Wnt Gene Expression Research Articles

Running Against the Wnt: How Wnt/β-Catenin Suppresses Adipogenesis.

Mesenchymal stem cells (MSCs) give rise to adipocytes, osteocytes, and chondrocytes and reside in various tissues, including bone marrow and adipose tissue. The differentiation choices of MSCs are controlled by several signaling pathways, including the Wnt/β-catenin signaling. When MSCs undergo adipogenesis, they first differentiate into preadipocytes, a proliferative adipocyte precursor cell, after which they undergo terminal differentiation into mature adipocytes. These two steps are controlled by the Wnt/β-catenin pathway, in such a way that when signaling is abrogated, the next step in adipocyte differentiation can start. This sequence suggests that the main role of Wnt/β-catenin signaling is to suppress differentiation while increasing MSC and preadipocytes cell mass. During later steps of MSC differentiation, however, active Wnt signaling can promote osteogenesis instead of keeping the MSCs undifferentiated and proliferative. The exact mechanisms behind the various functions of Wnt signaling remain elusive, although recent research has revealed that during lineage commitment of MSCs into preadipocytes, Wnt signaling is inactivated by endogenous Wnt inhibitors. In part, this process is regulated by histone-modifying enzymes, which can lead to increased or decreased Wnt gene expression. The role of Wnt in adipogenesis, as well as in osteogenesis, has implications for metabolic diseases since Wnt signaling may serve as a therapeutic target.

Interactive role of Wnt signaling and Zn in regulating testicular function of the common carp, Cyprinus carpio

Wnt signaling is conserved among all species and plays a significant role in various cellular processes including reproduction. The present study identified significant involvement of wnt4a, wnt5b, and wnt8a signaling in the testicular growth of common carp,Cyprinus carpio. Predominant expression of wnt4a, wnt5b, and wnt8a was found in the gonads and Wnt4a was localized in spermatocytes and interstitial cells. Ontogeny and testicular phase-wise analysis signified the importance of wnt isofoms analyzed in this study. Specific pathway activation of Wnt signaling revealed that Wnt4a and Wnt5b act through non-canonical while Wnt8a prefers the canonical pathway. The Wnt signaling regulates several steroidogenic enzyme and testis-related genes which was confirmed by the Wnt blockade experiments. Incidentally, zinc (Zn) is an essential trace element involved in the progression of spermatogenesis in teleosts. In adult male carp, a single administration of Zn at different doses elevated the expression of Wnt and Zn transporter genes and a single-dose (30 μg/g body weight [BW]) of Zn treatment elevated steroidogenic enzyme and testis-related genes which coincided with elevated androgens. Conversely, single-dose administration of Zn chelator to the Zn administered (30 μg/g BW) fish reversed the effects emphasizing a prominent role of Zn in the testicular function perhaps through Wnt signaling. Similar effects were observed in the in vitro experiments using the Zn chelator. Bioaccumulation of Zn and histological analysis revealed the importance of Zn in progression of spermatogenesis and sperm motility. Various assays related to cell viability and proliferation exhibited the role of Zn in promoting spermatogenic cell progression. Flow cytometric analysis confirmed Zn-induced elevation of Wnt and Zn transporter genes in germ and supporting cells. Furthermore, the effects of Zn are dose-related in carp. Taken together, it seems wnt4a, wnt5b, and wnt8a play an important role in testis and exposure of Wnt inhibitor, canonical as well as non-canonical activators, and Zn confirmed that Zn regulates Wnt signaling vis-à-vis promoting spermatogenesis in the common carp.

Inhibition of the Wnt Signalling Pathway: An Avenue to Control Breast Cancer Aggressiveness.

Breast cancer (BC) is the most common tumour in women. Although the introduction of novel therapeutic approaches in clinical practice has dramatically improved the clinical outcome of BC patients, this malignant disease remains the second leading cause of cancer-related death worldwide. The wingless/integrated (Wnt) signalling pathway represents a crucial molecular node relevantly implicated in the regulation of normal somatic stem cells as well as cancer stem cell (CSC) traits and the epithelial–mesenchymal transition cell program. Accordingly, Wnt signalling is heavily dysregulated in BC, and the altered expression of different Wnt genes is significantly associated with cancer-related aggressive behaviours. For all these reasons, Wnt signalling represents a promising therapeutic target currently under clinical investigation to achieve cancer eradication by eliminating CSCs, considered by most to be responsible for tumour initiation, relapse, and drug resistance. In this review, we summarized the current knowledge on the Wnt signalling pathway in BC and have presented evidence implicating the suitability of Wnt targeting in an attempt to improve the outcome of patients without affecting the normal somatic stem cell population.

