Akira Takasawa

Claudin-4-targeted therapy using Clostridium perfringens enterotoxin for prostate cancer

Clostridium perfringens enterotoxin (CPE) triggers lysis of epithelial cells through binding to tight‐junction proteins claudin‐3 (Cldn3) and Cldn4, which are over‐expressed in prostate cancer. We investigated the potential of Cldn‐targeted therapy using CPE.

Regulation of tight junctions by sex hormones in normal human endometrial epithelial cells and uterus cancer cell line Sawano

The number of patients with uterine endometrial carcinoma, the cause of which involves sex hormones, has recently been growing rapidly because of increases in life expectancy and obesity. Tight junction proteins claudin-3 and −4 are receptors of Clostridium perfringens enterotoxin (CPE) and increase during endometrial carcinogenesis. In the present study of normal human endometrial epithelial (HEE) cells and the uterus cancer cell line Sawano, we investigate changes in the expression of tight junction proteins including claudin-3 and −4, the fence and barrier functions of the tight junction and the cytotoxic effects of CPE by sex hormones. In primary cultured HEE cells, treatment with progesterone (P4) but not estradiol (E2), induced claudin-1, −3, −4 and −7 and occludin, together with the downregulation of the barrier function but not the fence function. In Sawano cells, claudin-3 and −4 were upregulated by E2 but not by P4, together with a disruption of both the barrier and fence function. In primary cultured HEE cells, claudin-3 and −4 were localized at the apicalmost regions (tight junction areas) and no cytotoxicity of CPE was observed. In Sawano cells, claudin-3 and −4 were found not only in the apicalmost regions but also at the basolateral membrane and the cytotoxicity of CPE was enhanced by E2. Thus, tight junctions are physiological regulated by sex hormones in normal HEE cells during the menstrual cycle suggesting that safer and more effective therapeutic methods targeting claudins in uterine cancer can be developed.

Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial–mesenchymal transition of pancreatic cancer

Protein kinase Cα (PKCα) is highly expressed in pancreatic cancer. However, the effects of PKCα on Snail and claudin-1, which play crucial roles in epithelial cell polarity during epithelial-mesenchymal transition (EMT), remain unclear. In this study, we investigated the mechanisms of regulation of Snail and claudin-1 via a PKCα signal pathway during EMT in pancreatic cancer cells and in normal human pancreatic duct epithelial cells (HPDEs). By immunostaining, overexpression of PKCα and downregulation of claudin-1 were observed in poorly differentiated human pancreatic cancer tissues and the pancreatic cancer cell line PANC-1. Treatment with the PKCα inhibitor Gö6976 transcriptionally decreased Snail and increased claudin-1 in PANC-1 cells. The PKCα inhibitor prevented upregulation of Snail and downregulation of claudin-1 during EMT induced by transforming growth factor-β1 (TGF-β1) treatment and under hypoxia in PANC-1 cells. The effects of the PKCα inhibitor were in part regulated via an extracellular signal-regulated kinase (ERK) signaling pathway. The PKCα inhibitor also prevented downregulation of the barrier function and fence function during EMT in well-differentiated pancreatic cancer cell line HPAC. In normal HPDEs, the PKCα inhibitor transcriptionally induced not only claudin-1 but also claudin-4, -7 and occludin without a change of Snail. Treatment with the PKCα inhibitor in normal HPDEs prevented downregulation of claudin-1 and occludin by TGF-β1 treatment and enhanced upregulation of claudin-1, -4, -7 and occludin under hypoxia. These findings suggest that PKCα regulates claudin-1 via Snail- and mitogen-activated protein kinase/ERK-dependent pathways during EMT in pancreatic cancer. Thus, PKCα inhibitors may be potential therapeutic agents against the malignancy of human pancreatic cancer cells.

Human equilibrative nucleoside transporter 1 and Notch3 can predict gemcitabine effects in patients with unresectable pancreatic cancer

Background:Pancreatic ductal carcinoma (PDC) is one of the most lethal human carcinomas. Expression patterns of some genes may predict gemcitabine (GEM) treatment efficacy. We examined predictive indicators of survival in GEM-treated patients by quantifying the expression of several genes in pre-treatment endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) samples from patients with PDC.Methods:The expressions of human equilibrative nucleoside transporter 1 (hENT1), deoxycitidine kinase, ribonucleoside reductase 1, ribonucleoside reductase 2 and Notch3 in EUS-FNA tissue samples from 71 patients with unresectable PDC were quantified using real-time reverse transcription–polymerase chain reactions and examined for correlations with GEM sensitivity.Results:The log-rank test detected no significant differences in overall survival between GEM-treated patients with low and high mRNA levels of all genes examined. However, low Notch3 mRNA expression was significantly associated with longer overall survival in a multivariate analysis for survival (P=0.0094). High hENT1 expression level was significantly associated with a longer time to progression (P=0.039). Interaction tests for GEM administration and hENT1 or Notch3 mRNA expression were statistically significant (P=0.0054 and 0.0047, respectively).Conclusion:hENT1 and Notch3 mRNA expressions in EUS-FNA specimens were the key predictive biomarkers of GEM effect and GEM sensitivity in patients with unresectable PDC.

