Elevated pulmonary levels of Axin2 in mice exposed to herbicide 2,4-D with or without endotoxin.

2,4-Dichlorophenoxyacetic acid (2,4-D), a member of the phenoxy family of herbicides is commonly used in agriculture for controlling broadleaf weeds but its uncontrolled and incoherent use has been linked to incidences of lung toxicity. The present study aimed to understand the molecular mechanisms behind the 2,4-D alone or in combination with endotoxin (lipopolysaccharide [LPS]) induced pulmonary toxicity. Blood and lung samples were collected from Swiss albino mice (n = 48) following chronic exposure to high (37 mg/kg; 1/10th of LD50 ) and low (18.5 mg/kg; 1/20th of LD50 ) doses of 2,4-D alone or in combination with endotoxin (80 µg/animal). Transcriptome analysis revealed Wnt Canonical signaling as one of the top dysregulated pathways in mice lung following exposure to 2,4-D with and without endotoxin (LPS) co-exposure. Global view of differentially expressed genes showed increased messenger RNA expression of Axin2 by 0.26, 2.58, 3.14, 2.59, and 2.97 folds following exposure to LPS, high dose alone or in combination with LPS and low dose alone or in combination with LPS, respectively. The microarray data were validated using quantitative polymerase chain reaction and immunohistochemistry. Furthermore, the plasma concentration of Axin2 was elevated in the high dose group as revealed by Sandwich ELISA. The data taken together suggest a role of Axin2 to activate the Canonical Wnt signaling pathway in 2,4-D and or endotoxin-induced lung damage in mice.

The effect of baicalein on Wnt/ß-catenin pathway and miR-25 expression in Saos-2 osteosarcoma cell line

Background/aim: Osteosarcoma is the most common primary bone malignancy that occurs frequently in children and adolescents. Baicalein, a flavonoid that has attracted great attention in recent years with its strong antitumor activity, shows a wide range of biological and pharmaceutical effects.MicroRNAs have been found to be involved in many critical processes in cancers. This study aimed to investigate the effect of baicalein and miR-25 on Wnt/ß-catenin signaling pathway of osteosarcoma cell line Saos-2. Materials and methods: Cell viability was assessed, and qRT-PCR and Western blot were performed to study the effects of baicalein on expression of Wnt/ß-catenin signaling pathway-realted genes (ß-catenin, GSK-3ß, and Axin2) of Saos-2 cells. Results: Our results indicated that baicalein can inhibit the proliferation (IC50 value 35 µM), regulate Wnt/ß-catenin pathway and also increase miR-25 expression of Saos-2. Baicalein and also miR-25 decreased the expression of ß-catenin and Axin2, while increasing the expression of GSK-3ß. Down regulation of miR-25 decreased the expression of GSK-3ß, while ß-catenin and Axin2 expression increased. Conclusion: These findings demonstrate that baicalein may target genes related to the Wnt/ß-catenin pathway by regulating miR-25 expression and may be a potential Wnt/ß-catenin pathway inhibitor for osteosarcoma therapy.

Curcumin and quercetin synergistically inhibit cancer cell proliferation in multiple cancer cells and modulate Wnt/ß-catenin signaling and apoptotic pathways in A375 cells.

BACKGROUND: Traditional therapy using natural products, especially flavonoids and alkaloids have been in practice for a long time. Among flavonoids, curcumin, quercetin, berberine, and epigallocatechin have been studied in greater detail in terms of their anticancer and anti-inflammatory activities. Although many studies focused on the PI3K, MAP kinase and NF-κB pathways, a thorough investigation of modulation of players in the apoptotic and Wnt/ß-catenin signaling pathway by curcumin and quercetin has not been done. Also, only few studies have been carried out on curcumin and quercetin co-treatment studies. HYPOTHESIS/PURPOSE: We hypothesized that the combination of natural products will have synergistic effects and the antiproliferative effect will be attenuated via apoptotic as well as Wnt/ß-catenin signaling pathways. STUDY DESIGN AND METHODS: To test our hypothesis, we compared potency of natural anticancer agents in four cancer cell lines, A549, HCT116, MCF7, and A375 by MTT and colony proliferation assays and investigated mechanism of anticancer activities by analyzing players in apoptotic and Wnt/ß-catenin signaling pathways in A375 cells treated with test agents individually or in combination. RESULTS: Epicatechins, up to 100 µM concentration, did not inhibit cancer cell proliferation, while curcumin inhibited proliferation in A549 and HCT116 cancer cell lines with an IC50 of 3 to 8.5 µM. Quercetin showed stronger inhibition of cell proliferation than berberine. Combination study with two most potent agents, curcumin and quercetin, in 4 cancer cell lines, suggested synergistic effect on cell proliferation with several fold decreases in IC50. Further investigation of the mechanism of action of curcumin and quercetin in melanoma cells, A375, suggested that inhibition of cell proliferation occurred through down-regulation of Wnt/ß-catenin signaling pathway proteins, DVL2, ß-catenin, cyclin D1, Cox2, and Axin2. In addition, both curcumin and quercetin induced apoptosis by down-regulating BCL2 and inducing caspase 3/7 through PARP cleavage. CONCLUSION: These results demonstrate that curcumin and quercetin inhibit cancer cell proliferation synergistically and Wnt/ß-catenin signaling and apoptotic pathways are partly responsible for antiproliferative activities.

