Gen Fujii

Activin receptor mRNA is expressed early in Xenopus embryogenesis and the level of the expression affects the body axis formation

Activin is a member of the transforming growth factor beta (TGF-beta) and possesses various activities in cellular control phenomena. During Xenopus embryonic development, activin is thought to act as a natural mesoderm-inducing factor. We isolated here the Xenopus activin receptor cDNA from Xenopus tadpole cDNA library and examined the expression of the Xenopus activin receptor gene during the course of early embryonic development. The Xenopus activin receptor has an 87% homology at the level of deduced amino acid sequence with the mouse activin receptor, and using the cDNA obtained, three bands of mRNA with different lengths were detected in Xenopus embryos throughout early embryogenesis. We synthesized activin receptor mRNA in vitro and tested the effect of the injection of the mRNA into Xenopus fertilized eggs on subsequent development. When the synthetic mRNA was injected into uncleaved fertilized eggs, embryos with reduced trunk structure were formed. However, when the mRNA was injected into the ventral blastomeres at the 16-cell stage, embryos with a secondary body axis were formed. These results indicate the importance of the function of activin receptor in the regulatory mechanism for body axis formation.

Podoplanin Expression Identified in Stromal Fibroblasts as a Favorable Prognostic Marker in Patients with Colorectal Carcinoma

Objective: The microenvironment of cancer plays a critical role in its progression. However, the molecular features of cancer-associated fibroblasts (CAFs) are less well understood than those of cancer cells. We investigated the clinicopathological significance of podoplanin expression in stromal fibroblasts in patients with colorectal cancer (CRC). Methods: We selected podoplanin as an upregulated marker in CAF from a DNA microarray experiment. Consequently, podoplanin was identified as an upregulated gene. Immunohistochemical podoplanin expression was investigated at the National Cancer Center Hospital, Tokyo, Japan, in 120 patients with advanced CRC, and its clinicopathological significance was examined. The biological function of podoplanin expression was also assessed by a coculture invasion assay with CRC cell lines such as HCT116 and HCT15. Results: Podoplanin expression was exclusively confined to stromal fibroblasts and absent in tumor cells. Podoplanin is absent in normal stroma except for lymphatic vessels. Staining was considered positive when over 30% of the cancer stroma was stained. Positive podoplanin expression was significantly correlated with a more distal tumor localization (p = 0.013) and a shallower depth of tumor invasion (p = 0.011). Univariate analysis revealed that negative podoplanin expression in stromal fibroblasts was significantly associated with reduced disease-specific survival (p = 0.0017) and disease-free survival (p < 0.0001). Multivariate analysis revealed that negative podoplanin expression (p = 0.016) and lymph node metastasis (p = 0.027) were significantly associated with disease-free survival. CRC cell invasion was augmented by co-culture with CAFs that were treated with siRNA for podoplanin. Conclusions: Our results suggest that a positive podoplanin expression in stromal fibroblasts could have a protective role against CRC cell invasion and is a significant indicator of a good prognosis in patients with advanced CRC, supported by biological analysis showing that podoplanin expression in CAFs is associated with decreased CRC cell invasion.

Deduced primary structure of a Xenopus proteasome subunit XC3 and expression of its mRNA during early development

Proteasome is a non-lysosomal proteinase complex ubiquitously distributed in eukaryotic cells. We isolated here the cDNA clone for one of the proteasome subunits (XC3) from Xenopus ovary cDNA libraries using rat RC3 cDNA as a prove. The cDNA is 885 bp long and encodes 234 amino acids. The deduced amino acid sequence is highly homologous (95.3%) to those of rat RC3 and human HC3 subunits. The mRNA for XC3 is one of the maternal mRNAs and detected at all the embryonic stages investigated, but its level changes in a characteristic way especially at the gastrula stage. We suggest that the highly conserved XC3 subunit plays an essential role in proteasome function and also that during Xenopus embryogenesis mRNA for XC3 subunit is replaced from maternal to newly-synthesized one probably around the gastrula stage.

