Yoshiharu Motoo

Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development

Pancreatic cancer is a disease with an extremely poor prognosis. Tumor protein 53-induced nuclear protein 1 (TP53INP1) is a proapoptotic stress-induced p53 target gene. In this article, we show by immunohistochemical analysis that TP53INP1 expression is dramatically reduced in pancreatic ductal adenocarcinoma (PDAC) and this decrease occurs early during pancreatic cancer development. TP53INP1 reexpression in the pancreatic cancer-derived cell line MiaPaCa2 strongly reduced its capacity to form s.c., i.p., and intrapancreatic tumors in nude mice. This anti-tumoral capacity is, at least in part, due to the induction of caspase 3-mediated apoptosis. In addition, TP53INP1−/− mouse embryonic fibroblasts (MEFs) transformed with a retrovirus expressing E1A/rasV12 oncoproteins developed bigger tumors than TP53INP1+/+ transformed MEFs or TP53INP1−/− transformed MEFs with restored TP53INP1 expression. Finally, TP53INP1 expression is repressed by the oncogenic micro RNA miR-155, which is overexpressed in PDAC cells. TP53INP1 is a previously unknown miR-155 target presenting anti-tumoral activity.

Expression of Clusterin in Human Pancreatic Cancer

Introduction Clusterin, also known as apolipoprotein J, has been implicated in numerous processes, including active cell death. Clusterin is reported to be overexpressed in breast and prostate cancers. However, its expression in pancreatic cancer is yet to be reported. Aim and Methodology To examine clusterin expression and apoptosis in 52 pancreatic tissues, including specimens from 33 cases of pancreatic cancer, by immunohistochemistry. Results Clusterin was expressed in 49% (16) of 33 cases of pancreatic cancer, 50% (13) of 26 cases of chronic pancreatitis, and 67% (4) of 6 cases of mucinous cystadenoma. It was not expressed in normal pancreas. Clusterin mRNA was also expressed in the pancreatic cancer cell lines. Clusterin was significantly more highly expressed at stage I and II (well-differentiated and moderately differentiated cancers). Clusterin and apoptosis were localized in the same cells in pancreatic cancer. However, clusterin expression was not significantly associated with apoptosis in any pancreatic disease. Clusterin-positive patients with pancreatic cancer survived significantly longer. Conclusion These results suggest that clusterin expression is induced in pancreatic cancer as well as chronic pancreatitis and that downregulation of clusterin may be involved in the progression of pancreatic cancer.

Clinical study of chronic pancreatitis with focal irregular narrowing of the main pancreatic duct and mass formation: comparison with chronic pancreatitis showing diffuse irregular narrowing of the main pancreatic duct.

Introduction Main pancreatic duct (MPD)-narrowed chronic pancreatitis (CP) may be an autoimmune abnormality. It also has been called autoimmune pancreatitis and sclerosing pancreatitis. It is unclear whether cases with focal pancreatographic changes are part of the same clinical entity as cases with diffuse MPD changes. Aim and Methodology We reviewed seven cases of chronic pancreatitis (CP) with focal narrowing of the main pancreatic duct (MPD), evidenced by endoscopic retrograde cholangiopancreatography (ERCP), and swelling of one or two segments of the pancreas, evidenced by ultrasonography (US) /computed tomography (CT), and indicated the clinicopathologic features of focal-type MPD-narrowed CP. Results The patient group comprised six men and one woman, and their age range was 28–75 years, with a mean of 63.7 years. Affected sites were in the head in two patients, the body in one patient, the tail in one patient, and the body and tail in three patients; ERP showed narrowing in six patients and obstruction in one. Stricture of the lower portion of the common bile duct (CBD) that caused obstructive jaundice was shown by ERC in two cases in which the pancreas head was affected. In all six patients, a dynamic study by CT or MRI homogeneously showed delayed enhancement of involved segments of the pancreas. Serum levels of pancreatic enzyme were elevated in five patients, but only one subject had pancreatitis-like epigastric pain. Serological evidence suggestive of autoimmune abnormality was detected in only three patients with hypergammaglobulinemia (≥2.0 g/dL) or positive titers of antinuclear antibody (ANA; ≥80). Histological assessment was available for five patients, who characteristically had dense lymphocytic or plasmocytic infiltration with severe fibrosis that caused luminal narrowing. The clinical, serologic, and histologic findings as described above were comparable to those for 12 CP patients with diffuse narrowing of the MPD, diagnosed during the same period. Surgical resection was performed in 5 patients, in 2 of whom a similar inflammatory process recurred in the remnant head of the pancreas, whereas pancreatitis no longer developed in the other 3 patients. One patient was initially treated with steroids, with clinical remission, although there was neither hypergammaglobulinemia nor positive ANA. Conclusion These results indicate that CP with focal narrowing of the MPD is part of the same clinical spectrum as CP with diffuse narrowing of the MPD, and whether the distribution is diffuse or focal seems to be related to the stage or the extent of the disease. It is therefore important to recognize the possible existence of this focal variant to avoid unnecessary surgery.

