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Biomedical subjects

Kazuhiko Nakahara

Publications and source records attributed to Kazuhiko Nakahara.

At least 19 recordsLinked to original sources
PubMed2007

Antiproliferative effect on human cancer cell lines after treatment with nimbolide extracted from an edible part of the neem tree (Azadirachta indica).

Nimbolide, a triterpenoid extracted from the flowers of the neem tree (Azadirachta indica), was found to have antiproliferative activity against some cancer cell lines. Treatment of cells with 0.5-5.0 microm concentrations of nimbolide resulted in moderate to very strong growth inhibition in U937, HL-60, THP1 and B16 cell lines. Flow cytometric analysis of U937 cells showed that nimbolide treatment (1-2.5 microm) resulted in cell cycle disruption by decreasing the number of cells in G0/G1 phase, with initial increases in S and G2/M phases. Cells exposed to a higher dose of nimbolide for a longer period displayed a severely damaged DNA profile, resulting in a remarkable increase in the number of cells in the sub-G1 fraction, with a reciprocal decrease of cells in all phases. Quantification of the expression of phosphatidylserine in the outer cell membrane showed that doses of nimbolide higher than 0.4 microm exerted remarkable lethality, with over 60% of cells exhibiting apoptotic features after exposure to 1.2 microm nimbolide. The antiproliferative effect of nimbolide and its apoptosis-inducing property raise hope for its use in anticancer therapy by enhancing the effectiveness of cell cycle disruption.

Antineoplastic Agents, Phytogenic↗
PubMed2006

Inhibition of colon cancer (HT-29) cell proliferation by a triterpenoid isolated from Azadirachta indica is accompanied by cell cycle arrest and up-regulation of p21.

Nimbolide, a natural triterpenoid present in the edible parts of the neem tree ( Azadirachta indica), was found to be growth-inhibitory in human colon carcinoma HT-29 cells. Nimbolide treatment of cells at 2.5 - 10 microM resulted in moderate to very strong growth inhibition. Flow cytometric analysis of HT-29 cells showed that nimbolide treatment (2.5 microM, 12 h) caused a 6.5-fold increase in the number of cells (55.6 %) in the G2/M phase compared with the control cells (8.8 %). At 48 h, the cell population in the G2/M phase decreased to 18 %, while that in the G0/G1 phase increased to 52.3 %. Western blot analysis revealed that nimbolide-mediated G2/M arrest was accompanied by the up-regulation of p21, cyclin D2, Chk2; and down-regulation of cyclin A, cyclin E, Cdk2, Rad17. At G0/G1 cell cycle arrest, modulation in the expression of the cell cycle regulatory molecules was also observed. We found that nimbolide-induced growth inhibition and cell cycle arrest were not associated with cellular differentiation. Quantification of cells with respect to the expression of phosphatidylserine in the outer cell membrane showed an increase in apoptotic cells by about 13 % after 48 h of nimbolide treatment.

Alkaline Phosphatase↗
PubMed2006

Apoptosis, necrosis and cell proliferation-inhibition by cyclosporine A in U937 cells (a human monocytic cell line).

The immunosuppressive drug cyclosporine A (CsA) has been used in both organ transplantation and the treatment of autoimmune disorders. However, the drug causes adverse effects in the kidney, liver and nervous system, characterized by cellular loss in the affected area. Apoptosis has been shown to play a role in CsA-induced cytotoxicity. Because permeabilization of the mitochondrial membrane is a common criterion in most apoptotic settings in vertebrate cells, here we evaluated whether CsA causes loss of mitochondrial function in the pathway leading to cellular cytotoxicity. We found that CsA caused a concentration- and time-dependent loss of cell viability in the U937 cell line. Treatment of cells at a dose of 10 microM CsA resulted in G0/G1 arrest with a concurrent decrease in the number of cells in the S and G2/M phases of the cell cycle. In mechanistic studies related to the loss of viability, treating cells with 10 microM CsA for 24 h resulted in both DNA fragmentation and an increase of annexin-V-positive cells. CsA treatment also increased activity of the cysteine protease caspase-3, decreased the mitochondrial membrane potential and induced the release of cytochrome c into the cytosol. Furthermore, CsA treatment increased the number of cells in the sub-G0/G1 peak, indicative of a reduction in DNA, although this increase was not observed when cells were pre-treated with a broad caspase inhibitor. In the study, we also found that a higher dose of CsA induces LDH release when the cells were incubated for a longer period. Taken together, these data suggest that the mode of cell death induced by CsA is dose- and time-dependent. Short-term incubation with lower doses of CsA arrests cell growth; this arrest overlaps with the occurrence of apoptosis and then with necrosis after longer treatment periods with higher doses of CsA.