The expression and regulation of Wnt1 in tooth movement–initiated mechanotransduction

The Wnt signaling pathway acts as a key regulator of skeletal development and its homeostasis. However, the potential role of Wnt1 in the mechanotransduction machinery of orthodontic tooth movement-initiated bone remodeling is still unclear. Hence, this study focused on the regulatory dynamics of the Wnt1 expression in both the periodontal ligament (PDL) and osteocytes invivo and invitro. The Wnt1 expression in the orthodontically moved maxillary first molar in mice was assessed at 0, 1, and 5days, on both the compression and tension sides. Primary isolated human PDL (hPDL) fibroblasts, as well as murine long-bone osteocyte-Y4 (MLO-Y4) cells, were exposed to continuous compressive force and static tensile force. The relative quantification of immunodetection showed that orthodontic tooth movement significantly stimulated the Wnt1 expression in both the PDL and alveolar osteocytes on the tension side on day 5, whereas the expression on the compression side did not change. This increase in the Wnt1 expression, shown invivo, was also noted after the application of 12% static tensile force in isolated hPDL fibroblasts and 20% in MLO-Y4 cells. In contrast, a compressive force led to the attenuation of the Wnt1 gene expression in both hPDL fibroblasts and MLO-Y4 cells in a force-dependent manner. In the osteocyte-PDL coculture system, recombinant sclerostin attenuated Wnt1 in PDL, whereas the antisclerostin antibody upregulated its gene expression, indicating that mechanically-driven Wnt1 signaling in PDL might be regulated by osteocytic sclerostin. Our findings provide that Wnt1 signaling plays a vital role in tooth movement-initiated bone remodeling via innovative mechanotransduction approaches.

Wnt Genes in Wing Pattern Development of Coliadinae Butterflies

Wnt genes are major developmental genes highly conserved across all animals. Yet, our understanding of the Wnt gene repertoire and their functions is still largely incomplete. In Lepidoptera, Wnt genes have been implicated in wing pattern development. For example, WntA has been shown as a driver of wing pattern diversification in nymphalid butterflies. In this study we characterize the Wnt gene repertoire in Zerene cesonia (Family: Pieridae), which diverged from nymphalids ~51 million years ago, to determine if Wnt genes may have a conserved role in wing development across distant butterfly lineages. We first show that Wnt gene content is highly conserved across butterflies, but that there is strikingly different expression across the eight Wnt genes during wing development of Z. cesonia and nymphalid butterflies. Surprisingly, while four Wnt genes (Wnt1, 6, 7 and 11) are expressed during wing development in Z. cesonia, the renown nymphalid wing patterning gene WntA was undetected. However, despite the differences in Wnt gene expression, Heparin injections yielded a similar disruption in wing color pattern development in both Z. cesonia and nymphalid butterflies. Interestingly, these assays also resulted in the loss of structurally-based ultraviolet wing patterns in Z. cesonia. Collectively, the study shows that pierids maintained the highly conserved nature of Wnt gene content in Lepidoptera, but might deploy these genes very differently from nymphalid butterflies during wing pattern development.

Functional variations between Mesenchymal Stem Cells of different tissue origins: A comparative gene expression profiling.

Mesenchymal Stem Cells (MSCs), regardless of the tissue sources, are considered as excellent candidates for cellular therapy as they are immune-privileged cells containing a multitude of therapeutic functions that aid in tissue regeneration and repair. For the effective application of these cells in cell therapy, it is important to understand and characterize their biological functions. The present study attempts to characterize the variations in multipotent function such as cell surface antigen levels, proliferation, differentiation and stemness (pluripotency) potential of MSCs isolated from foetal [wharton's jelly (WJ), foetal and maternal side of placenta (PF and PM)] and adult tissue sources [bone marrow (BM) and adipose tissue (AT)] using gene expression by real time PCR (qRT-PCR). Amongst the different tissue sources, PM, PF and AT-MSCs exhibited significant increase (p < 0.001, p < 0.001 and p < 0.01 respectively) in CD 73 expression and therefore could have a role in immunomodulation. WJ-MSCs exhibited superior proliferation potential based on growth curve, PCNA and Wnt gene expression. BM-MSCs were superior in exhibiting trilineage differentiation. Enhanced stemness potential (Oct 4 and Nanog) was observed for both BM and WJ-MSCs. In addition, BM and WJ-MSCs expressed high levels of CD 90 making them suitable in bone repair and regeneration. Thus to conclude, out of the five different sources tested, BM an adult source and WJ-MSCs a foetal source were superior in exhibiting most of the biological functions indicating that these sources may be suitable candidates for cell repair and regeneration studies.

