Tohru Onogawa

Identification of thyroid hormone transporters in humans: different molecules are involved in a tissue-specific manner.

We have recently identified that rat organic anion transporters, polypeptide2 (oatp2) and oatp3, both of which transport thyroid hormones. However, in humans the molecular organization of the organic anion transporters has diverged, and the responsible molecule for thyroid hormone transport has not been clarified, except for human liver-specific transporter (LST-1) identified by us. In this study we isolated and characterized a novel human organic anion transporter, OATP-E from human brain. The isolated complementary DNA encodes a polypeptide of 722 amino acids with 12 transmembrane domains. A rat counterpart, oatp-E, was also identified. Homology analysis and the phylogenetic tree analysis revealed that OATP-E/oatp-E is a subfamily of the organic anion transporter. Human OATP-E transported 3,3′,5-triiodo-l-thyronine (Km, 0.9μ m), thyronine, and rT3 in a Na+-independent manner. Although the clone was isolated from the brain, OATP-E messenger RNA was abundantly expressed in various peripheral tissues. The ...

Human liver-specific organic anion transporter-2 is a potent prognostic factor for human breast carcinoma.

Human liver‐specific organic anion transporter‐2 (LST‐2/OATP8/SLCO1B3) has been demonstrated to be expressed in various gastrointestinal carcinomas and also to play pivotal roles in the uptake of a wide variety of both endogenous and exogenous anionic compounds, including bile acids, conjugated steroids and hormones, into hepatocytes in the human liver. However, the biological significance of LST‐2 in human carcinomas remains unknown. In the present study, we examined the expression of LST‐2 in 102 cases of breast carcinoma using immunohistochemistry and correlated the findings with various clinicopathological parameters in order to examine the possible biological and clinical significance of LST‐2. LST‐2 immunoreactivity was detected in 51 cases (50.0%); of these 51 positive cases, LST‐2 immunoreactivity was inversely correlated with tumor size (P = 0.0289). In addition, LST‐2 immunoreactivity was significantly associated with a decreased risk of recurrence and improved prognosis by both univariate (P = 0.02 and P = 0.01) and multivariate (P = 0.03 and P = 0.01) analyses. In the estrogen receptor‐positive groups, the LST‐2‐positive patients showed good prognoses. Considering that LST‐2 transports estrone‐3‐sulfate, these results suggest that LST‐2 overexpression is associated with a hormone‐dependent growth mechanism of the breast cancer. The results of our present study demonstrate that LST‐2 immunoreactivity is a potent prognostic factor in human breast cancer. (Cancer Sci 2007; 98: 1570–1576)

Novel prognostic protein markers of resectable pancreatic cancer identified by coupled shotgun and targeted proteomics using formalin‐fixed paraffin‐embedded tissues

Pancreatic cancer is among the most lethal malignancies worldwide. We aimed to identify novel prognostic markers by applying mass spectrometry (MS)‐based proteomic analysis to formalin‐fixed paraffin‐embedded (FFPE) tissues. Resectable, node positive pancreatic ductal adenocarcinoma (PDAC) with poor (n = 4) and better (n = 4) outcomes, based on survival duration, with essentially the same clinicopathological backgrounds, and noncancerous pancreatic ducts (n = 5) were analyzed. Cancerous and noncancerous cells collected from FFPE tissue sections by laser microdissection (LMD) were processed for liquid chromatography (LC)‐tandem MS (MS/MS). Candidate proteins were identified by semiquantitative comparison and then analyzed quantitatively using selected reaction monitoring (SRM)‐based MS. To confirm the associations between candidate proteins and outcomes, we immunohistochemically analyzed a cohort of 87 cases. In result, totally 1,229 proteins were identified and 170 were selected as candidate proteins for SRM‐based targeted proteomics. Fourteen proteins overexpressed in cancerous as compared to noncancerous tissue showed different expressions in the poor and better outcome groups. Among these proteins, we found that three novel proteins ECH1, OLFM4 and STML2 were overexpressed in poor group than in better group, and that one known protein GTR1 was expressed reciprocally. Kaplan–Meier analysis showed high expressions of all four proteins to correlate with significantly worse overall survival (p < 0.05). In conclusion, we identified four proteins as candidates of prognostic marker of PDAC. The combination of shotgun proteomics verified by SRM and validated by immunohistochemistry resulted in the prognostic marker discovery that will contribute the understanding of PDAC biology and therapeutic development.

