Functional Expression of Choline Transporters in Microglia and Their Regulation of Microglial M1/M2 Polarization.

BACKGROUND: Microglia are key cells of the immune system in the central nervous system and are suggested to be deeply involved in the development of neurodegenerative diseases. It is well known that microglia have functional plasticity, with an inflammatory M1 phenotype and an anti-inflammatory M2 phenotype. Inhibition of choline transport in macrophages has been reported to suppress the secretion of inflammatory cytokines. However, the role of the choline transport system in regulating microglial M1/M2 polarization has not been fully elucidated to date. In this study, we investigated the mechanism of choline uptake in microglia, and its association with microglial M1/M2 polarization. METHODS: The immortalized mouse microglial cell line SIM-A9 was used for [3H]choline uptake and expression analysis of choline transporters. The association between the choline uptake system and the M1/M2 polarization of microglia was also analyzed. RESULTS: Choline transporter-like protein (CTL) 1 and CTL2 were highly expressed in SIM-A9 cells, and CTL1 and CTL2 were localized in the plasma membrane and mitochondria, respectively. Functional analysis of choline uptake demonstrated the existence of Na+-independent, pH-dependent, and intermediate-affinity choline transport systems. Choline uptake was concentration-dependently inhibited by hemicholinium-3 (HC-3), an inhibitor of choline uptake, and increased by lipopolysaccharide (LPS) and interleukin-4 (IL-4). Expression of the mRNA of M1 microglia markers IL-1ß and IL-6 was increased by LPS, and their effects were suppressed by choline deprivation and HC-3. In contrast, mRNA expression of the M2 microglial marker arginase-1 (Arg-1) was increased by IL-4, and the effect was enhanced by choline deprivation and HC-3. CONCLUSIONS: Our results suggest that inhibition of CTL1-mediated choline uptake in microglia preferentially induces M2 microglia polarization, which is a potential therapeutic approach for inflammatory brain diseases.

Molecular and Functional Analysis of Choline Transporters and Antitumor Effects of Choline Transporter-Like Protein 1 Inhibitors in Human Pancreatic Cancer Cells.

Choline, an organic cation, is one of the biofactors that play an important role in the structure and the function of biological membranes, and it is essential for the synthesis of phospholipids. Choline positron emission tomography-computed tomography (PET/CT) provides useful information for the imaging diagnosis of cancers, and increased choline accumulation has been identified in a variety of tumors. However, the molecular mechanisms of choline uptake and choline transporters in pancreatic cancer have not been elucidated. Here, we examined molecular and functional analyses of choline transporters in human pancreatic-cancer cell line MIA PaCa-2 and the elucidation of the action mechanism behind the antitumor effect of novel choline-transporter-like protein 1 (CTL1) inhibitors, Amb4269951 and its derivative Amb4269675. CTL1 and CTL2 mRNAs were highly expressed in MIA PaCa-2 cells, and CTL1 and CTL2 proteins were localized in the plasma membrane and the intracellular compartments, respectively. Choline uptake was characterized by Na+-independence, a single-uptake mechanism, and inhibition by choline-uptake inhibitor HC-3, similar to the function of CTL1. These results suggest that the uptake of extracellular choline in MIA PaCa-2 cells is mediated by CTL1. Choline deficiency and HC-3 treatment inhibited cell viability and increased caspase 3/7 activity, suggesting that the inhibition of CTL1 function, which is responsible for choline transport, leads to apoptosis-induced cell death. Both Amb4269951 and Amb4269675 inhibited choline uptake and cell viability and increased caspase-3/7 activity. Ceramide, which is increased by inhibiting choline uptake, also inhibited cell survival and increased caspase-3/7 activity. Lastly, both Amb4269951 and Amb4269675 significantly inhibited tumor growth in a mouse-xenograft model without any adverse effects such as weight loss. CTL1 is a target molecule for the treatment of pancreatic cancer, and its inhibitors Amb4269951 and Amb4269675 are novel lead compounds.

Lack of ACTPK1, an STY kinase, enhances ammonium uptake and use, and promotes growth of rice seedlings under sufficient external ammonium.