Expression of a Constitutively Active Form of Hck in Chondrocytes Activates Wnt and Hedgehog Signaling Pathways, and Induces Chondrocyte Proliferation in Mice.

Runx2 is required for chondrocyte proliferation and maturation. In the search of Runx2 target genes in chondrocytes, we found that Runx2 up-regulated the expression of hematopoietic cell kinase (Hck), which is a member of the Src tyrosine kinase family, in chondrocytes, that Hck expression was high in cartilaginous limb skeletons of wild-type mice but low in those of Runx2–/– mice, and that Runx2 bound the promoter region of Hck. To investigate the functions of Hck in chondrocytes, transgenic mice expressing a constitutively active form of Hck (HckCA) were generated using the Col2a1 promoter/enhancer. The hind limb skeletons were fused, the tibia became a large, round mass, and the growth plate was markedly disorganized. Chondrocyte maturation was delayed until E16.5 but accelerated thereafter. BrdU-labeled, but not terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive, chondrocytes were increased. Furthermore, Hck knock-down reduced the proliferation of primary chondrocytes. In microarray and real-time RT-PCR analyses using hind limb RNA from HckCA transgenic mice, the expression of Wnt (Wnt10b, Tcf7, Lef1, Dkk1) and hedgehog (Ihh, Ptch1, and Gli1) signaling pathway genes was upregulated. These findings indicated that Hck, whose expression is regulated by Runx2, is highly expressed in chondrocytes, and that HckCA activates Wnt and hedgehog signaling pathways, and promotes chondrocyte proliferation without increasing apoptosis.

WNT3A Promotes Neuronal Regeneration upon Traumatic Brain Injury.

The treatment of traumatic brain injury (TBI) remains a challenge due to limited knowledge about the mechanisms underlying neuronal regeneration. This current study compared the expression of WNT genes during regeneration of injured cortical neurons. Recombinant WNT3A showed positive effect in promoting neuronal regeneration via in vitro, ex vivo, and in vivo TBI models. Intranasal administration of WNT3A protein to TBI mice increased the number of NeuN+ neurons without affecting GFAP+ glial cells, compared to control mice, as well as retained motor function based on functional behavior analysis. Our findings demonstrated that WNT3A, 8A, 9B, and 10A promote regeneration of injured cortical neurons. Among these WNTs, WNT3A showed the most promising regenerative potential in vivo, ex vivo, and in vitro.

Expression of apoptosis and myogenesis related genes during prenatal life in two divergent breeds of pigs

