Short-term depression and long-term enhancement of ACh-gated channel currents induced by linoleic and linolenic acid

The effects of cis-unsaturated free fatty acids such as linoleic and linolenic acid on ACh-evoked currents were examined using normal and mutant nicotinic acetylcholine (ACh) receptors lacking protein kinase C (PKC) phosphorylation sites on the α and δ subunits expressed in Xenopus oocytes. These free fatty acids reduced ACh-gated channel currents during treatment and to a greater extent in Ca²⁺-free extracellular solution. After treatment, the currents were enhanced as the drug was washed out, but this effect was not observed in the absence of extracellular Ca²⁺. Linolenic acid was more potent of the current enhancement (300% of the control) than linoleic acid (190% of the control). The current enhancement induced by these free fatty acids was inhibited by the selective PKC inhibitor, GF109203X, while the current depression was not affected. Furthermore, these lipids decreased ACh-evoked currents in mutant ACh receptors to the same extent as in normal ACh receptors, but never enhanced the currents. These results indicate that linoleic and linolenic acid have biphasic actions on ACh receptor currents; a short-term depression and a long-term enhancement. The short-term depression may be due to an interaction with the ACh receptor channels, presumably at Ca²⁺ binding sites. The long-lasting enhancement appears to result from Ca²⁺-dependent PKC activation followed by PKC phosphorylation of the ACh receptors. © 1997 Elsevier Science B.V. All rights reserved.     

Intensive Chemotherapy Followed by Autologous Bone Marrow Transplantation for a Patient Having Wide-spread Embryonal Carcinoma in the Central Nervous System

A patient with an intracranial embryonal carcinoma, which wasresistant to cisplatin-based combined chemotherapy and radiotherapy,developed spinal seeding. Alpha fetoprotein (AFP), human chorionicgonadotropin (HCG) and the beta chain of HCG (HCGß)in serum or cerebrospinal fluid immediately returned to elevatedlevels despite sequential chemotherapy and resection. Accordingly,we treated this patient with spinal irradiation and intensivecombination chemotherapy (cisplatin etoposide, vinblastine,cyclophosphamide and actinomycin D) followed by autologous bonemarrow transplantation. With this treatment, the clinical manifestationsshowed a tendency to improve and, furthermore, the serum levelsof AFP and HCG returned to normal. These findings suggest autologousbone marrow transplantation to be a potential approach to thetreatment of patients with embryonal carcinomas.     

Immunogenotypes of Lymphoid Malignancies; the Rearrangement of T Cell Receptor β Chain Gene Can Occur before the γ Chain Gene Rearrangement

Immunoglobulin (Ig) and T cell receptor (TcR) gene rearrangements were analyzed in 101 cases of lymphoid malignancies in association with a surface phenotype study. In leukemias/lymphomas with mature phenotype, there is a good correlation between phenotypes and genotypes. However, in leukemias/lymphomas with immature phenotype, we found many discordances between phenotypes and genotypes, suggesting the stochastic nature of hematopoietic cell differentiation at the early stage. As for TcR β and γ chains, the rearrangement of γ chain gene is considered to occur slightly prior to that of β chain gene. However, we observed a mature T cell malignancy, adult T-cell leukemia, with rearranged β chain gene and germ line γ chain gene, showing the possible existence of another pathway of T cell differentiation.     

Absorption and Bioavailability of Diclofenac after Rectal Administration of Diclofenac-Na Gel Preparation in Rat and Man

In order to evaluate diclofenac-Na (DC-Na) micro-enema, DC-Na gel preparations were administered to rats and man. When DC-Na gel preparations were rectally administered at various pH (pH 5– 8) to rats, their bioavailability increased at higher pH. The bioavailability of DC-Na gel preparations (pH 8.0) in rats was significantly higher than that with conventional suppository bases, Witepsol H-15 and polyethylene glycol 1000 (PEG 1000). In man, the DC-Na gel preparation showed higher C_max and higher bioavailability than commercial suppository made with an oily base. DC-Na gel preparations containing 10% v/v oleic acid showed a prolonged action. The irritative effect of DC-Na gel preparation on rectal mucosa in rats was weaker than that of PEG 1000, but similar to that of Witepsol H-15. Therefore, the present results suggest that gel preparation is a favorable form for rectal administration of diclofenac-Na.     

