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Yasushi Adachi

Publications and source records attributed to Yasushi Adachi.

At least 19 recordsLinked to original sources
PubMed2006

Effects of low-dose irradiation on enhancement of immunity by dendritic cells.

Low-doses of irradiation have been reported to have beneficial effects, particularly anti-tumor effects. In this paper, we show the effects of the low-dose irradiation on T cell activation induced by dendritic cells (DCs). DCs, which had been pre-irradiated at 0.02-1.0 Gy from a (137)Cs source, were cultured with allogeneic T cells, and the proliferation of T cells was then examined. The 0.05Gy-pre-irradiated DCs showed the highest proliferation capacity of T cells. The 0.05Gy-irradiation does not augment the expression of major histocompatibility complexes (MHCs) or costimulatory molecules on DCs, as with non-irradiated DCs or 1Gy-irradiated DCs, but does augment the production of IL-2, IL-12 and IFN-gamma DCs. These results suggest that the low-dose irradiation augments T cell-activation capacity through cytokine production by DCs, which might shift naïve helper T cells to Th1 cells.

Animals↗
PubMed2006

Genetic and epigenetic profiling in early colorectal tumors and prediction of invasive potential in pT1 (early invasive) colorectal cancers.

Morphologically, early colorectal tumors are divided into two groups, protruded-type tumors and flat-type tumors. Although some studies have shown genetic alterations in protruded-type tumors, little is known about genetic and epigenetic alterations in flat-type tumors, as well as pT1 (early invasive) colorectal cancers (CRCs). In the current study, we compared the frequencies of genetic and epigenetic alterations of the RAS-RAF and Wnt signaling pathways in flat-type and protruded-type tumors. In addition, we investigated the relationship between those alterations and invasive potential of pT1 CRCs. Methylations of RASSF2, O-6-methylguanine-DNA methyltransferase (MGMT), Wnt inhibitory factor-1 (WIF-1), EPHB2, CDKN2A and MLH1 were detected in 44.3, 30.3, 81.4, 7.5, 43.6 and 13.4% of the 307 early colorectal tumors, respectively. Mutations of KRAS, BRAF, catalytic subunit alpha of phosphatidylinositol 3'-kinase (PIK3CA) and beta-catenin were detected in 25.4, 4.6, 1.6 and 9.4% of those tumors, respectively. Methylations of MGMT, WIF-1 and CDKN2A were detected in significantly higher percentages of protruded-type tumors than in flat-type tumors. Mutation of at least one gene was detected in a significantly higher percentage of flat-type tumors than in protruded-type tumors. RASSF2 methylation was correlated significantly with KRAS, BRAF or PIK3CA mutation. Multiple logistic analysis showed that lymphatic invasion and RASSF2 methylation with KRAS, BRAF or PIK3CA mutation were independent risk factors for venous invasion in pT1 CRCs. In conclusion, since genetic alterations of these pathways have frequently occurred in flat-type tumors, flat-type tumors seem to have a distinct genetic profile different from that of protruded-type tumors. RASSF2 methylation with oncogenic activation is a promising biomarker for predicting invasive potential of pT1 CRCs.

Aged↗
PubMed2006

Insulin-like growth factor-I receptor as a marker for prognosis and a therapeutic target in human esophageal squamous cell carcinoma.

Insulin-like growth factor (IGF)-I receptor (IGF-Ir) signaling is required for tumorigenicity and progression of many tumors but this pathway has not been well studied as a prognostic factor or potential therapeutic target in esophageal squamous cell carcinoma (ESCC). In this paper, the association between the expression of IGF-Ir and IGF-II ligand and prognosis was investigated immunohistochemically in 100 surgically resected ESCC. We then assessed the therapeutic effect of blocking IGF receptor signaling using dominant negative IGF-Ir (IGF-Ir/dn) in ESCC in vitro. Expression of IGF-Ir and IGF-II were detected in 60 and 50% of tumors, respectively, and were associated with invasion depth, metastasis, advanced tumor stage and recurrence. Patients with tumors expressing both IGF-Ir and IGF-II had a significantly shorter survival than those expressing either alone or neither in both single and multivariate analysis. IGF-Ir/dn suppressed proliferation and motility as well as upregulating chemotherapy-induced apoptosis through blocking ligand-induced Akt activation. We propose that detection of IGF-Ir/IGF-II in ESCC may be useful for the prediction of recurrence and poor prognosis and for selecting patients for IGF-Ir-targeted therapy. Therapeutic blockade of IGF-Ir may be a useful anticancer therapeutic for ESCC.

