Biomedical subjects

Hideaki Ijichi

Publications and source records attributed to Hideaki Ijichi.

18 recordsLinked to original sources

PubMed2006

Aggressive pancreatic ductal adenocarcinoma in mice caused by pancreas-specific blockade of transforming growth factor-beta signaling in cooperation with active Kras expression.

Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly lethal disease in humans. Transforming growth factor-beta (TGF-beta) signaling plays an important role in PDAC progression, as indicated by the fact that Smad4, which encodes a central signal mediator downstream from TGF-beta, is deleted or mutated in 55% and the type II TGF-beta receptor (Tgfbr2) gene is altered in a smaller subset of human PDAC. Pancreas-specific Tgfbr2 knockout mice have been generated, alone or in the context of active Kras (Kras(G12D)) expression, using the Cre-loxP system driven by the endogenous Ptf1a (pancreatic transcription factor-1a) locus. Pancreas-selective Tgfbr2 knockout alone gave no discernable phenotype in 1.5 yr. Pancreas-specific Kras(G12D) activation alone essentially generated only intraepithelial neoplasia within 1 yr. In contrast, the Tgfbr2 knockout combined with Kras(G12D) expression developed well-differentiated PDAC with 100% penetrance and a median survival of 59 d. Heterozygous deletion of Tgfbr2 with Kras(G12D) expression also developed PDAC, which indicated a haploinsufficiency of TGF-beta signaling in this genetic context. The clinical and histopathological manifestations of the combined Kras(G12D) expression and Tgfbr2 knockout mice recapitulated human PDAC. The data show that blockade of TGF-beta signaling and activated Ras signaling cooperate to promote PDAC progression.

Adenocarcinoma↗

PubMed2006

TRAIL-induced cell death cooperates with IFN-gamma activation in the graft-versus-tumor effect against colon tumors.

The graft-versus-tumor (GVT) effect that occurs following allogeneic bone marrow transplantation (BMT) and donor lymphocyte infusion (DLI) is currently being subjected to intensive investigation because of clinical evidence for GVT efficacy against leukemia. In this report, we investigate the efficacy and molecular mechanisms of GVT against solid tumors, using a modification of the mouse parent-to-F1 BMT model. Mouse Colon26 cells in which tumor necrosis factor related apoptosis-inducing ligand (TRAIL) receptor expression was stably knocked down were transplanted to investigate the role of the TRAIL-TRAIL receptor system in the GVT effect. In addition, Fas ligand-(FasL) deficient mice on a C57BL6 (B6) background were used as donors, to determine the significance of the Fas-FasL system for the antitumor effect. The group that received B6 DLI followed by preconditioning with 950 rad irradiation underwent tumor reduction associated with the induction of IFN-gamma, TRAIL and tumor-cell apoptosis. In vitro cultured Colon26 cells were resistant to TRAIL but susceptible to the combination of IFN-gamma and TRAIL in a TRAIL-dose-dependent manner. The infusion of lymphocytes from FasL-defective donors reduced the tumor progression, although efficacy was decreased in the TRAIL receptor knockdown tumors but not in wild-type ones, compared with infusion of B6-derived lymphocytes. The findings indicate that GVT activity against subcutaneous colon tumors is efficiently induced by preconditioning with irradiation and allogeneic DLI, and that TRAIL and IFN-gamma act cooperatively in the antitumor effect.

Animals↗

PubMed2005

p53-Independent negative regulation of p21/cyclin-dependent kinase-interacting protein 1 by the sonic hedgehog-glioma-associated oncogene 1 pathway in gastric carcinoma cells.

