Taichi Matsumoto

Simple and sensitive HPLC method for the fluorometric determination of methotrexate and its major metabolites in human plasma by post-column photochemical reaction.

A simple and sensitive HPLC method has been developed for the determination of methotrexate (MTX) and its major metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 2,4-diamino-N(10-) methylpteroic acid (DAMPA), in human plasma. After deproteinization of the plasma with 5% aqueous acetonitrile solution containing 5% trichloroacetic acid, MTX, 7-OH-MTX, DAMPA and 2,4-diaminopteroic acid (DAPA) as an internal standard were separated on a reversed-phase column, and the eluent was subsequently irradiated with UV light (245 nm), producing fluorescent photolytic degradation products. The analytes were then detected spectrofluorometrically at 452 nm with excitation at 368 nm. The extraction efficiencies of MTX, 7-OH-MTX and DAMPA from plasma at 100 pmol/mL were 81.5±5.4, 82.5±5.3 and 56.2±7.0%, respectively. The limits of quantification for MTX, 7-OH-MTX and DAMPA in plasma were 5 pmol (2.3 ng), 0.8 pmol (0.38 ng) and 10 pmol (3.4 ng)/mL, respectively. The within- and between-day variations for MTX, 7-OH-MTX and DAMPA were reliable (each was lower than 6.3%). This method was also used to monitor the concentrations of MTX and its metabolites in a patient on MTX therapy.

CD133+ Cancer Stem Cell–like Cells Derived from Uterine Carcinosarcoma (Malignant Mixed Müllerian Tumor)

Cancer stem cells (CSCs) that display tumor‐initiating properties have recently been identified. CD133, a surface glycoprotein linked to organ‐specific stem cells, has been described as a marker of CSCs in different tumor types. We herein identify and characterize CSCs in human uterine carcinosarcoma (malignant mixed Müllerian tumor), which is one of the most aggressive and therapy‐resistant gynecological malignancies and is considered to be of mesodermal origin. The CD133+ population was increased in uterine carcinosarcoma, and this population showed biphasic properties in the primary tumor. CD133+ cells predominantly formed spheres in culture and were able to differentiate into mesenchymal lineages. CD133+ cells were more resistant to cisplatin/paclitaxel‐induced cytotoxicity in comparison with CD133− cells. A real‐time polymerase chain reaction analysis of the genes implicated in stem cell maintenance revealed that CD133+ cells express significantly higher levels of Oct4, Nanog, Sox2, and Bmi1 than CD133− cells. Moreover, CD133+ cells showed a high expression level of Pax2 and Wnt4, which are genes essential for Müllerian duct formation. These CD133+ cells form serially transplantable tumors in vivo and the resulting CD133+ tumors replicated the EpCAM, vimentin, and estrogen and progesterone receptor expression of the parent tumor, indicating that CSCs likely differentiated into cells comprising the uterine carcinosarcoma tissue. Moreover, strong CD133 expression in both epithelial and mesenchymal elements in primary tumor demonstrated significant prognostic value. These findings suggest that CD133+ cells have the characteristics of CSCs and Müllerian mesenchymal progenitors. STEM CELLS 2011;29:1485–1495

Inhibition of glucose transporter 1 induces apoptosis and sensitizes multiple myeloma cells to conventional chemotherapeutic agents

Despite the recent development of anti-myeloma drugs, the prognosis of high-risk multiple myeloma remains poor. Therefore, new effective treatment strategies for this disease are needed. It has been reported that high intensity of 18-fluorodeoxyglucose positron emission tomography is high-risk factor in myeloma, suggesting that glucose uptake can be therapeutic target in high-risk myeloma. In this study, we addressed the utility of glucose transporter 1 (GLUT1) as a therapeutic target for myeloma with increased glucose uptake. We found myeloma cell lines with elevated glucose uptake activity via GLUT1 up-regulation. STF-31, a selective GLUT1 inhibitor, completely suppressed the glucose uptake activity and induced apoptosis in GLUT1 expressing myeloma cells. On the other hand, this agent little shows the cytotoxicity in normal peripheral blood mononuclear cells. Moreover, STF-31 synergistically enhanced the cell death induced by melphalan, doxorubicin, and bortezomib. GLUT1 may be promising therapeutic target in myeloma with elevated glucose uptake.

Importance of inducible multidrug resistance 1 expression in HL-60 cells resistant to gemtuzumab ozogamicin

Abstract Resistance to gemtuzumab ozogamicin (GO) hampers the effective treatment of refractory acute myeloid leukemia (AML). To clarify the mechanism of resistance to GO, HL-60 cells were persistently exposed to GO in order to establish GO-resistant HL-60 (HL-60/GOR) cells. Multidrug resistance 1 (MDR-1) was strongly expressed in HL-60/GOR cells, but not in HL-60 cells. Although withdrawal of GO after the chronic exposure of HL-60/GOR cells to this compound gradually decreased MDR-1 expression to trace levels, reintroducing GO restored high MDR-1 expression in HL-60/GOR cells, but not in HL-60 cells. These results indicate that HL-60/GOR cells acquired the ability to induce MDR-1 expression in response to GO. U0126, a MEK1/2 inhibitor, prevented GO-inducible MDR-1 expression and abrogated GO resistance in HL-60/GOR cells. These results suggest that in the clinical use of GO, inducible MDR-1 expression in tumor cells should be investigated before treatment with GO. If the cells are positive then MEK1/2 inhibitors may be effective in overcoming resistance to GO.