Wnt/ß-catenin signaling pathway is involved in regulating the migration by an effective natural compound brucine in LoVo cells.

BACKGROUND: Colorectal cancer remains the third most common malignancies and migration is one of the main factors for its high mortality rate. Brucine, a natural plant alkaloid, has been proved to possess a variety of pharmacological functions including anti-tumor activities. PURPOSE: The aim of this study was to investigate the inhibitory effect of brucine on the colorectal cancer and the underlying mechanism. METHODS: In this study, colony formation assay and transwell assay were used to investigate the effect of brucine on LoVo cells viability and migration. Immunofluorescence assay, western blot assay and Gelatin zymography assay were used to study the mechanism of brucine. Xenograft model in nude mice was induced to investigate the in vivo effect of brucine on LoVo cells. RESULTS: Brucine could significantly decrease the viability, inhibit the colony formation and induce the apoptosis of LoVo cells. Brucine could also suppress the migration of LoVo cells in a dose-dependent manner. Western blot analysis elucidated that the inhibition of migration was associated with the decreasing expression of matrix metalloproteinases including MMP2, MMP3 and MMP9. Moreover, we found that treatment of brucine could downregulate the expression of Frizzled-8, Wnt5a, APC and GSNK1A1, and increase the expression of AXIN1. Meanwhile, brucine also decreased the phosphorylation level of LRP5/6 and GSK3ß, and increased the level of p-ß-catenin. Xenografted model in nude mice study also revealed that oral administration of brucine could inhibit the growth and migration of LoVo cells by activating the expression of AXIN1 and p-ß-catenin. CONCLUSION: Brucine could suppress the migration of the colorectal cancer in vitro and in vivo and the effect was associated with the inhibition of the Wnt/ß-catenin signaling pathway.

Photoperiodic and Diurnal Regulation of WNT Signaling in the Arcuate Nucleus of the Female Djungarian Hamster, Phodopus sungorus.

The WNT pathway was shown to play an important role in the adult central nervous system. We previously identified the WNT pathway as a novel integration site of the adipokine leptin in mediating its neuroendocrine control of metabolism in obese mice. Here we investigated the implication of WNT signaling in seasonal body weight regulation exhibited by the Djungarian hamster (Phodopus sungorus), a seasonal mammal that exhibits profound annual changes in leptin sensitivity. We furthermore investigated whether crucial components of the WNT pathway are regulated in a diurnal manner. Gene expression of key components of the WNT pathway in the hypothalamus of hamsters acclimated to either long day (LD) or short day (SD) photoperiod was analyzed by in situ hybridization. We detected elevated expression of the genes WNT-4, Axin-2, Cyclin-D1, and SFRP-2, in the hypothalamic arcuate nucleus, a key energy balance integration site, during LD compared with SD as well as a diurnal regulation of Axin-2, Cyclin-D1, and DKK-3. Investigating the effect of photoperiod as well as leptin on the activation (phosphorylation) of the WNT coreceptor LRP-6-(Ser1490) by immunohistochemistry, we found elevated activity in the arcuate nucleus during LD relative to SD as well as after leptin treatment (2 mg/kg body weight). These findings indicate that differential WNT signaling may be associated with seasonal body weight regulation and is partially regulated in a diurnal manner in the adult brain. Furthermore, they suggest that this pathway plays a key role in the neuroendocrine regulation of body weight and integration of the leptin signal.

Effects of a grape-supplemented diet on proliferation and Wnt signaling in the colonic mucosa are greatest for those over age 50 and with high arginine consumption.