Curcumin Prevents Replication of Respiratory Syncytial Virus and the Epithelial Responses to It in Human Nasal Epithelial Cells

The human nasal epithelium is the first line of defense during respiratory virus infection. Respiratory syncytial virus (RSV) is the major cause of bronchitis, asthma and severe lower respiratory tract disease in infants and young children. We previously reported in human nasal epithelial cells (HNECs), the replication and budding of RSV and the epithelial responses, including release of proinflammatory cytokines and enhancement of the tight junctions, are in part regulated via an NF-κB pathway. In this study, we investigated the effects of the NF-κB in HNECs infected with RSV. Curcumin prevented the replication and budding of RSV and the epithelial responses to it without cytotoxicity. Furthermore, the upregulation of the epithelial barrier function caused by infection with RSV was enhanced by curcumin. Curcumin also has wide pharmacokinetic effects as an inhibitor of NF-κB, eIF-2α dephosphorylation, proteasome and COX2. RSV-infected HNECs were treated with the eIF-2α dephosphorylation blocker salubrinal and the proteasome inhibitor MG132, and inhibitors of COX1 and COX2. Treatment with salubrinal, MG132 and COX2 inhibitor, like curcumin, prevented the replication of RSV and the epithelial responses, and treatment with salubrinal and MG132 enhanced the upregulation of tight junction molecules induced by infection with RSV. These results suggest that curcumin can prevent the replication of RSV and the epithelial responses to it without cytotoxicity and may act as therapy for severe lower respiratory tract disease in infants and young children caused by RSV infection.

Behavior of tricellulin during destruction and formation of tight junctions under various extracellular calcium conditions.

Tricellulin is an important component of tricellular tight junctions (TJs) and is involved in the formation of tricellular contacts. However, little is known about its regulation during the assembly and disassembly of tricellular TJs. By using the well-differentiated pancreatic cancer cell line HPAC, which highly expresses tricellulin at tricellular contacts, we have investigated changes in the localization, expression and phosphorylation of tricellulin and in its TJ functions as a barrier and fence during the destruction and formation of TJs induced by changes in the extracellular calcium concentration. During both extracellular Ca2+ depletion caused by EGTA treatment and Ca2+ repletion after Ca2+ starvation, the expression of tricellulin increased in whole lysates and in Triton-X-100-insoluble fractions without any change in its mRNA. The increases in immunoreactivity revealed by Western blotting were prevented by alkaline phosphatase treatment. Immunoprecipitation assays showed that tricellulin was phosphorylated on threonine residues when it increased after Ca2+ depletion and repletion. In the early stage after Ca2+ repletion, tricellulin was expressed not only at tricellular contacts but also in the cytoplasm and at bicellular borders. In confocal laser microscopy, tricellulin was observed at the apical-most regions and basolateral membranes of tricellular contacts after Ca2+ repletion. Knockdown of tricellulin delayed the recovery of the barrier and fence functions after Ca2+ repletion. Thus, the dynamic behavior of tricellulin during the destruction and formation of TJs under various extracellular calcium conditions seems to be closely associated with the barrier and fence functions of TJs.

Expression of claudins 7 and 18 in pancreatic ductal adenocarcinoma: association with features of differentiation.

