Kazuhiro Kami

N-Cadherin Expression and Epithelial-Mesenchymal Transition in Pancreatic Carcinoma

Purpose: Loss of intercellular adhesion and increased cell motility promote tumor cell invasion. In the present study, E- and N-cadherin, members of the classical cadherin family, are investigated as inducers of epithelial-to-mesenchymal transition (EMT) that is thought to play a fundamental role during the early steps of invasion and metastasis of carcinomas. Cell growth factors are known to regulate cell adhesion molecules. The purpose of the study presented here was to investigate whether a gain in N-cadherin in pancreatic cancer is involved in the process of metastasis via EMT and whether its expression is affected by growth factors. Experimental Design: We immunohistochemically examined the expression of N- and E-cadherins and vimentin, a mesenchymal marker, in pancreatic primary and metastatic tumors. Correlations among the expressions of N-cadherin, transforming growth factor (TGF)β, and fibroblast growth factor 2 was evaluated in both tumors, and the induction of cadherin and vimentin by growth factors was examined in cultured cell lines. Results: N-cadherin expression was observed in 13 of 30 primary tumors and in 8 of 15 metastatic tumors. N-cadherin expression correlated with neural invasion (P = 0.008), histological type (P = 0.043), fibroblast growth factor expression in primary tumors (P = 0.007), and TGF expression (P = 0.004) and vimentin (P = 0.01) in metastatic tumors. Vimentin, a mesenchymal marker, was observed in a few cancer cells of primary tumor but was substantially expressed in liver metastasis. TGF stimulated N-cadherin and vimentin protein expression and decreased E-cadherin expression of Panc-1 cells with morphological change. Conclusion: This study provided the morphological evidence of EMT in pancreatic carcinoma and revealed that overexpression of N-cadherin is involved in EMT and is affected by growth factors.

In vivo antitumor effect of the mTOR inhibitor CCI‐779 and gemcitabine in xenograft models of human pancreatic cancer

Mammalian target of rapamycin (mTOR) is considered to be a major effector of cell growth and proliferation that controls protein synthesis through a large number of downstream targets. We investigated the expression of the phosphatidylinositol 3′‐kinase (PI3K)/mTOR signaling pathway in human pancreatic cancer cells and tissues, and the in vivo antitumor effects of the mTOR inhibitor CCI‐779 with/without gemcitabine in xenograft models of human pancreatic cancer. We found that the Akt, mTOR and p70 S6 kinase (S6K1) from the PI3K/mTOR signaling pathway were activated in all of the pancreatic cancer cell lines examined. When surgically resected tissue specimens of pancreatic ductal adenocarcinoma were examined, phosphorylation of Akt, mTOR and S6K1 was detected in 50, 55 and 65% of the specimens, respectively. Although CCI‐779 had no additive or synergistic antiproliferative effect when combined with gemcitabine in vitro, it showed significant antitumor activity in the AsPC‐1 subcutaneous xenograft model as both a single agent and in combination with gemictabine. Furthermore, in the Suit‐2 peritoneal dissemination xenograft model, the combination of these 2 drugs achieved significantly better survival when compared with CCI‐779 or gemcitabine alone. These results demonstrate promising activity of the mTOR inhibitor CCI‐779 against human pancreatic cancer, and suggest that the inhibition of mTOR signaling can be exploited as a potentially tumor‐selective therapeutic strategy.

Prognostic Implication of Para-aortic Lymph Node Metastasis in Resectable Pancreatic Cancer

BackgroundThe survival curve of patients who undergo surgical resection of pancreatic cancer displays a steep decline within 1 year and a relatively slow decline thereafter. The patients with a short survival time may have identifiable clinicopathologic factors that lead to rapid relapse.Study DesignWe analyzed clinicopathologic factors in 133 patients who underwent margin-negative pancreatoduodenectomy with extended radical lymphadenectomy for invasive ductal carcinoma of the pancreas to detect factors that could be responsible for the short survival.ResultsTumor size, invasion of the anterior pancreatic capsule, retroperitoneal invasion, portal venous invasion, major arterial invasion, and metastasis to the para-aortic lymph nodes were variables associated with survival time in univariate analysis. Metastasis to the para-aortic lymph nodes was the single independent factor with a significant association with mortality in multivariate analysis. Some 84% of the patients who had positive para-aortic lymph nodes died within 1 year, versus 46% of the patients with negative nodes.ConclusionsAlthough tumors that involve the para-aortic lymph nodes may technically be resectable, the expected postoperative survival time for most patients is less than 1 year. If para-aortic nodal metastasis is detected, alternative treatment strategies should be considered.