Ammonium influx into plant roots via the high-affinity transport system (HATS) is down-modulated under elevated external ammonium, preventing ammonium toxicity. In ammonium-fed Arabidopsis, ammonium transporter 1 (AMT1) trimers responsible for HATS activity are allosterically inactivated in a dose-dependent manner via phosphorylation of the conserved threonine at the carboxyl-tail by the calcineurin B-like protein 1-calcineurin B-like protein-interacting protein kinase 23 complex and other yet unidentified protein kinases. Using transcriptome and reverse genetics in ammonium-preferring rice, we revealed the role of the serine/threonine/tyrosine protein kinase gene OsACTPK1 in down-modulation of HATS under sufficient ammonium. In wild-type roots, ACTPK1 mRNA and protein accumulated dose-dependently under sufficient ammonium. To determine the function of ACTPK1, two independent mutants lacking ACTPK1 were produced by retrotransposon Tos17 insertion. Compared with segregants lacking insertions, the two mutants showed decreased root growth and increased shoot growth under 1 mm ammonium due to enhanced ammonium acquisition, via aberrantly high HATS activity, and use. Furthermore, introduction of OsACTPK1 cDNA fused to the synthetic green fluorescence protein under its own promoter complemented growth and the HATS influx, and suggested plasma membrane localization. Root cellular expression of OsACTPK1 also overlapped with that of ammonium-induced OsAMT1;1 and OsAMT1;2. Meanwhile, threonine-phosphorylated AMT1 levels were substantially decreased in roots of ACTPK1-deficient mutants grown under sufficient ammonium. Bimolecular fluorescence complementation assay further confirmed interaction between ACTPK1 and AMT1;2 at the cell plasma membrane. Overall, these findings suggest that ACTPK1 directly phosphorylates and inactivates AMT1;2 in rice seedling roots under sufficient ammonium.

Functional analysis of choline transporters in rheumatoid arthritis synovial fibroblasts.

OBJECTIVES: In this study, we examined the functional characteristics of choline uptake and sought to identify the transporters in rheumatoid arthritis synovial fibroblasts (RASFs). METHODS: The expression of choline transporters was evaluated by quantitative real-time PCR, western blotting, and immunocytochemistry. Time course, Na+-dependency, and kinetics of [3H]choline uptake were investigated. Effects of cationic drugs on the uptake of [3H]choline, cell viability, and caspase-3/7 activity were also examined. Finally, we investigated the influence of choline uptake inhibitor, hemicholinium-3 (HC-3), and choline deficiency on cell viability and caspase-3/7 activity. RESULTS: Choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein were highly expressed in RASFs and were localized to the plasma membrane. [3H]Choline uptake occurred via a Na+-independent and pH-dependent transport system. The cells have two different [3H]choline transport systems, high- and low-affinity. Various organic cations, HC-3 and choline deficiency inhibited both [3H]choline uptake and cell viability, and enhanced the caspase-3/7 activity. The functional inhibition of choline transporters could promote apoptotic cell death. In RASFs, [3H]choline uptake was significantly increased compared with that in OASFs without a change in gene expression. CONCLUSIONS: These results suggest that CTL1 (high-affinity) and CTL2 (low-affinity) are highly expressed in RASFs and choline may be transported by a choline/H+ antiport system. Identification of this CTL1- and CTL2-mediated choline transport system should provide a potential new target for RA therapy.

Identification and functional analysis of choline transporter in tongue cancer: A novel molecular target for tongue cancer therapy.

We examined the functional characteristics of choline uptake in human tongue carcinoma using the cell line HSC-3. Furthermore, we explored the possible correlation between the inhibition of choline uptake and apoptotic cell death. Both choline transporter-like protein 1 (CTL1) and CTL2 mRNAs and proteins were expressed, and were located in plasma membrane and mitochondria, respectively. Choline uptake was saturable and mediated by a single transport system, which is pH-dependent. Several cationic drugs inhibited cell viability and [(3)H]choline uptake. Choline uptake inhibitors and choline deficiency inhibited cell viability and increased caspase-3/7 activity. We conclude that extracellular choline is mainly transported via a CTL1 that relies on a directed H(+) gradient as a driving force. The functional inhibition of CTL1 by cationic drugs could promote apoptotic cell death. Furthermore, CTL2 may be the major site for the control of choline oxidation in mitochondria and hence for the supply of endogenous betaine and S-adenosyl methionine, which serves as a major methyl donor. Identification of this CTL1- and CTL2-mediated choline transport system provides a potential new target for tongue cancer therapy.