Farnesoid X receptor, hepatocyte nuclear factors 1α and 3β are essential for transcriptional activation of the liver-specific organic anion transporter-2 gene

BackgroundWe isolated the human liver-specific organic anion transporter gene, LST-2 (OATP8/SLCO1B3), which is exclusively expressed in the basolateral membrane of the hepatocytes. In this study, we analyzed the transcriptional regulation of the LST-2 gene in hepatocyte-derived cells and the effect of bile acid.MethodsTranscriptional activity of the LST-2 gene was measured using a human LST-2 promoter–luciferase reporter plasmid under various concentrations of bile acids. Electrophoresis mobility shift assays of farnesoid X receptor (FXR), hepatocyte nuclear factor (HNF) 1α, and HNF3β were performed.ResultsLuciferase analysis showed that the 5′-flanking region from −180 to −20 bp is responsible for LST-2 transcriptional activity. By site-directed mutation analysis, it was revealed that the consensus binding sites for FXR, HNF1α, and HNF3β play important roles in the transcriptional activity of the LST-2 gene. By electrophoresis mobility shift assay, we observed specific protein–DNA complexes of FXR, HNF1α, and HNF-3β. Luciferase activity was increased fivefold when chenodeoxycholate or deoxycholate were added. Northern blot analyses revealed that the expression of LST-2 was increased by addition of chenodeoxycholate or deoxycholate in a dose-dependent manner.ConclusionsThis study demonstrated that the transcription of the LST-2 gene is regulated by three transcription factors, FXR, HNF1α, and HNF3β. HNF1α and HNF3β might contribute to its liver-specific expression, and FXR might play a role in its transcriptional activation by bile acids.

Bile acids repress E‐cadherin through the induction of Snail and increase cancer invasiveness in human hepatobiliary carcinoma

Although some kinds of bile acids have been implicated in colorectal cancer development, the mechanism of cancer progression remains unexplored in hepatobiliary cancer. From our personal results using complementary DNA microarray, we found that chenodeoxycholic acid (CDCA) induced Snail expression in human carcinoma cell lines derived from hepatocellular carcinoma and cholangiocarcinoma. Snail expression plays an important role in the regulation of E‐cadherin and in the acquisition of invasive potential in many types of human cancers including hepatocellular carcinoma. We found that CDCA and lithocholic acid (LCA) induced Snail expression in a concentration‐dependent manner and down‐regulated E‐cadherin expression in hepatocellular carcinoma and cholangiocarcinoma cell lines. Moreover, Snail short interference RNA (siRNA) treatment reduced the down‐regulation of E‐cadherin by CDCA or LCA. Luciferase analysis demonstrated that the promoter region from –111 to –24 relative to the transcriptional start site was necessary for this induction and, at least in part, nuclear factor Y (NF‐Y) and stimulating protein 1 (Sp1) might be an inducer of Snail expression in response to bile acids. In addition, using an in vitro wound healing assay and invasion assay, we observed that CDCA and LCA induced cell migration and invasion. These results suggest that bile acids repress E‐cadherin through the induction of transcription factor Snail and increase cancer invasiveness in human hepatocellular carcinoma and cholangiocarcinoma. Inhibition of this bile acid‐stimulated pathway may prove useful as an adjuvant in the therapy of hepatocellular carcinoma. (Cancer Sci 2008; 99: 1785–1792)

Nm23/nucleoside diphosphate kinase-A as a potent prognostic marker in invasive pancreatic ductal carcinoma identified by proteomic analysis of laser micro-dissected formalin-fixed paraffin-embedded tissue.

BackgroundPancreatic cancer is among the most lethal malignancies worldwide. This study aimed to identify a novel prognostic biomarker, facilitating treatment selection, using mass spectrometry (MS)-based proteomic analysis with formalin-fixed paraffin-embedded (FFPE) tissue.ResultsThe two groups with poor prognosis (n = 4) and with better prognosis (n = 4) had been carefully chosen among 96 resected cases of pancreatic cancer during 1998 to 2007 in Tohoku University Hospital. Although those 2 groups had adjusted background (UICC-Stage IIB, Grade2, R0, gemcitabine adjuvant), there was a significant difference in postoperative mean survival time (poor 21.0 months, better 58.1 months, P = 0.0067). Cancerous epithelial cells collected from FFPE tissue sections by laser micro-dissection (LMD) were processed for liquid chromatography-tandem mass spectrometry (LC-MS/MS). In total, 1099 unique proteins were identified and 6 proteins showed different expressions in the 2 groups by semi-quantitative comparison. Among these 6 proteins, we focused on Nm23/Nucleoside Diphosphate Kinase A (NDPK-A) and immunohistochemically confirmed its expression in the cohort of 96 cases. Kaplan-Meier analysis showed high Nm23/NDPK-A expression to correlate with significantly worse overall survival (P = 0.0103). Moreover, in the multivariate Cox regression model, Nm23/NDPK-A over-expression remained an independent predictor of poor survival with a hazard ratio of 1.97 (95% CI 1.16-3.56, P = 0.0110).ConclusionsWe identified 6 candidate prognostic markers for postoperative pancreatic cancer using FFPE tissues and immunohistochemically demonstrated high Nm23/NDPK-A expression to be a useful prognostic marker for pancreatic cancer.