Endogenous decoy receptor 3 blocks the growth inhibition signals mediated by Fas ligand in human pancreatic adenocarcinoma

Many cancers are resistant to Fas‐mediated apoptosis despite the expression of Fas. To investigate the mechanisms by which Fas signals are attenuated, we focused on decoy receptor 3 (DcR3). DcR3 is a soluble receptor against Fas ligand belonging to the tumor necrosis factor receptor superfamily and overexpresses in some forms of cancers. Exogenous DcR3 inhibits Fas‐mediated apoptosis in Fas‐sensitive Jurkat cells. In our study, we examined the expression and function of DcR3 in pancreatic cancers. TaqMan RT‐PCR showed that DcR3 mRNA was highly expressed in pancreatic cancer cell lines (71%) and tissues (67%). Its expression significantly correlated with cancer invasion to veins. Western blotting showed that the DcR3 protein was produced and secreted in 4 of 6 cell lines. The protein expressions were compatible with the mRNA expression. Five of 7 pancreatic cancer cell lines became sensitive to agonistic anti‐Fas antibody (CH‐11) to various extents, without Fas upregulation, when exposed to CH‐11 for 48 hr after pretreatment with IFNγ. Four of 7 pancreatic cancer cell lines were inhibited from growing, compared to control cells, when cocultured with membrane‐bounded Fas ligand (mFasL) transfected lymphomas for 48 hr after pretreatment with IFNγ. DcR3 reduced this growth inhibition when added exogenously. Regression analysis showed that the DcR3 expression significantly correlated with the sensitivity to mFasL, and not to CH‐11. These results suggest that DcR3 is highly expressed in many pancreatic cancers and endogenous DcR3 blocks the growth inhibition signals mediated by mFasL. DcR3 can be a candidate target molecule for the therapeutic intervention.

Clinical Significance of Focal Adhesion Kinase in Resectable Pancreatic Cancer

Focal adhesion kinase (FAK) is a non-receptor, cytoplasmic protein tyrosine kinase that is involved in the regulation of cellular signaling, migration, apoptosis, and cell cycle progression. Previous reports have shown that FAK is expressed in various kinds of cancer tissues and cancer cell lines; however, no information is available about human pancreatic carcinoma specimens. Tissue such specimens were obtained from 50 patients who underwent pancreatic resection for pancreatic invasive ductal carcinoma at our institute from 1996 to 2002. Immunohistochemical analysis of FAK was performed in the resected specimens. Focal adhesion kinase expression in seven human pancreatic cancer cell lines was analyzed by reverse transcription polymerase chain reaction (PCR) analysis and Western blot analysis. Focal adhesion kinase expression was detected in 24 of 50 cases (48%). There was a statistically significant correlation between FAK expression and tumor size (P = 0.004), although FAK expression did not significantly correlate with other factors such as tumor histological grade, lymph node metastasis, distant metastasis, histological stage, and overall survival. Reverse transcription PCR analysis and Western blot analysis showed that FAK was expressed in all seven pancreatic cancer cell lines. Focal adhesion kinase expression was not directly related to clinicopathological factors except tumor size in pancreatic carcinoma. Focal adhesion kinase expression may not be a prognostic marker for pancreatic cancer patients.

Contribution of 18F‐fluorodeoxyglucose positron emission tomography to the diagnosis of early pancreatic carcinoma

BACKGROUND/PURPOSE Pancreatic carcinoma has a poor prognosis, and early detection is essential to allow potentially curative resection. Despite the wide array of diagnostic tools available, the detection of small pancreatic tumors remains difficult. The aim of this study was to investigate the contribution of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) to the diagnosis of early pancreatic cancer. METHODS FDG-PET was performed in 56 patients with pancreatic cancer who underwent curative surgery. The standardized uptake value (SUV) for FDG was calculated in each patient and the relationships between the SUV and various clinicopathological factors were analyzed. RESULTS The tumors ranged from 0.8 to 6.5 cm in diameter. When the cutoff value for the SUV was set at 2.5, 51 of the 56 patients (91%) had a positive FDG-PET study. The SUV did not show a significant difference in relation to tumor differentiation or pTS and pT factors. There was also no correlation between the SUV and the maximum tumor diameter (r = 0.22; P = 0.1). Five tumors had an SUV below the cutoff value, and all of these lesions had intermediate or scirrhous stroma rather than medullary stroma. CONCLUSIONS These results indicate that FDG-PET is useful for the detection of small early pancreatic cancers.

Analysis of E-, N-cadherin, alpha-, beta-, and gamma-catenin expression in human pancreatic carcinoma cell lines.

Objectives: Cadherins are cell surface glycoproteins that mediate Ca2+-dependent, homophilic cell-cell adhesion. The classic cadherins interact with either β-catenin or γ-catenin, which is bound to α-catenin that links the complex to the actin cytoskeleton. It has been reported that alteration in cadherins/catenins function or expression is found in the neoplastic process as a step in metastasis. The aim of this study was to analyze the expressions of E- and N-cadherins and catenins in human pancreatic cancer cell lines. Methods: We examined the expression of cadherins and catenins in 7 human pancreatic cancer cells by RT-PCR, Western blotting, and immunocytochemistry. The interactions between cadherins and β-catenin were assessed by immunoprecipitation. Results: E-cadherin was expressed in all cell lines except for MIAPaCa-2, whereas N-cadherin was expressed in Capan-2, CFPAC-1, BxPC-3, and PANC-1. The α-, β-, and γ-catenins were expressed and cadherins/β-catenin interactions were detected in all cadherin-expressing cells. Immunocytochemical analysis showed membranous expression of cadherins and catenins. Conclusion: The decreased or loss of cadherins and catenins expression could be involved in the tumor progression and metastasis, although these events may occur in in vivo conditions by interaction between cancer cells and extracellular matrices.