[Postoperative cholangitis of balloon-occluded transarterial chemoembolization (B-TACE) for hepatocellular carcinoma].

In 35 patients who underwent balloon-occluded transarterial chemoembolization (B-TACE) for hepatocellular carcinoma (HCC) since January 2013, 5 patients (14%) had postoperative cholangitis, 1 of whom required drainage of a liver abscess. Four of these patients(80%)were treated with cisplatin (CDDP)-epirubicin (EPI)-Lipiodol (Lp) emulsion, and 1 was treated with EPI-Lp emulsion.The balloon was located and inflated at the lobar level (C: conventional)in 3 patients (60%) and at the subsegmental or more distal level (SS: superselective) in 2 patients (40%). Chemical vascular damage was considered to cause the cholangitis.We conclude that it is necessary to determine the optimal drug for B-TACE to reduce vascular damage. Miriplatin may be useful because of its lower vascular damage compared with CDDP-Lp and EPI-Lp.

Functional expression of choline transporter-like protein 1 (CTL1) in small cell lung carcinoma cells: a target molecule for lung cancer therapy.

Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine and the neurotransmitter acetylcholine (ACh). Elevated levels of choline and up-regulated choline kinase activity have been detected in cancer cells. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism and a prerequisite for cancer cell proliferation. However, the uptake system for choline and the functional expression of choline transporters in lung cancer cells are poorly understood. We examined the molecular and functional characterization of choline uptake in the small cell lung carcinoma cell line NCI-H69. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na(+) from the uptake buffer strongly enhanced choline uptake. This increase in choline uptake under the Na(+)-free conditions was inhibited by dimethylamiloride (DMA), a Na(+)/H(+) exchanger (NHE) inhibitor. Various organic cations and the choline analog hemicholinium-3 (HC-3) inhibited the choline uptake and cell viability. A correlation analysis of the potencies of organic cations for the inhibition of choline uptake and cell viability showed a strong correlation (R=0.8077). RT-PCR revealed that choline transporter-like protein 1 (CTL1) mRNA and NHE1 are mainly expressed. HC-3 and CTL1 siRNA inhibited choline uptake and cell viability, and increased caspase-3/7 activity. The conversion of choline to ACh was confirmed, and this conversion was enhanced under Na(+)-free conditions, which in turn was sensitive to HC-3. These results indicate that choline uptake through CTL1 is used for ACh synthesis. Both an acetylcholinesterase inhibitor (eserine) and a butyrylcholinesterase inhibitor (ethopropazine) increased cell proliferation, and these effects were inhibited by 4-DAMP, a mAChR3 antagonist. We conclude that NCI-H69 cells express the choline transporter CTL1 which uses a directed H(+) gradient as a driving force, and its transport functions in co-operation with NHE1. This system primarily supplies choline for the synthesis of ACh and secretes ACh to act as an autocrine/paracrine growth factor, and the functional inhibition of CTL1 could promote apoptotic cell death. Identification of this new CTL1-mediated choline transport system provides a potential new target for therapeutic intervention.

Licochalcone E, a ß-Amyloid Aggregation Inhibitor, Regulates Microglial M1/M2 Polarization via Inhibition of CTL1-Mediated Choline Uptake.