Identification of K‐ras Oncogene Mutations in the Pure Pancreatic Juice of Patients with Ductal Pancreatic Cancers

Pancreatic cancer is detected on the basis of morphological changes delineated by means of various image‐diagnostic methods. However, differentiation between chronic pancreatitis and pancreatic cancer, especially at the early stage, is not always simple when based upon the morphological changes alone. Therefore, we attempted to elucidate K‐ras mutations in the sediment of pure pancreatic juice (PPJ) containing exfoliated ductal pancreatic cancer cells. PPJ was collected endoscopically from 20 patients with pancreatic cancer (PC) and 18 patients with chronic pancreatitis (CP). Polymerase chain reaction and allele specific oligonucleotide dot blot hybridization for K‐ras mutations were performed with the DNA extracted from these samples. A K‐ras mutation at codon 12 was identified in the PPJ of 11/20 (55%) of the patients with PC. On the other hand, the same mutation was not identified in the PPJ of any patient with CP. Moreover, K‐ras mutations at codons 13 and 61 were not recognized in the PPJ of any patient with either PC or CP. These findings suggested that the presence of a K‐ras mutation at codon 12 in PPJ would be useful in confirming the diagnosis of PC.

Detection of K-ras point mutations at codon 12 in pure pancreatic juice for the diagnosis of pancreatic cancer by PCR-RFLP analysis

The present study was undertaken to detect K-ras point mutations at codon 12 in pure pancreatic juice (PPJ) for the diagnosis of pancreatic cancer (PC) using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. PPJ was collected through a cannula under a duodenal fiberscope from 26 patients with PC and 32 patients with chronic pancreatitis (CP). DNA was extracted from PPJ and was used as the template for PCR. Analysis of PPJ by PCR-RFLP with Bst NI revealed that the incidence of K-ras point mutations at codon 12 was 81% (21/26) in patients with PC and 6% (2/32) in those with CP. With reference to the location of PC, the incidence of K-ras mutations was 79% (11/14) in the head, 86% (6/7) in the body, and 80% (4/5) in the tail of the pancreas. The incidence of K-ras mutants was 50% (1/2) in tumor size 1 (TS1; ≤2.0 cm in size), 71% (5/7) in TS2 (2.1 to ≤4.0 cm), 89% (8/9) in TS3 (4.1 to ≤6.0 cm), and 88% (7/8) in TS4 (>6.1 cm). These results suggested that analysis of K-ras point mutations at codon 12 in PPJ using the PCR-RFLP method is a promising new genetic test for the diagnosis of PC.

Deregulated GSK3β Sustains Gastrointestinal Cancer Cells Survival by Modulating Human Telomerase Reverse Transcriptase and Telomerase

Purpose: Glycogen synthase kinase-3 (GSK3) regulates multiple cell signaling pathways and has been implicated in glucose intolerance, neurodegenerative disorders, and inflammation. We investigated the expression, activity, and putative pathologic role of GSK3 in gastrointestinal, pancreatic, and liver cancers. Experimental Design: Colon, stomach, pancreatic, and liver cancer cell lines; nonneoplastic HEK293 cells; and matched pairs of normal and tumor tissues of stomach and colon cancer patients were examined for GSK3 expression and its phosphorylation at serine 9 (inactive form) and tyrosine 216 (active form) by Western immunoblotting and for GSK3 activity by in vitro kinase assay. The effects of small-molecule GSK3 inhibitors and of RNA interference on cell survival, proliferation, and apoptosis were examined in vitro and on human colon cancer cell xenografts in athymic mice. The effects of GSK3 inhibition on human telomerase reverse transcriptase (hTERT) expression and telomerase activity were compared between colon cancer and HEK293 cells. Results: Cancer cell lines and most cancer tissues showed increased GSK3 expression and increased tyrosine 216 phosphorylation and activity but decreased serine 9 phosphorylation compared with HEK293 cells and nonneoplastic tissues. Inhibition of GSK3 resulted in attenuated cell survival and proliferation and increased apoptosis in most cancer cell lines and in HT-29 xenografts in rodents but not in HEK293 cells. GSK3 inhibition in colon cancer cells was associated with decreased hTERT expression and telomerase activity. Conclusion: The results indicate that deregulated GSK3 sustains gastrointestinal cancer cells survival through modulation of hTERT and telomerase. (Clin Cancer Res 2009;15(22):68109)

Biliary papillomatosis with the point mutation of K-ras gene arising in congenital choledochal cyst.