OSTEOSARCOMA Ultrastructural and Immunohistochemical Studies on Alkaline Phosphatase-positive Tumor Cells Constituting a Variety of Histologic Types

The osteosarcomas were subclassified into osteoblastic, fibroblastic, chondroblastic and telangiectatic types and examined by electron microscopy. Their immunohistochemical reactions were also studied. In an overall survey of the above types, fibroblast-like cells revealed poorly developed cytoplasmic organelles with rather short, branching rough endoplasmic reticulum, mixed with osteoblast-like cells that were hardly distinguishable from the former. They appeared to be an early stage of an osteoblastic cell lineage from the distribution and development of their cell organelles and highly positive vimentin activity. The tumor cells in malignant cartilage varied in appearance from chondroblast-like to osteoblast-like cells. All types of tumor cells expressed alkaline phosphatase activity to a significant degree. Immunohistochemical staining showed a mixture of procollagen type I-positive cells among the cells positive for both procollagen type II and S-100 protein in the malignant cartilage. Irrespective of any ultrastructural differences between these various tumor cell types, they all revealed a significant degree of ALPase activity unlike other types of bone tumors, suggesting that the tumor cells which constitute the various types of osteosarcoma are derived from a common precursor cell.     

Clinical Response to Busramustine (KM-2210) in Chronic Lymphocytic Leukemia: A Pilot Evaluation of Estrogen Receptor in Relation to Its Therapeutic Effect

Busramustine (KM-2210), the benzoate of a 17ß-estradiol-chlorambucilconjugate,$$$ was administered to 11 patients with chronic lymphocyticleukemia (CLL) which included eight cases of B-cell CLL andthree cases of T-cell CLL. Four patients had received priorchemotherapy. Busramustine was given orally at an initial dailydose of 50–100 mg continuously, and the dose was modifiedaccording to hematological improvement. Two cases of B-cellCLL achieved clinical complete responses, six cases includingtwo of T-cell CLL and four of B-cell CLL achieved partial responsesand one case of B-cell CLL achieved improvement. The partialand complete response rate was 72.7%. Four patients showed estrogenreceptor activity of CLL cells ranging from 3.5 to 57.5 fmol/mgcytosol protein, but there seemed to be no correlation betweenthe estrogen receptor activity of the CLL cells and the therapeuticeffects of busramustine. Toxic effects included diarrhea (2/11)and estrogen-related symptoms including breast pain (4/11),genital bleeding (2/5), gynecomastia (2/6) and loss of libido(2/6). The findings of this preliminary study suggest that busramustineis effective in the treatment of CLL, irrespective of the presenceof the estrogen receptor.     

Clinical significance of pyrimidine nucleoside phosphorylase (PyNPase) in breast cancer

To clarify the clinical significance of pyrimidine nucleoside phosphorylase (PyNPase) activity in breast cancer, we examined the possible correlation of PyNPase activity to clinicopathological features and prognosis in twenty-one patients with primary breast cancer from April 2000 to December 2001. Flow signals of tumors were analyzed by Power Doppler sonography (PDUS), and maximal velocity (V(max)) was calculated. PyNPase activity of resected specimens was assayed by ELISA method. PyNPase activities in resected cancerous tissue were 156.9+/-63.5 unit/mg (mean+/-SD), which were significantly higher than that in normal tissue (19.0+/-18.1 unit/mg, p<0.0001). PyNPase activity was positively correlated with tumor size (r=0.496, p=0.026) and V(max) (r=0.498, p=0.021). The disease free survival rate was significantly lower in the high PyNPase activity group than in the low PyNPase activity group. In overall survival rate, there was no significant difference between the high and low PyNPase activity groups. In the multivariate analysis, PyNPase activity was an independent predictor of postoperative recurrence (p=0.032). We suggest that PyNPase activity is associated with progression and proliferation of breast cancer, and that it may be useful for prediction of the prognosis.     