A diet rich in fruits and vegetables, and a grape-derived compound, resveratrol, have been linked to a reduced incidence of colon cancer. In vitro and in vivo, resveratrol suppresses Wnt signaling, a pathway constitutively activated in over 85 % of colon cancers.Thirty participants were placed on a low resveratrol diet and subsequently allocated to one of three groups ingesting 1/3-to-1 lb (0.15-0.45 kg) of grapes per day for 2 weeks. Dietary information was collected via 24-h recall. Colon biopsies for biomarker analysis were obtained pre- and post-grape and evaluated for the expression of Wnt pathway target genes and for markers of proliferation by RT-PCR and immunohistochemistry.Participants lost an average of 2 · 6 lb (1.2 kg, p = 0 · 0018) during the period of grape ingestion. The expression of CyclinD1 (p < 0 · 01), AXIN2, CD133 (p = 0 · 02) and Ki67 (p = 0 · 002) were all reduced after grape ingestion. Individuals over 50 years of age and those with high dietary arginine consumption had increased basal expression of CyclinD1, AXIN2, cMYC and CD133 (p value range 0 · 04 to <0 · 001) that, following grape ingestion, were reduced to levels seen in younger participants.The reduction in Wnt signaling and mucosal proliferation seen following short-term ingestion of 1/3-1 lb (0.15-0.45 kg) of grapes per day may reduce the risk of mutational events that can facilitate colon carcinogenesis. The potential benefit is most marked for high-risk older individuals and individuals whose diet is high in arginine intake. Dietary grape supplementation may play a role in colon cancer prevention for high-risk individuals.

[Changes in Wnt pathway inhibiting factors in nitrosamine-induced esophageal precancerosis lesions and effect of gexia zhuyu decoction].

OBJECTIVE: To discuss the changes in Wnt pathway inhibiting factors in esophageal precancerosis lesions induced by methyl benzyl nitrosamine (MBNA) and the effect of Gexia Zhuyu decoction. METHOD: Wistar rats were subcutaneously injected with MBNA (3.5 mg x kg(-1) for twice per week to establish the model. Since the 1st day after the model establishment, they were orally administered with Gexia Zhuyu decoction (16, 8 mg x kg(-1)). At the 10th week, esophageal tissues were collected to observe the pathological changes of esophageal mucosa, detect SFRP1, sFRP4, Axin1, Axin2 and GSK-3ß mRNA levels.by fluorescent quantitation PCR analysis and ß-catenin protein level by Western blotting. RESULT: Being induced by MBNA, rats in the model group showed slight atypical hyperplasia in the histopathological examination. Compared with the normal group, Gexia Zhuyu decoction dose high and low groups showed no significant pathomorphological and histological changes. The model group showed lower gene transcription levels of esophageal tissues sFRP1, sFRP4, Axin1 and Axin2 (P < 0.05 or P < 0.01) and higher ß-catenin protein expression level (P < 0.01) than the normal control group. The Gexia Zhuyu decoction low dose group showed higher gene transcription levels of esophageal tissues sFRP1, sFRP4, Axin1 and Axin2 (P < 0.05 or P < 0.01) and lower ß-catenin protein expression level (P < 0.01) than the normal control group. CONCLUSION: Up-regulated ß-catenin protein level and down-regulated Wnt pathway could enhance Wnt pathway activity of MBNA-induced esophageal precancerous lesions. Gexia Zhuyu decoction could down-regulate the ß-catenin protein level and up-regulate the transcription level of Wnt pathway inhibiting factors, but could not block MBNA-induced esophageal precancerosis lesions.

A phytoestrogen diarylheptanoid mediates estrogen receptor/Akt/glycogen synthase kinase 3ß protein-dependent activation of the Wnt/ß-catenin signaling pathway.

Estrogen promotes growth in many tissues by activating Wnt/ß-catenin signaling. Recently, ASPP 049, a diarylheptanoid isolated from Curcuma comosa Roxb., has been identified as a phytoestrogen. This investigation determined the involvement of Wnt/ß-catenin signaling in the estrogenic activity of this diarylheptanoid in transfected HEK 293T and in mouse preosteoblastic (MC3T3-E1) cells using a TOPflash luciferase assay and immunofluorescence. ASPP 049 rapidly activated T-cell-specific transcription factor/lymphoid enhancer binding factor-mediated transcription activity and induced ß-catenin accumulation in the nucleus. Interestingly, the effects of ASPP 049 on the transcriptional activity and induction and accumulation of ß-catenin protein in the nucleus of MC3T3-E1 cells were greater compared with estradiol. Activation of ß-catenin in MC3T3-E1 cells was inhibited by ICI 182,780, suggesting that an estrogen receptor is required. In addition, ASPP 049 induced phosphorylations at serine 473 of Akt and serine 9 of GSK-3ß. Moreover, ASPP 049 also induced proliferation and expressions of Wnt target genes Axin2 and Runx2 in MC3T3-E1 cells. In addition, ASPP 049 increased alkaline phosphatase expression, and activity that was abolished by DKK-1, a blocker of the Wnt/ß-catenin receptor. Taken together, these results suggest that ASPP 049 from C. comosa induced osteoblastic cell proliferation and differentiation through ERα-, Akt-, and GSK-3ß-dependent activation of ß-catenin signaling. Our findings provide a scientific rationale for using C. comosa as a dietary supplement to prevent bone loss in postmenopausal women.