Keizo Dono

Prognostic Significance of Activated Akt Expression in Pancreatic Ductal Adenocarcinoma

Purpose: Akt is a serine/threonine kinase that plays a central role in tumorigenesis. Among the members of Akt family, Akt2 is associated with the development of human cancers. The present study was designed to clarify the prognostic significance of Akt2 and activated Akt expression in pancreatic ductal adenocarcinoma (PDAC). In addition, activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) and the proliferation activity of tumor cells detected by Ki-67 immunohistochemistry were examined. Experimental Design: Immunohistochemical analysis was performed on paraffin-embedded specimens from 65 patients with PDAC; 36 males and 29 females with ages ranging from 48 to 79 years (median, 66 years) of age. Expression levels of Akt2, phosphorylated Akt (p-Akt), and phosphorylated ERK 1/2 (p-ERK 1/2) were categorized as either weaker (low intensity) or equal to stronger (high intensity) compared with those in the endothelial cells of the same specimens. For Ki-67 immunohistochemistry, cases were divided into two groups: level 1, Ki-67 labeling index (LI), <20%; level 2, Ki-67 LI, ≥20%. Results: Twenty-six (42.6%), 28 (45.9%), 39 (63.9%), and 46 (75.4%) of the tumors showed high intensity of Akt2, p-Akt, and p-ERK 1/2 expression, and Ki-67 LI level 2, respectively. A significant positive correlation was observed between Akt2 and p-Akt expression (P < 0.01). Multivariate analysis revealed that p-Akt expression, Ki-67 LI, and histological differentiation are independent prognosticators for PDAC. Conclusions: p-Akt expression is a significant prognostic indicator for PDAC. Inhibition of Akt is a possible molecular approach for treatment of PDAC.

Involvement of ribonucleotide reductase M1 subunit overexpression in gemcitabine resistance of human pancreatic cancer.

Pancreatic cancer is the most lethal of all solid tumors partially because of its chemoresistance. Although gemcitabine is widely used as a first selected agent for the treatment of this disease despite low response rate, molecular mechanisms of gemcitabine resistance in pancreatic cancer still remain obscure. The aim of this study is to elucidate the mechanisms of gemcitabine resistance. The 81‐fold gemcitabine resistant variant MiaPaCa2‐RG was selected from pancreatic cancer cell line MiaPaCa2. By microarray analysis between MiaPaCa2 and MiaPaCa2‐RG, 43 genes (0.04%) were altered expression of more than 2‐fold. The most upregulated gene in MiaPaCa2‐RG was ribonucleotide reductase M1 subunit (RRM1) with 4.5‐fold up‐regulation. Transfection with RRM1‐specific RNAi suppressed more than 90% of RRM1 mRNA and protein expression. After RRM1‐specific RNAi transfection, gemcitabine chemoresistance of MiaPaCa2‐RG was reduced to the same level of MiaPaCa2. The 18 recurrent pancreatic cancer patients treated by gemcitabine were divided into 2 groups by RRM1 levels. There was a significant association between gemcitabine response and RRM1 expression (p = 0.018). Patients with high RRM1 levels had poor survival after gemcitabine treatment than those with low RRM1 levels (p = 0.016). RRM1 should be a key molecule in gemcitabine resistance in human pancreatic cancer through both in vitro and clinical models. RRM1 may have the potential as predictor and modulator of gemcitabine treatment.

Treatment of hepatocellular carcinoma with major portal vein thrombosis by combined therapy with subcutaneous interferon-alpha and intra-arterial 5-fluorouracil; role of type 1 interferon receptor expression.

We previously reported the beneficial effects of combination therapy of interferon (IFN)-α/5-fluorouracil (FU) for advanced hepatocellular carcinoma (HCC) with tumour thrombi in the major portal branches. This report describes the results of longer follow-up and includes more than double the number of patients relative to the original report, and evaluates the role of IFN-α/type 2 interferon receptor (IFNAR2) expression on the response to the combination therapy. The study subjects were 55 patients with advanced HCC and tumour thrombi in the major branches of the portal vein (Vp3 or 4). They were treated with at least two courses of IFN-α/5-FU without major complication. In the 55 patients, 24 (43.6%) showed objective response (eight (14.5%) showed complete response, 16 (29.1%) partial response), four (7.3%) showed no response, and 27 (49.1%) showed progressive disease. Immunohistochemically, IFNAR2 expression was detected in nine out of 13 (69.2%) patients. There was significant difference in the time-to-progression survival (P=0.0002) and the overall survival (P<0.0001) between IFNAR2-positive and -negative cases. There was a significant correlation between IFNAR2 expression and response to IFN-α/5-FU combination therapy in univariate analysis (P=0.0070). IFN-α/5-FU combination therapy is a promising modality for advanced HCC with tumour thrombi in the major portal branches and could significantly depend on IFNAR2 expression.