Apoptosis↗
PubMed2005

The herbal medicine inchin-ko-to (TJ-135) induces apoptosis in cultured rat hepatic stellate cells.

Use of herbal remedies in the treatment of various diseases has a long tradition in Eastern medicine and the liver diseases are not an exception. In their use, lack of elucidation of mechanism(s) as well as randomized, placebo-controlled clinical trials has been a problem. Recently, we and others reported that inchin-ko-to (TJ-135), one of herbal remedies, suppressed hepatic fibrosis in animal models. In the course of clarifying the mechanism, we directed our focus on hepatic stellate cells (HSCs), playing a pivotal role in hepatic fibrosis, and found that rat HSCs cultured with TJ-135 changed their morphology to star-like configuration with thin, slender and dendritic processes with fewer stress fibers, which might be the features in apoptosis. In fact, TJ-135 induced HSC apoptosis in a time- and concentration-dependent manner as judged by the nuclear morphology, quantitation of cytoplasmic histone-associated DNA oligonucleosome fragments and caspase 3 activity. In HSCs treated with TJ-135, increased expression of p53 and decreased expression of Bcl-2 and phosphorylated Akt and Bad were determined. HSC apoptosis is shown to be involved in the mechanisms of spontaneous resolution of rat hepatic fibrosis and the agent which induces HSC apoptosis has been shown to reduce experimental hepatic fibrosis in rats. Thus, the induction of HSC apoptosis could be the mechanism how TJ-135 works on the resolution of hepatic fibrosis. Our current data may shed light on the novel effect of the herbal remedy.

Animals↗
PubMed2005

Platelet release of beta-thromboglobulin and platelet factor 4 and serotonin in plasma samples.

OBJECTIVES: Platelet release of alpha granule-derived CXC chemokines and dense granule-derived serotonin in plasma samples was evaluated. METHODS: Concentrations of the CXC chemokines beta-TG and PF4 were assayed by an enzyme immunoassay; serotonin was measured by an HPLC method. RESULTS: Beta-TG and PF4 were more easily released than serotonin by in vitro procedures. Use of the anti-platelet cocktail CTAD and preservation of the samples at 4 degrees C were necessary to accurately measure beta-TG and PF4, but not serotonin. CONCLUSIONS: Assaying serotonin may be useful for assessing platelet activation in vivo as a laboratory test because of facile preparation of plasma samples.

Adenosine↗
PubMed2005

Mahanine, a carbazole alkaloid from Micromelum minutum, inhibits cell growth and induces apoptosis in U937 cells through a mitochondrial dependent pathway.

1 Mahanine, a naturally occurring carbazole alkaloid in some Asian vegetables, has been shown to exhibit antimutagenicity, antimicrobial activity, cytotoxicity, and other biological properties. 2 In the present study, we investigated the effect of mahanine on the activation of the apoptotic pathway in human leukemia U937 cells. Various end points were used to screen for apoptosis: Morphological changes in cells, the relative numbers of viable and apoptotic cells; translocation of membrane bound phosphatidylserine and DNA analysis. 3 We found that mahanine-induced apoptosis in U937 cells involved activation of caspases, including caspase-3, release of cytochrome c into cytosol, loss of mitochondrial membrane permeability, and decreased levels of cellular ATP. 4 Pretreatment of cells with cyclosporine A, prior to/concomitant with exposure to mahanine, effectively prevented the deleterious effects of the alkaloid on cellular integrity and viability. 5 As mitochondrial permeability is known to be important in the regulation of cytochrome c release, our observations indicate that mitochondria are the principal target of mahanine. More specifically, we propose that mahanine causes the mitochondrial membranes to lose their permeability, resulting in caspase-3 activation and apoptosis.