Midkine promoter-based conditionally replicative adenovirus therapy for midkine-expressing human pancreatic cancer.

BackgroundTo develop a novel therapeutic strategy for human pancreatic cancer using a midkine promoter-based conditionally replicating adenovirus.MethodsWe examined midkine mRNA expression and midkine protein expression by seven human pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, HPAC, MIAPaCa-2, PANC-1, and Suit-2), as well as by non-cancerous pancreatic tissue and pancreatic cancers. Midkine promoter activity was measured in cancer cell lines by the dual luciferase reporter assay. Adenoviral transduction efficiency was assessed by fluorescent staining of cancer cell lines using adenovirus type 5 containing the green fluorescent protein gene (Ad5GFP). Replication of adenovirus type 5 containing the 0.6 kb midkne promoter (Ad5MK) was assessed by the detection of E1 protein in cancer cell lines. The cytotoxicity of Ad5MK for cancer cells was evaluated from the extent of growth inhibition after viral infection. Infection and replication were also assessed in nude mice with subcutaneous Suit-2 tumors by intratumoral injection of Ad5MK, Ad5GFP, or vehicle. E1a mRNA expression in the treated tumors and expression of the replication-specific adenoviral hexon protein were evaluated. Finally, the anti-tumor activity of Ad5MK against intraperitoneal xenografts of Suit-2 pancreatic cancer cells was examined after intraperitoneal injection of the virus.ResultsBoth midkine mRNA expression and midkine protein expression were strong in AsPC-1 and CFPAC-1 cell liens, moderate in BxPC-3, HPAC, and Suit-2 cell lines, and weak in PANC-1 and MIAPaCa-2 cell lines. Expression of midkine mRNA was significantly stronger in pancreatic cancers than in non-cancerous pancreatic tissues. The relative luciferase activity mediated by the 0.6 kb midkne fragment in AsPC-1, PANC-1, and Suit-2 cell lines was approximately 6 to 20 times greater than that in midkne-negative MIAPaCa-2 cell lines. Pancreatic cancer cell lines exhibited a heterogeneous adenoviral transduction profile. E1A expression was higher in cell lines with strong midkine expression than in cell lines with weak midkine expression. Ad5MK showed much greater cytotoxicity for midkine-expressing Suit-2 and PANC-1 cell lines than for midkine-negative MIAPaCa-2 cell lines. In the Suit-2 subcutaneous xenograft model, expression of E1A was detected in Ad5MK-treated tumors, but not in untreated and Ad5GFP-treated tumors. In the Suit-2 intraperitoneal xenograft model, the Ad5MK group survived for significantly longer than the Ad5GFP, PBS, and untreated groups.ConclusionAd5MK has an anti-tumor effect against human pancreatic cancer cell lines that express midkine mRNA. Midkine promoter-based conditionally replicative adenovirus might be a promising new gene therapy for pancreatic cancer.

Herpes simplex virus hepatitis after pediatric liver transplantation

T. Hori, Y. Ogura, S. Okamoto, A. Nakajima, K. Kami, J. Iwasaki, Y. Yonekawa, K. Ogawa, F. Oike, Y. Takada, H. Egawa, J.H. Nguyen, S. Uemoto. Herpes simplex virus hepatitis after pediatric liver transplantation
Transpl Infect Dis 2010: 12: 353–357. All rights reserved

A New Technique for Intraoperative Continuous Biliary Drainage during Pancreatoduodenectomy

Background: The common hepatic duct is divided during the early stage of pancreatoduodenectomy. Complete and prolonged closure of the proximal common duct stump can cause liver damage in the course of this long operation, resulting in associated complications. Methods: We performed intraoperative continuous external bile drainage by a new method using a novel drainage clamp in 47 consecutive patients (drainage clamp group) and compared postoperative liver enzyme levels, inflammation markers, morbidity, and outcomes with those of a conventional clamp group (n = 40). Results: The drainage clamp group had significantly lower transaminase levels within the first 14 postoperative days than the conventional clamp group. The number of patients with elevated transaminase was significantly less in drainage clamp group than conventional clamp group (p < 0.001). There were no significant differences between these two groups in terms of mortality rates and postoperative morbidity. Conclusion: Intraoperative complete closure of the common hepatic duct contributed to postoperative elevated transaminase levels, and the continuous decompression of the hepatic duct during pancreatoduodenectomy is beneficial to patients by avoiding liver dysfunction. The novel drainage clamp is a safe and useful tool for pancreatoduodenectomy and other operative procedure where extrahepatic bile duct is dissected.