Adenoviridae↗
PubMed2006

Adenovirus expressing shRNA to IGF-1R enhances the chemosensitivity of lung cancer cell lines by blocking IGF-1 pathway.

RNA interference is a phenomenon whereby small double-stranded RNA knocks down the expression of a sequence-specific gene. Double-stranded siRNA transfection, as currently used, is considered to have transient and low transfection efficiency. We constructed an adenoviral vector-based short hair-pin(sh)RNA system to overcome the limitations of the genetic blockade of IGF-1R, one of most important cancer therapy targets. We constructed three different IGF-1R specific shRNAs (612, 801, and 3425) and generated three ad-shIGF-1Rs using BD Adeno-X expression system. We assessed the effect of ad-shIGF-1R on signal transduction, induction of apoptosis, and in vitro tumorigenicity of lung cancer cell lines. Western blot and FACS assays demonstrated that endogenous IGF-1R expression was efficiently suppressed after transduction of lung cancer cell lines with the three different ad-shIGF-1Rs. IGF-1R blockade by ad-shIGF-1R inhibited ligand induced phosphorylation of pAkt and pErk, and ad-shIGF-1R effectively blocked the in vitro tumorigenicity of lung cancer cell lines. Moreover, the transduction of a human lung cancer cell line with ad-IGF-1R(3425) enhanced chemosensitivity to anticancer drugs. We conclude that the adenoviral vector-based approach to the RNA interference of IGF-1R induced effective IGF-1R silencing in lung cancer cell lines as manifested by effective blocking of the downstream pathway of IGF-1R and by an antitumor effect. We believe that this system can be usefully applied to other cancer targets.

Adenoviridae↗
PubMed2006

Flat-type colorectal advanced adenomas (laterally spreading tumors) have different genetic and epigenetic alterations from protruded-type advanced adenomas.

Morphologically, colorectal adenomas can be divided into two groups, protruded-type and flat-type. However, the accurate frequencies of genetic and epigenetic alterations in flat-type colorectal advanced adenomas (laterally spreading tumors) have remained largely unknown. In the current study, we investigated genetic and epigenetic alterations in 101 flat-type colorectal advanced adenomas and 68 protruded-type colorectal advanced adenomas by using direct DNA sequencing and quantitative real-time PCR (MethyLight), respectively. KRAS mutation was detected in a significantly higher percentage of flat-type adenomas (35%) than in protruded-type adenomas (13%). When the samples were limited to the tumors in the distal colon, the difference of KRAS mutation was still significant. KRAS mutation in G-to-A transitions at codons 12 and 13 was detected in a significantly higher percentage of flat-type adenomas (26%) than in protruded-type adenomas (9%). BRAF and beta-catenin mutations were detected in 3 and 8% of the 101 flat-type adenomas, respectively. No significant difference was found between frequencies of those mutations in flat-type adenomas and protruded-type adenomas. Methylations of MGMT, CDKN2A (p16) and MLH1 were detected in 28, 33 and 9% of the 101 flat-type adenomas, respectively. CDKN2A methylation was detected in a significantly lower percentage of flat-type adenomas than in protruded-type adenomas (63%). Methylation of at least one gene was detected in a significantly lower percentage of flat-type adenomas (54%) than in protruded-type adenomas (78%). In conclusion, KRAS mutation was frequently detected in flat-type advanced adenomas and the mutational patterns in most of them with KRAS mutations were a transition from G-to-A. Therefore, these genetic alterations seem to play an important role in the development of flat-type advanced adenomas, especially in the distal colon. Epigenetic alterations infrequently occurred in flat-type advanced adenomas, suggesting that they have different genetic and epigenetic alterations from those of protruded-type advanced adenomas.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Mutational analysis of beta-catenin and the RAS-RAF signalling pathway in early flat-type colorectal tumours.