A simple high‐performance liquid chromatography for the determination of linezolid in human plasma and saliva

Linezolid is an antimicrobial agent for the treatment of multiresistant Gram-positive infections. A practical high-performance liquid chromatography method was developed for the determination of linezolid in human plasma and saliva. Linezolid and an internal standard (o-ethoxybenzamide) were extracted from plasma and saliva with ethyl acetate and analyzed on a Capcell Pak C18 MG column with UV detection at 254 nm. The calibration curve was linear through the range 0.5-50 µg/mL using a 200 μL sample volume. The intra- and interday precisions were all <6.44% for plasma and 5.60% for saliva. The accuracies ranged from 98.8 to 110% for both matrices. The mean recoveries of linezolid were 80.8% for plasma and 79.0% for saliva. This method was used to determine the plasma and saliva concentrations of linezolid in healthy volunteers who were orally administered a 600 mg dose of linezolid. Our liquid-liquid extraction procedure is easy and requires a small volume of plasma or saliva (200 μL). This small volume can be advantageous in clinical pharmacokinetic studies, especially if children participate.

Am80 inhibits stromal cell-derived factor-1-induced chemotaxis in T-cell acute lymphoblastic leukemia cells

C-X-C motif chemokine receptor 4 (CXCR4) and stromal cell-derived factor-1 (SDF-1) play a potent role in metastasis and infiltration of many types of tumors, including T-cell acute lymphoblastic leukemia (T-ALL), into the central nervous system or lymph nodes. Although higher levels of CXCR4 expression have been shown to correlate with shorter survival of patients, effective drugs affecting cell surface CXCR4 expression are still unknown. In the present study, we examined the effects of a synthetic retinoid Am80 on CXCR4 expression of cultured T-ALL cells, such as Jurkat. Am80 inhibited surface CXCR4 expression and SDF-1-induced chemotaxis by the acceleration of CXCR4 internalization via activation of conventional PKC. Am80 may be an effective drug to inhibit the extramedullary infiltration of T-ALL cells.

Simultaneous determination of cytosine arabinoside and its metabolite, uracil arabinoside, in human plasma using hydrophilic interaction liquid chromatography with UV detection

A practical high-performance liquid chromatography using a Cosmosil HILIC column and UV detection was developed for determining the concentrations of cytosine arabinoside (Ara-C) and uracil arabinoside (Ara-U), which is a major metabolite of Ara-C, in human plasma. This method was used to determine the plasma concentrations of Ara-C and Ara-U in a patient treated with high-dose Ara-C therapy for end-stage renal failure.

Stimulating muscarinic M1 receptors in the anterior cingulate cortex reduces mechanical hypersensitivity via GABAergic transmission in nerve injury rats

Cholinergic systems modulate synaptic transmission across the neuraxis and play an important role in higher brain function including cognition, arousal and nociception. The anterior cingulate cortex (ACC) is a fundamental brain region for nociception and chronic pain, and receives cholinergic projections mainly from basal forebrain. Recently, we found that the activation of muscarinic M1 receptors in the ACC produced antinociceptive behavior in response to mechanical stimulation. However, it has not been tested whether stimulating muscarinic receptors in the ACC can reduce mechanical hypersensitivity in animal models of chronic pain. Here, we tested whether the activation of muscarinic M1 receptors in the ACC can alleviate mechanical hypersensitivity in a nerve injury model. The activation of muscarinic M1/M4 receptors by McN-A-343 injected into the contralateral side of the ACC, but not into the ventral posterolateral nucleus, was found to dose-dependently reduce mechanical hypersensitivity 7 days following partial sciatic nerve ligation in rats. The reduction of mechanical hypersensitivity by McN-A-343, was blocked by a selective muscarinic M1 antagonist, but not a M4 receptor antagonist. Importantly, the nerve injury model did not change the protein expression of muscarinic M1 receptors in the ACC. Additionally, a type A γ-aminobutyric acid (GABAA) receptor agonist injected into the ACC reduced the mechanical hypersensitivity in this injury model. Finally, a GABAA receptor antagonist blocked the reduction of mechanical hypersensitivity by McN-A-343 in the injury model. Collectively, these results suggest that activations of muscarinic M1 receptors in the ACC reduce nerve injury-induced mechanical hypersensitivity through GABAergic transmission via GABAA receptors.

Involvement of GABA B receptor in the antihypersensitive effect in anterior cingulate cortex of partial sciatic nerve ligation model

The role of the GABAB receptor in the anterior cingulate cortex (ACC) of neuropathic pain is unclear. Injection of a GABAB receptor antagonist CGP35348 into the ACC induced mechanical hypersensitivity in normal rats. Activation of the GABAB receptor injected by a GABAB receptor agonist baclofen into the ACC attenuated mechanical hypersensitivity in partial sciatic nerve ligation (PSNL) rats. Co-microinjection of CGP35348 with a muscarinic M1 receptor agonist McN-A-343 into the ACC significantly inhibited McN-A-343-induced antihypersensitivity in PSNL rats. These results suggest that the GABAB receptor in the ACC contributes to mechanical hypersensitivity and is involved in muscarinic M1 receptor-mediated antihypersensitivity.