Fas and Fas ligand: Expression and soluble circulating levels in bile duct carcinoma.

Alteration of the Fas receptor (Fas)-/ligand (FasL) system including their soluble forms (sFas and sFasL) is thought to be one of the mechanisms preventing the immune system from rejecting the tumor cells. We investigated the tissue expression of Fas, FasL, and the alteration of sFas and sFasL in patients with bile duct carcinoma. Thirty-four samples were immunostained for Fas and FasL, and levels of sFas and sFasL in the serum of 62 patients were determined using an enzyme-linked immunoadsorbent assay. Patients with strongly positive Fas levels showed significantly better prognosis than those with weakly positive or negative Fas levels. The mean serum levels of sFas in healthy individuals were significantly higher in patients with carcinoma. However, the mean serum levels of sFasL were significantly lower in the patients with carcinoma. Serum levels of sFas and sFasL might be a significant and independent prognostic parameter in patients with bile duct carcinoma.

Induction of apoptosis by ionizing radiation and CI-1033 in HuCCT-1 cells.

CI-1033 is a quinazoline-based HER family tyrosine kinase inhibitor that is currently being evaluated as a potential anticancer agent. The present study examines the molecular mechanism by which CI-1033 induces apoptosis either as a single agent or in combination with radiation. Although CI-1033 alone did not induce apoptosis, the simultaneous exposure of cells to CI-1033 and radiation induced significant levels of apoptosis. The sequential treatment of cells with CI-1033 followed by radiation induced an even greater effect with 62.6% of cells undergoing apoptosis but this enhanced effect was not seen if cells were treated first with radiation and then CI-1033. The combination treatment induces apoptosis of HuCCT-1 via upregulation of FasL and Bid cleavage. These data suggest that modulation of the Fas-FasL pathway and activation of Bid could be useful for increasing the anti-tumor effect of CI-1033 in this type of cancer.

Gemcitabine suppresses malignant ascites of human pancreatic cancer: correlation with VEGF expression in ascites.

It has been reported that vascular endothelial growth factor (VEGF) is a potent angiogenic factor that also has the ability to increase vascular permeability. VEGF plays an important role in the development of malignant ascites in various cancers. Gemcitabine has been prescribed for patients with inoperable human pancreatic ductal carcinoma as a first-line chemotherapy. However, the response rates of patients with malignant ascites who were undergoing systemic chemotherapy were extremely limited. In the present study, we investigated the role of VEGF and the effects of gemcitabine on malignant ascites of human pancreatic ductal carcinoma. As an in vitro assay, the human pancreatic cancer cell line (SUIT-2) was incubated in DMEM supplemented with serially diluted concentrations of gemcitabine for 24 h. The expression levels of VEGF in culture media were assayed using an enzyme-linked immunosorbent assay (ELISA). As an in vivo assay, a cell suspension (1 x 10(7) cells in 100 microliters PBS) was injected into the intraperitoneal region. The mice were randomly divided into two groups (control and treated with gemcitabine). The mice were sacrificed four weeks after inoculation, the ascites volume was measured, and the extent of peritoneal dissemination was examined. The expression levels of VEGF and CD31 in peritoneal nodules were examined by immunohistochemistry. In addition, secreted VEGF protein levels were quantified using ELISA. The results show that VEGF levels in the culture medium decreased in response to gemcitabine in a dose-dependent manner. The ascites formation and peritoneal dissemination within mice were suppressed by the treatment with gemcitabine. Immunohistochemical analysis suggested that expression of VEGF and CD31 in peritoneal nodules was suppressed by gemcitabine treatment, and the VEGF protein level in ascites was significantly decreased by gemcitabine (p<0.05). These results suggest that gemcitabine controls malignant ascites and peritoneal dissemination, either directly or indirectly, via VEGF. Moreover, intraperitoneal administration of gemcitabine may be a useful therapeutic approach for patients with malignant ascites in pancreatic carcinoma.

Prognostic value of p53 and Ki-67 expression in resected or biopsy specimens of bile duct carcinoma.

For cases of inoperable bile duct carcinoma, we perform intraluminal irradiation using an 192iridium wire following endoprostheses implantation. However, the effectiveness of this procedure is uncertain, and may lead to decreased patient quality of life in some cases. Therefore, we obtained samples of bile duct carcinoma either by percutaneous transhepatic cholangioscope (PTCS) or by surgery, and studied whether expression levels of Ki-67 and p53 in these tissues could predict the effectiveness of radiotherapy (RT). Immunohistochemistry was used to determine the expression of p53 and Ki-67 in 40 resected and 18 biopsy specimens. All biopsy specimens were stage IVA according to UICC classification. Labeling indices were calculated as percentage of positively stained tumor cell nuclei of total tumor cells counted. Samples were divided into two groups according to labeling index (LI). In the resected specimens, Ki-67 LI was significantly higher in cases positive for lymphatic invasion than in negative cases (p<0.05), or advanced-stage cases (p<0.05). Also, mean Ki-67 LI was higher in tumors from cases with lymph node metastasis than without. In the biopsy specimens, a significant correlation between Ki-67 LI and the term of stent patency (p<0.05) was observed. However, there were no significant correlations between clinicopathological factors or stent patency and p53 immunoreactivity. Assessment of mean Ki-67 antigen expression, as measured by MIB-1 staining, in samples of hilar bile duct carcinoma appeared to be an important indicator of clinical behavior. Biopsy specimens obtained by PTCS may be very useful in predicting the effectiveness of RT and assist in the selection of appropriate therapies.