The activation of Hedgehog (Hh) signaling has been implicated in the growth of various tumor types, including gastric carcinoma. However, the precise mechanisms of Hh activation and suppression of tumor growth by the blockade of Hh signaling in gastric carcinoma cells remain unknown. The aim of this study was to elucidate the mechanism of abnormal Hh signaling and the key molecules contributing to dysregulated growth of gastric carcinoma. The Sonic hedgehog (Shh) ligand and its receptor Patched were expressed in all five gastric carcinoma cell lines examined (MKN1, MKN7, MKN45, MKN74, and AGS cells). The blockade of Hh signaling with anti-Shh antibody inhibited the growth of all five gastric carcinoma cell lines. Shh was overexpressed (mean, 12.8-fold) in 8 of 14 (57.0%) cancerous tissue samples from patients with gastric carcinoma as compared with expression in the surrounding noncancerous tissues. The disruption of glioma-associated oncogene 1 (Gli1) by small interfering RNA induced an increase in p21/cyclin-dependent kinase-interacting protein 1 (CIP1), interfered with the G1-S transition, and suppressed cell proliferation. The stimulation or inhibition of Hh signaling did not affect p53 activity and the induction of p21/CIP1 expression and the G1 arrest by inhibition of Hh signaling were not affected by the p53 status. These findings suggest that the overexpression of Shh contributes to constitutive Hh activation and that this signaling pathway negatively regulates p21/CIP1 through a Gli1-dependent and p53-independent mechanism in gastric carcinoma cells.

Adult↗

PubMed2005

Cancer-derived VEGF plays no role in malignant ascites formation in the mouse.

AIM: Vascular endothelial growth factor (VEGF) is a potent mediator of peritoneal fluid accumulation following tumor progression. This study investigated the role of VEGF secreted by cancerous cells in the formation of malignant ascites. METHODS: VEGF expression was eliminated by knockdown in the pancreas cancer cell-line PancO2 using vector-based short-hairpin type RNA interference (RNAi). Malignant ascites formation in the mouse was analyzed by intraperitoneal injection of PancO2 cells expressing VEGF or with expression knockdown. RESULTS: The VEGF knockdown PancO2 cell was successfully established. Knockdown of VEGF did not affect cancer cell proliferation in vitro or in vivo. The volume of ascites following peritoneal expansion of the tumor in VEGF knockdown cells and control cells did not differ statistically in this in vivo study. Moreover, the VEGF concentration in the ascites did not differ statistically. CONCLUSION: Malignant ascites formation might be mediated by VEGF production in noncancerous tissues, such as stromal compartments. An anti-VEGF strategy against malignant ascites could be applied to various tumors regardless of whether they secrete VEGF.

Animals↗

PubMed2005

Single small-interfering RNA expression vector for silencing multiple transforming growth factor-beta pathway components.

Although RNA interference (RNAi) is a popular technique, no method for simultaneous silencing of multiple targets by small-hairpin RNA (shRNA)-expressing RNAi vectors has yet been established. Although gene silencing can be achieved by synthetic small-interfering RNA (siRNA) duplexes, the approach is transient and largely dependent on the transfection efficiency of the host cell. We offer a solution: a simple, restriction enzyme-generated stable RNAi technique that can efficiently silence multiple targets with a single RNAi vector and a single selection marker. In this study, we succeeded in simultaneous stable knockdown of transforming growth factor beta (TGF-beta) pathway-related Smads--Smad2, Smad3 and Smad4--at the cellular level. We observed distinct phenotypic changes in TGF-beta-dependent cellular functions such as invasion, wound healing and apoptosis. This method is best suited for an analysis of complex signal transduction pathways in which silencing of a single gene cannot account for the whole process.

Cell Line↗

PubMed2005

Blockade of the stromal cell-derived factor-1/CXCR4 axis attenuates in vivo tumor growth by inhibiting angiogenesis in a vascular endothelial growth factor-independent manner.