We aimed to evaluate apoptosis and myogenesis related genes expression in embryos and fetuses from two divergent genetic groups of pigs: Piau breed and a commercial line. Thirty females (15 Piau and 15 commercial line) were selected at 120 days of age. Estrous cycle was observed and on the third estrus females were considered sexually mature. Gilts were inseminated with semen from males of the respective breed. Three females from each breed were slaughtered at five different gestational ages: 15, 30, 45, 60 and 90 days. Whole embryos (15 and 30 d) and samples of longissimus dorsi muscle from fetuses (45, 60 and 90 d) were collected for RNA extraction. Expression of apoptosis and myogenesis related genes (BAX, BCL2, FGF4, IHH, HHIP, SHH, SOX2, WNT1 and WNT4) were evaluated by quantitative real time PCR. There was significant effect of interaction between breeds and gestational ages for all genes evaluated (P < 0.05). The BCL2 gene expression differed throughout pregnancy in Piau group with lower expression on day 15. The IHH gene expression throughout pregnancy was lower on days 15 and 60 in Piau group and lower on day 90 in commercial line group; and the SHH gene expression throughout pregnancy was higher on day 30 and lower on day 60 in Piau group and lower on day 45 in commercial group. The WNT1 gene expression along pregnancy was lower on day 15 and higher on days 30 and 45 in Piau group, and it was higher on day 30 than days 15 and 60 in commercial group. WNT4 gene expression throughout pregnancy was lower on day 15 in Piau group, and it was lower on day 30 in commercial pigs. Piau group presented higher expression of the FGF4 gene on days 45, 60 and 90, and commercial group showed higher expression on day 15 and 90. SOX2 gene expression was lower on day 15 in Piau pigs and it was constant throughout pregnancy in commercial group. Overall, the expression of IHH, SHH, WNT1, WNT4 and FGF4 genes were higher in commercial than Piau pigs on day 15, besides expression of BCL2 gene in Piau embryos was lower on day 15; these results might indicate that the muscle precursor cells are allowed to proliferate for a longer time in commercial than in Piau embryos by the balance of proliferative and apoptotic genes. Therefore, the expression differential between breeds can stimulate proliferation and differentiation of cells in different ways, explaining the postnatal differences in the muscularity between pigs from Piau breed and a commercial line.

Molecular Mechanism of Runx2-Dependent Bone Development.

Runx2 is an essential transcription factor for skeletal development. It is expressed in multipotent mesenchymal cells, osteoblast-lineage cells, and chondrocytes. Runx2 plays a major role in chondrocyte maturation, and Runx3 is partly involved. Runx2 regulates chondrocyte proliferation by directly regulating Ihh expression. It also determines whether chondrocytes become those that form transient cartilage or permanent cartilage, and functions in the pathogenesis of osteoarthritis. Runx2 is essential for osteoblast differentiation and is required for the proliferation of osteoprogenitors. Ihh is required for Runx2 expression in osteoprogenitors, and hedgehog signaling and Runx2 induce the differentiation of osteoprogenitors to preosteoblasts in endochondral bone. Runx2 induces Sp7 expression, and Runx2, Sp7, and canonical Wnt signaling are required for the differentiation of preosteoblasts to immature osteoblasts. It also induces the proliferation of osteoprogenitors by directly regulating the expression of Fgfr2 and Fgfr3. Furthermore, Runx2 induces the proliferation of mesenchymal cells and their commitment into osteoblast-lineage cells through the induction of hedgehog (Gli1, Ptch1, Ihh), Fgf (Fgfr2, Fgfr3), Wnt (Tcf7, Wnt10b), and Pthlh (Pth1r) signaling pathway gene expression in calvaria, and more than a half-dosage of Runx2 is required for their expression. This is a major cause of cleidocranial dysplasia, which is caused by heterozygous mutation of RUNX2. Cbfb, which is a co-transcription factor that forms a heterodimer with Runx2, enhances DNA binding of Runx2 and stabilizes Runx2 protein by inhibiting its ubiquitination. Thus, Runx2/Cbfb regulates the proliferation and differentiation of chondrocytes and osteoblast-lineage cells by activating multiple signaling pathways and via their reciprocal regulation.

Investigation of grape sap on wingless/integrated and β-catenin genes expression with histological factors on the hair follicle in rat.

Background and purpose:Natural products are used to improve the damage caused by harmful reagents in various pathological situations. This study investigated the effect of grape sap as a natural product with antioxidant properties on follicle cell proliferation in bleomycin (as a chemotherapy agent with toxic effects on hair growth) treated rats skin.Experimental approach:The bleomycin treated rats were administrated grape sap. Wingless/integrated (wnt) and β-catenin gene expression as follicle proliferative markers were evaluated using real-time polymerase chain reaction. Furthermore, histological factors and total antioxidant capacity were evaluated.Findings / Results:The data showed that, grape sap increased the number of anagenic hair follicle in grape sap (100 mg/kg) group (P < 0.001), sebaceous glands (P < 0.001), blood vessel density (P < 0.001), and hair growth length (P < 0.001). Also, wnt and β-catenin gene expression was elevated. The data showed that wnt and β-catenin gene expression were elevated in grape sap treated animals versus bleomycin group (P < 0.01 and 0.001, respectively).Conclusion and implications:Our finding showed that grape sap can be effective in increasing hair growth a gains bleomycin toxic effects on skin hair growth.