Alkaloids↗
PubMed2005

Sphingosine 1-phosphate-related metabolism in the blood vessel.

Sphingosine 1-phosphate (Sph-1-P) is a bioactive lipid released from activated platelets and plays an important role in vascular biology. In this study, we investigated Sph-1-P-related metabolism in the blood vessel, mainly using radio-labeled Sph and Sph-1-P. Sph was metabolically stable in the plasma, while it was converted into Sph-1-P in the presence of activated platelets. When the mixture of Sph-1-P and plasma was fractionated on a gel-filtration column, all Sph-1-P co-eluted with protein fractions that coincide with lipoproteins and albumin by agarose gel electrophoresis. When evaluated by polyacrylamide gel electrophoresis, 7.2 +/- 3.8%, 53.3 +/- 6.4%, and 39.5 +/- 7.9% of the radioactivity of Sph-1-P added to plasma was recovered in the low-density lipoprotein (LDL), high-density lipoprotein (HDL), and albumin fractions, respectively. On the other hand, 5.2 +/- 3.2%, 38.4 +/- 5.5%, and 56.3 +/- 5.7% of the radioactivity of Sph-1-P converted from Sph in collagen-stimulated platelets and released into the plasma was recovered in the LDL, HDL, and albumin fractions, respectively. When Sph-1-P release from activated platelets was examined, a stronger response was observed in the presence of albumin than lipoproteins, suggesting efficient Sph-1-P extraction from platelets by albumin. Finally, Sph-1-P, which is stable in the plasma, was markedly degraded by an ectophosphatase activity in the presence of vascular endothelial cells or in whole blood. Although Sph-1-P is stable in the plasma, it is likely that the level of this bioactive lipid is dynamically controlled by various factors including release from platelets, distribution among plasma proteins, and degradation by ectophosphatase.

Adult↗
PubMed2004

Mechanism of mahanine-induced apoptosis in human leukemia cells (HL-60).

Mahanine, a carbazole alkaloid occurs in the edible part of Micromelum minutum, Murraya koenigii and related species has been found to induce apoptosis in human myeloid cancer cell (HL-60). Concentration of 10 microM mahanine caused a complete inhibition of cell proliferation and the induction of apoptosis in a time dependent manner. Mahanine-induced cell death was characterized with the changes in nuclear morphology, DNA fragmentation, activation of caspase like activities, poly(ADP-ribose) polymerase cleavage, release of cytochrome c into cytosol and stimulation of reactive oxygen species generation. The cell death was completely prevented by a pancaspase inhibitor benzyloxycarbonyl-L-aspart-1-yl-[(2,6-dichlorobenzoyl)oxy]methane (Z-Asp-CH(2)-DCB). Mahanine activated various caspases such as caspase-3, -6, -8 and -9 (like) activities but not caspase-1 like activity. More than 70% cell survival was observed in the presence of a caspase-3 inhibitor. In addition, co-treatment of cyclosporin A markedly increased the survival of mahanine-treated HL-60 cells. Flow cytometric analysis revealed that mahanine decreased the mitochondrial membrane potential of intact cells, and disrupted cell cycle progression by increasing the number of cells in sub-diploid region, concomitantly with the decrease of cells in diploid phases, particularly at late hours of apoptosis. The overall results suggest that mahanine down regulates cell survival factors by activation of caspase-3 through mitochondrial dependent pathway, and disrupts cell cycle progression.

Antineoplastic Agents↗
PubMed2004

Sphingosine 1-phosphate breakdown in platelets.