Lysocellin, a metabolite of the novel drug 'alopestatin', induces G1 arrest and prevents cytotoxicity induced by etoposide

We report here that lysocellin, a polyether antibiotic from a streptomycete, induces G1 phase arrest in human osteosarcoma MG63 cells. Lysocellin up-regulates p21WAF1/Cip1 and down-regulates cyclin D1 at the mRNA level. In addition, cyclin D1 is down-regulated by the proteasome-dependent signal pathway in MG63 cells. In drug combination studies, we found that lysocellin treatment weakened the cytotoxic activity of etoposide in MG63 cells using a colony-formation assay. To study the in vivo efficacy of lysocellin, we isolated a novel compound related to lysocellin from the same streptomycete, and found that the novel drug is converted to lysocellin in vivo and decreases etoposide-induced alopecia in a neonatal rat model. We raise the possibility that this novel drug, named 'alopestatin', may be a promising agent against alopecia.     

A novel synthetic drug, LB-18, closely related to lembehyne-A derived from a marine sponge, induces caspase-independent cell death to human neuroblastoma cells

Neuroblastoma is a common solid tumor of children that arises from the sympathetic nervous system. Much work has consequently focused on the possibility of inducing marked cell death in neuroblastoma, and the new effective drugs are required. We have newly synthesized LB-18, closely related to lembehyne A (LB-A), a polyacetylene derived from a kind of marine sponge. LB-A has been shown to induce p21/WAF1 and causes G1 phase arrest in mouse neuroblastoma Neuro2A cells; however, we show here that LB-18 causes cell death in human neuroblastoma KP-N-TK cells in a dose-dependent manner. TUNEL assay and flow cytometric analysis showed that the cell death caused by LB-18 was associated with the DNA damage but the pan-caspase inhibitor, zVAD-fmk, could not prevent the cell death. Western blot analysis and cleavage of the caspase-3 or -7 substrate assay showed that LB-18 could not activate caspases 3, 7, 8 and 9. These results suggest that LB-18 causes caspase-independent cell death in human neuroblastoma cells. In the future, LB-18 may be useful for cancer therapeutics, especially for neuroblastoma.     

Neuroendocrine cell differentiation of poorly differentiated colorectal adenocarcinoma correlates with liver metastasis

Poorly differentiated (PD) adenocarcinoma often retains the capacity for neuroendocrine (NE) cell differ-entiation; however, it is difficult to distinguish the NE cell differentiation by routine hematoxylin and eosin staining. It is important to detect the presence of NE cell differentiation in advanced colorectal carcinomas because these carcinomas have been shown to produce distant metastasis at the time of diagnosis and to have a particularly poor prognosis. In this study, the characteristics of PD adenocarcinoma with NE cell differentiation and its biological metastatic mechanisms were investigated. Forty-eight of 2204 colorectal cancer patients, diagnosed as having PD adenocarcinoma (2.2%) were enrolled in this study. Immunohistochemical analysis was performed with anti-chromogranin A anti-synaptophysin, anti-CD34, anti-D2-40, and anti-VEGF antibodies. The clinicopathological factors for PD adenocarcinoma with NE cell differentiation were compared with those for PD adenocarcinoma without NE cell differentiation. Microvessel density (MVD) was assessed using immunostained slides with anti-CD34 antibody and vascular endothelial growth factor (VEGF) expression in PD adenocarcinoma with NE cell differentiation was confirmed by in situ hybridization. By immunohistochemical staining for chromogranin A and synaptophysin, NE cell differentiation was detected in eight of 48 patients (16.7%) with PD adenocarcinoma. The frequency of liver metastasis at the time of diagnosis was significantly higher in patients having PD adenocarcinoma with NE cell differentiation (p=0.03). Moreover, MVD and VEGF expression level tended to be higher in patients having PD adenocarcinoma with NE cell differentiation (p=0.13 and 0.068, respectively). NE cell differentiation in PD adenocarcinoma may produce liver metastasis through microvessel formation in the tumor induced by VEGF. In PD colorectal adenocarcinoma, immunohistochemical analysis of NE markers is important for establishing the presence of NE cell differentiation and further study is necessary to evaluate the effectiveness of anti-angiogenic drugs to PD adenocarcinoma with NE cell differentiation.