Alzheimer's disease (AD) is thought to be a series of neuroinflammatory diseases caused by abnormal deposits of amyloid-ß (Aß) and tau protein in the brain as part of its etiology. We focused on Aß aggregation and M1 and M2 microglial polarity in microglia to search for novel therapeutic agents. It has been reported that the inhibition of choline uptake via choline transporter-like protein 1 (CTL1) in microglia preferentially induces M2 microglial polarity. However, the role of the choline transport system on the regulation of microglial M1/M2 polarity in AD is not fully understood. Licochalcones (Licos) A-E, flavonoids extracted from licorice, have been reported to have immunological anti-inflammatory effects, and Lico A inhibits Aß aggregation. In this study, we compared the efficacy of five Licos, from Lico A to E, at inhibiting Aß1-42 aggregation. Among the five Licos, Lico E was selected to investigate the relationship between the inhibition of choline uptake and microglial M1/M2 polarization using the immortalized mouse microglial cell line SIM-A9. We newly found that Lico E inhibited choline uptake and Aß1-42 aggregation in SIM-A9 cells in a concentration-dependent manner, suggesting that the inhibitory effect of Lico E on choline uptake is mediated by CTL1. The mRNA expression of tumor necrosis factor (TNF-α), a marker of M1 microglia, was increased by Aß1-42, and its effect was inhibited by choline deprivation and Lico E in a concentration-dependent manner. In contrast, the mRNA expression of arginase-1 (Arg-1), a marker of M2 microglia, was increased by IL-4, and its effect was enhanced by choline deprivation and Lico E. We found that Lico E has an inhibitory effect on Aß aggregation and promotes polarity from M1 to M2 microglia via inhibition of the CTL1 function in microglia. Thus, Lico E may become a leading compound for a novel treatment of AD.

Portal vein embolization can prevent intrahepatic metastases to non-embolized liver.

BACKGROUND/AIMS: To determine the efficacy of portal vein embolization (PVE) against unresectable hepatocellular carcinoma (HCC). METHODOLOGY: We conducted a comparative study using 17 patients with HCC determined to be unresectable and who received a combination of PVE and transarterial chemoembolization (TACE) (PVE group) and 22 HCC patients with tumors in the unilateral lobe, which were treated only with repeated TACE (TACE group) from January 2000 to December 2008. RESULTS: There were no significant differences in background factors except for gender between the two groups. The cumulative intrahepatic recurrence rates in the non-portal-embolized area (in the contralateral lobe for the TACE group) at 1 year and 3 years was 41.1% and 58.8% in the PVE group and 77.3% and 81.8% in the TACE group, respectively. The former was significantly lower (p<0.05). The cumulative overall survival rate at 1 year, 3 and 5 years was 88.2%, 38.2% and 38.2% in the PVE group, and 68.1%, 22.7% and 8.5% in the TACE group, respectively. The former was significantly higher (p<0.05). CONCLUSIONS: Although in patients with unresectable HCC, when HCC is localized in the portal-embolized area, PVE combined with TACE can prevent intrahepatic metastasis to the non-portal-embolized area and improve overall survival.

A Novel Plant-Derived Choline Transporter-like Protein 1 Inhibitor, Amb544925, Induces Apoptotic Cell Death via the Ceramide/Survivin Pathway in Tongue Squamous Cell Carcinoma.

BACKGROUND: Despite recent advances in the early detection and treatment of TSCC patients, recurrence rates and survival rates have not improved. The high frequency of lymph node metastasis is one of the causes, and the drug development of new therapeutic mechanisms such as metastasis control is desired. Choline transporter-like protein 1 (CTL1) has attracted attention as a target molecule in cancer therapy. In this study, we examined the antitumor effects of Amb544925, a plant-derived CTL1 inhibitor. METHODS: The TSCC cell line HSC-3 was used to measure [3H]choline uptake, cell survival, caspase activity, and cell migration. Xenograft model mice were prepared to verify the antitumor effect of Amb544925. RESULTS: Amb544925 inhibited cell viability and increased caspase-3/7 activity at concentrations that inhibited choline uptake. Amb544925 and ceramide increased SMPD4 expression and suppressed surivivin expression. Furthermore, Amb544925 and ceramide inhibited the migration of HSC-3 cells. In the xenograft model mice, Amb544925 suppressed tumor growth and CTL1 mRNA expression. CONCLUSIONS: The plant-derived CTL1 inhibitor Amb544925 is a lead compound of a new anticancer agent exhibiting antitumor effects and inhibition of cell migration through the ceramide/survivin pathway.

Laparoscopy-assisted resection of an undiagnosed liver tumor and ascending colon cancer via mini median laparotomy: report of a case.