Morphologically, early colorectal tumours can be divided into two groups, protruded-type and flat-type. However, little is known about genetic mechanisms of the latter. We investigated mutations of beta-catenin, KRAS, BRAF, and PIK3CA in 310 early colorectal tumours. beta-catenin mutation was detected in 7.1% of 310 tumours. beta-catenin mutation was detected in a significantly higher percentage of flat-type tumours with depressed areas (4/17, 23.5%) than in other tumours (18/293, 6.1%; p=0.0246). KRAS, BRAF, and PIK3CA mutations were detected in 21.6%, 5.4%, and 1.0% of 310 tumours, respectively. Concomitant mutations of beta-catenin and KRAS or BRAF were detected in seven tumours. Mutation of at least one gene was detected in a significantly higher percentage of flat-type tumour tissues (75/193, 38.9%) than in protruded-type tumour tissues (25/117, 21.4%; p=0.0014), and it was correlated significantly with size (p=0.0001). In conclusion, beta-catenin mutation seemed to play an important role in flat-type tumours, especially in those with depressed areas. The genetic abnormalities could arise and accumulate in the early stage of colorectal tumourigenesis, and seem to contribute to the development of flat-type tumour.

Aged↗
PubMed2006

The antioxidant role of a reagent, 2',7'-dichlorodihydrofluorescin diacetate, detecting reactive-oxygen species and blocking the induction of heme oxygenase-1 and preventing cytotoxicity.

Heme oxygenase-1 (HO-1) degrades heme into biliverdin, iron and CO. The enzyme participates in adaptive and protective responses to oxidative stress and various inflammatory stimuli, and is induced in response to reactive oxygen species (ROS). 2',7'-Dichlorodihydrofluorescin diacetate (DCFH-DA) is a common reagent used to detect ROS by the oxidation of 2',7'-dichlorodihydrofluorescin (DCFH) to fluorescent dichlorodihydrofluorescein. We previously found that rapid oxidation of DCFH occurred with heme-compounds as well as ROS [Ohashi, T. et al. (2002) FEBS Lett. 511, 21-27], and then examined the effect of DCFH-DA on the induction of HO-1 expression by arsenite, cadmium and hemin, which induce oxidative stress and cytotoxicity. We found suppression of the arsenite-, cadmium- and hemin-dependent induction of HO-1 with DCFH-DA. The suppression occurred at the transcriptional level since the promoter activity of the Maf-recognition site of the HO-1 gene decreased with the DCFH-DA treatment. DCFH abolished the phosphorylation of extracellular signal-regulated kinase, the nuclear translocation of a transcriptional activator Nrf2, and cell death. An antioxidant, N-acetylcysteine (NAC), also suppressed the induction by arsenite and cadmium, but not that by hemin, indicating that DCFH blocked a different site in the stress signal pathway from NAC. Considering that the oxidation of DCFH diminishes ROS generated by various stressors, our findings provide a potential strategy for protection of cells from toxic insults using DCFH-like molecules.

Acetylcysteine↗
PubMed2006

Overexpression of poly(ADP-ribose) polymerase-1 (PARP-1) in the early stage of colorectal carcinogenesis.