The interaction between the chemokine receptor CXCR4 and its specific ligand, stromal cell-derived factor-1 (SDF-1/CXCL12), mediates several cellular functions. In cancer, SDF-1-positive or CXCR4-positive cells of various lineages are detected within tumor tissues. Recent intensive research has indicated the possibility that blocking CXCR4 could reduce the metastatic potential of cancer cells. Here, we show that the inhibition of the SDF-1/CXCR4 axis decreases the growth of s.c. gastrointestinal tumors through the suppression of tumor neoangiogenesis. The neutralization of CXCR4 suppressed the growth in vivo of tumors derived from mouse Colon38 and PancO2 cells, whereas it did not affect the growth of Colon38 and PancO2 cells in vitro. This attenuation of tumor growth was found to be independent of the expression of CXCR4 by the cancer cells themselves, because CXCR4 knocked-down Colon38 cells grew similarly to control cells. Furthermore, CD31-positive tumor capillaries were reduced to 45% (P < 0.001) and intratumor blood flows were decreased to 65% (P < 0.01) by blockade of CXCR4. The vascular endothelial growth factor (VEGF) concentration in the tumors was not affected by the neutralization of CXCR4. Taken together with the detection of CXCR4-positive endothelial cells in the tumor tissues, the findings suggest that the antiangiogenic effects of the blockade of CXCR4 are related to a reduction of the establishment of tumor endothelium independently of VEGF inhibition. Our data indicate that the SDF-1/CXCR4 pathway might be a general target for anticancer strategies and that blocking this system could be cooperatively effective in combination with other antiangiogenic therapies, such as blockade of VEGF.

Animals↗

PubMed2005

Smad4 silencing in pancreatic cancer cell lines using stable RNA interference and gene expression profiles induced by transforming growth factor-beta.

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using the stable RNA interference (RNAi) method. Smad4 protein expression was reduced dramatically and TGF-beta-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-beta and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-beta. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-beta stimulation. Quantitative RT-PCR analysis on several of these genes produced results that corroborated the outcome of the microarray analysis. Most of the genes in the S4KD and control cells identified by the array differed, which suggests signaling pathways that differ according to Smad4 status. Of the identified genes, 246 have not been reported previously as genes that lie downstream of TGF-beta. Genes that are involved in cell proliferation, adhesion, and motility were found to be regulated differentially with respect to S4KD and control cells. Cell migration induced by TGF-beta was inhibited in the S4KD cells, which might be associated with a different regulation of integrin beta7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.

Cell Line, Tumor↗

PubMed2005

Reduced p38 mitogen-activated protein kinase in donor grafts accelerates acute intestinal graft-versus-host disease in mice.

The gastrointestinal tract is a major target of graft-versus-host disease (GVHD), which constitutes a life-threatening complication of bone marrow transplantation. GVHD is mainly caused by the activation of donor-derived lymphocytes, in which cytokine cascades play essential roles. Since p38 MAPK (p38) has been identified as a regulator of cytokine reactions and proposed as a molecular target for anti-inflammatory therapy, we investigated the contribution of p38 to the severity of murine intestinal GVHD. Unexpectedly, p38alpha(+/-) donor graft induced more acute GVHD-related mortality and more severe gut injury. The survival of p38alpha(+/-) donor-derived intestinal intraepithelial lymphocytes (IEL) was prolonged in vitro and in vivo, and TNF-alpha expression in the p38alpha(+/-) donor-derived IEL was also increased compared with wild-type cells. In contrast, the p38alpha(+/-) grafted mice resulted in decreased expansion of donor lymphocytes in mesenteric lymph nodes, and the up-regulation of IL-12p40 and IL-18 was diminished. These findings suggest that p38 has dichotomous effects for inflammatory response in vivo; not only regulates inflammatory cytokine expression and lymphocyte expansion, but also has distinct regulatory functions for IEL in intestinal GVHD. In conclusion, the inhibition of p38 may not be a suitable anti-inflammatory strategy for GVHD due to the associated intestinal injury.

Acute Disease↗

PubMed2004

Interaction of the hepatitis B virus X protein (HBx) with heat shock protein 60 enhances HBx-mediated apoptosis.