JTE-522, a cyclooxygenase-2 inhibitor, is an effective chemopreventive agent against rat experimental liver fibrosis1 ☆

BACKGROUND & AIMS The aim of this study was to assess the effects of cyclooxygenase (COX)-2 inhibition on rat experimental liver fibrogenesis. METHODS We investigated the inhibitory effects of a selective COX-2 inhibitor, JTE-522, on liver fibrosis induced by a choline-deficient, l-amino acid-defined diet (CDAA). Inhibitory effect was also tested in a second model of thioacetamide (TAA)-induced liver fibrosis. RESULTS CDAA induced liver fibrosis and preneoplastic foci at 12 weeks and cirrhosis at 36 weeks. Hepatocellular carcinoma was noted in 13 of 15 rats (87%). JTE-522 significantly inhibited fibrosis and development of preneoplastic lesions in a dose-dependent manner and completely inhibited generation of cirrhosis and hepatocellular carcinoma at both low and high doses (10 and 30 mg/kg body wt/day, respectively). JTE-522 administrated only from 12 weeks to 36 weeks also prevented cirrhosis and formation of hepatocellular carcinoma. JTE-522 itself did not cause local or systemic gross or histopathologic changes at 36 weeks. Mechanistic studies indicated that the CDAA model displayed up-regulation of several biomarkers, including COX-2, arachidonate metabolite (prostaglandin E(2)), serum aspartate aminotransferase, and c-myc expression. The model also showed an increased proportion of activated hepatic stellate cells, proliferating cell nuclear antigen index, and CD45-positive inflammatory cells in the liver. JTE-522 effectively diminished these changes. JTE-522 exhibited similar antifibrosis effects in the TAA model. CONCLUSIONS Our results suggest that COX-2 is involved in CDAA- and TAA-induced liver fibrosis. Our data also indicate that JTE-522 is a potent chemopreventive agent of rat liver fibrosis with low toxicity.

Elevated Expression of Valosin-Containing Protein (p97) in Hepatocellular Carcinoma Is Correlated With Increased Incidence of Tumor Recurrence

PURPOSE Valosin-containing protein (VCP; also known as p97) has been shown to be associated with antiapoptotic function and metastasis via activation of the nuclear factor-kappaB signaling pathway. In this study, association of VCP expression with recurrence of hepatocellular carcinoma (HCC) and patient survival was examined. PATIENTS AND METHODS VCP expression in 170 patients (139 male and 31 female) with ages ranging from 31 to 81 years (median, 61 years) was analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, in which staining intensity in tumor cells was categorized as weaker (level 1) or equal to or stronger (level 2) than that in endothelial cells. RESULTS Immunohistochemically, 57 patients (35.2%) showed level 1, and 105 patients (64.8%) showed level 2, VCP expression. Quantitative RT-PCR analysis revealed higher VCP mRNA expression in level 2 patients (n = 7) than level 1 (n = 4) (P <.05). Patients with VCP-level 2 HCC showed higher rate of portal vein invasion in the tumor (P <.01) and poorer disease-free and overall survival (P <.0001 and P <.05, respectively) compared with level 1 patients. Multivariate analysis revealed VCP expression level, tumor multiplicity, and degree of fibrosis in the noncancerous liver tissue to be independent prognosticators for disease-free and overall survival. VCP level was an indicator for disease-free survival in each early- (I and II) and advanced- (III and IV) stage group of pathologic tumor-node-metastasis classification (P <.001 and P <.01, respectively). CONCLUSION VCP expression level has prognostic significance for disease-free and overall survival of patients with HCC.

Expression pattern of angiogenic factors and prognosis after hepatic resection in hepatocellular carcinoma: importance of angiopoietin‐2 and hypoxia‐induced factor‐1a

Abstract: Background: Hepatocellular carcinoma (HCC) is a hypervascular tumor and angiogenesis plays an important role in its progression. Angiogenesis is regulated by a balance between pro and antiangiogenic molecules. The aim of this study was to investigate the expressions of angiogenic factors and elucidate their roles in angiogenesis in HCC.

Significance of RRM1 and ERCC1 expression in resectable pancreatic adenocarcinoma

The identification of molecular markers, useful for therapeutic decisions in pancreatic cancer patients, is crucial for advances in disease management. Gemcitabine, although a cornerstone of current therapy, has limited efficacy. RRM1 is a key molecule for gemcitabine efficacy and is also involved in tumor progression. We determined in situ RRM1 and excision repair cross complementation group 1 (ERCC1) protein levels in 68 pancreatic cancer patients. All had R0 resections without preoperative therapy. Protein levels were determined by automated quantitative analysis (AQUA), a fluorescence-based immunohistochemical method. The relationship between protein expressions and clinical outcomes, including response to gemcitabine at the time of disease recurrence, was determined. Patients with high RRM1 showed significantly better overall survival than patients with low expression (P=0.0196). There was a trend toward better overall survival for patient with high ERCC1 (P=0.0552). When both markers were considered together, patients with both high RRM1 and ERCC1 faired the best in terms of overall and disease-free survival (P=0.0066, P=0.0127). In addition, treatment benefit from gemcitabine in patients with disease recurrence was observed only in patients with low RRM1. The combination of RRM1 and ERCC1 expression is prognostic in pancreatic cancer patients after a complete resection. On disease recurrence, only patients with low RRM1 derive benefit from gemcitabine.