Colorectal carcinogenesis is initiated mainly by aberrant activation of the Wnt signaling pathway, caused by mutation of either APC or beta-catenin (CTNNB1) gene. Poly(ADP-ribose) polymerase-1 (PARP-1) is a highly conserved nuclear enzyme, which binds tightly to DNA and plays a role in DNA repair, recombination, proliferation and genomic stability. It has recently been shown that PARP-1 is a novel co-activator of TCF-4/beta-catenin-evoked gene transactivation and may play a role in colorectal carcinogenesis. The aim of this study was to examine the PARP-1 expression and determine whether it is correlated with the expression of beta-catenin and its target genes such as c-myc, cyclin D1 and matrix metalloproteinase (MMP)-7 in the early stage of sporadic colorectal carcinogenesis. Using the semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), 91 colorectal tumours, including 65 adenomas and 26 submucosal (pT1) cancers, were analysed for the expression of PARP-1, beta-catenin, c-myc, cyclin D1 and MMP-7. Immunohistochemical analysis of PARP-1 and beta-catenin was also performed. PARP-1 mRNA overexpression was detected in 64 (70.3%) of the 91 tumours. PARP-1 overexpression was significantly correlated with tumour size and histopathology. Overexpression of beta-catenin, c-myc, cyclin D1 and MMP-7 mRNA expression was observed in 39.6%, 78.0%, 83.5% and 72.5% of the 91 tumours, respectively. PARP-1 overexpression was correlated significantly with overexpression of beta-catenin, c-myc, cyclin D1 and MMP-7. Correlation of PARP-1 expression with beta-catenin overexpression was also demonstrated by immunohistochemistry. The results suggest that PARP-1, in conjunction with beta-catenin, c-myc, cyclin D1 and MMP-7, plays an important role in the early stage of colorectal carcinogenesis.

Adenocarcinoma↗
PubMed2006

Treatment and transfer of emphysema by a new bone marrow transplantation method from normal mice to Tsk mice and vice versa.

We have recently established a new bone marrow transplantation (BMT) method in which bone marrow cells are injected into the intrabone marrow (IBM). In the present study, we used an animal model for emphysema (tight-skin [Tsk] mouse) to examine whether IBM-BMT could be used to treat emphysema in Tsk mice. IBM-BMT was carried out from C3H mice into Tsk mice (8-10 weeks old) that had already shown emphysema. Six months after transplantation, the lungs of all the Tsk mice treated with IBM-BMT [C3H-->Tsk] showed similar structures to those of normal mice, whereas the [Tsk-->Tsk] mice showed emphysema, as seen in age-matched Tsk mice. Next, we attempted to transfer emphysema from Tsk mice to C3H mice by IBM-BMT. Six months after IBM-BMT, the [Tsk-->C3H] mice showed emphysema. These results strongly suggest that emphysema in Tsk mice originates from defects of stem cells in the bone marrow.

Animals↗
PubMed2006

Myocardium-targeted delivery of endothelial progenitor cells by ultrasound-mediated microbubble destruction improves cardiac function via an angiogenic response.

Application of ultrasound-mediated destruction of microbubbles (US + Bubble) to skeletal muscle creates capillary ruptures leading to leakage of the cell components. We studied whether US + Bubble combined with bone-marrow-derived mononuclear cells (BM-MNCs) infusion enables the targeted delivery of endothelial-lineage cells into the myocardium and improves cardiac function of the cardiomyopathy model due to the paucity of neocapillary formation. Pulsed US was applied to the anterior chest of BIOTO2 cardiomyopathy hamsters for 90 s after the intravenous injection of microbubble (Optison) followed by infusion of BM-MNCs. Cardiac samples from US + microbubble + BM-MNCs (US + Bubble + BM), US + Bubble, US + BM without Bubble, and saline infusion control groups were analyzed 12 weeks after treatment. Labeled BM-MNCs transplanted by US + Bubble were found to be mainly localized in the microvessels, but not by US stimulation without microbubble (121.2 +/- 24.5 vs. 2.80 +/- 1.30 cells/mm2, P < 0.001). Capillary densities in US + Bubble + BM group were increased 1.7-fold (P < 0.05) over the control, and neither US + Bubble nor US + BM enhanced neocapillary formation. 99mTc-Tetrofosmin scintigraphy revealed that blood perfusion area in the US + Bubble + BM group was 48% greater than the control (P < 0.01). US + Bubble stimulation induces the expression of adhesion molecules (VCAM-1 and ICAM-1) in capillaries, and the US + Bubble-mediated supply of BM-MNCs increased the myocardial content of VEGF and bFGF. The left ventricular wt/body wt, area of cardiac fibrosis, and apoptotic cell numbers in the US + Bubble + BM group significantly (P < 0.05) decreased by 82%, 73%, and 64% relative to the control, respectively. The cardiac function in myopathic hamsters (assessed by fractional shortening) was markedly improved 36% (P < 0.05) by US + Bubble + BM treatment. Targeted delivery of BM-MNCs by US + Bubble to the myocardium of the cardiomyopathic hamster increased the capillary densities and regional blood flow and inhibited cardiac remodeling, resulting in the prevention of heart failure. This non-invasive cell delivery system may be useful as a novel efficient approach for angiogenic cell therapy to the myocardium.