Understanding the function of the hepatitis B virus X protein (HBx) is fundamental to elucidating the underlying mechanisms of hepatitis and hepatocarcinogenesis caused by hepatitis B virus (HBV) infection. We identified heat shock protein 60 (Hsp60) as a novel cellular target of HBx by the combination of affinity purification and mass spectrometry. Physical interaction between HBx and Hsp60 was confirmed by standard immunoprecipitation and immunoblot methods. Analysis of HBx deletion constructs showed that amino acids 88-117 of HBx were responsible for the binding to Hsp60. Confocal laser microscopy demonstrated that HBx and Hsp60 colocalized in mitochondria. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL) revealed that the introduction of Hsp60 into cells facilitated HBx-induced apoptosis. These findings suggest the importance of the molecular chaperon protein Hsp60 to the function of HBV viral proteins.

Amino Acid Sequence↗

PubMed2004

Proteomic analysis of the TGF-beta signaling pathway in pancreatic carcinoma cells using stable RNA interference to silence Smad4 expression.

Smad4 is a tumor-suppressor gene that is lost or mutated in 50% of pancreatic carcinomas. Smad4 is also an intracellular transmitter of transforming growth factor-beta (TGF-beta) signals. Although its tumor-suppressor function is presumed to reside in its capacity to mediate TGF-beta-induced growth inhibition, there seems to be a Smad4-independent TGF-beta signaling pathway. Here, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using stable RNA interference. Smad4 protein expression and TGF-beta-Smad4 signaling were impaired in S4KD cells, and we compared the proteomic changes with TGF-beta stimulation using two-dimensional gel electrophoresis (2-DE) and mass spectrometry. We identified five proteins that were up-regulated and seven proteins that were down-regulated; 10 of them were novel targets for TGF-beta. These proteins function in processes such as cytoskeletal regulation, cell cycle, and oxidative stress. Introducing siRNA-mediated gene silencing into proteomics revealed a novel TGF-beta signal pathway that did not involve Smad4.

Blotting, Western↗

PubMed2004

Different effects of point mutations within the B-Raf glycine-rich loop in colorectal tumors on mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase and nuclear factor kappaB pathway and cellular transformation.

Recently, mutations in the B-Raf gene have been identified in a variety of human cancers, such as melanoma and colorectal carcinoma, and more than 80% of the B-Raf mutations have been V599E. Although other mutations have been reported, their functional consequences are poorly understood. In our earlier study, we demonstrated that colon tumor-associated B-Raf mutations within the kinase activation segment are not necessarily associated with an increase in mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase (MEK/Erk) or nuclear factor kappaB (NFkappaB) signaling activity or in NIH3T3-transforming ability. In this study, we examined the effect of colon tumor-associated mutations within the B-Raf glycine-rich loop (G loop) on MEK/Erk and NFkappaB signaling and on the transformation of NIH3T3 fibroblasts or IEC-6 intestinal epithelial cells. Of the six G loop mutations examined, only the B-Raf G468A significantly increased MEK/Erk and NFkappaB signaling and NIH3T3 transformation. Only this mutation induced transformed phenotypes of IEC-6 cells. In contrast, the B-Raf G468E mutation significantly decreased MEK/Erk signaling and NIH3T3 transformation and had no effect on NFkappaB signaling. The B-Raf F467C mutation moderately elevated MEK/Erk signaling and NIH3T3 transformation. The other three B-Raf mutations, R461I, I462S, and G463E, did not increase MEK/Erk or NFkappaB signaling or NIH3T3 transformation. Except for F467C, none of the tumors with B-Raf mutations examined in this study had K-Ras mutations. These results suggest that some of the B-Raf G loop mutations reported in colorectal tumors do not increase kinase or transforming activities but might contribute to carcinogenesis via other mechanisms or be irrelevant to carcinogenesis.

Amino Acid Sequence↗

PubMed2004

Smad4-independent regulation of p21/WAF1 by transforming growth factor-beta.