Biliary papillomatosis is a rare entity. A case of biliary papillomatosis associated with congenital choledochal cyst and intrahepatic gallstones is reported here. Percutaneous transhepatic cholangioscopy revealed multiple papillary lesions of the right intrahepatic duct and the common bile duct. Microscopically, the papillary mucosal lesion showed papillary proliferations of bile duct epithelial cells with mild atypia. Furthermore, a point mutation at codon 12 of the K-ras oncogene was found in the papillary lesion. To the best of our knowledge, this is the first case of biliary papillomatosis arising in congenital choledochal cyst. Although the pathogenesis of biliary papillomatosis in our case was unclear, biliary irritation associated with choledochal cyst may be related to biliary papillomatosis with point mutation at codon 12 of K-ras gene.

Serum tumor markers and molecular biological diagnosis in pancreatic cancer.

Abstract: Recent studies on genetic abnormalities in pancreatic ductal cancer have led to the investigation of tumor markers and genetic markers in both serum and pancreatic juice (PJ). Serum type 1 chain carbohydrate antigens such as CA19-9 are positive in nearly 80% of patients with pancreatic cancer (PCa), of which most are in advanced stage, whereas false-positive rates are relatively high at 20%–30% in benign hepatobiliary and pancreatic diseases. Although the prevalence of type 2 chain carbohydrate antigens, such as SLX, is relatively low, cancer specificity of these antigens is high. However, serum tumor markers have limited diagnostic value for early detection of PCa. In PJ collected endoscopically from patients with PCa, K-ras mutations (KRM) are detectable in > 80%, whereas KRM are observed in 20%–30% of PJ from patients with chronic pancreatitis (CP), reflecting benign mucous cell hyperplasia harboring KRM. Thus, a qualitative analysis of KRM in PJ is unsuitable for diagnosis of PCa. On the other hand, using an hybridization protection assay that can quantitatively determine KRM, KRM were positive in 66% of PCa but only in 40% of CP cases, indicating that qualitative analysis of KRM in PJ may be useful for differentiating PCa from CP. p53 Mutations are found in 4%–50% in PJ from patients with PCa but are not detectable in PJ from CP, suggesting that the specificity of p53 mutations is very high for PCa. Furthermore, p53 mutations were detected in 7 of 15 (47%) patients with PCa in which the PJ cytologic diagnosis was negative. Telomerase (TE) activity or its catalytic subunit, h-TERT, was reportedly positive >80% in PJ from PCa but was detected in <20% of PJ from CP. TE activity in PJ from CP originates from lymphocytes. The development and application of these new genetic and epigenetic markers with high specificity and sensitivity for PCa in serum and PJ will significantly improve our diagnostic accuracy.

Epithelial-to-mesenchymal transition in pancreatic adenocarcinoma.

Epithelial to mesenchymal transition (EMT) is a physiologic process that allows morphological and genetic changes of carcinoma cells from an epithelial to a mesenchymal phenotype, which is the basis of the high metastatic potential of pancreatic cancer cells. EMT is triggered by various tumor microenvironmental factors, including cytokines, growth factors, and chemotherapeutic agents. This review summarizes the state-of-the-art knowledge on the molecular mechanisms that support pancreatic cancer EMT and the evidences that support its involvement in invasiveness/aggressiveness, and the drug resistance of pancreatic cancer cells.

Traditional Japanese Medicine, Kampo: Its History and Current Status

Traditional Japanese medicine, Kampo, is used by over 80% of medical doctors in Japan. Owing to its high quality and safety, Kampo has been integrated into modern medicine, and there are 345 randomized controlled trials using Kampo in Japan as of 2010. Although there are a number of articles in top journals about basic science research, we can find only small numbers of high-quality clinical evidence. Since undergraduate education on Kampo has been established, integrative approach with the balanced combination of modern medicine and Kampo is expected to generate good clinical evidence in the near future.