We describe how we resected a hepatic angiomyolipoma and ascending colon cancer synchronously via laparoscopic left lateral segmentectomy and laparoscopic right colectomy, respectively. The patient was a 72-year-old man, admitted to our hospital after a liver tumor and ascending colon cancer were detected during a general health check. Computed tomography (CT) showed a hypervascular liver tumor mimicking hepatocellular carcinoma, 2 cm in diameter, in segment 3 of the liver. The ascending colon cancer was diagnosed as T2N0M0, Stage I. The left lateral liver and right colon were mobilized laparoscopically and hepatic transaction, followed by resection and anastomosis of the colon, were performed extracorporeally through the same 7-cm upper median incision. This type of laparoscopy-assisted combined resection is useful to obtain a pathological diagnosis of the liver tumor and to remove the entire tumor in a minimally invasive and cosmetic manner.

Functional Expression of Choline Transporters in Human Neural Stem Cells and Its Link to Cell Proliferation, Cell Viability, and Neurite Outgrowth.

Choline and choline metabolites are essential for all cellular functions. They have also been reported to be crucial for neural development. In this work, we studied the functional characteristics of the choline uptake system in human neural stem cells (hNSCs). Additionally, we investigated the effect of extracellular choline uptake inhibition on the cellular activities in hNSCs. We found that the mRNAs and proteins of choline transporter-like protein 1 (CTL1) and CTL2 were expressed at high levels. Immunostaining showed that CTL1 and CTL2 were localized in the cell membrane and partly in the mitochondria, respectively. The uptake of extracellular choline was saturable and performed by a single uptake mechanism, which was Na+-independent and pH-dependent. We conclude that CTL1 is responsible for extracellular choline uptake, and CTL2 may uptake choline in the mitochondria and be involved in DNA methylation via choline oxidation. Extracellular choline uptake inhibition caused intracellular choline deficiency in hNSCs, which suppressed cell proliferation, cell viability, and neurite outgrowth. Our findings contribute to the understanding of the role of choline in neural development as well as the pathogenesis of various neurological diseases caused by choline deficiency or choline uptake impairment.

[An elderly patient with advanced gastric cancer maintaining complete response for over 3 years by oral administration of UFT following short span of S-1].

We report a successful case of chemotherapy with oral fluoropyrimidines. The patient was an 81-year-old woman who complained epigastric discomfort. Endoscopy revealed a type 3 advanced gastric cancer, and the biopsy specimen was defined histologically as poorly-differentiated adenocarcinoma. She didn't hope for an operation, but agreed to receive chemotherapy. S-1 (80 mg/day) was administered for 14 days, followed by 7 days rest. This schedule induced grade 1 thrombocytopenia and fatigue after two weeks administration. Therefore, we reduced the administration dosage to 60 mg/ day. Almost complete response (CR) was observed after 8 weeks of S-1 administration. But she was admitted urgently to other emergency hospital for stumbling due to dizziness accompanied with vomiting and anorexia. We considered it was difficult to continue S-1 administration. Therefore, we changed S-1 to UFT-E and started from 300 mg/day. One month later, as the adverse effects were not recognized, we increased a dosage of UFT-E to 400 mg/day for the purpose of more dose intensity. After 6 months, CR was confirmed continuously. We reduced UFT-E to 300 mg/day, and CR has been continued for 3 years until now without any adverse events. There was no evidence regarding the best timing to syop anticancer administration. As the adverse effect was very mild and her quality of life improved, we continued UFT-E administration for a long time.

Functional Expression of Choline Transporter-Like Protein 1 in LNCaP Prostate Cancer Cells: A Novel Molecular Target.

Prostate cancer is one of the most common cancers in men. Choline PET or PET/CT has been used to visualize prostate cancer, and high levels of choline accumulation have been observed in tumors. However, the uptake system for choline and the functional expression of choline transporters in prostate cancer are not completely understood. In this study, the molecular and functional aspects of choline uptake were investigated in the LNCaP prostate cancer cell line along with the correlations between choline uptake and cell viability in drug-treated cells. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA were highly expressed in LNCaP cells. CTL1 and CTL2 were located in the plasma membrane and mitochondria, respectively. [3H]Choline uptake was mediated by a single Na+-independent, intermediate-affinity transport system in the LNCaP cells. The anticancer drugs, flutamide and bicalutamide, inhibited cell viability and [3H]choline uptake in a concentration-dependent manner. The correlations between the effects of these drugs on cell viability and [3H]choline uptake were significant. Caspase-3/7 activity was significantly increased by both flutamide and bicalutamide. Furthermore, these drugs decreased CTL1 expression in the prostate cancer cell line. These results suggest that CTL1 is functionally expressed in prostate cancer cells and are also involved in abnormal proliferation. Identification of this CTL1-mediated choline transport system in prostate cancer cells provides a potential new therapeutic target for the treatment of this disease.