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, the Smad4-null pancreatic cancer cell line BxPC-3 was transfected with either the Smad4 expression vector or the empty vector and incubated in the presence or absence of TGF-beta. The cells were analysed using a cDNA microarray, which included 2280 named genes to screen for target genes regulated by TGF-beta in either a Smad4-dependent or -independent manner. The microarray and subsequent quantitative RT-PCR analysis demonstrated that the Smad4-independent and -dependent signaling pathways driven by TGF-beta upregulated only one of the 2280 genes, respectively, suggesting that Smad4-independent signaling downstream of TGF-beta might be as widespread as Smad4-dependent signaling. In this study, we demonstrated that the cyclin-dependent kinase inhibitor p21/WAF1, which has been considered the major effector of the Smad-dependent growth inhibitory signal of TGF-beta, is upregulated in a Smad4-independent manner. The upregulation occurs through Smad2/3-dependent transcriptional activation of the p21/WAF1 promoter region. These results suggest a novel mechanism of gene regulation, that is, a novel signal mediator other than Smad4.

Active Transport, Cell Nucleus↗

PubMed2004

Vitamin K2 inhibits the growth and invasiveness of hepatocellular carcinoma cells via protein kinase A activation.

Hepatocellular carcinoma (HCC) is a common human malignancy. Its high mortality rate is mainly a result of high intrahepatic recurrence and portal venous invasion (PVI). We previously reported that the development of PVI is related to levels of des-gamma-carboxy prothrombin (DCP), a serum protein that increases at a notably higher rate in patients with HCC. Because DCP is produced by a vitamin K shortage, we examined the biological effects of extrinsic supplementation of vitamin K(2) in HCC cells in vitro and in vivo. Consequently, vitamin K(2) inhibits the growth and invasion of HCC cells through the activation of protein kinase A, which modulates the activities of several transcriptional factors and inhibits the small GTPase Rho, independent of suppression of DCP. In addition, administration of vitamin K(2) to nude mice inoculated with liver tumor cells reduced both tumor growth and body weight loss. In conclusion, similar to an acyclic retinoid--which was previously reported to prevent the recurrence of HCC--vitamin K(2), another lipid-soluble vitamin, may be a promising therapeutic means for the management of HCC.

Animals↗

PubMed2004

Rapid detection of mutations in the BRAF gene using real-time polymerase chain reaction and melting curve analysis.

The BRAF gene is mutated in 66% of melanomas and less frequently in various human cancers. More than 80% of these mutations are T to A transversions at nucleotide 1796 (T1796A), leading to a substitution of glutamic acid for valine at amino acid 599 (V599E). We established a new method for rapidly detecting V599E mutations using real-time polymerase chain reaction and melting curve analysis. Furthermore, we examined mutations in gastrointestinal cancer cell lines using this method. We found a mutation in 1 of 12 (8%) colorectal cancer cell lines, but no mutation was detected in 9 gastric cancer cell lines. These results suggest that the BRAF mutation is unlikely to be involved in gastric carcinogenesis.

Amino Acid Substitution↗

PubMed2004

[TGF-beta signaling pathway in pancreatic cancer cells].

TGF-beta is a multifunctional cytokine which regulates cell growth, extracellular matrix deposition, cell differentiation, and immunosuppression etc. The signal is mainly mediated through Smad pathway to inhibit epithelial cell growth. In 50% of pancreatic cancer, Smad4/DPC4 gene is deleted or mutated, which might cause pancreatic carcinogenesis and be associated with highly invasive and metastatic character of the disease. On the other hand, TGF-beta signal itself has recently been shown to act in favor of cancer cells, especially in the late phase of tumor progression. In Smad4-inactivated pancreatic cancer cells, TGF-beta signal regulates a number of genes involved in tumor suppression and progression. The regulated genes and signaling pathways of TGF-beta signal should be investigated to obtain an effective therapeutic target molecule for pancreatic cancer.