Screening of genes specifically activated in the pancreatic juice ductal cells from the patients with pancreatic ductal carcinoma.

Pancreatic ductal carcinoma (PDC) is one of the most intractable human malignancies. Surgical resection of PDC at curable stages is hampered by a lack of sensitive and reliable detection methods. Given that DNA microarray analysis allows the expression of thousands of genes to be monitored simultaneously, it offers a potentially suitable approach to the identification of molecular markers for the clinical diagnosis of PDC. However, a simple comparison between the transcriptomes of normal and cancerous pancreatic tissue is likely to yield misleading pseudopositive data that reflect mainly the different cellular compositions of the specimens. Indeed, a microarray comparison of normal and cancerous tissue identified the INSULIN gene as one of the genes whose expression was most specific to normal tissue. To eliminate such a "population-shift" effect, the pancreatic ductal epithelial cells were purified by MUC1-based affinity chromatography from pancreatic juice isolated from both healthy individuals and PDC patients. Analysis of these background-matched samples with DNA microarrays representing 3456 human genes resulted in the identification of candidate genes for PDC-specific markers, including those for AC133 and carcinoembryonic antigen-related cell adhesion molecule 7 (CEACAM7). Specific expression of these genes in the ductal cells of the patients with PDC was confirmed by quantitative real-time polymerase chain reaction analysis. Microarray analysis with purified pancreatic ductal cells has thus provided a basis for the development of a sensitive method for the detection of PDC that relies on pancreatic juice, which is routinely obtained in the clinical setting.

Expressions of angiogenic factors in pancreatic ductal carcinoma: a correlative study with clinicopathologic parameters and patient survival.

INTRODUCTION: It has been reported that angiogenic factors play an important role in proliferation and metastasis in various cancers.AIMTo investigate the expression of angiogenic factors and microvessel density (MVD) in pancreatic ductal carcinoma and to examine the correlations among expression of angiogenic factors, clinicopathologic parameters, and clinical prognosis. METHODOLOGY: Paraffin-embedded specimens from 55 patients with pancreatic ductal carcinoma were immunostained for vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), platelet-derived endothelial cell growth factor (PD-ECGF), and CD34. The correlations among the expression of individual angiogenic factors and MVD, the clinicopathologic features, and the clinical prognoses were analyzed statistically. RESULTS: Immunostaining demonstrated that 70.8% of pancreatic ductal carcinomas were positive for VEGF, 60.9% for FGF-2, and 57.2% for PD-ECGF. A significant correlation was observed between VEGF expression and MVD (p < 0.05) but not between FGF-2 or PD-ECGF and MVD. Although the expression of each angiogenic factor had no correlation with clinicopathologic features, the patients with tumors that showed high expression of VEGF and FGF-2 had significantly shorter survival times than those with low or no such expression (p < 0.05). CONCLUSIONS: These observations suggest that the expression of VEGF closely correlates with MVD and with a poor prognosis in pancreatic ductal carcinoma.

Irsogladine malate up-regulates gap junctional intercellular communication between pancreatic cancer cells via PKA pathway.

INTRODUCTION: Gap junctions (GJs) are intercellular channels that aid communication between coupling cells and may play a critical role in cell differentiation and growth. Connexins (Cxs) are structural proteins of GJs. Though several reports have demonstrated that Cx expression decreases in various malignant tumors, a pancreatic cancer cell line, PANC-1, was reported to express Cx43 mRNA. It is known that irsogladine malate (IM) can up-regulate gap junctional intercellular communication (GJIC). We examined the effects of IM on GJ between pancreatic cancer cells (PC cells) and the mechanism of GJ up-regulation. METHODOLOGY: GJIC between PC cells (PANC-1) was evaluated by dye transfer methods. The expression of Cx43 was estimated by Western blot analysis with immunoprecipitation sample and immunohistochemical analysis. Intracellular cAMP level was estimated by enzyme-linked immunoassay. RESULTS: IM increased cell coupling in a dose-dependent manner (0M-10 ). Western blot analysis of Cx43 revealed that PANC-1 cells expressed Cx43 protein. Treatment with IM was found to move localization of Cx43 immunoreactive spots from the cytoplasm to boundary lesions with neighboring cells, but no major change was seen in the phosphorylation state of Cx43. Intracellular cAMP level was increased by IM. The PKA inhibitor H-89 and adenylyl cyclase inhibitor SQ22536 inhibited the effects of IM. CONCLUSION: These results suggest that IM up-regulates GJIC between PC cells via regulation of the PKA pathway. It also suggests a useful adjuvant of IM to pancreatic cancer therapy.