Wnt Gene Expression During Early Embryogenesis in the Nymphalid Butterfly Bicyclus anynana

The Wnt signalling pathway is an important developmental pathway involved in many cellular processes such cell cycle, proliferation and differentiation. Wnt ligands are released by source cells and signal to target cells by binding to the Frizzled receptor family and triggering changes in downstream target gene expression. Wnt signalling evolved early in animal evolution and an early expansion in the repertoire of Wnt ligands, which is thought to achieve specificity and robustness is present. There are 13 known Wnt ligand families in metazoans but little is known about these ligands in many animal lineages. Understanding the roles of these important signalling molecules in a wider range of animals is crucial to understand the regulation and evolution of cell fate during development and how this can lead to diversification. Here, we analysed the Wnt repertoire among lepidopterans, where these ligands are understudied compared to other insect orders. To be able to explore Wnt gene roles during butterfly embryogenesis we established a staging system for the well-established butterfly model, Bicyclus anynana, and assayed the expression pattern of all eight lepidopteran Wnt genes during early butterfly development, compared to their homologues in Drosophila melanogaster and Tribolium castaneum. We detected expression of Wnt1, Wnt10 and WntA in segmental stripes as well as expression of Wnt7 in the nervous system. Overall, our study provides much needed new insights into butterfly embryogenesis and potential roles of Wnt genes in specifying cell fate in these animals as well as how this compares to other animals.

Higher Plasma Sclerostin and Lower Wnt Signaling Gene Expression in White Adipose Tissue of Prediabetic South Asian Men Compared with White Caucasian Men.

BackgroundSouth Asians generally have an unfavourable metabolic phenotype compared with white Caucasians, including central obesity and insulin resistance. The Wnt protein family interacts with insulin signaling, and impaired Wnt signaling is associated with adiposity and type 2 diabetes mellitus. We aimed to investigate Wnt signaling in relation to insulin signaling in South Asians compared with white Caucasians.MethodsTen Dutch South Asian men with prediabetes and overweight or obesity and 10 matched Dutch white Caucasians were included. Blood samples were assayed for the Wnt inhibitor sclerostin. Subcutaneous white adipose tissue (WAT) and skeletal muscle biopsies were assayed for Wnt and insulin signaling gene expression with quantitative reverse transcription polymerase chain reaction (Clinicaltrials.gov NCT02291458).ResultsPlasma sclerostin was markedly higher in South Asians compared with white Caucasians (+65%, P<0.01). Additionally, expression of multiple Wnt signaling genes and key insulin signaling genes were lower in WAT in South Asians compared with white Caucasians. Moreover, in WAT in both ethnicities, Wnt signaling gene expression strongly positively correlated with insulin signaling gene expression. In skeletal muscle, WNT10B expression in South Asians was lower, but expression of other Wnt signaling and insulin signaling genes was comparable between ethnicities. Wnt and insulin signaling gene expression also positively correlated in skeletal muscle, albeit less pronounced.ConclusionSouth Asian men with overweight or obesity and prediabetes have higher plasma sclerostin and lower Wnt signaling gene expression in WAT compared with white Caucasians. We interpret that reduced Wnt signaling could contribute to impaired insulin signaling in South Asians.

The CLK inhibitor SM08502 induces anti-tumor activity and reduces Wnt pathway gene expression in gastrointestinal cancer models.

The Wnt/β-catenin signaling pathway is aberrantly activated in colorectal (CRC) and many other cancers, and novel strategies for effectively targeting it may be needed due to its complexity. In this report, SM08502, a novel small molecule in clinical development for the treatment of solid tumors, was shown to reduce Wnt pathway signaling and gene expression through potent inhibition of CDC-like kinase (CLK) activity. SM08502 inhibited serine and arginine rich splicing factor (SRSF) phosphorylation and disrupted spliceosome activity, which was associated with inhibition of Wnt pathway-related gene and protein expression. Additionally, SM08502 induced the generation of splicing variants of Wnt pathway genes, suggesting that its mechanism for inhibition of gene expression includes effects on alternative splicing. Orally administered SM08502 significantly inhibited growth of gastrointestinal tumors and decreased SRSF phosphorylation and Wnt pathway gene expression in xenograft mouse models. These data implicate CLKs in the regulation of Wnt signaling and represent a novel strategy for inhibiting Wnt pathway gene expression in cancers. SM08502 is a first-in-class CLK inhibitor being investigated in a Phase 1 clinical trial for subjects with advanced solid tumors (NCT03355066).

