菅野 敦

Periostin is a secretory protein that has been suggested to function as a cell adhesion molecule and promote the invasiveness or growth rate of tumors. However, little is known about the association of its expression and epithelial to mesenchymal transition (EMT), which is considered to play a crucial role in cancer cell metastasis. Thus, the authors investigated whether periostin could be involved in the process of EMT and the role of this gene in pancreatic cancer development. The expression of periostin was observed mainly in stromal cells but very little in cancer cells by immunohistochemistry and real-time RT-PCR. In vitro, pancreatic stellate cells (PSCs) exhibited a much higher basal expression of periostin compared with cancer cells. Periostin secreted in the supernatant from 293T cells that expressed periostin (approximately 150 ng/ml) inhibited the migration of pancreatic cancer cells. Coculture assay revealed that periostin expression in PSC was induced by pancreatic cancer cells. To assess the direct role of periostin in pancreatic cancer cells, the authors generated pancreatic cancer cell lines that stably express periostin. The induced expression of periostin (to 150 ng/ml) altered the morphology of cancer cells, changing them from mesenchymal to epithelial phenotypes with the induction of epithelial markers and a reduction of mesenchymal markers, and showed reduced cell migration in vitro and formed smaller tumors as well as suppressed metastasis in vivo. On the other hand, high concentration of recombinant periostin (1 mu g/ml) promoted cell migration with AKT activation. The findings suggest that periostin has biphasic effect on the development of pancreatic cancer. (C) 2008 Wiley-Liss, Inc.

Sonic hedgehog (SHH) is frequently expressed in pre-cancerous lesions and carcinoma of the pancreas. A recent study revealed that its expression was higher in the intraductal papillary mucinous neoplasm (IPMN) of the pancreas than in the pancreatic carcinoma. However, the correlation between its signaling pathway and turnorigenesis of IPMN has not yet been well documented. We investigated the expression of mRNA and protein of SHH as well as its downstream transcription factor Gli1 in 19 microdissected lesions from 15 cases and in 75 lesions from 33 cases of the IPMN by one-step quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. SHH and Gli1 mRNAs were detected in all the examined lesions and 8 out of 19 lesions in IPMNs, respectively. SHH and Gli1 mRNAs were likely to be up-regulated from the adenoma and from borderline to carcinoma cells, respectively. Immunohistochemical analysis also reported that SHH and Gli1 expression was correlated with the grade of cell atypia. These findings suggested that HH signaling was activated in IPMNs and contributed to tumorigenesis in these types of neoplasms.

MSX2 is thought to be a regulator of organ development and a downstream target of the ras signaling pathway; however, little is known about the role of MSX2 in the development of pancreatic cancers, most of which harbor a K-ras gene mutation. Therefore, we examined whether the presence of MSX2 correlates with the malignant behavior of pancreatic cancer cells. BxPC3 pancreatic cancer cells that stably overexpress MSX2 showed a flattened and scattered morphology accompanied by a change in localization of E-cadherin and beta-catenin from membrane to cytoplasm. Cell proliferation rate, cell migration, and anchorage-independent cell growth were enhanced in MSX2-expressing cells. Injection of MSX2-expressing cells into the pancreas of nude mice resulted in a significant increase in liver metastases and peritoneal disseminations compared with injection of control cells. Microarray analysis revealed a significant induction of Twist 1 expression in cells that express MSX2. When MSX2 was inactivated in pancreatic cancer cells following transfection with an MSX2-specific small interfering RNA, Twist 1 was down-regulated. Immunohistochemistry of human pancreatic carcinoma tissue revealed that MSX2 was frequently expressed in cancer cells, and that increased expression of MSX2 significantly correlated with higher tumor grade, vascular invasion, and Twist I expression. These data indicate that MSX2 plays a crucial role in pancreatic cancer development by inducing changes consistent with epithelial to mesenchymal transition through enhanced expression of Twist 1.