Anticancer Activity of Amb4269951, a Choline Transporter-Like Protein 1 Inhibitor, in Human Glioma Cells.

Choline transporter-like protein 1 (CTL1) is highly expressed in glioma cells, and inhibition of CTL1 function induces apoptotic cell death. Therefore, CTL1 is a potential target molecule for glioma therapy. Here, we investigated the therapeutic mechanism underlying the antitumor effects of Amb4269951, a recently discovered novel CTL1 inhibitor, in the human glioma cell line U251MG, and evaluated its in vivo effects in a mouse xenograft model. Amb4269951 inhibited choline uptake and cell viability and increased caspase-3/7 activity. CTL1-mediated choline uptake is associated with cell viability, and the functional inhibition of CTL1 by Amb4269951 may promote apoptotic cell death via ceramide-induced suppression of the expression of survivin, an apoptotic inhibitory factor. Finally, Amb4269951 demonstrated an antitumor effect in a mice xenograft model by significantly inhibiting tumor growth without any weight loss. Amb4269951 is the lead compound in the treatment of glioma and exhibits a novel therapeutic mechanism. These results may lead to the development of novel anticancer drugs targeting the choline transporter CTL1, which has a different mechanism of action than conventional anticancer drugs against gliomas.

Protein kinase C promotes choline transporter­like protein 1 function via improved cell surface expression in immortalized human hepatic cells.

Choline is used to synthesize phospholipids and a lack of choline induces a number of liver­related diseases, including non­alcoholic steatohepatitis. The current study characterized the choline uptake system, at molecular and functional levels, in the immortalized human hepatic cell line, Fa2N­4, to identify the specific choline transporter involved in choline uptake. The present study also assesed whether choline deficiency or the inhibited choline uptake affected cell viability and apoptosis. Reverse transcription­quantitative polymerase chain reaction (PCR) revealed choline transporter­like protein 1 (CTL1) and CTL2 mRNA and protein expression in Fa2N­4 cells. [Methyl­3H]choline studies revealed choline uptake was saturable and mediated by a single transport system that functioned in a Na+­independent but pH­dependent manner, which was similar to CTL1. Hemicholinium­3 (HC­3), which is a choline uptake inhibitor, and choline deficiency inhibited cell viability, increased caspase­3 and ­7 activities, and increased fluorescein isothiocyanate­Annexin V immunofluorescent staining indicated apoptosis. Immunofluorescent staining also revealed CTL1 and CTL2 localized in plasma and mitochondrial membranes, respectively. [Methyl­3H]choline uptake was enhanced by a protein kinase C (PKC) activator, phorbol­12­myristate 13­acetate (PMA). Immunofluorescence staining and western blot analysis demonstrated increased CTL1 expression on the cell membrane following PMA treatment. The results of current study indicated that extracellular choline is primarily transported via CTL1, relying on a direct H+ gradient that functions as a driving force in Fa2N­4 cells. Furthermore, it was hypothesized that CTL1 and the choline uptake system are strongly associated with cell survival, and that the choline uptake system is modulated by PKC signaling via increased CTL1 expression on the cell surface. These findings provide further insights into the pathogenesis of liver disease involving choline metabolism.

Molecular and Functional Characterization of Choline Transporter-Like Proteins in Esophageal Cancer Cells and Potential Therapeutic Targets.