Animals↗

PubMed2003

Functional analysis of mutations within the kinase activation segment of B-Raf in human colorectal tumors.

Mutations in the B-Raf gene have been reported in a number of human cancers, including colorectal carcinoma. More than 80% of the B-Raf mutations were V599E. Although other mutations have been reported, their functional consequences were unclear. Here, we examined the effect of colon tumor-associated B-Raf mutations within the kinase activation segment, including V599E, on extracellular signal-regulated kinase (Erk) and nuclear factor kappaB (NFkappaB) signaling, and on the transformation of NIH3T3 fibroblasts. Among the six mutations examined, only the B-Raf V599E and K600E mutations greatly increased Erk and NFkappaB signaling, and the transformation of NIH3T3 cells. The B-Raf F594L mutation moderately elevated Erk signaling and NIH3T3 transformation, but did not significantly increase NFkappaB signaling. Although the basal kinase activity of the B-Raf T598I mutant was comparable with that of wild-type, its oncogenic Ras-induced kinase activity was decreased to 60% of wild-type activity. The B-Raf D593V and G595R mutants showed severely reduced kinase activity and affected neither NFkappaB signaling nor NIH3T3 transforming activity. These results suggest that the B-Raf activation segment mutations other than V599E reported in colorectal tumors do not necessarily contribute to carcinogenesis by increasing kinase and transforming activities.

14-3-3 Proteins↗

PubMed2002

Analysis of the beta-catenin/T cell factor signaling pathway in 36 gastrointestinal and liver cancer cells.

We investigated the frequency and mechanism of beta-catenin/T cell factor (Tcf) signaling activation in a panel of 36 human gastrointestinal and liver cancer cell lines. Reporter assay and electrophoretic mobility shift assay revealed that the beta-catenin/Tcf signaling was upregulated in 12 of 12 (100%) colorectal, 5 of 8 (68%) gastric, 2 of 7 (29%) hepatic, and none of 9 pancreatic cancer cell lines. The activation of the pathway was mainly due to the mutation of adenomatous polyposis coli (APC) or beta-catenin, and Tcf-4 was highly expressed in these cell lines with upregulated signaling. Nuclear beta-catenin was observed not only in the signaling-activated cell lines, but also in 14 of 25 (56%) primary gastric cancers, 15 of 20 (75%) colon cancers, 5 of 19 (26%) hepatocellular carcinomas, and none of 13 pancreatic cancers. The presence of signaling-upregulated gastric cancer cell lines with intact APC and beta-catenin suggests the involvement of other mechanisms than mutations of APC or beta-catenin.

Carcinoma, Hepatocellular↗

PubMedDate not supplied

"Duodenal intussusception" due to adenoma of the papilla of Vater.

The case of a 55-year-old woman with a pedunculate adenoma of the papilla of Vater is presented. Diagnostic imaging modalities including ultrasonography, CT scan, magnetic resonance of cholangiopancreatography, simultaneous duodenography and cholangiography, and angiography showed a giant tumor protruding intraluminally and moving forward in the duodenum by peristalsis. It had a duodenal intussusception-like appearance, with remarkable left-lower deviation of the common bile duct and major pancreatic duct in the papilla of Vater as far as the left side of the aorta. Episodes of jaundice or ileus were absent, probably because the tumor was mobile in the duodenum. As biopsy specimens showed no malignancy and intraductal ultrasonography in the common bile duct revealed no intraductal invasion of the tumor in the papilla of Vater, the patient underwent transduodenal papillectomy with papilloplasty with pancreatic ductoplasty. Pathological diagnosis of consecutive specimens was a papillary adenoma with moderate atypia and occasional tubular structure. There seems to be an exceptional subtype of the tumor in the papilla of Vater, like this case, demonstrating the duodenal intussusception-like appearance without prominent clinical symptoms.

Adenoma↗