We examined the formation of sphingolipid mediators in platelets, which abundantly store, and release extracellularly, sphingosine 1-phosphate (Sph-1-P). Challenging [(3)H]Sph-labeled platelet suspensions with thrombin or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a decrease in Sph-1-P formation and an increase in sphingosine (Sph), ceramide (Cer), and sphingomyelin formation. Sph conversion into Cer, and Cer conversion into sphingomyelin were not affected upon activation, suggesting that Sph-1-P dephosphorylation may initiate the formation of sphingolipid signaling molecules. In fact, Sph-1-P phosphatase (but not lyase) activity was detected in platelets, but this activity was not enhanced by thrombin or TPA. When quantified with [(3)H]acetic anhydride acetylation, followed by HPLC separation, the amounts of Sph-1-P and Sph decreased and increased, respectively, upon stimulation with thrombin or TPA, and these changes were attenuated by staurosporine. Under these TPA treatment conditions, over half of the [(3)H]Sph-1-P (formed in platelets incubated with [(3)H]Sph) was detected extracellularly, possibly due to its release from platelets, which was completely inhibited by staurosporine pretreatment. Furthermore, when TPA-induced Sph-1-P release was blocked by staurosporine after the stimulation, the extracellular [(3)H]Sph-1-P radioactivity decreased, suggesting that the Sph-1-P released may undergo dephosphorylation extracellularly. To support this, [(32)P]Sph-1-P, when added extracellularly to platelet suspensions, was rapidly degraded, possibly due to the ecto-phosphatase activity. Our results suggest the presence in anucleate platelets of a transmembrane cycling pathway starting with Sph-1-P dephosphorylation and leading to the formation of other sphingolipid mediators.

Blood Platelets↗
PubMed2004

Frequency distribution of thiopurine S-methyltransferase activity in red blood cells of a healthy Japanese population.

Thiopurine S-methyltransferase (TPMT), which exhibits a genetic polymorphism, plays an important role in the metabolism of thiopurine drugs such as mercaptopurine, thioguanine, and azathioprine. To determine the frequency distribution of TPMT activity in 157 Japanese subjects with different TPMT genotypes, ie, TPMT*1/*1 and TPMT*1/*3, the authors measured levels of 6-methylmercaptopurine formed from 6-mercaptopurine in red blood cells lysates by HPLC. The TPMT activities in our Japanese subjects ranged from 11.0 to 42.6 pmol/h/mgHb. Although the mean value of TPMT activities in 6 subjects with TPMT*1/*3C (20.3 +/- 8.1 pmol/h/mgHb) was 25% lower than that in 151 subjects with TPMT*1/*1 (27.0 +/- 5.1 pmol/h/mgHb), there was overlap. The ranges of TPMT activity in subjects with TPMT*1/*1 and those with TPMT*1/*3C were similar. The median values in TPMT*1/*3C and TPMT*1/*1 individuals were 20.1 (11.0-31.2) and 26.8 pmol/h/mgHb (15.7-42.7), respectively (Mann-Whitney U-test: median difference 6.7 pmol/h/mgHb, 95% CI 0-25.5, P < 0.05). This observation may have relevance for the use of 6-mercaptopurine and azathioprine as therapeutic agents in Japanese patients.

Asian People↗
PubMed2004

Close physical contact of the heart with diaphragm causes pseudo-asynergy of left ventricular inferior wall in normal subjects.