1995年1月〜2007年3月の膵管内乳頭状粘液腫瘍(IPMN)患者156例(男96例、女60例、平均年齢68.2±8.6歳)を対象に生命予後と適切な手術適応を検討した。分枝型149例、主膵型7例で平均観察期間は1637±127日、手術例は41例(悪性22例、良性19例)であった。この中で、分枝型IPMNを手術例41例と経過観察例115例に分け、後向きに調査した。(1)分枝型IPMNの国際診療ガイドラインの治療方針が予後を反映しているかどうかを調べるため、超音波内視鏡(EUS)で経過観察可能であった84例を対象に、結節のある拡張分枝30mm以上で手術適応、以下で非適応として予後を比較した。結果、有意差は認めず予後を反映していなかった。(2)良悪性を規定する因子を決定するため、分枝型IPMN手術例41例を対象に拡張分枝膵管径、壁在結節径、主膵管径をROC曲線を用いて最も高い正診率を示す値を出し、良性悪性群間で比較した。この値が経過観察例の予後を反映するかを調べた。その結果、拡張分枝膵管径は良悪性群間に有意差を認めたがcut off値32mmで分けた正診率は64%と低く、主膵管径でも良悪性群間に有意差を認めたがcut off値6mmで分けた正診率は55%と低値であった。結節隆起高も良悪性間に有意差を認め、cut off値6mmで分けた正診率は77%と最も高かった。(3)経過観察例の予後をもとに、手術例の結節隆起高から最も予後を反映する値を導き出し、経過観察例の予後を比較した。結果、結節隆起高6mmで経過観察例を分けた予後比較では有意差は認めなかったが、9mmより大きい群と小さい群で予後に有意差を認め、手術例の結節隆起高では9mm以上例は全て悪性であった。以上より確実に悪性である結節隆起高9mm以上を最初に手術適応とすることは予後の観点から妥当と考えられた。

膵癌早期発見のためには膵発癌機構を解明することが不可欠である。そのためには、遺伝子解析も作成法も容易である動物モデルの開発が必要と考えられる。我々は化学発癌物質DMBA投与によってマウス膵発癌モデルを作成し、それがヒト膵癌研究に応用できるか否かを検討した。DMBA処理2週後からマウス膵にtubular complexが観察され、1ヵ月でPanIN類似病変、2ヵ月後3ヵ月後にかけて癌病変の形成が認められた。癌は肉腫様形態を呈したがcytokeratin陽性、vimentin,chymotrypsin陰性で膵管由来の癌と考えられた。過形成病変および癌病変において、ヒト膵癌同様、悪性度の進行に伴って、smad4発現の消失、cyclin D1,p53発現の増強、Notchシグナルの活性化が認められた。しかし、ヒトで最も初期にまた最も高頻度に異常の認められるK-ras遺伝子の変異は確認されなかった。これらの事から、本マウスモデルは、多くのヒト膵癌とは初期の発癌過程は異なるが進展過程に関与する遺伝子異常には類似性がみられ、ヒトにおける発癌過程の後期から癌進展過程の研究に適用できる可能性が示唆された。(著者抄録)

Living pancreatic cancer tissues grown subcutaneously in nude mice are studied by in vivo microscope Raman spectroscopy. Comparing the spectra of living pancreatic cancer tissue to that of the dead same tissue, it is found that they are different each other. In the subtraction spectrum, Raman bands observed at 937, 1251, 1447 and 1671 cm-1 are appeared in negative direction and those observed at 966 and 1045 cm-1 are appeared in positive direction. The results strongly suggest that the spectral changes reflect the protein conformational changes in the tumor tissue with death of the host animal. The present result demonstrates the importance of in vivo, real time studies of biomedical tissues using Raman spectroscopy.

