Seiji Adachi

Use of Samples From Endoscopic Ultrasound–Guided 19-Gauge Fine-Needle Aspiration in Diagnosis of Autoimmune Pancreatitis

BACKGROUND & AIMS Histologic techniques are used to distinguish autoimmune pancreatitis (AIP) from pancreatic malignancies and to confirm the etiology of pancreatitis. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is a well-established technique used in the diagnosis of pancreatic cancer. However, it is unclear whether specimens obtained from pancreatic lesions by EUS-FNA are adequate for the histologic diagnosis of AIP, because the evaluation of tissue architecture and immunostaining assays usually require larger samples. METHODS We evaluated samples collected by EUS-FNA with a conventional 19-gauge needle by histologic analysis, looking for features of AIP. We analyzed data from 44 patients who were diagnosed with AIP and underwent EUS-FNA with a 19-gauge needle from January 2004 to September 2010. The FNA specimens were reviewed by histologic analysis; AIP was diagnosed based on the presence of lymphoplasmacytic sclerosing pancreatitis or immunoglobulin (Ig)G4-positive plasma cells in the infiltrate. RESULTS The specimen amount was inadequate from 3 patients. Among the remaining 41 patients, histopathologic analysis revealed lymphoplasmacytic sclerosing pancreatitis in 17 samples and IgG4-positive plasma cells in 5 (3 samples were positive for both); no samples had granulocytic epithelial lesions. Therefore, 19 patients (43%) were diagnosed with AIP based on histologic analysis. One patient had temporary abdominal pain. CONCLUSIONS EUS-FNA, with a 19-gauge needle, is a safe and reliable procedure for obtaining pancreatic samples for the histologic analysis of AIP. Although it does not have a high diagnostic yield, it might be useful in patients without typical features of AIP because it would allow patients to avoid surgery.

(-)-Epigallocatechin gallate inhibits growth and activation of the VEGF/VEGFR axis in human colorectal cancer cells.

(-)-Epigallocatechin gallate (EGCG), the major constituent of green tea, inhibits the growth of colorectal cancer cells by inhibiting the activation of various types of receptor tyrosine kinases (RTKs). The RTK vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis induces tumor angiogenesis in colorectal cancer. This study examined the effects of EGCG on the activity of the VEGF/VEGFR axis and the expression of hypoxia-inducible factor (HIF)-1alpha, which promotes angiogenesis by elevating VEGF levels, in human colorectal cancer cells. Total and phosphorylated (i.e., activated) form (p-VEGFR-2) of VEGFR-2 proteins were overexpressed in a series of human colorectal cancer cell lines. Within 3h, EGCG caused a decrease in the expression of HIF-1alpha protein and VEGF, HIF-1alpha, insulin-like growth factor (IGF)-1, IGF-2, epidermal growth factor (EGF), and heregulin mRNAs in SW837 colorectal cancer cells, which express a constitutively activated VEGF/VEGFR axis. A decrease was also observed in the expression of VEGFR-2, p-VEGFR-2, p-IGF-1 receptor, p-ERK, and p-Akt proteins within 6h after EGCG treatment. Drinking EGCG significantly inhibited the growth of SW837 xenografts in nude mice, and this was associated with the inhibition of the expression and activation of VEGFR-2. The consumption of EGCG also inhibited activation of ERK and Akt, both of which are downstream signaling molecules of the VEGF/VEGFR axis, and reduced the expression of VEGF mRNA in xenografts. These findings suggest that EGCG may exert, at least in part, growth-inhibitory effects on colorectal cancer cells by inhibiting the activation of the VEGF/VEGFR axis through suppressing the expression of HIF-1alpha and several major growth factors. EGCG may therefore be useful in the chemoprevention and/or treatment of colorectal cancer.