Retinoic acid disrupts osteogenesis in pre-osteoblasts by down-regulating WNT signaling

The skull bones are formed by osteoblasts by intramembranous ossification. WNT signaling is a regulator of bone formation. Retinoic Acid (RA) act as a teratogen affecting craniofacial development. We evaluated the effects of RA on the differentiation and mineralization of MC-3T3 cells, and on the expression of WNT components. MC-3T3 were cultured with or without 0.5 μM RA in osteogenic medium and mineralization was assessed by alizarin red staining. The expression of osteogenic marker genes and WNT genes was evaluated at several time points up to 28 days. RA significantly inhibited MC-3T3 mineralization (p < 0.01), without affecting ALP activity or Alp gene expression. Both parameters gradually increased in time. During culture, RA stimulated Runx2 expression at 14 and 28 days compared to the respective controls (p < 0.05). Also, RA significantly reduced Sp7 expression at days 14 and 21 (p < 0.05). Simultaneously, RA significantly reduced the expression of the WNT genes cMyc, Lef1, Lrp5, Lrp6 and Wnt11 compared to the controls (p < 0.05). In contrast, RA increased the expression of the WNT inhibitors Dkk1 at day 21 and Dkk2 at days 14 and 21 (p < 0.01). Our data indicate that RA disrupts osteogenic differentiation and mineralization by inhibiting WNT signaling.

Evaluation of antitumor effects of aspirin and LGK974 drugs on cellular signaling pathways, cell cycle and apoptosis in colorectal cancer cell lines compared to oxaliplatin drug

Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies. Despite recent advances in the treatment for CRC, resistance to chemotherapy drugs and recurrence of the tumor are among the main problems for treatment in this cancer. The MTT assay was performed to assess the cytotoxic effects of drugs on CRC cell lines (SW742 and SW480) and normal colon cells. Three-dimensional culture (spheroid) was also used to evaluate the effect of drugs on tumor cell masses. The rate of expression of genes was also evaluated using Real-Time PCR. The analysis of the results demonstrated that aspirin and LGK974 have cytotoxic effects on CRC cell lines, and in the IC50 dose, they disintegrate the cancerous cell masses. These drugs reduce the invasion and increase apoptosis in SW742 and SW480 cell lines. A decrease in the expression of WNT, AXIN, TCF and APC genes and an increase in the expression of β-catenin gene in the WNT signaling pathway were revealed. The genes involved in the MAPK signaling pathway such as ERK, JNK, KRAS and MEK showed a decrease in expression and a increase in expression of RAF gene. In the apoptotic pathway, increased expression of BAX and decreased expression of BCL-2 were reported. Also, decreased expression of P53, cyclin D1 and COX-2 was observed. This study demonstrates that aspirin and LGK974 could be effective in inhibiting the signaling pathways of WNT and MAPK, arresting cell cycle and inducing apoptosis in CRC cell lines.

Abstract 2976: A comparative study between the therapeutic efficacy of vitamin D3 and its analogue, paricalcitol, in the treatment of colon cancer in rat