Aim This study was undertaken to evaluate the expression of claudins 7 and 18 in pancreatic ductal adenocarcinoma. Methods and results Material tested included 111 operated samples and 47 additional biopsy samples consisting of 26 cases of pancreatitis, 3 cases of pancreatic intraepithelial neoplasia and 18 ductal adenocarcinomas. Samples were stained with antibodies to claudins 7 and 18 and analysed for membranous and cytoplasmic expression. Membrane bound claudin 7 and 18 expression was detected in 62 of 105 (59%) and 78 of 111 (70%) cases, respectively. Membrane bound claudin 7 and 18 were associated with large or intermediate neoplastic ducts (p=0.01, p=0.002, respectively). Well differentiated pancreatic adenocarcinomas displayed more cases with membrane bound claudin 7 or 18 immunopositivity (p=0.003, p=0.03, respectively). All pancreatic intraepithelial neoplasias studied expressed membrane bound claudin 18. Membrane bound claudin 7 or 18 positivity was not associated with survival (p=0.17, p=0.98). In the biopsy cases membrane bound claudin 18 had 100% specificity and 51% sensitivity for a tumour marker. Conclusion Claudin 7 and 18 expression is related to gland size of neoplastic cells and is especially found in tumours with intermediate and large ducts and well differentiated tumours. Membrane bound claudin 18, when present, is a useful marker for diagnosis of pancreatic cancer. Claudins 7 and 18 were not associated with patient survival or spread of tumours.

Epidermal growth factor modulates claudins and tight junctional functions in ovarian cancer cell lines

Ovarian adenocarcinomas, like human ovarian surface epithelial cells, form functional tight junctions. Tight junction molecules claudin-3 and claudin-4, which are the receptors of Clostridium perfringens enterotoxin (CPE), are abnormally upregulated in epithelial ovarian cancers of all subtypes including, mucinous cystadenocarcinoma and serous cystadenocarcinoma. Clostridium perfringens enterotoxin may be a novel tumor-targeted therapy for ovarian cancers. In epithelial ovarian cancers, overexpression of epidermal growth factor receptor has been observed and the exogenous ligand EGF induces epithelial–mesenchymal transition in ovarian surface epithelium. Epidermal growth factor (EGF) signaling modulates expression of claudins with changes of fence and barrier functions in various cell types. However, the regulation of tight junctions by EGF in ovarian cancers remains unclear. In the present study, to investigate the mechanisms of the regulation of tight junctions in ovarian cancers, ovarian cancer cell lines mucinous cystadenocarcinoma (MCAS) and serous cystadenocarcinoma (HUOA) were treated with EGF. Epidermal growth factor downregulated claudin-3 in MCAS and claudin-4 in HUOA by inducing degradation of the proteins with changes in structures and functions of tight junctions via the MEK/ERK or PI3K/Akt signaling pathway. In addition, in HUOA but not MCAS, EGF downregulated the cytotoxic effect of CPE via claudin-4. Thus, there were different mechanisms for regulation of claudins by EGF between subtypes of epithelial ovarian cancer cells in vitro. These results indicate that EGF may affect claudins and tight junctional functions in ovarian cancer cells during cancer progression.

NK/T-cell lymphoma of bilateral adrenal glands in a patient with pyothorax

Primary lymphoma of adrenal glands is rare, and non-B-cell lymphoma associated with pyothorax is also very rare. Here we report the first autopsy case of non-B-cell lymphoma in bilateral adrenal glands of a 79-year-old woman with pyothorax who had an aggressive clinical course. Immunohistochemically, tumor cells showed CD3+, CD45RO+, CD5-, CD7-, CD4-, CD8-, CD10-, CD20-, CD30-, CD79a-, CD138-, CD56-, granzyme B-, TIA-1+ and ALK-. In addition, tumor cells were strongly EBER1-positive by in situ hybridization. In genomic DNA of tumor cells, T-cell receptor rearrangements were not detected by southern blotting. We finally diagnosed this case as extranodal NK/T-cell lymphoma (nasal type).Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/8050621197741854.

A case of urothelial carcinoma, lipid cell variant.

The lipid cell variant of urothelial carcinoma is a rare variant of urinary bladder cancer, comprised of lipoblast‐like cells. In this report, we describe a case of the lipid cell variant of aggressive urothelial carcinoma. A 78‐year‐old man was admitted to the hospital because of gross hematuria. On cystoscopy, an ulcerative lesion, non‐papillary architecture, was observed in the lateral wall of the bladder. Transurethral resection was performed. Histopathological findings of the bladder tumor indicated neoplastic cells forming irregular solid nests and sheets. Lipoblast‐like neoplastic cells that had eccentric nuclei and cytoplasmic vacuoles were observed, not only in the resected specimen, but also in urine samples. On mucin histochemistry, the tumor cell cytoplasm contained no neutral or acidic mucus. The lipoblast‐like cells were positive for cytokeratins (AE1/AE3, CK7) and adipophilin, known as a protein associated with neutral lipid synthesis. In general, it is difficult to prove the existence of intracytoplasmic lipid in formalin‐fixed paraffin‐embedded materials. This is the first report in which the presence of lipid in vacuoles of the lipid cell variant has been verified by immunohistochemistry.