PCR）. The results showed that the VICs managed to differentiate into osteoblast-like cells after the stimulation with ox-LDL and the levels of proteins and genes of GSK-3β and β-catenin were increased significantly in VICs after stimulation for 48 h（P0.05）. It is suggested that Wnt/β-catenin signaling pathway may play a key role in the differentiation of VICs into osteoblast-like cells and make great contribution to aortic valve calcification.

Angelica sinensis polysaccharides ameliorate 5-flourouracil-induced bone marrow stromal cell proliferation inhibition via regulating Wnt/β-catenin signaling

Chemotherapy may cause cellular oxidative stress to bone marrow.Oxidative damage of bone marrow hematopoietic microenvironment is closely related to chronic myelosuppression after chemotherapeutic treatment.Angelica sinensis polysaccharides(ASP)are major effective ingredients of traditional Chinese medicine Angelica with multi-target anti-oxidative stress features.In the current study,we investigated the protective roles and mechanisms of ASP on chemotherapy-induced bone marrow stromal cell(BMSC)damage.The human bone marrow stromal cell line HS-5 cells were divided into control group,5-FU group,5-FU+ASP group,and 5-FU+LiCl group to investigate the mechanism of ASP to alleviate 5-FU-induced BMSC proliferation inhibition.The results showed that 5-FU inhibits the growth of HS-5 cells in a time and dose-dependent manner;however,ASP partially counteracted the 5-FU-induced decrease in cell viability,whereas Wnt signaling inhibitor Dkk1 antagonized the effect of ASP on HS-5 cells.ASP reversed the decrease in total cytoplasmicβ-catenin,p-GSK-3β,and CyclinD1 following 5-FU treatment and modulated nuclear expression ofβ-catenin,Lef-1,and C-myc proteins.Furthermore,ASP also enhanced the antioxidant capacity of cells and reduced 5-FU-induced oxidative stress,attenuated FoxO1 expression,thus weakened its downstream apoptosis-related proteins and G0/G1 checkpoint-associated p27^(Kip1) expression to alleviate 5-FU-induced apoptosis and to promote cell cycle progression.All the results above suggest that the protective role of ASP in 5-FU-treated BMSCs proliferation for the chemotherapy may be related to its activating Wnt/β-catenin signaling and keeping homeostasis betweenβ-catenin and FoxO1 under oxidative stress.The study provides a potential therapeutic strategy for alleviating chemotherapeutic damage on BMSCs.

RING finger and WD repeat domain 3 regulates proliferation and metastasis through the Wnt/β-catenin signalling pathways in hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC)exhibits high invasiveness and mortality rates,and the molecular mechanisms of HCC have gained increasing research interest.The abnormal DNA damage response has long been recognized as one of the important factors for tumor occurrence and development.Recent studies have shown the potential of the protein RING finger and WD repeat domain 3(RFWD3)that positively regulates p53 stability in response to DNA damage as a therapeutic target in cancers.AIM To investigate the relationship between HCC and RFWD3 in vitro and in vivo and explored the underlying molecular signalling transduction pathways.METHODS RFWD3 gene expression was analyzed in HCC tissues and adjacent normal tissues.Lentivirus was used to stably knockdown RFWD3 expression in HCC cell lines.After verifying the silencing efficiency,Celigo/cell cycle/apoptosis and MTT assays were used to evaluate cell proliferation and apoptosis.Subsequently,cell migration and invasion were assessed by wound healing and transwell assays.In addition,transduced cells were implanted subcutaneously and injected into the tail vein of nude mice to observe tumor growth and metastasis.Next,we used lentiviral-mediated rescue of RFWD3 shRNA to verify the phenotype.Finally,the microarray,ingenuity pathway analysis,and western blot analysis were used to analyze the regulatory network underlying HCC.RESULTS Compared with adjacent tissues,RFWD3 expression levels were significantly higher in clinical HCC tissues and correlated with tumor size and TNM stage(P<0.05),which indicated a poor prognosis state.RFWD3 silencing in BEL-7404 and HCC-LM3 cells increased apoptosis,decreased growth,and inhibited the migration in shRNAi cells compared with those in shCtrl cells(P<0.05).Furthermore,the in vitro results were supported by the findings of the in vivo experiments with the reduction of tumor cell invasion and migration.Moreover,the rescue of RFWD3 shRNAi resulted in the resumption of invasion and metastasis in HCC cell lines.Finally,gene expression profiling and subsequent experimental verification revealed that RFWD3 might influence the proliferation and metastasis of HCC via the Wnt/β-catenin signalling pathway.CONCLUSION We provide evidence for the expression and function of RFWD3 in HCC.RFWD3 affects the prognosis,proliferation,invasion,and metastasis of HCC by regulating the Wnt/β-catenin signalling pathway.