In our study, we found that bone morphogeretic protein 4 (BMP4) has a novel effect as an inducer of epithelial-mesenchymal transition (EMT) on Panc-I cells, a human pancreatic carcinoma cell line. BMP4-treated Panc-I cells showed loose cell contacts and a scattered, fibroblast-like appearance along with E-cadherin clownregulation, Vimentin upregulation and enhanced cell migration, which are characteristic of EMT. BMP4 treatment also induced homeobox gene MSX2 expression, which we previously showed to be associated with EMT in pancreatic carcinoma cells. BMP4 treatment activated the Smad signaling pathway, and extracellular signal-related kinase (ERK) and p38 mitogen-activated kinase (MAPK) pathways in these cells. MSX2 was markedly induced by BMP4 through the ERK and p38 MAPK pathways in collaboration with the Smad signaling pathway. The repression of E-cadherin, induction of Vimentin and enhanced cell migration disappeared when siRNA-based MSX2 downregulated pancreatic cancer cells were treated with BMP4. These findings indicate that BMP4 may be involved in pancreatic carcinoma development through the promotion of EMT and that MSX2 is indispensable to this process.

To establish pancreatic cancer in mice, dimethylbenzanthracene (DMBA) was administered into mice pancreata. The formation of tubular complex lesions was found in the pancreatic sections from 2 weeks after DMBA treatment. Abnormal tubular complex formations with ductal metaplasia were found from 1 month after the administration. By 3 months after DMBA injection into the pancreas, 6 of 10 mice showed visually recognizable tumors with precursor lesions of various types of cell atypia. In contrast, there were no visually or histologically detectable tumors in the placebo-treated animals. The expression profiles of smad 4, cyclin D1 and p53 in the DMBA-induced tumors were similar to those of human pancreatic cancer, suggesting that this would be a useful mouse model for studying the morphological and molecular mechanisms involved in pancreatic carcinogenesis. Immunohistochemical study using specific antibodies revealed that Notch-1 and Hes-1 were expressed in lesions ranging from tubular complexes to carcinoma in these chemically induced pancreatic tumors. Semiquantitative reverse transcription-polymerase chain reaction with microdissection demonstrated that Notch-1 expression was continuous from precursor lesions to carcinoma cells, whereas Pdx-1 expression was attenuated in carcinoma cells compared to precursor lesions. In addition, inhibition of the Notch signaling pathway by the gamma-secretase inhibitor N-(N-[3,5-difluorophenacetyl]-L-alanyl)-S-phenylglycine t-butyl ester reduced pancreatic cancer cell growth. Therefore, Notch signaling is required to form the tubular complexes and its continuous activation might lead to the transition from tubular complexes to premalignant or malignant lesions and carcinoma cell development in the pancreas.

Objective: Intraductal papillary mucinous neoplasms (IPMNs) of the pancreas show heterogeneous proliferations with latent malignancy. Mucins (MUC) are high-molecular-weight glycoproteins, with an aberrant expression profile in various malignancies. Recently, MUC4 and MUC5AC expressions have been demonstrated to correlate with the unfavorable and the favorable prognosis of pancreatic duct cell carcinoma, respectively. However, little is known about these mucin expressions in IPMNs. Methods: To clarify the role of MUC4 and MUC5AC expressions in IPMNs, the expression profiles of MUC4 and MUC5AC were investigated in 50 lesions from 17 specimens with 16 IPMNs by immunohistochemistry, using each of their specific antibodies. Results: The expression of MUC4 was found in the lesions ranging from adenoma to cancer lesions of IPMNs, whereas it was undetectable in normal and hyperplastic lesions. Frequent expression of MUC4 is found in the higher grade of IPMNs (borderline and cancer lesions; 16/18 lesions, 94%). The differences were independently significant (P < 0.001) when the cutoff point was set between adenoma and borderline IPMNs. Similarly, frequent expression of MUC5AC was detected in the lesions from adenoma to cancer of IPMNs (32/34, 94%), whereas no intense expression was detected in normal or hyperplastic lesions. The significant difference was found when the cutoff point was set between hyperplasia and adenoma of IPMNs (P < 0.001). Conclusions: These results indicated that the expressions of MUC4 and MUC5AC are potential markers to distinguish adenoma or above malignant lesions of IPMNs from lesser malignant ones, respectively.