Heat shock factor 1 accelerates hepatocellular carcinoma development by activating nuclear factor-κB/mitogen-activated protein kinase

Heat shock factor 1 (HSF1), a major transactivator of stress responses, has been implicated in carcinogenesis in various organs. However, little is known about the biological functions of HSF1 in the development of hepatocellular carcinoma (HCC). To clarify the functional role of HSF1 in HCC, we established HSF1-knockdown (HSF1 KD) KYN2 HCC cells by stably expressing either small hairpin RNA (shRNA) against HSF1 (i.e. HSF1 KD) or control shRNA (HSF1 control). Tumorigenicity was significantly reduced in orthotopic mice with HSF1 KD cells compared with those with HSF1 control cells. Reduced tumorigenesis in HSF1 KD cells appeared attributable to increased apoptosis and decreased proliferation. Tumor necrosis factor-α-induced apoptosis was increased in HSF1 KD cells and HSF1(-/-) mouse hepatocytes compared with controls. Decreased expression of IκB kinase γ, a positive regulator of nuclear factor-κB, was also observed in HSF1 KD cells and HSF1(-/-) mouse hepatocytes. Furthermore, expression of bcl-2-associated athanogene domain 3 (BAG3) was dramatically reduced in HSF1 KD cells and HSF1(-/-) mouse hepatocytes. We also found that epidermal growth factor-stimulated mitogen-activated protein kinase signaling was impaired in HSF1 KD cells. Clinicopathological analysis demonstrated frequent overexpression of HSF1 in human HCCs. Significant correlations between HSF1 and BAG3 protein levels and prognosis were also observed. In summary, these results identify a mechanistic link between HSF1 and liver tumorigenesis and may provide as a potential molecular target for the development of anti-HCC therapies.

Diagnostic and prognostic significance of the alternatively spliced ACTN4 variant in high-grade neuroendocrine pulmonary tumours

BACKGROUND High-grade neuroendocrine tumours (HGNTs) of the lung manifest a wide spectrum of clinical behaviour, but no method for predicting their outcome has been established. MATERIALS AND METHODS We newly established a monoclonal antibody specifically recognizing the product of the alternatively spliced ACTN4 transcript (namely, variant actinin-4), and used it to examine the expression of variant actinin-4 immunohistochemically in a total of 609 surgical specimens of various histological subtypes of lung cancer. RESULTS Variant actinin-4 was expressed in 55% (96/176) of HGNTs, but in only 0.8% (3/378) of non-neuroendocrine (NE) lung cancers. The expression of variant actinin-4 was significantly associated with poorer overall survival in HGNT patients (P=0.00021, log-rank test). Multivariate analysis using the Cox proportional hazards model showed that the expression of variant actinin-4 was the most significant independent negative predictor of survival in HGNT patients (hazard ratio (HR), 2.15; P=0.00113) after the presence of lymph node metastasis (HR, 2.25; P=0.00023). CONCLUSIONS The expression of variant actinin-4 is an independent prognostic factor for patients with HGNTs. This protein has a high affinity for filamentous actin polymers and likely promotes aggressive behaviour of cancer cells. The present clinical findings clearly support this notion.

Prevention and Intervention Trials for Colorectal Cancer

There have been a number of candidates for chemopreventive agents from synthetic drugs and natural compounds suggested to prevent colorectal cancer. However, they have shown modest efficacy in humans. The reason for this could be partly explained by the use of inappropriate models in vitro and in vivo, and the limitation of chemoprevention trials. In Japan, there are no cancer chemopreventive medicines, and few cancer chemoprevention trials to date. In contrast, an increase in the prevalence of colorectal cancer in Japan has forced us to develop more efficient chemopreventive strategies. It is now a good time to review in detail the current status and future prospects for chemoprevention of colorectal cancer with respect to the future development of chemopreventive medicines, particularly using synthetic drugs and natural compounds in Asian populations. The role and mode of action of available synthetic drugs, mainly aspirin and metformin, are reviewed. In addition, the possible impact of natural compounds with anti-inflammatory/immunosuppressive properties, such as ω3 polyunsaturated fatty acid and lactoferrin, are also reviewed.

Molecular cloning and expression of Xenopus p300/CBP

Transcriptional coactivators act as signal committers from transcriptional regulators to basal transcriptional machineries. We isolated the cDNA for p300/CBP, one of the most important transcriptional coactivators, of Xenopus. We also report its regulated expression, and the effects of microinjection of its truncated form. Xenopus p300/CBP (Xp300) encodes a 2483 amino acid protein which is highly homologous with human p300. Northern hybridization analyses indicated that Xp300 mRNA is stored in the oocyte, and is present throughout early embryogenesis of this species. In situ hybridization studies have revealed that Xp300 mRNA localization is ubiquitous throughout early embryogenesis, but that in later stages it is predominant in the neural region. Among adult tissues, Xp300 mRNA was clearly detected in some tissues, suggesting that Xp300 functions as a transcriptional regulator in various tissues. Microinjection of a carboxy-terminal-truncated form of Xp300 RNA into both cells of Xenopus two-blastomere stage embryos invoked the malformation of the embryos. The neural plates of Xp300 RNA-injected embryos were loose and the trunk area was heavily contracted. These results suggest that Xp300 is indispensable for normal development of the early embryo, especially in neural formation.

