Overexpression of the receptor tyrosine kinase EphA4 in human gastric cancers.

AIM: To clarify the expression and role of Ephrin receptor A4 (EphA4) in gastric cancer in relation to clinicopathological characteristics and the expression of fibroblast growth factor receptor 1 (FGFR1) and ephrin ligands. METHODS: Eleven gastric carcinoma cell lines, 24 paired surgical fresh specimens of gastric adenocarcinoma and adjacent nontumor tissue, 74 conventional formalin-fixed, paraffin-embedded tumor specimens, and 55 specimens spotted on tissue microarray (TMA) were analyzed. Reverse transcription-PCR (RT-PCR), real-time RT-PCR, immunohistochemistry, and cell growth assays were performed. RESULTS: Overexpression of EphA4 mRNA expression was observed in 8 (73%) of 11 gastric cancer cell lines and 10 (42%) of 24 gastric cancer tissues. Overexpression of EphA4, analyzed by immunohistochemistry, was observed in 62 (48%) of 129 gastric cancer tissues. EphA4 overexpression, at the protein level, was significantly associated with depth of invasion and recurrence. EphA4 overexpression was also correlated with FGFR1 overexpression. Patients with EphA4-positive cancer had significantly shorter overall survival periods than did those with EphA4-negative cancer (P = 0.0008). The mRNAs for ephrin ligands were coexpressed in various combinations in gastric cancer cell lines and cancer tissues. Downregulation of EphA4 expression by siRNA in EphA4-overexpressing gastric cancer cell lines resulted in a significant decrease in cell growth. CONCLUSION: Our results suggest that overexpression of EphA4 plays a role in gastric cancer.

Differential expression of angiogenesis-related genes in human gastric cancers with and those without high-frequency microsatellite instability.

Gastric cancers with and those without high-frequency microsatellite instability (MSI-H) represent distinctive pathways of carcinogenesis. The aim of this study was to clarify if expression of p53 related genes involved in angiogenesis is differentially regulated between these cancers. We systematically analyzed the expression of vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2), thrombospondin 1 (THBS1), and brain-specific angiogenesis inhibitor 1 (BAI1), and we correlated the results with microvessel count (MVC), MSI status, p53 mutations, and prostaglandin-endoperoxide synthase 2 (PTGS2) expression in gastric cancers. Expression of VEGFA in carcinoma cells was immunohistochemically seen in 46% of 200 cases. VEGFA positivity was significantly associated with higher MVC, vascular invasion, lymph node and distant metastasis, and advanced tumor stage. FGF2 positivity was significantly associated with poor differentiation, depth of invasion, and higher MVC. VEGFA and FGF2 positivities and MVC were lower in MSI-H cancers than in MSI-L or MSS cancers. VEGFA expression was associated with both p53 mutations and PTGS2 expression. Methylation of the THBS1 gene was detected in 6 of 11 cancer cell lines and in 44% of 200 cases. THBS1 methylation was significantly associated with distal location, vascular invasion, distant metastasis, MSI-H, wild-type p53, and higher MVC. The prognosis was worst in patients with cancers that were VEGFA-positive and THBS1 methylation-positive. Gastric cancers with MSI-H were characterized by lower MVC, low frequency of VEGFA, FGF2, and PTGS2 overexpression, and high frequency of THBS1 methylation. Our results suggest that gastric cancers with and those without MSI-H represent distinctive pathways of carcinogenesis, including aberrant expression of factors regulating angiogenesis. The difference may be associated with less aggressive phenotype of these cancers with MSI-H and affect future molecular targeted therapeutics.

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.

Testosterone levels and discounting delayed monetary gains and losses in male humans.

OBJECTIVES: Although impulsivity has been associated with androgens (e.g., testosterone), little is known regarding the relationship between testosterone levels and impulsivity in intertemporal choice (delay discounting). This study was aimed to examine the relationship between delay discounting of gains and losses and testosterone levels, which is of interest in neuroendocrinology and neuroeconomics. METHODS: We assessed degrees to which delayed monetary gains and losses were discounted (hyperbolic discounting rate) in healty male students (age: 22.4+/-2.67). Participants' salivary testosterone levels were also assessed by utilizing liquid chromatography/mass spectroscopy (LC/MS) method. RESULTS: Non-linear curve fitting analysis showed an inverted-U relationship between delay discounting of gains and salivary testosterone levels; while no relationship between salivary testosterone levels and delay discounting of losses was observed. CONCLUSIONS: The results indicate that (i) testosterone may enhance delay discounting rate of gains in non-impulsive subjects, (ii) testosterone may have an opposite (reducing) effect on delay discounting rate of gains in impulsive subjects, and (iii) testosterone is unrelated to subject's sensitivity to future bad outcomes. Implications for evaluating the effects of testosterone treatment and anti-androgenic therapy on impulsive behavior often observed in psychiatrics (e.g., pathological gambling, credit card debt, substance misuse, and needle-sharing) are discussed.