散瘀止痛续骨方通过Wnt3a/Lrp5信号通路促进肱骨骨折大鼠骨愈合

【目的】探讨散瘀止痛续骨方对肱骨骨折大鼠的治疗作用及机制。【方法】将40只SD大鼠随机分为正常组、模型组、辛伐他汀组及散瘀止痛续骨方组,每组10只。对模型组、辛伐他汀组、散瘀止痛续骨方组大鼠采用骨钳截断加克氏针髓内固定法建立肱骨骨折模型,正常组不做任何处理。建模成功后,辛伐他汀组给予灌胃20.0 mg·kg^(-1)·d^(-1)辛伐他汀,散瘀止痛续骨方组给予灌胃1.5 mg·kg^(-1)·d^(-1)散瘀止痛续骨方水煎液,正常组、模型组同期给予灌胃同体积生理盐水,连续给药21 d。给药结束后,X线摄片观察大鼠骨折愈合程度,双能X线骨密度仪检测肱骨骨密度,小动物骨骼强度测定仪测定肱骨骨强度,苏木素-伊红(HE)染色法观察肱骨组织病理形态,Western Blot法检测大鼠肱骨组织中Wnt3a、Lrp5蛋白表达。【结果】与正常组比较,模型组肱骨X线片和HE染色结果均可见明显骨折特征表现,各段肱骨骨密度,肱骨骨强度及肱骨组织Wnt3a、Lrp5蛋白表达水平均显著降低(P<0.05);与模型组比较,辛伐他汀组、散瘀止痛续骨方组肱骨X线片和HE染色结果得到明显改善,各段肱骨骨密度,肱骨骨强度及肱骨组织Wnt3a、Lrp5蛋白表达水平均显著升高(P<0.05),且散瘀止痛续骨方组的改善作用优于辛伐他汀组(P<0.05)。【结论】散瘀止痛续骨方可有效促进肱骨骨折大鼠骨愈合,其作用机制可能与激活Wnt3a/Lrp5信号通路有关。

5-methoxytryptophan induced apoptosis and PI3K/Akt/FoxO3a phosphorylation in colorectal cancer

BACKGROUND Colorectal cancer(CRC)is a highly prevalent malignancy worldwide,and new therapeutic targets urgently need to be found to prolong patient survival.5-methoxytryptophan(5-MTP)is a tryptophan metabolite found in animals and humans.However,the effects of 5-MTP on proliferation and apoptosis of CRC cells are currently unknown.AIM To investigate the effects of 5-MTP on the proliferation,migration,invasion,and apoptosis abilities of CRC cells.Additionally,we seek to explore whether 5-MTP has the potential to be utilized as a drug for the treatment of CRC.METHODS In order to evaluate the effect of 5-MTP on CRC cells,a series of experiments were conducted for evaluation.Colony formation assay and Cell Counting Kit 8 assays were used to investigate the impact of 5-MTP on the proliferation of CRC cell lines.Cell cycle assays were employed to examine the effect of 5-MTP on cellular growth.In addition,we investigated the effects of 5-MTP on apoptosis and reactive oxygen species in HCT-116 cells.To obtain a deeper understanding of how 5-MTP affects CRC,we conducted a study to examine its influence on the PI3K/Akt signaling pathway in CRC cells.RESULTS This article showed that 5-MTP promoted apoptosis and cell cycle arrest and inhibited cell proliferation in CRC cells.In many articles,it has been reported that PI3K/Akt/FoxO3a signaling pathway is one of the most important signaling pathways involved in internal regulating cell proliferation and differentiation. Nevertheless, 5-MTP combined with PI3K/Akt/FoxO3a signaling pathway inhibitors significantly promotedapoptosis and cell cycle arrest and inhibited cell proliferation in CRC cells compared with 5-MTP alone in ourstudy.CONCLUSIONTherefore, there is strong evidence that 5-MTP can be used as an effective medicine for CRC treatment.