Activation of Wnt/ β -catenin signalling pathway induces chemoresistance to interferon- α /5-fluorouracil combination therapy for hepatocellular carcinoma

Type I IFN receptor type 2 (IFNAR2) expression correlates significantly with clinical response to interferon (IFN)-α/5-fluorouracil (5-FU) combination therapy for hepatocellular carcinoma (HCC). However, some IFNAR2-positive patients show no response to the therapy. This result suggests the possibility of other factors, which would be responsible for resistance to IFN-α/5-FU therapy. The aim of this study was to examine the mechanism of anti-proliferative effects of IFN-α/5-FU therapy and search for a biological marker of chemoresistance to such therapy. Gene expression profiling and molecular network analysis were used in the analysis of non-responders and responders with IFNAR2-positive HCC. The Wnt/β-catenin signalling pathway contributed to resistance to IFN-α/5-FU therapy. Immunohistochemical analysis showed positive epithelial cell adhesion molecule (Ep-CAM) expression, the target molecule of Wnt/β-catenin signalling, only in non-responders. In vitro studies showed that activation of Wnt/β-catenin signalling by glycogen synthesis kinase-3 inhibitor (6-bromoindirubin-3′-oxime (BIO)) induced chemoresistance to IFN-α/5-FU. BrdU-based cell proliferation ELISA and cell cycle analysis showed that concurrent addition of BIO and IFN-α/5-FU significantly to hepatoma cell cultures reduced the inhibitory effects of the latter two on DNA synthesis and accumulation of cells in the S-phase. The results indicate that activation of Wnt/β-catenin signalling pathway induces chemoresistance to IFN-α/5-FU therapy and suggest that Ep-CAM is a potentially useful marker for resistance to such therapy, especially in IFNAR2-positive cases.

Interferon-α and 5-fluorouracil combination therapy after palliative hepatic resection in patients with advanced hepatocellular carcinoma, portal venous tumor thrombus in the major trunk, and multiple nodules

The authors reported previously the beneficial effects of interferon (IFN)‐α/5‐fluorouracil (5‐FU) combination therapy for patients with advanced hepatocellular carcinoma (HCC) who have tumor thrombi in the major portal branches. In this report, the authors describe the results from IFN/5‐FU chemotherapy for patients who underwent palliative hepatic resection for advanced HCC with tumor thrombus in the main trunk of the portal vein and multiple nodules in the whole liver. In addition, they evaluated the correlation between the response to such therapy and expression of IFN‐α type 2 receptor (IFNAR2).

Partial Contribution of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)/TRAIL Receptor Pathway to Antitumor Effects of Interferon-α/5-Fluorouracil against Hepatocellular Carcinoma

Purpose: Our purpose was to explore the contribution of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/TRAIL receptor pathway to antitumor effects of IFNα and 5-fluorouracil (5-FU) combination therapy for hepatocellular carcinoma (HCC). Experimental Design: Susceptibility of HCC cell lines to TRAIL and/or 5-FU was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effects of 5-FU, IFNα, or both on the expression of TRAIL receptors (R1, R2, R3, and R4) on HCC cells or TRAIL in peripheral blood mononuclear cells (PBMC) were examined by flow cytometry. IFNα-induced cytotoxic effects of PBMC on HCC cell lines were examined by 51Cr release assay. TRAIL expression in peripheral blood mononuclear cells and liver tissue from patients was examined by real-time reverse transcription-PCR or immunohistochemistry. Results: HLE and HepG2 were sensitive to TRAIL, but HuH7, PLC/PRF/5, and HLF were resistant. 5-FU had synergistic effect on TRAIL in HLF and additive effect in four other HCC cell lines. TRAIL receptors on HCC cells were up-regulated by 5-FU, and IFNα induced TRAIL on CD4+ T cells, CD14+ monocytes, and CD56+ NK cells. Treatment of effector cells by IFNα and target HCC cells by 5-FU enhanced the cytotoxicity of CD14+ monocytes and CD56+ NK cells against HCC cells via a TRAIL-mediated pathway. TRAIL mRNA overexpression was noted in PBMC of HCC patients who clinically responded to IFNα/5-FU combination therapy, and TRAIL+ mononuclear cells were found in cancer tissue of a responder. Conclusion: Our results suggest that modulation of TRAIL/TRAIL receptor-mediated cytotoxic pathway might partially contribute to the anti-HCC effect of IFNα and 5-FU combination therapy.