Advanced bile duct carcinoma in a 15-year-old patient with pancreaticobiliary maljunction and congenital biliary cystic disease

We report a case of advanced bile duct carcinoma arising in a 15-year-old female with pancreaticobiliary maljunction and congenital biliary cystic disease. Pancreaticoduodenectomy and partial resection of the liver was performed. Surgical and histopathological findings indicated advanced tubular adenocarcinoma, classified as final stage IVb according to the General rules for surgical and pathological studies on cancer of the biliary tract proposed by the Japanese Society of Biliary Surgery, 5th edition, and stage IV according to the American Joint Committee on Cancer (AJCC)/International Union Against Cancer (UICC), 6th edition. She underwent chemotherapy with gemcitabine HCl after discharge. She died of cachexia 14 months after the surgery. Although it is well known that biliary malignancies arise frequently in patients with pancreaticobiliary maljunction, it is uncommon for advanced bile duct carcinoma to occur in a 15-year-old female. We should pay attention to the possibility of biliary malignancy in patients with pancreaticobiliary maljunction and congenital biliary cystic disease, even when the patients are juveniles.

Peroxisome proliferator-activated receptor α activates cyclooxygenase-2 gene transcription through bile acid transport in human colorectal cancer cell lines

BackgroundEvidence is accumulating that bile acids are involved in colon cancer development, but their molecular mechanisms remain unexplored. Bile acid has been reported to be associated with induction of the cyclooxygenase-2 (COX-2) gene. Because the human liver-specific organic anion transporter-2 (LST-2/OATP8/OATP1B3) is expressed in gastrointestinal cancers and might transport bile acids to the intracellular space, we studied the molecular mechanisms by which bile acids induce the transcription of COX-2, and the role of LST-2 in colonic cell lines.MethodsTranscriptional activity of COX-2 was measured using a human COX-2 promoter-luciferase assay under various concentrations of bile acids. Electrophoresis mobility shift assays (EMSAs) for peroxisome proliferators-activated receptor (PPAR) α and cyclic AMP responsive element (CRE) were performed.ResultsThe COX-2 promoter was induced by lithocholic acid (LCA), deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA). Deletion and site-directed mutation analyses showed that CRE is the responsive element for LCA. An adenovirus expression system revealed that LST-2 is responsible for induction of COX-2. By EMSA using oligonucleotides of CRE, we observed formation of a specific protein-DNA complex, which was inhibited by a specific antibody against PPARα and CRE. A PPARα-specific agonist induced transcription of COX-2.ConclusionThese results indicate that COX-2 is transcriptionally activated by the addition of LCA, CDCA, and DCA and that LST-2 plays an important role by transporting bile acid to the intracellular space. Moreover, LCA-dependent COX-2 gene activation consists of a transcriptional complex including PPARα and CRE-binding protein. Thus, this induction of COX-2 may participate in carcinogenesis and progression of colorectal cancer cells.

An analysis of a second-line S-1 monotherapy for gemcitabine-refractory biliary tract cancer.

BACKGROUND/AIMS Gemcitabine is widely used as a first-line therapy for biliary tract cancer (BTC). However, few studies have been conducted to analyze second- line therapies. METHODOLOGY From 33 patients who had been administered gemcitabine following resection between May 2005 and August 2007, we retrospectively analyzed the safety and efficacy of S-1 in 11 cases who received S-1 as second-line therapy due to recurrence or relapse of the primary disease. RESULTS Among the adverse events (AEs) observed during S-1 administration, the most common was a decrease in the concentration of hemoglobin, followed by thrombocytopenia. No Grade 4 AEs or worse were detected. In addition, the AEs and their respective severity strongly resembled those of gemcitabine used as a first-line therapy. There were 7 cases that could be evaluated according to RECIST criteria, of which 1 was considered in the partial response and 3 as stable disease. The medians of time to progression after S-1 administration and survival after S-1 administration were 5.6 months and 31 months, respectively. CONCLUSIONS S-1 could be taken safely as a second-line therapy without provoking severe AEs. By preventing the cessation of S-1 administration due to its AEs, more continued S-1 administration could lead to a better prognosis for BTC.

Expression of Ins1 and Ins2 genes in mouse fetal liver

A possible cure for diabetes is explored by using non-pancreatic cells such as fetal hepatocytes. The expression of insulin and transcription factors for insulin is investigated in mouse fetal liver. We detected mRNAs for insulin I (Ins1) and insulin II (Ins2) and proinsulin- and mature insulin-positive cells in mouse fetal liver by reverse transcription plus the polymerase chain reaction and immunohistochemistry. Glucagon, somatostatin and pancreatic polypeptide were not expressed throughout development. Mouse Ins2 and Ins1 promoters were transiently activated in mouse fetal hepatocytes of embryonic days 13.5 and 16.5, respectively. Pancreatic and duodenal homeobox 1 (Pdx1) mRNA was not expressed during development of the liver. In contrast, mRNAs and proteins of neurogenic differentiation (NeuroD)/β cell E-box transactivator 2 (Beta2) and v-maf musculoaponeurotic fibrosarcoma oncogene homolog (MafA) were almost simultaneously expressed with insulin genes in the liver. Ins2 and Ins1 promoters were activated in hepatoma cells by the transfection of the expression vector for NeuroD/Beta2 alone and by the combination of NeuroD/Beta2 and MafA, respectively. These results indicate that the expression of NeuroD/Beta2 and MafA is linked temporally with the transcription of Ins2 and Ins1 genes in mouse fetal liver and suggest the potential usage of fetal hepatocytes to make insulin-producing β cells by introducing transcription factors.