A Comprehensive Overview of Skeletal Phenotypes Associated with Alterations in Wnt/β-catenin Signaling in Humans and Mice

The Wnt signaling pathway plays key roles in differentiation and development and alterations in this signaling pathway are causally associated with numerous human diseases. While several laboratories were examining roles for Wnt signaling in skeletal development during the 1990s, interest in the pathway rose exponentially when three key papers were published in 2001-2002. One report found that loss of the Wnt co-receptor, Low-density lipoprotein related protein-5 （LRPS）, was the underlying genetic cause of the syndrome Osteoporosis pseudoglioma （OPPG）. OPPG is characterized by early-onset osteoporosis causing increased susceptibility to debilitating fractures. Shortly thereafter, two groups reported that individuals carrying a specific point mutation in LRP5 （G171V） develop high-bone mass. Subsequent to this, the causative mechanisms for these observations heightened the need to understand the mechanisms by which Wnt signaling controlled bone development and homeostasis and encouraged significant investment from biotechnology and pharmaceutical companies to develop methods to activate Wnt signaling to increase bone mass to treat osteoporosis and other bone disease. In this review, we will briefly summarize the cellular mechanisms underlying Wnt signaling and discuss the observations related to OPPG and the high-bone mass disorders that heightened the appreciation of the role of Wnt signaling in normal bone development and homeostasis. We will then present a comprehensive overview of the core components of the pathway with an emphasis on the phenotypes associated with mice carrying genetically engineered mutations in these genes and clinical observations that further link alterations in the pathway to changes in human bone.

Efficacy of Yisui granule(益髓颗粒)on myelodysplastic syndromes in SKM-1 mouse xenograft model through suppressing Wnt/β-catenin signaling pathway

OBJECTIVE:To unmask the underlying mechanisms of Yisui granule(益髓颗粒,YSG)for the treatment of Myelodysplastic syndromes(MDS).METHODS:Our study used an SKM-1 mouse xenograft model of MDS to explore the anti-tumor potential of YSG and its safety,assess its effect on overall survival(OS),and evaluate whether its mechanism is associated with the demethylation of the secreted frizzled related protein 5(s FRP5)gene and suppressing Wnt/β-catenin pathway.Bisulfite amplicon sequencing was applied to detect the level of methylation of the s FRP5 gene;western blotting,immunofluorescence staining,and real-time Polymerase Chain Reaction were performed to detect DNA methyltransferase 1(DNMT1),s FRP5,and other Wnt/β-catenin pathway-related m RNA and protein expression.RESULTS:The results showed that high-dosage YSG exerted an anti-tumor effect similar to that of decitabine,improved OS,and reduced long-term adverse effects in the long term.Mechanically,YSG reduced the expression of DNMT1 methyltransferase,decreased the methylation,and increased the expression of the Wnt/β-catenin pathway antagonist-s FRP5.Furthermore,components of the Wnt/β-catenin pathway,including Wnt3a,β-catenin,c-Myc,and cyclin D1,were down-regulated in response to YSG,suggesting that YSG could treat MDS by demethylating the s FRP5 gene and suppressing the Wnt/β-catenin pathway.CONCLUSIONS:Our findings demonstrated that YSG could be used alone or in combination with decitabine to improve outcomes in the MDS animal model,providing an alternative solution for treating MDS.

Complement C3a signaling mediates production of angiogenic factors in mesenchymal stem cells

A major portion of the beneficial effect of mesenchymal stem cells (MSC) is due to the production of trophic and angiogenic factors by these cells, and one of the efforts to improve the therapeutic efficacy of these cells lies in enhancing this capacity. Since there is complement activation in all areas of tissue injury, and both C3a and C5a activate MSC, it was asked whether stimulation with C3a or C5a would upregulate the production of trophic factors by MSC. C3a caused significant up-regulation of various angiogenic factors, including VEGF, CXCL8/IL-8 and IL-6. In contrast there was no detectable production of the pro-inflammatory cytokines TNF-α and IL-1β in spite of nuclear translocation of NFκB. Although C5a also caused moderate up-regulation of angiogenic factors, the effect was borderline significant. Furthermore the production of angiogenic factors induced by C3a was of physiological relevance: Supernatants of MSCs cultured under serum-free conditions induced minimal tube formation of HUVECs as an in vitro measure of angiogenesis;tube formation was considerably enhanced, when supernatants from C3a-stimulated MSC were used, while C3a itself had no direct angiogenic effect on HUVECs. The signaling cascade responsible for the production of angiogenic factors by C3a or C5a could be defined as activation of the rho cascade which was necessary for nuclear translocation of NFκB p65 and of phospho-ERK1/2. Although rho was only transiently activated, inhibition of the rho or “downstream of it” of the NFκB pathway, prevented C3a-and C5a-induced up-regulation of angiogenic factors.