Angiogenesis Inducing Agents↗
PubMed2006

Mechanisms underlying acceleration of blood flow recovery in ischemic limbs by macrophage colony-stimulating factor.

Recently we reported that macrophage colony-stimulating factor (M-CSF) can mobilize endothelial progenitor cells (EPCs) from the bone marrow into the peripheral blood, resulting in an increase in the number of blood vessels and augmentation of blood flow in the ischemia-induced legs. M-CSF accelerates neovascularization of ischemic lesions resulting from the mobilization of EPCs. In the present paper, we analyze the mechanisms underling the mobilization of EPCs by M-CSF. M-CSF augments the production of vascular endothelial growth factor (VEGF) from the bone marrow cells, especially from myeloid lineage cells. In vivo administration of anti-VEGF antibody abrogates both the acceleration of the recovery of blood flow in the ischemia-induced limbs by M-CSF and the augmentation of the mobilization of EPCs induced by M-CSF. These results suggest that the M-CSF contributes to rapid recovery of blood flow in ischemic lesions by mobilization of EPCs from the bone marrow through augmentation of VEGF production in the bone marrow and that the VEGF is mainly produced by myeloid lineage cells.

Animals↗
PubMed2006

Granulocyte colony-stimulating factor-mobilized circulating c-Kit+/Flk-1+ progenitor cells regenerate endothelium and inhibit neointimal hyperplasia after vascular injury.

BACKGROUND: Granulocyte colony-stimulating factor (G-CSF) treatment was shown to inhibit neointimal formation of balloon-injured vessels, whereas neither the identification of progenitor cells involved in G-CSF-mediated endothelial regeneration with a bone marrow (BM) transplant experiment nor the functional properties of regenerated endothelium have been studied. METHODS AND RESULTS: Recombinant human G-CSF (100 microg/kg per day) was injected daily for 14 days starting 3 days before balloon injury in the rat carotid artery. Neointimal formation of denuded vessels on day 14 was markedly attenuated by G-CSF (39% versus the control; P<0.05). Endothelial cell-specific immunostaining revealed an enhancement of re-endothelialization (1.8-fold increase versus the control; P<0.05) and inhibition of extravasation of Evans Blue dye (47%; P=0.02). The regenerated endothelium exhibited acetylcholine-mediated vasodilatation in NO-dependent manner. G-CSF increased the circulating c-Kit+/Flk-1+ cells (9.1-fold; P<0.02), which showed endothelial properties in vitro (acetylated low-density lipoprotein uptake and lectin binding) and incorporated into the regenerated endothelium in vivo. A BM replacement experiment with green fluorescent protein (GFP)-overexpressing cells showed that BM-derived GFP+/CD31+ endothelial cells occupied 39% of the total luminal length in the G-CSF-mediated neo-endothelium (2% in the control). CONCLUSIONS: The G-CSF-induced mobilization of BM-derived c-Kit+/Flk-1+ cells contributes to endothelial regeneration, and this cytokine therapy may be a feasible strategy for the promotion of re-endothelialization after angioplasty.

Animals↗
PubMed2006

Primary relapse of acute lymphoblastic leukemia in a cervical smear: a case report.