(–)‐Epigallocatechin gallate suppresses the growth of human hepatocellular carcinoma cells by inhibiting activation of the vascular endothelial growth factor–vascular endothelial growth factor receptor axis

The receptor tyrosine kinase vascular endothelial growth factor (VEGF) receptor (VEGFR) plays an important role in tumor angiogenesis of hepatocellular carcinoma (HCC). (–)‐Epigallocatechin gallate (EGCG), the major biologically active component of green tea, inhibits growth in a variety of human cancer cells by inhibiting the activation of several types of receptor tyrosine kinases. In this study, we examined the effects of EGCG on the activity of the VEGF–VEGFR axis in human HCC cells. The levels of total and phosphorylated (i.e. activated) form of VEGFR‐2 protein (p‐VEGFR‐2) were observed to increase in a series of human HCC cell lines in comparison to the Hc normal human hepatocytes. EGCG preferentially inhibited the growth of HuH7 HCC cells, which express constitutive activation of the VEGF–VEGFR axis, in comparison to Hc cells. Treatment of HuH7 cells with EGCG caused a time‐ and dose‐dependent decrease in the expression of VEGFR‐2 and p‐VEGFR‐2 proteins. The production of VEGF from HuH7 cells was reduced by treatment with EGCG. Drinking of EGCG significantly inhibited the growth of HuH7 xenografts in nude mice and this was associated with inhibition of the activation of VEGFR‐2 and its related downstream signaling molecules, including ERK and Akt. EGCG drinking also decreased the expression of Bcl‐xL protein and VEGF mRNA in the xenografts. These findings suggest that EGCG can exert, at least in part, its growth‐inhibitive effect on HCC cells by inhibiting the VEGF–VEGFR axis. EGCG might therefore be useful in the treatment of HCC. (Cancer Sci 2009; 100: 1957–1962)

Cancer chemoprevention with green tea catechins by targeting receptor tyrosine kinases

Recent studies indicate that receptor tyrosine kinases (RTKs), which play important roles in cell proliferation, are one of the possible targets of green tea catechins (GTCs) in cancer cell growth inhibition. (-)-Epigallocatechin-3-gallate (EGCG), the major catechin in green tea, inhibits cell proliferation and induces apoptosis in various types of cancer cells, including colorectal cancer and hepatocellular carcinoma cells, by blocking the activation of the epidermal growth factor receptor (EGFR) family of RTKs. EGCG inhibits the activation of insulin-like growth factor-1 receptor (IGF-1R) and VEGFR2, the other members of the RTK family, and this effect is also associated with the anticancer and chemopreventive properties of this agent. EGCG suppresses the activation of EGFR in part by altering membrane lipid organization and causing the subsequent inhibition of the dimerization and activation of this receptor. Preliminary trials have shown that GTCs successfully prevent the development and progression of precancerous lesions, such as colorectal adenomas, without causing severe adverse effects. The present report reviews evidence indicating that GTCs exert anticancer and chemopreventive effects by inhibiting the activation of specific RTKs, especially EGFR, IGF-1R, and VEGFR2, and concludes that targeting RTKs and their related signaling pathways by using tea catechins could be a promising strategy for the prevention of human cancers.

Epigallocatechin gallate causes internalization of the epidermal growth factor receptor in human colon cancer cells

We recently found that the inhibitory effect of (-)-epigallocatechin gallate (EGCG) on epidermal growth factor (EGF) binding to the epidermal growth factor receptor (EGFR) is associated with alterations in lipid organization in the plasma membrane of colon cancer cells. Since changes in lipid organizations are thought to play a role in the trafficking of several membrane proteins, in this study we examined the effects of EGCG on cellular localization of the EGFR in SW480 cells. Treatment of the cells for 30 min with as little as 1 microg/ml of EGCG caused a decrease in cell surface-associated EGFRs and this was associated with internalization of EGFRs into endosomal vesicles. Similar effects were seen with a green fluorescent protein (GFP)-EGFR fusion protein. As expected, the EGFR protein was phosphorylated at tyrosine residues, ubiquitinated and partially degraded when the cells were treated with EGF, but treatment with EGCG caused none of these effects. The loss of EGFRs from the cell surface induced by treating the cells with EGF for 30 min persisted for at least 2 h. However, the loss of EGFRs from the cell surface induced by temporary exposure to EGCG was partially restored within 1-2 h. These studies provide the first evidence that EGCG can induce internalization of EGFRs into endosomes, which can recycle back to the cell surface. This sequestrating of inactivated EGFRs into endosomes may explain, at least in part, the ability of EGCG to inhibit activation of the EGFR and thereby exert anticancer effects.