Wnt3a:functions and implications in cancer

Wnt3a,one of Wnt family members,plays key roles in regulating pleiotropic cellular functions,including self-renewal,proliferation,differentiation,and motility.Accumulating evidence has suggested that Wnt3 a promotes or suppresses tumor progression via the canonical Wnt signaling pathway depending on cancer type.In addition,the roles of Wnt3 a signaling can be inhibited by multiple proteins or chemicals.Herein,we summarize the latest findings on Wnt3 a as an important therapeutic target in cancer.

Expression of hepatic Wnt5a and its clinicopathological features in patients with hepatocellular carcinoma

Backgroud： Wingless-type MMTV integration site family member 5a （Wnt5a） is involved in carcinogenesis.However, little data are available in Wnt5a signaling with hepatocellular carcinoma （HCC）. In thepresent study, we investigated the expression of hepatic Wnt5a in HCC and the role of Wnt5a in HCCprogression and outcome.

Targeting key signalling pathways in oesophageal adenocarcinoma:A reality for personalised medicine?

Cancer treatments are rapidly changing.Curative treatment for oesophageal adenocarcinoma currently involves surgery and cytotoxic chemotherapy or chemoradiotherapy.Outcomes for both regimes are generally poor as a result of tumor recurrence.We have reviewed the key signalling pathways associated with oesophageal adenocarcinomas and discussed the recent trials of novel agents that attempt to target these pathways.There are many trials underway with the aim of improving survival in oesophageal cancer.Currently,phase 2 and 3 trials are focused on MAP kinase inhibition,either through inhibition of growth factor receptors or signal transducer proteins.In order to avoid tumor resistance,it appears to be clear that targeted therapy will be needed to combat the multiple signalling pathways that are in operation in oesophageal adenocarcinomas.This may be achievable in the future with the advent of gene signatures and a combinatorial approach.

益骨汤含药血清通过经典Wnt信号通路促进成骨细胞增殖分化的研究

目的:在经典wnt信号通路抑制剂大鼠Dickkopf相关蛋白1(DKK1)的干预下,观察益骨汤含药血清对成骨细胞的经典Wnt信号通路的影响,探究益骨汤治疗骨质疏松的作用机制,为临床使用补肾活血中药防治骨质疏松症提供实验依据。方法:将40只雌性SD大鼠随机分成2组,益骨汤水提液组和空白对照组,连续灌胃10天,获取含药血清。从乳鼠颅盖骨中分离培养成骨细胞,实验分为4组,即A组(益骨汤含药血清组)、B组(DKK1组)、C组(DKK1+益骨汤含药血清组)、D组(空白血清对照组)。进行细胞增殖MTT检测、碱性磷酸酶活性检测,RT-PCR检测经典Wnt/β-catenin通路中β-链蛋白(β-catenin)、低密度脂蛋白受体相关蛋白5(LRP-5)、DKK1基因的表达情况。结果:根据MTT法动态观察各组成骨细胞增殖,发现成骨细胞增殖呈规律性:48 h至72 h成骨细胞呈对数增长期,96 h后细胞增殖逐渐受到抑制。同一时间点与B组比较,A、C、D各组成骨细胞的增殖和ALP活性显著较高,差异均有统计学意义(P<0.05)。同一时间点与D组比较,A组成骨细胞的增殖和ALP活性显著较高,差异均有统计学意义(P<0.05)。与B组比较,A、C、D组β-catenin mRNA、LRP-5 mRNA、TCF mRNA的表达均明显升高,DKK1 mRNA的表达明显降低,差异均有统计学意义(P<0.05)。与D组比较,A组β-catenin mRNA、LRP-5 mRNA、TCF mRNA的表达均明显升高,DKK1 mRNA的表达明显降低,差异均有统计学意义(P<0.05)。结论:益骨汤含药血清可能是通过抑制DKK1的高表达,从而上调经典Wnt/β-catenin信号中β-catenin、LRP-5、TCF基因的表达,达到促进成骨细胞增殖,起到抗骨质疏松作用。