Abstract Background: Vitamin D3 (VD) and its analogue, Paricalcitol (Pcal), have previously shown anti-tumorigenic activities against colorectal cancer (CRC). Hence, this study compared the therapeutic efficacy of both VD types with or without chemotherapy (5-FU) in azoxymethane (AOM)-induced CRC in rat. Methods: seventy male Wistar rats were equally allocated into 7 groups: Control, AOM, VD, Pcal, 5-FU, 5-FU/VD and 5-FU/Pcal. AOM was subcutaneously injected for 2 weeks (15mg/kg/week). The study duration was 25 weeks. VD (500 IU/rat/day; 3 days/week) and Pcal (2.5 μg/kg/day; 3 days/week) were injected subcutaneously from week-15 till the end of the study. 5-FU intraperitoneal injections started in week-21 (12 mg/kg/day; 4 successive days) and continued in week-22 (6 mg/kg/4 doses every other day). The colons were collected for gross and histopathological examination. The expression of VD signalling system, Wnt, β-catenin, NF-κB, COX-2, iNOS, VEGF, smad4, p21 and p27 proteins was measured by immune-histochemistry/fluorescence. The mRNA expression of Wnt, β-catenin, DKK-1 and COX-2 genes was measured by Quantitative RT-PCR. ELISA was used to measure the concentrations of TGF-β1, VEGF and COX-2 proteins in tissues. Results: The AOM and 5-FU groups showed marked inhibitions in the VD receptor and binding protein compared with controls (P &amp;lt; 0.05). VD and Pcal monotherapies significantly increased the expression of the VD molecules and the VD group showed the utmost elevations. Moreover, the tumour numbers and sizes were equally and significantly reduced in the 5-FU, VD and Pcal groups compared with the AOM group (P &amp;lt; 0.05). Interestingly, the 5-FU/VD and 5-FU/Pcal resulted in significantly lower numbers of tumours compared with the remaining study groups and the highest significant reduction was observed in the former group. At the molecular level, the 5-FU/VD markedly inhibited the pro-oncogenic molecules (Wnt, β-catenin, NF-κB, COX-2, iNOS &amp; VEGF) as well as significantly upregulated the anti-tumorigenic molecules (DKK-1, TGF-β1, smad4, p21 and p27) compared with the other study groups (P &amp;lt; 0.05). Conclusions: Vitamin D3 showed a better synergism than Pcal with 5-FU. The enhanced anti-tumorigenic actions of 5-FU/VD involved the modulation of the Wnt/β-catenin and TGF-β1/smad4 pathways, angiogenesis and cell cycle arrest. Further studies are required to elucidate the therapeutic significance of VD in clinical settings Citation Format: Mohammed Abbas Baghdadi, Mohammed Aslam, Jawwad Ahmad, Shakir Idris, Bassem Refaat. A comparative study between the therapeutic efficacy of vitamin D3 and its analogue, paricalcitol, in the treatment of colon cancer in rat [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2976.

UV irradiation-induced DNA hypomethylation around WNT1 gene: Implications for solar lentigines.

Wnt/β-catenin signalling promotes melanogenesis in melanocytes and also induces melanocytogenesis from melanocyte stem cells (McSCs). Previous study reported that WNT1, a ligand which activates Wnt/β-catenin signalling pathway, was more highly expressed in the epidermis at SLs than in normal skin areas, suggesting that WNT1 causes hyperpigmentation. To elucidate the mechanism by which WNT1 expression is increased in SLs, we examined the methylation of 5-carbon of cytosine (5mC), that is 5-methylcytosine (5mC) level, in a region within the WNT1 promoter; the methylation of the region was known to negatively regulate WNT1 gene expression. We used an immortalized cell line of human interfollicular epidermal stem cells to analyse the effect of UVB irradiation on DNA methylation level of WNT1 promoter and found that UVB irradiation caused demethylation of WNT1 promoter and promoted WNT1 mRNA expression. It was also found that UVB irradiation reduced the expression of DNA methyltransferase 1 (DNMT1), an enzyme responsible for maintaining methylation patterns during cell division. Pathological analysis of SLs and non-SL regions in the human skin revealed that both DNMT1 expression and 5mC level were decreased at SLs compared to non-SL skins. Furthermore, bisulphite sequencing showed that the methylated CpG level in WNT1 promoter was also lower at SLs than in non-SL skins. Thus, in the skin exposed to a high amount of UV rays, excessive expression of WNT1 is thought to be caused by the demethylation of WNT1 promoter, and the upregulated WNT1 promotes melanocytogenesis and melanogenesis, then resulting in SL formation.

Regulatory Effect of MiR-543 on Proliferation and Apoptosis of Human Leukemia Cell Line K562 and Its Mechanism