[A case of rheumatoid arthritis/Sjogren's syndrome with acute renal failure due to hyperuricemia associated with mizoribine therapy].

A 64-year-old woman was diagnosed as having rheumatoid arthritis in 1999 at a nearby hospital. She had been treated with etodolac, actarit, mizoribine (MZ) and prednisolone. On May 25, 2001, she noticed fever and nausea and was treated with diclofenac sodium and clindamycin. On May 31, a nasal bleeding, tarry stool, hyperuricemia, renal dysfunction and thrombocytopenia developed and she was admitted to our hospital. Administration of drugs except prednisolone was stopped and hemodialysis was carried out on June 1. Fever and nausea improved during several days. Hyperuricemia and renal dysfunction disappeared on June 11. The platelet count became normal after platelet transfusion and she was discharged from our hospital on July 2. She was also diagnosed as having Sjogren's syndrome. In our case, a delay in MZ discharge by transient renal dysfunction might have caused a hyperuricemia, following an aggravation of renal dysfunction. So, care should be taken about latent renal dysfunction during the use of MZ. Moreover, it may be necessary to consider a discontinuation of MZ and administration of hemodialysis in the case of transient renal dysfunction.

[A case of Sjögren's syndrome associated with chronic hepatitis C and sarcoidosis].

A 68-year-old female had been treated for chronic type C hepatitis at our department since June, 1992. In August of the same year, swelling of the right eyelid developed and she was diagnosed as having sarcoidosis on the basis of uveitis, skin lesions, and bilateral hilar lymphadenopathy (BHL) on chest X-ray. With steroid therapy, the symptom improved and BHL disappeared. In July, 2001, dryness of the mouth and dry eyes developed, and she was diagnosed as having Sjögren's syndrome (SjS). This case may be precious in discussing the pathophysiology of sarcoidosis and SjS including the association with hepatitis C virus infection.

[A case of gastric mucosal bridge with Behçet's disease].

A 69-year-old man visiting our hospital with an epigastralgia and tarry stool was diagnosed as having Behçet's disease on the basis of repetitious aphthous stomatitis, erythema nodosum and arthralgia in 1991. The next year, he suffered from double active ulcers in the antrum of the stomach, and he had been operated on for intestinal perforation. In 1994, endoscopic examination revealed the gastric mucosal bridge between the double ulcers. The double ulcer healed after an eradication therapy of H. pylori, but the gastric mucosal bridge has remained there on the gastrointestinal endoscopy. The gastric mucosal bridge with Behçet's disease has not been reported in Japan, being considered to be very rare.

The coalescence stability of protein-stabilized emulsions estimated by analytical photo-centrifugation.

Various protein solutions were studied in order to quantify the emulsifying activity of proteins, and to explore oil-water interfacial tension, oil particle size analysis, and oil phase separation behaviors in protein-stabilized oil-in-water (O/W) emulsions. Three proteins, bovine serum albumin (BSA), ß-lactoglobulin (ß-lg), and ß-casein (ß-ca), were employed to disperse hexadecane in various pH and ionic strength solutions in a wide range of oil-water ratios. It was confirmed that the volume mean oil droplet diameter, d(43), changed depending on the oil content, the pH, the ionic strength, and the used protein. In a dilute protein solution (0.01 %) at pH 7, droplet size increased with oil content in so-called surfactant-poor regimes (e.g., above 5%, 10%, and 20% oil content for BSA, ß-lg, and ß-ca emulsion, respectively) but remained constant at ca. 10 mm, 6 mm, and 20 mm, respectively, in lower oil content surfactant-rich regimes. In surfactant-poor regimes, the most important factor determining the oil drop size was the threshold amount of protein adsorption onto the oil-water interface. In surfactant-rich regimes, on the other hand, it is suggested that drop size may be governed mainly by the mechanical strength of protein films covering the oil drops during emulsification, and this was quantified by the critical osmotic pressure, P(CR). In this study, the P(CR) was measured conveniently in the oil phase separation experiments for protein-stabilized emulsions using analytical photo-centrifugal apparatus. The correlation between the P(CR) and oil droplet size prepared by emulsification at different pH and ionic strength media is discussed.