In this study, we examined the molecular and functional characterization of choline uptake in the human esophageal cancer cells. In addition, we examined the influence of various drugs on the transport of [3H]choline, and explored the possible correlation between the inhibition of choline uptake and apoptotic cell death. We found that both choline transporter-like protein 1 (CTL1) and CTL2 mRNAs and proteins were highly expressed in esophageal cancer cell lines (KYSE series). CTL1 and CTL2 were located in the plasma membrane and mitochondria, respectively. Choline uptake was saturable and mediated by a single transport system, which is both Na+-independent and pH-dependent. Choline uptake and cell viability were inhibited by various cationic drugs. Furthermore, a correlation analysis of the potencies of 47 drugs for the inhibition of choline uptake and cell viability showed a strong correlation. Choline uptake inhibitors and choline deficiency each inhibited cell viability and increased caspase-3/7 activity. We conclude that extracellular choline is mainly transported via a CTL1. The functional inhibition of CTL1 by cationic drugs could promote apoptotic cell death. Furthermore, CTL2 may be involved in choline uptake in mitochondria, which is the rate-limiting step in S-adenosylmethionine (SAM) synthesis and DNA methylation. Identification of this CTL1- and CTL2-mediated choline transport system provides a potential new target for esophageal cancer therapy.

[A long term survival case of advanced intrahepatic cholangiocarcinoma treated with multidisciplinary treatment].

A 56-year-old man was admitted to our hospital for liver tumor of 7 cm in diameter, located mainly in the anterior segment. He underwent a hepatic resection of anterior and median segment of the liver in January 2002. Pathologically, the tumor was moderately differentiated intrahepatic cholangiocarcinoma (ICC) with vascular invasion. Hepatic arterial chemotherapy as a neoadjuvant therapy was performed. In December 2002, bone metastases in the 12th thoracic spine and iliac bone were found. Therefore, he received radiotherapy (total of 30 Gy) and a monthly bisphosphonate infusion. In May 2003, multiple intrahepatic recurrences were diagnosed. Transarterial chemoembolization for twice and an additional radio-frequency ablation were performed. Four months later, lung metastases were found, thus, he was administered TS-1. He has been alive with PS 0 for 4 and half years after the hepatic resection. No new intrahepatic recurrences were found and bone and lung metastases remained to be stable. Although only a hepatic resection is a useful therapy for ICC, such a multidisciplinary treatment may have an effect to improve the prognosis of patients with ICC.

Functional expression of choline transporter like-protein 1 (CTL1) and CTL2 in human brain microvascular endothelial cells.

In this study, we examined the molecular and functional characterization of choline transporter in human brain microvascular endothelial cells (hBMECs). Choline uptake into hBMECs was a saturable process that was mediated by a Na(+)-independent, membrane potential and pH-dependent transport system. The cells have two different [(3)H]choline transport systems with Km values of 35.0 ± 4.9 µM and 54.1 ± 8.1 µM, respectively. Choline uptake was inhibited by choline, acetylcholine (ACh) and the choline analog hemicholinium-3 (HC-3). Various organic cations also interacted with the choline transport system. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA were highly expressed, while mRNA for high-affinity choline transporter 1 (CHT1) and organic cation transporters (OCTs) were not expressed in hBMECs. CTL1 and CTL2 proteins were localized to brain microvascular endothelial cells in human brain cortical sections. Both CTL1 and CTL2 proteins were expressed on the plasma membrane and mitochondria. CTL1 and CTL2 proteins are mainly expressed in plasma membrane and mitochondria, respectively. We conclude that choline is mainly transported via an intermediate-affinity choline transport system, CTL1 and CTL2, in hBMECs. These transporters are responsible for the uptake of extracellular choline and organic cations. CTL2 participate in choline transport mainly in mitochondria, and may be the major site for the control of choline oxidation.

[A case of advanced gastric cancer with liver and lung metastases markedly responded to TS-1-based sequential chemotherapy].

A 61-year-old man with multiple liver and lung metastases from advanced gastric cancer was admitted to our hospital. We selected the patient from the outpatient department and started a single administration of TS-1 as a first line chemotherapy. TS-1 was markedly effective and the CEA level decreased 4,528 ng/ml to 44 ng/ml only in three months. The clinical response was assessed to be partial response (PR) comparable to complete response (CR) according to the tumor regression effect. The effect had been continued for almost six months. Because the CEA level elevated again, we estimated that the tumor acquired a drug tolerance to TS-1. Therefore, we applied CDDP with TS-1 as a second line chemotherapy. Unfortunately, it was not effective. Then we combined paclitaxel (PTX) to TS-1. The CEA level was remarkably reduced again, but transiently. Thereafter, we continued a sequential administration of TS-1 plus other drugs (CPT-11, docetaxel and MMC). Over 2 years, the patient is alive with a good quality of life.