To investigate the regulatory effect of miR-543 on proliferation and apoptosis of human leukemia cell line K562 and its mechanism. 46 CML patients treated in our hospital from 2018.2-2018.9 was selected and enrolled in CML group, and 30 healthy persons were selected and enrolled in control group at the same time. After Lipofectamine 2000 liposome was used to transfer the miR-543 mimic and negative control (Scramble mimic) to K562 cells, CCK8 assay was used to detect the effect of miR-543 on proliferation of K562 cells; Luciferase reporter assay was used to report the targeting relationship of miR-543 to Wnt gene. Flow cytometry was used to detect the effect of miR-543 on apoptosis of K562 cells; Western blot method was used to detect the Wnt, β-catenin, BCL-2, c-MYC and BAX expression level. The level of miRNA-543 in CML patients was significantly lower than that in healthy controls (P＜0.05). The expression level of miR-543 in mimic group was significantly higher than that in blank control group (P＜0.05). The Wnt gene expression level in mimic group was significantly lower than that in blank control group (P＜0.05). The expression of luciferase of Wnt wild plasmid was decreased by miR-543 mimic (P＜0.05), and the luciferase activity of Wnt mutant plasmid was not inhibited by miR-543 mimic after mutation of binding site (P＜0.05). There was no significant difference in the proliferation ability of K562 cells between the blank control group and the negative control (Scramble mimic) group (P＞0.05). The proliferation level of K562 cells in mimic group was significantly lower than that in blank control group and negative control (Scramble mimic) group (P＜0.05). Apoptotic level of K562 cells in miR-543 mimic group was significantly higher than that in blank control group and negative control (Scramble mimic) group (P＜0.05). Western blot assay showed that Wnt, β-catenin, BCL-2 and c-MYC protein levels in miR-543 mimic group were significantly lower than those in blank control group and negative control (Scramble mimic) group (P＜0.05); BAX protein level in miR-543 mimic group was higher than that in blank control group and negative control (Scramble mimic) group (P＜0.05). miR-543 can target Wnt protein to inhibit the activity of Wnt signaling pathway, thereby inhibiting the proliferation of leukemia cells and increasing the level of apoptosis.

Effects of SM08502, a novel, oral small-molecule inhibitor of Wnt pathway signaling, on gene expression and antitumor activity in colorectal cancer (CRC) models.

e15185 Background: Aberrant activation of Wnt signaling contributing to tumorigenesis is most commonly associated with CRC (90% harbor Wnt pathway mutations). SM08502, a novel, oral Wnt signaling pathway inhibitor, was evaluated in preclinical CRC models. Methods: In vitro Wnt signaling: assessed using TOPflash β-catenin/TCF reporter assay in SW480 human CRC cells. In vitro Wnt pathway gene expression: measured by qRT-PCR in SW480 and Wnt3a-stimulated cells (HEK-293T, IEC-6), and with the Nanostring Wnt pathway array (180 genes) across a panel of 16 CRC cell lines. In vitro cell proliferation: 17 CRC cell lines were used to test cell viability following treatment. In vivo antitumor activity: Oral SM08502 was tested in CRC mouse xenografts (SW480, HCT 116) and a PDX model over 20-21 days (QD, QOD). 24-hr pharmacodynamic (PD) analysis of Wnt pathway gene expression was done in SW480 tumor explants from mice following one 25 mg/kg dose. Results: SM08502 inhibited Wnt pathway signaling (EC50 = 46 nM) in SW480 cells. Wnt pathway gene expression was inhibited by SM08502 (0.3-3 µM) in Wnt3a-stimulated cells ( AXIN2, LEF1) and SW480 ( AXIN2, CTNNB1, LEF1, MYC, TCF7, TCF7L2) at 24 hrs ( P &lt; .05 vs. vehicle) . Corresponding effects on protein expression were confirmed for all genes except CTNNB1, suggesting SM08502 acted independently of β-catenin. Nanostring array screening identified inhibition of LRP5, DVL2, BTRC, and ERBB2 by SM08502. Cell proliferation was inhibited in all 17 lines (avg. EC50 = 177 nM). In vivo, SM08502 was well tolerated and induced dose-dependent antitumor effects in xenografts and PDX models. Tumor growth inhibition for 25 mg/kg QD (max dose) was 83%, 56%, and 70% in SW480, HCT 116, and PDX, respectively. PD analysis showed significant inhibition ( P&lt; .05 vs. vehicle) of TCF7, MYC, LRP5, DVL2, and BTRC expression 8 hrs post treatment. Conclusions: In preclinical CRC models, SM08502 was a potent inhibitor of Wnt pathway signaling and gene expression. It showed strong antitumor activity in human tumor models with activating Wnt pathway mutations. The safety, tolerability, and PK of SM08502 are being evaluated in an ongoing phase 1 study (NCT03355066).