Pancreatic arteriovenous malformation (AVM) is a relatively rare disease. Based on our literature search, 51 cases of pancreatic AVM have been reported since 1968. The gastrointestinal bleeding is the most common presenting symptom (24/51 cases [47%]). There were only 6 cases of pancreatitis in these cases. We describe 2 cases of acute pancretitis with pancreatic AVM. The patients who were diagnosed with acute pancreatitis were admitted to our hospital. Pancreatitis was considered to be caused by pancreatic AVM by some modalities of diagnostic imaging. The respective pancreatic AVM lesions of patients were resected to prevent the recurrence of pancreatitis. They are asymptomatic after the surgery. Pancreatic AVM is thought to be the one of the reasons for acute pancreatitis.

膵管内乳頭粘液腫瘍(IPMN)の手術適応をめぐって,画像診断を駆使した報告を多数認めるが,いまだに統一した見解はない.IPMNは異型度が進行するにつれ,遺伝子発現のパターンが変化することが推測されている.著者等は,IPMNの異型度別におけるMUC4,MUC5AC,sonic hedgehog,Smad4の発現を免疫染色にて検討した.その結果, 1)MUC4は癌との境界病変以上での発現, 2)MUC5ACは腺腫以上での発現, 3)shhは腺腫以上での発現を認めた.一方Smad4は合併膵癌では発現を認めず,浸潤癌で発現が消失していた.またIPM-C症例の膵液からRNAを採取しRT-PCRを施行したところ,MUC4,MUC5ACの発現を確認でき,免疫染色でも発現を認めた.すなわち膵液のRNAからIPMNの異型度を診断できる可能性が示唆された(著者抄録)

We report a case of autoimmune pancreatitis (AIP) with hepatic inflammatory pseudotumor (IP). The patient was clinically diagnosed as having multiple metastatic tumors originated from cholangiocellular carcinoma as well as autoimmune pancreatitis and underwent left lobectomy of the liver. Histological examination showed a diffuse or dense lymphoplasmacytic infiltration with obliterating phlebitis but an absence of neoplastic proliferation both in the liver tumor and in the biopsy specimen of the pancreas. Abundant IgG4-positive plasma cells were seen in the lesions. This is the first case report that shows a simultaneous occurrence of hepatic IP and AIP, suggesting that these lesions appeared on the background of the recently proposed entity of IgG4-related systemic disease.

AIM: To evaluate the effect of MSX2 on gemcitabine-induced caspase-3 activation in pancreatic cancer cell line Panc-1. METHODS: Using V5-tagged MSX2 expression vector, stable transfectant of MSX2 was generated from Panc-1 cells (Px14 cells). Cell viability under gemcitabine administration was determined by MTT assay relative to control cell line (empty-vector transfected Panc-1 cells; P-3EV cells). Hoechst staining was used for the detection of apoptotic cell. Activation of caspase-3 was assessed using Western blotting analysis and direct measurement of caspase-3 specific activities. RESULTS: MSX2 overexpression in Panc-1 cells resulted in decreased gemcitabine-induced caspase-3 activation and increased cell viability under gemcitabine treatment in Px14 cells. CONCLUSION: MSX2 exerts repressive effects on gemcitabine-induced apoptotic pathway. This novel apoptosis-regulating function of MSX2 may provide a new therapeutic target for pancreatic cancer. (C) 2005 The WJG Press and Elsevier Inc. All rights reserved.

当科において自己免疫性膵炎(AIP)と考えられた14例のうち,日本膵臓学会の臨床診断基準に合致したのは7例(男6例・女1例,44〜78歳:A群),非合致は7例(男6例・女1例,55〜86歳:B群)であった.診断基準の必須項目である「膵管狭窄1/3以上」を満たさなかったのはB群のうち4例で,これらはIgG4高値を診断の根拠とした.うち1例は膵腫大も認めず,胆管病変が中心であった.診断基準で示された血液検査6項目の陽性率をみると,全体的に陽性率は高くなく,最も高かったのは必須項目ではない「IgG4 135mg/dl以上」であった.γグロブリン高値またはIgG高値を認めず,IgG4高値を認めた症例が3例あり,これらはIgG4の上昇の程度が軽度で,高IgG血症の基準を満たしていなかった.すなわち,高γグロブリン血症および高IgG血症はIgG4の上昇を反映しているものと考えられ,IgG4高値がAIPの診断に重要であることが示唆された