(−)-Epigallocatechin gallate downregulates EGF receptor via phosphorylation at Ser1046/1047 by p38 MAPK in colon cancer cells

We previously reported that (-)-epigallocatechin gallate (EGCG) in green tea alters plasma membrane organization and causes internalization of epidermal growth factor receptor (EGFR), resulting in the suppression of colon cancer cell growth. In the present study, we investigated the detailed mechanism underlying EGCG-induced downregulation of EGFR in SW480 colon cancer cells. Prolonged exposure to EGCG caused EGFR degradation. However, EGCG required neither an ubiquitin ligase (c-Cbl) binding to EGFR nor a phosphorylation of EGFR at tyrosine residues, both of which are reportedly necessary for EGFR degradation induced by epidermal growth factor. In addition, EGCG induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), a stress-inducible kinase believed to negatively regulate tumorigenesis, and the inhibition of p38 MAPK by SB203580, a specific p38 MAPK inhibitor, or the gene silencing using p38 MAPK-small interfering RNA (siRNA) suppressed the internalization and subsequent degradation of EGFR induced by EGCG. EGFR underwent a gel mobility shift upon treatment with EGCG and this was canceled by SB203580, indicating that EGCG causes EGFR phosphorylation via p38 MAPK. Moreover, EGCG caused phosphorylation of EGFR at Ser1046/1047, a site that is critical for its downregulation and this was also suppressed by SB203580 or siRNA of p38 MAPK. Taken together, our results strongly suggest that phosphorylation of EGFR at serine 1046/1047 via activation of p38 MAPK plays a pivotal role in EGCG-induced downregulation of EGFR in colon cancer cells.

Decreased level of β-endorphin-like immunoreactivity in cerebrospinal fluid of patients with senile dementia of Alzheimer type

β-Endorphin-like immunoreactivity in cerebrospinal fluid (CSF) was observed to decrease in patients with Huntingtons disease and dementia due to brain vascular disease. The greatest decrease was seen in patients with presenile and senile dementia of Alzheimer type (SDAT). The immunoreactivity significantly correlated with psychological functions when examined using a dementia rating scale (r = 0.51, p < 0.01, for all dementia, r = 0.65, p < 0.02, for only SDAT). These results suggest that a B-endorphin-like substance may be related in the pathophysiology of dementia.

Endoscopic Ultrasound-Guided Fine Needle Aspiration Biopsy for Diagnosis of Lymphoproliferative Disorders: Feasibility of Immunohistological, Flow Cytometric, and Cytogenetic Assessments

OBJECTIVES:In addition to morphology, immunophenotype and genetic abnormalities should be assessed during diagnosis and subclassification of lymphoproliferative disorders. The objective of this study was to evaluate the yield of endoscopic ultrasound-guided fine needle aspiration biopsy (EUS-FNAB) using a standard 19-gauge needle for diagnosis and subclassification of lymphoma, assessing the feasibility of immunohistological, flow cytometric, and cytogenetic assessments.METHODS:Two hundred forty patients with suspected lymphoma were referred for EUS-FNAB to our quaternary EUS center between June 2005 and December 2010. EUS-FNAB using a conventional 19-gauge needle was attempted for all patients, followed by histological assessments including immunohistological staining, flow cytometry, and cytogenetic analysis (G-band karyotyping). Among the patients, 152 were ultimately diagnosed with lymphoma. The primary outcome measure of this study was the sensitivity of histological assessment, including immunohistological staining, flow cytometry, and G-band karyotyping, for diagnosis and subclassification of lymphoma.RESULTS:Among the 152 patients ultimately diagnosed with lymphoma, 147 patients (96.7%) were diagnosed by EUS-FNAB, and classification in accordance with the WHO (World Health Organization) system was also possible for 135 patients (88.8%) on the basis of histological findings, including immunohistological staining. Flow cytometry showed abnormal or unusual cell populations in 121 (79.6%) of the 152 patients diagnosed with lymphoma, and in 114 (90.5%) of the 126 patients diagnosed with B-cell lymphoma. Specific cytogenetic abnormalities were detected in 21 (13.8%) of the lymphoma patients.CONCLUSIONS:EUS-FNAB using a standard 19-gauge needle has high diagnostic value for lymphoma. Immunophenotyping is usually possible, while cytogenetic abnormalities can be identified in a relatively limited number of patients.