Uterine cervix and corpus are rarely the initial site of relapse in leukemia or lymphoma. We report herein a case of uterine cervical relapse with B-cell acute lymphoblastic leukemia (ALL). The patient, a 60-yr-old woman, had a history of ALL that had been in remission for 2 yr after chemotherapy. She presented with a chief complaint of genital bleeding. In a routine cervico-vaginal Papanicolau smear, abundant atypical lymphoid cells with round-to-oval nuclei, scant cytoplasm, and high nuclear to cytoplasmic ratios was observed. The nuclei of these cells had fine and dark chromatin and thickened nuclear membranes, with one or several nucleoli being visible. Biopsy under colposcope was performed, and a diagnosis of relapse of ALL was confirmed. The ongoing genital bleeding presented a problem with clinical management of the patient. It was decided to proceed with hysterectomy to end that problem and thereafter proceed with therapy directed against the leukemia. Our results suggest that in patients with known extrauterine cancer, the presence of malignancy in uterine cellular samples provides information regarding the extent of the neoplasm.

Fatal Outcome↗
PubMed2006

Role of matrix metalloproteinase-7 (matrilysin) in human cancer invasion, apoptosis, growth, and angiogenesis.

Matrix metalloproteinase (MMP)-7, also known as matrilysin, is a "minimal domain MMP" that exhibits proteolytic activity against components of the extracellular matrix (ECM). Matrilysin is frequently overexpressed in human cancer tissues and is associated with cancer progression. Tumorigenesis is a multistep process involving cell growth, invasion, metastasis, and angiogenesis. Matrilysin has been shown to play important roles not only in degradation of ECM proteins, but also in the regulation of several biochemical processes such as activation, degradation, and shedding of non-ECM proteins. This minire-view provides a summary of the current literature on the roles of matrilysin in tumorigenesis with a focus on the roles of modifications of non-ECM proteins by matrilysin and other related MMPs in tumorigenesis. Proteolysis of insulin-like growth factor binding protein by matrilysin results in increased bioavailability of insulin-like growth factors and enhanced cellular proliferation. Matrilysin has also been implicated in the ectodomain shedding of several cell surface molecules. Heparin-binding epidermal growth factor precursor (proHB-EGF) is cleaved by matrilysin into mature HB-EGF, which promotes cellular proliferation. Membrane-bound Fas ligand (FasL) is cleaved into soluble FasL, which increases apoptosis of cells adjacent to tumor cells. E-cadherin is converted to soluble E-cadherin to promote invasion. Tumor necrosis factor (TNF)-alpha precursor is cleaved to release soluble TNF-alpha to increase apoptosis. We propose that these matrilysin-mediated pathways provide the necessary and logical mechanisms to promote cancer progression.

Animals↗
PubMed2006

G-CSF and/or M-CSF accelerate differentiation of bone marrow cells into endothelial progenitor cells in vitro.

It has been reported that granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF) can mobilize endothelial progenitor cells (EPCs) in bone marrow cells (BMCs) into peripheral blood (PB) in vivo. Previously, we also reported that macrophage-colony stimulating factor (M-CSF) can mobilize EPCs into PB, which results in the rapid recovery of blood flow in induced-ischemia limbs by augmenting the number of intramuscular capillaries in vivo. In the present study, we demonstrate that M-CSF and/or G-CSF can increase EPCs from lineage (CD3, B220, Gr-1, Mac-1, CD11c, Ter119, NK1.1 or CD31)-negative BMCs in vitro. Lineage-negative BMCs were cultured with or without M-CSF and/or G-CSF. Three days after culture with M-CSF and/or G-CSF, the number of Flk-1+/CD45-, Sca-1+/CD45-, CD31+/CD45- or CD146+/CD45- cells increased in comparison with no cytokines. When the cultured BMCs with or without G-CSF and/or M-CSF were intravenously injected into ischemia-induced hindlimbs of mice, the number of intramuscular capillaries in the ischemia-induced legs increased; BMCs cultured with G-CSF and/or M-CSF were more effective than those of cytokine non-treated BMCs. These results suggest that M-CSF and/or G-CSF can induce the differentiation of BMCs into EPCs, even in vitro.