Paradoxical outward movement of left ventricular (LV) inferior wall in systole is occasionally recognized in normal subjects and clinically important in terms of the differential diagnosis between physiological pseudo-asynergy and pathological asynergy. In this study, the potential mechanisms by which pseudo-asynergy of LV inferior wall (PLI) is observed in normal subjects were investigated. PLI was defined as the outward movement of LV inferior wall observed during more than 50% of systole. The incidence of PLI was evaluated in 7843 consecutive subjects in routine echocardiography. The effects of body position and artificial gravity on the manifestation of PLI were also examined. PLI was observed in 0.11% (9/7842) of subjects on left lateral position. Measurement of the angle formed by LV long-axis and the long-axis of the body on frontal plane revealed that hearts in subjects with PLI were in relatively horizontal position. PLI was observed on sitting position in 43% (40/92) of subjects without PLI on left lateral position. The subjects with sitting position-induced PLI exhibited significantly higher obesity index. PLI was also induced by artificial gravity in 67% (14/21) of healthy volunteers on supine position, and the degree of PLI correlated with the intensity of gravity. Although the incidence of PLI in routine echocardiography is relatively low, PLI can be induced in normal subjects by any condition that causes close contact of LV inferior wall to diaphragm. Thus, PLI should be taken into consideration in the differential diagnosis of abnormal LV inferior wall motion, especially when performing exercise echocardiography.

Diaphragm↗
PubMed2004

Venous sampling for fibroblast growth factor-23 confirms preoperative diagnosis of tumor-induced osteomalacia.

Tumor-induced osteomalacia (TIO) is a paraneoplastic disorder characterized by hypophosphatemia, phosphaturia, inappropriately low serum levels of 1,25-dihydroxyvitamin D for hypophosphatemia, and skeletal undermineralization. Patients with TIO suffer from severe muscle weakness and pain. Because surgical removal of the responsible tumors is the only satisfactory treatment for TIO, identification of the tumors is clinically essential. However, because they are predominantly slow-growing neoplasms of benign mesenchymal origin, localization of the responsible tumors is often very difficult. Moreover, even if a tumor is found in a patient with hypophosphatemic osteomalacia, we have had no way to know that the tumor is actually causing the disease. Fibroblast growth factor-23 (FGF-23) was recently identified as a causative factor for TIO and was shown to induce renal phosphate wasting. We have recently shown that the circulatory FGF-23 level was high in a patient with TIO and rapidly decreased after removal of the responsible tumor. For the first time, we describe a patient with adult-onset hypophosphatemic osteomalacia in whom a clinical diagnosis of TIO was confirmed before surgical removal of the tumor by localizing the responsible tumor using venous sampling for FGF-23 together with magnetic resonance imaging. This combinatorial procedure would be clinically useful for sporadic cases of hypophosphatemic rickets/osteomalacia.

Abdominal Neoplasms↗
PubMed2004

[Establishment of assay system for fibroblast growth factor (FGF)-23 and pathophysiological roles of FGF-23 in the development of hypophosphatemic diseases].

Rickets/osteomalacia is characterized by impaired mineralization of bone matrix. X-linked hypophosphatemic rickets/osteomalacia(XLH), autosomal dominant hypophosphatemic rickets/osteomalacia(ADHR) and tumor-induced rickets/osteomalacia(TIO) share common clinical features including impaired proximal tubular phosphate reabsorption. Fibroblast growth factor(FGF)-23 was positionally cloned as a responsible gene for ADHR. We have also cloned FGF-23 as a causative factor for TIO. FGF-23 is a polypeptide with 251 amino acids. FGF-23 is proteolytically cleaved between Arg179 and Ser180 and only full-length FGF-23 showed biological activity to induce hypophosphatemia. Using two monoclonal antibodies that recognize N-terminal and C-terminal portion of the processing site of FGF-23, sandwich enzyme-linked immunosorbent assay for FGF-23 was established. This assay detects only full-length FGF-23. Circulatory level of FGF-23 in healthy controls distributed between 10 to 50 pg/ml suggesting that FGF-23 is a physiological humoral factor. FGF-23 levels in patients with TIO were elevated and rapidly decreased after removal of responsible tumors for TIO. These results confirm that FGF-23 is a causative factor of TIO and indicate that measurement of FGF-23 is useful for diagnosis and management of TIO. Moreover, FGF-23 levels were elevated in most patients with XLH suggesting FGF-23 is also involved in the pathogenesis of XLH. Further analysis is required to clarify more detailed actions of FGF-23 and its roles in the development of other diseases with disordered phosphate metabolism.

Enzyme-Linked Immunosorbent Assay↗