Acyclic retinoid induces partial differentiation, down-regulates telomerase reverse transcriptase mRNA expression and telomerase activity, and induces apoptosis in human hepatoma-derived cell lines.

BACKGROUND/AIMS Acyclic retinoid (AR; all trans-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid) prevented hepatocarcinogenesis in animal models and in a randomized clinical trial by eradicating premalignant and latent malignant clones of transformed cells from the liver. We investigated the possible mechanism of this clonal deletion at the cellular level. METHODS Human hepatoma-derived cell lines, PLC/PRF/5, HuH-7, and JHH-7, were treated in vitro with AR. Secretion of albumin and that of lectin-reactive isoform of alpha-fetoprotein (AFP-L3) were measured as markers of differentiation and dedifferentiation of the cells, respectively. Telomerase reverse transcriptase (TERT) mRNA expression and telomerase activity were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and stretch PCR assay, respectively. Caspase activities were measured by colorimetric protease assay. Mitochondrial membrane permeability transition was examined by Rhodamine staining. RESULTS Production of albumin was recovered while that of AFP-L3 was reduced after exposure of the cells to 10 microM AR for 2 days. This differentiation was maintained for another 2 days without retinoid. In parallel, both TERT mRNA expression and telomerase activity were down-regulated. The cells subsequently died due to apoptosis after 4-6 experimental days. Serial increases in mitochondrial membrane permeability and caspase-9 and -3 activities induced apoptosis. CONCLUSIONS AR first induces differentiation and reduces telomerase activity. Subsequent apoptosis may contribute to the eradication of the clone.

(-)-Epigallocatechin gallate prevents carbon tetrachloride-induced rat hepatic fibrosis by inhibiting the expression of the PDGFRβ and IGF-1R.

Hepatic fibrosis is a major complication of various chronic liver diseases. Activated hepatic stellate cells (HSCs) play a critical role in the development of liver fibrosis and the axis of platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR), a member of receptor tyrosine kinases (RTKs), is closely associated with the activation of HSC. Insulin-like growth factor (IGF)-1 receptor (IGF-1R), which also belongs to RTKs, interacts with the PDGF/PDGFR axis, thereby cooperatively promoting hepatic fibrosis. We herein examined the effects of (-)-epigallocatechin gallate (EGCG), which inhibits the activation of several types of RTKs, on the development of rat liver fibrosis induced by carbon tetrachloride (CCl4). Drinking water with 0.1% EGCG significantly decreased the serum levels of both aspartate aminotransferase and alanine aminotransferase raised by CCl4, thus indicating an improvement of liver injury. In CCl4-injected rats, EGCG markedly attenuated hepatic fibrosis and decreased the amount of hydroxyproline in the experimental liver. The expression of PDGFRbeta and IGF-1R mRNAs in the liver was significantly lowered by the treatment with EGCG. EGCG also decreased the expression of PDGFRbeta and alpha-smooth muscle actin proteins, thus indicating the inhibition of HSC activation. These findings suggest that EGCG can exert, at least in part, an anti-fibrotic effect on the liver by targeting PDGFRbeta and IGF-1R. EGCG might therefore be useful in both the prevention and treatment of hepatic fibrosis.