Animals↗
PubMed2006

Synergistic effects of granulocyte-colony stimulating factor and macrophage-colony stimulating factor on recovery of donor hematopoietic cells in allogeneic bone marrow transplantation.

G-CSF and M-CSF are used clinically to augment hematopoiesis after bone marrow transplantation (BMT) and chemotherapy. In this paper, we examined the synergistic effect of G-CSF and M-CSF on hematopoietic recovery in allogeneic BMT as a model of human BMT. We performed BMT from eGFP-transgenic mice (C57BL/6 background; H-2b) into lethally-irradiated C3H (H-2k). From the day after BMT, G-CSF and/or M-CSF were injected for 5 consecutive days. Not only the numbers of day 12 CFU-S and spleen weight, but also white blood cell (WBC) counts in the peripheral blood (PB) and nuclear cells in the bone marrow (BM) increased in the mice treated with G-CSF and/or M-CSF 12 days after BMT. Moreover, the number of donor-type WBCs in the PB and donor-type nuclear cells in the BM also increased in the mice treated with G-CSF and/or M-CSF. The effects were pronounced when G-CSF and M-CSF were used together rather than independently. These results suggest that treatment with the combination of G-CSF and M-CSF has a synergistic effect on hematopoiesis in allogeneic BMT.

Animals↗
PubMed2005

Frequent epigenetic inactivation of Wnt inhibitory factor-1 in human gastrointestinal cancers.

Aberrant activation and upregulation of the Wnt pathway is a key feature of many cancers. Wnt antagonists have recently attracted wide attention. Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that can bind to Wnt proteins directly and inhibit Wnt signaling pathway. It has been reported that WIF-1 expression is down regulated in several solid tumors and that WIF-1 is silenced by promoter hypermethylation in lung and colorectal cancer. By using RT-PCR, bisulfite sequence analysis, and methylation-specific PCR, we analysed expression and methylation of WIF-1 in cancer cell lines and freshly resected cancer tissues of the esophagus, stomach, colorectum, and pancreas. Downregulation of WIF-1 mRNA expression was observed in 61 (91.0%) of 67 cancer cell lines, 16 (80.0%) of 20 esophageal, 23 (74.2%) of 31 gastric, 41 (82.0%) of 50 colorectal, and six (75.0%) of eight pancreatic cancer tissues. Downregulation of WIF-1 expression was also observed at protein level. No significant association between WIF-1 downregulation and clinicopathological characteristics was found, suggesting that downregulation of WIF-1 expression is an early event in carcinogenesis of these cancers. Indeed, downregulation of WIF-1 expression was observed in 32 (72.7%) of 44 colorectal adenoma tissues and 18 (78.2%) of 23 early mucosal or submucosal colorectal carcinoma tissues. CpG island hypermethylation in the WIF-1 promoter region correlated with downregulation of WIF-1 expression in cancer cell lines and tissues. Treatment with demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), restored WIF-1 expression in cancer cell lines. A combined treatment of 5-aza-dC and a histone deacetylase inhibitor, trichostatinA, restored WIF-1 expression synergistically, indicating the role of cytosine methylation and histone deacetylation in the silencing of the WIF-1 gene. Transfection of the WIF-1 gene construct into TE-1 esophageal cancer cell lines or SW48 colon cancer cell lines lacking WIF-1 expression resulted in a significant inhibition on colony formation, cell proliferation, anchorage-independent growth in soft agar. TOPflash assay showed WIF-1 inhibits Wnt canonical signaling in these cell lines. These results suggest tumor suppressive function of WIF-1, due to its ability to inhibit Wnt signaling. Our results suggest that WIF-1 silencing due to promoter hypermethylation is an important mechanism underlying aberrant activation of the Wnt signaling pathway in carcinogenesis of the digestive organs. Modulation of the Wnt pathway, through reversal of WIF-1 silencing by demethylating agents, is a potential target for treatment and/or prevention of gastrointestinal cancers.

Adaptor Proteins, Signal Transducing↗