Endoscopic management of familial adenomatous polyposis in patients refusing colectomy.

BACKGROUND AND STUDY AIMS Colectomy protects against colorectal cancer in familial adenomatous polyposis (FAP); however, some patients with FAP refuse surgery. The aim of this study was to evaluate the feasibility and safety of endoscopic management of these patients. PATIENTS AND METHODS A retrospective review of medical records was performed to identify adult patients with FAP who refused colectomy and were managed by repeated colonoscopies to remove numerous polyps between 2001 and 2012. Polyps were removed by hot snare polypectomy or endoscopic mucosal resection. Polyps of < 10 mm in size and without endoscopic features suggesting cancer were discarded without histological examination; the remaining polyps were examined histologically. RESULTS Of the 95 eligible patients, five (5.3 %) were excluded. The remaining 90 patients (median age at first visit 29 years [range 16 - 68 years]; 46 males) were followed for a median of 5.1 years (interquartile range [IQR] 3.3 - 7.3 years). During this period, a total of 55 701 polyps were resected without adverse events such as bleeding or perforation. The median numbers of endoscopic treatment sessions and polyps removed per patient were 8 (IQR 6 - 11) and 475 (IQR 211 - 945), respectively. Five patients had noninvasive carcinoma (Category 4.2 according to the revised Vienna classification), detected within 10 months from the start of the follow-up period. All of these patients were treated endoscopically, without signs of recurrence during a median follow-up of 4.3 years (IQR 2.0 - 7.1 years). No invasive colorectal cancer was recorded during the study period. Two patients (2.2 %) underwent colectomy because the polyposis phenotype had changed to dense polyposis. CONCLUSION Endoscopic management of FAP is feasible and safe in the medium term.

Sesamol suppresses cyclooxygenase-2 transcriptional activity in colon cancer cells and modifies intestinal polyp development in ApcMin/+ mice

Excessive prostaglandin production by cyclooxygenase-2 in stromal and epithelial cells is a causative factor of colorectal carcinogenesis. Thus, compounds which inhibit cyclooxygenase-2 transcriptional activity in colon epithelial cells could be candidates for anti-carcinogenic agents. A cyclooxygenase-2 transcriptional activity in the human colon cancer cell line DLD-1 has been measured using a β-galactosidase reporter gene system. Using this system, we demonstrated that the decrease in basal cyclooxygenase-2 transcriptional activities at 100 µM sesamol, one of the lignans in sesame seeds, was 50%. Other compounds in sesame seeds such as sesamin, sesamolin, ferulic acid, and syringic acid did not exhibit significant suppression of cyclooxygenase-2 transcriptional activity at up to 100 µM. In a following experiment, 6-week-old male Min mice, Apc-deficient mice, were divided into a non-treated and 500 ppm sesamol groups. At the age of 15 weeks, it was found that treatment with sesamol decreased the number of polyps in the middle part of small intestine to 66.1% of the untreated value. Moreover, sesamol suppressed cyclooxygenase-2 and cytosolic prostaglandin E2 synthase mRNA in the polyp parts. The present findings may demonstrate the novel anti-carcinogenetic property of sesamol, and imply that agents that can suppress cyclooxygenase-2 expression may be useful cancer chemopreventive agents.

Suppression of intestinal carcinogenesis in Apc-mutant mice by limonin

Limonoids in citrus fruits are known to possess multiple biological functions, such as anti-proliferative functions in human cancer cell lines. Therefore, we aimed to investigate the suppressive effect of limonin on intestinal polyp development in Apc-mutant Min mice. Five-week-old female Min mice were fed a basal diet or a diet containing 250 or 500 ppm limonin for 8 weeks. The total number of polyps in mice treated with 500 ppm limonin decreased to 74% of the untreated control value. Neoplastic cell proliferation in the polyp parts was assessed by counting PCNA positive cells, and a tendency of reduction was obtained by limonin treatment. Moreover, expression levels of c-Myc and MCP-1 mRNA in the polyp part were reduced by administration of limonin. We finally confirmed the effects of limonin on β-catenin signaling, and found limonin significantly inhibited T-cell factor/lymphocyte enhancer factor-dependent transcriptional activity in a dose-dependent manner in the Caco-2 human colon cancer cell line. Our results suggest that limonin might be a candidate chemopreventive agent against intestinal carcinogenesis.